BackgroundGerm line mutations in BRCA1 and BRCA2 (BRCA1/2) and other susceptibility genes have been identified as genetic causes of hereditary breast and ovarian cancer (HBOC). To identify the disease-causing mutations in a cohort of 120 Brazilian women fulfilling criteria for HBOC, we carried out a comprehensive screening of BRCA1/2, TP53 R337H, CHEK2 1100delC, followed by an analysis of copy number variations in 14 additional breast cancer susceptibility genes (PTEN, ATM, NBN, RAD50, RAD51, BRIP1, PALB2, MLH1, MSH2, MSH6, TP53, CDKN2A, CDH1 and CTNNB1).MethodsCapillary sequencing and multiplex ligation-dependent probe amplification (MLPA) were used for detecting point mutations and copy number variations (CNVs), respectively, for the BRCA1 and BRCA2 genes; capillary sequencing was used for point mutation for both variants TP53 R337H and CHEK2 1100delC, and finally array comparative genomic hybridization (array-CGH) was used for identifying CNVs in the 14 additional genes.ResultsThe positive detection rate in our series was 26%. BRCA1 pathogenic mutations were found in 20 cases, including two cases with CNVs, whereas BRCA2 mutations were found in 7 cases. We also found three patients with the TP53 R337H mutation and one patient with the CHEK2 1100delC mutation. Seven (25%) pathogenic mutations in BRCA1/2 were firstly described, including a splice-site BRCA1 mutation for which pathogenicity was confirmed by the presence of an aberrant transcript showing the loss of the last 62 bp of exon 7. Microdeletions of exon 4 in ATM and exon 2 in PTEN were identified in BRCA2-mutated and BRCA1/2-negative patients, respectively.ConclusionsIn summary, our results showed a high frequency of BRCA1/2 mutations and a higher prevalence of BRCA1 (64.5%) gene. Moreover, the detection of the TP53 R337H variant in our series and the fact that this variant has a founder effect in our population prompted us to suggest that all female breast cancer patients with clinical criteria for HBOC and negative for BRCA1/2 genes should be tested for the TP53 R337H variant. Furthermore, the presence of genomic structural rearrangement resulting in CNVs in other genes that predispose breast cancer in conjunction with BRCA2 point mutations demonstrated a highly complex genetic etiology in Brazilian breast cancer families.

Pathogenic variants in known breast cancer (BC) predisposing genes explain only about 30% of Hereditary Breast Cancer (HBC) cases, whereas the underlying genetic factors for most families remain unknown. Here, we used whole-exome sequencing (WES) to identify genetic variants associated to HBC in 17 patients of Brazil with familial BC and negative for causal variants in major BC risk genes (BRCA1/2, TP53, and CHEK2 c.1100delC). First, we searched for rare variants in 27 known HBC genes and identified two patients harboring truncating pathogenic variants in ATM and BARD1. For the remaining 15 negative patients, we found a substantial vast number of rare genetic variants. Thus, for selecting the most promising variants we used functional-based variant prioritization, followed by NGS validation, analysis in a control group, cosegregation analysis in one family and comparison with previous WES studies, shrinking our list to 23 novel BC candidate genes, which were evaluated in an independent cohort of 42 high-risk BC patients. Rare and possibly damaging variants were identified in 12 candidate genes in this cohort, including variants in DNA repair genes (ERCC1 and SXL4) and other cancer-related genes (NOTCH2, ERBB2, MST1R, and RAF1). Overall, this is the first WES study applied for identifying novel genes associated to HBC in Brazilian patients, in which we provide a set of putative BC predisposing genes. We also underpin the value of using WES for assessing the complex landscape of HBC susceptibility, especially in less characterized populations.

