MLH1 Expression Research Articles

Universal screening for mismatch repair deficiency in gastrointestinal cancers in a reference center in Mexico.

840 Background: Gastrointestinal (GI) cancers have a poor prognosis. With the availability of immunotherapy for tumors with deficient mismatch repair (dMMR), identifying these patients has become increasingly important. Universal dMMR screening across all GI cancers is uncommon, and its prevalence has not been well studied prospectively. In Mexico, the prevalence of dMMR is unknown, even though GI cancers account for a large portion of new cancer cases and cancer-related deaths. Our objective was to prospectively determine the prevalence of dMMR in GI cancers at a referral center in Mexico. Methods: Prospective universal screening for dMMR began in March 2024. Primary tumor sites included were esophagus, stomach, pancreas, biliary tract, small intestine, colon, and rectum. Histologies included were adenocarcinoma, squamous cell, or mixed. All consecutive patients were included regardless of age, clinical stage or medical history. MMR status was determined by immunohistochemistry, analyzing expression of MLH1, PMS2, MSH2, and MSH6. This abstract presents the results from the first six months of this universal screening. Results: A total of 161 GI cancers were included (see Table). Prevalence of dMMR was 9.3% (n=15). The highest prevalence was in gastric cancer (13.3%, 2/15), followed by colon (12.1%, 7/58), pancreatic (11.1%, 4/36), and bile duct (8.3%, 2/24) cancer. MMR status was not performed in 9.3% (n=15) of cases. Combined deficiency was found in 60% (9/15) for MLH1/PMS2 and 20% (3/15) for MSH2/MSH6. Isolated deficiency occurred in 20% (3/15) for PMS2 and 13.3% (2/15) for MSH2. No difference in age or clinical stage was observed between MMR status groups. Conclusions: The high prevalence of dMMR, along with the poor prognosis and availability of immunotherapy based treatments, supports the need for universal screening. The high prevalence of dMMR in pancreatobiliary cancers was notable. Implementation efforts in our institution are essential to obtain MMR status in all cases. Characteristics of population. pMMRn=131 (%) dMMRn=15 (%) Not performedn=15 (%) Totaln=161 p value Gender Male 63 (48.1) 10 (66.7) 7 (46.7) 80 (49.7) 0.38 Female 68 (51.9) 5 (33.3) 8 (53.3) 81 (50.3) Age Median (IQR) 65 (54 -72) 52 (43 -80) 65 (57 -71) 65 (53 -72) 0.52 Histology Adenocarcinoma 130 (99.2) 15 (100.0) 14 (93.3) 159 (98.8) N/A Mixed 1 (0.8) 0 (0.0) 0 (0.0) 1 (0.6) Squamous Cell 0 (0.0) 0 (0.0) 1 (6.7) 1 (0.6) Clinical Stage Unknown 12 (9.2) 1 (6.7) 1 (6.7) 14 (8.7) 0.49 Localized 24 (18.3) 2 (13.3) 2 (13.3) 28 (17.4) Locally advanced 40 (30.5) 8 (53.3) 3 (20.0) 51 (31.7) Metastatic 55 (42.0) 4 (26.7) 9 (60.0) 68 (42.2) Tumor site Esophageal 9 (6.9) 0 (0.0) 1 (6.7) 10 (6.2) N/A Gastric 13 (9.9) 2 (13.3) 0 (0.0) 15 (9.3) Small B. 1 (0.8) 0 (0.0) 0 (0.0) 1 (0.6) Bile duct 14 (10.7) 2 (13.3) 8 (53.3) 24 (14.9) Pancreas 29 (22.1) 4 (26.7) 3 (20.0) 36 (22.4) Colon 50 (38.2) 7 (46.7) 1 (6.7) 58 (36.0) Rectal 15 (11.5) 0 (0.0) 2 (13.3) 17 (10.6)

Prevalence of Microsatellite Instability in Gastric and Gastroesophageal Junction Cancer patients from a Latin American Country.

