Colorectal carcinoma (CRC) was traditionally thought of

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1 Testing for Defective DNA Mismatch Repair in Colorectal Carcinoma A Practical Guide Lawrence J. Burgart, MD Context. Significant bench and clinical data have been generated during the last decade regarding DNA mismatch repair in colorectal carcinoma. Objectives. To review clinically relevant aspects of defective DNA mismatch repair in colorectal carcinoma and to suggest testing algorithms for identification of these tumors in the sporadic and familial settings. Data Sources. This article is based on literature review and clinical testing experience of more than 2000 patient samples. Conclusions. Approximately 15% of colorectal carcinomas arise as a result of defective DNA mismatch repair. Ninety percent of these carcinomas are sporadic, arising as a result of methylation of the MLH1 gene promoter, silencing expression. These sporadic carcinomas have improved stage-specific prognosis and can be identified by demonstrating aberrant loss of expression with an MLH1 immunoperoxidase stain. Familial colorectal carcinomas with defective DNA mismatch repair (Lynch syndrome) are due to a germline defect in one of several DNA mismatch repair genes. The familial carcinomas are best identified with a combination of immunohistochemistry and molecular microsatellite analysis. This testing facilitates subsequent directed genetic testing of the proband and family members. (Arch Pathol Lab Med. 2005;129: ) Colorectal carcinoma (CRC) was traditionally thought of as a monolithic disease process. During the last decade, however, variability in the underlying genetic pathogenesis has been described and has become clinically relevant. 1 Colon cancer can now be split into (at least) 2 subsets based on molecular pathogenesis, the first characterized by chromosome instability and the second by genomic (genome-wide) instability. The chromosomal instability (CIN) pathway accounts for 80% to 85% of all colon cancers. The CIN pathway involves loss of tumor suppressor genes and activation of oncogenes, characteristically identified as loss of heterozygosity, and often manifests overt aneuploidy. Commonly involved genes include the adenomatous polyposis coli (APC) gene, K-ras, the deleted in colon cancer (DCC) locus, and p53. This pathway also underlies familial adenomatous polyposis, which accounts for approximately 0.5% of all colon cancers. This large group probably comprises at least 2 major subsets likely to become better understood in the next several years. 2 Genomic instability results from a loss of DNA mismatch repair (MMR) activity. 3 Normal DNA synthesis is associated with a low but finite error rate in terms of which nucleotide is put into the daughter strand, or when- Accepted for publication July 12, From the Mayo Clinic, Rochester, Minn. The author has no relevant financial interest in the products or companies described in this article. Corresponding author: Lawrence J. Burgart, MD, Abbott NW Hospital, 800 E 28th St, Minneapolis, MN ( lawrence. burgart@allina.com). Reprints not available from the author. ever a base is added or deleted. It is, of course, critical to have a high-fidelity repair system to maintain the integrity of the genetic code. One of these repair systems is a family of proteins that works together as the DNA MMR complex. These genes/proteins have unfortunate alpha-numeric names, with the 4 major genes being MLH1, MSH2, PMS2, and MSH6. If any one of these proteins is absent, DNA MMR function is severely compromised. In the absence of DNA MMR activity, genome-wide mutations quickly accrue over the course of several generations of cell division (schematically represented in Figure 1). DNA microsatellites (mononucleotide, dinucleotide, or tetranucleotide repeat sequences found throughout the genome) make excellent test sites to assay for genetic errors that occur as a result of defective DNA MMR. Accordingly, the standard molecular laboratory test for defective DNA MMR is amplification of several microsatellites to look for abnormal variation in length, which is referred to as microsatellite instability (MSI). MICROSATELLITE INSTABILITY IS AN EXQUISITELY SENSITIVE AND SPECIFIC MARKER FOR DEFECTIVE DNA MMR Defective DNA MMR accounts for 15% to 20% of sporadic colon cancers, which typically occur at an older age than sporadic CIN carcinomas, are more frequently present in the proximal colon, and affect women twice as often as men. Lynch syndrome is a familial syndrome due to heritable defective DNA MMR that accounts for 2% of all colon cancers, demonstrates autosomal-dominant inheritance, has no gender bias, and has a broad age distribution with peak in the fifth decade. Sporadic colon cancer with defective DNA MMR is es- Arch Pathol Lab Med Vol 129, November 2005 DNA Mismatch Repair in Colorectal Carcinoma Burgart 1385

