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Many variants that we inherit from our parents or acquire de novo or somatically are rare, limiting the precision with which we can associate them with disease. We performed exhaustive saturation genome editing (SGE) of BAP1, the disruption of which is linked to tumorigenesis and altered neurodevelopment. We experimentally characterized 18,108 unique variants, of which 6,196 were found to have abnormal functions, and then used these data to evaluate phenotypic associations in the UK Biobank. We also characterized variants in a large population-ascertained tumor collection, in cancer pedigrees and ClinVar, and explored the behavior of cancer-associated variants compared to that of variants linked to neurodevelopmental phenotypes. Our analyses demonstrated that disruptive germline BAP1variants were significantly associated with higher circulating levels of the mitogen IGF-1, suggesting a possible pathological mechanism and therapeutic target. Furthermore, we built a variant classifier with >98% sensitivity and specificity and quantify evidence strengths to aid precision variant interpretation.

Two independent exome sequencing initiatives aimed to identify new genes involved in the predisposition to nonpolyposis colorectal cancer led to the identification of heterozygous loss-of-function variants in NPAT, a gene that encodes a cyclin E/CDK2 effector required for S phase entry and a coactivator of histone transcription, in two families with multiple members affected with colorectal cancer. Enrichment of loss-of-function and predicted deleterious NPATvariants was identified in familial/early-onset colorectal cancer patients compared to non-cancer gnomAD individuals, further supporting the association with the disease. Previous studies in Drosophila models showed that NPATabrogation results in chromosomal instability, increase of double strand breaks, and induction of tumour formation. In line with these results, colorectal cancers with NPATsomatic variants and no DNA repair defects have significantly higher aneuploidy levels than NPAT-wildtype colorectal cancers. In conclusion, our findings suggest that constitutional inactivating NPATvariants predispose to mismatch repair-proficient nonpolyposis colorectal cancer.

Screening for Lynch syndrome (LS) in colorectal cancer (CRC) and endometrial cancer patients generally involves immunohistochemical staining of the mismatch repair (MMR) proteins. In case of MLH1 protein loss, MLH1promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1variants from somatic epimutations. Recently, multiple studies have reported that MLH1-PM and pathogenic constitutional MMR variants are not mutually exclusive. This study describes 6 new and 86 previously reported MLH1-PM CRCs or endometrial cancers in LS patients. Of these, methylation of the MLH1gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2variant carriers at a median age at diagnosis of 48.5 years [interquartile range (IQR), 39–56.75 years], 39 years (IQR, 29–51 years), 58 years (IQR, 53.5–67 years), and 68 years (IQR, 65.6–68.5 years), respectively. For 31 MLH1-PM CRCs in LS patients from the literature, only the B region of the MLH1gene promotor was tested, whereas for 13 cases in the literature the tested region was not specified. Collectively, these data indicate that a diagnosis of LS should not be excluded when MLH1-PM is detected. Clinicians should carefully consider whether follow-up genetic MMR gene testing should be offered, with age <60 to 70 years and/or a positive family history among other factors being suggestive for a potential constitutional MMR gene defect.

BRCA1-associated protein-1 (BAP1) is a recognised tumour suppressor gene. Germline BAP1pathogenic/likely pathogenic variants are associated with predisposition to multiple tumours, including uveal melanoma, malignant pleural and peritoneal mesothelioma, renal cell carcinoma and specific non-malignant neoplasms of the skin, as part of the autosomal dominant BAP1-tumour predisposition syndrome. The overall lifetime risk for BAP1carriers to develop at least one BAP1-associated tumour is up to 85%, although due to ascertainment bias, current estimates of risk are likely to be overestimated. As for many rare cancer predisposition syndromes, there is limited scientific evidence to support the utility of surveillance and, therefore, management recommendations for BAP1carriers are based on expert opinion. To date, European recommendations for BAP1carriers have not been published but are necessary due to the emerging phenotype of this recently described syndrome and increased identification of BAP1carriers via large gene panels or tumour sequencing. To address this, the Clinical Guideline Working Group of the CanGene-CanVar project in the United Kingdom invited European collaborators to collaborate to develop guidelines to harmonize surveillance programmes within Europe. Recommendations with respect to BAP1testing and surveillance were achieved following literature review and Delphi survey completed by a core group and an extended expert group of 34 European specialists including Geneticists, Ophthalmologists, Oncologists, Dermatologists and Pathologists. It is recognised that these largely evidence-based but pragmatic recommendations will evolve over time as further data from research collaborations informs the phenotypic spectrum and surveillance outcomes.

