In certain populations, founder pathogenic variants are a common cause of Lynch syndrome. Here, we report the identification of a novel founder variant, NM_000179.3 (MSH6):c.2061T > G (p.Cys687Trp). Our study examined 14 probands and 18 additional family members who carry this variant. With one exception, haplotype data are consistent with a single origin for all heterozygotes. We investigated the clinicopathological consequences of this variant and found that, as previously reported for other MSH6 pathogenic variants a) presence of the founder pathogenic variant is not reliably associated with loss of MSH6 expression or microsatellite instability and b) endometrial cancer is the most common associated phenotype. Based on our findings, we recommend reclassifying MSH6 c.2061T > G from missense variant of unknown significance to likely pathogenic according to CanVIG-UK guidelines.

PURPOSE:Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is an extremely rare, highly aggressive cancer (mean age of onset, 24 years). Nearly all cases are associated with somatic or germline pathogenic variants (GPVs) in SMARCA4. Early bilateral oophorectomy is recommended for unaffected females with a SMARCA4 GPV. However, the penetrance of SMARCA4 GPVs for SCCOHT is highly uncertain and subject to ascertainment bias. METHODS:Leveraging the early-onset, sex-specific, highly morbid nature of SCCOHT, we hypothesized that the penetrance for SCCOHT could be quantified from the deficit in SMARCA4 GPVs in females compared with males in UK Biobank, a population cohort for which recruitment was restricted to those age 40 to 69. We also analyzed pedigrees ascertained internationally by the Montreal-based SCCOHT-SMARCA4 Registry. RESULTS:We observed SMARCA4 GPVs in 8/210,182 (0.0038%) female and 18/179,210 (0.0100%) male participants in UK Biobank (P = .028), representing a male:female odds ratio of 2.64 (95% CI 1.09-7.02), implying a penetrance of 62% for SCCOHT (given the absence of other SMARCA4-related female-specific early morbid diseases). A deficit of GPVs in females in UK Biobank was also demonstrated for BRCA1 and TP53. CONCLUSION:Our findings support bilateral oophorectomy in early adulthood as a rational choice for at-risk females with SMARCA4 GPVs.

SMARCA4 loss leads BRCA1 recruitment and R-Loop accumulation associated with paused Pol II

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive cancer affecting young women driven by inactivating mutations in SMARCA4, a key SWI/SNF chromatin remodeling gene. To uncover its druggable vulnerabilities, we performed a compound screen and found that SCCOHT cells and tumors were sensitive to PARP inhibitors. Paradoxically, SCCOHT displayed BRCA-deficient traits despite retaining wild-type BRCA1 expression. Elevated R-loop in SCCOHT sequestered BRCA1, limiting its availability for DNA damage repair. Proximity-dependent biotin identification revealed that wild-type SMARCA4, but not its pathogenic variants, promoted RNA polymerase II (Pol II) elongation by mediating the assembly of the polymerase-associated factor 1 complex. Thus, SMARCA4 loss increased Pol II pausing, resulting in elevated R-loops and BRCA1 redistribution. The suppression of BRCA1 activity sensitized SMARCA4-deficient SCCOHT cells and tumors to PARP inhibitors, which was further enhanced by the addition of a CDK9 inhibitor targeting Pol II elongation. Cotargeting PARP/CDK9 also elicited synergistic effects against other undifferentiated ovarian cancer cells with SMARCA4 loss. These findings link SMARCA4 loss to perturbed Pol II elongation and compromised DNA repair by BRCA1, providing a therapeutic opportunity to target SCCOHT and other SWI/SNF-deficient ovarian cancers. SIGNIFICANCE:Pol II stalling induced by SMARCA4 loss leads to R-loop accumulation that sequesters BRCA1 to transcription complexes, underlying the sensitivity of SMARCA4-deficient ovarian cancers to inhibitors targeting PARPs and Pol II elongation.

SMARCA4-deficient ovarian cancer cells depend on BRCA1 for their survival

As part of a provincial ministry of health initiative and following the recommendations of the Réseau Québécois de Diagnostic Moléculaire, in-province genomic analyses are offered for defined clinical indications, including for pediatric patients (<18 years) who are diagnosed with cancer. These patients now undergo germline and tumor exome-based sequencing as a standard of care. As part of germline sequencing, two gene panels were developed, one for solid tumors (99 genes), and one for hematological malignancies (69 genes).

