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Originally published as JCO Early Release 10.1200/JCO.2006.09.4607 on January 16 2007 © 2007 American Society of Clinical Oncology.
Clinical Uses of Microsatellite Instability Testing in Colorectal Cancer: An Ongoing Challenge
GI Cancer Research Laboratory, Division of Gastroenterology, Department of Internal Medicine, Baylor University Medical Center and Sammons Cancer Center and the Baylor Research Institute, Dallas, TX Microsatellite instability (MSI) is the mutational signature found in colorectal cancers (CRCs) that evolve as a result of inactivation of the DNA mismatch repair (MMR) system. MSI can be found in approximately 15% of all CRCs. Approximately 3% of all CRCs are a consequence of Lynch Syndrome, and nearly all Lynch Syndrome CRCs have MSI.1 Another 12% of CRCs represent the noninherited form of DNA MMR inactivation that is induced by methylation of the promoter of the MLH1 gene, which silences gene expression.2 MSI is one of the premier molecular markers in CRC because it indicates the pathophysiologic genesis of the tumor, and it provides some clinical information that is of use in caring for this subset of CRC patients. Testing for MSI is within the grasp of a molecular diagnostic laboratory, and many investigators have proposed various clinical applications of this information. This issue of the Journal of Clinical Oncology includes two articles on testing the clinical utility of MSI3,4; one of which confirms its utility in identifying Lynch Syndrome families,3 the other casts doubt on its utility as a prognostic and predictive marker in sporadic CRC.4 These articles illustrate the utility of molecular diagnostics, but also note the challenges of using new technologies.
Valle and colleagues3 gathered 64 familial clusters of CRC from a Spanish national registry, all of whom met the Amsterdam-I criteria.5 Each family had a pedigree that included at least three members with CRC, and at least one affected member was younger than 50 years old. This was the classic algorithm to identify Lynch Syndrome families, and it was initially assumed to be very specific for this diagnosis. However, a registry of familial clusters of CRC (the Colon Cancer Family Registry, or Colon CFR) reported that only Kim and colleagues4 examined the two other clinical uses of MSI testing in CRC: its value in predicting prognosis, and the response to chemotherapy. This group used the potentially risky approach of mining data retrospectively from prior National Surgical Adjuvant Breast and Bowel Project (NSABP) Collaborative Studies (trials C-01 through C-04), an approach necessitated by the absence of control groups that were not treated with chemotherapy since the late 1980s. The nature of this retrospective study required using untreated controls from trials C-01 and C-02, and compared them with treated patients from trials C-03 and C-04—an approach that statisticians prefer to avoid. Patient selection, diagnostic and staging techniques, and other unknown variables, all change over time. Tissue blocks were available for only 20% of untreated patients, adding sampling issues to this complex analysis. Unlike using MSI to test for Lynch Syndrome, clinical predictive applications of MSI testing have been more controversial. It was noted in the initial discovery of MSI in CRC that patients with MSI tumors had better rates of survival.7 This has been confirmed by others,8-10 particularly in younger patients (in whom the difference is especially impressive11), but other reports have had mixed findings. This may be due, at least in part, to the fact that the age distribution of MSI CRC patients is bimodal. Lynch Syndrome patients make up 20% to 25% of the MSI group, and develop CRC at an average age in the mid-40s. The sporadic, methylation-related MSI-CRC patients make up the other 75% to 80%, and are typically older than other sporadic CRC patients, with an average age that is older than 70 years. Depending on the relative proportions of each, one might get a cohort with many comorbidities, on the one hand, or a young, healthier group on the other. Kim et al found an interesting reduction in tumor recurrence for the MSI group (relative risk = 0.68), which was not significant because of the wide confidence intervals. The relative risk for overall survival was modest at best (relative risk = 0.91 for MSI tumors). Because many tissue specimens were unavailable for analysis, the number of patients analyzed was relatively small, inviting the possibility of missing a significant association through a beta error. However, to be fair, this is not the only study that has failed to confirm the prognostic significance of MSI in CRC.12 Kim and colleagues also tested the value of MSI to predict the response to chemotherapy, a concept that has taken controversy to a substantially higher level. It has long been known that the DNA MMR system is involved in signaling a cell death response after sufficiently toxic DNA damage, and that DNA MMR–deficient cells are relatively tolerant to DNA damage.13,14 It was subsequently found that this is also true for CRC cells, and that restoration of the MMR system would restore sensitivity to several compounds that damage DNA,15,16 including chemotherapeutic agents such as fluorouracil (FU).17 Thus, the prediction was made that patients with MSI CRCs would be relatively resistant to the beneficial effects of FU-based regimens. The