| Abstract|| |
Background: Colorectal cancers often arise from benign polyps. Adenomatous polyps and serrated polyps progress step by step to adenocarcinoma and change into malignant cancers. Genetic and epigenetic changes have correlation with specific stages of polyp-adenocarcinoma progression and colorectal cancer histopathological changes. Aims: In this study we used immunohistochemistry (IHC) staining in sporadic colorectal polyps to assay functional status of MLH1, MSH2, MSH6, and PMS2 proteins, to track genetic/epigenetic roles of this issue in our patients. Materials and Methods: In this cross-sectional study we assessed all patients who were admitted with sporadic colorectal polyps and underwent polypectomy in endoscopy department during 2004-2008. Result: IHC results were abnormal in 6.8% cases for MLH1, in 4.5% cases for MSH2, in 3% for MSH6, and in 4.8% for PMS2. In all cases with abnormal PMS2, MLH1 was also reported as abnormal. Same results were reported for abnormal MSH2, which is accompanied with abnormal MSH6 in all cases (P values < 0.001). There is no significant difference between IHC staining results, gender, dysplasia grade, adenomatous type, and invasion. On the other hand, there was significant difference between IHC staining results, polyp location, and mean age of patients. The same significant difference was between adenomatous polyps and serrated adenoma polyps by MLH1 and PMS2 (P values < 0.05). Conclusion: According to our findings, maybe MMR dysfunction is the cause of sporadic colorectal polyps in younger age and its increasing risk of dysplasia progression and malignancy progression is only in serrated adenoma. Sporadic polyps in left colon had a higher risk to progress to malignancies, and abnormal IHC staining for MLH1 and PMS2 in serrated polyps is much more than in other adenomatous polyps.
Keywords: Mismatch Repair proteins, serrated adenoma, sporadic colorectal polyp
|How to cite this article:|
Molaei M, Yadollahzadeh M, Almasi S, Shivarani S, Fatemi SR, Zali MR. Sporadic colorectal polyps and mismatch repair proteins. Indian J Pathol Microbiol 2011;54:725-9
|How to cite this URL:|
Molaei M, Yadollahzadeh M, Almasi S, Shivarani S, Fatemi SR, Zali MR. Sporadic colorectal polyps and mismatch repair proteins. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Jun 16];54:725-9. Available from: http://www.ijpmonline.org/text.asp?2011/54/4/725/91505
| Introduction|| |
Colorectal cancer is the third cause of death, related to cancer in Iran with the prevalence of 8 in 100,000 cases.  Most of colorectal cancers arise from benign polyps, with a gradual histological progress from hyperplastic adenomatous polyps or serrated adenomas to adenocarcinoma. Genetic and/or epigenetic changes correlate with special stages of polyp progression to adenoma and histological changes in colorectal cancer. 
Mismatch repair (MMR) system is necessary for diagnosis and correction of insertion or deletion errors and mismatches in DNA that occur during DNA replication and coupling.  Germline mutation in MMR genes may cause microsatellite instability (MSI), which has an important role in special types of cancers in colorectal, endometrium, ovary, stomach, small intestine, pancreas, upper urinary tract, and brain. ,,
Loss of DNA MMR activity accelerates the rate of accumulation of mutations in genes responsible for restraining cell growth. This provides a reasonable hypothesis for rapid growth of adenomas and their transition to colorectal cancers.  Also, there is controversy in role of MMR system on the prognosis of the cancers. Some previous studies have reported an improved prognosis in sporadic colon cancers with MSI, whereas others have not.  Anyway in humans, mutS (MSH2, MSH3, and MSH6) and mutL (MLH1, MLH3, PMS1, and PMS2) have the major role in MMR system. 
One proper way to detect MMR dysfunction is immunohistochemistry (IHC) staining. So, in this study we use IHC staining for MLH1, MSH2, MSH6, and PMS2 proteins to assess function of MMR proteins in 400 cases with sporadic colorectal polyps who admitted at gastroenterology ward. To know the prevalence and genetic or epigenetic track in our population, this could improve programs for screening, diagnostic paths, and preventive efforts.
| Materials and Methods|| |
In this study 400 patients with sporadic colorectal polyps, FAP (familial adenomatous polyposis) or other polyposis disorders were not include], were assessed. All patients underwent polypectomy procedure in endoscopy department during 2004-2008. Needed data such as age, gender, polyps' location, dysplasia grade, type of polyp, and invasion were collected from patients' pathology reports. Then tissue slides embedded in paraffin blocks were prepared and used for IHC staining and histopathological assay. Colon was divided into three parts: right colon (cecum, ascending colon, and splenic flexure), left colon (transverse, descending, and sigmoid colon), and rectum (rectum and anus).
Sample sections were collected on the slides using polyelizine glue. After deparaffinization and rehydration for antigen retrieval, slides were treated in EDTA buffer (pH = 9) in microwave oven for 45 min. After the mentioned procedures, antiperoxidase solution was used to block endogenous tissue peroxidase on the ready slides.
Next, antibodies against MLH1 (BD Biosciences Pharmingen: California, United States; clone: G168-15, 1/100 dilution), MSH2 (BD Transduction laboratories: New York, United States; clone: FE11, 1/100 dilution), MSH6 (BD Biosciences Pharmingen: California, United States; clone 44, 1/1000 dilution), and PMS2 (BD Pharmingen: California, United States; clone A 16-4, 1/500 dilution) was poured on slices and incubated for 90 min at room temperature.
Between all stages tris buffered saline (TBS) was used for rising for 3 min. Then samples were treated with Envision (DAKO, Real Envision). Finally, to visualize immunoactivity, 3,2'-diaminobenzidine was used and samples were counter stained with hematoxylin.
Normal epithelial cells, stromal cells, or intramucosal lymphocytes in each slide were used as internal control. Also nuclear staining was accepted as normal staining in all cells [Figure 1]. In the staining procedure, if nucleus of tumoral cells were not well stained, staining was considered as abnormal, while other well-stained cells had normal staining [Figure 2].
|Figure 1: Normal nuclear (IHC) staining for MMR proteins both in stromal cells (gray arrows) and epithelial cells (black arrows) as brownish accumulation of dye in the nucleus of the mentioned cells, (IHC × 100)|
Click here to view
|Figure 2: Abnormal IHC staining, stromal cells (gray arrows) still show normal staining for MMR proteins, while there is no dye accumulation in nucleus of epithelial cells (black arrows), (IHC × 100)|
Click here to view
| Statistical Analysis|| |
Gathered data were analyzed by SPSS 16 software. We used independent t-test, Chi2, Fisher exact, and ANOVA for comparative analysis (statistical significant P value < 0.05).
| Results|| |
All over, 400 colorectal polyp samples of 400 cases were reviewed. The mean age of patients was 60.17 (SD = 14.08) with the range of 15-89 years old. 237 cases (59.2%) were male and 163 cases (40.8%) were female. Polyps were in the right colon in 136 cases (34.11), in the left side in 201 cases (50.4%), and in rectum in 62 cases (15.5%). Polyp types were detected as adenomatous in 385 cases (96.2%) and serrated adenomas in 15 cases (3.8%). Also type of adenomatous polyps was tubular in 240 cases (62.3%), tubulovillous in 116 cases (29.6%), and villous in 31 cases (8.1%). Dysplasia grade was low in 324 samples (81%) and high in 76 samples (19%). In only 24 polyp samples (6%) invasion was detected.
IHC staining was abnormal for MLH1 in 27 cases (6.8%), for MSH2 in 18 cases (4.5%), for MSH6 in 12 cases (3%), and for PMS2 in 19 cases (4.8%). In comparative analysis all of the cases with abnormal PMS2 had abnormal MLH1 (P value < 0.001). All cases with abnormal MSH6 had abnormal MSH2 (P value < 0.001). Comparison of assessed parameters by IHC staining result and tumor location is mentioned in [Table 1] and [Table 2], respectively. There was not clear information about the location of tumor in one case, which is omitted from analysis in [Table 2].
|Table 1: Comparison of parameters by IHC staining result in patients with sporadic colorectal polyps|
Click here to view
|Table 2: Comparison of variables by location of sporadic colorectal polyps (n = 399)|
Click here to view
| Discussion|| |
In this study, we scrutinized 400 colorectal polyps' samples from 400 cases with sporadic colorectal polyps. The mean age and sex ratio in our cases were the same as Balbinotti et al. study in Brazil.  The major location of tumor was left colon, then right colon, and finally rectum. But there are different results in other studies. ,, Nusko et al. mentioned that sporadic adenoma polyps in left colon have more risk to transform to malignancy, then it is expected that the prevalence of high-grade tumors in various reports was related to the prevalence of left colon polyps. In two similar studies (40.4%,  and 25%  ), the prevalence of high-grade dysplasia was more than in our study (19%), which confirmed Nusko's theory. 
Similar to Rijken study  in this research 62.2% of adenomatous polyps were tubular, whereas Balbinotti  reported more than 80% tubular adenomatous polyps, and Percinel  reported 19%. On the other hand, the prevalence of serrated adenomas in our study was about two times more than Balbinotti report  (3.8% against 1.8%). But in Percinel  report serrated adenoma polyps were more than our findings (10% in contrast of 3.8%).
