MSH2 Expression Research Articles

BET protein inhibition sensitizes glioblastoma cells to temozolomide treatment by attenuating MGMT expression

Bromodomain and extra-terminal tail (BET) proteins have been identified as potential epigenetic targets in cancer, including glioblastoma. These epigenetic modifiers link the histone code to gene transcription that can be disrupted with small molecule BET inhibitors (BETi). With the aim of developing rational combination treatments for glioblastoma, we analyzed BETi-induced differential gene expression in glioblastoma derived-spheres, and identified 6 distinct response patterns. To uncover emerging actionable vulnerabilities that can be targeted with a second drug, we extracted the 169 significantly disturbed DNA Damage Response genes and inspected their response pattern. The most prominent candidate with consistent downregulation, was the O-6-methylguanine-DNA methyltransferase (MGMT) gene, a known resistance factor for alkylating agent therapy in glioblastoma. BETi not only reduced MGMT expression in GBM cells, but also inhibited its induction, typically observed upon temozolomide treatment. To determine the potential clinical relevance, we evaluated the specificity of the effect on MGMT expression and MGMT mediated treatment resistance to temozolomide. BETi-mediated attenuation of MGMT expression was associated with reduction of BRD4- and Pol II-binding at the MGMT promoter. On the functional level, we demonstrated that ectopic expression of MGMT under an unrelated promoter was not affected by BETi, while under the same conditions, pharmacologic inhibition of MGMT restored the sensitivity to temozolomide, reflected in an increased level of γ-H2AX, a proxy for DNA double-strand breaks. Importantly, expression of MSH6 and MSH2, which are required for sensitivity to unrepaired O6-methylguanine-lesions, was only briefly affected by BETi. Taken together, the addition of BET-inhibitors to the current standard of care, comprising temozolomide treatment, may sensitize the 50% of patients whose glioblastoma exert an unmethylated MGMT promoter.

Correlation of PD-L1 immunohistochemical expression with microsatellite instability and p53 status in endometrial carcinoma.

Endometrial carcinoma (EC) is the most common gynaecological cancer worldwide. The Cancer Genome Atlas molecular grouping of a given case of EC could be assessed by POLE gene mutation, mismatch repair (MMR) 'to reflect microsatellite instability' and p53 status, which has proved to be of prognostic value. Programmed cell death receptor 1 (PD-1) and its ligand (PD-L1) are playing a progressively important role in tumour immunology and cancer treatment. To investigate PD-L1 immunohistochemical expression in EC in relation to MMR and p53 status. Associations between marker expression and different histopathological parameters were also investigated. This retrospective study was performed on archival biopsies of 170 cases of EC using a tissue microarray model. Immunohistochemical staining was applied using antibodies against PD-L1, MLH1, MSH2 and p53. The percentages of positivity were as follows: PD-L1, 19.6%; MLH1, 79.5%; MSH2, 78.5%; and p53 mutant, 13.8%. There was significant correlation between MLH1 expression and MSH2 expression (p=0.008). Tumour grade was significantly correlated with stage (p=0.005) and p53 mutant expression (p=0.008). Combined PD-L1 positivity and MMR deficiency showed significant correlation with the presence of lymphovascular space invasion (p=0.014). MSH2 negativity was significantly associated with poorer overall survival (p=0.014). A panel of immunohistochemical markers (PD-L1, MLH1, MSH2 and p53) could help to predict the prognosis and plan the treatment of patients with EC. MMR deficiency seems to be a good predictor for PD-L1 status, and therefore the response to potential PD-1/PD-L1 inhibitor therapy.

Differential operation of MLH1/MSH2 and FANCD2 crosstalk in chemotolerant bladder carcinoma: a clinical and therapeutic intervening study.

