CpG Island Methylation in Aberrant Crypt Foci of the Colorectum
2002; Elsevier BV; Volume: 160; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)61128-5
ISSN1525-2191
AutoresAnnie On-On Chan, Russell R. Broaddus, Patrick Houlihan, Jean‐Pierre J. Issa, Stanley R. Hamilton, Asif Rashid,
Tópico(s)Colorectal Cancer Screening and Detection
ResumoAberrant crypt foci (ACF) are postulated to be the earliest precursor lesion in colorectal carcinogenesis, and CpG island methylation has been described as an important molecular pathway. We therefore studied methylation in ACF from patients with familial adenomatous polyposis (FAP) or sporadic colorectal cancer. We assessed methylation status of the p16 tumor suppressor gene, MINT1 (methylated in tumor 1), MINT2, MINT31, O6-methylguanine-DNA methyltransferase gene, and hMLH1 mismatch repair gene. We compared methylation to ACF histopathology, K-ras proto-oncogene mutation, loss of heterozygosity at chromosome 1p, and microsatellite instability. Methylation was present in 34% (21 of 61) of ACF, including both FAP and sporadic types, but was more frequent in sporadic ACF [53% (18 of 34) versus 11% (3 of 27), P = 0.002], especially dysplastic sporadic ACF [75% (3 of 4) versus 8% (2 of 24), P = 0.004]. MINT31 was more frequently methylated in heteroplastic ACF than dysplastic ACF [35% (11 of 31) versus 7% (2 of 30), P = 0.01]. Strong associations of ACF methylation with K-ras mutation (P = 0.007) and with loss of chromosome 1p (P = 0.04) were observed, but methylation was the only molecular abnormality identified in 16% (10 of 61) of ACF. Our findings suggest that methylation in ACF is an early event in the pathogenesis of a subset of colorectal carcinomas, and that ACF from FAP patients and patients with sporadic colorectal cancer have distinct epigenetic changes that reflect differences in molecular pathogenesis. Aberrant crypt foci (ACF) are postulated to be the earliest precursor lesion in colorectal carcinogenesis, and CpG island methylation has been described as an important molecular pathway. We therefore studied methylation in ACF from patients with familial adenomatous polyposis (FAP) or sporadic colorectal cancer. We assessed methylation status of the p16 tumor suppressor gene, MINT1 (methylated in tumor 1), MINT2, MINT31, O6-methylguanine-DNA methyltransferase gene, and hMLH1 mismatch repair gene. We compared methylation to ACF histopathology, K-ras proto-oncogene mutation, loss of heterozygosity at chromosome 1p, and microsatellite instability. Methylation was present in 34% (21 of 61) of ACF, including both FAP and sporadic types, but was more frequent in sporadic ACF [53% (18 of 34) versus 11% (3 of 27), P = 0.002], especially dysplastic sporadic ACF [75% (3 of 4) versus 8% (2 of 24), P = 0.004]. MINT31 was more frequently methylated in heteroplastic ACF than dysplastic ACF [35% (11 of 31) versus 7% (2 of 30), P = 0.01]. Strong associations of ACF methylation with K-ras mutation (P = 0.007) and with loss of chromosome 1p (P = 0.04) were observed, but methylation was the only molecular abnormality identified in 16% (10 of 61) of ACF. Our findings suggest that methylation in ACF is an early event in the pathogenesis of a subset of colorectal carcinomas, and that ACF from FAP patients and patients with sporadic colorectal cancer have distinct epigenetic changes that reflect differences in molecular pathogenesis. Aberrant crypt foci (ACF) in colorectal mucosa are the earliest known morphological precursors to colorectal cancer (CRC).1Nucci MR Robinson CR Longo P Campbell P Hamilton SR Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.Hum Pathol. 1997; 28: 1396-1407Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 2Pretlow TP Brasitus TA Fulton NC Cheyer C Kaplan EL K-ras mutations in putative preneoplastic lesions in human colon.J Natl Cancer Inst. 1993; 85: 2004-2007Crossref PubMed Scopus (223) Google Scholar, 3Heinen CD Shivapurkar N Tang Z Groden J Alabaster O Microsatellite instability in aberrant crypt foci from human colons.Cancer Res. 1996; 56: 5339-5341PubMed Google Scholar, 4Yamashita N Minamoto T Ochiai A Onda M Esumi H Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon.Gastroenterology. 