The most common malignancies that develop in carriers of BAP1germline mutations include diffuse malignant mesothelioma, uveal and cutaneous melanoma, renal cell carcinoma, and less frequently, breast cancer, several types of skin carcinomas, and other tumor types. Mesotheliomas in these patients are significantly less aggressive, and patients require a multidisciplinary approach that involves genetic counseling, medical genetics, pathology, surgical, medical, and radiation oncology expertise. Some BAP1carriers have asymptomatic mesothelioma that can be followed by close clinical observation without apparent adverse outcomes: they may survive many years without therapy. Others may grow aggressively but very often respond to therapy. Detecting BAP1germline mutations has, therefore, substantial medical, social, and economic impact. Close monitoring of these patients and their relatives is expected to result in prolonged life expectancy, improved quality of life, and being cost-effective.

Universal tumor screening in endometrial carcinoma (EC) is increasingly adopted to identify individuals at risk of Lynch syndrome (LS). These cases involve mismatch repair-deficient (MMRd) EC without MLH1promoter hypermethylation (PHM). LS is confirmed through the identification of germline MMR pathogenic variants (PV). In cases where these are not detected, emerging evidence highlights the significance of double-somatic MMR gene alterations as a sporadic cause of MMRd, alongside POLE/POLD1exonuclease domain (EDM) PV leading to secondary MMR PV. Our understanding of the incidence of different MMRd EC origins not related to MLH1-PHM, their associations with clinicopathologic characteristics, and the prognostic implications remains limited.

To determine impact of risk-reducing hysterectomy and bilateral salpingo-oophorectomy (BSO) on gynecological cancer incidence and death in heterozygotes of pathogenic MMR (path_MMR) variants.

AimsPrevious estimates of the prevalence of mismatch repair (MMR) deficiency and Lynch syndrome in small bowel cancer have varied widely. The aim of this study was to establish the prevalence of MMR deficiency and Lynch syndrome in a large group of small bowel adenocarcinomas.MethodsTo this end, a total of 400 small bowel adenocarcinomas (332 resections, 68 biopsies) were collected through the Dutch nationwide registry of histopathology and cytopathology (Pathologisch-Anatomisch Landelijk Geautomatiseerd Archief (PALGA)). No preselection criteria, such as family history, were applied, thus avoiding (ascertainment) bias. MMR deficiency status was determined by immunohistochemical staining of MMR proteins, supplemented by MLH1promoter hypermethylation analysis and next generation sequencing of the MMR genes.ResultsMMR deficiency was observed in 22.3% of resected and 4.4% of biopsied small bowel carcinomas. Prevalence of Lynch syndrome was 6.2% in resections and 0.0% in biopsy samples. Patients with Lynch syndrome-associated small bowel cancer were significantly younger at the time of diagnosis than patients with MMR-proficient and sporadic MMR-deficient cancers (mean age of 54.6 years vs 66.6 years and 68.8 years, respectively, p<0.000).ConclusionsThe prevalence of MMR deficiency and Lynch syndrome in resected small bowel adenocarcinomas is at least comparable to prevalence in colorectal cancers, a finding relevant both for treatment (immunotherapy) and family management. We recommend that all small bowel adenocarcinomas should be screened for MMR deficiency.