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3 sentially always due to the same, very specific DNA alteration. 4 All of these MSI-positive CRCs result from methylation of the MLH1 gene promoter, which stops transcription of both MLH1 alleles. Therefore, not only do all sporadic CRCs due to defective DNA MMR have MSI, but they also are essentially all due to lack of MLH1 expression. Based on the above discussion, CRCs can be divided into 4 subtypes (schematically represented in Figure 2): (1) familial colon cancer due to chromosomal stability (familial adenomatous polyposis), (2) sporadic colon cancers due to CIN (ordinary-type colon cancer), (3) familial colon cancer due to defective DNA MMR (Lynch syndrome), and (4) sporadic colon cancer due to defective DNA MMR. IS IT IMPORTANT TO IDENTIFY SPORADIC CRC DUE TO DEFECTIVE DNA MMR? Sporadic colon cancers with defective DNA MMR have a significantly improved prognosis over CIN-pathway CRCs. 5 7 In fact, stage III CRCs with defective DNA MMR have a survival curve superimposable on that of stage II CIN cancers. This equates to half as much mortality in stage III CRC with defective DNA MMR through stage II CRC with CIN. Unfortunately, there are few data on whether this is dependent or independent of treatment, and the available data are conflicting Currently, treatment decisions are not made based on DNA MMR status, despite in vitro data suggesting that 5-FU based therapies do not work in these cancers. 9,11 15 However, as chemotherapeutic regimens become refined and as panels of prognostic markers are put in place to determine which stage II and III CRC patients should receive therapy, these data will become essential. IF MY CLINICIANS ARE INTERESTED IN THIS PROGNOSTIC INFORMATION, WHAT IS THE BEST TEST TO PERFORM? The candidate tests for detection of defective DNA MMR in colon cancer include histology, immunohistochemistry, and molecular MSI testing. Characteristic histologic features do exist for CRC with defective DNA MMR. 16,17 The most specific morphologic feature is a cribriform/solid (medullary) architecture without anaplastic cytology (Figure 3). Mucinous colon cancers (colloid and signet ring) also have an increased incidence of defective DNA MMR, but are much less specific than the medullary pattern. The suboptimal aspect of using the cribriform/ solid architectural pattern as a test for defective DNA MMR is poor sensitivity. The sensitivity is approximately 50%; half of all CRCs with defective DNA MMR have ordinary-type morphology with gland formation and desmoplasia. Tumor-infiltrating lymphocytes are more sensitive for the detection of defective DNA MMR. 18,19 Tumorinfiltrating lymphocytes have to be intimately associated with the colon cancer cells to count for this assessment. Figure 3. Colon carcinoma with defective DNA mismatch repair demonstrating characteristic solid/cribriform (medullary) architecture and numerous tumor-infiltrating lymphocytes (hematoxylin-eosin, original magnification 600). Figure 4. Colon carcinoma with defective DNA mismatch repair due to aberrant loss of nuclear MLH1 expression in the neoplastic cells, as demonstrated by this immunoperoxidase stain. Note the internal positive control in adjacent benign colonocytes, lamina propria stroma, and tumor-infiltrating lymphocytes (MLH1 immunoperoxidase, original magnification 400). This does not include peritumoral or Crohn-like inflammation. Unfortunately, there is a sensitivity-specificity trade-off, which results in approximately 80% sensitivity with 80% specificity when you have approximately 5 tumor-infiltrating lymphocytes per high-power field. Therefore, this is a good but not perfect marker for the detection of MSI-positive colon cancers. For sporadic CRC with defective DNA MMR, immu- Figure 1. Schematic representation of normal versus defective DNA mismatch repair. DNA replication normally has a low, finite rate of erroneous base placement in the daughter strand, which is corrected by a complex of DNA repair enzymes. If the repair mechanism is inactivated by gene mutation or promoter methylation, genomic instability results from accumulation of genome-wide mutations. Genomic instability is readily measured in the laboratory by polymerase chain reaction analysis of DNA microsatellites. Figure 2. Schematic representation of relative frequency, by age, of the major categories of familial and sporadic colorectal carcinoma. FAP indicates familial adenomatous polyposis; CIN, chromosomal instability; and MSI, colorectal carcinoma with defective DNA mismatch repair resulting in microsatellite instability. Arch Pathol Lab Med Vol 129, November 2005 DNA Mismatch Repair in Colorectal Carcinoma Burgart 1387