1995; 108: 434-440Abstract Full Text PDF PubMed Scopus (136) Google Scholar, 5Otori K Konishi M Sugiyama K Hasebe T Shimoda T Kikuchi-Yanoshita R Mukai K Fukushima S Miyaki M Esumi H Infrequent somatic mutation of the adenomatous polyposis coli gene in aberrant crypt foci of human colon tissue.Cancer. 1998; 83: 896-900Crossref PubMed Scopus (59) Google Scholar, 6Takayama T Ohi M Hayashi T Miyanishi K Nobuoka A Nakajima T Satoh T Takimoto R Kato J Sakamaki S Niitsu Y Analysis of K-ras, APC, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar A role for ACF in colorectal carcinogenesis is supported by the presence of histopathological intraepithelial neoplasia (dysplasia) in some ACF, and the expression of markers of proliferation and of tumor-associated antigens and lectin-binding moieties.1Nucci MR Robinson CR Longo P Campbell P Hamilton SR Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.Hum Pathol. 1997; 28: 1396-1407Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 2Pretlow TP Brasitus TA Fulton NC Cheyer C Kaplan EL K-ras mutations in putative preneoplastic lesions in human colon.J Natl Cancer Inst. 1993; 85: 2004-2007Crossref PubMed Scopus (223) Google Scholar This role is further corroborated by the presence in some ACF of genetic alterations that are present in colorectal carcinomas, such as alterations in the adenomatous polyposis coli (APC) tumor suppressor gene, K-ras proto-oncogene mutations, and microsatellite instability (MSI).1Nucci MR Robinson CR Longo P Campbell P Hamilton SR Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.Hum Pathol. 1997; 28: 1396-1407Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 2Pretlow TP Brasitus TA Fulton NC Cheyer C Kaplan EL K-ras mutations in putative preneoplastic lesions in human colon.J Natl Cancer Inst. 1993; 85: 2004-2007Crossref PubMed Scopus (223) Google Scholar, 3Heinen CD Shivapurkar N Tang Z Groden J Alabaster O Microsatellite instability in aberrant crypt foci from human colons.Cancer Res. 1996; 56: 5339-5341PubMed Google Scholar, 4Yamashita N Minamoto T Ochiai A Onda M Esumi H Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon.Gastroenterology. 1995; 108: 434-440Abstract Full Text PDF PubMed Scopus (136) Google Scholar, 5Otori K Konishi M Sugiyama K Hasebe T Shimoda T Kikuchi-Yanoshita R Mukai K Fukushima S Miyaki M Esumi H Infrequent somatic mutation of the adenomatous polyposis coli gene in aberrant crypt foci of human colon tissue.Cancer. 1998; 83: 896-900Crossref PubMed Scopus (59) Google Scholar, 6Takayama T Ohi M Hayashi T Miyanishi K Nobuoka A Nakajima T Satoh T Takimoto R Kato J Sakamaki S Niitsu Y Analysis of K-ras, APC, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar The histopathology of human ACF is variable but can be subclassified into two broad categories: dysplastic and heteroplastic.1Nucci MR Robinson CR Longo P Campbell P Hamilton SR Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.Hum Pathol. 1997; 28: 1396-1407Abstract Full Text PDF PubMed Scopus (122) Google Scholar Dysplastic ACF resemble adenomas and are more common in familial adenomatous polyposis (FAP), which is because of germline mutation of the APC gene, than in patients with sporadic colorectal neoplasia. In addition to dysplasia, these ACF are characterized by abnormal epithelial proliferation in the upper aspects of the crypts, lack of K-ras mutations, and presence of APC mutations in dysplastic ACF from FAP patients but not patients with sporadic CRCs.5Otori K Konishi M Sugiyama K Hasebe T Shimoda T Kikuchi-Yanoshita R Mukai K Fukushima S Miyaki M Esumi H Infrequent somatic mutation of the adenomatous polyposis coli gene in aberrant crypt foci of human colon tissue.Cancer. 