MSH2 Expression Research Articles

Value of histopathology in predicting microsatellite instability in colorectal cancer

Background: Identifying colorectal cancers (CRCs) with high levels of microsatellite instability (MSI-H) is clinically important. Few studies reported the role of histology in identifying MSI-H colorectal carcinomas. Hence the present study was undertaken to assess the value of histopathology in predicting MSI-H and its correlation with histopathologic prognostic features in CRC. Methods: Total 100 histo-pathologically confirmed cases of CRC were enrolled in the study. Tumour’s histopathological typing, staging, and sizing was conducted based on WHO criteria. Tumour grade, extent of mucin production, tumour growth pattern and presence of a Crohn’s like inflammatory infiltrate were determined. For immunohistochemistry (IHC) we used monoclonal antibodies (ES05 or 25D12). Results: Among 100 tumour samples, loss of MLH1 or MSH2 expression was detected in 13(13%) cases (MSI tumours) while 87(87%) tumours showed normal expression for MLH1 and MSH2 immuno-reactivity (MSS tumours). In terms of MSI/MSS status of tumour samples, statistical analysis showed that abnormal MMR protein expression was associated with tumour site, side of colon with cancer, size of tumour, tumour infiltrating lymphocytes and crohn’s like inflammatory infiltrate. Some histopathological and clinical features that appeared highly specific but much less sensitive in predicting MSI include stage, and some features were more sensitive and less specific which include tumour differentiation, CLR, TIL, dirty necrosis and pushing margin. Conclusion: MSI is an important prognostic factor in CRC and an important predictive factor of CRC chemotherapeutic treatment and outcome efficacy. Also result of the study is not consistent in describing various histopathological and clinical features in predicting MSI, CRC.

P-84 Genotype-fenotype correlation in patients with Lynch syndrome and colorectal cancer

Lynch syndrome is a hereditary condition predisposing to cancer defined by a germline mutation in one of several DNA mismatch repair (MMR) genes or loss of expression of MSH2 due to deletion in the EPCAM gene. This condition causes approximately 3% of colorectal cancers (CCR) and 2% of endometrial cancers (CE). In this study, we aim to characterize the germline mutations and clinical characteristics of patients diagnosed with Lynch syndrome at a tertiary hospital in Madrid (Spain).

MSH2-deficient prostate tumours have a distinct immune response and clinical outcome compared to MSH2-deficient colorectal or endometrial cancer.

Recent publications have shown patients with defects in the DNA mismatch repair (MMR) pathway driven by either MSH2 or MSH6 loss experience a significant increase in the incidence of prostate cancer. Moreover, this increased incidence of prostate cancer is accompanied by rapid disease progression and poor clinical outcomes. We show that androgen-receptor activation, a key driver of prostate carcinogenesis, can disrupt the MSH2 gene in prostate cancer. We screened tumours from two cohorts (recurrent/non-recurrent) of prostate cancer patients to confirm the loss of MSH2 protein expression and identified decreased MSH2 expression in recurrent cases. Stratifying the independent TCGA prostate cancer cohort for MSH2/6 expression revealed that patients with lower levels of MSH2/6 had significant worse outcomes, in contrast, endometrial and colorectal cancer patients with lower MSH2/6 levels. MMRd endometrial and colorectal tumours showed the expected increase in mutational burden, microsatellite instability and enhanced immune cell mobilisation but this was not evident in prostate tumours. We have shown that loss or reduced levels of MSH2/MSH6 protein in prostate cancer is associated with poor outcome. However, our data indicate that this is not associated with a statistically significant increase in mutational burden, microsatellite instability or immune cell mobilisation in a cohort of primary prostate cancers.

WRN Germline Mutation Is the Likely Inherited Etiology of Various Cancer Types in One Iranian Family.