In Colombia, gastric cancer is fifth in incidence (12.8 cases per 100,000) and third in mortality (9.9 cases per 100,000). Microsatellite instability (MSI), a phenotype in gastric cancer treatment, lacks comprehensive exploration in Colombian and Hispanic/Latino populations. Data scarcity hinders immunotherapy approval in middle-income countries. Characterize the prevalence of MSI phenotype in Colombian patients with gastric and gastroesophageal junction cancer. We measured MLH-1, MSH-2, MSH-6, and PMS-2 expression in tumor pathology by immunohistochemistry markers. We conducted descriptive analysis and Fisher's test to identify associations for MSI expression. Final sample size was 106 patients, mean age of 62.5 years (25-93 ± 14.2). Prevalence of MSI was 12.26% (n = 13). We found an association between older age and positive MSI (p = 0.0042), as well as with non-diffuse histologic subtypes (p = 0.019). Prior studies report 22% MSI phenotype prevalence in gastric tumors, mostly in developed countries, excluding Hispanic/Latino populations. Identifying the prevalence of MSI in our population as 12.26% could pave the way for approving immune blockade drugs as a treatment option for these patients in Latin American countries. Our data could be utilized to conduct cost-utility studies in support of this.

DNA mismatch repair (MMR) genes expression in lung cancer and its correlation with different clinicopathologic parameters.

Lung cancer (LC) is a crucial rapidly developing disease. In Egypt, it is one of the five most frequent cancers. Little is known about the impact of deleted mismatch repair genes and its correlation to clinicopathological characteristics. This study evaluates immunohistochemical expression of the mismatch repair genes (PMS2), (MSH2), (MLH1) & (MSH6) & its correlation with clinicopathologic parameters & prognosis of LC. Age was higher with lost MLH1 & PMS2 but HTN was higher with lost four markers. Smoking was associated with expression of MLH1 & PMS2. A progressive course was associated with lost MSH2 & MSH6. Suprarenal metastasis was associated with lost all markers but bone metastasis was associated with lost MSH2 & MSH6. All the markers were significantly correlated with each other, with perfect correlations between MSH6 & MSH2 and between MLH & PMS2. Median overall survival among cases with lost markers was significantly lower than patients with preserved markers. We recommend evaluation of the four proteins as a biomarker that could guide LC therapy. In-depth biological research is imperative to elucidate the precise roles and mechanisms of these markers. This will advance management strategies and even guide immune checkpoint inhibitor therapy for LC.

Prevalence of Mismatch Repair Deficiency in Advanced Solid Tumors (Colorectal Cancer and Non-Colorectal Cancer) in One Mexican Institution.

Background/Objectives: Mismatch repair (MMR) status is an important prognostic and predictive indicator in cancer, distinguishing proficient (pMMR) tumors from deficient (dMMR) ones. This study aimed to determine the prevalence of dMMR in colorectal (CRC) and selected non-CRC solid tumors (gastric, esophageal, and endometrial cancers). Methods: This retrospective study was conducted at a private health institution in Mexico City, analyzing patients diagnosed with colorectal, gastric, esophageal, or endometrial cancer from January 2017 to December 2020. dMMR prevalence was assessed using available status information and tissue samples for immunohistochemistry (IHC). Data were analyzed via SPSS, presenting results in frequencies and percentages. Results: Most solid tumors exhibited MSH2, MSH6, and MLH1 expression above 90%, with slightly lower levels in endometrial cancer. Esophageal cancer showed 100% pMMR. dMMR prevalence was found to be 12.7% for CRC, 8.3% for gastric, and 18.5% for endometrial cancers. Prevalence rates were similar across genders (11.1% in women and 12.9% in men), with the highest prevalence in the 41-50 age group (20%) and the lowest in the 31-40 age group (7.7%). Conclusions: This study offers valuable insights into the frequency of dMMR mutations in a cohort of the Mexican population, providing a basis for further research on their prevalence in Mexico.

Recurrent GRHL fusions in a subset of sebaceoma: microscopic and molecular characterisation of eight cases.

