PMS2 Mutations Research Articles

SNP association study in PMS2-associated Lynch syndrome

Lynch syndrome (LS) patients are at high risk of developing colorectal cancer (CRC). Phenotypic variability might in part be explained by common susceptibility loci identified in Genome Wide Association Studies (GWAS). Previous studies focused mostly on MLH1, MSH2 and MSH6 carriers, with conflicting results. We aimed to determine the role of GWAS SNPs in PMS2 mutation carriers. A cohort study was performed in 507 PMS2 carriers (124 CRC cases), genotyped for 24 GWAS SNPs, including SNPs at 11q23.1 and 8q23.3. Hazard ratios (HRs) were calculated using a weighted Cox regression analysis to correct for ascertainment bias. Discrimination was assessed with a concordance statistic in a bootstrap cross-validation procedure. Individual SNPs only had non-significant associations with CRC occurrence with HRs lower than 2, although male carriers of allele A at rs1321311 (6p21.31) may have increased risk of CRC (HR = 2.1, 95% CI 1.2–3.0). A polygenic risk score (PRS) based on 24 HRs had an HR of 2.6 (95% CI 1.5–4.6) for the highest compared to the lowest quartile, but had no discriminative ability (c statistic 0.52). Previously suggested SNPs do not modify CRC risk in PMS2 carriers. Future large studies are needed for improved risk stratification among Lynch syndrome patients.

Germline Genetic Features of Young Individuals With Colorectal Cancer

The incidence of colorectal cancer (CRC) in individuals younger than 50 years is increasing. We sought to ascertain the proportion of young CRC cases associated with genetic predisposition. We performed a retrospective study of individuals diagnosed with CRC at an age younger than 50 years, evaluated by the clinical genetics service at a single tertiary care cancer center from 1998 through 2015. We collected data on patient histories, tumor phenotypes, and results of germline DNA sequencing. For subjects with uninformative clinical evaluations, germline DNA samples were (re)sequenced using a research-based next-generation sequencing multigene panel. The primary outcome was identification of a pathogenic germline mutation associated with cancer predisposition. Of 430 young CRC cases, 111 (26%) had a first-degree relative with CRC. Forty-one of the subjects with CRC (10%) had tumors with histologic evidence for mismatch repair deficiency. Of 315 subjects who underwent clinical germline sequencing, 79 had mutations associated with a hereditary cancer syndrome and 21 had variants of uncertain significance. Fifty-six subjects had pathogenic variants associated with Lynch syndrome (25 with mutations in MSH2, 24 with mutations in MLH1, 5with mutations in MSH6, and 2 with mutations in PMS2) and 10 subjects had pathogenic variants associated with familial adenomatous polyposis. Thirteen subjects had mutations in other cancer-associated genes (8 in MUTYH, 2 in SMAD4, 1 in BRCA1, 1 in TP53, and 1 in CHEK2), all identified through multigene panel tests. Among 117 patients with uninformative clinical evaluations, next-generation sequence analysis using a multigene panel detected actionable germline variants in 6 patients (5%). Only 43 of the 85 subjects with germline mutations associated with a hereditary cancer syndrome (51%) reported a CRC diagnosis in a first-degree relative. Approximately 1 in 5 individuals diagnosed with CRC at age younger than 50 years carries a germline mutation associated with cancer; nearly half of these do not have clinical histories typically associated with the identified syndrome. Germline testing with multigene cancer panels should be considered for all young patients with CRC.

Genetic anticipation in Swedish Lynch syndrome families.

