Whole-exome sequencing of non-BRCA1/BRCA2 mutation carrier cases at high-risk for hereditary breast/ovarian cancer.

The current study aimed to identify new breast and/or ovarian cancer predisposition genes. For that, whole-exome sequencing (WES) was performed in the germline DNA of 52 non-BRCA1/BRCA2/TP53 mutation carrier women at high-risk for hereditary breast and ovarian cancer (HBOC). All variants were classified using information from population and disease specific databases, in silico prediction tools and the American College of Medical Genetics and Genomics (ACMG) criteria. Loss of heterozygosity (LOH) of tumor samples and segregation analyses were performed whenever possible. The variants identified were investigated in a second, independent cohort of 17 BC cases. Pathogenic/Likely Pathogenic variants were identified in known cancer genes such as CHEK2, MUTYH, PMS2, and RAD51C. Rare and potentially pathogenic variants were identified in DNA repair genes (FAN1, POLQ, and RAD54L) and other cancer-related genes such as DROSHA and SLC34A2. Interestingly, the variant c.149T>G in the FAN1 gene was identified in two unrelated families, and exhibited LOH in the tumor tissue of one of them. In conclusion, this is the largest Brazilian WES study involving families at high-risk for HBOC which has brought novel insights into the role of potentially new genetic risk factors for hereditary breast and ovarian cancer.

Oncogenetics service and the Brazilian public health system: the experience of a reference Cancer Hospital.

The identification of families at-risk for hereditary cancer is extremely important due to the prevention potential in those families. However, the number of Brazilian genetic services providing oncogenetic care is extremely low for the continental dimension of the country and its population. Therefore, at-risk patients do not receive appropriate assistance. This report describes the creation, structure and management of a cancer genetics service in a reference center for cancer prevention and treatment, the Barretos Cancer Hospital (BCH). The Oncogenetics Department (OD) of BCH offers, free of charge, to all patients/relatives with clinical criteria, the possibility to perform i) genetic counseling, ii) preventive examinations and iii) genetic testing with the best quality standards. The OD has a multidisciplinary team and is integrated with all specialties. The genetic counseling process consists (mostly) of two visits. In 2014, 614 individuals (371 families) were seen by the OD. To date, over 800 families were referred by the OD for genetic testing. The support provided by the Oncogenetics team is crucial to identify at-risk individuals and to develop preventive and personalized behaviors for each situation, not only to the upper-middle class population, but also to the people whose only possibility is the public health system.

Whole-Exome Sequencing Identifies Pathogenic Germline Variants in Patients with Lynch-Like Syndrome.

Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, characterized by germline pathogenic variants in mismatch repair (MMR)-related genes that lead to microsatellite instability. Patients who meet the clinical criteria for LS and MMR deficiency and without any identified germline pathogenic variants are frequently considered to have Lynch-like syndrome (LLS). These patients have a higher risk of CRC and extracolonic tumors, and little is known about their underlying genetic causes. We investigated the germline spectrum of LLS patients through whole-exome sequencing (WES). A total of 20 unrelated patients with MMR deficiency who met the clinical criteria for LS and had no germline variant were subjected to germline WES. Variant classification was performed according to the American College of Medical Genetics and Genomics (ACMG) criteria. Pathogenic/likely pathogenic variants were identified in 35% of patients in known cancer genes such as MUTYH and ATM. Besides this, rare and potentially pathogenic variants were identified in the DNA repair gene POLN and other cancer-related genes such as PPARG, CTC1, DCC and ALPK1. Our study demonstrates the germline mutational status of LLS patients, a population at high risk of colorectal cancer.

New insights on familial colorectal cancer type X syndrome.

Familial colorectal cancer type X (FCCTX) is a heterogeneous colorectal cancer predisposition syndrome that, although displays a cancer pattern similar to Lynch syndrome, is mismatch repair proficient and does not exhibit microsatellite instability. Besides, its genetic etiology remains to be elucidated. In this study we performed germline exome sequencing of 39 cancer-affected patients from 34 families at risk for FCCTX. Variant classification followed the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic/likely pathogenic variants were identified in 17.65% of the families. Rare and potentially pathogenic alterations were identified in known hereditary cancer genes (CHEK2), in putative FCCTX candidate genes (OGG1 and FAN1) and in other cancer-related genes such as ATR, ASXL1, PARK2, SLX4 and TREX1. This study provides novel important clues that can contribute to the understanding of FCCTX genetic basis.

