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Abstract
Context: Globally, colorectal carcinoma (CRC) ranks the third most commonly diagnosed malignant disease, one of the leading causes of cancer deaths. Aims: To study the spectrum of clinicopathological characteristics of sporadic colorectal carcinoma and to assess mismatch repair gene deficiency by the expression pattern of the proteins assessed by immunohistochemistry. Setting and Design: Observational study conducted in a tertiary care hospital in West Bengal. Materials and Methods: Fifty-two surgically resected specimens of CRC received from January 2018 to May 2019 were studied for clinical, morphological, MSI status. Statistical Analysis Used: IBM SPSS 23. Results: A total of 50% of the cases belonged to younger and 50% to the older population, with male predominance being 53.8%. The most common histologic type was adenocarcinoma (88.5%). The majority was found to be well-differentiated carcinoma (50%). The majority cases were of the T3 stage accounting to 38.5%. A total of 24 out of 52 cases (46.15%) had an absent expression of at least one mismatch repair (MMR) protein. A significant correlation was found between the young age group and microsatellite instability (MSI) with a P value of 0.001. A significant association was found between MSI and tumor differentiation with P value of 0.018. A significant association was found between MSH6 and histological type with P value of 0.012. A significant association was found between MSI and tumor stage with P value of 0.032. Conclusions: This study shows a significantly higher number of sporadic colon cancers involving the young age group, and younger cases showed significant association with MSI. This alarming trend needs validation by studies involving larger populations and can be helpful prognostically as well as in formulating chemotherapeutic regimens.
Keywords: Colorectal carcinoma, microsatellite instability, sporadic, young age
How to cite this article: Introduction 
GLOBOCAN 2008 estimate shows colorectal carcinoma (CRC) is the third most commonly diagnosed cancer and the fourth leading cause of death in the world.[1] GLOBOCAN 2018 shows new colorectal cancer cases in India to be 20,064 (3.4%). CRC is a heterogeneous disease caused by the interaction of genetic and environmental factors with significant differences between clinical presentation, prognosis, and individual treatment response. CRC development is associated with various driver mutations and genetic and epigenetic tumor signatures.[2] Microsatellite instability (MSI) is a significant genetic marker that can be useful in the diagnosis, prognosis, and prediction of chemotherapeutic treatment efficacy. Sporadic CRCs with high-frequency MSI (MSI-H) are most often caused by epigenetic inactivation of the MLH1 gene by somatic promotor hypermethylation.[3],[4],[5],[6]
Study setting and design
This study is an observational cross-sectional study conducted at the department of pathology in a government medical college in Kolkata. Ethical committee approval was obtained from the Institutional Ethical Committee.
[TAG:2]Materials and Methods[/TAG:2]
A total of 52 resected specimens of CRC received in the department of pathology from January 2018 to May 2019 were studied for their clinical, morphological, and MSI status. The cases studied were taken consecutively. Clinical presentation and site of tumor involvement were noted. Tumors were categorized as adenocarcinoma, mucinous carcinoma, signet ring cell carcinoma. The presence of intratumoral lymphocytes (ITL) and tumor budding was noted in all the cases. Tumors were grouped as well-differentiated, moderately differentiated, and poorly differentiated carcinoma. Vascular emboli (LVI) were searched in the tumors and adjacent tissues. Perineural invasion (PNI) and lymph nodal involvement were also searched.
Ethical committee
The study was performed after approval from Institutional Ethical Committee.
Inclusion criteria
All newly diagnosed cases of colorectal carcinoma undergoing preoperative biopsy and potentially curative resection in our institution.
Exclusion criteria
All cases having a history of preoperative chemotherapy or radiotherapy or family history of colorectal carcinoma and clinical evidence of infection.
No familial genetic studies were performed.
MSI status
To assess the microsatellite MSI status, immunohistochemistry was done using a four antibody panel of mismatch repair (MMR) proteins namely MHL1, MSH2, MSH6, PMS2 (Bio Gene X, TSIIC SEZ for Aerospace and Precision Engineering, Adibatla R. R District Telengana 501510) on paraffin-embedded tissue blocks. Immunohistochemistry (IHC) was done manually. The sections were deparaffinized in xylene, rehydrated in alcohol, and washed in distilled water. Antigen retrieval was done using Tris ethylenediaminetetraacetic acid (EDTA) buffer in a microwave at 800 watts for 10 min for two cycles. Primary antibodies (MLH1, MSH2, MSH6, PMS2) were added which were ready to use monoclonal antibodies.