Sebaceous neoplasms constitute a group of adnexal tumours, including sebaceous adenoma, sebaceoma and sebaceous carcinoma. Although mismatch repair deficiency may be observed, the nature of the genetic alterations contributing to the development of most of these tumours is still unknown. In the present study, we describe the clinical, microscopic, and molecular features of eight sebaceomas with GRHL gene rearrangement. Among these sebaceomas, four occurred in women and four in men; the median age was 63 years (range = 29-89). The tumours were located in the head and neck area in all cases. Microscopic examination revealed a well-demarcated lesion located in the dermis with focal extension into the subcutaneous tissue (three cases). The neoplasms displayed macronodular (eight cases), cribriform (seven cases) and organoid (six cases) growth patterns, occurring in combination. The tumours were mainly composed of immature basophilic cells associated with scattered mature sebocytes. Numerous small infundibular cysts were present in seven cases. Mitotic activity was low (none/one to four mitoses/mm2). Immunohistochemistry showed positivity for androgen receptor and p63. Preserved expression of MLH1, PMS2, MSH2 and MSH6 was observed in all cases. RNA-sequencing revealed RCOR1::GRHL2 (three cases), BCL6::GRHL1 (two cases), a BCOR::GRHL2 (one case), RCOR1::GRHL1 (one case) and TLE1::GRHL1 (one case) fusion transcript. Methylation analysis demonstrated that GRHL-fused sebaceomas form an independent cluster and highlight the proximity of such tumours with poromas with folliculo-sebaceous differentiation. In conclusion, we report recurrent fusions of the GRHL genes in a distinctive subset of sebaceomas harbouring infundibulocystic differentiation, a frequent organoid growth pattern and lack of mismatch repair deficiency.

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability.

TAR DNA-binding protein 43 (TDP43) is increasingly recognized for its involvement in neurodegenerative diseases, particularly amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP43 proteinopathy, characterized by dysregulated nuclear export and cytoplasmic aggregation, is present in most ALS/FTD cases and is associated with a loss of nuclear function and genomic instability in neurons. Building on prior evidence linking TDP43 pathology to DNA double-strand breaks (DSBs), this study identifies a novel regulatory role for TDP43 in the DNA mismatch repair (MMR) pathway. We demonstrate that depletion or overexpression of TDP43 affects the expression of key MMR genes, including MLH1, MSH6, MSH2, MSH3, and PMS2. Specifically, TDP43 modulates the expression of MLH1 and MSH6 proteins through alternative splicing and transcript stability. These findings are validated in ALS mice models, patient-derived neural progenitor cells and autopsied brain tissues from ALS patients. Furthermore, MMR depletion showed a partial rescue of TDP43-induced DNA damage in neuronal cells. Bioinformatics analysis of TCGA cancer database reveals significant correlations between TDP43 and MMR gene expressions and mutational burden across various cancer subtypes. These results collectively establish TDP43 as a critical regulator of the MMR pathway, with broad implications for understanding the genomic instability underlying neurodegenerative and neoplastic diseases.

Solitary Loss of PMS2 in a patient with Colon carcinoma

Abstract Introduction/Objective In the United States, there are about 150,000 new cases of Colorectal cancer (CRC) and about 50,000 CRC related deaths each year. Hereditary nonpolyposis colorectal cancer (Lynch Syndrome) accounts for 2% - 4% of CRC. It is characterized by germline mutations in mismatch repair (MMR) genes - MLH1, MSH2, MSH6 and PMS2. Patients with Lynch syndrome have up to an 80% lifetime risk (LTR) of CRC and up to 60% LTR of endometrial cancer. Lynch syndrome patients also have an associated LTR of ovarian cancer (38%), upper urologic tract cancer (18%), gastric cancer (13%), small bowel cancer (6%), hepatobiliary tract cancer (4%), and brain tumors (3% - usually glioblastoma). Methods/Case Report A 76-year-old man with history of moderately differentiated colorectal adenosarcoma involving the transverse colon and a recently diagnosed grade group 5 (Gleason 4 + 5) prostate adenocarcinoma, currently being managed at an outside hospital. The case was sent to UAB for DNA MMR/microsatellite instability (MSI) status evaluation testing. Immunostaining showed loss of PMS2 and intact expression of MLH1, MSH2 and MSH6. These results were confirmed by the IdyIIa MSI assay which detected microsatellite instability in 5 of 7 markers and negative for BRAF V600E variant confirming microsatellite instable tumor. Results (if a Case Study enter NA) NA Conclusion Solitary loss of PMS2 in colonic adenocarcinoma is uncommon and may be caused by an underlying germline mutation of MLH-1 or PMS2, thus could represent Lynch Syndrome. Our patient’s age doesn’t favor Lynch syndrome. Next step would be to perform MLH-1 promoter methylation to confirm sporadic nature of carcinoma. Somatic methylation of the MLH1 promoter occurs in approximately 80% of CRC with defective MMR. Prostate cancer is not currently considered a part of Lynch syndrome.

Immunostaining for Mismatch Repair Complex Proteins Impacts on Clinical-Pathological Characteristics and Prognosis of Adenoid Cystic Carcinoma of Salivary Glands.