Germline variants in the DNA mismatch repair (MMR) gene PMS2cause 1–14% of all Lynch Syndrome cancers. Correct variant analysis of PMS2is complex due to the presence of multiple pseudogenes and the occurrence of gene conversion. The analysis complexity increases in highly fragmented DNA from formalin-fixed paraffin-embedded (FFPE) tissue. Here we describe a reliable approach to detect true PMS2variants in fragmented DNA. A custom NGS panel designed for FFPE tissue was used targeting four MMR genes, POLEand POLD1. Amplicon design for PMS2was based on the position of paralogous sequence variants (PSVs) that distinguish PMS2from its pseudogenes. PMS2variants in exons 1–11 can be correctly curated based on this information. For exons 12–15 this is less reliable as these undergo gene conversion. Using this method, we screened PMS2variants in 125 MMR-deficient tumors. Of the 125 tumors tested, six were unexplained MMR-deficient tumors with solitary PMS2 protein expression loss. In these six tumors two unclassified variants (class 3) and five variants likely affecting function (class 4/5) were detected in PMS2. One microsatellite unstable tumor with positive staining for all MMR proteins was found to carry a frameshift PMS2variant (class 5). No class 4 or class 5 PMS2variants were detected in tumors with other patterns of MMR protein expression loss.

This study aimed to determine the prevalence of APC-associated familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) in a large cohort, taking into account factors as adenoma count and year of diagnosis. All application forms used to send patients in for APCand MUTYHvariant analysis between 1992 and 2017 were collected (n= 2082). Using the data provided on the application form, the APCand biallelic MUTYHprevalence was determined and possible predictive factors were examined using multivariate multinomial logistic regression analysis in SPSS. The prevalence of disease causing variants in the APCgene significantly increases with adenoma count while MAP shows a peak prevalence in individuals with 50–99 adenomas. Logistic regression analysis shows significant odds ratios for adenoma count, age at diagnosis, and, interestingly, a decline in the chance of finding a variant in either gene over time. Moreover, in 22% (43/200) of patients with FAP-related extracolonic manifestations a variant was identified. The overall detection rates are above 10% for patients with >10 adenomas aged <60 and >20 adenomas aged <70. Patients with variants outside these criteria had FAP-related extracolonic manifestations, colorectal cancer aged <40, somatic KRASc.34G > T variant in the tumor or a first-degree relative with >10 adenomas. Therefore, APCand MUTYHtesting in patients with >10 adenomas aged <60 and with >20 adenomas aged <70 is advised. Almost all FAP and MAP patients not meeting these criteria showed other characteristics that can be used as an indication to prompt genetic testing.

Constitutional mismatch repair deficiency (CMMRD) is a rare childhood cancer predisposition syndrome caused by biallelic germline mutations in one of four mismatch-repair genes. Besides very high tumour risks, CMMRD phenotypes are often characterised by the presence of signs reminiscent of neurofibromatosis type 1 (NF1). Because NF1 signs may be present prior to tumour onset, CMMRD is a legitimate differential diagnosis in an otherwise healthy child suspected to have NF1/Legius syndrome without a detectable underlying NF1/SPRED1germline mutation. However, no guidelines indicate when to counsel and test for CMMRD in this setting. Assuming that CMMRD is rare in these patients and that expected benefits of identifying CMMRD prior to tumour onset should outweigh potential harms associated with CMMRD counselling and testing in this setting, we aimed at elaborating a strategy to preselect, among children suspected to have NF1/Legius syndrome without a causative NF1/SPRED1 mutation and no overt malignancy, those children who have a higher probability of having CMMRD. At an interdisciplinary workshop, we discussed estimations of the frequency of CMMRD as a differential diagnosis of NF1 and potential benefits and harms of CMMRD counselling and testing in a healthy child with no malignancy. Preselection criteria and strategies for counselling and testing were developed and reviewed in two rounds of critical revisions. Existing diagnostic CMMRD criteria were adapted to serve as a guideline as to when to consider CMMRD as differential diagnosis of NF1/Legius syndrome. In addition, counselling and testing strategies are suggested to minimise potential harms.

