Mismatch repair protein deficiency and its implications on distant metastasis in colorectal cancer: A comprehensive analysis.

BACKGROUND: While previous studies have indicated variability in distant metastatic potential among different mismatch repair (MMR) states in colorectal cancer (CRC), their findings remain inconclusive, especially considering potential differences across various ethnic backgrounds. Furthermore, the gene regulatory networks and the underlying mechanisms responsible for these variances in metastatic potential across MMR states have yet to be elucidated. METHODS: We collected 2058 consecutive primary CRC samples from the South West of China and assessed the expression of MMR proteins (MLH1, MSH2, MSH6, and PMS2) using immunohistochemistry. To explore the inconsistencies between different MMR statuses and recurrence, we performed a meta-analysis. To delve deeper, we employed Weighted Gene Co-expression Network Analysis (WGCNA), ClueGo, and iRegulon, pinpointing gene expression networks and key regulatory molecules linked to metastasis and recurrence in CRC. Lastly, both univariate and multivariate Cox regression analyses were applied to determine the impact of core regulatory molecules on metastasis. RESULTS: Of the samples, 8.2% displayed deficient MMR (dMMR), with losses of MLH1 and PSM2 observed in 40.8% and 63.9%, respectively. A unique 24.3% isolated loss of PMS2 without concurrent metastasis was identified, a result that diverges from established literature. Additionally, our meta-analysis further solidifies the reduced recurrence likelihood in dMMR CRC samples compared to proficient MMR (pMMR). Two gene expression networks tied to distant metastasis and recurrence were identified, with a majority of metastasis-related genes located on chromosomes 8 and 18. An IRF1 positive feedback loop was discerned in the metastasis-related network, and IRF1 was identified as a predictive marker for both recurrence-free and distant metastasis-free survival across multiple datasets. CONCLUSION: Geographical and ethnic factors might influence peculiarities in MMR protein loss. Our findings also highlight new gene expression networks and crucial regulatory molecules in CRC metastasis, enhancing our comprehension of the mechanisms driving distant metastasis.

Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques.

BACKGROUND: Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. METHODS: We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. RESULTS: Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. CONCLUSIONS: These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.

Association of a novel frameshift variant and a known deleterious variant in MMR genes with Lynch syndrome in Chinese families.

BACKGROUND: Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome. This condition is characterized by germline variants in DNA mismatch repair (MMR) genes, including MLH1, MSH2, MSH6, and PMS2. In this study, we analyzed the molecular defects and clinical manifestations of two families affected with CRC and proposed appropriate individual preventive strategies for all carriers of the variant. METHODS: We recruited two families diagnosed with CRC and combined their family history and immunohistochemical results to analyze the variants of probands and those of other family members by using whole exome sequencing. Subsequently, gene variants in each family were screened by comparing them with the variants available in the public database. Sanger sequencing was performed to verify the variant sites. An online platform ( https://www.uniprot.org ) was used to analyze the functional domains of mutant proteins. RESULTS: A novel frameshift variant (NM_001281492, c.1129_1130del, p.R377fs) in MSH6 and a known deleterious variant (NM_000249.4:c.1731G > A, p.S577S) in MLH1 were identified in the two families with CRC. Using bioinformatics tools, we noted that the frameshift variant reduced the number of amino acids in the MSH6 protein from 1230 to 383, thereby leading to no MSH6 protein expression. The silent variant caused splicing defects and was strongly associated with LS. 5-Fluorouracil-based adjuvant chemotherapy is not recommended for patients with LS. CONCLUSIONS: The novel frameshift variant (MSH6, c.1129_1130del, p.R377fs) is likely pathogenic to LS, and the variant (MLH1, c.1731G > A, p.S577S) has been further confirmed to be pathogenic to LS. Our findings underscore the significance of genetic testing for LS and recommend that genetic consultation and regular follow-ups be conducted to guide individualized treatment for cancer-afflicted families, especially those with a deficiency in MMR expression.

