Abstracts from the 57th European Society of Human Genetics (ESHG) Conference: Hybrid Posters (2025)

P01.001.A Hypoxia-mimetic agent DFO inhibits cancer cell proliferation and induces DNA damage

David Diez-Castro ^1;2, Luis Perez-Romasanta³, Rogelio González-Sarmiento^1;2;4, Ana-Belén Herrero^1;2;4

¹Institute of Biomedical Research of Salamanca, Institute of Biomedical Research of Salamanca, Salamanca; ²Faculty of Medicine, Departament of Medicine, Salamanca; ³Hospital University of Salamanca, Oncology service, Salamanca; ⁴Institute of Molecular and Cellular Biology of Cancer (IBMCC), University-CSIC, Salamanca

Background/Objectives: Hypoxia is common in solid tumour microenvironments and has been associated with radiotherapy resistance and poor clinical outcomes. Replicating hypoxic conditions in vitro requires incubation at 1% O₂, 5% CO₂ and 94% N₂. This nitrogen-induced hypoxia is costly and time-consuming since it needs 24 hours to establish hypoxia. An alternative technique to replicate hypoxic conditions is the use of mimetics like deferoxamine (DFO). This molecule is an iron chelating agent that facilitates the accumulation of hypoxia-inducible factors and has been shown to induce apoptosis in breast tumor cell lines¹.

Methods: MCF7 (breast), VCAP (prostate), CAL33 (head and neck) and OVCAR-8 (ovarian) cancer cell lines were used to test the effect of DFO on proliferation using MTT and annexin-V/propidium iodide assays. DNA damage, specifically double strand break (DSB) formation, was tested by γH2AX foci quantification via immunofluorescence assays.

Results: DFO inhibited cell proliferation and apoptosis in a dose- and time-dependent manner. These effects were more marked than the one achieved through nitrogen-induced hypoxia. Additionally, DFO induces DNA damage through DSB formation.

Conclusion: Although DFO is usually used as a hypoxia-mimetic agent because it is cheaper and less time-consuming than nitrogen-induced hypoxia, our results show that it decreases cell proliferation and induces DNA damage through DSB formation. Therefore, its antitumoral effect should be further explored.

Grants: Study was financed by Gerencia Regional de Salud, JCyL (GRS2171/A/2020)

1. 1.

   Lynn, J. V. et al. The Role of Deferoxamine in Irradiated Breast Reconstruction: A Study of Oncologic Safety. Plast. Reconstr. Surg. 143, 1666–1676 (2019).

Conflict of Interest: None declared

P01.002.B Mapping of Splicing Regulatory Elements-rich intervals and identification of spliceogenic variants in ATM exon 7

Inés Llinares-Burguet ¹, Lara Sanoguera-Miralles¹, Elena Bueno-Martínez¹, Alicia García-Álvarez¹, Eladio A. Velasco-Sampedro¹

¹Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Consejo Superior de Investigaciones Científicas – Universidad de Valladolid (CSIC-UVa), Splicing and Genetic Susceptibility to Cancer Laboratory, Valladolid, Spain

Background/Objectives: Alternative splicing regulates gene expression patterns and is controlled by splicing regulatory elements (SRE). Mutations in SREs might affect splicing and be involved in disease susceptibility. Our purpose was to study the alternatively spliced exon 7 of the breast cancer susceptibility gene ATM.

Methods: A minigene with exons 4 to 9 of ATM (MG_ATM_4-9) was used in this work. The enhancer/silencer profile in ATM exon 7 was bioinformatically analysed by HexPlorer to identify the potential regions to study. Selected sequences were deleted in MG_ATM_4-9 by site-directed mutagenesis. In the region with more impact on exon 7 inclusion, we introduced 3 internal overlapping microdeletions. Additionally, we tested all possible variants localised in critical SRE intervals and variants with HexPlorer score below -40 were selected. Thus, 19 variants were introduced into the minigene and functionally tested.

Results: We identified two minimal regions (12/13-nt) involved in exon recognition, located in the 3’-end exon [△(E7): 50-75%]. Of the 19 variants analysed, seventeen (89%) impaired splicing, seven of which had strong impact on splicing, producing exon 7 skipping (59-69%) and <30% of the minigene full-length transcript.

Conclusion: The functional SRE mapping by exonic microdeletions was a useful approach to identify enhancer-rich sequences and SRE variants that impair splicing. The identification of spliceogenic variants is a helpful tool in the genetic counselling consultation, facilitating the early detection and diagnostic of diseases.

Grants: Predoctoral fellowship from the Junta de Castilla y León (2022-2025); ISCIII (PI20/00225 and PI23/00047).

Conflict of Interest: None declared

P01.003.C Rare Germline Variants in glioma: A genomic analysis of 125 individuals from Northern Sweden

Adam Rosenbaum ¹, Anna Dahlin¹, Carl Wibom¹, Beatrice Melin¹

¹Umeå universitet, Department of Diagnostics and Intervention, Umeå, Sweden

Background/Objectives: The etiology of glioma, the most common malignant CNS tumor, remains inadequately understood. Although germline predisposition, including common and rare variants, have been studied, the role of rare germline variants to glioma predisposition remains insufficiently explored.

Methods: We conducted whole genome sequencing on germline samples from 125 individuals from Northern Sweden, diagnosed with various subtypes of glioma. Of these, 46 were diagnosed before 40 years of age, and 12 familial gliomas from 5 families. We studied rare variants in established cancer predisposition genes, as well as a broader investigation of other genes with rare likely pathogenic variants.

Results: Our study reveals 20 rare coding variants in 18 genes, including TP53. Several are likely pathogenic and reside within known cancer predisposition genes, suggesting a link to glioma development. We identified rare variants in genes not previously associated with glioma, providing possible new insights into the etiology of glioma. Comparing the frequency of rare coding mutation among our cases and 300 individuals of similar lineage 7 genes have a significantly increased burden of rare coding variants. These genes are awaiting validation.

Conclusion: Our findings show that the germline genetic predisposition to glioma still holds some explanatory potential to the development of the disease. Continuing studying this aspect of predisposition may unlock new understanding of glioma development.

Grants: Swedish Cancer Society (CAN2018/390 to BM)

Swedish Research Council (2019-01566 to BM)

Umeå University Hospital Grant (7003839 to BM)

Northern Sweden Cancer Foundation (AMP 23-1141 to AR)

Conflict of Interest: None declared

P01.007.C Large genomic rearrangement: tandem duplication and triplication in BRCA1 gene causative for hereditary breast and ovarian cancer

Bernardus Aldrige Allister ¹, Jonathan Lühmann¹, Lena Wendeburg¹, Tim Ripperger¹, Bernd Auber¹, Frank Dechend², Carmela Beger³, Stefanie Tölle³, Julia von Ehr⁴, Nataliya Di Donato¹, Doris Steinemann¹

¹Hannover medical school (MHH), Department of Human Genetics, Hannover, Germany; ²MVZ Reproduction Medicine and Human Genetics, Hildesheim, Germany; ³MVZ Labor Krone GbR, Bad Salzuflen / Bielefeld, Germany; ⁴Frauenärzte am Schloss, Wolfenbüttel, Germany

Background/Objectives: Large genomic rearrangements (LGRs) include copy number variations like duplications or triplications of coding or non-coding genomic regions within the human genome. Here, we report on two LGRs in two families with the suspicion of hereditary breast and/or ovarian cancer (HBOC) syndrome, a pathogenic BRCA1 tandem duplication targeting exon 18-19 and a for the first time a pathogenic BRCA1 tandem triplication of exon 2.

Methods: MLPA, Optical Genome Mapping (OGM), cDNA analysis and Sanger sequencing have been performed to identify these rearrangements and to characterize their localization and orientation as well as to predict their pathogenicity for HBOC development.

Results: We show that the duplication of exon 18-19 in BRCA1, is a tandem duplication: ogm[GRCh38] dup(17)(q21.31q21.31)(43057052_43063373). Validation by sanger sequencing of the cDNA shows that this tandem duplication of exon 18-19 generate a premature termination codon at the second codon of the duplicated exon 18. In the second index patient, four copies of the BRCA1 promotor region, exon 1a,1b and exon 2 were detected via MLPA and OGM enabled us to resolve this LGR as a BRCA1 exon 2 tandem triplication: ogm[GRCh38] trip(17)(q21.31q21.31)(43117155_43124115). cDNA analysis shows that this LGR causes an inclusion of a portion of intron 2 sequence and provokes a premature termination codon exactly at the junction between exon 2 and the intronic sequence of intron 2.

Conclusion: Due to a predicted premature termination codon we classify both LGRs as pathogenic variants. This signifies that LGRs such as tandem duplications and triplications can be causative for HBOC.

Grants:

Conflict of Interest: None declared

P01.008.D Atypical cancer risk profile in carriers of Italian founder BRCA1 variant p.His1673del: implications for classification and clinical management

Giovanni Innella ^1;2, Cristina Fortuno³, Laura Caleca⁴, Bing-Jian Feng⁵, Courtney Carroll⁵, Michael T. Parsons³, Sara Miccoli², Marco Montagna⁶, Daniele Calistri⁷, Laura Cortesi⁸, Barbara Pasini⁹, Siranoush Manoukian¹⁰, Daniela Giachino¹¹, Laura Matricardi⁶, Maria Cristina Foti⁶, Valentina Zampiga⁷, Claudia Piombino⁸, Elena Barbieri⁸, Francesca Vignolo⁹, Jacopo Azzolini¹⁰, Rita Danesi¹², Valentina Arcangeli¹², Sandrine Caputo¹³, Nadia Boutry-Kryza¹⁴, Vincent Goussot¹⁵, Susan Hiraki¹⁶, Marcy Richardson¹⁷, Simona Ferrari², Paolo Radice⁴, Amanda B. Spurdle³, Daniela Turchetti^1;2

¹Università di Bologna, Dipartimento di Scienze Mediche e Chirurgiche, Bologna, Italy; ²2.IRCCS Azienda Ospedaliero-Universitaria di Bologna, Medical Genetics Unit, Bologna, Italy; ³QIMR Berghofer Medical Research Institute, Population Health, Brisbane, Australia; ⁴Fondazione IRCCS Istituto Nazionale dei Tumori, Unit of Predictive Medicine: Molecular Bases of Genetic Risk, Department of Experimental Oncology, Milano, Italy; ⁵University of Utah, Salt Lake City, United States; ⁶Veneto Institute of Oncology IOV - IRCCS, Immunology and Molecular Oncology Unit, Padova, Italy; ⁷IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” IRST, SC Laboratorio di Bioscienze, Meldola, Italy; ⁸Centro Oncologico Modenese, Azienda Ospedaliero-Universitaria Policlinico di Modena, Dipartimento di Oncologia ed Ematologia, Modena, Italy; ⁹Azienda Ospedaliero-Universitaria Città della Salute e della Scienza di Torino, SC Genetica Medica U, Torino, Italy; ¹⁰Fondazione IRCCS Istituto Nazionale dei Tumori, Unit of Medical Genetics, Department of Medical Oncology and Hematology, Milano, Italy; ¹¹Azienda Ospedaliero-Universitaria San Luigi Gonzaga, Regione Gonzole, Counselling Genetico, Orbassano, Italy; ¹²IRCCS Istituto Romagnolo per lo Studio dei Tumori “Dino Amadori” IRST, SC Epidemiologia Clinica e Sperimentale, Meldola, Italy; ¹³, Institut Curie, Paris, France and Paris Sciences Lettres Research University, Department of Genetics, Paris, France; ¹⁴Hospices Civils de Lyon, Service de génétique, Lyon, France; ¹⁵Centre de Lutte Contre le Cancer Georges François Leclerc, Département de Biologie et Pathologie des Tumeurs, Dijon, France; ¹⁶GeneDx, Gaithersburg, United States; ¹⁷Ambry Genetics, Aliso Viejo, United States

Background/Objectives: BRCA1:c.5017_5019del(p.His1673del) is a founder variant relatively frequent in Northern Italy. Despite previous suggestion of pathogenicity, variant classification in public databases is still conflicting, needing additional evidence.

Methods: Maximum likelihood penetrance of breast/ovarian and other cancer types was estimated using full pedigree data from 53 informative Italian families. The effect of the variant on BRCA1-ABRAXAS1 interaction was assessed using a GFP-fragment reassembly-based PPI assay. Results were combined with additional data from multiple sources to classify the variant according to ACMG/AMP classification rules specified for BRCA1/2.

Results: Variant-carriers displayed increased risk for ovarian cancer (HR = 33.0, 95%CI = 7.0-155.0; cumulative risk at age 70 = 27.6%, 95%CI = 12.6-40.0%) but not for breast cancer (HR = 0.7, 95%CI = 0.2-2.2). An increased risk of uterine cancer (HR = 8.0, 95%CI = 1.03-61.6) emerged, warranting further evaluation. Likelihood ratio in favor of pathogenicity was 98898642.82 under assumption of standard BRCA1 breast and ovarian penetrance, and 104240832.84 after excluding breast cancer diagnoses (based on penetrance results). Functional analysis demonstrated that the variant abrogates the BRCA1-ABRAXAS1 binding, supporting the PS3 code assignment within the ACMG/AMP rule-based model. Collectively, these findings allowed to classify the variant as pathogenic.

Conclusion: Pathogenicity of BRCA1:c.5017_5019del(p.His1673del) has been confirmed; however, breast cancer risk in Italian families is not increased, unlike in families from other countries and in carriers of most BRCA1 pathogenic variants. Haplotype analysis is underway to explore the hypothesis of potential in-cis modifiers. Nevertheless, the knowledge of atypical risk profiles for this and other variants will pave the way for personalized management based on specific genotype.

Grants:

Conflict of Interest: Giovanni Innella: None declared, Cristina Fortuno National Breast Cancer Foundation, Australia (IIRS-21-102), Laura Caleca Italian Association for Cancer Research (AIRC; IG22093), Bing-Jian Feng: None declared, Courtney Carroll: None declared, Michael T. Parsons National Institutes of Health grant U24 5U24CA258058-02, Sara Miccoli: None declared, Marco Montagna: None declared, Daniele Calistri: None declared, Laura Cortesi: None declared, Barbara Pasini: None declared, Siranoush Manoukian: None declared, Daniela Giachino: None declared, Laura Matricardi: None declared, Maria Cristina Foti: None declared, Valentina Zampiga: None declared, Claudia Piombino: None declared, Elena Barbieri: None declared, Francesca Vignolo: None declared, Jacopo Azzolini: None declared, Rita Danesi: None declared, Valentina Arcangeli: None declared, Sandrine Caputo: None declared, Nadia Boutry-Kryza: None declared, Vincent Goussot: None declared, Susan Hiraki: None declared, Marcy Richardson: None declared, Simona Ferrari: None declared, Paolo Radice Italian Association for Cancer Research (AIRC; IG22093), Amanda B. Spurdle NHMRC Investigator Fellowship (APP177524), Daniela Turchetti: None declared

P01.009.A Further elucidating the genetic landscape of glioma predisposition in a European familial glioma cohort

Frank Brand ¹, Amir H. Akbarzadeh¹, Christine A. M. Weber¹, Lily S. Rose¹, Robert Geffers², Gunnar Schmidt¹, Bernd Auber¹, Michael Friese³, Mareike Müller⁴, Michael Lalk⁵, Isabel Eckert⁶, Arya Nabavi⁵, Paul Kremer⁷, Amir Samii⁸, Guido Reifenberger^9;10, Joachim K. Krauss¹¹, Bettina Wiese^6;11, Christian Hartmann¹², Ruthild G. Weber¹

¹Hannover Medical School, Department of Human Genetics, Hannover, Germany; ²Helmholtz Centre for Infection Research, Genome Analytics Research Group, Braunschweig, Germany; ³Asklepios Klinik Nord - Heidberg, Department of Pathology and Neuropathology, Hamburg, Germany; ⁴Heinrich Heine University, Medical Faculty, Department of Neurosurgery, Düsseldorf, Germany; ⁵KRH Klinikum Nordstadt, Department of Neurosurgery, Hannover, Germany; ⁶Diakovere Krankenhaus gGmbH, Henriettenstift, Department of Neurology, Hannover, Germany; ⁷Asklepios Klinik Nord - Heidberg, Department of Neurosurgery, Hamburg, Germany; ⁸International Neuroscience Institute, Department of Neurosurgery, Hannover, Germany; ⁹Heinrich Heine University, Medical Faculty, Institute of Neuropathology, Düsseldorf, Germany; ¹⁰German Cancer Consortium (DKTK), Partner Site Essen/Düsseldorf and German Cancer Research Center (DKFZ), Heidelberg, Germany; ¹¹Hannover Medical School, Department of Neurosurgery, Hannover, Germany; ¹²Hannover Medical School, Institute of Pathology, Department of Neuropathology, Hannover, Germany

Background/Objectives: Familial occurrence of glioma, the most common type of malignant brain tumor, is observed in about 5% of cases. Studies on the genetic landscape of glioma predisposition are still scarce.

Methods: Leukocyte DNA of 122 glioma patients from 115 tumor families with at least one glioma patient each were analyzed by whole-exome sequencing. Data were analyzed using two approaches: (1) variants in established cancer predisposition genes (CPGs) or suspected glioma risk genes (n = 154) were extracted and classified, (2) the frequency of rare (MAF < 0.01%) or deleterious variants in all genes in the glioma cohort was compared with that in a control cohort (n = 257 families). Exome data of >70 additional patients are pending and will be included in our analysis.

Results: Both approaches associated BRCA2 variants, including the pathogenic variants c.316+5G > C, p.(K944*), p.(I1470Kfs*11), most strongly with familial glioma. BRCA2 variants were significantly more frequent in the glioma than in the control cohort (P = 0.0088). In addition, rare deleterious variants in APC, ATM, and EGFR were recurrently observed in the glioma cohort. In agreement with Choi et al., Sci. Adv., 2023, our familial glioma cases carried rare deleterious variants in known CPGs, e.g. ATM, PMS2, and POLE, and novel CPGs, e.g. SLC4A7 and WDR7. Furthermore, we identified 31 genes, not previously associated with cancer predisposition, that were recurrently affected in the glioma but not at all in the control cohort.

Conclusion: Our study on a large European familial glioma cohort implicates known CPGs, particularly BRCA2 and ATM, and novel CPGs in glioma risk.

Conflict of Interest: None declared

P01.010.B Analysis of aberrant splicing in capture RNA-seq data as a supplement to DNA germline testing to increase diagnostic yield

Florentine Scharf¹, Thomas Keßler¹, Evgenia Vibe ¹, Oliver Klaas¹, Jonas Ingermann¹, Caroline Heintz¹, Anna Benet-Pages^1;2, Sabrina Angerbauer¹, Martin Wendlandt¹, Tobias Wohlfrom¹, Verena Steinke-Lange^1;3, Andreas Laner¹, Ariane Hallermayr^1;3, Julia Romic-Pickl^1;3, Morghan Lucas^1;3, Elke Holinski-Feder^1;3

¹MGZ – Medizinisch Genetisches Zentrum, Munich, Germany; ²Institute of Neurogenomics, Helmholtz Research Center, München, Germany; ³Medizinische Klinik und Poliklinik IV, Campus Innenstadt, Klinikum der Universität München

Background/Objectives: Hereditary tumor syndromes comprise 5-10% of cancer cases, necessitating accurate identification of causal genetic variants for effective patient management. The diagnostic yield of whole-exome sequencing (WES), the gold standard for molecular diagnostics, ranges from 15-25% in these syndromes. High-throughput functional studies, such as RNA sequencing (RNA-seq), play a crucial role in reclassifying variants of uncertain significance (VUS) and unveiling pathomechanisms like aberrant splicing, particularly when initial variant detection fails.

Methods: We developed a cost-efficient, high-throughput RNA-seq approach to analyze RNA phenotypes where we complement polyA mRNA or hexamer capture with enrichment of 49 cancer-associated genes from PAXgene-derived RNA samples.

Results: We achieved ultra-high coverage sequencing data of ~3,500X mean target coverage and, on average, 80% of exons covered with >50 read depth using hexamer capture and target enrichment. In almost 80% of positive controls for aberrant splicing, we detected a difference of >20% in the PSI (percent-spliced in) score compared to negative controls.

Conclusion: Our workflow provides high-quality RNA-seq data, which allows the assessment of splicing events. Our data on the comparisons between positive and negative controls substantiate the possibility of an automated high throughput pipeline. This work represents the basis for implementing targeted RNA-seq in cancer diagnostics, in which we want to establish the parallel analysis of WES and RNA-seq to increase the diagnostic yield and turnaround time for patients with hereditary tumor syndromes.

Grants: N/A

Conflict of Interest: None declared

P01.011.C Identification of shared genetic variants among different cancers and between adenomyosis and cancer utilizing members of the same family

Sevcan Aydın ¹, nura fitnat topbas selcuki², pinar yalcin bahat³, engin oral⁴, Feyza Tuncer⁵

¹Istanbul University, Graduate School of Health Sciences, Istanbul, Türkyie; ²University of Health Sciences Turkey, Istanbul Sisli Hamidiye Etfal Training and Research Hospital, Obstetrics and Gynecology, Istanbul, Türkyie; ³University of Health Sciences Turkey, Istanbul Kanuni Sultan Suleyman Training and Research Hospital, Department, Obstetrics and Gynecology, Istanbul, Türkyie; ⁴Biruni University Faculty of Medicine, Obstetrics and Gynecology, Istanbul, Türkyie; ⁵Istanbul University, Aziz Sancar Institute of Experimental Medicine, Genetics, Istanbul, Türkyie

Background/Objectıves: Adenomyosis is a benign uterine condition characterized by the localization of endometrial-like tissue in myometrium. Its pathogenesis remains elusive, but it carries cancer-like characteristics. We aimed to identify shared genetic variants both between different cancers and adenomyosis and cancer, utilizing a family having women affected by both conditions.

Methods: Five women from the same family were subjected to gynecological examinations via transvaginal ultrasonography, amongst two receieved pathologically confirmed diagnosis of ovarian mucinous adenocarcinoma and clear cell renal cell carcinoma (ccRCC). Whole exome sequencing was performed on all women utilizing Illumina NextSeq550. Bioinformatic analyses were performed on Genomize SEQ and Pairend platforms, MAF < %1 was applied to identify rare and novel variants. Cases with cancer versus all cases were evaluated in parallel, for which one member was utilized as a control, who manifested neither of the conditions.

Result: Pathological evaluations excluded adenomyosis in the case diagnosed with ovarian mucinous adenocarcinoma, while the case with ccRCC received adenomyosis diagnosis. A novel genetic variant in NTN1 gene was identified across all cases, in addition to rare variants in 10 genes shared among cases with cancers.

Conclusıon: NTN1 gene might be one of the many links between the pathogenesis of cancer and adenomyosis, fitting in with its attributed roles in cell migration and angiogenesis. The impact of the novel variant in NTN1 merits clarification functionally. Further studies are in need to elucidate the diversifying mechanisms among two similar conditions evolving towards either malignity or benignity.

Grant Reference:Istanbul University Scientific Research Projects Coordination Unit(TSA-2023-38950)

Conflict of Interest: None declared

P01.012.D Reproductive factors and sex hormones levels on differentiated thyroid cancer risk: A Mendelian Randomization study

See Hyun PARK ^1;2, Mojgan Karimi², Cloe Domenighetti², Thérèse Truong²

¹Paris Saclay Universite, Gif-sur-Yvette, France; ²INSERM, Exposome and Heredity, Villejuif

Consortium: EPITHYR consortium, and The European Prospective Investigation into Cancer and Nutrition (EPIC-Europe)

Background: Differentiated thyroid carcinoma (DTC) is the most common type of thyroid cancer, occurring three times more frequently in women than in men. However, the underlying biological mechanisms driving this sex-specific discrepancy remain poorly understood. To analyze the causal role of sex hormones and reproductive factors in the risk of DTC, we implemented a two-sample Mendelian Randomization (MR) analysis using genome-wide association studies (GWAS) summary statistics.

Methods: GWAS on DTC were derived from a meta-analysis of 6 studies including 7,705 cases and 963,612 controls of European population, while GWAS summary statistics on sex hormones, reproductive factors, and gynecological factors related to hysterectomy were retrieved from publicly available literature. We used the inverse-variance weighted method to estimate odds ratio, with additional multiple sensitivity analyses to ensure the suitability of the MR analysis, and conducted multivariable MR to account for potential confounding variables.

Results: We showed that endometrial cancer was associated with DTC (OR = 1.15, p = 9 × 10^-3). We also observed an association between SHBG levels and increased risk of DTC, however this association lost significance upon adjustment for obesity-related factors. Putative causal associations was observed with uterine fibroids; nonetheless, this association was not supported by additional sensitivity analyses. No significant associations were found for levels of sex hormones, age at menopause, menarche and first birth, nor with endometriosis.

Conclusion: Using the largest GWAS on DTC to date, our findings do not support a significant influence of sex hormones and reproductive factors in the observed differences in DTC risk between women and men.

Grants: Funded by doctoral school of Université Paris-Saclay

Conflict of Interest: None declared

P01.013.A Evolution of genomic profiles in primary and recurrent astrocytic gliomas

Libuse Lizcova ¹, Halka Lhotska¹, Karolina Janeckova¹, Lucie Hodanova¹, Karla Svobodova¹, Hana Cechova², Tatiana Aghova¹, Sarka Ransdorfova², Lenka Pavlistova¹, Filip Kramar³, David Netuka³, Zuzana Zemanova¹

¹General University Hospital and First Faculty of Medicine, Charles University in Prague, Center of Oncocytogenomics,Institute of Medical Biochemistry and Laboratory Diagnostics, Prague, Czech Republic; ²Institute of Hematology and Blood Transfusion, Prague, Czech Republic; ³First Faculty of Medicine of Charles University and Military University Hospital Prague, Department of Neurosurgery and Neurooncology, Prague

Background/Objectives: Astrocytic gliomas, including grades 2-4 astrocytoma and grade 4 glioblastoma, are characterized by diverse biological behaviors and recurrent lesions. During disease progression, these tumors undergo genomic evolution with newly acquired genetic properties. However, the mechanisms and evolutionary dynamics underlying tumor progression and relapse remain poorly understood.

Methods: Genomic profiles of 35 paired glioma samples (primary and recurrent; 15x astrocytoma, 20x glioblastoma) were analyzed using combination of cytogenomic methods: I-FISH (Abbott Molecular, MetaSystems), aCGH/SNP (Agilent), MLPA, MS-MLPA (MRC-Holland) and targeted NGS panel (Invitae).

Results: Primary tumors displayed common aberrations like IDH1/IDH2 and TERT mutations, CNAs of chromosome 7, 10, and CDKN2A/2B and EGFR genes, persisting in recurrent lesions. All but two patients experienced recurrences with newly acquired genetic/epigenetic changes with a high frequency of CNAs leading to complex rearrangements. Divergent clonal evolution was observed in 22 cases, while 11 cases displayed linear evolution. Aberrations of chromosomal regions 4p, 6p, 8q, 9q, 11p and 11q were recurrently identified in recurrences. cnLOH was proved in 22 cases, mainly on 7p and 17p. Disease progression occurred in 28 patients.

Conclusions: This study highlights heterogeneity in tumor evolution driven by genomic/microenvironmental imbalances or treatment. Recurrence may arise from major tumor clone or multiple subclones within the primary tumor. Cytogenomic analyses of recurrent tissues contribute to understanding these processes and identification of alterations associated with glioma progression. These biomarkers could subsequently serve as resource for precision oncology targeting cancer dynamics in astrocytic gliomas.

Grants: MH CZ AZV-NU21-04-00100, MH CZ-DRO-0064165 and GAUK 159020.

Conflict of Interest: None declared

P01.014.B PAX5 alterations in a consecutive childhood B-ALL cohort treated on ALL IC-BFM 2009 protocol

Klementina Črepinšek ^1;2, Janez Jazbec^2;3, Marko Kavčič^2;3, Tomaž Prelog³, Tine Tesovnik^1;2, Barbara Jenko Bizjan^1;2, Robert Sket^1;2, Jernej Kovač^1;2, Marusa Debeljak^1;2

¹University Children’s Hospital, UMC Ljubljana, Clinical Institute for Special Laboratory Diagnostics, Ljubljana, Slovenia; ²University of Ljubljana, Faculty of Medicine, Ljubljana; ³University Children’s Hospital, UMC Ljubljana, Department of Oncology and Haematology, Ljubljana

Background/Objectives: In this study, we aimed to identify patients within our B-ALL cohort with altered PAX5. Our objective was to characterize the types of genetic changes, determine their origin (somatic/germline), and analyze the clinical outcomes associated with them.

Methods: A consecutive cohort of 99 patients with B-ALL treated at the Children’s Hospital of the UMC Ljubljana according to the ALL IC-BFM 2009 protocol was included in our study. We used RNA sequencing data for gene expression analysis, fusion gene detection and variant identification, multiplex-ligation dependent probe amplification for copy number variation assessment, and Sanger sequencing to detect germline variants.

Results: PAX5 was impacted in a 33.3% of our patients, with the genetic alterations ranging from CNVs and rearrangements to SNVs. The most common were CNVs, which were found in 33.7% of patients, followed by point mutations in 5.2% and gene rearrangements in 4.1%. We identified 8 patients with a PAX5-associated genetic subtypes that were previously classified as “B-other”, and they showed intermediate outcomes. We showed a trend of poorer survival in hyperdiploid cases carrying duplications in PAX5 compared to other hyperdiploid cases. We also report an interesting case of a patient with PAX5::FKBP15 and a pathogenic variant in PTPN11 who underwent an early relapse with a monocytic switch.

Conclusion: In conclusion, this study provides valuable insights into the presence, frequency, and prognostic significance of diverse PAX5 alterations in B-ALL patients, highlighting the complexity of genetic factors and their impact on patient outcomes.

Grants: Young Research Fellowship SRA#54654, UMC-tertiary grants: TP20230039

Conflict of Interest: None declared

P01.015.C Rare mutations in FH and POLE genes in a patient with Ewing sarcoma and familial cancer burden

Dimitar Bakalov ¹, Svilen Maslyankov², Kalina Belemezova³, Radka Tafradjiiska-Hadjiolova¹, Zafer Sabit¹, Ivanka Dimova⁴

¹Medical University of Sofia, Department of Physiology and Pathophysiology, Sofia, Bulgaria; ²Medical University of Sofia, Department of Surgery, Sofia, Bulgaria; ³Medical University of Sofia, Department of Medical Biology, Sofia, Bulgaria; ⁴Medical University of Sofia, Department of Medical Genetics, Sofia, Bulgaria

Background/objectives: Ewing sarcoma is a rare and aggressive form of bone and soft tissue cancer, predominantly affecting young individuals. We explore the underlying genetic factors contributing to Ewing sarcoma in a 22-year-old patient, having familial cancer burden (colon, kidney, breast, bladder, melanoma).

Methods: Whole exome sequencing was performed on genomic DNA from the affected individual. Comprehensive bioinformatic analyses were employed to prioritize and validate variants, with a particular focus on genes implicated in cancer susceptibility.

Results: The analysis revealed two very rare heterozygous missense variants of unknown significance: c.809 A > G (p.Tyr270Cys) in the FH gene (frequency of 3e-5) and c.1015 G > A (p.Asp339Asn) in POLE gene (frequency of 8e-5) in the patient’s germline. FH encodes fumarate hydratase, a key enzyme in the tricarboxylic acid cycle, which is associated with autosomal dominant “Leiomyomatosis and renal cancer” phenotype. POLE is essential for DNA replication and repair, and is associated with autosomal dominant “Colorectal cancer, succeptibility to, 12” phenotype.

Conclusion: This study contributes to the growing body of knowledge surrounding the genetic landscape of Ewing sarcoma, highlighting the role of FH and POLE gene variants. Additionally, the severe family history of colon, urothelial, breast and skin cancers in first- and second-degree relatives raises intriguing questions about shared genetic predisposition. Further investigations into the potential synergistic effects of these variants and their implications for familial cancer risk are warranted. This case underscores the importance of genetic counseling in individuals with Ewing sarcoma and multidisciplinary approach to personalized cancer care.

Grant: Project № BG-RRP-2.004-0004-C01

Conflict of Interest: None declared

P01.016.D Liquid biopsy integrating machine learning for prostate cancer detection

Ondrej Pös ^1;2, Zuzana Hanzlikova^2;3, Jaroslav Budiš^1;2;4, Jakub Styk^1;2, Matej Hrnčiar², Werner Krampl^1;2;5, Tomáš Sládeček^1;2, Jozef Sitarcik^1;2, Pavol Misenko², Silvia Bokorova^1;2, Diana Rusnakova^1;2, Lydia Lukyova^1;5, Monika Kubanova¹, Terezia Duranova¹, Tatiana Sedlackova^1;2, Tomas Szemes^1;2;5

¹Comenius University Science Park, Bratislava, Slovakia; ²Geneton Ltd., Bratislava, Slovakia; ³Faculty of Informatics and Information Technologies, Slovak University of Technology, Bratislava, Slovakia; ⁴Slovak Center of Scientific and Technical Information, Bratislava, Slovakia; ⁵Faculty of Natural Sciences, Comenius University, Department of Molecular Biology, Bratislava, Slovakia

Background/Objectives: Early-stage cancer manifests with minimal or no noticeable symptoms, leading to a diagnostic delay and subsequent less effective treatment. Since analysis of cell-free DNA (cfDNA) may serve to identify early neoplastic changes, liquid biopsy-based tests hold potential for non-invasive cancer screening.

Methods: The plasma of 60 prostate cancer patients and 369 control individuals were collected to analyze cfDNA by low-coverage whole-genome next-generation sequencing. Data were processed using an in-house bioinformatics pipeline to detect genetic variability and characterize qualitative and quantitative sequencing metrics. A total of 379 samples (49 patients, 330 controls) were used to train the model from decision trees using gradient boosting to facilitate prostate cancer prediction. Subsequently, a testing set of 50 samples (11 patients, 39 control) was used to assess the accuracy of the model.

Results: The robust combination and evaluation of 671 attributes, including genomic variability and sequencing metrics, by ensemble learning model, have shown a sensitivity of 81.8% and specificity of 100.0% on the testing dataset. The ROC AUC of 97.4% suggests a high prediction capability of the test.

Conclusion: Here, we emphasize the decent potential of liquid biopsy test enhanced by artificial intelligence to evaluate an extensive set of cfDNA sequencing metrics and genomic attributes for non-invasive prostate cancer screening.

Grants: The work was supported by the OPII project ITMS: 313010Q927 (GenoScan LBquant), co-financed by ERDF. Support was also provided by Horizon Europe Framework Programme Grant agreement ID: 101087124 (ADDIT-CE) and by Slovak Research and Development Agency grants APVV-21-0296 (INCAM) and APVV-20-0472 (Sepmin).

Conflict of Interest: Ondrej Pös The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Zuzana Hanzlikova The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Jaroslav Budiš The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Jakub Styk The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Matej Hrnčiar The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Werner Krampl The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Tomáš Sládeček The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Jozef Sitarcik The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Pavol Misenko The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Silvia Bokorova The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Diana Rusnakova The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Lydia Lukyova: None declared, Monika Kubanova: None declared, Terezia Duranova: None declared, Tatiana Sedlackova The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company., Tomas Szemes The author is an employee of Geneton Ltd. and is involved in numerous research and development efforts to adapt novel approaches to liquid biopsy screening applications. However, the employee does not hold any financial interest in the company.

P01.017.A Development and validation of an expanded comprehensive genomic profiling assay with enhanced variant sensitivity for tumor biopsies

Stephanie Constantinou ¹, Alexia Eliades¹, Kyriakos Tsangaras¹, Chrysa Soteriou¹, chrystalla lazarou¹, Achilleas Achilleos¹, charalambos loizides¹, Christos Lemesios¹, Chrystalla Havadjia¹, sarah barbour¹, aris pallaris¹, chrysovalando soteriou¹, Louisa Constantinou¹, Haris Kkoufou¹, Michalis Spyrou¹, antonis antoniou¹, Antonia Matsentidou¹, Melina Vaki¹, george manoli¹, Christos Prokopi¹, Styliana Georgiou¹, Elena Kypri¹, Marios Ioannides¹, George Koumbaris¹, Philippos Patsalis^1;2

¹Medicover Genetics, Nicosia, Cyprus; ²University of Nicosia Medical School, Department of Basic and Clinical Sciences, Nicosia, Cyprus

Background/Objectives: Comprehensive genomic profiling using next-generation sequencing (NGS) has become increasingly important in the classification and management of cancer, enabling a growing number of patients to benefit from targeted therapies. Such approved therapies include ones developed for less prevalent gene alterations, thus emphasising the need for assessing a broader panel of genes for multiple alteration classes with an enhanced sensitivity. Herein, we describe the development of an expanded 392-gene panel, offering high analytical performance.

Methods: DNA isolated from a set of formalin-fixed, paraffin-embedded (FFPE) tissue samples comprising reference materials, non-malignant and tumor samples was subjected to library preparation and hybrid capture enrichment with our expanded 392-gene panel. Enriched libraries were subsequently sequenced by NGS on a NovaSeq platform and data was analysed using proprietary bioinformatic pipelines.

Results: A set of FFPE samples comprising benign, malignant and reference material samples, was used to evaluate the assay’s analytical performance. The assay demonstrated high sensitivity and specificity for all classes of genomic alterations, including single nucleotide variants (SNVs), small insertions and deletions (Indels), copy number alterations (CNAs), translocations, as well as the immuno-oncology biomarkers, microsatellite instability (MSI) and tumor mutational burden (TMB). Notably, the developed assay can detect mutations down to 1% variant allele frequency (VAF).

Conclusion: Validation results demonstrate the development of a tissue-based NGS assay for the assessment of an expanded panel of genomic alterations and therapy-associated biomarkers that can detect less prevalent variants with enhanced sensitivity.

Conflict of Interest: Stephanie Constantinou Author employed by Medicover Genetics, Alexia Eliades Author employed by Medicover Genetics, Kyriakos Tsangaras Author employed by Medicover Genetics, Chrysa Soteriou Author employed by Medicover Genetics, chrystalla lazarou Author employed by Medicover Genetics, Achilleas Achilleos Author employed by Medicover Genetics, charalambos loizides Author employed by Medicover Genetics, Christos Lemesios Author employed by Medicover Genetics, Chrystalla Havadjia Author employed by Medicover Genetics, sarah barbour Author employed by Medicover Genetics, aris pallaris Author employed by Medicover Genetics, chrysovalando soteriou Author employed by Medicover Genetics, Louisa Constantinou Author employed by Medicover Genetics, Haris Kkoufou Author employed by Medicover Genetics, Michalis Spyrou Author employed by Medicover Genetics, antonis antoniou Author employed by Medicover Genetics, Antonia Matsentidou Author employed by Medicover Genetics, Melina Vaki Author employed by Medicover Genetics, george manoli Author employed by Medicover Genetics, Christos Prokopi Author employed by Medicover Genetics, Styliana Georgiou Author employed by Medicover Genetics, Elena Kypri Author employed by Medicover Genetics, Marios Ioannides Author employed by Medicover Genetics, George Koumbaris Author employed by Medicover Genetics, Philippos Patsalis Author employed by Medicover Genetics

P01.019.C Increased prevalence of pathogenic and likely pathogenic CHEK2 variants in the Balearic Islands Hereditary Cancer cohorts.

Maria Antònia Caro-Miró ¹, Catalina Lladó-Sampol², Antònia Perelló-Martorell², Evelin Horvath², Paloma de la Torre-Rubio³, DAMIAN HEINE SUÑER¹, Victor Asensio-Landa¹, Laura Torres-Juan¹, María Carmen Prado-Farnos¹, Susana Renee Avella-Klaassen¹, Iciar Martínez¹, Jesús Alarcón Company², Antonia Obrador-Hevia¹

¹Hospital Universitari Son Espases, Molecular Diagnosis and Clinical Genetics Unit, Palma, Spain; ²Hospital Universitari Son Espases, Oncology, Palma, Spain; ³Hospital Universitari Son Espases, Digestology, Palma, Spain

Background/Objectives: CHEK2 has been identified as an intermediate-risk gene associated with breast, prostate, and colon cancer (OMIM: 609265).

In this study, we examined the frequency and mutational spectrum of CHEK2 variants within the Balearic Islands’ hereditary cancer (HC) cohort, comparing it to the Catalonian population databases in order to investigate potential founder mutations.

Methods: The research assessed the prevalence of pathogenic/likely pathogenic (P/LP) CHEK2 variants and included two Balearic Island cohorts: 1559 patients from the HC cohort sequenced by the TruSight Hereditary Cancer panel (Illumina®) between 2016 and 2022, and 3352 non-HC patients sequenced via Whole Exome Sequencing (WES) (Illumina®). Frequencies from the genetically closest population, Catalonia, were used for comparison.

Results:

Conclusion: The prevalence of P/LP variants in the HC cohort was found to be 2.2% (2.2% in BOP cancer patients and 2.4% in colorectal cancer patients), significantly higher than the genetically closest population of Catalonia. This possibly suggests the presence of a founder effect of c.1427C > T and c.279G > A variants, enriched in Mallorca and Ibiza patients, respectively.

Grants:

Conflict of Interest: None declared

P01.020.D Deciphering the Gene Expression Signature of High-Grade B-cell Lymphomas with 11q Aberrations

Emil Chteinberg ¹, Gioia Di Stefano², Henry Löffler-Wirth³, Annette M. Staiger^4;5, Sina Hillebrecht¹, Rabea Wagener¹, Susanne Bens¹, Kathrin Oehl-Huber¹, Sonja Dahlum¹, Dmitriy Abramov⁶, Birgit Burkhardt⁷, Abner Louissaint,Jr.⁸, Katrin Kurz⁵, Heike Horn⁴, Anja Mottok^1;9, Raffaella Santi², Ilske Oschlies¹⁰, Wolfram Klapper¹⁰, Kristian Schafernak¹¹, Yanming Zhang¹², Andreas Rosenwald⁹, Hans Binder³, Megan Limm¹², German Ott⁵, Lorenzo Leoncini¹³, Sebastien Hergalant¹⁴, Coral DelVal^1;15;16, Reiner Siebert¹

¹Ulm University and Ulm University Medical Center, Institute of human genetics, Ulm, Germany; ²University of Florence, Careggi University Hospital and Department of Health Sciences, 2 Pathology Section, Florence, Italy; ³Leipzig University, Interdisciplinary Centre for Bioinformatics (IZBI), Leipzig, Germany; ⁴University of Tübingen, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany; ⁵Robert-Bosch-Hospital, Department of Clinical Pathology, Stuttgart, Germany; ⁶Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Department of Pathology, Moscow, Russian Federation; ⁷Münster University Hospital, Pediatric Hematology and Oncology, Münster, Germany; ⁸Massachusetts General Hospital, Department of Pathology, Boston, United States; ⁹Julius-Maximilians-University Würzburg, Department of Pathology, Würzburg, Germany; ¹⁰University of Kiel, Department of Pathology, Kiel, Germany; ¹¹Phoenix Children’s Hospital, Department of Pathology and Laboratory Medicine, Phoenix, United States; ¹²Memorial Sloan Kettering Cancer Center, Department of Pathology and Laboratory Medicine, New York, United States; ¹³University of Siena, Department of Medical Biotechnologies, Section of Pathology, Siena, Italy; ¹⁴University De Lorraine, Inserm UMR 1256 Nutrition-Génétique et Exposition aux Risques Environnementaux (NGERE), Nancy, France; ¹⁵University of Granada, Andalusian Research Institute in Data Science and Computational Intelligence, Department of Computer Science and Artificial Intelligence, Granada, Spain; ¹⁶Instituto de Investigación Biosanitaria ibs.GRANADA, Granada, Spain

Consortium: ICGC-MMML-Seq Consortium

Background/Objectives: The 5^th edition of the WHO Classification of Haematolymphoid Tumours describe disease entities defined by genetic abnormalities. For instance, the diagnosis of Burkitt lymphoma (BL) requires an IG::MYC juxtaposition, e.g. due to t(8;14)(q24;q32). Some high-grade B-cell lymphomas (HGBL) lacking the MYC translocation exhibit immunophenotypic similarities to BL but share similarities at the mutational level with diffuse large B-cell lymphomas (DLBCL). A subset of these HGBL (HGBL-11q) show characteristic chromosomal aberration defined by gains in 11q23.2-11q23.3 and losses in the 11q24.1-qter region. Here, we identify differential transcriptomic features of such HGBL-11q relative to BL and DLBCL.

Methods: Gene expression profiling was conducted using the HTG EdgeSeq Pan B-Cell Lymphoma Panel using RNA obtained from formal-fixed paraffin-embedded (FFPE) tissues of 7 BL, 26 HGBL-11q, 132 DLBCL, and two reactive tonsils. The panel was verified against RNAseq data available from overlapping samples of the ICGC-MMML-Seq cohort, leaving 206 genes for subsequent clustering and DESeq2 analyses. A two-step cumulative gene expression classifier was developed to segregate HGBL-11q from BL and DLBCL.

Results: Using unsupervised UMAP analysis of the 206 genes, we observed that HGBL-11q clustered apart from BLs and DLBCLs. DESeq2 revealed 48 and 38 genes differentially expressed between HGBL-11q and DLBCL or BL, respectively. A two-step classifier was trained to distinguish HGBL-11q from DLBCLs and BLs, respectively. PostHoc genomic analyses of cases classified as HGBL-11q showed two cases not yet assigned to this entity to carry typical 11q aberrations.

Conclusion: Gene expression profiling of FFPE tissues was effective in identifying HGBL-11q among other mature aggressive lymphomas.

Grants: None

Conflict of Interest: None declared

P01.021.A Germline ESR1 variants may be causative of juvenile papillomatosis of the breast

Zahra Firoozfar ¹, Shahryar Alavi¹, Mohammadreza Dehghani², Mohammad Yahya Vahidi Mehrjardi³

¹Palindrome, Isfahan, Iran; ²Yazd Reproductive Sciences Institute, Shahid Sadoughi University of Medical Sciences, Yazd, Iran; ³Diabetes Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Objectives: Germline variants are causative of 10% of cancer incidences. Juvenile papillomatosis of the breast (JPB), also known as Swiss cheese disease, is a benign condition occurs in individuals with a family history of breast carcinoma and its incidence in males is rare. Here, we report a case with several affected males by JPB or gynecomastia, harbouring a significant germline mutation in the ESR1 gene.

Methods: WES was performed on 200 Iranian individuals with hereditary cancers. Sequence raw data was analysed using GATK, and GRCh38 as reference genome. We also called copy number variations using PalinDepth, our proprietary algorithm. Variants were filtered based on the ACMG guideline, then were manually curated to find the best match with the patients’ phenotype.

Results: Germline ESR1 D538G mutation (chr6:g.152098791A > G) was detected in heterozygous state in a 5 years old boy suffering from JPB, with several males in the pedigree are affected with gynecomastia. No other significant exome variant was detected. Sanger sequencing showed that another affected family member is heterozygous for the ESR1 variant.

Conclusion: ESR1 D538G mutant is well described in sporadic breast cancer where ESR1 somatic mutations lead to malignancies. Recently it has been shown that ESR1 variants could increase the risk of hereditary breast cancer. Interestingly, we detected D538G mutation in a patient with familial breast anomalies in males. Further experiments could validate plausible effects of germline D538G mutation, investigating if this could trigger hereditary breast cancer in females.

Grants:

Conflict of Interest: None declared

P01.022.B Cascade testing for hereditary breast cancer: what is achievable in a South African clinical service?

Tabitha Osler¹, Mardelle Schoeman², Jennifer Edge³, Willem Pretorius², Felix Rabe⁴, Christopher Mathew⁵, Michael Urban ^6;7

¹Sydney Brenner Institute for Molecular Bioscience, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; ²Division of Molecular Biology and Human Genetics, University of Stellenbosch, Cape Town, South Africa; ³Department of Surgery, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; ⁴Faculty of Medicine and Health Sciences, University of Stellenbosch, Cape Town, South Africa; ⁵Sydney Brenner Institute for Molecular Bioscience, University of the Witwatersrand, Johannesburg, South Africa; ⁶Division of Human Genetics, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; ⁷Department of Obstetrics and Gynaecology, Faculty of Medicine and Health Sciences, University of Stellenbosch, Cape Town, South Africa

Background/Objectives: A main reason for genetic counselling and testing people with possible hereditary breast cancer is to identify probands and their asymptomatic family members who carry pathogenic variants, by the process of cascade testing. This allows prevention by radiographic screening and risk-reducing surgery, especially in female relatives. We investigated uptake in a South African regional breast cancer service.

Methods: Pedigrees and laboratory electronic records in 83 consecutive female breast cancer patients with pathogenic variants associated with high or moderate penetrance of hereditary breast cancer were used to assess family member testing. Electronic appointment, clinical and radiology records were used in test-positive relatives to identify preventive interventions conducted by a year later.

Results: 1173 family members were considered eligible for testing, 55% female. Relatives of 39/83 (47%) probands took up testing, with 111 tested and 40 testing positive (3 in ATM, 17 in BRCA1, 17 in BRCA2, 3 in PALB2), including 31 females. Family member testing was associated with gender and degree of relationship. In female relatives, the proportion tested dropped from 25% in first degree to 8% in second degree relatives, and in males from 14% to 0.8%. Prophylactic measures undertaken in guideline-eligible asymptomatic female relatives included radiographic screening in 5/20 (25%), risk-reducing mastectomy in 9/19 (47%), and bilateral salpingo-oophorectomy in 3/13 (23%).

Conclusion: Compared to international evidence uptake of pre-symptomatic testing is low; in test-positive female relatives, uptake of screening is low but of risk-reducing surgery appears similar. We will explore further with a qualitative study.

Grants: Cancer Association of South Africa Research Grant 2022

Conflict of Interest: None declared

P01.023.C Piperine exerts antitumor effects in head and neck cancer

Juliana Prado Gusson-Zanetoni¹, Luana Cardoso¹, Stefanie Oliveira de Sousa¹, Laura Luciana De Melo Moreira Silva¹, Tiago Henrique², Eloiza Tajara², Claudia Regina Bonini Domingos¹, Sonia Maria Oliani¹, Eny Maria Goloni-Bertollo², Flavia Cristina Rodrigues-Lisoni ¹

¹São Paulo State University (UNESP), Institute of Biosciences, Humanities and Exact Science, Department of Biological Science, São José do Rio Preto; ²School of Medicine of São José do Rio Preto (FAMERP), Molecular Biology Department, São José do Rio Preto, Brazil

Background/Objectives: Head and neck cancer (HNC) is promoted by genomic instability that affects cell growth, metabolism and inflammation. Treatments for patients with this cancer are generally very invasive and the use of natural products has been used as an adjuvant. Among these, piperine (1-piperoylpiperidine) stands out, which can be isolated from the fruits or roots of black pepper (Piper nigrum), as it has pharmacological effects such as antioxidants, anti-inflammatory and immunomodulatory. Therefore, the aim of the present study was to investigate the molecular mechanisms of action of piperine in relation to its potential antitumor effect on head and neck squamous cells carcinoma (HNSCC).

Methods: Parameters related to neoplastic potential including proliferation, cytotoxicity, migration, apoptosis, cell cycle, clonogenicity, genotoxicity were verified in HNSCC (HEp-2 and SCC-25) treated with piperine. Furthermore, molecular analysis of the cytokines, proteins and genes were investigated to understand how piperine modulates the inflammatory pathways.

Results: The results of the tests indicated that piperine modified the morphology, inhibited growth/viability and the formation of cell colonies, promoted genotoxicity by triggering apoptosis and cell cycle arrest in G2/M and S. The decrease in cell migration also was observed, probably due to the decrease in the expression of MMP2/9 genes. Furthermore, piperine reduced the expression of molecules related to inflammation such as PTGS2, PTGER4, ERK, p38 and cytokines.

Conclusion: These results suggest that piperine exerts antitumor effects on HNSCC and may be a promising natural treatment by regulating signaling pathways associated with inflammation and cancer.

Grants: Fapesp, CNPq.

