MSH6 Variants Research Articles

Genetic identification and characterization of Lynch syndrome in a multi-ethnic biobank.

1520 Background: Lynch syndrome (LS), caused by germline pathogenic variants in mismatch repair (MMR) genes, results in increased risk of colorectal, endometrial, and other cancers. LS has a prevalence of ~1 in 440 in European ancestry populations; prevalence data in other populations are limited. We identified and characterized carriers of pathogenic MMR gene variants in the multi-ethnic Bio Me Biobank in New York City. Methods: Exome sequence data from ~31,000 Bio Me participants were evaluated for known (per ClinVar) and predicted (loss-of-function) pathogenic variants in MMR genes. Population groups were defined by genetic ancestry. Participant questionnaires and electronic health records (EHRs) of carriers were reviewed for personal or family history of malignancy. Results: We identified 48 carriers of 33 distinct pathogenic variants in PMS2 (48%), MLH1 (27%), MSH6 (15%), and MSH2 (10%), for an estimated prevalence of ~1/640 in the Bio Me Biobank. Prevalence was higher among individuals of Non-Jewish European (N = 14; 1/400) and African (N = 14; 1/490) ancestries, compared to Puerto Rican (N = 8; 1/640), Ashkenazi Jewish (N = 6; 1/690), and other/mixed (N = 6) ancestries. Carriers had a median age of 56 (range 27 to 77) years and were 50% female. Overall rate of malignancy among carriers was 38%, with the lowest rate in PMS2 (26%) and the highest rate in MSH6 (57%) variant carriers. We found a high prevalence of endometrial cancer (21% of female carriers) and a lower prevalence of colorectal cancer (4% of all carriers). Only 2 carriers (4%) had a diagnosis of LS in their EHRs, and only 1 carrier met Amsterdam diagnostic criteria for LS. Conclusions: These data show that ~0.15% of participants in a multi-ethnic biobank are carriers of pathogenic MMR gene variants and suggest that the prevalence is higher in European and lower in non-European ancestry populations. Notably, most carriers do not have a clinical diagnosis of LS and do not meet diagnostic criteria for LS. Carriers demonstrate variable rates of cancer, which may contribute to under-diagnosis of LS. Genomic screening for pathogenic MMR variants may lead to earlier diagnosis of LS and improved outcomes.

Improving cascade genetic testing for families with inherited pancreatic cancer (PDAC) risk: The genetic education, risk assessment and testing (GENERATE) study.

TPS4162 Background: 4-10% of PDAC patients harbor pathogenic germline variants in cancer susceptibility genes, including APC, ATM, BRCA1, BRCA2, CDKN2A, EPCAM, MLH1, MSH2, MSH6, PALB2, PMS2, STK11, and TP53. For families with such pathogenic variants, the greatest potential impact of germline testing is to identify relatives with the same pathogenic variant (cascade testing), thereby providing the opportunity for early detection and cancer interception of PDAC and other associated malignancies. Numerous factors limit cascade testing in real-world practice, including family dynamics, widespread geographic distribution of relatives, access to genetic services, and misconceptions about the importance of germline testing, such that the preventive benefits of cascade testing are often not fully realized. The primary aim of this study is to analyze two alternative strategies for cascade testing in families with inherited PDAC susceptibility. Methods: 1000 individuals (from approximately 200 families) with a confirmed pathogenic germline variant in any of the above genes in a 1st/2nd degree relative and a 1st/2nd degree relative with PDAC will be remotely enrolled through the study website (www.generatestudy.org) and randomized between two different methods of cascade testing (individuals with prior genetic testing will be ineligible): Arm 1 will undergo pre-test genetic education with a pre-recorded video and live interactive session with a genetic counselor via a web-based telemedicine platform (Doxy.me), followed by germline testing through Color Genomics; Arm 2 will undergo germline testing through Color Genomics without dedicated pre-test genetic education. Color Genomics will disclose results to study personnel and directly to participants in both arms. Participants in both arms will have the option of pursuing additional telephone-based genetic counseling through Color Genomics. The primary outcome will be uptake of cascade testing. Secondary outcomes will include participant self-reported genetic knowledge, cancer worry, distress, decisional preparedness, familial communication, and screening uptake, which will be measured via longitudinal surveys. Enrollment will begin February, 2019. Clinical trial information: NCT03762590.