4 Figure 5. Approximately colorectal carcinomas are diagnosed in the United States each year. Approximately 30% of colon cancer patients have a positive family history in a first- or second-degree relative. Approximately 2% of colon cancers are due to Lynch syndrome (heritable defective DNA mismatch repair). nohistochemistry is an excellent assay (Figure 4). 3 MLH1 immunoperoxidase staining has essentially 100% sensitivity and specificity for defective DNA MMR in the sporadic setting due to the homogeneous underlying molecular pathogenesis (MLH1 promoter methylation). One would have to do this immunostain on all colon cancers to have near-100% sensitivity, but molecular microsatellite analysis would be redundant and therefore unnecessary. Immunohistochemical reagents are available for MLH1, MSH2, MSH6, and PMS2 expression. The vast majority of all cell types (except those very terminally differentiated) normally demonstrate nuclear expression of each of these DNA MMR enzymes. This provides an extremely useful internal positive control when assaying for aberrant loss of a DNA MMR protein (Figure 4). IS IT IMPORTANT TO PERFORM IMMUNOHISTOCHEMISTRY AND/OR MOLECULAR MICROSATELLITE ANALYSIS TO DETECT LYNCH SYNDROME PROBANDS? Approximately 2% of the CRCs diagnosed in the United States each year occur in patients with Lynch syndrome (Figure 5) This means there are approximately 3000 CRCs per year in Lynch syndrome families. In addition, Lynch syndrome families are at risk for endometrial, upper gastrointestinal, upper urinary tract, ovarian, and pancreaticobiliary carcinomas. Identifying these families allows for sorting of at-risk family members. This autosomal-dominant disease results in 50% of family members having an 80% to 100% lifetime risk of carcinoma development, with the other half having normal cancer risk. Therefore, identification of Lynch syndrome probands can impact decision making (eg, screening, prophylactic surgery) tremendously. It is reasonable to clarify some terminology at this point. Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is a clinically defined term based on strong family history of colon cancer. The original HNPCC definition, referred to as the Amsterdam Criteria, includes the following: (1) 3 involved relatives, 2 of whom are first-degree relatives; (2) successive generations involved; and (3) 1 involved patient younger than 50 years. In only 60% of the families with a strong family history of colorectal carcinoma, even to the extent of meeting these criteria, the colorectal carcinoma is due to heritable DNA mismatch repair. 24,25 Therefore, 40% of HNPCC families remain unexplained as to their genetic basis. Disease in those HNPCC families with heritable DNA MMR is referred to as Lynch syndrome. 21 Approximately 30% of colon cancer patients have at least 1 first- or second-degree relative also with colon cancer. Immunohistochemical findings in Lynch syndrome cases are much more complex than in sporadic MSI-positive CRCs. While 100% of Lynch syndrome related carcinomas have MSI, only approximately 95% of them will have abnormal immunohistochemistry findings (loss of expression of a DNA MMR enzyme). The rough distribution of abnormal DNA MMR gene and subsequent loss of protein expression within Lynch syndrome families is as follows: MLH1, 40%; MSH2, 40%; MSH6, 5%; and PMS2, 5%. A small percentage of Lynch syndrome families develop MSI-positive carcinomas with intact staining of all 4 of these enzymes due either to a missense mutation affecting function but not antigenicity, or involvement of another minor protein in the complex. Based on the above information, recommended testing for Lynch syndrome includes a combination of molecular microsatellite analysis and immunohistochemistry for MLH1, MSH2, PMS2, and MSH6. This allows for highsensitivity detection of MSI within the tumors and identification of which enzyme is abnormal within that family, in turn guiding the subsequent germline mutation analysis. Both microsatellite analysis and immunohistochemistry are performed on paraffin-embedded tissue. The microsatellite analysis requires both tumoral and nontumoral tissue for comparison. WHICH CRC PATIENTS SHOULD BE TESTED FOR LYNCH SYNDROME? The rule of thumb is any patient with CRC arising with positive family history and/or young age. A series of National Institutes of Health sponsored consensus conferences have established the Bethesda Guidelines for selecting patients to test Arch Pathol Lab Med Vol 129, November 2005 DNA Mismatch Repair in Colorectal Carcinoma Burgart

