Clinical features and impact of p53 status on sporadic mismatch repair deficiency and Lynch syndrome in uterine cancer.

The clinical features of sporadic mismatch repair deficiency (MMRd) and Lynch syndrome (LS) in Japanese patients with endometrial cancer (EC) were examined by evaluating the prevalence and prognostic factors of LS and sporadic MMRd in patients with EC. Targeted sequencing of five LS susceptibility genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) was carried out in 443 patients with EC who were pathologically diagnosed with EC at the National Cancer Center Hospital between 2011 and 2018. Pathogenic variants in these genes were detected in 16 patients (3.7%). Immunohistochemistry for MMR proteins was undertaken in 337 of the 433 (77.9%) EC patients, and 91 patients (27.0%) showed absent expression of at least one MMR protein. The 13 cases of LS with MMR protein loss (93.8%) showed a favorable prognosis with a 5-year overall survival (OS) rate of 100%, although there was no statistically significant difference between this group and the sporadic MMRd group (p = 0.27). In the MMRd without LS group, the 5-year OS rate was significantly worse in seven patients with an aberrant p53 expression pattern than in those with p53 WT (53.6% vs. 93.9%, log-rank test; p = 0.0016). These results suggest that p53 abnormalities and pathogenic germline variants in MMR genes could be potential biomarkers for the molecular classification of EC with MMRd.

Targeted genotyping for recurring variants in cancer susceptibility genes in non-Ashkenazi Jewish patients with breast cancer diagnosed ≥50 years.

PURPOSE: Several recurring pathogenic variants (PVs) in BRCA1/BRCA2 and additional cancer susceptibility genes are described in the ethnically diverse Israeli population. Since 2019, testing for these recurring PVs is reimbursed unselectively for all patients with breast cancer (BC) in Israel. The aim was to evaluate the yield of genotyping for these PVs in non-Ashkenazi Jewish (AJ) patients with BC diagnosed ≥age 50 years. METHODS: Clinical and genotyping data of all patients with BC undergoing oncogenetic counseling at the Oncology Institute at Sheba Medical Center from June 2017 to December 2023 were reviewed. RESULTS: Of 2706 patients with BC (mean age at diagnosis, 54 years; range, 20-92 years) counseled, 515 patients of non-AJ (all four grandparents) descent, diagnosed ≥age 50 years of age were genotyped, 55 with triple-negative BC (TNBC) and 460 with non-TNBC. One of the recurring PVs in BRCA1/BRCA2 were detected in 12.7% (7/55) of TNBC patients and 0.65% (3/460) of non-TNBC. One patient with non-TNBC had PMS2 PV. Low-penetrance variants were found in 2.5% of genotyped TNBC and in 3.7% of patients with non-TNBC, including CHEK2 c.499G>A (n = 3), APC c.3920T > A (n = 4), and heterozygous MUTYH c.1187G>A (n = 5). Following first-pass genotyping, 146 patients performed multigene panel testing, none carried a BRCA1/BRCA2 PV, and only 5/127 non-TNBC (3.9%) harbored PVs in CHEK2 (n = 2, c.846+1G>C and c.592+3A>T), ATM c.103C>T (n = 2), and NBN c.966C>G (n = 1). CONCLUSIONS: The observed low rates of PV detection in non-AJ non-TNBC cases ≥age 50 years at diagnosis, mostly for clinically insignificant variants, questions the justification of unselected genotyping in this subset of patients.

Molecular and Clinicopathologic Characterization of Mismatch Repair-Deficient Endometrial Carcinoma Not Related to MLH1 Promoter Hypermethylation.