Suspected and confirmed germline variants from tumor-only somatic sequencing of 864 gastrointestinal malignancies.

e13131 Background: Targeted tumor-only somatic sequencing informs therapies and is becoming a routine part of cancer care. It also identifies patients with possible germline variants who require confirmatory genetic testing. The aim was to identify patients with suspected and confirmed germline variants whose GI tumors underwent somatic sequencing. Methods: 864 patients with GI tumors who had Foundation One (FO) somatic sequencing from 4/2003-3/2018 were evaluated. Inclusion criteria for suspected germline variants were: a) allele frequency ≥ 35% in hereditary cancer genes and b) pathogenic variants by FO and/or ClinVar. Variants in commonly mutated somatic genes ( TP53, KRAS, CDKN2A) were excluded in patients over age 40. Recommendation of genetic evaluation and germline test results were recorded. Patient, family, and tumor characteristics were compared using univariate analysis. Results: 199 of 864 patients had suspected germline pathogenic variants. 50 patients were recommended genetic evaluation, and 26 patients underwent genetic testing. A germline pathogenic variant was confirmed in 15 patients. Among all patients suspected to have germline variants, 8% were confirmed by genetic testing. Patients under age 40 and those with family cancer history were more often referred for testing (Table). Patients with variants in BRCA1, MLH1, MSH2, PMS2, POLE and TP53 were more often referred for testing. Conclusions: A quarter of patients carried a somatic pathogenic variant with allele frequency ≥35% in a hereditary cancer gene. 25% of these patients were recommended for genetic evaluation. Younger patients and those with family history were more often referred. 8% of patients with suspected germline variants were confirmed by genetic testing. These results provide “real world” experience in using somatic only tumor testing to identify patients with germline pathogenic variants who then might benefit from future cancer screening and genetic testing in family members. Comparison of characteristics by recommendation to undergo genetic testing based on somatic tumor sequencing results. [Table: see text]

Next-generation sequencing of primary prostate cancer tumors to reveal actionable opportunities for clinical management.

e16582 Background: The Strata Trial (NCT03061305) is a multi-institutional precision oncology collaboration structured as an observational protocol that aims to match patients to genomically guided therapies. Methods: Selected University of North Carolina (UNC) metastatic prostate cancer (mPC) patients were enrolled on this IRB-approved study. Formalin fixed paraffin-embedded primary tumor specimens, without matched germline controls, were sent for targeted next generation sequencing (NGS) to detect actionable variants, including: mutations in 87 genes, copy number variations in 31 genes, gene fusions in 46 gene drivers, and microsatellite instability (MSI) status. mPC-related genes of specific interest included: AR, ATM, BRCA1/2, ERG, MSH2, MSH6, PTEN, RB1, and TP53. Results: Of the 108 cases sequenced, 6 (6%) failed testing. Of the 102 mPC patients with sequence data, the median age was 69 (47-86), 60 (59%) were white, 35 (34%) were black, 1 (1%) was Asian, and 6 (6%) declined to identify race. NGS data revealed 122 variants in 27 genes: 73 patients (71%) had at least one variant. Among those 73 patients, 38 (52%) had only 1 variant, 24 (33%) had 2 variants, 8 (11%) had 3 variants, and 3 (4%) had 4 variants. TMPRSS2-ERG fusions occurred most frequently (51%), followed by TP53 variants (38%), and PTEN variants (16%). Only 8% of patients had variants in DNA damage repair genes, including ATM (3%), BRCA2 (3%) and MSH2 (2%). Two patients with MSI high tumors were treated with pembrolizumab, while 4 patients with deep BRCA2 or ATM deletions were eligible for trials of PARP inhibition. Conclusions: Our UNC experience shows that a high proportion of primary prostate cancer tumors from mPC patients have genomic variants, and two patients were treated based on these data. Limited actionability may reflect the landscape of currently FDA approved mPC treatments available clinical trials, or due to short duration of follow-up after enrollment on the Strata Trial.

Methylation Tolerance-Based Functional Assay to Assess Variants of Unknown Significance in the MLH1 and MSH2 Genes and Identify Patients With Lynch Syndrome.

