Mis-Match Repair (MMR) Protein Expression and Microsatellite Instability (MSI) Analysis in Sporadic Adenocarcinoma Diagnosed on Colonoscopic Biopsy

Introduction

Colorectal cancer (CRC) being the third most frequent cancer in men and the second most common in women worldwide, is a major contributor to cancer-related mortality.¹ The highest age-adjusted incidence rate for CRCs was recorded as 4.1 for men and 5.2 for women in India, where it ranks ninth among the most frequent cancers in both sexes.² The most frequent pathway for the development of colorectal neoplasia is chromosomal instability (75%), followed by the microsatellite instability (MSI) pathway (15%).^3,4 The CpG island methylator phenotype, which results in epigenetic gene silencing in 10% of CRCs, is the third main route.^4-6 Lynch syndrome (LS) or hereditary nonpolyposis colorectal cancer (HNPCC) accounts for about 15% of the MSI-related malignancies, while the remaining 85% are sporadic.^3,4 

DNA mismatch repair (MMR) is a vital step in the cell cycle to correct mistakes in DNA replication. Loss of MMR pathway function results from mutations in MMR genes. Because the MMR system is ineffective at repairing replication-associated errors, mismatch mutations can persist throughout the genome, but especially in microsatellite regions, which are repetitive DNA structures. This instability in the microsatellite region causes the phenomenon of microsatellite instability (MSI), which leads to the rapid accumulation of new mutations and the emergence of carcinoma. MLH1, MSH2, MSH6, and PMS2 are the four DNA mismatch repair genes that are most frequently mutated and they function as a heterodimer. MSH2 and MSH6 are heterodimer where MSH2 is the dominant one i.e. loss of MSH2 leads to degradation of MSH6, but not the vice versa. Similarly MLH1 and PMS2 are heterodimer where MLH1 is the dominant one i.e. loss of MLH1 leads to degradation of PMS2, but not the vice versa. Defective MMR gene can be detected via immunohistochemistry and its absence is a surrogate marker for microsatellite instability. Testing for MSI (thus classifying microsatellite stable, MSI–H and MSI–L) is done by molecular testing of at least five loci recommended by National cancer institute using Polymerase Chain Reaction (PCR) or next-generation sequencing (NGS) technique.⁷

In 1996, Leach et al. developed monoclonal antibodies that identified the MSH2 protein in DNA mismatch-proficient cell lines 1, and this sparked a number of studies on the use of immunohistochemical detection of DNA mismatch repair (MMR) proteins in the recognition of colorectal tumors with microsatellite instability (MSI).⁸ The fundamental flaw in MSI has been discovered to be faulty DNA MMR function as a result of either germline mutation of one of multiple MMR genes, including MLH1, PMS2, MSH2, and MSH6 or abnormal methylation of the promoter of MLH1. Also, detection of MMR using Immunohistochemistry (IHC) is cost effective and easily available. Specific staining is performed on tumor tissue for each of the four mismatch repair proteins; mutations in MMR genes typically lead to nonfunctional proteins that do not stain by IHC suggestive of defective DNA mismatch repair within the tumor.

Detection of CRC with dMMR proteins has clinical importance in terms of prognostic value with better prognosis and reduced recurrence rates,⁹ as a predictive marker of response to 5FU chemotherapy specially in colorectal carcinomas and lynch syndrome screening tool.¹⁰

Immunohistochemical (IHC) analysis of MMR proteins is a promising approach to identify patients with CRC and MSI. This study aimed to evaluate MSI status using the IHC expression pattern of four MMR proteins in CRC endoscopic samples.

Materials and Methods

Forty-two colonoscopic endoscopic biopsies pathologically diagnosed as CRC were collected from the Department of Pathology, between June 2019 and June 2021. All samples satisfied the following criteria: (1) sporadic colon or rectal cancer confirmed by pathological diagnosis, (2) no preoperative therapy. Biopsies with inadequate tissue were excluded from the study. The patients’ demographic records, clinical details were retrieved from our digital archive. The study protocol was approved by the institutional review board, and written informed consent was obtained from all the participants.