We aimed to understand the crosstalk between mismatch repair (MMR) and FA-BRCA pathway in primary bladder carcinoma (BlCa) samples as well as in chemotolerant cell line. We analysed the genetic alterations of MLH1 and MSH2 (MMR-related genes) and after that we correlated it with the nuclear translocation of FANCD2 protein. Next, we evaluated this crosstalk in T24 BlCa cell line in response to doxorubicin treatment. In primary BlCa tumors, infrequent genetic deletion (17-20%) but frequent promoter methylation (28-55%) of MLH1 and MSH2 was observed, where MLH1 was significantly (p < 0.05) more methylated among the early staged samples (NMIBC). However, MSH2 was significantly more altered among the NMIBC samples, signifying the importance of MMR pathway during the early pathogenesis of the disease. Furthermore, BlCa samples with underexpressed MLH1/MSH2 protein possessed cytoplasmic FANCD2 protein; encouraging that inefficiency of MMR proteins might restrict FANCD2 nuclear translocation. Next, we analysed publicly available data in GEO2R tool where we observed that in response to chemotherapeutic drugs, expression of MLH1, MSH2 and FANCD2 were diminishing. Validating this result in doxorubicin tolerant T24 cells, we found that expression of MLH1 and MSH2 was gradually decreased with increasing dose of doxorubicin. Interestingly, FANCD2 mono-ubiquitination (L-form) was also reduced in chemotolerant T24 cells. The crosstalk between MMR and FA-BRCA pathway was substantiated in the primary BlCa tumors. Further, in response to doxorubicin, this crosstalk was found to be hampered due to under-expression of MLH1 and MSH2 gene, thereby rendering chemotolerance.

Associations of dietary, lifestyle, and other participant characteristics with APC, β-catenin, E-cadherin, and MSH2 expression in the normal mucosa of sporadic colorectal adenoma patients

Abnormal expression of Wnt pathway and DNA mismatch repair proteins is common during colorectal carcinogenesis. To investigate cross-sectional associations of lifestyle, dietary, and other participant characteristics with the expression of such proteins, we measured APC, β-catenin, E-cadherin, and MSH2 colorectal crypt expression in biopsies of normal-appearing colorectal mucosa from 104 sporadic colorectal adenoma patients using automated immunohistochemistry and quantitative image analysis. We used multivariable general linear models to compare adjusted mean biomarker expression across categories of participant characteristics. Example findings include that among women relative to men, mean APC expression in whole crypts, the upper 40% of crypts (differentiation zone), and the lower 60% of crypts (proliferation zone) was 322.9% higher (p&amp;lt;0.01), 296.7% higher (p&amp;lt;0.01), and 399.1% higher (p&amp;lt;0.01), respectively. Among participants with higher alcohol consumption, APC expression in the crypt differentiation zone was estimated to be 15.9% lower (p=0.08). Among those with higher total meat consumption, β-catenin expression in whole crypts and the crypt proliferation zone was estimated to be 20.5% higher (p=0.07) and 19.6% higher (p=0.06), respectively, and MSH2 expression in the crypt differentiation zone was estimated to be 64.4% lower (p=0.10). Among those with a higher body mass index, MSH2 expression in the crypt differentiation zone was estimated to be 87.5% lower (p=0.15). These pilot study findings suggest that being male, higher adiposity, and higher alcohol and meat consumption may be unfavorably associated with biomarkers of colorectal carcinogenesis pathway proteins in the normal-appearing colorectal mucosa of sporadic colorectal adenoma patients and support further investigation in larger studies.

A Novel Epigenetic Marker Predicts Treatment Response in Glioblastoma and Provides a Target for Overcoming Treatment Resistance

<h3>Purpose/Objective(s)</h3> Glioblastoma (GBM) is a highly aggressive primary brain tumor. A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. An important hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways in tumor cells. However, the effect of epigenetic signaling on chemotherapy response in GBM remains to be elucidated. SETD2 is a unique histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. <h3>Materials/Methods</h3> We have performed next-generation sequencing of the SETD2 gene and immunohistochemical staining if its product H3K36me3 in 129 GBM patient samples that was supplemented with TCGA database analysis of additional 279 GBM samples. We used primary patient-derived GBM lines to investigate the mechanism of chemotherapy resistance in SETD2-mutant GBM. <h3>Results</h3> We demonstrate that inactivating SETD2 mutations occur in up to 13% of GBM patients. Our investigation reveals that SETD2 mutations are associated with reduced progression-free (Median PFS 0.93 vs. 0.44 years, p=0.0022) and overall survival (Median survival 1.69 vs. 1.30 years, p=0.0044) in patients with methylated MGMT (methyl-guanine methyl transferase) promotor who received temozolomide suggesting that mutation of SETD2 is a predictive marker for treatment outcomes in GBM. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels in patient samples and a profound resistance to temozolomide in primary patient-derived GBM lines. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that plays a critical role in converting primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells us due to disrupted MMR. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the <i>MSH6</i> gene in GBM. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. <h3>Conclusion</h3> Our findings establish SETD2 mutation as a novel molecular marker predictive of treatment response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting an epigenetic signaling pathway.

