Microsatellite Instability Testing in Colorectal Carcinoma: A Practical Guide

Abstract

As health care reform progresses, the pressure to more closely integrate clinical service lines such as colorectal cancer (CRC) management has intensified. The practicing gastroenterologist may find that they are not equipped to understand pathology information required for coordinated team-based care of their patients. This is especially true in the case of molecular classification of colorectal cancer, something that has become a standard component of comprehensive oncologic care and has been incorporated into many gastroenterology (GI) practice pathology services. Molecular characterization and classification of colorectal cancer not only provides insight into the pathogenesis of cancer but has prognostic and therapeutic implications and is important for the gastroenterologist to understand and manage well. This review is a practical guide to the most common molecular tests used in what has become standard GI practice.Molecular Classification of Colorectal CancerThe molecular classification of colon cancer is based on the cumulative study of precursor lesions (such as adenomas and sessile serrated polyps), inherited colon cancer syndromes (such as familial adenomatous polyposis syndrome and Lynch syndrome/hereditary nonpolyposis colon cancer), and molecular profiling of colorectal cancers. Broadly, colorectal cancers are divided into 2 general groups based on genomic differences: chromosomal instability, accounting for 75% to 80% of all colorectal cancers, and microsatellite instability (MSI), accounting for 15% to 20% of all colorectal cancers.1Markowitz S.D. Bertagnolli M.M. Molecular origins of cancer: molecular basis of colorectal cancer.N Engl J Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1367) Google Scholar, 2Al-Sohaily S. Biankin A. Leong R. et al.Molecular pathways in colorectal cancer.J Gastroenterol Hepatol. 2012; 27: 1423-1431Crossref PubMed Scopus (154) Google Scholar Inherited colorectal susceptibility syndromes are estimated to account for approximately 1% to 2% of the MSI cancers and less than 1% of chromosomal instability cancers.Microsatellite Instability PathwayMSI is defined by changes of microsatellite length (repetitive noncoding DNA sequences) resulting from deficient mismatch repair (dMMR) during DNA replication.1Markowitz S.D. Bertagnolli M.M. Molecular origins of cancer: molecular basis of colorectal cancer.N Engl J Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1367) Google Scholar, 3Zhang X. Li J. Era of universal testing of microsatellite instability in colorectal cancer.World J Gastrointest Oncol. 2013; 5: 12-19Crossref PubMed Google Scholar, 4Zaanan A. Meunier K. Sangar F. et al.Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications.Cell Oncol (Dordr). 2011; 34: 155-176Crossref PubMed Scopus (38) Google Scholar The protein complex responsible for mismatch repair function is a tetramer composed of 2 heterodimers: MLH1/PMS2 and MSH2/MSH6.4Zaanan A. Meunier K. Sangar F. et al.Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications.Cell Oncol (Dordr). 2011; 34: 155-176Crossref PubMed Scopus (38) Google Scholar The expression of each protein in a heterodimer is dependent on its partner, such that if one protein is absent, the partner protein consequently is degraded. When this occurs, the heterodimer is not available to form a functional tetramer and dMMR, as manifested by MSI, is the result.Most dMMR/MSI cancers occur sporadically and are associated with the loss of MLH1 expression owing to epigenetic silencing of the MLH1 gene promoter via CpG island methylation.1Markowitz S.D. Bertagnolli M.M. Molecular origins of cancer: molecular basis of colorectal cancer.N Engl J Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1367) Google Scholar, 2Al-Sohaily S. Biankin A. Leong R. et al.Molecular pathways in colorectal cancer.J Gastroenterol Hepatol. 