Approximately 75% of patients with suspected Lynch syndrome carry variants in MLH1 or MSH2, proteins encoded by these genes are required for DNA mismatch repair (MMR). However, 30% of these are variants of unknown significance (VUS). A assay that measures cell response to the cytotoxic effects of a methylating agent can determine the effects of VUS in MMR genes and identify patients with constitutional MMR-deficiency syndrome. We adapted this method to test the effects of VUS in MLH1 and MSH2 genes found in patients with suspected Lynch syndrome. We transiently expressed MLH1 or MSH2 variants in MLH1- or MSH2-null human colorectal cancer cell lines (HCT116 or LoVo), respectively. The MMR process causes death of cells with methylation-damaged DNA bases, so we measured proportions of cells that undergo death following exposure to the methylating agent; cells that escaped its toxicity were considered to have variants that affect function of the gene product. Using this assay, we analyzed 88 variants (mainly missense variants), comprising a validation set of 40 previously classified variants (19 in MLH1 and 21 in MSH2) and a prospective set of 48 VUS (25 in MLH1 and 23 in MSH2). Prediction scores were calculated for all VUS according to the recommendations of the American College of Medical Genetics and Genomics, based on clinical, somatic, in silico, population, and functional data. The assay correctly classified 39 of 40 variants in the validation set. The assay identified 12 VUS that did alter function of the gene product and 28 VUS that did not; the remaining 8 VUS had intermediate effects on MMR capacity and could not be classified. Comparison of assay results with prediction scores confirmed the ability of the assay to discriminate VUS that affected the function of the gene products from those that did not. Using an assay that measures the ability of the cells to undergo death following DNA damage induction by a methylating agent, we were able to assess whether variants in MLH1 and MSH2 cause defects in DNA MMR. This assay might be used to help assessing the pathogenicity of VUS in MLH1 and MSH2 found in patients with suspected Lynch syndrome.

Constitutional Mosaic Epimutations - a hidden cause of cancer?

Silencing of tumor suppressor genes by promoter hypermethylation is a key mechanism to facilitate cancer progression in many malignancies. While promoter hypermethylation can occur at later stages of the carcinogenesis process, constitutional methylation of key tumor suppressors may be an initiating event whereby cancer is started. Constitutional BRCA1 methylation due to cis-acting germline genetic variants is associated with a high risk of breast and ovarian cancer. However, this seems to be a rare event, restricted to a very limited number of families. In contrast, mosaic constitutional BRCA1 methylation is detected in 4-7% of newborn females without germline BRCA1 mutations. While the cause of such methylation is poorly understood, mosaic normal tissue BRCA1 methylation is associated with a 2-3 fold increased risk of high-grade serous ovarian cancer (HGSOC). As such, BRCA1 methylation may be the cause of a significant number of ovarian cancers. Given the molecular similarities between HGSOC and basal-like breast cancer, the findings with respect to HGSOC suggest that constitutional BRCA1 methylation could be a risk factor for basal-like breast cancer as well. Similar to BRCA1, some specific germline variants in MLH1 and MSH2 are associated with promoter methylation and a high risk of colorectal cancers in rare hereditary cases of the disease. However, as many as 15% of all colorectal cancers are of the microsatellite instability (MSI) “high” subtype, in which commonly the tumors harbor MLH1 hypermethylation. Constitutional mosaic methylation of MLH1 in normal tissues has been detected but not formally evaluated as a potential risk factor for incidental colorectal cancers. However, the findings with respect to BRCA1 in breast and ovarian cancer raises the question whether mosaic MLH1 methylation is a risk factor for MSI positive colorectal cancer as well. As for MGMT, a promoter variant is associated with elevated methylation across a panel of solid cancers, and MGMT promoter methylation may contribute to an elevated cancer risk in several of these malignancies. We hypothesize that constitutional mosaic promoter methylation of crucial tumor suppressors may trigger certain types of cancer, similar to germline mutations inactivating the same particular genes. Such constitutional methylation events may be a spark to ignite cancer development, and if associated with a significant cancer risk, screening for such epigenetic alterations could be part of cancer prevention programs to reduce cancer mortality in the future.

Genetic, Epigenetic, and Immunologic Profiling of MMR-Deficient Relapsed Glioblastoma.