To evaluate the influence of MMR proteins on clinicopathological characteristics and prognosis of salivary gland adenoid cystic carcinoma (ACC). The solid pattern of ACC showed lower expression for MSH2 (p = 0.039). Significant imbalance in MSH2/MSH6 immunostaining was observed in all histological patterns (p < 0.001), and imbalance in PMS2/MLH1 immunostaining was observed in the cribriform pattern (p = 0.011). The presence of capsule was associated with high expression of MSH6 (p = 0.019), MLH1 (p = 0.045) and PMS2 (p = 0.009). The absence of cribriform pattern (p = 0.002) and capsule pattern (p = 0.025), as well as low expression for MSH6 (p = 0.006) and PMS2 (p = 0.037) were associated with lower overall survival. In multivariate analysis, loss of MSH2 (p = 0.039) and MLH1 (p = 0.017) were significantly associated with worse overall survival. Twenty-four ACC were clinical-pathologically evaluated and we perform immunohistochemistry for MSH2, MSH6, PMS2 and MLH1. Percentage counting of positive cells was performed in 10 fields of each histological pattern (cribriform, tubular and solid) and the averages of the 30 fields were considered for evaluation with other clinical-pathological variables (Kruskal-Wallis/Dunn, Friedman/Dunn, chi-square, Log-Rank Mantel-Cox tests and Cox regression; SPSS v20.0, p < 0.05). Salivary glands' ACC shows imbalance of the MMR complex and loss of expression of its components is associated with the overall survival of these patients.

Establishment and Characterization of Testis Organoids with Proliferation and Differentiation of Spermatogonial Stem Cells into Spermatocytes and Spermatids.

Organoids play pivotal roles in uncovering the molecular mechanisms underlying organogenesis, intercellular communication, and high-throughput drug screening. Testicular organoids are essential for exploring the genetic and epigenetic regulation of spermatogenesis in vivo and the treatment of male infertility. However, the formation of testicular organoids with full spermatogenesis has not yet been achieved. In this study, neonatal mouse testicular cells were isolated by two-step enzymatic digestion, and they were combined with Matrigel and transplanted subcutaneously into nude mice. Histological examination (H&E) staining and immunohistochemistry revealed that cell grafts assembled to form seminiferous tubules that contained spermatogonial stem cells (SSCs) and Sertoli cells, as illustrated by the co-expression of PLZF (a hallmark for SSCs) and SOX9 (a marker for Sertoli cells) as well as the co-expression of UCHL1 (a hallmark for SSCs) and SOX9, after 8 weeks of transplantation. At 10 weeks of transplantation, SSCs could proliferate and differentiate into spermatocytes as evidenced by the expression of PCNA, Ki67, c-Kit, SYCP3, γ-HA2X, and MLH1. Notably, testicular organoids were seen, and spermatids were observed within the lumen of testicular organoids after 16 weeks of transplantation, as shown by the presence of TNP1 and ACROSIN (hallmarks for spermatids). Collectively, these results implicate that we successfully established testicular organoids with spermatogenesis in vivo. This study thus provides an excellent platform for unveiling the mechanisms underlying mammalian spermatogenesis, and it might offer valuable male gametes for treating male infertility.

The Relationship Between DNA Mismatch Repair Status and Clinicopathologic Characteristics in Colon Cancer.

DNA mismatch repair (MMR) proteins are essential for repairing genetic mutations that occur during DNA replication. Deficiency of MMR proteins results in a phenotype called microsatellite instability (MSI), which occurs in Lynch syndrome as well as sporadic colorectal cancers (CRC), and it is associated with several clinicopathological features. We aimed to investigate the association of the loss of MMR proteins with clinicopathologic considerations in our CRC series. In this retrospective study, DNA MMR protein status in CRC is evaluated in a total of 200 colorectal resection specimens by immunohistochemistry (IHC) for MLH1, MSH2, MSH6 and PMS2 protein expression. The BRAF mutation was investigated by the real-time PCR in cases with loss of MLH1 protein expression. The relationship between MMR status and clinicopathological parameters was investigated statistically. Loss of MMR protein expression was detected in 26 of 200 CRC cases. The BRAFV600E mutation was detected in 2 of the cases with MLH1 loss and accepted as sporadic. The remaining 24 cases (12%) were identified as Lynch syndrome candidates. There were statistical differences observed regarding the presence of tumor-infiltrating lymphocytes (P < .001), Crohn's-like reaction (P = .001), expansile growth (P < .001), tumor heterogeneity (P < .001), mucinous differentiation (P < .001), and presence of metastatic lymph nodes (P = .045) between sporadic cases with preserved MMR and Lynch candidates. However, difference in the survival rates between sporadic cases and Lynch candidates was not significant. Immunohistochemical staining for MMR is a practical method for predicting MSI phenotype as well as Lynch candidates. MMR expression status was found to be associated with certain clinicopathological features some of which also have prognostic significance.