2012; 27: 1423-1431Crossref PubMed Scopus (154) Google Scholar The precursor lesion of sporadic dMMR/MSI cancers is believed to be the sessile serrated polyp, an epithelial proliferation characterized by the V600E BRAF mutation. Therefore, sporadic dMMR/MSI cancers also frequently harbor the V600E BRAF mutation.5Lochhead P. Kuchiba A. Imamura Y. et al.Microsatellite instability and BRAF mutation testing in colorectal cancer prognostication.J Natl Cancer Inst. 2013; 105: 1151-1156Crossref PubMed Scopus (324) Google ScholarApproximately 1% to 2% of dMMR/MSI cancers occur in the setting of Lynch syndrome as a result of a hereditary gene defect in 1 of the 4 MMR genes.6Ward R.L. Hicks S. Hawkins N.J. Population-based molecular screening for Lynch syndrome: implications for personalized medicine.J Clin Oncol. 2013; 31: 2554-2562Crossref PubMed Scopus (67) Google Scholar, 7Umar A. Boland C.R. Terdiman J.P. et al.Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2430) Google Scholar The most frequently mutated gene in Lynch syndrome patients is MSH2 (40%), followed by MLH1 (30%). MSH6 and PMS2 are mutated at lesser frequencies, approximately 15% each. In contrast to the sporadic setting, dMMR/MSI cancer in Lynch syndrome patients arises from adenomas without BRAF mutations. Therefore, cancers in Lynch syndrome patients will have a wild-type BRAF gene.5Lochhead P. Kuchiba A. Imamura Y. et al.Microsatellite instability and BRAF mutation testing in colorectal cancer prognostication.J Natl Cancer Inst. 2013; 105: 1151-1156Crossref PubMed Scopus (324) Google ScholarTechnical Aspects of TestingMethods of Deficient Mismatch Repair/Microsatellite Instability DetectiondMMR is detected by immunohistochemistry (IHC) and MSI is detected by polymerase chain reaction (PCR).3Zhang X. Li J. Era of universal testing of microsatellite instability in colorectal cancer.World J Gastrointest Oncol. 2013; 5: 12-19Crossref PubMed Google Scholar, 8Laghi L. Bianchi P. Malesci A. Differences and evolution of the methods for the assessment of microsatellite instability.Oncogene. 2008; 27: 6313-6321Crossref PubMed Scopus (82) Google Scholar PCR involves extraction of DNA from a tumor followed by DNA amplification of microsatellite markers, and determination of the amplified microsatellite lengths as compared with nontumor DNA from the same patient. Although laboratories vary with regard to the number of microsatellites tested, most use a standard set of 5 microsatellite markers. A tumor is classified as MSI-high if 2 or more of the 5 microsatellite markers show instability, as MSI-low if only 1 of 5 markers is unstable, and as microsatellite stable (MSS) if the microsatellite markers show no expansion.3Zhang X. Li J. Era of universal testing of microsatellite instability in colorectal cancer.World J Gastrointest Oncol. 2013; 5: 12-19Crossref PubMed Google Scholar, 8Laghi L. Bianchi P. Malesci A. Differences and evolution of the methods for the assessment of microsatellite instability.Oncogene. 2008; 27: 6313-6321Crossref PubMed Scopus (82) Google ScholarThe IHC method uses antibodies directed against each MMR protein to detect the expression of the proteins in the tumor cells.3Zhang X. Li J. Era of universal testing of microsatellite instability in colorectal cancer.World J Gastrointest Oncol. 2013; 5: 12-19Crossref PubMed Google Scholar In cancers with dMMR/MSI, loss of nuclear expression of MMR proteins is seen in the cancer cells. In contrast, non-neoplastic cells, such as lymphocytes or adjacent colonic mucosa, show preserved nuclear expression of the MMR proteins, irrespective of the hereditary or sporadic setting. The non-neoplastic cells therefore serve as an important internal control for the IHC procedure. Most laboratories test each of the 4 MMR proteins. The majority of dMMR/MSI cancers show loss of expression of both MMR proteins in a heterodimer (either MLH1/PMS2 or MSH2/MSH6) in the cancer cells, with preserved expression of the other heterodimer. In sporadic dMMR/MSI cancers, loss of MLH1/PMS2 expression is characteristic, whereas in Lynch syndrome either heterodimer may be lost.3Zhang X. Li J. Era of universal testing of microsatellite instability in colorectal cancer.World J Gastrointest Oncol. 2013; 5: 12-19Crossref PubMed Google Scholar, 6Ward R.L. Hicks S. Hawkins N.J. Population-based molecular screening for Lynch syndrome: implications for personalized medicine.J Clin Oncol. 