BRCA1/2variant analysis in tumor tissue could streamline the referral of patients with epithelial ovarian, fallopian tube, or primary peritoneal cancer to genetic counselors and select patients who benefit most from targeted treatment. We investigated the sensitivity of BRCA1/2variant analysis in formalin-fixed, paraffin-embedded tumor tissue using a combination of next-generation sequencing and copy number variant multiplex ligation-dependent probe amplification. After optimization using a training cohort of known BRCA1/2mutation carriers, validation was performed in a prospective cohort in which screening of BRCA1/2tumor DNA and leukocyte germline DNA was performed in parallel. BRCA1promoter hypermethylation and pedigree analysis were also performed. In the training cohort, 45 of 46 germline BRCA1/2variants were detected (sensitivity, 98%). In the prospective cohort (n = 62), all six germline variants were identified (sensitivity, 100%), together with five somatic BRCA1/2variants and eight cases with BRCA1promoter hypermethylation. In four BRCA1/2variant–negative patients, surveillance or prophylactic management options were offered on the basis of positive family histories. We conclude that BRCA1/2formalin-fixed, paraffin-embedded tumor tissue analysis reliably detects BRCA1/2variants. When taking family history of BRCA1/2variant–negative patients into account, tumor BRCA1/2variant screening allows more efficient selection of epithelial ovarian cancer patients for genetic counseling and simultaneously selects patients who benefit most from targeted treatment.

Diagnosis of Lynch Syndrome (LS) caused by a pathogenic germline MSH6variant may be complicated by discordant immunohistochemistry (IHC) and/or by a microsatellite stable (MSS) phenotype. The aim of this study was to identify the various causes of the discordant phenotypes of colorectal and endometrial carcinomas (CRC/EC) in MSH6-associated LS. Data were collected from Dutch family cancer clinics. Carriers of a (likely) pathogenic MSH6variant diagnosed with CRC or EC were categorized based on an MSI/IHC test outcome that might fail to result in a diagnosis of LS (e.g. retained staining of all four mismatch repair proteins, with or without an MSS phenotype, and other staining patterns). When tumor tissue was available, MSI and/or IHC were repeated. Next-generation sequencing (NGS) was performed in cases with discordant staining patterns. Data were obtained from 360 families with 1763 (obligate) carriers. MSH6variant carriers with CRC or EC (n=590) were included, consisting of 418 CRCs and 232 ECs. Discordant staining was reported in 77 cases (36% of MSI/IHC results). Twelve patients gave informed consent for further analysis of tumor material. Upon revision, two out of three MSI/IHC cases were found to be concordant with the MSH6variant, and NGS showed that four discordant IHC results were actually sporadic rather than LS-associated tumors. In one case, somatic events explained the discordant phenotype. The use of reflex IHC MMR testing, the current standard in most Western countries, may lead to misdiagnosis of germline MSH6variant carriers. The pathologist should point out that further diagnostics for inheritable colon cancer, including LS, should still be considered in case of a strong positive family history. Germline DNA analysis of the MMR genes, preferably as part of a larger gene panel, should therefore be considered in potential LS patients.

Purpose:Lynch syndrome (LS), a heritable disorder with an increased risk of primarily colorectal cancer (CRC) and endometrial cancer (EC), can be caused by mutations in the PMS2 gene. We wished to establish whether genotype and/or parent-of-origin effects (POE) explain (part of) the reported variability in severity of the phenotype.Methods:European PMS2 mutation carriers (n = 381) were grouped and compared based on RNA expression and whether the mutation was inherited paternally or maternally.Results:Mutation carriers with loss of RNA expression (group 1) had a significantly lower age at CRC diagnosis (51.1 years vs. 60.0 years, P = 0.035) and a lower age at EC diagnosis (55.8 years vs. 61.0 years, P = 0.2, nonsignificant) compared with group 2 (retention of RNA expression). Furthermore, group 1 showed slightly higher, but nonsignificant, hazard ratios (HRs) for both CRC (HR: 1.31, P = 0.38) and EC (HR: 1.22, P = 0.72). No evidence for a significant parent-of-origin effect was found for either CRC or EC.Conclusions:PMS2 mutation carriers with retention of RNA expression developed CRC 9 years later than those with loss of RNA expression. If confirmed, this finding would justify a delay in surveillance for these cases. Cancer risk was not influenced by a parent-of-origin effect.Genet Med 18 4, 405–409.

Lynch syndrome (LS), a heritable disorder with an increased risk of primarily colorectal cancer (CRC) and endometrial cancer (EC), can be caused by mutations in the PMS2gene. We wished to establish whether genotype and/or parent-of-origin effects (POE) explain (part of) the reported variability in severity of the phenotype.