In-depth characterization of recurrent glioblastoma (rGBM) might contribute to a better understanding of the mechanisms behind tumor progression and enable rGBM treatment with targeted drugs.Experimental Design: In this study, GBM samples were collected at diagnosis and recurrence from adult patients treated with Stupp protocol. Expression of mismatch repair (MMR) proteins was evaluated by IHC, followed by whole exome sequencing (WES) of tumor samples showing loss of MSH6 reactivity. Established genetic, epigenetic, and immunologic markers were assessed by standard methods and correlated with loss of MMR proteins and patient survival. Expression of MMR proteins was partially or completely lost in 25.9% rGBM samples. Specifically, 12 samples showed partial or total MSH6 expression reduction. Conversely, 96.4% of GBM samples at diagnosis expressed MMR markers. WES disclosed lack of variants in MMR genes in primary samples, whereas two MSH6-negative rGBM samples shared a c.3438+1G>A* splicing MSH6 variant with a potential loss of function effect. MSH6-negative rGBM specimens had high tumor mutational burden (TMB), but no microsatellite instability. In contrast, GBM samples with partial loss of MMR proteins disclosed low TMB. MMR-deficient rGBM showed significant telomere shortening and MGMT methylation and are characterized by highly heterogeneous MHC class I expression. Multilevel profiling of MMR-deficient rGBM uncovered hypermutated genotype uncoupled from enriched expression of immune-related markers. Assessment of MHC class I expression and TMB should be included in protocols aiming to identify rGBM patients potentially eligible for treatment with drugs targeting immune-checkpoint inhibitors.

Impact of gene-specific germline pathogenic variants on presentation of endometrial cancer in Lynch syndrome.

Lynch syndrome is a risk factor for developing endometrial carcinoma. Our aim was to evaluate the impact of gene-specific germline pathogenic variants on clinical features of patients affected by endometrial cancer. Patients with a diagnosis of endometrial cancer and with a germline pathogenic variant in mismatch repair genes were reviewed. Patients were classified on the basis of classes of risk according to the ESGO-ESGO-ESTRO (European Society of Medical Oncology/European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology) guidelines. One-way analysis of variance (ANOVA) and Kruskal-Wallis test were performed to compare three groups of continuous parametric and non-parametric variables, respectively. χ2 test was used to analyze proportions. Overall, 68 patients with endometrial cancer and Lynch syndrome were evaluated. Ten (14.7%) patients were excluded because of absence of information about the gene involved in Lynch syndrome, thus leaving 58 (85.3%) patients available for the final analysis. MLH1, MSH2, and MSH6 pathogenic variants were observed in 19 (32.7%), 33 (56.9%), and six (10.3%) patients, respectively. Mean±SD age at endometrial cancer diagnosis was 51±6.4, 43.5±7.4, and 60.3±8.8 years (p=0.0002). Prevalence of non-endometrioid endometrial cancer was 15.7%, 24.2%, and 0% in the MLH1, MSH2, and MSH6 groups, respectively (p=0.345). According to the ESMO-ESGO-ESTRO classification, low, intermediate, and high risk endometrial cancer accounted for 47.3%, 10.5%, and 42.1% of the MLH1 group, 57.6%, 3%, and 39.4% of the MSH2 group, and 50%, 50%m and 0% of the MSH6 group (p=0.009). Patients with MLH1 and MSH2 pathogenic variants are at a higher risk of early onset of endometrial cancer than patients with MSH6 pathogenic variants.

Heterogenous loss of mismatch repair (MMR) protein expression: a challenge for immunohistochemical interpretation and microsatellite instability (MSI) evaluation.

Immunohistochemistry (IHC) for mismatch repair (MMR) proteins is used to identify MMR status: being diffusely positive (intact/retained nuclear staining) or showing loss of nuclear tumour staining (MMR protein deficient). Four colonic adenocarcinomas and a gastric adenocarcinoma with associated dysplasia that displayed heterogenous IHC staining patterns in at least one of the four MMR proteins were characterised by next‐generation sequencing (NGS). In order to examine a potential molecular mechanism for these staining patterns, the respective areas were macrodissected, analysed for microsatellite instability (MSI) and investigated by NGS and multiplex ligation‐dependent probe amplification (MLPA) analysis of MLH1, MSH2, MSH6 and PMS2 genes, including MLH1 methylation analysis. One colonic adenocarcinoma showed heterogenous MSH6 IHC staining and molecular analysis demonstrated increasing allelic burden of two MSH6 frameshift variants (c.3261delC and c.3261dupC) in areas with MSH6 protein loss compared to areas where MSH6 was retained. Two colonic adenocarcinomas with heterogenous MLH1 staining showed no differences in sequence variants. In one of these cases, however, MLH1 was hypermethylated in the area of MLH1 loss. Another colon carcinoma with heterogenous PMS2 staining (but with retained MSH6) showed both MSH6 c.3261dupC and 3260_3261dupCC where PMS2 protein was lost and only c.3261dupC where PMS2 was retained. The gastric carcinoma showed complete loss of MSH6 in dysplastic foci, while the underlying invasive carcinoma showed retention of MSH6. Both these areas, however, were MSI‐high and showed the same MSH6 variant: c.3261delC. The gastric dysplasia additionally showed MSH6 c.3261dupC. In four of the five cases where MMR protein was lost, these areas were MSI‐high. Heterogenous MMR IHC (focal and/or zonal within the same tumour or between invasive and dysplastic preinvasive areas) is not always due to artefact and is invariably related to MSI‐high status in the areas of loss. An interesting aspect to this study is the presence of MSH6 somatic mutations irrespective of whether MSH6 IHC staining was intact or lost.

