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Development of Sporadic Microsatellite Instability in Colorectal Tumors Involves Hypermethylation at Methylated-In-Tumor Loci in Adenoma

2010; Elsevier BV; Volume: 177; Issue: 5 Linguagem: Inglês

10.2353/ajpath.2010.091103

1525-2191

Michiel F.G. de Maat, Norihiko Narita, Anne Benard, Tetsunori Yoshimura, Christine Kuo, Rob A.�E.�M. Tollenaar, Noel F.C.C. de Miranda, Roderick R. Turner, Cornelis J.�H. van de Velde, Hans Morreau, Dave S.�B. Hoon,

Colorectal Cancer Screening and Detection

Microsatellite instability (MSI) and genomic hypermethylation of methylated-in-tumor (MINT) loci are both strong prognostic indicators in a subgroup of patients with sporadic colorectal cancer (CRC). The present study was designed to determine whether the methylation of MINT loci during the progression of adenoma to CRC is related to MSI in CRC cases. Methylation index (MI) was measured by absolute quantitative assessment of methylated alleles at seven MINT loci in primary CRC with contiguous adenomatous and normal tissues of 79 patients. Results were then validated in primary CRC tissues from an independent group of 54 patients. Increased MI of both MINT loci 1 and 31 was significantly associated with MSI in CRC and was specific for adenoma. Total MI and the number of methylated loci were threefold (P = 0.02) and fivefold (P = 0.004) higher, respectively, in adenomas associated with microsatellite-stable CRC versus microsatellite-unstable CRC. MINT MI was found to be correlated with mismatch repair protein expression, MSI, BRAF (V600E) mutation status, mut-L homologue 1 methylation status, and disease-specific survival in the second independent validation group of patients. MI of specific MINT loci may be prognostic indicators of colorectal adenomas that will develop into sporadic microsatellite-unstable CRCs. Increased MINT locus methylation appears to precede MSI and may have utility in defining clinical pathology in the absence of features of malignant invasive tumors. Microsatellite instability (MSI) and genomic hypermethylation of methylated-in-tumor (MINT) loci are both strong prognostic indicators in a subgroup of patients with sporadic colorectal cancer (CRC). The present study was designed to determine whether the methylation of MINT loci during the progression of adenoma to CRC is related to MSI in CRC cases. Methylation index (MI) was measured by absolute quantitative assessment of methylated alleles at seven MINT loci in primary CRC with contiguous adenomatous and normal tissues of 79 patients. Results were then validated in primary CRC tissues from an independent group of 54 patients. Increased MI of both MINT loci 1 and 31 was significantly associated with MSI in CRC and was specific for adenoma. Total MI and the number of methylated loci were threefold (P = 0.02) and fivefold (P = 0.004) higher, respectively, in adenomas associated with microsatellite-stable CRC versus microsatellite-unstable CRC. MINT MI was found to be correlated with mismatch repair protein expression, MSI, BRAF (V600E) mutation status, mut-L homologue 1 methylation status, and disease-specific survival in the second independent validation group of patients. MI of specific MINT loci may be prognostic indicators of colorectal adenomas that will develop into sporadic microsatellite-unstable CRCs. Increased MINT locus methylation appears to precede MSI and may have utility in defining clinical pathology in the absence of features of malignant invasive tumors. Epigenetic changes in epithelial cells, such as DNA methylation of CpG islands, have been related to the genesis and progression of some gastrointestinal cancers.1Haydon AM Jass JR Emerging pathways in colorectal-cancer development.Lancet Oncol. 