Conflict of Interest: None declared

P01.024.D Germline Pathogenic Variants In Pakistani Patients Evaluated At A Hereditary Breast Cancer Clinic

Fizza Akbar ¹, Shamila Ladak², aushna saleem³, alizeh fatimi², bassim zahid², zahraa siddiqui², uzair ansari⁴, Salman Kirmani¹

¹Aga Khan University Hospital, Division of women and child health, Karachi, Pakistan; ²Aga Khan University Hospital, Medical College, Karachi, Pakistan; ³Albert Einstein College of Medicine, United States; ⁴Aga Khan University Hospital, Paediatrics and Child Health, Karachi, Pakistan

Background/Objectives: Hereditary breast cancer (HBC) accounts for 5-15% of all breast cancer cases. These are attributed to pathogenic (P) and likely pathogenic (LP) germline variants in breast cancer predisposition genes. This study describes the spectrum of germline variants causing HBC in Pakistan and explores clinical correlations with P/LP variants.

Methods: Retrospective chart review of 751 patients from HBC clinic who proceeded with multi-gene panel testing between 2017 and 2023. Descriptive and inferential statistical analyses were done.

Results: Eighteen percent (137/751) tested positive, 36% (273/751) had a VUS and 45% (341/751) had a negative result. Sixty percent of positive patients had P/LP variants in BRCA1/2, while the other 40% were attributed to other genes. Six recurrent BRCA1 variants reported were in 20 unrelated patients, with BRCA1 c.1961dup (p.Y655Vfs*18) being the most common, present in eight patients. CHEK2 (5.4%), FANCM (5.4%), PALB2 (4.7%) were the next most prominent genes. Patients with positive results had a younger age at diagnosis at 42, IQR (34-50). Having a positive result showed a positive association with triple-negative breast cancer (TNBC). Of the patients who tested positive for BRCA1/2, 84% had a diagnosis of TNBC. Of all patients with positive results, 12% of patients had a negative family history of cancer.

Conclusion: Our findings give unique insights into the genetic factors causing HBC in the Pakistani population. As more patients are tested and protocol-driven referrals are made across the country, our insight into the genetic architecture of hereditary breast cancer in our population will continue to increase.

Grants: Not-funded

Conflict of Interest: None declared

P01.025.A Recurrent variant in the 3’UTR of the MSH6 gene in four unrelated Italian families with Lynch syndrome phenotype

Domizia Pasquetti ¹, Federica Francesca L’Erario², Roberta Pietrobono¹, Maria Grazia Pomponi², Arianna Panfili³, Emanuela Lucci Cordisco^1;2, Maurizio Genuardi^1;2

¹Section of Genomic Medicine, Department of Life Sciences and Public Health, Catholic University of Sacred Heart and Unit of Medical, Rome, Italy; ²UOC Genetica Medica, Fondazione Policlinico Universitario “A.Gemelli” IRCCS, Rome, Italy; ³Direzione Scientifica, Fondazione Policlinico Universitario “A.Gemelli” IRCCS, Roma

Background/Objectives: Pathogenic variants in MSH6 account for 12%-35% of Lynch syndrome (LS). Despite classification guidelines, variants of uncertain significance (VUS) in MMR genes represents a diagnostic dilemma. We discuss here the c.*23_*26dup MSH6 variant, that is reported as a VUS in ClinVar but considered as pathogenic in a previous published study.

Methods: Genetic counseling involved four unrelated Italian families, with germline NGS analysis performed in four subjects and targeted variant analysis in two. IHC of MMR proteins was conducted on four endometrial tumors (EC) and one colorectal cancer (CRC); MSI was performed on one of two CRCs.

Results: All the six individuals were heterozygotes for MSH6 c.*23_*26dup. EC was diagnosed in all four female carriers (median age: 45.7 years), showing no MSH2 and MSH6 protein expression. CRC occurred in two male carriers at ages 62 and 32; IHC and MSI revealed no MSH2 and MSH6 expression, with MSI-H status in one tested CRC.

Conclusion: The clinical phenotypes of c.*23_*26dup MSH6 carriers are highly suggestive of LS, with significantly high penetrance for early-onset gynecological tumors. The MSH6 variant could be pathogenic and cause LS in these families. However, the lack of MSH2 expression suggests involvement of the MSH2 gene. Under this assumption, the MSH6 c.*23_*26dup variant could be a linked marker of an undetected MSH2 alteration. The previously reported families were from Northern Italy, while our families are from Central Italy. The geographic origin and the phenotypic similarities across the families suggest that they might derive from a common founder.

Conflict of Interest: None declared

P01.026.B A case of MYC and BCL2- positive high-grade B-cell lymphoma with concurrent 11q gain-loss aberration

Gulleyla Kilic ¹, Olga Meltem Akay², Emre Osmanbasoglu², Dicle Ballikaya¹, Tuba Akan³, İbrahim Öner Doğan⁴, Hülya Kayserili^1;5

¹Koc University Hospital, Genetic Diseases Evaluation Center, Istanbul, Türkyie; ²Koc University Medical School, Hematology, Istanbul, Türkyie; ³Koc University Hospital, Flow Cytometry, Istanbul, Türkyie; ⁴Koc University Medical School, Pathology, Istanbul, Türkyie; ⁵Koc University Medical School, Medical Genetics, Istanbul, Türkyie

Background/Objectives:High-grade B-cell lymphoma (HGBCL, previously known as double-hit lymphoma) is an aggressive type of B-cell non-Hodgkin lymphoma characterized by rearrangements in MYC and BCL2, or/and less commonly, BCL6 gene. Rare cases of HGBCL with concurrent MYC rearrangement and 11q aberrations have been described¹. In this study we provide further evidence that 11q aberrations can also be found in HGBCL with MYC and BCL2 rearrangements (HGBCL-MYC-BCL2-11q)². Clinical, immunohistochemical and detailed cytogenetic characterization of the case is discussed.

Methods:A 36-year-old woman with three-month history of cervical lymphadenopathy,

pancytopenia and elevated LDH was referred to our hospital. The diagnosis of HGBCL-MYC-BCL2-11q was established based on histopathologic/immunohistochemical examination of the bone marrow sample with flow cytometry, classical cytogenetic and interphase/metaphase fluorescence in situ hybridization analyses.

Results: A bone marrow examination showed diffuse infiltration of neoplastic B-cells with CD19 + , HLA-DR + , CD10 + , CD38 + , CD20 + , CD22 + , TdT- and kappa light chain expression. iFISH analysis showed abnormal BCL2, cMYC, IGH, MLL (KMT2A) and TEL/AML1 signals. Conventional cytogenetic analysis defined a highly complex karyotype with a derivative chromosome 1, trisomy 7, t(8;14), triplication 11q, deletion 11q, partial trisomy 12p, t(14;18), trisomy 18 and monosomy X. The complex karyotype was further clarified by additional selected FISH probes for chromosomes 1,8,11,14,18 and X. She received GMALL-B-ALL 2002 protocol treatment and expired 7 months after the diagnosis.

Conclusion: Our findings highlight the importance of clarifying the complex karyotype in HGBCL patients. Future studies are needed to define the prognosis, treatment response and survival of MYC and BCL2-positive HGBCL patients with 11q aberrations.

Grants:-

Conflict of Interest:-

References:¹Shestakova et al.,2023;PMID:36997031.²Gebauer et.al.,2021;PMID:34500469.

Conflict of Interest: None declared

P01.027.C A pilocytic astrocytoma with wovel ATG16L1::NTRK2 fusion responsive to larotrectinib: A case report with genomic and functional analysis

Lily Deland ^1;2, Simon Keane², Thomas Olsson Bontell^3;4, Tomas Sjöberg Bexelius^5;6, Esther De La Cuesta⁷, Inga Gudinaviciene⁸, Jonas A Nilsson^9;10, Helena Carén¹¹, Helena Mörse¹², Frida Abel^1;2

¹Sahlgrenska University Hospital, Clinical Genetics & Genomics, Center for Medical Genomics, Gothenburg, Sweden; ²Gothenburg University, Institute of Bimedicine, Laboratory Medicine, Gothenburg, Sweden; ³Sahlgrenska University Hospital, Dept. of Clinical Pathology, Dept. of Clinical Pathology, Gothenburg, Sweden; ⁴Gothenburg University, Institute of Neuroscience & Physiology, Sahlgrenska Academy, Gothenburg, Sweden; ⁵Karolinska University Hospital, Section for Pediatric Oncology, Highly Specialized Pediatric Medicine 2, Astrid Lindgren’s Children’s Hospital, Stockholm, Sweden; ⁶Karolinska Institute, Department of Women’s and Children’s Health, Stockholm, Sweden; ⁷Bayer Pharmaceuticals U.S., Global Medical Affairs – Oncology, Whippany, United States; ⁸Laboratory Medicine Region Skåne, Department of Genetics and Pathology, Lund, Sweden; ⁹Gothenburg University, Sahlgrenska Center for Cancer Research, Institute of Clinical Sciences, Department of Surgery, Gothenburg, Sweden; ¹⁰Harry Perkins Institute of Medical Research, Perth, Australia; ¹¹Gothenburg University, Institute of Biomedicine, Sahlgrenska Center for Cancer Research, Department of medical biochemistry and cell biology, Gothenburg, Sweden; ¹²Skåne University Hospital, Pediatric Cancer Center, Lund, Sweden

Background/Objectives: The outcome of pilocytic astrocytoma depends heavily on the success of surgery. In the cases where surgery alone is not curative, next generation sequencing approaches can be used to identify treatment targets for precision medicine. Here we report a pediatric pilocytic astrocytoma case that underwent incomplete surgical resection due to the location of the tumor.

Clinical genetics analyses demonstrated that the tumor did not carry a BRAF fusion or a BRAF mutation. After post-operative surveillance according to the low-grade glioma protocol, recurrent tumor progressions resulted in multiple chemotherapy regimens.

Methods: RNA sequencing by an open-ended targeted approach was performed and detected the presence of a novel ATG16L1::NTRK2 fusion gene. The rearrangement was subsequently confirmed by interphase fluorescent in situ hybridization in tumor cells. Functional analysis of the fusion was performed by in-vitro transient transfection of HEK293 cells. The ATG16L1::TRKB fusion protein was found to activate both the MAPK-pathway, and the PI3K-pathway, through increased phosphorylation of ERK, AKT and S6.

Results: As a result of the identification of a NTRK fusion, the patient was enrolled in a phase I/II clinical trial of the nonselective TRK inhibitor larotrectinib. The patient responded well without significant side-effects. Eight months after start of treatment, tumor lesions were no longer detectable consistent with complete response. Presently, 14 months later, complete remission is sustained.

Conclusion: Our findings highlight the importance of screening for other MAPK-drivers in BRAF-negative low-grade gliomas, since rare fusion genes may serve as targets for precision oncology therapy.

Conflict of Interest: Lily Deland: None declared, Simon Keane: None declared, Thomas Olsson Bontell: None declared, Tomas Sjöberg Bexelius: None declared, Esther De La Cuesta Medical doctor at Bayer Oncology, clinical trials with larotrectinib., Inga Gudinaviciene: None declared, Jonas A Nilsson: None declared, Helena Carén: None declared, Helena Mörse: None declared, Frida Abel: None declared

P01.028.D The neurofibromin-lysosome axis in breast cancer

Antonella Delicato^1;2, Pilar Chacon Millan^1;2, Bruno Achutti Duso³, Alessia Castiglioni³, Eleonora Messuti³, Sara Galavotti³, Diego Medina^2;4, Luca Mazzarella³, Manuela Morleo ^1;2

¹University of Campania “Luigi Vanvitelli”, Department of Precision Medicine, Naples, Italy; ²Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli, Italy; ³IEO European Institute of Oncology - IRCCS, Department of Experimental Oncology, Milan, Italy; ⁴University of Naples ‘Federico II’, Department of Medical and Translational Science, Naples, Italy

Background/Objectives: Breast cancer is the most frequent cancer in women. Neurofibromin (NF1) loss-of-function mutations are associated with poor breast cancer survival. Loss of NF1 (a negative regulator of the Ras-MAPK pathway) leads to constitutive mTORC1 signaling by activation of the Ras-MAPK pathway contributing to neoplastic transformation. NF1 interacts with LAMTOR1, a lysosomal protein crucial for mTORC1 activation on lysosomes, while changes in lysosome function and spatial distribution are closely related to cancer development and progression. We hypothesize that neurofibromin has a direct functional role in lysosomal metabolism and mTOR activation in breast cancer.

Methods:We generated NF1 knockout (NF1-KO) human breast cancer cells (SKBR3, BT474, HCC1954) using CRISPR-Cas technology and developed cell-based assays by high-content imaging approaches for lysosomal phenotype screening. mTOR signaling was analyzed by Western blot upon nutrient deprivation and then upon refeeding.

Results: mTOR activity decreases under nutrient deprivation and is reactivated under nutrient refeeding in the three breast cancer cell lines. However, loss of NF1 abrogates the decrease in mTOR signaling under nutrient deprivation. Moreover, lysosomes appear more condensed and increase in number in NF1-KO cells.

Conclusions: These results demonstrate that mTOR is constitutively active and lysosomal activity is altered in three different NF1-KO breast cancer cell models. In-depth investigation of specific lysosomal changes and NF1-dependent molecular mechanisms will provide a deeper understanding of the dynamic changes in cancer development, providing more opportunities for cancer diagnosis and treatment.

Grants:M6C2/2.1 “Strengthening and enhancing biomedical research in the NHS”, funded by the Union European NextGenerationEU, CUP B63C22002140001

Conflict of Interest: None declared

P01.029.A Treatment-related clonal competition and disrupted molecular processes in chronic lymphocytic leukemia

Kristyna Zavacka ^1;2, Jakub Porc^1;3, Petr Taus¹, Karol Pál¹, Sarka Pavlova^1;2;3, Jitka Malcikova^1;2;3, Kamila Stranska^1;2;3, Marcela Zenatova², Boris Tichy¹, Anna Panovska², Michael Doubek^1;2;3, Sarka Pospisilova^1;2;3, Karla Plevova^1;2;3

¹Central European Institute of Technology, Masaryk University, Center of Molecular Medicine, Brno, Czech Republic; ²University Hospital Brno, Department of Internal Medicine – Hematology and Oncology, Brno, Czech Republic; ³Medical faculty, Masaryk University, Institute of Medical Genetics and Genomics, Brno, Czech Republic

Background/Objectives: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in Western countries, is characterized by a highly variable clinical course, which is closely related to dynamic changes in leukemic cell clones over time. The expansion of subclones with different gene mutations, many of which are non-recurrent, makes CLL challenging to treat. We aimed to describe the clonal evolution in CLL, focusing on specific treatments and different stages of the disease. We particularly studied factors influencing the clonal development of TP53-mutated subclones, as defects in the TP53 gene are the strongest predictive and prognostic marker in CLL and drive chemoimmunotherapy resistance.

Methods: We investigated a cohort of 62 CLL patients with detailed clinical characterization and known patterns of TP53 mutation evolution. Whole-exome sequencing was employed to identify somatic variants at two to six different timepoints during the disease course of each patient. Subsequently, we analyzed abnormal molecular pathways and mutational signatures in defined patient groups and tracked their changes over time.

Results: Our analysis revealed striking competition among co-existing CLL subclones of individual patients. We observed progressive evolution of specific gene mutations, highlighting both their mutual exclusivity and co-occurrence. Moreover, we observed several impaired pathways and mutational signatures associated with different disease stages, treatment outcomes, and TP53 status.

Conclusion: Our study provides insights into the molecular aspects underlying clonal evolution in CLL. These findings enhance our understanding of distinct disease phenotypes and suggest potential targets for personalized treatment strategies in CLL.

Grants: AZV NU21-08-00237, NPO-NUVR LX22NPO5102, MUNI/A/1558/2023, and MHCZ-DRO FNBr65269705.

Conflict of Interest: None declared

P01.030.B Joint data analysis of more than 29,000 individuals with HBOC as part of the NASGE initiative

Jan Henkel¹, Andreas Laner ^1;2, Melanie Locher^1;2, Tobias Wohlfrom¹, Birgitta Neitzel¹, Kerstin Becker^1;2, Teresa Neuhann^1;2, Angela Abicht^1;2;3, Verena Steinke-Lange^1;2;4, Birgit Eichhorn⁵, Winfried Schmidt⁶, Daniel Berner⁷, Anna Teubert^2;8, Anne Holtorf⁹, Sarah Heinrich⁹, Gabriele Wildhardt¹⁰, Martin Schulze¹¹, Laura von der Heyden^2;7, Daniela Steinberger¹⁰, Saskia Kleier^2;6, Peter Lorenz^2;5, Ralf Glaubitz^2;8, Saskia Biskup^2;11, Elke Holinski-Feder^1;2;4

¹MGZ - Medical Genetics Center, München, Germany; ²Nationale Allianz für seltene genetische Erkrankungen; ³Neurologische Klinik und Poliklinik mit Friedrich-Baur-Institut, München, Germany; ⁴Medizinische Klinik und Poliklinik IV, Campus Innenstadt, Klinikum der Universität München, München, Germany; ⁵MVZ Mitteldeutscher Praxisverbund Humangenetik, Dresden, Germany; ⁶Gemeinschaftspraxis für Humangenetik und Genetische Labore - DNA Diagnostik Hamburg, Hamburg, Germany; ⁷MVZ genetikum GmbH, Neu-Ulm, Germany; ⁸AMEDES Medizinische Dienstleistungen GmbH, Germany; ⁹Medicover Genetics, MVZ Martinsried GmbH, Planegg, Germany; ¹⁰MVZ diagnosticum Frankfurt, Zentrum für Humangenetik, Frankfurt am Main, Germany; ¹¹Zentrum für Humangenetik Tübingen, Tübingen, Germany

Consortium: NASGE - Nationale Allianz für seltene genetische Erkrankungen

Background/Objectives: Although there are recommendations at national and international level as to which genes should be analyzed in the context of a hereditary breast and ovarian cancer (HBOC), the individual composition of the corresponding gene panels varies, resulting in a different diagnostic yield.

Methods: We performed a retrospective NGS dataset analysis of suspected HBOC patients who had been tested at different German diagnostic laboratories that are part of the NASGE network. These results were compared with the most common panel recommendations and an internal HBOC core gene panel. Additionally, we analyzed the data concerning other potential tumor risk syndromes (TRS) not caused by pathogenic variants in the core panel genes.

Results: We collected 29,317 HBOC datasets and at least one pathogenic variant (class 4/5) was reported in about 4250 datasets, resulting in an overall diagnostic yield of 14.5%. The diagnostic yield of pathogenic variants (class 4/5) varied depending on the analyzed panel (between 5 and 26 genes) from 9.0% to 13.8% with the internal panel having a yield of 12.7%. Notably, in about 1% of cases, a pathogenic variant outside the established HBOC core genes was identified, potentially indicating the presence of other TRS.

Conclusion: These results are consistent with previous observations that a significant proportion of patients with HBOC predisposition were not detected by the guideline-based gene panels and suggest that expanded diagnostics compared to currently recommended multigene panels may identify additional patients at high risk for developing cancer.

Grants:

Conflict of Interest: None declared

P01.031.C RNA-seq of a cohort of cancer patients demonstrates its capacity to reclassify uncertain variants by unveiling aberrant splicing events

Florentine Scharf¹, Thomas Keßler¹, Evgenia Vibe¹, Oliver Klaas¹, Jonas Ingermann¹, Caroline Heintz¹, Sabrina Angerbauer¹, Tobias Wohlfram¹, Andreas Laner¹, Ariane Hallermayr^1;2, Julia Romic-Pickl^1;2, Morghan Lucas^1;2, Verena Steinke-Lange ^1;2, Elke Holinski-Feder^1;2

¹MGZ – Medizinisch Genetisches Zentrum, Munich, Germany; ²Medizinische Klinik und Poliklinik IV, Campus Innenstadt, Klinikum der Universität München, Munich, Germany

Background/Objectives: Around 5-10% of cancers are caused by hereditary tumor syndromes. For the current state-of-the-art method of gene panel testing via whole-exome sequencing (WES), the diagnostic yield ranges between 15-25% for these syndromes. Additional high-throughput methods are therefore required to increase the diagnostic yield. RNA-seq can guide the reclassification of variants of unclear significance (VUS) and detect previously unknown causative variants by revealing aberrant splicing at the RNA level.

Methods: We developed a cost-efficient, high-throughput targeted RNA-seq approach to analyze aberrant splicing events.

Results: Over 100 cases underwent RNA-seq, previously examined by NGS exome sequencing without revealing a clear pathogenic variant. Of these, 144 had a VUS, while 4 showed no initial concerning variants. RNA-seq led to the reclassification of 4 variants as likely benign and 8 as (likely) pathogenic, an increase in diagnostic yield by 8%. Of the 8 (likely) pathogenic reclassified variants, 2 were in BRCA1/2 genes. Among reclassified cases, 9 variants affected an intronic splice site, and 3 were outside of obvious splice-relevant regions. For one case with no initially reported variants, RNA-seq identified the use of a cryptic exon in the APC gene, guiding subsequent intronic region analysis.

Conclusion: RNA-seq offers clinically relevant insights on VUS and cases where no variant was detected, aiding clear diagnoses for unresolved cases. Overall, ~8% of cases previously unsolved by DNA exome sequencing were successfully reclassified by our RNA-seq approach, significantly improving diagnostic yield and optimizing patient care for hereditary cancer syndromes.

Grants: N/A

Conflict of Interest: None declared

P01.032.D The role of liquid biopsy by rapid NGS in cancer management

Mustafa Ozen ¹, Serhat Seyhan², Emre Ilhan², Rabia Aksoy²

¹Istanbul University-Cerrahpasa, Medical Genetics, Istanbul, Türkyie; ²Medroyal Genetic Assessment Center, Medical Genetics, Istanbul, Türkyie

Background/Objectives: Liquid biopsy (LB) is a promising technique for early detecting and monitoring cancer; however, there are still room to improve in sensitivity and duration of the test. Here, we report data in 20 cancer patients referred to us for LB. We propose that LB by rapid NGS technique is crucial for cancer management.

Methods: At least 10 ml of whole blood is collected in EDTA containing vacutainers. Plasma is seperated immediately and stored in -80 until further use. Total nucleic acid (TNA) is isolated from the plasma samples using MagMAX ^TM Cell-Free TNA isolation kit. Total of 10 ng TNA is subjected to Genexus NGS platform where library preperation and sequencing are done automatically in 24 hours by using Oncomine Precision assay. This assay detects actionable mutations in 50 genes in DNA and RNA level. The data is analysed initially by Genexus platform and further analysed by Oncomine software.

Results: Out of 20 patients analysed, 10 patients are dignosed with Lung cancer, 3 had suspicion for lung cancer, 2 had pancreas cancer, 1 patient in each cancer type of breast, bronchial, hepatocellular and colon cancers. We were able to detect actionable mutations in 70 % of our patients. Most of the mutations were SNV in the following genes analyzed: EGFR, p53, KRAS, PTEN and BRAF. One patient had EML4-ALK fusion and one AR amplification.

Conclusion: LB by rapid NGS is an important tool for cancer management. İt is critical to include DNA and RNA in the analysis to precisely detect mutations.

Grants: N/A

Conflict of Interest: None declared

P01.033.A Meta-analysis of GRIN2A variants contradicts a role in tumorigenesis

Robin-Tobias Jauss ¹, Johannes Lemke^1;2, Vincent Strehlow¹

¹Institute of Human Genetics, University of Leipzig Medical Center, Leipzig; ²Center for Rare Diseases, University of Leipzig Medical Center, Leipzig

Background/Objectives: GRIN2A has previously been identified as frequently mutated in tumor samples, leading to a hypothesised involvement of GRIN2A in tumorigenesis. Pathogenic GRIN2A germline variants on the other hand lead to neurodevelopmental disorders, and extensive phenotyping of affected individuals revealed no evidence for tumor burden.

Methods: All publicly available GRIN2A variants were obtained from ClinVar, Cosmic and gnomAD to account for germline variation, somatic variation and variants identified in the general population, respectively. Functional consequences, mutational hotspots and gene expression were assessed for each dataset to draw conclusions on potential pathomechanisms of tumorigenesis.

Results: Pathogenic germline variants in GRIN2A expose a genotype-phenotype correlation with distinct clustering in functionally relevant domains. Somatic GRIN2A variants are indeed unique when compared to pathogenic germline variants, but exhibit a homogeneous distribution and no enriched functional consequences in specific domains of GRIN2A. Expression of GRIN2A is lower in tumor samples compared to non-diseased tissues.

Conclusion: The homogeneous distribution and low expression of somatic variants implies a lack of mutational clustering and functional relevance, which refutes the hypothesised major role of GRIN2A in tumorigenesis.

Grants: None

Conflict of Interest: None declared

P01.036.D First mutational analysis of TERT promoter and CTNNB1 in hepatocellular carcinoma, cirrhotic, and non-cirrhotic liver tissues from Brazilian patients.

Mariana Leonardo Terra¹, José Junior Barros¹, Antônio Hugo José Fróes Marques Campos^2;3, Vera Pannain⁴, Natalia Araujo ¹

¹Oswaldo Cruz Institute - FIOCRUZ, Laboratory of Molecular Virology and Parasitology, Rio de Janeiro, Brazil; ²A.C.Camargo Cancer Center, Department of Anatomic Pathology, São Paulo, Brazil; ³Rede D’Or São Luiz, Division of Pathology, São Paulo, Brazil; ⁴Federal University of Rio de Janeiro, Clementino Fraga Filho University Hospital, Rio de Janeiro, Brazil

Background/Objectives: Hepatocellular carcinoma (HCC) is a major global health burden, with poor prognosis especially at later stages. HCC often arises in cirrhotic livers due to specific risk factors. Its development involves complex genetic mutations. TERT and CTNNB1 somatic mutations are prevalent in HCC but unexplored in Brazilian patients. This study aims to investigate the association of TERT promoter mutations (C228T and C250T) and CTNNB1 exon 3 mutations with HCC development in Brazil.

Methods: Eighty-five liver tissue samples, comprising 26 tumor/surrounding tissue pairs, and 16 HCC, 9 cirrhotic and 8 non-cirrhotic unpaired samples, were analyzed. DNA extracted from cryopreserved or formalin-fixed tissues underwent mutation analysis through Sanger sequencing of PCR fragments.

Results: C250T frequency in HCC tissues was notably low (2.4%) and absent in cirrhotic and non-cirrhotic tissues. C228T was more prevalent in HCC (45.2%) and cirrhotic tissues (19%) than non-cirrhotic (0%) tissues, exhibiting a gradual increase with liver disease progression. Significantly different C228T frequencies were observed in HCC vs. non-cirrhotic (P = 0.0001) and cirrhotic vs. non-cirrhotic (P = 0.0485) tissues. Paired analysis demonstrated exclusive C228T presence in HCC in 46.2% of pairs. While C228T showed higher prevalence in HCCs linked to HCV infection (66.7%), statistical significance was not reached. Preliminary CTNNB1 analysis showed mutations occurred more frequently in HCC (23.8%) than cirrhotic (9.1%) and non-cirrhotic (0%) tissues, with concordance observed with C228T mutation in 66.7% of cases.

Conclusion: TERT promoter mutation C228T, and potentially CTNNB1 mutations, emerge as promising biomarkers for HCC in Brazilian patients, facilitating early detection, risk stratification, and offering potential therapeutic targets for this aggressive cancer.

Conflict of Interest: None declared

P01.039.C Importance of detecting mosaic variants in a case suggestive of Gardner’s syndrome.

Jesús Cabanes Madrid¹, Blanca Luis Sánchez², José Manuel Sánchez Zapardiel ¹, Beatriz Hidalgo Calero¹, Montserrat de Miguel¹, Ricardo Blázquez Martín¹, Marina Ibáñez Vizcaíno¹, Francisca Luengo Sainz de Baranda¹, Victoria Carrero Blázquez¹, Luis Robles Díaz³

¹Hereditary Cancer Unit, Hospital 12 de Octubre. Madrid, Department of Clinical Analysis-Clinical Biochemestry, Madrid, Spain; ²Hospital Puerta del Mar, Cádiz, Department of Clinical Analysis-Clinical Biochemestry, Cádiz; ³Familiar Cancer Unit, Hospital 12 de Octubre, Madrid, Department of Medical Oncology, Madrid, Spain

Background/Objectives: A 19-year-old male was found to have 14 adenomatous polyps and was diagnosed with multiple osteomas in the left ethmoid sinus and frontal sinus. These findings pointed to Gardner’s syndrome and the patient was referred to the Family Cancer Unit.

Methods: Next Generation sequencing (Illumina) was carried out using the Hereditary Cancer Solution kit (Sophia Genetics). The results were analyzed with Sophia-DDM and the variant allele frequency (VAF) detected were confirmed from different tissues by high depth NGS and Sanger sequencing.The segregation was carried out in both parents.

Results: The variant c.4537G > T p.(Glu1513*) in the APC gene are located on mutation cluster region (MCR) and generates a premature stop codon which causes a truncated protein. According to American College of Medical Genetics (ACMG) criteria it was classified as pathogenic. The variant has been identified by high depth NGS in blood and buccal swab at a VAF 10% an 9% respectively, and by sanger sequencing in different sections of colon biopsy at a VAF mean of 10-30%. The results obtained confirm the presence of this variant in mosaicism and “de novo” origin.

Conclusion: Unusual percentage of allele frequencies may be due to artefacts or false positives from sequencing errors. However, mosaic variants are also found in this range and it’s important screening them, paying special attention to genes related to the patient’s phenotype. Otherwise, it would result in inadequate genetic counselling for the patient and their relatives.

Grants: Project “PI19/00340” to L.R., funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union.

Conflict of Interest: None declared

P01.040.D Germline predisposition in paediatric melanoma patients undergoing matched tumor-normal sequencing

Alexandra Liebmann ¹, Jakob Admard¹, Hannah Wild², Michael Abele², Irina Bonzheim³, Ewa Bien⁴, Stephan Forchhammer⁵, Dominik Schneider⁶, Christopher Schroeder¹, Stephan Ossowski¹, Ines Brecht²

¹University Hospital Tübingen, Institute of Medical Genetics and Applied Genomics, Tübingen, Germany; ²University Children’s Hospital Tübingen, Paediatric Hematology and Oncology, Tübingen, Germany; ³University Hospital Tübingen, Institute of Pathology and Neuropathology, Tübingen, Germany; ⁴Medical University of Gdansk, Poland, Department of Paediatrics, Hematology, Oncology, Gdansk, Poland; ⁵University Hospital Tübingen, Center for Dermatooncology, Department of Dermatology, Tübingen, Germany; ⁶Dortmund Municipal Hospital, Dortmund, Clinic of Paediatrics, Dortmund, Germany

Background/Objectives: Genomic characterisation has led to an improved understanding of adult melanoma. However, the aetiology of melanoma in children is still unclear. The aim of this study was to further characterize paediatric melanoma and to identify putative causative germline alterations possibly relevant for prevention and surveillance.

Methods: The cohort of 26 paediatric melanoma patients was grouped into different categories: spitzoid melanoma (SM), conventional melanoma (CM) and others (OT). Tumor-normal exome sequencing was performed and analyzed using an in-house bioinformatics pipeline (megSAP). Germline variants were called with freebayes and annotated with VEP.

Results: No (likely) pathogenic variants were found in the high-risk melanoma genes CDK4 and CDKN2A. In all patients with CM (n = 10) germline variants were found in low to moderate melanoma risk genes: 8 CM patients carried at least one MC1R variant, one patient carried the MITF E318K variant, another patient carried variants in PTEN (c.800del, p.Lys267Arg*9) and in BRCA2 (c.9253dup, p.Thr3085Asn*26). In the SM group (n = 12), 41.7% (n = 5) patients carried at least one MC1R variant. In the OT (n = 4) group, 2 patients carried a MC1R variant. The MC1R variants detected were V92M (n = 6), R151C (n = 5), R160W (n = 4), V60L (n = 4), D84E (n = 1), R163Q (n = 1), K278E (n = 1).

Conclusion: Genetic risk factors may play a more important role in paediatric melanoma than previously anticipated. It can be envisioned to develop combinatorial risk models including genetic and environmental factors to identify individuals at risk and to establish prevention measures.

Grants: Fortüne (2529-0-0), German Childhood Cancer Foundation (DKS 2013/18 DKS 2018/37), DFG (Project-ID286/2020B01-428994620)

Conflict of Interest: None declared

P01.041.A Validation of the BOADICEA model for epithelial tubo-ovarian cancer risk prediction in the UK Biobank

Xin Yang ¹, yujia wu¹, Lorenzo Ficorella¹, naomi wilcox¹, Joe Dennis¹, jonathan tyrer¹, Timothy Carver¹, nora pashayan¹, marc tischkowitz², paul pharoah³, Douglas F. Easton^1;4, Antonis C. Antoniou¹

¹University of Cambridge, Department of Public Health and Primary Care, Cambridge, United Kingdom; ²University of Cambridge, Department of Medical Genetics, Cambridge, United Kingdom; ³Cedars-Sinai Medical Center, Department of Computational Biomedicine, Los Angeles, United States; ⁴University of Cambridge, Department of Oncology, Cambridge, United Kingdom

Background/Objectives: To assess the clinical validity of the multifactorial BOADICEA model for predicting 10-year epithelial tubo-ovarian cancer (EOC) risk in a large, independent prospective cohort.

Methods: We evaluated the model discrimination and calibration in the prospective UK Biobank cohort comprising 199,429 women (733 incident EOCs) of European ancestry without previous cancer history. We predicted 10-year EOC risk incorporating data on questionnaire-based risk factors (QRFs), family history, a 36-SNP polygenic risk score (PRS) and pathogenic variants (PV) in six EOC susceptibility genes (BRCA1, BRCA2, RAD51C, RAD51D, BRIP1 and PALB2).

Results: Individually, QRFs, the PRS and PVs (each combined with age), gave similar discrimination (AUC~0.64-0.65). Discrimination was considerably greater using the multifactorial model that included the PRS, QRFs, FH and PV status (AUC = 0.68, 95%CI:0.66-0.70). This model was well calibrated in deciles of predicted risk with calibration slope = 0.99 (95%CI:0.98-1.01). The model had similar discriminative ability in women younger or older than 60 years. The AUC was higher when analyses were restricted to PV carriers (0.76, 95%CI:0.69-0.82); the model remained well calibrated in all risk groups. Using relative risk (RR) thresholds, the full model classified 97.7%, 1.7%, 0.4% and 0.2% women in the RR < 2.0, 2.0≤ RR < 2.9, 2.9 ≤ RR < 6.0 and RR ≥ 6.0 categories respectively, identifying 9.1% of incident EOC among those with RR ≥ 2.0.

Conclusion: BOADICEA, implemented in CanRisk (www.canrisk.org), provides a valid individualized 10-year EOC risks which can facilitate decision making in clinical EOC risk management.

Grants: Cancer Research UK (PPRPGM-Nov20\100002).

Conflict of Interest: None declared

P01.043.C Histopathological phenotyping of cancers in PTEN Hamartoma Tumor Syndrome for improved recognition

Ane Jordan Schei-Andersen ¹, Linda Hendricks¹, Rachel van der Post², Arjen Mensenkamp¹, Jolanda Schieving³, Muriel A Adank^4;5, Floor Duijkers^5;6, Mirjam de Jong^5;7, Marjolijn Jongmans^5;8, Liselotte P van Hest^5;9, Yvette Van Ierland^5;10, Tjitske Kleefstra^1;5, Edward Leter^5;11, Maartje Nielsen^5;12, Janneke Schuurs-Hoeijmakers¹, Nicoline Hoogerbrugge¹, Janet Vos¹

¹Radboud University Medical Center, Department of Human Genetics, Nijmegen, Netherlands; ²Radboud University Medical Center, Department of Pathology, Nijmegen, Netherlands; ³Radboud University Medical Center, Department of Pediatric Neurology, Nijmegen, Netherlands; ⁴The Netherlands Cancer Institute (NKI), Department of Clinical Genetics, Amsterdam, Netherlands; ⁵PTEN Study Group, Netherlands; ⁶Amsterdam UMC, locatie AMC, Department of Human Genetics, Amsterdam, Netherlands; ⁷University Medical Center Groningen, Department of Genetics, Groningen, Netherlands; ⁸UMC Utrecht, Princess Máxima Center for Pediatric Oncology and Department of Genetics, Utrecht, Netherlands; ⁹Vrije Universiteit Amsterdam, Department of Clinical Genetics, Amsterdam, Netherlands; ¹⁰Erasmus MC, Department of Clinical Genetics, Rotterdam, Netherlands; ¹¹Maastricht UMC + , Department of Clinical Genetics, Maastricht, Netherlands; ¹²Leiden University Medical Center (LUMC), Department of Clinical Genetics, Leiden, Netherlands

Consortium: PTEN Study Group

Background/Objectives: PTEN Hamartoma Tumor Syndrome (PHTS) has a broad clinical spectrum including benign and malignant tumors with varying age of onset. Many patients remain unrecognized, unaware of their increased cancer risk. We assessed the cancer spectrum and histopathological cancer characteristics to determine whether this could improve PHTS recognition.

Methods: Genetic testing results and pathology reports were collected for patients tested for germline PTEN variants between 1997-2020 from the diagnostic laboratory and the Dutch nationwide pathology databank (Palga). Cancer spectrum and age of onset were assessed in patients with (PTENpos) and without (PTENneg) a germline PTEN variant. Histopathological cancer characteristics were assessed in a nested cohort.

Results: 341 PTENpos patients (56% females) and 2,882 PTENneg patients (66% females) were included. PTENpos presented mostly with female breast (BC, 30%), endometrial (EC, 6%), thyroid (TC, 4%) or colorectal cancer (4%). Besides PHTS-related cancers, PTENpos had non-serous ovarian cancer (3%). PTENpos were significantly younger at cancer onset (43 vs. 47years) and developed more often (46% vs. 18%) a second BC. PTEN detection rates were highest for BC < 40 years (9%), TC < 20 years (15%) and EC < 50 years (28%). Histopathological characteristics were similar between groups.

Conclusion: No histopathological cancer characteristics were distinctive for PHTS. However, PTENpos were significantly younger at cancer onset. Therefore early-onset BC, EC or TC warrants consideration of PHTS diagnostics either by pre-screening for other PHTS features or direct germline testing.

Grants: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No955534

Conflict of Interest: None declared

P01.044.D Evaluation of the results of cytogenetic, molecular cytogenetic and molecular analysis in non-Hodgkin lymphoma

Can Berk Leblebici ¹, Nüket Yürür Kutlay¹, Arzu Vicdan¹, Sadiye Ekinci¹, Hatice Ilgın Ruhi¹, Timur Tuncalı¹, Halil Gürhan Karabulut¹, Şule Altıner¹, Ezgi Gökpınar İli¹

¹Ankara University Medical School, Medical Genetics, Turkiye

Background/Objectives: Non-Hodgkin lymphomas (NHL) are the most common lymphoma type in adults. In this study, the results of cytogenetic, fluorescence in situ hybridization (FISH) and next generation sequencing (NGS) analysis of patients diagnosed with NHL is presented.

Methods: The results of genetic analysis performed on BM or PB samples of patients with NHL in our department between 2022-2023 were evaluated retrospectively.

Results: Sixty patients (28 women 32 men), their average age was 58, were included in the study. There were a total of 12 types of NHL and the most frequent one was diffuse large B-cell lymphoma. Loss-of-function changes in the DNMT3A gene in B-cell-derived lymphomas and gain-of-function changes in STAT5B and STAT3 genes in T-cell-derived lymphomas were detected as the most common mutations.

The number of detected pathological findings by the method used is summarized in the table below:

Conclusion: Lymphoma pathogenesis is complex. Genetic test results make important contributions to the evaluation of parameters related to diagnosis, treatment and prognosis in cases with NHL. Pathological NGS findings may be related to the pathogenesis of lymphoma but the interpretation may be difficult. In this study, evaluation will be made regarding the genetic test results of NHL cases.

References:

PMID:35732829

PMID:37672206

Grants: No

Conflict of Interest: None declared

P01.045.A Utility of whole body MRI surveillance in children and adults with cancer predisposition syndromes

Frances Victoria Que ¹, Shao-Tzu Li¹, Jeanette Yuen¹, Tarryn Shaw¹, Hui Xuan Goh¹, Nur Diana Binte Ishak¹, Zewen Zhang¹, Jianbang Chiang¹, Lee Lian Chew², Choon Hua Thng², Joanne Ngeow^1;3;4

¹National Cancer Centre Singapore, Cancer Genetics Service, Division of Medical Oncology, Singapore, Singapore; ²National Cancer Centre Singapore, Division of Oncologic Imaging, Singapore, Singapore; ³Duke-NUS Medical School, Oncology Academic Clinical Program, Singapore, Singapore; ⁴Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore

Background/Objectives: Surveillance improves outcomes by detecting cancer early in individuals with cancer predisposition syndromes (CPS). Whole body MRI (WB-MRI) provides head-to-thigh imaging in one sitting without radiation exposure and is recommended for individuals with CPS associated with increased risk of multi-focal tumors/cancers and/or susceptibility to radiation. This study evaluated the diagnostic performance of WB-MRI as a screening tool for Li-Fraumeni syndrome, Constitutional Mismatch Repair Deficiency syndrome, Hereditary Paraganglioma-Pheochromocytoma syndrome and other CPS.

Methods: A review of patients with CPS seen at the Cancer Genetics Service, National Cancer Center Singapore was conducted. Patients who underwent screening WB-MRI from 2014 to 2022 were identified to determine the sensitivity and specificity of WB-MRI in detecting cancer early.

Results: Of 103 patients with CPS who were indicated to have WB-MRI surveillance, 54 underwent the procedure (52% uptake rate): 28 (52%) were female, median age was 41 years (range 1-65 years); 21 (39%) had Li-Fraumeni syndrome, six (11%) had Hereditary Paraganglioma-Pheochromocytoma syndrome, and seven (13%) had Von Hippel Lindau syndrome. WB-MRI screening led to a diagnosis of cancer in seven (13%) patients (renal cell carcinoma, prostate adenocarcinoma, osteosarcoma, neuroendocrine tumor); four of these seven patients (57%) were treated with curative intent. Twelve (22%) patients required additional investigations, with eight (15%) having benign findings. The sensitivity and specificity of WB-MRI in our cohort was 64% and 81% respectively, with a false-positive rate of 19% and false-negative rate of 36%.

Conclusion: WB-MRI is an effective screening tool in patients with specific CPS, demonstrating a high specificity and low false positive rate.

Grants: none

Conflict of Interest: None declared

P01.046.B Targeted transcriptomics validates metastasis-relevant genes from case-case GWAS risk variants and functional annotation in sporadic colorectal carcinomas.

Peh Yean Cheah ¹, Kuen Kuen Lam¹, Lai Fun Thean¹, Michelle Su Mei Lo¹, Emile Tan¹, Choong Leong Tang¹, Iain Bee Huat Tan²

¹Singapore General Hospital; ²National Cancer Centre Singapore, Singapore

Background/Objectives: Colorectal cancer (CRC) is the third highest incidence cancer and leading cause of cancer mortality worldwide. Metastasis to distal organ is the major cause of cancer mortality. Currently, the underlying genetic factors are unclear. This study aims to identify metastasis-relevant genes and pathways for better management of metastasis-prone patients.

Methods: A case-case genome-wide association study comprising 2677 sporadic Chinese CRC cases with clinically determined metastasis status (1282 metastasis-positive vs 1395 metastasis-negative) was performed using the Human SNP6 microarray platform and analyzed with the correlation/trend test based on the additive model. 647,545 single nucleotide polymorphism (SNP) variants with association testing -log10p-value ≥ 5 were imported into Functional Mapping and Annotation (FUMA) and Ingenuity (IPA) software for functional annotation and pathway analysis.

Results: About 196 candidate genes were identified for both overall and disease subtypes stratified by gender, tumor stage, site, and metastasis organs. Targeted high throughput transcriptomics was performed on 200 (100 metastasis-positive vs 100 metastasis-negative) left-sided stage III tumors using the Fluidigm platform. About a third of the candidate genes identified by the combined case-case GWAS and in silico analysis were validated; half of these are significant in the female subset only. This reaffirms that there is female-specific metastasis-relevant genes contributing to sexual dimorphism in CRC metastasis.

Conclusion: Combining genome-wide association testing with in silico functional annotation and wet-bench validation identified metastasis-relevant genes that could serve as features to develop subtype-specific metastasis-risk signatures for tailored management of CRC patients.

Grants:

Conflict of Interest: None declared

P01.047.C Exploring Germline Variations and Predisposition in Childhood Lymphoma

TUĞÇE SUDUTAN^1;2, Khusan Khıdzhaev^1;2, Yucel Erbilgin², didem altındirek^1;2, sumeyye kocaağa^1;2, merve sarıtaş^1;2, süheyla ocak³, ayça kıykım⁴, deniz tuğcu⁵, hikmet gülşah tanyıldız⁵, rejin kebudi⁶, tülin tiraje celkan⁷, Muge Sayitoglu ²

¹Istanbul University Institute of Graduate Studies in Health Sciences, Istanbul, Türkyie; ²Istanbul University Aziz Sancar Experimental Medicine Research Institute, Department of Genetics, Istanbul, Türkyie; ³Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Department of Pediatric Hematology-Oncology,Department of Children’s Health and Disease, Istanbul, Türkyie; ⁴Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Department of Pediatric Allergy and Immunology, Department of Children’s Health and Disease, Istanbul, Türkyie; ⁵Istanbul University Istanbul Medical Faculty, Department of Pediatric Hematology-Oncology, Istanbul, Türkyie; ⁶Istanbul University Oncology Institute, Department of Pediatric Hematology-Oncology, Istanbul, Türkyie; ⁷İstinye University, İstinye Medical Faculty, Department of Pediatric Hematology-Oncology, Istanbul, Türkyie

Background/Objectives: Approximately 10% of cancer patients harbour germline variations predisposing to cancer. Increased lymphoma development risk has been reported in several syndromes and patients with familial history. We aimed to investigate germline predisposition genes in paediatric lymphoma cases/families with at least one indication for genetic counselling.

Methods: Whole exome sequencing was performed in 22 paediatric lymphoma cases (Hodgkin’s lymphoma (n = 13), diffuse large B-cell lymphoma (n = 3), Burkitt’s lymphoma (n = 3), T-cell anaplastic lymphoma (n = 2), lymphoproliferation (n = 1)) with indications for genetic counselling. Segregation analyses were performed for candidate genes.

Results: Almost 60% of cases were associated with immunodeficiency without differential clinical diagnosis, and germline variations in genes related to primary immunodeficiency such as CD70, RAG2, LRBA, NBN, ITK and TNFSF9. In other lymphoma families, germline variations in inherited cancer susceptibility genes like PRF1, BRIP1, SMAD4, KDR, ALK have been observed. In addition to the major predisposition genes, each case was found to carry numerous heterozygous variants of germline cancer genes (NOTCH1, FLT3, BCR, JAK2, CCDN1,etc.). Two new candidate genes/variants thought to be responsible for germline predisposition to lymphoma have also been identified.

Conclusion: Implementation of next-generation sequencing has recently enabled discovering rare genetic variants predisposing paediatric patients to lymphoid neoplasms. It is valuable for identifying individuals at risk in families for early diagnosis, genetic counselling and follow-up. It is also important to evaluate patients for indications for genetic counselling in order not to miss rare hereditary cases in the clinic.

Grants: Scientific Research Projects Coordination Unit of Istanbul University (TSA-2023-39470) and Ministry of Industry and Technology-Istanbul Development Agency(ISTKA) (Tr10/22/TNH/0001)

Conflict of Interest: None declared

P01.048.D Barriers and facilitators to sustainment of the Tumor-First workflow for hereditary epithelial ovarian cancer detection

Vera M. Witjes ¹, Yvonne H.C.M. Smolders¹, Joanne A. De Hullu², Margreet G.E.M. Ausems³, Marian J.E. Mourits⁴, Marjolijn Ligtenberg^1;5, Nicoline Hoogerbrugge¹, Julie E.M. Swillens⁶

¹Radboud university medical center, Department of Human Genetics, Nijmegen, Netherlands; ²Radboud university medical center, Department of Obstetrics and Gynecology, Nijmegen, Netherlands; ³University Medical Center Utrecht, Division Laboratories, Pharmacy and Biomedical Genetics, Department of Genetics, Utrecht, Netherlands; ⁴University of Groningen, University Medical Center Groningen, Department of Obstetrics and Gynecology, Groningen, Netherlands; ⁵Radboud university medical center, Department of Pathology, Nijmegen, Netherlands; ⁶Radboud university medical center, Scientific Center for Quality of Healthcare (IQ Healthcare), Nijmegen, Netherlands

Background/Objectives: Around 10-15% of women with epithelial ovarian cancer (OC) have a genetic predisposition, making genetic testing essential. To optimize detection of heredity, we have implemented the “Tumor-First” workflow nationwide. This workflow implies that OC tumor DNA is examined for presence of pathogenic variants (e.g. in BRCA1/2) serving to stratify germline testing and to provide information on the effectiveness of PARP-inhibitors. Implementation was successful with the uptake of tumor DNA testing exceeding 80% and positive experiences. However, with the everchanging field of genetic testing, we aimed to determine the sustainability of the Tumor-First workflow.

Methods: We used a qualitative study design. 30 healthcare professionals from 7 medical disciplines were interviewed to discuss factors important for sustainment of the Tumor-First workflow. Barriers and facilitators of sustainment were analyzed using the framework of Flottorp.

Results: Main facilitators of sustainment were support, motivation, and awareness amongst healthcare professionals, presence of leading figures in the field, practical advantages of the workflow and (financial) embeddedness within the healthcare system. Main barriers were concerns about sustainment of nationwide uniformity considering ongoing developments in the field and the nearing end of project-related nationwide coordination. Without coordination, developments in tumor sequencing techniques and the rise of homologous-recombination-deficiency testing might lead to heterogeneity amongst laboratories in procedures and workflows.

Conclusion: As the field of genetic testing undergoes rapid developments, nationwide coordination is needed to sustain a uniform workflow. These results provide guidance on implementation and sustainment of innovations in the field of genetic testing.

Grants: Dutch Cancer Society (12732)

Conflict of Interest: None declared

P01.049.A Risk reducing surgery, cancer incidence and mortality in the Swedish BRCA cohort

Anna Rosén¹, Annika Idahl², Ylva Bengtsson^3;4, Åke Borg³, Hans Ehrencrona^5;6, Linda Hartman⁷, Per Karlsson⁸, Ylva Karlsson⁹, Ekaterina Kuchinskaya^10;11, Svetlana Bajlica Lagercrantz¹², Malin Sund¹³, Anna von Wachenfeldt Väppling¹⁴, Rebecka Wiberg¹³, Anna Öfverholm⁸, Barbro Numan Hellquist¹, Niklas Loman ^3;4

¹Umeå University, Diagnostics and Intervention, Oncology, Umeå, Sweden; ²Umeå University, Clinical Sciences, Obstretics and Gynecology, Umeå, Sweden; ³Lund University, Division of Oncology, Department of Clinical Sciences, Lund, Sweden; ⁴Skåne University Hospital, Dept of Hematology, Oncology and Radiation physics, Lund, Sweden; ⁵Lund University, Laboratory Medicine, Clinical Genetics, Lund, Sweden; ⁶Skåne University Hospital, Office of Medical services, Pathology and Molecular Diagnostics, Clinical genetics, Lund, Sweden; ⁷Lund University, Centre of Mathematical Sciences, Lund, Sweden; ⁸Sahlgrenska Academy, University of Gothenburg, Clinical sciences, Oncology, Gothenburg, Sweden; ⁹Uppsala University Hospital, Immunology, Genetics and Pathology, Uppsala, Sweden; ¹⁰Linköping University, Clinical Genetics, Linköping, Sweden; ¹¹Linköping University, Biomedical and Clinical Sciences, Linköping, Sweden; ¹²Karolinska Institutet, Oncology-Pathology, Stockholm, Sweden; ¹³Umeå University, Diagnostics and Intervention, Surgery, Umeå, Sweden; ¹⁴Södersjukhuset, Breast Center, Stockholm, Sweden

Background/objectives: Women carrying germline pathogenic variants in BRCA1 and BRCA2 are offered surveillance, risk-reducing bilateral mastectomy (RRBM) and salpingoophorectomy (RRSO) to reduce cancer morbidity and mortality. The aim of this study is to evaluate cancer incidence and mortality in relation to risk-reducing surgery in a nationwide cohort of healthy BRCA-carriers.