BackgroundFamilial cancers comprise a considerable distribution of colorectal cancers (CRCs), of which only about 5% occurs through well-established hereditary syndromes. It has been demonstrated that deleterious variants at the newly identified cancer-predisposing genes could describe the etiology of undefined familial cancers.MethodsThe present study aimed to identify the genetic etiology in a 32-year-old man with early onset familial CRC employing several molecular diagnostic techniques. DNA was extracted from tumoral and normal formalin-fixed-paraffin-embedded (FFPE) blocks, and microsatellite instability (MSI) was evaluated. Immunohistochemistry staining of MMR proteins was performed on tumoral FFPE blocks. Next-generation sequencing (NGS), multiplex ligation-dependent amplification (MLPA) assay, and Sanger sequencing were applied on the genomic DNA extracted from peripheral blood. Data analysis was performed using bioinformatics tools. Genetic variants interpretation was based on ACMG.ResultsMSI analysis indicated MSI-H phenotype, and IHC staining proved no expressions of MSH2 and MSH6 proteins. MLPA and NGS data showed no pathogenic variants in MMR genes. Further analysis of NGS data revealed a candidate WRN frameshift variant (p.R389Efs*3), which was validated with Sanger sequencing. The variant was interpreted as pathogenic since it met the criteria based on the ACMG guideline including very strong (PVS1), strong (PS3), and moderate (PM2).Conclusion WRN is a DNA helicase participating in DNA repair pathways to sustain genomic stability. WRN deficient function may contribute to CRC development that is valuable for further investigation as a candidate gene in hereditary cancer syndrome diagnosis.

Atorvastatin Enhances Inhibitory Effects of Irradiation on Tumor Growth by Reducing MSH2 Expression Both in Prostate Cancer Cells and Xenograft Tumor Models.

Prostate cancer (PCa) is the fourth most common tumor in males. This study aimed to investigate effects of atorvastatin (AS) on PCa cells proliferation and clarify the associated mechanisms. PCa cell lines were cultured and treated with irradiation (IR) (4 Gy), AS (6 μg/ml), transfected with Bcl-2 siRNA, and then divided into different groups. Xenograft tumor mouse model was established. Bcl-2 and MSH2 gene transcription and protein expression were evaluated using RT-PCR assay and western blot assay. Plate clone formation assay was employed to examine colony formation. MTT assay was used to detect cell viabilities. Flow cytometry analysis was utilized to verify apoptosis. Co-immunoprecipitation and immuno-fluorescence assay were used to identify interaction between Bcl-2 and MSH2. IR significantly reduced colony formation, enhanced Bcl-2 and reduced MSH2 gene transcription in PCa cells compared to un-treated cells (p<0.05). AS significantly strengthened radio-therapeutic effects of IR on colony formation, decreased cell apoptosis and increased Bcl-2 gene transcription/protein expression in PCa cells compared to single IR treatment cells (p<0.05). AS combining IR down-regulated MSH2 gene transcription/protein expression in PCa cells compared to single IR treatment cells (p<0.05). Bcl-2 interacted with MSH2 both in PCa cells and tumor tissues administrating with AS. AS enhanced reductive effects of IR on tumor size of Xenograft tumor mice. Atorvastatin administration enhanced inhibitory effects of IR either on PCa cells or tumor size of Xenograft tumor mice. The inhibitory effects of atorvastatin were mediated by reducing MSH2 expression and triggering interaction between Bcl-2 and MSH2, both in vitro and in vivo levels.

CD63 and Dna Mismatch Repair Protein Expression in Prostate Cancer

Abstract Protein expression levels in immunohistochemistry and molecular biomarkers have been reported for their ability to predict recurrence, progression, development of metastases, and patient survival. The molecular features in low- and high-grade prostate cancer can differ and influence treatment decision and prognosis. The objective of the current study was to compare the expression of exosomal biomarkers CD63 and mismatch repair proteins (MSH2, MSH6, MLH1, and PMS2) by immunohistochemistry (IHC) in tissue of patients with prostate cancer and benign hyperplasia. Altogether, 62 patients with prostate acinar adenocarcinoma and 20 patients with prostate benign hyperplasia were enrolled in this retrospective study. CD63, MSH2, MSH6, MLH1, and PMS2 expression was analysed by immunohistochemistry. The obtained results showed that CD63 expression was significantly higher in patients with Grade III–V prostate cancer compared to Grade I–II, respectively; 2.23 (1–3) vs 0.92 (0–2) score, p = 0.001. In addition, a significant positive correlation between CD63 expression and grade groups was revealed (Rho = +0.54; p &lt; 0.0001). Furthermore, progression-free survival was significantly higher in patients with low CD63 expression, compared to high CD63 expression (p = 0.0007). MMR expression was absent in 14 patients (four patients with Grade I–II cancer and 10 patients with Grade III–cancer). MMR was present in all cases of benign prostate hyperplasia (mild to moderate staining). The conclusion was that high grade prostate cancer (Grade groups III–V) was characterised by increased CD63 expression, which correlated with progression-free survival.