2013; 31: 2554-2562Crossref PubMed Scopus (67) Google Scholar Occasionally, unusual IHC patterns exist, usually in the setting of Lynch syndrome, such as isolated loss of MSH6 in 10% of cancers or isolated loss of PMS2 in approximately 5% of cancers.6Ward R.L. Hicks S. Hawkins N.J. Population-based molecular screening for Lynch syndrome: implications for personalized medicine.J Clin Oncol. 2013; 31: 2554-2562Crossref PubMed Scopus (67) Google ScholarPolymerase Chain Reaction vs ImmunohistochemistryThe results obtained from PCR and IHC studies are complementary but provide different information.3Zhang X. Li J. Era of universal testing of microsatellite instability in colorectal cancer.World J Gastrointest Oncol. 2013; 5: 12-19Crossref PubMed Google Scholar, 7Umar A. Boland C.R. Terdiman J.P. et al.Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2430) Google Scholar The PCR method does not detect which protein in the mismatch repair tetramer is deficient. Therefore, PCR cannot distinguish between sporadic or Lynch syndrome associated dMMR/MSI cancer. IHC, on the other hand, provides specific mismatch repair protein expression data and can suggest etiology. Loss of MSH2/MSH6 suggests Lynch syndrome, whereas loss of MLH1/PMS2, although seen in Lynch syndrome, is characteristic of the more common sporadic dMMR/MSI cancer.6Ward R.L. Hicks S. Hawkins N.J. Population-based molecular screening for Lynch syndrome: implications for personalized medicine.J Clin Oncol. 2013; 31: 2554-2562Crossref PubMed Scopus (67) Google Scholar When present, abnormal IHC results also can be used to guide gene sequencing in patients with a high risk of Lynch syndrome. If the nuclear protein expression of all 4 MMR proteins is intact, the tumor is assumed to be MSS, with rare exceptions, and PCR may not be needed except in patients at high risk for Lynch syndrome.IHC is inexpensive, is widely available in most pathology laboratories, and can be performed on both biopsy specimens and resection specimens, usually within 1 to 2 days. In the majority of cases, interpretation of IHC expression is straightforward and requires little training, with false-negative results rarely occurring. The latter occurs in less than 10% of Lynch syndrome patients with mutations that lead to protein dysfunction with preserved immunoreactivity.4Zaanan A. Meunier K. Sangar F. et al.Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications.Cell Oncol (Dordr). 2011; 34: 155-176Crossref PubMed Scopus (38) Google Scholar PCR analysis is performed on tissue removed from a tissue block containing an adequate tumor sample (at least 30% of the tissue within the block consisting of tumor) for DNA extraction, as well as accompanying normal tissue for comparison. Biopsy samples may not contain sufficient tumor volume for PCR, whereas most resections are sufficient. The turnaround time for PCR is 5 days to 2 weeks.When to Test and Which SpecimenIt now generally is accepted that all patients with colorectal cancer should be tested for MSI/dMMR using either IHC, PCR, or both at some point during the evaluation and treatment of their cancer, regardless of their age.3Zhang X. Li J. Era of universal testing of microsatellite instability in colorectal cancer.World J Gastrointest Oncol. 2013; 5: 12-19Crossref PubMed Google Scholar, 9Ngeow J. Eng C. Population-based universal screening for Lynch syndrome: ready, set... how?.J Clin Oncol. 2013; 31: 2527-2529Crossref PubMed Scopus (10) Google Scholar Timing of testing and which specimen is used for molecular testing vary widely among medical centers and GI pathology services. Published guidelines, such as the 2004 Revised Bethesda guidelines and the Revised American College of Gastroenterology 2008 guidelines, are aimed at detecting Lynch syndrome patients postoperatively.7Umar A. Boland C.R. Terdiman J.P. et al.Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2430) Google Scholar, 10Rex D.K. Johnson D.A. Anderson J.C. et al.American College of Gastroenterology guidelines for colorectal cancer screening 2009 [corrected].Am J Gastroenterol. 