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant cancer predisposition syndrome characterized by cutaneous leiomyomas, uterine leiomyomas, and aggressive renal cancer. Germline variants in the fumarate hydratase (FH) gene predispose to HLRCC. Identifying germline pathogenic FH variants enables lifetime renal cancer screening and genetic testing for family members. In this report, we present a FH missense variant (c.1039T>C (p.S347P)), initially classified as a variant of uncertain significance. Clinical assessment, histopathological findings, molecular genetic studies, and enzymatic activity studies support the re-classification of the FH c.1039T>C variant to "pathogenic" based on ACMG/AMP criteria. Further insights into pathological recognition of FH-deficient renal cancer are discussed and should be recognized. This study has shown how (a) detailed multi-disciplinary analyses of a single variant can reclassify rare missense variants in FH and (b) careful pathological review of renal cancers is obligatory when HLRCC is suspected.

Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome associated with germline pathogenic/likely pathogenic variants in TP53. Genotype-phenotype correlations are progressively being characterized in LFS with certain TP53 variants associated with attenuated penetrance and phenotypes. We report on a family harboring a TP53 p.R181H variant presenting with a restricted cancer phenotype in adulthood. The proband was a female with breast cancer at the age of 71 years who had three first degree relatives also diagnosed with breast cancer after the age of 40 years (mother, two sisters). Of the nine individuals harboring the variant (6 genetically confirmed, 3 obligate heterozygous), six have not developed malignancies at this time (age range: 36-42). No childhood-onset cancers were reported in this family. A concomitant literature review identified 51 additional individuals harboring the p.R181H variant in TP53, presenting a tumor phenotype dominated by breast cancer. Rare occurrences of other adult-onset cancers (prostate, colorectal and thyroid) and only few childhood onset cancer were documented. These observations are consistent with functional analysis showing that p.R181H retains partial p53 function and suggesting possible reduced cancer penetrance, particularly in the pediatric setting.

Heterozygous germline pathogenic variants (GPVs) in SMARCA4, the gene encoding the ATP-dependent chromatin remodelling protein SMARCA4 (previously known as BRG1), predispose to several rare tumour types, including small cell carcinoma of the ovary, hypercalcaemic type, atypical teratoid and malignant rhabdoid tumour, and uterine sarcoma. The increase in germline testing of SMARCA4 in recent years has revealed putative GPVs affecting SMARCA4 in patients with other cancer types. Here we describe 11 patients with neuroblastoma (NBL), including 4 previously unreported cases, all of whom were found to harbour heterozygous germline variants in SMARCA4 Median age at diagnosis was 5 years (range 2 months-26 years); nine were male; and eight of nine cases had tumour location information in the adrenal gland. Eight of the germline variants were expected to result in loss of function of SMARCA4 (large deletion, truncating and canonical splice variants), while the remaining four were missense variants. Loss of heterozygosity of the wild-type SMARCA4 allele was found in all eight cases where somatic testing was performed, supporting the notion that SMARCA4 functions as a classic tumour suppressor. Altogether, these findings strongly suggest that NBL should be included in the spectrum of SMARCA4-associated tumours.

Small cell carcinoma of the ovary of hypercalcaemic type (SCCOHT) is a rare and aggressive ovarian neoplasm that is most common in the second and third decades. Molecular studies have established inactivating SMARCA4 alterations as the driver of SCCOHT, these being present in over 95% of these neoplasms. SMARCA4 alterations almost always result in loss of immunoreactivity with SMARCA4 (BRG1) antibody, and this is an extremely useful adjunct in the diagnosis of SCCOHT. Herein, we report 7 cases of SCCOHT (2 from the same patient) with retention of nuclear immunoreactivity with SMARCA4, but with SMARCA4 alterations identified on molecular testing. All cases exhibited loss of SMARCA2 (BRM) immunoreactivity. In addition, following the identification of diffuse TLE1 immunoreactivity in one of these cases (which did not exhibit an SS18 gene rearrangement characteristic of synovial sarcoma), we stained a total of 63 cases of SCCOHT (14 on whole tissue sections: 49 on tissue microarray) with this marker and 7 of 14 (50%) and 22 of 49 (45%) were positive on whole sections and tissue microarray, respectively. Most cases were focally positive but occasional cases exhibited diffuse immunoreactivity. Our observations highlight the importance of SMARCA2 immunohistochemical staining and molecular testing in suspected cases of SCCOHT that exhibit retained SMARCA4 immunoreactivity. Th common expression of TLE1 in these neoplasms represents a potential diagnostic pitfall since synovial sarcoma may be considered in the differential, especially in cases with retained SMARCA4 immunohistochemistry.