Among hereditary colorectal cancer predisposing syndromes, Lynch syndrome (LS) caused by mutations in DNA mismatch repair genes MLH1, MSH2, MSH6 or PMS2 is the most common. Patients with LS have an increased risk of early onset colon and endometrial cancer, but also other tumors that generally have an earlier onset compared to the general population. However, age at first primary cancer varies within families and genetic anticipation, i.e. decreasing age at onset in successive generations, has been suggested in LS. Anticipation is a well-known phenomenon in e.g neurodegenerative diseases and several reports have studied anticipation in heritable cancer. The purpose of this study is to determine whether anticipation can be shown in a nationwide cohort of Swedish LS families referred to the regional departments of clinical genetics in Lund, Stockholm, Linköping, Uppsala and Umeå between the years 1990–2013. We analyzed a homogenous group of mutation carriers, utilizing information from both affected and non-affected family members. In total, 239 families with a mismatch repair gene mutation (96 MLH1 families, 90 MSH2 families including one family with an EPCAM–MSH2 deletion, 39 MSH6 families, 12 PMS2 families, and 2 MLH1+PMS2 families) comprising 1028 at-risk carriers were identified among the Swedish LS families, of which 1003 mutation carriers had available follow-up information and could be included in the study. Using a normal random effects model (NREM) we estimate a 2.1 year decrease in age of diagnosis per generation. An alternative analysis using a mixed-effects Cox proportional hazards model (COX-R) estimates a hazard ratio of exp(0.171), or about 1.19, for age of diagnosis between consecutive generations. LS-associated gene-specific anticipation effects are evident for MSH2 (2.6 years/generation for NREM and hazard ratio of 1.33 for COX-R) and PMS2 (7.3 years/generation and hazard ratio of 1.86). The estimated anticipation effects for MLH1 and MSH6 are smaller.

Increased Sensitivity of Diagnostic Mutation Detection by Re-analysis Incorporating Local Reassembly of Sequence Reads.

Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations. We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases. ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2×101-bp) and NextSeq500 (2×151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools. This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.

Screening for germline mutations in mismatch repair genes in patients with Lynch syndrome by next generation sequencing.

Lynch syndrome (LS) is an autosomal dominant disorder, with high penetrance that affects approximately 3% of the cases of colorectal cancer. Affected individuals inherit germline mutations in genes responsible for DNA mismatch repair, mainly at MSH2, MLH1, MSH6 and PMS2. The molecular screening of these individuals is frequently costly and time consuming due to the large size of these genes. In addition, PMS2 mutation detection is often a challenge because there are 16 different pseudogenes identified until now. In the present work we evaluate a molecular screening strategy based in next generation sequencing (NGS) in order to optimize the mutation detection in LS patients. We established 16 multiplex PCRs for MSH2, MSH6 and MLH1 and 5 Long-Range PCRs for PMS2, coupled with NGS. The strategy was validated by screening 66 patients who filled Bethesda and Amsterdam criteria for LS from health institutions of Brazil. The mean depth of coverage for MSH2, MSH6, MLH1 and PMS2 genes was 7.988, 36.313, 11.899 and 4.772 times, respectively. Ninety-four variants were found in exons and flanking intron/exon regions for the four MMR genes. Twenty-five were pathogenic or VUS and found in 32 patients (7 in MSH2, 5 in MSH6, 12 in MLH1 e 1 in PMS2). All variants were confirmed by Sanger sequencing. The strategy was efficient to reduce time consuming and costs to identify genetic changes at these MMR genes, reducing in three times the number of PCR reactions performed per patient and was efficient in identifying variants at PMS2 gene.

Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify PMS2 mutation carriers

Until recently, no prediction models for Lynch syndrome (LS) had been validated for PMS2 mutation carriers. We aimed to evaluate MMRpredict and PREMM5 in a clinical cohort and for PMS2 mutation carriers specifically. In a retrospective, clinic-based cohort we calculated predictions for LS according to MMRpredict and PREMM5. The area under the operator receiving characteristic curve (AUC) was compared between MMRpredict and PREMM5 for LS patients in general and for different LS genes specifically. Of 734 index patients, 83 (11%) were diagnosed with LS; 23 MLH1, 17 MSH2, 31 MSH6 and 12 PMS2 mutation carriers. Both prediction models performed well for MLH1 and MSH2 (AUC 0.80 and 0.83 for PREMM5 and 0.79 for MMRpredict) and fair for MSH6 mutation carriers (0.69 for PREMM5 and 0.66 for MMRpredict). MMRpredict performed fair for PMS2 mutation carriers (AUC 0.72), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers (AUC 0.51). The only statistically significant difference between PMS2 mutation carriers and non-mutation carriers was proximal location of colorectal cancer (77 vs. 28%, p < 0.001). Adding location of colorectal cancer to PREMM5 considerably improved the models performance for PMS2 mutation carriers (AUC 0.77) and overall (AUC 0.81 vs. 0.72). We validated these results in an external cohort of 376 colorectal cancer patients, including 158 LS patients. MMRpredict and PREMM5 cannot adequately identify PMS2 mutation carriers. Adding location of colorectal cancer to PREMM5 may improve the performance of this model, which should be validated in larger cohorts.