1998; 83: 896-900Crossref PubMed Scopus (59) Google Scholar, 6Takayama T Ohi M Hayashi T Miyanishi K Nobuoka A Nakajima T Satoh T Takimoto R Kato J Sakamaki S Niitsu Y Analysis of K-ras, APC, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar In contrast, heteroplastic ACF resemble hyperplastic polyps histopathologically, lack dysplasia, have proliferation mainly in the lower aspects of the crypts, have frequent K-ras mutations, and lack APC mutations.1Nucci MR Robinson CR Longo P Campbell P Hamilton SR Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.Hum Pathol. 1997; 28: 1396-1407Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 5Otori K Konishi M Sugiyama K Hasebe T Shimoda T Kikuchi-Yanoshita R Mukai K Fukushima S Miyaki M Esumi H Infrequent somatic mutation of the adenomatous polyposis coli gene in aberrant crypt foci of human colon tissue.Cancer. 1998; 83: 896-900Crossref PubMed Scopus (59) Google Scholar, 6Takayama T Ohi M Hayashi T Miyanishi K Nobuoka A Nakajima T Satoh T Takimoto R Kato J Sakamaki S Niitsu Y Analysis of K-ras, APC, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar Thus, ACF are phenotypically and genotypically diverse, but reflect their molecular pathogenesis. Recent studies have shown that methylation of CpG islands is a molecular defect common in CRC.7Toyota M Ahuja N Ohe-Toyota M Herman JG Baylin SB Issa J-PJ CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2178) Google Scholar CpG islands are 0.5- to 2-kb regions rich in cytosine-guanine dinucleotides and are present in the 5′ region of approximately half of all human genes.8Bird AP CpG-rich islands and the function of DNA methylation.Nature. 1986; 321: 209-213Crossref PubMed Scopus (3204) Google Scholar CpG island methylation (CIM) is a mechanism for suppression of transcription of genes in physiological and pathological settings including neoplasia.9Baylin SB Herman JG Graff JR Vertino PM Issa J-PJ Alterations in DNA methylation: a fundamental aspect of neoplasia.Adv Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar The recently discovered CpG island methylator phenotype (CIMP) is a novel pathway characterized by methylation of multiple CpG islands in colorectal carcinomas and adenomas, including genes known to be important in tumorigenesis such as the p16 tumor suppressor gene and hMLH1 mismatch repair gene.10Toyota M Ohe-Toyota M Ahuja N Issa J-PJ 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 (410) Google Scholar, 11Rashid A Shen L Morris JS Issa JPJ Hamilton SR CpG island methylation in colorectal adenomas.Am J Pathol. 2001; 159: 1129-1135Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar In addition, CIMP is associated with K-ras mutations through methylation of MGMT (O6-methylguanine-DNA methyltransferase).12Esteller M Toyota M Sanchez-Cespedes M Capella G Peinado MA Watkins DN Issa J-PJ Sidransky D Baylin SB Herman JG Inactivation of the DNA repair gene O6methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.Cancer Res. 2000; 60: 2368-2371PubMed Google Scholar Dysplastic ACF are recognized precursors to CRC, but the relationship of heteroplastic ACF is less certain.1Nucci MR Robinson CR Longo P Campbell P Hamilton SR Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.Hum Pathol. 1997; 28: 1396-1407Abstract Full Text PDF PubMed Scopus (122) Google Scholar, 2Pretlow TP Brasitus TA Fulton NC Cheyer C Kaplan EL K-ras mutations in putative preneoplastic lesions in human colon.J Natl Cancer Inst. 1993; 85: 2004-2007Crossref PubMed Scopus (223) Google Scholar, 3Heinen CD Shivapurkar N Tang Z Groden J Alabaster O Microsatellite instability in aberrant crypt foci from human colons.Cancer Res. 1996; 56: 5339-5341PubMed Google Scholar, 4Yamashita N Minamoto T Ochiai A Onda M Esumi H Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon.Gastroenterology. 1995; 108: 434-440Abstract Full Text PDF PubMed Scopus (136) Google Scholar, 5Otori K Konishi M Sugiyama K Hasebe T Shimoda T Kikuchi-Yanoshita R Mukai K Fukushima S Miyaki M Esumi H Infrequent somatic mutation of the adenomatous polyposis coli gene in aberrant crypt foci of human colon tissue.Cancer. 