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© 2003 American Society for Clinical Oncology Automated, Multiplex Assay for High-Frequency Microsatellite Instability in Colorectal Cancer
From the Departments of Surgery and Pathology, Memorial Sloan-Kettering Cancer Center, New York; and the Department of Microbiology, Weill Medical College of Cornell University, Ithaca, NY. Address reprint requests to Philip B. Paty, MD, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021; email: patyp{at}mskcc.org.
Purpose: In a series of hereditary nonpolyposis colorectal cancer (HNPCC) patients, we evaluated the sensitivities of the individual microsatellites recommended by the National Cancer Institute (NCI) consensus workshop for detection of high-frequency microsatellite instability (MSI-H). On the basis of this evaluation, we developed a three-marker assay that assigns microsatellite instability (MSI) in a multiplex polymerase chain reaction. Methods: Individual marker sensitivity was assessed in 18 HNPCC tumors. Multiplex and NCI assays were then assessed in a series of 120 patients with early-onset colon cancer. Results: The sensitivity of microsatellite markers BAT25, BAT26, D2S123, D5S346, and D17S250 for ASI in HNPCC cancers was 100%, 94%, 72%, 50%, and 50%, respectively. The three most accurate markers were combined and optimized in a multiplex assay that assigned MSI-H whenever at least two of three markers revealed ASI. In early-onset colon cancers, the prevalence of MSI-H determined by the multiplex assay and by the NCI assay was 16% and 23%, respectively. The additional MSI-H tumors and patients with MSI-H identified by the NCI assay lacked the traits characteristic of MSI-H seen in tumors and patients identified by the multiplex assay: retention of heterozygosity (NCI additional 22% v multiplex 84%; P = .003), characteristic tumor morphology (0% v 64%; P = .006), and 5-year cancer survival rate (44% v 100%; P = .0003). Conclusion: The multiplex assay identifies colon cancers with MSI-H by assessing three highly accurate microsatellite markers. This assay identifies a smaller MSI-H cohort with more homogeneous clinical features and is superior as a marker of favorable prognosis. It merits prospective evaluation as a marker of prognosis and as a screening test for HNPCC.
TWO MAJOR pathways of genomic instability have been identified in colorectal cancer: a chromosomal instability (CIN) pathway and a high-frequency microsatellite instability (MSI-H) pathway.1–5 CIN is found in the majority of cancers and is characterized by aneuploidy, loss of heterozygosity (LOH), chromosomal amplifications, and translocations. MSI-H is found in 10% to 20% of primary colorectal cancers (CRCs) and is characterized by diploidy and mutation or methylation silencing of mismatch repair (MMR) genes.6–17 In cancers with MSI-H, the MMR pathway is defective and cancer cells are unable to repair slippage mutations that occur during DNA replication. The cancers accumulate unrepaired slippage events throughout the genome but particularly in microsatellites.2,12,13 Routine evaluation of tumors for the presence or absence of MSI-H at the time of clinical presentation with CRC has significant potential for improving patient management. MSI-H is present in more than 85% of CRCs that occur in families with hereditary nonpolyposis colon cancer (HNPCC).13,18 MSI-H is also found in approximately 10% to 15% of cancers not associated with HNPCC, many of which have lost MMR function because of methylation silencing of the MLH1 gene.10,11 At present, MSI-H testing remains primarily a research tool, but it can be helpful in the clinical assessment and management of CRC patients. MSI-H may serve as a screening test for patients with heritable mutations in MMR genes that cannot be identified accurately by clinical presentation or family history alone.13,19–22 In addition, retrospective studies have shown that MSI-H is an important prognostic marker because CRCs with MSI-H have a favorable prognosis after surgical resection.14,15 Moreover, the response of CRCs with MSI-H to treatment with chemotherapeutic agents may be different from that of microsatellite stable (MSS) cancers.23–28 Thus, the currently available data present a strong rationale for