Mismatch Repair Deficient (dMMR) Colorectal Carcinoma in a Pakistani Cohort: Association With Clinical and Pathological Parameters

Introduction Microsatellite instability (MSI) is an important pathway in colorectal carcinoma (CRC) pathogenesis. MSI occurs due to mutations in mismatch repair (MMR) genes that include MutL protein homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2), MutS homolog 2 (MSH2), and MutS homolog 6 (MSH6). CRC with MSI is termed MMR deficient (dMMR) CRC. Conversely, CRC with intact MMR genes is called microsatellite stable (MSS) or MMR proficient (pMMR). In this study, we compared the clinicopathological features of dMMR CRC with pMMR CRC. Methods It was a retrospective study conducted in the Department of Histopathology, Liaquat National Hospital, Karachi, Pakistan, from March 2020 to February 2022, over a duration of two years. Biopsy-proven cases of CRC with upfront surgical resection were included in the study. Microscopic examination was performed to evaluate tumor type, grade, and extent of invasion, presence of necrosis, perineural invasion (PNI), lymphovascular invasion (LVI), peritumoral lymphocytes (PTL), intratumoral lymphocytes (ITL), and nodal metastasis. Immunohistochemical staining was performed using antibodies, namely, MLH1, PMS2, MSH2, and MSH6. Any loss of nuclear expression in tumor cells was termed dMMR or microsatellite instable, whereas the intact nuclear expression in tumor cells was labeled as MSS or pMMR. Results A total of 135 cases of CRC were included in the study. The mean age at diagnosis was 46.76 ± 17.74 years, with female predominance (60.7%). The loss of MLH1, PMS2, MSH2, and MSH6 expression was noted in 39.3%, 34.1%, 17.8%, and 16.3% cases, respectively. Overall, 59.3% of CRCs were pMMR, while 40.7% were dMMR. A significant association of MMR status was noted with respect to age, PNI, LVI, tumor grade, tumor (T) and nodal (N) stage, mucinous differentiation, and ITL. dMMR CRC was significantly above 50 years than pMMR CRC. The frequency of PNI and LVI was lower in dMMR CRC than in pMMR CRC. Conversely, the higher grade (grade 3) and higher T-stage (T4) were associated with dMMR CRC. Alternatively, the frequency of higher N stage (N2b) was more commonly seen in pMMR CRC. Moreover, mucinous differentiation and ITL were significantly associated with dMMR CRC. Conclusion A significant proportion of CRC patients in our population demonstrated dMMR status. dMMR CRC had a higher histological grade with a higher frequency of mucinous differentiation and higher T-stage. Conversely, the presence of LVI, PNI, and higher N stages were associated with pMMR CRC.

Approximately 10-15% of CRCs are dMMR, among which 80% are sporadic owing to MLH1 promoter hypermethylation or v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations, whereas 20% are due to autosomal dominant germline mutations, termed Lynch syndrome (LS) [7]. MSI screening for CRC is initially performed through immunohistochemical (IHC) analysis for MLH1, PMS2, MSH2, and MSH6. The loss of one or two markers mandates MLH1 promoter methylation status and BRAF mutation analysis. The absence of BRAF mutation and MLH1 promoter hypermethylation prompts next-generation sequencing for germline mutations. Previous studies have shown that dMMR CRC is associated with certain histological features, such as mucinous histology, peritumoral lymphocytes (PTL), and intratumoral lymphocytes (ITL) [6]. CRC in the Pakistani population was found to be at a higher tumor (T) and nodal (N) stage than western CRC [8,9]. Clinicopathological features of dMMR CRC have not been widely studied in our population; therefore, we conducted this IHC-based MSI analysis in CRC to better understand the pathogenesis and pathological features of dMMR CRC.

Materials And Methods
It was a retrospective study conducted in the Department of Histopathology, Liaquat National Hospital, Karachi, Pakistan, from March 2020 to February 2022, over a duration of two years. Biopsy-proven cases of CRC with upfront surgical resection were included in the study. Cases with incomplete clinical or pathological records were excluded. Similarly, CRC with neoadjuvant chemotherapy before surgical resection or systemic metastasis at the time of diagnosis was excluded from the analysis. Institutional Review Board (IRB) approval was not needed as it was a retrospective study, and the institution don't mandate IRB approval for retrospective studies.
Surgical specimens were received in the histopathology laboratory, followed by overnight fixation. Gross dimensions of the tumor were recorded, and representative sections were submitted from the tumor along with adjacent normal mucosa and surgical resection margins. Lymph nodes were dissected from the mesenteric tissue and submitted for microscopic examination. Haematoxylin and eosin-stained microscopic sections were examined for tumor type, grade, and extent of invasion, presence of necrosis, perineural invasion (PNI), lymphovascular invasion (LVI), T-and N-stage, PTL, ITL, and nodal metastasis. Histological tumor typing was performed according to the World Health Organization (WHO) classification of the tumors of the digestive tract. Mucinous differentiation was labelled when there were extracellar mucin pools, whereas signet ring differentiation was called when tumor cells show intracytoplasmic mucin vacuole pushing the nucleus to the periphery. Tumors showing more then 50% mucinous and signet ring differentiation were termed mucinous and signet ring adenocarcinoma, respectively. Tumors were graded according to the proportion of gland formation. Well-differentiated/grade 1 tumors had more than 90% gland formation, whereas grade 2 and grade 3 tumors were labeled when tumor showed 6-50% and less than 50% gland formation, respectively. T and N staging was performed according to the American Joint Committee on Cancer (AJCC) staging system. PTL and ITL were categorized into none, mild to moderate, and marked according to the College of American Pathologist's (CAP) guidelines. PTL is labelled when tumor periphery showed lymphoid follicles with germinal center formation. ITL corresponds to lymphocytes within tumor cells. More than three lymphocytes per high-power field were required for marked ITL categorization.