2002; 3: 83-88Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 2Herman JG Epigenetic changes in cancer and preneoplasia.Cold Spring Harb Symp Quant Biol. 2005; 70: 329-333Crossref PubMed Scopus (26) Google Scholar Aberrations in DNA methylation are considered to be as important as genetic alterations in gastrointestinal tumor initiation and progression. In colorectal cancer (CRC), both hypomethylation and hypermethylation of promoter-region related CpG islands have been correlated with clinical and histopathological parameters.3Esteller M Epigenetics in cancer.N Engl J Med. 2008; 358: 1148-1159Crossref PubMed Scopus (2883) Google Scholar, 4Tanemura A Terando AM Sim MS van Hoesel AQ de Maat MF Morton DL Hoon DS CpG island methylator phenotype predicts progression of malignant melanoma.Clin Cancer Res. 2009; 15: 1801-1807Crossref PubMed Scopus (150) Google Scholar A number of tumor-related gene promoter regions are methylated in premalignant dysplastic lesions such as hyperplastic polyps,5Wynter CV Walsh MD Higuchi T Leggett BA Young J Jass JR Methylation patterns define two types of hyperplastic polyp associated with colorectal cancer.Gut. 2004; 53: 573-580Crossref PubMed Scopus (141) Google Scholar aberrant crypt foci,6Chan AO Broaddus RR Houlihan PS Issa JP Hamilton SR Rashid A CpG island methylation in aberrant crypt foci of the colorectum.Am J Pathol. 2002; 160: 1823-1830Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 7Luo L Chen WD Pretlow TP CpG island methylation in aberrant crypt foci and cancers from the same patients.Int J Cancer. 2005; 115: 747-751Crossref PubMed Scopus (35) Google Scholar and adenomas.8de Maat MF Umetani N Sunami E Turner RR Hoon DS Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles.Mol Cancer Res. 2007; 5: 461-471Crossref PubMed Scopus (26) Google Scholar, 9Jass JR Serrated adenoma of the colorectum and the DNA-methylator phenotype.Nat Clin Pract Oncol. 2005; 2: 398-405Crossref PubMed Scopus (164) Google Scholar, 10Wynter CV Kambara T Walsh MD Leggett BA Young J Jass JR DNA methylation patterns in adenomas from FAP, multiple adenoma and sporadic colorectal carcinoma patients.Int J Cancer. 2006; 118: 907-915Crossref PubMed Scopus (25) Google Scholar Furthermore, progressive genomic and epigenomic aberrations may be linked in CRC progression.11Baylin SB Ohm JE Epigenetic gene silencing in cancer: a mechanism for early oncogenic pathway addiction?.Nat Rev Cancer. 2006; 6: 107-116Crossref PubMed Scopus (1391) Google Scholar For instance, it has been reported that widely increased methylation in sporadic CRCs overlaps with microsatellite instability (MSI).12Ahuja N Mohan AL Li Q Stolker JM Herman JG Hamilton SR Baylin SB Issa JP Association between CpG island methylation and microsatellite instability in colorectal cancer.Cancer Res. 1997; 57: 3370-3374PubMed Google Scholar, 13Herman JG Umar A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1693) Google Scholar, 14Veigl ML Kasturi L Olechnowicz J Ma AH Lutterbaugh JD Periyasamy S Li GM Drummond J Modrich PL Sedwick WD Markowitz SD Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers.Proc Natl Acad Sci USA. 1998; 95: 8698-8702Crossref PubMed Scopus (557) Google Scholar, 15Weisenberger DJ Siegmund KD Campan M Young J Long TI Faasse MA Kang GH Widschwendter M Weener D Buchanan D Koh H Simms L Barker M Leggett B Levine J Kim M French AJ Thibodeau SN Jass J Haile R Laird PW CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer.Nat Genet. 2006; 38: 787-793Crossref PubMed Scopus (1525) Google Scholar Studies to pinpoint the onset of MSI have been reported in hereditary nonpolyposis colorectal cancers that carry germline mutations (mt) in mismatch repair (MMR) genes,13Herman JG Umar A Polyak K Graff JR Ahuja N Issa JP Markowitz S Willson JK Hamilton SR Kinzler KW Kane MF Kolodner RD Vogelstein B Kunkel TA Baylin SB Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci USA. 1998; 95: 6870-6875Crossref PubMed Scopus (1693) Google Scholar, 14Veigl ML Kasturi L Olechnowicz J Ma AH Lutterbaugh JD Periyasamy S Li GM Drummond J Modrich PL Sedwick WD Markowitz SD Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers.Proc Natl Acad Sci USA. 1998; 95: 8698-8702Crossref PubMed Scopus (557) Google Scholar, 16Shibata D When does MMR loss occur during HNPCC progression?.Cancer Biomark. 