Universal tumor screening in endometrial carcinoma (EC) is increasingly adopted to identify individuals at risk of Lynch syndrome (LS). These cases involve mismatch repair-deficient (MMRd) EC without MLH1 promoter hypermethylation (PHM). LS is confirmed through the identification of germline MMR pathogenic variants (PV). In cases where these are not detected, emerging evidence highlights the significance of double-somatic MMR gene alterations as a sporadic cause of MMRd, alongside POLE/POLD1 exonuclease domain (EDM) PV leading to secondary MMR PV. Our understanding of the incidence of different MMRd EC origins not related to MLH1-PHM, their associations with clinicopathologic characteristics, and the prognostic implications remains limited. In a combined analysis of the PORTEC-1, -2, and -3 trials (n = 1254), 84 MMRd EC not related to MLH1-PHM were identified that successfully underwent paired tumor-normal tissue next-generation sequencing of the MMR and POLE/POLD1 genes. Among these, 37% were LS associated (LS-MMRd EC), 38% were due to double-somatic hits (DS-MMRd EC), and 25% remained unexplained. LS-MMRd EC exhibited higher rates of MSH6 (52% vs 19%) or PMS2 loss (29% vs 3%) than DS-MMRd EC, and exclusively showed MMR-deficient gland foci. DS-MMRd EC had higher rates of combined MSH2/MSH6 loss (47% vs 16%), loss of >2 MMR proteins (16% vs 3%), and somatic POLE-EDM PV (25% vs 3%) than LS-MMRd EC. Clinicopathologic characteristics, including age at tumor onset and prognosis, did not differ among the various groups. Our study validates the use of paired tumor-normal next-generation sequencing to identify definitive sporadic causes in MMRd EC unrelated to MLH1-PHM. MMR immunohistochemistry and POLE-EDM mutation status can aid in the differentiation between LS-MMRd EC and DS-MMRd EC. These findings emphasize the need for integrating tumor sequencing into LS diagnostics, along with clear interpretation guidelines, to improve clinical management. Although not impacting prognosis, confirmation of DS-MMRd EC may release patients and relatives from burdensome LS surveillance.

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.

A Validated Highly Sensitive Microsatellite Instability Assay Accurately Identifies Individuals Harboring Biallelic Germline PMS2 Pathogenic Variants in Constitutional Mismatch Repair Deficiency.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) is a rare and extraordinarily penetrant childhood-onset cancer predisposition syndrome. Genetic diagnosis is often hampered by the identification of mismatch repair (MMR) variants of unknown significance and difficulties in PMS2 analysis, the most frequently mutated gene in CMMRD. We present the validation of a robust functional tool for CMMRD diagnosis and the characterization of microsatellite instability (MSI) patterns in blood and tumors. METHODS: The highly sensitive assessment of MSI (hs-MSI) was tested on a blinded cohort of 66 blood samples and 24 CMMRD tumor samples. Hs-MSI scores were compared with low-pass genomic instability scores (LOGIC/MMRDness). The correlation of hs-MSI scores in blood with age of cancer onset and the distribution of insertion-deletion (indel) variants in microsatellites were analyzed in a series of 169 individuals (n = 68 CMMRD, n = 124 non-CMMRD). RESULTS: Hs-MSI achieved high accuracy in the identification of CMMRD in blood (sensitivity 98.5% and specificity 100%) and detected MSI in CMMRD-associated tumors. Hs-MSI had a strong positive correlation with whole low-pass genomic instability LOGIC scores (r = 0.89, P = 2.2e-15 in blood and r = 0.82, P = 7e-3 in tumors). Indel distribution identified PMS2 pathogenic variant (PV) carriers from other biallelic MMR gene PV carriers with an accuracy of 0.997. Higher hs-MSI scores correlated with younger age at diagnosis of the first tumor (r = -0.43, P = 0.011). CONCLUSIONS: Our study confirms the accuracy of the hs-MSI assay as ancillary testing for CMMRD diagnosis, which can also characterize MSI patterns in CMMRD-associated cancers. Hs-MSI is a powerful tool to pinpoint PMS2 as the affected germline gene and thus potentially personalize cancer risk.

Expression patterns of mismatch repair proteins in cervical cancer uncover independent prognostic value of MSH-2.