The interpretation was done using the College of American Pathologists (CAP) protocol for IHC. Any nuclear staining was taken as “no loss of expression” and only complete absence of nuclear staining was taken as “loss of expression” provided internal controls were negative. If internal non-neoplastic tissues showed invalid negative staining, the procedure was routinely repeated. Loss of any of the markers was taken as microsatellite instability positive and called as microsatellite instability low (MSI-L) for <30% loss of markers, loss of more than one marker was taken as microsatellite instability-high (MSI-H) for >30% loss of the markers, and no loss of the markers was taken as microsatellite stable (MSS).
Results A total of 52 cases were studied.
A total of 50% of the cases belonged to the younger population i.e., aged <40 yrs (26 out of 52 cases) and 50% of the cases belonged to >40 yrs of age, with male predominance accounting to 53.8% (28 out of 52 cases). The most common presentation was per rectal bleeding comprising 42.3% followed by bowel obstruction comprising 36.5% of the cases. The majority of the cases in the study comprised left-sided tumors i.e., rectal tumors accounting to 73.1% (38 out of 52 cases), and right-sided tumors comprised 26.9% of the cases.
The most common histologic type in the study was found to be adenocarcinoma accounting for 88.5% of the cases (46 out of 52 cases). Other histologic types found in the study were mucinous carcinoma comprising four cases, and signet ring cell carcinoma comprising two cases. The majority of the cases in our study were found to be well-differentiated carcinoma comprising 50% of the cases followed by moderately differentiated carcinoma accounting to 40.4% of the cases, and only 9.6% of the cases were poorly differentiated carcinoma. Most cases in the study were found to be of higher T stage with T3 stage accounting to 38.5% followed by T4 stage accounting to 34.6%. A total of 19.2% of the cases belonged to the T2 stage and only 7.7% of the cases belonged to the T1 stage. The majority of the cases in the study were found to be of the N1 stage comprising 59.6% of the cases followed by the N0 stage 26.9% of the cases. Only 3.8% of the cases belonged to the N2 stage, and in 9.6% of thecases, the N stage could not be determined. A total of 33 out of 52 cases had intratumoral lymphocytes (ITL) accounting to 63.5%. Lymphovascular invasion (LVI) was found in 23 cases and not found in 29 out of 52 cases accounting to 55.8% of the cases, and PNI was found in 19 cases and not found in the majority of cases i.e., 33 out of 52 accounting to 63.5%. Tumor budding was found in 24 cases, and it was not found in 28 cases.
MSI status
Assessment of MSI status by MLH1, MSH2, MSH6, and PMS2 by IHC:
A total of 24 cases out of 52 cases (46.15%) had absent expression of at least one MMR protein (MLH1, MSH2, MSH6, PMS2) i.e., MSI positive with 19 MSI-H (36.53%, 19/52), 5 MSI-L (9.61%, 5/52), and the rest 28 cases were MSS [Figure 1]a and [Figure 1]b. Twelve cases (23.07%, 12/52) showed absent expression of all the four MMR proteins. Three tumors (5.7%, 3/52) displayed absent coexpression of MLH-1 and PMS2. Two MLH1 negative tumors showed intact PMS2 expression, and two PMS2 negative tumors showed intact MLH1 expression. Two (3.8%, 2/52) cases showed absent coexpression of MSH2 and MSH6. All the MSH6 tumors showed loss of expression of MSH2 except one which showed intact expression of MSH2. Two cases showed loss of MSH2 with an intact expression of MSH6. One case showed loss of expression of MLH1, MSH2, MSH6 with intact PMS2. Isolated loss of MLH1 expression was seen in one case. Isolated loss of PMS2 was seen in one case. Two cases showed isolated loss of MSH2, and one case showed isolated loss of MSH6. One case showed absent expression of MSH2 and PMS2. Eighteen out of 26 cases i.e., 69.23% had MSI who were aged <40 yrs. A significant correlation was found between the young age group and microsatellite instability with a P value of 0.001 as shown in [Table 1].Twelve out of 24 female cases were MSI positive which accounted for 50%, and 12 out of 28 male cases were MSI positive accounting to 42.86%. However, no significant correlation was found between sex predilection and MSI. Fifteen out of 38 left-sided tumors had mismatch repair deficiency (MMRd) accounting to 39.47%, and 9 out of 14 right-sided tumors had MMRd accounting to 64.29%. Grossly, right-sided tumors showed more loss of MMR proteins although no significant correlation was found with MSI. Eleven out of 19 cases (57.89%) with bowel obstruction had MMRd, 5 out of 7 cases (71.43%) with mass felt per abdomen had MMRd. Mass per abdomen