Abstract

Introduction:?Around 80% of colorectal carcinoma are associated with chromosomal instability while rest of 20% are euploid, possessing defect in mismatch repair system (MMR) quintessential for surveillance and correction of errors introduced into microsatellites. The microsatellite instability (MSI) phenotype has three major clinical applications: prognosis of colorectal cancer (CRC), prediction of response to 5 fluorouracil, and irinotecan, and genetic assessment of Lynch syndrome.?Materials and Methods:?We analyzed all Stage II and Stage III colorectal cancer (CRC) for MSI, who presented at Army Hospital, Research and Referral, New Delhi, from January 2014 to December 2016. Although patients of Stage II CRC were taken throughout the study period, Stage III CRC was included in last 1? years to compare the prevalence of MSI in these two subsets of patients.?Results:?26.2% of Stage II and 11.3% of Stage III patients were found to be MSI-high (MSI-H) (P?= 0.04). Nineteen (86%) of 22 MSI-H patients were below 30 years of age (P?= 0.01). Of 22 MSI-H patients, 18 had right-sided tumors (P?= 0.03) and only three patients had rectal tumors. Most common pattern of MSI-H tumors was loss of expression of MLH1 and PMS2, seen in 15 of 16 (88%) of Stage II and three of 6 (50%) of Stage III CRC (P?= 0.04).?Conclusion:?We conclude higher prevalence of MSI-H tumors in Stage II, as compared to Stage III CRC, which was demonstrated slightly higher in our study compared to published literature. MSI-H tumors tend to occur with high frequency in younger population, with right-sided colonic tumors, histopathology characterized by mucinous subtype with high prevalence of tumor infiltrating lymphocytes. Loss of expression of two MMR proteins, namely, PMS2 and MLH1 has been identified in most of MSI-H patients of our study, of which 86% were <30>70 years) patients with MSI-H tumors, and in younger patients, MSI-H status was associated with loss of MLH1, MSH2, and MSH6.

Keywords

Colorectal carcinoma - microsatellite instability - mismatch repair

Introduction

Colorectal cancers (CRCs) are the third most common malignancy in men (10.0% of all cancer cases) and the second most common malignancy in women (9.4% of all cancer cases) worldwide.[1] It remains the fourth most common cause of death due to cancer. In India, the annual incidence rates for colon cancer in men are 4.4 per 100000, while that in women is 3.9 per 100000.[2] Around 80% of CRC are associated chromosomal instability, while rest 20% are euploid, defective in mismatch repair (MMR) system quintessential for surveillance and correction of errors introduced into microsatellites. This defective MMR system creates microsatellite instability (MSI), noted in 12%?15% of all CRC, of which 2%?3% are caused due to germline mutational inactivation of MMR genes and rest due to epigenetic mutational inactivation of MMR system. These are classified as MSI-high (MSI-H), where> 30% of the microsatellite marker panel is mutated and MSI-low (MSI-L) if <30>

The MMR genes are highly conserved from bacteria to humans. Hereditary nonpolyposis colorectal cancer (HNPCC) is a common, autosomal dominant syndrome characterized by early onset (average age at onset <45 href="https://www.thieme-connect.com/products/ejournals/html/10.4103/ijmpo.ijmpo_35_17#JR_3" xss=removed>3],[4] Cancers with MSI account for approximately 15% of all CRCs (usually MLH1 methylation), and for HNPCC germline mutations, there are three key DNA MMR genes (i.e., MSH2, MLH1 and in attenuated cases, MSH6) that are responsible for these cancers. A few candidate genes (e.g., PMS2 and MLH3) are still awaiting additional validation regarding their role in the etiology of CRCs with MSI.[3],[4] MSI has been observed in gastric, endometrial, ovarian, and sebaceous carcinomas as well as glioblastoma and lymphomas apart from CRC.[5] Several population-based studies have reported that the prevalence of MSI-H in CRC ranges from 15% to 20%.[6] MSI is more common among Stage II (~20%) than Stage III (~12%) CRC and is even less frequent among Stage IV CRC (~4%).[7]

Microsatellite instability -high and microsatellite instability -low

MSI is detected by PCR amplification of specific microsatellite repeats. The presence of instability is determined by comparing the length of nucleotide repeats in tumor cells and normal cells. Normal DNA is typically extracted from adjacent normal mucosa.[13] A consensus reference panel, known as the Bethesda panel, established a panel of microsatellite markers with appropriate sensitivity and specificity to diagnose MSI CRC.[14] Three categories of MSI have been established based on the following criteria: MSI-H, indicating instability at two or more loci (or> 30% of loci if a larger panel of markers is used); MSI-L, indicating instability at one locus (or in 10%?30% of loci in larger panels), and microsatellite stable (MSS), indicating no loci with instability (or <10>

Diagnosis of microsatellite instability

The principal use of MSI testing in the clinic is to identify patients with Lynch syndrome. Approximately 15% of all colorectal tumors have MSI and 75% to 80% of this group have acquired methylation of MLH1; only ~2% to 3% of all CRCs have germline mutations in one of the MMR genes.[15] MSI identifies MMR deficient CRC with approximately 93% sensitivity; most insensitivity is caused by mutations in MSH6.[16]

Immunohistochemical (IHC) analysis of MMR proteins recently has become a popular alternative to detect MSI. Antibodies against MLH1, MSH2, MSH6, and PMS2 provide insight into the functionality of the MMR system. Lack of expression of one or more of these proteins is diagnostic of deficient MMR. The limitation of IHC is that staining can be heterogeneous throughout the tumor, which affects the sensitivity of the test.

