|Year : 2016 | Volume
| Issue : 4 | Page : 513-517
Microsatellite instability in stage II colorectal cancer: An Indian perspective
AP Dubey, S Vishwanath, P Nikhil, A Rathore, A Pathak
Department of Medical Oncology, Army Hospital (Research and Referral), New Delhi, India
|Date of Web Publication||21-Apr-2017|
Department of Medical Oncology, Army Hospital (Research and Referral), New Delhi
Source of Support: None, Conflict of Interest: None
INTRODUCTION: Around 80% of colorectal carcinoma are associated with chromosomal instability (CIN) while rest of 20 % are euploid, possessing defect in mis match repair system (MMR) quintessential for surveillance and correction of errors in introduced into microsatellites. MATERIALS AND METHODS: We analyse all stage II CRC for MSI who presented at MDTC at Army hospital (research and refrral) new delhi during last 2 years (Jan 14 to Dec 2015). RESULTS: We found that 22.2% patients out of 45 patients with stageII CRC being MSI-. high. We also noticed all suchcases were associated with loss of expression of PMS2 & MLH1, that was in contrast other studies where loss of MLH1 and [email protected], MSH6 were seen more commonly. CONCLUSION: MSI occurs in a significant proportion of colorectal cancers in young (<50 years old) patients. Young age at colorectal cancer diagnosis, proximal tumor location, family history of colorectal cancer were independent predictors of MSI status in our patients. In a proportion of these young patients with MSI tumors, loss of expression of proteins by 2 MMR genes PMS2 and hMLH1 has been identified.
Keywords: Colorectal cancer, microsatellite instability, mismatch repair
|How to cite this article:|
Dubey A, Vishwanath S, Nikhil P, Rathore A, Pathak A. Microsatellite instability in stage II colorectal cancer: An Indian perspective. Indian J Cancer 2016;53:513-7
|How to cite this URL:|
Dubey A, Vishwanath S, Nikhil P, Rathore A, Pathak A. Microsatellite instability in stage II colorectal cancer: An Indian perspective. Indian J Cancer [serial online] 2016 [cited 2021 Apr 15];53:513-7. Available from: https://www.indianjcancer.com/text.asp?2016/53/4/513/204772
| » Introduction|| |
Colorectal cancers (CRCs) are the third most common malignancy in men (10.0% of all cancer cases) and second most common malignancy in women (9.4% of all cancer cases) worldwide. It remains the fourth most common cause of death due to cancer. In India, the annual incidence rates for colon cancer in men are 4.4/100,000 while that in women are 3.9/100,000. Broadly, the risk factors for CRC can be genetic and environmental/lifestyle-related factors. Most CRCs are sporadic although genetic factors increase the risk considerably. Around 80% of CRC are associated chromosomal instability (CIN) 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 CRCs, 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% of the marker panel is mutated. MMR status should be assessed in all patients who present with stage II CRC and irrespective of stage, in those fulfilling the Bethesda guidelines as they have both prognostic and therapeutic implications.
Mismatch repair system
The MMR genes are highly conserved from bacteria to humans. The yeast homologs of the bacterial mutS and mutL genes were cloned and given the names Mut S homolog (MSH) and Mut L homolog (MLH). Then, additional homologous copies of these genes were cloned from yeast, giving rise to the terms MSH1 through MSH6 and MLH1 through MLH3. Another MutL homolog, called postmeiotic segregation-1 (PMS1), was also identified in yeast. Each of these genes has been associated with specific repair functions in nuclear DNA, during meiosis, or in mitochondrial DNA. MLH1 and PMS2 mediate long-patch excision in postreplication MMR. The most frequent errors associated with microsatellites are base-base mismatches and insertion-deletion loops generating frameshift mutations and protein truncations. MSI arises from defects in DNA MMR system.
MSI has been observed in gastric, endometrial, ovarian, and sebaceous carcinomas as well as glioblastoma and lymphomas apart from CRC. Several population-based studies have reported that the prevalence of MSI-H in CRC ranges from 15% to 20%. MSI is more common among stage II (~20%) than stage III (~12%) CRC and is even less frequent among stage IV CRC (~4%).
The tumors with “ubiquitous somatic mutations at simple repeated sequences,” also simply called microsatellites, were more likely to arise in the proximal colon, less likely to be invasive, less likely to have mutations in KRAS or p53, more likely to appear poorly differentiated, and came from younger patients.