MLH1 Promotor Hypermethylation in Colorectal and Endometrial Carcinomas from Patients with Lynch Syndrome.

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, MLH1 promotor hypermethylation (MLH1-PM) testing is performed to indirectly distinguish the constitutional MLH1 variants 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 MLH1 gene promotor C region was reported in 30 MLH1, 6 MSH2, 6 MSH6, and 3 PMS2 variant 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 MLH1 gene 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.

Functional and phenotypic consequences of an unusual inversion in MSH2.

Lynch syndrome is an autosomal dominant disorder that usually results from a pathogenic germline variant in one of four genes (MSH2, MSH6, MLH1, PMS2) involved in DNA mismatch repair. Carriers of such variants are at risk of developing numerous cancers during adulthood. Here we report on a family suspected of having Lynch syndrome due to a history of endometrial adenocarcinoma, ovarian clear cell carcinoma, and adenocarcinoma of the duodenum in whom we identified a germline 29 nucleotide in-frame inversion in exon 3 of MSH2. We further show that this variant is almost completely absent at the protein level, and that the associated cancers have complete loss of MSH2 and MSH6 expression by immunohistochemistry. Functional investigation of this inversion in a laboratory setting revealed a resultant abnormal protein function. Thus, we have identified an unusual, small germline inversion in a mismatch repair gene that does not lead to a premature stop codon yet appears likely to be causal for the observed cancers.

A Comprehensive Study of Heterogeneous Mismatch Repair Expression in Solid Tumors Reveals Different Immunohistochemical Patterns and Distinct Genetic Mechanisms.

OBJECTIVE: Immunohistochemistry is routinely performed to detect mismatch repair deficiency in solid tumors. Heterogeneous MMR expression (MMR-het) has been reported occasionally but not systemically studied. METHODS: In this study, we depicted MMR-het patterns of 40 tumors of different anatomical sites and analyzed MMR genetic alterations and tumor mutational burdens (TMB) through comprehensive genomic profiling. RESULTS: The MMR-het patterns were classified into 4 subgroups: "single-loss" (3 cases), "MLH1/PMS2 double-loss" (16 cases), "MSH2/MSH6 double-loss" (8 cases), and "triple/tetra-loss" (13 cases). Seventeen MMR-het cases exhibited histological heterogeneity, in which MMR protein loss was generally confined to either poorly differentiated or well-differentiated tumor areas. All "single-loss" tumors had MMR somatic mutations and coexisting POLE exonuclease domain mutations. "MLH1/PMS2 double-loss" tumors unexceptionally harbored MLH1 hypermethylation without MMR germline mutations. In the "MSH2/MSH6 double-loss" subgroup, 4 cases had MSH2/MSH6 germline mutations, while another 4 cases had multiple MSH2/MSH6 somatic mutations. Additional POLE exonuclease domain mutations were identified in 2 cases. Tumors in the "triple/tetra-loss" subgroup generally had MLH1 abnormalities (8 MLH1 hypermethylation, 4 MLH1 germline mutation, 1 MLH1 double somatic mutations), and coexistent somatic mutations on MSH2/MSH6 . Thirty-one cases (83.8%) were TMB-H, and all POLE -mutated cases exhibited ultra-high TMB (111.4 to 524.2 mut/Mb). CONCLUSION: Our findings highlighted the importance of accurately interpreting heterogeneous MMR protein staining patterns for developing a more efficient personalized genetic investigation strategy.

Prevalence and characteristics of patients with upper urinary tract urothelial carcinoma having potential Lynch syndrome identified by immunohistochemical universal screening and Amsterdam criteria II.