Methods: The Swedish BRCA-cohort includes Swedish BRCA-carriers identified 1994-2018. Their data was linked with nationwide Cancer -, Patient-, and Cause of Death registers. Cancer-free women were followed from the date of testing, until death or end of follow-up (Aug 2022).

Results: The 837 women cumulated to 9329 person years, whereof 4972 before/never RRBM and 4357 after RRBM, 6102 before/never RRSO and 3227 after RRSO. Preliminary results indicate that at the end of follow-up, 74 (16%) developed breast cancer (median age 47, range 24-72), 16 (4%) ovarian cancer (median age 54, range 30-73), and 44 (median age 66, range 29-97) had died. In total, 386 carriers (46%) had undergone RRBM (median age 39, range 20-72) and 400 (48%) RRSO (median age 44, range 21-77).

Conclusions: This study will summarize cancer incidence and mortality and the effect of risk reducing interventions, on a national level in Sweden during the first 20 years of clinical BRCA1 and BRCA2 -testing. Updated data on age-dependent cancer incidence as well as the effect of risk reducing surgery on mortality will be presented.

Funding: The Swedish Breast Cancer Association

Conflict of Interest: Anna Rosén: None declared, Annika Idahl: None declared, Ylva Bengtsson: None declared, Åke Borg: None declared, Hans Ehrencrona: None declared, Linda Hartman: None declared, Per Karlsson Coinventor Prelude DX and Exact Sciences (not related to the current study), Astrazeneca, Novartis, Seagen, Ylva Karlsson: None declared, Ekaterina Kuchinskaya: None declared, Svetlana Bajlica Lagercrantz: None declared, Malin Sund: None declared, Anna von Wachenfeldt Väppling: None declared, Rebecka Wiberg: None declared, Anna Öfverholm Lecture for oncology health care professionals on request from Pfizer and Astra, Barbro Numan Hellquist: None declared, Niklas Loman: None declared

P01.050.B Expression analysis of ultraconserved regions in childhood acute lymphoblastic leukemia

Maria Augusta Poersch¹, Gabriela Canalli Kretzschmar², Saloe Bispo², Daniela Fiori Gradia¹, Roberto Rosati², Jaqueline Carvalho de Oliveira ¹

¹Federal University of Parana, Genetics, Curitiba - PR, Brazil; ²Pelé Pequeno Príncipe Research Institute, Curitiba - PR, Brazil

Background/Objectives: The ultraconserved regions (UCRs) are genomic elements that exhibit complete sequence conservation across human, rat, and mouse genomes and are implicated in various cancers. However, their role in childhood acute lymphoblastic leukemia (ALL) remains underexplored. The present study aims to identify differentially expressed transcripts associated with UCRs across ALL subtypes.

Methods: Using RNA sequencing data from St. Jude Research Hospital (804 subjects aged 0-15), differential expression analysis was conducted with DESeq2, including 240 annotated transcripts. Comparisons spanned T-ALL versus B-ALL, high-risk versus low-risk subtypes, and distinct expression profiles of hypodiploid, high hyperdiploid, and recurrent genetic abnormalities: ETV6-RUNX1, rearrangement of KMT2A, iAMP21, BCR-ABL1, and TCF3-PBX.

Results: The differential expression analyses revealed 40 significantly deregulated transcripts, with the T-ALL vs. B-ALL comparison displaying the highest diversity (16 transcripts). Noteworthy oncogenes, including ZNF521, EBF1, EBF3, PBX1, and EPHA7, exhibit subtype-specific expression patterns. ZNF521 is more expressed in high-risk ALL, while EBF1 was found to be overexpressed in B-ALL. PBX1 shows increased expression in the TCF3-PBX1 subtype, and EPHA7 demonstrates higher expression in B vs. T ALL. Furthermore, regulatory genes such as HOXA2, HOXA3, and HOXA5 were found to be downregulated in BCR-ABL1 positive patients and upregulated in the KMT2A-rearrangement.

Conclusion: This comprehensive analysis identifies UCR-related transcripts deregulated in ALL subtypes, including critical oncogenes. In conclusion, our study sheds light on the crucial role of T-UCR-associated transcripts in childhood ALL, improving the understanding of the disease and offering valuable insights for personalized treatment approaches in the future.

Grants: CNPq, CAPES, Fundação Araucária, PRPPG-UFPR.

Conflict of Interest: None declared

P01.051.C Enhanced cytogenomic analysis of complex karyotype in myelodysplastic syndrome using optical genome mapping

Andriana Valkama ¹, Sandra Vorimo¹, Anna Tervasmäki¹, Hannele Räsänen², Eeva-Riitta Savolainen², Katri Pylkäs^1;2, Tuomo Mantere¹

¹Laboratory of Cancer Genetics and Tumor Biology, Translational Medicine Research Unit, Medical Research Center Oulu and Biocenter Oulu, University of Oulu, Oulu, Finland; ²Northern Finland Laboratory Centre Nordlab, Oulu, Finland

Background/Objectives: Myelodysplastic syndrome with complex karyotype (CK-MDS) is a clinically challenging disease. In the clinical workup of MDS, cytogenetic analyses detecting structural variants (SVs) are crucial for prognostication and guiding treatment decisions. However, the current standard of care (SOC) testing, relying on karyotyping, often yields ambiguous results in cases with a complex karyotype.

Methods: We evaluated the SV detection by optical genome mapping (OGM) for 15 CK-MDS cases compared with SOC and explored the use of OGM as a tool for novel discoveries in combination with exome and transcriptome sequencing.

Results: Abnormalities were reported in 73 chromosomes within the cohort using karyotyping. The concordance with OGM, defined as the presence of large SVs in the same chromosomes, was 89% (65/73). The concordance between OGM and FISH was 100% (20/20). Importantly, OGM provided additional important information for all the analyzed CK-MDS cases. This included the detection of SVs >5Mb in chromosomes that were not specified by karyotyping to harbor abnormalities (93% of cases) and smaller SVs overlapping with leukemia-associated genes (53% of cases). Our analysis also confirmed the presence of three in-frame gene-fusions, including NUP98::PRRX2, and two novel fusions.

Conclusion: This study supports the application of OGM for CK-MDS cases to enhance SV detection and acquiring gene-level information. OGM could be effective as either a first-tier test or in combination with karyotyping. Furthermore, our results underscore the utility of OGM as a research tool in cancer samples with complex genomic rearrangements.

Grants: Sigrid Juselius Foundation [220111], Academy of Finland [338374].

Conflict of Interest: None declared

P01.052.D Developing the UK infrastructure for the identification and management of patients with germline predisposition to haematological malignancy.

Olga Tsoulaki¹, Beverley Speight², James Drummond², Clair Engelbrecht³, Jude Fitzgibbon⁴, Shreyans Gandhi³, Angela Hamblin⁵, Helen Hanson^6;7, Steven Hardy⁸, Jamshid Khorashad⁹, Austin Kulasekararaj³, Joanna Large³, Paula Page¹⁰, Nicholas Parkin³, Ana Rio-Machin⁴, Polly Talley¹¹, Kate Tatton-Brown¹², Roochi Trikha³, beth torr¹³, Clare Turnbull¹³, Sarah Westbury¹⁴, Christopher Wragg¹⁵, Terri McVeigh⁹, Katie Snape ¹

¹St George’s University of London, London, United Kingdom; ²Addenbrooke’s Hospital, East Anglian Medical Genetics Service, Cambridge, United Kingdom; ³Kings College Hospital NHS Foundation Trust, Dept of Haematology, London; ⁴Barts Cancer Institute, Queen Mary University, Centre for Haemato-Oncology, London; ⁵Oxford University Hospitals NHS Foundation Trust, Dept of Haematology, Oxford; ⁶; ⁷Royal Devon University Healthcare NHS Foundation Trust, Department of Clinical Genetics, Exeter, United Kingdom; ⁸Genomics and Rare Disease, National Disease Registration Service, Leeds; ⁹Royal Marsden NHS Foundation Trust, Centre for Molecular Pathology, London, United Kingdom; ¹⁰Birmingham Women’s and Children’s NHS Foundation Trust, West Midlands Regional genetics Laboratory, Birmingham, United Kingdom; ¹¹St James’s University Hospital, North East and Yorkshire Genomic Laboratory Hub, United Kingdom; ¹²NHS England, National Genomics Education, Birmingham; ¹³Institute of Cancer Research, Division of Genetics and Epidemiology, London; ¹⁴University of Bristol, School of Cellular and Molecular Medicine, Bristol, United Kingdom; ¹⁵North Bristol NHS Trust, Bristol Genetics Laboratory, Bristol

Background/Objectives: Use of genomic technologies in the investigation of haematological malignancies has led to an increase in the identification of disease associated genetic variants which may also confer inherited susceptibility. We report a collaborative approach supported by the CanGene-CanVar research programme and UK Cancer Genetics Group to develop a clinical and educational infrastructure to improve clinical pathways for the assessment and management of germline predisposition to haematological malignancies across the UK National Health Service (NHS).

Methods:

1. 1.

   An online National consensus group meeting was convened with stakeholders across clincial and laboratory services alongside policy makers from NHS England. Stakeholders completed pre-meeting questionnaires, followed by in-meeting polling on consensus statements. Consensus was deemed to be reached when ≥80% respondents selected ‘Agree/Strongly Agree’ or ‘Yes’ in response to the statement posed.

2. 2.

   An online survey based gap and training needs analysis (TNA) was undertaken across the relevant healthcare workforce to identify and analyse related training needs mapped to clinical pathways.

Results: 146 stakeholders registered for participation in the consensus meeting. Consensus was reached on 28 statements across the whole clinical-laboratory pathway. 272 healthcare workers participated in the TNA. This showed significant variation across the workforce in knowledge and skills required to deliver these clinical pathways.

Conclusion: A national clinical/scientific collaboration enabled the publication of consensus guidelines. Identification of training needs highlighted a requirement for educational resources. We present updates on the UK national educational and clinical infrastructure to iteratively improve clinical care pathways for patients with germline predisposition to haematological malignancies.

Grants: CanGene-CanVar (C61296/A27223)

Conflict of Interest: None declared

P01.053.A Identifying genetic vulnerabilities and trascriptional programs in TERT-promoter-mutant glioblastoma

Kevin Tu ^1;2, Connor Stewart^2;3, Peter Hendrickson², Joshua Regal^2;4, So Young Kim², David Ashley², Matthew Waitkus², Zachary Reitman²

¹Cancer Research UK Cambridge Institute, United Kingdom; ²Duke University, Durham, United States; ³Emory University, Atlanta, United States; ⁴Roswell Park Comprehensive Cancer Center, Buffalo, United States

Background/Objectives: Mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are early somatic mutations that occur in >80% of glioblastomas (GBMs). TERT promoter mutations (TPMs) create a neomorphic E-twenty-six (ETS) transcription factor binding site which reactivates TERT expression and drives GBM immortality. Directly targeting TERT and transcription factors has historically proven challenging. Here, we seek to identify potential targets upstream of TPMs using pooled genetic screens.

Methods: We generated T98G-TERT-ON, a TPM + GBM cell line with inducible TERT expression. We performed a minature pooled CRISPR/Cas9 screen in T98G-TERT-ON against TERT and GABPB1L, a critical ETS subunit that binds to TPM+ promoters. We annotated TPM status for >400 cell lines and 500 primary GBMs, then conducted comparative analyses between TPM status using genetic dependency data and/or gene expression profiles.

Results: We did not detect a dependency for TERT or GABPB1L in the CRISPR/Cas9 screen in T98G-TERT-ON. Similarly, TERT was not a dependency in TPM + GBM cell lines within the computational cohort. The ETS subunits GABPB1 and GABPA were dependencies within both TPM+ and TPM- cell lines. TPM+ cell line expression profiles were regulated by multiple ETS transcription factors. Moreover, in primary GBMs, TERT expression was strongly associated with the expression of ETS transcription factors ELF5, ETV1, ETV5, ERF, ETV4, ETV2, ETS2, GABPB1, SPI1.

Conclusion: Our findings suggest multiple ETS-factor regulate TERT interchangeably and/or redundantly in a cellular context-dependent manner, posing challenges in targeting TPMs through upstream regulators. Approaches to target other aspects of TPM biology merit attention.

Grants: NCI-K08256045l; U19CA264385; P30CA014236; P50CA190991

Conflict of Interest: Kevin Tu: None declared, Connor Stewart: None declared, Peter Hendrickson: None declared, Joshua Regal: None declared, So Young Kim: None declared, David Ashley: None declared, Matthew Waitkus: None declared, Zachary Reitman ZJR is listed as an inventor for intellectual property related to genetic testing for TERT and other alterations in brain tumors that is managed by Duke Office of Licensing and Ventures and has been licensed to Genetron Health.

P01.054.B First case of congenital diffuse colonic hamartomatous polyposis, genetically confirmed as Peutz-Jeghers syndrome

Didac Casas-Alba ¹, Diana Salinas-Chaparro¹, Antonio Federico Martínez-Monseny¹, Loreto Martorell Sampol¹, Jordi Genoves¹, Daniel Castillo¹, Laura Martí¹, Adrián Alcalá San Martín¹, Cristina Hernando-Davalillo¹, Moira Garraus Oneca², Juan Pablo Muñoz², Victor Vila Miravet³, Silvia Planas Roman⁴, Miguel Bejarano Serrano⁵, Pedro Palazon Bellver⁵, Ruth del Rio Florentino⁶, Lluisa Hernandez Platero⁷, Sara Bobillo Perez⁷, Marina Pons Espinal⁸, Aina Martinez Planas⁸, Francesc Palau¹, Hector Salvador²

¹Sant Joan de Déu Barcelona Hospital, Genetics Department, Esplugues de Llobregat, Spain; ²Sant Joan de Déu Barcelona Hospital, Department of Oncology and Hematology, Esplugues de Llobregat, Spain; ³Sant Joan de Déu Barcelona Hospital, Pediatric Gastroenterology Hepatology and Nutrition Unit, Esplugues de Llobregat, Spain; ⁴Sant Joan de Déu Barcelona Hospital, Department of Pathology, Esplugues de Llobregat, Spain; ⁵Sant Joan de Déu Barcelona Hospital, Department of Pediatric Surgery, Esplugues de Llobregat, Spain; ⁶Sant Joan de Déu Barcelona Hospital, Neonatal Unit, Esplugues de Llobregat, Spain; ⁷Sant Joan de Déu Barcelona Hospital, Pediatric Intensive Care Unit Service, Esplugues de Llobregat, Spain; ⁸Sant Joan de Déu Barcelona Hospital, Paediatrics Department, Esplugues de Llobregat, Spain

Background: Pathogenic variants in the STK11 gene cause Peutz-Jeghers syndrome (PJS) (OMIM#175200). PJS-type hamartomatous polyps have been reported in only three newborns without genetic confirmation, with the youngest confirmed case documented at 6 months. Infant cases involved either the small bowel or stomach.

Methods: We present a 5-month-old male, second child of non-consanguineous parents, with an unremarkable familial history. Prenatal ultrasounds revealed an arachnoidal cyst and polyhydramnios. At birth, he presented marked abdominal distension. An abdominal ultrasound revealed diffuse thickening of the colon, a hepatic arteriovenous malformation, and multiple hepatic lesions suggesting hemangiomas. No mucocutaneous pigmentation was observed. After inconclusive studies, the condition worsened with subocclusion episodes, leading to subtotal colectomy at 2 months. Pathological study revealed diffuse colon involvement with hamartomatous polyposis.

Results: Clinical exome sequencing identified two heterozygous variants in STK11. First, an exon 1 deletion, further confirmed by MLPA, spanning at least 1300 bp and including the promoter region and start codon. This deletion, previously reported in PJS, was classified as pathogenic following ACMG criteria. Second, a missense variant of unknown significance (NM_000455.5:c.525G > C/p.Lys175Asn). Digital PCR and direct sequencing validated the patient’s results and showed that the variants were absent in both parents. Microsatellite marker genotyping further supported declared paternity and maternity. Long-range PCR revealed a cis configuration. Array-CGH was normal.

Conclusion: This is the first case of congenital presentation of diffuse colonic hamartomatous polyposis, which was genetically confirmed as PJS. The codominant trans configuration hypothesis was ruled out, leaving other explanations for early onset severity under consideration.

Conflict of Interest: None declared

P01.055.C Genetic and Epigenetic characterization of B-cell neoplasms with IG enhancer hijacking

Cosima Drewes ¹, Cristina López², Nnamdi Okeke¹, Sina Hillebrecht¹, Petra Schuetz¹, Anja Fischer¹, Anja Mottok¹, Susanne Bens¹, Christof Schneider³, Stephan Stilgenbauer³, Eugen Tausch³, Reiner Siebert¹

¹Institute of Human Genetics, Ulm University and Ulm University Medical Center, Ulm, Germany; ²Institut d’Investigaciones Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; ³Division of CLL, Department of Internal Medicine III, Ulm University Medical Center, Ulm, Germany

Background/Objectives: Oncogene translocations leading to enhancer hijacking from one of the immunoglobulin (IG) loci, such as IGH::MYC, IGH::BCL2, or IGH::CCND1, are pathognomonic aberrations of several lymphoma entities. In Chronic Lymphocytic Leukemia (CLL), e.g. BCL2 and BCL3 are oncogenes recurrently targeted by IG translocations. We here aimed to comprehensively characterize CLL with IG translocations at the genetic and epigenetic level.

Methods: B-cell neoplasms with IG translocations and respective partner oncogenes as well as recurrent imbalances were identified by FISH (n = 325). DNA methylation and CNV analyses were performed using Infinium® MethylationEPIC (EPIC) BeadChip arrays (n = 313). The IGHV mutation status was determined by Sanger Sequencing (n = 284).

Results: We investigated 313 B-cell neoplasms, including 305 CLLs, with IG translocation affecting various partners. (IGH::BCL2: n = 43, IG::BCL3: n = 89, IG::MYC: n = 41, IGH::unknown partner: n = 140). From the IGH::BCL2-translocated CLLs, 87% (33/38) carried a mutated IGHV, 37% (16/43) trisomy 12 and 35% (15/43) 13q deletion. In contrast, IG::BCL3-translocated CLLs exhibited in 96% (72/75) unmutated IGHV and in 59% (50/84) trisomy 12, whereas 13q deletion was rare (12%, 11/84). In unsupervised DNA methylation analyses, IGHV mutation status segregated subgroups of CLLs with best discrimination applying a threshold of ≥99% for unmutated IGHV. Moreover, despite confounding by the IGHV mutation status, also IGH::BCL2 and IG::BCL3-translocated CLLs segregated between them and also from other CLLs. IGH::MYC-translocated CLL and IGH::unknown partner CLL built no specific groups.

Conclusions: Our genetic and epigenetic analyses suggest that IGH::BCL2 and IG::BCL3-translocated CLLs form two distinct subgroups of CLL based on DNA methylation, IGHV mutation status, and CNVs.

Grants: SFB1074 B10

Conflict of Interest: None declared

P01.056.D Exome sequencing identified rare recurrent copy number variants and hereditary breast cancer susceptibility

Tuomo Mantere ¹, Timo Kumpula¹, Sandra Vorimo¹, Taneli Mattila², Luke O’Gorman³, Galuh Astuti³, Anna Tervasmäki¹, Susanna Koivuluoma¹, Tiina Mattila¹, Mervi Grip⁴, Robert Winqvist¹, Outi Kuismin⁵, Jukka Moilanen⁵, Alexander Hoischen³, Christian Gilissen³, Katri Pylkäs¹

¹Laboratory of Cancer Genetics and Tumor Biology, Research Unit of Translational Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland; ²Department of Pathology, Oulu University Hospital and University of Oulu, Oulu, Finland; ³Department of Human Genetics and Radboud Institute of Medical Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands; ⁴Department of Surgery, Oulu University Hospital and University of Oulu, Oulu, Finland; ⁵Department of Clinical Genetics, Medical Research Center Oulu and PEDEGO Research Unit, Oulu University Hospital and University of Oulu, Oulu, Finland

Background/Objectives: Copy number variants (CNVs) are a major source of genetic variation and can disrupt genes or affect gene dosage. They are known to cause or contribute to the predisposition to various diseases. However, the role of CNVs in inherited breast cancer (BC) susceptibility has not been thoroughly investigated.

Methods: We performed a whole-exome sequencing-based analysis of rare CNVs in 98 high-risk Northern Finnish BC cases. After filtering, selected candidate alleles were validated and characterized using a combination of orthogonal methods, including optical genome mapping and long-read sequencing. The variants were genotyped in geographically matched cases and controls, comprising 278 hereditary and 1983 unselected BC cases, as well as 1229 controls.

Results: Analysis revealed three recurrent alterations: a 31 kb deletion co-occurring with a retrotransposon insertion (delins) in RAD52, a 13.4 kb deletion in HSD17B14 and a 64 kb partial duplication of RAD51C. The RAD52 delins and HSD17B14 deletion both showed significant enrichment among cases with indications of hereditary disease susceptibility. RAD52 delins was identified in 7/278 cases (P = 0.034, OR = 2.86) and HSD17B14 deletion in 8/278 cases (P = 0.014, OR = 3.28), the frequency of both variants in the controls being 11/1229. The RAD51C duplication was identified in 2/278 of hereditary cases and 2/1229 controls (P = 0.157, OR = 4.45).

Conclusion: This study suggests a role for RAD52 and HSD17B14 in hereditary BC susceptibility, highlighting the importance of studying CNVs alongside single nucleotide variants in the search for genetic factors underlying hereditary disease predisposition.

Grants: Academy of Finland [307808], Cancer Foundation of Finland and Sigrid Jusélius foundation.

Conflict of Interest: None declared

P01.057.A Identification of genetic tumor risk syndromes in individuals with non-small cell lung cancer within the German National Network Genomic Medicine Lung Cancer (nNGM)

Rebecca Grüneis ^1;2;3;4;5;6, Marie Arlt^1;2;3;4;5;6, Oliver Kutz^1;2;3;4;5;6, Judith Böthig^1;2;3;4;5;6, Sarah Wölffling^1;2;3;4;5;6, Tim Hutschenreiter^1;2;3;4;5;6, Karl Hackmann^1;2;3;4;5;6, Doreen William^1;2;3;4;5;6, André Reis⁷, Arndt Hartmann^8;9, Stefan Gattenlöhner¹⁰, Axel Hillmer^11;12;13, Arne Jahn^1;2;3;4;5;6, Reinhard Büttner^11;12;14, Evelin Schröck^1;2;3;4;5;6

¹Institute for Clinical Genetics, University Hospital Carl Gustav Carus at TUD Dresden University of Technology and Faculty of Medicine of TUD Dresden University of Technology, Dresden, Germany; ²ERN GENTURIS, Hereditary Cancer Syndrome Center Dresden, Germany; ³National Center for Tumor Diseases (NCT), NCT/UCC Dresden, a partnership between German Cancer Research Center (DKFZ), Faculty of Medicine and University Hospital Carl Gustav Carus, TUD Dresden University of Technology and Helmholtz-Zentrum Dresden-Rossendorf (HZDR), Germany; ⁴German Cancer Consortium (DKTK), Dresden, Germany; ⁵German Cancer Research Center (DKFZ), Heidelberg, Germany; ⁶Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; ⁷Institute of Human Genetics, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany; ⁸Institute of Pathology, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany; ⁹Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), Universitätsklinikum Erlangen, FAU Erlangen-Nürnberg, Erlangen, Germany; ¹⁰Institute for Pathology, Justus Liebig University, Gießen, Germany; ¹¹Institute of Pathology, University Hospital and Medical Faculty, University of Cologne, Cologne, Germany; ¹²Network Genomic Medicine, Cologne, Germany; ¹³Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany; ¹⁴Lung Cancer Group Cologne, Department I for Internal Medicine, University Hospital Cologne, Cologne, Germany

Background/Objectives: Very recent studies reported pathogenic germline variants in up to 10% of non-small cell lung cancer (NSCLC) patients. Possible implications are (experimental) targeted therapy or interventions, adjusted cancer surveillance, and predictive testing for family members. The “national Network Genomic Medicine (nNGM) Lung Cancer” offers multicentric somatic gene panel testing for patients with advanced NSCLC throughout Germany. Within this network the Task Force 5 “Genetic Tumor Risk” investigates pathogenic germline variants and further oncogenic somatic alterations by parallel whole-exome/ whole-genome sequencing.

Methods: Inclusion criteria favor never-smokers, young age of onset, multiple cancer diseases and a family cancer history. Whole-genome sequencing of control samples (University Hospital Dresden) and whole-exome sequencing of formalin-fixed paraffin-embedded lung cancer tissues (University Hospital Cologne) were performed. Germline variants were assessed in 141 cancer predisposition genes and somatic variants were evaluated to identify the relevance of germline variants for tumorigenesis and alternative drivers. Clinical information and follow-up information were collected.

Results: 103 individuals have been included. Median age of onset was 57.5 years (IQR: 47.8;67.5), 30% (n = 31) were never-smokers, 43% (n = 44) had multiple tumor diseases, and 73% (n = 75) a positive family cancer history. Prospective germline sequencing of 72 individuals revealed 10 pathogenic heterozygous variants in two dominant (BRCA2, RAD51D) and six recessive cancer predisposition genes.

Conclusion: This ongoing prospective, multicentric study identifies pathogenic germline variants in selected NSCLC patients and investigates associations with clinical and molecular parameters. An increased cohort size and extended bioinformatic analysis will allow assessment of pathogenic germline variant yields and their clinical implications for NSCLC patients.

Grants: DKH 13006795

Conflict of Interest: None declared

P01.058.B Whole exome sequencing revealed GATA2 rare mutations in Bulgarian patients with personal/family history of breast cancer

Svilen Maslyankov ¹, Kalina Belemezova², Tsvetan Popov^2;3, Dimitar Bakalov², Radka Tafradzhyiska², Irina Miteva³, Ivanka Dimova²

¹Second surgery clinic, Department of Surgery, Medical Faculty, Medical University Sofia, Sofia, Bulgaria; ²Medical University Sofia; ³Second surgery clinic, Department of Surgery, Medical Faculty, Medical University Sofia

Background/Objectives: The significance of GATA2 mutations in cancer risk is an area of ongoing research, and our understanding of the role of this gene in cancer continues to evolve. While GATA2 mutations are associated with an increased risk of certain hematological malignancies, they are not as commonly implicated in solid tumors, such as breast or ovarian cancers. We aimed to characterize the GATA2 genetic variants, revealed in BRCA1/2 negative Bulgarian women with strong indications for familial breast cancer.

Methods: After DNA isolation from peripheral venous blood, whole exome sequencing was performed using Illumina technology.

Results: In two women rare genetic variants in GATA2 gene were discovered. The variant c.829A > G (p.Ser277Gly) was revealed in a woman with strong family history for breast cancer. The variant c.669G > A (p.Met223Ile) was found in 42-years old woman affected by bilateral invasive breast cancer from non-specific type (NST), which was Her2-positive; the woman had grand-mother and aunt affected by gynecological cancer. Both variants have been reported in patients with leukemia syndromes.

Conclusion: A comprehensive evaluation of the individual’s personal and family history is essential in determining the most appropriate genetic testing strategy. The landscape of genetic testing for hereditary cancer is continually evolving, and new genes associated with breast cancer risk may be identified over time. GATA2 is a key transcriptional regulator of hematopoiesis involved in the development of monocytes, mast cells, NK cells, and megakaryocytes. Its deficiency could be predisposing factor to breast cancer development as well.

Funding: Project № BG-RRP-2.004-0004-C01.

Conflict of Interest: None declared

P01.059.C Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 by hybrid minigenes

Lara Sanoguera-Miralles ¹, Inés Llinares-Burguet¹, Elena Bueno-Martínez¹, Lobna Ramadane-Morchadi², Cristiana Stuani³, Alberto Valenzuela-Palomo¹, Alicia García-Álvarez¹, Pedro Pérez-Segura², Emanuele Buratti³, Miguel de la Hoya², Eladio A. Velasco-Sampedro¹

¹Instituto de Biomedicina y Genética Molecular de Valladolid (IBGM), Consejo Superior de Investigaciones Científicas – Universidad de Valladolid (CSIC-UVa), Splicing and Genetic Susceptibility to Cancer Laboratory, Valladolid, Spain; ²Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), Molecular Oncology Laboratory, Madrid, Spain; ³International Centre of Genetic Engineering and Biotechnology (ICGEB), Molecular Pathology Laboratory, Trieste, Italy

Background/Objectives: Exon recognition is controlled by a large set of splicing regulatory elements (SREs), such as splicing enhancers and silencers. Variants at these motifs may impair splicing and lead to disease-associated loss-of-function transcripts. Our aim was to investigate the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2 with a view to identifying spliceogenic SRE variants.

Methods: We used a CHEK2 minigene with exons 6–10 that produced the expected minigene full-length transcript and replicated naturally occurring events, in particular the skipping of exons 8 and 10. By site-directed mutagenesis, 12 internal microdeletions were incorporated in order to map SRE-rich intervals of both exons by splicing assays in MCF-7 cells. Then, 87 variants located in critical SRE intervals were introduced into the wild-type minigene and functionally tested.

Results: We identified three minimal regions (10-, 11- and 15-nt) essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Of the 87 variants tested in these intervals, thirty-eight (44%) impaired splicing, four of which (c.883G > A, c.883G > T, c.884A > T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full-length transcript.

Conclusion: Any exonic change is capable of disrupting splicing, since thirty-three out of the 38 spliceogenic variants were annotated as missense, three as nonsense and two as synonymous. Moreover, c.883G>A, c.883G>T and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP-based criteria.

Grants: Predoctoral fellowship from the AECC-Scientific Foundation, Sede Provincial de Valladolid (2019–2023); ISCIII (PI20/00225); Consejería de Educación, Junta de Castilla y León.

Conflict of Interest: None declared

P01.060.D Molecular cytogenetic characterization of Polyploid Giant Cancer Cells (PGCCs) in cytarabine-resistant AML cells

Pei-Yi Chen ^1;2, Yu-Xuan Lin², Chia-Ling Wu¹, Jui-Hung Yen²

¹Laboratory of Medical Genetics, Genetic counseling center, Hualien Tzu Chi Hospital, Hualien, Taiwan; ²Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien, Taiwan

Background/Objectives: Polyploid giant cancer cells (PGCCs) are cells with multiple nuclei or a single giant nucleus containing multiple sets of chromosomes. These cells exhibit cancer stem cell characteristics, regulate the cancer microenvironment, and are highly associated with drug resistance, metastasis, and cancer relapse. While PGCCs have been extensively studied in solid tumors, their role in liquid tumors remains unclear. We generated two AML PGCC cell lines derived from MV4-11 and MOLM13 cells through prolonged exposure to cytarabine. This study aims to further investigate the cytogenetic characteristics of these AML PGCCs.

Methods: Cell viability was assessed using the MTT assay. Cell morphology was observed through cytospin/Giemsa staining and immunofluorescence analysis. The chromosomal polyploidy level was measured using karyotyping, FISH, and flow cytometry. Molecular characteristics of the PGCCs were assessed using microarray analysis, RT-q-PCR, and western blot analysis.

Results: Drug resistance was confirmed by a pronounced increase in cell viability in the derived cells after cytarabine treatment. Immunofluorescence staining revealed that a portion of the resistant cells displayed multiple nuclei and giant cell size, resembling the so-called PGCCs. Interphase FISH was conducted to demonstrate a significant increase in the polyploid cell population detected in these PGCC cell lines. Microarray analysis showed differential gene expression and signaling pathways associated with the PGCCs compared to the parent cells.

Conclusion: We successfully established AML PGCCs and assessed their biological features. Our data provide information to elucidate the characteristics of PGCCs in liquid tumors, including phenotypes, genotypes, and crucial molecular mechanisms.

Grants: NSTC-112-2320-B-320-002-MY3 and NSTC-112-2320-B-303-003

Conflict of Interest: None declared

P01.061.A Importance of a bioinformatics pipeline for the detection of Alu in the NGS panel of hereditary cancer genes

Ricardo Blázquez Martín ¹, José Manuel Sánchez Zapardiel¹, Celia Amil Manjón¹, Pilar Duarte García¹, Marina Ibáñez Vizcaíno¹, Francisca Luengo Sainz de Baranda¹, Victoria Carrero Blázquez¹, Beatriz Hidalgo Calero¹, Montserrat de Miguel¹, Juan de Dios García Díaz²

¹Hereditary Cancer Unit, Hospital 12 de Octubre. Madrid, Department of Clinical Analysis-Clinical Biochemestry, Madrid, Spain; ²Clinical Genetic Unit, Hospital Principe de Asturias, Madrid, Department Internal Medicine, Alcalá de Henares, Spain

Background/Objectives: Alu elements are the most abundant transposable elements, they are short interspersed elements (SINEs) ~300 nucleotides in length. Transposable elements are rare sequences of DNA that can move themselves to new positions within the genome and may be linked to genetic diseases and susceptibility to certain disorders, including cancers. In our case, it is a case of a family with a multiple history of breast, ovarian, pancreatic and prostate cancer at an early age. After a non-informative study, it was reprocessed with the multigene panel we are currently working with.

Methods: Next-generation sequencing was performed using the Hereditary Cancer Solution kit (Sophia Genetics) and sequencing on the NextSeq platform (Illumina). Results were analyzed using Sophia-DDM software.

Results: The bioinformatic pipeline detected an Alu element in exon 5 of PALB2 gene, likely resulting in a premature stop codon and subsequent production of a truncated or ausent protein by NMD mechanism. According to American College of Medical Genetics (ACMG) criteria it was classified as pathogenic. However, the exact location, the complete sequence and the generated break points of this Alu element could not be determined and an orthogonal method is required.

Conclusion: It is important to have a bioinformatics pipeline capable of identifying Alu elements in families with a long history of cancer and at very young ages that have been previously uninformative. An accurate characterisation of these elements and a correct genetic counselling is crucial for a correct clinical management for patients and their families.

Conflict of Interest: None declared

P01.062.B Rare genetic phenomena in TP53 can lead to a misdiagnosis of Li-Fraumeni Syndrome

Vasiliki Dellatola ¹, Paraskevi Apostolou¹, Jan Traeger-Synodinos², MARIA TZETIS², DRAKOULIS YANNOUKAKOS¹, Irene Konstantopoulou¹, Florentia Fostira¹

¹Human Molecular Genetics Laboratory, Institute of Nuclear & Radiological Sciences and Technology, Energy & Safety, National Center for Scientific Research “Demokritos”, Athens, Greece; ²Laboratory of Medical Genetics, St. Sophia’s Children’s Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece

Background/Objectives: Next-generation sequencing of germline DNA extracted from whole blood can reveal TP53 Pathogenic/Likely Pathogenic (P/LP) variants in low Variant Allele Frequencies (VAF ≤30%). These might be attributed to mosaicism, circulating tumor DNA, or clonal hematopoiesis, a process induced by advanced age and chemotherapy. Herein, we aimed to differentiate these rare phenomena in a selected group of patients with cancer diagnoses focusing on association to Li-Fraumeni Syndrome (LFS).

Methods: This study included 14 patients with TP53 P/LP variants of low VAF (7-30%) and a diagnosis of breast (5), ovarian (4), colorectal (3), endometrial (1), and prostate cancer (1). The mean age at diagnosis was 58.6 years (range:28-78 years). Re-sampling of blood/saliva (mesoderm) and re-analysis was performed >3 months after initial testing. If the TP53 variant was still evident, DNA from hair follicles (ectoderm) was analyzed through Sanger sequencing. Parameters considered involved patient age, time at genetic testing, personal history, and disease burden.

Results: Through re-sampling, the TP53 variant under investigation was not present in 85.7% (12/14) of patients, indicating either clonal hematopoiesis or the presence of circulating tumor DNA. Two patients (14.3%) proved to have the underlying TP53 variant in mosaicism, predisposing for LFS, upon validation in both mesodermal and ectodermal tissues. Notably, both individuals had breast cancer and one met the genetic testing criteria for LFS.

Conclusion: Accurate delineation of TP53 variants origin and their association with LFS is imperative for tailored clinical interventions. This is of high importance in regions lacking systematized genetic centers, such as Greece.

Conflict of Interest: Vasiliki Dellatola: None declared, Paraskevi Apostolou: None declared, Jan Traeger-Synodinos: None declared, MARIA TZETIS: None declared, DRAKOULIS YANNOUKAKOS: None declared, Irene Konstantopoulou: None declared, Florentia Fostira AstraZeneca, Roche Pharmaceuticals, Takeda, GSK and AstraZeneca

P01.063.C Surveillance in children with PTEN Hamartoma Tumor Syndrome: the yield of yearly thyroid ultrasound in a Dutch expertise centre

Esther Bormans¹, Janneke Schuurs-Hoeijmakers ², Petra van Setten³, Linda Hendricks^2;4, Meggie Drissen^2;4, Martin Gotthardt⁵, Hedi Claahsen³, Nicoline Hoogerbrugge^2;4, Jolanda Schieving¹

¹Radboud University Medical Centre, Amalia Children’s Hospital, Department of Pediatric Neurology, Nijmegen, Netherlands; ²Radboud University Medical Centre, Department of Human Genetics, Expertise centre for PHTS, Nijmegen, Netherlands; ³Radboud University Medical Centre, Amalia Children’s Hospital, Department of Pediatric Endocrinology, Nijmegen, Netherlands; ⁴Radboud University Medical Centre, Radboud Institute for Medical Innovation, Nijmegen, Netherlands; ⁵Radboud University Medical Centre, Department of Medical Imaging, Nuclear Medicine, Nijmegen, Netherlands

Consortium: -

Background/Objectives: Children with PTEN hamartoma tumor syndrome (PHTS) are at increased risk for developing thyroid abnormalities, including differentiated thyroid carcinoma (DTC). The Dutch PHTS guideline recommends ultrasound surveillance starting from age 18. The literature describes PHTS patients who developed DTC before age 18, the Dutch PHTS expertise centre has initiated annual ultrasound surveillance starting from age 12. We describe the yield of thyroid ultrasound surveillance in these children with PHTS.

Methods: A retrospective single centre cohort study was conducted. Pediatric PHTS patients who received thyroid ultrasound surveillance before age 18 between 2016–2023 were included. Patients’ medical records have been reviewed. Primary outcomes included prevalence and time to develop thyroid nodules ≥10mm, nodular growth, goiter, thyroiditis and DTC. Descriptive statistics and Kaplan-Meier analyses were performed.

Results: Forty-three patients were included. Two patients (5%) were diagnosed with DTC at ages 12 and 17. Both DTCs were identified as minimally invasive follicular carcinoma at stages pT3NxMx and pT1NxMx. A total of 84% were diagnosed with thyroid abnormalities at a median age of 12 years (range 9-18, table 1). Most common findings were benign, including nodular disease (74%), goiter (30%) and autoimmune thyroiditis (12%). Nodular growth was observed in 14 patients (33%) resulting in (hemi)thyroidectomy in 7 patients (16%).

Conclusion: Thyroid ultrasound surveillance resulted in the detection of DTC in 2/43 PHTS patients before age 18. These findings support the recommendation to initiate thyroid ultrasound surveillance in children from age 10 – 12 onwards, preferably within an expertise centre.

Grants: None.

Conflict of Interest: None declared

P01.064.D Chloroquine-induced DNA damage synergizes with DNA repair inhibition to cause head and neck cancer cell death

Diego Iglesias-Corral ^1;2;3, marta lópez alonso², Lucía garcía collado², Nuria Arroyo-Garrapucho^1;2;3, Juan-Luis García^1;3, Rogelio González-Sarmiento^1;2;3, Ana-Belén Herrero^1;2;3

¹Biomedical Research Institute of Salamanca (IBSAL), IIMD-07, Salamanca, Spain; ²University of Salamanca, Faculty of Medicine, Department of Medicine, Unit of Molecular Medicine, Salamanca, Spain; ³Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain

Background/Objectives: Head and neck cancer (HNC) is a heterogeneous group of malignancies that accounts for 450.000 deaths annually. Tumor heterogenicity, along with the emergence of resistances to conventional treatments highlight the necessity for new therapies. Chloroquine (CQ) is a well-known autophagy inhibitor that induces DNA double-strand breaks (DSBs) through reactive oxygen species (ROS) production. We have previously described in in vitro studies that the combination of CQ with inhibitors of homologous recombination (HR) and nonhomologous end-joining (NHEJ) DNA repair pathways is a promising approach for the treatment of different tumors.

Methods: HNC cell lines 32816 (generated in our laboratory) and CAL-33 were used. The combination of CQ with the HR inhibitor, Panobinostat (LBH) or NHEJ inhibitors (KU-57788, NU-7026) were tested. Antitumoral activity was evaluated by proliferation and apoptosis assays and synergistic interaction between the drugs was calculated using the Chou-Talalay method. DSB induction was tested by γH2AX foci quantification via immunofluorescence assays. To evaluate HR efficiency, cells were transfected with a GFP-reporter construct, then treated with LBH for 48 h. HR proficient cells (GFP + ) were quantified by flow cytometry.

Results: All drugs tested inhibited cell proliferation and induced apoptosis in the studied cell lines. The combination of CQ with either HR or NHEJ inhibitors exhibited a synergistic effect on apoptosis induction that was largely prevented by antioxidant addition.

Conclusion: Our studies suggest that the combination of CQ and inhibitors of DNA-repair pathways could be a new therapeutic approach against HNC.

Grants: Study funded by PI20/01569.

Conflict of Interest: None declared

P01.065.A A Canadian Lab’s Experience Using Optical Genome Mapping To Clinically Genotype Hematological Neoplasms

Zeid Hamadeh ^1;2, Eric McGinnis^1;2, Tara Spence^1;2

¹The University of British Columbia, Medicine, Vancouver, Canada; ²Vancouver General Hospital, Cytogenomics, Vancouver, Canada

Background/Objectives: Diagnosis and management of hematologic malignancies relies on detection of acquired genetic abnormalities in cancer cells. We report the added yield of optical genome mapping (OGM) observed in our retrospectively selected clinical validation cohort of hematologic neoplasms.

Methods: We comprehensively assessed 83 patients by both standard-of-care (SOC) techniques and OGM to validate OGM as a routine clinical test for acute myeloid leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, and chronic lymphocytic leukemia. Samples included 61 bone marrow aspirates (BMAs) and 22 peripheral bloods received by our laboratory for routine karyotyping and/or FISH.

Results: We observed high concordance with SOC methods with a sensitivity of 97.6%, specificity of 100%, and accuracy of 98.6%. OGM identified or clarified findings either cryptic or not resolvable using SOC, decreasing the proportion of with normal karyotype by 23% of BMAs, identifying chromothripsis in 13% of all cases, identifying cytogenetically visible or recurring variants in 46% of BMAs, and generating prognostically or diagnostically relevant findings in 42% of BMAs. Additional findings included, but were not limited to, three KMT2A rearrangements, a KMT2A partial tandem duplication, a RUNX1::MECOM rearrangement, a NUP214::ABL1 fusion, and two submicroscopic TP53 deletions.

Conclusion: OGM shows significant power in detecting or resolving abnormalities previously inadequately characterized by SOC testing, permitting refinement of genomics-based diagnosis and risk stratification and potentially altering treatment and prognostication for a portion of patients. Future assessment will be needed in a prospective unselected cohort to definitively establish clinical utility of OGM.

Conflict of Interest: None declared

P01.066.B Role of ATG7 loss in taxane-resistant head and neck cancer

Nerea Gestoso-Uzal ^1;2;3, Laura Molinero-Sicilia^2;3, Mercedes León-López^2;3, Ana-Belén Herrero^1;2;3, Juan-Luis García^2;3, Juan Jesús Cruz-Hernández^1;2;3, Rogelio González-Sarmiento^1;2;3

¹Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain; ²Biomedical Research Institute of Salamanca (IBSAL), University Hospital of Salamanca-USAL- CSIC, Salamanca, Spain; ³Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain

Background/Objective: Head and neck squamous cell carcinomas (HNSCC) include heterogeneous tumours in the cervicofacial area. Combating therapy resistance is one of the main challenges in HNSCC. The main aim of this work was to characterize the molecular mechanisms underlying taxane resistance in HNSCC.

Methods: Taxane resistance was induced in two HNSCC cell lines, 32816 and 32860, by gradually increasing paclitaxel concentration. Cell lines were characterized by aCGH and expression microarrays. Autophagy was analyzed by western blot. CRISPR-Cas9 technology was used to generate ATG7 knockout cell lines. The efficacy of its design was tested by Sanger sequencing, RT-qPCR and western blot.

Results: A 32816 taxane-resistant stable cell line was established, while 32860 cells remained sensitive to taxanes after twenty cycles of treatment. The aCGH study showed a differential deletion in 3p26.6-3p21.1 in 32860 cell line. Expression microarrays identified a significant underexpression of ATG7, a gene located in this region and involved in autophagy. Moreover, the study of the autophagy signalling pathway suggested a blockade of autophagy in 32860 cells, that may be responsible for the 32860 response to taxane resistance induction. To get a deeper understanding of the role of ATG7, we generated ATG7 knockout cell lines from 32816 and confirmed the blockade of autophagy caused by ATG7 loss in them. Further studies are being performed to establish the response of ATG7 knockout cells to taxane resistance induction.

Conclusion: The loss of ATG7 causes a blockade of autophagy in HNSCC that may be associated with the therapeutic response to taxanes.

Grants: This project was funded by FIS-FEDER:PI18/01476.

Conflict of Interest: None declared

P01.067.C digitalMLPA: Multiplex NGS-based assays to detect a wide range of structural variants implicated in hereditary cancer predisposition syndromes

Sebastiaan Lamers ¹, Maria Giovanna Maturo², Janina Luitz², Jonas de Groot³, Martin Loden⁴, Lisette Stolk¹

¹MRC Holland, Cytogenetics, Amsterdam, Netherlands; ²MRC Holland, IVD Development, Amsterdam, Netherlands; ³MRC Holland, Genetic Disorders, Amsterdam, Netherlands; ⁴MRC Holland, Oncogenetics, Amsterdam, Netherlands

Background/Objectives: Hereditary cancer predisposition syndromes (HCPS) account for 5-10% of all cancers. Among these syndromes is Lynch syndrome, a disorder that predisposes carriers of DNA mismatch repair (MMR) gene sequence mutations and copy number variants (CNVs) to early-onset cancer. A recently identified paracentric inversion involving MMR gene MSH2 exon 2-6 may account for a subset of unexplained Lynch syndrome cases.

Methods: An updated version of SALSA^® digitalMLPA™ Probemix D001 Hereditary Cancer Panel 1 and an extended assay, SALSA^® digitalMLPA™ Probemix D002 Hereditary Cancer Panel 2 are in development. The assays incorporate probes for the identification of CNVs and multiple targeted mutations in more than 50 genes linked to HCPS, of which 28 are targeted by both panels. Specifically, two probes were integrated in both panels detecting the 3’ and 5’ breakpoints of the MSH2 exon 2-6 inversion. These probemixes were tested on more than 50 positive samples carrying aberrations in different target regions, including a sample carrying the MSH2 exon 2-6 inversion.

Results: Both assays successfully identified CNVs and targeted mutations in all target regions in the tested samples. Additionally, the MSH2 exon 2-6 inversion was detected with the two inversion-specific probes.

Conclusion: These assays are suitable for detection of a wide range of CNVs involved in HCPS. Furthermore, the integration of probes targeting mutations like the MSH2 exon 2-6 inversion in SALSA^® digitalMLPA™ assays underscores the potential of the digitalMLPA technique in uncovering a broad spectrum of genetic factors involved in HCPS.

Grants:

Conflict of Interest: Sebastiaan Lamers MRC Holland, Maria Giovanna Maturo MRC Holland, Janina Luitz MRC Holland, Jonas de Groot MRC Holland, Martin Loden MRC Holland, Lisette Stolk MRC Holland

P01.068.D Comprehensive molecular profiling of breast cancer subtypes in a Turkish population: Insights from next-generation sequencing analysis

Drenushe Zhurı ¹, Engin Atli¹, Hazal Sezginer Guler¹, Fulya Dusenkalkan¹, Nilay Ozupek¹, Selma Demir¹, Sinem YALÇINTEPE¹, HAKAN GURKAN¹

¹Trakya University, Medical Genetics, Edirne, Türkyie

Background/Objectives: Breast cancer is a heterogeneous disease with diverse molecular subtypes that have implications for prognosis and treatment. The most commonly recognized immunohistochemical classification of breast cancer is based on the expression of the hormone receptors estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2). As a result, the following four breast cancer subtypes are commonly recognized as luminal A, luminal B, HER2-positive, and triple-negative. This study presents the findings from a single-center experience of next-generation sequencing analysis of breast cancer subtypes in the Thrace region.

Methods: We retrospectively analyzed the NGS data of 397 patients with breast cancer who applied to our center between 2020 January-2023 December. After the isolation of DNA, the NextSeq550-Illumina system and the Qiaseq Targeted (Qiagen) Amplicon panel kit were used for molecular analysis. BRCA1/BRCA2 deletion/duplications were evaluated with the Multiplex ligation-dependent probe amplification method.

Results: As a result of analysis for ninety-three targeted cancer genes, we detected eighty-three pathogenic/likely pathogenic (P/LP) variations in seventy-four cancer cases. Twenty-one P/LP variants (25%) in nineteen Luminal A breast cancer cases. Forty-four P/LP variants (53%) in thirty-nine Luminal B breast cancer cases and nineteen P/LP variants (22%) in HER2-positive cases. BRCA1/BRCA2 gene deletion was detected only in one HER2-positive case.

Conclusion: Clinical and pathological characteristics were correlated with molecular subtypes identified by NGS. The diagnosis rate was 18.63% in this study. Our results shed light on the distribution of breast cancer subtypes in the Thrace region and provide insights into the genomic landscape of these tumors in this population.

Conflict of Interest: None declared

P01.069.A Prevalence and consequences of APC mosaicism in patients with colorectal adenomas

Diantha Terlouw¹, Manon Suerink², Monique van Leerdam³, Frederik Hes⁴, Demi van Egmond², Dina Ruano², Anja Wagner⁵, Floris Groenendijk⁶, Sanne ten Broeke⁷, Arjen Mensenkamp⁸, Iris Nagtegaal⁹, Carli Tops¹⁰, Alexandra Langers³, Tom van Wezel², Hans Morreau², Maartje Nielsen ¹⁰

¹Lumc, Clinical genetics, Leiden, Netherlands; ²Lumc, Pathology, Leiden, Netherlands; ³Lumc, Gastroenterology and Hepatology, Leiden, Netherlands; ⁴UZ Brussel, Clinical Genetics, Jette, Belgium; ⁵Erasmus University Medical Center, Clinical Genetics, Rotterdam, Netherlands; ⁶Erasmus University Medical Center, Pathology, Rotterdam, Netherlands; ⁷University Medical Center Groningen, Clinical genetics, Groningen, Netherlands; ⁸Radboud University Medical Center, Molecular genetics, Nijmegen, Netherlands; ⁹Radboud University Medical Center, Pathology, Nijmegen, Netherlands; ¹⁰Lumc, Clinical Genetics, Leiden, Netherlands

Background/Objectives: Although germline pathogenic APC variants are detected in up to 80% of adenomatous polyposis patients, a substantial proportion remains unexplained

Methods: APC mosaicism was analyzed in 458 patients with a broad spectrum of phenotypes in three university medical centers in the Netherlands.

Results: The mosaicism detection rate was 11.1% (51 out of 458) in the entire cohort. This rate was 17.1% (46 out of 269) in patients with ≥10 adenomas before 60 years or with ≥20 adenomas before 70 years and 2.6% (5 out of 189) in patients falling outside Dutch polyposis testing guidelines. Overall, the odds of finding APC mosaicism increased significantly with adenoma count and a younger age at diagnosis. Interestingly, 28% (9 out of 32) of mosaic patients undergoing an esophagogastroduodenoscopy were diagnosed with gastroduodenal adenomas. Moreover, none of the children tested inherited the mosaic variant. In one patient without children, the mosaic variant was detected in semen. Patients having a recurrent colibactin-associated c.835-8A>G variant in some but not all lesions were phenotypically similar to non-mosaic patients.

Conclusion: APC mosaicism was found in over 10% of unexplained polyposis patients. We recommend testing in -at least- all patients with negative germline results with (1) multiple adenomas before 50 years, (2) ≥20 adenomas before 60 years, or (3) ≥30 adenomas before 70 years. Regular colonoscopy and at least one gastroduodenoscopy should be offered; the frequency of follow-up should depend on the findings. Furthermore, consider germline testing for offspring, especially when mosaicism exceeds the colon.