OBJECTIVE: Although early-detected cervical cancer is associated with good survival, the prognosis for late-stage disease is poor and treatment options are sparse. Mismatch repair deficiency (MMR-D) has surfaced as a predictor of prognosis and response to immune checkpoint inhibitor(s) in several cancer types, but its value in cervical cancer remains unclear. This study aimed to define the prevalence of MMR-D in cervical cancer and assess the prognostic value of MMR protein expression. METHODS: Expression of the MMR proteins MLH-1, PMS-2, MSH-2, and MSH-6 was investigated by immunohistochemical staining in a prospectively collected cervical cancer cohort (n=508) with corresponding clinicopathological and follow-up data. Sections were scored as either loss or intact expression to define MMR-D, and by a staining index, based on staining intensity and area, evaluating the prognostic potential. RNA and whole exome sequencing data were available for 72 and 75 of the patients and were used for gene set enrichment and mutational analyses, respectively. RESULTS: Five (1%) tumors were MMR-deficient, three of which were of neuroendocrine histology. MMR status did not predict survival (HR 1.93, p=0.17). MSH-2 low (n=48) was associated with poor survival (HR 1.94, p=0.02), also when adjusting for tumor stage, tumor type, and patient age (HR 2.06, p=0.013). MSH-2 low tumors had higher tumor mutational burden (p=0.003) and higher frequency of (frameshift) mutations in the double-strand break repair gene RAD50 (p<0.01). CONCLUSION: MMR-D is rare in cervical cancer, yet low MSH-2 expression is an independent predictor of poor survival.

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.

Next-generation sequencing and immunohistochemistry approaches for microsatellite instability detection in endometrial cancer.

The purpose was to determine the effectiveness of immunohistochemical observation, a quick and easy method for determining Microsatellite instability MSI and/or other types of endometrial cancer (EC) based on modern classification. As molecular study takes more cost we want to compare both methods and show the possibility of doing IHC instead of molecular study. This study was designed to establish a small gene panel of Next Generation Sequencing (NGS) of endometrial cancer patients targeting 4 mismatch repair genes; MSH6, MSH2, MLH1 and PMS2. Using the DNBSEQ-G400 Platform, the Human Core Exome kit and Python software for analysis were used. At the same time, the Dako kit was used to perform IHC for six primary antibodies used to detect each of MSH6, MSH2, MLH and PMS2. The primary antibodies were applied on 5 µm formalin-fixed paraffin-embedded (FFPE). Results showed that histopathological examinations of all patients were at stage I endometrioid endometrial carcinoma. The FIGO classifications were Ia and Ib. Microsatellite instability (MIS) was observed through the IHC study. The molecular studies detected several polymorphisms that have clinical significance and some of them have conflicting interpretations. In conclusion, we considered that a simple immunoreaction staining procedure can be used as an alternative method for MSI phenotype detection rather than any type of more expensive and complex method of NGS.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

In vitro data suggest a role for PMS2 Kozak sequence mutations in Lynch syndrome risk.

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Heterozygous loss-of-function variants in PMS2 are linked to LS. While these variants are not directly cancer causing, reduced PMS2 function results in the accumulation of somatic variants and increased cancer risk over time due to DNA mismatch repair dysfunction. It is reasonable that other types of genetic variation that impact the expression of PMS2 may also contribute to cancer risk. The Kozak sequence is a highly conserved translation initiation motif among higher eukaryotes and is defined as the nine base pairs upstream of the translation start codon through the first four bases of the translated sequence (5'-[GTT]GCATCCATGG-3'; human PMS2: NM_000535.7). While Kozak sequence variants in PMS2 have been reported in ClinVar in patients with suspected hereditary cancer, all variants upstream of the translation start site are currently classified as variants of undetermined significance (VUSs). We hypothesized that variants significantly disrupting the Kozak sequence of PMS2 would decrease PMS2 protein expression, contributing to increased cancer risk over time. Using a dual-luciferase reporter plasmid and site-directed mutagenesis, we generated the wild-type human PMS2 and the ClinVar VUSs within the PMS2 Kozak sequence. Besides the c.1A>C variant, which is already known to be pathogenic, we implicate six additional variants as American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) pathogenic supporting (PP) variants and classify ten as benign supporting (BP). In summary, we present a method developed for the classification of human PMS2 Kozak sequence variants that can contribute to the re-classification of VUSs identified in patients.

Open-Source Bioinformatic Pipeline to Improve PMS2 Genetic Testing Using Short-Read NGS Data.

