Abstract
BackgroundMultigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene.MethodsWhole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53, or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate.ResultsSixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2, in vitro expression of which caused downstream signaling pathway activation.ConclusionTumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype.
Highlights
Hereditary forms of cancer have been described for decades
Patients and germline DNA samples Patients were eligible for inclusion if they had a personal history of breast and/or ovarian cancer, met the criteria for clinical genetic counseling and testing based on the guidelines of the Belgian Society of Human Genetics and were negative for BRCA1, BRCA2, TP53, and CHEK2 pathogenic mutations
As the overexpression of wild-type ERBB2 has an oncogenic effect which could prevent us from seeing the effect of the mutations, we artificially reduced the number of ERBB2 proteins expressed in each cell, by transfecting a mix (5% ERBB2 plasmid and 95% of empty lentiviral vector) of plasmids into the HEK293T cells using jetPEI® (Polyplus, France) according to the manufacturer’s instructions
Summary
Hereditary forms of cancer have been described for decades. Evidence-based guidelines for screening are applied for suspected hereditary breast and ovarian cancer (HBOC) syndrome, Lynch syndrome, and other conditions [1, 2]. Screening multiple genes simultaneously by massively parallel sequencing is cost-effective and has replaced single-gene sequencing in hereditary breast cancer (HBC). It can reveal mutations in clinically validated genes in up to 5% of cases without BRCA1 or BRCA2 mutations [3]. Its use will probably expand, as recent publications question the validity of established screening criteria given the high number of germline mutations identified in cancer types unrelated to the initial syndrome or in patients lacking family history [4, 5]. Multigene panel testing has a major drawback: the likelihood of identifying a variant of unknown significance (VUS) far exceeds that of discovering a pathogenic mutation, especially as the number of genes tested increases [6]. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene
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