Abstract 4269: Risk of different cancers among first-degree relatives of pancreatic cancer patients and impact of probands’ germline mutation on sibling cancer risk

Abstract Background: There have been varying reports on risk of different cancers, other than pancreatic cancer (PC), among first-degree relatives (FDRs) of PC patients. Because the pattern and scope of aggregation of PC with other malignancies in families of PC patients are not entirely clear, we investigated risk of 15 common malignancies among FDRs of unselected PC probands. Methods: The study included 17,181 FDRs with more than 336,000 person-years at risk. The FDRs were identified through sequentially enrolled PC probands (n=2,305) in the Mayo Clinic prospective pancreatic cancer patient registry from 2000-2016. Data on family history of cancer were provided by the probands at the time of enrollment in structured risk factor questionnaires. Standardized incidence ratios (SIRs) and 95% confidence intervals (CIs) were calculated by comparing cases of each cancer type observed among the FDRs with those expected using data from the Surveillance Epidemiology and End Results Program (SEER). Stratified analyses were performed among siblings of the probands based on whether the related proband tested positive for a mutation in ATM, BARD1, BRCA1, BRCA2, CDKN2A, CHEK2, MUTYH, NBN, PALB2, or PMS2; or tested negative for mutation in a total of 25 sequenced cancer susceptibility genes. Results: Compared to the SEER reference population, risk of PC was two-fold higher than expected among the FDRs (SIR=2.04, 95% CI: 1.78-2.31) and 12-fold higher than expected among FDRs with history of PC in at least two blood relatives (SIR=11.99, 95% CI: 10.48-13.64). Siblings of mutation-positive probands had higher risk of PC (SIR=13.57, 95% CI: 6.19-25.76) than siblings of mutation-negative probands (SIR=8.91, 95% CI=6.73-11.57). For other cancer types, primary liver cancer was elevated among female FDRs (SIR=2.10, 95% CI: 1.34-3.12), whereas breast (SIR=3.16, 95% CI: 1.63-5.52) and ovarian (SIR =6.61, 95% CI: 1.33-19.31) cancers were elevated only among siblings of the mutation-positive probands. There also were suggestions of lower than expected risk of other malignancies, such as bladder, colorectal and prostate cancers, among the FDRs as compared with the SEER population. Conclusions: These findings confirm familial aggregation of PC with breast and ovarian cancers, and further suggest a potential aggregation of PC and primary liver cancer among female FDRs of PC probands. The elevated risks of breast cancer and ovarian cancer among siblings of the mutation-positive probands suggests a strong influence of genetic susceptibility shared with the related proband, possibly due to an inherited mutation in BRCA1, BRCA2, or PALB2. These findings lend support to genetic counseling and targeted screening of certain cancers in high-risk families. Citation Format: Samuel O. Antwi, Sarah E. Fagan, Kari G. Chaffee, William R. Bamlet, McWilliams R. Robert, Ann L. Oberg, Gloria M. Petersen. Risk of different cancers among first-degree relatives of pancreatic cancer patients and impact of probands’ germline mutation on sibling cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4269. doi:10.1158/1538-7445.AM2017-4269

Abstract 4870: Mutational landscape and timing of resistant clone emergence in 104 Chinese pediatric patients with relapsed acute lymphoblastic leukemia