Testing strategies to reduce morbidity and mortality from Lynch syndrome

Objective: Lynch syndrome (LS) has an autosomal dominant inheritance pattern and is associated with increased risk for colorectal cancer (CRC) and other cancers. Various strategies are used to identify patients at risk and offer surveillance and preventive programs, the cost effectiveness of which is much dependent on the prevalence of LS in a population. Universal testing (UT) is proposed as an effective measure, targeting all newly diagnosed CRC patients under a certain age.Materials and methods: LS cases were identified in a cohort of 572 consecutive CRC patients. Immunohistochemistry was performed in 539 cases, using antibodies against mismatch repair proteins MLH1, PMS2, MSH2, and MSH6. Microsatellite instability and gene mutation screening were performed in 57 cases.Results: In total 11 pathogenic variants were detected, identifying LS in 1.9% of new CRC cases. Comparing the results with current clinical methods, 2 pathogenic variants were found with Amsterdam criteria and 9 when using either Bethesda guidelines or our institution’s prior clinical criteria. Pathogenic variants in MSH6 were the most common in our series. We also found different outcomes using different age cut offs.Conclusion: Our study demonstrates that UT of tumors before age on onset at 75 years would most likely be cost-efficient and essentially equivalent to applying the Bethesda guidelines or our institution’s prior clinical criteria on all new CRC.

A retrospective study of extracolonic, non-endometrial cancer in Swedish Lynch syndrome families

BackgroundLynch Syndrome is an autosomal dominant cancer syndrome caused by pathogenic germ-line variants in one of the DNA-mismatch-repair (MMR) genes MLH1, MSH2, MSH6 or PMS2. Carriers are predisposed to colorectal and endometrial cancer, but also other cancer types. The purpose of this retrospective study was to characterize the tumour spectrum of the Swedish Lynch syndrome families.MethodsData were obtained from genetically verified 235 Lynch families from five of the six health care regions in Sweden. The material was stratified for gender, primary cancer, age and mutated gene and the relative proportions of specific cancer types were compared to those in the general population.ResultsA total of 1053 family members had 1493 cancer diagnoses of which 1011 were colorectal or endometrial cancer. Individuals with pathogenic variants in MLH1 and MSH2 comprised 78% of the cohort. Among the 482 non-colorectal/non-endometrial cancer diagnoses, MSH2 carriers demonstrated a significantly increased proportion of urinary tract, gastric, small bowel, ovarian and non-melanoma skin cancer compared to the normal population. MLH1 carriers had an elevated proportion of gastrointestinal cancers (gastric, small bowel, pancreas), while MSH6 carriers had more ovarian cancer than expected. Gastric cancer was predominantly noted in older generations.ConclusionLynch syndrome confers an increased risk for multiple cancers other than colorectal and endometrial cancer. The proportions of other cancers vary between different MMR genes, with highest frequency in MSH2-carriers. Gender and age also affect the tumour spectrum, demonstrating the importance of additional environmental and constitutional parameters in determining the predisposition for different cancer types.

Duodenal tumor risk in Lynch syndrome

Background and aimsLynch syndrome (LS) is associated with an increased risk of small bowel tumors but routine screening is not recommended in international guidelines. The aim of our study was to determinate the prevalence of duodenal tumors in a French cohort of LS patients. MethodsPatients carrying a germline pathogenic variant in a MMR gene, supported by our local network, in which at least one upper endoscopy had been performed, were included. We registered the occurrence of duodenal lesions in those patients. Results154 LS patients were identified including respectively 85 MSH2 and 41 MLH1 mutated patients respectively. Seven out of 154 (4.5%) had at least one duodenal lesion. Median age at diagnosis was 58 years (range: 49–73). The twelve lesions locations were: descending duodenum (n = 7), genu inferius (n = 2), duodenal bulb (n = 1), ampulla (n = 1), fourth duodenum (n = 1). Three lesions were invasive adenocarcinomas. The incidence rate of duodenal lesions in patients with MSH2 or MLH1 pathogenic variants was respectively 7.1% (6 out of 85) and 2.4% (1 out of 41) emphasizing a trend toward increased risk of developing duodenal lesion in MSH2 mutated patients: OR: 5.17, IC95% (0.8–60.07), p = 0.1307. ConclusionRegarding this high prevalence rate, especially in MSH2 patients, regular duodenal screening during upper endoscopy should be considered in routine in LS patients.

