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.

Biomarker characterization in endometrial cancer in Europe: first survey data analysis from 69 pathological academic and hospital labs.

Introduction: Endometrial carcinoma (EC) is the commonest gynecological cancer affecting women in Western populations. To predict patient risk, the 2020 edition of the World Health Organization (WHO) Classification of Tumors of the Female Genital Tract stressed the importance of integrated histo-molecular classification of the disease. This survey analysis poses attention on the most frequently used immunohistochemical and molecular markers adopted in daily categorization of ECs in European laboratories. Methods: We analyzed data collected through questionnaires administered to 40 Italian, 20 Spanish, 3 Swiss and 6 United Kingdom (UK) laboratories. We collected information regarding daily practice in EC evaluation, specifically concerning mismatch repair status (MMR) and microsatellite instability (MSI). Summary and descriptive statistical analyses were carried out to evaluate the current practice of each laboratory. Results: The results show that MMR status is mainly evaluated by using immunohistochemistry (IHC) on most EC samples. The most frequent approach for the analysis of MMR status is IHC of four proteins (PMS2, MSH6, MSH2, MLH1). MSI analysis by molecular methods is uncommon but useful as a supplemental tool in specific conditions. MLH1 promoter hypermethylation and BRAF V600 mutations analysis are performed in case of negative expression of MLH1/PMS2. Other markers (mainly p53 followed by POLE and PTEN) are investigated in particular in Spain and Switzerland in a consistent number of cases. Conclusion: Guidelines consultation and standardization of laboratory procedures are efficient means for EC prognostic risk stratification and improving the quality of care.

MRE11A: a novel negative regulator of human DNA mismatch repair.

BACKGROUND: DNA mismatch repair (MMR) is a highly conserved pathway that corrects DNA replication errors, the loss of which is attributed to the development of various types of cancers. Although well characterized, MMR factors remain to be identified. As a 3'-5' exonuclease and endonuclease, meiotic recombination 11 homolog A (MRE11A) is implicated in multiple DNA repair pathways. However, the role of MRE11A in MMR is unclear. METHODS: Initially, short-term and long-term survival assays were used to measure the cells' sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Meanwhile, the level of apoptosis was also determined by flow cytometry after MNNG treatment. Western blotting and immunofluorescence assays were used to evaluate the DNA damage within one cell cycle after MNNG treatment. Next, a GFP-heteroduplex repair assay and microsatellite stability test were used to measure the MMR activities in cells. To investigate the mechanisms, western blotting, the GFP-heteroduplex repair assay, and chromatin immunoprecipitation were used. RESULTS: We show that knockdown of MRE11A increased the sensitivity of HeLa cells to MNNG treatment, as well as the MNNG-induced DNA damage and apoptosis, implying a potential role of MRE11 in MMR. Moreover, we found that MRE11A was largely recruited to chromatin and negatively regulated the DNA damage signals within the first cell cycle after MNNG treatment. We also showed that knockdown of MRE11A increased, while overexpressing MRE11A decreased, MMR activity in HeLa cells, suggesting that MRE11A negatively regulates MMR activity. Furthermore, we show that recruitment of MRE11A to chromatin requires MLH1 and that MRE11A competes with PMS2 for binding to MLH1. This decreases PMS2 levels in whole cells and on chromatin, and consequently comprises MMR activity. CONCLUSIONS: Our findings reveal that MRE11A is a negative regulator of human MMR.

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.

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.

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.

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.

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.

Neurofibromatosis type 1 mosaicism in patients with constitutional mismatch repair deficiency.

