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Journal of Clinical Oncology, Vol 24, No 12 (April 20), 2006: pp. 1960-1962 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.05.3207
Is It Time to Abandon Microsatellite Instability As a Pre-Screen for Selecting Families for Mutation Testing for Mismatch Repair Genes?Academic Unit of Medical Genetics, Regional Genetics Service, St Mary's Hospital, Manchester, UK
Department of Colorectal Surgery, Manchester Royal Infirmary, Manchester, UK To the Editor: We have been prompted to write in response to a recent article on the use of molecular tumor testing to prioritise early onset colorectal cancer patients for germline mismatch repair (MMR) gene testing.1 We broadly agree with the conclusions of Southey et al that immunohistochemistry (IHC) testing of tumors for the four main MMR proteins (MSH2, MLH1, MSH6, PMS2) should be the ideal prescreen, but would go further in suggesting the abandonment of microsatellite instability (MSI) testing. Approximately 3% of colorectal cancer is due to inherited mutations in the two main MMR genes MSH2 and MLH1,2 with an additional 0.3% to 0.6% due to MSH6. The implications to the individual and the family are serious, with an increased cancer risk throughout the GI tract (stomach, biliary tree, small intestine), uterus, ovaries, and ureter. Screening intervals for colonoscopy should be at least once every 2 years.2 Identifying a pathogenic mutation provides a defin-itive genetic test that may obviate the need for screening in more than 50% of unaffected family members. Mutation screening is expensive, and it is impractical to test all incident cases of colorectal cancer. Guidelines for testing in the United States suggest at least a 10% likelihood threshold for mutation testing.3 Clinical guidelines for testing using the Amsterdam criteria (AC; I or II; Table 1) will detect families with a 50% to 60% likelihood of an identifiable MSH2 or MLH1 mutation.4 Mutation testing in lower risk families has been guided by MSI testing of tumors using the Bethesda criteria (BC; Table 1)
Recently some authors have suggested that the sensitivity of MSI is so high that all families should have MSI as a prescreen before mutation testing.5 In order to reduce the detection rate for AC families below 10%, MSI sensitivity of at least 86% would be necessary calculated by Bayes' theorum. We were interested to see that the sensitivity of MSI high (MSH) in the Australian study1 was only 72% and 2 of 4 (50%) of the AC families without MSH had mutations in MSH6. Our own experience with false-negative results on MSI for MSH2 and MLH1 led us to audit our hereditary nonpolyposis colorectal cancer testing service to establish whether MSI could be relied on in a service setting (Fig 1).
In total, 232 pathology blocks (fulfilling AC or BC) were tested for MSI using the five-panel set approved by the National Cancer Institute (Bethesda, MD).5 Of the 232, 31 were MSH (13%). Of these, 17 of 31 have been shown to have a pathogenic mutation, six in families fulfilling BC only. Nineteen samples were MSI low, of which three had pathogenic mutations, one of which fulfilling BC only. One hundred and eighty-two (78%) were microsatellite stable (MSS). Of the MSS samples, 50 underwent mutation screening for a variety of reasons. In nine of these (18%), pathogenic mutations in MLH1/MSH2 were demonstrated. One of these families fulfilled BC only. Thereby, had we only proceeded to mutation screening for MSH, 12 mutations would have been missed (41%), most of these in families fulfilling AC. In families fulfilling BC only, six of eight samples with mutations demonstrated MSH tumors. Of 21 AC samples with mutations, ten were MSS or low including three from different family members with a MLH1 exon 13 duplication. This family also had normal MLH1 IHC in their tumors. One family with a pathogenic mutation fulfilling BC demonstrates the difficulty of MSI, as the father's colon tumor (diagnosed at 44) was MSH, but his son's rectal tumor (diagnosed at 36) was MSS. Within the service as a whole, 187 samples have been fully tested for MSH2 and MLH1 using single strand conformation polymorphism and sequencing for variant bands plus multiple ligation-dependent probe amplification to identify large rearrangements.6 A total of 