Abstract Relapsed pediatric acute lympoblastic leukemia (ALL), occurring in 15-20% of cases, is one of the leading causes of cancer-related death in children, conferring a dismal survival rate of less than 40% primarily due to drug resistance. We performed whole-genome sequencing (WGS) and RNA-seq of matched diagnosis and relapse tumors as well as WGS of remission DNA from 104 pediatric ALL patients treated at Shanghai Children’s Medical Center (n=89), Institute of Hematology &amp; Blood Disease Hospital in Tianjin (n=14) and the Second Hospital of Anhui Medical University (n=1) between 2003 and 2014. The median age at diagnosis was 6.29 (range, 0.23-16.05) years. Relapse occurred during chemotherapy in 85 patients and after completion of therapy in 19 patients. Somatic mutation rate in ALL increased significantly from an average of 0.23 per Mb at diagnosis to 0.65 per Mb at relapse. We identified 34 potential driver genes at diagnosis and 45 at relapse based on significance of mutation recurrence and pathogenicity. The four most significant relapse-specific driver genes harbor known point mutations on purine metabolism genes NT5C2 (n=13) and PRPS1/PRPS2 (n=7), novel alterations affecting glucocorticoids receptor NR3C1 (n=13) and folylpolyglutamate synthetase FPGS (n=8). Functional characterization showed that all NR3C1 mutations resulted in significant loss of transcription activator function and subcellular mislocalization. Ectopic expression of mutant NR3C1 in ALL cell lines consistently led to prednisone resistance. Five driver genes (TP53, CREBBP, SMARCA4, WHSC1 and NIPBL) showed the highest increase (2-5 fold) of mutation frequency from diagnosis to relapse with mutations collectively present in 28% of relapsed ALL. Truncation mutations in DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 occurred exclusively at relapse in four ALL with the highest mutation burden. Targeted ultra-deep sequencing at 5,000X of 268 somatic alterations was performed on 214 serial bone-marrow samples collected during ALL therapy for 17 cases. The resulting mutant allele fraction (MAF) values showed high concordance with minimum residual disease (MRD) measured by flow cytometry, indicating suitability for quantifying leukemia burden. Temporal change of MAF in the serial samples also allows us to track clonal identity and emergence of drug-resistant clones during and after therapy. For example, one case had deep sequencing performed on 11 somatic lesions from 10 bone barrow samples collected during the 15 months between diagnosis and relapse. We found that a minor clone present in 0.5-1% of ALL blasts at diagnosis emerged five months prior to relapse, from which two new subclones arose harboring NT5C2 mutations L406&amp;gt;SSF and D407&amp;gt;DRD. Preliminary data generated from serial bone marrow samples provides the first insight into the use of somatic alterations for monitoring leukemia progress during relapse. Citation Format: Benshang Li, Yongjin Li, Shuhong Shen, Xiaofan Zhu, Xiaotu Ma, Ningling Wang, Yanling Liu, Yu Liu, Hui Zhang, Ting-Nien Lin, Michael Rusch, Michael Edmonson, John Easton, Yingchi Zhang, Jingliao Zhang, Cheng Cheng, Jingyan Tang, James R. Downing, Ching-Hon Pui, Jun J. Yang, Jinghui Zhang. Mutational landscape and timing of resistant clone emergence in 104 Chinese pediatric patients with relapsed acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4870. doi:10.1158/1538-7445.AM2017-4870

Constitutional mismatch repair deficiency and Lynch syndrome among consecutive Arab Bedouins with colorectal cancer in Israel.

We assessed the molecular characteristics and the frequency of mutations in mismatch-repair genes among Bedouin patients with colorectal cancer (CRC) in Israel. Bedouin patients with a diagnosis of CRC at a major hospital in the southern part of Israel were deemed eligible for this study. The primary screening method was immunohistochemical staining for mismatch-repair proteins (MLH1, MSH2, MSH6, and PMS2). For subjects with abnormal immunohistochemical staining, we performed microsatellite instability (MSI) analyses, and for tumors with a loss of MLH1 expression we also performed BRAF testing. In MSI high cases we searched further for germline mutations. Of the 24 patients enrolled, four subjects (16.7%) had MSI high tumors: one subject was found to harbor a biallelic PMS2 mutation, one subject had Lynch syndrome (LS) with MSH6 mutation and two subjects had a loss of MLH1/PMS2 proteins/BRAF wild type/normal MLH1 sequence. Ten patients (41.7%) were younger than 50 at the time of diagnosis and none had first degree relatives with CRC. In conclusion, in this cohort of 24 consecutive Arab Bedouins with CRC, one patient was found to harbor a constitutional mismatch repair deficiency, one patient had LS with MSH6 mutation, and two patients had unresolved loss of MLH1/PMS2 proteins/BRAF wild type phenotype.

Functional analysis of rare variants in mismatch repair proteins augments results from computation-based predictive methods

ABSTRACTThe cancer-predisposing Lynch Syndrome (LS) arises from germline mutations in DNA mismatch repair (MMR) genes, predominantly MLH1, MSH2, MSH6, and PMS2. A major challenge for clinical diagnosis of LS is the frequent identification of variants of uncertain significance (VUS) in these genes, as it is often difficult to determine variant pathogenicity, particularly for missense variants. Generic programs such as SIFT and PolyPhen-2, and MMR gene-specific programs such as PON-MMR and MAPP-MMR, are often used to predict deleterious or neutral effects of VUS in MMR genes. We evaluated the performance of multiple predictive programs in the context of functional biologic data for 15 VUS in MLH1, MSH2, and PMS2. Using cell line models, we characterized VUS predicted to range from neutral to pathogenic on mRNA and protein expression, basal cellular viability, viability following treatment with a panel of DNA-damaging agents, and functionality in DNA damage response (DDR) signaling, benchmarking to wild-type MMR proteins. Our results suggest that the MMR gene-specific classifiers do not always align with the experimental phenotypes related to DDR. Our study highlights the importance of complementary experimental and computational assessment to develop future predictors for the assessment of VUS.