first clinical test of the hypothesis provided unexpectedly contrary results. A retrospective Australian study concluded that patients with Dukes' C MSI CRCs derived a specific survival benefit from FU.18 The problem with this study was that the patients had not been randomly assigned into treatment groups, but rather had been selected for treatment before this regimen became the standard of care. The clinicians apparently exercised good judgment when selecting their patients, as the median age in the treatment group was 13 years younger than those not treated—a group that presumably included the patients with the worst comorbidities. Another nonrandomized study design led to a similar report from Finland.19 These articles triggered additional studies, but most of them challenged the initial reports and demonstrated that chemotherapy did not provide benefit in the MSI subset of CRCs.8,20-23 In the largest of these studies,20 a significantly better prognosis was observed with MSI, and there was a (nonsignificant) two-fold mortality hazard associated with treating MSI CRC patients with chemotherapy, which was a particularly disturbing finding that might reflect the adverse effects of chemotherapy on the brisk immunologic response observed in these tumors.24 Other studies have reported neither benefit nor harm from FU-based chemotherapy in MSI CRCs.25 In retrospect, it appears that the initial retrospective nonrandomized studies had inadvertently selected a biased treatment group with a different natural history and reached conclusions that were not sustained by subsequent studies. Good investigation has the ability to correct prior errors, as occurred in this instance. Kim et al4 also had to deal with the definition of MSI at the technical level. MSI is defined, by convention, as a mutation in more than 30% of microsatellite sequences tested.26 One major technical challenge is that the DNA polymerase used to amplify the microsatellite sequences in vitro introduces mutations that must be distinguished from those generated biologically in the tumor. Accurately defining MSI requires some experience in the interpretation. If one were to overestimate the proportion of CRCs with MSI, this would distort the composition of the patient groups, which might obscure a relatively small survival benefit if the sample size were small. The proportion of tumors with MSI in the study by Kim et al was at the high end of the spectrum (21.4% in the untreated group). A check on the accuracy of the assignment to the MSI group can be estimated by looking at the results with the two mononucleotide repeats, BAT25 and BAT26, which are thought to be 90% to 95% sensitive for MSI.27 Kim et al reported mutations in 82.6% and 78.3% at these loci respectively, raising the possibility that a proportion (perhaps 10% to 15%) of the tumors identified as MSI may have been false positives. Kim at al were unable to confirm that MSI testing is useful for predicting survival or response to chemotherapy, but the wide confidence intervals made it impossible to reject the possibility that modest effects on either might have been overlooked. It may be the case that technical considerations—added to the major challenge of the retrospective design—made it difficult to detect the truth. The history of this field demonstrates that issues of design and technique can overwhelm attempts to predict outcomes, in part because the differences may not be large enough to detect without having a large number of patients and an unassailable approach to MSI testing. The article by Valle et al3 illustrates the clinical utility of MSI testing in familial clusters of CRCs, and confirms the important point that the Amsterdam criteria will identify both true Lynch Syndrome and a second group of familial cancer that is not caused by defective DNA MMR activity—Syndrome X. The article by Kim and colleagues illustrates that the proposed ability of MSI testing to predict prognosis is perhaps modest in magnitude, and it may require a very large number of case subjects to prove the point. They could find neither benefit nor harm from treating MSI CRCs with adjuvant chemotherapy. More importantly, this work underscores the point that one must be very attentive to study design and technical issues when attempting to dissect the conflicting trends of a biologic process that may confer a relative resistance to our therapeutic interventions, against the background of a possibly beneficial natural history conferred by the same process. At the very least, nearly all of the reports subsequent to the initial nonrandomized studies from Australia and Finland have failed to demonstrate a positive therapeutic response to FU in MSI CRCs. Whether or not it is hazardous to give chemotherapy to such patients with MSI CRCs is still unproven, but good judgment requires solid evidence of benefit before we offer treatment to patients not the absence of proof that would make them worse. While the jury is out on this final issue, it may be prudent to exercise caution when contemplating adjuvant chemotherapy for patients with these types of CRCs. Furthermore, MSI should be taken into account in future trials of chemotherapy for CRC. The magnitude of benefit for adjuvant chemotherapy is still relatively small for stage III CRCs (and arguably not present at all for stage II CRCs). And in the case of MSI CRCs, most investigators have suggested that the natural history of the