routine assessment of MSI-H in primary CRCs, particularly for patients who enter chemoprevention or adjuvant therapy trials. The accuracy of MSI-H as a marker to identify colon cancers that have lost MMR function is variable and is dependent on the technical details of the assay. Molecular diagnosis of MSI-H using polymerase chain reaction (PCR) assays, although not technically demanding, has not been fully standardized.29 MSI-H analyses have varied with regard to the microsatellite markers used, the method for detecting allele size instability (ASI), and the number and proportion of microsatellite markers with size instability required for a positive diagnosis of MSI-H. To standardize the molecular definition of MSI-H, in 1997 a National Cancer Institute (NCI) workshop recommended a panel of five microsatellite markers. These loci were selected for their efficacy in identifying colorectal adenocarcinomas with loss of hMSH2 or hMLH1 expression.29,30 The panel consisted of two mononucleotide microsatellites (BAT25, BAT26) and three dinucleotide microsatellites (D2S123, D5S346, and D17S250).31 MSI-H tumors are defined by the NCI workshop as having at least two of five microsatellite markers demonstrating instability. Low-frequency microsatellite instability (MSI-L) and MSS are defined by size instability in one or zero markers, respectively.31 The NCI panel of microsatellite markers, or variations of it, has been used for correlative studies of survival and molecular profiling in CRC and for screening patients for HNPCC tumors. However, no rigorous evaluation of the accuracy of these markers has been published, despite evidence that ASI, especially among dinucleotide markers, may occur in tumors that retain an intact DNA MMR pathway.32 It was our aim to examine the individual sensitivities of the markers for microsatellite slippage events in HNPCC tumors and their specificity in a larger series of all CRCs. To facilitate more informative clinical correlative studies, we wished to develop a rapid, automated MSI assay. We reasoned that MSI analysis would be made simpler, faster, less costly, and possibly more accurate if only the most sensitive microsatellite markers were used and they could be combined in a single, automated reaction. In this article, we compare results of an optimized, multiplex, three-marker MSI assay to the established five-marker assay recommended by the 1997 NCI workshop.
Tumor Samples Tumor and normal tissue was collected under Institutional Review Board protocol from patients having surgery for CRC at Memorial Sloan-Kettering Cancer Center, New York, NY. The operations were performed between November 1992 and November 2000. Tissue was obtained at time of surgical resection and snap frozen in liquid nitrogen. DNA was extracted and purified using a proteinase K, phenol-chloroform, LiCl, and EtOH protocol. Tissue from 18 patients with HNPCC, defined by Amsterdam Criteria, served as positive controls for MSI.33 These positive controls were used to evaluate each microsatellite marker’s sensitivity for detecting ASI. To compare the performance of the NCI and multiplex assays, a series of 120 early-onset (age  60 years) colon cancer patients were identified from our frozen tumor bank (stage I, n = 14; stage II, n = 27; stage III, n = 35; and stage IV, n = 44).
PCR
ASI Analysis
LOH Analysis
Multiplexing
Morphology
Statistics
There was successful PCR amplification of all microsatellite loci in 100% of samples for both uniplex and multiplex reactions.
HNPCC Specimens
Early-Onset Cancer Specimens When the NCI panel of five loci was used, ASI was seen in at least two loci in 23% of specimens (28 of 120), at one locus in 11% (13 of 120), and at no loci in 66% (79 of 120; Table 2  ). By NCI guidelines, cancers with ASI in at least two loci would be classified as MSI-H, cancers with ASI in one locus would be classified as MSI-L, and cancers with ASI at no loci would be classified as MSS.
At least one LOH event was observed in 55% (66 of 120) of early-onset cancers. LOH was observed in 36% (10 of 28) of the tumors with MSI-H by NCI criteria (MSI-H/NCI) and in 61% (56 of 92) of the tumors with MSS by NCI criteria (MSS/NCI). LOH was observed in 46% (six of 13) of the tumors with MSI-L by NCI criteria (MSI-L/NCI).