Inhibition of colorectal cancer genomic copy number alterations and chromosomal fragile site tumor suppressor FHIT and WWOX deletions by DNA mismatch repair.

Homologous recombination (HR) enables precise DNA repair after DNA double strand breaks (DSBs) using identical sequence templates, whereas homeologous recombination (HeR) uses only partially homologous sequences. Homeologous recombination introduces mutations through gene conversion and genomic deletions through single-strand annealing (SSA). DNA mismatch repair (MMR) inhibits HeR, but the roles of mammalian MMR MutL homologues (MLH1, PMS2 and MLH3) proteins in HeR suppression are poorly characterized. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) carrying Mlh1, Pms2, and Mlh3 mutations have higher HeR rates, by using 7,863 uniquely mapping paired direct repeat sequences (DRs) in the mouse genome as endogenous gene conversion and SSA reporters. Additionally, when DSBs are induced by gamma-radiation, Mlh1, Pms2 and Mlh3 mutant MEFs have higher DR copy number alterations (CNAs), including DR CNA hotspots previously identified in mouse MMR-deficient colorectal cancer (dMMR CRC). Analysis of The Cancer Genome Atlas CRC data revealed that dMMR CRCs have higher genome-wide DR HeR rates than MMR proficient CRCs, and that dMMR CRCs have deletion hotspots in tumor suppressors FHIT/WWOX at chromosomal fragile sites FRA3B and FRA16D (which have elevated DSB rates) flanked by paired homologous DRs and inverted repeats (IR). Overall, these data provide novel insights into the MMR-dependent HeR inhibition mechanism and its role in tumor suppression.

Development and Validation of the PREMM5 Model for Comprehensive Risk Assessment of Lynch Syndrome.

Purpose Current Lynch syndrome (LS) prediction models quantify the risk to an individual of carrying a pathogenic germline mutation in three mismatch repair (MMR) genes: MLH1, MSH2, and MSH6. We developed a new prediction model, PREMM5, that incorporates the genes PMS2 and EPCAM to provide comprehensive LS risk assessment. Patients and Methods PREMM5 was developed to predict the likelihood of a mutation in any of the LS genes by using polytomous logistic regression analysis of clinical and germline data from 18,734 individuals who were tested for all five genes. Predictors of mutation status included sex, age at genetic testing, and proband and family cancer histories. Discrimination was evaluated by the area under the receiver operating characteristic curve (AUC), and clinical impact was determined by decision curve analysis; comparisons were made to the existing PREMM1,2,6 model. External validation of PREMM5 was performed in a clinic-based cohort of 1,058 patients with colorectal cancer. Results Pathogenic mutations were detected in 1,000 (5%) of 18,734 patients in the development cohort; mutations included MLH1 (n = 306), MSH2 (n = 354), MSH6 (n = 177), PMS2 (n = 141), and EPCAM (n = 22). PREMM5 distinguished carriers from noncarriers with an AUC of 0.81 (95% CI, 0.79 to 0.82), and performance was similar in the validation cohort (AUC, 0.83; 95% CI, 0.75 to 0.92). Prediction was more difficult for PMS2 mutations (AUC, 0.64; 95% CI, 0.60 to 0.68) than for other genes. Performance characteristics of PREMM5 exceeded those of PREMM1,2,6. Decision curve analysis supported germline LS testing for PREMM5 scores ≥ 2.5%. Conclusion PREMM5 provides comprehensive risk estimation of all five LS genes and supports LS genetic testing for individuals with scores ≥ 2.5%. At this threshold, PREMM5 provides performance that is superior to the existing PREMM1,2,6 model in the identification of carriers of LS, including those with weaker phenotypes and individuals unaffected by cancer.