When considering family history, hereditary breast cancer (HBC) syndromes correspond to nearly 5-10% of all breast cancer (BC) cases. However, pathogenic variants in known moderate- and high-risk BC genes explain only ~30% of familial BC cases. Recent advances in sequencing technology resulted in an increasing number of BC genes being revealed in previously negative families by using extended gene panels and whole-exome sequencing (WES). WES offers the opportunity to concomitantly investigate several rare risk genes as well as to identify new BC-predisposing genes. Thus, in this study we used WES to disclose variants contributing to BC increased risk in patients that were negative for mutations in three major BC genes (BRCA1/2 and TP53) and met stringent clinical criteria indicating a genetic predisposition to BC. We selected 17 women (two of them sisters) who fulfilled one or more of the following criteria: early onset BC (<36 years); bilateral BC; breast and other primary related tumor (ovary, fallopian tube, or primary peritoneal tumors). Firstly, we used WES data to search for rare variants in 27 well-established and emerging HBC predisposing genes. This analysis identified two patients harboring truncating mutations, one in ATM (p.Tyr2334Glnfs*4 – pathogenic in ClinVar database) and one in BARD1 (Tyr739Leufs*2 – not described, probably pathogenic according to ACMG guidelines). Additionally, we identified nine variants of unknown clinical significance in these genes, occurring in seven patients. For the remaining 15 patients with no putative pathogenic mutations in HBC genes, we first applied quality filters to exclude false-positive results (included variants: variant base coverage ≥5X, variant frequency ≥25%, QD>2, FS<6). Remaining variants were excluded if present in population databases with a minor allele frequency (MAF) > 0.01 or present in five BRCA1 mutated patients sequenced in the same platforms. Next, using a functional-based variant prioritization, we selected candidate variants according to the predicted impact in the protein function, the affected gene, and segregation with the phenotype (in the family-based study of the two sisters). Candidate variants included all loss-of-function variants as well as missense and in-frame indels occurring in a specific list of 651 genes of interest (DNA repair and cancer-related genes) and if predicted to be damaging by at least 3/6 prediction software. The resulting 244 selected variants were submitted for technical validation by targeted NGS and, of these, 220 were validated (89%). Using the same custom panel, we evaluated 25 control samples of healthy Brazilian women for filtering common polymorphisms in our population, resulting in a final 139 variants in 129 genes (89 LOF and 50 missense variants). For two families---one with WES of two affected sisters and target NGS of one affected aunt; one with WES of the affected daughter and target NGS of the affected mother---we were able to perform cosegregation analysis in other family members and 11 out of 23 variants were found to be segregating with the disease. These 11 distinct genes as well as 82 genes harboring LOF variants were evaluated in an independent cohort of 34 WES of high-risk BC patients. Several rare, possibly damaging variants were identified in this cohort, providing additional evidence of the potential role in BC predisposition of some new genes. Of those, we highlight ATRX, FANCC, TET2, ERCC1, PTPRD, and ROS1 genes, which are involved in DNA repair and other tumorigenic pathways. Overall, our results provide a set of putative BC-predisposing genes and underpin WES as a useful tool for assessing the complex landscape of HBC predisposition. The discovery and validation of new HBC genes in different populations will continue to provide insights into disease mechanisms, eventually leading to the development of more effective therapies and improved management of affected families. Citation Format: Giovana T. Torrezan, Fernanda GSR Almeida, Marcia CP Figueiredo, Bruna DF Barros, Claudia A. Paula, Renan Valieris, Jorge ES Souza, Elisa N. Ferreira, Felipe CC Silva, Amanda F. Nobrega, Maria Isabel Achatz, Edenir I. Palmero, Rodrigo F. Ramalho, Sandro J. Souza, Dirce M. Carraro. Complex landscape of germline variants in hereditary and early-onset breast cancer ascertained through whole exome sequencing [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A37.

Lynch syndrome (LS), former known as Hereditary Non Polyposis Colorectal cancer (HNPCC), accounts for 3-5% of all colorectal cancers (CRC) and is inherited in an autossomal dominant fashion. This syndrome is characterized by early CRC onset, high incidence of tumors in the ascending colon, excess of synchronous and metachronous tumors, and accelerated transition adenoma-carcinoma (2-3 years). Nowadays, LS is regarded of patients who carry deleterious germline mutations in one of the five Mismatch repair genes, such as MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), post-meiotic segregation increased 1 (PMS1) or post-meiotic segregation increased 2 (PMS2). In order to characterize Brazilian patients suspect for LS, we assessed the frequency of germline point mutations in MSH6, PMS1 and PMS2 in patients negative for point mutations in MLH1 and MSH2 by capillary sequencing. We also assessed chromosomal deletions in MLH1 and MSH2 and MSH6 generating a complete characterization of MMR genes. The analysis of the five MMR genes revealed 46 carriers of pathogenic mutations, including 25 in MSH2 (54%), 16 in MLH1 (35%), 4 in MSH6 (9%) and one in PMS2 (2%) gene In addition, from the 70 negative patients, we selected one mutation negative family that met the stringent Amsterdam criteria (AC) for whole exome sequencing using the SOLID platform. For this family, we selected 5 close blood relatives, including 3 affected and 2 unaffected individuals in order to facilitate the identification of new susceptibility genes with autosomal dominant pattern. The results revealed two candidate genes, c-KIT and WDR27 that segregated with the disease. Additional analysis are under evaluation. Citation Format: Felipe C. Silva, Giovana T. Torrezan, Márcia CP Figueiredo, Jose Roberto O. Ferreira, Érika M. Santos, Wilson T. Nakagawa, Samuel Aguiar-Junior, Benedito M. Rossi, Fabio O. Ferreira, Dirce M. Carraro. Molecular characterization of Brazilian patients suspected for Lynch syndrome. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 18. doi:10.1158/1538-7445.CANSUSC14-18