Mismatch Repair Gene Variants Research Articles

Constitutional mismatch repair deficiency mimicking Lynch syndrome is associated with hypomorphic mismatch repair gene variants

Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are distinct cancer syndromes caused, respectively, by mono- and bi-allelic germline mismatch repair (MMR) variants. LS predisposes to mainly gastrointestinal and genitourinary cancers in adulthood. CMMRD predisposes to brain, haematological, and LS-spectrum cancers from childhood. Two suspected LS patients with first cancer diagnosis aged 27 or 38 years were found to be homozygous for an MMR (likely) pathogenic variant, MSH6 c.3226C>T (p.(Arg1076Cys)), or variant of uncertain significance (VUS), MLH1 c.306G>A (p.(Glu102=)). MLH1 c.306G>A was shown to cause leaky exon 3 skipping. The apparent genotype-phenotype conflict was resolved by detection of constitutional microsatellite instability in both patients, a hallmark feature of CMMRD. A hypomorphic effect of these and other variants found in additional late onset CMMRD cases, identified by literature review, likely explains a LS-like phenotype. CMMRD testing in carriers of compound heterozygous or homozygous MMR VUS may find similar cases and novel hypomorphic variants. Individualised management of mono- and bi-allelic carriers of hypomorphic MMR variants is needed until we better characterise the associated phenotypes.

Abstract PO5-08-05: Breast Cancer in PMS2 related Lynch Syndrome: evidence of a possible association?

Abstract Background: Lynch syndrome (LS) is a hereditary cancer predisposition syndrome caused by pathogenic variants (PVs) in mismatch repair genes, MLH1, MSH2, MSH6, and PMS2. LS is associated with increased risk of colorectal, and endometrial cancers but breast cancer risk is uncertain, with some studies suggesting MSH6 and PMS2 PVs may have a higher risk for breast cancer. Our study outlines the incidence and clinical characteristics of breast cancer in PMS2-related LS identified at a large academic center. Methods: Patients with PMS2 PVs identified between July 2009 and May 2023 were selected from a database at the Nancy and James Grosfeld Cancer Genetic Center at Beaumont Health. Data on demographics, genetic testing, cancer types, and breast cancer characteristics were retrospectively analyzed. Results: A total of 108 patients from 74 families were found to carry a pathogenic or likely pathogenic variant in PMS2, of whom the majority were female (75%) and Caucasian (85%). The median age at genetic testing was 52y (19 to 95y). The most common variant was c.137G > T (p.Ser46Ile), seen in 15 of 74 (20%) families. Forty-seven patients (46%) had a personal history of cancer, with a median age at diagnosis of 58y. Of these forty-seven patients, sixteen (34%) had multiple malignancies, including one patient with six separate cancers. The most common malignancies were breast (19; 40%), colon (16; 34%), followed by uterine cancer (7), pancreas (4), and prostate (3). The median age at breast cancer diagnosis was 53y, with a range of 41-78y. The most common histopathology was invasive ductal carcinoma (54%), followed by DCIS (31%), and invasive lobular carcinoma (15%). The majority of patients had breast tumors with stage 0 or 1 (75%), grade 2 (63%), ER-positive (92%), PR positive (67%), and HER2-negative (88%). One patient had triple-negative breast cancer. Sixty-seven percent of breast cancers were less than two centimeters, and 92% were lymph node-negative. Fifty-nine percent received radiation therapy and 53% received chemotherapy. Conclusion: Our study outlines the incidence and clinical characteristics of breast cancer in PMS2-related LS. Breast cancer was the most common malignancy in our cohort of PMS2 PV carriers, accounting for 40% of all cancers, supporting a possible association between PMS2 and breast cancer. The majority of PMS2-related breast cancers were invasive ductal type, early stage, intermediate grade, and hormone receptor-positive. Further studies with larger diverse cohorts of patients with LS are needed to better characterize a possible association in order to provide tailored breast cancer screening and management guidelines for this population. Citation Format: Ujjwal Karki, Tara Rangarajan, Dana Zakalik. Breast Cancer in PMS2 related Lynch Syndrome: evidence of a possible association? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-08-05.

Null Mismatch Repair Proteins Expression Reveals the Temporal Molecular Events in Lynch Syndrome-Related Cancers.