1998; 83: 896-900Crossref PubMed Scopus (59) Google Scholar, 6Takayama T Ohi M Hayashi T Miyanishi K Nobuoka A Nakajima T Satoh T Takimoto R Kato J Sakamaki S Niitsu Y Analysis of K-ras, APC, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis.Gastroenterology. 2001; 121: 599-611Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar However, recent studies have proposed a hyperplastic polyp/serrated adenoma-carcinoma sequence as an alternative pathway to the usual adenoma-carcinoma sequence.13Rashid A Houlihan PS Booker S Peterson GM Giardiello FM Hamilton SR Phenotypic and molecular characteristics of hyperplastic polyposis.Gastroenterology. 2000; 119: 323-332Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 14Jass JR Cottier DS Pokos V Parry S Winship IM Mixed epithelial polyps in association with hereditary non-polyposis colorectal cancer providing an alternative pathway of cancer histogenesis.Pathology. 1997; 29: 28-33Crossref PubMed Scopus (73) Google Scholar, 15Iino H Jass JR Simms LA Young J Leggett B Ajioka Y Watanabe H DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer?.J Clin Pathol. 1999; 52: 5-9Crossref PubMed Scopus (313) Google Scholar, 16Jass JR Iino H Ruszkiewicz A Painter D Solomon MJ Koorey DJ Cohn D Furlong KL Walsh MD Palazzo J Edmonston TB Fishel R Young J Leggett BA Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum.Gut. 2000; 47: 43-49Crossref PubMed Scopus (228) Google Scholar, 17Jass JR Serrated route to colorectal cancer: back street or super highway?.J Pathol. 2001; 193: 283-285Crossref PubMed Scopus (185) Google Scholar In addition, CIMP is present in hyperplastic polyps from patients with hyperplastic polyposis and colorectal neoplasia.18Chan AO Issa J-PJ Morris JS Hamilton SR Rashid A Concordant CpG island methylation in hyperplastic polyposis.Am J Pathol. 2002; 160: 529-536Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Therefore in the present study, we characterized ACF for the presence of CIM and compared the findings with histopathological and molecular alterations of importance in colorectal tumorigenesis, including K-ras mutation, loss of heterozygosity (LOH) of chromosome 1p, and MSI. ACF were isolated from the grossly normal mucosa in 10 colectomy specimens from patients with sporadic CRCs and from the nonpolyploid mucosa in 2 colectomy specimens from FAP patients with numerous polyps but no cancer. The specimens were collected in surgical pathology at the M. D. Anderson Cancer Center. The colons or rectums were opened longitudinally and rinsed of their luminal contents. The mucosa was dissected from the underlying muscularis propria, and the entire mucosal sheet was pinned flat on wax and fixed in formalin for 2 to 4 hours. The fixed colonic sheet was stained with 0.2% methylene blue for several minutes, and the ACF were visualized using an Olympus dissecting microscope (Olympus Optical Company, Ltd., Tokyo, Japan). ACF were marked with India ink, excised from the mucosal sheet in small strips, and submitted for routine histological processing. The mucosal strips were embedded longitudinally in paraffin blocks, sectioned onto glass slides, and stained with hematoxylin and eosin (H&E). The H&E-stained ACF were characterized by light microscopy (Figure 1) of coded specimens by two gastrointestinal pathologists (RB and AR) unaware of the source of the specimens (Figure 1). The ACF were classified as dysplastic, heteroplastic, or mixed (features of both dysplastic and heteroplastic ACF).1Nucci MR Robinson CR Longo P Campbell P Hamilton SR Phenotypic and genotypic characteristics of aberrant crypt foci in human colorectal mucosa.Hum Pathol. 