Mosaic BRCA1 promoter methylation contribution in hereditary breast/ovarian cancer pedigrees.

PURPOSE: Mosaic BRCA1 promoter methylation (BRCA1meth) increases the risk of early-onset breast cancer, triple-negative breast cancer and ovarian cancer. As mosaic BRCA1meth are believed to occur de novo, their role in family breast/ovarian cancer has not been assessed. PATIENTS: Blood-derived DNA from 20 unrelated affected cases from families with aggregation of breast/ovarian cancer, but with no germline pathogenic variants in BRCA1/2, PALB2 or RAD51C/D, were screened by methylation-sensitive high-resolution melting. CpG analysis was performed by pyrosequencing on blood and buccal swab. Two probands carried a pathogenic variant in a moderate-penetrance gene (ATM and BARD1), and 8 of 18 others (44%) carried BRCA1meth (vs none of the 20 age-matched controls). Involvement of BRCA1 in tumourigenesis in methylated probands was demonstrated in most tested cases by detection of a loss of heterozygosity and a homologous recombination deficiency signature. Among the eight methylated probands, two had relatives with breast cancer with detectable BRCA1meth in blood, including one with high methylation levels in two non-tumour tissues. CONCLUSIONS: The high prevalence of mosaic BRCA1meth in patients with breast/ovarian cancer with affected relatives, as well as this first description of a family aggregation of mosaic BRCA1meth, shows how this de novo event can contribute to hereditary breast/ovarian cancer pedigrees.

First report of medulloblastoma in a patient with MUTYH-associated polyposis.

AIMS: The mutY DNA glycosylase encoded by the MUTYH gene prevents G:C → T:A transversions through the base excision repair DNA repair system. Germline biallelic pathogenic variants in MUTYH cause an adenomatous polyposis called MUTYH-associated polyposis (MAP), an autosomal recessive disease (OMIM: 608456), with an increased risk of colorectal cancer. Digestive lesions in this context show an excess of G:C → T:A transversions, individualising a specific mutational signature associated with MUTYH deficiency called signature SBS36. Predisposition to other tumours in patients with germline biallelic pathogenic variants in MUTYH is suspected but remains unclear. We report the first case of medulloblastoma in a patient with MAP, carrying the homozygous pathogenic variant c.1227_1228dup, p.(Glu410Glyfs*43) in MUTYH. METHODS: Whole exome sequencing was performed on the medulloblastoma to enlighten single nucleotide variants of interest, microsatellite status and mutational signature. The objective was to determine the involvement of MUTYH deficiency in the oncogenesis of this medulloblastoma. RESULTS: The medulloblastoma has the mutational signature SBS36 and driver pathogenic variants in CTNNB1, PTCH1 and KDM6A corresponding to G:C → T:A transversions, suggesting a role of MUTYH deficiency in oncogenesis. CONCLUSIONS: Therefore, medulloblastoma could be a rare manifestation associated with germline biallelic pathogenic variants in MUTYH.

MSH3: a confirmed predisposing gene for adenomatous polyposis.

BACKGROUND: The MSH3 gene is part of the DNA mismatch repair system, but has never been shown to be involved in Lynch syndrome. A first report of four patients from two families, bearing biallelic MSH3 germline variants, with a phenotype of attenuated colorectal adenomatous polyposis raised the question of its involvement in hereditary cancer predisposition. The patients' tumours exhibited elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), a hallmark of MSH3 deficiency. METHODS: We report five new unrelated patients with MSH3-associated polyposis. We describe their personal and familial history and study the EMAST phenotype in various normal and tumour samples, which are relevant findings based on the rarity of this polyposis subtype so far. RESULTS: All patients had attenuated colorectal adenomatous polyposis, with duodenal polyposis in two cases. Both women had breast carcinomas. EMAST phenotype was present at various levels in different samples of the five patients, confirming the MSH3 deficiency, with a gradient of instability in polyps depending on their degree of dysplasia. The negative EMAST phenotype ruled out the diagnosis of germline MSH3 deficiency for two patients: one homozygous for a benign variant and one with a monoallelic large deletion. CONCLUSION: This report lends further credence to biallelic MSH3 germline pathogenic variants being involved in colorectal and duodenal adenomatous polyposis. Large-scale studies may help clarify the tumour spectrum and associated risks. Ascertainment of EMAST may help with the interpretation of variants of unknown significance. We recommend adding MSH3 to dedicated diagnostic gene panels.