BACKGROUND: This study aimed to identify patients with upper urinary tract urothelial carcinoma (UTUC) having potential Lynch syndrome (pLS) by immunohistochemistry (IHC) of DNA mismatch repair gene-related proteins (MMRPs) and Amsterdam criteria II and explore their clinical characteristics. METHODS: We retrospectively collected the clinical data of 150 consecutive patients with UTUC who underwent surgical resection at our institution between February 2012 and December 2020, and immunohistochemistry (IHC) of four MMRPs (MLH1, MSH2, MSH6, and PMS2) on all UTUC specimens was performed. Patients who tested positive for Amsterdam criteria (AMS) II and/or IHC screening were classified as having pLS and others as non-pLS, and their characteristics were explored. RESULTS: In this study, 5 (3%) and 6 (4%) patients were positive for AMS II and IHC screening, respectively. Two patient were positive for both AMS II and IHC screening, resulting in 9 (6%) patients with pLS. The pLS group was predominantly female (67% vs. 36%; p = 0.0093) and had more right-sided tumors (100% vs. 43%; p = 0.0009) than the non-pLS group. Of the 6 patients who were positive for IHC screening, 4 showed a combined loss of MSH2/MSH6 (n = 3) and MLH1/PMS2 (n = 1). Other two patients showed single loss of MSH6 and PSM2. CONCLUSIONS: AMS II and IHC screening identified pLS in 6% of patients with UTUC. The IHC screening-positive group tends to have relatively high rate of combined loss, but some patients have single loss. AMS II may overlook patients with LS, and a universal screening may be required for patients with UTUC as well as those with colorectal and endometrial cancer.

Mismatch Repair Protein Deficiency Does Not Affect Disease Free Survival in Type I Endometrial Carcinoma.

BACKGROUND: This study aimed to analyze the correlation between the 3-year disease-free survival (DFS) and mismatch repair (MMR) protein levels in patients with type 1 endometrial carcinoma. Many studies have reported different results regarding the role of MMR in the prognosis of endometrial carcinoma; therefore, we aimed to identify this association in our hospital. METHODS: This observational study employed a historical cohort design and included patients with type 1 endometrial carcinoma who underwent surgery at Dr. Soetomo Hospital between January 2017 and December 2019. Medical records and paraffin blocks meeting these criteria were obtained. MMR proteins (MLH1 and MSH2) were assessed using immunohistochemistry. RESULTS: A total of 46 patients with type 1 endometrial carcinoma were analyzed. We observed MMR deficiency (dMMR) in 12 patients (26.1%) and MMR proficiency (pMMR) in 34 patients (73.9%). Of the 12 patients with dMMR, nine cases (75%) were diagnosed as stage I and 7 (58.33%) as low grade. The 3-year DFS in patients with dMMR and pMMR was 83.3% and 67.6%, respectively (Hazard Ratio 2.31, 95% CI 0.5135-10.475, p=0.27). Higher stages had a 5.42 times increased risk of recurrence (95% CI 1.3378-21.9358, p=0.018). Higher histopathological grades were also associated with 8.65 times increased risk of recurrence (95% CI 2.5020-29.8738, p=0.001). CONCLUSION: Patients with dMMR had a better DFS compared to those with pMMR; however, the difference was not statistically significant. The tumor stage and histopathological grade were independent risk factors for recurrence.

Key role of phosphorylation sites in ATPase domain and Linker region of MLH1 for DNA binding and functionality of MutLα.

MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.

Improving genetic testing following abnormal mismatch repair immunohistochemistry results in endometrial cancer.