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

ABSTRACT 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.

MMR status of colorectal carcinomas at a tertiary cancer care centre in Central India

Microsatellite instability (MSI) and mismatch repair (MMR) deficiency are important biomarkers in colorectal cancer (CRC) that have clinical implications for patient management and treatment. MMR deficiency can lead to MSI, which is characterized by alterations in the length of microsatellite DNA sequences. Clinical applications of MMR deficiency in colorectal adenocarcinoma are as a prognostic marker as well as Predictive marker for adjuvant chemotherapy and immunotherapy. To assess the MMR status of colorectal adenocarcinomas.: This was a retrospective study of 127 colorectal adenocarcinomas cases. All the samples were either biopsies or surgically resected tumor tissues from patients of Carcinoma Colon and rectum. Immunohistochemistry was performed on Roche Ventana Benchmark XT autostainer.: MLH-1, PMS2, MSH2 and MSH6 expression was retained in the tumor cells in 106 cases and it was lost in 21 cases. Among various clinicopathological variables, tumor site and AJCC pStage were found to be significantly associated with dMMR tumors. Patients of colorectal adenocarcinomas were benefited by MMR testing as it influenced the adjuvant treatment in all Stage IIA cases as well as all metastatic cases. Incorporating MMR testing into routine clinical practice can help optimize patient management and treatment strategies in CRC.

Analysis of clinical characteristics of mismatch repair status in colorectal cancer: a multicenter retrospective study

BackgroundMicrosatellite instability (MSI) caused by DNA mismatch repair (MMR) deficiency is of great significance in the occurrence, diagnosis and treatment of colorectal cancer (CRC).AimThis study aimed to analyze the relationship between mismatch repair status and clinical characteristics of CRC.MethodsThe histopathological results and clinical characteristics of 2029 patients who suffered from CRC and underwent surgery at two centers from 2018 to 2020 were determined. After screening the importance of clinical characteristics through machine learning algorithms, the patients were divided into deficient mismatch repair (dMMR) and proficient mismatch repair (pMMR) groups based on the immunohistochemistry results and the clinical feature data between the two groups were observed by statistical methods.ResultsThe dMMR and pMMR groups had significant differences in histologic type, TNM stage, maximum tumor diameter, lymph node metastasis, differentiation grade, gross appearance, and vascular invasion. There were significant differences between the MLH1 groups in age, histologic type, TNM stage, lymph node metastasis, tumor location, and depth of invasion. The MSH2 groups were significantly different in age. The MSH6 groups had significant differences in age, histologic type, and TNM stage. There were significant differences between the PMS2 groups in lymph node metastasis and tumor location. CRC was dominated by MLH1 and PMS2 combined expression loss (41.77%). There was a positive correlation between MLH1 and MSH2 and between MSH6 and PMS2 as well.ConclusionsThe proportion of mucinous adenocarcinoma, protruding type, and poor differentiation is relatively high in dMMR CRCs, but lymph node metastasis is rare. It is worth noting that the expression of MMR protein has different prognostic significance in different stages of CRC disease.