Metastatic colorectal carcinoma to the right atrium: a case report and review of the literature

BackgroundCardiac metastasis due to colon cancer is extraordinarily uncommon. Given the rarity of diagnosis, there is paucity of evidence and hence, no established guidelines for evaluation or clinical management strategy.Clinical presentationWe present the case of a 59 year old male with a previously treated colonic carcinoma who presented with new onset exertional dyspnea. He was noted to be having a right atrial mass on an echocardiogram performed at his cardiologist’s office. Further workup with CT angiogram of the chest confirmed a right atrial mass measuring 4.0 cm. Serum CEA was normal. Biopsies of the right atrial mass demonstrated metastatic moderately differentiated colonic adenocarcinoma. Mismatch repair protein expression analysis by immunohistochemistry showed no loss of MLH1, MSH2, MSH6 or PMS2 expression. Next generation sequencing for RAS and BRAF mutations was negative. Patient received treatment with FOLFIRINOX/ bevacizumab with noted reduction in size of mass.ConclusionTo the best of our knowledge, this is the first report of next generation sequencing results available on a biopsy of metastatic colorectal cancer to the heart with the largest literature review of 31 reported cases of metastatic colorectal cancer to the heart. It will help direct clinical management and also adds evidence to the potential efficacy of treatment of this rare aggressive disease with chemotherapy in combination with VEGF inhibitors.

Phenotype-Genotype Correlation in Colorectal Cancer: A Real-Life Study

Background and Aims: Colorectal cancer (CRC) is a heterogeneous disease with distinctive genetic pathways, such as chromosomal instability, microsatellite instability and methylator pathway. Our aim was to correlate clinical and genetic characteristics of CRC patients in order to understand clinical implications of tumour genotype. Methods: Single-institution retrospective cohort of patients who underwent curative surgery for CRC, from 2012 to 2014. RAS and BRAF mutations were evaluated with the real-time PCR technique Idylla®. Mismatch repair deficiency (dMMR) was characterized by absence of MLH1, MSH6, MSH2 and/or PMS2 expression, evaluated by tissue microarrays. Overall survival (OS) and disease-free survival (DFS) were assessed using survival analysis. Results: Overall, 242 patients were included (males 57.4%, age 69.3 ± 12.9 years; median follow-up 49 months). RAS-mutated tumours were associated with reduced DFS (p = 0.02) and OS (p = 0.045) in stage I–III CRC. BRAF-mutated tumours were more predominant in females and in the right colon, similarly to dMMR tumours. BRAF status did not influence OS (4 years)/DFS (3.5 years) in stage I–III disease. However, after relapse, length of survival was 3.5 months in BRAF-mutated tumours in contrast to 18.6 months in BRAF wild-type tumours (p = NS). No germline mutations in mismatch repair genes were so far identified in the patients with dMMR tumours. Molecular phenotype (RAS, BRAF and MMR) did not influence OS in metastatic patients. Our small sample size may be a limitation of the study. Conclusion: In our cohort, RAS-mutated tumours were associated with worse DFS and OS in early-stage CRC, whereas the remaining molecular variables had no prognostic influence.

Expression of Mismatch Repair Proteins in Merkel Cell Carcinoma.

Simple SummaryMerkel cell carcinoma (MCC) is a rare and highly malignant skin cancer with neuroendocrine differentiation. About 80% are Merkel cell polyomavirus (MCPyV) positive. The aim of this work was to immunohistochemically investigate the expression of mismatch repair proteins (MSH2, MSH6, MLH1, and PMS2) in MCC (n = 56). In a second step, tumors with a low expression were tested for microsatellite instability. Microsatellite instability in MCC could have an impact on immune checkpoint inhibitor therapy (ICI) outcome. This study showed a significant association between low expression of mismatch repair proteins and a negative MCPyV status. Microsatellite instability was detected in only one case. Future studies will establish whether this subset of MCC patients respond better to ICI.We aimed to assess for the first time the mismatch repair (MMR) protein expression in Merkel cell carcinoma (MCC). Immunohistochemistry was performed for MLH1, MSH2, MSH6, and PMS2 on patients’ tumor tissue (n = 56), including neighbored healthy control tissue. In cases with low-level MMR expression (<10th percentile), we performed multiplex PCR in combination with high-resolution capillary electrophoresis in order to confirm microsatellite instability (MSI). Microscopic evaluation revealed a high median expression for all MMR proteins studied (91.6–96.3%). However, six patients (56/10.7%) had low-level MLH1 expression, six (55/10.9%) had low-level MSH2 expression, five (56/8.9%) had low-level MSH6 expression, and six (54/11.1%) had low-level PMS2 expression. Together, we observed nine (56/16.1%) patients who had low-level MMR expression of at least one protein. Of the patients with low-level MMR expression, MSI evaluation was possible in five cases, revealing one case with high-level MSI. In all MMR proteins assessed, low-level expression was significantly (p = 0.0004 to p < 0.0001) associated with a negative Merkel cell polyomavirus (MCPyV) status. However, the expression profiles of the MMR proteins did not correlate with clinical outcome measures such as disease relapse or death (p > 0.05). MCC appears to be a malignancy characterized by low-level MMR rather than completely deficient MMR in a subset of cases, predominantly affecting MCPyV-negative tumors. Future studies will establish whether this subset of MCC patients respond better to immune checkpoint inhibitor therapy.