The immunohistochemical assessment of mismatch repair (MMR) proteins represents a pivotal screening tool for identifying Lynch syndrome (LS)-related cancers, as the loss of their expression often indicates MMR dysfunction associated with genetic or epigenetic alterations. Frequently, LS-related colorectal cancers present germline pathogenic variants in the MLH1 or MSH2 genes, which result in the simultaneous immunohistochemical loss of MLH1 and PMS2 or MSH2 and MSH6 proteins expression, respectively. Less commonly observed is the single involvement of the MSH6 or PMS2 proteins expression, indicative of the presence of germline pathogenic variants in the corresponding genes. Extremely rarely reported are the null immunohistochemistry phenotypes represented by the complete loss of expression of all MMR proteins. The molecular mechanisms contributing to the raising of this latter uncommon immunohistochemical phenotype are derived from the combination of pathogenic germline variants in MMR genes with the somatic hypermethylation of the MLH1 gene promoter. This study focuses on elucidating the molecular cascade leading to the development of the null immunohistochemical phenotype, providing valuable insights into understanding the sequential molecular events driving the LS-associated tumorigenesis, which may have pivotal implications in the clinical management of patients with LS-related cancers.

Abstract 7329: Novel MAVE models for MLH1, MSH2, and PMS2 have high accuracy

Abstract Lynch syndrome, the most common cause of hereditary colorectal cancer, is due to pathogenic germline variants in mismatch repair (MMR) genes. Variants of uncertain significance (VUS) in MMR genes hinder diagnoses and clinical management. Multiplex assays of variant effects (MAVEs) are high-throughput cellular assays designed to provide functional data on variant activity. Successful MAVE experiments accurately define the pathogenicity of single nucleotide variants and small insertions-deletions which can then be applied to clinical variant classification (VC). Here we report internally generated MAVE datasets for three MMR genes (MSH2, MLH1 and PMS2) and estimate their impact on patients. Three separate pools of targeted variants were generated for three MMR genes. Pooled variants were introduced into cell populations such that each cell contained one variant copy of an MMR gene. Single-cell RNA expression profiles were generated for the cells which were then normalized and filtered. Supervised machine learning was used to find patterns in gene expression that were different between cells harboring established pathogenic (P) and benign (B) variants and to assess the accuracy of variant predictions. VC impact was estimated for all datasets by simulating VCs with the added MAVE evidence compared to the original VCs. Variants with an original VC that was different after MAVE evidence were counted as impacted. Overall results are shown in Table 1, including the number of variants successfully targeted, the number of known P and B variants used for training and testing, the area under the receiver operating characteristic curve (AUROC), the distribution of calibrated predictions, and the estimated patient impact for each model. Table 1. Gene MSH2 model MLH1 model PMS2 model Variants successfully targeted (n) 223 212 214 Known pathogenic (P) and benign (B) variants used to train the model 29 P 31 P 14 P 72 B 55 B 71 B Known pathogenic (P) and benign (B) variants used to test the model 14 P 14 P 7 P 32 B 22 B 31 B AUROC 0.9996 0.9642 0.9918 # of variants strongly predicted P (PPV ≥ 95%) or B (NPV≥ 95%) 44 P 52 P 26 P 177 B 160 B 183 B # of variants with uncertain prediction (PPV < 80%, NPV < 80%) 2 0 5 Estimated # of impacted variants 4 LP>P 0 LP>P 4 LP>P 3 VUS>LP 5 VUS>LP 1 VUS>LP 26 VUS>LB 17 VUS>LB 17 VUS>LB 31 LB>B 7 LB>B 6 LB>B The MSH2, MLH1, and PMS2 MAVE models provide a high-quality source of functional evidence for VC with the potential to impact many patients in the future. Citation Format: Wolfgang Michael Korn, Samskruthi Padigepati, David Stafford, Flavia Facio, Britt Johnson, Keith Nykamp, Jason Reuter. Novel MAVE models for MLH1, MSH2, and PMS2 have high accuracy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7329.

Abstract 258: An organoid model for investigating Lynch syndrome tumorigenesis

Abstract Lynch syndrome (LS) is the most common inherited predisposition for developing colon cancer and is caused by germline variants in mismatch repair (MMR) genes. Still, the mechanism of tumorigenesis in these patients is unknown. An LS patient inherits one defective copy of an MMR gene. At some point in their lifetime, the remaining wild-type allele is lost in a cell rendering it entirely MMR deficient. MMR deficiency increases mutational rate and the chance of acquiring mutations in an oncogene or tumor suppressor that drive tumor initiation. However, loss of MMR in cells also leads to increased resistance to certain forms of DNA damage. Thus, our question is whether this phenotype imparts a selective advantage in colonic cells that may lead to clonal expansion of MMR-deficient cells as a first step towards tumorigenesis in LS patients. To test this, we are using stem cell-derived human colonic organoids and CRISPR gene editing to examine competition between MMR-deficient (MMRd) and MMR-proficient (MMRp) colonic cells over time. We have initially observed that MMRd stem cells are much more efficient at generating colonic organoids that grow larger and survive longer, consistent with our hypothesis of a selective advantage. To mimic the spontaneous loss of heterozygosity that occurs in LS patients’ colonic crypts, we are forming organoids from differentially labeled MMRd and MMRp stem cells. Live fluorescent imaging of individual organoids is used to quantify changes in cell population over time. Preliminary data indicate that MMRd cells can outgrow MMRp cells resulting in a shift in the ratio of green to red cells over a two-week time period while challenged with a DNA-damaging agent. These results suggest that loss of MMR function imparts an immediate selective advantage in colonic cells in response to DNA damage. Citation Format: Caroline Guild, Christopher Heinen, Samantha Nadeau. An organoid model for investigating Lynch syndrome tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 258.