Grants: Dutch Cancer Society (11292).

Conflict of Interest: Diantha Terlouw Dutch Cancer Society Project number: 11292, Manon Suerink: None declared, Monique van Leerdam: None declared, Frederik Hes: None declared, Demi van Egmond: None declared, Dina Ruano: None declared, Anja Wagner: None declared, Floris Groenendijk: None declared, Sanne ten Broeke: None declared, Arjen Mensenkamp: None declared, Iris Nagtegaal: None declared, Carli Tops: None declared, Alexandra Langers: None declared, Tom van Wezel: None declared, Hans Morreau: None declared, Maartje Nielsen: None declared

P01.070.B A Recurrent POT1 Founder Germline Variant Associated with Early Onset Melanoma, Various Malignancies and High Tumor Burden

Aasem Abu Shtaya ^1;2, Inbal Kedar², Lily Bazak², lina basel-salmon², Sarit Farage-Barhom², Ori Segol¹, Michal Naftali³, Marina Eskin-Schwartz⁴, Ohad Shmuel Birk⁴, Shirley Polager-Modan⁵, Nitzan Keidar⁶, Gili Reznick Levi⁷, Ahmad Mahamid⁸, Noy Azulay², Yael Goldberg²

¹Carmel Medical Centre, Unit of Gastroenterology, Haifa, Israel; ²Rabin Medical Center, Recanati Genetics Institute, Petah Tikva, Israel; ³Clalit Genomic Center, Petach Tikba, Israel; ⁴Soroka Medical Center, Genetics Institute, Be’er Sheva, Israel; ⁵Carmel Medical Centre, Genetics Institute, Haifa, Israel; ⁶Schneider- Children’s Medical Center, Pediatric Genetic Unit, Petah Tikva, Israel; ⁷Rambam Health Care Campus, Genetics Institute, Haifa, Israel; ⁸Carmel Medical Centre, Department of Surgery, Haifa, Israel

Background/Objectives: POT1 is a key component of the shelterin complex that plays a critical role in telomere protection and length regulation. Germline variants in the POT1 gene have been implicated in predisposition primarily to malignant melanoma and chronic lymphocytic leukemia. This study reports the identification of POT1 p.I78T, a founder variant among Ashkenazi Jews, and describes a unique clinical landscape in carriers.

Methods: A directed database search was conducted for individuals referred for genetic counselling in 2018 to 2023 who were found to carry the POT1(NM_015450.3) c.233T>C:p.(I78T) germline variant. Demographic, clinical, genetic, and pathology data of patients and family members were analyzed.

Results: Ten carriers of the POT1:p.(I78T) variant aged 25 to 67 years from ten unrelated families of AJ descent were identified. Carriers had a total of 28 primary malignancies (range 1-6); eight carriers aged 25-63 years had recurrent malignant melanoma, three carriers (30%) had desmoid tumors; three (30%) had Papillary thyroid cancer, and four women (57% of female carriers) had breast cancer. Additional tumors included CLL, sarcoma, endocrine tumors and colonic polyps. Review of a local genome database yielded an allelic frequency of the variant of 0.06% among all ethnicities and of 0.25% in Ashkenazi Jews. A shared haplotype was found in all probands carrying the variant, indicating that a common founder was likely in these families.

Conclusion: POT1:p.(I78T) is a recurrent, founder disease-causing variant associated with early-onset melanoma in addition to other solid malignancies, with a high tumor burden. Genetic testing of POT1 should be included in germline panels for various cancer types.

Conflict of Interest: None declared

P01.071.C Analysis of molecular genetic testing yields for hereditary forms of cancer 2020-2023

Ilona Pirushka ¹, Baiba Lace², Ieva Mičule^3;4, Arvids Irmejs⁵, Peteris Loža⁵, jelena maksimenko⁵

¹Children’s Clinical University Hospital, Clinic of Medical Genetics and Prenatal Diagnosis, Riga, Latvia; ²Riga East Clinical University Hospital, Rīga, Latvia; ³Riga Stradins University, Rīga, Latvia; ⁴Children’s Clinical University Hospital, Rīga, Latvia; ⁵Pauls Stradiņš Clinical University Hospital, Rīga, Latvia

Objectives:The aim of this study was to determine the clinical and molecular characteristics of patients from 2020 to 2023 who underwent hereditary cancer multigene panel testing.

Methods:The study enrolled 247 adults: 5% (n = 13) men and 95% (n = 234) women with: breast cancer 89% (n = 221), ovarian cancer 6% (n = 14), colorectal cancer 3% (n = 8), testicular cancer 0,8% (n = 2), melanoma 0,8% (n = 2). 17 adults underwent cascade testing. Patients with cancer (n = 247) were tested by gene panels, predictive testing for patients’ relatives (n = 17) - by Sanger sequencing. All breast and ovarian cancer patients had initial testing for BRCA1/2 founder mutations before NGS testing.

Results:The mean age at the time of NGS was 46 years. Genetic testing identified a pathogenic/likely pathogenic variant in 32 % of patients (n = 78). Test result was negative in 64% (n = 158) of patients. 11 VUS were identified in 4% (n = 9) of patients.

The most identified germline mutations were in BRCA1 (n = 36; 46%) BRCA2, (n = 17; 22%), CHEK2(n = 9;12%). Pathogenic/likely pathogenic variants in ATM, PALB2, RAD51C, FANCM, TP53,MLH1, PMS2 were also identified. 33% (n = 15) from all positive BRCA1/2 variants were founder mutations. The cascade screening for the asymptomatic persons was positive in 88% (n = 15), negative in 12% (n = 2).

Conclusion:We propose that genetic testing in Latvia, utilizing a panel of 11 BRCA1/BRCA2 gene founder mutations, provides an inexpensive, efficient platform for cancer prevention/treatment. This panel may be expanded or replaced by multigene hereditary cancer panel in the future.

Conflict of Interest: None declared

P01.072.D Genome sequencing in patients with hereditary breast and ovarian cancer

Ulrike Faust ¹, Dennis Witt¹, Antje Stäbler¹, Silja Gauß¹, Marc Sturm¹, Benita Menden¹, Olga Kelemen¹, Leon Schütz¹, Ines Gruber², Kristin Bosse¹, Stephan Ossowski¹, Tobias Haack¹, Olaf Riess¹, Christopher Schroeder¹

¹Institute of Medical Genetics and Applied Genomics, Tübingen, Germany; ²Department for Women’s Health, Tübingen, Germany

Background/Objectives: Multi-gene panel or exome sequencing are the current standard for hereditary breast and ovarian cancer (HBOC) testing. However, a pathogenic variant in the HBOC genes is only found in 15% to 25% of high-risk families. The implementation of genome sequencing (GS) has the potential to increase the diagnostic sensitivity through detection of structural variants, alterations in regulatory regions, as well as the integration of polygenic-risk-scores (PRS) and analysis of actionable genes.

Methods: The study involved 819 patients with breast and/or ovarian cancer, fulfilling criteria for HBOC testing and 4810 healthy controls. GS was performed and analyzed in our accredited diagnostic laboratory following standard procedures. Our pipeline was designed to incorporate the Breast Cancer PRS 313 (Mavaddat et al., 2019) and Ovarian Cancer PRS (OCAC 36 by canrisk.org).

Results: Causative variants were found in 12.8% of cases across 9 HBOC genes. The in-depth characterization of a complex BARD1-structural variant confirmed the advantages of GS over other methods. The raw PRS of our breast cancer cohort as well as of our ovarian cancer cohort was significantly higher than in our local controls. Additionally, 20 pathogenic variants in actionable genes were found in 18 patients.

Conclusion: GS offers advantages over current standard multi-gene panel or exome sequencing. We were able to benefit from additional structural variants and increased process standardization. PRS might explain up to an additional 20% of breast cancer cases. Finally, GS has a higher flexibility regarding re-analysis of candidate genes, regulatory regions, and structural variants.

Grants:

Conflict of Interest: Ulrike Faust: None declared, Dennis Witt: None declared, Antje Stäbler: None declared, Silja Gauß: None declared, Marc Sturm: None declared, Benita Menden: None declared, Olga Kelemen: None declared, Leon Schütz: None declared, Ines Gruber: None declared, Kristin Bosse: None declared, Stephan Ossowski S.O. received reimbursement for travel expenses and payment for conference presentations from Illumina Inc. and Oxford Nanopore Technologies., S.O. received reimbursement for travel expenses and payment for conference presentations from Illumina Inc. and Oxford Nanopore Technologies., Tobias Haack: None declared, Olaf Riess: None declared, Christopher Schroeder Illumina Inc. Institutional grant for the GE-MED subproject, medical writing

P01.073.A Unique mutation spectrum in the Israeli Arab population suspected of Hereditary Breast and Ovarian Cancer or MUTYH Associated Polyposis syndrome

Gili Reznick Levi ¹, Elizabeth E Half^2;3, Tamar Paperna¹, Hanna Segev¹, Yael Goldberg^4;5, Karin Weiss^1;3

¹Rambam Health Care Campus, The Genetics Institute, Haifa, Israel; ²Rambam Health Care Campus, Institute of Gastroenterology, Haifa, Israel; ³The Rappaport Faculty of Medicine, Technion, Haifa, Israel; ⁴Raphael Recanati Genetic Institute, Rabin Medical Center, Petach Tikva, Israel; ⁵Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Background/Objectives: Most of the Founder Pathogenic Variants (PVs) in genes related to cancer predisposition syndromes in Israel were described in the Jewish population.

This work aims to describe the mutation spectrum of Hereditary Breast and Ovarian Cancer (HBOC) in Christian Arabs and MUTYH associated polyposis (MAP) in Israeli Arabs.

Methods: Electronic records of all Arab patients presenting to the Oncogenetic clinic at Rambam Health Care Campus between 2013- 2020 were reviewed for personal or family history suspected of HBOC or polyposis syndromes.

Results: 35 unrelated Christian Arab patients, with a personal or family history of breast and\or ovarian cancer, underwent BRCA1/BRCA2 (14/35) testing or multi-gene panel testing (21/35). A recurrent BRCA2 exon 5-11 duplication was found in 5/33 (15.2%) patients for whom copy number variant (CNVs) analysis was performed. 37 Arab polyposis patients from 30 unrelated families underwent APC and MUTYH testing; 8(26.6%) carried bi-allelic MUTYH PVs. The major variant detected was p.(Glu452del) seen in 75% of positive cases. Interestingly, Druze and Muslims shared the same haplotype.

Conclusions: We identified a high prevalence of a BRCA2 duplication among Christian Arabs with personal/ family history of HBOC in Israel, emphasizing the importance of CNV analysis in this sub-population. MAP is quite frequent (27%) among Arab polyposis cases in Northern Israel. PV spectrum is unique, with high frequency of the founder variant p.(Glu452del). Our results impact the genetic testing strategy in the Israeli Arab population suspected of HBOC or MAP.

Conflict of Interest: None declared

P01.074.B Compilation and reclassification of BRCA2 VUS found in the Genetic Counselling Unit of Salamanca in the last 23 years

Paloma Martín-Bejarano Soto ^1;2;3, Eva María Sánchez-Tapia^1;2;3, Paula García-Vallés^1;2;3, Mercedes León-López^1;2;3, Teresa Martín-Gómez⁴, emilio fonseca sánchez⁴, Rogelio González-Sarmiento^1;2;3, Ana-Belén Herrero^1;2;5

¹Molecular Medicine Unit, Department of Medicine, University of Salamanca, Salamanca, Spain; ²Biomedical Research Institute of Salamanca (IBSAL), Salamanca, Spain; ³Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanc, Spain; ⁴Medical Oncology Service, University Hospital of Salamanca, Salamanca, Spain; ⁵Institute of Molecular and Cellular Biology of Cancer (IBMCC), University of Salamanca-CSIC, Salamanca, Spain

Background/Objectives: BRCA1/2 genetic testing is key for improving clinical management and for patients with hereditary breast/ovarian cancer. Variants found in these genetic tests are classified according to their impact on the protein function and thus on the risk of developing breast cancer. However, the identification of variants of unknown significance (VUS) limits the clinical utility of genetic testing as the impact on protein function is unknown. VUS are mainly missense variants that cannot be classified due to insufficient experimental and clinical data.

Methods: The variants identified in the Genetic Counselling Unit (GCU) of the University Hospital of Salamanca between 2000-2023 have been compiled. They were searched for re-evaluation in the aggregators Varsome and ClinVar, which are based on the use of the classification guidelines specified by the ACMG/AMP.

Results: Of the 79 VUS detected in over 2000 studies performed in the last 23 years, 62 (76.4 %) were reclassified according to Varsome and 23 (26.6%) according to ClinVar (Table 1). Of the 79 variants, most (91.1%) are missense variants and belong to 94 families.

Table 1. Updated classification of the 79 VUS according to Varsome and ClinVar.

Conclusion: This study demonstrates the need for updating VUS detected in GCU due to their important clinical impact. Furthermore, it highlights the need to generate methods that allow the reclassification of these VUS as a priority.

Grants: This project was funded by IP20.

Conflict of Interest: None declared

P01.075.C Investigation of variant distribution and frequency in triple negative breast cancer patients living in the Trakya Region of Turkey: A Single Center Experience

HAKAN GURKAN ¹, Engin Atli¹, Nilay Ozupek¹, Bilge Nihan Satkın², Drenusha Zhuri¹, Hazal Sezginer Guler¹, Selma Demir¹, Sernaz Topaloğlu³, Atakan Sezer⁴, Ebru Taştekin⁵, Nermin Tunçbilek⁶

¹Trakya University Faculty of Medicine, Department of Medical Genetics, Edirne, Türkyie; ²Trakya University Faculty of Engineering, Department of Genetics and Bioengineering, Edirne, Türkyie; ³Trakya University Faculty of Medicine, Department of Medical Oncology, Edirne, Türkyie; ⁴Trakya University Faculty of Medicine, Department of General Surgery, Edirne, Türkyie; ⁵Trakya University Faculty of Medicine, Department of Medical Pathology, Edirne, Türkyie; ⁶Trakya University Faculty of Medicine, Department of Radiology, Edirne, Türkyie

Background/Objectives: Approximately 15% to 20% of all breast cancers are triple negative breast cancers (TNBCs). TNBC is most common in premenopausal women aged < 40 years and is highly aggressive, and poorer clinical outcome than those of hormone receptor positive and HER2-enriched breast cancers. In fact, >85% of breast cancers developing in the context of BRCA1 germline pathogenic variant carriers display a triple-negative phenotype, and 11% to 19% of patients with triple-negative disease harbor BRCA1 germline or somatic mutations.

Methods: The aim of our study is to investigate the distribution and frequency of variants in TNBC patients living in the Trakya Region of Turkey. 45 patients who applied to our Genetic Diseases Evaluation Center with a diagnosis of TNBC between October 2019 and October 2023 were included in the study. The average age of the patients is 53.27 years. Targeted Breast NGS panel (94 genes) and BRCA1/2 MLPA were studied in patients using the NGS method.

Results: Pathogenic/likely pathogenic (P/LP) variant was detected in 16 patients (35.5%), and VUS variant was detected in 16 patients (35.5%). 10 of the 16 P/LP variants (62.5%) were located in BRCA1. NM_007294.3(BRCA1):c.5266dupC (p.Gln1756Profs) variant was detected in four patients (40%). Other P/LP variants were detected in the BRCA2 (2), CHEK2 (1), MUTYH (1), PALB2 (1) and MUC16 (1) genes.

Conclusion: Unlike the literature, we detected a higher rate (62.5%) of P/LP variants in the BRCA1 gene in our patients with triple-negative phenotype. This result may be related to ethnic stratification and/or variants with a founder effect.

Grants: No grants

Conflict of Interest: None declared

P01.077.A Addressing the ‘leaky pipe’ in colorectal cancer genetics referrals: The critical need for optimization

Nur Diana Binte Ishak ¹, Shao-Tzu Li¹, jianglei wu¹, Jianbang Chiang¹, Joanne Ngeow^1;2

¹National Cancer Centre Singapore, Cancer Genetics Service; ²Lee Kong Chian School of Medicine, Nanyang Technological University

Background/Objectives: Colorectal cancer significantly burdens Singapore, with over 1,865 cases diagnosed annually. Lynch syndrome, the most common hereditary colorectal cancer, affects 1 in 300 Singaporeans. Despite high prevalence, referrals to Cancer Genetics Service (CGS) remain low, highlighting a gap in Lynch syndrome detection. This study delves into referral discrepancies and barriers to timely hereditary cancer syndrome diagnosis among colorectal cancer patients in Singapore.

Methods: We evaluated patients seen at the National Cancer Centre Singapore from 2017 to 2021, identifying 1,716 colorectal cancer cases diagnosed under age 50 or with a second Lynch syndrome-related cancer. Appropriate CGS referrals were determined based on age at cancer diagnosis, deficient mismatch repair protein expression, polyp pathology (adenomatous, hamartomatous, juvenile polyps), and presence of desmoid tumours.

Results: We found that only 15% of the 878 colorectal cancer patients under the age of 50 were referred to CGS, while the referral rate for patients with a second Lynch syndrome-associated cancer was even lower at 9.8%. While most non-referred cases had MMR evaluation, 20% were not referred in spite of tumour pathology, indicating missed opportunities for hereditary cancer risk detection.

Conclusion: This gap in referrals underscores the urgent need for increased awareness and improved referral practices among medical professionals. Prioritizing early detection through timely referrals is essential not only for initiating preventive strategies and enhancing patient outcomes but also for identifying at-risk relatives and substantially reducing the community’s cancer burden. Proactively addressing these gaps is crucial, ensuring effective management of hereditary colorectal cancer syndromes towards a more preventive healthcare model.

Grants:NA

Conflict of Interest: None declared

P01.078.B An interdisciplinary approach to counsel children with cancer predisposition and their families for genetic testing and surveillance

Nicola Dikow ¹, Anna Lisa Nitschke^1;2, Steffen Hirsch^1;2;3, Joachim Kunz^2;4;5, Kerstin Grund¹, Peggy Lüttich⁶, Christian Sutter¹, Katrin Hinderhofer¹, Cornelis M. van Tilburg^2;4;6, Olaf Witt^2;4;6, Andreas Kulozik^2;4;6, David Jones^2;5, Maja Hempel¹, Till Milde^2;4;6, Christian Schaaf¹, Stefan Pfister^2;4;6, Kristian Pajtler^2;4;6

¹Heidelberg University, Institute of Human Genetics, Heidelberg, Germany; ²Hopp Children’s Cancer Center Heidelberg (KiTZ), and National Center for Tumor Diseases (NCT), Heidelberg, Germany; ³German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Division of Pediatric Neurooncology, Heidelberg, Germany; ⁴German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany, Clinical Cooperation Unit Pediatric Oncology, Heidelberg; ⁵German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Division of Pediatric Glioma Research, Heidelberg; ⁶Heidelberg University Hospital, Heidelberg, Germany, Department of Pediatric Oncology, Hematology, Immunology & Pulmonology

Background: The knowledge of cancer predisposition syndromes (CPS) is the prerequisite for surveillance programs, that have proven benefits in an increasing number of CPS. Our interdisciplinary expert panel discusses, evaluates and counsels children with suspected CPS and their families. Here, we report the diagnostic rates and surveillance recommendations.

Patients and Methods: All individuals seen in the interdisciplinary consultation (2018 - 2022) were included for retrospective analysis. Data on medical and family history, physical examination, molecular and histological tumor characteristics, diagnosis and surveillance recommendations were retrieved from medical records.

Results: Of 144 individuals (median age 9 years), 99 had symptoms of CPS (including 52 with a tumor diagnosis); 45 were clinically unaffected and presented for predictive genetic testing of familial genetic variants. A CPS was diagnosed prior to consultation in 33/99 patients and unknown in 66/99. For 57/66 patients, molecular analysis was performed. A CPS was detected in 26 patients (45,5%), and in 8/10 (80%) of those with more than one type of cancer. The most frequently affected genes were NF1, TP53, MEN1, APC and PTEN.

Surveillance recommendations could be given for almost all individuals with a CPS diagnosis.

Conclusion: Our interdisciplinary approach contributes to the identification of CPS in pediatric patients and families at risk and highlights the need for personalized surveillance recommendations established by an expert panel, especially in rare CPS. Our approach is intended to lay the foundation for more systematic CPS assessment and the establishment of standardized surveillance concepts.

Conflict of Interest: None declared

P01.079.C Hematological malignancies as a secondary finding on diagnostic exome sequencing: should we consider reporting them?

Gaber Bergant ¹, Aleš Maver¹, Borut Peterlin¹

¹University Medical Center Ljubljana, Clinical Institute of Genomic Medicine, Ljubljana, Slovenia

Background/Objectives: The detection of somatic secondary findings (SFs) during diagnostic next-generation sequencing presents significant challenges in the context of patients with suspected rare disorders with no existing international guidelines. The differentiation between somatic and germline variants is a complex aspect of genetic analysis, with implications for diagnostic accuracy and patient care.

Methods: We conducted a detailed analysis of four cases where patients underwent diagnostic exome sequencing for suspected rare disorders in which we detected somatic secondary findings associated with hematological malignancies.

Results: Our investigation revealed the presence of somatic SFs in the patient samples, which were associated with hematological malignancies. We found that these somatic SFs could potentially influence the primary diagnostic interpretation, underscoring the difficulty in distinguishing them from germline variants. The analysis highlights the complexity of interpreting exome sequencing data when somatic variants are present.

Conclusion: Our findings underscore the urgent need for clear guidelines on how to analyze and report somatic secondary findings in diagnostic exome sequencing. This work emphasizes the importance of increased awareness and a systematic approach to the reporting of SFs that are unrelated to germline genomic variations. The presence of somatic SFs has significant implications for patient care, and addressing this challenge is crucial for the advancement of precision medicine.

Grants: None

Conflict of Interest: None declared

P01.080.D Differences in polygenic score distributions in European ancestry populations: implications for breast cancer risk prediction

Kristia Yiangou ¹, Nasim Mavaddat², Joe Dennis², Maria Zanti¹, Jacques Simard³, Antonis C. Antoniou², Douglas F. Easton^2;4, Kyriaki Michailidou¹

¹The Cyprus Institute of Neurology and Genetics, Biostatistics Unit, Nicosia, Cyprus; ²University of Cambridge, Centre for Cancer Genetic Epidemiology, Department of Public Health and Primary Care, Cambridge, United Kingdom; ³Centre Hospitalier Universitaire de Québec – Université Laval Research Center, Genomics Center, Québec City, Québec, Canada; ⁴University of Cambridge, Centre for Cancer Genetic Epidemiology, Department of Oncology, Cambridge, United Kingdom

Consortium: Breast Cancer Association Consortium (BCAC)

Background/Objectives: Polygenic risk scores (PRSs) provide a promising tool for personalized breast cancer (BC) risk prediction. A 313-variant PRS (PRS-313) has been constructed and is being used in several clinical trials of risk-based screening. However, specific evaluation of its distribution across different European populations has not been performed which could be potentially important for accurate risk estimation.

Methods: We explored the distribution of PRS-313 across European ancestry populations, using data from 94,072 females without BC from 21 countries participating in the Breast Cancer Association Consortium (BCAC) and from 225,105 white females without BC participating in the UK Biobank. We calculated the mean PRS by country in BCAC and by country of birth in UK Biobank. We investigated the implications of PRS distribution variability across the countries in BC risk prediction and potential approaches to account for the observed distribution variability.

Results: The mean PRS differed markedly by country in both BCAC and UK Biobank; being highest in the south-east (e.g. Cyprus, Greece, Italy) and lowest in the north-west (e.g. Ireland). Using the PRS distribution to define risk categories leads to risks over- or underestimation in a proportion of individuals from south-eastern and north-western countries, respectively. Adjustment for the leading ancestry informative principal components explained the differences in PRS distribution in both datasets.

Conclusion: PRS distributions differ across European ancestry populations. Country-specific distributions are required to provide well calibrated risk estimates. These approaches could also be used for the evaluation of PRSs for other diseases.

Grants: None

Conflict of Interest: None declared

P01.081.A Stress granules: from rare disorder to potential novel cancer treatment

Johannes Lederbauer ¹, Davor Lessel¹, Prof. Dr. rer. nat. Hans-Jürgen Kreienkamp²

¹University Hospital Salzburg, Institute of Human Genetics, Salzburg, Austria; ²University Hospital Hamburg-Eppendorf, Institute of Human genetics, Hamburg, Germany

Background/Objectives: Stress granules (SGs) are cytoplasmic aggregates that assemble upon stress to block the translation and thereby provide a protective mechanism. However, hyper-assembly of SGs is observed in many cancers and thought to cause chemotherapy resistance. Recently, we linked the RNA helicase DHX30 to a rare, neurodevelopmental syndrome, and found that underlying, heterozygous missense variants cause an increased propensity to trigger SG formation.

Methods: We utilized DHX30-deficient cellular and animal models, different SG inducing agents including different chemotherapeutics and analysis on protein and RNA level to further investigate the role of DHX30 in SG assembly.

Results: Our in depth analyses identified DHX30 as the first essential gene for general SG assembly. Mechanistic analyses revealed that DHX30 interacts via RNA with two main nucleators of SGs, G3BP1 and Caprin1. Notably, expression of both proteins was significantly reduced in DHX30 deficient models upon several stressors. Overexpression of both proteins in DHX30 deficient cells led to formation of SGs thus providing a mechanistic understanding. In addition, highly prevalent, somatic, KRAS missense variants (p.Gly12/Asp/Cys/Val) have been shown to enhance SG formation in pancreatic tumours. Interestingly, DHX30 silencing mitigated SG hyper-assembly in KRAS-mutant cancer cells. Similar results were obtained when KRAS-mutant plasmid were overexpressed in DHX30 deficient cell lines.

Conclusion: By analysing a rare Mendelian disorder, we identified DHX30 as the first essential SG assembly gene. Furthermore, we show that molecular findings from such rare disorders can be translated to common diseases. Indeed, we identified a potential novel molecular target to treat KRAS-mutated cancers.

Grants: Fritz Thyssen Stiftung

Conflict of Interest: None declared

P01.083.C How does polygenic risk score (PRS) modify breast cancer (BC) risk estimation in carriers versus non-carriers of a moderate pathogenic variant (PV)?

Laura Duran-Lozano ¹, Alejandra Rezqallah^1;2, Adrià López-Fernández^1;2, Mònica Pardo³, Eduard Perez^1;2, Esther Darder⁴, Rosa Alfonso⁵, Ana Raquel Jimenez-Macedo⁶, Mireia Cartro⁷, Mara Cruellas^1;2, Estela Carrasco López^1;2, Adriana Bareas¹, Maite Torres⁸, Anna Vallmajo⁹, Victor Navarro¹⁰, Ariadna Roque⁸, Lidia Feliubadaló⁸, Conxi Lazaro⁸, Noemi Tuset⁹, Martín Espinosa-Bravo², Teresa Ramon y cajal⁵, Joan Brunet⁸, Judith Balmaña^1;2

¹Vall d’Hebron Institute of Oncology (VHIO), Hereditary Cancer Genetics Group; ²Vall d’Hebron Hospital, Medical Oncology, Barcelona, Spain; ³Hospital del Mar, Medical oncology service. Genetic Counselling Unit, Barcelona, Spain; ⁴Catalan Institute of Oncology, Genetic Counselling Unit. Hereditary Cancer Program, Spain; ⁵Hospital de Sant Pau, Familial Cancer Clinic. Medical Oncology, Barcelona, Spain; ⁶Consorci Sanitari de Terrassa, Genetic Counselling Unit, Spain; ⁷Corporació Sanitària Parc Taulí, Genetic Counselling Unit, Spain; ⁸Catalan Institute of Oncology, Genetic Counselling Unit. Hereditary Cancer Program., Spain; ⁹Hospital Universitari Arnau de Vilanova, Genetic Counselling in Familial Cancer Unit. Medical Oncology Service., Lleida, Spain; ¹⁰Vall d’Hebron Institute of Oncology (VHIO), Oncology Data Science (ODySey) Group, Spain

Consortium: PRiSma

Background/Objectives: BC risk is a multifactorial phenotype, and incorporation of PRS in risk estimation can lead to higher accuracy accounting for a bigger proportion of the genetic risk fraction. We aimed to explore the risk distribution in different populations of women at risk and investigate the effect of incorporating PRS in their risk estimation.

Methods: PRS genotyping was performed using the PRISMA₂₆₈ array. BC risk was estimated with the BOADICEA.v6 model using personal and family history, breast density (BD) and then compared to estimations including PRS in the same model.

We estimated the 10-year risk in two populations of healthy women: (1) 133 women carrying a germline PV in ATM, BARD1, CHEK2, PALB2 or RAD51C/D and (2) 593 women with a BC family history and negative genetic test. Patients were classified in average, moderate, or high risk following the NICE guidelines. t-test and X2 were used to assess differences in BC risk estimation between carriers and non-carriers and the change of distribution when adding PRS.

Results: There were no statistically significant differences in 10-year BC risk between carriers and non-carriers (7.43% vs 4.10%, p = 0.55). Adding PRS led to re-classification of 28% and 22% of carriers and non-carriers, respectively(p = 0.16). Overall, 11-15% increased their risk group and 11-13% decreased their risk group.

Conclusion: Risk stratification for carriers of moderate penetrance variants is comparable to non-carriers with family history. Incorporation of PRS₂₆₈ leads to change in risk group in 20-30% of healthy individuals with a family history, regardless of their carrier status.

Grants: ISCIII-PI19/01195, ISCIII-PI23/01047, BCRF

Conflict of Interest: None declared

P01.084.D Cytotoxic effect of thymoquinone with tamoxifen on hormone-positive breast cancer cells

Vildan Betul Yenigun ¹, Ebru Kanimdan¹, Ayfer Sendul², Abdurrahim Kocyigit²

¹Bezmialem Vakıf University, Vocational School of Health Services, Istanbul, Türkyie; ²Bezmialem Vakıf University, Medical Biochemistry, Istanbul, Türkyie

Background/Objectives: Breast cancer is the most common cancer among women. Hormonal therapy is frequently used for hormone receptor-expressing breast cancers which is the most common form. Tamoxifen (TAM) is an estrogen receptor antagonist which competes with estrogen to bind to its receptor. Thymoquinone (TQ) is the main bioactive component of black cumin essential oil with anti-cancer properties. Recent studies have shown that using TQ alone or with other chemotherapeutics is effective on cancer cells. Therefore, this study aimed to examine the possible synergistic effect of the combined use of TAM and TQ on breast cancer.

Methods: ER+ breast cancer cells (MCF-7) were used in this study to investigate the combination effect of TAM and TQ. MTT cell viability test was used to calculate both drugs’ half-maximal inhibitory concentrations (IC50) at 24 h and observe the cell viability after they were co-administered. Apoptosis was detected by Annexin V/PI in addition to Acridine Orange/Ethidium Bromide (AO/EB) staining.

Results: Statistical difference in cell death was observed starting from 25 µM TQ with an IC₅₀ value of 74,7 µM in 24 hours. TAM induced statistical cell death starting from 10 µM concentration. Combining TQ with TAM showed apparent synergism, decreasing the IC₅₀ value of TAM. Combination index analysis showed additive effect with CI values of 1,07. Apoptosis was also increased with a non-toxic dose of TAM (5 µM) and TQ combination.

Conclusion: The results of this study showed that TQ could synergistically increase the efficacy of TAM on ER+ breast cancer cells.

Conflict of Interest: None declared

P01.085.A Towards characterizing the DNA methylome of classic Hodgkin lymphoma

Nadine Matscheko ¹, Anja Fischer¹, Selina Glaser¹, Cosima Drewes¹, Björn Brändl^2;3, Franz-Josef Müller³, Helene Kretzmer², Ole Ammerpohl¹, Reiner Siebert¹

¹Institute of Human Genetics, Ulm University and Ulm University Medical Center, Ulm, Germany; ²Max Planck Institute for Molecular Genetics, Department of Genome Regulation, Berlin, Germany; ³Zentrum für Integrative Psychiatrie gGmbH, Universitätsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany

Background/Objectives: Classic Hodgkin’s lymphoma (cHL) is a B-cell tumor characterized by B-cell identity loss, chromosomal instability, and aberrant long tandem repeat expression. Malignant mono-nucleated Hodgkin and multi-nucleated Reed-Sternberg cells (HRS) constitute only minorities in tumor samples, making (epi-)genetic alteration screening challenging. Here, cHL cell lines offer promising alternatives and were examined for HRS-specific DNA methylation compared to B-cell lymphoma cell lines (BCL) and normal B-cell populations (BCs).

Methods: Bisulfite-converted DNA from 10 cHL, 45 BCL cell lines, and 110 BCs was investigated using Illumina HumanMethylation450K and EPIC BeadChips. Significantly differentially methylated cytosines (DMCs/CpGs) in cHL cell lines were identified (FDR ≤ 0.001, Δβ_mean ≥ 0.4). Moreover, 6 cHL and 10 BCL cell lines were sequenced with Oxford Nanopore (ONT), data was processed with ONT softwares dorado and modkit, and DMCs were identified (FDR ≤ 0.001).

Results: Array data-based analysis identified 4493 hyper- and 264 hypomethylated CpGs in cHL cell lines compared to BCL cell lines and BCs. Associated genes were enriched for B-cell receptor signaling, transcription, and cytoskeleton functions. Comparing ONT data of cHL and BCL cell lines, the former revealed 40,432 hypermethylated and 17,192 hypomethylated DMCs, with 10.08% and 1.22% overlap, respectively, with array-identified DMCs. Thereby, hypermethylated sites were enriched for promoter-sequences.

Conclusion: Compared to BCs and BCL cell lines, cHL cell lines predominantly exhibit hypermethylated, promoter-localized DMCs. However, the contribution of differentially methylated B-cell receptor signaling genes to B-cell identity loss is still unclear. Furthermore, investigating affected cytoskeleton functions would be worthwhile as they may affect cytokinesis, potentially leading to multi-nucleated HRS.

Grants: None.

Conflict of Interest: None declared

P01.086.B Assessment of pathogenic germline variants in patients with luminal B subtype breast cancer.

Andressa Almeida ¹, Karina Miranda Santiago¹, Fabiana Makdissi¹, José Rocha¹, Giovana Torrezan¹, Dirce Carraro¹

¹A.C. Camargo Cancer Center, Brazil

Background/Objectives:This study aimed to examine the prevalence of germline pathogenic variants (GPVs) in luminal B breast cancer and correlate these with patient clinical data.

Methods:A retrospective study was conducted on an unselected series of 69 Brazilian women diagnosed with luminal B breast cancer, with samples available at the biobank of the A.C.Camargo Cancer Center. Participants underwent genetic sequencing of a 112-gene panel related to hereditary cancer predisposition, with clinical data from medical records review..

Results:Of the 69 patients, 20% (14/69) presented GPVs in the genes: CHEK2 (4 patients), BRCA2 (2), TP53 (2), ATM (1), BARD1 (1), BLM (1), BRIP1 (1), LZTR1 (1), MUTYH (1) and RAD51C (1). GPVs carriers showed an earlier cancer diagnosis compared to non-carriers (p-value 0,021), ranging between 31 and 72 years, median of 39.5 years. 57% (8/14), were diagnosed before 45 years of age, 21% (3/14) between 46 and 60 years, and 14% (2/14) after 60 years. HER2 overexpression was observed in 64% (9/14) of cases, and the ductal histological subtype was predominant in 85% (12/14) of cases.

Conclusion:We identified a high incidence of GPVs in luminal B breast cancer, and an earlier onset of cancer in these patients.The research revealed genetic diversity, including a low incidence of variants in the BRCA1 and BRCA2 genes.Despite a small sample, variants in other genes and high HER2 overexpression offer new insights, highlighting the need for research on less studied breast cancer subtypes.Future research should delve into how genetic variants, HER2 overexpression, and histological subtypes interrelate to clarify hereditary breast cancer’s origins.

Grants:FAPESP and CNPq

Conflict of Interest: None declared

P01.087.C miRNA Profiling Identifies Potential Risk Biomarkers in Familial Prostate Cancer Cases

Lucía Chica Redecillas ^1;2, Juan Miguel Guerrero-González², Sergio Cuenca López¹, Elena Arance², Fernando Marín-Benesiu^1;2, Carmen María Morales-Álvarez^1;2, Maria Jesus Alvarez Cubero^1;2;3, Luis Javier Martínez-González^1;2

¹Faculty of Medicine - University of Granada, Department of Biochemistry and Molecular Biology III and Immunology, Faculty of Medicine, Granada, Spain; ²Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government (GENYO), Granada, Spain; ³Biosanitary Research Institute (ibs. GRANADA), University of Granada, Spain

Background/Objectives: About 5-10% of prostate cancer (PC) cases have a hereditary component. Men with a family history of this disease face a twofold increased risk, with a tendency for more aggressive cancer. Current guidelines for early diagnosis and patient stratification are inadequate. Present study aims to identify potential miRNA biomarkers for the detection and stratification of high-risk familial PC.

Methods: We analysed miRNA expression in two PC multicase families. We used the edgeR package to study the transcriptome and only considered results with FDR < 0.05 as significant. To ensure the validity of the results, we compared them with The Cancer Genome Atlas (TCGA) database.

Results: Eight miRNAs significantly expressed in hereditary PC patients were identified and validated by TCGA: hsa-miR-205-5p, hsa-miR-197-3p, hsa-miR-144-5p, hsa-miR-423-3p, hsa-miR-101-3p, hsa-miR-501-5p, hsa-miR-671-3p and hsa-miR-744-5p. Enrichment analysis revealed the involvement of target genes of identified miRNAs, such as SOX9, ZEB2, FOXO3 and LRRK2, in crucial pathways related to cancer development, highlighting the Wnt signaling pathway. Construction of miRNA-mRNA network also revealed a high interconnection among identified miRNAs with genes involved in the Wnt pathway.

Conclusion: miRNAs have the potential to be significant cancer biomarkers. Specifically, hsa-miR-205-5p, hsa-miR-101-3p and hsa-miR-144-5p play important roles in crucial pathways in PC. Our study underscores the importance of the Wnt signaling pathway in the development of hereditary PC. These findings are consistent with our previous research on this disease.

Grants: Ministerio de Ciencia e Innovación (No.PID2019-110512RA-I00/MCIN/AEI/10.13039/501100011033) and Urology Research Foundation (FIU);

Conflict of Interest: None declared

P01.088.D Germline variants of homology-directed repair or mismatch repair genes in cervical cancer

Lara Kokemüller ¹, Dhanya Ramachandran¹, Peter Schürmann¹, Geffers Robert², Gerd Böhmer³, Hans Georg Strauss⁴, Christine Hirchenhain⁵, Schmidmayr Monika⁶, Florian Müller⁷, Peter Fasching⁸, Norman Häfner⁹, Alexander Luyten^10;11, Matthias Jentschke¹, Peter Hillemanns¹, Thilo Dörk¹

¹Hannover Medical School, Department of Gynaecology, Hannover, Germany; ²Helmholtz Centre for Infection Research, Braunschweig, Germany; ³IZD Ärztliche Partnerschaft Böhmer & Partner, Hannover, Germany; ⁴Martin-Luther-University Halle-Wittenberg, Gynecology Department, Halle (Saale), Germany; ⁵University Hospital Carl Gustav Carus Dresden, Gynecology Department, Dresden, Germany; ⁶Technical University of Munich, Gynecology Department, München, Germany; ⁷Martin Luther Hospital, Berlin, Germany; ⁸University of Erlangen-Nuremberg, Department of Gynecology and Obstetrics, Erlangen, Germany; ⁹Friedrich Schiller University Jena, Department of Gynecology, Jena, Germany; ¹⁰MARE Klinikum, Dysplasia Unit, Department of Gynecology and Obstetrics, Kronshagen, Germany; ¹¹Hospital of the city of Wolfsburg, Wolfsburg, Germany

Consortium: Cervigen consortium

Background/Objectives: While cervical cancer is associated with HPV infection, it appears to be influenced by genomic risk factors which have remained largely obscure. Pathogenic variants in genes of homology-directed repair (HDR) or mismatch repair (MMR) are known to predispose to diverse malignancies including breast and ovarian cancer (HDR) or colon and endometrial cancer (MMR). We here investigate the spectrum of HDR and MMR germline variants in cervical cancer, stratified by HPV status and histological subgroups.

Methods: We performed targeted next-generation sequencing of eleven HDR genes and five MMR genes on genomic DNA samples from 555 German patients with cervical dysplasia or invasive cancer. Variants were classified as pathogenic or likely pathogenic based on ClinVar categories and additional ESM1b and AlphaMissense prediction.

Results: 5 % of our patients carried a pathogenic or likely pathogenic germline variant. These included eight patients (1.4%) with truncating variants in BRCA1, BRCA2, BARD1 or BRIP1. While one patient with HPV-negative adenocarcinoma carried a pathogenic MMR gene variant (in MSH6), HDR germline variants were associated with HPV-positive squamous invasive cancer. Age at diagnosis of invasive cancer tended to be lower in HDR variant carriers (38.8 ys) compared to non-carriers (46.3 ys).

Conclusion: Our study indicates a potential risk-modifying role of HDR germline variants in a small fraction of cervical cancer patients but no association with HPV-negative status.

Grants: Supported by grants from the Bruno and Helene Jöster Foundation and the Comprehensive Cancer Center of Lower Saxony (CCC-N).

Conflict of Interest: Lara Kokemüller: None declared, Dhanya Ramachandran: None declared, Peter Schürmann: None declared, Geffers Robert: None declared, Gerd Böhmer: None declared, Hans Georg Strauss: None declared, Christine Hirchenhain: None declared, Schmidmayr Monika: None declared, Florian Müller: None declared, Peter Fasching Peter A. Fasching conducts research funded by Amgen, Novartis and Pfizer., Peter A. Fasching Honoraria from Roche, Novartis and Pfizer., Norman Häfner: None declared, Alexander Luyten: None declared, Matthias Jentschke: None declared, Peter Hillemanns: None declared, Thilo Dörk: None declared

P01.089.C Comparative sequencing study of mismatch repair and homology-directed repair genes in endometrial cancer and breast cancer patients from Kazakhstan

Ying Zheng¹, Natalia Vdovichenko ¹, Peter Schürmann¹, Dhanya Ramachandran¹, Geffers Robert², Lisa-Marie Speith¹, Natalia Bogdanova^1;3, Julia Enßen¹, Natalia Dubrowinskaja¹, Tatyana Yugai⁴, Zura Berkutovna Yessimsiitova⁵, Nurzhan Turmanov¹, Peter Hillemanns¹, Thilo Dörk¹

¹Hannover Medical School, Gynaecology Research Unit, Hannover; ²Helmholtz Institute for Infection Research, Genome Analytics, Braunschweig; ³Hannover Medical School, Radiation Oncology Research Unit, Hannover; ⁴Rahat Clinics, Almaty, Kazakhstan; ⁵al-Farabi Kazakh National University, Department of Biodiversity and Bioresources, Almaty, Kazakhstan

Background/Objectives: Endometrial cancer has been associated with pathogenic variants in mismatch repair (MMR) genes, especially in the context of the hereditary Lynch Syndrome. More recently, pathogenic variants in genes of homology-directed repair (HDR) have also been suggested to contribute to a subset of endometrial cancers although their causal role is debated.

Methods: In the presented targeted sequencing study, we investigated the relative distribution of pathogenic MMR or HDR gene variants in a hospital-based series of 351 endometrial cancer patients from the Oncology Clinic in Almaty, Kazakhstan. In a direct comparison, we also sequenced 179 breast cancer patients from the same population with the same panel of 16 genes. Identified variants were classified according to ClinVar, ESM1b and AlphaMissense prediction tools.

Results: We found 10 endometrial cancer patients (2.8 %) carrying pathogenic or likely pathogenic variants in MMR genes while 14 endometrial cancer patients (4.0 %) carried pathogenic variants in HDR genes. In the breast cancer series, we found nine carriers (4.8%) of pathogenic or likely pathogenic variants in MMR genes while 14 patients (7.4%) harbored pathogenic or likely pathogenic HDR gene variants. One patient who developed breast cancer first and endometrial cancer later carried a novel frameshift variant in MSH6.

Conclusion: Our results indicate that pathogenic MMR and HDR gene variants occur at substantial frequencies in both breast and endometrial cancer patients from the Kazakh population.

Grants: Funded by the Wilhelm Sander Foundation.

Conflict of Interest: None declared

P01.090.A Genomic Risk Variants and HPV Infection Modulate Gene Expression at the Human Leukocyte Antigen Locus in Cervical Cancer

Rieke Eisenblätter ¹, Finja Seifert¹, Theresa Beckhaus¹, Peter Schürmann¹, Gerd Böhmer², Hans Georg Strauss³, Christine Hirchenhain⁴, Schmidmayr Monika⁵, Florian Müller⁶, Peter Fasching⁷, Norman Häfner⁸, Alexander Luyten^9;10, Matthias Jentschke¹, Peter Hillemanns¹, Thilo Dörk¹, Dhanya Ramachandran¹

¹Hannover Medical School, Department of Gynecology, Hannover, Germany; ²IZD Ärztliche Partnerschaft Böhmer & Partner, Hannover, Germany; ³Martin-Luther-University Halle-Wittenberg, Department of Gynaecology, Halle (Saale), Germany; ⁴University Hospital Carl Gustav Carus Dresden, Department of Gynaecology, Dresden, Germany; ⁵Technical University of Munich, Department of Gynaecology, München, Germany; ⁶Martin Luther Hospital, Berlin, Germany; ⁷University of Erlangen-Nuremberg, Department of Gynecology and Obstetrics, Erlangen, Germany; ⁸Friedrich Schiller University Jena, Department of Gynecology, Jena, Germany; ⁹MARE Klinikum, Dysplasia Unit, Department of Gynecology and Obstetrics, Kronshagen, Germany; ¹⁰Hospital of the city of Wolfsburg, Wolfsburg, Germany

Consortium: Cervigen consortium

Background/Objectives: Human papillomavirus (HPV) infection triggers cervical cancer development, and genome-wide association studies have identified multiple genomic variants at the human leukocyte antigen (HLA) locus (6p21.32-33). The targets and mode of action at this locus are not well understood. We here aim to refine these associations in an independent case-control series and investigate their functional relevance by eQTL analysis in cervical specimens.

Methods: We genotyped candidate variants at the HLA locus in a German series of invasive cervical cancers, dysplasias, and healthy controls (n > 1,000 in each group). We then used stepwise conditional logistic regression analyses to identify independent signals and tested these variants for their impact on 36 HLA gene transcripts in 200 cervical tissue samples.

Results: The HLA region contained at least four independently associated risk variants for cervical cancer. In addition to previous findings from our cohort, rs17190106 associated with overall cervical disease and invasive cancer, and rs535777 associated with adenocarcinoma. We identified mRNA transcripts upregulated in HPV-positive samples. One variant, rs9272117, affected the expression of multiple genes across the HLA region.

Conclusion: We confirm the presence of independent cervical cancer risk signals at 6p21 in our hospital-based cohort. We identify genomic variants that modulate gene transcript levels together with HPV, indicating that highly controlled gene regulation underlies cervical cancer susceptibility at the HLA locus. These genes may act in concert to modulate the host immune response towards HPV infection.

Grants: Supported by the Bruno and Helene Jöster Foundation and the Comprehensive Cancer Center of Lower Saxony (CCC-N).

Conflict of Interest: Rieke Eisenblätter: None declared, Finja Seifert: None declared, Theresa Beckhaus: None declared, Peter Schürmann: None declared, Gerd Böhmer: None declared, Hans Georg Strauss: None declared, Christine Hirchenhain: None declared, Schmidmayr Monika: None declared, Florian Müller: None declared, Peter Fasching Peter A. Fasching conducts research funded by Amgen, Novartis and Pfizer., Peter A. Fasching received Honoraria from Roche, Novartis and Pfizer., Norman Häfner: None declared, Alexander Luyten: None declared, Matthias Jentschke: None declared, Peter Hillemanns: None declared, Thilo Dörk: None declared, Dhanya Ramachandran: None declared

P01.091.B Germline PTEN pathogenic variant in a patient with Kaposiform haemangioendothelioma

Hannah Massey ¹, Jennie Murray¹, larry hayward¹

¹Western General Hospital, United Kingdom

Background/Objectives: Kaposiform haemangioendothelioma (KHE) is a rare vascular neoplasm usually presenting in childhood. Its high morbidity and mortality is secondary to compression, invasion, and coagulopathy. The genetics of KHE remain poorly understood. Studies have detected somatic variants in GNA14 and germline variants in PIKC3A contributing to tumorigenesis via the Ras and mTOR pathways respectively. We present the first case of a germline change in PTEN identified in the context of Kaposiform haemangioendothelioma.

Methods: A 28-year-old female with metastatic KHE was enrolled in the IMAGINE study (Integrating Medically Actionable Genomics Into Early Phase Trials).

Results: A PTEN heterozygous c.277C>Tp.(His93Tyr) pathogenic variant was detected initially in tumour and subsequently saliva indicating a constitutional genetic change. Reverse phenotyping revealed the patient had a large head circumference, palmar keratosis and papillomas on the hand, feet, and gums, in keeping with PTEN-Hamartoma tumour syndrome (PHTS).

Conclusion: We report the first case of a constitutional PTEN pathogenic variant in association with a KHE. PHTS is a well characterised tumour predisposition syndrome with an 85% lifetime tumour risk. While over 50% of patients with pathogenic PTEN variants have vascular anomalies no malignant vascular tumours have previously been reported. Pathogenic variants in PTEN lead to loss of protein function with a subsequent upregulation of mTOR signalling. Upregulation of mTOR signalling is commonly observed in cancer. This raises the question as to whether mTOR inhibitors could be used in the treatment of KHE.

Grants: None

Conflict of Interest: None declared

P01.092.C Genomic risk loci for cervical cancer: Associations with disease severity and HPV type

Theresa Beckhaus ¹, Linda Kachuri², Finja Seifert¹, Rieke Eisenblätter¹, Dandan Liao¹, Peter Schürmann¹, Gerd Böhmer³, Hans Georg Strauss⁴, Christine Hirchenhain⁵, Schmidmayr Monika⁶, Florian Müller⁷, Peter Fasching⁸, Norman Häfner⁹, Alexander Luyten^10;11, Matthias Jentschke¹, Peter Hillemanns¹, Francis Stephen¹², John Witte², Thilo Dörk¹, Dhanya Ramachandran¹

¹Hannover Medical School, Department of Gynecology, Hannover, Germany; ²Stanford University, Department of Epidemiology and Population Health, Stanford, United States; ³IZD Ärztliche Partnerschaft Böhmer & Partner, Hannover, Germany; ⁴Martin-Luther-University Halle-Wittenberg, Department of Gynaecology, Halle (Saale), Germany; ⁵University Hospital Carl Gustav Carus Dresden, Department of Gynaecology, Dresden, Germany; ⁶Technische Universität München, Department of Gynaecology, München, Germany; ⁷Martin Luther Hospital, Berlin, Germany; ⁸Friedrich-Alexander University Erlangen-Nuremberg Department of Law (Juridicum), Department of Gynecology and Obstetrics, Erlangen, Germany; ⁹Friedrich Schiller University Jena, Department of Gynecology, Jena, Germany; ¹⁰MARE Klinikum, Dysplasia Unit, Department of Gynecology and Obstetrics, Kronshagen, Germany; ¹¹Hospital of the city of Wolfsburg, Wolfsburg, Germany; ¹²University of California, San Francisco, Department of Neurological Surgery, San Francisco, United States

Consortium: Cervigen consortium

Background/Objectives: Persistent infection by high-risk human papillomavirus triggers the development of cervical cancer and dysplasia. Genome-wide association studies for cervical cancer have so far identified multiple genomic risk variants at the human leukocyte antigen (HLA) locus (6p21.32-33), as well as variants on chromosome 2 (PAX8), 5 (near CLPTM1L) and 17 (GSDMB).

Methods: We directly genotyped reported variants, as well as 15 novel variants derived from a HPV seropositivity GWAS, in a German case-control series of invasive cervical cancer, dysplasia, and healthy controls (N > 1,000 in each group). We performed unadjusted logistic regression analyses stratified by histology and HPV type. Selected variants were further investigated for their impact on RNA transcript levels in cervical specimens.