Clinicopathological and molecular characterization of Brazilian families at risk for Lynch syndrome.

Lynch syndrome (LS), is the most common hereditary colorectal cancer syndrome. However, it is poorly characterized in Brazil. Therefore, we aimed to determine the spectrum of pathogenic variants in Mismatch Repair (MMR) genes and investigate the MLH1 promotor methylation role as a second hit in LS tumors. Tumor screening through microsatellite instability and immunohistochemistry for MMR proteins was performed in 323 cases who met clinical criteria. BRAF-V600E and MLH1 promoter methylation were analyzed for all MLH1-deficient tumors. Patients with MMR deficient tumor proceeded to germline genetic testing. MMR deficient tumors were detected in 41% of patients recruited. About half of patients carried a pathogenic germline variant. Two recurrent variants in MLH1 and three novel pathogenic variants were identified. Furthermore, pathogenic germline variants with concomitant somatic MLH1 hypermethylation were found in 6% of cases. Predictive genetic testing was offered to 387 relatives. Overall, 127 tumors were diagnosed in 100 LS patients, from 62 unrelated families. Our molecular data provide new information about the spectrum of MMR mutations, which contributes to a better characterization of LS in Brazil. Furthermore, we call attention to the possibility of failure in the diagnosis of germline MLH1 mutation carriers when somatic MLH1 hypermethylation is used to rule out LS.

Using Co-segregation and Loss of Heterozygosity Analysis to Define the Pathogenicity of Unclassified Variants in Hereditary Breast Cancer Patients.

The use of gene panels introduces a new dilemma in the genetics field due to the high frequency of variants of uncertain significance (VUS). The objective of this study was to provide evidence that may help in the classification of these germline variants in terms of their clinical impact and association with the disease in question. A total of 52 unrelated women at-risk for HBOC and negative for BRCA1/BRCA2 pathogenic variants were evaluated through a gene panel comprising 14 breast and/or ovarian cancer susceptibility genes. Of the 453 germline variants identified, 15 variants (classes 3, 4, and 5) in the ATM, BRIP1, CHEK2, MRE11A, MUTHY, PALB2, RAD50, and RAD51C genes were evaluated via databases, co-segregation studies and loss of heterozygosity in the tumor. The co-segregation analysis allowed the establishment of an association with the presence of variants and the risk of cancer for variant c.316C>T in the BRIP1 gene. Four variants of uncertain significance showed loss of heterozygosity in the tumor (ATM c.4709T>C; CHEK2 c.1036C>T; PALB2 c.1001A>G, and RAD50 c.281T>C), which is an indication of pathogenicity. Thus, the present study provides novel evidence that favors the association of variants in moderate-risk genes with the development of hereditary breast cancer.

The Brazilian TP53 mutation (R337H) and sarcomas.

Sarcomas represent less than 1% of all solid neoplasms in adults and over 20% in children. Their etiology is unclear, but genetic susceptibility plays an important role in this scenario. Sarcoma is central in Li-Fraumeni Syndrome (LFS), a familial predisposition cancer syndrome. In Brazil, the high prevalence of p.Arg337His mutations in the TP53 gene brings about a unique condition: a cluster of LFS. In the present work, we studied 502 sarcoma patients not selected by age or family history in an attempt to assess the impact of the so-called "Brazilian germline TP53 mutation" (p.Arg337His) on this tumor type. We found that 8% of patients are carriers, with leiomyosarcoma being the main histologic type of sarcoma, corresponding to 52.5% of the patients with the mutated TP53 gene. These findings emphasize the importance of genetic counseling and can better guide the management of sarcoma patients.

TP53 variants of uncertain significance: increasing challenges in variant interpretation and genetic counseling.