Splicing analysis of 24 potential spliceogenic variants in MMR genes and clinical interpretation based on refined ACMG/AMP criteria.

Lynch syndrome (LS) is a common hereditary cancer syndrome caused by heterozygous germline pathogenic variants in DNA mismatch repair (MMR) genes. Splicing defect constitutes one of the major mechanisms for MMR gene inactivation. Using RT-PCR based RNA analysis, we investigated 24 potential spliceogenic variants in MMR genes and determined their pathogenicity based on refined splicing-related American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria. Aberrant transcripts were confirmed in 19 variants and 17 of which were classified as pathogenic including 11 located outside of canonical splice sites. Most of these variants were previously reported in LS patients without mRNA splicing assessment. Thus, our study provides crucial evidence for pathogenicity determination, allowing for appropriate clinical follow-up. We also found that computational predictions were globally well correlated with RNA analysis results and the use of both SPiP and SpliceAI software appeared more efficient for splicing defect prediction.

Neoadjuvant nivolumab (N) + ipilimumab (I) in cisplatin-ineligible patients with upper tract urothelial cancer (UTUC): Updated results.

645 Background: Perioperative platinum-based chemotherapy for UTUC improves pathologic responses and disease-free survival (DFS). We report the updated results from our on-going phase II neoadjuvant trial of N+I for cisplatin-ineligible patients (pts) with UTUC (NCT03520491). Methods: Cisplatin-ineligible pts with histologically confirmed high-grade UTUC and/or radiographically invasive UTUC with positive selective urine cytology were eligible. Pts were treated with I 3mg/kg + N 1mg/kg (weeks 0, 6), and “N” 3mg/kg (week 3) prior to radical nephroureterectomy (NU). The primary endpoint (EP) was pathologic complete response (pCR, ypT0pN0) and secondary EPs included <ypT2pN0 rate, DFS, and toxicity. Next generation sequencing of pre-treatment tumors was correlated with pathologic response. Results: Seventeen pts (76% male) were enrolled between 2/2021-6/2023 with median age 73 (range 59-85). Primary sites included ureter in 10 pts, renal pelvis in 5 and both in 2. Median tumor diameter was 3.0 cm, (range: 1.5 - 4.2 cm), with 8 pts (47%) having hydronephrosis. To date, 11 pts (65%) have received all planned treatment, 6 pts had N+/-I stopped early due to treatment-related toxicities, 2 pts are pending NU, 13 pts have undergone NU, 1 declined surgery, and another progressed on N+I. A pCR was seen in 4/15 pts (27%) and <ypT2pN0 in 9/15 (60%). Median time from last treatment to NU was 1.6 months (range 0.5-4.9). Three pts developed metastatic recurrence at 3.9, 10.3 and 15.6 months after treatment initiation. CTCAE grade ≥3 treatment-related adverse events occurred in 5 pts (29%). Four pts died during follow up: two of metastatic disease, one of complications related to a bowel leak (7.2 months after N+I initiation and 4.7 months after NU), and one from complications of a fall unrelated to disease or treatment (9.2 months after N+I initiation and 4.7 months after NU). Median tumor mutational burden (TMB) in 9 pts with pre-treatment sequencing was 13.2 mutations/mb (range 4.1-106.3), with 4/5 pts with TMB>10 <ypT2pN0 at NU. Pathogenic germline variants in mismatch repair genes were confirmed in 4 pts (MSH2 in 3, MLH1 in 1) and MSI-high tumors without germline alterations in 2 pts, with 5 of 6 completing NU (1 pending surgery); all 5 achieved pCR (n=3) or ypTaN0 (n=2) and remain alive and disease-free at last follow-up (6, 13.4, 17, 17.9 and 31 months). Conclusions: Updated findings from an on-going phase II trial of cisplatin-ineligible pts with UTUC receiving neoadjuvant N+I shows clinical activity in line with our initial results. All pts with pathogenic germline variants in mismatch repair genes or MSI-high tumors without germline alteration who underwent NU achieved <ypT2pN0 and remain free of disease. Clinical trial information: NCT03520491 .

