Association of clinicopathological features with DNA mismatch repair status among colorectal cancer patients presenting to a Tertiary Care Cancer Hospital

Gayathri G Nair; Vijayalakshmi Nair; Usha Mary Abraham

doi:10.4103/ijamr.ijamr_243_20

ORIGINAL ARTICLE

Year : 2022 | Volume : 9 | Issue : 2 | Page : 107-112

Association of clinicopathological features with DNA mismatch repair status among colorectal cancer patients presenting to a Tertiary Care Cancer Hospital

Gayathri G Nair¹, Vijayalakshmi Nair², Usha Mary Abraham²
¹ Department of Pathology, MES Medical College, Perinthalmanna, Kerala, India
² Department of Pathology, Amala Institute of Medical Sciences, Thrissur, Kerala, India

Date of Submission	20-Oct-2020
Date of Decision	23-Aug-2022
Date of Acceptance	28-Aug-2022
Date of Web Publication	29-Oct-2022

Correspondence Address:
Dr. Gayathri G Nair
Assistant Professor, Department of Pathology, MES Medical College & Hospital, Palachode P.O, Malappuram - 679 338, Kerala
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/ijamr.ijamr_243_20

Abstract

Background: About 12%–15% of sporadic colorectal cancers (CRCs) display a defect in the DNA mismatch repair (MMR) system resulting in microsatellite instability (MSI). Many authors have described certain clinicopathological predictors of MSI and confirmed with ancillary studies. The purpose of this study was to determine the clinicopathological features and their association with MMR deficiency (dMMR) among CRC patients. Materials and Methods: A cross-sectional study was designed among patients presented with CRC. A predesigned proforma was used to document the particulars of the patient along with histological parameters to be assessed. Cases were analyzed for dMMR using MLH1 and MSH2 immunostains and categorized into dMMR and MMR-proficient. The association of clinicopathological features with MMR status was statistically analyzed. Results: Sixty-four CRC cases were analyzed in the study. Thirteen out of 64 cases showed dMMR. Most of the dMMR tumors were located in the right-sided colon (P < 0.001). Three patients with a family history of CRC exclusively had dMMR (P = 0.01). Mucinous (P = 0.04), signet ring cell differentiation (P = 0.04), and lack of dirty necrosis (P < 0.001) showed a significant difference between deficient and proficient MMR categories. Gender, mean tumor-infiltrating lymphocytes per hpf, Crohn's-like reaction, and tumor stage did not show any significant difference between the two categories. Conclusions: Clinicopathological features such as family history, tumor location, tumor size, histologic type, tumor differentiation, mucinous, signet ring cell component, and dirty necrosis are associated with MMR status in CRC.

Keywords: Colorectal cancer, DNA mismatch repair, immunostains, microsatellite instability, mismatch repair deficiency

How to cite this article:
Nair GG, Nair V, Abraham UM. Association of clinicopathological features with DNA mismatch repair status among colorectal cancer patients presenting to a Tertiary Care Cancer Hospital. Int J Adv Med Health Res 2022;9:107-12

How to cite this URL:
Nair GG, Nair V, Abraham UM. Association of clinicopathological features with DNA mismatch repair status among colorectal cancer patients presenting to a Tertiary Care Cancer Hospital. Int J Adv Med Health Res [serial online] 2022 [cited 2023 Apr 2];9:107-12. Available from: https://www.ijamhrjournal.org/text.asp?2022/9/2/107/360034

Introduction

Colorectal cancer (CRC) is the fourth most common cancer among men and third among women worldwide and the majority of them are sporadic.^[1] Fearon and Vogelstein proposed that inactivation of the adenomatous polyposis coli gene led to the initial appearance of adenoma.^[2] Since then, our understanding of the molecular pathogenesis of CRC has improved. Three major pathways involved in carcinogenesis are^[1] chromosomal instability pathway,^[2] microsatellite instability (MSI) pathway, and^[3] CpG island methylation phenotype pathway. The DNA mismatch repair (MMR) system involves mainly four genes – MLH1, MSH2, MSH6, and PMS2. These gene products form heterodimers, i.e., MSH2 with MSH6 and MLH1 with PMS2 which induces ATP-dependent conformational change to mismatched bases thus repairing the DNA.^[3] Microsatellites are more prone to errors during replication.^[4] Therefore, in deficient MMR (dMMR) tumors, the accumulation of mutated microsatellites results in MSI.^[5],[6] About 15% of sporadic CRC were identified to be microsatellite instable.^[7] Lynch syndrome (1%–3% of all CRCs) is an autosomal dominant condition and is associated with germline mutations in MMR genes.^[8] The most common tumors associated with this syndrome are CRC and endometrial carcinomas.