and bowel obstruction exhibit significantly higher loss of MLH1. Fifteen out of 26 well-differentiated cases were MSI positive i.e., 57.69% [Figure 2], 5 out of 21 moderately differentiated cases had MMRd, and 4 out of 5 cases of poorly differentiated cases had MMRd as shown. A significant association was found between MSI and tumor differentiation as shown in [Table 2]. Nineteen out of 46 adenocarcinomas were found to have MMRd accounting to 41.3%, 3 out of 4 mucinous carcinomas were MSI positive (75%), and 2 out of 2 signet ring cell carcinoma had MMRd accounting to 100% [Figure 3]a and [Figure 3]b. A significant association was found between MSH6 and histological type with a P value of 0.012 [Table 3]. One out of 4 T1 cases were found to have MSI positive accounting to 25%. One out of 10 T2 cases (10%) were found to have MMRd. Eleven out of 20 T3 cases (55%) were found to have MMRd. Eleven out of 18 T4 cases (46.15%) were found to be MSI positive. A significant association was found between MSI and tumor stage as shown [Table 4]. One out of 5 cases (20%) of Nx tumors had MMRd, 7 out of 14 cases of N0 tumors had MSI (50%), 15 out of 31 cases of N1 tumors had MSI positive (48.59%), 1 out of 2 cases of N2 tumors (50%) had MMRd. No significant association was found between MSI and N stage with a P value of 0.759. Neither significant association was found between the M stage and MMRd. Seven out of 23 cases of LVI-positive tumors had MMRd, and 16 out of 23 cases (69.57%) of the LVI-positive cases were MSS. MSI exhibited a negative association with LVI. No significant association was found between MSI and PNI status of the tumor. Eighteen of the 33 (54.55%) ITL positive tumors were found to have MSI, and 15 of the 33 (45.45%) ITL positive tumors were found to be MSS. Though we have got a higher number of ITL positive cases in MSI, statistically significant association could not be established. Studies with a greater number of cases could be helpful. Thirteen out of 24 cases that had tumor budding were found to be MSS accounting to 54.17% i.e., tumor budding was found more in MSS tumors.
Figure 1: Photomicrograph of MSI negative (MSS) well differentiated adenocarcinoma (a) MLH 1 positivity (b) MSH 2 positivity (× 100)
Figure 2: Photomicrograph of MSI positive well differentiated adeno carcinoma showing MLH 1 negativity (× 40)
Figure 3: Photomicrograph of MSI-H Signet ring cell carcinoma with (a) MSH 2 negative (× 400) (b) MSH 6 IHC negative stain (× 100)
Table 1: Correlation between young age group and microsatellite instability Discussion Colorectal cancer incidence rises sharply after the age of 45, and 90% of cases occur in persons over the age of 50.[7] In the study conducted by Sudarshan et al.,[8] 39.05% of the cases were presented before the age of 40 years. Similar results were found in the study conducted by Patil et al.,[9] 33% of the cases were <40 yrs. In this study, 69.23% (18/26) cases were MSI +ve among those aged <40 yrs out of which 42.85% were MSI-H. A significant association was found between MSI-positive tumors and young age group <40 yrs with P = 0.001.
Among carcinoma of the colon and rectum, rectal carcinoma is more common. Men show a proportionately higher incidence of rectal cancer than women.[10] In this study, the majority of the cases were left-sided tumors i.e., rectal tumors (73.1%, 38/52) and only 26.9% were right-sided tumors. A higher incidence of rectal cancer was in males comprising 52.63% of the cases (20/38).
Colorectal carcinomas with MSI-H in the study conducted by Yuan et al.[5] display a predilection for the right colon. In this study, 64.28% (9/14) of the proximal colon tumors were MSI +ve, and 57.14% were MSI-H. More than 90% of colorectal carcinomas originate from epithelial cells of the colorectal mucosa and are adenocarcinomas.[11] In this study, we got three histological subtypes of which the commonest being adenocarcinoma comprising 88.46% of the cases (46 out of 52 cases), whereas mucinous carcinoma comprised 7.7% (4 cases) and signet ring cell carcinoma 3.8% (2 cases).
Studies have shown a significant difference between MMRd and MSS tumors with MSI-H associated with mucinous and signet ring differentiation.[12],[13],[14],[15] In this study, all of the signet ring cell carcinoma (100%) showed MSI-H, and 75% of the mucinous carcinoma (3 out of 4 cases) showed MMRd of which 50% were MSI-H. This shows a strong trend of MSI in mucinous and signet ring cases although difficult to conclude with such a small number of cases. Statistically, a significant association could only be established between the loss of MSH6 and mucinous and signet ring cell histological subtype with P = 0.012.