2006; 2: 29-35PubMed Google Scholar, 17Cunningham JM Christensen ER Tester DJ Kim CY Roche PC Burgart LJ Thibodeau SN Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability.Cancer Res. 1998; 58: 3455-3460PubMed Google Scholar but as of yet, no study has examined this process in sporadic CRC. Spontaneous regression of colorectal polyps is known to occur. Therefore, a critical issue in molecular analysis of colorectal precursor tumor lesions is whether a lesion with low or intermediate adenomatous dyplasia would develop into an invasive cancer. The DNA extracted from such lesions may not contain key premalignant aberrations. We recently described an on-slide sodium bisulfite modification (SBM) technique for gene methylation analysis in small (1 to 2 mm2) tissue areas isolated from a single section of paraffin-embedded archival tissue (PEAT).18Umetani N de Maat MF Sunami E Hiramatsu S Martinez S Hoon DS Methylation of p16 and Ras association domain family protein 1a during colorectal malignant transformation.Mol Cancer Res. 2006; 4: 303-309Crossref PubMed Scopus (13) Google Scholar On-slide SBM allows comparison of gene methylation in the primary CRC, the contiguous adenoma lesion, and normal epithelium when these three tissue types are present on the same tissue section. Simultaneous assessment of methylation and MSI changes in CRC, adenoma, and normal tissues from the same patient using the on-slide SBM technique would provide an accurate analysis model to test development of these epigenetic and genetic events during CRC formation. In a previous study, our group demonstrated the technical feasibility of using methylated-in-tumor (MINT) loci 1, 2, 12, and 31 to detect methylation differences between areas of the same colorectal tissue section.8de Maat MF Umetani N Sunami E Turner RR Hoon DS Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles.Mol Cancer Res. 2007; 5: 461-471Crossref PubMed Scopus (26) Google Scholar MINT loci are conservative human genomic sequences that adhere to the CpG island definition.19Toyota M Ohe-Toyota M Ahuja N Issa JP Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotype.Proc Natl Acad Sci USA. 2000; 97: 710-715Crossref PubMed Scopus (403) Google Scholar, 20de Maat MF van de Velde CJ van der Werff MP Putter H Umetani N Klein-Kranenbarg EM Turner RR van Krieken JH Bilchik A Tollenaar RA Hoon DS Quantitative analysis of methylation of genomic loci in early-stage rectal cancer predicts distant recurrence.J Clin Oncol. 2008; 26: 2327-2335Crossref PubMed Scopus (27) Google Scholar They are found in noncoding genomic regions, and their gene regulatory purpose or other functional attributes are unknown. We have previously shown the clinical utility of methylation levels of specific MINT loci in rectal cancer20de Maat MF van de Velde CJ van der Werff MP Putter H Umetani N Klein-Kranenbarg EM Turner RR van Krieken JH Bilchik A Tollenaar RA Hoon DS Quantitative analysis of methylation of genomic loci in early-stage rectal cancer predicts distant recurrence.J Clin Oncol. 2008; 26: 2327-2335Crossref PubMed Scopus (27) Google Scholar and melanoma.4Tanemura A Terando AM Sim MS van Hoesel AQ de Maat MF Morton DL Hoon DS CpG island methylator phenotype predicts progression of malignant melanoma.Clin Cancer Res. 2009; 15: 1801-1807Crossref PubMed Scopus (150) Google Scholar Other groups have studied MINT locus methylation in colorectal20de Maat MF van de Velde CJ van der Werff MP Putter H Umetani N Klein-Kranenbarg EM Turner RR van Krieken JH Bilchik A Tollenaar RA Hoon DS Quantitative analysis of methylation of genomic loci in early-stage rectal cancer predicts distant recurrence.J Clin Oncol. 2008; 26: 2327-2335Crossref PubMed Scopus (27) Google Scholar, 21Park SJ Rashid A Lee JH Kim SG Hamilton SR Wu TT Frequent CpG island methylation in serrated adenomas of the colorectum.Am J Pathol. 2003; 162: 815-822Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 22Toyota M Ahuja N Ohe-Toyota M Herman JG Baylin SB Issa JP CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2121) Google Scholar and gastric cancers.20de Maat MF van de Velde CJ van der Werff MP Putter H Umetani N Klein-Kranenbarg EM Turner RR van Krieken JH Bilchik A Tollenaar RA Hoon DS Quantitative analysis of methylation of genomic loci in early-stage rectal cancer predicts distant recurrence.J Clin Oncol. 