Differential diagnosis between constitutional mismatch repair deficiency (CMMRD) and neurofibromatosis type 1 (NF1) is crucial as treatment and surveillance differ. We report the case of a girl with a clinical diagnosis of sporadic NF1 who developed a glioblastoma. Immunohistochemistry for MMR proteins identified PMS2 loss in tumour and normal cells and WES showed the tumour had an ultra-hypermutated phenotype, supporting the diagnosis of CMMRD. Germline analyses identified two variants (one pathogenic variant and one classified as variant(s) of unknown significance) in the PMS2 gene and subsequent functional assays on blood lymphocytes confirmed the diagnosis of CMMRD. The large plexiform neurofibroma of the thigh and the freckling were however more compatible with NF1. Indeed, a NF1 PV (variant allele frequencies of 20%, 3% and 9% and in blood, skin and saliva samples, respectively) was identified confirming a mosaicism for NF1. Retrospective analysis of a French cohort identified NF1 mosaicism in blood DNA in 2 out of 22 patients with CMMRD, underlining the existence of early postzygotic PV of NF1 gene in patients with CMMRD whose tumours have been frequently reported to exhibit somatic NF1 mutations. It highlights the potential role of this pathway in the pathogenesis of CMMRD-associated gliomas and argues in favour of testing MEK inhibitors in this context.

Specific Pathology Features Enrich Selection of Endometrial Carcinomas for POLE Testing.

Identification of ultramutated/ POLE -mutated endometrial carcinomas ( POLEM ECs) has important implications given its association with better prognosis. However, POLE mutation testing is not widely available. Our objective was to evaluate POLEM ECs versus POLE wild-type ( POLEWT ) ECs, within a cohort of consultation cases with features suggestive of an ultramutated phenotype. Consultation cases of EC that had undergone POLE hotspot mutation testing over a 3.5-year period were included. Tumor morphology and immunohistochemistry were reviewed for both groups. Chi-square test and t test were used for statistical analysis. Of 25 consultation cases, 12 harbored a POLE mutation (48%) and 13 were wild-type (52%). Patients with POLEM ECs were younger (59 vs. 71.3 y; P =0.01). Ambiguous histomorphology (5/12 vs. 1/13; P =0.04) and the presence of more than rare bizarre nuclei (8/12 vs. 2/12; P =0.01) differed significantly between POLEM and POLEWT ECs, respectively. In the POLEM group, one case (1/12) demonstrated PMS2 loss, and one (1/12) showed subclonal MLH1/PMS2 loss. Among POLEWT ECs, 3/13 (23%) showed MLH1/PMS2 loss. p53 was subclonally overexpressed in 4/10 POLEM and 1/13 POLEWT cases ( P =0.06). Mutant p53 patterns were seen in 1/10 POLEM versus 6/13 of POLEWT ECs, respectively ( P =0.06). Within our cohort, the specificity of ambiguous histomorphology, bizarre nuclei, subclonal biomarker expression, and marked tumor-infiltrating lymphocytes for POLEM EC was 83%, 80%, 80%, and 71%, respectively. Where universal POLE testing is not available, these data suggest that morphologic screening (particularly ambiguous histomorphology and the presence of more than rare bizarre nuclei) can be useful for selective enrichment of ECs for POLE testing.

PMS2 or PMS2CL? Characterization of variants detected in the 3' of the PMS2 gene.

PMS2 germline pathogenic variants are one of the major causes for Lynch syndrome and constitutional mismatch repair deficiencies. Variant identification in the 3' region of this gene is complicated by the presence of the pseudogene PMS2CL which shares a high sequence homology with PMS2. Consequently, short-fragment screening strategies (NGS, Sanger) may fail to discriminate variant's gene localization. Using a comprehensive analysis strategy, we assessed 42 NGS-detected variants in 76 patients and found 32 localized on PMS2 while 6 on PMS2CL. Interestingly, four variants were detected in either of them in different patients. Clinical phenotype was well correlated to genotype, making it very helpful in variant assessment. Our findings emphasize the necessity of more specific complementary analyses to confirm the gene origin of each variant detected in different individuals in order to avoid variant misinterpretation. In addition, we characterized two PMS2 genomic alterations involving Alu-mediated tandem duplication and gene conversion. Those mechanisms seemed to be particularly favored in PMS2 which contribute to frequent genomic rearrangements in the 3' region of the gene.