Rare germline mutation and MSH2-&MSH6 + expression in a double primary carcinoma of colorectal carcinoma and endometrial carcinoma: a case report

BackgroundMultiple primary malignancies are rare in cancer patients, and risk factors may include genetics, viral infection, smoking, radiation, and other environmental factors. Lynch syndrome (LS) is the most prevalent form of hereditary predisposition to double primary colorectal and endometrial cancer in females. LS, also known as hereditary nonpolyposis colorectal cancer (HNPCC), is a common autosomal dominant condition. Pathogenic germline variants in the DNA mismatch repair (MMR) genes, namely MLH1, MSH2, MSH6, and PMS2, and less frequently, deletions in the 3’ end of EPCAM cause LS. It manifested itself as loss of MMR nuclear tumor staining (MMR protein deficient, dMMR).Case presentationThis case study describes a double primary carcinoma in a 49-year-old female. In June 2022, the patient was diagnosed with highly to moderately differentiated endometrioid adenocarcinoma. The patient’s mother died of esophageal cancer at age 50, and the father died of undefined reasons at age 70. Immunohistochemical stainings found ER (++), PR (++), P53 (+), MSH2 (-), MSH6 (+), MLH1 (+), and PMS2 (+). MMR gene sequencing was performed on endometrial tumor and peripheral blood samples from this patient. The patient carried two pathogenic somatic mutations in the endometrial tumor, MSH6 c.3261dupC (p.Phe1088LeufsTer5) and MSH2 c.445_448dup (p.Val150fs), in addition to a rare germline mutation MSH6 c.133G > C (p.Gly45Arg). Two years ago, the patient was diagnosed with moderately differentiated adenocarcinoma in the left-half colon. Immunohistochemical stainings found MSH2(-), MSH6(+), MLH1(+), and PMS2(+) (data not shown).ConclusionsIn the case of a patient with double primary EC and CRC, a careful evaluation of the IHC and the genetic data was presented. The patient carried rare compound heterozygous variants, a germline missense mutation, and a somatic frameshift mutation of MSH6, combined with a novel somatic null variant of MSH2. Our study broadened the variant spectrum of double primary cancer and provided insight into the molecular basis for abnormal MSH2 protein loss and double primary carcinoma.

Evolutionary origin of germline pathogenic variants in human DNA mismatch repair genes

BackgroundMismatch repair (MMR) system is evolutionarily conserved for genome stability maintenance. Germline pathogenic variants (PVs) in MMR genes that lead to MMR functional deficiency are associated with high cancer risk. Knowing the evolutionary origin of germline PVs in human MMR genes will facilitate understanding the biological base of MMR deficiency in cancer. However, systematic knowledge is lacking to address the issue. In this study, we performed a comprehensive analysis to know the evolutionary origin of human MMR PVs.MethodsWe retrieved MMR gene variants from the ClinVar database. The genomes of 100 vertebrates were collected from the UCSC genome browser and ancient human sequencing data were obtained through comprehensive data mining. Cross-species conservation analysis was performed based on the phylogenetic relationship among 100 vertebrates. Rescaled ancient sequencing data were used to perform variant calling for archeological analysis.ResultsUsing the phylogenetic approach, we traced the 3369 MMR PVs identified in modern humans in 99 non-human vertebrate genomes but found no evidence for cross-species conservation as the source for human MMR PVs. Using the archeological approach, we searched the human MMR PVs in over 5000 ancient human genomes dated from 45,045 to 100 years before present and identified a group of MMR PVs shared between modern and ancient humans mostly within 10,000 years with similar quantitative patterns.ConclusionOur study reveals that MMR PVs in modern humans were arisen within the recent human evolutionary history.

High amount of fertility reducing tumors and procedures, but no evidence for premature ovarian failure in female Lynch syndrome patients.

Lynch syndrome (LS; HNPCC) patients carry heterozygous pathogenic germline variants in mismatch repair (MMR) genes, which have also been shown to play an important role in meiosis. Therefore, it was hypothesized, that LS might be associated with a higher risk for premature ovarian failure (POF) or earlier menopause. Data on medical gynaecological history, cancer diagnoses and therapy were collected from 167 female LS patients and compared to a population-based control cohort. There was no difference between the age of menopause in patients compared to controls and no evidence for a higher risk of POF in LS patients. However, around one third (35%) of the probands have already had premenopausal cancer and mostly cancer-related treatment affecting fertility before the age of 45 years. Therefore, childbearing time might still be limited in these patients, especially due to the premenopausal cancer risk. LS patients should be informed in time about the elevated premenopausal cancer risks and the possible impact on family planning. This is particularly relevant since the average childbearing age has increased during the last decades.