2008; 26: 2327-2335Crossref PubMed Scopus (27) Google Scholar, 21Park SJ Rashid A Lee JH Kim SG Hamilton SR Wu TT Frequent CpG island methylation in serrated adenomas of the colorectum.Am J Pathol. 2003; 162: 815-822Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 22Toyota M Ahuja N Ohe-Toyota M Herman JG Baylin SB Issa JP CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2121) Google Scholar, 23Abraham SC Park SJ Cruz-Correa M Houlihan PS Half EE Lynch PM Wu TT Frequent CpG island methylation in sporadic and syndromic gastric fundic gland polyps.Am J Clin Pathol. 2004; 122: 740-746Crossref PubMed Scopus (18) Google Scholar, 24Toyota M Ahuja N Suzuki H Itoh F Ohe-Toyota M Imai K Baylin SB Issa JP Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype.Cancer Res. 1999; 59: 5438-5442PubMed Google Scholar Methylation of MINT loci has also been linked to MSI in CRC.25Whitehall VL Wynter CV Walsh MD Simms LA Purdie D Pandeya N Young J Meltzer SJ Leggett BA Jass JR Morphological and molecular heterogeneity within nonmicrosatellite instability-high colorectal cancer.Cancer Res. 2002; 62: 6011-6014PubMed Google Scholar, 26Samowitz WS Albertsen H Herrick J Levin TR Sweeney C Murtaugh MA Wolff RK Slattery ML Evaluation of a large, population-based sample supports a CpG island methylator phenotype in colon cancer.Gastroenterology. 2005; 129: 837-845Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar However, whether this was present in the precursor adenoma is an important question that our analysis model may answer. The objective was to determine whether MINT locus hypermethylation is associated with MMR during early stages of CRC development. Methylation at MINT loci was quantified by using the on-slide SBM technique combined with absolute quantitative assessment of methylated alleles (AQAMA). We further evaluated MSI status, KRAS mt at codons 12 and 13, BRAF V600E mt, and methylation status of the MutL homolog 1 MMR gene (MLH1) in primary CRC as well as the contiguous precursor lesion to rigorously assess whether these events occur at early stages of CRC development. Our hypothesis was that MINT locus methylation, MLH1 promoter region methylation, and MSI in adenoma tissue are higher in those adenomas adjacent to MSI-high (MSI-H) compared with microsatellite-stable (MSS) invasive CRC. For the first phase of the study, we identified patients whose resected CRC specimen contained histopathologically confirmed areas of adenoma as well as invasive cancer in our cancer registry database. Excluded were any cases without available PEAT specimens. Consecutive patients were identified in reverse chronological order until an adequate sample size was reached. The final cohort of 115 patients who underwent surgical resection of CRC between 1996 and 2009 at the Saint John's Health Center was selected for the first phase of the study. A single H&E section was prepared and mounted; 7-μm sections were consecutively cut and mounted on silane-coated glass slides for DNA studies. Areas of adenoma (serrated and nonserrated), carcinoma, and normal tissue as well as the adenoma type were identified by a surgical pathologist (R.R.T.) with expertise in CRC.8de Maat MF Umetani N Sunami E Turner RR Hoon DS Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles.Mol Cancer Res. 2007; 5: 461-471Crossref PubMed Scopus (26) Google Scholar, 27de Maat MF van de Velde CJ Umetani N de Heer P Putter H van Hoesel AQ Meijer GA van Grieken NC Kuppen PJ Bilchik AJ Tollenaar RA Hoon DS Epigenetic silencing of cyclooxygenase-2 affects clinical outcome in gastric cancer.J Clin Oncol. 