MSI can be screened using immunohistochemistry (IHC) and by polymerase chain reaction. Detection by IHC is quick and relatively simple. A complete absence of nuclear staining within tumor cells with concurrent positive staining in internal control defines MSI. dMMR tumors were proximally located, more common among females, and more frequent in the <40 years age group. This association was found to be significant among first-degree relatives with CRC.^[9] Recent researches are going on to find a significant association between the presence of lymph node metastasis, lymphovascular (LVI) and perineural invasion (PNI), and changes in adjacent mucosa such as ulcerative colitis (UC) with MSI.^[10] Only very few studies on clinicopathological predictors of MSI in CRC have been done in South India. Dubey et al.^[11] and Paraf et al.^[12] included LVI and PNI in their studies but failed to establish a significant association. Sugai et al.^[13] analyzed surrounding normal mucosa for DNA methylation and found that the normal crypts surrounding cancer tissue did not show evidence of a decreasing gradient of methylation with increasing distance from the tumor, irrespective of MMR status, and tumor location. The assessment of MSI has significance in both prognostic and therapeutic aspects. MSI CRC is found to have a better overall prognosis compared to microsatellite stable (MSS) cancers. Further, MSI CRCs are less responsive to 5-fluorouracil-based chemotherapy compared to MSS cancers. The aim of this study was to analyze the association of clinicopathological features with MSI using MLH1 and MSH2 immunostains.

Materials and Methods

Study setting and design

A cross-sectional study was designed in the department of pathology among subjects who had undergone surgery for CRC within a period of 18 months. All the patients who had undergone surgery for CRC within the study period were included in the study. Subjects who have undergone preoperative chemoradiotherapy or were treated with local excision were excluded from the study. Informed consent was taken from patients recruited for the study. The study was conducted after getting clearance from the institutional research committee and institutional ethics committee (AIMSIEC/57/2017).

Study procedure

A predesigned proforma was used to document the particulars of the patient (age, gender, and family history of CRC) along with histological parameters (location, size, and differentiation of tumor, mucinous and signet ring cell component, mean tumor-infiltrating lymphocytes (TIL)/hpf, Crohn's-like reaction, dirty necrosis, etc). Histological diagnosis was rendered on properly oriented, formalin-fixed, and paraffin-embedded hematoxylin and eosin (H and E) stained sections. All the cases were then subjected to immunohistochemical evaluation using markers – MLH1 (ES05 and Dako) and MSH2 (FE11 and Dako). Analysis of MLH1 and MSH2 was done as per the standard procedure. Normal colonic mucosa and stromal and lymphoid cells served as the internal controls. The absence of staining of either MLH1 or MSH2 or both was considered dMMR [Figure 1], and intact nuclear staining was considered MMR-proficient [Figure 2]. All the slides were evaluated by two pathologists. The association of clinicopathological features with these two categories was analyzed.

Figure 1: Photomicrographs showing mismatch repair deficient CRC. (a) Signet ring cell carcinoma (H and E, ×100). Immunostains showing loss of expression of (b) MLH1 and (c) MSH2 (×100). CRC: Colorectal cancer, H and E: Hematoxylin and Eosin

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Figure 2: Photomicrographs showing mismatch repair proficient CRC. (a) Moderately differentiated adenocarcinoma with predominant glandular histology (H and E, ×100). Immunostains showing expression of (b) MLH1 and (c) MSH2 (×100; Internal control is on the right side). H and E: Hematoxylin and Eosin, CRC: Colorectal cancer

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Statistical analysis

The data were entered into a Microsoft Excel worksheet and coded accordingly. It was then analyzed using IBM SPSS version 23 statistical software (Chicago, IL, US). The variables were evaluated using Chi-square/Fisher's exact test. Tumor size was compared using an unpaired t-test. P < 0.05 was considered significant.