Mononucleotide Repeat Versus Dinucleotide Repeat Size Instability
Multiplexing
Multiplex Assay Versus NCI Assay There were fewer cancers identified as having MSI by the multiplex assay compared with the NCI assay (Table 2  ). When the NCI assay was used, the prevalence of MSI (MSI/NCI) was 23% (n = 28) in 120 early-onset colon cancers. Using the multiplex assay, the prevalence of MSI (MSI/multiplex) was 16% (n = 19). All clinical stages of cancer were present in each MSI cohort (MSI/multiplex: I, n = 4; II, n = 10; III, n = 3; IV, n = 2; and MSI/NCI: I, n = 5; II, n = 11; III, n = 6; IV, n = 6). LOH was observed in three of 19 (16%) MSI/multiplex patients and in 63 of 101 (63%) MSS/multiplex patients (P = .0001). LOH was observed in 10 of 28 (36%) MSI/NCI patients and in 56 of 92 (61%) MSS/NCI patients (P = .03). The additional patients identified as MSI-H by the NCI assay had a significantly higher prevalence of LOH compared with the MSI/multiplex patients (78% v 16%; P = .003).
Morphology
Survival
BAT40 The mononucleotide BAT40 was assessed in all tumors to determine if use of a third mononucleotide marker in place of D2S123 might improve the accuracy of MSI-H assessment. In the HNPCC patients, BAT40 had intermediate sensitivity for ASI (evident in 13 of 18 patients), which was comparable to that of D2S123 (evident in 13 of 18 patients). In the 120 early-onset colon cancers, BAT40 showed ASI in 57% (16 of 28) of MSI-H/NCI patients, in 31% (four of 13) of MSI-L/NCI patients, and in 6% (five of 79) of MSS/NCI patients. If BAT40 was substituted for D2S123 in the multiplex assay, only one tumor among the 120 early-onset cancers would have changed its MSI designation (from MSI-H to MSS; Table 3 ). There was a discrepancy between BAT25 (ASI) and BAT26 (allele size stability [ASS]) in this patient, with D2S123, D5S346, and D17S250 all showing ASI, whereas BAT40 showed ASS. In the two other patient cases of disagreement between BAT25 and BAT26, BAT40 agreed with the remaining markers in both patients (Table 3). There were four MSI-L patients, defined by ASI in one dinucleotide marker, that also had ASI of BAT40. None of these four patients had MSI morphology, two had LOH, and two had died of disease at last follow-up.
MSI-H has particular clinical importance in the assessment of CRC patients. Each year, there are an estimated 20,000 patients diagnosed with CRCs with MSI-H in the United States.38 Patients with HNPCC represent approximately 5% of all patients with CRC,39 and patients with sporadic MSI account for an additional 10% to 15%.14 In both instances, MSI indicates disruption of the MMR pathway within the cancer cells. The availability of an MSI test that is rapid and highly accurate in identifying the presence or absence of MMR deficiency in colorectal tumors is crucial for successful use of MSI testing in clinical and research applications. Clinical uses include screening patients with early-onset, familial, or metachronous colon cancer for germline mutation testing for HNPCC, as well as individualizing prognosis for all patients with colon cancer. The research applications are numerous and include studies of cancer surveillance, chemoprevention, cancer progression, molecular profiling, and adjuvant therapy.
Developments in PCR technology, fluorescent labeling of DNA fragments, and computer-aided detection and analysis of gel banding patterns have improved the use of microsatellites in DNA diagnostics.40,41 These advances prompted us to examine the molecular diagnosis of MSI in CRC as outlined by the NCI workshop in 1997. Our goal was a simpler, more rapid, and more accurate method of MSI-H assessment. Because the value of an individual microsatellite marker is based on its ability to report accurately the status of the MMR pathway within tumors, we evaluated the performance of the five microsatellite markers used in the NCI assay in reporting ASI in HNPCC cancers and in reporting ASS in sporadic MSS cancers. We found marker sensitivities for ASI ranged from 100% to 50% (Table 1