Results: We replicated risk loci in the HLA region as well as at PAX8 and GSDMB at p < 0.05 in our independent case-control series, although the GSDMB locus was only associated with cervical dysplasia. We found evidence that HPV16 and HPV18 seropositivity variants at chromosome 6 and 14 associate with cervical cancer risk in an HPV type-specific manner. rs9357152 may exert its effect through inducing HLA-DRB1 in the presence of HPV. Genotyping of further suggestive HPV-type specific signals from the seropositivity GWAS provided evidence of association at p < 0.05 for seven additional cervical cancer risk regions.

Conclusion: Our results support the view that several genomic risk loci exist for cervical dysplasia and cancer, and some of them may be HPV-type specific.

Grants: Supported by the Bruno and Helene Jöster Foundation and the Comprehensive Cancer Center of Lower Saxony (CCC-N).

Conflict of Interest: Theresa Beckhaus: None declared, Linda Kachuri: None declared, Finja Seifert: None declared, Rieke Eisenblätter: None declared, Dandan Liao: None declared, Peter Schürmann: None declared, Gerd Böhmer: None declared, Hans Georg Strauss: None declared, Christine Hirchenhain: None declared, Schmidmayr Monika: None declared, Florian Müller: None declared, Peter Fasching Peter A. Fasching conducts research funded by Amgen, Novartis and Pfizer., Peter A. Fasching received Honoraria from Roche, Novartis and Pfizer., Norman Häfner: None declared, Alexander Luyten: None declared, Matthias Jentschke: None declared, Peter Hillemanns: None declared, Francis Stephen: None declared, John Witte: None declared, Thilo Dörk: None declared, Dhanya Ramachandran: None declared

P01.093.D Precision medicine from the renal cancer genome

Yasser Riazalhosseini ¹

¹McGill University, Human Genetics, Montreal, Canada

Background/Objectives: Renal cell carcinomas (RCC) are characterized by their inherent resistance to chemotherapy and radiation and their largely diverse clinical outcomes. Leveraging genomic diversity among individual tumors, our research during the past decade has concentrated on developing personalized medicine approaches for the most common and the most aggressive type of RCC, clear cell renal carcinoma (ccRCC).

Methods: We generated the largest dataset of ccRCC including somatic genomic and clinical annotations for over 940 ccRCC patients, and developed a genomic classifier, based on mutational status of 12 RCC-relevant genes, which is able to stratify patients according to their risk of relapse after nephrectomy and/or death due to RCC.

Results: The performance of this classifier, which is independent from tumor stage and patient age, is validated for 75% of ccRCC patients whose tumors are affected by mutations of VHL gene. We have expanded the application of our 12-gene RCC assay to liquid biopsy analysis, aiming at developing a non-invasive approach for the application of our classifier, and establishing early detection of relapse in high-risk patients. Additionally, we observe a different genomic evolution trajectory of disease progression in ccRCCs that do not harbor VHL mutations. Our ongoing research focuses on understanding the distinct biology of these tumors using single-cell and spatial genomics, and our cohort of patient-derived organoid models of aggressive ccRCCs to identify novel therapeutic targets.

Conclusion: At the conference, I will present this decade-long endeavor and will discuss how our Pan-Canadian ‘Precision RCC’ program leverages this legacy to establish personalized medicine in RCC.

Grants:

Conflict of Interest: None declared

P01.094.A Predicting mutation risk at nucleotide scale using transformer neural networks

Marcell Veiner ^1;2, Iván Galván-Femenía¹, Daniel Naro^1;3, Fran Supek¹

¹IRB Barcelona - Institute for Research in Biomedicine, Genome Data Science, Barcelona, Spain; ²Universitat de Barcelona, Faculty of Medicine and Health Sciences, Barcelona, Spain; ³Universitat Politècnica de Catalunya · Barcelona Tech - UPC, Barcelona, Spain

Background/Objectives: Somatic mutation rates in cancer vary across the human genome at different scales. At the megabase- and gene-scale variation is well-studied, the mutation rate heterogeneity at sub-gene scales remains less explored. The aim of this contribution is to evaluate somatic mutation rates of Single Nucleotide Variants (SNV) in the human genome at base-pair resolution (1-300bp), and associate DNA sequence features to each mutational process.

Methods: We separated ~230M SNVs obtained from 12,166 pan-cancer whole-genome sequences into de novo mutational signatures using non-negative matrix factorisation, and fit to known COSMIC signatures. Subsequently, we trained a transformer neural network called MutFormer to differentiate between mutated vs trinucleotide-matched non-mutated sites on 18 SBS signatures, and to guide the discovery process of novel DNA determinants.

Results: Our models were validated on an independent cohort, achieving AUCs ranging from 0.55 on flat signatures (SBS5) to 0.86 on signatures dominated by a few mutation types (SBS17a/b), suggesting wider sequence context for these signatures (5-10bp on each side of the mutation). We found several DNA features shaping the mutational landscape, some as a wider sequence context (e.g. AAAKAATAAAW, SBS10a), while some by being in the vicinity of the mutation (CCWGG, SBS17b). Our method was also applied to model the germline mutation rate, achieving similarly high AUCs.

Conclusion: Overall, our transformers provide a state-of-the-art method for inferring the risk of SNVs occurring given the nearby DNA sequence, with implications to disease gene discovery and evolutionary biology.

Grants: “la Caixa” Foundation (ID LCF/BQ/DR23/12000017), ERC StG “HYPER-INSIGHT” 757700.

Conflict of Interest: None declared

P01.095.B Dyskeratosis congenita segregation in a large Middle Eastern family reveals a novel pathogenic TERC variant

Michal Barzily Rokni ¹, Chezi Ganzel^2;3, Malka BenUziyahu¹, Adi Zisman¹, Rachel Beeri¹, Ephrat Levy-Lahad^1;3

¹Shaare Zedek Medical Center, Medical Genetics Institute, Jerusalem, Israel; ²Shaare Zedek Medical Center, Department of Hematology, Jerusalem, Israel; ³Hebrew University of Jerusalem, Faculty of Medicine, Jerusalem, Israel

Background: Dyskeratosis congenita (DC) is a rare, heterogeneous genetic disorder caused by defective telomere maintenance resulting in shortened telomeres. TERC pathogenic variants (PVs) (the telomerase RNA component) gene cause autosomal dominant DC. The highly variable expression of the disease, even in the same family, makes diagnosis and surveillance recommendations a challenge.

Objectives: To determine the pathogenicity of a TERC variant of unknown significance (VUS), using segregation in an extensive Muslim Arab family (over 100 members).

Results: A 50-year-old man with myelodysplastic syndrome (MDS) reported family history of four brothers and a sister who died of leukemia. Exome-based panel testing identified a TERC gene variant, n.97T>G (rs1777963567). This variant has been reported once in ClinVar, as a VUS. It is not present in any database, and is located within the template/pseudoknot domain of the TERC RNA component, which is required for RNA or RNP stability. We reclassified the VUS as a novel, likely pathogenic variant after: (1) Identifying clinical features of DC in the patient, including: dysplastic nails, oral leukoplakia, pulmonary fibrosis, prematurely gray hair and liver disease. (2) Determining shortened telomere length in the patient’s blood. (3) Segregation analysis revealing all affected siblings (four) as heterozygous for the variant and non-affected siblings (five) as negative for the variant. Genetic counseling services and surveillance recommendations were offered to all at risk family members, of which only a few responded.

Conclusion: We report n.97T>G as a novel PV in the TERC gene causing DC, in order to further aid identification of and treatment for DC families.

Conflict of Interest: None declared

P01.096.C A rare homozygous germline missense variant of TP53 causing Li-Fraumeni syndrome

Fuad Chowdhury ¹, Melanie Finkbeiner², Katie Girgulis², Ryan Lamont³, Jillian Parboosingh³, Lucie Pecheux⁴, Renee Perrier¹

¹University of Calgary and Alberta Children’s Hospital, Department of Medical Genetics, Calgary, Canada; ²University of Calgary and Alberta Children’s Hospital, Department of Pediatric Hematology-Oncology, Calgary, Canada; ³Molecular Diagnostic Laboratory, Department of Medical Genetics, Calgary, Canada; ⁴University of Alberta and Stollery Children’s Hospital, Department of Pediatric Hematology-Oncology, Edmonton, Canada

Background: Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome caused by heterozygous variants in TP53 often through loss-of-function mechanisms. Subsequent somatic loss-of-heterozygosity is frequently observed in tumour samples of many cancer types, while bi-allelic germline variants are extremely rare in individuals with LFS. Most of these individuals are homozygous for the lowly-penetrant R337H founder variant.

Methods: Clinical panel or single-gene sequencing was performed on proband peripheral blood DNA to identify TP53 variants. Subsequently, first-degree relatives were tested for inheritance of the TP53 variant, when possible.

Results: We report two previously healthy and unrelated girls diagnosed with adrenocortical carcinoma (ACC). Both presented with several months of virilization and clinical evidence of androgen excess. The first patient is a 5-year-old girl of Pakistani ancestry with consanguineous parents who is homozygous for a NM_000546.6(TP53):c.328C>T (p.R110C) germline variant of uncertain significance (VUS). The second is a 2-year-old girl presenting additionally with hypercortisolism and she is heterozygous for the same TP53 variant. Neither family had a history of LFS-spectrum cancers.

Conclusions: The presence of a germline R110C variant in two unrelated girls with a rare ACC provides strong clinical evidence that TP53 p.R110C can cause LFS. Pathogenic variants affecting the same residue have been described. Furthermore, previous in vitro functional characterization of p53-R110C demonstrated reduced transcriptional activity, induction of apoptosis, and altered subcellular localization. Moreover, this report provides further clinical evidence of the pathogenicity of hypomorphic germline TP53 variants when homozygous and supports surveillance for relatives with at least one R110C variant.

Grants: None.

Conflict of Interest: None declared

P01.097.D Systematic application of ClinGen InSiGHT APC-specific ACMG/AMP variant classification criteria alleviates the burden of variants of uncertain significance in ClinVar and LOVD databases

Xiaoyu Yin^1;2;3, Marcy Richardson⁴, Andreas Laner⁵, Xuemei Shi⁶, Elisabet Ognedal⁷, Valeria Vasta⁸, Thomas van Overeem Hansen^9;10, Marta Pineda^11;12;13, Deborah Ritter^14;15, Johan T. den Dunnen¹⁶, Emadeldin Hassanin^17;18, Lyman Lin¹⁹, Ester Borras²⁰, Karl Krahn²¹, Margareta Nordling^22;23, Alexandra Martins²⁴, Khalid Mahmood²⁵, Emily Nadeau²⁶, Victoria Beshay²⁷, Carli Tops¹⁶, Maurizio Genuardi²⁸, Tina Pesaran⁴, Ian M. Frayling^29;30;31, Gabriel Capellá^11;12;13, Andrew Latchford^29;32, Sean V. Tavtigian^33;34, Carlo Maj^17;35, Sharon E Plon^14;15, Marc Greenblatt²⁶, Finlay A. Macrae^1;2, Isabel Spier ^3;11;36, Stefan Aretz^3;11;36

¹Department of Colorectal Medicine and Genetics, Royal Melbourne Hospital, Parkville, Australia; ²Department of Medicine, University of Melbourne, Parkville, Australia; ³Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, Germany; ⁴Ambry Genetics, Aliso Viejo, California, USA; ⁵Medical Genetics Center Munich, MGZ Munich, Germany; ⁶Greenwood Genetic Center, Greenwood, South Carolina, USA; ⁷Western Norway Familial Cancer Center, Haukeland University Hospital, Bergen, Norway; ⁸Northwest Genomics Center, Department of Genome Sciences, University of Washington, Seattle; ⁹Department of Clinical Genetics, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark; ¹⁰Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark; ¹¹European Reference Network on Genetic Tumour Risk Syndromes (ERN GENTURIS) – Project ID No 739547; ¹²Hereditary Cancer Program, Catalan Institute of Oncology – ONCOBELL, IDIBELL, Barcelona, Spain; ¹³Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Instituto Salud Carlos III, Madrid, Spain; ¹⁴Baylor College of Medicine, Houston, Texas, USA; ¹⁵Texas Children’s Cancer Center, Texas Children’s Hospital, Houston, Texas, USA; ¹⁶Department of Human Genetics, Leiden University Medical Center, Leiden, Netherlands; ¹⁷Institute for Genomic Statistics and Bioinformatics, University Hospital Bonn, Bonn, Germany; ¹⁸Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg; ¹⁹St Vincents Hospital Melbourne, East Melbourne, Australia; ²⁰Invitae Corporation, San Francisco, California, USA; ²¹GeneDx, Gaithersburg, Maryland, USA; ²²Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden; ²³Department of Clinical Genetics, Linköping University Hospital, Linköping, Sweden; ²⁴Univ Rouen Normandie, Inserm U1245, F-76000 Rouen, France; ²⁵Colorectal Oncogenomics Group, Department of Clinical Pathology, University of Melbourne, Melbourne, Australia; ²⁶Department of Medicine, Larner College of Medicine, University of Vermont, Burlington, Vermont, USA; ²⁷Peter MacCallum Cancer Centre, Melbourne, Australia; ²⁸Fondazione Policlinico Universitario A. Gemelli IRCCS, and Dipartimento di Scienze della Vita e Sanità Pubblica, Università Cattolica del Sacro Cuore, Rome, Italy; ²⁹Polyposis Registry, St Mark’s Hospital, London, UK; ³⁰Inherited Tumour Syndromes Research Group, Institute of Cancer & Genetics, Cardiff University, UK; ³¹National Centre for Colorectal Disease, St Vincent’s University Hospital, Dublin, Ireland; ³²Department of Surgery and Cancer, Imperial College, London UK; ³³Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, USA; ³⁴Department of Oncological Sciences, School of Medicine, University of Utah, Salt Lake City, Utah, USA; ³⁵Centre for Human Genetics, University of Marburg, Germany.; ³⁶National Center for Hereditary Tumor Syndromes, University Hospital Bonn, Bonn, Germany

Consortium: ClinGen InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel

Background/Objectives: To resolve the interpretative challenges of variants of uncertain significance (VUS), gene-specific ACMG/AMP variant classification criteria were developed and validated for the APC gene by the ClinGen-InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP) (https://cspec.genome.network/cspec/ui/svi/doc/GN089).

Methods: A streamlined algorithm using the APC-specific criteria was developed and applied to reassess all APC variants in ClinVar and the reference APC gene variant database in the Leiden Open Variation Database (LOVD).

Results: A total of 10,228 unique APC variants were analysed. 94% and 96% of the ClinVar and LOVD variants, respectively, with an initial classification of (Likely) Benign or (Likely) Pathogenic remained in their original categories. 41% of ClinVar and 61% of LOVD VUS were reclassified into clinically actionable classes, the vast majority as (Likely) Benign. The total number of VUS was reduced by 37% from 6142 to 3865. For 36 promising APC variants that remained VUS despite evidence for pathogenicity, a data mining-driven work-up was undertaken to curate additional evidence which allowed the further reclassification of 18 VUS as (Likely) Pathogenic.

Conclusion: The application of APC-specific classification criteria substantially reduced the number of VUS in ClinVar and LOVD. The study also demonstrates the feasibility of a systematic approach to variant classification with large datasets, which serves as a generalisable model for other gene-/disease-specific variant interpretation initiatives. It also allows for the prioritization of VUS that will benefit from in-depth evidence collection. The reclassified promising APC variants will be subjected to VCEP approval and made publicly available through ClinVar and LOVD for widespread clinical use.

Grants: U24CA258119

Conflict of Interest: Xiaoyu Yin: None declared, Marcy Richardson: None declared, Andreas Laner: None declared, Xuemei Shi: None declared, Elisabet Ognedal: None declared, Valeria Vasta: None declared, Thomas van Overeem Hansen: None declared, Marta Pineda: None declared, Deborah Ritter: None declared, Johan T. den Dunnen: None declared, Emadeldin Hassanin: None declared, Lyman Lin: None declared, Ester Borras: None declared, Karl Krahn: None declared, Margareta Nordling: None declared, Alexandra Martins: None declared, Khalid Mahmood: None declared, Emily Nadeau: None declared, Victoria Beshay: None declared, Carli Tops: None declared, Maurizio Genuardi: None declared, Tina Pesaran: None declared, Ian M. Frayling: None declared, Gabriel Capellá: None declared, Andrew Latchford: None declared, Sean V. Tavtigian: None declared, Carlo Maj: None declared, Sharon E Plon Sharon Plon is a member of the scientific advisory panel of Baylor Genetics Laboratories., Marc Greenblatt: None declared, Finlay A. Macrae: None declared, Isabel Spier: None declared, Stefan Aretz: None declared

P01.098.A Using the homologous recombination deficiency mutational signature to look for the missing heritability in breast cancer

Jose Camacho Valenzuela ^1;2, nancy hamel^2;3, Thibaut Matis^4;5, paz polak⁶, Carla Daniela Robles Espinoza^7;8, William Foulkes^1;2;3

¹McGill University, Human Genetics, Montréal, Canada; ²MUHC Research Institute, Cancer Research Program, Montréal, Canada; ³Lady Davis Institute at the Jewish General Hospital, Montréal, Canada; ⁴Institute Bergonié, Cancer genetics unit, Bordeaux, France; ⁵Bric Bordeaux Institute Of Oncology - Inserm U1312 University Bordeaux - Institute D’oncologie De Bordeaux., Bordeaux, France; ⁶Icahn School of Medicine at Mount Sinai, Oncological sciences, New York, United States; ⁷Laboratorio Internacional de Investigación sobre el Genoma Humano (LIIGH) UNAM, Juriquilla, Mexico; ⁸Wellcome Sanger Institute, Experimental cancer genetics, Hinxton, United Kingdom

Background/Objectives: Approximately 50% of hereditary breast cancer is explained by germline variants in well-known Breast Cancer Susceptibility Genes (BCSGs), predominantly BRCA1/2. The remaining 50% remains unexplained. We hypothesized that analyzing DNA from normal tissue and breast tumor from the same patient would allow us to identify other Homologous Recombination Repair (HRD)-related BCSGs, should they exist.

Methods: Focusing on matched normal/tumor exome data of breast cancer patients from The Cancer Genome Atlas, we developed a combined multidimensional pipeline to correlate the presence of Mutational Signature 3 (Sig3) with germline cancer predisposition variants, accounting for secondary tumor events. We evaluated genes enriched with variants in patients with Sig3 in their tumors, including non-cancer genes.

Results: We confirmed a significant association of BRCA1/2 with Sig3 (p < 0.05). We also observed an association for some HRD genes, specifically RAD51C (p < 0.05), PALB2 and RAD51D, though the last two did not reach significance. We did not identify positive correlation with Sig3 for previously unrelated genes. Somatic biallelic loss of BRCA1/2 (~70%) and methylation in BRCA1/RAD51C (~20%) explain most cases lacking germline BRCA1/2 variants. BARD1 and PALB2 germline variants with somatic second hits explained some of the remaining non-BRCA1/2 cases. We are further investigating the non-BRCA cases with Sig3.

Conclusion: Somatic and epigenetic events in well-known HRD genes appear to explain approximately 90% of HRD cases without BRCA1/2 germline variants. Other non-BRCA1/2 genes likely explain only a small proportion of cases. An integrated germline/tumor approach is a powerful genomic strategy to understand non-BRCA1/2 cases.

Grants: CIHR (FDN-148390).

Conflict of Interest: None declared

P01.099.B Landscape of Germline Variants in Cases of Ovarian Cancer: Insights from a Single-Center Study

Ece ÇİÇEK ¹, Drenushe Zhurı¹, Nilay Ozupek¹, Hazal Sezginer Guler¹, umut çiçek¹, HAKAN GURKAN¹

¹Trakya University, Medical Genetics, Edirne, Türkyie

Background/Objectives: Ovarian cancer stands as one of the predominant gynecological malignancies globally. Diverse risk factors have been elucidated in connection with ovarian cancer, encompassing advanced age, genetic predisposition, familial history, nulliparity, and others. Existing research underscores that over 20% of ovarian cancer cases exhibit a hereditary genetic basis, with approximately 70% of these genetic anomalies manifesting as germline mutations in the BRCA1/2 genes. In this study, we aimed to investigate the mutational spectrum of ovarian cancer in a single-center patient group.

Methods: Between January 2022 and September 2023, our study enrolled 60 patients diagnosed with ovarian cancer who applied to our Genetic Diseases Evaluation Center. DNA samples extracted from the peripheral blood of patients underwent analysis utilizing the The QIAseq Human Breast Cancer Panel (93 genes, DHS-001Z; Qiagen).

Results: In fifteen cases, comprising 25% of the study cohort, sixteen pathogenic/likely pathogenic (P/LP) variants were identified, with one of them being novel. Additionally, seventeen cases presented 22 variants of unknown clinical significance (VUS). These cases were categorized into two groups: (1) carriers of BRCA1/2 variant and (2) non-carriers of BRCA1/2 variant, with 50% of the P/LP variants detected in BRCA1/2 genes, primarily within group1.

Conclusion: The mutations most frequently observed in cases of ovarian cancer are typically found in the BRCA genes. However, our study’s findings indicate a lack of correlation with previously reported mutation rates in genes other than BRCA1/2. This observation suggests the potential to offer novel insights into the mutational spectrum of ovarian cancer.

Conflict of Interest: None declared

P01.100.C Mutational landscape of glioblastoma tissue and derived tumoroid exomes

Vita Rovite ¹, Sigita Hasnere², Raitis Pečulis¹, Aija Gerina², Olesja Rogoza¹, Omkar Suhas Vinchure³, Vaibhav Jadhav³, Jay Gopalakrishnan³

¹Latvian Biomedical Research and Study centre, Molecular and Functional Genomics, Riga, Latvia; ²P. Stradins Clinical University Hospital, Oncology, Riga, Latvia; ³Institute for Human Genetics Heinrich-Heine-Universität Universitätsklinikum Düsseldorf, Laboratory for Centrosome and Cytoskeleton Biology, Düsseldorf, Germany

Background/Objectives: Glioblastoma (GB) is the most frequent malignant brain tumor. GB patients’ median survival is <24 months, and there is no cure. Glioma-PerMed project is consortium based effort to establish rapid and efficient glioma invasion assays and machine learning algorithms for predicting the GB invasion in ex-vivo, personalized diagnostics, drug discovery, and patient follow-up in the clinics. Aim of this study was to charcterize mutational landscape of GB tissue and derived tumoroid models.

Methods: Five GB tissue and seven tumoroids was obtained at Jay Gopalakrishnan lab in University of Dusseldorf, Germany. Sequencing of the obtained biological samples was done at Latvian Biomedical Research and Study cente using MGIEasy Exome Capture V4 Probe Set and MGIEasy Exome Universal Library Prep Set on DNBSEQ-G400 sequencer. Exome sequencing reads were trimmed using fastp v0.23.4, afterwards reads were aligned to human genome reference GRCh38.p14 using bwa mem v0.7.17. Tumor tissue - tumoroid sample pair variant differences were detected with Strelka v2.9.10.

Results: GB tissue samples contained from 149124 to 209524 variants while tumoroids from 18169 to 186502 variants. Specifically, we assessed the presence of mutations in 79 genes realted to GB pathogenesis. In tumour tissue we found mutations in 34-30 genes, while tumoroids this varied from 16 - 45 genes.

Conclusion: In our sample set tumour tissues most often we detected mutations in known glioma associted genes, for example, TP53, PDGFRA, ATR, and less number of cases IDH1, EGFR and PTEN mutations.

Grants: ERA PerMed ES RTD/2023/20

Conflict of Interest: None declared

P01.102.A Germline mutations in HIC1 are associated with serrated polyposis syndrome

Xavier Domínguez-Rovira ¹, Coral Arnau-Collell¹, Gemma Gonfaus¹, Gemma Llargués-Sistac¹, Jenifer Muñoz¹, Ainara Llopis¹, Yasmin Soares de Lima¹, Cristina Herrera-Pariente¹, Leticia Moreira¹, Teresa Ocaña¹, Marcos Díaz-Gay², Miriam Cuatrecasas³, Sabela Carballal¹, Anael Lopez-Novo⁴, Guerau Fernandez⁵, Antoni Castells¹, Luis Bujanda⁶, Gabriel Capellá⁷, Joaquin Cubiella⁸, Daniel Rodriguez-Alcade⁹, Laura Valle⁷, Francesc Balaguer¹, Clara Ruiz-Ponte⁴, Laia Bonjoch¹, Sergi Castellví-Bel¹

¹Hospital Clínic, FRCB-IDIBAPS, CIBEREHD, Gastroenterology, Barcelona, Spain; ²Moores Cancer Center at UC San Diego, La Jolla, San Diego, United States; ³Hospital Clínic de Barcelona, Pathology, Barcelona, Spain; ⁴Fundación galega de medicina xenómica, Grupo de Medicina Xenómica-USC, IDIS, CIBERER, Santiago de Compostela, Spain; ⁵Sant Joan de Déu Barcelona Hospital, Barcelona, Spain; ⁶Donostia Hospital, CIBEREHD, Gastroenterology, San Sebastián, Spain; ⁷Catalan Institute of Oncology, Barcelona, Spain; ⁸Complexo Hospitalario Universitario de Ourense - CHUO, Gastroenterology, Ourense, Spain; ⁹Hospital Universitario de Móstoles, Digestive diseases section, Móstoles, Spain

Background/Objectives: Serrated polyposis syndrome (SPS) is characterized by multiple and/or large serrated polyps and colorectal cancer (CRC) risk. HIC1 (Hypermethylated in cancer 1) is a transcriptional repressor that functions as a tumor suppressor gene, previously suggested as a new candidate gene to germline predisposition to SPS (1). We aimed to provide functional evidence to evaluate HIC1 as a new hereditary gene for SPS.

Methods: Gene-panel sequencing of 211 SPS patients was performed to validate the findings in a discovery cohort of 39 patients from 16 SPS families. Two cellular models for HIC1, a CRISPR/Cas9 knock-out model and an overexpression model, were produced. We assessed the DNA damage response and the SIRT1 regulatory pathway upon HIC1 genetic variation.

Results: Three predicted pathogenic variants were identified within the HIC1 gene (p.Ala37Val, p.Gln432Arg and p.Gly471Arg) in the discovery and validation cohorts. Functional characterization revealed higher p-H2AX levels (a marker for ongoing DNA damage) and impaired HIC1 binding to the SIRT1 promoter in cellular models carrying the identified genetic variants when compared with the wild-type sequence.

Conclusion: Our results indicate that HIC1 genetic variants located in the zinc finger region can lead to disruption of protein function. Our results support HIC1 as a new gene associated with SPS predisposition, although additional cohorts should be evaluated to further investigate its hereditary role.

References:

(1) Soares de Lima Y et al. Cancers 2021; 13(4):1–21.

Grants: FIS-FEDER 20/00113 and 23/00189, Marató TV3-202008-10, Horizon Europe Twinning Project STEPUPIORS, AECC PRYGN211085CAST, CIBEREHD, CERCA Program and AGAUR GRC2021SGR01185.

Conflict of Interest: None declared

P01.103.B Breast Cancer Risk Polygenic Score (PGS) optimisation through a novel SNP selection algorithm

Carla Debernardi ¹, ANGELO SAVOCA¹, Elisabetta Casalone¹, Kristina Zguro², Giulia Brunelli², Zhiyu Yang³, Brooke Wolford⁴, Alessia Russo¹, Samuli Ripatti⁵, Giuseppe Matullo¹

¹University of Turin, Department of Medical Sciences, Turin, Italy; ²University of Siena, Department of Medical Biotechnologies, Med Biotech Hub and Competence Center, Siena, Italy; ³University of Helsinki, Helsinki; ⁴HUNT Center for Molecular and Clinical Epidemiology, Department of Public Health and Nursing, NTNU, Norwegian University of Science and Technology, Trondheim, Norway; ⁵Institute for Molecular Medicine Finland, FIMM, HiLIFE, University of Helsinki, Helsinki, Finland; Clinicum, Department of Public Health, University of Helsinki, Helsinki, Finland; Broad Institute of MIT and Harvard,Cambridge, MA, USA., Finland

Background/Objectives: Many Polygenic Scores (PGS) have been described for Breast Cancer (BC), differing by number and selection of SNPs. This variability leads to a wide range of estimated PGS in the same individual and therefore to inconsistent risk stratification. The aim of this study is to optimise the computation of BC risk scores by the selection of the variants involved.

Methods: Starting from the 100 PGSs available for BC on the PGS Catalog, we aligned all their variants to obtain the most represented SNPs associated to disease risk. TheSNPs selection approach was based on the association with Breast Cancer and on the concordance between the different PGSs. Standardization and embedding operations were performed on the effect weights to create a new PGS.

Results: The newly created PGS has 4,606 SNPs. We tested this PGS on Genomic England, UK Biobank and FinnGen data. In all the data sets tested the combined approach to estimate the effect weights shows the best performance, distributions between BC patients and controls were significantly different (Kolmogorov–Smirnov test p-value < 0.0001). Across cohorts, women in the highest 1% of the score had a two to three-fold increased risk of develop BC compared with women in the middle quintile Lifetime risk of BC for women in the lowest and highest quintiles of the PRS was 10% and more than 25%, respectively.

Conclusion: These finding and optimization approach strengthen the standardisation of PGS that could help clinicians to better stratify patients risk of developing BC.

Grants: INTERVENE, HORIZON Project 2020 n.101016775

Conflict of Interest: None declared

P01.104.C Innovative Prostate Cancer Classifier: Validating an Explainable Artificial Intelligence Tool

Patricia Porras-Quesada ^1;2, Alberto Ramírez-Mena², Verónica Arenas-Rodríguez^1;2, Beatriz Álvarez-González³, Jesús Alcalá-Fdez⁴, Luis Javier Martínez-González^1;2, Maria Jesus Alvarez Cubero^1;2;5

¹Faculty of Medicine - University of Granada, Biochemistry and Molecular Biology III and Immunology Department, Granada, Spain; ²Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain; ³Faculty of Medicine - University of Granada, Legal Medicine and Toxicology Department, Granada, Spain; ⁴E.T.S. of Computer and Telecommunication Engineering, Computer Science and Artificial Intelligence Department. Andalusian Research Institute in Data Science and Computational Intelligence (DaSCI). University of Granada, Granada, Spain; ⁵IBS, Biosanitary Research Institute (ibs. GRANADA), University of Granada, Granada, Spain

Background/Objectives: Although prostate cancer (PC) is the second most frequent tumour worldwide, its diagnosis focuses on inconclusive or not necessarily cancer-specific strategies. Therefore, a classifier is proposed to predict the occurrence of PC and to provide a tool to assist the pathologist in the decision-making process.

Methods: Different machine learning methods based on eXplainable Artificial Intelligence were trained using gene expression data from 550 samples available on ‘The Cancer Genome Atlas’ repository. Our model was validated in 463 samples from four external cohorts. Additionally, a set of SHapley Additive exPlanations was provided to help clinicians to understand each prediction. To analyze reliability and feasibility of applying the classifier in clinical practice, a gene expression analysis by qPCR was performed in 60 fresh prostate tissue samples (20 control individuals and 40 PC patients stratified by tumor aggressiveness).

Results: An in-depth analysis showed that the Random Forest algorithm combined with undersampling procedure was the best performing approach with robust statistical significance (sensitivity:0.90; specificity:0.8; AUC:0.84) across all databases. Classifier validation in prostate tissue samples revealed an increase in DLX1, TDRD1, AMACR, HPN, HOXC6 and OR51E2 expression when comparing high vs. low aggressive PC patients and PC patients vs. controls.

Conclusion: Proposed tool has shown good performance in 4 independent external cohorts of different ancestries and the explanations provided are consistent with the results obtained in the experimental validation with biological samples. In the near future, highlighted genes expression, in combination with our model, could be applied to liquid biopsy to improve PC screening.

Grants: Regional Government of Andalusia (PIP-0043-2022).

Conflict of Interest: None declared

P01.105.D Risk management for breast cancer in BRCA mutation carriers; compliance to the recommended surveillance.

Toine Werker ¹, Simone Salemink¹, Wendy Van Zelst-Stams¹, Arjen Mensenkamp¹, Angela van Remortele¹, Maaike Haadsma¹

¹Radboud University Medical Center, Human Genetics, Nijmegen, Netherlands

Background/Objectives: Due to their highly increased risk of breast cancer, women with a pathogenic mutation in the BRCA1- or BRCA2-gene are eligible for preventive mastectomy or specific breast cancer surveillance protocols to warrant early detection. This study investigates the extent to which BRCA mutation carriers follow the Dutch breast cancer guideline recommendations.

Methods: We performed a cross-sectional study at the department of Human Genetics of the Radboud University Medical Center, Nijmegen, The Netherlands, among presymptomatic women aged 25 to 75 with a pathogenic mutation in the BRCA1- or BRCA2-gene, identified between 2012 and 2021. Women were asked about their current surveillance status through a survey.

Results: 256 out of 709 (36,1%) women responded and the 200 women without a history of breast cancer were included. In total, 86% (172/200) of them were compliant to the recommended guidelines. Of these women, 85 (42.5%) underwent preventive mastectomy. In the 115 other women, eligible for surveillance, in 75.7% (87/115) breast screening was performed according to the guideline. Women non-compliant to guideline surveillance were more often BRCA1 mutation carriers (p = 0.045), of older age (p = 0.019), and had a longer time since mutation detection (p = 0.031).

Conclusion: This study, not previously conducted among Dutch BRCA mutation carriers, reveals that the majority of women were compliant with recommendations concerning their breast cancer risk. However, nearly one-fourth of those eligible for surveillance do not receive guideline-compliant breast screening. We believe that guideline compliance can be improved by closer collaboration between involved healthcare providers.

Grants: Not applicable.

Conflict of Interest: None declared

P01.106.A Clinical and Molecular Characterization of Fumarate Hydratase (FH) germline carriers in Omani families.

Abeer Alsaegh ¹, Reem Abdulrahim¹, Chantel Van Wyk², Ilse Crous², Sara Al Kiyumi², Amira Al Balushi², Suad Al Malki², Bushra Al Muhairi²

¹Sultan Qaboos Comprehensive Cancer Care and Research Center, Genomics, Oman; ²Sultan Qaboos Comprehensive Cancer Care and Research Center

Backgroud/Objecties: Although there are several families described with FH mutations associated with Hereditary Leiomyomatosis and Renal Cell Cancer, the prevalence and full spectrum of the phenotype is still unknown. The objective of this study is to collect and describe the clinical and molecular phenotype in Omani families with pathogenic mutations in FH gene.

Methods: Retrospective hospital chart review of individuals referred to clinical genetics service from 2021 to 2023 to Sultan Qaboos Comprehensive Cancer Care and Research Center.

Results: A total of 30 FH mutation carriers were identified in this cohort. The majority were diagnosed with renal cell carcinoma (19/30). There was also (2/30) patients diagnosed with Uterine leiomyosarcoma, (5/30) diagnosed with pre- menopausal young onset breast cancer, (2/30) diagnosed with bladder carcinoma and only 2/30 with the typical skin lesions for cutaneous leiomyomas. Uterine fibroids were only reported in (4/30).

Conclusion: In our cohort we seem to identify more carriers diagnosed with breast and bladder cancer and all have the common founder mutation in FH gene (c.698G>T). In view of these results, patients with this mutation may benefit from early breast cancer screening in addition to other common cancers associated with this syndrome.

Grant: no grant was obtained to do this study

Conflict of Interest: None declared

P01.107.B ATM c.7475T>G, p.(Leu2492Arg) variant confers a moderate risk for breast cancer

Ivana Maleva Kostovska ¹, Predrag Noveski¹, Sanja Kiprijanovska¹, Simona Jakovchevska¹, Dijana Plaseska-Karanfilska¹

¹Macedonian Academy of Sciences & Arts, Research Center for Genetic Engineering and Biotechnology “Georgi D. Efremov”, Skopje, Macedonia

Background/Objectives: Case-control and family-based studies have identified ATM as a moderate breast cancer (BC) susceptibility gene. While germline protein truncating variants (PTVs) in ATM are linked to a 2-4 fold elevated BC risk, the risk posed by missense variants remains uncertain, with the majority classified as variants of uncertain significance (VUS). Our study aimed to comprehensively evaluate the prevalence of ATM variants among BC cases and general population.

Methods: Sequencing of BC cases (n = 971) was performed using TruSight Cancer NGS sequencing panel, while ATM variants in the control samples (n = 871) were derived from WES conducted in our laboratory. Additionally, 280 BC cases were screened for the ATM c.7475T>G variant with allele specific PCR.

Results: The observed ATM mutation rate was 1.9% (18/971) in BC cases and 0.5% (4/871) in the control cohort, with the majority of pathogenic variants (80%, 12/15) being PTVs. VUS variants were identified in 53 BC cases (5.5%) and 41 controls (4.7%). The most common VUS c.7475T>G, p.(Leu2492Arg) was identified in 1.6% of the BC patients and 0.6% of the controls (20/1251 vs. 5/871), thus carrying a moderate increased risk for BC (OR 2.8; 95%CI 1.05-7.52, p = 0.03). This variant has higher allele frequency in our population compared to gnomAD (0.59% vs. 0.01%), is located in the FAT domain, has CADD score of 26.7 and is classified as VUS in ClinVar.

Conclusion: This is the first study to demonstrate moderate BC risk in ATM c.7475T>G carriers, emphasizing the importance of studying ATM VUS in different populations where some VUS exhibit higher frequencies.

Conflict of Interest: None declared

P01.108.C Investigating the Impact of IDH1 Mutation on Monocyte Differentiation Transcriptome in Early Glioma Development

Ebru BAKIR ¹, Yavuz Oktay¹

¹izmir Biomedicine and Genome Center, Molecular Biology and Genetics, İzmir, Türkyie

Background/Objectives: Gliomas, the most prevalent primary brain tumors, often harbor IDH1 mutations, notably the R132H alteration, leading to the production of the oncometabolite 2-hydroxyglutarate (2-HG). This study aims to investigate the effects of secreted factors from astrocytes mimicking early IDH1 R132H gliomas on monocyte differentiation, shedding light on the molecular mechanisms underlying glioma development.

Methods: Immortal human astrocytes expressing IDH1 R132H were utilized to generate conditioned media (CM) mimicking early glioma stages. Primary monocytes were exposed to these CM during differentiation to macrophages. Transcriptome-wide analysis of differentiated macrophages was conducted via RNA sequencing to identify differentially expressed genes in response to the IDH1 mutation.

Results: RNA sequencing revealed 124 differentially expressed genes associated with the IDH1 mutation. Gene ontology analysis indicated interference with the p53 signaling pathway and elevated signatures related to negative regulation of complement activation in IDH1 R132H-exposed cases. Additionally, pathways associated with Th1 and Th2 cell differentiation, as well as cytotoxic T cell homeostasis, were significantly enriched.

Conclusion: Secreted factors from astrocytes in early glioma formation significantly impact monocyte differentiation transcriptome, potentially shaping the tumor microenvironment. The findings underscore the role of IDH1 mutation in modulating immune responses and highlight potential therapeutic targets for glioma treatment.

Grants: TÜBİTAK 1001 Project no:214S097

Conflict of Interest: None declared

P01.109.D Characterization of the transgenic animal models Danio Rerio and Caenorhabditis Elegans to examine pathophysiology associated with SMARCB1 variants

Elena Tacchetto^1;2, Lisa Buson², valeria morbidoni¹, Cristina Cerqua¹, Valerio Magnaghi³, Laura Papi⁴, Eva Trevisson ^1;2

¹IRP – Istituto di Ricerca Pediatrica Città della Speranza, Padova, Italy; ²Università degli Studi di Padova, Salute della Donna e del Bambino, Roma, Italy; ³Università degli Studi di Milano, Dipartimento di scienze farmaceutiche e molecolari, Milano, Italy; ⁴Università di Firenze, Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, Firenze, Italy

Background/ Objectives: SMARCB1 is a nuclear protein and core subunit of the BAF chromatin-remodeling complex. SMARCB1 variants are associated with tumor predisposition syndromes and developmental disorders. We developed and characterized two transgenic models, C.elegans and zebrafish, to examine the pathophysiology associated with SMARCB1.

Methods: We analyzed the expression of SMARCB1 orthologues. In C.elegans, snfc-5 was analyzed by q-RT PCR. In D.rerio there are two paralogs: smarcb1a and smarcb1b, whose spatial and temporal expression was analyzed. We employed CRISPR/Cas9 to obtain our transgenic animals: we generated smarcb1a-/-, smarcb1b-/- and smarcb1ab + /- lines in D. rerio. In C. elegans, we modeled mutations associated with distinct human phenotypes. We performed a series of experiments to characterize the transgenic lines, as brood size and longevity and we are studying nociception. In zebrafish, we are performing phenotypical observation, survival curves and histological analysis.

Results: In zebrafish the two isoforms were detected from the first stages of embryonic development and ubiquitously. With an in-vivo analysis, we observed that smarcb1 reaches the highest levels of expression in the central nervous system. In C. elegans, the single isoform snfc-5 is expressed from the egg stage to the adulthood. Our data suggest that smarcb1 is a maternal determinant. The phenotypical analysis of our transgenic lines, suggest that SMARCB1 plays an important role in survival, embryonic development and tissue maintenance.

Conclusion: Our findings indicate that SMARCB1 plays key roles in different pathways and developmental stages. The integration of this multidisciplinary approach could help to identify novel therapeutic targets.

Grants: PRINN2017

Conflict of Interest: Elena Tacchetto PRINN2017, Lisa Buson: None declared, valeria morbidoni: None declared, Cristina Cerqua: None declared, Valerio Magnaghi PRINN2017, Laura Papi PRINN2017, Eva Trevisson PRINN2017

P01.110.A KMT2C mutations associated with colorectal and lung cancer development

Dimitar Dimitrov ¹, Nelly Miteva-Marcheva¹, Hristo Ivanov¹, Aleksandar Linev¹, Vili Stoyanova¹

¹Medical University of Plovdiv, Department of Pediatrics and Medical Genetics, Plovdiv, Bulgaria

Background: Worldwide, cancer is a leading cause of death. We conducted a study which aim was to investigate mutations associated with tumorigenesis in patients with colorectal and lung cancer.

Methods: A total of 31 cancer patients (12 with lung cancer, 19 with colorectal carcinoma) were enrolled, from whom ctDNA samples were collected by liquid biopsy and subjected to targeted sequencing with a panel of 484 cancer-related genes followed by bioinformatic analysis.

Results: Sequencing revealed diverse genetic variants, and pathogenic or likely pathogenic variant were found in the KMT2C gene in 100% of the patients (KMT2C c.2961C>G, KMT2C c.8390del, KMT2C c.2578C>T, KMT2C c.2573G>T and KMT2C c.2917A>G).

Discussion: The KMT2C gene is part of the KMT2 family. Dysregulation or mutation of KMT2 family have been observed frequently in various types of human cancers including lung and colorectal cancers. Most KMT2C mutations lead to protein reduction, promoting tumorigenesis by dysregulating enhancer activity. Similar to our study, a research by Chatarina Larsson et al. found that the KMT2C variant c.8390del occurs in 25-48% of MSI CRC cases, also this variant leads to inactivation of KMT2C, promoting the development of colorectal cancer through transcriptional dysregulation in several pathways known to be involved to cancer.

Conclusion: Our findings underscore the potential of KMT2C mutations as significant contributors to cancer development and as promising targets for future therapeutic strategies. Further research is needed to fully understand the implications of these mutations in cancer progression and treatment.

Grants: Project № BG-RRP-2.004-0007-С01 Strategic Research and Innovation Program for the Development of MU - PLOVDIV–(SRIPD-MUP)

Conflict of Interest: None declared

P01.111.B Parafibromin immunohistochemical staining of tumors from patients with hyperparathyroidism-jaw tumor syndrome

Ana Blatnik ¹, Olga Blatnik², Primož Drev², Vita Setrajcic Dragos³, Anja Zagožen Klasinc³, Iva Opalič¹, Srdjan Novaković³, Mateja Krajc¹

¹Institute of Oncology Ljubljana, Department of Clinical Cancer Genetics, Ljubljana, Slovenia; ²Institute of Oncology Ljubljana, Department of Pathology, Ljubljana, Slovenia; ³Institute of Oncology Ljubljana, Department of Molecular Diagnostics, Ljubljana, Slovenia

Background/Objectives: Germline pathogenic variants (PV) in CDC73 gene are associated with hyperparathyroidism-jaw tumor syndrome (HPT-JT), characterized by parathyroid adenomas/carcinomas, ossifying fibromas of the maxilla/mandible, renal lesions, and benign and malignant uterine tumors. CDC73 encodes a putative tumor suppressor parafibromin. In our study, we assessed the results of parafibromin immunostaining of tumors from HPT-JT patients.

Methods: We identified carriers of CDC73 PVs in the Slovenian National Registry of Tested Individuals from Families with Hereditary Cancer and obtained their tumor specimens. Nuclear expression of parafibromin was assessed in tumor tissue sections using a rabbit monoclonal antibody, according to standard operating procedures.

Results: Of 3283 cancer predisposition carriers, 6 (5 female, 1 male) were carriers of CDC73 PVs. One was asymptomatic, 3 developed parathyroid adenomas, 4 had uterine tumors, and 2 were treated for breast ductal carcinoma in situ (DCIS). Notwithstanding technical difficulties, immunostaining was successful in 1 parathyroid adenoma, 3 uterine tumors and both DSICs. Loss of parafibromin was demonstrated in the parathyroid adenoma. All 3 uterine tumors (2 fibroepithelial polyps and 1 atypical adenomyoma/adenosarcoma) showed loss of parafibromin in the mesenchymal component and normal staining in the epithelial component. Both DCIS samples had normal parafibromin expression.

Conclusion: Our parafibromin immunostaining results are compatible with what is currently known about HPT-JT tumor spectrum. Breast cancers in our patients do not appear to be associated with germline CDC73 variants. Epithelial-mesenchymal uterine lesions displayed loss of parafibromin in the stromal component which metastasized to the lungs and proved to be fatal for one patient.

Conflict of Interest: None declared

P01.112.C Functional analysis of genetic variants in genes involved in cancer helps establish pathogenicity and improves diagnostics for familial tumour syndromes.

Rick van Minkelen ¹, Mark Nellist¹, Marianne Hoogeveen-Westerveld¹, Hannie Douben¹, Leontine M A van Unen¹, Esmee Kasteleijn¹, Evelien Kroon¹, Heidi de Gruyter¹, Guido Breedveld¹, Margreethe van Vliet¹, Yvette Van Ierland¹, Berna Beverloo¹, Anja Wagner¹, Tjakko Van Ham¹

¹Erasmus Medical Centre, Clinical Genetics, Rotterdam, Netherlands

Background/Objectives: The identification of a pathogenic DNA variant in an individual with a genetic predisposition to cancer helps establish a diagnosis, enabling appropriate treatment and monitoring, and facilitating informed reproductive decision-making. However, in many individuals, variants of uncertain clinical significance (VUS) are identified. In these cases the genetic specialists are unable to provide the patient with certainty regarding their affection and/or carrier status.

Methods: To improve variant classification we apply in vitro functional testing to interrogate the effects of diagnostically relevant genetic variants on the expression and activity of components of the MTOR and RAS signalling pathways. Defects in MTOR or RAS signalling are common features of many sporadic tumours, and are associated with a diverse group of Mendelian disorders, particularly familial tumour syndromes such as tuberous sclerosis complex (TSC1, TSC2) and neurofibromatosis (NF1).

Results: Using our functional approach, we have so far been able to test >700 variants in genes involved in MTOR (AKT3, DEPDC5, MTOR, NPRL2, NPRL3, PIK3CA, PIK3R2, PTEN, TSC1 and TSC2) and RAS signalling (HRAS, NF1, SPRED1). Evidence to support pathogenicity was obtained for 73/114 clinically relevant variants in NF1, 63/197 in TSC1 and 291/543 in TSC2.

Conclusion: Our work demonstrates that a range of specific biochemical assays can be applied in a diagnostic setting to help establish variant pathogenicity, and provide an accurate molecular diagnosis.

Grants: None

Conflict of Interest: None declared

P01.114.A Pathogenic insertion of a mitochondrial DNA fragment in the PALB2 gene associated with hereditary breast and ovarian cancer syndrome.

Alejandro Moles-Fernandez ^1;2, Miriam Masas^1;2, Jordi Leno^1;2, Eduard Perez^3;4, Berta Campos^1;2, Judith Balmaña^3;4, Orland Diez¹, Elena García-Arumí^1;2;5, Eduardo Tizzano^1;2

¹Vall d’Hebron Barcelona Hospital Campus, Vall d’Hebron Hospital Universitari, Department of Clinical and Molecular Genetics, Barcelona, Spain; ²Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, Vall d’Hebron Hospital Universitari, Medicine Genetics Group, Barcelona, Spain; ³Vall d’Hebron University Hospital and Vall d’Hebron Institute of Oncology, Medical Oncology Department, Barcelona, Spain; ⁴Vall d’Hebron Institute of Oncology, Vall d’Hebron Barcelona Hospital Campus, Hereditary Cancer Genetics Group, Barcelona, Spain; ⁵Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, Vall d’Hebron Hospital Universitari, Research Group on Neuromuscular and Mitochondrial Disorders, Barcelona, Spain

Background/Objectives: Hereditary breast and ovarian cancer syndrome (HBOCS) has been related to deleterious alterations in genes involved in homologous recombination, such as BRCA1, BRCA2, and PALB2. Point, copy number variants, and even the insertion of transposable elements, such as Alu sequences, have been identified as mechanisms of alteration of these, and others genes related to monogenic diseases. However, in very few cases, the insertion of mitochondrial DNA fragments (mtDNA) in coding sequences of disease-related nuclear genes has been described.

Methods: Massive parallel sequencing (MPS) of a gene panel associated with HBOCS in germline DNA was performed. The variant found was confirmed by PCR and sequencing of the DNA fragment of interest. The sequence was aligned with the nuclear and mitochondrial genome.

Results: The patient, with a family history compatible with suspected hereditary cancer, was diagnosed with invasive ductal carcinoma of the left breast at the age of 55 years. The pathogenic variant c.2515_2516ins70, p.(Thr839Ilefs*14), in the PALB2 gene was detected in heterozygosity. This 70-nucleotide insertion was localized into exon 6 of PALB2 and 65 nucleotides of this, were coincident with an mtDNA fragment comprising the sequence of the mitochondrial MT-TA gene, mitochondrial alanine tRNA.

No other pathogenic variants or variants of unknown significance were identified in the patient. Following the ACMG variant classification guidelines, this variant was classified as pathogenic.

Conclusion: To our knowledge, this is the first germline pathogenic variant caused by an mtDNA insertion in an HBOCS susceptibility gene.

Grants: FIS PI19/ 01772. La Marató TV3-Foundation (590/C/2020).

Conflict of Interest: None declared

P01.115.B DDX41 germline predisposition to Myeloid Neoplasms: two siblings with the same double-hit germline/somatic non-canonical genomic constellation

Chiara Minotti ^1;2, Carmelo Gurnari³, Giorgia Silvestrini³, Enrico Attardi¹, Giorgia Ranucci³, Camilla Page³, Valentina Ferradini^1;2, Giuseppe Novelli^1;2, Maria Teresa Voso³, Federica Sangiuolo^1;2, Enrica Marchionni^1;2

¹Tor Vergata University of Rome, Medical Genetics Section, Dep. of Biomedicine and Prevention, Rome, Italy; ²Tor Vergata Hospital, Medical Genetics Unit, Rome, Italy; ³Tor Vergata University of Rome, Dep. of Biomedicine and Prevention, Rome, Italy

Up to 15% of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome/Neoplasms (MDS) patients harbor inherited predisposition traits. Germline mutations of DDX41 (MIM*608170), encoding for an RNA helicase, accounts for about 80% of MDS/AML with a documented germline predisposition. General disease ontogenesis encompasses the acquisition of a second somatic hit on the contralateral allele (most typically p.R525H). DDX41-mutant MDS/AML are characterized by male predominance, late onset, hypoplastic bone marrow, and indolent disease course.