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.

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.

Glioma arising in the setting of mismatch repair deficiency-rare or are we missing it?

Germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, PMS2) can be mono-allelic or biallelic, resulting in a Lynch syndrome (LS) or constitutional mismatch repair deficiency (CMMRD) syndrome respectively. Glioma arising in the setting of MMR deficiency is uncommon. We describe two pediatric patients with high-grade glioma (HGG) and associated MMR protein deficiency. On histomorphology both cases showed HGG with astrocytic morphology and prominent multinucleated tumor cells. On immunohistochemistry, the first case was negative for IDH1 p.R132H showed loss of ATRX and p53 positivity. The second case was positive for IDH1 p.R132H and p53, but showed retained expression of ATRX. The histomorphology in both cases and additionally IDH mutation with retained ATRX in the second case, prompted us to test for MMR protein deficiency which was carried out by immunohistochemistry (IHC). One case revealed an immunostaining pattern suggestive of CMMRD while the other was suggestive of LS. Both the cases showed good response to surgery and radio-chemotherapy in the follow-up available. Our cases highlight the importance of testing for MMR proteins by simple IHC, in the setting of appropriate clinical scenario, histopathological and immunohistochemical findings. The recognition of these tumors is extremely important to guide further treatment and prompt family screening.

Characterization of mismatch-repair/microsatellite instability-discordant endometrial cancers.

BACKGROUND: Mismatch-repair (MMR)/microsatellite instability (MSI) status has therapeutic implications in endometrial cancer (EC). The authors evaluated the concordance of testing and factors contributing to MMR expression heterogeneity. METHODS: Six hundred sixty-six ECs were characterized using immunohistochemistry (IHC), MSI testing, and mut-L homolog 1 (MLH1) methylation. Select samples underwent whole-transcriptome analysis and next-generation sequencing. MMR expression of metastatic/recurrent sites was evaluated. RESULTS: MSI testing identified 27.3% of cases as MSI-high (n = 182), MMR IHC identified 25.1% cases as MMR-deficient (n = 167), and 3.8% of cases (n = 25) demonstrated discordant results. A review of IHC staining explained discordant results in 18 cases, revealing subclonal loss of MLH1/Pms 1 homolog 2 (PMS2) (n = 10) and heterogeneous MMR IHC (mut-S homolog 6 [MSH6], n = 7; MLH1/PMS2, n = 1). MSH6-associated Lynch syndrome was diagnosed in three of six cases with heterogeneous expression. Subclonal or heterogeneous cases had a 38.9% recurrence rate (compared with 16.7% in complete MMR-deficient cases and 9% in MMR-proficient cases) and had abnormal MMR IHC results in all metastatic recurrent sites (n = 7). Tumors with subclonal MLH1/PMS2 demonstrated 74 differentially expressed genes (determined using digital spatial transcriptomics) when stratified by MLH1 expression, including many associated with epithelial-mesenchymal transition. CONCLUSIONS: Subclonal/heterogeneous MMR IHC cases showed epigenetic loss in 66.7%, germline mutations in 16.7%, and somatic mutations in 16.7%. MMR IHC reported as intact/deficient missed 21% of cases of Lynch syndrome. EC with subclonal/heterogeneous MMR expression demonstrated a high recurrence rate, and metastatic/recurrent sites were MMR-deficient. Transcriptional analysis indicated an increased risk for migration/metastasis, suggesting that clonal MMR deficiency may be a driver for tumor aggressiveness. Reporting MMR IHC only as intact/deficient, without reporting subclonal and heterogeneous staining, misses opportunities for biomarker-directed therapy. PLAIN LANGUAGE SUMMARY: Endometrial cancer is the most common gynecologic cancer, and 20%-40% of tumors have a defect in DNA proofreading known as mismatch-repair (MMR) deficiency. These results can be used to guide therapy. Tests for this defect can yield differing results, revealing heterogeneous (mixed) proofreading capabilities. Tumors with discordant testing results and mixed MMR findings can have germline or somatic defects in MMR genes. Cells with deficient DNA proofreading in tumors with mixed MMR findings have DNA expression profiles linked to more aggressive characteristics and cancer spread. These MMR-deficient cells may drive tumor behavior and the risk of spreading cancer.