IntroductionSmall cell carcinoma of the ovary hypercalcemic type (SCCOHT) is a highly aggressive ovarian malignancy that occurs in young women and is defined by inactivating mutations in SMARCA4. While current treatment modalities only show limited success, emerging evidence suggests that a subset of patients may respond to immunotherapy. Here we set out to assess if methylation profiling can stratify SCCOHT into clinically meaningful subgroups.MethodsWe collected a multicenter series of clinically annotated SCCOHT. Tumor samples were analysed using the Illumina EPIC and 450k BeadChip. Focal copy number score (FCS) was computed from segmented array data using CNApp. Statistical analyses included the Chi-Squared tests and one-way ANOVA.ResultsOur cohort included 27 SCCOHT. The age at diagnosis ranged from 7 – 47 years (n=25, median of 25 years) and 45% of tumors where this information was available (n=20) presented with low stage disease. Clustering analysis of DNA methylation data identified two distinct tumor clusters (C1, n=15 and C2, n=12). C1 was associated with a trend towards younger patient age when compared to C2 (23.7 years vs. 30.2 years). Tumors assigned to C1 also showed a trend towards a lower mean FCS as compared to tumors from C2 (7.4 vs. 12.75). There was no difference in clinical stage between the two clusters.Conclusion/ImplicationsBased on a small series our data suggests that there are two distinct DNA methylation clusters of SCCOHT. Analyses of larger cohorts of SCCOHT are warrented to understand, if the clusters identified correlate with patient survival and/or response to (immuno-)therapy.

Small cell carcinoma of the ovary of hypercalcaemic type (SCCOHT) is a rare and aggressive ovarian neoplasm that is most common in the second and third decades. Molecular studies have established inactivating SMARCA4 alterations as the driver of SCCOHT, these being present in over 95% of these neoplasms. SMARCA4 alterations almost always result in loss of immunoreactivity with SMARCA4 (BRG1) antibody, and this is an extremely useful adjunct in the diagnosis of SCCOHT. Herein, we report 7 cases of SCCOHT (2 from the same patient) with retention of nuclear immunoreactivity with SMARCA4, but with SMARCA4 alterations identified on molecular testing. All cases exhibited loss of SMARCA2 (BRM) immunoreactivity. In addition, following the identification of diffuse TLE1 immunoreactivity in one of these cases (which did not exhibit an SS18 gene rearrangement characteristic of synovial sarcoma), we stained a total of 63 cases of SCCOHT (14 on whole tissue sections: 49 on tissue microarray) with this marker and 7 of 14 (50%) and 22 of 49 (45%) were positive on whole sections and tissue microarray, respectively. Most cases were focally positive but occasional cases exhibited diffuse immunoreactivity. Our observations highlight the importance of SMARCA2 immunohistochemical staining and molecular testing in suspected cases of SCCOHT that exhibit retained SMARCA4 immunoreactivity. Th common expression of TLE1 in these neoplasms represents a potential diagnostic pitfall since synovial sarcoma may be considered in the differential, especially in cases with retained SMARCA4 immunohistochemistry.