Features and applications of microsatellite instability

CRC displaying MSI-H tends to be right sided and diagnosed at lower pathological stages compared with MSS cancers. Sporadic MSI-H cases are generally diagnosed in the elderly> 70 years of age, whereas familial cases in younger <50 href="https://www.thieme-connect.com/products/ejournals/html/10.4103/ijmpo.ijmpo_35_17#JR_17" xss=removed>17] CRC with MSI-H generally have high-histological grades, mucinous phenotypes with prominent numbers of tumor infiltrating lymphocytes, a lack of necrotic cellular debris within the lumen of the neoplastic glands in the colorectal mucosa (dirty necrosis), and a Crohn's-like host response.[12] The MSI phenotype has three major clinical applications: prognosis of CRC, prediction of response to 5 fluorouracil (5FU) and irinotecan, and genetic assessment of Lynch syndrome.

Sporadic MSI-H CRC cases are more frequently associated with BRAF mutations than in hereditary cases, and are caused by hyper methylation of MLH.[8],[9] Strong association has been proved in studies between sporadic MSI and the presence of the V600E BRAF mutation. BRAF mutation profoundly reduces the probability of a diagnosis of Lynch syndrome, though it does not entirely exclude the possibility.[10]KRAS mutations, however, are more likely to be observed in MSS (CIN) cancers than MSI tumor.?PTEN, a tumor suppressor gene?is not only mutated but also epigenetically silenced with higher frequency in MSI-H tumors.[11]

Prognostic and predictive value of microsatellite instability in colorectal cancer

MSI tumors had a more favorable prognosis and are less prone to lymph node spread and metastasis than MSS tumors. The prognostic value of MSI is more prominent in Stage II than Stage III CRC cases.[18] Tumors with MSI-H have greater numbers of tumor-infiltrating lymphocytes that are activated and cytotoxic;[19] which by itself is an independent factor associated with longer survival.[20] Patients receiving 5FU had no advantage over those who did not receive 5FU and this treatment might even be harmful for Stage II cases displaying MSI-H. Adjuvant combined chemotherapy with a 5FU-based regimen remains the standard of care in patients diagnosed with Stage III disease, regardless of MSI status. MSI-H cells are especially sensitive to irinotecan compared with their proficient counterparts.[21]

Aims and objectives

The aim of this study was to analyze the frequency and clinicopathological characteristics of MSI-H colorectal Stage II and Stage III cancers at a tertiary care center.

Materials and Methods

We analyzed all Stage II and Stage III CRC patients who visited malignant disease treatment center at Army Hospital Research and Referral from January 2014 to December 2016. Data of Stage II CRC were taken throughout the 3 years of study whereas patients of Stage III CRC were included only during the last 1? years for comparing the relative prevalence of MSI-H between these two subsets of patients. Data were collected in a predesigned computerized format containing details about patient characteristics, risk factors, clinical presentation, imaging findings, tumor stage, histology, MSI, genetic mutation testing, and treatment received.

Results

Of the 114 patients evaluated, 61 had Stage II colorectal tumors (CRC), rest 53 had Stage III CRC [Table 1]. Median age of patients with MSI-H CRC was 35.2 years. Of 22 MSI-H patients, 19 (87%) were <30 class="i" xss=removed>P?= ?0.018) [Table 2]. There was no significant difference between males and females in the prevalence of MSI-H tumors [Table 3]. Of 61 Stage II CRC, 48 (79%) had right-sided tumors and 13 (21%) had left-sided lesions. Sixteen (26.2%) patients had profile of MSI-H tumors in the form of lack of expression of two or more MMR proteins on immunohistochemistry (IHC). Of 16 Stage II MSI-H CRC cases, 12 (75%) were right-sided and rest 4 (25%) of cases were left-sided tumors. Of the sixteen Stage II MSI-H cases, 14 (87%) had colon cancer, and rest two had rectal carcinoma (P?= 0.04) [Table 4]. Fifty-three patients of Stage III CRC, who presented to this institute for 1? years, were evaluated for MSI. Six (11.3%) patients tested positive for MSI-H, of which 4 (67%) were right-sided CRC and rest two (33%) were left-sided CRC. Of 6 MSI-H Stage III CRC, five were colonic tumors and one was rectal carcinoma (P?= ?0.04). The incidence of MSI had a striking association with age at CRC diagnosis (87% MSI-H <30 xss=removed href="https://www.thieme-connect.com/products/ejournals/html/10.4103/ijmpo.ijmpo_35_17#TB_5" xss=removed>Table 5]. The median age at diagnosis of MSI-H case was 35.2 years. Family history of CRC in first- and second-degree relatives was associated with significantly higher occurrence of MSI-H tumors: 23% (n?= 5) compared with 5% (n?= 4) in patients with MSI-L/MSS tumors in our study (P?= 0.03) [Table 6].

