INTRODUCTION Hereditary breast cancer is estimated to account for approximately 10% of all breast cancer cases. In addition, an estimated 15-20% of those affected by breast cancer have a positive family history. Despite the fact that BRCA1 and BRCA2 are the two most significant genes in hereditary breast cancer predisposition, twenty years of analysis has highlighted the fact, that mutations in these two highly penetrant genes, are only present in approximately 20% of high risk families. Other genes, mutations in which are associated with high risk of breast cancer, were identified because of the strong association with familial cancer syndromes, in which breast cancer is one of the defining components. Technological advances in molecular biology and especially DNA sequencing, commonly designated as “Next Generation Sequencing – NGS” have aided in the concentrated efforts to identify new genes responsible for the missing heritability, allowing the application of this knowledge in the diagnostic setting. AIM The aim of this study was to investigate the extent and nature of mutations in 26 genes implicated in hereditary cancer predisposition in families of Romanian descent. MATERIALS & METHODS In total, 297 Romanian families have been analyzed by our group in the past three years. Genomic DNA was enriched for targeted regions of 26 genes involved in hereditary predisposition to cancer (ATM, BARD1, BLM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM (only intron 8, exon 9 and 3'UTR), FAM175A, MEN1, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53, XRCC2). Sequencing was carried out using the Illumina NGS technology. Reads were aligned to the reference sequence (GRCh38), and sequence changes were identified and interpreted in the context of a single clinically relevant transcript. The presence of large genomic rearrangements was investigated by use of MLPA. All clinically significant observations were confirmed by orthogonal technologies. RESULTS In total, a pathogenic mutation was identified in 79 of the 297 families (26.6%) analyzed. Clinically significant mutations were identified in 17 of the genes included in the panel. The most commonly mutated genes in the Romanian population were BRCA1 and BRCA2, accounting for 50% of the mutations identified, followed by PALB2 (12%), CHEK2 (9.4%) and ATM, NBN and RAD50 which accounted for 3.5% of the mutations each. Of note is that 7 of the 79 affected families (8.8%) carried clinically significant mutations in two different genes. CONCLUSIONS Our results support the clinical significance of analysis of a panel of genes involved in hereditary cancer predisposition. In this series of patients, analysis of this panel allowed for the identification of 14% additional pathogenic variants. This is especially true in those cases where more than one pathogenic variant was identified. Citation Format: Tsoulos N, Apessos A, Agiannitopoulos K, Pepe G, Tsaousis G, Kambouri S, Eniu DT, Ungureanu A, Banu E, Ciule L, Blidaru A, Chiorean A, Stanculeanu DL, Mateescu D, Nasioulas G. Analysis of hereditary cancer syndromes by use of a panel of genes: More answers than questions [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-03-03.

Background: Hereditary cancer predisposition syndromes are responsible for approximately 5-10% of all diagnosed cancer cases. In the past, single-gene analysis of specific high risk genes was used for the determination of the genetic cause of cancer heritability in certain families. The application of Next Generation Sequencing (NGS) technology has facilitated multigene panel analysis and is widely used in clinical practice, for the identification of individuals with cancer predisposing gene variants. The purpose of this study was to investigate the extent and nature of variants in genes implicated in hereditary cancer predisposition in individuals referred for testing in our laboratory.Methods: In total, 1197 individuals from Greece, Romania and Turkey were referred to our laboratory for genetic testing in the past 4 years. The majority of referrals included individuals with personal of family history of breast and/or ovarian cancer. The analysis of genes involved in hereditary cancer predisposition was performed using a NGS approach. Genomic DNA was enriched for targeted regions of 36 genes and sequencing was carried out using the Illumina NGS technology. The presence of large genomic rearrangements (LGRs) was investigated by computational analysis and Multiplex Ligation-dependent Probe Amplification (MLPA). Results: A pathogenic variant was identified in 264 of 1197 individuals (22.1%) analyzed while a variant of uncertain significance (VUS) was identified in 34.8% of cases. Clinically significant variants were identified in 29 of the 36 genes analyzed. Concerning the mutation distribution among individuals with positive findings, 43.6% were located in the BRCA1/2 genes whereas 21.6, 19.9, and 15.0% in other high, moderate and low risk genes respectively. Notably, 25 of the 264 positive individuals (9.5%) carried clinically significant variants in two different genes and 6.1% had a LGR. Conclusions:In our cohort, analysis of all the genes in the panel allowed the identification of 4.3 and 8.1% additional pathogenic variants in other high or moderate/low risk genes, respectively, enabling personalized management decisions for these individuals and supporting the clinical significance of multigene panel analysis in hereditary cancer predisposition.

Several tumor types have been efficiently treated with PARPis, which are now approved for the treatment of ovarian, breast, prostate, and pancreatic cancer. The BRCA1/2 genes and mutations in many additional genes involved in the HR pathway may be responsible for the HRD phenomenon. In the present study, an NGS assay was used to analyze 513 genes associated with targeted and immuno-oncology therapies in 406 samples. In addition, the %gLOH values of 24 samples were also calculated using the Affymetrix technology to compare the results obtained by the two methodologies. HR variations occurred in 20.93% of the malignancies, while BRCA1/2 gene alterations in 5.17%. %LOH was highly correlated with alterations in the BRCA1/2 genes since 76.19% (16/21) of the BRCA1/2 positive tumors had a high %LOH value (p=0.007). Moreover, the LOH status was highly correlated with the TP53 and KRAS statuses, but there was no association with the TMB value. Lin's Concordance Correlation Coefficient for the 24 evaluable samples simultaneously examined by both assays was 0.87, indicating a nearly perfect agreement. In conclusion, the addition of gLOH analysis could assist the detection of additional patients eligible for PARPis