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Background: Breast cancer is the most frequently diagnosed cancer in women and about 10% of breast cancer cases are hereditary. BRCA1 and BRCA2 are the genes most frequently associated with Hereditary Breast Cancer, although there are numerous other genes, such as PALB2, CHEK2 and ATM, that require to be considered as well. Germline Copy Number Variation (CNV) is one mutation type that is an important contributor to hereditary breast cancer. Nowadays, next-generation sequencing (NGS) technologies has contributed to multi-gene panel analysis used in clinical practice. Methods: In total, 1418 individuals were tested for breast cancer predisposition, using a solution-based capture approach. Targeted NGS was performed with a panel of 36 genes. The capture-based approach allowed for computational analysis of CNVs from NGS data. Results: We investigate the performance of the CNV module of the commercial software suite SeqPilot (JSI Medical Systems) and the non-commercial tool panelcn.MOPS. Both algorithms are specifically developed for CNV analysis of sequencing data reporting 99-100% sensitivity and up to 100% specificity for the prediction of CNVs up to the level of a single gene exon. All CNVs detected with these algorithms were then verified experimentally using the MLPA technique as an orthogonal assay. At least one pathogenic/likely pathogenic variant was identified in 289 samples (20.4%). CNVs accounted for 10.4% (30/289), referring to the deletion of one or more exons of a gene. Interestingly, 50% of deletions were single exon and approximately 36% of CNVs were detected in genes other than BRCA1/2. In specific, of the 30 CNVs detected, 60% occurred in BRCA1, 3.3% in BRCA2, 20% in CHEK2, 6.7% in FANCA, 6.7% in PMS2, and 3.3% in ATM. The majority of CNVs in BRCA1 were deletions of exons 19, 22, and 22-23 whereas deletions of exons 9-10 were the most common deletions in CHEK2. Detailed information of all CNVs detected is provided in Table 1. Conclusions: Our results suggest that CNV analysis should not be restricted to BRCA1/2 due to the significant proportion of CNVs (36%) in additional breast cancer predisposition genes. Furthermore, in silico CNV detection tools provide a cost-effective and feasible methodology for the identification of CNVs from NGS experiments. This outlines the clinical utility of comprehensive genetic testing that includes full sequencing and CNV analysis in hereditary breast cancer facilitating personalized management decisions for patients. Table 1.Pathogenic Copy Number Variations (CNVs) identified in this studyGeneHGVS nomenclatureOther nomenclature# detectedATMNM_000051:c.(-30+1_-29-1)_(331+1_332-1)deldeletion of exons 2-41BRCA1NM_007294:c.(5467+1_5468-1)-(*1_?)deldeletion of exon 237BRCA1NM_007294:c.(5406+1_5407-1)_(*1_?)deldeletion of exons 23-245BRCA1NM_007294:c.(5193+1_5194-1)-(5277+1_5278-1)deldeletion of exon 196BRCA2NM_000059:c.(6841+1_6842-1)_(7007+1_7008-1)deldeletion of exons 12-131CHEK2NM_007194:c.(908+1_909-1)_(1095+1_1096-1)deldeletion of exons 9-104CHEK2NM_007194:c.(792+1_793-1)_(846+1_847-1)deletion of exon 72FANCANM_000135:c.(1626+1_1627-1)_ (2852+1_2853-1)deldeletion of exons 18-291FANCANM_000135:c.(893+1_894-1)_(1359+1_1360-1)deldeletion of exons 11-141PMS2NM_000535: c.(903+1_904-1)_(988+1_989-1)deldeletion of exon 91PMS2NM_000535:c.(705+1_706-1)_(2006+1_2007-1)deldeletion of exons 7-111 Citation Format: Nikolaos Tsoulos, Konstantinos Agiannitopoulos, Georgia Pepe, Eirini Papadopoulou, Georgios N Tsaousis, Despina Apostolopoulou, Angeliki Meintani, Vassileios Venizelos, Christos Markopoulos, Rodoniki Iosifidou, Sofia Karageorgopoulou, Christos Christodoulou, Ioannis Natsiopoulos, Konstantinos Papazisis, Maria Vasilaki-Antonatou, Eleftherios Kabletsas, Amanta Psyrri, Stylianos Giassas, Dimitrios Ziogas, Efthalia Lalla, Anna Koumarianou, Christos Papadimitriou, Vahit Ozmen, Sualp Tansan, Kerim Kaban, Tahsin Ozatlı, Dan Tudor Eniu, Angelica Chiorean, Alexandru Blidaru, George Nasioulas. Different CNVs account for 10.4% of pathogenic variants in 1418 patients referred for hereditary breast cancer testing [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-09-10.

