DNA Mismatch Repair Deficiency in Colorectal Adenocarcinoma by a Two-Antibody Immunohistochemical Approach and its Association with Clinicopathological Features among Bangladeshi Patients

Research Article

Ann Hematol Onco. 2023; 10(3): 1426.

DNA Mismatch Repair Deficiency in Colorectal Adenocarcinoma by a Two-Antibody Immunohistochemical Approach and its Association with Clinicopathological Features among Bangladeshi Patients

Shiraj-Um-Mahmuda S¹; Begum F²; Rahman MM³; Islam T4; Shabnam US5; Afroz S6; Emita U7; Islam KBMS8*

1Lecturer, Department of Pathology, Dhaka Medical College, Bangladesh

2Professor, Department of Pathology, Faculty of Basic Science, Bangabandhu Sheikh Mujib Medical University, Bangladesh

3Associate Professor, Department of Pathology, Faculty of Basic Science, Bangabandhu Sheikh Mujib Medical University, Bangladesh

4Specialist (Pathology), Square Hospital, Bangladesh

5Medical officer, Department of Pathology and Microbiology, National Institute of Diseases of the Chest and Hospital, Bangladesh

6Medical officer, Department of Histology, National Institute of Cancer Research and Hospital, Bangladesh

7Clinical Pathologist, Khulna Medical College Hospital, Bangladesh

8Professor, Department of Medicine and Public Health, Faculty of Animal Science and Veterinary Medicine, Sher-e-Bangla Agricultural University, Bangladesh

*Corresponding author: KBM Saiful Islam Department of Medicine and Public Health, Faculty of Animal Science and Veterinary Medicine, Sher-e-Bangla Agricultural University, Dhaka-1207, Bangladesh. Tel: +8802-44814101 Email: [email protected]

Received: April 21, 2023 Accepted: May 15, 2023 Published: May 22, 2023


Background: Defective DNA Mismatch Repair (dMMR) genes cause dMMR/Microsatellite Instability (MSI)-related Colorectal Cancers (CRC) in humans which are different from Microsatellite Stable (MSS) tumors in terms of biological behavior, therapeutic response, and prognosis. We aimed to determine the frequency of dMMR CRCs by a two-antibody (PMS2 and MSH6) immunohistochemical approach and to evaluate their association with clinicopathological parameters to document the ever first report of such cases in Bangladesh.

Methods: Fifty histopathologically confirmed resected bowel specimens of CRC were studied over a period of two years at a tertiary-level hospital in Bangladesh. Histopathological parameters like morphologic variants, histologic subtypes, grade, stage, Lymphovascular Invasion (LVI), intratumoral lymphocytic infiltrate, and Crohn-like peritumoral reaction were assessed. Immunohistochemistry using PMS2 and MSH6 was performed on representative paraffin blocks by DAKO EnVision method. Expression of both of the markers was evaluated in classified groups.

Results: Mean age of study population was 48.60±14.6 years with a male to female ratio of 1.8:1. dMMR was recorded in 32% of cases. Expression of PMS2 and MSH6 were lost in 20% and 12% of cases, respectively. dMMR status was significantly associated with mucinous histology (p=0.014), lower pN staging (p=0.042), low LVI (p=0.002), exhibited intra-tumoral lymphocytosis (p=0.001), and Crohn like peritumoral reaction (p=0.001). No significant association with gender, age, right-sided location, histologic type, pT stage or grade was observed.

Conclusion: Frequency of dMMR CRCs was comparatively higher in the Bangladeshi population than in other races. Identification of dMMR tumors by their protein expression pattern using at least two, preferably four antibodies is proposed for routine screening of CRC cases.

Keywords: Mismatch repair deficiency; Microsatellite instability; Colon; Immunohistochemistry; PMS2; MSH6 etc.

Key points: Around one third CRC cases of Bangladesh are dMMR/MSI-related CRCs

• dMMR/MSI-related CRCs are more prevalent in Bangladeshi male patients below 50 years of age than that in female.