S2204 A Rare Case of Medullary Carcinoma of the Colon With Lymphatic Metastasis

Introduction: Medullary carcinoma of the colon (MAC) is a rare cancer encompassing only .03% of all colorectal carcinomas. We present a rare case of MAC occurring in an older adult with a strong tobacco use history. Case Description/Methods: A 76-year-old female with PMHx of tobacco use disorder, COPD, and known pulmonary nodules presented to the ED for surveillance of her pulmonary nodules. A review of systems was positive for chronic shortness of breath and cough. A CT scan of the chest revealed enlarging lung nodules and mediastinal adenopathy. A PET scan revealed a hypermetabolic 2.5 x 2.5 cm soft mass in the distal ascending colon and hypermetabolic lymph nodes in the right upper quadrant (Figure). Colonoscopy revealed an infiltrative 2 cm mass at the hepatic flexure, and pathology revealed an invasive, poorly differentiated adenocarcinoma with normal expression of MSH2 and MSH6 and absence of MLH1 and PMS2. These findings were consistent with medullary carcinoma of the colon. Endoscopic ultrasound with biopsy of peripancreatic and porta-hepatis lymph nodes favored metastatic medullary carcinoma. The patient underwent a right hemicolectomy and follows closely outpatient. Discussion: MAC is a rare cancer associated with a high level of microsatellite instability and deficient mismatch repair proteins. It is a non-gland forming cancer composed of large polygonal eosinophilic cells growing in solid sheets and infiltrated with small lymphocytes. The majority of reported cases revealed a lack of MLH-1 and PMS2, just as our patient did. They instead express markers that are not commonly associated with colorectal cancers, such as calretinin, CK7, SATB2, and CDH17. MACs were previously difficult to differentiate from poorly differentiated adenocarcinomas due to their unique tumor marker expression and histopathology. The current standard for diagnosis of MAC therefore requires distinct microscopic morphology and molecular staining patterns. There are no current treatment guidelines due to the rarity of MACs, although MACs do have a favorable prognosis when compared to other adenocarcinomas and surgical resection and FOLFOX chemotherapy have been used successfully in some studies. Although MACs have been sporadically reported in case reports, further studies are required to determine guidelines for both diagnosis and treatment of this rare disease.Figure 1.: Endoscopic Visualization with Final Needle Aspiration of Abnormal Lymph Node.

To Test or Re-Test, that is the Question: Comparison of the Mismatch Repair Deficiency between Primary and Recurrent Sites of Uterine Cancers (088)

To Test or Re-Test, that is the Question: Comparison of the Mismatch Repair Deficiency between Primary and Recurrent Sites of Uterine Cancers (088)

The association of tumor mutational burden, microsatellite stability, and mismatch repair deficiency in an endometrial cancer patient cohort (194)

The association of tumor mutational burden, microsatellite stability, and mismatch repair deficiency in an endometrial cancer patient cohort (194)

Clinicopathological prognostic factors for gallbladder carcinoma: a retrospective study.