We report on a 68-year old patient with an initial diagnosis of hypoplastic MDS (2% blasts). At the age of 76, he received a diagnosis of MDS-IB2 (10.5% blasts). At that time, a 94 myeloid genes NGS panel on his bone marrow aspirate detected the c.1547A>G [p.Tyr516Cys] heterozygous pathogenic variant in DDX41 (NM_016222.4) at a Variant Allele Frequency (VAF) of 48%. Given the suspicion of a germline origin, Sanger sequencing confirmed the presence of this variants on fingernail DNA.

Notably, the patient’s older brother received a diagnosis of MDS at 69 years of age. Segregation analysis, performed on DNA extracted from CD3+ lymphocytes used as a germline reference, disclosed the presence of the known heterozygous variant.

Interestingly, both siblings progressed to MDS upon acquisition of the same non-canonical DDX41 somatic variant p.Thr227Met (VAF of 10% and 3.5%), whose functional characterization is ongoing.

Recognition of germline predisposition in MDS/AML is crucial for disease surveillance/therapy, and donor selection for hematopoietic stem cell transplantation. Genetic counselling of affected patients and their unaffected relatives is advisable to establish a proper multidisciplinary management.

Conflict of Interest: None declared

P01.117.D Clinical utility of RNA-based targeted next-generation sequencing for the molecular diagnostics of non-small cell lung cancer

Magdalena Pelc ¹, Urszula Lechowicz¹, Joanna Moes-Sosnowska¹, Adam Szpechcinski¹, Paulina Skronska¹, Aneta Zdral¹, Adriana Rozy¹, Emil Wojda², Mateusz Polaczek², Małgorzata Szołkowska³, Tadeusz Orłowski⁴, Pawel Sliwinski⁵, Reanata Langfort³, Joanna Chorostowska-Wynimko¹

¹Institute of Tuberculosis and Lung Diseases, Department of Genetics and Clinical Immunology, Warsaw, Poland; ²Institute of Tuberculosis and Lung Diseases, III Department of Lung Diseases and Oncology, Warsaw, Poland; ³Institute of Tuberculosis and Lung Diseases, Department of Pathology, Warsaw, Poland; ⁴Institute of Tuberculosis and Lung Diseases, Department of Surgery, Warsaw, Poland; ⁵Institute of Tuberculosis and Lung Diseases, II Department of Lung Diseases, Warsaw, Poland

Background/Objectives: In recent years, the number of druggable somatic alterations with relevant targeted therapeutic agents has significantly increased in non-small cell lung cancer (NSCLC). Therefore, a robust, time- and cost-effective testing methods should be applied in molecular diagnostics of NSCLC. We evaluated the utility of RNA-based targeted next-generation sequencing (NGS) for the detection of clinically significant genetic variants in NSCLC patients.

Methods: RNA-based targeted NGS (FusionPlex_Lung assay, Archer) was performed in 312 tumor specimens (~75% adenocarcinoma; mainly advanced) from 309 NSCLC patients (mean age of 71 ± 8.4 years) in search of pathogenic single nucleotide molecular variants (SNVs, including indels) and/or actionable gene fusions.

Results: Genetic changes were detected in 171 NSCLC samples (~55%) from 169 patients. Sequencing results from 14 samples (~4,5%) did not meet quality criteria for further analysis. Pathogenic SNVs were detected in 160 tumor specimens from 158 patients, including single nucleotide changes or small deletions/insertions in KRAS (n = 106 samples, 105 patients), EGFR (n = 31 samples, 30 patients), BRAF (n = 14), MET (n = 1), RET (n = 1) genes and splicing mutations resulting in MET exon 14 skipping (n = 8). In four cases a co-occurrence of two molecular variants was noted. Actionable gene fusions occurred in 11 patients, comprising EML4::ALK (n = 7), CD74::ROS1 (n = 2), SDC4::ROS1 (n = 1), KIF5B::RET (n = 1).

Conclusion: Targeted RNA-based NGS assay enables robust, specific and sensitive detection of the clinically relevant molecular alterations essential to predict the potential benefit of NSCLC patients from targeted therapies, as it simultaneously tests for both SNVs and gene fusions within single analysis.

Grants: IGICHP 3.3/22

Conflict of Interest: None declared

P01.118.A Validation of RNA-based targeted next-generation sequencing assay for molecular variants detection in non-small cell lung cancer

Urszula Lechowicz ¹, Magdalena Pelc¹, Adam Szpechcinski¹, Joanna Moes-Sosnowska¹, Paulina Skronska¹, Adriana Rozy¹, Aneta Zdral¹, Mateusz Polaczek², Emil Wojda², Małgorzata Szołkowska³, Tadeusz Orłowski⁴, Pawel Sliwinski⁵, Reanata Langfort³, Joanna Chorostowska-Wynimko¹

¹The Institute of Tuberculosis and Lung Diseases, Department of Genetics and Clinical Immunology, Warsaw, Poland; ²The Institute of Tuberculosis and Lung Diseases, III Department of Lung Diseases and Oncology, Warsaw, Poland; ³The Institute of Tuberculosis and Lung Diseases, Department of Pathology, Warsaw, Poland; ⁴The Institute of Tuberculosis and Lung Diseases, Department of Surgery, Warsaw, Poland; ⁵The Institute of Tuberculosis and Lung Diseases, II Department of Lung Diseases, Warsaw, Poland

Background/Objectives: The accurate molecular characterization of non-small cell lung cancer (NSCLC) is mandatory for the optimal care of patients. We have validated RNA-based targeted next-generation sequencing (NGS) assay for the detection of important genetic variants in different types of NSCLC specimens.

Methods: 379 tumor specimens from 372 patients were analyzed using the Archer FusionPlexLung (AFPL) with reference to conventional IVD qPCR and fluorescence in situ hybridization (FISH) tests and DNA-based targeted NGS assay (TruSight_Tumor15, Illumina). A minimum required number of reads per sample was 0.5 million and an Internal Sample Quality Control index ≥10.

Results: Valid molecular results were obtained in 312 NSCLC samples; 17.8% specimens did not meet the acceptance criteria either for NGS procedure or data analysis. Actionable variants were detected in 171 (54.8%) samples. Concentrations ≥10ng of RNA and Cp at PreSeq <29 were established as borderline values allowing a sample to be qualified for further NGS analyses. The validation process allowed to set the reporting thresholds for SNVs at the lowest allele frequency of ≥4,9%, with at least 10 altered reads at ≥100 reads coverage. The reporting a gene fusion at ≥10% fusion site reads and supporting reads ≥5, with unique origins ≥3 in NGS resulted in very good concordance with FISH and qPCR. The median values for Total Fragments, RNA QC and RNA fragment length were 1371234, 194 and 123, respectively.

Conclusion: RNA-based NGS offers a diagnostic solution ensuring precise identification of variants across numerous genes that hold therapeutic significance in NSCLC.

Grants: IGICHP_3.3/22

Conflict of Interest: None declared

P01.119.B POU5F1B is a human-restricted ROCK inhibitor-sensitive oncoprotein that restructures membrane nanodomains to increase cell adhesion.

Laia Simó Riudalbas ¹, Sandra Offner², Abrami Laurence², Eduard Unterauer³, Julien Duc², Evarist Planet², Ralf Jungmann³, Alexandre Reymond¹, Gisou van der Goot², Didier Trono²

¹Center for Integrative Genomics, Lausanne, Switzerland; ²École polytechnique fédérale de Lausanne, Lausanne, Switzerland; ³Max Planck Institute of Biochemistry, Planegg, Germany

Background/Objectives: POU5F1B arose by retrotransposition of POU5F1/OCT4 in the last common ancestor of modern hominidae. In human the encoded protein is endowed with oncogenic properties. It promotes colorectal cancer growth and metastasis. Here, we aim at investigating the molecular mechanisms by which POU5F1B exerts its pro-oncogenic effect.

Methods: Detergent resistant membranes (DRMs) were isolated from colorectal cancer cell lines to study cholesterol-rich nanodomains. Acyl-RAC capture assay and radiolabeling 3H-palmitic acid incorporation were used to study protein S-acylation. We used LC-MS/MS and high-resolution microscopy to investigate protein composition and cell focal adhesions, respectively.

Results: We found that the oncogenic action of POU5F1B requires ubiquitination of lysine residues present only in the human isoform. This post-translational modification is essential for POU5F1B to localize to the cytoplasm and become S-acylated by the DHHC17 S-acyltransferase. This leads to POU5F1B association with DRMs, where it triggers the accumulation of signaling molecules and integrins, thereby stimulating cell focal adhesion and modifying the mechanic properties of cancer cells and their interactions with the extracellular matrix. The stability of POU5F1B depends on Rho-associated protein kinase (ROCK), a regulator of cytoskeletal dynamics and cell motility. Consistently, we identified ROCK inhibitors as POU5F1B destabilizers in a high-throughput drug screening.

Conclusion: We identified POU5F1B as the first ever described human-lineage specific cancer-promoting protein. It acts by an unprecedented mechanism, the remodeling of membrane nanodomains. Its action can be blocked by ROCK inhibitors. These results have interesting evolutionary and therapeutic implications.

Grants: Swiss Cancer Research Foundation KFS-4968-02-2020 and KFS-5823-02-2023.

Conflict of Interest: Laia Simó Riudalbas 2 patent applications:

EP23154002.2

EP20190150151 20190103, Swiss Cancer Research Foundation grants:

KFS-4968-02-2020

KFS-5823-02-2023, Sandra Offner: None declared, Abrami Laurence: None declared, Eduard Unterauer: None declared, Julien Duc: None declared, Evarist Planet: None declared, Ralf Jungmann: None declared, Alexandre Reymond: None declared, Gisou van der Goot: None declared, Didier Trono: None declared

P01.121.D DNA methylation biomarkers of thymomas and thymic carcinomas

Vanessa Nicolì¹, Marianna Giangreco¹, Eleonora Pardini¹, Iacopo Petrini¹, Diana Bacchin², Vittorio Aprile², Franca Melfi³, Marco Lucchi², Roberta Ricciardi⁴, michelangelo maestri⁴, Andrea Stoccoro¹, Lucia Migliore¹, Fabio Coppedè ¹

¹University of Pisa, Translational Research and of New Surgical and Medical Technologies, Pisa, Italy; ²University of Pisa, Division of Thoracic Surgery, Dept. of Surgical, Medical and Molecular Pathology and Critical Care Medicine, Italy; ³University Hospital of Pisa, Minimally Invasive and Robotic Thoracic Surgery, Robotic Multispecialty Center of Surgery, Italy; ⁴University of Pisa, Neurology Unit., Dept. of Clinical and Experimental Medicine, Italy

Background/object: Thymic epithelial tumors (TETs) are the most frequent neoplasms of the anterior mediastinum and include thymomas (TMs), thymic carcinomas (TCs), and rare neuroendocrine tumors. Epigenetic modifications are increasingly observed in these tumors, indicating the need for further characterization of their distribution among different subtypes, stages and histotypes.

Methods: We reviewed the literature related to DNA methylation studies in TETs, including candidate gene association studies and genome-wide investigations. We then designed a panel of genes among the most promising epigenetic biomarkers of TETs and evaluated their methylation levels in 71 bioptic TET samples consecutively collected over a 3-year period, including 64 TMs and 7 TCs. Blood samples were collected at thymectomy from each patient and screened for peripheral epigenetic biomarkers of TET subtypes.

Results: TCs showed hypermethylation of GHSR and ELF3 genes and reduced IL1RN methylation levels compared to TMs. We also observed a significant hypomethylation of the RAG1 promoter region in TMs, but not in TCs, compared to healthy thymic tissues. No difference in the methylation levels of the investigated genes was seen among TM stages and subtypes. No changes in the blood methylation levels of the investigated genes were seen among TET subtypes.

Conclusion: The present study revealed TET methylation biomarkers and a set of genes that can discriminate between TMs and TCs.

Grant References: THYMOGENE project, Bando Salute Tuscany Region.

Conflict of Interest: None declared

P01.123.B Identification and validation of new DNA methylation panels for the differentiation between hepatocellular carcinoma and cholangiocarcinoma

Tina Draškovič ¹, Nina Hauptman¹

¹Institute of Pathology, Faculty of Medicine, University of Ljubljana, Department of Molecular Genetics, Ljubljana, Slovenia

Background/Objectives: Differentiating between hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) is sometimes challenging. The aim of our study was to identify and validate novel DNA methylation biomarkers and to develop panels that can differentiate between HCC, CCA and their paired normal tissue.

Methods: Using bioinformatics, we identified new differentially methylated promoter regions and designed panels for the diagnosis and differentiation between HCC and CCA. DNA was isolated from formalin-fixed, paraffin-embedded tissue samples of 15 HCC, 15 CCA and their paired normal tissue. After DNA isolation and DNA bisulfite conversion, the methylation status of the selected regions was determined by dPCR. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated based on the hyper- or hypomethylation of each sample.

Results: The design panel for HCC includes the promoter regions of the genes RNF135 and EFNB2, which successfully differentiate HCC from CCA with a sensitivity of 87%, a specificity of 100%, a PPV of 1 and an NPV of 0.88. The design panel for CCA includes the promoter regions of the genes RNF125, HOXC4 and ABLIM1, which differentiate CCA from HCC with a sensitivity of 67%, a specificity of 100%, a PPV of 1 and an NPV of 0.75. Both panels are 100% cancer specific.

Conclusion: Our results show that the DNA methylation status of the selected promoter regions and the designed panels successfully diagnose and differentiate HCC and CCA with high sensitivity, specificity, PV and NPV and are cancer specific.

Grants: Slovenian Research and Innovation Agency (P3-0054, J3-3070 and PhD research funding).

Conflict of Interest: None declared

P01.124.C Genetic landscape of hereditary hematological malignancies (HHM) in adults

Dora Csaban ¹, Livia Varga¹, Zoltan Orfi¹, Lenke Tanko¹, Nora Kondor¹, Andras Bors¹, Jozsef Harasztdombi¹, János Dolgos¹, Judit Reichardt¹, Beata Koller¹, János Fábián¹, Andrea Varkonyi¹, Viktor Lakatos¹, Laszlo Gopcsa¹, Valyi-Nagy Istvan¹, Peter Remenyi¹, Hajnalka Andrikovics¹

¹Central Hospital of Southern Pest, National Institute of Hematology and Infectious Diseases, Budapest, Hungary

Background:Due to advancements in sequencing technologies, more insights are gained in the background of hereditary hematological malignancies (HHM), which were recently recognized as novel clinical entity in WHO and European LeukemiaNet Classifications.

Methods:Peripheral blood or bone marrow samples were used for isolation of tumour derived DNA from 455 patients with haematological malignancy (ALL, AML, MDS, MPN). Next generation sequencing (NGS) targeted panel contained genes associated with somatic and germline hematologic malignancies. The presumed germline pathogenic variants were confirmed by Sanger sequencing from independent samples (hair follicles or potential hematopoietic stem cell donor family members).

Results:In our study with ‘tumour only’ testing strategy, 58 variants were presumed to be hereditary pathogenic alterations, of which were confirmed in 9 cases (1.98%): DDX41 (n = 3), RUNX1 (n = 3), TP53 (n = 1), TERT (n = 1), PTPN11 (n = 1). With the exception of DDX41 (age at diagnosis: 60-69 year), HMM patients were younger than average patients with haematological malignancies (17-49 years). All patients showed unobtrusive personal and family history without any malignancies. Cascade screening of family members identified carrier status in 16 out of 50 cases, and no clinical symptoms were observed in several older relatives.

Conclusion:Our results highlight that HHM occurs not only in young individuals. The prevalence of HMM might be underestimated due to its low penetrance. Germline genetic information influence the choice of targeted therapy and the selection of hematopoietic stem cell donor from the family. Additionally, it draws attention to the inclusion of asymptomatic carrier relatives in hematological/oncological care.

Conflict of Interest: None declared

P01.125.D A Novel Association Of CHEK2 Splice Site Mutation With Early Onset Gastric Cancer

AHMED AL-OMAR¹, Süleyman Alıcı², İlknur Sunar ¹, Ayse Busra Akalin³, Ibrahim Akalin¹

¹Metagentech Center for Genetic Diseases, istanbul, Türkyie; ²Hisar Intercontinental Hospital, Medical Oncology, istanbul, Türkyie; ³Bahcesehir University, Faculty of Medicine, istanbul, Türkyie

Background/Objectives: CHEK2 is a tumor suppressor gene that arrests cell cycles and leads to apoptosis after DNA damage. Germline mutations in CHEK2 gene increase risk for breast and other types of cancer. There are different types of pathogenic variants such as short insertions/deletions, single nucleotide variations, large genomic rearrangements, and splicing variants.

Methods: A 31-year-old female patient with a history of gastric cancer was admitted to the hospital and operated for total gastrectomy. Pathological evaluation revealed T4N3 MSI stabile, lymph node and extra peritoneal metastatic adenocarcinoma. Biochemistry laboratory results showed elevated levels of ALT, AST, GGT and alkaline phosphatase. The patient received 6 cycle FLOT chemotherapy. External radiotherapy was applied with capecitabine. The patient was referred for genetic testing for hereditary cancer genes.

Result: Exome sequencing analysis revealed unknown significance of c.-185-6del heterozygous splice site mutation in CHEK2 gene, responsible for autosomal dominantly inherited “Cancer Predisposition Syndrome, CHEK2-related, Li-Fraumeni Syndrome 2, AD(?) [MIM#609265]”.

Conclusion: The mutation was identified in gnomAD database and splice site CHEK2 mutations were attributed to be responsible for gastric cancer development under 55-year-old patients. Here we for the first time report a CHEK2 splice site variation (c.-185-6del) supposed to be pathogenic in gastric cancer patients up to date.

Grants:

Conflict of Interest: None declared

P01.126.A Harnessing cDNA analysis to enhance variant classification in cancer predisposition genes

Liesbeth Claeys ^1;2, Suzanne Vanhauwaert^1;2;3, Annelies De Jaegher^1;2, Kim De Leeneer^1;2;3, Bram Parton^1;2, Ilse Coene^1;2, Martine De Bleeckere^1;2, Katia De Pauw^1;2, Arne De Visscher^1;2, Elke D’Haese^1;2, Tjoïlina Reyniers^1;2, Hélène Verhaeghe^1;2, Bruce Poppe^1;2;3, Robin de Putter^1;2;3, Kathleen Claes^1;2;3

¹Ghent University Hospital, Center for Medical Genetics Ghent, Ghent, Belgium; ²Ghent University, Department of Biomolecular Medicine, Ghent, Belgium; ³Ghent University and Ghent University Hospital, Cancer Research Institute Ghent (CRIG), Ghent, Belgium

Background/Objectives: In clinical practice a panel of 107 genes linked to familial cancer syndromes, comprising various subpanels tailored to clinical requests, is routinely employed. This study aims to assess the utility of cDNA analysis in enhancing molecular diagnostics.

Methods: SeqCap EZ HyperCap technology was applied for target enrichment, followed by NovaSeq6000 sequencing. Digital MLPA and ExomeDepth were utilized for exon-level CNV detection. RNA extraction from short term lymphocyte cultures, supplemented with puromycin as an NMD inhibitor, enabled RT-PCR and Sanger sequencing. A total of 44 cases spanning 16 genes were investigated.

Results: Among 9871 patients screened, (likely) pathogenic variants (class 4/5) were detected in 10% (n = 1038), with 6% (n = 585) harboring variants of unknown clinical significance (VUS, class 3).

Algorithms predicted aberrant splicing for 25 variants, with 12 corroborated by cDNA-analysis, upgrading them to class 4. Nine variants were downgraded to class 2 while 4 others remained class 3. Notably, an aberrant transcript was confirmed in 4 cases with an SVA element insertion in PMS2. Twelve exon CNVs were further investigated by cDNA testing. Only one of five affecting PMS2 exons 13-15 was confirmed. Four exon-CNVs in other genes were confirmed. In three other cases cDNA analysis discerned disease relevant from pseudogene artifacts.

Conclusion: We demonstrate the multifaceted benefits of cDNA analysis in familial cancer syndrome diagnostics: it enhances variant classification, validates exon CNVs, and distinguishes gene sequences from (non-expressed) pseudogenes. The integration of cDNA analysis enriches the molecular diagnostic arsenal, refining patient management strategies.

Conflict of Interest: None declared

P01.127.B Structural variant in LRRFIP2 detected by whole-genome sequencing as the cause of an unsolved Lynch-like syndrome family

Gemma Llargués-Sistac ¹, Laia Bonjoch¹, Jenifer Muñoz¹, xavier domínguez-rovira¹, Teresa Ocaña¹, Maria Isabel Alvarez², Celia Badenas², Miriam Cuatrecasas³, Antoni Castells¹, Francesc Balaguer¹, Leticia Moreira¹, Guerau Fernandez⁴, Sergi Castellví-Bel¹

¹Hospital Clínic, FRCB-IDIBAPS, CIBEREHD, Gastroenterology, Barcelona, Spain; ²Hospital Clinic, FRCB-IDIBAPS, CIBERER, Biochemistry and Molecular Genetics, Barcelona, Spain; ³Hospital Clínic, IDIBAPS, Pathology, Barcelona; ⁴Hospital San Joan de Déu, CIBERER, Barcelona, Spain

Background/Objectives: Lynch Syndrome is the most common form of hereditary colon cancer, caused by germline mutations in four genes of the DNA-mismatch repair pathway (MLH1, MSH2, MSH6 and PMS2). Current diagnostic approaches identify causative genetic variants in less than 50% of cases. We present an unsolved case from an Equatorian family with Lynch-like syndrome, with tumor loss of MLH1 and PMS2 expression and BRAF wild-type, and a familial history of colorectal and stomach cancer, but no underlying genetic variants detected through standard diagnostic tests.

Methods: We performed germline whole-genome sequencing (WGS) of DNA from blood samples of two siblings to identify the causative mutational event. To further underpin the molecular mechanism involved, subsequent transcriptome sequencing (RNA-seq) and quantitative PCR in one of the siblings was performed.

Results: In both siblings, a tandem duplication of 37 kilobases was found in the LRRFIP2 gene, contiguous to MLH1. RNA-seq data confirmed an altered expression of LRRFIP2 and MLH1. Results were validated with a quantitative PCR for both genes.

Conclusions: WGS allowed the identification of a duplication in the LRRFIP2 gene that affects the expression of MLH1, explaining the clinical presentation of the patient and their family. This case highlights the added value of using WGS and RNA-seq to current standard diagnostic approaches to help solve cases that present with complex genomic rearrangements that otherwise would have not been detected with current genetic testing.

Grants: AECC PRYGN211085CAST, FIS-FEDER 20/00113 and 23/00189, Marató TV3-202008-10, Horizon Europe Twinning Project STEPUPIORS, CIBEREHD, CERCA Program and AGAUR GRC2021SGR01185.

Conflict of Interest: None declared

P01.128.C Liquid Biopsy in Glioblastoma Management – preliminary results

domingos roda¹, pedro veiga², Luís Miguel Pires², alexandra mascarenhas², Francisco Caramelo^3;4, Clara Romero⁵, inês pinto⁶, Tomás Dinis⁶, Pedro Abreu⁵, Pedro Teles⁵, José Luís Alves⁷, Joana Barbosa de Melo^2;4;8, Isabel Marques Carreira^2;4;8, Ilda Patrícia Ribeiro ^2;4;8

¹Joaquim Chaves Saúde (JCS), Algarve Radiation Oncology Unit, Faro, Portugal; ²University of Coimbra, Cytogenetics and Genomics Laboratory, Institute of Cellular and Molecular Biology, Faculty of Medicine, Coimbra, Portugal; ³Laboratory of Biostatistics and Medical Informatics, iCBR - Faculty of Medicine, University of Coimbra, Coimbra, Portugal, Coimbra, Portugal; ⁴University of Coimbra, Coimbra Institute for Clinical and Biomedical Research (iCBR) and Center of Investigation on Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, Coimbra, Portugal., Coimbra, Portugal; ⁵Centro Hospitalar e Universitário do Algarve, CHUA, Serviço de Neurocirurgia, Faro, Portugal; ⁶Centro Hospital e Universitário de Coimbra, Serviço de Radioterapia, Coimbra, Portugal; ⁷Centro Hospitalar e Universitário de Coimbra, CHUC, Serviço de Neurocirurgia, Coimbra, Portugal; ⁸University of Coimbra, Center for Innovative Biomedicine and Biotechnology (CIBB), Coimbra, Portugal and Clinical Academic Center of Coimbra (CACC), Coimbra, Portugal., Coimbra, Portugal

Background/Objectives:Glioblastoma (GBM) represents the most aggressive form of nervous system cancer. The objectives of this pilot study were to characterize the genomic profile of GBM tumor tissue samples and to investigate the potential of liquid biopsies for diagnosing and monitoring patients.

Methods: A total of 110 samples of plasma were collected from 45 GBM patients before and during treatment. Plasma samples from 10 controls were also collected. After isolating cfDNA, concentrations were determined, compared between patients and controls, and monitored throughout the patients’ clinical course. Tumor tissue samples were analyzed using array Comparative Genomic Hybridization (aCGH).

Results: aCGH analysis showed frequent gains and losses of chromosomes 1, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 17, 20, 21, 22, and Y. Amplifications involving the EGFR and MET genes and deletions of CDKN2A, PTEN, RB1 and NF1 were common alterations observed in our cohort. The levels of plasma cfDNA revealed that GBM patients have higher plasma baseline cfDNA levels than healthy controls. In some of the patients a substantial increase in plasma cfDNA levels was observed during follow up, which is being correlating with clinical events.

Conclusion: Our findings demonstrate the feasibility of isolating cfDNA from the plasma of GBM patients. Furthermore, monitoring cfDNA levels during treatment may provide valuable insights into disease progression and treatment response. Integrating the analysis of liquid and tissue biopsies enables a comprehensive characterization of the tumor profile. A larger cohort and an extended follow-up period are required for further validation.

Conflict of Interest: None declared

P01.129.D Rare variants modulate phenotype in NF1 carriers’

Elena Pasquinelli^1;2, Chiara Fallerini ^2;3, Samantha Minetto^2;3, Giulia Rollo^2;3, Margherita Baldassarri^2;3;4, Alessandra Renieri^2;3;4

¹Medical Genetics, University of Siena, Italy, Department of Medical Biotechnologies, Siena; ²Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, University of Siena, Siena, Italy, Department of Medical Biotechnologies, Siena, Italy; ³Medical Genetics, University of Siena, Italy, Department of Medical Biotechnologies, Siena, Italy; ⁴Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy, Siena, Italy

Background/Objectives: Neurofibromatosis type 1, is a rare genetic disorder predisposing to a pleiotropic phenotype ranging from psychosocial issues, congenital malformations, benign tumors of the nervous systems or aggressive solid cancer. We hypothesize that this phenotypic variability is due to additional rare variants in other genes, acting as an oligogenic threshold model.

Methods: Clinical and Exome sequencing data were retrieved in a retrospective nested-study from 32 patients with pathogenic/like pathogenic NF1 variants.

Results: NF1 carriers with solid cancers show a mean of 4.12 variants in cancer driver genes per subject while NF1 carriers without solid cancers have 2.16 variants per subject with Mann-Whitney test significant at p < 0.05. Furthermore, we present that less frequently (macrocephaly or cognitive disorder) or rarely (connective disorder or polydactyly) ancillary characteristics could be linked to rare variants in additional genes. NF1 carriers with cognitive impairment have a mean of 2.7 additional variants, with at least one of them being a P/LP variant in a well known dominant neurodevelopmental disorder gene. In inherited cases, we detect in probands that NF1 inherited variant combined with other rare variants inherited from both parents act reinforcing the NF1 susceptibility with an additive trend.

Conclusion: In conclusion, we have highlighted that rare variants may modulate phenotypic outcome in neurofibromatosis. These results are relevant for both clinical policy and clinical practice: we propose to use the rare variant capture for amelioration screening program in NF1 carriers and identification of specific gene cards for personalized treatment.

Grants: INTERVENE, HORIZON Project 2020 n.101016775

Conflict of Interest: None declared

P01.130.A Germline testing for patients with pancreatic adenocarcinoma: a retrospective study in the department of genetics at Amiens University Hospital, France

Luana Giovannangeli ¹, Emilie Lacot-Leriche¹, Gilles Morin¹, Bénédicte Bonte¹, Florence Amram¹, Florence Jobic¹, Emma Lachaier¹

¹CHU Amiens-Picardie, Genetics, Amiens, France

Background/Objectives: In 2019, the American Guidelines recommended universal germline testing for patients with Pancreatic Cancer (PC). Literature reports pathogenic variations in 10-15% of PC. In France, due to limited access to oncogenetics, germline testing is usually restricted to patients with familial history, age under 50 years or somatic alteration in BRCA which can lead to PARP-inhibitor therapy. In our department, we applied universal testing. This study aimed to determine the benefit of universal germline testing compared to French practices.

Methods: We conducted a retrospective study (2022-2023) in our department of genetics in Amiens, France. Patients with pancreatic adenocarcinoma (locally advanced or metastatic) regardless of familial history or age were included. The analyzed genes were, at least: EPCAM, ATM, BRCA1, BRCA2, TP53, PALB2, CDKN2A/p14, CDKN2A/p16, and MMR ones.

Results: Among 45 patients, one was excluded (neuroendocrine cancer). According to American Guidelines: 5 had pathogenic variations (1 BRCA1, 3 BRCA2, 1 ATM), 11 had variants of unknown significance (VUS) and 28 had negative testing. According to French practices, 8 had criteria for germline testing. Among them, 1 had pathogenic variation (BRCA2), 3 VUS, and 4 negative results.

Conclusion: According to French practices, 4 pathogenic variations would not have been highlighted. Despite a restricted panel of genes with follow-up recommendation compared to the American panel, the rates of pathogenic variants and VUS remain high. Our study confirms the relevance of universal testing for PC. Universal testing improves analytical performance but also increase consultation activity, approximatively 20 per year in our department.

Conflict of Interest: None declared

P01.132.C The advantage of targeted next-generation sequencing over qPCR in testing for druggable EGFR variants in non-small cell lung cancer

Adam Szpechcinski ¹, Joanna Moes-Sosnowska¹, Urszula Lechowicz¹, Paulina Skronska¹, Magdalena Pelc¹, Małgorzata Szołkowska², Emil Wojda³, Piotr Rudzinski⁴, Krystyna Maszkowska-Kopij⁵, Mateusz Polaczek³, Renata Langfort², Pawel Sliwinski⁶, Tadeusz Orłowski⁴, Joanna Chorostowska-Wynimko¹

¹The Institute of Tuberculosis and Lung Diseases, Department of Genetics and Clinical Immunology, Warszawa, Poland; ²The Institute of Tuberculosis and Lung Diseases, Department of Pathology, Warszawa, Poland; ³The Institute of Tuberculosis and Lung Diseases, III Department of Lung Diseases and Oncology, Warszawa, Poland; ⁴The Institute of Tuberculosis and Lung Diseases, Department of Thoracic Surgery, Warszawa, Poland; ⁵The Institute of Tuberculosis and Lung Diseases, Outpatient Clinic, Warszawa, Poland; ⁶The Institute of Tuberculosis and Lung Diseases, II Department of Lung Diseases, Warszawa, Poland

Background/Objectives: Targeted therapies in non-small cell lung cancer (NSCLC) are dedicated to patients with tumors exhibiting features of oncogene addiction, including activation of epidermal growth factor receptor (EGFR) by SNVs/indels in exons 18-21 of the gene. Nowadays, diagnostic techniques based on qPCR and direct DNA sequencing are being replaced by next-generation sequencing (NGS) for actionable variants detection. In this study, we evaluated the diagnostic usefulness of targeted NGS panel for druggable EGFR variants testing in clinical NSCLC material previously analyzed by IVD-certified qPCR test with respect to DNA reference material.

Methods: The EGFR variants were tested in tumor tissue specimens from 59 NSCLC patients by the NGS ‘TruSight Tumor 15’ assay (Illumina) and the qPCR ‘Cobas EGFR mutation test’ (Roche Diagnostics). The sensitivity and specificity of NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of EGFR variants.

Results: In DNA reference material, NGS assay demonstrated sufficient lower detection limit for diagnostic applications (VAF < 5%); all EGFR variants were correctly identified. NGS showed high repeatibility of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 59%. The majority of discordant results concerned false-positive detection of EGFR exon 20 insertions by qPCR. TP53 was frequently co-mutated gene in EGFR-positive NSCLC patients.

Conclusion: The NGS showed a number of superior features over the qPCR in EGFR variant detection (exact identification of EGFR variants, calculation of allelic frequency, high analytical sensitivity) which might enhance the basic diagnostic report.

Grants: IGIChP_3.11/23

Conflict of Interest: None declared

P01.134.A Exome sequencing identifies candidate genes for platinum response in ovarian cancer patients

Lisa-Marie Speith ¹, Lara Thomys¹, Donna-Lucil Sperber¹, Peter Schürmann¹, Dhanya Ramachandran¹, Robert Geffers², Britta Wieland¹, Mu Yang¹, Kristine Bousset¹, Clemens Liebrich³, Tjoung-Won Park-Simon¹, Peter Hillemanns¹, Thilo Dörk¹

¹Hannover Medical School, Gynaecology Research Unit, Hannover; ²Helmholtz Institute for Infection Research, Genome Analytics, Braunschweig, Germany; ³Wolfsburg Clinics, Gynaecology and Obstetrics, Wolfsburg, Germany

Background/Objectives: Carriers of pathogenic germline variants in BRCA1 or BRCA2 have an improved response after standard platinum treatment for ovarian cancer. In the present study we used genomic DNA from long-term survivors to identify and characterize additional genes that may be involved in the development of an extraordinary platinum response.

Methods: We performed exome sequencing in 30 selected patients with high-grade serous ovarian cancer who responded particularly well to carboplatin chemotherapy and were alive and tumor-free more than two years after the end of therapy. Candidate genes harboring truncating variants were ranked through pathway enrichment analyses and literature search. Selected candidate genes were further investigated through silencing or inhibitor studies in different ovarian cell lines using flow cytometry and survival assays.

Results: We identified truncating germline variants in nine genes associated with DNA replication in 7/30 patients (23%). Four of the nine genes were also known to be involved in R-loop suppression. Analyses of large case-control sequencing data sets indicated that none of them was associated with overall risk for ovarian cancer. In functional studies using a pair of platinum-sensitive and platinum-resistant A2780 cell lines, the inhibition or down-regulation of two of the candidate genes modulated platinum response and differentially induced cell death.

Conclusion: Germline variants in replication-associated genes may contribute to the platinum sensitivity of tumors in long-survivors. Inhibition of such genes may open an avenue for improving platinum therapy in patients with resistant tumors.

Grants: Supported by the German Research Foundation (Do761/15-1).

Conflict of Interest: None declared

P01.135.B Whole Exome Sequencing (WES), SNP-array and Shallow Whole Genome Sequencing (sWGS) identifies recurrent genomic alterations and molecular heterogeneity in multiple myeloma.

Federica Cannas ^1;2, Baran Bayindir¹, Alessia Mascia³, Chiara Cocco¹, Laura Serventi¹, Roberta Murru⁴, Ludovica Martorana⁴, Annalisa Azzena⁴, Maria Valentina Licheri⁴, Stefano Mocci^1;2, Celeste Sanna¹, Michela Murgia¹, Meropi Plousiou¹, Giulia Nutile¹, Federico Marongiu¹, Maximilian Frick¹, Daniele Derudas⁵, Erika Giuressi⁴, Andrea Perra³, Sabrina Giglio^1;2;4

¹University of Cagliari, Medical Genetics, Department of Medical Sciences and Public Health, University of Cagliari, Cagliari, Italy; ²University of Cagliari, Centre for Research University Services (CeSAR, Centro Servizi di Ateneo per la Ricerca), University of Cagliari, Monserrato (CA), Italy; ³University of Cagliari, Unit of Oncology and Molecular Pathology, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy; ⁴Medical Genetics, R. Binaghi Hospital, Local Public Health and Social Care Unit (ASSL) of Cagliari, Cagliari, Italy; ⁵UO of Hematology and Bone Marrow Transplant Centre, A. Businco Hospital, Cagliari, Italy

Background/Objectives: multiple myeloma (MM) is a hematologic disease caused by clonal proliferation of plasma cells. We examined the genetic aspects of MM, including sequence-related genetic alterations and genomic instability, to demonstrate the superiority of sWGS, a high-throughput technology, as a valid alternative compared to classical cytogenetics. Indeed, although FISH is the current gold standard to detect cytogenetic events in MM, it is costly, labor-intensive and does not show the exact genomic background of this disorder.

Methods: we analyzed 100 patients at various stages of MM. We performed SNP-array and FISH to detection of copy number aberrations (CNA) and balanced translocations, respectively. Additionally, we performed sWGS (0.1X) and WES on each sample.

Results: SNP-arrays suggested genomic instability in approximately 30% of patients, often associated with gain-of-function variants in several oncogenes (NRAS, KRAS, BRAF) and loss-of-function variants in TP53, directly related to the risk of progression, survival, and resistance to therapy. Moreover, we detected variants in DNMT3A gene correlated with tumor progression. Furthermore, sWGS detected CNA and LOH with superior sensitivity compared to array and, with a specific bioinformatic pipeline, was able to identify balanced translocations, demonstrating sWGS is superior to FISH, which is not able to identify all CNA and LOH.

Conclusion: sWGS has proven to be a superior method for identifying biallelic events and rearrangements with heterogeneous breakpoints. WES and sWGS, to be used as a first diagnostic approach, can be helpful in addressing various aspects related to targeted therapy, therapy failure and relapse, providing crucial information on clinical decision-making.

Conflict of Interest: None declared

P01.136.C Whole Exome Sequencing (WES) and Shallow Whole Genome Sequencing (sWGS) of tissue and Cell-Free DNA (cf-DNA) for the assessment of Homologous Recombination Deficiency (HRD) in Ovarian Cancer

Giulia Nutile ¹, Baran Bayindir¹, Federica Cannas^1;2, Chiara Cocco¹, Laura Serventi¹, Roberta Murru³, Ludovica Martorana³, Mirella Casula³, Gaia Maria Tosone¹, Michela Lorrai¹, Caterina Mereu¹, Stefano Mocci^1;2, Nicola Orrù³, Federico Marongiu¹, Maximilian Frick¹, Erika Giuressi³, Andrea Perra⁴, Daniela Fanni¹, Sabrina Giglio^1;2;3

¹University of Cagliari, Medical Genetics, Department of Medical Sciences and Public Health, University of Cagliari, Cagliari, Italy; ²University of Cagliari, Centre for Research University Services (CeSAR, Centro Servizi di Ateneo per la Ricerca), University of Cagliari, Monserrato (CA), Italy; ³Medical Genetics, R. Binaghi Hospital, Local Public Health and Social Care Unit (ASSL) of Cagliari, Cagliari, Italy; ⁴Unit of Oncology and Molecular Pathology, Department of Biomedical Sciences, University of Cagliari, Cagliari, Italy

Background/Objectives: pathogenetic variants in the genes BRCA1/BRCA2 have a significant role in developing ovarian cancer (OC) and are essential for genome integrity by repairing double-stranded DNA breaks via the homologous recombination repair (HRR) pathway. Our aim is to identify whether the HR disruption correlates with oncogene alterations and, ideally, with therapy responses.

Methods: a total of 96 women affected by OC (13 in paclitaxel-platinum with or without bevacizumab, 3 in PARP-inhibitors) were enrolled. We performed: on 66 samples a multigene panel on FFPE tissue and HRS-test to identify HRD; on 30 samples WES (germinal and somatic) and SNP-array; on all 96 samples shallow whole-genome sequencing (sWGS), which results were compared to the reference assay SOPHiA. Furthermore, sWGS was also performed on cf-DNA in 50 cases.

Results: we found germinal variants in BRCA1/BRCA2 on 5.21% and 6.25% of subjects, respectively. Moreover, we detected several variants in suppressor genes and oncogenes, including mismatch repair (MMR) genes, likewise causative variants in HRR-related genes like BARD1 (2,08%), ATM (2,08%), CHEK2 (1,04%). Somatic pathogenic variants were: 71,88% in TP53, 16,67% in BRCA1 and 11,46% in BRCA2. Almost all cases (98%) presented genomic instability highlighted both on tissue and cf-DNA.

Conclusion: BRCA1/BRCA2 mutation status is the main genetic biomarker of HRD but is not sufficient to describe the total HRD status, which can be driven by somatic events and further genetic alterations. Therefore, a bioinformatics pipeline to evaluate the HRR system status in OC has been set, based on sWGS, to support therapeutics and follow-up strategies.

Conflict of Interest: None declared

P01.137.D Rare Capillary Malformation-Arteriovenous Malformation Syndrome (CM-AVM): Intrafamilial phenotypic heterogeneity

stavroula psoni¹, STYLIANI AMENTA², Emmanouel Manolakos³, Eirini Tsoutsou ⁴, Christina Kanaka-Gantenbein⁵, HELEN FRYSSIRA⁶

¹Pediatrician-Medical Geneticist, Private Practice; ²MITERA Maternity Hospital, Clinical Genetics; ³ATG-Access to Genome Labs, Athens, Greece; ⁴General Hospital of Athens “G. Gennimatas”, Thalassemia Unit; ⁵“Aghia Sofia” Children’s Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece, Pediatrics-Pediatric Endocrinology, First Pediatric Clinic; ⁶Medical School, National and Kapodistrian University of Athens, Greece, Medical Genetics

Background/Objectives: Capillary Malformation-Arteriovenous Malformation Syndrome (CM-AVM) (OMIM:608354) syndrome is a rare autosomal dominant genetic disease characterized by broad phenotypic variability and caused by mutations in the RASA1 gene (p210RasGAP-protein-activator-1). CM-AVM comprises a wide phenotypic spectrum with atypical capillary and fast-flow vascular malformations being the most prominent clinical features. The CMs are usually multifocal and surrounded by pale halo with central red dot and can be observed in the brain, face or extremities. We describe a new CM-AVM case.

Methods: The 3.5 year-old female proband was offspring of non-consaguineous parents and presented with short stature, lordosis, genu valgum, macrocephaly and multiple nodular macule like café-au-lait skin lesions on the trunk and extremities. The family history revealed propensity for malignancies on the maternal ancestors. Hormonal, biochemical testing and internal organs imaging were normal. The karyotype was 46, XX.

Whole Exome Sequencing (WES) was subsequently performed on DNA extracted from the proband and her parents after obtaining signed informed consent, using standard procedures.

Results: WES identified in heterozygous state the RASA1 mutation c.261_262del (p.Gly89ArgfsTer22) which comprises a 2bp deletion at exon-1 leading to frameshift and premature stop codon. The proband’s mother was a carrier, although not presenting any skin lesions.

Conclusion: Upregulation of the Ras/MAPK signaling pathway, caused by haploinsufficiency of the Ras21-protein-activator-1, disrupts the normal growth regulation, cell differentiation and proliferation which in turn leads to angiogenesis overdrive. Given the CM-AVM phenotypic variability, clinical genetic evaluation and testing is essential for the diagnosis, follow-up and counseling of the patients and their families.

Grants: None

Conflict of Interest: None declared

P01.138.A Deciphering the molecular landscape of diffuse gastric cancers in truncating CTNNA1 germline variant carrier patients

Silvana Lobo ^1;2;3, Alexandre Dias^1;2;3, Marta Ferreira^1;2;4, Nelson Martins¹, Rita Barbosa-Matos^1;2;3, João Fonseca^1;2;5, J. Garcia-Pelaez^1;2;5, Ana Maria Pedro¹, Irene Gullo^1;6, André Oliveira¹, Celina São José¹, Chrystelle Colas^7;8, Robert Hüneburg^8;9;10, Jacob Nattermann^8;9;10, Lise Boussemart^11;12, Liselotte P van Hest¹³, Leticia Moreira^14;15;16, Carolyn Horton¹⁷, Dana Farengo Clark¹⁸, Sigrid Tinschert^19;20, Golmard Lisa^7;8, Isabel Spier^8;9;21, Adrià López-Fernández^8;22, Daniela Oliveira^23;24;25, Magali Svrcek^26;27, Pierre Bourgoin²⁷, Helene Delhomelle^7;8, Jeremy Davis²⁸, Birthe Zäncker²⁹, Conxi Lazaro^8;30;31, Joana Guerra^3;32, Manuel Teixeira^3;8;32, Kasmintan Schrader^33;34, Verena Steinke-Lange^8;35;36, Sérgio Sousa^23;24;25, Manuela Balsinha⁶, Stefan Aretz^8;9;21, Judith Balmaña^8;22, Melyssa Aronson³⁷, Augusto Perazzolo Antoniazzi³⁸, Edenir Palmero^39;40, Lizet Van der Kolk⁴¹, Annemieke Cats⁴², Jolanda M van Dieren⁴², Sergi Castellvi-Bel^14;15;16, Bryson Katona¹⁸, Rachid Karam¹⁷, Florence Coulet⁴³, Paulo Pereira^1;44, Patrick Benusiglio^45;46, carla oliveira^1;5;8

¹i3S – Instituto de Investigação e Inovação em Saúde, Porto, Portugal; ²IPATIMUP – Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal; ³Institute of Biomedical Sciences Abel Salazar of the University of Porto, Porto, Portugal; ⁴Faculty of Science of the University of Porto, Porto, Portugal; ⁵Faculty of Medicine of the University of Porto, Porto, Portugal; ⁶Centro Hospitalar Universitário São João, Porto, Portugal; ⁷Institut Curie, University Paris Sciences Lettres, Department of Genetics, Paris, France; ⁸Full Member of the European Reference Network on Genetic Tumor Risk Syndromes (ERN GENTURIS) – Project ID No 739547; ⁹National Center for Hereditary Tumor Syndromes, University Hospital Bonn, Bonn, Germany; ¹⁰University Hospital Bonn, Department of Internal Medicine I, Bonn, Germany; ¹¹Nantes Université, Univ Angers, CHU Nantes, INSERM, Immunology and New Concepts in ImmunoTherapy (INCIT), Nantes, France; ¹²Nantes University Hospital, Department of Dermatology, Nantes, France; ¹³Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands; ¹⁴Hospital Clínic Barcelona, Department of Gastroenterology, Barcelona, Spain; ¹⁵Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain; ¹⁶Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain; ¹⁷Ambry Genetics, Aliso Viejo, California, United States; ¹⁸University of Pennsylvania Perelman School of Medicine, Division of Gastroenterology and Hepatology, Philadelphia, Pennsylvania, United States; ¹⁹Medical University Innsbruck, Division of Human Genetics, Innsbruck, Austria; ²⁰IFLb Laboratoriumsmedizin Berlin GmbH, Windscheidstraße, Berlin-Charlottenburg, Berlin, Germany; ²¹Institute of Human Genetics, Medical Faculty, University of Bonn, Bonn, Germany; ²²Hereditary Cancer Group, Medical Oncology Department Hospital Vall d’Hebron, and Vall d’Hebron Institute of Oncology, Barcelona, Spain; ²³Paediatric Hospital, Coimbra Hospital and University Centre, Medical Genetics Unit, Coimbra, Portugal; ²⁴Faculty of Medicine, University of Coimbra, University Clinic of Genetics, Coimbra, Portugal; ²⁵Clinical Academic Centre of Coimbra, Coimbra, Portugal; ²⁶Sorbonne Université, UPMC Univ Paris 06, INSERM, UMRS 938, SIRIC CURAMUS, Equipe Instabilité Des Microsatellites Et Cancer, Equipe Labellisée Par La Ligue Contre Le Cancer, Centre de Recherche Saint Antoine, Paris, France; ²⁷Sorbonne Université, Laboratoire D’anatomie Et Cytologie Pathologiques, Hôpital Saint-Antoine, AP-HP, Paris, France; ²⁸National Cancer Institute, National Institutes of Health, Surgical Oncology Program, Maryland, United States; ²⁹Institut für Klinische Genetik, Universitätsklinikum Carl Gustav Carus Dresden, Dresden, Germany; ³⁰Hereditary Cancer Program, Catalan Institute of Oncology, Bellvitge Institute for Biomedical Research, Barcelona, Spain; ³¹Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain; ³²Portuguese Oncology Institute of Porto, Department of Laboratory Genetics, Porto, Portugal; ³³Faculty of Medicine, University of British Columbia, Department of Medical Genetics, Vancouver, Canada; ³⁴BC Cancer, Department of Molecular Oncology, Vancouver, Canada; ³⁵Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Munich, Germany; ³⁶Medizinisch Genetisches Zentrum, Munich, Germany; ³⁷Zane Cohen Centre, Sinai Health System, Toronto, Canada; ³⁸Barretos Cancer Hospital, Cancer Genetics Departament, Barretos, Brazil; ³⁹Barretos Cancer Hospital, Medical Genetics Unit, Barretos, Brazil; ⁴⁰National Cancer Institute, Department of Genetics, Brazil; ⁴¹Netherlands Cancer Institute, Department of Clinical Genetics, Amsterdam, Netherlands; ⁴²Netherlands Cancer Institute, Department of Gastrointestinal Oncology, Amsterdam, Netherlands; ⁴³Unité fonctionnelle d’Onco-angiogénétique et génomique des tumeurs solides, Département de Génétique médicale, Hôpital Pitié-Salpêtrière, AP-HP, Sorbonne Université, Paris, France; ⁴⁴IBMC – Instituto de Biologia Molecular e Celular, Porto, Portugal; ⁴⁵Unité fonctionnelle d’Oncogénétique clinique, Département de Génétique, Groupe Hospitalier Pitié-Salpêtrière, AP-HP, Sorbonne Université, Paris, France; ⁴⁶Chirurgie générale et digestive, Hôpital Saint-Antoine, AP-HP, Sorbonne Université, Paris, France

Background/Objectives: We previously demonstrated that carriers of CTNNA1-truncating variants present increased lifetime-risk of developing diffuse gastric cancer (DGC). These carriers are 14-fold more likely to develop DGC than carriers of CTNNA1-missense variants. Herein, we aim at understanding if CTNNA1 germline transcripts bearing premature termination codons (PTC) are targeted for nonsense-mediated mRNA decay (NMD), and to disclose the transcriptomic profile of CTNNA1-deficient tumors.

Methods: We CRISPR/Cas9 edited gastric cancer cell lines mimicking CTNNA1 truncating variants (CTNNA1-PTC) and assessed whether NMD is triggered, and obtained the transcriptomic profile of CTNNA1-deficient DGC tumors using RNA-seq from CTNNA1 germline carriers.

Results: CTNNA1-PTC cells lose CTNNA1/ αE-catenin mRNA/protein expression. NMD blockade significantly increased (~13-fold) their CTNNA1 mRNA expression, compared to untreated cells. As CTNNA1 expression recovered wild-type mRNA levels, NMD is likely the mechanism responsible for CTNNA1 mRNA decay. Immunohistochemistry showed αE-catenin protein loss in DGC tumors, but not in normal tissue. Transcriptome analysis of 11 DGC tumors revealed 14 significantly downregulated and 940 upregulated genes in comparison to matched normal tissue (p < 0.01; differential expression: 2-fold increase/decrease in tumors vs normal). Upregulated signaling pathways involve cell adhesion, actin cytoskeleton, cell migration, angiogenesis and immune response, as well as JAK/STAT, EGFR or TGF-β oncogenic pathways.

Conclusion: We demonstrated that NMD acts over CTNNA1 PTC-bearing transcripts, being responsible for mRNA decay of germline mutant alleles. CTNNA1-mutant tumors present a transcriptional landscape recapitulating the behavior of poorly cohesive, invasive, proliferative and migratory cancers. CTNNA1-deficient DGC overexpress molecules and pathways worth exploring as therapy targets.

Grants:FCT_PhDfellowship(Ref:2020.05773.BD);ERN-GENTURIS (ProjectID:No.739547);LEGOH Project(Ref:PTDC/BTM-TEC/6706/2020).