Li-Fraumeni syndrome (LFS) and Li-Fraumeni Like (LFL) are autosomal dominant cancer predisposition syndromes caused by pathogenic germline variants in the TP53 gene. Recent studies have shown that the incorporation of next-generation sequencing by using multigene panels in clinical practice has resulted in the frequent identification of variants of uncertain significance (VUS). Given that there is no established medical management for VUS carriers, the identification of these variants may cause confusion and anxiety for both patients and practitioners. Herein, we aimed to verify VUS frequency and review VUS classification and interpretation in 1844 patients submitted for comprehensive germline TP53 testing independent of clinical criteria. Variant characterization was done assessing clinical information whenever available, variant frequency in population databases, pathogenicity predictions using in silico tools and previous functional studies. All variants were classified based on the guidelines proposed by the American College of Medical Genetics and Genomics (2015) and by the Sherloc framework (2017). Of the twelve VUS (0.65%) identified in TP53, two were classified as likely pathogenic and two were classified as likely benign after re-evaluation, potentially resulting in significant management modification for the proband and relatives. This report cases highlights the challenges and impact of TP53 variant interpretation especially when there is no clear LFS/LFL phenotype.

Clinical and Molecular Characterization of Surgically Treated Oropharynx Squamous Cell Carcinoma Samples.

A better understanding of the clinical and molecular features of oropharyngeal squamous cell carcinomas (OPSCC) may help in the development of strategies for a better patient management, improving survival rates. This retrospective study conducted a clinical and molecular characterization of surgically treated OPSCC samples. Paraffin-embedded samples from a series of cases were screened for high-risk (HR) human papillomavirus (HPV) infection, methylation of a 5-gene panel, p53 expression, and TP53 mutation. The study was conducted at Barretos Cancer Hospital. Twenty-five surgically treated OPSCC with available tissue were included in the study. Samples were classified according to HPV status and molecular features and some of these characteristics were associated to clinical data. Twenty percent of the cases were HR-HPV positive and 62.5% presented TP53 mutations. DAPK hypermethylation was associated with HPV status (p = 0.023), while methylated CCNA1 was inversely related to TP53 mutations in primary tumors (p = 0.042) and associated with a better disease-free survival (22.3% vs. 100.0%; p = 0.028) and overall survival (8.0% vs. 100.0%; p = 0.012). The results show differences regarding molecular and clinical characteristics in the oropharynx cases identified that should be validated in more cases to confirm whether these differences are able to classify patients according to outcome and help in a more thorough patient management.

Prevalence of BRCA1/BRCA2 mutations in a Brazilian population sample at-risk for hereditary breast cancer and characterization of its genetic ancestry.

BACKGROUND: There are very few data about the mutational profile of families at-risk for hereditary breast and ovarian cancer (HBOC) from Latin America (LA) and especially from Brazil, the largest and most populated country in LA. RESULTS: Of the 349 probands analyzed, 21.5% were BRCA1/BRCA2 mutated, 65.3% at BRCA1 and 34.7% at BRCA2 gene. The mutation c.5266dupC (former 5382insC) was the most frequent alteration, representing 36.7% of the BRCA1 mutations and 24.0% of all mutations identified. Together with the BRCA1 c.3331_3334delCAAG mutation, these mutations constitutes about 35% of the identified mutations and more than 50% of the BRCA1 pathogenic mutations. Interestingly, six new mutations were identified. Additionally, 39 out of the 44 pathogenic mutations identified were not previously reported in the Brazilian population. Besides, 36 different variants of unknown significance (VUS) were identified. Regarding ancestry, average ancestry proportions were 70.6% European, 14.5% African, 8.0% Native American and 6.8% East Asian. MATERIALS AND METHODS: This study characterized 349 Brazilian families at-risk for HBOC regarding their germline BRCA1/BRCA2 status and genetic ancestry. CONCLUSIONS: This is the largest report of BRCA1/BRCA2 assessment in an at-risk HBOC Brazilian population. We identified 21.5% of patients harboring BRCA1/BRCA2 mutations and characterized the genetic ancestry of a sample group at-risk for hereditary breast cancer showing once again how admixed is the Brazilian population. No association was found between genetic ancestry and mutational status. The knowledge of the mutational profile in a population can contribute to the definition of more cost-effective strategies for the identification of HBOC families.