BackgroundGallbladder carcinoma (GBC) is an uncommon neoplasm with poor long-term survival. Worldwide the incidence rates vary according to geographic area. The multifactorial aetiology and the rarity of the disease limits the studies to improve outcomes in patients, since the treatment remains mostly surgical. The aim of this study was to identify clinicopathological prognostic factors for survival in patients with GBC submitted to surgery in our institution—a tertiary centre in Portugal. Also, to assess the expression of possible biomarkers (HER2, CD44 and ALDH1) in GBC, as well as the frequency of microsatellite instability (MSI) tumours.MethodsClinicopathological characteristics of 41 consecutive patients that underwent surgical resection for GBC (2008–2019) at our hospital were retrospectively reviewed. Clinicopathological factors were assessed and an immunohistochemical (IHC) analysis was done. Microsatellite stability (MSS) was considered if there was maintenance of nuclear expression of MLH1, MSH2, MSH6 and PMS2. Human epidermal growth factor receptor 2 (HER2) expression was evaluated according to the rules applied for gastric cancer and expression of CD44 and ALH1 was evaluated in order to detect cancer stem cells (CSC). Survival analysis was conducted using Kaplan-Meier and Cox regression was used to find prognostic factors.ResultsIncidence of GBC in our cohort of patients was 0.45%, most commonly affecting females. Median overall survival (OS) was 23 months with a 39.6% 5-year survival rate. Stage > II [hazard ratios (HR) =8.58; P=0.007], lymphovascular invasion (LVI) (HR =4.06; P=0.045) and hepatic resection (HR =0.288; P=0.034) independently influenced survival. HER2 positivity and high expression of CD44 or ADLH1 did not show significant influence in survival (P=0.649, P=0.868 and P=0.914, respectively), although HER2 and ALDH1 positive patients showed a tendency to a shorter OS, compared to negative patients. We found no relation between these biomarkers expression and disease stage. All analysed samples had MSS.ConclusionsGBC patients with a worse prognosis can be identified. The overexpression of HER2 could select patients for targeted therapy and prompt tissue sampling in unresectable patients.

PMS2 Expression With Combination of PD-L1 and TILs for Predicting Survival of Esophageal Squamous Cell Carcinoma.

BackgroundDNA mismatch repair (MMR) deficiency (dMMR) has been recognized as an important biomarker for immunotherapy in esophageal squamous cell carcinoma (ESCC), along with programmed death ligand 1 (PD-L1) expression and/or tumor-infiltrated lymphocytes (TILs). However, in ESCC, MMR protein assessment has not been well studied at present.MethodsA total of 484 ESCC tissues treated between 2007 and 2010, in our hospital, were enrolled. Immunohistochemical expression of MLH1, MSH2, MSH6, PMS2, and PD-L1 on tissue microarray specimens and clinicopathological features, including TILs, were analyzed retrospectively.ResultsOut of the 484 studied cases, loss of MLH1, MSH2, MSH6, and PMS2 expression were found in 6.8%, 2.1%, 8.7%, and 4.8% patients, respectively. dMMR was found in 65 patients, 37 cases involved in one MMR protein, 17 cases involved in two proteins, 7 cases involved in three proteins, and 4 cases involved in four proteins. There was no significant survival difference between pMMR (MMR-proficient) and dMMR patients (P>0.05). However, 224 patients with low PMS2 expression had better DFS and OS than 260 patients with high PMS2 expression (P=0.006 for DFS and 0.008 for OS), which was identified as an independent prognostic factor in multivariate analyses. Positive PD-L1 expression was detected in 341 (70.5%) samples. In stage I-II disease, patients with PD-L1 expression had better DFS and OS than those without PD-L1 expression(P<0.05), which was not found in stage III-IV disease. With the ITWG system, 40.1% of cases were classified as high TILs. Patients in the high-TILs group tended to have better DFS (P=0.055) and OS (P=0.070) than those in the low-TILs group and the differences were statistically significant in pMMR, high MSH6, or PMS2 expression cases (P<0.05). Also, high PMS2 expression patients with both PD-L1 expression and high TILs, had similar DFS and OS compared with low PMS2 expression patients (P>0.05), which were much better than other high PMS2 expression patients.ConclusionThe expression level of MMR proteins could also be used as a prognostic factor in ESCC and PMS2 expression outperformed other MMR proteins for predicting survival. The combination of PD-L1 expression and TILs may lead to more efficient risk stratification of ESCC.

Fusobacterium nucleatum impairs DNA mismatch repair and stability in patients with squamous cell carcinoma of the head and neck.

