Intermediate Methylation Epigenotype and Its Correlation to KRAS Mutation in Conventional Colorectal Adenoma
2011; Elsevier BV; Volume: 180; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.10.010
ISSN1525-2191
AutoresKoichi Yagi, Hirokazu Takahashi, Kiwamu Akagi, Keisuke Matsusaka, Yasuyuki Seto, Hiroyuki Aburatani, Atsushi Nakajima, Atsushi Kaneda,
Tópico(s)Epigenetics and DNA Methylation
ResumoA subset of colorectal cancer shows significant accumulation of aberrant promoter methylation. Previously, we developed two groups of methylation markers that classified colorectal cancer into three epigenotypes: i) high-, ii) intermediate-, and iii) low-methylation epigenotypes. High-methylation epigenotype, with methylation of both group 1 and group 2 markers, correlates to BRAF-mutation(+). Intermediate-methylation epigenotype, with methylation of group 2 markers, but not group 1, correlates to KRAS-mutation(+). To gain insight into epigenotype development in colorectal carcinogenesis, especially intermediate-methylation epigenotype and its correlation to KRAS-mutation(+) in adenoma, we analyzed methylation levels of group 1 and group 2 markers quantitatively by matrix assisted laser desorption ionization-time of flight mass spectrometry, in 51 adenomas, 13 aberrant crypt foci, and 26 normal mucosa samples, and we compared them to 149 previously analyzed colorectal cancer samples. Three serrated adenomas were all BRAF-mutation(+), showing great methylation of group 1 and group 2 markers, thus high-methylation epigenotype. Forty-eight conventional adenomas were not methylated in group 1 markers and were classified into two clusters with higher and lower methylation of group 2 markers, thus into intermediate- and low-methylation epigenotypes, respectively. Adenoma with intermediate-methylation epigenotype correlated to KRAS-mutation(+). Methylation levels of group 2 markers in adenoma were higher than aberrant crypt foci and normal samples, but similar to cancer. These data suggested that epigenotype development occur at an earlier stage than carcinoma formation, and already be completed at the adenoma stage. Intermediate methylation epigenotype and its correlation to KRAS-mutation(+) were developed in conventional adenoma. A subset of colorectal cancer shows significant accumulation of aberrant promoter methylation. Previously, we developed two groups of methylation markers that classified colorectal cancer into three epigenotypes: i) high-, ii) intermediate-, and iii) low-methylation epigenotypes. High-methylation epigenotype, with methylation of both group 1 and group 2 markers, correlates to BRAF-mutation(+). Intermediate-methylation epigenotype, with methylation of group 2 markers, but not group 1, correlates to KRAS-mutation(+). To gain insight into epigenotype development in colorectal carcinogenesis, especially intermediate-methylation epigenotype and its correlation to KRAS-mutation(+) in adenoma, we analyzed methylation levels of group 1 and group 2 markers quantitatively by matrix assisted laser desorption ionization-time of flight mass spectrometry, in 51 adenomas, 13 aberrant crypt foci, and 26 normal mucosa samples, and we compared them to 149 previously analyzed colorectal cancer samples. Three serrated adenomas were all BRAF-mutation(+), showing great methylation of group 1 and group 2 markers, thus high-methylation epigenotype. Forty-eight conventional adenomas were not methylated in group 1 markers and were classified into two clusters with higher and lower methylation of group 2 markers, thus into intermediate- and low-methylation epigenotypes, respectively. Adenoma with intermediate-methylation epigenotype correlated to KRAS-mutation(+). Methylation levels of group 2 markers in adenoma were higher than aberrant crypt foci and normal samples, but similar to cancer. These data suggested that epigenotype development occur at an earlier stage than carcinoma formation, and already be completed at the adenoma stage. Intermediate methylation epigenotype and its correlation to KRAS-mutation(+) were developed in conventional adenoma. Colorectal cancer arises as a consequence of genetic alteration and epigenetic alteration.1Grady W.M. Carethers J.M. Genomic and epigenetic instability in colorectal cancer pathogenesis.Gastroenterology. 