The study conducted by Yuan et al.[5] showed 32.3% of CRCs associated with MSI-H had poor differentiation as compared to MSS CRCs which was found to be (19.6%). In this study, 50% (26/52) of the cases were well-differentiated carcinoma, 40.4 (21/52) of the cases were moderately differentiated, and 9.62% (5/52) of the cases were poorly differentiated.
Among the well-differentiated tumors, 57.69%, (15/26) had MMRd out of which 50% were MSI-H. Among the moderately differentiated tumors, only 23.80% (5/21) were MSI +ve and among poorly differentiated tumors, 80% (4/5) had MMRd. Three out of 5 cases (60%) of the poorly differentiated had MSI-H. A significant association was found between MSI and poor tumor differentiation with P = 0.018.
Most cases in our study (38 cases) comprised T3 and T4 stage of the disease, which accounted for 38.46% (20/52) and 34.61% (18/52), respectively i.e., late T stage comprised 73.1% of all the cases. Only 14 cases belonged to the early T stage, T1 comprising 7.7% of the cases (4 cases) and T2 comprising 19.21% of the cases (10 cases). In the study conducted by Patil et al.[9], 50.7% of the cases were T3, and 28.8% were T4. The patients with MSI-H colorectal carcinoma in a study showed to have relatively lower stage compared with patients with MSS tumor, with 32.4% of MMRd CRCs and 52.5% of MSS CRCs at III/IVTNM stage.[5] In this study, 57.89% (22/38) of late T stage were MSI +ve out of which 44.73% (17/38) had MSI-H. As we have got a low number of T1 and T2 cases as compared to T3 and T4, the association between MSI and late T (T3 and T4) stage should be interpreted with caution despite a significant P- value of 0.032 in this study population. MMRd tumors are less prone to metastasize to lymph nodes or distant organs as compared to MSS ones, with only 30.9% and 2.9% of tumors have lymph nodes and distant metastasis, respectively.[5] Sixteen of the 24 MSI cases had lymph node positivity (66.66%). In this study, maximum cases were N1 stage (59.61%, 31/52). Among the N1 cases, 48.38%, (15/31 had MMRd of which 32.25% cases were MSI-H. Among the N0 cases, 50%, (7/14 had MMRd. No association was found between the N stage as well as the M stage with MSI.
Lymphocytic infiltration is generally recognized as a striking characteristic of MMRd CRCs, as showed by previous studies issued,[12],[14],[15] The majority of cases in this study had intratumoral lymphocytic infiltrate comprising 65.38% of the cases (33/52). Eighteen of the 33 (54.55%) ITL positive tumors were found to be MSI, and 15 of the 33 (45.45%) cases were found to be MSS. Though we have got a higher number of ITL positive cases in MSI, statistically significant association could not be established. Studies with a greater number of cases could be helpful.
MMRd tumors have a less tendency to metastasize to lymph nodes or distant organs compared to MSS,[5],[16],[17],[18] and that possibly explains for low LVI. In this study, 7 out of 23 cases of LVI positive tumors had MMRd, and 16 out of 23 cases (69.57%) of the LVI positive cases were MSS. Significantly higher positive LVI patients exhibited no loss of the MMR proteins. Therefore, MSI exhibited a negative association with LVI with P = 0.043.
In a study conducted by Graham et al.[19], high tumor budding was associated with advanced pathologic stage (P < 0.001) and microsatellite stability (P = 0.005). Eleven out of 24 cases who had tumor budding were found to be MSI positive accounting to 45.83%. Whereas 13 out of these 24 cases were found to be MSS, accounting to 54.17%. In this study, tumor budding was found more in MSS tumors which is in concordance with previous studies. MSI accounted for approximately 15% of all colorectal carcinomas. Most of which were sporadic MSI i.e., 12%.[5]
Conclusion This study shows a significantly higher number of sporadic colon cancers involving the young age group, and younger cases showed significant association with MSI. This alarming trend needs validation by studies involving larger populations and can be helpful prognostically as well as in formulating chemotherapeutic regimens. The present study also suggests a trend towards mucinous and signet ring carcinomas being associated with the loss of MMR protein, which needs to be corroborated with further studies involving a larger population. There is positive association of MSI with poor tumor differentiation. To conclude, MSI is associated with distinct clinicopathological parameters and hence would be useful in the prognostic evaluation and determining therapeutic approach.
Financial support and sponsorship
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Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_531_21
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