2009; 104: 739-750Crossref PubMed Scopus (1164) Google Scholar However, testing of biopsy material before surgical resection has been advocated by some and has become standard practice in many institutions. At least 2 updated guidelines are in process, one by the US Multi-Society Task Force on Colorectal Cancer and one by the American Gastroenterological Association Institute (John I. Allen, MD, personal communication).Testing of cancer in the setting of neoadjuvant chemoradiation treatment can be challenging because marked therapy responses limit the amount of cancerous tissue available for DNA extraction. Residual tumor volume may be sufficient for IHC analysis. Pitfalls exist; neoadjuvant therapy has been reported to induce MSH6 loss in 20% of colon cancer.11Bao F. Panarelli N.C. Rennert H. et al.Neoadjuvant therapy induces loss of MSH6 expression in colorectal carcinoma.Am J Surg Pathol. 2010; 34: 1798-1804Crossref PubMed Scopus (98) Google Scholar In this setting, comparison with PCR results or prior biopsy samples may be needed.In addition to testing cancer tissue, dMMR/MSI testing also can be performed on adenomatous tissue in patients with a high risk of having Lynch syndrome based on clinical criteria.12Pino M.S. Mino-Kenudson M. Wildemore B.M. et al.Deficient DNA mismatch repair is common in Lynch syndrome-associated colorectal adenomas.J Mol Diagn. 2009; 11: 238-247Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar However, in this setting, the interpretation of intact MMR expression by IHC should not be used as evidence against the possibility of Lynch syndrome because MMR loss is speculated to occur as a late event in the adenoma-carcinoma sequence.Initiation of dMMR/MSI testing is best achieved through a coordinated effort between clinicians (gastroenterologists, oncologists, surgeons), pathologists, and genetic counselors. Decisions regarding which test to use (IHC, PCR, or both) are center-dependent. There is a trend at many centers to begin with IHC for dMMR, reserving PCR analysis for specific situations (Figure 1).Figure 1MMR IHC algorithm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Clinical Aspects of TestingIndications for Mismatch Repair/Microsatellite Instability TestingTissue testing for dMMR/MSI serves 2 clinically important functions: to screen for Lynch syndrome and to provide prognostic information regardless of syndrome status. In addition, although beyond the scope of this review, MSI testing is also increasingly used in other investigative contexts, such as to predict therapeutic response to 5-fluorouracil. For these reasons, and because the Amsterdam and Bethesda criteria have failed to serve as effective screens for Lynch syndrome, determination of dMMR/MSI now is recommended in all colorectal tumors.13Palomaki G.E. McClain M.R. Melillo S. et al.EGAPP supplementary evidence review: DNA testing strategies aimed at reducing morbidity and mortality from Lynch syndrome.Genet Med. 2009; 11: 42-65Abstract Full Text Full Text PDF PubMed Scopus (391) Google ScholarRelevance to Lynch SyndromedMMR/MSI detection serve solely as a screen for Lynch syndrome, and are not by themselves diagnostic. In addition, each test will miss 5% to 15% of all cases of Lynch syndrome. Therefore, a negative tumor screen should not negate a referral to genetic counseling if the personal and/or family history is suggestive of a hereditary cancer syndrome.14Overview of testing for Lynch syndrome/HNPCC. Updated 2012. Available at: http://www.nchpeg.org/index.php?option=com_docman&task=cat_view&gid=58&Itemid=. Accessed October 2013.Google Scholar If IHC shows loss of MLH1 and PMS2 expression, BRAF analysis should be performed. If a V600E BRAF mutation is present and there are no other risk factors (Supplementary Table 1), the patient most likely does not have Lynch syndrome. Outside of this scenario, patients whose tumors are MSI-high and/or show abnormal IHC should be referred to a certified genetic counselor for informed consent to undergo diagnostic germline testing for mutations in the Lynch genes (Figure 1). Informed consent before tumor testing has been raised as an ethical