2008; 135: 1079-1099Abstract Full Text Full Text PDF PubMed Scopus (718) Google Scholar Gene mutations (eg, KRAS, p53, and APC) are well-known genetic alterations that occurred in colorectal cancer, which were demonstrated in the model of "adenoma-carcinoma sequence" by Bert Vogelstein.2Vogelstein B. Fearon E.R. Hamilton S.R. Kern S.E. Preisinger A.C. Leppert M. Nakamura Y. White R. Smits A.M. Bos J.L. Genetic alterations during colorectal-tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (6128) Google Scholar Epigenetic alteration, such as DNA methylation or loss of imprinting, is also important in colorectal carcinogenesis, and aberrant promoter methylation is a major epigenetic mechanism for gene silencing to be involved in the initiation and progression of cancer.3Jones P.A. Baylin S.B. The epigenomics of cancer.Cell. 2007; 128: 683-692Abstract Full Text Full Text PDF PubMed Scopus (3780) Google Scholar, 4Kaneda A. Feinberg A.P. Loss of imprinting of IGF2: a common epigenetic modifier of intestinal tumor risk.Cancer Res. 2005; 65: 11236-11240Crossref PubMed Scopus (115) Google Scholar As for accumulation of aberrant methylation, Toyota et al5Toyota M. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2178) Google Scholar reported in 1999 that a subset of colorectal cancer shows significantly frequent CpG island methylation [ie, the so-called CpG island methylator phenotype (CIMP)]. CIMP+ colorectal cancer significantly correlates to microsatellite instability and BRAF mutation.6Weisenberger D.J. Siegmund K.D. Campan M. Young J. Long T.I. Faasse M.A. Kang G.H. Widschwendter M. Weener D. Buchanan D. Koh H. Simms L. Barker M. Leggett B. Levine J. Kim M. French A.J. Thibodeau S.N. Jass J. Haile R. Laird P.W. 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 (1582) Google Scholar Colorectal adenoma is known as a precursor lesion of colorectal cancer. Serrated adenomas were reported to show CIMP+ and frequent BRAF mutation, and DNA methylation was thus considered to be an early event in the serrated pathway, which explains carcinogenesis from serrated adenoma to colorectal cancer with microsatellite instability.7Kambara T. Simms L.A. Whitehall V.L. Spring K.J. Wynter C.V. Walsh M.D. Barker M.A. Arnold S. McGivern A. Matsubara N. Tanaka N. Higuchi T. Young J. Jass J.R. Leggett B.A. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.Gut. 2004; 53: 1137-1144Crossref PubMed Scopus (633) Google Scholar, 8Park S.J. Rashid A. Lee J.H. Kim S.G. Hamilton S.R. Wu T.T. Frequent CpG island methylation in serrated adenomas of the colorectum.Am J Pathol. 2003; 162: 815-822Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar Adenomas without serrated features (nonserrated adenomas) were classified as conventional adenomas, which correspond to tubular, tubulovillous, and villous adenomas.9Snover D.C. Update on the serrated pathway to colorectal carcinoma.Hum Pathol. 2011; 42: 1-10Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar Existence of methylation phenotype in the conventional adenomas and its correlation to KRAS mutation were largely unknown. In our previous study, we epigenotyped colorectal cancer by the two-way hierarchical clustering method using highly quantitative DNA methylation data, and we identified three clusters of colorectal cancer with distinct methylation epigenotypes.10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar High-methylation epigenotype (HME) correlated to BRAF-mutation(+) and microsatellite instability,10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar as CIMP was previously reported.6Weisenberger D.J. Siegmund K.D. Campan M. Young J. Long T.I. Faasse M.A. Kang G.H. Widschwendter M. Weener D. Buchanan D. Koh H. Simms L. Barker M. Leggett B. Levine J. Kim M. French A.J. Thibodeau S.N. Jass J. Haile R. Laird P.W. 