Dysbiosis of the laryngeal microbiota has been demonstrated to the development of head and neck squamous cell carcinoma (HNSCC), but the association of Fusobacterium and Fusobacterium nucleatum (F. nucleatum) with DNA mismatch repair (MMR) and microsatellite instability (MSI) has not been investigated. The abundance of Fusobacterium and F. nucleatum, the status of deficient MMR (dMMR) and MSI, and MMR-related gene expression were analyzed in 171 HNSCC tissues, 61 paired para-tumor tissues, and 60 vocal cord polyp tissues. The molecular mechanism of F. nucleatum and MMR-related gene expression were investigated in two human HNSCC cell lines (Tu 686 and FD-LSC-1). Our results demonstrated that a high Fusobacterium abundance was detected in the HNSCC tissues and was exaggerated in the recurrent patients. We further found that a high Fusobacterium abundance was detected in the HNSCC tissues with dMMR and MSI. The Fusobacterium abundance was negatively correlated with the expression of MLH1, MSH2, and MSH6 in the HNSCC tissues. The Fusobacterium abundance was closely associated with the F. nucleatum abundance in the HNSCC tissues. F. nucleatum increased miR-205-5p expression to suppress MLH1, MSH2, and MSH6 expression via the TLR4- and MYD88-dependent innate immune signaling pathway, resulting in dMMR, DNA damage, and cell proliferation in HNSCC. F. nucleatum impacts HNSCC epigenetic changes in tissues with dMMR to promote DNA damage and cell proliferation by suppressing MMR-related gene expression via the TLR4/MYD88/miR-205-5p signaling pathway, which is valuable in the development of efficient strategies for HNSCC prevention and treatment. This study clearly indicates that Fusobacterium induced head and neck squamous cell carcinoma (HNSCC) aggressiveness to affect poor prognosis in HNSCC patients by epigenetic alteration of DNA mismatch repair (MMR) and microsatellite instability. Moreover, the research has shown that Fusobacterium nucleatum ( F. nucleatum ) impacts HNSCC epigenetic changes in tissues with deficient MMR to promote DNA damage and cell proliferation by suppressing MMRrelated gene expression via the TLR4/MYD88/miR-205-5p signaling pathway.

Expression and methylation status of MMR and MGMT in well-differentiated pancreatic neuroendocrine tumors and potential clinical applications.

Recent studies claim that immune checkpoint inhibitors are effective in defective mismatch repair (dMMR) cancers. This raises the question of whether similar therapies are effective in PanNETs (pancreatic neuroendocrine tumors); however, in general, assessment of MMR status in PanNETs has been inconsistent in previous studies. MGMT (O6-methylguanine-DNA methyltransferase) is potentially important for guiding temozolomide (TMZ) therapy in glioblastoma. The number of reports on MGMT expression and promoter methylation in PanNETs are limited. In this study we assessed the expression of MGMT and MMR proteins MSH2, MSH6, MLH1 and PMS2 in a series of PanNETs by IHC. The methylation status of MGMT and MMR genes in a subset of PanNETs was further assessed by MS-MLPA analysis. Survival curves were constructed using the Kaplan-Meier method, and differences were assessed using the log-rank test. Multivariate Cox proportional hazards regression models were used to determine the prognostic value of the variables. According to evaluation criteria for mismatch repair defects, none of PanNETs shown nuclear staining loss for MSH2, MSH6, MLH1, and PMS2. MGMT low-intensity PanNETs were more commonly found in higher grade, higher Ki67 index and non-functional tumors (P < 0.05). In multivariate analysis, stage III-IV and low-intensity MGMT were shown to be independent risk factors for progression of PanNETs in the entire cohort, non-functioning subgroup and G2 subgroup (P < 0.05 for all). MGMT promoter methylation tended to be higher in the group with low expression of MGMT, However, methylation of MGMT did not statistically correlate with low expression of MGMT (P = 0.153). In conclusion, our study suggests that decreased expression of MGMT but not MMR is associated with a higher risk of progression of pancreatic neuroendocrine tumors.