5 There are instances in which it can be difficult to differentiate between familial and sporadic defective DNA MMR colon cancer. For example, a 50-year-old with a colon cancer showing high levels of MSI and loss of MLH1 expression can be ambiguous as to germline versus somatic abnormality if the family history is not clear or unavailable. Germline analysis is helpful in these situations, but is also less than 100% sensitive. Recently, a specific B- raf mutation in codon 600 has been identified in a majority of sporadic (methylated) defective DNA mismatch repair CRC cases, which rarely is present in Lynch syndrome carcinomas. 26,27 Therefore, B-raf mutational analysis may soon become part of the testing algorithm in this situation. In summary, 15% to 20% of CRCs are due to defective DNA MMR. Ninety percent of these cases will be caused by silencing of MLH1 expression due to methylation of the gene promotor. These sporadic CRCs with defective DNA MMR have a significantly improved prognosis, but therapy is unchanged. Lynch syndrome proband identification is a very active testing program taking advantage of both microsatellite analysis and immunohistochemistry on paraffin-embedded tissue with subsequent gene mutation analysis (peripheral blood DNA) to sort at-risk family members. References 1. Hamilton SR. The molecular genetics of colorectal neoplasia. Gastroenterology. 1993;105: Burgart LJ. Colorectal polyps and other precursor lesions: need for an expanded view. Gastroenterol Clin North Am. 2002;31: Thibodeau SN, French AJ, Cunningham JM, et al. Microsatellite instability in colorectal cancer: different mutator phenotypes and the principal involvement of hmlh1. Cancer Res. 1998;58: Cunningham JM, Christensen ER, Tester DJ, et al. Hypermethylation of the hmlh1 promoter in colon cancer with microsatellite instability. Cancer Res. 1998;58: Hemminki A, Mecklin JP, Jarvinen H, Aaltonen LA, Joensuu H. Microsatellite instability is a favorable prognostic indicator in patients with colorectal cancer receiving chemotherapy. Gastroenterology. 2000;119: Coleman LW, Curtin K, Schaffer D, Leppert M, Slattery ML, Samowitz WS. Microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level. Mod Pathol. 2001;14:83A. 7. 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. 2004;53: Elsaleh H, Joseph D, Grieu F, et al. Association of tumour site and sex with survival benefit from adjuvant chemotherapy in colorectal cancer. Lancet. 2000; 355: Claij N, te Riele H. Microsatellite instability in human cancer: a prognostic marker for chemotherapy? Exp Cell Res. 1999;246: 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. 2003;349: Fink D, Aebi S, Howell SB. The role of DNA mismatch repair in drug resistance. Clin Cancer Res. 1998;4: Anthony DA, McIlwrath AJ, Gallagher WM, Edin AR, Brown R. Microsatellite instability, apoptosis, and loss of p53 function in drug-resistant tumor cells. Cancer Res. 1996;56: Kat A, Thilly WG, Fank W-H, et al. An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair. Proc Natl Acad Sci U S A. 1993;90: Branch P, Aquilina G, Bignami M, Karran P. Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage. Nature. 1993;362: Meyers M, Wagner MW, Hwang HS, Kinsella TJ, Boothman DA. Role of the hmlh1 DNA mismatch repair protein in fluoropyrimidine-mediated cell death and cell cycle responses. Cancer Res. 2001;61: Kim H, Jen J, Vogelstein B, Hamilton SR. Clinical and pathological characteristics of sporadic colorectal carcinomas with DNA replication errors in microsatellite sequences. Am J Pathol. 1994;145: Krishna M, Burgart LJ, French AJ, Moon-Tasson L, Halling KC, Thibodeau SN. Histopathologic features associated with microsatellite instability in colorectal carcinomas. Gastroenterology. 1996;110:A Smyrk TC, Watson P, Kaul K, Lynch HT. Tumor-infiltrating lymphocytes are a marker for microsatellite instability in colorectal carcinoma. Cancer. 2001;91: Alexander J, Watanabe T, Wu TT, Rashid A, Li S, Hamilton SR. Histopathologic identification of colon cancer with microsatellite instability. Am J Pathol. 2001;158: Mecklin J-P, Järvinin H, Hakkiluoto A, et al. Frequency of hereditary nonpolyposis colorectal cancer: a prospective multicenter study in Finland. Dis Colon Rectum. 1995;38: Umar A, Boland CR, Terdiman JP, et al. Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst. 2004;96: Lynch HT, Smyrk TC, Watson P, et al. Genetics, natural history, tumor spectrum, and pathology of hereditary nonpolyposis colorectal cancer: an updated review. Gastroenterology. 1993;104: Hampel H, Frankel WL, Martin E, et al. Screening for the Lynch syndrome (hereditary nonpolyposis colorectal cancer). N Engl J Med. 2005;352: Kievit W, de Bruin JH, Adang EM, et al. Current clinical selection strategies for identification of hereditary non-polyposis colorectal cancer families are inadequate: a meta-analysis. Clin Genet. 2004;65: Lipton LR, Johnson V, Cummings C, et al. Refining the Amsterdam Criteria and Bethesda Guidelines: testing algorithms for the prediction of mismatch repair mutation status in the familial cancer clinic [published correction appears in J Clin Oncol. 2005;23:3652]. J Clin Oncol. 2004;22: Domingo E, Laiho P, Ollikainen M, et al. BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet. 2004;41: McGivern A, Wynter CV, Whitehall VL, et al. Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer. Fam Cancer. 2004;3: Arch Pathol Lab Med Vol 129, November 2005 DNA Mismatch Repair in Colorectal Carcinoma Burgart 1389