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

BackgroundLynch 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.MethodsWe 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.ResultsA 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.ConclusionsThe 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.

Evaluation of mismatch-repair and microsatellite-instability status in a Chinese colorectal cancer Cohort

BackgroundImmunohistochemistry (IHC) and traditional polymerase chain reaction (PCR) are the methods of choice in clinical practice to identify the mismatch repair (MMR) and microsatellite instability (MSI) status in colorectal cancer (CRC). In some previous researches, the concordance rate between two methods was different and discordance existed in about 1 %–9.7 %. MethodsWe retrospectively reviewed 406 patients received surgical CRC resections and tests of both MMR IHC and MSI PCR from January 2019 to April 2022 in Shanghai Changzheng Hospital. The incidence of deficient mismatch repair (dMMR) or microsatellite instability-high (MSI-H) CRCs, the concordance rate between two methods, and the reasons for discordant results were evaluated with clinicopathological data, immunochemical staining, whole-exome sequencing, and MLH1 methylation analysis. ResultsAmong 406 patients, the incidence of MSI-H CRCs was 7.88 %. Nearly a quarter of the cases under reexamination of IHC was initial misinterpreted. Besides, the concordance rate between MMR IHC and MSI PCR was 99.26 % (401 of 404) and the Kappa value was 0.945 (p < 0.001). Finally, some somatic variants of MMR and POLE genes which may explain the discordance were identified. ConclusionThe incidence rate of MSI-H in Chinese patients with CRC might be relatively low owing to tumor location. Although MSI and IHC analyses are highly concordant, both MMR IHC and MSI PCR tests should be simultaneously performed and MMR IHC should be interpreted by experienced pathologists. In the future, further studies on discordant results should be carried out to improve the personalized management of CRC.

Genotype-phenotype correlations in carriers of the PMS2 founder variant c.1831dup.

Lynch syndrome represents one of the most common cancer predispositions worldwide and is caused by germline pathogenic variants (PV) in DNA mismatch repair (MMR) genes. We repeatedly identified a PV in the MMR gene PMS2, c.1831dup, accounting for 27% of all Swiss PMS2 PV index patients identified. Notably, 2/18 index patients had been diagnosed with colorectal cancer (CRC) before age 30. In this study, we investigated if this PV could (i) represent a founder variant by haplotype analysis and (ii) be associated with a more severe clinical phenotype. Haplotype analysis identified a shared common region of about 0.7 Mb/1.3 cM in 13 (81%) out of 16 index patients. Genotype-phenotype correlations, combining data from the 18 Swiss and 18 literature-derived PMS2 c.1831dup PV index patients and comparing them to 43 Swiss index patients carrying other PMS2 PVs, indicate that the PMS2 c.1831dup variant may be associated with earlier (<50 y) age at CRC diagnosis (55% vs. 29%, respectively; p = 0.047). Notably, 30% (9/30) of cancers from c.1831dup carriers displayed atypical MMR protein expression patterns on immunohistochemistry. Our results suggest that the PMS2 c.1831dup PV represents a, probably ancient, founder mutation and is possibly associated with an earlier CRC diagnosis compared to other PMS2 PVs.

Diffuse Large B-Cell Lymphoma Has a Low Frequency of dMMR and High Frequencies of DNA Mismatch Repair Protein High Expression Associated with Lower T-Cell Infiltration