Prevalence of NTRK1/3 fusions in mismatch repair-deficient (dMMR)/microsatellite instable (MSI) tumors of patients with metastatic colorectal cancer (mCRC).

e15537 Background: Inhibitors of tropomyosin receptor kinase (TRK) have shown promising activity against neurotrophic TRK ( NTRK) fusion-driven cancers, regardless of tumor histotype or cell of origin. NTRK gene fusions are observed in less than 1% of colorectal cancers (CRCs). CRCs harboring wild-type BRAF and KRAS and MisMatch Repair deficiency (dMMR)/MicroSatellite Instability (MSI) due to MLH1 hypermethylation have been associated with NTRK fusions in small cohorts of non-metastatic tumors. We aimed at evaluating the incidence of NTRK fusions among dMMR/MSI metastatic CRCs (mCRC) for which there is a need for innovative therapies, as well as the associated clinical characteristics of these patients (pts) carrying NTRK fusion-positive tumors. Methods: Tumor samples of dMMR/MSI mCRC pts, paired primary and metastasis or primary alone, were obtained from a French multicenter retrospective cohort and from a single-center cohort of patients treated by immune checkpoint inhibitors (ICI) (Saint-Antoine Hospital, Paris). Clinico-pathological data including KRAS and BRAFV600E status, MMR proteins and MLH1 methylation status were available for all pts. All samples were screened for TRK expression by immunohistochemistry (IHC) using a pan-TRK antibody (clone EPR17341, Abcam; positivity: 1% of labeled tumor cells)) and for NTRK1 and NTRK3 gene rearrangements, by fluorescent in situ hybridation (FISH). A threshold of 15% nuclei positive for a break apart signal was considered positive for gene rearrangement. Results: A total of 158 pts with dMMR/MSI mCRCs (paired samples: n=39; primary only: n=119) were screened. Tumor samples of 10 patients (6.3%) harbored NTRK fusion genes by FISH ( NTRK1=8; NTRK3=2). Only four of these 10 patients had TRK immunoreactivity. One patient showed a discordance between metastasis harboring NTRK1 fusion (+) and primary tumor being negative. Eight tumors were sporadic with MLH1 hypermethylation. The remaining 2 cases were related to a MMR gene germline mutation (Lynch syndrome) with concurrent loss of MSH2 and MSH6 expression and isolated loss of MSH6 respectively. One Lynch-related tumor was KRAS mutated, one sporadic MLH1-negative tumor was BRAF V600E mutated. Four patients out of 91 treated by ICI had tumors with NTRK fusions. Three have shown radiological response according to iRECIST criteria (two complete responses, one partial response with 25 to 54 months of follow up) and one had primary resistance to ICI. Conclusions: Frequency of NTRK1/3 fusions is 6.3% in our dMMR/MSI mCRCs population. These fusions are not restricted to sporadic cases. The diagnostic accuracy of pan-TRK IHC is low. Optimal testing algorithms for theragnostics remain to be defined in this setting.

Cytoplasmic MSH2 Related to Genomic Deletions in the MSH2/EPCAM Genes in Colorectal Cancer Patients With Suspected Lynch Syndrome.