IHC procedure and interpretation

Four µm thick sections were taken on positively charged slides. After overnight incubation at 37ºC and deparaffinization with repeated washes of xylene (10 min each), rehydration of tissues with graded alcohol was done. The slides were then washed with running water (10 min) and distilled water (5 min). Antigen retrieval with citrate or EDTA buffer at 95ºC was done and cooled at room temperature. Peroxide block was carried out for 10–15 min and washed in Phosphate buffer solution (PBS) for 5 min and incubated with Monoclonal Rabbit Anti-Human MutS Protein Homolog 6 Clone EP49 for MSH6, Monoclonal Mouse Anti-Human MutS Protein Homolog 2 Clone FE11 for MSH2, Monoclonal Mouse Anti-Human Mut L Protein Homolog 1 Clone ES05 for MLH1, Monoclonal Rabbit Anti-Human post meiotic segregation increased 2, clone EP51 for PMS2 for 45 min. Subsequently, tissues were incubated with Poly Excel Target Binder for 15 to 20 min and washed with PBS for five minutes. Again, incubation was done with Poly Excel Poly H.R.P (Horse Radish Peroxidase) for 15 to 20 min, washed with PBS for five minutes and developed with DAB (Diaminobenzidine) Chromogen for 5-8 minutes. Sections were counterstained with haematoxylin after washing with running water. Slides were mounted and observed under light microscope for interpretation. Normal colonic mucosa, lymphocytes and stromal cells were used as positive control.

The H&E slides for morphological features and the IHC results were interpreted by the consultant pathologists using a Nikon microscope. Reporting was done following the College of American Pathologists (CAP) protocol of biomarker testing for MMR protein. Any amount of intact nuclear staining by IHC was considered as positive (intact expression), cases with weak and focal nuclear positive were also considered as positive, but noted separately. The loss of nuclear staining by IHC with positive internal control (lymphocytes, normal colonic mucosa and stromal cells) was considered as loss of expression. All cases were categorized into one of the following six patterns: MLH1- PMS2-, MSH2+MSH6-, MSH2-MSH6-, MLH1+PMS2-, MLH1-PMS2-MSH2-MSH6, MLH1+ PMS2-MSH2+ MSH6-.

Furthermore, if all four markers showed intact nuclear expression, the biopsy was classified as Microsatellite Stable (MSS). If only one marker showed loss of expression, it was labelled as deficient MMR (dMMR) and categorized as Microsatellite instability -Low (MSI-L), and if two or more makers showed loss of expression, the tissue was dMMR and categorized as Microsatellite instability - High (MSI-H).

Descriptive statistics were used to summarize the demographic characteristics of the study population. All statistical studies were performed using IBM SPSS Version 29. The frequencies and percentages of positive and negative MMR protein expression were calculated.

Results

Forty-two colonoscopic biopsies with colorectal adenocarcinoma cases were studied. There were 27 males and 15 females (Figure 1) with a mean age of 56.65±15.4 years. Out of the 42 cases, 22 (52.3%) were mismatch repair proficient (pMMR), while the remaining 20 (47.6%) were mismatch repair deficient (dMMR). MSI-L accounted for six cases (14%) and MSI-H accounted for the remaining 14 cases (33%) (Figure 2). The mean age for dMMR cases was 53.5 years, while that of pMMR was 54 years.

Mismatch repair protein expression for MLH1, PMS2, MSH2 and MSH6 on Immunohistochemistry has been depicted in Table 1.

The pattern of expression was further classified into one of the following patterns: MLH1- PMS2-, MSH2+ MSH6-, MSH2-MSH6-, MLH1+PMS2-, MLH1 PMS2- MSH2-MSH6-, MLH1+PMS2-MSH2+ MSH6- as depicted in Figure 3. MLH1+PMS2- was the most common pattern accounting for 20% for dMMR cases and MLH1-PMS2-MSH2-MSH6- or the “Null IHC/ Pattern” was the least common with 10% of dMMR cases.

Figure 4 showcases a 62-year-old male with colorectal adenocarcinoma with pMMR immunohistochemistry status and was Microsatellite stable (MSS) with positive IHC expression for all four MMR proteins.

Figure 5 represents a 68-year-old male with colorectal adenocarcinoma with dMMR Immunohistochemistry status and was MSI-H with negative IHC expression of all four MMR proteins.