In the test group of patients with early-onset CRC (age The NCI consensus group defined MSI-L as one of five markers with ASI. In contrast to MSI-H tumors, MSI-L tumors do not have loss of MSH2 or MLH1 expression.42 Phenotypically MSI-L tumors appear to be indistinguishable from MSS tumors.43 Recently, evidence has emerged indicating that MSI-L tumors represent a third pathway of colorectal carcinogenesis. Much controversy remains regarding the genetic origin and background of MSS and MSI-L tumors.44–47 The analysis of MSI-L is difficult because the greater the number of microsatellites that are interrogated, the larger the MSI-L group becomes.48 Our assay does not attempt to distinguish MSS from MSI-L. Given that these groups are clinically indistinguishable with our current knowledge, the distinction of these groups remains a research endeavor. Our purpose was to improve the accuracy and usefulness of MSI-H assessment. Published reports indicate that mononucleotide markers have greater sensitivity and specificity for MMR deficiency than dinucleotide markers;29,32 this conclusion is supported by our own analysis. In HNPCC tumors, BAT25 and BAT26 were highly sensitive for microsatellite instability (ASI in 33 of 36 inquiries), whereas in sporadic tumors presumed to be competent in MMR (MSS and MSI-L), ASI was rare (with ASI in one of 184 inquiries). In contrast, dinucleotide instability was only moderately sensitive for microsatellite instability in HNPCC tumors (ASI in 31 of 54 inquiries), and was also present at low frequency in MSS and MSI-L tumors (ASI in 12 of 276 inquiries). However, the high sensitivity and specificity of mononucleotide markers for MSI-H did not extend to BAT40. Using BAT40, we found ASI in 13 of 18 inquiries in HNPCC tumors and in nine of 92 inquiries in MSS and MSI-L tumors. This 10% rate of ASI in MSS/MSI-L tumors indicates poor specificity for MSI-H, second only to D17S250 among the six markers tested. A multiplex assay designed with only the mononucleotide markers (BAT25, BAT26, and BAT40) would not have reclassified any tumors as MSI-H; one tumor with four of five markers positive by the NCI assay would have been dubiously reclassified as MSS. In contrast, we have shown that D2S123 is 72% sensitive for HNPCC and 99% specific for MSS, and others have shown that it is 100% reproducible.29 To exclude D2S123 in favor of a less reliable mononucleotide would ignore these experimental data. These findings do not support the use of BAT40 in our MSI-H assay. Investigators have argued that BAT26 alone may be sufficient to identify MSI-H.49,50 However, no single microsatellite marker is likely to have perfect accuracy for identifying colon cancers with DNA MMR deficiency.49–53 We recorded one HNPCC tumor and two sporadic MSI-H tumors with ASS at BAT26. On the other hand, evaluating many microsatellite markers to increase sensitivity can leave the assay vulnerable to loss of specificity. The multiplex assay addresses this problem by retaining the three most sensitive markers and requiring two markers with ASI for a positive result and two markers with ASS for a negative result. By relying on concordance of at least two markers, a high degree of accuracy is obtained. If microsatellite analysis is to be useful for detection of MMR deficiency, the MSI assay must achieve high specificity. If the true prevalence of MMR deficiency in a series of CRCs is 12% and if an MSI-H assay has a sensitivity of 100% but a specificity of only 85%, more than half of the patients identified with MSI-H will not have true MMR deficiency. This large number of misclassified patients would result in high costs of germline mutation screening and would severely handicap investigators in correlative studies. A simple, rapid, and highly accurate MSI-H assay has the potential to improve the management of CRC patients both in routine practice and in clinical research. The multiplex assay presented here is well suited for this task.
Supported by a grant from the National Cancer Institute (2 P01 CA65930-05A2) and by the philanthropy of Marie and William Bianco. This work was presented, in part, as a poster at the May 2002 meeting of the American Society of Clinical Oncology under the title, "An Objective, Multiplex Microsatellite Instability (MSI) Assay for Colorectal Adenocarcinoma."