Results

A total of 64 CRC cases were included in the study. The distribution of clinicopathologic features is shown in [Table 1]. In the current study, 13 out of 64 cases (20.3%) were dMMR. Among the 13 dMMR cases, loss of MLH1 expression alone was noted in one case, and loss of MSH2 alone in five cases. Seven tumors showed loss of nuclear expression of both MLH1 and MSH2. The age of the study population ranged from 32 to 93 years with a mean age of 62.1 (±12.4) years. The majority of the patients (31.3%) belonged to the age group of 50–59 years. There were 30 females (46.9%) and 34 males (53.1%). Age and gender showed no significant difference between the two categories. Only three cases had a positive family history of CRC; all of them were exclusively dMMR.

Table 1: Distribution of clinicopathological features

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Even though the majority of the cases were in the left-sided colon, 9 of 13 dMMR cases were located in the proximal colon (P < 0.001). The size of the tumor ranged from 2 to 12 cm in greatest dimension with a mean of 4.9 (±2.2) cm. A significant association was noted between tumor size and MMR status (P < 0.042). Larger tumors were noted more in association with dMMR tumors. All of the 64 cases (100%) showed an infiltrative growth pattern and therefore, a significant association with MMR status could not be tested. The majority of cases had glandular histology (82.8%). A combination of glandular and solid patterns was exclusively exhibited in dMMR cases. Thirty-nine cases (60.9%) were moderately differentiated. The undifferentiated tumors were signet ring cell carcinomas. A significant association was noted between tumor differentiation and MMR status in the current study (P = 0.017).

Ten of the 64 cases (15.6%) were mucinous carcinomas (mucinous components comprising >50% of the tumor). Ten out of 13 cases with mucinous components were dMMR cancers. A mean TIL/hpf of >2 was noted only in one case which was dMMR cancer. The remaining cases had a mean TIL of 2 or less. The mean TIL/hpf between dMMR and MMR was not significantly different (P = 0.203). All the 13 dMMR tumors lacked dirty or garland necrosis and thus the results showed a significant difference between the two categories. Only 10 out of 64 cases showed Crohn's-like reaction; this was not statistically significant (P = 0.345). A significant association between dMMR (P = 0.508) and LVI/PNI (P = 0.914) could not be established in our study. None of the cases exhibited any changes in adjacent mucosa such as UC. The majority of the dMMR tumors were without lymph node involvement (69%). Among the 13 dMMR tumors, the maximum number of cases (30.7%) belonged to Stage II A. There was no significant difference (P = 0.105) in tumor stage between MMR and MMR-proficient groups.

Discussion

Among the 64 cases, 13 (20.3%) showed dMMR of which 1 case showed loss of MLH1 (7.6%) expression alone and 5 cases showed loss of MSH2 (44%) expression alone. Seven tumors showed loss of nuclear expression of both MLH1 and MSH2. A recent study by Quintanilla-Guzman et al.,^[14] among 47 CRC patients showed loss of MLH1 expression in 20/47, loss of MSH2 in 1/47, and loss of both MLH1 and MSH2 expression in 3/47 cases. A high MSH2 loss evidenced in this study may be due to the small sample size. Rai et al. reported 44% loss of MSH2 and 33.3% loss of MLH1 among CRC patients in South India.^[15] Since the loss of MLH1 or MSH2, or both, will result in the concurrent loss of latter proteins, determination of MLH1 and MSH2 alone may be sufficient to screen dMMR status in tumors.^[16] Despite the population in this study being small, almost equal numbers of cases from both genders were included over a wide age range. It was observed that the majority of the dMMR cases occurred in age groups above 50 years (69%) in contrary to the observations made by Greenson et al.,^[17] Huang et al.,^[18] and Jenkins et al.^[19] This may be due to the increased number of younger patients included in their study which increased the probability of MSI.