Colorectal Cancer (CRC) is one of the most common cancers affecting humans globally. It is the second most common malignancy in women and the third most common malignancy in men counting 9.4% and 10.6% of all cancer cases, respectively [1]. The global burden of colorectal cancer is expected to increase by 60% by 2030. Its incidence shows a 10-fold variation across the world [2]. The prevalence of colorectal cancer is lower in Asia than in Western countries. But the incidence has been alarmingly increasing in countries of Asia-Pacific region during the last two decades due to the westernization of lifestyles [3]. In Bangladesh 5-year prevalence of colon and rectal cancer are 3.28 and 3.1 per 100,000 populations respectively [1]. CRCs develop through a series of events leading to the transformation of normal mucosa to adenoma and then to carcinoma. Three distinct molecular pathways of colorectal carcinogenesis including Chromosomal Instability (CIN), Microsatellite Instability (MSI) and CpG Island Methylation (CIMP) have been recognized with overlap between these pathways [4]. Microsatellite instability has been detected in 15% and 90% of cases of sporadic CRC and CRC secondary to Hereditary Non-Polyposis Colorectal Cancer (HNPCC), respectively [5].

The DNA replication process is not error-free. DNA Mismatch Repair System (MMR) is the cellular post-replication process that preserves DNA homeostasis and guarantees of genomic stability [6]. At least five different MMR proteins including MSH2, MLH1, PMS1, MSH6, and PMS2 are required to perform DNA mismatch repair [7]. Any inherited or somatic mutation or epigenetic silencing of any of these genes lead to MSI and the tumors associated with this are referred to as MSI high or MSI-H tumors [8].

Clinicopathologic presentation, biological behavior, treatment options, therapeutic response and prognosis of MSI-H colorectal cancers show some differences from Microsatellite Stable (MSS) tumors of the same stage [9-11]. There are two methods for screening of MSI/dMMR cases. One is to detect the amplified microsatellite loci by PCR and another is the detection of proteins encoded by DNA Mismatch Repair Genes (MMR) including MLH1, PMS2, MSH2 and MSH6 by Immunohistochemistry (IHC). IHC is a specific, sensitive, fast and cost-effective tool for detecting MSI/dMMR colon cancers. The predictive value of IHC using all four antibodies is virtually equivalent to that of MSI testing by PCR [12].

Mismatch repair proteins form functional heterodimer complexes during repair, MLH1 with PMS2, (MutLa heterodimer) and MSH2 with MSH6 (MutS a heterodimer). MLH1 and MSH2 are the obligatory partners which stabilize the secondary partners PMS2 and MSH6, respectively to protect from proteolytic degradation. As a result, loss of the MLH1 protein leads to PMS2 degradation while loss of MSH2 leads to loss of MSH6. However, the converse is not true because the obligatory partners can bind with other minor proteins. Based on this concept, a “two-stain” method using only the MSH6 and PMS2 proteins has been developed and employed by several studies that demonstrated this 2-antibody approach is as effective as using the 4-antibody panel with the further reduction of time and resources [13-16].

The detection of dMMR status is becoming more and more important for patients’ survival because of its crucial therapeutic, prognostic and predictive implications. In another study, we documented an increased trend towards young age Colorectal Carcinoma (CRC) in the Bangladeshi population over recent years [17]. However, neither the dMMR status in Bangladeshi CRC patients is documented nor their morphological features are studied in the Bangladeshi population yet. Therefore, the study aimed to determine the frequency of dMMR CRC cases by a two-antibody immunohistochemical approach and to evaluate their association with several clinical and histopathological parameters to document the ever first report of such cases in Bangladesh.


Ethical Approval

Advanced approval was obtained from the local Ethics Committee (Institutional Review Board) of Bangabandhu Sheikh Mujib Medical University (BSMMU), Bangladesh for the study. All participants were informed about the nature & purpose of the study and prior written consent was obtained.

Exclusion Criteria

Clinically suspected colorectal carcinoma subsequently proved to be non-epithelial tumors of the colon were excluded from this study. Patients with a history of pre-operative chemo and/or radiation therapy, tumors composed mostly of mucin and a very small number of cells, cases with lost expression of immunomarkers in both internal control and tumor cells were also excluded from the study.

Study Design, Period and Sample

A cross-sectional, descriptive, hospital-based, study was conducted at the Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka, Bangladesh for a period of two years (from March 2019 to February 2021). A total of 50 Paraffin blocks of large bowel resection specimens histologically diagnosed as adenocarcinoma were taken as the samples for the study. All the slides of the study cases were retrieved and reviewed. Then, representative paraffin-fixed tissue blocks were selected that showed both tumor and adjacent non-neoplastic control tissue. Demographic and clinical information was obtained from patients’ attendants using a pre-tested questionnaire.