2007; 25: 4887-4894Crossref PubMed Scopus (69) Google Scholar To further validate the findings of the first phase of the study, PEAT blocks of operative specimens were obtained from 54 patients who underwent surgical resection of CRC at the Leiden University Medical Center (LUMC) between 1990 and 2001. All specimens had been previously analyzed for MMR sufficiency at LUMC's pathology department. From each PEAT block, a single section was cut for H&E staining, and 7-μm sections were consecutively cut on coated slides for on-slide SBM. Tumor areas were identified and marked by an expert CRC pathologist (H.M.). Study protocols for assessment of patient specimens were approved by the institutional review boards at the LUMC and at Saint John's Health Center. DNA from PEAT was modified in situ by sodium bisulfite according to our previously reported protocol.18Umetani N de Maat MF Sunami E Hiramatsu S Martinez S Hoon DS Methylation of p16 and Ras association domain family protein 1a during colorectal malignant transformation.Mol Cancer Res. 2006; 4: 303-309Crossref PubMed Scopus (13) Google Scholar AQAMA of MINT loci 1, 2, 3, 12, 17, 25, and 31 was performed, and data were analyzed in a manner as previously described.8de Maat MF Umetani N Sunami E Turner RR Hoon DS Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles.Mol Cancer Res. 2007; 5: 461-471Crossref PubMed Scopus (26) Google Scholar, 28Umetani N de Maat MF Mori T Takeuchi H Hoon DS Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase.Biochem Biophys Res Commun. 2005; 329: 219-223Crossref PubMed Scopus (33) Google ScholarMLH1 methylation status was analyzed by capillary-array-electrophoresis methylation-specific PCR.27de Maat MF van de Velde CJ Umetani N de Heer P Putter H van Hoesel AQ Meijer GA van Grieken NC Kuppen PJ Bilchik AJ Tollenaar RA Hoon DS Epigenetic silencing of cyclooxygenase-2 affects clinical outcome in gastric cancer.J Clin Oncol. 2007; 25: 4887-4894Crossref PubMed Scopus (69) Google Scholar, 29Hoon DS Spugnardi M Kuo C Huang SK Morton DL Taback B Profiling epigenetic inactivation of tumor suppressor genes in tumors and plasma from cutaneous melanoma patients.Oncogene. 2004; 23: 4014-4022Crossref PubMed Scopus (203) Google Scholar, 30Mori T Kim J Yamano T Takeuchi H Huang S Umetani N Koyanagi K Hoon DS Epigenetic up-regulation of C-C chemokine receptor 7 and C-X-C chemokine receptor 4 expression in melanoma cells.Cancer Res. 2005; 65: 1800-1807Crossref PubMed Scopus (108) Google Scholar Primer sequences were selected based on previous literature and correlation with MLH1 protein expression determined.31Arnold CN Goel A Boland CR Role of hMLH1 promoter hypermethylation in drug resistance to 5-fluorouracil in colorectal cancer cell lines.Int J Cancer. 2003; 106: 66-73Crossref PubMed Scopus (242) Google Scholar, 32Arnold CN Goel A Compton C Marcus V Niedzwiecki D Dowell JM Wasserman L Inoue T Mayer RJ Bertagnolli MM Boland CR Evaluation of microsatellite instability, hMLH1 expression and hMLH1 promoter hypermethylation in defining the MSI phenotype of colorectal cancer.Cancer Biol Ther. 2004; 3: 73-78Crossref PubMed Scopus (40) Google Scholar, 33Ashktorab H Smoot DT Carethers JM Rahmanian M Kittles R Vosganian G Doura M Nidhiry E Naab T Momen B Shakhani S Giardiello FM High incidence of microsatellite instability in colorectal cancer from African Americans.Clin Cancer Res. 2003; 9: 1112-1117PubMed Google Scholar, 34Ashktorab H Smoot DT Farzanmehr H Fidelia-Lambert M Momen B Hylind L Iacosozio-Dononue C Carethers JM Goel A Boland CR Giardiello FM Clinicopathological features and microsatellite instability (MSI) in colorectal cancers from African Americans.Int J Cancer. 2005; 116: 914-919Crossref PubMed Scopus (71) Google Scholar, 35Brim H Mokarram P Naghibalhossaini F Saberi-Firoozi M Al-Mandhari M Al-Mawaly K Al-Mjeni R Al-Sayegh A Raeburn S Lee E Giardiello F Smoot DT Vilkin A Boland CR Goel A Hafezi M Nouraie M Ashktorab H Impact of BRAF. MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study.Mol Cancer. 