Colombian consensus for the molecular diagnosis of endometrial cancer

Objective: The Cancer Genome Atlas research program (TCGA) developed the molecular classification for endometrial cancer with prognostic and therapeutic utility, which was replaced by the ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer) classification by consensus and international guidelines due to its high cost. This article aims to present national recommendations from an expert consensus that allows unification and implementation of the molecular classification for women with endometrial cancer nationwide, with a rational use of resources and technology. Methods: Consensus of 36 experts in clinical oncology, oncological gynecology, pathology, and genetics, with clinical practice in the national territory. The leader group performed a literature review and structuring of questions rated 1 to 9 points. A modified nominal group technique was used. There was a face-to-face meeting with master presentations, deliberative dialogue, and Google Forms (Google LLC, Mountain View, CA, USA) questionnaire voting with analysis and discussion of responses. The non-consensual responses led to a second round of voting. The final manuscript was finally prepared and revised. Results: Seven recommendations were formulated integrating the panelist responses based on evidence, but adjusted to the Colombian context and reality. Recommendation 1. The molecular classification is recommended in all the endometrial cancers using the immunohistochemistry markers as subrogated results from the molecular profile initially proposed in the TCGA classification. Recommendation 2. The sequential test strategy is recommended, starting with the immunohistochemistry markers (p53, MLH1, MSH 2, MSH6, PMS2) simultaneously in all the patients, defining to request POLE (DNA polymerase epsilon) (if available) according to the risk classification based on the surgical piece. Recommendation 3. It is recommended, that the gynecologist oncologist should be the one to request the POLE (if available) according to the final pathology report. This test must be requested for all endometrial cancers stage I-II, except in low risk (stage IA low grade endometrioid histology without linfovascular invasion normal p53) and, stages III-IV without residual disease, without affecting the request of subrogated immunohistochemistry molecular markers upon histology. The consensus proposes that the POLE is requested after the immunohistochemistry and according to the categories in the risk classification established by the 2020 ESGO/ESTRO/ESP guidelines. Recommendation 4. It is recommended to perform immunohistochemistry for hormonal receptors for all women with endometrial cancer and the HER2 in patients with p53abn, simultaneously with the others immunohistochemistry markers. Recommendation 5. It is recommended to perform the immunohistochemistry markers (p53, MLH1, MSH2, MSH6 y PMS2) in an initial endometrial biopsy or curettage when the specimen is adequate and available. In case the initial immunohistochemistry is inconclusive, or there are histological discrepancies between the initial and definitive pathology, it is recommended to repeat the molecular profile in the surgical pathology. The immunohistochemistry markers must be reported in the pathology report according to the CAP (College of American Pathologists) recommendations, independently of the type of sample. Recommendation 6. It is recommended to perform MLH1 promoter methylation testing in patients who exhibit loss of expression of MLH1 in immunohistochemistry whether it is accompanied or not with loss of expression of PMS2. All the patients with deficient MMR (mismatch repair), should be sent for genetic counseling to rule out Lynch syndrome. Recommendation 7. It is recommended to consider the molecular classification in addition to the classical histopathological criteria when making adjuvant judgments, as incorporated by the classification of prognostic groups of the 2020 ESGO/ESTRO/ESP guidelines. Conclusions: It is necessary to implement the molecular classification of endometrial cancer in clinical practice in accordance to the Colombian context, due to its prognostic and probably predictive value. This will enable the characterization of the Colombian population in order to offer individualized guided treatments. This is an academic and nonregulatory document.

Erk Inhibition as a Promising Therapeutic Strategy for High IL-8-Secreting and Low SPTAN1-Expressing Colorectal Cancer.

Tumor recurrence and drug resistance are responsible for poor prognosis in colorectal cancer (CRC). DNA mismatch repair (MMR) deficiency or elevated interleukin-8 (IL-8) levels are characteristics of CRCs, which have been independently correlated with treatment resistance to common therapies. We recently demonstrated significantly impaired therapeutical response and increased IL-8 release of CRC cell lines with reduced expression of MMR protein MLH1 as well as cytoskeletal non-erythrocytic spectrin alpha II (SPTAN1). In the present study, decreased intratumoral MLH1 and SPTAN1 expression in CRCs could be significantly correlated with enhanced serum IL-8. Furthermore, using stably reduced SPTAN1-expressing SW480, SW620 or HT-29 cell lines, the RAS-mediated RAF/MEK/ERK pathway was analyzed. Here, a close connection between low SPTAN1 expression, increased IL-8 secretion, enhanced extracellular-signal-regulated kinase (ERK) phosphorylation and a mesenchymal phenotype were detected. The inhibition of ERK by U0126 led to a significant reduction in IL-8 secretion, and the combination therapy of U0126 with FOLFOX optimizes the response of corresponding cancer cell lines. Therefore, we hypothesize that the combination therapy of FOLFOX and U0126 may have great potential to improve drug efficacy on this subgroup of CRCs, showing decreased MLH1 and SPTAN1 accompanied with high serum IL-8 in affected patients.