The molecular diagnosis of mismatch repair-deficient cancer syndromes is hampered by difficulties in sequencing the PMS2 gene, mainly owing to the PMS2CL pseudogene. Next-generation sequencing short reads cannot be mapped unambiguously by standard pipelines, compromising variant calling accuracy. This study aimed to provide a refined bioinformatic pipeline for PMS2 mutational analysis and explore PMS2 germline pathogenic variant prevalence in an unselected hereditary cancer (HC) cohort. PMS2 mutational analysis was optimized using two cohorts: 192 unselected HC patients for assessing the allelic ratio of paralogous sequence variants, and 13 samples enriched with PMS2 (likely) pathogenic variants screened previously by long-range genomic DNA PCR amplification. Reads were forced to align with the PMS2 reference sequence, except those corresponding to exon 11, where only those intersecting gene-specific invariant positions were considered. Afterward, the refined pipeline's accuracy was validated in a cohort of 40 patients and used to screen 5619 HC patients. Compared with our routine diagnostic pipeline, the PMS2_vaR pipeline showed increased technical sensitivity (0.853 to 0.956, respectively) in the validation cohort, identifying all previously PMS2 pathogenic variants found by long-range genomic DNA PCR amplification. Fifteen HC cohort samples carried a pathogenic PMS2 variant (15 of 5619; 0.285%), doubling the estimated prevalence in the general population. The refined open-source approach improved PMS2 mutational analysis accuracy, allowing its inclusion in the routine next-generation sequencing pipeline streamlining PMS2 screening.

Cascade screening in HBOC and Lynch syndrome: guidelines and procedures in a UK centre.

In the 33 years since the first diagnostic cancer predisposition gene (CPG) tests in the Manchester Centre for Genomic Medicine, there has been substantial changes in the identification of index cases and cascade testing for at-risk family members. National guidelines in England and Wales are usually determined from the National Institute of healthcare Evidence and these have impacted on the thresholds for testing BRCA1/2 in Hereditary Breast Ovarian Cancer (HBOC) and in determining that all cases of colorectal and endometrial cancer should undergo screening for Lynch syndrome. Gaps for testing other CPGs relevant to HBOC have been filled by the UK Cancer Genetics Group and CanGene-CanVar project (web ref. https://www.cangene-canvaruk.org/ ). We present time trends (1990-2020) of identification of index cases with germline CPG variants and numbers of subsequent cascade tests, for BRCA1, BRCA2, and the Lynch genes (MLH1, MSH2, MSH6 and PMS2). For BRCA1/2 there was a definite increase in the proportion of index cases with ovarian cancer only and pre-symptomatic index tests both doubling from 16 to 32% and 3.2 to > 8% respectively. A mean of 1.73-1.74 additional family tests were generated for each BRCA1/2 index case within 2 years. Overall close to one positive cascade test was generated per index case resulting in > 1000 risk reducing surgery operations. In Lynch syndrome slightly more cascade tests were performed in the first two years potentially reflecting the increased actionability in males with 42.2% of pre-symptomatic tests in males compared to 25.8% in BRCA1/2 (p < 0.0001).

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

BACKGROUND: Multiple 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 PRESENTATION: This 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). CONCLUSIONS: In 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.

Mismatch repair protein deficiency in triple-negative breast carcinomas.

BACKGROUND: Breast cancer, particularly triple-negative breast cancer (TNBC), poses a significant global health burden. Chemotherapy was the mainstay treatment for TNBC patients until immunotherapy was introduced. Studies indicate a noteworthy prevalence (0.2% to 18.6%) of mismatch repair protein (MMRP) deficiency in TNBC, with recent research highlighting the potential of immunotherapy for MMRP-deficient metastatic breast cancer. This study aims to identify MMRP deficiency in TNBC patients using immunohistochemistry. METHODS: A retrospective cohort study design was used and included TNBC patients treated between 2015 and 2021 at King Hussein Cancer Center. Immunohistochemistry was conducted to assess MMRP expression. RESULTS: Among 152 patients, 14 (9.2%) exhibited deficient MMR (dMMR). Loss of PMS2 expression was observed in 13 patients, 5 of whom showed loss of MLH1 expression. Loss of MSH6 and MSH2 expression was observed in one patient. The median follow-up duration was 44 (3-102) months. Despite the higher survival rate (80.8%, 5 years) of dMMR patients than of proficient MMR patients (62.3%), overall survival did not significantly differ between the two groups. CONCLUSION: Approximately 9% of TNBC patients exhibit dMMR. dMMR could be used to predict outcomes and identify patients with TNBC who may benefit from immunotherapy.