49 point mutations and 11 large rearrangements have been identified resulting in detection rate of 32% for pathogenic mutations. Of these 60 mutations, 77%4-6 were from 80 families fulfilling AC and 14 from BC families. Only limited testing has so far been carried out for MSH6. Using the same panel of five markers a United States group identified 14 of 14 samples with germline mutations as being MSH and recommended that MSI and the BC should be used as the sole criteria for testing. The sensitivity of MSI in our audit fell substantially below the 86% required to obviate mutation screening. Other groups have also reported sensitivities below this level, including the Australian report. While the detection of MSH was lower in our tumors than is found in selected series, it represents transition from research to a service setting. It has been suggested that protein staining alone or in combination with MSI increases sensitivity, but interpretation of absence of staining is subjective and certain mutations are associated with a stable protein product. Sensitivities as low as 72% have been reported for antibody studies.5 Therefore, neither MSI nor IHC alone reliably produces sensitivities sufficient to reduce the detection rate in Amsterdam families below 10%. Indeed even in the Australian study two of 18 patients with mutations were scored as having absent protein only after some debate. Multiple tumors from the same family or more markers could be used, but this increases the cost, timescale to finding a mutation, and difficulties with consent. In order to undertake MSI testing tumor sections need to be cut by a trained pathologist and at least 70% of cells analyzed should be tumor cells, only then can the DNA be extracted and tested for MSI.7 The same pathologist could carry out IHC saving considerable time. Given the high detection rate (58% in our series) in Amsterdam families these clinical criteria alone should remain sufficient to test for germline mutations. While sensitivity for IHC may be sufficient in certain laboratories (and it has the advantage of targetting which gene to screen), MSI is not sensitive enough in a service setting for all families particularly given the results for MSH6.1 Any laboratory would have to prove that it produces sensitivity above 90% on a large sample set before abandonment of AC. While MSI is a useful prescreen in BC families, it is surpassed by IHC. We would, therefore, advocate abandonment of MSI testing as a prescreen before abandoning AC. Authors' Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest.
REFERENCES
1. Southey MC, Jenkins MA, Mead L, et al: Use of molecular tumor characteristics to prioritize mismatch repair gene testing in early onset colorectal cancer. J Clin Oncol 23:1-6, 2005 2. Dunlop MG, British Society for Gastroenterology, Association of Coloproctology for Great Britain and Ireland: Guidance on gastrointestinal surveillance for hereditary non-polyposis colorectal cancer, familial adenomatous polypolis, juvenile polyposis, and Peutz-Jeghers syndrome. Gut 51:V21-7, 2002 (suppl 5)[CrossRef][Medline] 3. American Society of Clinical Oncology. Statement of the American Society of Clinical Oncology: Genetic testing for cancer susceptibility. J Clin Oncol 14: 1730-1736, 1996 4. Park JG, Vasen HF, Park YJ, et al: Suspected HNPCC and Amsterdam criteria II: Evaluation of mutation detection rate, an international collaborative study. Int J Colorectal Dis 17:109-114, 2002[CrossRef][Medline] 5. Wahlberg SS, Schmeits J, Thomas G, et al: Evaluation of microsatellite instability and immunohistochemistry for the prediction of germ-line MSH2 and MLH1 mutations in hereditary nonpolyposis colon cancer families. Cancer Res 62:3485-3492, 2002 6. Taylor CF, Charlton RS, Burn J, et al: Genomic deletions in MSH2 or MLH1 are a frequent cause of hereditary non-polyposis colorectal cancer: Identification of novel and recurrent deletions by MLPA. Hum Mutat 22:428-433, 2003[CrossRef][Medline] 7. Baudhuin LM, Burgart LJ, Leontovich O, et al: Use of microsatellite instability and immunohistochemistry testing for the identification of individuals at risk of Lynch syndrome. Familial Cancer 4:255-265, 2005[CrossRef][Medline]
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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