Abstract 1634: Reduction or loss of Msh2 confers resistance to temozolomide in glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor. The only intervention that has improved the survival rate of GBM patients over the past several decades has been combining temozolomide (TMZ) with radiotherapy (RT), which increased median survival by only ~2.5 months to where it currently stands at ~15 months. Unfortunately, all GBM patients eventually die due to tumor recurrence. Intrinsic or acquired resistance to TMZ is a significant contributing factor to tumor progression, and various mechanisms have been suggested, including deficiencies in DNA mismatch repair (MMR) genes such as MSH2. Utilizing patient GBM samples from the TCGA, we have identified a correlation between low MSH2 expression and shorter patient survival. To test the biological significance of Msh2 in GBM growth and resistance to TMZ and immunotherapy, we have generated immunocompetent genetically engineered mouse models (GEMMs) of GBM with germline or somatic loss of Msh2. To generate tumors with reduced or abrogated Msh2, we utilized GEMMs based on the RCAS/tv-a gene transfer system in combination with mice that exhibited heterozygous and homozygous loss of Msh2. When PDGFB was overexpressed in combination with the silencing of Tp53 to induce tumors, tumor-bearing mice with reduced or deficient Msh2 demonstrated shorter survival time compared to wild-type tumor-bearing mice. In addition, our results indicate Msh2 deficiency increases the malignancy of gliomas. Our data demonstrate that two weeks of TMZ treatment at a clinically relevant dose of 25 mg/kg provides a significant survival advantage in WT-tumor-bearing mice, while no efficacy was observed in tumor-bearing mice with either heterozygous or homozygous loss of Msh2. We are currently evaluating the role of Msh2 in the efficacy of immune checkpoint inhibitors in glioblastoma. Citation Format: Montserrat Puigdelloses Vallcorba, Alice Buonfiglioli, Gonzalo Piñero, Zhihong Chen, Dolores Hambardzumyan. Reduction or loss of Msh2 confers resistance to temozolomide in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1634.

Exosomal CTCF Confers Cisplatin Resistance in Osteosarcoma by Promoting Autophagy via the IGF2-AS/miR-579-3p/MSH6 Axis.

Cancer-derived exosomes participate in carcinogenesis and progression of cancers, including metastasis and drug-resistance. Of note, CTCF has been suggested to induce drug resistance in various cancers. Herein, we aim to investigate the role of cisplatin- (CDDP-) resistant osteosarcoma- (OS-) derived exosomal CTCF in OS cell resistance to CDDP and its mechanistic basis. Differentially expressed transcription factors, long noncoding RNAs (lncRNAs), miRNAs, and genes in OS were retrieved using bioinformatics approaches. Exosomes were extracted from CDDP-resistant OS cells and then cocultured with parental OS cells, followed by lentiviral transduction to manipulate the expression of CTCF, IGF2-AS, miR-579-3p, and MSH6. We assessed the in vitro and in vivo effects on malignant phenotypes, autophagy, CDDP sensitivity, and tumor formation of OS cells. It was established that CTCF and IGF2-AS were highly expressed in CDDP-resistant OS cells, and the CDDP-resistant OS cell-derived exosomal CTCF enhanced IGF2-AS transcription. CDDP-resistant OS-derived exosomes transmitted CTCF to OS cells and increased CDDP resistance in OS cells by activating an autophagy-dependent pathway. Mechanistically, CTCF activated IGF2-AS transcription and IGF2-AS competitively bound to miR-579-3p to upregulate MSH6 expression. Additionally, the promoting function of exosomal CTCF-mediated IGF2-AS/miR-579-3p/MSH6 in OS cell resistance to CDDP was confirmed in vivo. Taken together, CDDP-resistant OS-derived exosomal CTCF enhanced resistance of OS cells to CDDP via activating the autophagy-dependent pathway, providing a potential therapeutic consideration for OS treatment.