1997; 28: 1396-1407Abstract Full Text PDF PubMed Scopus (122) Google Scholar The size of ACF was measured from the H&E-stained slides. Lesional and nonlesional tissues were microdissected from the H&E-stained sections of ACF and mucosa without coverslip. Genomic DNA was extracted and prepared from the microdissected ACF as described previously.19Moskaluk CA Kern SE Microdissection and polymerase chain reaction amplification of genomic DNA from histological tissue sections.Am J Pathol. 1997; 150: 1547-1552PubMed Google Scholar The methylation status of the p16 tumor suppressor gene, MINT1 (methylated in tumor 1), MINT2, MINT31, MGMT gene, and hMLH1 mismatch repair gene was determined by bisulfite treatment of DNA followed by methylation-specific PCR as described.12Esteller M Toyota M Sanchez-Cespedes M Capella G Peinado MA Watkins DN Issa J-PJ Sidransky D Baylin SB Herman JG Inactivation of the DNA repair gene O6methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.Cancer Res. 2000; 60: 2368-2371PubMed Google Scholar, 20Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5272) Google Scholar The six loci selected for methylation analysis are unmethylated (<1% methylation) in normal tissues.7Toyota M Ahuja N Ohe-Toyota M Herman JG Baylin SB Issa J-PJ CpG island methylator phenotype in colorectal cancer.Proc Natl Acad Sci USA. 1999; 96: 8681-8686Crossref PubMed Scopus (2178) Google Scholar MINT1 and MINT2 correspond to CpG islands that are in the 5′ region of cDNAs with open reading frames that have no known protein homology (JP Issa, unpublished data). MINT31 is 2-kb upstream of the CACNA1G, a T-type calcium channel gene.21Toyota M Ho C Ohe-Toyota M Baylin SB Issa J-PI Inactivation of CAGNA1G, a T-type calcium channel gene, by aberrant methylation of its 5′ CpG island in human tumors.Cancer Res. 1999; 59: 4535-4541PubMed Google Scholar In brief, 2 μg of microdissected genomic DNA were denatured with 2 mol/L of NaOH at 37°C for 10 minutes, followed by incubation with 3 mol/L of sodium bisulfite (pH 5.0) at 50°C for 16 hours in darkness. After treatment, DNA was purified using the DNA Cleanup Kit (Promega, Madison, WI) as recommended by the manufacturer, incubated with 3 mol/L of NaOH at room temperature for 5 minutes, precipitated with 10 mol/L ammonium acetate and 100% ethanol, washed with 70% ethanol, and finally resuspended in 20 μl of distilled water. The primers and polymerase chain reaction (PCR) conditions for p16 were the same as reported by Herman and colleagues.20Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5272) Google Scholar The primers and PCR conditions for the MINT loci, MGMT, and hMLH1 are listed in Table 1. In brief, 2 μl of bisulfite-treated DNA was used as template for PCR reactions using primers specific for methylated and unmethylated alleles. DNA from the RKO colon cancer cell line (American Type Culture Collection, Manassas, VA) and water were used as positive and negative controls, respectively. PCR products from methylated and unmethylated reactions were electrophoresed on 6% acrylamide gels and visualized by ethidium bromide staining.Table 1Primers and PCR Conditions for MINT 1, MINT 2, MINT 31, MGMT, and hMLH1LocusAlleleSense primersAntisense primersCycling conditionsMINT1Methylated5′-AATTTTTTTATATATATTTTCGAAGC-3′5′-AAAAACCTCAACCCCGCG-3′95°C for 10 minutes, 37 cycles ofUnmethylated5′-AATTTTTTTATATATATTTTTGAAGTGT-3′5′-AACAAAAAACCTCAACCCCACA-3′95°C for 30 seconds, 55°C for 45 secondsMINT2Methylated5′-TTGTTAAAGTGTTGAGTTCGTC-3′5′-AATAACGACGATTCCGTACG-3′95°C for 10 minutes, 40 cycles ofUnmethylated5′-GATTTTGTTAAAGTGTTGAGTTTGTT-3′5′-CAAAATAATAACAACAATTCCATACA-3′95°C for 30 seconds, 60°C for 45 secondsMINT31Methylated5′-TGTTGGGGAAGTGTTTTTCGGC-3′5′-CGAAAACGAAACGCCGCG-3′95°C for 10 minutes, 38 cycles ofUnmethylated5′-TAGATGTTGGGGAAGTGTTTTTTGGT-3′5′-TAAATACCCAAAAACAAAACACCACA-3′95°C for 30 seconds, 60°C for 45 secondsMGMTMethylated5′-GGTCGTTTGTACGTTCGC-3′5′-GACCGATACAAACCGAACG-3′95°C for 10 minutes, 38 cycles ofUnmethylated5′-GTAGGTTGTTTGTATGTTTGT-3′5′-AACCAATACAAACCAAACA-3′95°C for 30 seconds, 55°C for 45 secondshMLH1Methylated5′-GATAGCGATTTTTAACGC-3′5′-TCTATAAATTACTAAATCTCTTCG-3′95°C