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 (1582) Google Scholar In microsatellite stable colorectal cancer, intermediate methylation epigenotype (IME) correlated to KRAS-mutation(+) and a lack of BRAF mutation, and low methylation epigenotype (LME) correlated to lack of BRAF/KRAS mutation. These three epigenotypes and their strong correlation to different oncogene mutations suggested distinct molecular genesis of colorectal cancer.10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar The two-way hierarchical clustering in our study also classified DNA methylation markers into two groups (ie, group 1 and group 2 markers).10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar, 11Kaneda A. Yagi K. Two groups of DNA methylation markers to classify colorectal cancer into three epigenotypes.Cancer Sci. 2011; 102: 18-24Crossref PubMed Scopus (68) Google Scholar Group 1 markers included most of the previously established CIMP markers5Toyota M. Ahuja N. Ohe-Toyota M. Herman J.G. Baylin S.B. Issa J.P. CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2178) Google Scholar, 6Weisenberger D.J. Siegmund K.D. Campan M. Young J. Long T.I. Faasse M.A. Kang G.H. Widschwendter M. Weener D. Buchanan D. Koh H. Simms L. Barker M. Leggett B. Levine J. Kim M. French A.J. Thibodeau S.N. Jass J. Haile R. Laird P.W. 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 (1582) Google Scholar, 12Nosho K. Irahara N. Shima K. Kure S. Kirkner G.J. Schernhammer E.S. Hazra A. Hunter D.J. Quackenbush J. Spiegelman D. Giovannucci E.L. Fuchs C.S. Ogino S. Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample.PLoS One. 2008; 3: e3698Crossref PubMed Scopus (264) Google Scholar and were characterized to be methylated specifically in HME/CIMP+ colorectal cancer. Group 2 markers are methylated in both HME and IME, but not in LME. Therefore, colorectal cancer methylated in group 1 markers (and also inevitably group 2 markers) is regarded as HME, and colorectal cancer without group 1 marker methylation (but methylated in group 2 markers) is regarded as IME.11Kaneda A. Yagi K. Two groups of DNA methylation markers to classify colorectal cancer into three epigenotypes.Cancer Sci. 2011; 102: 18-24Crossref PubMed Scopus (68) Google Scholar Whereas BRAF mutation and CIMP marker methylation were reported in serrated adenomas,7Kambara T. Simms L.A. Whitehall V.L. Spring K.J. Wynter C.V. Walsh M.D. Barker M.A. Arnold S. McGivern A. Matsubara N. Tanaka N. Higuchi T. Young J. Jass J.R. Leggett B.A. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum.Gut. 2004; 53: 1137-1144Crossref PubMed Scopus (633) Google Scholar, 8Park S.J. Rashid A. Lee J.H. Kim S.G. Hamilton S.R. Wu T.T. Frequent CpG island methylation in serrated adenomas of the colorectum.Am J Pathol. 2003; 162: 815-822Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar methylation accumulation in conventional adenomas, or association between the methylation and KRAS mutation, has not been clarified yet. We therefore analyzed methylation status in 48 conventional adenomas using 15 group 2 markers, as well as three group 1 markers, by bisulfite-PCR-based highly quantitative method, matrix assisted laser desorption ionization-time of flight mass spectrometry (MassARRAY). Herein we report that epigenotype development is earlier than cancer stage and already completed at adenoma stage, and that IME and its correlation