Comprehensive population-wide analysis of Lynch syndrome in Iceland reveals founder mutations in MSH6 and PMS2

Lynch syndrome, caused by germline mutations in the mismatch repair genes, is associated with increased cancer risk. Here using a large whole-genome sequencing data bank, cancer registry and colorectal tumour bank we determine the prevalence of Lynch syndrome, associated cancer risks and pathogenicity of several variants in the Icelandic population. We use colorectal cancer samples from 1,182 patients diagnosed between 2000–2009. One-hundred and thirty-two (11.2%) tumours are mismatch repair deficient per immunohistochemistry. Twenty-one (1.8%) have Lynch syndrome while 106 (9.0%) have somatic hypermethylation or mutations in the mismatch repair genes. The population prevalence of Lynch syndrome is 0.442%. We discover a translocation disrupting MLH1 and three mutations in MSH6 and PMS2 that increase endometrial, colorectal, brain and ovarian cancer risk. We find thirteen mismatch repair variants of uncertain significance that are not associated with cancer risk. We find that founder mutations in MSH6 and PMS2 prevail in Iceland unlike most other populations.

Analysis of 100,000 human cancer genomes reveals the landscape of tumor mutational burden

BackgroundHigh tumor mutational burden (TMB) is an emerging biomarker of sensitivity to immune checkpoint inhibitors and has been shown to be more significantly associated with response to PD-1 and PD-L1 blockade immunotherapy than PD-1 or PD-L1 expression, as measured by immunohistochemistry (IHC). The distribution of TMB and the subset of patients with high TMB has not been well characterized in the majority of cancer types.MethodsIn this study, we compare TMB measured by a targeted comprehensive genomic profiling (CGP) assay to TMB measured by exome sequencing and simulate the expected variance in TMB when sequencing less than the whole exome. We then describe the distribution of TMB across a diverse cohort of 100,000 cancer cases and test for association between somatic alterations and TMB in over 100 tumor types.ResultsWe demonstrate that measurements of TMB from comprehensive genomic profiling are strongly reflective of measurements from whole exome sequencing and model that below 0.5 Mb the variance in measurement increases significantly. We find that a subset of patients exhibits high TMB across almost all types of cancer, including many rare tumor types, and characterize the relationship between high TMB and microsatellite instability status. We find that TMB increases significantly with age, showing a 2.4-fold difference between age 10 and age 90 years. Finally, we investigate the molecular basis of TMB and identify genes and mutations associated with TMB level. We identify a cluster of somatic mutations in the promoter of the gene PMS2, which occur in 10% of skin cancers and are highly associated with increased TMB.ConclusionsThese results show that a CGP assay targeting ~1.1 Mb of coding genome can accurately assess TMB compared with sequencing the whole exome. Using this method, we find that many disease types have a substantial portion of patients with high TMB who might benefit from immunotherapy. Finally, we identify novel, recurrent promoter mutations in PMS2, which may be another example of regulatory mutations contributing to tumorigenesis.

Homozygous germ-line mutation of the PMS2 mismatch repair gene: a unique case report of constitutional mismatch repair deficiency (CMMRD)

BackgroundConstitutional mismatch repair deficiency syndrome results from bi-allelic inheritance of mutations affecting the key DNA mismatch repair genes: MLH1, MSH2, MSH6 or PMS2. Individuals with bi-allelic mutations have a dysfunctional mismatch repair system from birth; as a result, constitutional mismatch repair deficiency syndrome is characterised by early onset malignancies. Fewer than 150 cases have been reported in the literature over the past 20 years. This is the first report of the founder PMS2 mutation - NM_000535.5:c.1500del (p.Val501TrpfsTer94) in exon 11 and its associated cancers in this family.Case presentationThe proband is 30 years old and is alive today. She is of Pakistani ethnic origin and a product of consanguinity. She initially presented aged 24 with painless bleeding per-rectum from colorectal polyps and was referred to clinical genetics. Clinical examination revealed two café-au-lait lesions, lichen planus, and a dermoid cyst. Her sister had been diagnosed in childhood with an aggressive brain tumour followed by colorectal cancer. During follow up, the proband developed 37 colorectal adenomatous polyps, synchronous ovarian and endometrial adenocarcinomas, and ultimately a metachronous gastric adenocarcinoma. DNA sequencing of peripheral lymphocytes revealed a bi-allelic inheritance of the PMS2 mutation NM_000535.5:c.1500del (p.Val501TrpfsTer94) in exon 11. Ovarian tumour tissue demonstrated low microsatellite instability. To date, she has had a total abdominal hysterectomy, bilateral salpingo-oophorectomy, and a total gastrectomy. Aspirin and oestrogen-only hormone replacement therapy provide some chemoprophylaxis and manage postmenopausal symptoms, respectively. An 18-monthly colonoscopy surveillance programme has led to the excision of three high-grade dysplastic colorectal tubular adenomatous polyps. The proband’s family pedigree displays multiple relatives with cancers including a likely case of ‘true’ Turcot syndrome.ConclusionsConstitutional mismatch repair deficiency syndrome should be considered in patients who present with early onset cancer, a strong family history of cancer, and cutaneous features resembling neurofibromatosis type I. Immunohistochemistry analysis of tumour and normal tissue is sensitive and specific for identifying patients with mismatch repair deficiency and should direct DNA sequencing of lymphocytic tissue to establish a diagnosis. Microsatellite instability status appears to be of little value in identifying patients who may have constitutional mismatch repair deficiency syndrome.