Some grades of atypia or low-grade dysplasia were seen in all the serrated adenomas' samples. Because of the same clinical behavior as tubular adenocarcinoma with low-grade dysplasia, they have been categorized as low-grade dysplasia. It is the same as result of the study by Percinel. 
Abnormal IHC in our study for MLH1 and MSH2 (6.8% and 4.5%) were less than in Balbinotti's  report (20% and 15.5%). In all cases with abnormal PMS2, MLH1 was also abnormal, and in cases with abnormal MSH6, MSH2 was also abnormal. This correlation may be due to mutLα and mutSα hetrodimmers formation by these couples of MMR proteins,  which caused PMS2 and MSH6 genes products to be unstable without their hetrodimmers. , On the other hand impairment in both hetrodimmers of mutLα and mutSα could be a sign of hereditary mutation and an introduction for familial disease.  This led to genetic consultation and other screenings and possibly the examination of family members seemed necessary. Although it was not recommended for sporadic polyps up to now.
Similar to Balbinotti's  report in our study there is no any significant difference between IHC result groups by sex, dysplasia grade, type of adenomatous polyps, and invasion. In contrast to the mentioned report in our study, IHC findings have correlation with polyp locations and age of patients. , There were also significant differences between adenomatous polyps and serrated polyps by immune reactivity for MLH1 and PMS2 (all P values < 0.05).
The prevalence of abnormal IHC staining for four MMR proteins in right colon polyps was more than in left colon and rectum polyps. Published researches showed that abnormal IHC and MMR dysfunction were detected more in polyps of right colon comparing with left colon in patients with hereditary nonpolyposis colorectal cancer (HNPCC), and accelerated changes of adenoma to carcinoma in patients with HNPCC are related to polyp sites, which is more prevalent in right colon. , In contrast, in patients with sporadic colorectal polyps, risk of malignant transformation is more in left colon, but the cause of this opposite findings is not yet clear. ,,,
Although MMR gene dysfunction in HNPCC patients is not possibly a beginner for malignancy progression, but they represent in very primitive dysplasia stages, and seem to be a promoter of progression to high-grade dysplasia.  On the other hand, there was not any significant difference between dysplasia grades by MMR proteins IHC staining. Although prevalence of abnormal IHC for MMR in left colon was less, but high-grade dysplasia in left colon especially in rectum was high, which confirmed higher risk of progression to malignancies in sporadic polyps detected in left colon comparing to hereditary polyps. 
MMR genes impairment (especially in MLH1 and MSH2) may lead to MSI. In sporadic colorectal polyps increased risk of malignancies related to MSI is limited to serrated adenomas.  In our study similarly to previous reports, prevalence of abnormal MLH1 and PMS2 proteins in serrated adenoma was much more than other adenomatous polyps (P values are 0.002 and 0.004, respectively), which is possibly due to CpG (Cytosine - phosphodiester - Guanine) island. ,,, However, all cases with abnormal MSH6 were detected with adenomatous polyps, but MSH2's and MSH6's IHC staining had no significant difference between serrated adenomas and other adenomatous polyps. MMR proteins impairment in HNPCC cases may lead to lesions (especially, malignant lesions) in younger age. However, our cases were diagnosed as sporadic polyps, but in abnormal IHC staining group mean age was significantly lower than in normal IHC staining group (P values < 0.05).
| Conclusions|| |
According to our findings, sporadic polyps in left colon had a higher risk to progress to malignancies because of higher prevalence of high-grade dysplasia and villous type of adenomatous. In our study there is no significant difference between IHC result groups by sex, dysplasia grade, type of adenomatous polyps, and invasion, but have significant difference by polyp locations and age of patients. Abnormal IHC staining for MLH1 and PMS2 in serrated polyps is much more than in other adenomatous polyps. Possibly, MMR dysfunction is the cause of sporadic colorectal polyps in younger age and its increasing risk of dysplasia progression and malignancy progression is only in serrated adenoma.  But fortunately all of our serrated adenomas' samples had low-grade dysplasia, the same as Percinel  study. Further studies about this topic are required to reveal genetic or epigenetic track in our population to throw more light on the improvement of programs for screening, diagnostic paths and preventive efforts.
