Tumor analysis of MMR genes in Lynch-like syndrome: Challenges associated with results interpretation.

BACKGROUND: Up to 70% of suspected Lynch syndrome patients harboring MMR deficient tumors lack identifiable germline pathogenic variants in MMR genes, being referred to as Lynch-like syndrome (LLS). Previous studies have reported biallelic somatic MMR inactivation in a variable range of LLS-associated tumors. Moreover, translating tumor testing results into patient management remains controversial. Our aim is to assess the challenges associated with the implementation of tumoral MMR gene testing in routine workflows. METHODS: Here, we present the clinical characterization of 229 LLS patients. MMR gene testing was performed in 39 available tumors, and results were analyzed using two variant allele frequency (VAF) thresholds (≥5% and ≥10%). RESULTS AND DISCUSSION: More biallelic somatic events were identified at VAF ≥ 5% than ≥10% (35.9% vs. 25.6%), although the rate of nonconcordant results regarding immunohistochemical pattern increased (30.8% vs. 20.5%). Interpretation difficulties question the current utility of the identification of MMR somatic hits in the diagnostic algorithm of suspected LS cases.

Biological basis of extensive pleiotropy between blood traits and cancer risk.

BACKGROUND: The immune system has a central role in preventing carcinogenesis. Alteration of systemic immune cell levels may increase cancer risk. However, the extent to which common genetic variation influences blood traits and cancer risk remains largely undetermined. Here, we identify pleiotropic variants and predict their underlying molecular and cellular alterations. METHODS: Multivariate Cox regression was used to evaluate associations between blood traits and cancer diagnosis in cases in the UK Biobank. Shared genetic variants were identified from the summary statistics of the genome-wide association studies of 27 blood traits and 27 cancer types and subtypes, applying the conditional/conjunctional false-discovery rate approach. Analysis of genomic positions, expression quantitative trait loci, enhancers, regulatory marks, functionally defined gene sets, and bulk- and single-cell expression profiles predicted the biological impact of pleiotropic variants. Plasma small RNAs were sequenced to assess association with cancer diagnosis. RESULTS: The study identified 4093 common genetic variants, involving 1248 gene loci, that contributed to blood-cancer pleiotropism. Genomic hotspots of pleiotropism include chromosomal regions 5p15-TERT and 6p21-HLA. Genes whose products are involved in regulating telomere length are found to be enriched in pleiotropic variants. Pleiotropic gene candidates are frequently linked to transcriptional programs that regulate hematopoiesis and define progenitor cell states of immune system development. Perturbation of the myeloid lineage is indicated by pleiotropic associations with defined master regulators and cell alterations. Eosinophil count is inversely associated with cancer risk. A high frequency of pleiotropic associations is also centered on the regulation of small noncoding Y-RNAs. Predicted pleiotropic Y-RNAs show specific regulatory marks and are overabundant in the normal tissue and blood of cancer patients. Analysis of plasma small RNAs in women who developed breast cancer indicates there is an overabundance of Y-RNA preceding neoplasm diagnosis. CONCLUSIONS: This study reveals extensive pleiotropism between blood traits and cancer risk. Pleiotropism is linked to factors and processes involved in hematopoietic development and immune system function, including components of the major histocompatibility complexes, and regulators of telomere length and myeloid lineage. Deregulation of Y-RNAs is also associated with pleiotropism. Overexpression of these elements might indicate increased cancer risk.

RNA assay identifies a previous misclassification of BARD1 c.1977A>G variant.

Case-control studies have shown an association of BARD1 with hereditary breast and/or ovarian cancer (HBOC) predisposition. BARD1 alternatively spliced isoforms are abundant and some are highly expressed in different cancer types. In addition, a number of BARD1 germline pathogenic variants have been reported among HBOC patients. In previous reports, BARD1 c.1977A>G variant has been classified as pathogenic since it produces a frameshift transcript lacking exons 2 to 9. In the present study, we sought to validate the mRNA splicing results previously published and to contribute with new evidence to refine the classification of this substitution according to ACMG/AMP guidelines. The presence of the variant was screened in patients and controls. RT-PCR was performed in order to compare the transcriptional profiles of two variant carriers and ten non-carrier controls. In addition, allele-specific expression was assessed. No differences in variant frequency were detected between patients and controls. The RNA assay confirmed the presence of the shorter transcript lacking exons 2-9, but it was detected both in carriers and non-carriers. Furthermore, allelic imbalance was discarded and no significant differences in the proportion of full-length and shorter transcript were detected between carriers and controls. The shorter transcript detected corresponds to BARD1 isoform η, constituted by exons 1, 10 and 11. Our results support that this transcript is a constitutive splicing product rather than an aberrant transcript caused by BARD1 c.1977A>G variant, and for this reason this variant should be considered as likely benign following ACMG/AMP guidelines.

Paired Somatic-Germline Testing of 15 Polyposis and Colorectal Cancer-Predisposing Genes Highlights the Role of APC Mosaicism in de Novo Familial Adenomatous Polyposis.