Germline pathogenic variants in DICER1 predispose individuals to develop a variety of benign and malignant tumors. Accurate variant curation and classification is essential for reliable diagnosis of DICER1-related tumor predisposition and identification of individuals who may benefit from surveillance. Since 2015, most labs have followed the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) sequence variant classification guidelines for DICER1 germline variant curation. However, these general guidelines lack gene-specific nuances and leave room for subjectivity. Consequently, a group of DICER1 experts joined ClinGen to form the DICER1 and miRNA-Processing Genes Variant Curation Expert Panel (VCEP), to create DICER1- specific ACMG/AMP guidelines for germline variant curation. The VCEP followed the FDA-approved ClinGen protocol for adapting and piloting these guidelines. A diverse set of 40 DICER1 variants were selected for piloting, including 14 known Pathogenic/Likely Pathogenic (P/LP) variants, 12 known Benign/Likely Benign (B/LB) variants, and 14 variants classified as variants of uncertain significance (VUS) or with conflicting interpretations in ClinVar. Clinically meaningful classifications (i.e., P, LP, LB, or B) were achieved for 82.5% (33/40) of the pilot variants, with 100% concordance among the known P/LP and known B/LB variants. Half of the VUS or conflicting variants were resolved with four variants classified as LB and three as LP. These results demonstrate that the DICER1-specific guidelines for germline variant curation effectively classify known pathogenic and benign variants while reducing the frequency of uncertain classifications. Individuals and labs curating DICER1 variants should consider adopting this classification framework to encourage consistency and improve objectivity.

Background: Lung cancer is the leading cause of cancer death in both men and women. Quebec has the highest lung cancer mortality out of all provinces in Canada, believed to be caused by higher smoking rates. Molecular testing for lung cancer is standard of care due to the discovery of actionable driver mutations that can be targeted with tyrosine kinase inhibitors. To date, no detailed molecular testing characterization of Quebec patients with lung cancer using next generation sequencing (NGS) has been performed. Materials and methods: The aim of this study was to describe the genomic landscape of patients with lung cancer (n = 997) who underwent NGS molecular testing at a tertiary care center in Quebec and to correlate it with clinical and pathology variables. Results: Compared to 10 other NGS studies found through a structured search strategy, our cohort had a higher prevalence of KRAS mutations (39.2%) compared to most geographical locations. Additionally, we observed a significant positive association between decreasing age and a higher proportion of KRAS G12C mutations. Conclusion: Overall, it remains important to assess institutional rates of actionable driver mutations to help guide governing bodies, fuel clinical trials and create benchmarks for expected rates as quality metrics.

The endoribonuclease DICER1 plays an essential role in the microRNA (miRNA) biogenesis pathway, cleaving precursor miRNA (pre-miRNA) stem-loops to generate mature single-stranded miRNAs. Germline pathogenic variants (GPVs) in DICER1 result in DICER1 tumor predisposition syndrome (DTPS), a mainly childhood-onset tumor susceptibility disorder. Most DTPS-causing GPVs are nonsense or frameshifting, with tumor development requiring a second somatic missense hit that impairs the DICER1 RNase IIIb domain. Interestingly, germline DICER1 missense variants that cluster in the DICER1 Platform domain have been identified in some persons affected by tumors that also associate with DTPS. Here, we demonstrate that four of these Platform domain variants prevent DICER1 from producing mature miRNAs and as a result impair miRNA-mediated gene silencing. Importantly, we show that in contrast to canonical somatic missense variants that alter DICER1 cleavage activity, DICER1 proteins harboring these Platform variants fail to bind to pre-miRNA stem-loops. Taken together, this work sheds light upon a unique subset of GPVs causing DTPS and provides new insights into how alterations in the DICER1 Platform domain can impact miRNA biogenesis.

Inactivating mutations in SMARCA4 and concurrent epigenetic silencing of SMARCA2 characterize subsets of ovarian and lung cancers. Concomitant loss of these key subunits of SWI/SNF chromatin remodeling complexes in both cancers is associated with chemotherapy resistance and poor prognosis. Here, we discover that SMARCA4/2 loss inhibits chemotherapy-induced apoptosis through disrupting intracellular organelle calcium ion (Ca 2+ ) release in these cancers. By restricting chromatin accessibility to ITPR3 , encoding Ca 2+ channel IP3R3, SMARCA4/2 deficiency causes reduced IP3R3 expression leading to impaired Ca 2+ transfer from the endoplasmic reticulum to mitochondria required for apoptosis induction. Reactivation of SMARCA2 by a histone deacetylase inhibitor rescues IP3R3 expression and enhances cisplatin response in SMARCA4/2-deficient cancer cells both in vitro and in vivo. Our findings elucidate the contribution of SMARCA4/2 to Ca 2+ -dependent apoptosis induction, which may be exploited to enhance chemotherapy response in SMARCA4/2-deficient cancers.

Supplementary Data from Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer