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Year : 2019  |  Volume : 56  |  Issue : 4  |  Page : 309--314

Microsatellite instability in young patients with mucinous colorectal cancers – characterization using molecular testing, immunohistochemistry, and histological features

Nitty Skariah Mathews1, Dipti Masih1, Rohin Mittal2, Benjamin Perakath2, Dhananjayan Sakthi1, Grace Rebekah3, Rekha Pai1, Anna B Pulimood1,  
1 Department of General Pathology, Christian Medical College, Vellore, Tamil Nadu, India
2 Department of Colorectal Surgery, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Biostatistics, Christian Medical College, Vellore, Tamil Nadu, India

Correspondence Address:
Nitty Skariah Mathews
Department of General Pathology, Christian Medical College, Vellore, Tamil Nadu
India

Abstract

CONTEXT: The incidence of colorectal cancers (CRCs) in young Indian patients is higher than the international average. CRCs in young patients are commonly of mucinous type and show microsatellite instability (MSI). AIMS: To ascertain the MSI status of mucinous CRCs in patients ≤40 years of age by molecular testing and to correlate this with immunohistochemical (IHC) analysis and tumor histology. SUBJECTS AND METHODS: Archived formalin-fixed paraffin embedded tissue blocks of 30 young mucinous CRC patients were retrieved. MSI testing was done using two mononucleotide markers – BAT26 and NR24. IHC analysis was done using MLH1, MSH2, and MSH6. Histological features of all cases were studied. Data were analyzed using the SPSS software and the Pearson's chi-square test and Fisher's exact test. RESULTS: Eight out of 30 cases (26.7%) showed MSI by molecular testing. IHC identified seven of these cases. Histological features showing a statistically significant association with MSI were the presence of a well-differentiated adenocarcinoma component (P = 0.003), peritumoral lymphocytes (P = 0.002) and tumor budding (P = 0.021). CONCLUSION: The detection of defective mismatch repair (MMR) proteins using IHC for MLH1, MSH2, and MSH6 and molecular testing using BAT26 and NR24 appears to be a good protocol to detect CRCs with MSI. Histology could be useful in identifying cases that require screening for presence of MMR protein defects.



How to cite this article:
Mathews NS, Masih D, Mittal R, Perakath B, Sakthi D, Rebekah G, Pai R, Pulimood AB. Microsatellite instability in young patients with mucinous colorectal cancers – characterization using molecular testing, immunohistochemistry, and histological features.Indian J Cancer 2019;56:309-314


How to cite this URL:
Mathews NS, Masih D, Mittal R, Perakath B, Sakthi D, Rebekah G, Pai R, Pulimood AB. Microsatellite instability in young patients with mucinous colorectal cancers – characterization using molecular testing, immunohistochemistry, and histological features. Indian J Cancer [serial online] 2019 [cited 2019 Dec 12 ];56:309-314
Available from: http://www.indianjcancer.com/text.asp?2019/56/4/0/267596


Full Text



 Introduction



Incidence of colorectal cancer (CRC) among the young is increasing worldwide. Studies from India have reported an increased incidence of CRCs in young patients compared to the international average.[1] CRCs in young patients (less than 40 years of age) are more likely to present at an advanced stage, with regional or distant metastases. Histologically, they are more likely to be of mucinous or signet-ring cell type. CRCs are categorized as mucinous adenocarcinomas if more than 50% of the lesion is composed of pools of extracellular mucin that contain malignant cells as acinar structures, layers of tumor cells, or individual tumor cells including signet-ring cells.[2]

Genomic and epigenetic instability are hallmark features of colorectal carcinogenesis. Inactivation of mismatch repair (MMR) genes are responsible for about 15% of all CRCs. Microsatellite instability (MSI) is the molecular fingerprint of a deficient MMR system involved in keeping microsatellites at germline length.[3] Immunohistochemistry can identify protein products of the MMR genes and help establish mutations in the genes. Certain histopathological features also raise suspicion for a deficient MMR system. This study aimed at characterizing MSI in a unique cohort of mucinous CRCs in young patients. We also aimed to establish a standard test protocol to detect MSI tumors with high precision in a resource-limited setting.

 Subjects and Methods



Patients with mucinous CRC, ≤40 years of age, diagnosed, and treated between 2003 and 2012 were identified from the Department of Pathology database. Patients receiving preoperative chemotherapy were excluded from the study. Archived formalin-fixed paraffin-embedded tissue blocks and histopathology slides of these patients were retrieved for confirmation of diagnosis and molecular, immunohistochemical (IHC) and histopathological studies. Demographic data and clinical outcomes were collected from a prospectively maintained surgical database. The study was approved by the Institutional Review Board.

For molecular testing of MSI, two mononucleotide markers BAT 26 and NR 24 as described by Xicola et al. were used.[4] DNA was extracted from the tumor and adjacent normal tissue when available using a commercial extraction kit (MN kit, Germany). Quantification of DNA was done using the NanoDrop1000 spectrophotometer (Nanodrop Technologies, Thermoscientific, USA). The final elute was stored at -70°C. The polymerase chain reaction (PCR) was performed in a thermal cycler (Veriti Thermal Cycler; Applied Biosystems, USA) with an initial denaturation step at 95°C for 45 s, annealing at 58°C for 1 min for BAT 26 and at 62°C for 1 min for NR 24, and an extension at 72°C for 1 min. The thermal cycling was performed for 38 cycles with a final extension at 72°C for 10 min. The DNA from the normal and tumor sections were amplified separately for each target. The amplified PCR products along with control sample were run on 2% agarose gel and were stained with Safe View (Sigma-Aldrich, India) and the image was recorded using the Gel Documentation System (XR+ Gel Imaging System, BioRad, USA).

Using a GeneScan based fluorescence capillary electrophoresis procedure, the amplified PCR product, both from normal tissue and the tumor, were processed using the ABI 3130 Genetic Analyzer (Applied Biosystems, USA). The GeneScan of each tumor sample was correlated with that of its corresponding normal tissue if available, or with the negative controls, to determine the pattern of MSI.

Immunohistochemistry was performed using a Ventana Benchmark XT autostainer. Mouse monoclonal antibodies against hMLH1 (M1 prediluted, Roche Diagnostics, Mannheim, Germany), hMSH2 (G219–1129; 1:50 dilution, Cell Marque, Rocklin, CA, USA), and hMSH6 (44; 1:50 dilution; Cell Marque, Rocklin, CA, USA) were used in this study. A standard staining protocol was used for these markers. Normal epithelium within the tumor and stromal cells throughout the tissue served as internal controls for each section.

At least three haematoxylin and eosin (H and E) stained sections of all tumors were reviewed independently by two pathologists. A CH-20i microscope (Olympus Corporation, Japan) was used for the study. The features studied were:

Margin of tumor (infiltrating or expanding)Presence of a precursor lesion (tubulovillous, villous, tubular, or serrated adenomas)Presence of >10% nonmucinous tumor including well, moderately or poorly differentiated adenocarcinomas, signet-ring cells and a medullary component defined as >10% sheets of small- and medium-sized tumor cells with eosinophilic cytoplasm, often with abundant intraepithelial lymphocytesPercentage of tumor area showing a mucinous phenotypePresence of tumor-infiltrating lymphocytes (TILs) defined as a section of tumor with at least four undisputable intraepithelial lymphocytes in a single 40× fieldPresence of peritumoral lymphocytes based on finding a mantle or cap of chronic inflammatory cells deep to the point of tumor spreadvii. Crohn's-like reaction defined as at least three nodular lymphocytic aggregates in a 4× field deep to the advancing front of the tumorTumor budding defined as single or group of <5 detached cells, usually but not always at the invasive frontDirty necrosis, defined as glandular lumina filled with inspissated eosinophilic mucus, and cellular and nuclear debris.

Statistics

Data were analyzed using the SPSS software (SPSS 16.0; IBM Bangalore). Continuous variables were reported as mean ± SD (standard deviation) and analyzed using the t test. Categorical variables were reported as percentage (%) ± range and analyzed using the Pearson's Chi-square test or Fisher's exact test. P- values of ≤0.05 was considered significant. The Kaplan–Meier estimate was used to plot the survival curve and determine time to recurrence, and the Mann–Whitney's U-test was used to estimate median follow-up.

 Results



Thirty patients were included in the study; 23 surgical resections (20 with tumor and adjacent normal tissue and 3 with tumour only) and 7 mucosal biopsies. Twenty one were male. Mean age was 29.8 years and 73.3% were left-sided tumours [Table 1].{Table 1}

On molecular testing, 8/30 (26.7%) were positive for MSI [Figure 1]; six were resected tumors; and two mucosal biopsies. Three showed instability only for BAT26, three only for NR24 and two samples showed instability for both the markers [Table 2].{Figure 1}{Table 2}

On IHC testing for MLH1, MSH2 and MSH6, 7 out of 30 samples (23.3%) showed absence of staining for at least 1 MMR protein, when compared to its normal internal control [Table 2]. Four had exclusive absence of MLH1 protein expression, one of MSH2 expression and the remaining two of both MSH2 and MSH6 when compared to their controls [Figure 2] and [Figure 3]. There were no cases with exclusive absence of staining for MSH6.{Figure 2}{Figure 3}

[Figure 4] demonstrates some of the histomorphological features that were assessed in all 30 specimens. Six out of 8 MSI tumors and at least 17 out of 22 microsatellite stability (MSS) tumors had assessment of all required histological features. The rest were endoscopic mucosal biopsies with limited ability to assess detailed histological features. [Table 3] demonstrates the differences between the MSI and MSS group based on histology. We found that well-differentiated adenocarcinoma, peritumoural lymphocytes and tumor budding were much more common in the MSI group.{Figure 4}{Table 3}

Clinical correlation with microsatellite instability

Clinical details of MSI and MSS patients are presented in [Table 4]. There were no differences in age, gender and tumour distribution between both groups. There were more early stage tumors in the MSI group. Six of the 8 MSI patients and 13 of 22 MSS patients had follow-up data with a mean follow-up of 19 and 36 months, respectively. MSI status of the tumors was not known at the time of initiating chemotherapy. On follow-up, 5 patients in the MSI group and 18 in the MSS got adjuvant chemotherapy. There were no recurrences in the MSI group and four recurrences in the MSS group (P = 0.315). All patients with recurrences had received adjuvant chemotherapy.{Table 4}

 Discussion



The concept of MSI in CRCs has been extensively studied over the past two decades. Approximately 15% of all CRCs display MSI, 12% being sporadic and 3% associated with Lynch syndrome.[3],[5] MSI high/MMR deficient tumors have been shown to have a better outcome when compared to MSS/MMR proficient tumours. MSI high/MMR deficient tumors also do not benefit from adjuvant fluoropyrimidine-based therapy.[6],[7],[8] Assessment of MSI/MMR status, therefore, has implications for prognostication as well as management of patients.

The incidence of CRC in a younger population is rising globally, with increasing numbers of these patients being managed in Indian centers over the past years.[1],[9] The incidence of mucinous tumors is also higher in young patients.[9],[10] Studies on MSI status in a cohort of mucinous colorectal tumors reveal a 29%–36% MSI-H status.[11],[12],[13] Our findings in young, mucinous CRCs is in keeping with this, with a 26.7% MSI rate. To the best of our knowledge, there are no prior studies from India exclusively studying MSI status of mucinous CRCs in young patients. Better understanding of CRCs in this cohort could aid in better management and outcome.

Conventionally MSI testing is performed using five markers,[14] which makes the test expensive. We used two mononucleotide repeat markers (BAT26 and NR24) for molecular testing chosen rationally based on an elaborate multicentre study by Xicola et al.[4] This study compared the performance of these two markers against the pentaplex panel and found that the sensitivity and specificity of the trimmed-down panel was similar to the pentaplex panel. However, by using two marker testing systems, we will not be able to identify MSI-L tumors, as this identifies only MSI-H tumours. Studies have shown that MSI-L and MSS are possibly similar phenotypically and the exact implications of the MSI-L category is not yet clear.[14] Therefore, in the absence of data indicating the utility of identifying the MSI-L category, a simplified testing system involving two markers appears justified for routine use and is cost-effective in resource poor settings. Our results support the fact that a 2-mononucleotide marker system may be sufficient, at least as a screening test for MSI.

Mutations of the MMR genes including MLH1, MSH2, MSH6, and PMS2 are an important pathway in colorectal carcinogenesis. The genes found to be mutated most commonly are MLH1 and MSH2. Malhotra et al., found 20% of their 30 CRC cases to have absent expression of either MLH1 or MSH2 by IHC.[15] In contrast, Dubey et al., in their study of stage 2 colon cancer, showed loss of expression of either PMS2 or MLH1 by IHC in all MSI cases.[16] We used antibodies to MLH1, MSH2, and MSH6 in our study because as a panel, they have a sensitivity and specificity over 90% in detecting MMR gene mutations.[17],[18],[19] This panel gave us a sensitivity of 87.5% when compared to MSI testing. The one false negative case could be attributed to protein truncating or missense mutations of MLH1 gene, which renders the protein antigenically intact leading to positive staining and false-labelling as MMR proficient.[20] It is well established that MSH2 and MSH6 are heterodimers. However, we had one case in which there was isolated loss of MSH2 expression by IHC. Though rare, there are infrequent cases reported in the literature showing isolated loss of MSH2 protein expression with intact MSH6,[19],[21],[22] the plausible explanation being the presence of a secondary mutation in the intragenic C8 mononucleotide repeat region in exon 5 of MSH6.[23],[24] Addition of PMS2 could also potentially increase the sensitivity of IHC testing. The positive and negative predictive value of IHC testing when compared with MSI testing in our study was 100% and 95.65%, respectively. These results are comparable to available international data.