OBJECTIVES: Although universal mismatch repair (MMR) immunohistochemistry (IHC) in endometrial cancer began at our institution in July 2015, not all eligible patients were referred for genetic testing (GT). In April 2017, genetic counselors obtained IHC data and contacted physicians to approve genetic counseling referrals (GCRs) for Lynch Syndrome (LS) in eligible patients. We assessed if this protocol increased frequency of GCRs and GT in patients with abnormal MMR IHC. METHODS: We retrospectively (7/2015-5/2022) identified patients with abnormal MMR IHC at a large urban hospital. GCRs and GT were compared between cases from 7/2015-4/2017 (pre-protocol) and 5/2017-5/2022 (post-protocol) with chi-square and Fisher's exact tests. RESULTS: Of 794 patients with IHC testing, 177 (22.3%) had abnormal MMR results with 46 (26.0%) meeting criteria for LS screening with GT. Of 46 patients, 16 (34.8%) were identified prior to and 30 (65.2%) after the protocol initiation. GCRs significantly increased from 11/16 (68.8%) to 29/30 (96.7%) in the pre-protocol versus post-protocol groups, p = 0.02. There was no statistically significant difference in GT between groups (10/16, 62.5% vs 26/30, 86.7%, p = 0.07). Of 36 patients who underwent GT, 16 (44.4%) had LS: MSH6, 9; MSH2, 4; PMS2, 2; MLH1, 1. CONCLUSIONS: Increased frequency of GCRs was observed following the change in protocol, which is important as LS screening has clinical implications for patients and their families. Despite this additional effort, approximately 15% who met criteria did not undergo GT; further efforts such as universal germline testing in patients with endometrial cancer should be considered.

Concordance in detection of microsatellite instability by PCR and NGS in routinely processed tumor specimens of several cancer types.

BACKGROUND: Microsatellite instability (MSI) occurs in several cancer types and is commonly used for prognosis and as a predictive biomarker for immune checkpoint therapy. METHODS: We analyzed n = 263 formalin-fixed paraffin-embedded (FFPE) tumor specimens (127 colorectal cancer (CRC), 55 endometrial cancer (EC), 33 stomach adenocarcinoma (STAD), and 48 solid tumor specimens of other tumor types) with a capillary electrophoresis based multiplex monomorphic marker MSI-PCR panel and an amplicon-based NGS assay for microsatellite instability (MSI+). In total, n = 103 (39.2%) cases with a known defect of the DNA mismatch repair system (dMMR), determined by a loss in protein expression of MSH2/MSH6 (n = 48, 46.6%) or MLH1/PMS2 (n = 55, 53.4%), were selected. Cases with an isolated loss of MSH6 or PMS2 were excluded. RESULTS: The overall sensitivity and specificity of the NGS assay in comparison with the MSI-PCR were 92.2% and 98.8%. With CRC cases a nearly optimal concordance was reached (sensitivity 98.1% and specificity 100.0%). Whereas EC cases only show a sensitivity of 88.6% and a specificity of 95.2%, caused by several cases with instability in less than five monomorphic markers, which could be difficult to analyze by NGS (subtle MSI+ phenotype). CONCLUSIONS: MSI analysis of FFPE DNA by NGS is feasible and the results show a high concordance in comparison with the monomorphic marker MSI-PCR. However, cases with a subtle MSI+ phenotype, most frequently manifest in EC, have a risk of a false-negative result by NGS and should be preferentially analyzed by capillary electrophoresis.

Mutation-specific Mismatch Repair-deficient Benign Endometrial Glands in Endometrial Biopsies and Curettings Are a Biomarker of Lynch Syndrome and Associate With Endometrial Carcinoma Development.

Endometrial carcinoma is the most common extraintestinal cancer in Lynch syndrome (LS). Recent studies have demonstrated mismatch repair (MMR) deficiency can be detected in benign endometrial glands in LS. We performed MMR immunohistochemistry in benign endometrium from endometrial biopsies and curettings (EMCs) from a study group of 34 confirmed LS patients and a control group of 38 patients without LS who subsequently developed sporadic MLH1-deficient or MMR-proficient endometrial carcinoma. MMR-deficient benign glands were only identified in patients with LS (19/34, 56%) and were not identified in any control group patient (0/38, 0%) ( P < 0.001). MMR-deficient benign glands were identified as large, contiguous groups in 18 of 19 cases (95%). MMR-deficient benign glands were identified in patients with germline pathogenic variants in MLH1 (6/8, 75%), MSH6 (7/10, 70%), and MSH2 (6/11, 55%) but not in patients with variants in PMS2 (0/4). MMR-deficient benign glands were seen in all EMC samples (100%) but in only 46% of endometrial biopsy samples ( P =0.02). Patients with MMR-deficient benign glands were significantly more likely to have endometrial carcinoma (53%) compared with LS patients with only MMR-proficient glands (13%) ( P =0.03). In conclusion, we demonstrated that MMR-deficient benign endometrial glands are frequently identified in EMB/EMC in women with LS and are a specific marker for LS. Women with LS with MMR-deficient benign glands were more likely to have endometrial carcinoma suggesting that MMR-deficient benign glands may be a biomarker of increased risk of endometrial carcinoma development in LS.