Background: DNA mismatch repair (MMR) deficiency (dMMR), which leads to genomic and microsatellite instability, is a biomarker that predicts response to immunotherapies and has variable prognostic effects for chemotherapies in solid tumors. The dMMR can be detected using gene sequencing or immunohistochemistry for four essential MMR proteins: MSH6, MSH2, MLH1, and PMS2. Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma. Previous studies have suggested the role of MMR gene variants in DLBCL lymphomagenesis, yet the prognostic role of dMMR in DLBCL has not been well studied. Methods: We performed targeted next-generation sequencing and immunohistochemistry for MSH6, MSH2, MLH1, and PMS2 in a large cohort of DLBCL patients treated with standard chemoimmunotherapy. Gene expression profiling (GEP) was performed using the Affymetrix GeneChip Human Genome HG-U133 Plus 2.0 microarray (data in GSE31312). Fluorescent multiplex immunohistochemistry (mIHC) was performed using MultiOmyx multiplexing immunofluorescence staining protocols and antibodies against 13 immune markers. We investigated the frequencies of dMRR and MMR protein expression and correlated the mutation status and expression levels with patient survival and DLBCL biology, including the number of mutated genes, GEP data, previously analyzed DLBCL biomarkers, and immune cell immunophenotypes. Results: MMR gene mutations and loss of MMR protein expression were infrequent in DLBCL (Figure A) and did not show a significant prognostic impact. MMR proteins were commonly expressed in DLBCL samples and the germinal centers of reactive tonsil controls (Figure B), with higher mean and median percentages of tumor cells expressing MSH6 and MLH1 proteins than in the solid tumor samples used for comparison. High expression of MMR proteins was associated with Ki-67, MYC, and p53 overexpression as determined by immunohistochemistry. GEP analysis identified significantly upregulated genes involved in the mitotic cell cycle and DNA metabolism and prominent downregulated immune gene signatures in DLBCL with high MMR protein expression. Fluorescent mIHC confirmed the associations with decreased T cell abundance in MSH6/MSH2/MLH1/PMS2 highly expressing DLBCL (Figure C) independent of p53 and MYC expression status, whereas MSH6/ MLH1 mutations were associated with increased T cell frequencies. High versus low expression of MSH6, MLH1, and PMS2 showed significant unfavorable prognostic effects in DLBCL when the optimal cutoffs were used for high expression. However, when the median percentages were used as cutoffs, the prognostic effects were not significant; nonetheless, MSH6 and PMS2 high expression showed significant adverse prognostic effects in the MYC¯ DLBCL subset. Interestingly, when we used Ki-67, MYC + or BCL2 + percentages in DLBCL assessed by immunohistochemistry to subtract MMR protein percentages and then correlated the differences to survival, we found significant favorable prognostic impact by higher percentage expression differences between MMR proteins and BCL2, or by intermediately higher differences between MMR proteins and MYC. For MSH2/MLH1/PMS2, the higher percentages compared with MYC/BCL2 showed no significant correlations with T cell infiltration levels, whereas the higher differences between MSH6 and MYC/BCL2 were associated with significantly lower T cell frequencies (Figure D). Conclusions: This study revealed that high expression of MMR proteins is a common feature of DLBCL associated with lower tumor-infiltrating T cells, MYC and p53 overexpression, and context-dependent prognostic effects. In contrast, dMMR and loss of MMR proteins are infrequent and have no significant prognostic effects in DLBCL treated with standard chemoimmunotherapy. These results may have implications for understanding DLBCL biology and the low efficacy of PD-1 blockade immunotherapy in DLBCL.

MTHFR C677T and A1298C polymorphism’s effect on risk of colorectal cancer in Lynch syndrome

Lynch syndrome (LS) is characterised by an increased risk of developing colorectal cancer (CRC) and other extracolonic epithelial cancers. It is caused by pathogenic germline variants in DNA mismatch repair (MMR) genes or the EPCAM gene, leading to a less functional DNA MMR system. Individuals diagnosed with LS (LS individuals) have a 10–80% lifetime risk of developing cancer. However, there is considerable variability in the age of cancer onset, which cannot be attributed to the specific MMR gene or variant alone. It is speculated that multiple genetic and environmental factors contribute to this variability, including two single nucleotide polymorphisms (SNPs) in the methylenetetrahydrofolate reductase (MTHFR) gene: C677T (rs1801133) and A1298C (rs1801131). By decreasing MTHFR activity, these SNPs theoretically reduce the silencing of DNA repair genes and increase the availability of nucleotides for DNA synthesis and repair, thereby protecting against early-onset cancer in LS. We investigated the effect of these SNPs on LS disease expression in 2,723 LS individuals from Australia, Poland, Germany, Norway and Spain. The association between age at cancer onset and SNP genotype (risk of cancer) was estimated using Cox regression adjusted for gender, country and affected MMR gene. For A1298C (rs1801131), both the AC and CC genotypes were significantly associated with a reduced risk of developing CRC compared to the AA genotype, but no association was seen for C677T (rs1801133). However, an aggregated effect of protective alleles was seen when combining the alleles from the two SNPs, especially for LS individuals carrying 1 and 2 alleles. For individuals with germline pathogenic variants in MLH1, the CC genotype of A1298C was estimated to reduce the risk of CRC significantly by 39% (HR = 0.61, 95% CI 0.42, 0.89, p = 0.011), while for individuals with pathogenic germline MSH2 variants, the AC genotype (compared to AA) was estimated to reduce the risk of CRC by 26% (HR = 0.66, 95% CI 0.53, 0.83, p = 0.01). In comparison, no association was observed for C677T (rs1801133). In conclusion, our study suggests that combining the MMR gene information with the MTHFR genotype, including the aggregated effect of protective alleles, could be useful in developing an algorithm that estimates the risk of CRC in LS individuals.