BackgroundA large proportion of patients with Lynch syndrome (LS) have MSH2 abnormalities, but genotype-phenotype studies of MSH2 mutations in LS are still lacking. The aim of this study was to comprehensively analyze the clinicopathological characteristics and molecular basis of colorectal cancer (CRC) in patients with uncommon MSH2 cytoplasmic expression.MethodsWe retrospectively reviewed 4195 consecutive cases of CRC patients diagnosed between January 2015 and December 2017 at the Cancer Hospital Chinese Academy of Medical Sciences. Of the 4195 patients with CRC, 69 were indicated to have abnormal MSH2 expression through tumor immunohistochemical staining. Genetic tests, such as next-generation sequencing, large genomic rearrangement (LGR) analysis, microsatellite instability status analysis and genomic breakpoint analysis, were performed. Clinicopathological and molecular characteristics and clinical immunotherapy response were analyzed.ResultsForty-five of 69 patients were identified to have LS with pathogenic germline mutations in MSH2 and/or EPCAM. Of these LS patients, 26.7% were confirmed to harbor large genomic rearrangements (LGRs). Of note, three tumors from two unrelated family pedigrees exhibited a rare cytoplasmic MSH2 staining pattern that was found in LS patients with EPCAM/MSH2 deletions. RNA analysis showed that two novel mRNA fusions of EPCAM and MSH2 resulted in the predicted protein fusion with MSH2 cytoplasmic localization. Analyses of genomic breakpoints indicated that two novel deletions of EPCAM and MSH2 originated from Alu repeat-mediated recombination events. Our study also provides clinical evidence for the beneficial effect of the PD-1 inhibitor pembrolizumab for CRC patients that exhibit cytoplasmic MSH2 staining.ConclusionOur study demonstrates that the rare cytoplasmic MSH2 staining pattern should be fully recognized by pathologists and geneticists. Given the specific genotype-phenotype correlation in LS screening, we advocate that all CRC patients with cytoplasmic MSH2 staining in histology should be screened for LGRs of EPCAM and MSH2.

A Co-Occurrence of Familial Non-Medullary Thyroid Cancer and Lynch Syndrome

Abstract Background: Lynch syndrome (LS) is an autosomal dominant disease caused by germline mutations in mismatch repair genes (MMR), leading to the early manifestation of tumors characterized by microsatellite instability (MSI) in &amp;gt;3 family members across at least 2 generations. MSI is a rare event in thyroid cancer (TC), occurring in up to 2.5% of sporadic cases. There is limited data on germline MMR variants’ role in familial non-medullary thyroid cancer (FNMTC). The goal of this study was to analyze the potential clinical and molecular association between LS and FNMTC. Material and Methods: We performed a cohort study analyzing the demographic, clinical, and pathologic data of 43 kindreds with FNMTC. We performed a high-throughput whole exome sequencing (WES) of peripheral-blood DNA samples of 168 participants (54 affected by FNMTC and 140 unaffected). The GATK pipeline was used in variant analysis. The NIH Institutional Review Board approved the study. Results: The study included 383 family members (104 affected, 279 unaffected) aged 43.5 [7-99] years, with 2-9 members per family affected by FNMTC. FNMTC was more prevalent in women (68.3%) and characterized by a median tumor size of 1 [0.2-5] cm, multifocal growth in 44%, gross extrathyroidal extension in 11.3%, central neck lymph node metastases in 40.3%, lateral neck lymph node metastases in 12.9% of patients, and no distant metastases. Family history screening revealed one family of Caucasian descent meeting the clinical criteria for FNMTC and LS diagnosis with 5 members affected by FNMTC and 8 individuals by Lynch-like tumors (3 with colorectal cancer/colon polyps, 2 with endometrial or ovarian tumors, 1 with kidney cancer, 1 with keratoacanthoma and 1 with unspecified Lynch-like tumors with detailed pathology report unavailable). We performed whole exome sequencing of 10 members from this family (3 affected and 7 unaffected) and remaining 158 study participants and detected exclusively in this family, a heterozygous missense variant rs373226409, in MSH2 gene c2120G&amp;gt;A (pCys707Tyr) in three adults affected by LS-like manifestations and two unaffected children under the age of 18 with clear segregation across three generations. This variant appears to be relatively rare with a minor allele frequency (MAF) of 0.0006 in Caucasians; however, it is more common in the South Asian population at 0.003 MAF. Immunostaining performed on the TC tumor tissue of one of the affected family members revealed intact nuclear expression of MSH2, suggestive of no major effect of the variant on MSH2 expression. Five out of seven in-silico models predicted the variant to be functionally deleterious. Conclusion: The co-occurrence of LS and FNMTC is a rare event, presenting in 2% (1/43) of families in our cohort. A common genetic association between LS and FNMTC has not been identified, and the MSH2 variant observed in this family is unlikely to be an etiologic factor.