MMR markers correlate with clinical outcome in patients with esophageal squamous cell carcinoma.

BACKGROUND: The DNA mismatch repair system is one of the defense mechanisms in the body, and the inactivation of mismatch repair plays a pivotal role in secondary carcinogenesis and progression. However, the significance of mismatch repair in esophageal squamous cell carcinoma (ESCC) has not been established. In this study, we explored the diagnostic and prognostic significance of mismatch repair markers, mutL homologue 1 (MLH1), post-meiotic segregation increased 2 (PMS2), mutS homologue 2 (MSH2), and mutS homologue 6 (MSH6), in patients with ESCC. METHODS: We used a notation based on the proportion of immunoreactivity/expression for immunohistochemistry (PRIME notation), which allows the comparison of mismatch repair expression by assigning a score to PRIME notation. MLH1, PMS2, MSH2, and MSH6 were examined immunohistochemically in 189 surgically resected ESCC specimens. RESULTS: A total of 100/189 patients with ESCC (53%) received preoperative chemotherapy. The rates of ESCC cases with decreased mismatch repair status were 13.2%, 15.3%, 24.8%, and 12.6% for MLH1, PMS2, MSH2, and MSH6, respectively. The decreased status of individual mismatch repair markers was significantly correlated with worse prognosis in patients with ESCC. Additionally, MSH2, MSH6, and PMS2 were significantly associated with response to preoperative chemotherapy. Multivariate analysis revealed that MLH1, PMS2, and MSH2 are independent prognostic factors. CONCLUSION: Our results suggest that mismatch repair is a prognostic biomarker for ESCC and could contribute to the selection of appropriate adjuvant therapy for patients with ESCC.

Endometrial Carcinomas With Subclonal Loss of Mismatch Repair Proteins: A Clinicopathologic and Genomic Study.

Subclonal loss of mismatch repair (MMR) proteins has been described in a small subset of endometrial carcinomas (ECs), but the genomic basis for this phenomenon has received limited attention. Herein, we retrospectively evaluated all ECs with MMR immunohistochemistry (n=285) for subclonal loss, and in those (n=6), performed a detailed clinicopathologic and genomic comparison of the MMR-deficient and MMR-proficient components. Three tumors were FIGO stage IA, and one each stage IB, II, and IIIC2. Patterns of subclonal loss were as follows: (1) 3 FIGO grade 1 endometrioid carcinomas with subclonal MLH1/PMS2, MLH1 promoter hypermethylation, and no MMR gene mutations; (2) POLE -mutated FIGO grade 3 endometrioid carcinoma with subclonal PMS2, and PMS2 and MSH6 mutations limited to the MMR-deficient component; (3) dedifferentiated carcinoma with subclonal MSH2/MSH6, as well as complete loss of MLH1/PMS2, MLH1 promoter hypermethylation, and PMS2 and MSH6 mutations in both components; (4) dedifferentiated carcinoma with subclonal MSH6, and somatic and germline MSH6 mutations in both components, but with a higher allele frequency in MMR-deficient foci. Recurrences occurred in 2 patients, one consisted of the MMR-proficient component from a FIGO 1 endometrioid carcinoma, while the other was from the MSH6 -mutated dedifferentiated endometrioid carcinoma. At the last follow-up (median: 44 mo), 4 patients were alive and disease-free and 2 were alive with disease. In summary, subclonal MMR loss reflects subclonal and often complex genomic and epigenetic alterations, which may have therapeutic implications and therefore must be reported when present. In addition, subclonal loss can occur in both POLE -mutated and Lynch syndrome-associated ECs.