Immunohistochemical analysis
IHC staining was performed using antibodies, namely, MLH1, PMS2, MSH2, and MSH6 antibodies on representative tumor blocks that contained adjacent normal mucosa for the documentation of internal positive control. Any loss of nuclear expression in tumor cells was termed dMMR, whereas intact nuclear expression in all tumor cells was labeled as MSS or pMMR [10].

Statistical analysis
Data analysis was performed using IBM SPSS Statistics for Windows, Version 26.0 (Released 2019; IBM Corp., Armonk, NY). The mean was calculated for patient age, while frequencies and percentages were calculated for other clinicopathological variables. Chi-square and Fisher's exact tests were applied to determine the association of various clinicopathological features with MMR status. A p-value of <0.05 was considered significant.

Results
A total of 135 cases of CRC were included in the study. The mean age at diagnosis was 46.76 ± 17.74 years, with female predominance (60.7%). Rectosigmoid was the most common tumor location (67.4%), followed by cecum and splenic flexure (11.9%). The most common tumor type was adenocarcinoma, not otherwise specified (65.2), followed by mucinous carcinoma (24.4%). The most common tumor grade was grade 2 (67.4%), while PNI and LVI were present in 33.3% and 23.7% cases, respectively. Nodal metastasis was seen    Table 2 shows the association of the MMR status of CRC with clinicopathological variables. A significant association of MMR status was noted with respect to age, PNI, LVI, tumor grade, T and N-stage, mucinous differentiation, and ITL. dMMR CRC was significantly above 50 years than pMMR CRC. The frequency of PNI and LVI was lower in dMMR CRC than in pMMR CRC. Conversely, the higher grade (grade 3) and higher Tstage (T4) were associated with dMMR CRC. Alternatively, the frequency of higher N-stage (N2b) was more commonly seen in pMMR CRC. Moreover, mucinous differentiation and ITL were significantly associated with dMMR CRC.

Discussion
We found a relatively high dMMR CRC in our population (40.7%). MLH1 was the most frequently deficient MMR marker in our cohort of cases, followed by PMS2, MSH2, and MSH6. dMMR CRCs were associated with mucinous histology and ITL. With respect to prognostic parameters, dMMR CRCs were associated with higher grade and T-stage, whereas the frequency of PNI, LVI, and higher N-stage was lower.
There has been an interest in dMMR CRC recently owing to differences in prognosis and the role of immunotherapy. dMMR CRC can be due to germline mutations owing to LS or sporadic BRAF mutations or MLH1 promoter hypermethylation. They found a significant association of dMMR status with age, gender, and tumor site, whereas no significant association was noted with T-and N-stages [11]. Conversely, Liang et al. [12] in a study including 61 CRC patients concluded that there was a significant association of dMMR status with age (<55 years), female gender, location (right colon), tumor size (>5 cm), T-stage (T4), high grade, and mucinous differentiation. We also found a significant association of dMMR CRC with T4 stage, higher tumor grade, and mucinous differentiation, however, the association of dMMR CRC with younger age was not established in our study. Similarly, Liang et al. [12] did not find any association of dMMR status with LVI, as noted in our study. Sacdalan et al. [13] also reported aggressive histological features (poor differentiation, mucinous histology) of dMMR CRC in Filipino patients. Concordant with our findings, mucinous and poor differentiation were found in dMMR CRC [14].
Other histological features of dMMR CRC include the presence of immune cell infiltration (PTL and ITL). PTL corresponds to a Crohn's-like inflammatory reaction at the periphery of the tumor, whereas ITL is the presence of inflammatory cells within the cancer cells. This unique feature of dMMR CRC was attributed to elevated mutational burden and neoantigen overload that results in an immune reaction against tumor cells [15]. We found a significant association of dMMR CRC with ITL, whereas no significant association was established with PTL.
Despite aggressive histological features, there is a survival benefit in CRC with dMMR status. Previous studies have reported better overall survival in dMMR CRC than pMMR CRC, especially in early-stage CRC [16]. We did not evaluate survival in our study but found a significant association of dMMR status with lack of PNI, LVI, and lower N-stage signifying prognostically better pathological features in dMMR CRC.
Another significance of dMMR status is the predictive role of immunotherapy. There is a promising role of immunotherapy in dMMR metastatic CRC, while its role in pMMR CRC is being sought [17]. We did not evaluate the predictive value of dMMR status in our study.

Limitations
The main limitation of our study was the lack of clinical follow-up to evaluate the overall and disease-free survival of dMMR CRC. Second, molecular studies for BRAF, MLH1 promoter hypermethylation, and nextgeneration sequencing for germline mutations for LS were not performed. Additionally, there is an increasing interest in the role of immunotherapy in dMMR CRC that we did not evaluate in our study. Moreover, this was a single-center study; therefore, the sample size was also limited.

Conclusions
In this study, we evaluated dMMR status in CRC and found that a significant proportion of CRC cases were dMMR in our study population. We noted that poor differentiation (high grade) and mucinous histology were associated with dMMR CRC. Moreover, dMMR CRC was of higher T-stage than pMMR in our study. Conversely, dMMR CRC was associated with better prognostic features, such as lower N-stage and lower frequency of PNI and LVI. Due to these distinctive features of dMMR CRC, evaluating the dMMR status in