| References|| |
|1.||Mousavi S, Gouya M, Ramazani R, Davanlou M, Hajsadeghi N, Seddighi Z. Cancer incidence and mortality in Iran. Ann Oncol 2009;20:556-63. |
|2.||Petko Z, Ghiassi M, Shuber A, Gorham J, Smalley W, Washington MK, et al. Aberrantly methylated CDKN2A, MGMT, and MLH1 in colon polyps and in fecal DNA from patients with colorectal polyps. Clin Cancer Res 2005;11:1203-9. |
|3.||Iyer R, Pluciennik A, Burdett V, Modrich P. DNA mismatch repair: Functions and mechanisms. Chem Rev 2006;106:302-23. |
|4.||Brooks-Wilson A, Kaurah P, Suriano G. Germline E-cadherin mutations in hereditary diffuse gastric cancer: Assessment of 42 new families and review of genetic screening criteria. J Med Genet 2004;41:508-17. |
|5.||Pharoah P, Guilford P, Caldas C. International Gastric Cancer Linkage Consortium. Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology 2001;121:1348-53. |
|6.||Lynch H, Grady W, Suriano G, Huntsman D. Gastric Cancer: New Genetic Developments. J Surg Oncol 2005;90:114-33. |
|7.||Lynch HT, Lynch JF, Lynch PM, Attard T. Hereditary colorectal cancer syndromes: Molecular genetics, genetic counseling, diagnosis and management. Fam Cancer 2008;7:27-39. |
|8.||Samowitz WS, Curtin K, Ma KN, Schaffer D, Coleman LW, Leppert M, et al. Microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level. Cancer Epidemiol Biomarkers Prev 2001;10:917-23. |
|9.||Gill S, Lindor NM, Burgart LJ, Smalley R, Leontovich O, French AJ, et al. Isolated Loss of PMS2 Expression in Colorectal Cancers: Frequency, Patient Age, and Familial Aggregation. Clin Cancer Res 2005;11:6466- 71. |
|10.||Balbinotti RA, Ribeiro U, Sakai P, Safatle-Ribeiro AV, Balbinotti SS, Scapulatempo C, et al. hMLH1, hMSH2 and cyclooxygenase-2 (cox-2) in sporadic colorectal polyps. Anticancer Res 2007;27:4465-71. |
|11.||Rijcken FE, Hollema H, Kleibeuker JH. Proximal adenomas in hereditary non-polyposis colorectal cancer are prone to rapid malignant transformation. Gut 2002;50:382-6. |
|12.||Percinel S, Savas B, Ensari A, Kuzu I, Kuzu MA, Bektas M, et al. Mucins in the colorectal neoplastic spectrum with reference to conventional and serrated adenomas. Turk J Gastroenterol 2007;18:230-8. |
|13.||Nusko G, Mansmann U, Altendorf H. Risk of invasive carcinoma in colorectal adenomas assessed by size and site. Int J Colorectal Dis 1997;12:267-71. |
|14.||Sheridan TB, Fenton H, Lewin MR, Burkart AL, Iacobuzio-Donahue CA, Frankel WL, et al. Sessile serrated adenomas with low- and high-grade dysplasia and early carcinomas: An immunohistochemical study of serrated lesions "caught in the act". Am J Clin Pathol 2006;126:564-71. |
|15.||Young J, Barker M, Fraser L, Walsh M, Spring K, Biden K, et al. Mutation searching in colorectal cancer studies: Experience with a denaturing high-pressure liquid chromatography system for exon-by-exon scanning of tumour suppressor genes. Pathology 2002;34:529-33. |
|16.||Chaves P, Cruz C, Lage P. Immunohistochemical detection of mismatch repair gene proteins as a useful tool for the identification of colorectal carcinoma with the mutator phenotype. J Pathol 2000;191:355-60. |
|17.||Marcus V, Madlensky L, Gryfe R. Immunohistochemistry for hMLH1 and hMSH2: A practical test for DNA mismatch repair-deficient tumors. Am J Surg Pathol 1999;23:1248-55. |
|18.||Ruschoff J, Aust D, Hartmann A. Colorectal serrated adenoma: Diagnostic criteria and clinical implications. Verh Dtsch Ges Pathol 2007;91:119-25. |
|19.||Wynter C, Walsh M, Higuchi T. Methylation patterns define two types of hyperplastic polyp associated with colorectal cancer. Gut 2004;53:573-80. |
|20.||Torlakovic E, Skovlund E, Snover D. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003;27:65-81. |
|21.||Oh K, Redston M, Odze RD. Support for hMLH1 and MGMT silencing as a mechanism of tumorigenesis in the hyperplasticadenoma- carcinoma (serrated) carcinogenic pathway in the colon. Hum Pathol 2005;36:101-11. |
Research Center for Gastroenterology and Liver disease Shahid Beheshti University of Medical Sciences, Taleghani Hospital, Tehran 19857, P.O. Box 19835-187
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]