Rare germline variants in pancreatic cancer and multiple primary cancers: an autopsy study.

BACKGROUND: There is a lack of information on rare germline variants of pancreatic cancer-predisposing genes. Risk genes for multiple primary cancers may overlap with those for pancreatic cancer. METHODS: A retrospective study of autopsy cases with a negative family history in the Japanese single nucleotide polymorphism for geriatric research database examined rare germline variants in the protein-coding regions of 61 genes. Targeted sequencing of these genes was performed and classified for pathogenicity using the American College of Medical Genetics and Genomics guidelines. Polyphen-2, SIFT and LoFtool algorithms were used to predict damage to protein function. RESULTS: Of the 189 subjects used (90 cancer and 99 non-cancer controls), 72 patients had pancreatic cancer (23 had multiple primary cancers) and 18 had no pancreatic cancer in multiple primary cancers. APC, BRCA2, BUB1B, ENG and MSH6 were associated with cancer predisposition, and pathogenic/likely pathogenic (P/LP) variants occurred in 6% [pancreatic cancer (4/72); all-cancer (5/90)] and 54% (49/90) carried only variants of uncertain significance (VUS) among cancer patients. Of these VUS, in pancreatic cancer patients, four DNA mismatch repair (MMR) genes ( MLH1, MSH2, MSH6 and PMS2 ), and POLQ in men were significantly associated (odds ratio = 3.83; P = 0.025; P = 0.027, respectively). The most abundant predictor of functionally damaging variants was POLQ . CONCLUSIONS: The frequency of P/LP variants in patients with sporadic pancreatic cancer suggests the need for genetic evaluation of individuals with no family history. VUS of MMR genes ( MLH1, MSH2, MSH6 and PMS2 ) and POLQ may be useful in predicting genetic trends in the potential risk of pancreatic cancer, especially in individuals lacking P/LP.

Universal screening for Lynch syndrome in operated colorectal cancer by immunohistochemistry: a cohort of patients in Liaoning province, China.

OBJECTIVE: Lynch syndrome (LS) is the most common hereditary colorectal cancer syndrome worldwide. Due to the decreasing family size in Liaoning province. The Bethesda and Amsterdam II criteria have lower sensitivity and specificity and are not suitable for the local population. Immunohistochemistry screening for mutations in DNA mismatch repair (MMR) in newly diagnosed colorectal cancer can improve the detection rate of LS. METHODS: All newly diagnosed colorectal cancer patients who underwent surgery between January 2018 and June 2020 at Cancer Hospital of China Medical University and Shengjing Hospital of China Medical University from Liaoning China were included retrospectively, and the ratio of universal LS screening by immunohistochemistry, MMR protein deficiency (dMMR) ratio, MLH1 loss, MSH2 loss, MSH6 loss, and PMS2 loss was analyzed. The clinicopathological characteristics of patients with pMMR and dMMR were analyzed. RESULTS: A total of 7019 colorectal cancer patients underwent surgery and 4802 (68.41%) patients were screened by immunohistochemistry for MMR, 258 (5.37%) cases were reported to have a loss of MMR expression. In the dMMR group, a higher number of patients were under 50 years old, more tumors were located at the right colon, less patients have lymph node metastasis, more tumors were stage II, and histological types of mucinous carcinoma or signet ring carcinoma were more common, compared with the pMMR group. Only 2.71% dMMR patients meet Amsterdam criteria II, 2.71% of patients meet Revised Bethesda guidelines, and 17.83% meet Chinese LS criteria. Twenty-five dMMR patients were confirmed by next-generation sequencing and five families were confirmed as Lynch family. CONCLUSION: These data imply that universal screening for LS by immunohistochemistry may be effective in Liaoning province.

Identification PMS1 and PMS2 as potential meiotic substrates of CDK2 activity.