Molecular features of microsatellite instability tumors
The MSI phenotype is strongly associated with mutations in BRAF, MRE11A, and KRAS. The majority of CRC is characterized by CIN that arises through loss or gain of chromosome arms, chromosomal translocations, or gene amplification. MSI CRC tends to show a stable karyotype (CIN negative), and genetic instability in this subtype of CRC primarily reflects a defective MMR system.
BRAF mutations in CRC are more frequent in sporadic MSI-H tumors than in hereditary cases and are caused by hypermethylation of MLH. Strong association exists between sporadic MSI and the presence of the V600E BRAF mutation. Although a BRAF mutation profoundly reduces the probability of a diagnosis of Lynch syndrome, it does not entirely exclude the possibility. KRAS mutations, however, are more likely to be observed in microsatellite stable (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. High numbers of tumor-infiltrating lymphocytes observed in MSI-H tumors  are consistent with the strong immune response that MSI-H tumors elicit in the host.
Besides MSI-H/MMR, there is no validated molecular marker approved for the use in adjuvant settings. Although BRAF mutation portends a poor survival in patients with stage II tumors without MSI-H status, it does not help in tailoring treatment.
Microsatellite instability-high and microsatellite instability-low
MSI is detected by polymerase chain reaction 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. A consensus reference panel, known as the Bethesda panel, established a panel of microsatellite markers with appropriate sensitivity and specificity to diagnose MSI CRC. 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 MSS, indicating no loci with instability (or <10% of loci in larger panels).
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%–80% of this group have acquired methylation of MLH1; only ~2%–3% of all CRCs have germline mutations in one of the MMR genes. MSI identifies MMR-deficient CRC with approximately 93% sensitivity; most insensitivity is caused by mutations in MSH6.
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 elderly >70 years of age, whereas familial cases in younger <50 years of age. CRC with MSI-H generally has 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-like host response. 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.
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. Tumors with MSI-H have greater numbers of tumor-infiltrating lymphocytes that are activated and cytotoxic; which by itself is an independent factor associated with longer survival. 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.
It is a hereditary disease caused by inactivating germline mutations in MMR genes. These patients develop tumors at an early age (<30 years), frequently have multiple tumors, apart from those arising from the colon, rectum, endometrium, stomach, ovary, urinary tract, small intestine, and other sites. If germline mutations are harbored in MMR genes, the cumulative risk of developing CRC is 60%–70% in men and 30%–40% in women; the cumulative risk of developing endometrial cancer is 40%–80%. Patients with Lynch syndrome have lower stage disease when they are identified compared to patients with other types of CRC. Most MMR gene deletions and nonsense mutations in Lynch syndrome have pathologic consequences, but missense mutations are not always interpretable, therefore labeling them as “variants of uncertain significance.” It is also known that the missense mutation in MLH1 (c415G→C; D132H), increases risk for CRC, but does not always cause MSI in CRC cells.
Microsatellite instability in sporadic colorectal cancer
Only about 3% of all CRCs come from Lynch syndrome families. Approximately 15% of all CRCs have MSI-H, and most CRCs with MSI-H are sporadic. The majority of CRC with MSI-H has a loss of expression of MLH1 and PMS2 protein. The characteristic features of sporadic CRC with MSI-H include the absence of significant familial clustering, absence of MLH1 and PMS2 proteins, and frequent mutation (usually V600E) in BRAF. The tumors are frequently diploid (74%) and have a better prognosis than those with non-MSI tumors. Sporadic CRCs with MSI-H are older on a comparison note with Lynch syndrome patients.
Aims and objectives
The aim of this study is to analyze the frequency and clinicopathologic characteristics of MSI-H colorectal stage II cancers at a tertiary care center.
| » Materials and Methods|| |
We analyzed all stage II CRC patients who visited malignant disease treatment center at Army Hospital Research and Referral during January 2014 to December 2015. 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 [Table 1].
| » Results|| |
Of the 45 stage II colorectal tumors studied, 10 (22.2%) showed MSI-H. The findings correlated with those found in Ho et al. study (22.6%). Of ten cases, eight (80%) were men and rest two (20%) women (34.7% vs. 9%, P = 0.03). The incidence of MSI had a striking association with age at CRC diagnosis (70% MSI-H in below age 30 years, P = 0.04). The median age at diagnosis of MSI-H case was 35.2 years while that in Ho et al. study was 39.9 years which was comparable. Among the tumors of patients diagnosed before the age of 30 years, 36.8% (n = 7) showed MSI-H, in contrast to only 11.5% (n = 3) in those aged 31 or above at cancer diagnosis (P = 0.04). This finding also substantially correlated with those found in Ho et al. study (43.1% vs. 15.1%, P = 0.0005).