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome responsible for 1% of colorectal cancers (CRCs). Up to 90% of classic FAPs are caused by inactivating mutations in APC, and mosaicism has been previously reported in 20% of de novo cases, usually linked to milder phenotypic manifestations. This study aimed to explore the prevalence of mosaicism in 11 unsolved cases of classic FAP and to evaluate the diagnostic yield of somatic testing. Paired samples of colorectal polyps, tumors, and/or mucosa were analyzed using a custom next-generation sequencing panel targeting 15 polyposis and CRC-predisposing genes. Whenever possible, the extension of mosaicism to blood or sperm was also examined. Of 11 patients with classic adenomatous polyposis, a mosaic pathogenic variant in APC was identified in 7 (64%). No other altered genes were identified. In two of seven patients (29%), mosaicism was found restricted to colonic tissues, whereas in five of seven patients (71%), it was extended to the blood. Germline affectation was confirmed in one patient. We report the first analysis at a somatic level of 15 genes associated with CRC susceptibility, which highlights the role of APC mosaicism in classic FAP etiology. The results further reinforce the importance of testing target tissues when blood test results are negative.

BARD1 Pathogenic Variants are Associated with Triple-Negative Breast Cancer in a Spanish Hereditary Breast and Ovarian Cancer Cohort.

Only a small fraction of hereditary breast and/or ovarian cancer (HBOC) cases are caused by germline variants in the high-penetrance breast cancer 1 and 2 genes (BRCA1 and BRCA2). BRCA1-associated ring domain 1 (BARD1), nuclear partner of BRCA1, has been suggested as a potential HBOC risk gene, although its prevalence and penetrance are variable according to populations and type of tumor. We aimed to investigate the prevalence of BARD1 truncating variants in a cohort of patients with clinical suspicion of HBOC. A comprehensive BARD1 screening by multigene panel analysis was performed in 4015 unrelated patients according to our regional guidelines for genetic testing in hereditary cancer. In addition, 51,202 Genome Aggregation Database (gnomAD) non-Finnish, non-cancer European individuals were used as a control population. In our patient cohort, we identified 19 patients with heterozygous BARD1 truncating variants (0.47%), whereas the frequency observed in the gnomAD controls was 0.12%. We found a statistically significant association of truncating BARD1 variants with overall risk (odds ratio (OR) = 3.78; CI = 2.10-6.48; p = 1.16 × 10-5). This association remained significant in the hereditary breast cancer (HBC) group (OR = 4.18; CI = 2.10-7.70; p = 5.45 × 10-5). Furthermore, deleterious BARD1 variants were enriched among triple-negative BC patients (OR = 5.40; CI = 1.77-18.15; p = 0.001) compared to other BC subtypes. Our results support the role of BARD1 as a moderate penetrance BC predisposing gene and highlight a stronger association with triple-negative tumors.

ERCC3, a new ovarian cancer susceptibility gene?

BACKGROUND: Hereditary breast and ovarian cancer syndrome (HBOC) is an inherited disorder with an increased risk of breast cancer (BC) and ovarian cancers (OC). Mutations in BRCA1-BRCA2 explains less than a half of cases. In the last decade several genes with different penetrance have been associated with an increased risk of BC or OC. A recurrent heterozygous ERCC3 truncating mutation increases the risk for breast cancer in patients with Ashkenazi Jewish ancestry. Our study aimed to investigate the role of ERCC3 truncating variants in a cohort of patients with suspicion of HBOC. PATIENTS AND METHODS: ERCC3 screening by multigene-panel analysis in 1311 unrelated patients after our regional consensus for genetic testing in hereditary cancer was done. In addition, 453 Spanish cancer-free individuals and 51,343 GnomAD non-Finnish, non-cancer European individuals were used as control populations. RESULTS: We identified 13 patients with heterozygous ERCC3 truncating variants (0.99%). Five of them also carried a mutation in a high- /moderate-penetrance HBOC gene (BRCA1, BRCA2, CHEK2, and TP53) being Multilocus Inherited Neoplasia Alleles syndrome (MINAS) patients. The frequency in 453 Spanish controls was of 0.22%; similar to that observed in 51,343 non-Finnish European GnomAD population (0.24%). We found an almost statistically significant association of truncating ERCC3 variants with BC (odds ratio [OR] = 2.25, confidence interval [CI] = 0.6-5.93, P = 0.11), and we observed for the first time a significant association with OC (OR = 4.74, CI = 1-14.34, P = 0.028), that holds even after removing MINAS cases. CONCLUSIONS: To our knowledge, this is the largest HBOC series comprehensively analysed for ERCC3 mutations, and the first study identifying ERCC3 as a cancer risk for OC.

Improving Genetic Testing in Hereditary Cancer by RNA Analysis: Tools to Prioritize Splicing Studies and Challenges in Applying American College of Medical Genetics and Genomics Guidelines.

RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.

Comprehensive analysis and ACMG-based classification of CHEK2 variants in hereditary cancer patients.