Male breast cancer (MBC) is a very rare disorder affecting approximately 1 in 833 men. Genetic predisposition is one of the most important risk factors of MBC with BRCA2 being the most commonly mutated gene in males diagnosed with breast cancer. However, a large part of MBC heritability is still unexplained. This study sought to add to the data already available on the genetics of MBC.Our study initially involved comprehensive analysis of BRCA1 and BRCA2, followed by analysis of 43 genes implicated in cancer predisposition in a series of 100 Greek patients diagnosed with MBC between 1995-2015.Pathogenic variants were identified in 13 patients, with BRCA2 being the most commonly affected gene, followed by BRCA1, RAD50, RAD51B, and MSH3.In agreement with previous reports, BRCA2 is the most important genetic factor of MBC predisposition, while the remaining known cancer predisposition genes are each very rarely involved, rendering conclusions as to their cumulative effect difficult to draw.

Background Classification of splicing variants (SVs) in genes associated with hereditary cancer is often challenging. The aim of this study was to investigate the occurrence of SVs in hereditary cancer genes and the clinical utility of RNA analysis. Material and methods 1518 individuals were tested for cancer predisposition, using a Next Generation Sequencing (NGS) panel of 36 genes. Splicing variant analysis was performed using RT-PCR and Sanger Sequencing. Results In total, 34 different SVs were identified, 53% of which were classified as pathogenic or likely pathogenic. The remaining 16 variants were initially classified as Variant of Uncertain Significance (VUS). RNA analysis was performed for 3 novel variants. Conclusion The RNA analysis assisted in the reclassification of 20% of splicing variants from VUS to pathogenic. RNA analysis is essential in the case of uncharacterized splicing variants, for proper classification and personalized management of these patients.

Background/Aim: The use of multi-gene panels for germline testing in breast cancer enables the estimation of cancer risk and guides risk-reducing management options. The aim of this study was to present data that demonstrate the different levels of actionability for multi-gene panels used in genetic testing of breast cancer patients and their family members. Materials and Methods: We performed an analysis in our clinical database to identify breast cancer patients undergoing genetic testing. We reviewed positive results in respect of risk estimation and management, cascade family testing, secondary findings and information for treatment decision-making. Results: A total of 415 positive test reports were identified with 57.1%, 18.1%, 10.8% and 13.5% of individuals having pathogenic/likely pathogenic variants in high, moderate, low and with insufficient evidence for breast cancer risk genes, respectively. Six point seven percent of individuals were double heterozygotes. Conclusion: Germline findings in 92% of individuals are linked to evidence-based treatment information and risk estimates for predisposition to breast and/or other cancer types. The use of germline findings for treatment decision making expands the indication of genetic testing to include individuals that could benefit from targeted treatments.

Next-generation sequencing (NGS) technology is used to evaluate hereditary cancer risks of patients worldwide; however, information concerning the germline multigene mutational spectrum among patients with breast cancer (BC) with consanguineous marriage (CM) is limited. Therefore, this prospective study aimed to determine the molecular characteristics of patients with BC who were tested with multigene hereditary cancer predisposition NGS panel and to show the effect of CM on cancer-related genes. Patients with BC with or without CM and family history (FH) of BC treated in our breast center were selected according to The National Comprehensive Cancer Network (NCCN) criteria for hereditary BC. In these patients, the analysis of a panel of 33 genes involved in hereditary cancer predisposition was performed after genetic counseling by using NGS. The pathogenic variant (PV) and the variant of uncertain significance (VUS) were found to be 15.8 and 47.4%, respectively. PVs were identified in 10/33 genes in 34 patients; 38.2% in BRCA1/2 genes; 6, 24, and 14% in other high, moderate and low-risk genes, respectively. The CM rate was 17.7% among the 215 patients with BC. The PV rate was 13.2% in patients with CM and 16.4% in patients without CM (P=0.80). When PV and VUS were evaluated together, the PV+VUS ratio was significantly higher in patients with CM and FH of BC than patients without CM and FH of BC (88.2 vs. 63.3%, P=0.045). Analysis of multigene panel provided 9.76% additional PVs in moderate/low-risk genes. The PV rate was similar in patients with BC with or without CM. A high PV+VUS ratio in patients with CM and FH of BC suggests that genes whose importance are unknown are likely to be pathogenic genes later.

Background Carriers with pathogenic variants in MSH2 have increased risk to develop colorectal, endometrium, ovarian, and other types of cancer. The PALB2 is associated with breast, ovarian, pancreatic, and prostate cancer. We describe the case of a 42‐year‐old female diagnosed with endometrial cancer at the age of 42 years with a strong family history of colorectal cancer, which was referred to our private diagnostic laboratory for genetic testing. Methods In this study, we performed next‐generation sequencing (NGS) using an amplicon based 26 genes panel. The presence of multi‐exonic copy number variations (CNVs) was investigated by computational analysis and Multiplex Ligation‐dependent Probe Amplification (MLPA). Results A gross deletion of the genomic region encompassing exons 11–16 of the MSH2 and the loss‐of‐function variant c.757_758delCT, p.(Leu253Ilefs*3) in the PALB2 were identified in the proband. Conclusions Multigene analysis using NGS technology allows the identification of pathogenic variants in genes that would normally not be tested based on the patient diagnosis. In our case these results explained not only the personal and/or family history of cancer but also allowed the surveillance for prevention of other cancer types. Moreover, the detection of large genomic rearrangements should be routinely included in hereditary cancer testing., We describe the case of a 42‐year‐old female diagnosed with endometrial cancer with a strong family history of colorectal cancer, which was referred to our private diagnostic laboratory for genetic testing. By next‐generation sequencing (NGS) and Multiplex Ligation‐dependent Probe Amplification (MLPA) analysis we identified a gross deletion of the genomic region encompassing exons 11–16 of the MSH2 gene and the loss‐of‐function variant c.757_758delCT, p.(Leu253Ilefs*3) in the PALB2 gene in our proband.

Background CHEK2 is involved in the DNA damage repair response Fanconi anemia (FA)-BRCA pathway. An increased risk for breast and other cancers has been documented in individuals who carry a single pathogenic CHEK2 variant. As for other genes involved in cancer predisposition, different types of pathogenic variants have been observed, including single nucleotide variations, short insertions/deletions, large genomic rearrangements and splicing variants. Splicing variants occurring in the splicing acceptor or donor site result in alternative mature mRNA produced and can cause intron retention, exon skipping, or creation of alternative 3′ and 5′ splice site. Thus, the pathogenicity of this type of alterations should always be explored experimentally and their effect in the mRNA and consequently the protein produced, should be defined. The aim of this study was the delineation of the effect of a splicing variant in the CHEK2 gene. Case presentation A healthy 28-year-old woman with a family history of breast and ovarian cancer was referred for genetic testing. The variant c.793-1G > A (rs730881687) was identified by Next Generation Sequencing (NGS) using a solution-based capture method, targeting 33 cancer predisposition genes (SeqCap EZ Probe library, Roche NimbleGen). Experimental analysis in patient-derived leukocytes using RT-PCR of mRNA followed by cDNA sequencing revealed the deletion of one base from the alternative transcript created (r.793del). This resulted in a frameshift leading to premature termination codon within exon 7 (p.(Asp265Thrfs*10)). Conclusions This finding suggests that the CHEK2 splicing variant c.793-1G > A is a deleterious variant. Our case shows that RNA analysis is a valuable tool for uncharacterized splice site variants in individuals referred for testing and facilitates their personalized management.

Gastrointestinal malignancies are among the leading causes of cancer-related deaths worldwide. Like all human malignancies they are characterized by accumulation of mutations which lead to inactivation of tumor suppressor genes or activation of oncogenes. Advances in Molecular Biology techniques have allowed for more accurate analysis of tumors' genetic profiling using new breakthrough technologies such as next generation sequencing (NGS), leading to the development of targeted therapeutical approaches based upon biomarker-selection. During the last 10 years tremendous advances in the development of targeted therapies for patients with advanced cancer have been made, thus various targeted agents, associated with predictive biomarkers, have been developed or are in development for the treatment of patients with gastrointestinal cancer patients. This review summarizes the advances in the field of molecular biomarkers in tumors of the gastrointestinal tract, with focus on the available NGS platforms that enable comprehensive tumor molecular profile analysis.

Background The application of the Next Generation Sequencing (NGS) technology has facilitated multigene panel testing for hereditary breast cancer (BC) in clinical practice. We performed a retrospective analysis of individuals referred for testing in our lab aiming to investigate the contribution of included genes and evaluate current genetic testing guidelines in BC. Methods In total, 1141 BC patients and 184 unaffected individuals with family history (FH) of BC were referred from physicians for testing using a multigene panel. Genomic DNA was enriched for targeted regions of 33 genes and sequencing was carried out using the Illumina NGS technology. The presence of large genomic rearrangements (LGRs) was investigated computationally and by Multiplex Ligation-dependent Probe Amplification (MLPA). Results A pathogenic variant (PV) was identified in 22% (291/1325) of analyzed individuals and in specific in 23.2% of BC patients and 14.1% of unaffected individuals (P = 0.006). Among individuals with PVs, 49.1% were located in the BRCA1/2 genes whereas 8.6%, 22.7% and 19.6% occurred in other high, moderate and low-risk genes respectively. Notably, 21 of the 291 positive individuals (7.2%) carried clinically significant variants in two different genes and 6.5% had a LGR. A retrospective analysis of positive individuals showed that 88.3% of BC patients met the NCCN criteria for further genetic risk evaluation compared to 80.8% of unaffected individuals with FH of BC (P = 0.269). In BRCA-positive cases, NCCN criteria were met in 92.3% of the referrals compared to 81.8% in individuals positive for other genes (P = 0.008). Conclusions Extended multigene panel testing in hereditary BC facilitates the detection of nearly twice as many individuals that could benefit from personalized management. In our cohort, the currently used selection criteria for HBOC failed to identify only 12.7% of individuals positive for pathogenic variants, suggesting strong selection strategies from physicians. However, our results indicate that selection criteria perform better for the identification of BRCA-positive BC patients and should be revised to facilitate towards the inclusion of BC patients with PVs in other genes. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Germline mutations in the BRCA1 and BRCA2 genes are associated with hereditary predisposition to breast and ovarian cancer. Sensitive and accurate detection of BRCA1 and BRCA2 mutations is crucial for personalized clinical management of individuals affected by breast or ovarian cancer, and for the identification of at-risk healthy relatives. We performed molecular analysis of the BRCA1 and BRCA2 genes in 898 Greek families, using Sanger sequencing or Next Generation Sequencing for the detection of small insertion/deletion frameshift, nonsynonymous, truncating and splice-site alterations and MLPA for the detection of large genomic rearrangements. In total, a pathogenic mutation was identified in 12.9% of 898 families analyzed. Of the 116 mutations identified in total 9% were novel and 14.7% were large genomic rearrangements. Our results indicate that different types of mutational events in the BRCA1 and BRCA2 genes are responsible for the hereditary component of breast/ovarian cancer in the Greek population. Therefore the methodology used in the analysis of Greek patients must be able to detect both point and small frameshift mutations in addition to large genomic rearrangements across the entire coding region of the two genes.