for 10 minutes, 40 cycles of 95°C for 30 seconds, 53°C for 45 secondsUnmethylated5′-AGAGTGGATAGTGATTTTTAATGT-3′5′-ACTCTATAAATTACTAAATCTCTTCA-3′ Open table in a new tab The first exon of K-ras was amplified in 50-μl volumes using 2 μl of genomic DNA, 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L potassium chloride, 2 mmol/L magnesium chloride, 0.8 mmol/L dNTP mix, 2.25 U Ampli Taq Gold (Applied Biosystems, Foster City, CA), 0.125 U pfu DNA Polymerase (Stratagene, La Jolla, CA), and 20 pmol of forward and reverse primers (5′-GGCCGGTAGTGTATTAACCTTATG TGTGACAT-3′ and 5′-CCGCGGCCGGCGGCCAAAACAAGATTTACCTCTATTGTTGG-3′; Life Technologies, Rockville, MD). PCR reactions were performed using the following cycling conditions: denaturation at 95°C for 10 minutes; 14 cycles of 95°C for 20 seconds, 59°C to 52°C in 0.5°C decrements/cycle for 60 seconds, and 72°C for 60 seconds; 25 cycles of 95°C for 20 seconds, 52°C for 60 seconds, and 72°C for 60 seconds; and extension at 72°C for 10 minutes on a GeneAmp PCR System 9700 (Applied Biosystems). PCR products were purified using 10 U of exonuclease I and 2 U of shrimp alkaline phosphatase (Amersham Life Science, Indianapolis, IN), incubated at 37°C for 15 minutes, and inactivated by incubating at 80°C for 15 minutes. DNA sequencing was performed in 20-μl volumes comprised of 2 μl of purified PCR product, 8 μl ABI Prism BigDye Terminator Cycle Sequencing Kit (Applied Biosystems), and 5 pmol of forward primer using the following cycling conditions: 25 cycles of 95°C for 20 seconds, 52°C for 60 seconds, and 72°C for 60 seconds. After spin-column purification (Princeton Separations, Adelphia, NJ), the reaction products were sequenced by capillary electrophoresis using an ABI Prism 3700 DNA Analyzer (Applied Biosystems). Mutations were confirmed by sequencing using the reverse primer. LOH and MSI were determined by fluorescently labeled PCR amplification using fluorescent dye-labeled and unlabeled primers (Applied Biosystems). The 5′ oligonucleotide was end-labeled with 6-FAM (BAT-25, D1S199, and D1S507), HEX (BAT-26, D1S468, and D1S255), or NED (D1S214) fluorescent dye. PCR was performed in 15-μl reaction volumes containing 40 ng of DNA, 9 μl ABI Prism True Allele PCR Premix (Applied Biosystems), and 5 pmol of each primer. PCR was performed using the following cycling conditions: denaturation at 95°C for 12 minutes; 10 cycles (94°C for 15 seconds, 55°C for 15 seconds, 72°C for 30 seconds), 32 cycles (89°C for 15 seconds, 55°C for 15 seconds, 72°C for 30 seconds), and extension at 72°C for 10 minutes. A 1.0-μl aliquot of each fluorescent-labeled PCR product was combined with 12 μl of formamide and 0.5 μl of Genescan 400HD (ROX) size standard (Applied Biosystems). The samples were then subjected to capillary electrophoresis on an ABI 3700 DNA Analyzer using Genescan Analysis software (Applied Biosystems). Loss of chromosome 1p was determined by using five dinucleotide-repeat microsatellite markers on the short arm of chromosome 1 (in order from centromere to telomere D1S255, D1S199, D1S507, D1S214, and D1S468). Complete or partial loss of chromosome 1p was based on the pattern of loss of the five dinucleotide microsatellite markers evaluated. Loss of a marker was considered to be present when the PCR assay showed absence or decrease in intensity by more than 50% of a band from a tumor sample as compared with the paired control nonneoplastic sample. MSI status was determined by using the five markers on the short arm of chromosome 1p and two mononucleotide-repeat microsatellite markers, BAT 25 and BAT 26.13Rashid A Houlihan PS Booker S Peterson GM Giardiello FM Hamilton SR Phenotypic and molecular characteristics of hyperplastic polyposis.Gastroenterology. 