Germline mutations in seemingly sporadic pancreatic cancer.

312 Background: Genetic predisposition underlies approximately 10% of pancreatic cancer (PC). Germline mutations in BRCA1, BRCA2, PALB2, ATM, TP53, MLH1, MSH2, MSH6, PMS2, PRSS1, APC, BMPR1A, STK11, CDKN2A, and SMAD4 are associated with PC susceptibility. Since young onset is not a hallmark of hereditary PC, we have hypothesized that seemingly sporadic cases may carry germline mutations in these genes. Methods: To investigate the prevalence of germline mutations in these 15 genes among sporadic PC cases, we analyzed lymphocyte DNA next generation sequencing data from 296 PC cases ascertained over 1 year from two PC referral centers in Toronto and Montreal. Results: We identified 21 mutations in 5 of the 15 genes (10 in BRCA2, 2 in BRCA1, 5 in ATM, 1 in p16, 3 in PRSS1) in 19 individuals. One individual carried two BRCA2 mutations that are usually observed together, and another individual carried both a PRSS1 and ATM mutation, however, in the absence of pancreatitis, PRSS1 mutations are not known to increase risk of PC. Incidences of 1.7% and 4.1% in ATM and BRCA-1 or -2 were observed. Approximately half of these mutations (4/9 for BRCA2, 1/2 for BRCA1, and 1/1 for p16) were known founder mutations in the French Canadian, Ashkenazi Jewish, Dutch or Asian populations. Since the role of ATMin PC predisposition remains under investigation, we obtained further evidence by testing for loss of the wild-type allele in cases with available tumor tissue. We observed loss of the wild-type allele in the 2 cases tested. Interestingly, the final pathology for the resected specimen from one of these two cases favored an ampullary cancer rather than a PC diagnosis. Conclusions: Since there are screening implications for the relatives of carriers and potentially treatment considerations for patients using therapies targeting DNA repair defects, these findings suggest a role for reflex testing of incident cases for at least population-specific recurrent mutations. Our findings also suggest that ATM germline mutations may give rise to ampullary cancer, underlie 1.4% of incident PC cases and imply broader testing considerations. For patients, testing may direct therapy choices if these tumors are targetable using agents that exploit DNA repair defects.

Approach to Lynch Syndrome for the Gastroenterologist.

Lynch syndrome is an autosomal-dominant hereditary cancer syndrome. Mutations in mismatch repair genes, including MLH1, MSH2, MSH6, and PMS2, are implicated in the pathogenesis of the syndrome through microsatellite instability (MSI) and a rapid adenoma-carcinoma sequence. The primary methodologies for diagnosis include clinical criteria (Amsterdam I/II, Revised Bethesda Guidelines), computational models, and genetic testing (MSI, immunohistochemistry, germline testing). Universal genetic testing of colorectal cancers has gained popularity as a method to identify high-risk individuals and to offer appropriate cancer surveillance, psychological reassurance, and family planning. Management includes short-interval surveillance with colonoscopy in those without colorectal cancer and colectomy for those with cancer. Long-term chemoprevention with aspirin may improve mortality.