BACKGOUND: Hereditary cancer predisposition syndromes are believed to be responsible for approximately 5-10% of all diagnosed cancer cases. In the past, single genes analysis of certain high risk genes was used for the determination of the genetic cause of cancer heritability in certain families. The selection of genes was mainly based on the family history of the individuals analyzed and included only highly associated genes (e.g. the BRCA1 and BRCA2 genes for families with breast cancer history. Nowadays though, 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 predisposition gene mutations. AIM: The aim of this study was to investigate the extent and nature of mutations in 36 genes implicated in hereditary cancer predisposition in individuals referred for testing in our lab. MATERIALS & METHODS: In total, 1197 individuals were referred for testing in our lab in the past four years from Greece, Romania and Turkey. The analysis of genes involved in hereditary cancer predisposition was performed using two NGS approaches. The first 451 individuals were analyzed using an amplicon based sequencing method (26 gene panel), while the following 746 individuals were analyzed using a capture based method (33 gene panel). Genomic DNA was enriched for targeted regions of 36 genes involved in hereditary predisposition to cancer included in both versions of the panel (APC, BMPR1A, BRCA1, BRCA2, CDH1, CDK4, CDKN2A, EPCAM, MEN1, MLH1, MSH2, MSH6, MUTYH, PALB2, PMS2, PTEN, RET, SMAD4, STK11, TP53, VHL, ATM, BRIP1, CHEK2, NBN, RAD51C, RAD51D, BARD1, BLM, CHEK1, ABRAXAS1 (FAM175A), MRE11 (MRE11A), NF1, RAD50, RAD51B, XRCC2). Sequencing was carried out using the Illumina NGS technology. Reads were aligned to the reference sequence (GRCh37), 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 computational analysis of NGS results and the use of MLPA for 13 genes. All clinically significant observations were confirmed by orthogonal technologies. RESULTS: In total, a pathogenic mutation was identified in 259 of the 1197 individuals (21.6%) analyzed while a VUS was identified in 35.7% of the cases. Clinically significant mutations were identified in 29 of the genes analyzed. Concerning the mutation distribution among individuals with positive findings, 44.7% of them were located in BRCA1/2 genes whereas 20.9%, 19.9%, and 14.5% in high, moderate and low risk genes respectively. In addition to BRCA1 and BRCA2 genes other highly mutated genes were CHEK2 (10.6%), PALB2 (7.1%), MUTYH (7.1%) and ATM (4.3%). Of note is that 25 of the 259 positive individuals (9.7%) carried clinically significant mutations in two different genes and 5.8% had a large genomic rearrangement (LGR). CONCLUSIONS: Our results support the clinical significance of analysis of a panel of genes involved in hereditary cancer predisposition. In our cohort, analysis of this panel allowed for the identification of 8.3% additional pathogenic variants in moderate/low risk genes, enabling personalized management of these individuals. Citation Format: Tsoulos N, Tsaousis GN, Papadopoulou E, Agiannitopoulos K, Pepe G, Kambouri S, Apessos A, Diamantopoulos N, Floros T, Iosifidou R, Katopodi O, Koumarianou A, Markopoulos C, Papazisis K, Venizelos V, Xanthakis I, Xepapadakis G, Banu E, Eniu DT, Negru S, Stanculeanu DL, Ungureanu A, Ozmen V, Tansan S, Tekinel M, Yalcin S, Nasioulas G. Analysis of hereditary cancer syndromes by using a panel of genes: Novel and multiple pathogenic mutations [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-03-07.

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.