MSH6 and PMS2 germ-line pathogenic variants implicated in Lynch syndrome are associated with breast cancer

PurposeAn association of Lynch syndrome (LS) with breast cancer has been long suspected; however, there have been insufficient data to address this question for each of the LS genes individually. MethodsWe conducted a retrospective review of personal and family history in 423 women with pathogenic or likely pathogenic germ-line variants in MLH1 (N = 65), MSH2 (N = 94), MSH6 (N = 140), or PMS2 (N = 124) identified via clinical multigene hereditary cancer testing. Standard incidence ratios (SIRs) of breast cancer were calculated by comparing breast cancer frequencies in our study population with those in the general population (Surveillance, Epidemiology, and End Results 18 data). ResultsWhen evaluating by gene, the age-standardized breast cancer risks for MSH6 (SIR = 2.11; 95% confidence interval (CI), 1.56–2.86) and PMS2 (SIR = 2.92; 95% CI, 2.17–3.92) were associated with a statistically significant risk for breast cancer whereas no association was observed for MLH1 (SIR = 0.87; 95% CI, 0.42–1.83) or MSH2 (SIR = 1.22; 95% CI, 0.72–2.06). ConclusionOur data demonstrate that two LS genes, MSH6 and PMS2, are associated with an increased risk for breast cancer and should be considered when ordering genetic testing for individuals who have a personal and/or family history of breast cancer.

A multi-gene panel beyond BRCA1/BRCA2 to identify new breast cancer-predisposing mutations by a picodroplet PCR followed by a next-generation sequencing strategy: a pilot study

By analyzing multiple gene panels, next-generation sequencing is more effective than conventional procedures in identifying disease-related mutations that are useful for clinical decision-making. Here, we aimed to test the efficacy of an 84 genes customized-panel in BRCA1 and BRCA2 mutation-negative patients. Twenty-four patients were enrolled in this study. DNA libraries were prepared using a picodroplet PCR-based approach and sequenced with the MiSeq System. Highly putative pathogenic mutations were identified in genes other than the commonly tested BRCA1/2: 2 pathogenic mutations one in TP53 and one in MUTYH; 2 missense variants in MSH6 and ATM, respectively; 2 frameshift variants in KLLN, and ATAD2, respectively; an intronic variant in ANPEP, and 3 not functionally known variants (a frameshift variant in ATM a nonsense variant in ATM and a missense variant in NFE2L2). Our results show that this molecular screening will increase diagnostic sensitivity leading to a better risk assessment in breast cancer patients and their families. This strategy could also reveal genes that have a higher penetrance for breast and ovarian cancers by matching gene mutation with familial and clinical data, thereby increasing information about hereditary breast and ovarian cancer genetics and improving cancer prevention measures or therapeutic approaches.

Whole exome sequencing of breast cancer (TNBC) cases from India: association of MSH6 and BRIP1 variants with TNBC risk and oxidative DNA damage.

Whole exome sequencing in triple negative breast cancer cases (n = 8) and targeted sequencing in healthy controls (n = 48) revealed BRIP1 rs552752779 (MAF: 75% vs. 6.25%, OR 45.00, 95% CI 9.43-243.32), ERBB2 rs527779103 (MAF: 62.5% vs. 7.29%, OR 21.19, 95% CI 5.11-94.32), ERCC2 rs121913016 (MAF: 56.25% vs. 7.29%, OR 16.34, 95% CI 4.02-70.41), MSH6 rs2020912 (MAF: 56.25% vs. 1.04%, OR 122.13, 95% CI 12.29-2985.48) as risk factors for triple negative breast cancer. Construction of classification and regression tree followed by smart pruning identified MSH6 and BRIP1 variants as the major determinants of TNBC (Triple Negative Breast Cancer) risk. Except for ERBB2, all other genes regulate DNA repair and chromosomal integrity. In TNBC cases, two likely pathogenic variations i.e. NCOR1 rs562300336 and PIM1 rs746748226 were observed at frequencies of 18.75% and 12.5%, respectively. Among the 24 variants of unknown significance, MMP9 rs199676062, SYNE1 rs368709678, AURKA rs373550419, ABCC4 rs11568694 have variant allele frequency ≥ 62.5%. These genes regulate metastasis, nuclear modeling, cell cycle and cellular detoxification, respectively. To conclude, aberrations in DNA mismatch repair, nucleotide excision repair or BRCA1 associated genome surveillance mechanism contribute towards triple negative breast cancer.