Pathological Analysis

Selected cases were evaluated elaborately and parameters including gross feature, histological type, tumor grading, staging, lymphovascular invasion, Crohn’s-like peri-tumoral reaction and intratumoral lymphocytic infiltrate were assessed. One representative section from each case was selected for immunohistochemical staining with PMS2 and MSH6.

Histopathological Features

Selected Hematoxylin and Eosin (H&E) slides cases were independently reviewed by two accredited histopathologists of BSMMU. Evaluation of tumor features and host response were done using the following criteria:

Tumor Features

Mucinous histology: Extracellular mucin accumulation bounded either by tumor epithelium or stroma. Tumors were sub-grouped as mucinous histology being none, 1–50%, and >50% of tumor area involved [18].

Signet ring differentiation: Presence of tumor cells with intracytoplasmic mucin and peripherally displaced crescent-shaped nucleus, whether present within extracellular mucin pools or invading the stroma. These tumors were subcategorized by- no signet ring cell, signet ring cell involving 1-50% and >50% of the tumor area [19].

Medullary pattern: Sheets, trabeculae, or nests of small to medium-sized tumor cells showing syncytial pattern, frequent mitosis, and abundant stromal lymphocytic infiltration.

Features of the Host’s Immune Response

Crohn-like peri-tumoral reaction: characterized by the pronounced lymphoid reaction to tumor, composed of lymphoid follicles at tumor edges, not associated with either mucosa or pre-existing lymph node. Two or more large lymphoid aggregates in a section were required for the presence of this feature [20].

Intra-tumoral lymphocytic infiltrate: marked by the presence of small round lymphocytes within neoplastic epithelial cells. Subgrouping of this category was done into none, mild to moderate (up to two Intra-Epithelial Lymphocytes (IEL)/HPF) and marked (≥ 3 IEL/HPF) by a semi-quantitative method [21].

Immunohistochemical Study

Immunohistochemical study was performed by a two-antibody panel of MMR proteins containing MSH6 and PMS2 using the DAKO EnVision method on the representative paraffin-fixed tissue blocks. Three to four μm thick tissue sections were deparaffinized in xylene, rehydrated in alcohol, and washed in distilled water. All the antibodies were ready-to-use monoclonal antibodies provided in liquid form in a buffer containing stabilizing protein and 0.015 mol/L sodium azide (PMS2 clone, EP51; MSH6 clone, EP49). The formalin-fixed, paraffin-embedded tissue sections were pretreated with heat-induced epitope retrieval (HIER) at 97°C for 35–40 min at high pH (50×). The slides were then incubated with PMS2 and MSH6 antibodies. Normal/intact staining pattern was defined as the presence of unequivocal nuclear staining (staining intensity at least similar to control) in any percentage of malignant cells, while nuclear staining in adjacent non-neoplastic tissue (lymphocytes, basal colonic crypt cells, and some stromal cells) was considered as a positive internal control. Negative staining was defined as the complete absence of nuclear staining in malignant cells where internal control was positive. Hence, carcinoma was considered dMMR when there was the absence of nuclear staining for at least one of the selected proteins. Tumors in which internal control and tumor cells both fail to express the markers were excluded from the study.

• MMR status was assigned on the basis of IHC testing as below:Deficient MMR (dMMR): Cases showing absence of detectable staining in 100% tumor nuclei with one or both of the IHC markers tested.

• Proficient MMR (pMMR): Normal expression of both markers in any percentage of tumor nuclei detected by immunohistochemistry.

Statistical Analysis

The statistical analysis was carried out using the Statistical Package for Social Sciences version 22.0 for Windows (SPSS Inc., Chicago, Illinois, USA). The result was calculated by using descriptive statistical formulas and presented in Tables, Figures, and Diagrams. The frequency of different entities was expressed as percentage. The association of expressions of selected markers with clinicopathologic parameters was evaluated with unpaired T-Test and Fisher’s exact test.


Out of total 50 CRC cases, 32% (n=16) cases showed loss of expression of at least one MMR protein (dMMR). Expressions of PMS2 and MSH6 proteins were lost in 20% (n=10) cases and 12% (n=6) cases, respectively. No tumor showed combined loss of both markers (Figure 1).