MSI-H tumors were more frequently right-sided (i.e. located proximal to the splenic flexure): Eight (80%) of MSI-H tumors were at the right colon compared with only 22.8% (n = 8) of MSS tumors in our study (P = 0.0008). PETACC-03 trial also found a slightly lower percentage of MSI-H tumors in the right colon (63.6%). None of the MSI-H cases were smokers. Baseline carcinoembryonic antigen level was elevated in 40% (n = 4) of the cases with MSI-H in our study.
Overall, mucinous adenocarcinoma was the predominant histology noted in six (60%) of all MSI-H cases, followed by well-differentiated and moderately differentiated adenocarcinoma accounting for 20% of cases each. Tumor-infiltrating lymphocytes were noted in 80% of MSI-H cases. None of the MSI-H cases had unfavorable histological features, namely, lymphovascular or perineural invasion.
Patients with MSI-H tumors had an increased incidence of synchronous CRCs accounting to 20% (n = 2); however, this association in our study was not found statistically significant (P = 0.3).
Family history of CRC in the first- and second-degree relatives was associated with significantly higher occurrence of MSI-H tumors: 40% (n = 4) compared with 11% (n = 4) in patients with MSI-L/MSS tumors in our study (P = 0.03).
For the exceptionally young patients (<30-year-old), the MSI rate was high, and there was a trend for MSI tumors to be located at the proximal colon (60%, n = 6). For patients in the remaining age group, despite the fact that MSI rate diminished with increasing age, there was again a strong association of MSI-H status with proximal colonic cancers (20%, n = 2). When considering MSI-H tumors alone, the predilection for proximal site distribution between those younger or older than 30 years was found statistically significant (P = 0.005).
On multivariate analysis, age at CRC diagnosis (</> 30 years), site of tumor (proximal or distal), and family history of CRC stood out as independent factors predicting MSI-H tumor occurrence. Ho et al. study also found the presence of synchronous tumors to be an independent factor predicting MSI-H positivity, which however was not found in our study. All (100%) patients tested positive for loss of PMS2 and MLH1 protein expression on IHC. Findings of Ho et al. study deferred with our study, with hMLH1 and hMSH2 being the predominant MMR protein losses observed.
| » Discussion|| |
MSI-H is more common among stage II (~20%) than stage III (~12%) CRC  and is even less frequent among stage IV CRC (~4%). We found a similar percentage of patients in stage II CRC to be MSI-H in our study (22.2%, n = 10). Similar to a previous report by Liu et al. (1995), we also found that the MSI-H rate in our tumors diminished as age at cancer diagnosis increased. More than half of the patients with MSI-H were younger than 30 years. The MSI-H rate fell rapidly for those more than 40 years. The MSI rate decreased even further for those older than 50 years (Chan et al., 1999b). In our study, patients aged <40 years had higher incidence of MSI-H tumors, which was higher than those found in study by Ho et al. (70% vs. 43.1%, respectively). Comparing the present and the previous studies, we have proven by statistical analysis that age is an important determinant of the prevalence of MSI-H.
Recognizable clinicopathological profile of MSI has been established from clinical studies. CRC with MSI-H has high histological grade, predominant mucinous phenotype, with a significant proportion of cases showing chron's-like host response, and numerous tumor-infiltrating lymphocytes on histology. In our study, out of ten patients, 60% had mucinous histology and 80% had tumor-infiltrating lymphocytes.
For the whole group of young patients (<30 years), MSI-H was significantly associated with tumor location at the proximal colon, in accordance with previous results (Lothe et al., 1993; Thibodeau et al., 1993; Kim et al., 1994; Liu et al., 1995; and Senba et al., 1998).
Positive family history of CRC was also an independent predictor for MSI tumor in our young patients. The group of patients who had synchronous cancers in colorectum was too small in our study, to comment on the statistically insignificant association found in relation to MSI-H. All patients tested positive for PMS2 and MLH1 MMR protein losses (n = 10).
In view of smaller study population of stage II CRC (n = 45), the association of clinical features with loss of MMR proteins cannot be determined with heightened certainty. Even so, the preliminary data suggest that patients with MMR protein losses are younger and have a stronger family history of CRC and a predominantly proximal colon location. However, we could not observe any association of loss of MMR protein expression with synchronous cancers in our study population.