DNA methylation status of MutS genes in ameloblastoma.

Ameloblastoma is an odontogenic epithelial tumour with a low expression of mismatch repair system components. We aimed to investigate the methylation status of the genes MSH2, MSH3 and MSH6 (MutS group) in conventional ameloblastomas. The ameloblastoma and dental follicle samples (n=10 each) were collected from 20 different patients. Each ameloblastoma sample was sectioned into two fragments: one was paraffin-embedded while the other one, likewise the dental follicle samples, was fixed in RNAlater and frozen at -196°C. All frozen samples were investigated for the MutS genes methylation levels, using the enzymatic restriction digestion and quantitative real-time PCR (qPCR) assay. The ameloblastoma paraffin-embedded samples were submitted to immunohistochemical reactions for MutS proteins detection and digitally quantification. Correlation analyses were performed between the immunohistochemical results and the respective gene methylation percentage. There are no significant differences between the MutS genes methylation levels in the ameloblastoma and the dental follicle. However, a strong negative correlation was found between MSH2 and MSH6 gene methylation status and their respective proteins expressions evaluated by immunohistochemistry. Our results show that the genes methylations is in part responsible for decreasing the expression of MSH2 and MSH6 genes in ameloblastoma.

CircLIFR synergizes with MSH2 to attenuate chemoresistance via MutS\u03b1/ATM-p73 axis in bladder cancer

BackgroundCisplatin (CDDP) has become a standard-of-care treatment for muscle-invasive bladder cancer (MIBC), while chemoresistance remains a major challenge. Accumulating evidence indicates that circular RNAs (circRNAs) are discrete functional entities. However, the regulatory functions as well as complexities of circRNAs in modulating CDDP-based chemotherapy in bladder cancer are yet to be well revealed.MethodsThrough analyzing the expression profile of circRNAs in bladder cancer tissues, RNA FISH, circRNA pull-down assay, mass spectrometry analysis and RIP, circLIFR was identified and its interaction with MSH2 was confirmed. The effects of circLIFR and MSH2 on CDDP-based chemotherapy were explored by flow cytometry and rescue experiments. Co-IP and Western blot were used to investigate the molecular mechanisms underlying the functions of circLIFR and MSH2. Biological implications of circLIFR and MSH2 in bladder cancer were implemented in tumor xenograft models and PDX models.ResultsCircLIFR was downregulated in bladder cancer and expression was positively correlated with favorable prognosis. Moreover, circLIFR synergizing with MSH2, which was a mediator of CDDP sensitivity in bladder cancer cells, positively modulated sensitivity to CDDP in vitro and in vivo. Mechanistically, circLIFR augmented the interaction between MutSα and ATM, ultimately contributing to stabilize p73, which triggered to apoptosis. Importantly, MIBC with high expression of circLIFR and MSH2 was more sensitive to CDDP-based chemotherapy in tumor xenograft models and PDX models.ConclusionsCircLIFR could interact with MSH2 to positively modulate CDDP-sensitivity through MutSα/ATM-p73 axis in bladder cancer. CircLIFR and MSH2 might be act as promising therapeutic targets for CDDP-resistant bladder cancer.

Mismatch repair protein expression of colorectal cancer: a retrospective analysis of 3 428 cases