to KRAS mutation are developed in conventional adenoma. Clinical colorectal adenoma, aberrant crypt foci, and normal mucosa samples were obtained from the patients who underwent endoscopic examination at Yokohama City University Hospital with written informed consents, and kept at −80°C until use. Colorectal cancer samples were obtained from the patients who underwent surgery at Saitama Cancer Center, with written informed consents, and their DNA was extracted in our previous study.10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar All of the aberrant crypt foci and adenoma samples were microscopically examined for determination of its lesion contents by two independent pathologists. Then, 51 adenoma (2 traditional serrated adenomas, 1 sessile serrated adenoma, and 48 conventional adenomas) and 13 aberrant crypt foci samples that contained at least 40% of lesion cells, were prepared (Figure 1A). We analyzed methylation and oncogene mutation statuses of 48 conventional adenoma samples, and compared them with 3 serrated adenomas, 13 aberrant crypt foci, 26 normal mucosa samples, and 149 previously analyzed colorectal cancers.10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar DNA of clinical samples was extracted using QIAamp DNA Micro Kit (QIAGEN, Hilden, Germany). This study was certified by the Ethics Committee in Tokyo University, Yokohama City University and Saitama Cancer Center. Bisulfite conversion of genomic DNA was performed as previously described.13Kaneda A. Wakazono K. Tsukamoto T. Watanabe N. Yagi Y. Tatematsu M. Kaminishi M. Sugimura T. Ushijima T. Lysyl oxidase is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in human gastric cancers.Cancer Res. 2004; 64: 6410-6415Crossref PubMed Scopus (149) Google Scholar After sonication of genomic DNA in 30 seconds by Bioruptor (Cosmobio Co. Ltd., Tokyo, Japan), 500 ng of DNA was denatured in 0.3 N NaOH, and then subjected to 15 cycles of 30 seconds at 95°C and 15-minute incubation in 3.6 M sodium bisulfite and 0.6 mmol/L hydroquinone at 50°C. The samples were desalted with the Wizard DNA Clean-Up system (Promega, Madison, WI), desulfonated in 0.3 N NaOH at room temperature for 5 minutes, and then purified by ethanol precipitation. Finally, genomic DNA was diluted by 40 μL of distilled water. Quantitative methylation analysis was performed using MALDI-TOF mass spectrometry (MassARRAY, Sequenom, San Diego, CA).10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar, 14Ehrich M. Nelson M.R. Stanssens P. Zabeau M. Liloglou T. Xinarianos G. Cantor C.R. Field J.K. van den Boom D. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry.Proc Natl Acad Sci USA. 2005; 102: 15785-15790Crossref PubMed Scopus (703) Google Scholar Bisulfite-treated DNA was amplified by PCR, the PCR product was transcribed by in vitro transcription, and the RNA was cleaved by RNaseA. Unmethylated cytidine was converted to Uridine by bisulfite treatment [ie, thymidine in the PCR product], and finally adenosine in the in vitro transcription product. Methylated cytidine was not converted (ie, cytidine in the PCR product), and finally guanosine in the in vitro transcription product. Since RNaseA cleaves RNA at the 3′ site of both thymidine and cytidine, thytidine-specific cleavage was possible by containing deoxycytidine instead of cytidine in the in vitro transcription mixture. Methylation status was determined by mass difference between adenine and guanine in a cleaved RNA product. Quantitative methylation was calculated for each cleaved product. This analytic unit containing several CpG sites in a cleaved product was called the "CpG unit." Primers were designed in the previous study10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar to include no CpG site or only one CpG site in 5′ regions of primers, which is listed in Table 1. Three group 