Mainstream Model of Genetic Testing for Prostate Cancer at a Large Tertiary Cancer Centre.

BACKGROUND: An estimated 20% to 30% of men with advanced prostate cancer carry a mutation in DNA damage repair genes, of which half are estimated to be germline. Eligibility criteria for germline genetic testing expanded significantly for Ontario patients in May 2021 and many centers adopted a "mainstream" model, defined as oncologist-initiated genetic testing. METHODS: We conducted a retrospective chart review to report on the first-year mainstream experience of a large tertiary oncologic center, the Sunnybrook Odette Cancer Centre. All patients who underwent mainstream at the discretion of their treating physician were included. A subset underwent somatic profiling as part of clinical trial screening. Descriptive statistics were used to report baseline clinicopathologic characteristics and treatments received. RESULTS: Between May 1, 2021, and May 30, 2022, 174 patients with prostate cancer underwent mainstream germline genetic testing with a 19-gene panel. Median age was 75 (IQR 68-80), and 82% of patients were diagnosed with either de novo metastatic or high-risk localized prostate adenocarcinoma. Fourteen patients (8%; 95% CI 4%-12%) were found to have a deleterious germline mutation, including pathogenic or likely pathogenic variants in BRCA1/2, ATM, CHEK2, PMS2, RAD51C, HOXB13, and BRIP1. Forty-nine patients (28%; 95% CI 21%-35%) were found to have a variant of uncertain significance. Thirty-four patients also had next-generation sequencing (NGS) of their somatic tissue. Among this subset, 8 of 34 (23%) had an alteration in homologous recombination repair (HRR) genes. Of the 14 patients with a germline mutation, none had a prior personal history of malignancy and 6 (43%) did not have any first- or second-degree relatives with history of prostate, pancreatic, breast, or ovarian cancer. CONCLUSION: We report on the real-world characteristics of prostate cancer patients who underwent mainstream germline genetic testing. Personal history and family history of cancer cannot reliably stratify patients for the presence of pathogenic germline variants.

DNA mismatch repair defect and intratumor heterogeneous deficiency differently impact immune responses in diffuse large B-cell lymphoma.

Deficient (d) DNA mismatch repair (MMR) is a biomarker predictive of better response to PD-1 blockade immunotherapy in solid tumors. dMMR can be caused by mutations in MMR genes or by protein inactivation, which can be detected by sequencing and immunohistochemistry, respectively. To investigate the role of dMMR in diffuse large B-cell lymphoma (DLBCL), MMR gene mutations and expression of MSH6, MSH2, MLH1, and PMS2 proteins were evaluated by targeted next-generation sequencing and immunohistochemistry in a large cohort of DLBCL patients treated with standard chemoimmunotherapy, and correlated with the tumor immune microenvironment characteristics quantified by fluorescent multiplex immunohistochemistry and gene-expression profiling. The results showed that genetic dMMR was infrequent in DLBCL and was significantly associated with increased cancer gene mutations and favorable immune microenvironment, but not prognostic impact. Phenotypic dMMR was also infrequent, and MMR proteins were commonly expressed in DLBCL. However, intratumor heterogeneity existed, and increased DLBCL cells with phenotypic dMMR correlated with significantly increased T cells and PD-1+ T cells, higher average nearest neighbor distance between T cells and PAX5+ cells, upregulated immune gene signatures, LE4 and LE7 ecotypes and their underlying Ecotyper-defined cell states, suggesting the possibility that increased T cells targeted only tumor cell subsets with dMMR. Only in patients with MYC¯ DLBCL, high MSH6/PMS2 expression showed significant adverse prognostic effects. This study shows the immunologic and prognostic effects of genetic/phenotypic dMMR in DLBCL, and raises a question on whether DLBCL-infiltrating PD-1+ T cells target only tumor subclones, relevant for the efficacy of PD-1 blockade immunotherapy in DLBCL.