Genetic insights: High germline variant rate in an indigenous African cohort with early-onset colorectal cancer

IntroductionThe increase in incidence of colorectal cancer in young patients of African ancestry coupled with increased aggressiveness has warranted investigation of the heritable nature of these cancers. Only a limited number of published reports of hereditary colorectal cancer in indigenous African populations have been reported and no systematic screening of these groups has been performed previously. We aimed to investigate causative germline variants and to establish the incidence of pathogenic/likely pathogenic germline variants in the known colorectal cancer genes in indigenous African colorectal cancer patients using a next-generation sequencing (NGS) multigene panel.Materials and methodsPatients were selected from two hospitals in Cape Town and Johannesburg, South Africa. Patients with unresolved molecular diagnosis with an age of onset below or at 60 years were selected. Germline DNA samples were analyzed using a 14-gene NGS panel on the Ion Torrent platform. Variant calling and annotation were performed, and variants were classified according to the American College of Medical Genetics and Genomics guidelines. Observed variants were verified by Sanger sequencing and/or long-range PCR.ResultsOut of 107 patients, 25 (23.4%) presented with a pathogenic/likely pathogenic germline variant (PGV). Fourteen PGVs in at least one mismatch repair (MMR) gene were identified and verified in 12 patients (11.2%). Of these MMR gene variants, five were novel. The remaining 10 PGVs were in the APC, BMPR1A, MUTYH, POLD1, and TP53 genes.ConclusionThe high incidence of PGVs associated with early-onset colorectal cancer in indigenous African patients has important implications for hereditary colorectal cancer risk management. These findings pave the way for personalized genetic screening programs and cascade testing in South Africa. The next step would involve further screening of the unresolved cases using tools to detect copy number variation, methylation, and whole exome sequencing.

DNA Mismatch Repair Gene Variant Classification: Evaluating the Utility of Somatic Mutations and Mismatch Repair Deficient Colonic Crypts and Endometrial Glands.

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. Lynch syndrome specific tumor features were evaluated for their ability to support the ACMG/InSiGHT framework in classifying variants of uncertain clinical significance (VUS) in the MMR genes. Twenty-eight CRC or EC tumors from 25 VUS carriers (6xMLH1, 9xMSH2, 6xMSH6, 4xPMS2), underwent targeted tumor sequencing for the presence of microsatellite instability/MMR-deficiency (MSI-H/dMMR) status and identification of a somatic MMR mutation (second hit). Immunohistochemical testing for the presence of dMMR crypts/glands in normal tissue was also performed. The ACMG/InSiGHT framework reclassified 7/25 (28%) VUS to likely pathogenic (LP), three (12%) to benign/likely benign, and 15 (60%) VUS remained unchanged. For the seven re-classified LP variants comprising nine tumors, tumor sequencing confirmed MSI-H/dMMR (8/9, 88.9%) and a second hit (7/9, 77.8%). Of these LP reclassified variants where normal tissue was available, the presence of a dMMR crypt/gland was found in 2/4 (50%). Furthermore, a dMMR endometrial gland in a carrier of an MSH2 exon 1-6 duplication provides further support for an upgrade of this VUS to LP. Our study confirmed that identifying these Lynch syndrome features can improve MMR variant classification, enabling optimal clinical care.

The Prevalence and Molecular Landscape of Lynch Syndrome in the Affected and General Population.

Lynch syndrome (LS) is the most frequent genetically pre-disposed colorectal cancer (CRC) syndrome, accounting for 2-3% of all CRC cases. In Estonia, ~1000 new cases are diagnosed each year. This retroactive and prospective study aimed to estimate the prevalence of LS and describe disease-causing variants in mismatch repair (MMR) genes in a diagnostic setting and in the Estonian general population. LS data for the diagnostic cohort were gathered from 2012 to 2022 and data for the general population were acquired from the Estonian Biobank (EstBB). Furthermore, we conducted a pilot study to estimate the improvement of LS diagnostic yield by raising the age limit to >50 years for immunohistochemistry analysis of MMR genes. We estimated LS live birth prevalence between 1930 and 2003 in Estonia at 1:8638 (95% CI: 1: 9859-7588). During the study period, we gathered 181 LS individuals. We saw almost a six-fold increase in case prevalence, probably deriving from better health awareness, improved diagnostic possibilities and the implementation of MMR IHC testing in a broader age group. The most common genes affected in the diagnostic and EstBB cohorts were MLH1 and PMS2 genes, respectively. The LS diagnosis mean age was 44.8 years for index cases and 36.8 years (p = 0.003) for family members. In the MMR IHC pilot study, 29% had LS.