1 markers were randomly chosen and used, because three were enough to confirm hypermethylation status of group 1 markers (≅CIMP markers) in serrated adenoma. As many as 15 group 2 markers showing a variety of average methylation levels10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar were chosen, because investigation on IME existence in conventional adenomas was the major purpose of this study, and such number of markers were necessary for hierarchical clustering and demonstration of methylation development in precursor lesions. All of the primers were validated for their accuracy for quantitative analysis by calculating correlation coefficient (r2) of the standard curve using methylation control samples (0, 25, 50, 75, and 100% methylation) at each CpG unit. CpG units with R2 ≤ 0.9 were excluded, and primer pairs whose amplicon contained three or more CpG units with R2 > 0.9 were used in this study.Table 1Methylation Marker Genes and Primer SequencesGene symbolsPrimer sequencesPositions (TSS = +1)GroupForwardReverseForward 5′Reverse 5′ADAMTS15′-GTTTTTTGGGGTTTTAATGT-3′5′-CTCCRACACCACTAACTCCTC-3′+440+6472COL4A25′-TAGYGTAGGATGAGGGAGGT-3′5′-CRCCTTATACAAACTAAAACTACAC-3′+302+5512DFNA55′-GGTTAGATTTTTTAGAAGTTTTAGA-3′5′-AATCCACACTCCACTATAAATAAC-3′+170+4352EFEMP15′-TAGGAGTTGGTTAGAAGTTGG-3′5′-ACRACTAATTCTCTTTTATCTTATCA-3′−167+532ELMO15′-AATGTGTTTTTGGTTAGTAGGAG-3′5′-AAATAACTCTACCTCTATCCTATACC-3′−89+612FBN25′-GGATATTGGAAAGTTGTAAAAG-3′5′-CCRCCCTCTCTCTTACTAAC-3′−176+132HAND15′-GGGAAAGTTTATAGTGGAGAGAG-3′5′-CAAATCATCACTCCTTAAAAATC-3′−1216−9502IGFBP35′-GTTTTTTGTTTGGATTTTATAGTT-3′5′-AAACAACACCAACAAAATCAAC-3′+38+1892IGFBP75′-GAGAAGGTTATTATTTAGGTTAGTAA-3′5′-ACTACCAACTCTTTCCCTCC-3′+455+6512LOX5′-TGGTATTGTTTGGTGGAGAT-3′5′-AAACTCAACAAACTAAACACCTA-3′+128+4531MINT315′-GGTGGTGTAGTTTTAGGAGAG-3′5′-AACACTTCCCCAACATCTAC-3′NANA1NEUROG15′-AGTTTGGGGTTGTTATTTTGT-3′5′-CTTAAAAAATCCTAAAACCAATC-3′−42+1602RUNX35′-GAGTAGTGGGGATGGGAGGT-3′5′-CCRTTAAAAATCATTCCTACAAAAC-3′+771+9021SFRP15′-GTTTTGTTTTTTAAGGGGTGTTGAG-3′5′-ACACTAACTCCRAAAACTACAAAAC-3′−186+222STOX25′-GGTTTTAGGTTGGGGTAGTT-3′5′-GGTTTTAGGTTGGGGTAGTT-3′+291+5682THBD5′-TAGTTTTTTTTATTAGGATTTTTTT-3′5′-CCCAAACATATTACCCAAAC-3′−99+1692TSPYL55′-GGAAGAGATGAAATGGTAGTAT-3′5′-TCAAAAACACRCTATAACCCTA-3′−183+852UCHL15′-YGGTAGAAATAGTTTAGGGAAG-3′5′-TACTCCATACACTCAAAAAACAC-3′−234+512Positions of 5′ end of the primers were shown, regarding transcription start site as + 1 bp. NA, not applicable, because MINT31 is not a gene. Open table in a new tab Positions of 5′ end of the primers were shown, regarding transcription start site as + 1 bp. NA, not applicable, because MINT31 is not a gene. Mutation at BRAF 1799 and KRAS 34, 35, and 38 were analyzed by genotyping assay on MassARRAY platform.15Spring K.J. Zhao Z.Z. Karamatic R. Walsh M.D. Whitehall V.L. Pike T. Simms L.A. Young J. James M. Montgomery G.W. Appleyard M. Hewett D. Togashi K. Jass J.R. Leggett B.A. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy.Gastroenterology. 2006; 131: 1400-1407Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar First, PCR amplification primers and a post-PCR extension primer were designed using MassARRAY Assay Design 3.0 software (Sequenom), as listed in Table 2. BRAF 1799 and KRAS 38 mutation were analyzed in a single reaction by multiplex PCR. The PCR amplification was performed in 5 μL volumes containing 0.5 units of Taq polymerase, 5 ng of genomic DNA, 0.5 pmol of PCR primer, and 2.5 nmol of deoxyribonucleotide triphosphates. PCR reactions were cycled at 94°C for 15 minutes, followed by 45 cycles of 94°C for 20 seconds, 56°C for 30 seconds, and 72°C for 1 minute. Shrimp alkaline phosphatase treatment was performed at 37°C for 20 minutes and 85°C for 5 minutes. Post-PCR primer extension was performed using 5.6 pmol of extension primer. Extension reaction were cycled at 94°C for 30 