2008; 7: 68-79Crossref PubMed Scopus (72) Google Scholar For MSI assessment in the first phase of the study, a tissue section from each of the specimens was deparaffinized and stained with hematoxylin to identify adenoma and CRC cells for DNA isolation, as previously described.8de Maat MF Umetani N Sunami E Turner RR Hoon DS Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles.Mol Cancer Res. 2007; 5: 461-471Crossref PubMed Scopus (26) Google Scholar Normal epithelial cells were harvested from a separate tissue block of the same specimen (ie, from noninvolved resection margins). As biomarkers for MSI assessment, we used three quasi-monomorphic mononucleotide repeats (BAT25, BAT26, and BAT40) and two microsatellite, dinucleotide repeats (D2S123 and D5S346) found in the revised Bethesda guidelines to interrogate the specimens.36Umar A Boland CR Terdiman JP Syngal S de la Chapelle A Ruschoff J Fishel R Lindor NM Burgart LJ Hamelin R Hamilton SR Hiatt RA Jass J Lindblom A Lynch HT Peltomaki P Ramsey SD Rodriguez-Bigas MA Vasen HF Hawk ET Barrett JC Freedman AN Srivastava S Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2461) Google Scholar Forward primers were dye-labeled for automated high-throughput multiplex detection by capillary array electrophoresis37Fujimoto A Takeuchi H Taback B Hsueh EC Elashoff D Morton DL Hoon DS Allelic imbalance of 12q22–23 associated with APAF-1 locus correlates with poor disease outcome in cutaneous melanoma.Cancer Res. 2004; 64: 2245-2250Crossref PubMed Scopus (80) Google Scholar (CEQ 8000XL; Beckman Coulter, Fullerton, CA). Forward primer sequences for BAT25, BAT26, BAT40, D2S123, and D5S346 were as follows: 5′-CCTCGCCTCCAAGAATGTAA-3′, 5′-GCAGTCAGAGCCCTTAACCTT-3′, 5′-AAGATTAACTTCCTACACCACAACC-3′, 5′-TGGCCAGAGAA ATTAGACACA-3′, and 5′-TTCAGGGAATTGAGAGTTACAGG-3′, respectively; corresponding reverse primer sequences were as follows: 5′-TGCTTTTGGTTACCACACTTCA-3′, 5′-CCATTTAAAGCTAGTTATCTAATCCA-3′, 5′-GTAGAGCAAGACCACCTTGT-3′, 5′-TCT GACTTGGATACCATCTATCTATCT-3′ and 5′-TCACTCTAGTGATAAATCGGGAAA-3′. Differences in PCR product fragment length among different tissue categories were visualized by the CEQ software (Beckman Coulter). PCR products from the five amplified microsatellite regions in adenoma and cancer were compared with the reference normal epithelium. KRAS (codons 12 and 13) and BRAF (V600E) mt were assessed by a peptide nucleic acid clamp-based quantitative real-time PCR assay as previously described.38Shinozaki M O'Day SJ Kitago M Amersi F Kuo C Kim J Wang HJ Hoon DS Utility of circulating B-RAF DNA mutation in serum for monitoring melanoma patients receiving biochemotherapy.Clin Cancer Res. 2007; 13: 2068-2074Crossref PubMed Scopus (128) Google Scholar, 39Kim J Reber HA Dry SM Elashoff D Chen SL Umetani N Kitago M Hines OJ Kazanjian KK Hiramatsu S Bilchik AJ Yong S Shoup M Hoon DS Unfavourable prognosis associated with K-ras gene mutation in pancreatic cancer surgical margins.Gut. 2006; 55: 1598-1605Crossref PubMed Scopus (77) Google Scholar These assays were performed in triplicate and carried out at least twice to confirm accuracy. A gene was considered mutated when the results were uniformly positive in the triplicates under the optimal conditions. Respective normal and positive PEATs as well as cell line controls were included in each assay. The diagnostic techniques used by the LUMC's pathology department for MSI status and MMR protein expression assessment are described previously.40de Jong AE van Puijenbroek M Hendriks Y Tops C Wijnen J Ausems MG Meijers-Heijboer H Wagner A van Os TA Brocker-Vriends AH Vasen HF Morreau H Microsatellite instability, immunohistochemistry, and additional PMS2 staining in suspected hereditary nonpolyposis colorectal cancer.Clin Cancer Res. 2004; 10: 972-980Crossref PubMed Scopus (188) Google Scholar Briefly, MSI status was assessed by using MSI Analysis System (Promega Corp., Madison, WI; five mononucleotide and two pentanucleotide repeats).41Murphy KM Zhang S Geiger T Hafez MJ Bacher J Berg KD Eshleman JR Comparison of the microsatellite instability analysis system and the Bethesda panel for the determination of microsatellite instability in colorectal cancers.J Mol Diagn. 2006; 8: 305-311Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar Immunostaining of MMR proteins was performed with anti-MLH1 (clone G168-728; 1:50; BD Biosciences, Franklin Lakes, NJ) and anti-PMS2 (clone A16-4; 1:50; BD Biosciences). Immunohistochemistry (IHC) staining was performed on PEAT sections (4 μm in thickness) from tissue microarray (TMA). IHC staining patterns of these MMR proteins were evaluated by using normal epithelial, stromal, or inflammatory cells, or the centers of lymphoid follicles as internal controls, as previously published.42Hendriks Y Franken P Dierssen JW De Leeuw W Wijnen J Dreef E Tops C Breuning M Brocker-Vriends A Vasen H Fodde R Morreau H Conventional and tissue microarray immunohistochemical expression analysis of mismatch repair in hereditary colorectal tumors.Am J Pathol. 