Applicability of the FDA-approved Immunohistochemical Panel for Identification of MMRd Phenotype in Uterine Endometrioid Carcinoma.

Recently, the US Food and Drug Administration (FDA) approved the Ventana MMR RxDx Panel as the first immunohistochemical companion diagnostic test for identification of tumors with mismatch repair (MMR) status. The aim of this study was to investigate the accuracy of this test in comparison with polymerase chain reaction (PCR)-based microsatellite instability (MSI) analysis. We assessed the MMR/MSI concordance rate in 140 cases of endometrioid carcinoma. MMR status was evaluated by immunohistochemistry (MMR-IHC), and MSI status was evaluated by PCR-based analysis (MSI-PCR). Potential molecular mechanisms responsible for MSH6 staining variations were also analyzed. Immunohistochemistry showed that 34 tumors (24.3%) were MMRd; these included 26 with combined MLH1/PMS2 loss, 2 with combined MSH2/MSH6 loss, and 6 with isolated MSH6 loss. Heterogeneous MSH6 loss was found in 10 tumors and was recognized only in tumors with combined MLH1/PMS2 loss. Eight of 10 tumors with heterogeneous MSH6 loss harbored MSH6 C8 tract instability, suggesting a secondary somatic event after MLH1/PMS2 loss. MSI-PCR revealed that 102 tumors were MSS, 4 were MSI-low, and 34 were MSI-high. Consequently, MMR-IHC and MSI-PCR showed perfect concordance (kappa=0.080, P <0.0001). However, 10 of the 34 MSI-high tumors, including the 6 tumors with isolated MSH6 loss, showed only minimal microsatellite shift by MSI-PCR, which may have been erroneously interpreted as MSS or MSI-low. On the basis of these findings, we consider that the FDA-approved immunohistochemical panel can detect MMR variations consistently and is more accurate than MSI-PCR for determining the applicability of immune checkpoint inhibitors for treatment of endometrioid carcinomas.

[Comprehensive assessment of mismatch repair and microsatellite instability status in molecular classification of endometrial carcinoma].

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.

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.

[Pan-cancer analysis of ubiquitin-specific protease 7 and its expression changes in the carcinogenesis of scar ulcer].