seconds, followed by 40 cycles of 94°C for 5 seconds, 5 cycles of 52°C for 5 seconds, and 80°C for 5 seconds, and 72°C for 3 minutes. Reaction products were transferred to a SpectroCHIP (Sequenom), and mass difference was analyzed using MALDI-TOF mass spectrometry, to see the extended base at the possible mutation site.Table 2Primer Sequences Used for Mutation AnalysisMutation sitesPrimer sequencesExtend primersKRAS_34 Forward5′-ACGTTGGATGAGGCCTGCTGAAAATGACTG-3′5′-ACTCTTGCCTACGCCAC-3′ Reverse5′-ACGTTGGATGTAGCTGTATCGTCAAGGCAC-3′KRAS_38 Forward5′-ACGTTGGATGAGGCCTGCTGAAAATGACTG-3′5′-AGGCACTCTTGCCTACG-3′ Reverse5′-ACGTTGGATGTAGCTGTATCGTCAAGGCAC-3′KRAS_35 Forward5′-ACGTTGGATGAGGCCTGCTGAAAATGACTG-3′5′-CACTCTTGCCTACGCCA-3′ Reverse5′-ACGTTGGATGTAGCTGTATCGTCAAGGCAC-3′BRAF_1799 Forward5′-ACGTTGGATGTCTTCATGAAGACCTCACAG-3′5′-CCCACTCCATCGAGATTTC-3′ Reverse5′-ACGTTGGATGTTCAAACTGATGGGACCCAC-3′ Open table in a new tab Correlation between epigenotypes and clinicopathological factors, except age and tumor size were analyzed by Fisher's exact test. Age and tumor size were analyzed by Student's t-test. Unsupervised two-way hierarchical clustering was performed based on Euclid distance correlation and average linkage clustering algorithm in sample and marker directions using GeneSpring 7.3.1 software (Agilent Technology, Santa Clara, CA). We analyzed mutation statuses of BRAF and KRAS in 51 colorectal adenomas (3 serrated and 48 conventional adenomas) and 13 aberrant crypt foci, using MALDI-TOF mass spectrometry (Figure 1). All of the three serrated adenoma samples (including two traditional and one sessile serrated adenomas) showed BRAF mutation (100%) and no KRAS mutation (0%). Among 48 conventional adenoma samples, 11 showed KRAS mutation (23%) and none showed BRAF mutation (0%). In 13 aberrant crypt foci, 5 showed KRAS mutation (38%) and none showed BRAF mutation (0%). Among group 1 and group 2 markers we established in the previous study,10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar we analyzed methylation rates of three group 1 markers and 15 group 2 markers in adenoma, aberrant crypt foci, and normal mucosa samples. Methylation frequency of 18 markers in adenoma samples was summarized in Figure 2A. Three top-ranking samples with the most frequent methylation were serrated adenomas; each sample showed methylation in two of the three group 1 markers as well as very frequent methylation of group 2 markers, ranging 93 to 100%. The three serrated adenoma samples were thus considered as HME, and all of the three showed BRAF mutation whereas none of conventional adenoma showed BRAF mutation (P = 4.8 × 10−5) (Figure 1). In conventional adenoma, group 1 markers were hardly methylated, suggesting that there was no HME case. Group 2 marker methylation varied from 0% to 93% (Figure 2A). There was significant correlation between frequent methylation of group 2 markers and KRAS mutation (P = 2.7 × 10−3, Wilcoxon rank sum test). When number of methylation markers and oncogene mutation status were compared, the three BRAF-mutation(+) serrated adenoma samples showed markedly frequent methylation (Figure 2B). KRAS-mutation(+) adenoma showed significantly frequent methylation of group 2 markers (9.7 ± 3.6 markers) than BRAF/KRAS-mutation(−) adenoma (6.0 ± 3.1 markers, P = 7.8 × 10−3, Student's t-test). To analyze whether conventional adenoma can be classified into some clusters using DNA methylation information, two-way unsupervised hierarchical clustering method was applied in the analysis using quantitative methylation data (Figure 3). Conventional adenoma was classified into two major clusters; a cluster with higher methylation rate of group 2 markers was considered as IME, and the other with lower methylation rate was LME. IME adenoma showed significant correlation to KRAS-mutation(+) (P = 4.7 × 10−4, Fisher's