Discussion

The annual incidence of CRC worldwide raises a serious public health concern. Familial CRC represents 20% of all CRC and Lynch syndrome represents 3.5% of all CRC. These figures along with the recognition of the role of genomic instability in initiation and progression in CRC, raises the need for molecular screening of all CRC for Lynch syndrome.¹ According to a study by Hashmi et al. (2017), the gold standard for identifying the MSI phenotype is still PCR amplification of microsatellite repeats, although this method is impractical for use in ordinary pathology labs.¹¹ As a result, Hampel (2018) advised IHC be used to screen for Lynch syndrome because it is equally sensitive to PCR, is less expensive, is more widely available, and predicts the nonworking gene, which helps to limit the number of genes that need to be sequenced.¹²

The pathogenesis of colorectal cancer is still unclear, but it could be generally defined in two molecular pathways of genomic instability. One is chromosome instability, which is involved in certain oncogenes and tumour suppressor genes, such as APC, KRAS, and TP53. Another way is microsatellite instability (MSI), which is due to mis-match repair system defect, and it accounts for approximately 15% of all colorectal carcinomas. Microsatellites are simple repetitive sequences of DNA that are scattered throughout the genome. These sequences are stably inherited, vary from individual to individual, and have a low alteration rate. Instability within these sequences has been recognized as a marker for genome-wide mutations and DNA repair deficiencies. MSI has been detected in cancers associated with hereditary non-polyposis colorectal cancer syndrome (HNPCC), as well as in a variety of sporadic cancers, including colorectal cancers associated with ulcerative colitis (UC).^13,14

Mismatch repair proteins are byproducts of MMR genes that are involved in the repair of mismatched bases that may have occurred during DNA replication, genetic recombination, chemical or physical damage.¹⁵ The effects of these mutations include a failure to recognize and repair mismatched nucleotides, as well as insertion/deletion loops induced by slippage of DNA polymerase.¹⁶ As a result, in tumors attributable to MMR protein deficiency, there are insertion and deletion mutations in stretches of short tandem DNA repeats (microsatellites) as well as nucleotide substitutions throughout the genome.¹⁷ Such CRC tends to display high levels of microsatellite instability (MSI-H), whereas in tumors with proficient MMR proteins, they may have either low levels of microsatellite instability (MSI-L) or microsatellite stable (MSS).¹⁷

In our study, out of the 42 colonic adenocarcinoma cases, 20 cases i.e. 47.6% of colonic adenocarcinoma were dMMR. In similar studies, Kalita Lachit et al. found dMMR in 27.1% of CRC.⁷ Pandey et al. using two markers MLH1 and MSH2 found 15.7% of colorectal carcinoma cases with dMMR.¹⁸ Singh et al. performed two marker MLH1 and MSH2 and observed MMR deficient in 63.3% cases.¹⁹ Nayak et al. performed IHC in total 236 cases of colorectal carcinomas using all four MMR proteins and found 22.9% cases with dMMR.²⁰

A study conducted by Kumar A et al. in North India on pattern of mismatch repair protein loss and its clinicopathological correlation in colorectal cancer demonstrated high frequency of MMR protein loss (29%) in colorectal cancer in North Indian patients which was more common in right colon cancers and the sensitivity of IHC to detect MMR loss was 90-92% with use of all four antibodies (MLH1, MSH2, MSH6, PMS2).²¹

Regarding pattern of MMR protein loss, in our study, the loss of nuclear expression of PMS2 was the most common, followed by loss of MSH6, MSH 2 and MLH1 expression which is similar to most other studies.^22,23

In two cases, there was absence of all four MMR proteins; such “null IHC/null pattern” phenotype is very rare. Wang et al. reported one CRC case which was associated with concurrent somatic inactivation of MLH1 and MSH2.²⁴ Hagen et al. also reported one such “null pattern” IHC in a CRC which was associated with germline MSH2 mutation and somatic MLH1 hypermethylation.²⁵

As a useful predictive and prognostic biomarker in CRC, immunohistochemical analysis of MMR proteins is reasonably simple to incorporate into routine testing in laboratories. Before assessing MMR proteins by IHC, tissue fixation and processing must follow established protocols to prevent antigen loss owing to procedural mistakes. In order to effectively manage patients, screen for Lynch syndrome, and find families with predisposed germ line mutations. There is a need for more extensive testing of MMR protein using IHC as a result of the introduction of immunological check point blocking therapy.

Conclusion

Our findings and the previous reports point out high incidence of MSI in our population and highlights the importance of molecular screening of patients with colorectal cancer for MSI using immunohistochemistry. The limitation of this study was the small sample size. A larger, multicenter study comparing the MMR protein expression using immunohistochemistry and comparing it with the gold standard MSI genetic analysis by polymerase chain reaction is recommended.

Conflict of interest

Nil

Source of funding

The study received funding from RGUHS research grant.