issue15Chubak B. Heald B. Sharp R.R. Informed consent to microsatellite instability and immunohistochemistry screening for Lynch syndrome.Genet Med. 2011; 13: 356-360Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar; however, it is not currently required because tumor testing is simply a screen for Lynch syndrome. It is advised that clinicians prepare patients for the possibility that if their tumor screen is positive, they then will be referred for genetic counseling and testing.Tumor screening and genetic testing for Lynch syndrome is critical in preventing additional primary malignancies in the patient, and in testing and providing appropriate surveillance and risk reduction to family members. However, a recent study showed that surgeons referred fewer than half of their patients with high-risk tumors for genetic testing.6Ward R.L. Hicks S. Hawkins N.J. Population-based molecular screening for Lynch syndrome: implications for personalized medicine.J Clin Oncol. 2013; 31: 2554-2562Crossref PubMed Scopus (67) Google Scholar This represents not only a substantial liability risk for clinicians and their institutions, but a waste of health care dollars and a potentially life-saving lost opportunity for patients and their families. At some centers genetic counselors review all MSI testing, including follow-up methylation/BRAF testing, to ensure correct interpretation and to increase the likelihood that patients will receive the necessary genetic counseling and testing they need.9Ngeow J. Eng C. Population-based universal screening for Lynch syndrome: ready, set... how?.J Clin Oncol. 2013; 31: 2527-2529Crossref PubMed Scopus (10) Google Scholar, 16Lynch P.M. Tumor-based screening for hereditary nonpolyposis colorectal cancer: does age-based selection optimize yield?.J Oncol Pract. 2013; 9: 180-181Crossref PubMed Scopus (1) Google ScholarThe cost of dMMR/MSI testing is variable and depends on methods used (IHC and/or PCR). With the decreasing prices of germline gene panels that include not only Lynch syndrome genes but many other genes associated with hereditary cancer syndromes, it soon may be less expensive and more accurate to offer all patients diagnosed with CRC at age 50 years or younger, and those with a personal or family history suggestive of a hereditary cancer syndrome, genetic counseling and testing and to reserve routine tumor testing for those diagnosed with CRC at older than age 50 with no risk factors. In fact, with the cost of genome-wide analysis expected to decrease to less than $1000 within a few years, gastroenterologists may be increasingly confronted with a patient who brings their tumor analysis, showing an alteration in MMR genes. Thus, education about the implications of these findings will become increasingly important.Relevance of Mismatch Repair/Microsatellite Instability Testing to PrognosisThe diagnosis of dMMR/MSI colorectal cancer has important clinical implications regarding prognosis, and has emerged as an essential component of the evaluation and management of patients with colorectal cancer, especially those with stage II colon cancer.Retrospective studies have shown that dMMR/MSI colorectal cancer is associated with a favorable prognosis independent of classic clinical prognostic factors, including stage.17Popat S. Hubner R. Houlston R.S. Systematic review of microsatellite instability and colorectal cancer prognosis.J Clin Oncol. 2005; 23: 609-618Crossref PubMed Scopus (1420) Google Scholar, 18Gryfe R. Kim H. Hsieh E.T. et al.Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer.N Engl J Med. 2000; 342: 69-77Crossref PubMed Scopus (1163) Google Scholar Patients with dMMR/MSI tumors are less likely to have lymph node and distant metastatic disease than patients with MMR-proficient tumors, and the prevalence of the dMMR/MSI phenotype decreases with advancing stage at diagnosis from more than 20% in stage II to less than 4% in stage IV. In one of the largest pooled analyses of more than 7600 colorectal cancer cases, of which 16.7% were dMMR/MSI, the hazard ratio for overall survival associated with dMMR/MSI was 0.65, and this benefit was confirmed in all stages.17Popat S. Hubner R. Houlston R.S. Systematic review of microsatellite instability and colorectal cancer prognosis.J Clin Oncol. 