Potential genetic biomarker of Saudi Arabian patients with colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths globally. We implemented a comprehensive literature review regarding CRC genetics studies to offer a perception into the genes associated with CRC recognized in Saudi patients. Definite genetic variants in ABCB1, ADIPOQ, CTNNB1, SFRP3, LRP6, CYP19A1, PARP-1, TDG genes exhibited significant protection against CRC development in Saudi population. Whereas, other gene mutations in ABCB1, ABCC1, CASR, IL-17F, NOTCH1, NOTCH4, PRNCR1, TDG, TLR2, TLR4, TLR-9, TSLP, TSLPR and TNF-α genes showed irrelevant correlation with CRC risk in Saudi Arabia. On the other hand, specific mutations in ABCC1, ADIPOQ, CYP1A1, KIR, IL-17A, MMP2, NOTCH3, PRNCR1, RETN, TDG, TLR2, BRAF, PARP-1, TLR4, TLR-9, TNF-α, TSLP and XRCC1 genes demonstrated a substantial augmented CRC risk development in Saudi patients. Furthermore, ATR, ATM, BMI1, CCAT1, Chk1, Chk2, COX-2, FoxM1, FSCN1, Ki67, MALAT1, miR-29, miR-34a, miR-92, miR-182-5, PANDAR, PIK3CA, TIGAR over-expression revealed a robust association with CRC in Saudi Arabia (KSA). Moreover, gene alterations in APC, EGFR, FBXW7, TP53, PTEN, K-ras genes were concomitant in CRC. As well as, lower expression of MLH1, MSH2, MSH6, PMS2, EPCAM and MUTYH genes were recognized in LS patients and future CRC Saudi patients. These gene mutations may be used as diagnostic and/or prognostic genetic markers in CRC Saudi patients and could offer a potential therapeutic target for CRC management.

Assessment of Microsatellite Instability in Endometrioid Carcinoma by Immunohistochemistry At Armed Forces Institute of Pathology, Rawalpindi

Objective: To assess microsatellite instability in endometrioid carcinoma by immunohistochemistry expression of MLH1, PMS2, MSH2 and MSH6.&#x0D; Study design: Case series.&#x0D; Place and Duration of Study: Department of Histopathology, Armed Forces Institute of Pathology, Rawalpindi, from Jun to Dec 2018.&#x0D; Methodology: Thirty-two cases of endometrioid carcinoma were included in the study. Patient age and menopausal status were recorded. The grade of the tumour was ascertained by microscopic examination. Immunohistochemistry for MLH1, PMS2, MSH2 and MSH6 assessed microsatellite instability.&#x0D; Results: In our study, eight females (25%) had premenopausal status while 24 (75%) had postmenopausal status. Out of 32 cases, 25 (78.1%) females had grade-I tumours, 4 (12.5%) had grade-II tumours, and 3 (9.45%) had grade-III endometrioid carcinoma. MMR status was proficient in 8 cases (25%) while deficient in 24 cases (75%). Among the 24 cases of MMR deficient endometrioid carcinoma, loss of expression of MLH1 was most frequent n=15 (62.5%), followed by PMS2 n=14 (58.3%) MSH2 n=7 (29.2%). MSH6 was retained in all cases. The most common pattern was a combined loss of MLH1/PMS2 in 12 cases (50%).&#x0D; Conclusion: As depicted by the high percentage of MMR deficient tumours, microsatellite instability is observed in many endometrioid carcinomas. Patients suffering from endometrioid carcinoma should be screened for Lynch syndrome by testing for MMR status.

Percent Agreement Between Immunohistochemistry and Next-Generation Sequencing in Testing Patients for Mismatch Repair Deficiency.