A mosaic pathogenic variant in MSH6 causes MSH6-deficient colorectal and endometrial cancer in a patient classified as suspected Lynch syndrome: a case report

Germline pathogenic variants in the DNA mismatch repair (MMR) genes (Lynch syndrome) predispose to colorectal (CRC) and endometrial (EC) cancer. However, mosaic variants in the MMR genes have been rarely described. We identified a likely de novo mosaic MSH6:c.1135_1139del p.Arg379* pathogenic variant in a patient diagnosed with suspected Lynch syndrome/Lynch-like syndrome. The patient developed MSH6-deficient EC and CRC at 54 and 58 years of age, respectively, without a detectable germline MMR pathogenic variant. Multigene panel sequencing of tumor and blood-derived DNA identified an MSH6 somatic mutation (MSH6:c.1135_1139del p.Arg379*) common to both the EC and CRC, raising suspicion of mosaicism. A droplet digital polymerase chain reaction (ddPCR) assay detected the MSH6 variant at 5.34% frequency in normal colonic tissue, 3.49% in saliva and 1.64% in blood DNA, demonstrating the presence of the MSH6 variant in all three germ layers. This study highlights the utility of tumor sequencing to guide sensitive ddPCR testing to detect low-level mosaicism in the MMR genes. Further investigation of the prevalence of MMR mosaicism is needed to inform routine diagnostic approaches and genetic counselling.

A comprehensive analysis of clinical characterization in 149 Chinese patients with Lynch syndrome.

10606 Background: Lynch syndrome (LS) caused by germline pathogenic variants (PVs) in mismatch repair (MMR) genes, which increased risk of malignancy in different organs. LS-related tumors included colorectal, endometrial, gastric, ovarian, pancreas, urinary system, biliary tract, central nervous system, skin and small intestinal carcinomas (CRC, EC, GC, OC, PC, UST, BTC, CNST, SC, SIC). As sequencing technology becomes more economical and widespread, more MMR PVs were detected in LS-unrelated tumors. The aim of this study was to explore the clinical characterization in Chinese real-world LS solid tumor. Methods: We applied next-generation sequencing (NGS) screening to detect the germline mutation in MMR (include MLH1, MSH2, MSH6, PMS2), tumor mutation burden (TMB) and microsatellite instability (MSI) in Chinese solid tumor patients (pts). In the cohort, lung cancer (LC) was the most common cancer type, followed by CNST, CRC, hepatic carcinoma (HC). Results: 149 LS pts were observed in our cohort, among which 81.2% (121/149) carried LS-related tumors (44 CRC, 47 CNST, 9 EC, 9 BTC, 4 UST, 3 OC, 2 GC, 2 SC, 1 SIC), 18.8% (28/149) carried with LS-unrelated tumors(18 LC, 5 HC, 3 sarcoma, 1 ampullary carcinoma, 1 peritoneal cancer), and the age showed no significant difference between LS-related tumors and LS-unrelated tumors (average age: 48 vs 53, p=0.08). The distribution of MMR PVs were as follows: MLH1 (37, 24.8%), MSH2 (46, 30.9%), MSH6 (34, 22.8%), PMS2 (32, 21.5% ), and comparative analysis showed that LS-related tumors tended to carry MLH1 and MSH2 PVs (60.3%, 73/121), while LS-unrelated tumors tended to carry MSH6 and PMS2 PVs (67.9%, 19/28) ( p&lt;0.05). 46.3% LS pts had MSI-H, concentrated in MLH1 or MSH2 PVs carriers. MSI-H showed a higher detection rate in LS-related tumors other than LS-unrelated tumors(56.1% vs 3.6%, p&lt;0.005). Among LS-related tumors, the top 3 prevalence of MSI-H were CRC (93.2%, 41/44), BTC (55.6%, 5/9) and EC (44.4%, 4/9), which were lower in CNST (31.9%, 15/47). In addition, we found that the rate of TMB-H in LS-related tumors was also higher than LS-unrelated tumors ( p&lt;0.001). Conclusions: In our dates, 18.8%(28/149) LS pts carried with LS-unrelated tumors, which emphasized the importance of germline genetic assessment for LS in pan-cancer. In addition, MSI-H, TMB-H and MMR PVs distributions were significantly different in LS-related tumors and LS-unrelated tumors. The differences about LS clinical characterization associated with MMR genes require further study.