Conflict of Interest: Silvana Lobo: None declared, Alexandre Dias: None declared, Marta Ferreira: None declared, Nelson Martins: None declared, Rita Barbosa-Matos: None declared, João Fonseca: None declared, J. Garcia-Pelaez: None declared, Ana Maria Pedro: None declared, Irene Gullo: None declared, André Oliveira: None declared, Celina São José: None declared, Chrystelle Colas: None declared, Robert Hüneburg: None declared, Jacob Nattermann: None declared, Lise Boussemart: None declared, Liselotte P van Hest: None declared, Leticia Moreira: None declared, Carolyn Horton Full Time Paid Employee at Ambry Genetics, Dana Farengo Clark: None declared, Sigrid Tinschert: None declared, Golmard Lisa: None declared, Isabel Spier: None declared, Adrià López-Fernández: None declared, Daniela Oliveira: None declared, Magali Svrcek: None declared, Pierre Bourgoin: None declared, Helene Delhomelle: None declared, Jeremy Davis: None declared, Birthe Zäncker: None declared, Conxi Lazaro: None declared, Joana Guerra: None declared, Manuel Teixeira: None declared, Kasmintan Schrader: None declared, Verena Steinke-Lange: None declared, Sérgio Sousa: None declared, Manuela Balsinha: None declared, Stefan Aretz: None declared, Judith Balmaña: None declared, Melyssa Aronson: None declared, Augusto Perazzolo Antoniazzi: None declared, Edenir Palmero: None declared, Lizet Van der Kolk: None declared, Annemieke Cats: None declared, Jolanda M van Dieren: None declared, Sergi Castellvi-Bel: None declared, Bryson Katona: None declared, Rachid Karam Full Time Paid Employee at Ambry Genetics, Florence Coulet: None declared, Paulo Pereira: None declared, Patrick Benusiglio AstraZeneca, BMS and MSD (Merck pharmaceuticals), carla oliveira: None declared

P01.139.B Challenges of multigene panel testing of breast cancer patients in Turkey

Deniz Agirbasli ¹, Mehmet Erinc Onal², Didem Gulhan², Aysel Kalayci¹

¹Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Medical Genetics, Istanbul, Türkyie; ²Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine, Istanbul, Türkyie

Background/Objectives: Hereditary breast cancer represents 5-10% of all breast cancer cases. Germline pathogenic/likely pathogenic (P/LP) variants in BRCA1/2 genes are responsible for only 15% of the familial cases. Next-generation sequencing (NGS) based-multigene panels for detecting germline variants have emerged as a valuable tool in genetic assessment.

Methods: The multigene NGS panel results of 207 women with breast cancer who applied to our center between 2021-2022 were analyzed retrospectively.

Results: The mean age of cases at diagnosis was 45,93 ± 9,98. Out of 180 patients, the most common histologic types were ductal (n = 150, 83%) and lobular carcinoma, respectively (n = 15, 8,3%), followed by mixed type (n = 8, 4,4%), tubular (n = 4, 2,2%), mucinous (n = 2, 1,1%), metastatic spindle cell carcinoma (n = 1, 0,5%). No identifiable variants were detected in 110 patients (53%). Number of pathogenic variants detected were 24 (11,5%), variants of uncertain significance (VUS) were 61 (29,5%), and likely pathogenic variants were 14 (%6,7). 19 patients carried multiple variants (9,1%). Most common P/LP mutations were detected in CHEK2 (n = 8, 3.8%), BRCA1 (n = 6, 2,8%), BRCA2 (n = 6, 2.8%), ATM (n = 4, 1.9%), PALB2 (n = 3, 1,4%) genes followed by BARD1, BRIP1, XRCC2, RAD50, NBN, BLM.

Conclusion: The results of multigene panels will diversify genetic counseling as well as prevention and treatment selection. Although no clinical decisions can be made on the basis of variants of uncertain significance, genetic re-evaluation should be performed at regular intervals, and functional studies should be performed. Current family history risk assessment tools must be improved for patients with a strong family history of cancer as in our cohort.

Conflict of Interest: None declared

P01.140.C Prevalence and impact of CDKN2A/2B Gene Deletions in Pediatric Acute Lymphoblastic Leukemia: A SNPa study.

Luís Miguel Pires ¹, Pedro Veiga¹, Mariana Val¹, Susana Isabel Ferreira¹, Nuno Lavoura¹, Marta Pinto¹, Ana Jardim¹, Paula Gameiro², Maria João Martins², Margarida Coucelo³, Sónia Silva⁴, Manuel Brito⁴, Ximo Duarte⁵, Ilda Patrícia Ribeiro^1;6;7, Joana Barbosa de Melo^1;6;7, Isabel Marques Carreira^1;6;7

¹Laboratory of Cytogenetics and Genomics, Faculty of Medicine, University of Coimbra, Coimbra, Portugal; ²Laboratório de Hemato-Oncologia, Instituto Português de Oncologia de Lisboa, Lisboa, Portugal; ³Unidade Funcional de Hematologia Molecular, Centro Hospitalar e Universitário de Coimbra EPE, Hospital Pediátrico, Coimbra, Portugal; ⁴Oncologia Pediátrica, Centro Hospitalar e Universitário de Coimbra EPE, Hospital Pediátrico, Coimbra, Portugal; ⁵Serviço de Pediatria e do Adolescente, Instituto Português de Oncologia de Lisboa, Lisboa, Portugal; ⁶Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, Coimbra, Portugal; ⁷Institute for Clinical and Biomedical Research (iCBR) and Center of Investigation on Environment Genetics and Oncobiology (CIMAGO), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Background/Objectives: The CDKN2A/2B gene is a tumor suppressor and its deletion is one of the most frequent events in acute lymphoblastic leukemia (ALL), acting as a secondary cooperating factor and playing a critical role in cell cycle regulation and chemosensitivity. CDKN2A/B deletions are detected in approximately 20-25% of pediatric B-cell precursor ALL cases and 40-50% of T-cell precursor ALL patients. It is indicative of poor prognosis in pediatric and adult B-ALL. We present the incidence of deletions in CDKN2A/2B and other specific genetic alterations in ALL key-genes in a Portuguese cohort of pediatric ALL used for risk stratification.

Methods: 105 ALL samples were analyzed by Array Comparative Genomic Hybridization combined with Single Nucleotide Polymorphism array (SNPa). The analysis included the evaluation of ploidy, whole-genome alterations, loss of heterozygosity and copy number alterations affecting ALL critical genes, including CDKN2A/B.

Results: The results of our study demonstrate that the most frequent genetic alteration was the deletion of the CDKN2A/B gene, present in 34% of the samples, followed by deletions in ETV6, PAX5 and EGR. CDKN2A/B homozygous deletion is more prevalent than heterozygous deletion present in 23% of the cases. On the other hand, is relevant that in 64% of the cases in which the CDKN2A/B deletion was found, another genetic alteration was present (PAX5 or ETV6 deletion).

Conclusion: The presence of CDKN2A/2B homozygous deletions can represent a new ALL subgroup, with different outcomes. Risk stratification considering new sub-groups of genetic alterations, can improve survival rates and therapeutic response.

Conflict of Interest: None declared

P01.141.D Next generation sequencing results in acute myeloid leukemia diagnostics

Zoltan Orfi ¹, Dora Csaban¹, Andras Bors¹, Livia Varga¹, Nora Meggyesi¹, Lenke Tanko¹, Adrienn Borsy¹, Andras Kozma¹, Nora Kondor¹, János Dolgos¹, Laszlo Gopcsa¹, Jozsef Harasztdombi¹, Viktor Lakatos¹, Gabor Mikala¹, Judit Reichardt¹, János Fábián¹, Peter Remenyi¹, Valyi-Nagy Istvan¹, Hajnalka Andrikovics¹

¹Central Hospital of Southern Pest, National Institute of Hematology and Infectious Diseases, Budapest, Hungary

Background/Objectives: Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder of myeloid stem cells. In favor of increasing the precision of diagnosis, prognosis and in order to select most suitable targeted therapy, a comprehensive genetic mutation screening for acquired genetic abnormalities is necessary. Massively parallel sequencing effectively enables the identification of measurable residual disease marker(s). According to international recommendations (ELN, NCCN) more than 30 genes should be screened besides recurrent cytogenetic abnormalities.

Methods: At our institute, 175 bone marrow samples from AML patients were analyzed by cytogenetics and next-generation sequencing (NGS targeted panel of 23-68 genes or WES).

Results: Recurrent cytogenetic abnormalities were detected in 20 cases [t(15;17)(q24.1;q21.2)/PML::RARA; t(8;21)(q22;q22.1)/RUNX1::RUNX1T1; inv(16)(p13.1q22)/CBFB::MYH11; t(11q23;v)/KMT2A; inv(3)(q21.3q26.2)/MECOM]. Recurrent WHO defining genetic mutations were found in CEBPA (n = 6; 3%), NPM1 (n = 67; 38%) in normal karyotype AML. Mutations in TP53 (n = 10; 6%), in MDS related mutations (n = 48; 27%) or MDS type cytogenetics (n = 4; 2%) were also observed. No WHO defining category were identified in 20 cases (11%). In the NPM1 group the following frequent mutation associations were observed: DNMT3A, FLT3, IDH1/2, NRAS, PTPN11, RAD21 and WT1.

Conclusion: The frequency of identified gene mutations in our study group is in agreement with the international literature. Genetic profiling with NGS facilitates and complements the diagnosis, prognosis of complex disease clusters and provides valuable information about targeted therapeutic possibilities and significantly enhances the identification of hereditary malignancies as well. Thus, NGS combined with cytogenetics is now an indispensable tool for patients with curative myeloid malignancies.

Conflict of Interest: None declared

P01.142.A BRG1 and HIF1A functional crosstalk defines transcription of ABC transporters in paclitaxel resistantcancer cells

Karolina Gronkowska ¹, Sylwia Michlewska², Tomasz Płoszaj³, Kinga Kołacz¹, Agnieszka Robaszkiewicz¹

¹University of Lodz, Department of General Biophysics, Lodz; ²University of Lodz, Laboratory of Microscopic Imaging and Specialized Biological Techniques, Lodz; ³Medical University of Lodz, Department of Clinical Genetics, Lodz

Background/Objectives: Cancer drug irresponsiveness to chemotherapy is followed by substantial chromatin remodeling at the regulatory elements of genes, which confer drug resistance. Based on TCGA database we delineated strong correlation between mRNA level of SWI/SNF subunit BRG1 encoded by frequently mutated SMARCA4 and ABC transporters, which remove or sequester anticancer drugs inside cancer cell organelles. Therefore, we asked if overexpression of BRG1 is responsible for the limited drug responsiveness of cancers, which developed resistance during paclitaxel therapy.

Methods: ChIP- and RNA-Seq samples were sequenced with Illumina550, data analysis and integration were performed in Galaxy, gene mutations were detected with Sanger sequencing, genes were silenced with siRNA, co-immunoprecitpiated proteins were detected by western blot, colocalization was quantified by confocal microscopy.

Results: The tested SMARCA4^+/+ TNBC samples showed redistribution of BRG1 around TSS of ABC genes responsible for anticancer drug detoxication in paclitaxel-resistant phenotypes without substantial enrichment of the enzyme, whereas mutations gained during the therapy reverted BRG1 truncation caused by pre-existed deletions in SMARCA4^del/del in non-small lung cancer cells, thereby causing recruitment of BRG1 to ABCC5 and ABCC10 gene promoters. Motif spacing analysis of BRG1 binding sites allowed to identify inter alia HIF1A as possible co-factor of BRG1-based SWI/SNF complex. This transcription factor co-occurred in the nucleus and formed immunoprecipitable complex with BRG1 and p300 acetyltransferase in paclitaxel resistant cancer phenotypes. The deficiency of BRG1 or HIF1A declined ABC gene overexpression and restored paclitaxel-resistant cancer cell responsiveness to numerous chemotherapeutics.

Conclusion: HIF1A defines BRG1-SWI/SNF activity in paclitaxel-resistant cancer cells.

Grants: LIDER/22/0122/L-10/18/NCBR/2019.

Conflict of Interest: None declared

P01.143.B Analysis of aberrantly expressed microRNAs in advanced laryngeal carcinoma showed most targetable pathways

Silva Kyurkchiyan ¹, Veronika Petkova¹, Gergana Stancheva¹, Diana Popova², Radka Kaneva¹, Todor Popov²

¹Medical University - Sofia, Molecular Medicine Center, Department of Medical Chemistry and Biochemistry, Sofia, Bulgaria; ²Medical University - Sofia, UMHAT “Tsaritsa Yoanna – ISUL”, Department of ENT, Sofia, Bulgaria

Background/Objectives: Advance laryngeal squamous cell carcinoma (LSCC) is still tumor burden, associated with poor survival. miRNAs were known regulatory molecules. They were suggested as biomarkers to seek new treatments. The aim of our study is to evaluate miRNAs, which were deeply and significantly involved in deregulation of major pathways in advanced LSCC.

Methods: In total 131 patients, diagnosed with advanced LSCC were enrolled. The patients were separated in two: training (n = 60) and validation (n = 71) groups. Global microRNA profiling was done, and most significantly miRNAs were analyzed additionally in patient cohort via RT-qPCR. Promising miRNA results were used in in silico, validation and correlation analysis. SPSSv.27 and data bases were used in statistical and bioinformatic analysis. P-value <0.05 was taken as appropriate.

Results: Several miRNAs were most often co-expressed, namely miR-93-5p, miR-145-5p and miR-210-3p. Their expressions were validated in in vivo advanced LSCC samples and further used in bioinformatic and enrichment analysis. Our results highlight the significant roles played by miR-93-5p, miR-145-5p, and miR-210-3p in the processes, regulating pathways linked to cell cycle, hypoxia, metabolism, apoptosis, angiogenesis, and metastasis. miR-93-5p/miR-145-5p/miR-210-3p appear to correlate with biomarkers of those pathways: three HIFα isoforms, VEGFA, VEGFR1, VEGFR2, TP53, BCL-2. Moreover, these miRNAs are actively involved in regulating processes that affect currently used chemo- and target therapies.

Conclusion: Our findings suggest that investigating key regulators of hypoxia, apoptosis, autophagy, and metabolic deregulations could be essential in the management of LSCC.

Grants: European Union-Next Generation EU/National Recovery and Resilience Plan of the Republic of Bulgaria/project №BG-RRP-2.004-0004-C01/group3.1.5 and project D01-302/17.12.2021 by MES, Bulgaria.

Conflict of Interest: None declared

P01.144.C Incidental detection of acquired cryptic chromosomal rearrangements (CCRs) in patients who underwent germline genomic testing.

Moneeb Othman ¹, Peter Bauer¹, Boodor Al-Kawlani¹, Uros Hladnik¹, Omid Paknia¹

¹Centogene AG, Rostock, Germany

Background/Objectives: Acquired genetic changes contributing to cancer, such as complex genomic rearrangements and cryptic copy number abnormalities (CNAs) can be incidentally detected by NGS-based copy number variant (CNV) analysis. We report here two cases referred to our laboratory. First case is a 13-year-old girl with abnormalities in bone marrow cell morphology, acute leukemia, and pancytopenia. The second case is a 12-year-old boy abnormalities in multiple cell lineages in the bone marrow, hepatosplenomegaly, leukocytosis, eosinophilia, monocytosis and myelofibrosis.

Methods:WES-based CNV analysis was performed for both cases, and the results were confirmed by chromosomal microarray analysis.

Results: In the first case, a terminal pathogenic gain of 7.3 Mb within the 9q34.12q34.3 chromosomal cytobands, encompassing the ABL1 gene and a proximal pathogenic gain of 7.5 Mb within the 22q11.1q11.23 chromosomal cytobands, encompassing the BCR gene, have been detected. The unbalanced rearrangement results in the gain of a second copy of the Philadelphia chromosome and is predicted to lead to an abnormal BCR-ABL1 fusion. In the second case, an interstitial deletion of 876 kb encompassing partially FIP1L1 and PDGFRA genes and the entire CHIC2 gene was identified within the 4q12 chromosomal cytoband was detected. The deletion was predicted to result in an abnormal FIP1L1-PDGFRA fusion. A FIP1L1-PDGFRA fusion gene usually results from a cryptic interstitial deletion in 4q12.

Conclusion: The incidental detection of acquired genetic alterations in patients undergoing germline genomic testing highlights the potential of NGS-based CNV analysis in uncovering unsuspected genomic rearrangements. Therefore, detecting gene fusions in leukemia is significantly valuable for diagnosis, prognosis and potential treatment strategies.

Conflict of Interest: None declared

P01.145.D RNA-based gene alteration and expression analysis in NSCLC with known KRAS G12C status

Joanna Moes-Sosnowska ^1;1, Urszula Lechowicz¹, Magdalena Pelc¹, Adam Szpechcinski¹, Paulina Skronska¹, Adriana Rozy¹, Aneta Zdral¹, Mateusz Polaczek², Emil Wojda², Małgorzata Szołkowska³, Tadeusz Orłowski⁴, Pawel Sliwinski⁵, Renata Langfort³, Joanna Chorostowska-Wynimko¹

¹The Institute of Tuberculosis and Lung Diseases, Department of Genetics and Clinical Immunology, Warsaw, Poland; ²The Institute of Tuberculosis and Lung Diseases, III Department of Lung Diseases and Oncology, Warsaw, Poland; ³The Institute of Tuberculosis and Lung Diseases, Department of Pathology, Warsaw, Poland; ⁴The Institute of Tuberculosis and Lung Diseases, Department of Surgery, Warsaw, Poland; ⁵The Institute of Tuberculosis and Lung Diseases, II Department of Lung Diseases, Warsaw, Poland

Background/Objectives: The KRAS c.34G>T, p.(Gly12Cys) (G12C) variant is frequently present in non-small cell lung cancer (NSCLC) targeted with KRAS inhibitors. However, the reliability of that treatment is not satisfactory. To better understand NSCLC biology and identify potential biomarkers, it is important to analyze the prevalence of KRAS variants and their relationship to other oncogenesis-related genes. Therefore, we aimed to compare the expression of 14genes among KRAS-positive and negative tumors using RNA-based next-generation sequencing (NGS).

Methods: KRAS variants and expression levels of 14genes (ALK,BRAF,EGFR,FGFR1-3,KRAS,MET,NRG1,NTRK1-3,RET, and ROS1) were evaluated in 160 tumors (formalin-fixed, paraffin-embedded(FFPE) biopsies and surgically resected tissues) including 126(79%) adenocarcinomas, 24(15%) large cell lung cancer and 10(6%) not otherwise specified (NOS) with the use of RNA-FusionPlex Lung panel(Archer).

Results: Overall, 58(17.4%) of the 160 samples harbored 58 clinically significant KRAS variants, of which 95% were found in adenocarcinomas. The G12C was the most frequent variant among all KRAS-positive tumors(36%). KRAS-negative tumors characterized with variants: EGFR (n = 13); BRAF(n = 7); CD74::ROS1(n = 1); EML4::ALK(n = 3) and METex14 skipping(n = 6). The gene expression analysis revealed significantly higher median expression of the NTRK2(TrkB) in tumors with the G12C variant (p = 0.016) and significantly lower median expression of the KRAS in tumors carrying the c.37G>T, p.(Gly13Cys) variant (p = 0.011).

Conclusion: This study adds new data to the genomic characterization of KRAS-positive tumors, especially those with G12C variants. Our study revealed higher levels of NTRK2 in G12C-positive tumors which can be associated with unfavorable prognosis since NTRK2 signaling may contribute to metastasis, and is associated with intratumoral hypoxia. Further research is needed to confirm these observations.

Grants: project 3.9/2022

Conflict of Interest: None declared

P01.146.A deepHRD, a clinically validated NGS- and AI-based method for detection of genomic instability in ovarian cancer

Juliette Albuisson ¹, Gwladys Le Dain², Sandy Chevrier¹, Anthony Comte¹, Romain Boidot¹, Valentin Derangère¹, Vincent Goussot¹, Caroline Truntzer²

¹Centre Georges François Leclerc, Département de Biologie et Pathologie des Tumeurs, DIJON, France; ²Centre Georges François Leclerc, Plateforme de transfert en Biologie Cancérologique, DIJON, France

Homologous Recombination Repair Deficiency (HRD) leads to tumor genomic instability (GIS) and sensitivity to PARP inhibitors in ovarian cancers (OC), and the identification of HRD status is considered a major therapeutic issue.

We developed an NGS- and AI-based HRD detection tool that allows the detection of GIS in tumor samples. First, sequencing of a SNP and CNV containing genomic backbone (2.8 Mb size) is performed in a standard Illumina NGS platform. Second an AI-based algorithm analyzes NGS results in tumor sample is applied.

We performed several steps of validation: technical validation was done comparing Myriad Mychoice GIS score/class and our own GIS score/class in 177 tumor samples analyzed in our Centre. Second, technical sensitivity was assessed using a tumor sample with GIS score just above threshold and decreasing tumor purity. Third, clinical validation was done in 360 tumor samples from PAOLA-2 study.

Our method showed very good technical performance (sensitivity and specificity 0.94 and 0.95 respectively). Clinical performance was good (Sensitivity 0.93, specificity 0.84, using combined BRCA sequencing results and deepHRD scoring, PFS 54.3 [40.2-60.6] vs 18.7 [14.3-24.7] in GIS+ vs GIS- respectively). In all 43 Myriad Mychoice inconclusive results, deepHRD showed a similar performance. Technical sensitivity could be confirmed with GIS detection in samples with as less as 20% of tumor purity.

Technology transfer is currently under progress in our centre and will be soon available either through collaboration or commercial use.

Conflict of Interest: Juliette Albuisson Grant Astra-Zeneca, Gwladys Le Dain: None declared, Sandy Chevrier: None declared, Anthony Comte: None declared, Romain Boidot: None declared, Valentin Derangère: None declared, Vincent Goussot: None declared, Caroline Truntzer: None declared

P01.148.C Impact of rare and low-frequency genetic variants in pleural mesothelioma

Elisabetta Casalone ¹, elton jalis herman¹, Cecilia Di Primio¹, Carla Debernardi¹, Rebecca Filomena¹, ANGELO SAVOCA¹, Miriam Rosselli¹, Dario Mirabelli², Giuseppe Matullo^1;3

¹University of Turin, Medical Sciences, Turin, Italy; ²University-Hospital and Center for Cancer Prevention, Cancer Epidemiology, Turin, Italy; ³AOU Città della Salute e della Scienza, Medical Genetics, Turin, Italy

Consortium: EPIC network

Background: Previous Genome Wide Association Studies (GWASs) in pleural mesothelioma (PM) identified low risk genetic variants conferring a limited increase risk of the diseases and showing evidence of interaction with asbestos exposure. Other sequencing studies on cancer gene panels in PM cases identified 5-10% of germline mutations increasing susceptibility to PM even at low levels of asbestos exposure. Here, we conducted a whole exome sequencing (WES) analysis aimed at identifying genes with rare variants contributing to PM risk.

Methods: WES screening was performed on DNA isolated from blood of 129 pre-clinical PM and 129 non-cancer individuals. Alignment of the sequencing reads, variant calling and annotation were performed using JuliaOmixTM software (GenomeUp, Italy). SNVs with a read depth > 20X and a m.a.f. < 0.01 in GnomAD database, were taken into account for downstream analyses.

Results: In the group of cases we identified pathogenic rare variants in cancer predisposing genes such as ACVR1B, B2M, DNMT3A, HRAS, NPM1, PTPN11, STAG2. Sequence Kernel association test performed in about 1 million of rare variants across 17758 genes showed a significant enrichment in genes involved in DNA methylation, chromosome replication and segregation.

Conclusion: WES sequencing technologies allowed us to identify rare germline mutations in novel genes potentially contributing, together with asbestos exposure, to increase risk of PM development. Functional analysis will be performed to test pathogenic effects and the potential impact of the mutational processes in somatic cells.

Grants: AIRC under IG 2018—[ID.21390 project—P.I. GM]

Conflict of Interest: None declared

P01.149.D Major impact on modifiable colo-rectal cancer risk factors on at risk individuals through personalised prevention in clinical genetics practice

marie-gabrielle delorme guinand¹, anna serova-erard¹, laury nicolas¹, noemie demare², simon rey¹, martine duclos³, François Cornélis ¹

¹CHU Clermont-Ferrand, 63000, Clermont-Ferrand, France; ²Avicenne Hopital, Assistance Publique des Hopitaux de Paris, Paris, France; ³CHU Observatoire National de l’Activité Physique et de la Sédentarité, 63000, Clermont-Ferrand, France

Introduction : In EU-27 countries in 2020, colorectal cancer (CCR) was the 2^nd cause of cancer death. Major modifiable risk factors are the lack of physical activity (PA) and of dietary fiber intake (DFI). The majority of outpatients in clinical genetics showing no germline pathogenic variants are affected with a multifactorial disease, for which correction of risk factors is essential. Our aim was to evaluate the impact of personalised advice following standardized evaluation of PA and DFI.

Patients & Methods : Genetics adult outpatients were systematically evaluated using the International PA Questionnaire short form and a in-house questionnaire for DFI, taking 5 minutes on average. At high CCR risk individuals had personal or familial history of CCR or colon adenomas.

Results : Evaluation of PA and FDI revealed 51.3% (n = 559, since 2018) and 74.5% (n = 212, since 2021), respectively, of individuals below the OMS recommendation, who were given personalised advice. At the second evaluation, 9 months later (in mean, range 3-12 months), out of those, 42.7% and 24.1%, respectively, had corrected to meet the OMS target. For the subgroup of individuals at high CCR risk (n = 92), 48.9% and 81.6% were below the targets at the first evaluation and, out of those, 35.6% and 24.1%, respectively, had corrected at the second evaluation.

Conclusion : Systematic personalised prevention in clinical genetics practice has a major impact on modifiable colo-rectal cancer risk factors such as PA and, to a lesser extents, DFI, with a similar magnitude on individuals at high or low CCR risk.

Conflict of Interest: None declared

P01.150.A Assessment of cancer predisposition syndromes in a pediatric oncology center from Midwest-Brazil

Flávia Delgado Martins ¹, Renata Sandoval²

¹Hospital da Criança de Brasília José Alencar, Pediatric oncology, Brasília; ²Hospital da Criança de Brasília José Alencar, Genetic service, Brasília

Background/Objectives: Pediatric cancer can be a sentinel for cancer predisposition syndromes (CPS). Understanding the association of specific tumor subtypes with recognizable clinical patterns may help to identify carriers, and to inform care. Here we provide descriptive data from pediatric cancer patients with referral for genetic evaluation.

Methods: We retrospectively reviewed medical charts of childhood cancer patients who were referred to the oncogenetics service between January 2018 and July 2023. Cases without pathology and/or imaging reports supporting a malignancy diagnosis were excluded. Referral indications, clinical data and germline genetic tests results (when available) were assessed.

Results: Among 129 patients who were referred to genetic evaluation, 96 met study criteria. A suspicious family history of cancer was a recurrent referral indication (n = 47). Phenotypic-driven diagnosis was possible for 37 cases (38.5%): 14 Neurofibromatosis type 1, 12 Tuberous Sclerosis Complex and 11 polyposis syndromes. Among patients who performed germline testing, 85% (17/21) harbored a pathogenic/likely pathogenic germline variant in a CPS gene.

Conclusion: In this highly selected cohort we found almost 40% of CPSs identified by a phenotype-driven approach and among those who performed germline testing 85% of positive results. This preliminary data allowed the creation of an institutional registry of CPSs and future prospective studies.

Conflict of Interest: None declared

P01.151.B Reflex CRC Initiative - Advancing Colorectal Cancer Care through Comprehensive Genetic Testing

Christoforos Pappas ¹, Hannes Olauson², Felix Haglund³, Johan Lindberg⁴, Caroline Beergrehn⁵, Emma Tham¹, Annika Sjövall¹

¹Karolinska Institute, Department of Molecular Medicine and Surgery, Stockholm, Sweden; ²Karolinska Institute, Department of Laboratory Medicine, Stockholm, Sweden; ³Karolinska Institute, Department of Oncology-Pathology, Stockholm, Sweden; ⁴Karolinska Institute, Department of Medical Epidemiology and Biostatistics, Stockholm, Sweden; ⁵Capio St Göran Hospital, Department of Surgery, Stockholm, Sweden

Background/Objectives: Colorectal cancer management is evolving towards personalized medicine, with genetic testing playing a crucial role in treatment decisions. The Reflex Testing for Colorectal Cancer initiative aimed to integrate comprehensive genetic testing into routine care. Specifically, the aim was to promptly determine treatment predictive genetic factors and hereditary cancer risk syndromes that would lead to potential prophylactic surgery as well as identify DPYD deficiency that affects oncological treatment.

Methods: The study, conducted from October 2021 to December 2023, included 370 patients diagnosed with colorectal cancer. Genetic analysis was performed on DNA extracted from blood and from tumor tissue taken from biopsy during endoscopy, using a gene panel of 385 genes (GMCK panel) designed to identify variants relevant to treatment prediction, prognosis, and heredity for all solid tumors.

Results: In the first 100 cases (48 females, 52 males), 7% were diagnosed before 50 years and 18% between 50 and 60 years of age. The tumor localization varied, with 23% in the right colon, 9% in the transverse colon, 27% in the left colon and 41% in the rectum.15% showed high microsatellite instability. Additionally, four patients (4%) were identified with Lynch syndrome (disease-associated variants in MLH1(x2) or MSH6(x2)). Regarding treatment-predictive biomarkers, pathogenic variants were found in KRAS in 44% and in BRAF in 20%.

Conclusion: The analysis of paired tumor and blood data can identify both somatic and germline aberrations that are of clinical significance and thus enable personalized therapeutic decisions at diagnosis.

Grants: Regional Cancer Center Stockholm-Gotland funded this project.

Conflict of Interest: Christoforos Pappas Karolinska University Hospital, Hannes Olauson Karolinska University Hospital, Felix Haglund Illumina (reagents), ROCHE Global (reagents), Cancerfonden, Stockholm Cancer Society, Merck, Swedish Sarcoma Society, Karolinska University Hospital and Karolinska Institutet, Johan Lindberg Karolinska University Hospital, Caroline Beergrehn Capio Saint Göran Hospital, Emma Tham Cancerfonden, Barncancerfonden, Region Stockholm, Japan-Sweden Foundation, Karolinska University Hospital, Annika Sjövall Karolinska University Hospital

P01.153.D Recessive effect of human polymerase delta proofreading deficiency discovered through mutational analysis of POLD1-mutated normal and cancer cells

Maria A. Andrianova¹, Vladimir B. Seplyarskiy^2;3, Mariona Terradas⁴, Ana Beatriz Sánchez-Heras^5;6, Pilar Mur⁴, José Luis Soto^5;7, Gemma Aiza⁴, Emma Borràs Angosto⁸, Fyodor A. Kondrashov^9;10, Alexey S. Kondrashov¹¹, Georgii A. Bazykin^2;3, Laura Valle ^4;12

¹Institute for Research in Biomedicine (IRB Barcelona), the Barcelona Institute of Science and Technology, Barcelona, Spain; ²Harvard Medical School, Department of Biomedical Informatics, Boston (MA), United States; ³Brigham and Women’s Hospital, Harvard Medical School, Division of Genetics, Boston (MA), United States; ⁴Catalan Institute of Oncology, IDIBELL, Hereditary Cancer Group, Oncobell Program, Hospitalet de Llobregat (Barcelona), Spain; ⁵Foundation for the Promotion of Health and Biomedical Research of Valencia Region (FISABIO), Elche Health Department, Elche, Spain; ⁶Elche University Hospital, Medical Oncology Department, Cancer Genetic Counseling Unit, Elche, Spain; ⁷Elche University Hospital, Molecular Genetics Unit, Elche, Spain; ⁸Consorci Sanitari de Terrassa, Molecular Genetics Unit, Terrassa (Barcelona), Spain; ⁹Institute of Science and Technology Austria, Klosterneuburg, Austria; ¹⁰Okinawa Institute of Science and Technology Graduate University, Evolutionary and Synthetic Biology Unit, Okinawa, Japan; ¹¹University of Michigan, Department of Ecology and Evolutionary Biology, Ann Arbor (MI), United States; ¹²Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain

Background: Constitutional heterozygous pathogenic variants in the exonuclease domain of POLE and POLD1, which affect the proofreading activity of corresponding polymerases, cause a cancer predisposition syndrome (polymerase proofreading-associated polyposis), characterized by increased risk of gastrointestinal polyposis, colorectal cancer, endometrial cancer and other tumour types. The connection between the disruption of polymerase proofreading activity and cancer development is through an increase in the somatic mutation rate.

Methods: We studied an extended family with multiple members heterozygous for the pathogenic POLD1 variant c.1421T>C p.(Leu474Pro). Genome sequencing was performed to evaluate mutational patterns of patient-derived fibroblasts colonies and of de novo mutations obtained by parent-offspring comparisons. Exome sequencing data from tumours developed by patients with the hereditary cancer syndrome were also analysed.

Results: Heterozygous POLD1 L474P just subtly increases somatic and germline mutation burden in non-tumour tissues. In contrast, tumours developed in individuals with a heterozygous mutation of the exonuclease domain of POLD1, including L474P, have extremely high mutation rates (>100 mut/Mb) associated with tumour mutational signature SBS10d. To explain these observations, also common to other POLD1 exonuclease domain pathogenic variants, we show, for the first time, that POLD1 proofreading deficient tumours developed in the context of the hereditary cancer syndrome require somatic inactivation of the wildtype allele in the target tissue (e.g. colon or endometrial epithelium), usually through loss of heterozygosity, resulting in the elimination of all exonuclease-proficient copies of the gene.

Conclusion: These results provide strong evidence that POLD1 pathogenic variants have a recessive effect on mutation rate in somatic cells.

Conflict of Interest: None declared

P01.154.A Germline testing in routine care of ovarian cancer patients in Estonia 2009-2023

Mikk Tooming ^1;2, Kadri Rekker², Kadri Toome², Olga Fjodorova², Hanno Roomere², Ülle Murumets², Piret Laidre², Katrin Ounap^1;2, Tiina Kahre^1;2

¹Institute of Clinical Medicine, Department of Clinical Genetics, Tartu, Estonia; ²Genetics and Personalized Medicine Clinic, Tartu University Hospital, Tartu, Estonia

Background/Objectives: Germline testing in routine care of ovarian cancer (OC) patients in Estonia has evolved from single-gene testing to targeted NGS gene panels during the years 2009 to 2023. This study aimed to investigate the prevalence of (likely) pathogenic variants (PV) in Estonian OC patients.

Methods: The study included 761 OC patients referred from tertiary hospitals between 2009 and 2023 to the Genetics and Personalized Medicine Clinic at Tartu University Hospital for genetic testing. Individuals were analyzed using either APEX microarray, BRCA1/2 Sanger sequencing, BRCA1/2 multiplex ligation-dependent probe amplification analysis or Illumina TruSight Cancer/Hereditary Cancer (94 or 113 genes) gene panels.

Results: Most common histological OC type was serous carcinoma 559/761 (73%). The mean age of OC onset was 56 years (4-91y), and the mean age of testing was 60 years (10-91y). Bilateral OC occurred in 42/761 (5.5%) patients. Overall, 207/761 (27%) OC patients carried PV. BRCA1/2 PV was found in 158/761 (21%) patients. Additionally, 49/761 (6.4%) patients had PV in non-BRCA genes associated with OC, including ATM, BRIP1, DICER1, PALB2, PMS2, RAD51C 25/761 (3.3%), and secondary findings in BARD1, CHEK2, FANCM, MITF, MUTYH, PPM1D, TP53 24/761 (3.1%). BRCA1 was the most prevalently mutated gene, PV detected in 123/207 (59%) of patients, followed by BRCA2 35/207 (17%), CHEK2 13/207 (6.2%), and BRIP1 11/207 (5.3%).

Conclusion: The diagnostic efficacy of OC patients in Estonia was ~27%. The detection rate with the NGS gene panel is ~6% higher than focusing on BRCA1/2 gene testing alone.

Grants: Estonian Research Council grant PRG471/PRG2040

Conflict of Interest: None declared

P01.155.B The co-regulation of circular RNAs and messenger RNAs in colorectal cancer: the fate of an unbalanced couple?

Corentin Levacher¹, Philippe Ruminy², Camille Charbonnier³, Joanna Delfosse¹, Edwige Kasper⁴, Charbonnier Françoise¹, Mathieu Viennot², Jacques Mauillon⁵, Nathalie Parodi⁵, Stéphanie Baert-Desurmont⁴, Claude Houdayer ⁴

¹Univ Rouen Normandie, Inserm U1245, Normandie Univ, F-76000 Rouen, France; ²Univ Rouen Normandie, Inserm U1245, Normandie Univ, Centre Henri Becquerel, F-76000 Rouen, France; ³Univ Rouen Normandie, Inserm U1245, Normandie Univ, CHU Rouen, Department of Biostatitics, F-76000 Rouen, France; ⁴Univ Rouen Normandie, Inserm U1245, Normandie Univ, CHU Rouen, Department of Genetics, F-76000 Rouen, France; ⁵Univ Rouen Normandie, Normandie Univ, CHU Rouen, Department of Genetics, F-76000 Rouen, France

Background/Objectives: Circular RNAs (circRNAs) are emerging players in human diseases, with plausible function as part of competing endogenous networks. We hypothesized that circRNAs and mRNAs are co-regulated with a mandatory balance which, when broken, may favor disease development.

Methods: This hypothesis was tested in colorectal cancer (CRC) patients, thanks to the French multicenter Oligogenic Determinism of Colorectal Cancer collection (Baert-Desurmont et al., 2016) which contains whole blood RNA samples from 716 patients suspected of CRC predisposition excluding Lynch syndromes and polyposis, and 249 matched controls. The circRNA-mRNA couple was studied by SEALigHTS (Splice and Expression Analyses by exon Ligation and High-Throughput Sequencing) (Levacher et al., 2023) using a panel of 23 genes involved in CRC predisposition, i.e. 935 probes designed at exon ends, enabling the exploration of all exon-exon junctions. Following reverse transcription and probing on cDNA, nearby probes are ligated and the number of ligations quantified using unique molecular identifiers and sequencing.

Results: We described the landscape of circRNAs for these 23 genes i.e. 258 circular junctions, including 64 novel ones. The circRNA/mRNA ratios were calculated for each gene and ranged from 0.5% to 17% for MLH1 and POLD1, respectively. The circRNA/mRNA ratio was 1.5-fold higher in patients compared to controls (p < 2 × 10-16), irrespective of age of cancer onset or gender. This increase was mainly driven by POLD1 (p = 5.2 × 10-8) and did not reflect a competitive mechanism.

Conclusion: Overall, the unbalanced circRNA/mRNA couple observed in this large cohort opens new avenues in circRNA importance and CRC genetics.

Grants: RIN RECHERCHE

Conflict of Interest: None declared

P01.156.C Empowering Hereditary Cancer risk assessment: an unforeseen local geographic pattern revealed by Copy Number Variation analysis

Lia Bonamici ¹, Marco Marino², Lucia Artuso², Enrico Tagliafico^2;3, Elena Tenedini^2;3

¹University of Modena and Reggio Emilia, Department of Biomedical, Metabolic and Neural Sciences, Modena, Italy; ²Modena University Hospital, Department of Laboratory Medicine and Pathology, Modena, Italy; ³University of Modena and Reggio Emilia, Department of Medical and Surgical Sciences, Modena, Italy

Background/Objectives: Next Generation Sequencing (NGS) multi-gene panels (MGP) are extensively employed in molecular oncology and recommended for individuals with personal or familial criteria for hereditary cancer testing. Analysis algorithms for NGS data, which allow the simultaneous detection of sequence (SNV) and copy number variants (CNVs), have significantly increased the detection rates and cost-effectiveness of the tests, while maintaining high accuracy and reliability. CNVs, consisting of large deletions or duplications, potentially occur in all the genes currently examined for SNVs and may play an important role in tumor development. Despite this, they remain less characterized.

Methods: In this study, we analyzed results from 2989 patients affected mainly by breast or ovarian cancer, who underwent testing with a 26-gene panel for hereditary cancer.

Results: The cohort carried either variants of undetermined significance (42%) or Pathogenic/Likely Pathogenic (13.3%). According to previous studies, SNVs were ubiquitous across all tested genes, with pathogenic variants being more frequent in BRCA1, BRCA2, CHEK2, and MUTYH. Conversely, CNVs, observed in 1.8% of the patients, affected only ten genes. Among these, PALB2 showed over 50% of the total variants. Moreover, the pathogenic deletion of exon 11, accounted for the 85.7% of the PALB2 CNVs. The identification of a common place of origin for patients with exon 11 deletion, suggests a potential geographic effect contributing to this phenomenon.

Conclusion: These data provide insights into the relevance of CNVs detection, which can reveal valuable information in variant-associated cancer risk assessment and reveal unforeseen prevalence and distributions of variants across different regions and ethnic groups.

Grants:

Conflict of Interest: None declared

P01.157.D Homozygous substitution of threonine 191 by proline in polymerase η causes Xeroderma pigmentosum variant

Roberto Ricciardiello¹, Giulia Forleo¹, Lina Cipolla¹, Geraldine Van Winckel², Caterina Marconi², Thierry Nouspikel², Thanos Halazonetis³, Omar Zgheib ², Simone Sabbioneda¹

¹Istituto di Genetica Molecolare “Luigi Luca Cavalli-Sforza”, CNR, Pavia, Italy; ²Geneva University Hospitals, Division of Genetic Medicine, Geneva, Switzerland; ³University of Geneva, Department of Molecular and Cellular Biology, Geneva, Switzerland

Consortium: The submitted abstract has not yet been presented at a conference, but was recently published in:

Ricciardiello, R., Forleo, G., Cipolla, L. et al. Homozygous substitution of threonine 191 by proline in polymerase η causes Xeroderma pigmentosum variant. Sci Rep 14, 1117 (2024). https://doi.org/10.1038/s41598-023-51120-1

Background/Objectives: DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη function is associated with the human disease Xeroderma pigmentosum variant (XPV). To date, no missense variant has been reported as being pathogenic or likely pathogenic, owing to the lack of functional studies adhering to ACMG guidelines for variant interpretation.

Methods: We hereby report the case of a 60-year-old woman known for XPV and carrying a Polη Thr191Pro variant in homozygosity. We further characterize the variant in vitro by primer extension assays and in vivo by UV survival assays and cell cycle analysis.

Results: We provide evidence that the substitution abrogates polymerase activity and results in UV sensitivity through deficient damage bypass. This is the first functional molecular characterization of a missense variant of Polη, whose reported pathogenic variants have thus far been loss of function truncation or frameshift mutations.

Conclusion: Our work allows the upgrading of Polη Thr191Pro from ‘variant of uncertain significance’ to ‘likely pathogenic mutant’, bearing direct impact on molecular diagnosis and genetic counseling. Furthermore, we have established a robust experimental approach that will allow a precise molecular analysis of further missense mutations possibly linked to XPV. Finally, our work provides insight into critical Polη residues that may be targeted to develop small molecule inhibitors for cancer therapeutics.

Grants: Associazione Italiana per la Ricerca sul Cancro Investigator Grant [S.S.]

MUR PRIN 2017 [S.S.]

MUR/PNRR Next Generation EU PRIN 2022 [S.S.]

NUTRAGE, CNR [S.S.]

Swiss National Science Foundation [T.D.H.]

Conflict of Interest: None declared

P01.158.A CRISPR/Cas9-mediated Generation of reference iPSCs expressing the Philadelphia translocation

Md Faruq Hossain ¹, Lisa Hagenau¹, Lars Jensen¹, Anna-Maria Pielka², Susanne Thümecke², Björn Nowack², Andreas Kuß¹

¹Interfaculty Institute for Genetics and Functional Genomics, Department of Functional Genomics, Greifswald, Germany; ²SensID GmbH, Rostock, Germany

Background/Objectives: Next Generation Sequencing (NGS), particularly RNA-sequencing (RNAseq), has become vital for research and diagnostics. Reliable reference materials are essential for ensuring comparability in results. Here CRISPR/Cas9 technology was used to develop reference cell lines, including a human iPSC line, expressing the well-studied BCR-ABL1 fusion transcript. This fusion arises from a chromosomal translocation involving the ABL and BCR proto-oncogenes.

Methods: CRISPR/Cas9 technology was employed to create a fusion gene in different cell types including human iPSCs, blood and bladder cancer cells by targeting the breakpoint region of two introns associated with the rearrangement. Co-transfection of CRISPR guide RNAs and Cas9 induced double-strand breaks within both genes, leading to DNA misrepair and subsequent gene fusion.

Results: The BCR-ABL1 fusion gene was successfully incorporated into the wildtype human iPSC line MHHi001-A, confirmed by sequencing. Notably, the pluripotency and differentiation capabilities of the genetically modified iPSCs into the three germ layers remained uncompromised with no observed genomic aberrations or off-target modifications. Further cell lines that were modified in this fashion include the Jurkat cell line and human bladder cancer cells with a pre-existing FGFR3-TACC3 fusion on chromosome 4. The latter results in a cell line with two distinct fusion transcripts per cell.

Conclusions: The newly created genetically modified cell lines, particularly the human iPSC featuring the BCR-ABL1 fusion gene, can serve as valuable reference materials for NGS-based RNA analysis. Their utilization significantly improves the precision and reliability of NGS applications, addressing a crucial need in research and diagnostics.

Grants: TBI-V-1-423-VBW-144 (Mecklenburg Vorpommern).

Conflict of Interest: None declared

P01.159.B Systematic review of multiple primary renal tumours (MPRT)

Huairen Zhang ¹, Avgi Andreou¹, Rupesh Bhatt², James Whitworth¹, Bryndis Yngvadottir¹, Eamonn Maher^1;3

¹School of Clinical Medicine, University of Cambridge, Department of Medical Genetics, Cambridge, United Kingdom; ²Queen Elizabeth Hospital Birmingham, Department of Urology, Birmingham, United Kingdom; ³Aston Medical School, Birmingham, United Kingdom

Background: In a subset of patients with renal tumours, multiple primary lesions may occur. Predisposition to multiple primary renal tumours (MPRT) is a well-recognised feature of some inherited renal cancer syndromes. The diagnosis of MPRT should therefore provoke a thorough assessment for clinical and genetic evidence of disorders associated with predisposition to renal tumourigenesis.

Method: To define the clinical and genetic characteristics of MPRT, a systematic literature review was undertaken.

Result: A total of 7562 patients from 448 articles were identified with MPRT. Compared to all patients with renal cell carcinoma (RCC), people with MPRT were more likely to be male (63% versus 71.7%) and have an earlier age at diagnosis (< 46 years, 19% versus 34.5%). In 60.1% of cases MPRT were synchronous, and MPRT lesions had similar histology in 78.9% of individuals enrichment of multiple papillary RCC (16.8%). 10.2% of patients with MPRT had a family history of cancer or were diagnosed with a hereditary RCC associated syndrome with von Hippel Lindau (VHL) disease being the most common one (71.3%), followed by Birt Hogg Dube syndrome (14.6%). Individuals with a known or likely genetic cause were, on average, younger (43.3 years versus 56.7 years). In rare cases intrarenal metastatic RCC can phenocopy MPRT.

Conclusion: Based on these findings we suggest an investigative pathway for individuals with MPRT and approaches to cases in which routine germline genetic testing does not provide a diagnosis.

Grants: VHL alliance UK, NIHR Cambridge Biomedical Research Centre, Cancer Research UK

Conflict of Interest: None declared

P01.160.C Deciphering copy number variations within the complex genomic region of the PMS2 gene using Optical Genome Mapping

Anne Holtorf¹, Jasmin Maier ¹, Wilena Telman¹, Charlotte Singruen¹, Sarah Heinrich¹, Nina Wohlrab¹, Sophie Stoesslein¹, Siegel Corinna¹, Konstanze Hörtnagel¹

¹Medicover Genetics, MVZ Martinsried GmbH, Martinsried, Germany

Background/Objectives: The PMS2 gene is one of four DNA mismatch repair (MMR) genes and is critical for correcting errors after DNA replication and homologous recombination. Inactivation of PMS2 leads to the accumulation of mutations, predisposing carriers to cancer and causing constitutional mismatch repair deficiency syndrome (CMMRDS). However, PMS2 sequence analysis is complicated because the gene is located within a complex genomic region on chromosome 7, which contains a pseudogene with almost 100% sequence homology (PMS2CL). Particularly challenging is the identification of copy number variations (CNVs) within this genomic region, with the added complexity of the inability to distinguish between PMS2 and PMS2CL using conventional laboratory techniques such as MLPA. Here we report on the possibility of using Optical Genome Mapping (OGM) as a new tool for detecting CNVs in this complex region of the genome.

Methods: One colon cancer patient (MSI-H, loss of PMS2) consented to germline testing. Next-generation sequencing was performed, including CNV analysis and additional MLPA. Subsequently, OGM was done to follow up on MLPA results.

Results: MLPA results suggested a deletion on chromosome 7, possibly encompassing PMS2 exons 12-15. However, further investigation using long-range PCR to determine whether this deletion was in PMS2 or its pseudogene remained inconclusive. Fortunately, OGM results revealed a heterozygous deletion of 77 kb including exons 12-15 of the coding PMS2 gene, confirming Lynch syndrome.

Conclusion: OGM is a promising tool for identifying and deciphering structural variants in complex genomic regions, enabling diagnosis for patients and their families.

Grants: -

Conflict of Interest: None declared

P01.161.D DNA methylation patterns in families with rhabdoid tumor predisposition syndrome (RTPS)

Matej Boros ¹, Karolina Nemes^2;3, Sebastian Bühner^2;3, Susanne Bens¹, Nnamdi Okeke¹, Sonja Dahlum¹, Selina Glaser¹, Nadine Matscheko¹, Anja Fischer¹, Britt König¹, Ole Ammerpohl¹, Pascal Johann^2;3, Martin Hasselblatt⁴, Michael C. Frühwald^2;3, Reiner Siebert¹

¹Institute of Human Genetics, Ulm University & Ulm University Medical Center, Ulm, Germany, Ulm, Germany; ²Pediatrics and Adolescent Medicine, Swabian Children’s Cancer Center, University Medical Center Augsburg, Augsburg, Germany; ³Bavarian Cancer Research Center (BZKF), Augsburg, Germany; ⁴Institute of Neuropathology, University Hospital Münster, Münster, Germany

Background/Objectives: Rhabdoid Tumor Predisposition Syndrome (RTPS) is due to germline SMARCB1 or SMARCA4 pathogenic variants. It confers a high risk for malignant rhabdoid tumors in early childhood. Despite a grim prognosis with 5-year overall survival rates of around 40%, the molecular mechanisms underlying mutation origin, tumor penetrance,or unfavorable prognosis are incompletely understood. We investigated familial DNA methylation patterns in RTPS.

Methods: 90 children with RTPS from 16 countries were identified in the EU-RHAB database (01/2004 - 01/2021). We analyzed a subcohort of 49 well-characterized RTPS1 and 2 RTPS2 patients, along with 44 mutation-negative parents and 4 mutation-negative siblings. DNA methylation was assessed in blood DNA using the Infinium MethylationEPIC v1.0 array and analyzed using ANOVA (limma R package) as well as UMAP- and PCA-based clustering.

Results: Comparing patients with parents or siblings group identified 12,238 CpGs (4,662 genes) and 355 CpGs (214 genes) differentially methylated CpGs, respectively, with 128 CpGs and 72 genes shared among these analyses. Only 1 CpG was differently methylated between parent and sibling controls. Sibling samples clustered with RTPS patients and both segregated apart from parents. Age was a predominant driver of variability between the observed clusters whereas sex, type of SMARCB1/SMARCA4 mutation, or tumor DNA methylation subtype were unrelated to the observed clusters.

Conclusion: DNA methylation analyses of patients and relatives did not show a specific blood-born epigenotype of RTPS patients. DNA methylation heterogeneity in the families was strongly confounded by age, emphasizing the need for age-matched control populations.

Grant: Supported by Deutsche Krebshilfe.

