Tumor Sequencing Is Useful to Refine the Analysis of Germline Variants in Unexplained High-Risk Breast Cancer Families

Cédric Van Marcke; Raphaël Helaers; Anne De Leener; Ahmad Merhi; Céline A. Schoonjans; Jérôme Ambroise; Christine Galant; Paul Delrée; Françoise Rothé; Isabelle Bar; Elsa Khoury; Pascal Brouillard; Jean-Luc Canon; Peter Vuylsteke; Jean-Pascal Machiels; Martine Berlière; Nisha Limaye; Miikka Vikkula; François P. Duhoux


Breast Cancer Res. 2020;22(36) 

In This Article

Abstract and Introduction


Background: Multigene 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.

Methods: Whole-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.

Results: Sixty-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.

Conclusion: Tumor 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.


Hereditary forms of cancer have been described for decades. Evidence-based guidelines for screening are now 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] However, 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] Indeed, several converging arguments are required to define pathogenicity of a variant.[7,8]

Taking VUS into consideration is a daily clinical challenge. It has a major impact on the preventive screening or treatment strategy; therefore, misinterpretation of a VUS can be physically or psychologically harmful.[9] Functional testing helps reclassify VUS and is trending in translational studies,[10] but feasibility on a clinical scale is sparse and not yet implemented.[11] Large international consortia like ENIGMA aim to reclassify variants by gathering genotypic and phenotypic data from various sources, recognizing that the rarity of the variants is the main issue.[12]

Current variant classification guidelines do not include analysis of matched tumor samples. Yet, the two-hit theory for inherited cancer predisposition conferred by heterozygous, germline mutations in tumor suppressor genes postulates that the normal allele is locally lost or outcompeted by the mutant allele, due to a second, somatic variation in the same gene. These may be copy number alterations (CNAs), pathogenic point mutations, small insertions/deletions (INDELs), or epigenetic modifications that reduce the expression or function of the normal allele, or increase that of the germline mutant.[13] We therefore hypothesized that matched tumor sequencing could serve as an argument to study the implication of germline variants in the development of cancer in HBC patients, based on the presence of somatic events in the same gene.