PD-L1+ diffuse large B-cell lymphoma with extremely high mutational burden and microsatellite instability due to acquired PMS2 mutation.

We present a unique case of a single patient presenting with two mutationally distinct, PD-L1+ diffuse large B-cell lymphomas (DLBCLs). One of these DLBCLs demonstrated exceptionally high mutational burden (eight disease-associated variants and 41 variants of undetermined significance) with microsatellite instability (MSI) and an acquired PMS2 mutation with loss of PMS2 protein expression, detected postchemotherapy. This report, while highlighting the extent of possible tumor heterogeneity across separate clonal expansions as well as possible syndromic B-cell neoplasia, supports the notion that, although rare, PD-L1 expression and associated states permissive of high mutational burden (such as mismatch repair gene loss of function/MSI) should be more routinely considered in DLBCLs. Appropriate testing may be predictive of outcome and inform the utility of targeted therapy in these genetically diverse and historically treatment-refractory malignancies.

Comprehensive assessment of mismatch repair and microsatellite instability status in molecular classification of endometrial carcinoma / 中华妇产科杂志

Objective: To explore the concordance and causes of different mismatch repair (MMR) and microsatellite instability (MSI) detection results in endometrial carcinoma (EC) molecular typing. Methods: A total of 214 EC patients diagnosed from January 2021 to April 2023 were selected at the Department of Pathology, Peking University Third Hospital. The immunohistochemistry (IHC) results of MMR protein were reviewed. Tumor specific somatic mutations, MMR germline mutations, microsatellite scores and tumor mutation burden (TMB) were detected by next-generation sequencing (NGS) with multi-gene panel. Methylation-specific PCR was used to detect the methylation status of MLH1 gene promoter in cases with deficient MLH1 protein expression. In cases with discrepant results between MMR-IHC and MSI-NGS, the MSI status was detected again by PCR (MSI-PCR), and the molecular typing was determined by combining the results of TMB and MLH1 gene promoter methylation. Results: (1) In this study, there were 22 cases of POLE gene mutation subtype, 55 cases of mismatch repair deficient (MMR-d) subtype, 29 cases of p53 abnormal subtype, and 108 cases of no specific molecular profile (NSMP). The median age at diagnosis of MMR-d subtype (54 years old) and the proportion of aggressive histological types (40.0%, 22/55) were higher than those of NSMP subtype [50 years old and 12.0% (13/108) respectively; all P<0.05]. (2) Among 214 patients, MMR-IHC test showed that 153 patients were mismatch repair proficient (MMR-p), 49 patients were MMR-d, and 12 patients were difficult to evaluate directly. MSI-NGS showed that 164 patients were microsatellite stable (MSS; equal to MMR-p), 48 patients were high microsatellite instability (MSI-H; equal to MMR-d), and 2 patients had no MSI-NGS results because the effective sequencing depth did not meet the quality control. The overall concordance between MMR-IHC and MSI-NGS was 94.3% (200/212). All the 12 discrepant cases were MMR-d or subclonal loss of MMR protein by IHC, but MSS by NGS. Among them, 10 cases were loss or subclonal loss of MLH1 and (or) PMS2 protein. Three discrepant cases were classified as POLE gene mutation subtype. In the remaining 9 cases, 5 cases and 3 cases were confirmed as MSI-H and low microsatellite instability (MSI-L) respectively by MSI-PCR, 6 cases were detected as MLH1 gene promoter methylation and 7 cases demonstrated high TMB (>10 mutations/Mb). These 9 cases were classified as MMR-d EC. (3) Lynch syndrome was diagnosed in 27.3% (15/55) of all 55 MMR-d EC cases, and the TMB of EC with MSH2 and (or) MSH6 protein loss or associated with Lynch syndrome [(71.0±26.2) and (71.5±20.1) mutations/Mb respectively] were significantly higher than those of EC with MLH1 and (or) PMS2 loss or sporadic MMR-d EC [(38.2±19.1) and (41.9±24.3) mutations/Mb respectively, all P<0.01]. The top 10 most frequently mutated genes in MMR-d EC were PTEN (85.5%, 47/55), ARID1A (80.0%, 44/55), PIK3CA (69.1%, 38/55), KMT2B (60.0%, 33/55), CTCF (45.5%, 25/55), RNF43 (40.0%, 22/55), KRAS (36.4%, 20/55), CREBBP (34.5%, 19/55), LRP1B (32.7%, 18/55) and BRCA2 (32.7%, 18/55). Concurrent PTEN, ARID1A and PIK3CA gene mutations were found in 50.9% (28/55) of MMR-d EC patients. Conclusions: The concordance of MMR-IHC and MSI-NGS in EC is relatively high.The discordance in a few MMR-d EC are mostly found in cases with MLH1 and (or) PMS2 protein loss or MMR protein subclonal staining caused by MLH1 gene promoter hypermethylation. In order to provide accurate molecular typing for EC patients, MLH1 gene methylation, MSI-PCR, MMR gene germline mutation and TMB should be combined to comprehensively evaluate MMR and MSI status.

