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Originally published as JCO Early Release 10.1200/JCO.2005.05.032 on August 22 2005 © 2005 American Society of Clinical Oncology.
First Amsterdam, Then Bethesda, Now Melbourne?University of Southern California/Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA Should we use immunohistochemistry (IHC) for mismatch repair (MMR) proteins instead of microsatellite instability (MSI) analysis to identify potential candidates for mutational analyses in patients with early-onset colorectal cancer? As oncologists, we occasionally find ourselves facing a young patient diagnosed with colon cancer. In this situation, the patient and often the relatives take the next logical step and find themselves concerned about a potentially higher risk for colon cancer in their family. At the same time, this newly diagnosed patient is overwhelmed with emotions, information, and pending decisions regarding the next step in treatment, such as surgery, radiation, or chemotherapy. In most cases, a complex discussion with a genetic counselor about genetic predisposition, counseling, and testing seems inappropriate when the patient is struggling with the new diagnosis and treatment discussions. When the patient is able to address the issue of genetic predisposition, we arrange for genetic counseling to establish the risk and discuss the risks, limitations, and potential benefits of genetic testing. For example, patients who are at risk for hereditary nonpolyposis colorectal cancer (HNPCC) and are candidates for genetic counseling and testing are well characterized and categorized in accordance with the Amsterdam and Bethesda criteria used by oncologists and genetic counselors (Tables 1 and 2). These criteria are used to identify patients who are at risk for HNPCC or who should be candidates for testing for MSI, the landmark finding for patients with HNPCC. The American Gastroenterology Association has developed guidelines for genetic counselors and geneticists for patients who meet Amsterdam or Bethesda criteria (Fig 1). This decision tree helps the clinician decide when to test for MSI and when to use germline testing for mMSH2/mMLH1. These guidelines use MSI testing and germline testing, and not any IHC, for the known MMR mutations. Within the process of genetic counseling, the sequence of tests necessary to identify a family with HNPCC is complicated and is limited by the sensitivity of the technologies used. Despite what is known, there are ongoing questions. Should we use MSI or sequencing? How sensitive is MSI analysis followed by sequencing of MSH1 and MLH2 for patients younger than age 45 years? Does immunohistochemistry play a role? These questions are especially relevant because not all HNPCCs have MSI, and we must not forget that HNPCC can also be caused by other MMR gene mutations such as PMS2 and MSH6.
To begin addressing questions like these, we must begin with what we already know. First, HNPCC is caused by germline mutations of the DNA MMR genes (hMLH1, hMSH2, hPMS1, hPMS2, hMSH6). However, in the original family found to have an hPMS1 mutation, it was later shown that the mutation did not segregate with cancer and an MSH2 gene mutation was later identified. Thus, the role of PMS1 is questionable, and has not been shown to be clinically significant. Genetic testing in HNPCC is indicated for affected individuals in families meeting the Amsterdam criteria, for affected individuals meeting the modified Bethesda criteria, and for first-degree adult relatives of those with known mutation.1 We usually test for MSI using the Bethesda criteria first, given that MSI provides evidence for the presence of germline mutation in an MMR gene. However, MSI testing requires tumor tissue of individuals putatively affected with HNPCC. A result of high MSI in tumor DNA prompts the patient testing for a germline mutation, usually by sequencing for mutations in the hMSH2 and hMLH1 genes. In addition, mutation analysis by polymerase chain reaction (PCR) –based full gene sequencing with Southern blot analysis for large deletions is now available to increase sensitivity. What are the limitations to these approaches? We are not screening for all MMR gene mutations, and we are finding that MSI analysis does not provide us with even a clue about which MMR gene is mutated. We do not screen for the mismatch genes PMS2 and MSH6. In fact, MSH6 is found in HNPCC families and has been shown not to be associated with MSI because the inactivation of MSH6 is due to methylation rather than a germline mutation. With this in mind, two critical questions arise. First, is it possible to detect lack of MMR protein expression, using IHC, in the presence of MSH6 methylation? Second, would the results of IHC of MMR repair proteins provide us with any indication about which gene (hMSH2, hMLH1, hPMS2, or hMSH6) to target for germline analysis? Recent data suggest that IHC for hMLH1 and hMSH2 protein expression is an inexpensive adjunct analysis of colorectal tumors, and when used in conjunction with MSI, can help to determine strategies for germline testing. Detection of lack of tumor expression of either hMSH2 or hMLH1 by IHC is correlated with MSI in the tumor. Interestingly, the majority of MSI observed in sporadic colorectal cancer patients seems to be caused by somatic hypermethylation of the hMLH1 promoter.2,3 It has also been observed that absent hMSH2 IHC expression is associated with germline hMSH2 mutation, and a minority of absent hMLH1 IHC expression is associated with germline hMLH1 mutation.3 IHC for these two genes is now in the transition stages of becoming routine clinical practice, which further supports the added value of using IHC for the MMR proteins. In this issue, Southey et al4 report that in 131 unselected cases, the IHC of four MMR proteins (MLH1, MSH2, PMS2, and MSH6) provided a highly sensitive strategy for identifying MMR gene mutations in early-onset colorectal cancer. Half of the detected mutations would have been missed had only the Amsterdam criteria been used. The use of IHC demonstrated that it is highly sensitive for detecting carriers of germline mutations, but also allows direction of the sequencing to genes harboring the mutation. Consequently, the IHC method potentially serves to reduce the high costs of germline testing. Carriers of germline mutations can be identified with the same sensitivity using the traditional Bethesda criteria; however, the IHC approach further identifies the specific mutated genes to be sequenced. Another key advantage to using IHC is that pathologists can easily perform the testing. Conversely, MSI analysis requires a molecular laboratory, which is more expensive and does not provide gene-specific information. The IHC approach suggested by Southey et al4 still leaves the question unanswered about when to include a genetic counselor. Should genetic counseling be arranged before IHC is ordered or after the results are received? Given that the intention of the test is the identification of a genetic predisposition, the patient should be seen by a genetic counselor and be informed about the risks, limitations, and benefits associated with this process. Rather than being burdensome, the knowledge a genetic counselor provides regarding the IHC method should make the testing process and the interpretation of the results more effective and informative for the patient. This begs the question about whether we still need testing for MSI. Evaluation for MSI is suggested, by consensus panel5 and expert opinion,6,7 as the first step in the genetic work-up of pedigrees suspected to be affected by HNPCC. Individuals with low-MSI or microsatellite stable tumors are unlikely to harbor germline MMR gene mutations, suspending the need for additional genetic evaluation. However, some studies and reported laboratory experience show that low-MSI tumors are associated with MSH6 mutations. With families or individuals meeting any of the first three of the modified Bethesda criteria, germline testing without initial MSI analysis or in the presence of microsatellite stability may be considered. Recently, microsatellite stability has been found in the tumors of patients with hMSH6 germline mutations, yet the clinical implications of this finding are unresolved and remain an ongoing area of research. Would IHC have been able to identify these mutations by the lack of MSH6 protein expression? Additional studies are necessary to determine the value of IHC and MSI in the identification of individuals harboring germline mutations. The sensitivity of MSI and IHC may be dependent on the onset of colorectal cancer. According to the findings of Southey et al,4 the highest rate of gene mutation was present in those individuals younger than 30 years of age with colorectal cancer and positive MSI tumor tissue (63%). The range of positivity varied widely, depending on testing methodology and the number of genes tested (Table 3). The positivity decreased substantially for individuals from families with suspected HNPCC, or for those individuals meeting less stringent criteria (0% to 47%). Thirty percent positivity was noted for individuals meeting the Bethesda criteria when two genes (hMLH1 and hMSH2) were analyzed, whereas 10% tested positive when hMSH6 alone was analyzed. The occurrence of MSI substantially increased the likelihood of detecting a germline mutation, as noted in individuals with colorectal cancer younger than the age of 50 and, separately, younger than the age of 30, with and without MSI testing.
The role of MSI analysis has led to the development of the Bethesda criteria (Table 2), which sets forth clinical indications for use of this assay and standard measures for tumor analysis. However, the data from Southey4 suggest that the use of IHC on the four MMR genes may be sufficient for identifying the mutation carriers in patients with early-onset disease. IHC can be implemented easily by pathologists, and testing for the four MMR proteins strongly predicts for germline MMR mutation status for particular genes. This tumor-based strategy may be a more efficient way to identify individuals with HNPCC as a result of MMR gene mutations. Genetic testing does allow for identification of individuals who are at increased risk and who should therefore be screened more frequently, which has been shown to reduce cancer incidence and to ultimately result in clinical benefit for the at-risk HNPCC population.8 Author's Disclosures of Potential Conflicts of Interest The author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Dollar Amount Codes (A) < $10,000 (B) $10,000-99,999 (C)
REFERENCES 1. Giardiello FM, Brensinger JD, Petersen GM: AGA technical review on hereditary colorectal cancer and genetic testing. Gastroenterology 121:198-213, 2001[Medline] 2. Cunningham JM, Kim CY, Tester DJ: The frequency and mechanisms of defective DNA mismatch repair in unselected colorectal carcinomas. Proc Am Assoc Cancer Res 40:1611, 1999
3. Cunningham JM, Christensen ER, Tester DJ, et al: Hypermethylationof the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 58:3455-3460, 1998
4. Southey MC, Jenkins MA, Mead LA, et al: Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early-onset colorectal cancer. J Clin Oncol 23:6524-6532, 2005 5. National Comprehensive Cancer Network: NCCN Colorectal Cancer Screening Practice Guidelines: National Comprehensive Cancer Network. Oncology (Huntingt) 13:152-179, 1999
6. Giardiello FM: Genetic testing in hereditary colorectal cancer. JAMA 278:1278-1281, 1997
7. O'Reilly S, Johnson KA, Brensinger JD, et al: Hereditary nonpolyposis colorectal cancer. Ann Oncol 8:1151-1156, 1997 8. Jarvinen HJ, Mecklin JP, Sistonen P: Screening reduces colorectal cancer rate in families with hereditary non polyposis colorectal cancer. Gastroenterology 108:1405-1411, 1995[CrossRef][Medline]
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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