2005; 23: 609-618Crossref PubMed Scopus (1420) Google Scholar In patients with stage II and III colon cancer, recurrence-free survival and overall survival are increased significantly in patients with dMMR/MSI tumors compared with those with MMR-proficient/MSS tumors.19Sargent D.J. Marsoni S. Monges G. et al.Defective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancer.J Clin Oncol. 2010; 28: 3219-3226Crossref PubMed Scopus (1123) Google Scholar, 20Sinicrope F.A. Foster N.R. Thibodeau S.N. et al.DNA mismatch repair status and colon cancer recurrence and survival in clinical trials of 5-fluorouracil-based adjuvant therapy.J Natl Cancer Inst. 2011; 103: 863-875Crossref PubMed Scopus (387) Google Scholar These observations, in the aggregate, support the hypothesis that dMMR/MSI tumors have reduced metastatic potential and a favorable biology compared with MMR-proficient/MSS tumors.Despite its prognostic value, MMR status is not incorporated into widely used calculators for the assessment of risk of recurrence in stage II and III colorectal cancer and does not figure into colonoscopy surveillance recommendations after cancer resection. Nonetheless, MMR/MSI status should be assessed routinely and considered in risk assessment in all patients with stage II colon cancer because the favorable prognosis of the dMMR/MSI phenotype is a key determinant in the decision to use adjuvant chemotherapy in these patients.Although BRAF mutation confers a worse prognosis compared with BRAF wild type in dMMR/MSI colorectal cancer, the prognosis of dMMR/MSI colorectal cancer remains superior to MMR-proficient/MSS colorectal cancer, irrespective of BRAF status.5Lochhead P. Kuchiba A. Imamura Y. et al.Microsatellite instability and BRAF mutation testing in colorectal cancer prognostication.J Natl Cancer Inst. 2013; 105: 1151-1156Crossref PubMed Scopus (324) Google Scholar However, BRAF status in dMMR/MSI colorectal cancer is not yet incorporated routinely into risk assessment or treatment decisions.ConclusionsIn a previous article in this “Practice Management: The Road Ahead” section of Clinical Gastroenterology and Hepatology, the authors described how gastroenterologists might develop a seamless CRC clinical service line including risk assessment for hereditary CRC syndromes.21Braden G.L. Allen J.I. Organizing your clinical service line: colon cancer prevention.Clin Gastroenterol Hepatol. 2013; 11: 2-5Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar In addition, with rapid movement to value-based reimbursement and the potential to create a bundled colonoscopy payment methodology,22Ketover S.R. Bundled payment for colonoscopy.Clin Gastroenterol Hepatol. 2013; 11: 454-457Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar careful consideration of how molecular testing and analysis will be incorporated into our standard CRC prevention practice will be important and challenging. Hopefully, this article will add to the knowledge of practicing gastroenterologists in a direct and useful way. As health care reform progresses, the pressure to more closely integrate clinical service lines such as colorectal cancer (CRC) management has intensified. The practicing gastroenterologist may find that they are not equipped to understand pathology information required for coordinated team-based care of their patients. This is especially true in the case of molecular classification of colorectal cancer, something that has become a standard component of comprehensive oncologic care and has been incorporated into many gastroenterology (GI) practice pathology services. Molecular characterization and classification of colorectal cancer not only provides insight into the pathogenesis of cancer but has prognostic and therapeutic implications and is important for the gastroenterologist to understand and manage well. This review is a practical guide to the most common molecular tests used in what has become standard GI practice. Molecular Classification of Colorectal CancerThe molecular