Cyclin dependent-kinase 2 (CDK2) plays important functions during the mitotic cell cycle and also facilitates several key events during germ cell development. The majority of CDK2's known meiotic functions occur during prophase of the first meiotic division. Here, CDK2 is involved in the regulation of meiotic transcription, the pairing of homologous chromosomes, and the maturation of meiotic crossover sites. Despite that some of the CDK2 substrates are known, few of them display functions in meiosis. Here, we investigate potential meiotic CDK2 substrates using in silico and in vitro approaches. We find that CDK2 phosphorylates PMS2 at Thr337, PMS1 at Thr331, and MLH1 in vitro. Phosphorylation of PMS2 affects its interaction with MLH1 to some degree. In testis extracts from mice lacking Cdk2, there are changes in expression of PMS2, MSH2, and HEI10, which may be reflective of the loss of CDK2 phosphorylation. Our work has uncovered a few CDK2 substrates with meiotic functions, which will have to be verified in vivo. A better understanding of the CDK2 substrates will help us to gain deeper insight into the functions of this universal kinase.

Resolution of sequence divergence for repeat-mediated deletions shows a polarity that is mediated by MLH1.

Repeat-mediated deletions (RMDs) are a type of chromosomal rearrangement between two homologous sequences that causes loss of the sequence between the repeats, along with one of the repeats. Sequence divergence between repeats suppresses RMDs; the mechanisms of such suppression and of resolution of the sequence divergence remains poorly understood. We identified RMD regulators using a set of reporter assays in mouse cells that test two key parameters: repeat sequence divergence and the distances between one repeat and the initiating chromosomal break. We found that the mismatch repair factor MLH1 suppresses RMDs with sequence divergence in the same pathway as MSH2 and MSH6, and which is dependent on residues in MLH1 and its binding partner PMS2 that are important for nuclease activity. Additionally, we found that the resolution of sequence divergence in the RMD product has a specific polarity, where divergent bases that are proximal to the chromosomal break end are preferentially removed. Moreover, we found that the domain of MLH1 that forms part of the MLH1-PMS2 endonuclease is important for polarity of resolution of sequence divergence. We also identified distinctions between MLH1 versus TOP3α in regulation of RMDs. We suggest that MLH1 suppresses RMDs with sequence divergence, while also promoting directional resolution of sequence divergence in the RMD product.

Diagnostic and therapeutic challenges of glioblastoma as an initial malignancy of constitutional mismatch repair deficiency (CMMRD): two case reports and a literature review.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) results from a biallelic germline pathogenic variant in a mismatch repair (MMR) gene. The most common CMMRD-associated malignancies are brain tumors; an accurate diagnosis is challenging when a malignant brain tumor is the only tumor at presentation. We describe two cases of glioblastoma as the initial CMMRD malignancy and discuss current diagnostic and therapeutic challenges. CASE PRESENTATION: Two children with brain tumors without remarkable family history had biallelic pathogenic germline variants in PMS2. Patient 1: A 6-year-old girl presented biallelic PMS2 germline pathogenic variants. Glioblastomas at the left frontal lobe and right temporal lobe were resistant to immune-checkpoint inhibitor, temozolomide, and bevacizumab. Patient 2: A 10-year-old boy presented biallelic PMS2 germline variants. His glioblastoma with primitive neuroectodermal tumor-like features responded to chemoradiotherapy, but he developed advanced colon cancer and acute lymphocytic leukemia. In both patients, only a monoallelic PMS2 germline variant was detected by conventional gene tests. PMS2 immunohistochemistry showed lack of staining at both the tumors and normal tissue as vascular endothelial cells. Further gene tests revealed large genomic deletion including the entire PMS2 gene, confirming biallelic PMS2 germline variants. CONCLUSION: Conventional multi-gene panel tests are insufficient for detecting large deletions of MMR genes, resulting in misdiagnoses of CMMRD as Lynch syndrome. PMS2 variants have low cancer penetrance; family histories may thus be absent. Long-range gene analyses or immunohistochemical staining of MMR proteins in normal tissue should be considered for pediatric brain tumors with a single allele MMR variant when CMMRD is suspected.