Identificación del fenotipo de inestabilidad microsatelital en carcinoma colorrectal mediante el análisis de la expresión de proteínas reparadoras del ADN: Revisión narrativa / Identification of microsatellite instability phenotype in colorectal carcinoma through expression analysis of DNA mismatch repair proteins: Narrative review

El carcinoma colorrectal (CCR) es de las primeras causas de mortalidad del mundo, presentando Guatemala una incidencia anual de 7.4/millón de habitantes. El síndrome de Lynch se caracteriza clínicamente por un inicio temprano del CCR con lesiones causadas por alteraciones en genes que codifican proteínas reparadoras.Los microsatélites son regiones del ADN con una unidad repetitiva de uno o más nucleótidos y son susceptibles a errores durante la replicación de ADN de los enterocitos. Existe un sistema de reparación que corrige estos errores. Cuando las proteínas reparadoras de este sistema están mutadas o ausentes, dichos errores del ADN persisten. Estas proteínas reparadoras se expresan en el núcleo de las células colónicas normales y son detecta-bles utilizando estudios de inmunohistoquímica (IHQ). Los genes MLH1 y MSH2 pueden encontrarse mutados en el 90% de los casos de cáncer colorrectal y el resto corresponde a MSH6 y PMS2. Esta vía oncogénica se caracteriza por alteración del sistema de reparación de errores durante la replicación del ADN, controlado por los genes MMR (mismatch repair), principalmente MLH1, MSH2, MSH6 y PMS2. Se realizó una revisión extensa de la literatura en PubMed, Springer y JAMA, usando las palabras clave: fenotipo de CCR, Síndrome de Lynch e inestabilidad microsatelital, detectándose 55 artículos. El objetivo de esta revisión es describir la importancia de la identificación del fenotipo del CCR por medios de IHQ y de pruebas moleculares para el eficaz tratamiento con inmunoterapia anti-PD1/PD-L1.

Colorectal cancer (CRC) is one of the leading causes of mortality in the world. In Guatemala it's an important cause of morbidity (7.4 per million inhabitants). Lynch syndrome is clinically characterized by an early onset of nonpolyposis colorectal carcinoma, with multiple lesions and neoplasms. The syndrome is caused by mutations in genes encoding DNA mismatch repair proteins. The microsatellites are regions of the DNA that repeat between one or more nucleotides and are susceptible to errors during replication, these are corrected by a repair system, when genes are mutated, the errors persist. The genes encoding repair proteins are expressed in the nuclei of normal colonic cells which can be observed using immunohistochemical studies. The MLH1, MSH2 genes are found to be mutated in 90% of the cases and the rest corresponds to the MSH6 and PMS2 genes. This oncogenic pathway characteristically consists of an alteration in the DNA repair system that is controlled by mismatch repair genes (MMR). An extensive research was conducted on PubMed, Springer and JAMA, using the keyword: CRC phenotype, Lynch syndrome and microsatellite instability. 55 articles were found. This review«s objective is to understand the mechanisms of nonpolyposis colorectal cancer and the importance of identifying patients with a mutant phenotype as a predictive factor for the efficacy of the anti-PD1/PDL1 immunotherapy and for prognosis.