classification of colon cancer is based on the cumulative study of precursor lesions (such as adenomas and sessile serrated polyps), inherited colon cancer syndromes (such as familial adenomatous polyposis syndrome and Lynch syndrome/hereditary nonpolyposis colon cancer), and molecular profiling of colorectal cancers. Broadly, colorectal cancers are divided into 2 general groups based on genomic differences: chromosomal instability, accounting for 75% to 80% of all colorectal cancers, and microsatellite instability (MSI), accounting for 15% to 20% of all colorectal cancers.1Markowitz S.D. Bertagnolli M.M. Molecular origins of cancer: molecular basis of colorectal cancer.N Engl J Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1367) Google Scholar, 2Al-Sohaily S. Biankin A. Leong R. et al.Molecular pathways in colorectal cancer.J Gastroenterol Hepatol. 2012; 27: 1423-1431Crossref PubMed Scopus (154) Google Scholar Inherited colorectal susceptibility syndromes are estimated to account for approximately 1% to 2% of the MSI cancers and less than 1% of chromosomal instability cancers. The molecular classification of colon cancer is based on the cumulative study of precursor lesions (such as adenomas and sessile serrated polyps), inherited colon cancer syndromes (such as familial adenomatous polyposis syndrome and Lynch syndrome/hereditary nonpolyposis colon cancer), and molecular profiling of colorectal cancers. Broadly, colorectal cancers are divided into 2 general groups based on genomic differences: chromosomal instability, accounting for 75% to 80% of all colorectal cancers, and microsatellite instability (MSI), accounting for 15% to 20% of all colorectal cancers.1Markowitz S.D. Bertagnolli M.M. Molecular origins of cancer: molecular basis of colorectal cancer.N Engl J Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1367) Google Scholar, 2Al-Sohaily S. Biankin A. Leong R. et al.Molecular pathways in colorectal cancer.J Gastroenterol Hepatol. 2012; 27: 1423-1431Crossref PubMed Scopus (154) Google Scholar Inherited colorectal susceptibility syndromes are estimated to account for approximately 1% to 2% of the MSI cancers and less than 1% of chromosomal instability cancers. Microsatellite Instability PathwayMSI is defined by changes of microsatellite length (repetitive noncoding DNA sequences) resulting from deficient mismatch repair (dMMR) during DNA replication.1Markowitz S.D. Bertagnolli M.M. Molecular origins of cancer: molecular basis of colorectal cancer.N Engl J Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1367) Google Scholar, 3Zhang X. Li J. Era of universal testing of microsatellite instability in colorectal cancer.World J Gastrointest Oncol. 2013; 5: 12-19Crossref PubMed Google Scholar, 4Zaanan A. Meunier K. Sangar F. et al.Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications.Cell Oncol (Dordr). 2011; 34: 155-176Crossref PubMed Scopus (38) Google Scholar The protein complex responsible for mismatch repair function is a tetramer composed of 2 heterodimers: MLH1/PMS2 and MSH2/MSH6.4Zaanan A. Meunier K. Sangar F. et al.Microsatellite instability in colorectal cancer: from molecular oncogenic mechanisms to clinical implications.Cell Oncol (Dordr). 2011; 34: 155-176Crossref PubMed Scopus (38) Google Scholar The expression of each protein in a heterodimer is dependent on its partner, such that if one protein is absent, the partner protein consequently is degraded. When this occurs, the heterodimer is not available to form a functional tetramer and dMMR, as manifested by MSI, is the result.Most dMMR/MSI cancers occur sporadically and are associated with the loss of MLH1 expression owing to epigenetic silencing of the MLH1 gene promoter via CpG island methylation.1Markowitz S.D. Bertagnolli M.M. Molecular origins of cancer: molecular basis of colorectal cancer.N Engl J Med. 2009; 361: 2449-2460Crossref PubMed Scopus (1367) Google Scholar, 2Al-Sohaily S. Biankin A. Leong R. et al.Molecular pathways in colorectal cancer.J Gastroenterol Hepatol. 2012; 27: 1423-1431Crossref PubMed Scopus (154) Google Scholar The precursor lesion of sporadic dMMR/MSI cancers is believed to be