Molecular Background of Colorectal Tumors From Patients With Lynch Syndrome Associated With Germline Variants in PMS2

Germline variants in mismatch repair genes MLH1, MSH2 (EPCAM), MSH6, or PMS2 cause Lynch syndrome. Patients with these variants have an increased risk of developing colorectal cancers (CRCs) that differ from sporadic CRCs in genetic and histologic features. It has been a challenge to study CRCs associated with PMS2 variants (PMS2-associated CRCs) because these develop less frequently and in older patients than CRCs with variants in other mismatch repair genes. We analyzed 20 CRCs associated with germline variants in PMS2, 22 sporadic CRCs, 18 CRCs with germline variants in MSH2, and 24 CRCs from patients with germline variants in MLH1. Tumor tissue blocks were collected from Dutch pathology departments in 2017. After extraction of tumor DNA, we used a platform designed to detect approximately 3,000 somatic hotspot variants in 55 genes (including KRAS, APC, CTNNB1, and TP53). Somatic variant frequencies were compared using the Fisher exact test. None of the PMS2-associated CRCs contained any somatic variants in the catenin-β1 gene (CTNNB1), which encodes β-catenin, whereas 14 of 24 MLH1-associated CRCs (58%) contained variants in CTNNB1. Half the PMS2-associated CRCs contained KRAS variants, but only 20% of these were in hotspots that encoded G12D or G13D. These hotspot variants occurred more frequently in CRCs associated with variants in MLH1 (37.5%; P= .44) and MSH2 (71.4%; P= .035) than in those associated with variants in PMS2. In a genetic analysis of 84 colorectal tumors, we found tumors from patients with PMS2-associated Lynch syndrome to be distinct from colorectal tumors associated with defects in other mismatch repair genes. This might account for differences in development and less frequent occurrence.

Cancer prevention by aspirin in children with Constitutional Mismatch Repair Deficiency (CMMRD)

Constitutional MisMatch Repair Deficiency (CMMRD) is caused by homozygous or compound heterozygous germline variants in one of the mismatch repair (MMR) genes (MSH2, MSH6, PMS2, MLH1). This syndrome results in early onset colorectal cancer, leukemia and lymphoma, brain tumors and other malignancies. Children with CMMRD are at high risk of developing multiple cancers and cancer surveillance does not guarantee detection of cancer at a curable stage. The development of a preventive treatment strategy would be a major step forward. Long-term daily use of acetylsalicylic acid (ASA) has been shown to reduce cancer risk in individuals with Lynch syndrome (LS). LS is caused by heterozygous germline variants of MSH2, MSH6, PMS2 and MLH1 and characterized by an increased risk of developing colorectal and endometrial cancer at adult age. Here we discuss the potential use of ASA for cancer prevention in patients with CMMRD.

Using Somatic Mutations from Tumors to Classify Variants in Mismatch Repair Genes

Present guidelines for classification of constitutional variants do not incorporate inferences from mutations seen in tumors, even when these are associated with a specific molecular phenotype. When somatic mutations and constitutional mutations lead to the same molecular phenotype, as for the mismatch repair genes, information from somatic mutations may enable interpretation of previously unclassified variants. To test this idea, we first estimated likelihoods that somatic variants in MLH1, MSH2, MSH6, and PMS2 drive microsatellite instability and characteristic IHC staining patterns by calculating likelihoods of high versus low normalized variant read fractions of 153 mutations known to be pathogenic versus those of 760 intronic passenger mutations from 174 paired tumor-normal samples. Mutations that explained the tumor mismatch repair phenotype had likelihood ratio for high variant read fraction of 1.56 (95% CI 1.42-1.71) at sites with no loss of heterozygosity and of 26.5 (95% CI 13.2-53.0) at sites with loss of heterozygosity. Next, we applied these ratios to 165 missense, synonymous, and splice variants observed in tumors, combining in a Bayesian analysis the likelihood ratio corresponding with the adjusted variant read fraction with pretest probabilities derived from published analyses and public databases. We suggest classifications for 86 of 165 variants: 7 benign, 31 likely benign, 22 likely pathogenic, and 26 pathogenic. These results illustrate that for mismatch repair genes, characterization of tumor mutations permits tumor mutation data to inform constitutional variant classification. We suggest modifications to incorporate molecular phenotype in future variant classification guidelines.