Objective: To explore the biological role and clinical significance of ubiquitin-specific protease 7 (USP7) in the carcinogenesis of scar ulcer. Methods: A retrospective observational study combined with bioinformatics analysis was used. The RNA expression profile data of USP7 in tumor and/or its corresponding paracancular normal tissue were obtained from The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus database, and the RNA sequencing data were transformed by log2. The variations of USP7 gene were analyzed by cBioPortal database. The USP7 mRNA expression in tumor and adjacent normal tissue in TCGA database were obtained by using the "Gene_DE" module in TIMER 2.0 database. The survival rates of patients with high and low USP7 expression in cutaneous melanoma (SKCM), cervical squamous cell carcinoma (CESC), lung squamous cell carcinoma (LUSC), and head and neck squamous cell carcinoma (HNSC) were analyzed using the Gene Expression Profile Interactive Analysis 2 (GEPIA2) database, and the Kaplan-Meier survival curves were drawn. Sangerbox database was used to analyze the correlation of USP7 expression in pan-cancer with microsatellite instability (MSI) or tumor mutation burden (TMB) pan-cancer. Through the "correlation analysis" module in the GEPIA2 database, the correlation of USP7 expression in pan-cancer with the expression levels of five DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS2, and EPCAM) and three essential DNA methyltransferases (DNMT)--DNMT1, DNMT3A, and DNMT3B were evaluated. The USP7 expression in CESC, HNSC, LUSC, and SKCM and its correlation with infiltration of immune cells (B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells) were analyzed by the "Immune-Gene" module in TIMER 2.0 database. The "Similar Genes Detection" module of GEPIA2 database was used to obtain the top 100 protein sets with similar expression patterns to USP7. Intersection analysis was performed between the aforementioned protein sets and the top 50 protein sets that were directly physically bound to USP7 obtained by using the STRING database. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis were performed for the two protein sets mentioned above using the DAVID database. The samples of normal skin, hypertrophic scar, scar ulcer, and scar carcinoma with corresponding clinicopathologic features were collected from the Department of Pathology of Tongren Hospital of Wuhan University & Wuhan Third Hospital from October 2018 to October 2022, and the USP7 expression in tissue was detected by immunohistochemical method, with the number of samples of 6. Data were statistically analyzed with Log-rank test, one-way analysis of variance, and Bonferroni test. Results: In pan-cancer, the main gene variations of USP7 were mutation and amplification, and the top 3 tumors with the highest variation frequency (>6%) were bladder urothelial carcinoma, SKCM, and endometrial carcinoma. The main mutation of USP7 gene in pan-cancer was missense mutation. In SKCM with the highest mutation frequency, the main type of mutation was missense mutation in USP7_ICP0_bdg domain. USP7 mRNA expression in breast invasive carcinoma, bile duct carcinoma, colon carcinoma, esophageal carcinoma, HNSC, renal chromophobe cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, LUSC, prostate carcinoma, and gastric carcinoma was significantly higher than that in corresponding paracancer normal tissue (P<0.05). USP7 mRNA expression in glioblastoma multiforme, renal clear cell carcinoma, renal papillary cell carcinoma, and thyroid carcinoma was significantly lower than that in corresponding paracancular normal tissue (P<0.05). In addition, USP7 mRNA expression in SKCM metastases was much higher than that in primary tumor tissue (P<0.05). Survival curves showed no significant difference in survival rate between patients with high USP7 expression and patients with low USP7 expression in CESC, HNSC, LUSC, and SKCM (Log-rank P>0.05, with hazard ratios of 1.00, 0.99, 1.00, and 1.30, respectively). USP7 expression in colon cancer, colorectal cancer, thymic cancer, and thyroid cancer was negatively correlated with TMB (with Pearson correlation coefficients of -0.26, -0.19, -0.19, and 0.11, respectively, P<0.05). USP7 expression in glioma, CESC, lung adenocarcinoma, mixed renal carcinoma, and LUSC was positively correlated with MSI expression (with Pearson correlation coefficients of 0.22, 0.14, 0.15, 0.08, and 0.14, respectively, P<0.05), and USP7 expression in colon cancer, colorectal cancer, invasive breast cancer, prostate cancer, HNSC, thyroid cancer, and diffuse large B-cell lymphoma were significantly negatively correlated with MSI expression (with Pearson correlation coefficients of -0.31, -0.27, -0.13, -0.19, -0.16, -0.18, and -0.53, respectively, P<0.05). The expression of USP7 in CESC was positively correlated with that of both MSH2 and MSH6 (with Spearman correlation coefficients of 0.51 and 0.44, respectively, P<0.05), and the expression of USP7 in HNSC was positively correlated with the expression of EPCAM, MLH1, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.39, 0.14, 0.49, 0.54, and 0.41, respectively, P<0.05), and the expression of USP7 in LUSC was positively correlated with the expression of EPCAM, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.20, 0.36, 0.40, and 0.34, respectively, P<0.05), and the expression of USP7 in SKCM was positively correlated with the expression of EPCAM, MLH1, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.11, 0.33, 0.42, 0.55, and 0.34, respectively, P<0.05). The expression of USP7 in CESC, HNSC, LUSC, and SKCM was significantly positively correlated with the expression of DNMT1, DNMT3A, and DNMT3B (with Spearman correlation coefficients of 0.42, 0.34, 0.22, 0.45, 0.52, 0.22, 0.36, 0.36, 0.22, 0.38, 0.46, and 0.21, respectively, P<0.05). The expression of USP7 in CESC, HNSC, LUSC, and SKCM was positively correlated with CD4+ T cell infiltration (with Partial correlation coefficients of 0.14, 0.22, 0.13, and 0.16, respectively, P<0.05). Being similar to the pattern of USP7 expression and ranked among top 100 protein sets, the top 5 proteins were C16orf72, BCLAF1, UBN, GSPT1, ERI2 (with Spearman correlation coefficients of 0.83, 0.74, 0.73, and 0.72, respectively, all P values<0.05). The top 50 protein sets that directly physically bind to USP7 overlapped with the aforementioned protein set by only one protein, thyroid hormone receptor interaction factor 12. KEGG enrichment analysis showed that USP7 related genes were involved in cell cycle, spliceosome, cell senescence, and p53 signal pathway. GO enrichment analysis showed that USP7 related genes were involved in transcriptional regulation, protein ubiquitination, DNA repair, and cytoplasmic pattern recognition receptor signal pathways. Analysis of clinical samples showed that USP7 expression was significantly higher in hypertrophic scars (0.35±0.05), scar ulcers (0.43±0.04), and scar cancers (0.61±0.03) than in normal skin (0.18±0.04), P<0.05. Conclusions: USP7 may be a clinical biomarker for the progression of cicatricial ulcer cancer.