A larger tumor size, proximal location, and histological heterogeneity were noted to be associated with dMMRCRC. Dubey et al.,^[11] Huang et al.,^[18] and Greenson et al.^[17] found that right-sided location is a strong predictor of MSI. Poor differentiation and undifferentiated tumors showed an increased association with dMMR tumors in our study.

The molecular classification of CRC will help to identify patient subgroups at varying risk of recurrence and death and for whom personalized approaches to therapy may be beneficial. CRC with MSI tumors has consistently been associated with better stage-adjusted survival compared to MSS tumors, and hence may respond differently to 5-FU-based adjuvant chemotherapy.^[20] This variability underscores the need for robust prognostic and predictive biomarkers to guide therapeutic decision-making including the use of adjuvant chemotherapy. To increase the identification of MSI among CRCs, it is recommended that all resected CRCs should be analyzed for MMR status.^[4] In this study, we have attempted to recognize the unique clinicopathological features of MSI tumors which will help in their identification and can then be subjected to ancillary studies.

Ten out of 13 cases with mucinous components (8 cases with >50% and 2 cases with <50% mucinous component) were dMMR. Three of 4 cases with signet ring cell carcinoma (involving >50% of the tumor area) were found to be dMMR. Greenson et al.^[21] established that any percentage of mucinous and signet ring cell differentiation was a significant predictor of MSI. TILs can activate CD3+CD8+T lymphocytes and are seen as small blue mononuclear cells that typically have a halo around them. We counted lymphocytes infiltrating between tumor cells in five consecutive 40× fields, after scanning for areas with maximum TILs following which we calculated the mean. The genetic instability in dMMR CRC leads to the production of mutated peptides that function as tumor-specific antigens. For an antigen-specific response, these antigens must be presented in an HLA-restricted manner. In dMMR CRC, there is an increased frequency of mutations in β₂-microglobulin which is necessary for major histocompatibility complex Class I presentation.^[22] This may be responsible for the emergence of immunoresistant neoplastic cell clones that can escape tumor-specific CD8+ cytotoxic T lymphocyte recognition.^[23],[24] Researchers have observed a value of >2 mean TIL/hpf as a predictor for MSI.^[11] Only one case in our study showed > 2 TIL/hpf. Paraf et al.^[12] identified TIL using CD3 and found a significant association which must have contributed to their significant result. In our study, TILs were counted on routine H and E sections alone, and no immunostaining was performed. This could be a probable explanation for the insignificant result obtained.

All 13 dMMR tumors lacked dirty or garland necrosis. More than 90% of dMMR tumors did not show Crohn's-like reaction. A lack of dirty necrosis and the presence of Crohn's-like reaction are considered strong independent predictors of MSI in CRC.^[16],[25] LVI, PNI, and lymph node metastasis were not as important in elucidating the histologic characteristics of MSI CRC.^[26] We tried to analyze these parameters and their association with MMR in this study, though a significant association could not be established. More research is needed to delineate the role of these three characteristics since they are usually associated with a poor prognosis and influence the treatment protocol.^[10] Another histologic feature, yet to be systematically assessed, changes in adjacent mucosa especially UC because the risk of developing CRC is high among patients with long-standing UC. None of the cases in our study showed any such changes.

Most authors observed that MSI tumors were more common in Stage II with an improved prognosis. In our study, the prognosis of the patients was not assessed. Sixty-four cases were distributed among stages from I to IV A with the majority in Stage III B (29.7%), followed by Stage II A (20.3%). The study population comprised predominantly of MMR-proficient tumors. Among the 13 dMMR tumors, the maximum number of cases (30.7%) belonged to Stage II A. Huang et al.^[18] and Raut et al.^[27] found that low stage is a statistically significant predictor of MSI compared to MSS. We were able to establish a significant association of various parameters with two IHC markers MLH1 and MSH2, which are considered obligate proteins. Our sample size of 64 cases was small; larger, multicentric studies are needed to confirm our findings.

Conclusions

In this study, significant associations were identified between many clinicopathologic parameters and MMR status in CRCs. The potential utility of clinicopathological features, combined with IHC, as a screening tool to predict the MMR status must be evaluated in future studies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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