Objective: To analyze the expression of mismatch repair (MMR) proteins in colorectal cancers (CRC) and to evaluate the feasibility and potential pitfalls of immunohistochemistry (IHC) analysis for MMR. Methods: The IHC sections for MMR proteins were reviewed in 3 428 cases of resected CRC without neoadjuvant therapy at Tianjin Medical University Cancer Institute and Hospital from July 2014 to October 2018. For the cases with unclear MMR IHC results during the initial review, IHC staining was repeated and microsatellite instability (MSI) analysis was performed. Relationships between the expression of MMR proteins and MSI status as well as the clinicopathological parameters were analyzed. Results: IHC staining for MMR was repeated in 28 (0.8%) cases due to poor quality of original IHC sections. Inconsistent results between the original diagnosis and re-diagnosis were found in 119 (3.5%) cases, mainly resulting from PMS2 and MLH1. Finally, 261 (7.6%) cases of CRC showed mismatch repair deficiency (dMMR), mainly from the deficiency of both MLH1 and PMS2 (43.3%,113/261). In the 14 cases with MSI results, the concordant of MSI and MMR was 13 cases. In the 29 dMMR cases with next generation sequencing (NGS) results, the concordant of MSI-high and dMMR was 93.1%(27/29). The cases with inconsistent results between MSI and MMR showed negative expression of MSH6 or PMS2. Twenty-one CRC showed negative expression of MLH1 and partially positive (or weak positive) expression of PMS2, or negative expression of MSH2 and partially positive (or weak positive) expression of MSH6. Among the 19 cases with MSI results, 16 cases were MSI-high, two cases were MSI-low, and one case was microsatellite stable. Compared with mismatch repair proficiency (pMMR), dMMR was more frequently detected in female patients younger than 50 years old, with family history, at early stage (Ⅰ-Ⅱ) CRC, and in the tumors from right colon,with poor differentiation, or mucinous adenocarcinoma/signet ring cell carcinoma (all P<0.05). Conclusions: At present, IHC staining is a clinically effective and convenient method to detect MMR expression, but the operating process and result assessment remain variable and need to be standardized. MSI analysis can be performed in the difficult-to-evaluate cases for MMR to enhance prognostic evaluation and treatment option.

Universal Screening for Lynch Syndrome in Uterine Cancer Patients: A Quality Improvement Initiative

(Abstracted from Gynecol Oncol 2021;160:169–174) The Society of Gynecologic Oncology (SGO) recommends systematic clinical screening for Lynch syndrome through immunohistochemistry (IHC) testing of tumors identified in patients younger than 60 years or of any age and a thorough review of personal and family history. Testing targets expression of mismatch repair genes MLH1, MLH2, PMS2, and MSH6 to evaluate microsatellite instability.

Histologic heterogeneity and syndromic associations of non-ampullary duodenal polyps and superficial mucosal lesions

BackgroundDuodenal polyps and superficial mucosal lesions (DP/SMLs) are poorly characterised. AimsTo describe a series of endoscopically-diagnosed extra-ampullary DPs/SMLs. MethodsThis is a retrospective study conducted in a tertiary referral Endoscopy Unit, including patients who had DPs or SMLs that were biopsied or removed in 2010–2019. Age, gender, history of familial polyposis syndromes, DP/SML characteristics were recorded. Histopathological, immunohistochemical and molecular analyses were performed. Results399 non-ampullary DP/SMLs from 345 patients (60.6% males; median age 67 years) were identified. Gastric foveolar metaplasia represented the most frequent histotype (193 cases, 48.4%), followed by duodenal adenomas (DAs; 77 cases, 19.3%). Most DAs (median size 6 mm) were sessile (Paris Is; 48%), intestinal-type (96.1%) with low-grade dysplasia (93.5%). Among syndromic DAs (23%), 15 lesions occurred in familial adenomatous polyposis 1, two were in MUTYH-associated polyposis and one was in Peutz-Jeghers syndrome (foveolar-type, p53-positive, low-grade dysplasia). Only one (3.3%) tubular, low-grade DA showed mismatch repair deficiency (combined loss of MLH1 and PMS2, heterogeneous MSH6 expression), and it was associated with a MLH1 gene germline mutation (Lynch syndrome). ConclusionDPs/SMLs are heterogeneous lesions, most of which showing foveolar metaplasia, followed by low-grade, intestinal-type, non-syndromic DAs. MMR-d testing may identify cases associated with Lynch syndrome.

DDRE-04. THE COMBINED TREATMENT OF L-ASPARAGINASE AND 6-DIAZO-5-OXO-L-NORLEUCINE INHIBIT THE PROLIFERATION OF TEMOZOLOMIDE-SENSITIVE OR RESISTANT GLIOBLASTOMA CELLS