Variants of FasL and ABCC5 are predictive of outcome after chemotherapy-based treatment in osteosarcoma

ObjectivesPrevious pharmacogenetics studies showed that genetic variants could be indicative of the response to chemotherapy. We aimed to investigate whether variants of FasL, MSH2, ABCC5, CASP3 and CYP3A4 are associated with the outcome after chemotherapy-based treatment in osteosarcoma. Methods132 osteosarcoma patients who had completed the neoadjuvant chemotherapy in our center were included. 5-year progression-free survival (PFS) was assessed from the initial treatment to the earliest sign of disease progression or death from any cause. 5 SNPs were genotyped using TaqMan SNP Genotyping Assay, including rs763110 of FasL, rs4638843 of MSH2, rs939338 of ABCC5, rs2720376 of CASP3 and rs4646437 of CYP3A4. Patients were classified into two groups according to the 5-year PFS (event/no event). The chi-square test was used to analyze difference of genotype frequency. Logistic regression analysis was used to determine the independent predictors of the PFS rate. ResultsThe overall 5-year PFS was 61.4% (81/132). Genotype TT/CT of rs763110 and genotype GG/AG of rs939338 were significantly associated with the event of 5-year PFS (p = 0.028 for rs763110; p = 0.039 for rs939338). Patients with no risk allele showed a 5-year PFS of 73.7% (42/57), which was significantly higher than a PFS of 54.2% (26/48) for patients with one risk allele and 48.1% (13/27) for patients with two different risk alleles (p = 0.03). Logistic regression analysis showed that allele T of FasL rs763110 and allele G of ABCC5 rs939338 were independent risk factors of the 5-year PFS. The ORs were 2.14 (95%CI = 1.13–3.35, p = 0.01) for rs763110 and 1.73 (95%CI = 1.05–2.52, p = 0.03) for rs939338, respectively. ConclusionsThe association of variants of FASL and ABCC5 with survival outcome after chemotherapy was validated in patients with osteosarcoma. Our findings may provide a new insight into a more personalized treatment for patients with osteosarcoma.

Germline pathogenic variants in PALB2 and other cancer-predisposing genes in families with hereditary diffuse gastric cancer without CDH1 mutation: a whole-exome sequencing study

SummaryBackgroundGermline pathogenic variants in the E-cadherin gene (CDH1) are strongly associated with the development of hereditary diffuse gastric cancer. There is a paucity of data to guide risk assessment and management of families with hereditary diffuse gastric cancer that do not carry a CDH1 pathogenic variant, making it difficult to make informed decisions about surveillance and risk-reducing surgery. We aimed to identify new candidate genes associated with predisposition to hereditary diffuse gastric cancer in affected families without pathogenic CDH1 variants.MethodsWe did whole-exome sequencing on DNA extracted from the blood of 39 individuals (28 individuals diagnosed with hereditary diffuse gastric cancer and 11 unaffected first-degree relatives) in 22 families without pathogenic CDH1 variants. Genes with loss-of-function variants were prioritised using gene-interaction analysis to identify clusters of genes that could be involved in predisposition to hereditary diffuse gastric cancer.FindingsProtein-affecting germline variants were identified in probands from six families with hereditary diffuse gastric cancer; variants were found in genes known to predispose to cancer and in lesser-studied DNA repair genes. A frameshift deletion in PALB2 was found in one member of a family with a history of gastric and breast cancer. Two different MSH2 variants were identified in two unrelated affected individuals, including one frameshift insertion and one previously described start-codon loss. One family had a unique combination of variants in the DNA repair genes ATR and NBN. Two variants in the DNA repair gene RECQL5 were identified in two unrelated families: one missense variant and a splice-acceptor variant.InterpretationThe results of this study suggest a role for the known cancer predisposition gene PALB2 in families with hereditary diffuse gastric cancer and no detected pathogenic CDH1 variants. We also identified new candidate genes associated with disease risk in these families.FundingUK Medical Research Council (Sackler programme), European Research Council under the European Union's Seventh Framework Programme (2007–13), National Institute for Health Research Cambridge Biomedical Research Centre, Experimental Cancer Medicine Centres, and Cancer Research UK.