1. Fearon E, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell 61:759–767, 1990[CrossRef][Medline] 2. Ionov Y, Peinado M, Malkhosyan S, et al: Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363:558–561, 1993[CrossRef][Medline] 3. Lengauer C, Kinzler K, Vogelstein B: Genetic instability in colorectal cancers. Nature 396:643–649, 1998[CrossRef][Medline] 4. Lengauer C, Kinzler K, Vogelstein B: Genetic instability in human cancers. Nature 386:643–649, 1997[CrossRef][Medline]
5. Toyota M, Ohe-Yoyota M, Ahuja N, et al: Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype. Proc Natl Acad Sci U S A 97:710–715, 2000 6. Sugai T, Haban W, Nakamura S, et al: Genetic alterations in DNA diploid, aneuploid and multiploid colorectal carcinomas identified by the crypt isolation technique. Int J Cancer 88:614–619, 2000[CrossRef][Medline] 7. Liu B, Parsons R, Papadopoulos N, et al: Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat Med 2:169–174, 1996[CrossRef][Medline] 8. Kinzler K, Vogelstein B: Lessons from hereditary colorectal cancer. Cell 87:159–170, 1996[CrossRef][Medline] 9. Bapat B, Madlensky L, Temple L: Family history characteristics, tumor microsatellite instability and germline MSH2 and MLH1 mutations in hereditary colorectal cancer. Hum Genet 104:167–176, 1999[CrossRef][Medline]
10. Kane M, Loda M, Gaida G, 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
11. Herman J, Umar A, Polyak K, et al: Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci U S A 95:6870–6875, 1998
12. Thibodeau S, Bren G, Schaid: Microsatellite instability in cancer of the proximal colon. Science 260:816–819, 1993
13. Aaltonen L, Peltomaki P, Leach F, et al: Clues to the pathogenesis of familial colorectal cancer. Science 260:812–816, 1993
14. Gryfe R, Kim H, Hsieh E, et al: Tumor microsatellite instability and clinical outcome in young patients with colorectal cancer. N Engl J Med 342:69–77, 2000
15. Samowithz W, Curtin K, Ma K, et al: Microsatellite instability in sporadic colon cancer is associated with an improved prognosis at the population level. Cancer Epidemiol Biomarkers Prev 10:917–923, 2001
16. Lothe R, Peltomaki P, Meling G, et al: Genomic instability in colorectal cancer: Relationship to clinicopathological variables and family history. Cancer Res 53:5849–5852, 1993
17. Diep C, Thorstensen L, Meling G, et al: Genetic tumor markers with prognostic impact in Dukes’ stages B and C colorectal cancer patients. J Clin Oncol 21:820–829, 2003
18. Aaltonen L, Peltomäki P, Mecklin J-P, et al: Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Res 54:1645–1648, 1994
19. Katballe N, Christensen M, Wikman F, et al: Frequency of hereditary non-polyposis colorectal cancer in Danish colorectal cancer patients. Gut 50:43–51, 2002 20. Reyes C, Allen B, Terdiman F, et al: Comparison of selection strategies for genetic testing of patients with hereditary nonpolyposis colorectal carcinoma. Cancer 95:1848–1856, 2002[CrossRef][Medline] 21. Samowitz W, Slattery M, Kerber R: Microsatellite instability in human colonic cancer is not a useful indicator of familial colorectal caner. Gastroenterology 109:2031–2033, 1995[CrossRef][Medline] 22. Liu B, Nicolaides N, Markowitz S, et al: Mismatch repair gene defects in sporadic colorectal cancers with microsatellite instability. Nat Genet 9:48–55, 1995[CrossRef][Medline] 23. Magrini R, Bhonde M, Hanski M, et al: Cellular effects of CPT-11 on colon carcinoma cells: Dependence on p53 and hMLH1 status. Int J Cancer 101:23–31, 2002[CrossRef][Medline]
24. Jacob S, Aguado M, AFallik D, et al: The role of the DNA mismatch repair system in the cytotoxicity of the topoisomerase inhibitors camptothecin and etoposide to human colorectal cancer cells. Cancer Res 61:6555–6562, 2001 25. Hemminki A, Mecklin J, 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] 26. Wright C, Dent O, Barker M, et al: Prognostic significance of extensive microsatellite instability in sporadic clinicopathological stage C colorectal cancer. Br J Surg 87:1197–1202, 2000[CrossRef][Medline]
27. Watanabe T, Wu T, Catalano P, et al: Molecular predictors of survival after adjuvant chemotherapy for colon cancer. N Engl J Med 344:1196–1206, 2001 28. Ribic C, Sargent D, Moore M, et al: Tumor microsatellite instability (MSI) and the benefit of 5-FU based chemotherapy in stage II&III colon cancer: A pooled molecular reanalysis of randomized chemotherapy trials. Proc Am Soc Clin Oncol 21:128a, 2002