2003; 162: 469-477Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar TMAs contained three cores punched from each primary tumor. Individual cores of the TMA were scored as either positive (showing nuclear staining in at least some tumor cells) or negative. Cases were considered positive if at least one TMA tissue core showed nuclear staining; if not, cases were considered negative. Cases in which both tumor and internal control stained negative were not included in the study. Cases were scored by two independent reviewers (H.M. and N.F.C.C. de M.); in case of a discrepancy, both reviewers reassessed the slides for consensus. KRAS43van Eijk R, van Puijenbroek M, Chhatta AR, Gupta N, Vossen RH, Lips EH, Cleton-Jansen AM, Morreau H, van Wezel T: Sensitive and specific KRAS somatic mutation analysis on whole-genome amplified DNA from archival tissues. J Mol Diagn 12:27–34Google Scholar and BRAF44Sanchez-de-Abajo A de la Hoya M van Puijenbroek M Tosar A Lopez-Asenjo JA Diaz-Rubio E Morreau H Caldes T Molecular analysis of colorectal cancer tumors from patients with mismatch repair proficient hereditary nonpolyposis colorectal cancer suggests novel carcinogenic pathways.Clin Cancer Res. 2007; 13: 5729-5735Crossref PubMed Scopus (44) Google Scholar mt were detected by means of sequencing as previously described. Significance of changes in methylation index (MI) at individual MINT loci and total MI was evaluated with nonparametric tests for related and independent sample sets (Wilcoxon's rank-sum test; Mann-Whitney's U-test). Fisher's exact test (two-tailed) was used to assess significance of intergroup differences in the prevalence of MLH1 methylation, BRAF, or KRAS mt. Spearman's rank correlation coefficient between quantitative MINT MI and MLH1 MI was assessed as a nonparametric measure of correlation. Correlations with clinical parameters were tested with Pearson's χ2 test and, for ordinal variables, with Mann-Whitney's U-test or Kruskal-Wallis' test. Postoperative distant recurrence probability and disease-free and overall survival were estimated with Kaplan-Meier plots, and significance was assessed with the log-rank test. Cox regression models considered the following variables that were entered in a stepwise manner: age, nodal stage, MSI status, and tumor differentiation. P < 0.05 (two-sided) was considered significant. SPSS (SPSS Inc., Chicago, IL) statistical software version 16.0.1 was used for all analyses. In the first phase of the study, we assessed operative specimens from 115 patients who underwent open resection of CRC and whose specimen, according to the pathology report, contained adenoma as well as cancer (see Table 1 for patient characteristics). Fifty of these specimens included normal tissue. Twenty-seven of the 115 patients were excluded from the study because the adenomas contained high-grade dysplasia and/or had carcinoma without evidence of submucosal invasion. Of the remaining 88 specimens that contained low- or medium-grade adenomatous dysplasia, 79 still had invasive carcinoma tissue after reviewing the cut sections. MSI status was analyzed in normal and cancer tissue of the 79 patients by using five established genomic markers.36Umar A Boland CR Terdiman JP Syngal S de la Chapelle A Ruschoff J Fishel R Lindor NM Burgart LJ Hamelin R Hamilton SR Hiatt RA Jass J Lindblom A Lynch HT Peltomaki P Ramsey SD Rodriguez-Bigas MA Vasen HF Hawk ET Barrett JC Freedman AN Srivastava S Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2461) Google Scholar Specimens from nine patients (11%) showed instability in ≥4 biomarkers; these patients comprised the MSI-H group. Specimens from 65 patients (83%) did not show a shift in any of the markers, and specimens from five (6%)