tumor may be such that an additional benefit from FU may be difficult to achieve for stage II disease.11 Our search for a reliable marker that predicts outcome (prognosis) and response to intervention (prediction) is still young, and we still have much to learn. AUTHOR'S DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST The author indicated no potential conflicts of interest. ACKNOWLEDGMENTS This editorial was supported in part by a grant from the National Institutes of Health (Grant No. R-01 CA72851). NOTES published online ahead of print at www.jco.org on January 16, 2007. REFERENCES 1. Boland CR: Evolution of the nomenclature for the hereditary colorectal cancer syndromes. Fam Cancer 4:211-218, 2005[CrossRef][Medline] 2. Kane MF, Loda M, Gaida GM, et al: Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 57:808-811, 1997 3. Valle L, Perea J, Carbonell P, et al: Clinicopathologic and pedigree differences in Amsterdam I–positive hereditary nonpolyposis colorectal cancer families according to tumor microsatellite instability status. J Clin Oncol 25:781-786, 2007 4. Kim GP, Colangelo LH, Wieand S, et al: Prognostic and predictive roles of high-degree microsatellite instability in colon cancer: A National Cancer Institute–National Surgical Adjuvant Breast and Bowel Project Collaborative Study. J Clin Oncol 25:767-772, 2007 5. Vasen HF, Mecklin JP, Khan PM, et al: The International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC). Dis Colon Rectum 34:424-425, 1991[CrossRef][Medline] 6. Lindor NM, Rabe K, Petersen GM, et al: Lower cancer incidence in Amsterdam-I criteria families without mismatch repair deficiency: Familial colorectal cancer type X. JAMA 293:1979-1985, 2005 7. Thibodeau SN, Bren G, Schaid D: Microsatellite instability in cancer of the proximal colon. Science 260:816-819, 1993 8. Benatti P, Gafa R, Barana D, et al: Microsatellite instability and colorectal cancer prognosis. Clin Cancer Res 11:8332-8340, 2005 9. Parc Y, Gueroult S, Mourra N, et al: Prognostic significance of microsatellite instability determined by immunohistochemical staining of MSH2 and MLH1 in sporadic T3N0M0 colon cancer. Gut 53:371-375, 2004 10. Sinicrope FA, Rego RL, Halling KC, et al: Prognostic impact of microsatellite instability and DNA ploidy in human colon carcinoma patients. Gastroenterology 131:729-737, 2006[CrossRef][Medline] 11. Gryfe R, Kim H, Hsieh ET, et al: Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 342:69-77, 2000 12. Barnetson RA, Tenesa A, Farrington SM, et al: Identification and survival of carriers of mutations in DNA mismatch-repair genes in colon cancer. N Engl J Med 354:2751-2763, 2006 13. Branch P, Aquilina G, Bignami M, et al: Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature 362:652-654, 1993[CrossRef][Medline] 14. Kat A, Thilly WG, Fang WH, et al: An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair. Proc Natl Acad Sci U S A 90:6424-6428, 1993 15. Hawn MT, Umar A, Carethers JM, et al: Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint. Cancer Res 55:3721-3725, 1995 16. Carethers JM, Hawn MT, Chauhan DP, et al: Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine. J Clin Invest 98:199-206, 1996[Medline] 17. Aebi S, Kurdi-Haidar B, Gordon R, et al: Loss of DNA mismatch repair in acquired resistance to cisplatin. Cancer Res 56:3087-3090, 1996 18. Elsaleh H, Joseph D, Grieu F, et al: Association of tumour site and sex with survival benefit from adjuvant chemotherapy in colorectal cancer. Lancet 355:1745-1750, 2000[CrossRef][Medline] 19. Hemminki A, Mecklin JP, Jarvinen H, et al: Microsatellite instability is a favorable prognostic indicator in patients with colorectal cancer receiving chemotherapy. Gastroenterology 119:921-928, 2000[CrossRef][Medline] 20. Ribic CM, Sargent DJ, Moore MJ, et al: Tumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancer. N Engl J Med 349:247-257, 2003 21. Carethers JM, Smith EJ, Behling CA, et al: Use of 5-fluorouracil and survival in patients with microsatellite-unstable colorectal cancer. Gastroenterology 126:394-401, 2004[CrossRef] 22. Jover R, Zapater P, Castells A, et al: Mismatch repair status in the prediction of benefit from adjuvant fluorouracil chemotherapy in colorectal cancer. Gut 55:848-855, 2006 23. Carethers JM: Prospective evaluation of fluorouracil chemotherapy based on the genetic makeup of colorectal cancer. Gut 55:759-761, 2006 24. Jass JR: HNPCC and sporadic MSI-H colorectal cancer: A review of the morphological similarities and differences. Fam Cancer 3:93-100, 2004[CrossRef][Medline] 25. de Vos tot Nederveen Cappel WH, Meulenbeld HJ, Kleibeuker JH, et al: Survival after adjuvant 5-FU treatment for stage III colon cancer in hereditary nonpolyposis colorectal cancer. Int J Cancer 109:468-471, 2004[CrossRef][Medline] 26. Boland CR, Thibodeau SN, Hamilton SR, 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 58:5248-5257, 1998 27. Suraweera N, Duval A, Reperant M, et al: Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR. Gastroenterology 123:1804-1811, 2002[CrossRef][Medline] Related Articles
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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60% of these classic clusters can be linked to a germline mutation in a DNA MMR gene. The other 40% of families had microsatellite stable (MSS) tumors, and these families have been given the temporary designation "Syndrome X"; the genetic basis of this syndrome is being explored.