O09 Microsatellite-unstable sporadic sebaceous and duodenal tumours are associated with loss of mismatch protein expression

Abstract Introduction and aims Lynch syndrome (LS) is a common heritable cancer predisposition syndrome. Tumours from patients with LS demonstrate microsatellite instability (MSI-high), a genetic signature detectable by low-cost DNA sequencing assays that is currently utilized in colorectal cancer screening. The utility of MSI assays for LS detection has not been extensively studied in unselected sebaceous skin tumours or duodenal tumours, where currently immunohistochemical (IHC) expression of mismatch repair (MMR) proteins is used. In addition, it has recently been suggested that cutaneous squamous cell carcinoma (cSCC) could be linked to LS; however MSI status has not been studied in patients with early-onset cSCC. Methods We investigated MSI status in archival formalin-fixed paraffin-embedded cases with ethical approval. We examined 110 sebaceous tumours (STs) (45 female patients, 65 male patients; median age 77 years), 50 duodenal tumours (DTs) (28 female patients, 22 male patients; median age 64 years), and 148 cSCCs selected from patients under 50 years of age (69 female patients, 79 male patients; median age 49 years). Immunohistochemistry (IHC) was performed on each MSI-high sample to determine the expression of MMR proteins MLH1, MSH2, MSH6 and PMS2. Samples that passed quality control were included in the analysis. Results A total of 42 of 106 STs (39.6%) were MSI-high. All STs had loss of at least one MMR protein. Overall, 10 of 41 (24%) DTs were MSI-high. All DTs had loss of at least one MMR protein. We found that 12 of 148 (8.1%) cSCCs were MSI-high and 5 of 12 SCCs exhibited loss of at least one MMR protein expression (41%). Conclusions MSI-high status correlated well with loss of MMR protein expression in STs and DTs samples. Future studies will help establish MSI status in tumours initially selected by MMR loss on IHC, informing its use as an adjunct to MMR IHC in LS detection in patients with STs and DTs.

A Prospective Study to Evaluate the Prevalence of Microsatellite Instability in Endometrial Carcinoma by using Immunohistochemistry for Mismatch Repair Proteins as a Surrogate Marker

Abstract Aim Use of immunohistochemistry for mismatch repair (MMR) proteins to identify the prevalence of microsatellite instability (MSI) in cases of endometrial carcinoma and its subsequent correlation with various histopathological parameters. Materials and Methods The expression of MMR proteins, viz PMS2, MLH1, MSH2, and MSH6, were assessed in 114 endometrial cancer cases by immunohistochemistry using Dako EnVision FLEX system, on paraffin blocks of tumor tissue fixed in 10% formalin. Results We studied 114 endometrial cases for MMR protein expression, of which the majority were of endometrioid histologic subtype (n = 93, 81.6%), whereas the remainder comprised serous carcinoma (n = 12, 10.5%), clear cell carcinoma (n = 1, 0.9%), carcinosarcoma (n = 5, 4.4%), and dedifferentiated uterine carcinoma (n = 3, 2.6%). Twenty-one (18%) of these cases were found to be deficient for MMR proteins, of which 20 were of endometrioid histologic subtype and only 1 was dedifferentiated uterine carcinoma. Loss of MMR protein expression occurred in pairs of either PMS2 and MLH1 or MSH2 and MSH6. Conclusion MSI is one of the major molecular pathways contributing to tumorigenesis in endometrial carcinomas. Immunohistochemistry for MMR proteins is a highly sensitive and cost-effective alternative for molecular testing for MSI. It is also a great tool for screening patients for Lynch syndrome. Immunohistochemical testing for MMR should be offered to all patients of endometrial cancers.

Investigation of sperm hsa-mir-145-5p and MLH1 expressions, seminal oxidative stress and sperm DNA fragmentation in varicocele.