CHEK2 variants are associated with intermediate breast cancer risk, among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1848 prospective patients with HC suspicion. We refined ACMG-AMP criteria and applied different combined rules to classify CHEK2 variants and define risk alleles. We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; two can also be considered "established risk-alleles" and one as "likely risk-allele." The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary nonpolyposis colorectal cancer patients). Here, we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this study would be useful for variant classification of other genes with low effect variants.

Exploring the Role of Mutations in Fanconi Anemia Genes in Hereditary Cancer Patients.

Fanconi anemia (FA) is caused by biallelic mutations in FA genes. Monoallelic mutations in five of these genes (BRCA1, BRCA2, PALB2, BRIP1 and RAD51C) increase the susceptibility to breast/ovarian cancer and are used in clinical diagnostics as bona-fide hereditary cancer genes. Increasing evidence suggests that monoallelic mutations in other FA genes could predispose to tumor development, especially breast cancer. The objective of this study is to assess the mutational spectrum of 14 additional FA genes (FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, FANCP, FANCQ, FANCR and FANCU) in a cohort of hereditary cancer patients, to compare with local cancer-free controls as well as GnomAD. A total of 1021 hereditary cancer patients and 194 controls were analyzed using our next generation custom sequencing panel. We identified 35 pathogenic variants in eight genes. A significant association with the risk of breast cancer/breast and ovarian cancer was found for carriers of FANCA mutations (odds ratio (OR) = 3.14 95% confidence interval (CI) 1.4-6.17, p = 0.003). Two patients with early-onset cancer showed a pathogenic FA variant in addition to another germline mutation, suggesting a modifier role for FA variants. Our results encourage a comprehensive analysis of FA genes in larger studies to better assess their role in cancer risk.

Does multilocus inherited neoplasia alleles syndrome have severe clinical expression?

IMPORTANCE: Genetic testing of hereditary cancer using comprehensive gene panels can identify patients with more than one pathogenic mutation in high and/or moderate-risk-associated cancer genes. This phenomenon is known as multilocus inherited neoplasia alleles syndrome (MINAS), which has been potentially linked to more severe clinical manifestations. OBJECTIVE: To determine the prevalence and clinical features of MINAS in a large cohort of adult patients with hereditary cancer homogeneously tested with the same gene panel. PATIENTS AND METHODS: A cohort of 1023 unrelated patients with suspicion of hereditary cancer was screened using a validated panel including up to 135 genes associated with hereditary cancer and phakomatoses. RESULTS: Thirteen (1.37%) patients harbouring two pathogenic mutations in dominant cancer-predisposing genes were identified, representing 5.7% (13/226) of patients with pathogenic mutations. Most (10/13) of these cases presented clinical manifestations associated with only one of the mutations identified. One case showed mutations in MEN1 and MLH1 and developed tumours associated with both cancer syndromes. Interestingly, three of the double mutants had a young age of onset or severe breast cancer phenotype and carried mutations in moderate to low-risk DNA damage repair-associated genes; two of them presented biallelic inactivation of CHEK2. We included these two patients for the sake of their clinical interest although we are aware that they do not exactly fulfil the definition of MINAS since both mutations are in the same gene. CONCLUSIONS AND RELEVANCE: Genetic analysis of a broad cancer gene panel identified the largest series of patients with MINAS described in a single study. Overall, our data do not support the existence of more severe manifestations in double mutants at the time of diagnosis although they do confirm previous evidence of severe phenotype in biallelic CHEK2 and other DNA repair cancer-predisposing genes.

Novel genetic mutations detected by multigene panel are associated with hereditary colorectal cancer predisposition.

Half of the high-risk colorectal cancer families that fulfill the clinical criteria for Lynch syndrome lack germline mutations in the mismatch repair (MMR) genes and remain unexplained. Genetic testing for hereditary cancers is rapidly evolving due to the introduction of multigene panels, which may identify more mutations than the old screening methods. The aim of this study is the use of a Next Generation Sequencing panel in order to find the genes involved in the cancer predisposition of these families. For this study, 98 patients from these unexplained families were tested with a multigene panel targeting 94 genes involved in cancer predisposition. The mutations found were validated by Sanger sequencing and the segregation was studied when possible. We identified 19 likely pathogenic variants in 18 patients. Out of these, 8 were found in MMR genes (5 in MLH1, 1 in MSH6 and 2 in PMS2). In addition, 11 mutations were detected in other genes, including high penetrance genes (APC, SMAD4 and TP53) and moderate penetrance genes (BRIP1, CHEK2, MUTYH, HNF1A and XPC). Mutations c.1194G>A in SMAD4, c.714_720dup in PMS2, c.2050T>G in MLH1 and c.1635_1636del in MSH6 were novel. In conclusion, the detection of new pathogenic mutations in high and moderate penetrance genes could contribute to the explanation of the heritability of colorectal cancer, changing the individual clinical management. Multigene panel testing is a more effective method to identify germline variants in cancer patients compared to single-gene approaches and should be therefore included in clinical laboratories.