A retrospective study of consistency between immunohistochemistry and polymerase chain reaction of microsatellite instability in endometrial cancer.

Objectives: Identification of endometrial cancers (EC) with mismatch repair deficiency (dMMR) or microsatellite instability-high (MSI-H) is essential for Lynch syndrome screening and treatment stratification. We aimed to assess the utility of immunohistochemistry (IHC) staining for MMR protein expression and polymerase chain reaction (PCR)-based MSI assays in EC and the correlation between MMR/MSI status and various clinicopathological parameters. Methods: We reviewed the clinical and pathological information of 333 patients with EC. MMR protein expression was assessed as retained or lost to determine MMR status by IHC staining, and MSI status was identified by PCR capillary electrophoresis (PCR-CE) testing with a National Cancer Institute (NCI) panel. The correlation of MMR/MSI status with clinicopathological features was determined by statistical analysis. Discrepant results were further analyzed using an alternative PCR-CE MSI (Promega panel) method, MLH1 promoter methylation assays, and next-generation sequencing (NGS). Results: Among the EC patients, the overall percentage of dMMR was 25.2%, and the overall percentage of MSI-H was 24%. Among the dMMR patients, 50 (59.5%) showed loss of MLH1 and PMS2 expression, 19 (22.6%) loss of MSH2 and MSH6 expression, and seven (8.3%) and eight (9.5%) loss of PMS2 and MSH6 expression, respectively. The dMMR subgroup was significantly younger than the pMMR subgroup, especially for <60-years-old patients (p = 0.038). In addition, we identified a strong correlation between MMR/MSI status and high-grade endometrioid or nonendometrioid components (p = 0.004 or p = 0.003). IHC staining and PCR-CE assay results showed a high level of overall concordance (98.8%, Cohen's κ = 0.98). Four patients were found to have dMRR/MSS in both examinations. We reanalyzed them with additional methods. One case showed MLH1 promotor methylation, and the other three cases harbored MSH6 germline pathogenic variations. One of the cases with MSH6 deficiency was reanalyzed as MSI-H by alternative PCR-CE assay or NGS testing. Conclusions: This study indicates that the combined use of MMR-IHC and PCR-CE MSI analyses may effectively avoid misdiagnoses of EC patients with dMMR/MSI-H. However, use of PCR-CE alone to evaluate MMR/MSI status may lead to missed diagnosis, especially for EC patients with MSH6 deficiency and presenting MSS.