exact test) (Figure 3). As for other clinicopathological factors, adenoma size in IME was significantly larger than LME (P = 0.043, Student's t-test). Tumor location did not show significant difference (P = 0.31, Fisher's exact test) (Table 3).Table 3Clinical and Molecular Characteristics of Adenoma according to Three EpigenotypesClinical or molecular featuresHMEIMELMEP value (IME versus LME)Number of samples3 (6%)13 (26%)34 (68%)Sex Male2 (66%)12 (92%)23 (68%)0.14 Female1 (33%)1 (8%)11 (32%)Age Mean ± SD72 ± 11.166.7 ± 5.666.9 ± 10.50.93Tumor size Average (mm) ± SD13.3 ± 2.913.6 ± 5.99.7 ± 4.20.043⁎P value between IME and LME < 0.05 (calculated by Fisher's exact test, except age and tumor size by Student's t-test). >10 mm2 (66%)7 (54%)10 (29%)0.18 ≤10 mm1 (33%)6 (46%)24 (71%)Tumor location Proximal1 (33%)3 (23%)14 (41%)0.32 Distal2 (67%)10 (77%)20 (59%)BRAF mutation (+)3 (100%)0 (0%)0 (0%)1 (−)0 (0%)13 (100%)34 (100%)KRAS mutation (+)0 (0%)8 (62%)3 (9%)4.7 × 10−4⁎P value between IME and LME < 0.05 (calculated by Fisher's exact test, except age and tumor size by Student's t-test). (−)3 (100%)5 (38%)31 (91%)HME, high methylation epigenotype; IME, intermediate methylation epigenotype; LME, low methylation epigenotype; SD, standard deviation.Proximal location is the cecum to the transverse colon. Distal location is the descending colon to the rectum. P value between IME and LME < 0.05 (calculated by Fisher's exact test, except age and tumor size by Student's t-test). Open table in a new tab HME, high methylation epigenotype; IME, intermediate methylation epigenotype; LME, low methylation epigenotype; SD, standard deviation. Proximal location is the cecum to the transverse colon. Distal location is the descending colon to the rectum. The methylation rates of markers were compared according to epigenotypes (Figure 4). In group 1 markers, the three serrated adenoma samples showed higher methylation rate than IME and LME adenomas, confirming that the serrated adenoma is HME (Figure 4A). In most group 2 markers, the methylation rate was generally highest in HME, lowest in LME, and at an intermediate level in IME (Figure 4B). We had classified group 2 markers into H>I>L and H=I>L types in a previous study of colorectal cancer,10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar, 11Kaneda A. Yagi K. Two groups of DNA methylation markers to classify colorectal cancer into three epigenotypes.Cancer Sci. 2011; 102: 18-24Crossref PubMed Scopus (68) Google Scholar whereas similar methylation patterns were confirmed in adenoma (eg, ELMO1, STOX2, NEUROG1, HAND1, and IGFBP7 as H>I>L type, and THBD, FBN2, ADAMTS, and COL4A2 as H=I>L type. In the previous analysis of colorectal cancer, we developed a two-step panel method to classify HME, IME, and LME easily without hierarchical clustering.10Yagi K. Akagi K. Hayashi H. Nagae G. Tsuji S. Isagawa T. Midorikawa Y. Nishimura Y. Sakamoto H. Seto Y. Aburatani H. Kaneda A. Three DNA methylation epigenotypes in human colorectal cancer.Clin Cancer Res. 2010; 16: 21-33Crossref PubMed Scopus (200) Google Scholar Because group 1 markers are methylated specifically in HME, and group 2 markers are methylated commonly in HME and IME, HME cases should be extracted as frequently methylated samples using the first panel containing three to five group 1 markers. The remaining cases could be divided into IME and LME by the second panel containing three to five group 2 markers.11Kaneda A. Yagi K. Two groups of DNA methylation markers to classify colorectal cancer into three epigenotypes.Cancer Sci. 2011; 102: 18-24Crossref PubMed Scopus (68) Google Scholar Here we developed a two-step marker panel in the similar manner, with 94% accuracy (Figure 4C). The first panel containing three group 1 markers (LOX, MINT31
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