Our criteria for histological assessment was based on a paper published by Young et al.[19] In their publication, a contiguous traditional adenoma was seen in 16 out of 80 hereditary nonpolyposis colon cancer (HNPCC) cases (20%) and only 1 out of 42 sporadic MSI-H cases (2%) (P < 0.001). The sporadic MSI-H group showed a strong association with contiguous serrated adenomas. We found residual adenomas in four of eight MSI tumours (50%), all four were tubulovillous adenomas with high-grade dysplasia. Although differentiation between HNPCC and sporadic tumours was not practicable in this study, the finding of 50% tumours with a precursor tubulovillous adenoma points towards some of these cases being HNPCC. Five out of 22 MSS cases had adjacent lesions; four were tubulovillous adenomas, three with high-grade dysplasia and one with low-grade dysplasia, and one was a case of ulcerative colitis with low-grade dysplasia.

Six of the eight tumours with MSI in our study were located on the left side and two on the right. Another study from India which had 10.8% MSI cases (5 out of 46), also showed a left-sided predominance with four left-sided tumours and only one right-sided tumour.[25] It has been shown that MSI-positive right-sided tumours are predominantly seen with HNPCC and left-sided tumours with MSI are mostly sporadic. The higher frequency of left-sided tumors in our series, together with an absence of family history, points to a predominant sporadic etiology.

From the results of our study, we also note that certain histological features of mucinous CRCs can be used as pointers toward an underlying deficient MMR or MSI-H status. Histological features like a well-differentiated adenocarcinoma, peritumoral lymphocytes, and tumor budding were significantly associated with the MSI status of a tumour, in concordance with other world series. Although features like TILs, medullary differentiation and Crohn's-like reaction were not significant in our series (likely due to a small sample size), other series have shown that these are also predictive of MSI tumours.

MSI-H tumours have been shown, in many series, to have a better prognosis and survival when compared to MSS tumors. This is similar to our findings, where all four recurrences were in the MSS group, with none in the MSI group. This better outcome was independent of all prognostic factors including tumour stage.

Studies worldwide have shown that MSI-H CRCs do not respond well to 5FU (5-Fluorouracil) as a chemotherapeutic agent.[26] However, a France-based group using FOLFOX (5FU-Oxaliplatin) therapy has shown that disease-free survival is significantly higher in the MMR deficient group when compared to MMR proficient tumors.[27] This relationship between the MSI status and response to 5FU is critical in deciding postoperative chemotherapy for these patients. In our study five MSI patients in stages II and III received 5FU as single adjuvant chemotherapeutic agent. This is probably due to the fact that MSI status was not known at the time of being offered chemotherapy. None of them developed recurrence, metastasis, or died during the follow-up period.

In summary, clinical and histologic features can raise a high suspicion for the presence of MMR gene mutations. The first step to confirm this should be IHC testing for MMR proteins. If this shows deficiency in MMR protein expression, MSI status is established with fair degree of certainty. However, if IHC shows MMR proficient status, MSI testing with a 2-marker panel should be undertaken to rule out any missed MMR gene mutations. In a resource poor setting, this protocol will help us identify most patients.

This study has several limitations including small sample size, high loss to follow-up, and nonavailability of resection specimens to assess histopathological parameters in some cases. Nevertheless, this study has helped us generate useful information with regard to the MSI pattern among a subset of patients that are common, yet not extensively studied in the Indian setting. These results can be used as baseline for larger cohort studies.

 Conclusion



In a resource limited setting, a combination of histopathological examination, IHC testing for MMR protein status with hMLH1, hMSH2, and hMSH6, and MSI testing with BAT26 and NR24, together form a cost-effective diagnostic panel for identifying MSI in CRCs.

Financial support and sponsorship

Institutional Review Board (IRB Min. No. 7989 dated 08.09.2012).

Conflicts of interest

There are no conflicts of interest.

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