Glioblastoma is one of the aggressive brain tumors with a 5-year survival rate of < 10%. The standard treatment is maximal safe resection, followed by radiation therapy and temozolomide (TMZ). Clinically, the resistance to TMZ is a big problem. Cancer cells have been revealed to show different metabolism from normal cells. The object of this study is to evaluate whether cancer metabolism, especially asparagine, could be a new target of treatment in primary and recurrent glioblastoma. Glioblastoma cells (U251 and U87) were treated with L-asparaginase and/or 6-diazo-5-oxo-L-norleucine (DON). L-asparaginase converts asparagine into aspartate and depletes asparagine. DON is a glutamine analog that inhibits several glutamine-utilizing enzymes, including asparagine synthetase. L-asparaginase or DON suppressed the proliferation of U251, and U87 cells in a dose-dependent manner. Combined treatment with these drugs had a synergistic antiproliferative effect in these cell lines. The effect was counteracted by exogenous asparagine. The combined treatment induced greater apoptosis and autophagy than did single-drug treatment. Several clones of TMZ-resistant U251 were obtained after long treatment of TMZ to U251. The expression of MSH6, one of the mismatch repair proteins, was suppressed in these resistant clones. The synergistic effect of L-asparaginase and DON was detected in these U251-derived TMZ-resistant clones. These results suggest that the combination of L-asparaginase and DON could be a new therapeutic option for patients with primary and recurrent glioblastoma.

Molecular profiling of appendiceal serrated lesions, polyps and mucinous neoplasms: a single-centre experience

PurposeNon-neuroendocrine neoplasms of the appendix are a phenotypically heterogeneous group of lesions; a comprehensive molecular characterization of these tumors is still lacking.MethodsA total of 52 samples taken from 49 patients was evaluated: 18 sessile serrated lesions (SSL; 3 with dysplasia), 2 high-grade tubular adenomas, 1 tubulo-villous adenoma,1 hyperplastic polyp, 18 low-grade appendiceal mucinous neoplasms (LAMN), 3 high-grade appendiceal mucinous neoplasms (HAMN) and 9 mucinous adenocarcinomas. Hotspot mutational profiling of the RNF43, SMAD4, KRAS, NRAS, BRAF and PIK3CA genes was performed. Expression of p53, MLH1, PMS2, MSH2, and MSH6 was evaluated by immunohistochemistry.ResultsKRAS was the most frequently mutated gene (53.9% of cases), followed by RNF43 (15.4%), and BRAF (13.5%). In particular: KRAS was mutated in 44.4% of adenocarcinomas, 66.7% of HAMNs, 61.1% of LAMNs, 53.3% of SSL without dysplasia and in 66.7% of SSL with dysplasia; RNF43 was mutated in 33.3% of adenocarcinomas, 66.7% of HAMNs, 11.1% of LAMNs and in 6.7% of SSL without dysplasia; BRAF was mutated in 11.1% of adenocarcinomas, 26.7% of SSL without dysplasia and in 5.6% of LAMNs. Only a case of high-grade tubular adenoma showed mismatch repair deficiency, while immunohistochemical expression of p53 was altered in 21.1% of cases.ConclusionsThe histological phenotypic similarities between appendicular mucinous lesions and serrated colon lesions do not reflect a similar genetic landscape. Mismatch repair deficiency is a rare event during appendiceal mucinous carcinogenesis.

Mismatch Repair Deficiency and Microsatellite Instability in Triple-Negative Breast Cancer: A Retrospective Study of 440 Patients.

PurposeTo investigate the status of mismatch repair (MMR) and microsatellite instability (MSI) in triple-negative breast cancer (TNBC) and to examine correlations between MMR/MSI status and clinicopathological parameters.MethodsWe retrospectively collected tissue samples from 440 patients with TNBC and constructed tissue microarrays. Protein expression of MLH1, MSH2, MSH6, and PMS2 was detected by immunohistochemistry (IHC). We also analyzed 195 patient samples using MSI polymerase chain reaction (PCR) testing. Correlations between MSI status and clinicopathological parameters and prognosis were analyzed.ResultsThe median age of the cohort was 49 years (range: 24–90 years) with a median follow-up period of 68 months (range: 1–170 months). All samples were positive for MLH1, MSH2, MSH6, and PMS2, except for one sample identified as MMR-deficient (dMMR) by IHC, with loss of MSH2 and intact MSH6 expression. MSI PCR revealed no case with high-frequency MSI (MSI-H), whereas 14 (7.2%) and 181 (92.8%) samples demonstrated low-frequency and absence of MSI events, respectively. The dMMR sample harbored low-frequency instability, as revealed by MSI PCR, and a possible EPCAM deletion in the tumor, as observed from next-generation sequencing. No correlations were detected between MMR or MSI status and clinicopathological parameters, programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression, or survival.ConclusionsThe incidence of dMMR/MSI-H is extremely low in TNBC, and rare discordant MSI PCR/MMR IHC results may be encountered. Moreover, MMR/MSI status may be of limited prognostic value. Further studies are warranted to explore other predictive immunotherapy biomarkers for TNBC.