References

1. LOBOCAN Available from: http://www.globocan.iarc.fr/factsheets/cancers/colorectal.asp. [Last accessed on 2017 Feb 16].
2. CRP. Three-Year Report of the Population Based Cancer Registries ? 2009-2011. National Cancer Registry Programme. Bangalore, India: Indian Council of Medical Research (ICMR); 2013
3. ulligan KM, Meyer-Gauen G, Lyons-Weiler J, Hays JB.?Evolutionary origin, diversification and specialization of eukaryotic MutS homolog mismatch repair proteins. Nucleic Acids Res 2000; 28: 463-71
4. iricny J.?The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 2006; 7: 335-46
5. oland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW.?et al.?A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: Development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998; 58: 5248-57
6. d">6?Ligtenberg MJ, Kuiper RP, Chan TL, Goossens M, Hebeda KM, Voorendt M.?et al.?Heritable somatic methylation and inactivation of MSH2 in families with lynch syndrome due to deletion of the 3' exons of TACSTD1. Nat Genet 2009; 41: 112-7
7. oth AD, Tejpar S, Delorenzi M, Yan P, Fiocca R, Klingbiel D.?et al.?Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: Results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol 2010; 28: 466-74
8. earon ER, Vogelstein B.?A genetic model for colorectal tumorigenesis. Cell 1990; 61: 759-67
9. d">9?Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE.?et al.?Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002; 418: 934
10. Deng G, Bell I, Crawley S, Gum J, Terdiman JP, Allen BA.?et al.?BRAF mutation is frequently present in sporadic colorectal cancer with methylated hMLH1, but not in hereditary nonpolyposis colorectal cancer. Clin Cancer Res 2004; 10: 191-5
11. d">11?Parsons DW, Wang TL, Samuels Y, Bardelli A, Cummins JM, DeLong L.?et al.?Colorectal cancer: Mutations in a signalling pathway. Nature 2005; 436: 792
12. d">12?Greenson JK, Bonner JD, Ben-Yzhak O, Cohen HI, Miselevich I, Resnick MB.?et al.?Phenotype of microsatellite unstable colorectal carcinomas: Well-differentiated and focally mucinous tumors and the absence of dirty necrosis correlate with microsatellite instability. Am J Surg Pathol 2003; 27: 563-70
13. d">13?Veitenhansl M, Stegner K, Hierl FX, Dieterle C, Feldmeier H, Gutt B.?et al.?40th EASD annual meeting of the European association for the study of diabetes: Munich, germany, 5-9 september 2004. Diabetologia 2004; 47: A1-A464
14. Umar A, Boland CR, Terdiman JP, Syngal S, de la ChapelleA, R?schoff J.?et al.?Revised bethesda guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst 2004; 96: 261-8
15. Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P.?et al.?Screening for the lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med 2005; 352: 1851-60
16. Shia J.?Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part I. The utility of immunohistochemistry. J Mol Diagn 2008; 10: 293-300
17. d">17?Poynter JN, Haile RW, Siegmund KD, Campbell PT, Figueiredo JC, Limburg P.?et al.?Associations between smoking, alcohol consumption, and colorectal cancer, overall and by tumor microsatellite instability status. Cancer Epidemiol Biomarkers Prev 2009; 18: 2745-50
18. Roth AD, Tejpar S, Delorenzi M, Yan P, Fiocca R, Klingbiel D.?et al.?Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: Results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol 2010; 28: 466-74
19. d">19?Phillips SM, Banerjea A, Feakins R, Li SR, Bustin SA, Dorudi S.?et al.?Tumour-infiltrating lymphocytes in colorectal cancer with microsatellite instability are activated and cytotoxic. Br J Surg 2004; 91: 469-75
20. Ogino S, Nosho K, Irahara N, Meyerhardt JA, Baba Y, Shima K.?et al.?Lymphocytic reaction to colorectal cancer is associated with longer survival, independent of lymph node count, microsatellite instability, and cpG island methylator phenotype. Clin Cancer Res 2009; 15: 6412-20
21. d">21?Rodriguez R, Hansen LT, Phear G, Scorah J, Spang-Thomsen M, Cox A.?et al.?Thymidine selectively enhances growth suppressive effects of camptothecin/irinotecan in MSI+ cells and tumors containing a mutation of MRE11. Clin Cancer Res 2008; 14: 4576-83
22. Wang L, Cunningham JM, Winters JL, Guenther JC, French AJ, Boardman LA.?et al.?BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Res 2003; 63: 5209-12
23. Sinicrope FA, Rego RL, Halling KC, Foster N, Sargent DJ, La PlantB.?et al.?Prognostic impact of microsatellite instability and DNA ploidy in human colon carcinoma patients. Gastroenterology 2006; 131: 729-37