Germline PMS 2 mutation screened by mismatch repair protein immunohistochemistry of colorectal cancer in Japan

Germline PMS2 gene mutations were detected by RT‐PCR/direct sequencing of total RNA extracted from puromycin‐treated peripheral blood lymphocytes (PBL) and multiplex ligation‐dependent probe amplification (MLPA) analyses of Japanese patients with colorectal cancer (CRC) fulfilling either the revised Bethesda Guidelines or being an age at disease onset of younger than 70 years, and screened by mismatch repair protein immunohistochemistry of formalin‐fixed paraffin embedded sections. Of the 501 subjects examined, 7 (1.40%) showed the downregulated expression of the PMS2 protein alone and were referred to the genetic counseling clinic. Germline PMS2 mutations were detected in 6 (85.7%), including 3 nonsense and 1 frameshift mutations by RT‐PCR/direct sequencing and 2 genomic deletions by MLPA. No mutations were identified in the other MMR genes (i.e. MSH2,MLH1 and MSH6). The prevalence of the downregulated expression of the PMS2 protein alone was 1.40% among the subjects examined and IHC results predicted the presence of PMS2 germline mutations. RT‐PCR from puromycin‐treated PBL and MLPA may be employed as the first screening step to detect PMS2 mutations without pseudogene interference, followed by the long‐range PCR/nested PCR validation using genomic DNA.

Germline multi-gene hereditary cancer panel testing in an unselected endometrial cancer cohort

AbstractHereditary endometrial carcinoma is associated with germline mutations in Lynch syndrome genes. The role of other cancer predisposition genes is unclear. We aimed to determine the prevalence of cancer predisposition gene mutations in an unselected endometrial carcinoma patient cohort. Mutations in 25 genes were identified using a next-generation sequencing-based panel applied in 381 endometrial carcinoma patients who had undergone tumor testing to screen for Lynch syndrome. Thirty-five patients (9.2%) had a deleterious mutation: 22 (5.8%) in Lynch syndrome genes (three MLH1, five MSH2, two EPCAM-MSH2, six MSH6, and six PMS2) and 13 (3.4%) in 10 non-Lynch syndrome genes (four CHEK2, one each in APC, ATM, BARD1, BRCA1, BRCA2, BRIP1, NBN, PTEN, and RAD51C). Of 21 patients with deleterious mutations in Lynch syndrome genes with tumor testing, 2 (9.5%) had tumor testing results suggestive of sporadic cancer. Of 12 patients with deleterious mutations in MSH6 and PMS2, 10 were diagnosed at age >50 and 8 did not have a family history of Lynch syndrome-associated cancers. Patients with deleterious mutations in non-Lynch syndrome genes were more likely to have serous tumor histology (23.1 vs 6.4%, P=0.02). The three patients with non-Lynch syndrome deleterious mutations and serous histology had mutations in BRCA2, BRIP1, and RAD51C. Current clinical criteria fail to identify a portion of actionable mutations in Lynch syndrome and other hereditary cancer syndromes. Performance characteristics of tumor testing are sufficiently robust to implement universal tumor testing to identify patients with Lynch syndrome. Germline multi-gene panel testing is feasible and informative, leading to the identification of additional actionable mutations.

Phenotypic Heterogeneity by Germline Mismatch Repair Gene Defect in Lynch Syndrome Patients.

Lynch syndrome is the most common form of hereditary colorectal cancer, being also responsible for endometrial andother types of cancers. It is associated with germline mutations in DNA mismatch repair genes and microsatellite instability. MLH1 andMSH2 mutations have a "classical" Lynch syndrome phenotype, with MSH2 having a higher association with extracolonic cancer. MSH6and PMS2 mutations have an atypical phenotype. Clinical expression is heterogeneous, with correlation between mismatch repairmutated gene and phenotypic patterns. We retrospectively analyzed data from patients fulfilling Amsterdam criteria or having mismatch repair genemutations, between September 2012 and October 2015. We identified 28 patients. Seventeen had colorectal cancer with right colon predominance. Five developed endometrialcancer (median age of diagnosis - 53), with no MSH6 mutations. Five developed other cancers. All mutated mismatch repair casesstudied had microsatellite instability. Most cases had MSH2 mutations despite MLH1 being described in the literature as the most frequently mutated.Interestingly, colorectal cancer patients showed no tendency for high inflammatory infiltrate. Despite the high incidence of synchronousand metachronous tumours, most patients underwent a partial colectomy. Prophylactic hysterectomy and adnexectomy was performedin menopausal/perimenopausal patients. A standardized registration of patient's data may lead to better management and knowledge about Lynch syndrome.Use of Bethesda Guidelines might identify new cases non-identified by Amsterdam criteria. Microsatellite instability analysis must beperformed in a much larger scale. The genotypic/phenotypic correlation described in the literature was not verified in our study withstatistical significance, perhaps due to small data sample and insufficient clinical registration.