Our dedicated oncology center at Delhi is the referral center for all cases of CRCs over India, serving the beneficiaries of armed forces throughout the nation, with its resource-rich setting providing all the appropriate investigations, and treatment available for its patients. Hence, the data genuinely reflect the prevalence of MSI tumors in stage II CRC patients of Northern India.
Knowledge of the association of various clinical features with MSI tumors and loss of MMR protein expression may facilitate selection of suitable patients for genetic testing. Besides, such an association may have an impact on the clinical management of young CRC patients and their families. Of the exceptionally young patients (<30 years) and the patients aged 30–49 years with proximal cancers, about half will have MSI tumors and may harbor germline losses in MMR protein expression. Therefore, total abdominal colectomy should be the surgical treatment of choice for these patients in view of their high chance of metachronous CRC development. In addition, first-degree relatives of these patients should be offered CRC screening. Similar clinical management should also be considered for a young CRC patient who has a strong family history of CRC or a personal history of metachronous cancer. For patients with proven germline loss of MMR protein expression, their family members should be offered predictive genetic testing.
| » Conclusion|| |
MSI occurs in a significant proportion of CRCs in patients <50-year-old. Young ages at CRC diagnosis, proximal tumor location, and family history of CRC were independent predictors of MSI status in our patients. Loss of expression of two MMR proteins, namely, PMS2, and MLH1 has been identified in all MSI-H patients of our study, of which 70% were <30 years of age, and all of them were below 50 years of age. This is in contrast to observation in previous studies where loss of PMS2 and MLH1 proteins was observed in older (>70 years) patients with MSI-H tumors, and in younger patients, MSI-H status was associated with loss of MLH1, MSH2, and MSH6 (Lynch syndrome).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| » References|| |
NCRP. Three-Year Report of the Population Based Cancer Registries- 2009-2011. National Cancer Registry Programme, Indian Council of Medical Research (ICMR), Bangalore, India; 2013.
Culligan 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.
Jiricny J. The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 2006;7:335-46.
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.
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.
Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-67.
Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934.
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(1 Pt 1):191-5.
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.
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.
Laghi L, Bianchi P, Malesci A. Differences and evolution of the methods for the assessment of microsatellite instability. Oncogene 2008;27:6313-21.
Umar A, Boland CR, Terdiman JP, Syngal S, de la Chapelle A, Rüschoff J, et al.
Revised bethesda guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst 2004;96:261-8.
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.
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.
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.
Roth AD, Tejpar S, Delorenzi M, Yan P, Fiocca R, 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.
Phillips SM, Banerjea A, Feakins R, Li SR, Bustin SA, Dorudi S. Tumour-infiltrating lymphocytes in colorectal cancer with microsatellite instability are activated and cytotoxic. Br J Surg 2004;91:469-75.
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.
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;14:5476-83.
Peltomäki P. Lynch syndrome genes. Fam Cancer 2005;4:227-32.
Watson P, Vasen HF, Mecklin JP, Bernstein I, Aarnio M, Järvinen HJ, et al.
The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome. Int J Cancer 2008;123:444-9.
Stoffel E, Mukherjee B, Raymond VM, Tayob N, Kastrinos F, Sparr J, et al.
Calculation of risk of colorectal and endometrial cancer among patients with Lynch syndrome. Gastroenterology 2009;137:1621-7.
Aarnio M, Mustonen H, Mecklin JP, Järvinen HJ. Prognosis of colorectal cancer varies in different high-risk conditions. Ann Med 1998;30:75-80.
Lipkin SM, Rozek LS, Rennert G, Yang W, Chen PC, Hacia J, et al.
The MLH1 D132H variant is associated with susceptibility to sporadic colorectal cancer. Nat Genet 2004;36:694-9.
Hampel H, Frankel WL, Martin E, Arnold M, Khanduja K, Kuebler P, et al.
Feasibility of screening for Lynch syndrome among patients with colorectal cancer. J Clin Oncol 2008;26:5783-8.
Ward R, Meagher A, Tomlinson I, O'Connor T, Norrie M, Wu R, et al.
Microsatellite instability and the clinicopathological features of sporadic colorectal cancer. Gut 2001;48:821-9.
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.
Sinicrope FA, Rego RL, Halling KC, Foster N, Sargent DJ, La Plant B, et al.
Prognostic impact of microsatellite instability and DNA ploidy in human colon carcinoma patients. Gastroenterology 2006;131:729-37.