Hereditary cancer predispositions: Comparison of multigene panel sequencing on fresh-frozen breast/ovarian tumor versus blood.

In breast or ovarian cancer (BC/OC) patients with evocative personal and/or family history, multigene panel sequencing is performed on blood to diagnose hereditary predispositions. Additionally, BRCA1/BRCA2 testing can be performed on tumor sample for therapeutic purpose. The accuracy of multigene panel tumor analysis on BC/OC to detect predisposing germline pathogenic variants (gPV) has not been precisely assessed. By comparing sequencing data from blood and fresh-frozen tumor we show that tumor genomic instability causes pitfalls to consider when performing tumor testing to detect gPV. Even if loss of heterozygosity increases germline signal in most cases, somatic copy number variants (CNV) can mask germline CNV and collapse point gPV variant allele frequency (VAF). Moreover, VAF does not allow an accurate distinction between germline and somatic pathogenic variants.

APC germline pathogenic variants and epithelial ovarian cancer: causal or coincidental findings?

APC germline pathogenic variants result in predisposition to familial adenomatous polyposis and extraintestinal tumours such as desmoid fibromatosis, medulloblastomas and thyroid cancers. They have also been recently involved in ovarian microcystic stromal tumours. APC inactivation has been described at the tumour level in epithelial ovarian cancers (EOCs). Here, we report the identification of APC germline pathogenic variants in two patients diagnosed with premenopausal EOC in early 30s, with no other pathogenic variant detected in the known ovarian cancer predisposing genes. Subsequent tumour analysis showed neither a second hit of APC inactivation nor ß-catenin activation. Both tumours did not have a homologous recombination (HR) deficiency, pointing towards the implication of other genes than those involved in HR. APC may contribute to the carcinogenesis of EOC in a multifactorial context. Further studies are required to clarify the role of APC in predisposition to EOC.

The PI3K/mTOR Pathway Is Targeted by Rare Germline Variants in Patients with Both Melanoma and Renal Cell Carcinoma.

Background: Malignant melanoma and RCC have different embryonic origins, no common lifestyle risk factors but intriguingly share biological properties such as immune regulation and radioresistance. An excess risk of malignant melanoma is observed in RCC patients and vice versa. This bidirectional association is poorly understood, and hypothetic genetic co-susceptibility remains largely unexplored. Results: We hereby provide a clinical and genetic description of a series of 125 cases affected by both malignant melanoma and RCC. Clinical germline mutation testing identified a pathogenic variant in a melanoma and/or RCC predisposing gene in 17/125 cases (13.6%). This included mutually exclusive variants in MITF (p.E318K locus, N = 9 cases), BAP1 (N = 3), CDKN2A (N = 2), FLCN (N = 2), and PTEN (N = 1). A subset of 46 early-onset cases, without underlying germline variation, was whole-exome sequenced. In this series, thirteen genes were significantly enriched in mostly exclusive rare variants predicted to be deleterious, compared to 19,751 controls of similar ancestry. The observed variation mainly consisted of novel or low-frequency variants (<0.01%) within genes displaying strong evolutionary mutational constraints along the PI3K/mTOR pathway, including PIK3CD, NFRKB, EP300, MTOR, and related epigenetic modifier SETD2. The screening of independently processed germline exomes from The Cancer Genome Atlas confirmed an association with melanoma and RCC but not with cancers of established differing etiology such as lung cancers. Conclusions: Our study highlights that an exome-wide case-control enrichment approach may better characterize the rare variant-based missing heritability of multiple primary cancers. In our series, the co-occurrence of malignant melanoma and RCC was associated with germline variation in the PI3K/mTOR signaling cascade, with potential relevance for early diagnostic and clinical management.