Conflict of Interest: Matej Boros: None declared, Karolina Nemes: None declared, Sebastian Bühner: None declared, Susanne Bens: None declared, Nnamdi Okeke: None declared, Sonja Dahlum: None declared, Selina Glaser: None declared, Nadine Matscheko: None declared, Anja Fischer: None declared, Britt König: None declared, Ole Ammerpohl: None declared, Pascal Johann: None declared, Martin Hasselblatt: None declared, Michael C. Frühwald MCF received grant support from the Deutsche Krebshilfe (70113981), Reiner Siebert RS received grant support from the Deutsche Krebshilfe (70114040)

P01.162.A Cancer prognosis and treatment results in patients with PTEN Hamartoma Tumor Syndrome (PHTS) – a European cohort study

Linda Hendricks^1;2;3, Katja Verbeek ^1;2;3, Janneke Schuurs-Hoeijmakers^1;3, Robin de Putter⁴, Hilde Brems⁵, Sien van Daele⁵, Violetta Anastasiadou⁶, Lenka Foretova⁷, Patrick Benusiglio⁸, Anna Gerasimenko^9;10, Chrystelle Colas^11;12, Marie-Charlotte Villy¹¹, Claude Houdayer¹³, Maud Brauchaud¹³, Robert Hüneburg^14;15, Stefan Aretz^14;16, Arne Jahn^17;18;19, Verena Steinke-Lange²⁰, Giovanni Innella^21;22, Daniela Turchetti^21;22, Valeria Barili²³, Maurizio Genuardi^24;25, Arianna Panfili²⁴, Margherita Baldassarri^26;27;28, Arvids Irmejs^29;30, Mirjam de Jong³¹, Thera Links³², Edward Leter³³, Daniëlle Bosch³⁴, Stephany Donze³⁴, Rachel van der Post³⁵, Arjen Mensenkamp^1;2;3, harm westdorp³⁶, Hildegunn Høberg Vetti^37;38, Marianne Tveit Haavind³⁷, Kjersti Jørgensen³⁹, Lovise Maehle³⁹, Siri Briskemyr⁴⁰, Juliette Dupont⁴¹, Ana Blatnik⁴², Judith Balmaña⁴³, Maite Torres⁴³, Joan Brunet⁴⁴, Roser Lleuger-Pujol⁴⁴, Emma Tham^45;46, Marc Tischkowitz⁴⁷, D Gareth Evans⁴⁸, Zerin Hyder⁴⁸, Nicoline Hoogerbrugge^1;2;3, Janet Vos^1;2;3

¹Radboud university medical center, Radboudumc expert center for PHTS, Nijmegen, Netherlands; ²Radboud university medical center, Radboud Institute for Medical Innovation, Nijmegen, Netherlands; ³Radboud university medical center, Human Genetics, Nijmegen, Netherlands; ⁴Ghent University Hospital, Center for Medical Genetics, Ghent, Belgium; ⁵University of Leuven, Human Genetics, Leuven, Belgium; ⁶Archbishop Makarios III Children’s Hospital, Karaiskakio Foundation, Nicosia, Cyprus; ⁷Masaryk Memorial Cancer Institute, Cancer Epidemiology and Genetics, Brno, Czech Republic; ⁸Hôspital Pitié-Salpêtrière, AP-HP, Sorbonne Université, UF d’oncogénétique Clinique, department de Génétique, Paris, France; ⁹APHP Sorbonne Université, GH Pitié Salpêtrière et Trousseau, Département de Génétique, Centre de référence “déficiences intellectuelles de causes rares”, Paris, France; ¹⁰Sorbonne Université, Hôpital de la Pitié Salpêtrière, Institut du Cerveau - Paris Brain Institute - ICM, Inserm, CNRS, Paris, France; ¹¹Institut Curie, Service de Génétique, Paris, France; ¹²Inserm U830, DNA Repair and Uveal Melanoma (D.R.U.M.), Equipe Labellisée Par la Ligue Nationale Contre le Cancer, Paris, France; ¹³Univ Rouen Normandie, Inserm U1245 and CHU Rouen, Department of Genetics, Rouen, France; ¹⁴University Hospital of Bonn, Center for Hereditary Tumor Syndromes, Bonn, Germany; ¹⁵University Hospital Bonn, Department of Internal Medicine I, Bonn, Germany; ¹⁶University of Bonn, Institute of Human Genetics, Medical Faculty, Bonn, Germany; ¹⁷Technische Universität Dresden, Institute for Clinical Genetics, Faculty of Medicine Carl Gustav Carus, Dresden, Germany; ¹⁸Hereditary Cancer Syndrome Center Dresden, ERN-GENTURIS, Dresden, Germany; ¹⁹German Cancer Consortium (DKTK) and National Center for Tumor Diseases (NCT), Dresden, Germany; ²⁰Klinikum der Universität München, Medical Genetics Center, Germany; Arbeitsgruppe Erbliche Gastrointestinale Tumore, Medizinische Klinik und Poliklinik IV - Campus Innenstadt, München, Germany; ²¹University of Bologna, Department of Medical and Surgical Sciences, Center for Studies on Hereditary Cancer, Bologna, Italy; ²²IRCCS Azienda Ospedaliero-Universitaria di Bologna, Unit of Medical Genetics, Bologna, Italy; ²³University of Parma, Medical Genetics, Department of Medicine and Surgery, Parma, Italy; ²⁴Fondazione Policlinico Universitario A. Gemelli IRCCS, Department of Laboratory and Infectious Diseases, Rome, Italy; ²⁵Università Cattolica del Sacro Cuore, Medical Genetics Section, Department of Life Sciences and Public Health, Rome, Italy; ²⁶University of Siena, Medical Genetics, Siena, Italy; ²⁷University of Siena, Med Biotech Hub and Competence Center, Department of Medical Biotechnologies, Siena, Italy; ²⁸Azienda Ospedaliero-Universitaria Senese, Genetica Medica, Siena, Italy; ²⁹Riga Stradins University, Institute of Oncology, Riga, Latvia; ³⁰Pauls Stradins Clinical University Hospital, Breast Unit, Riga, Latvia; ³¹University of Groningen, University Medical Center Groningen, Department of Genetics, Groningen, Netherlands; ³²University of Groningen, University Medical Center Groningen, Department of Endocrinology, Groningen, Netherlands; ³³Maastricht University Medical Center, Department of Clinical Genetics, Maastricht, Netherlands; ³⁴Erasmus MC Rotterdam, Department of Clinical Genetics, Rotterdam, Netherlands; ³⁵Radboud university medical center, Department of Pathology, Nijmegen, Netherlands; ³⁶Radboud university medical center, Department of Medical Oncology, Nijmegen, Netherlands; ³⁷Haukeland University Hospital, Western Norway Familial Cancer Center, Department of Medical Genetics, Bergen, Norway; ³⁸VID Specialized University, Faculty of Health Studies, Bergen, Norway; ³⁹Oslo University Hospital, Department of Medical Genetics, Oslo, Norway; ⁴⁰University Hospital of North Norway, Department of Medical Genetics, Tromsø, Norway; ⁴¹Centro Hospitalar Universitário Lisboa Norte, Lisbon, Portugal; ⁴²Institute of Oncology Ljubljana, Department of Clinical Cancer Genetics, Ljubljana, Slovenia; ⁴³Vall d’Hebron Hospital Universitari, Medical Oncology Department, Barcelona, Spain; ⁴⁴Catalan Institute of Oncology, IDIBELL-IDIBGI, Hereditary Cancer Program, Barcelona-Girona, Spain; ⁴⁵Karolinska University Hospital, Department of Clinical Genetics, Stockholm, Sweden; ⁴⁶Karolinska Institutet, Department of Molecular Medicine and Surgery, Stockholm, Sweden; ⁴⁷University of Cambridge, Department of Medical Genetics, National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge, United Kingdom; ⁴⁸University of Manchester, Manchester Centre for Genomic Medicine, St Mary’s Hospital, Division of Evolution and Genomic Sciences, School of Biological Sciences, Manchester, United Kingdom

Background/Objectives: PTEN Hamartoma Tumor Syndrome (PHTS) patients have hereditary cancer risks reaching 76% for breast cancer (BC), comparable to BRCA1/2, 22% for endometrial cancer (EC), and 21% for thyroid cancer (TC). Cancer prognosis in PHTS is unknown, and we aimed to provide this for PHTS-associated cancers.

Methods: This European cohort study included adult PHTS patients with data from medical files, registries, and/or questionnaires. Survival and risk factors were assessed using Kaplan-Meier and Cox regression analyses and were compared with sporadic cancer and population mortality using standardized mortality (SMR) and relative survival rates (RSR). Survival bias was addressed using left-truncation.

Results: Overall, 147 female BC patients were included (73% PHTS-indexes), median age of 41 years(IQR:35-49). The 10y-OS was 77%(95%CI:66-90), decreasing per increasing stages 0-IV: 90%(95%CI:73-100), 83%(95%CI:64-100), 74%(95%CI:48-100), 51%(95%CI:26-100) and 0%(95%CI:0-0). Relative survival after BC was comparable to sporadic BC until 2 years (2y-RSR = 1.1;95%CI = 1.1-1.1), gradually increasing to 5y-RSR = 1.7(95%CI = 1.6-1.7). For TC (N = 56, median age of 37(IQR:26-46)) and EC patients (N = 35, median age of 46(IQR:36-55)), the 10y-OS was 87%(95%CI:74-100) and 64%(95%CI:38-100). Overall and cancer-specific mortality in female patients was increased compared to the general population (SMR = 3.7;95%CI:2.6-5.0 and SMR = 2.7;95%CI:1.6-4.4).

Conclusion: This study showed comparable cancer prognosis in PHTS to the general population. The increased overall mortality in the total PHTS population was presumably related to higher cancer incidence. These results, together with the high survival observed in early-stage cancer, emphasize the importance of early PHTS recognition for the potential benefit of early cancer detection in patients with high hereditary cancer risks.

Grants: PTEN Research Foundation

Conflict of Interest: Linda Hendricks: None declared, Katja Verbeek: None declared, Janneke Schuurs-Hoeijmakers: None declared, Robin de Putter: None declared, Hilde Brems: None declared, Sien van Daele: None declared, Violetta Anastasiadou: None declared, Lenka Foretova: None declared, Patrick Benusiglio: None declared, Anna Gerasimenko: None declared, Chrystelle Colas: None declared, Marie-Charlotte Villy: None declared, Claude Houdayer: None declared, Maud Brauchaud: None declared, Robert Hüneburg: None declared, Stefan Aretz: None declared, Arne Jahn: None declared, Verena Steinke-Lange: None declared, Giovanni Innella: None declared, Daniela Turchetti: None declared, Valeria Barili: None declared, Maurizio Genuardi: None declared, Arianna Panfili: None declared, Margherita Baldassarri: None declared, Arvids Irmejs: None declared, Mirjam de Jong: None declared, Thera Links: None declared, Edward Leter: None declared, Daniëlle Bosch: None declared, Stephany Donze: None declared, Rachel van der Post: None declared, Arjen Mensenkamp AstraZeneca, harm westdorp: None declared, Hildegunn Høberg Vetti: None declared, Marianne Tveit Haavind: None declared, Kjersti Jørgensen: None declared, Lovise Maehle: None declared, Siri Briskemyr: None declared, Juliette Dupont: None declared, Ana Blatnik: None declared, Judith Balmaña: None declared, Maite Torres: None declared, Joan Brunet GlaxoSmithKline, Roser Lleuger-Pujol: None declared, Emma Tham: None declared, Marc Tischkowitz: None declared, D Gareth Evans: None declared, Zerin Hyder: None declared, Nicoline Hoogerbrugge: None declared, Janet Vos: None declared

P01.163.B Deciphering the molecular complexity of the IKZF1plus profile using Optical Genome Mapping

Jonathan Lühmann ¹, Winfried Hofmann¹, Anke Katharina Bergmann¹, Anja Möricke², Gunnar Cario², Martin Schrappe², Brigitte Schlegelberger¹, Martin Zimmermann³, Martin Stanulla³, Doris Steinemann¹

¹Hannover Medical School, Department of Human Genetics, Hannover, Germany; ²ALL-BFM Study Group, Christian-Albrechts University Kiel and University Medical Center Schleswig-Holstein, Department of Pediatrics, Kiel, Germany; ³Hannover Medical School, Pediatric Hematology and Oncology, Hannover, Germany

Background/Objectives: Acute lymphoblastic leukemia (ALL), the most frequent pediatric cancer, has witnessed improved survival rates exceeding 90% through genetic marker-based stratification and novel therapies. The IKZF1^plus profile, characterized by IKZF1 deletion (IKZF1^del) alongside additional deletions, emerged as a poor prognostic marker in retrospective analyses of AIEOP-BFM ALL 2000/2009 trials and is used for high-risk stratification in the ongoing AIEOP-BFM ALL 2017 trial. A deeper genome-wide characterization of leukemic cells is necessary for a better understanding of the molecular response.

Methods: In total, 142 patients with IKZF1 deletion from the AIEOP-BFM ALL 2000/2009 trial were re-analyzed by means of Optical Genome Mapping (OGM) enabling the simultaneous detection of structural variants and aneuploidies. Genomic profiles were correlated with patient outcome with respect to event-free survival (EFS), cumulative incidence of relapse (CIR) and overall survival (OS).

Results: About half of the patients in this study exhibited well-known and new genomic alterations in addition to IKZF1^del/plus. In 53 patients gene fusions (ABL-class, CRLF2, PAX5, JAK2, ZNF384) have been detected. Eighteen patients displayed established prognostic markers namely high hyperdiploidy, ETV6::RUNX1, or iAMP21. Our data demonstrate the genomic complexity and heterogeneity of the IKZF1^del/plus patient group. In this cohort EFS, CIR and OS are similar in IKZF1^del and IKZF1^plus patients and mainly dependent on the accompanying good or poor prognostic markers.

Conclusion: Through the utilization of OGM, we reveal the complex genomic landscape in ALL and demonstrate that the significance of the IKZF1^plus profile is not as prominent as previously anticipated.

Grants: Deutsche Kinderkrebsstiftung (German Childhood Cancer Foundation)

Conflict of Interest: None declared

P01.164.C Functional assessment of variants of uncertain significance in PTEN with a predicted effect on splicing

Elke van Veen ¹, Arjen Mensenkamp¹, Janneke Schuurs-Hoeijmakers¹, Nicoline Hoogerbrugge¹, Richarda de Voer¹

¹Radboud University Medical Center, Department of Human Genetics, Nijmegen, Netherlands

Background: Pathogenic variants in PTEN are associated with PTEN hamartoma tumour syndrome (PHTS). However, in a number of patients variants of uncertain significance (VUSs) are identified. To assign these VUSs a meaningful pathogenicity classification, the functional effect of these variants needs to be assessed. Here, we selected PTEN VUSs identified in PHTS-like patients with a predicted effect on splicing to assess their functional effect.

Methods: VUSs were identified in an ERN-GENTURIS PHTS cohort (n = 510). All variants were classified as a VUS using the PTEN-specific ACMG guidelines and had a spliceAI score of >0.1, with no published functional data to date were included. Amplicons spanning one or two exons that harboured the identified VUSs were designed and cloned into a minigene vector using gateway cloning. Site-directed mutagenesis was used to generate the variants of interest and splice assays with mutant and wild type constructs were performed.

Results: Eight PTEN VUSs were identified for further functional testing. We identified five missense variants (c.77C>A, p.(Thr26Asn); c.89C>A, p.(Pro30Gln); c.94A>T, p.(Ile32Phe); c.801G>T, p.(Lys267Asn); c.929A>G, p.(Asp310Gly)) and three intronic variants (c.80-1G>C; c.165-18T>A; c.253+5G>C). One additional variant (c.334C>G, p.(Leu112Val)) was included in the assay as a positive control. These nine variants were distributed over seven exon-intron regions, captured in five amplicons ranging between ~2.5-5.2 kb in size.

Conclusion: Upon assessment, coding variants may also exert an effect on splicing and thus impact the function of PTEN. Splice effect analysis will help to further classify the pathogenicity of PTEN VUSs.

Grants: This work is funded by HORIZON-MSCA-2021-PF-01.

Conflict of Interest: None declared

P01.165.D Complex translocation leading to13q interstitial deletion in a Moroccan child with retinoblastoma and intellectual disability

ZHOUR EL AMRANI ¹, siham chafai elalaouia², Wafaa Jdioui³, aziza sbiti⁴, ilham ratbi⁵, Thomas Liehr⁶, abdelaziz sefiani⁷, Abdelhafid Natiq⁸

¹Research team in genomics and molecular epidemiology of genetic diseases, Genomics Center of Human Pathologies, Faculty of Medicine and Pharmacy, Medical Genetics, RABAT, Morocco; ²Faculty Of Medicine And Pharmacy Of Rabat, Medical Genetics, Rabat, Morocco; ³Centre des consultations et des explorations externes, Hopital d’enfants, CHU Ibn SinaConsultation de génétique, Rabat, Medical Genetics, Morocco; ⁴National Institute D’hygiène Du Maroc, Medical Genetics, Rabat, Morocco; ⁵Faculty Of Medicine And Pharmacy Of Rabat, Rabat, Morocco; ⁶Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, Germany; ⁷National Institute D’hygiène, Medical Genetics, Morocco; ⁸Faculty Of Medicine And Pharmacy Of Rabat, Medical Genetics, Morocco

Background/Objectives: Retinoblastoma (RB) is the most common malignant intraocular tumor in children; it affects their eyes often even prenatally. RB may be sporadic or familial, due to germinal mutation in RB1 gene or by abnormal chromosomal abnormalities involving RB1 gene, located in 13q14. Monosomy of subband 13q14 as a partial deletion can also be responsible for RB with additional symptoms. The latter may be RB associated with psychomotor retardation, macrocephaly, broad forehead, thick earlobes, and bulbous nose.

Methods: We present here the case of a boy from a consanguineous marriage with bilateral retinoblastoma, intellectual disability and facial dysmorphic features. Classical and molecular cytogenetics(FISH) were used to recognize genotype-phenotype association.

Results: The karyotype showed a three way translocation involving chromosomes 5, 12 and 13. Further molecular cytogenetics analysis revealed a deletion of 13q14 involving the tumor suppressor gene RB1.

Conclusion: This case highlights the impact of classical and molecular cytogenetics in diagnosis of rare genetic syndromes and for the genetic counselling of patients and their families. Pure molecular karyotyping analyses would miss the underlying chromosomal mechanism leading to the rearrangement.

Grants: this work does not receive any funding, and I would like to apply for a fellowship to attend the European Human Genetics Conference.

Conflict of Interest: None declared

P01.166.A Deep intronic insertions in patients at high risk for hereditary breast and ovarian cancer

Bernardus Aldrige Allister¹, Winfried Hofmann ¹, Jonathan Lühmann¹, Bernd Auber¹, Nataliya Di Donato¹, Monika M. Golas^2;2;3, Doris Steinemann¹

¹Hannover medical school (MHH), Department of Human Genetics, Hannover, Germany; ²University of Augsburg, Human Genetics, Faculty of Medicine, Augsburg, Germany; ³University Hospital Augsburg, Comprehensive Cancer Center Augsburg, Augsburg, Germany

Background/Objectives: 13 core genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, PALB2, PTEN, RAD51C, RAD51D, STK11, and TP53) are routinely tested in patients with the suspicion of the hereditary breast and ovarian cancer (HBOC). Non-coding variants such as Alu-elements, the most prevalent mobile-element insertion (MEI) in the human genome are increasingly recognized as a cause of the genetic disorders. However, their relevance in HBOC predisposition may be underestimated.

Methods: We perform whole genome and whole transcriptome sequencing on 134 high risk HBOC patients (Asian:9, European:125), who are tested negative for pathogenic variants in HBOC core genes. The data was analyzed using an in-house-NGS-megSAP-pipeline coupled with the MEI detection tool Mobster.

Results: We detected eight deep intronic Alu-element insertions in eight different European patients: 7 in BRCA2, 1 in PTEN. The Alu-element insertion in BRCA2 intron 13 (NC_000013.11:g.32348295_32348296insAluYa5) was annotated in seven unrelated patients by Mobster. This Alu-element insertion is present within the International Genome Sample Resource with different population frequencies: European:4/628 = 0,64%, Asian:56/1172 = 4,78%, African:12/885 = 1,36%, American:13/485 = 2,68%. cDNA analysis, showed that this AluYa5 insertion causes BRCA2 in-frame exon 12 skipping.

Conclusion: High population frequency of the insertion suggests that the possible hypomorphic-effect of exon 12 deletion requires additional analysis of its pathogenicity. However, our findings suggested that deep intronic Alu-element insertion could play a role in causing disease in unresolved cases of HBOC.

Grants: Deutsche Forschungsgemeinschaft (DFG)

Conflict of Interest: None declared

P02.001.C Heterozygote missense variations in PROKR2 present partial penetrance for causing Hypogonadotropic Hypogonadism

Florence Abou ^1;2, moran gershoni³, Netanel Waldenberg⁴, Naama Steiner⁵, Noga Fuchs Weizman⁶, Shimi Barda⁶, Sandra Kleiman⁶, Shlomi Barak^4;7, Ruti Parvari^1;2

¹Ben-Gurion University of the Negev, National Institute of Biotechnology in the Negev, Be’er sheva, Israel; ²Ben-Gurion University of the Negev, The Shraga Segal Department of Microbiology, Immunology and Genetics Faculty of Health Sciences, Be’er sheva, Israel; ³Institute of Animal Science, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel; ⁴The Multidisciplinary Center for Female and Male Infertility, Male fertility, Tel Aviv, Israel; ⁵Soroka University Medical Center, Fertility and IVF Unit, Department of Obstetrics and Gynecology, Be’er Sheva, Israel; ⁶, Male Fertility Clinic and Sperm Bank, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, affiliated with the Sackler Faculty of Medicine, Tel Aviv, Israel; ⁷Assuta Ashdod University Hospital, Reproductive Services, Ashdod, Israel

Background: Hypogonadotropic hypogonadism (HH), with a prevalence of 1–10 per 100,000 individuals, is characterized by reduced function of the gonads due to insufficient stimulation by the gonadotropin-releasing hormone (GnRH) from the hypothalamus. Kallman syndrome is a form of HH characterized by delayed or absent puberty and an impaired sense of smell.

Heterozygote mutations in the PROKR2 (prokineticin receptor-2) gene were associated with HH. PROKR2 and its ligand PROK2 have a role in the cellular migration of both GnRH neurons and the Olfactory interneurons.

Objective: Identify the genetic causes of infertility in males.

Methods: Whole Exome Sequencing of peripheral blood DNA and bioinformatic analysis.

Results: In one infertile man not presenting HH, we identified a previously reported heterozygote variant in PROKR2 (p.L173R) associated with Kallman syndrome, which was suggested to be a founder mutation. The variation occurs in up to 0.5% in various control populations (gnomAD). In a cohort of 83 individuals, none present with HH, we identified two additional missense variations (p.R286C and p.R85C) reported in the literature, each in a different infertile patient. The population frequencies of these variants are up to 4.6% and 0.27% respectively in specific control populations. Overall, 23 variations in PROKR2 were reported to be associated with Kallman syndrome or HH, and six additional pathogenic variations appear in Clinvar. Most are more common in the population than the prevalence of HH.

Conclusion: Clinical manifestations of p.L173R, p.R286C, and p.R85C, together with other reported variations in PROKR2 suggest complex inheritance and low penetrance in the disease phenotype.

Grants: ISF 2844/21.

Conflict of Interest: None declared

P02.002.D Detection and interpretation of mosaicism in PGT-A programs

Julia Gontar¹, Nadiya Kazachkova¹, Nataliia Buderatska², Ihor Ilyin³, Olga Parnytska², Yuriy Herevych³, Eduard Kapustin³, Sergiy Lavrynenko², Yana Lakhno¹, Olena Fedota ⁴

¹LLC “Medical Center IGR”, Diagnostic Laboratory, Kyiv, Ukraine; ²LLC “Medical Center IGR”, Embriologic Laboratory, Kyiv, Ukraine; ³LLC “Medical Center IGR”, IVF Deparment, Kyiv, Ukraine; ⁴LLC “AMS”, Research Department, Kharkiv, Ukraine

Background/Objectives: Mosaicism interpretation is a PGT-A problem. Сomprehensive chromosome screening methods differ in the results information. Genetic analysis methodology depends on the tasks, economic expediency and family features. The study of approaches to optimizing mosaicism detection in PGT-A is relevant.

Methods: The study involved 3382 embryos samples obtained from 668 women (33.7 ± 5.4 years) treated with IVF/ICSI cycles. For PGT-A a trophectoderm biopsy was performed on the post-fertilization hours 120 or 144. 1134 samples were diagnosed by NGS, 2248 samples - using FISH in chromosomes 9, 13, 15, 16, 17, 18, 21, 22, X, Y, 70 samples - by both methods.

Results: According to NGS or FISH, variants were established: norma – 43.7% and 43.5% (p > 0.05), aneuploidy – 53.7% and 26.4%, mosaicism – 1.7% and 25.8%, polyploidy – 0 and 4.3%. Thus, the proportions of embryos with abnormalities show a difference (p < 0.001). After FISH-detected mosaicism, such combinations were established: diploidy/polyploidy – 13,4%, disomy/monosomy/trisomy – 1,6%, trisomy/monosomy – 1,2% on the same chromosome. These mosaicisms cannot be identified by NGS, but may have clinical implications. Disomy/monosomy and disomy/trisomy were detected by NGS – 1,1% and 0,6% and FISH – 5,7% and 4,0%.

Using both NGS/FISH, the same result was obtained for 78.6% of embryos: true match – 64.3%, N/N – 45.7%, An/An – 18.6%; formal – aneuploidy on different chromosomes, An/An – 7.1%, various combinations – An/mos 4.3%, An or mos/An+mos – 2.9%. 21.4% of samples were different: An/N – 8.6%, N/An – 1.4%, N/mos – 4.3%, N/mos or An – 5.7%, mos or An/N – 1.4%.

Conclusion: Optimizing of PGT-A, especially for families with biological limitations, may require a combination of several methods.

Grants: Absent

Conflict of Interest: None declared

P02.003.A Characterisation of novel variants for endometriosis risk identified in participants within the genomics England database

Elpida Fragouli¹, Amelia Warren ¹, Demetra Andreou¹, Anna Mantzouratou¹

¹Bournemouth University, Life and Environmental Sciences, Bournemouth, United Kingdom

Background/Objectives: Endometriosis, altough multifactorial, tends to affect women of the same family with heritability between 47-51%. Studies have identified 40+ single nucleotide polymorphisms (SNPs) mostly associated with advanced endometriosis. To date no variants have been associated with earlier endometriosis stages. Genomic England database (GED) cantains whole genomes information and medical records from thousands of participants. The aim of this is to identify endometriosis risk variants in selected populations within GED and investigate the pathways these variants could act on.

Methods: Previous studies identified genomic areas associated with an increased endometriosis risk. Of these, 5 genes were selected for furher invesigation, IDO1, IL6, CNR1, TACR3 and KISS1R. Participant data with endometriosis were extractred from selected participant panels, filtered according to specific inclusion criteria and classified to stages according to the degree of medical interventions presented in the clinical data. A database was created and populated with targeted genomic similarity queries within our participant cohorts.

Results: Fifty-seven patients were identified as appropriate candidates for the study. Deferential variant frequency in the GED endometriosis cohort was seen in 10 SNPs compared with that in the general GED population. Two variants were significanly increased in frequency in the endometriosis cohort and assosciatied with IDO1 and CNR1 genes. Both variants are not yet characterised, one is novel, and not previously associated with the condittion.

Conclusion: Targeted investigations in GED identified new variations for IDO1 and CNR1 genes with significantly increased frequency in endometriosis patients. Their further characterisation could reveal novel pathways about the onset of endometriosis.

Grants: Bournemouth University

Conflict of Interest: None declared

P02.004.B The “Goldilocks” panel: Determining the optimal number of genes to include for equitable, pan-ancestry Carrier Screening.

Mia Gruzin ^1;2, Matthew Hobbs¹, Sarah Poll³, Jaysen Knezovich⁴, Swaroop Aradhya³, Leslie Burnett^1;2;5

¹Garvan Institute of Medical Research, Darlinghurst, Australia; ²School of Clinical Medicine, UNSW Medicine and Health, St Vincent’s Clinical Healthcare Campus, Darlinghurst, Australia; ³Invitae Corporation, San Francisco, United States; ⁴Blue Jay Consulting Services, Brisbane, Australia; ⁵Northern Clinical School, Faculty of Medicine and Health, University of Sydney, St Leonards, Australia

Background/Objectives: Clinical utility of reproductive carrier screening varies across diverse populations because of the preponderance of genomic data from European individuals in public databases, and the heterogeneity of available carrier tests. We have defined the size and composition of an optimal (“Goldilocks”) carrier panel with clinical utility across diverse populations, regardless of ancestry.

Methods: Using gnomAD (v4.0) and ClinVar data for ~1,300 serious, childhood-onset autosomal recessive (AR) and X-linked (XL) monogenic conditions, we modelled various in silico screening panels, validating with real-world data from >380,000 individuals. To compare targeted “hotspot” variant analysis against full gene sequencing, we studied differences in carrier yield between specific ancestry groups for CFTR.

Results: A gene panel of ~500 prioritised genes (“Goldilocks”) achieved 99% of maximum possible carrier yield, irrespective of genetic ancestry. Smaller panels provided diminished carrier yield and inequitable benefit to specific ancestry groups. Real-world carrier screening data from >75 countries validated our model. Using CFTR as an example, compared to full gene sequencing, “hotspot” analysis provided unequal yield between specific ancestry groups, missing >10% of the most prevalent pathogenic variants for patients with self-reported Asian, Hispanic, and Middle Eastern ancestry.

Conclusion: An optimally-sized, globally equitable, pan-ancestry Goldilocks panel will identify carrier risk for 99% of most-severe AR/XL conditions. This panel may be analysed virtually from exome or genome sequencing, allowing for ongoing refinement based on emerging evidence. To maximise clinical utility across diverse ancestry groups, full gene sequencing approaches are preferred over targeted “hotspot” variant analyses.

Grants: Not applicable.

Conflict of Interest: Mia Gruzin Former contractor at Invitae Australia., Matthew Hobbs: None declared, Sarah Poll Shareholder at Invitae (NVTA)., Current employee at Invitae Corp., Jaysen Knezovich Former employee at Invitae Australia., Swaroop Aradhya Shareholder at Invitae (NVTA)., Current employee at Invitae Corp., Leslie Burnett Shareholder at Invitae (NVTA)., Former employee of Invitae Australia., 1. Member, Community Genetics Advisory Board, Wolper Jewish Hospital - Honorary; 2. Consultant, Virtus Health Specialist Diagnostics; 3. Consultant, 23 Strands.

P02.005.C Copy number variation sequencing for the products of conception: what is the optimal testing strategy

Shuyuan Li ¹, yiyao chen¹

¹Hengshan Road, Shanghai, China

Background/Objectives: Copy number variation sequencing (CNV-seq) is crucial in prenatal diagnosis, but its limitations in detecting polyploidy, maternal cell contamination (MCC), and uniparental disomy (UPD) restrict its application in the analysis of products of conception (POCs). This study aimed to investigate an optimal genetic testing strategy for POCs in the era of CNV-seq.

Methods: CNV-seq and quantitative fluorescent polymerase chain reaction (QF-PCR) were performed in all 4,211 spontaneous miscarriage cases. Different testing strategies were compared and the optimal testing strategies were proposed.

Results: Of the 4211 cases, 2561 (60.82%) exhibited clinically significant chromosomal abnormalities. CNV-seq alone, without QF-PCR, might misdiagnose 311 (7.39%) cases, including 278 polyploidy, 13 UPD, and 20 MCC. In 20 MCC cases identified by QF-PCR, CNV-seq successfully pinpointed the cause of miscarriage in 13 cases. Furthermore, in cases where QF-PCR suggested polyploidy, CNV-seq improved the diagnostic accuracy in 54 (1.28%) hypo/hypertriploidy cases. After comparing four different strategies, the sequential approach (initiating with CNV-seq followed by QF-PCR if necessary) emerged as advantageous, reducing approximately 70% of the cost associated with QF-PCR while maintaining result accuracy.

Conclusion: We propose an initial CNV-seq followed by QF-PCR if needed—an efficient and cost-effective strategy for the genetic analysis of POCs.

Grants: This work was supported by the Shanghai Municipal Science and Technology Commission (22Y11906700) and Clinical Research Plan of IPMCH (IPMCH2022CR1-02).

Conflict of Interest: None declared

P02.007.A A homozygous HIPK4 variant causes a familial form of male infertility

Sophie Adina Koser ¹, Avinash S. Gaikwad¹, Andela Kovacevic², Isabella Aprea³, Claudia Krallmann⁴, Birgit Stallmeyer¹, Johanna Raidt³, Heymut Omran³, Sabine Kliesch⁴, Hubert Schorle², Corinna Friedrich¹, Frank Tüttelmann¹

¹Institute of Reproductive Genetics (IRG), University of Münster, Münster, Germany; ²Institute of Pathology, Department of Developmental Pathology, University of Bonn, Bonn, Germany; ³Department of General Paediatrics, University of Münster, Münster, Germany; ⁴Centre of Reproductive Medicine and Andrology (CeRA), Department of Clinical and Surgical Andrology, University of Münster, Münster, Germany

Background/Objectives: Oligoasthenoteratozoospermia (OAT) is a highly variable phenotype of male infertility that is characterised by reduced sperm count and motility, and abnormal sperm morphology. Its genetic basis is not well understood. Exome sequencing in familial cases offers the opportunity to discover novel candidate genes also for this infertility phenotype.

Methods: Four infertile brothers with varying OAT were recruited at the CeRA. Exome sequencing including the parents and a fertile brother enabled segregation analysis expecting a shared autosomal recessive or X-linked cause. Candidate variants were validated by Sanger sequencing and further analysed via heterologous expression in HEK293T cells.

Results: Three infertile brothers were homozygous for a start loss variant (c.1A>G) in HIPK4, which is highly expressed in spermatids. Heterologous expression demonstrated that a downstream start codon in frame was used, likely resulting in an N-terminally truncated protein disrupting a part of HIPK4’s kinase domain. Interestingly, the fourth brother carried compound heterozygous variants in DNAH17 (c.[1076_1077dup];[7752 + 2T>A]) known to cause multiple morphological abnormalities of the sperm flagella (MMAF).

Conclusion: In contrast to previous studies reporting HIPK4 as associated with azoospermia, we identify HIPK4 as likely cause for familial OAT. Importantly, published male knockout mice mimic the patients’ OAT phenotype with predominantly abnormal sperm heads in mice and men. Our work highlights the utility of exome-based segregation analysis and illustrates the possibility of different monogenic causes within the same family.

Grants: This work was supported by the DFG Clinical Research Unit 326 ‘Male Germ Cells’ (CRU326) and the Medical Faculty Münster’s ‘CareerS’ programme.

Conflict of Interest: None declared

P02.008.B Towards a diagnostic gene panel for impaired sperm morphology and motility causing male infertility

Clara Barkhaus ¹, Johanna Kuß¹, Birgit Stallmeyer¹, Frank Tüttelmann¹

¹Institute of Reproductive Genetics (IRG), University of Münster, Münster, Germany

Background/Objectives: Male infertility is a clinically and genetically extremely heterogeneous disease affecting approximately 7% of men. Morphological defects of spermatozoa (teratozoospermia), often associated with impaired sperm motility (asthenozoospermia), are a common clinical phenotype. Several candidate genes have been described to cause the disorder. To establish a diagnostic gene panel for affected men, it is necessary to separate validated disease genes from genes with limited evidence.

Methods: To identify candidate genes for impaired sperm motility and morphology, we performed a Pubmed search using the terms, ‘asthenozoospermia’, ‘teratozoospermia’, ‘multiple abnormalities of the sperm flagella’ and ‘disease gene’, followed by a standardised evaluation of all described candidate genes based on the ClinGen gene-disease clinical validity curation.

Results: We identified 128 genes in which genetic variants were associated with teratozoospermia, asthenozoospermia or a combination of both. Of these, 50 genes were reported with autosomal recessive inheritance. For 41 genes, a gene-disease relationship was described in at least 2 independent reports and/or for more than 3 different variants. According to the ClinGen framework, more than 30 of these genes reached sufficient evidence to be included in a diagnostic gene panel.

Conclusion: Male infertility due to impaired sperm motility and/or morphology is a common and heterogeneous disease. Analysis of genes with a validated gene-disease correlation should be implemented in the diagnostic workflow of affected men.

Grants: This work was supported by the DFG Clinical Research Unit 326 ‘Male Germ Cells’ (CRU326).

Conflict of Interest: None declared

P02.010.D Expanded carrier screening for monogenic disorders as part of the preconception care

Martina Hajduskova ¹, Martina Hruba¹, Lucie Solcova¹, Anna Rykovska¹, Monika Pittrova¹, Petr Losan¹

¹Genetika Plzen, s.r.o., Laboratory of reproductive genetics, Plzeň

Background/Objectives: Clinical genetics aims to minimize the risk of inheriting monogenic recessive disorders (MRDs). This is achieved by screening for multiple MRDs and identifying asymptomatic carriers in a population. Such pan-ethnic expanded carrier screening (ECS) with follow-up care may improve preventive preconception measures.

Methods: A new ECS at Genetika Plzen targets 128 autosomal and 23 X-linked MRDs (103 genes), reflecting the international guidelines (ESHG, ACOG, ACMG). Our ECS employs an NGS panel with a hybridization enrichment using custom probes (Agilent Technologies), the MiSeq V3 Platform (Illumina), and the Varsome Clinical software (Saphetor SA). For all genes, the entire coding sequence including UTRs and exon-intron junctions are examined, both SNVs and CNVs are assessed. Only variants of pathogenicity classes 4 and 5 are reported.

Results: During 2021-2023, we screened 406 subjects, 198 couples and 10 individuals, mostly living in the Czech Republic. 238 individuals (59%) carried at least one class-4/-5 gene variant. We identified 16 couples (8%) with increased reproductive risk, i.e., either the female partner (for X-linked MRDs) or both partners (autosomal MRDs) carrying a clinically significant variant in the affected gene. As follow-up care, they were offered preimplantation/prenatal diagnostics.

Conclusion: Within preconception care, we have successfully established ECS to significantly reduce the risk of having a child suffering from a severe MRD (the ECS detection rate of up to 99% is achieved for most genes). Our results show that ECS detects significant percentage of couples at reproductive risk even in a population without high prevalence of particular MRDs.

Conflict of Interest: None declared

P02.011.A Association of IL-6 polymorphism -174G>C with the Premature Ovarian Insufficiency in Bulgarian patients

Ivelina Oprova¹, Eliana Dimova ², Kalina Belemezova², Mariela Hristova-Savova², Petya Andreeva¹, Ivanka Dimova³

¹Shterev Hospital, Department of Infertility and IVF, Sofia, Bulgaria; ²Shterev Hospital, Department of Medical genetics, Sofia, Bulgaria; ³Medical University Sofia, Department of Medical genetics, Sofia, Bulgaria

Background/Objectives: Primary premature ovarian insufficiency (POI) represents the premature exhaustion of the ovarian reserve in women under the age of 40. Interleukin-6 is implicated in the pathophysiological characteristics associated with this condition. We explored the frequency in IL6 gene polymorphism -174 G>C among patients with POI, compared to non-selected European individuals (controls), with the aim of establishing a correlation between this genetic factor and the disease.

Methods: Twenty-four patients with POI (median age of 33.5 years) were subjected to our survey. IL 6 gene polymorphism -174 G>C was examined by Real-Time PCR Genotyping kit (DNA Technology LLC). The allele and genotype frequencies were compared to the data from 503 controls (data collected from ensembl.org). For the purpose of our study, we used the Chi-Square Test (p < 0.05 was considered significant).

Results: The genotype frequencies observed among the 24 cases and 503 controls were as follows: G/G - 62.5 % and 36 %, G/C - 33.3 % and 44,9 %, and C/C - 4.2 % and 19,1 %. The G and C allele frequencies were 79.2 % (cases) and 58,4 % (controls), and 20.8 % and 41,6 %, respectively. The genotype G/G and G allele distribution revealed significant differences between POI patients and controls (p values < 0.05).

Conclusion: We discovered an association of IL 6 –174 G/G genotype and G allele with POI in Bulgarian patients. IL-6 is involved in inflammation and apoptosis and dysregulation of these processes could contribute to the accelerated loss of ovarian follicles seen in POI.

Conflict of Interest: None declared

P02.012.B Fractonation of high-quality spermatozoa promotes peripherial positioning of sex chromosomes

Zuzanna Graczyk ¹, Jagoda Kostyk¹, Julia Pospieszna^1;2, Zuzanna Myślicka¹, Marzena Kamieniczna¹, Marta Olszewska¹, Maciej Kurpisz¹

¹Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland; ²Poznan University of Medical Sciences, Department of Toxicology, Collegium Pharmaceuticum, Poznan, Poland

Background/Objectives: Sperm chromosomes are non-randomly organized in three-dimensional nuclear space. This nuclear organization is one of the epigenetic mechanisms that can regulate genome functioning. After fertilization, chromosomes undergo chromatin remodeling, transcriptional activation and replication.

The aim of this study was to determine whether selection of spermatozoa with good motility or proper chromatin density, defines specific positioning of chromosomes.

Methods: Semen samples from 5 normozoospermic males were collected and processed for fractionation via swim up (to select viable and motile spermatozoa) or Percoll gradient (to select sperm with properly packed chromatin). Fluorescence in situ hybridization (FISH) was applied to analyse the positioning of chromosomes 4, 7, 8, 9, 18, X and Y. At least n ≥ 100 of spermatozoa per subpopulation/chromosome were analysed (Leica DM5500 microscope, filter set: DAPI, SpG, SpO, SpAq, 630x magnification, motorized stage, LASX Navigator software).

Results: In spermatozoa with good motility or proper chromatin density, chromosome 4 moves towards the acrosome, compared to non-fractionated sperm. In motile sperm fraction chromosomes 18, X, and Y shift to the nuclear periphery, while in sperm with condensed chromatin, chromosomes 8, 9, and Y move to the nuclear periphery, compared to non-fractionated sperm.

Conclusion: High-quality sperm selectioning promotes repositioning of sex chromosomes towards nuclear periphery, which is the first region that interacts with the ooplasm during fertilization. Then, sex chromosomes must undergo remodelling of chromatin due to Meiotic Sex Chromosome Inactivation, which is crucial in the early stages of embryo development.

Grants: National Science Centre 2020/38/E/NZ2/00134

Conflict of Interest: None declared

P02.013.C Genetic and functional evidence for TDRD6 as male infertility-associated gene

Farah Ghieh ¹, Erik Schüftan¹, Sabine Kliesch², Frank Tüttelmann¹, Birgit Stallmeyer¹

¹Institute of Reproductive Genetics (IRG), University of Münster, Münster, Germany; ²Center of Reproductive Medicine and Andrology (CeRA), University Hospital Münster, Münster, Germany

Background/Objectives: Tudor domain-containing protein 6 (TDRD6) is essential for spermatogenesis and male fertility in mice. It localises to the chromatoid body (CB), a subcellular structure in round spermatids, and interacts with key proteins of the piRNA pathway. The function of TDRD6 in humans is unexplored warranting investigations whether TDRD6 is also a disease gene for male infertility.

Methods: Exome/genome data of 2,362 infertile men with severe oligo- or azoospermia from the Male Reproductive Genomics (MERGE) study were screened for rare (MAF <0.01 in gnomAD) biallelic high impact variants in TDRD6 (loss-of-function or missense with CADD >20). Compound heterozygosity was established by segregation analysis or long-read sequencing. Testicular phenotypes and the impact of TDRD6 on the chromatoid body architecture were determined by immunohistochemical staining of marker proteins.

Results: We identified five infertile men with biallelic variants in TDRD6 who shared a common reproductive phenotype of oligoasthenoteratozoospermia (OAT). One patient carried a homozygous missense variant and four patients were compound heterozygous for a loss-of-function variant and a missense variant. Two missense variants were predicted to destabilise TDRD6 protein structure. In two variant carriers, the subcellular localisation of CB marker protein DDX4 was impaired while the expression profile of piRNA pathway factors PIWIL1 and MAEL was unaffected.

Conclusion: This study establishes TDRD6 as candidate gene for OAT-related human male infertility and provides functional evidence for a role of TDRD6 in CB formation during late spermatogenesis.

Grants: This work was supported by the IZKF Münster (Tüt4/011/23).

Conflict of Interest: None declared

P02.014.D Gennet Endometrium Receptivity Test

Štěpán Chvojka ¹, Lucie Dohnalova¹, Romana Vlckova¹, Simona Suhajova¹, Hana Dvorackova¹, Martina Bittoova¹, Monika Koudová¹

¹GNTlabs by GENNET, Czech Republic

Introduction: Nowadays in vitro fertilization treatment is a complex process which thanks to new knowledge and technological development is constantly being expanded to include additional examinations with one common goal - to increase the success of this treatment and thus enable the couple to have a healthy child. One of them is the examination of endometrial receptivity.

Methods: The endometrium is normally examined using ultrasound, with the development of Next Generation Sequencing (NGS) methods it is possible to assess the endometrium from another point of view, that of molecular genetics. The endometrium becomes receptive to the embryo only during a short period of time in the menstrual cycle. NGS techniques in combination with prediction algorithms make it possible to determine this period and determine the “implantation window”, the time when the endometrium is most receptive to embryo transfer. This should ideally be between 19-21 day of the cycle for every fertile woman. However according to studies, up to 30% of women have a shifted implantation window.

Results: In cooperation with the Yikon company we have implemented the GERT (Gennet Endometrium Receptivity Test) in our laboratory which evaluates a total of 175 genes involved in the receptivity. Examination is indicated in women with repeated failure of euploid embryo implantation (after PGT-A). The result of the test is to determine whether the patient’s endometrium is in the pre-, post- or receptive phase.

Conclusion: Based on the results the optimal time of the personalized embryo transfer is then recommended to the patients.

Conflict of Interest: None declared

P02.015.A Investigating the role of solute carrier family of genes in adenomyosis pathogenesis utilizing whole exome sequencing in a sporadic patient cohort

Nimet Eser¹, nura fitnat topbas selcuki², Ipek Yildiz Ozaydin³, engin oral⁴, Feyza Tuncer ⁵

¹Istanbul University, Graduate School of Health Sciences, Istanbul, Türkyie; ²Istanbul Sisli Hamidiye Etfal Training and Research Hospital, Department of Obstetrics and Gynecology, Istanbul, Türkyie; ³Istanbul Kanuni Sultan Suleyman Training and Research Hospital, Department of Pathology, Istanbul, Türkyie; ⁴Biruni University Faculty of Medicine, Department of Obstetrics and Gynaecology, Istanbul, Türkyie; ⁵Istanbul University, Aziz Sancar Institute of Experimental Medicine, Department of Genetics, Istanbul, Türkyie

Background/Objectives: Adenomyosis and endometriosis are gynecological benign conditions characterized by the ectopic localization of endometrial tissue and stroma, which manifest with similar symptomology. Definite diagnosis is accomplished through histopathological confirmation of disease with surgical material. So far, genetic studies focused on endometriosis with limited attention to adenomyosis. We aimed to identify shared genetic variants among sporadic adenomyosis patients utilizing whole exome sequencing (WES).

Methods: Ten women, who received histopathological diagnosis of adenomyosis were recruited. Genomic DNA isolated from peripheral blood was subjected to WES on Illumina NovaSeq 6000 system. Pairend NGS Cloud platform was utilized in bioinformatic analysis, followed by filtering for MAF < 1%. Variants were prioritized in accordance with functional relevance and impact determined by in silico prediction tools.

Results: Remarkable accumulation of rare genetic variants in the members of the solute carrier family of genes (SLCs) were detected in our cohort. Among them, three of our patients carried rare variants of SLC12A1 and SLC12A2 which were prominent due to abnormal expression levels determined in a previous study. Moreover, nine of our patients harbored rare variants in genes (SIX4, BSND, WNK2, SGK1, SLC12A9) shown to influence the expression of SLC12A1 and SLC12A2.

Conclusion: This is the first study to assert the importance of SLCs in the pathogenesis of adenomyosis through WES analysis. Prospective work includes assessing the expressions of SLC12A1 and SLC12A2 utilizing the genomic materials recovered from these patients’ formalin-fixed paraffin-embedded hysterectomy tissues.

Grants: Istanbul University Scientific Research Projects Coordination Unit(TYL-2023-40298)

Conflict of Interest: None declared

P02.016.B Estimation of carrier frequencies of autosomal recessive genetic conditions based on gnomAD v4.0 data

Ronja Hotakainen ¹, Timo Järvinen¹, Kaisa Kettunen¹, Anna-Kaisa Anttonen^1;2;3, Eveliina Salminen^2;3

¹University of Helsinki and Helsinki University Hospital, Laboratory of Genetics, Helsinki, Finland; ²Helsinki University Hospital, Department of Clinical Genetics; ³University of Helsinki, Department of Medical and Clinical Genetics, Helsinki, Finland

Background/Objectives: A comprehensive strategy in the screening and prevention of rare diseases would benefit the current healthcare system. While monogenic rare diseases are relatively rare on their own, they contribute to around 10% of infant deaths and 20% of pediatric hospitalizations. The American College of Medical Genetics and Genomics recommends screening of autosomal recessive and X-linked conditions with a population frequency of ≥ 1/200, for all pregnant patients and those planning a pregnancy. Previous studies have used gnomAD v2.1.1 and v3.1.2 to estimate the gene carrier frequencies (GCF). In November 2023 gnomAD v4.0 was released, being nearly 5x larger than previous versions v2 and v3. The new 4.0 release provides added global diversity since it includes around 138 000 individuals of non-European ancestry.

Methods: We utilized the newest gnomAD v4.0 to calculate the GCF for gnomAD v4.0 populations. The analysis was performed for OMIM autosomal recessive genes utilizing ClinVar P/LP and Conflicting variants with > 80% P and/or LP classifications. The gnomAD v4.0 data is available for download separately as exomes and genomes. We combined these data to calculate the combined GCF and at-risk couple rates (ACR) per population.

Results: The number of genes with GCF ≥ 1/200 varied greatly per population, whereas the at-risk couple rates were more similar between populations, Ashkenazi Jewish having the highest ACR of 6.16%.

Conclusion: This work highlights the effects of increased samples sizes in gnomAD v4.0 on GCFs.

Grants: This work was supported by Helsinki University Hospital Diagnostic Center Research fund.

Conflict of Interest: None declared

P02.017.C Success rate of mosaic embryos in ART cycles

Jose A. Ortiz ^1;1, Ruth Morales¹, Belen Lledó¹, Alba Cascales¹, Adoracion Rodriguez-Arnedo², Andrea Bernabeu³, Rafael Bernabeu³

¹Instituto Bernabeu, Molecular Biology and Genetics, Alicante; ²Instituto Bernabeu, Reproductive Biology, Alicante; ³Instituto Bernabeu, Reproductive Medicine, Alicante

Background/Objectives: After embryo biopsy and PGT-A analysis, embryos can be classified as euploid, aneuploid or mosaic in which normal and chromosomally altered cells are combined. The transfer of mosaic embryos has been controversial, but currently the recommendation of international scientific societies is to transfer them as they have been shown to result in healthy children and the risk of congenital anomaly is similar to spontaneous pregnancies. The aim of the study is to analyse the clinical outcomes of mosaic embryos compared to euploid embryos.

Methods: Observational and retrospective study. A total of 9385 embryos from 2899 PGT-A cycles were included (January-2017 to October-2023). The trophectoderm biopsies were analysed by NGS. Clinical data from euploid and mosaic embryos were compared using binary-logistic regression (Rv.4.3.1).

Results: The analysis of embryos through PGT-A revealed that 54.17% were aneuploid, and 9.97% showed mosaicism. A total of 2166 embryos were transferred, of which 274 showed mosaicisms. No differences were observed in the clinical outcomes between euploid and mosaic groups. The pregnancy, biochemical and clinical-pregnancy loss and ongoing-pregnancy rates were 54.3 vs. 51.8, 9.0 vs. 8.8, 14.0 vs. 11.2 and 38.8 vs. 37.9 percent, respectively. Furthermore, when data are analysed according to the percentage or type of mosaicism (segmental vs. whole chromosome) or the number of chromosomes involved, no differences in clinical outcomes are observed.

Conclusion: Mosaic embryos have a high implantation potential, comparable to that of euploid embryos. Non-transfer of these embryos would reduce the chances of successful ART.

Grants: Not applicable.

Conflict of Interest: None declared

P02.018.D Possible exacerbation of semen phenotype in infertile men by an additive contribution of 2 genetic factors

Sandra Kleiman ^1;2, Noga Weizman¹, Shimi Barda¹, Ofer Lehavi¹, Eli Arama³, Shmuel Pietrokovski³, moran gershoni⁴

¹Tel Aviv Sourasky Medical Center; ²Sackler Faculty of Medicine, Tel Aviv University; ³Weizmann Institute of Science; ⁴Institute of Animal Science, ARO-The Volcani Center