29. Bocker T, Diermann J, Friedl W, et al: Microsatellite instability analysis: A multicenter study for reliability and quality control. Cancer Res 57:4739–4743, 1997
30. Dietmaier W, Wallinger S, Bocker T, et al: Diagnostic microsatellite instability: Definition and correlation with mismatch repair protein expression. Cancer Res 57:4749–4756, 1997
31. Boland D, Thibodeau S, Hamilton S, 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
32. Perucho M: Correspondence re: Boland DR, 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 59:249–256, 1999 33. Vasen H, Mecklin J, Khan P, et al: The International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC). Dis Colon Rectum 34:424–425, 1991[CrossRef][Medline] 34. Utemani N, Sasaki S, Watanabe T, et al: Diagnostic primer sets for microsatellite instability optimized for a minimal amount of damaged DNA from colorectal tissue samples. Ann Surg Oncol 7:276–280, 2000[CrossRef][Medline]
35. Alexander J, Watanabe T, Wu T, et al: Histopathological identification of colon cancer with microsatellite instability. Am J Path 158:527–535, 2001
36. Ward R, Meagher A, Tomlinson I, et al: Microsatellite instability and the clinicopathological features of sporadic colorectal cancer. Gut 48:821–829, 2001
37. Guidoboni M, Gafa R, Viel A, et al: Microsatellite instability and high content of activated cytotoxic lymphocytes identify colon cancer patients with a favorable prognosis. Am J Path 159:297–304, 2001 38. American Cancer Society: Cancer Facts and Figures—2002. Atlanta, GA, American Cancer Society, 2002 39. Lynch H, Lanspa S, Smyrk T, et al: Hereditary nonpolyposis colorectal cancer (Lynch syndromes I & II): Genetics, pathology, natural history, and cancer control, part I. Cancer Genet Cytogenet 53:143–160, 1991[CrossRef][Medline]
40. Berg KD, Glaser CL, Thompson RE, et al: Detection of microsatellite instability by fluorescence multiplex polymerase chain reaction. J Mol Diagn 2:20–28, 2000 41. Sutter C, Gebert J, Bischoff P, et al: Molecular screening of potential HNPCC patients using a multiplex microsatellite PCR system. Mol Cell Probes 13:157–165, 1999[CrossRef][Medline]
42. Lindor N, Burgart L, Leontovich O, et al: Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 20:1043–1048, 2002
43. Thibodeau S, French A, Cunningham J, et al: Microsatellite instability in colorectal cancer: Different mutator phenotypes and the principal involvement of hMLH1. Cancer Res 58:1713–1718, 1998
44. Whitehall V, Wynter C, Walsh M, et al: Morphological and molecular heterogeneity within nonmicrosatellite instability-high colorectal cancer. Cancer Res 62:6011–6014, 2002
45. Whitehall V, Walsh M, Young J, et al: Methylation of O-6-methylguanine DNA methyltransferase characterizes a subset of colorectal cancer with low-level microsatellite instability. Cancer Res 61:827–830, 2001
46. Kambara T, Matsubara N, Nakagawa H, et al: High frequency of low-level microsatellite instability in early colorectal cancer. Cancer Res 61:7743–7746, 2001
47. Kolodner R, Tytell J, Schmeits J, et al: Germ-line msh6 mutations in colorectal cancer families. Cancer Res 59:5068–5074, 1999 48. Laiho P, Launonen V, Lahermo P, et al: Low-level microsatellite instability in most colorectal carcinomas is not a useful clinical indicator of familial colorectal cancer. Cancer Res 62:116–1170, 2002
49. Hoang J, Cottu P, Thuille B, et al: BAT-26, an indicator of the replication error phenotype in colorectal cancers and cell line. Cancer Res 57:300–303, 1997
50. Loukola A, Eklin K, Laiho P, et al: Microsatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (HNPCC). Cancer Res 61:4545–4549, 2001 51. 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]
52. Aaltonen LA, Salovaara R, Kristo P, et al: Incidence of hereditary nonpolyposis colorectal cancer and the feasibility of molecular screening for the disease. N Engl J Med 338:1481–1487, 1998
53. Samowitz WS, Slattery ML, Potter JD, et al: BAT-26 and BAT-40 Instability in colorectal adenomas and carcinomas and germline polymorphisms. Am J Pathol 154:1637–1641, 1999 Submitted November 26, 2002; accepted May 29, 2003.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 or Fisher’s exact tests were applied to the results as appropriate. All reported P values are two-sided, and P values of 