Mechanisms by which varicocele causes infertility are not clear and few studies have reported that some miRNAs show expression alterations in men with varicocele. Recently, sperm promoter methylation of MLH1 has been shown to be higher in men diagnosed with varicocele. This study aimed to assess the potential effects of miR-145, which was determined to target MLH1 mRNA in silico on sperm quality and function in varicocele. Sperm miR-145 and MLH1 expressions of six infertile men with varicocele (Group 1), nine idiopathic infertile men (Group 2), and nine fertile men (control group) were analyzed by quantitative PCR. Sperm DNA fragmentation was evaluated by TUNEL and the levels of seminal oxidative damage and total antioxidant capacity were analyzed by ELISA. Our results have shown that sperm expression of miR-145 was decreased in Group 1 compared to Group 2 (P = 0.029). MLH1 expression was significantly higher in Group 2 than the controls (P = 0.048). Total antioxidant level and sperm DNA fragmentations of Group 1 and Group 2 were decreased (P = 0.001 and P = 0.011, respectively). Total antioxidant capacity was positively correlated with sperm concentration (ρ = 0.475, P = 0.019), total sperm count (ρ = 0.427, P = 0.037), motility (ρ = 0.716, P < 0.0001) and normal morphological forms (ρ = 0.613, P = 0.001) and negatively correlated with the seminal oxidative damage (ρ=-0.829, P = 0.042) in varicocele patients. This is the first study investigating the expressions of sperm miR-145 and MLH1 in varicocele patients. Further studies are needed to clarify the potential effect of miR-145 on male fertility.

Histone H3K9 Lactylation Confers Temozolomide Resistance in Glioblastoma via LUC7L2-Mediated MLH1 Intron Retention.

Temozolomide (TMZ) resistance remains the major obstacle in the treatment of glioblastoma (GBM). Lactylation is a novel post-translational modification that is involved in various tumors. However, whether lactylation plays a role in GBM TMZ resistance remains unclear. Here it is found that histone H3K9 lactylation (H3K9la) confers TMZ resistance in GBM via LUC7L2-mediated intron 7 retention of MLH1. Mechanistically, lactylation is upregulated in recurrent GBM tissues and TMZ-resistant cells, and is mainly concentrated in histone H3K9. Combined multi-omics analysis, including CUT&Tag, SLAM-seq, and RNA-seq, reveals that H3K9 lactylation is significantly enriched in the LUC7L2 promoter and activates LUC7L2 transcription to promote its expression. LUC7L2 mediates intron 7 retention of MLH1 to reduce MLH1 expression, and thereby inhibit mismatch repair (MMR), ultimately leading to GBM TMZ resistance. Of note, it is identified that a clinical anti-epileptic drug, stiripentol, which can cross the blood-brain barrier and inhibit lactate dehydrogenase A/B (LDHA/B) activity, acts as a lactylation inhibitor and renders GBM cells more sensitive to TMZ in vitro and in vivo. These findings not only shed light on the mechanism of lactylation in GBM TMZ resistance but also provide a potential combined therapeutic strategy for clinical GBM treatment.

Aberrant PRDM2 methylation as an early event in serrated lesions destined to evolve into microsatellite-instable colorectal cancers.

Up to 30% of colorectal cancers (CRCs) develop from sessile serrated lesions (SSLs). Within the serrated neoplasia pathway, at least two principally distinct oncogenetic routes exist generating microsatellite-stable and microsatellite-instable CRCs, respectively. Aberrant DNA methylation (DNAm) is found early in the serrated pathway and might play a role in both oncogenetic routes. We studied a cohort of 23 SSLs with a small focus (<10 mm) of dysplasia or cancer, 10 of which were MLH1 deficient and 13 MLH1 proficient. By comparing, for each SSL, the methylation status of (1) the region of dysplasia or cancer (SSL-D), (2) the nondysplastic SSL (SSL), and (3) adjacent normal mucosa, differentially methylated probes (DMPs) and regions (DMRs) were assessed both genome-wide as well as in a tumor-suppressor gene-focused approach. By comparing DNAm of MLH1-deficient SSL-Ds with their corresponding SSLs, we identified five DMRs, including those annotating for PRDM2 and, not unexpectedly, MLH1. PRDM2 gene promotor methylation was associated with MLH1 expression status, as it was largely hypermethylated in MLH1-deficient SSL-Ds and hypomethylated in MLH1-proficient SSL-Ds. Significantly increased DNAm levels of PRDM2 and MLH1, in particular at 'critical' MLH1 probe sites, were to some extent already visible in SSLs as compared to normal mucosa (p = 0.02, p = 0.01, p < 0.0001, respectively). No DMRs, nor DMPs, were identified for SSLs destined to evolve into MLH1-proficient SSL-Ds. Our data indicate that, within both arms of the serrated CRC pathway, the majority of the epigenetic alterations are introduced early during SSL formation. Promoter hypermethylation of PRDM2 and MLH1 on the other hand specifically initiates in SSLs destined to transform into MLH1-deficient CRCs suggesting that the fate of SSLs may not necessarily result from a stochastic process but possibly is already imprinted and predisposed.