Genomic Instability Signature of Palindromic Non-Coding Somatic Mutations in Bladder Cancer.

Numerous pan-genomic studies identified alterations in protein-coding genes and signaling pathways involved in bladder carcinogenesis, while non-coding somatic alterations remain weakly explored. The goal of this study was to identify clinical biomarkers in non-coding regions for bladder cancer patients. We have previously identified in bladder tumors two non-coding mutational hotspots occurring at high frequencies (≥30%). These mutations are located close to the GPR126 and PLEKHS1 genes, at the guanine or the cytosine of a TGAACA core motif flanked, on both sides, by a stretch of palindromic sequences. Here, we hypothesize that such a pattern of recurrent non-coding mutations could be a signature of somatic genomic instability specifically involved in bladder cancer. We analyzed 26 additional mutable non-coding sites with the same core motif in a cohort of 103 bladder cancers composed of 44 NMIBC cases and 59 MIBC cases using high-resolution melting (HRM) and Sanger sequencing. Five bladder cancers were additionally analyzed for protein-coding gene mutations using a targeted NGS panel composed of 571 genes. Expression levels of three members of the APOBEC3 family genes were assessed using real-time quantitative RT-PCR. Non-coding somatic mutations were observed for at least one TGAACA core motif locus in 62.1% (64/103) of bladder tumor samples. These non-coding mutations co-occurred in the bladder tumors but were absent in prostate tumor, HPV-positive Head and Neck Squamous Cell Carcinoma, and high microsatellite instability (MSI-H) colorectal tumor series. This signature of palindromic non-coding somatic mutations, specific to bladder tumors, was not associated with patients' outcome and was more frequent in females. Interestingly, this signature was associated with high tumor mutational burden (TMB) and high expression levels of APOBEC3B and interferon inducible genes. We identified a new type of somatic genomic instability targeting the TGAACA core motif loci flanked by palindromic sequences in bladder cancer. This mutational signature is a promising candidate clinical biomarker for the early detection of relapse and a major low-cost alternative to the TMB to monitor the response to immunotherapy for bladder cancer patients.

Comparison of Pathogenicity Prediction Tools on Somatic Variants.

Genomic sequencing is increasingly used in managing patients with cancer. Interpretation of somatic variants and their pathogenicity is often complex. Pathogenicity prediction tools are commonly used as part of the expert interpretation of somatic variants, but most of these tools were initially developed for germline variants. Our aim was to benchmark their performance for somatic variants. A gold standard list was assembled of 4319 somatic single-nucleotide variants, classified as oncogenic (n = 2996) or neutral (n = 1323), based on their presence in curated databases or on their allele frequency in the general population. These variants were annotated with the most commonly used prediction tools [Database for Nonsynonymous SNPs' Functional Predictions (dbNSFP) and Universal Mutation Database Predictor (UMD-Predictor)] and computed performance calculations. Stratification of the prediction tools based on Matthews correlation coefficient and area under the receiver operating characteristic curve allowed the identification of the top-performing ones, namely, Combined Annotation-Dependent Depletion (CADD), Eigen or Eigen Principal Components (Eigen-PC), Polymorphism Phenotyping version 2 (PolyPhen-2), Protein Variation Effect Analyzer (PROVEAN), UMD-Predictor, and Rare Exome Variant Ensemble Learner (REVEL). Interestingly, Sorting Intolerant From Tolerant (SIFT), which is a commonly used prediction tool for somatic variants, was ranked in the second performance category. Combining tools two by two only marginally improved performances, mainly because of the occurrence of discordant predictions.