The presence of mismatch repair deficiency is frequently assessed in gastrointestinal and gynecologic neoplasms by surgical pathologists using immunohistochemical methods. Targeted next-generation sequencing (NGS) covering some genes in the mismatch repair complex is used with increasing frequency, however, the percent positive and negative agreement of immunohistochemical methods and NGS of mismatch repair genes is not well-described in the literature. We sought to compare performance of immunohistochemistry (IHC) and NGS of mismatch repair genes on our institutional targeted panel. We evaluated the concordance of immunohistochemical and panel-based gene sequencing methods in a retrospective cohort study of patients evaluated at our center with both immunohistochemical and panel-based sequencing. Our NGS panel covers only MLH1 and MSH2, whereas our immunohistochemical panel assesses for expression of MLH1, PMS2, MSH2, and MSH6. We identified 68 unique patients with both immunohistochemical evaluation of mismatch repair protein expression and NGS panel sequencing, of which 67 were suitable for analysis given the patterns of immunohistochemical loss of expression observed. The percent positive agreement for NGS with IHC was 50%, albeit with very rare positive cases (n=2/4). Percent negative agreement was also high at 100% (n=63/63). One case with loss of MLH1, PMS2, and MSH6 expression by IHC and no pathogenic variants by NGS exhibited MLH1 promoter hypermethylation. Percent negative agreement between immunohistochemical and NGS gene sequencing is high, although firm conclusions regarding percent positive agreement between NGS and IHC are limited by low numbers of positive cases in our cohort. In general, we consider the findings to support continued use of immunohistochemical methods to screen for the presence of mismatch repair deficiency and consider additional testing by NGS likely to add little diagnostic value in the context of intact immunohistochemical expression of mismatch repair proteins.

FOCUS4 biomarker laboratories: from the benefits to the practical and logistical issues faced during 6 years of centralised testing

AimsFOCUS4 was a phase II/III umbrella trial, recruiting patients with advanced or metastatic colorectal cancer, between 2014 and 2020. Molecular profiling of patients’ formalin-fixed, paraffin-embedded tumour blocks was undertaken at...

Lynch Syndrome-Associated Endometrial Cancer With Combined EPCAM-MSH2 Deletion: A Case Report.

BackgroundLynch syndrome (LS), an autosomal dominant disorder, is characterized by germline pathogenic variants in DNA mismatch repair (MMR) genes like MSH2. EPCAM deletions cause a minority (3%) of LS cases. However, there are only a few reports of LS-associated endometrial cancer (LS-EC) induced by the inactivation of the MSH2 gene due to EPCAM deletions.Case PresentationWe present the case of a 45-years old woman diagnosed with endometrial cancer (EC). Definitive surgery revealed meso-differentiated endometrioid adenocarcinoma, stage IA without lymph-vascular space invasion. Four months later, she received radiation therapy (125I radioactive seeds implantation), and platinum-containing regimen combined chemotherapy because of vaginal stump metastasis of EC. After five years, we performed immunohistochemistry (IHC) on pelvic mass because of presacral metastatic lymph node. IHC showed the absence of MSH2 and MSH6 protein expression in the pelvic mass tissue. Peripheral blood was used for genetic testing based on her cancer diagnosis and family history of cancer in close relatives. Genetic testing revealed deletions of exon 8 and 9 in EPCAM and deletions of exon 1 and 8 in MSH2; thus, we diagnosed the presence of LS. The patient underwent interstitial brachytherapy (BT) of the presacral metastatic lymph node.ConclusionThis case highlights that patients with LS-EC who are carriers of combined EPCAM-MSH2 deletion might experience better oncologic outcomes even with early recurrence.

Diffuse Large B-Cell Lymphoma, Epstein–Barr Virus -Positive Kappa Monotypic Plasma Cell Proliferation and Invasive Carcinoma, Developing in a Child With Defective Mismatch Repair

Constitutional mismatch repair deficiency (CMMRD) syndrome is characterized by biallelic mutations in a mismatch repair gene and is associated with development of childhood cancers and symptoms resembling neurofibromatosis type 1, like café-au-lait spots. We describe the extremely rare case of a 12-year-old male presenting with several light brown macular lesions on the skin, gastrointestinal diffuse large B-cell lymphoma, adenomatous polyposis throughout the gastrointestinal tract and an intra-abdominal invasive carcinoma derived from upper gastrointestinal system. All neoplasia, as well as normal tissues, showed loss of Msh6 expression with immunohistochemistry. Molecular studies showed pathogenic homozygous p.F1088Sfs*2 mutation in MSH6. Furthermore, signs consistent with immunodeficiency, namely decreased levels of IgG and IgA in the serum, nodular lymphoid hyperplasia and EBV-associated plasma cell proliferation with monotypic kappa light chain expression in the ileum, were also noted. Our case depicts the phenotypic diversity of CMMRD syndrome and emphasizes its association with immunodeficiency, raising awareness to a feature not widely recognized.