2000; 119: 323-332Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar Specimens with high levels of MSI (MSI-high) were defined by shifts of bands as compared to control DNA in at least 30% of evaluable markers, and low levels of MSI (MSI-low) by shifts in less than 30% of evaluable microsatellite markers, as in previous studies.13Rashid A Houlihan PS Booker S Peterson GM Giardiello FM Hamilton SR Phenotypic and molecular characteristics of hyperplastic polyposis.Gastroenterology. 2000; 119: 323-332Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar Fisher’s exact test and chi-square test were used for comparing associations among genetic alterations and between genetic alterations and clinicopathological factors. Student’s t-test was used to compare the means of size, age, and methylation. The patient demographic data in relationship to ACF characteristics are summarized in Table 2. Twenty-seven ACF were identified from two FAP patients and 34 ACF from 10 patients with sporadic colorectal carcinomas. The histopathological type of ACF was strongly associated with the patient groups: 89% (24 of 27) of ACF from FAP patients were dysplastic, and only 11% (3 of 27) were heteroplastic. In contrast, 82% (28 of 34) of ACF from the patients with sporadic CRC were heteroplastic, only 12% (4 of 34) were dysplastic, and 6% (2 of 34) were mixed having both dysplastic and heteroplastic regions (P = 0.000001 for sporadic versus FAP patients).Table 2Demographic Data and Characteristics of Patients with ACFHistology of ACF, no.PatientAgeSexSite of ACFHeteroplasticDysplasticMixedOther clinical associations150FL270FAP217MR1170FAP349ML700Sporadic CRC479FR100Sporadic CRC569MR001Sporadic CRC661ML801Sporadic CRC764FL310Sporadic CRC865FL210Sporadic CRC937FL200Sporadic CRC1045FL500Sporadic CRC1160FL010Sporadic CRC1258MR010Sporadic CRCF, female; M, male; L, left, colorectum; R, right, colon. Open table in a new tab F, female; M, male; L, left, colorectum; R, right, colon. Methylation of the p16 gene, MINT31, MINT2, MINT1, MGMT gene, and hMLH1 gene was present in 4% (2 of 56), 21% (13 of 61), 5% (3 of 59), 8% (5 of 59), 12% (6 of 51), and 3% (2 of 61) of the ACF, respectively (examples in Figure 2A and summarized in Figure 3). Thirty-four percent (21 of 61) of the ACF were methylated in at least one locus: 2% (1 of 61) of ACF were methylated at three loci, 13% (8 of 61) at two loci, and 20% (12 of 61) at one locus. The p16 gene was methylated in two ACF, the hMLH1 gene in two ACF, and the MGMT gene in six ACF (Figure 3). Six of eight ACF with methylation of p16, hMLH1, or MGMT had methylation of at least one additional locus.Figure 2A: Methylation analysis of CpG islands in ACF. Examples of methylation of p16, MINT1, MINT31, and MGMT are shown. Methylation-specific PCR using primers for methylated (M) and unmethylated (U) alleles of bisulfite-treated DNA was performed. Loci examined and ACF numbers are indicated above each gel. MW represents lane with molecular weight marker. B: Nucleotide sequencing of the K-ras gene in ACF. A G-to-A mutation of codon 12 is indicated by the arrowhead. C: Allelic loss of chromosome 1p in ACF. The lane from normal DNA (N) has two alleles at marker D1S199 and the lane from the tumor DNA (T) shows loss of one of the alleles.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3CIM, loss of chromosome 1p, K-ras mutation, and histopathology of ACF from FAP patients and patients with sporadic CRC.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Methylation of the six markers was analyzed in 36 samples of mucosa adjacent to the ACF (Figure 3). Methylation at one locus was found in only three mucosal samples. Two of these samples were methylated at MINT31 and MINT1, respectively, and the adjacent ACF were concordantly methylated for the same loci. The third sample of mucosa was methylated at MINT1, and was discordant with the methylation status of the adjacent ACF. K-ras mutations were present in 25% (15 of 61) of ACF (examples in Figure 2B and summarized in Figure 3). All 15 mutations were present at codon 12, and none were found in codon 13. G-to-A transition at the second nucleotide of codon 12 was present in seven ACF, G-to-T tran
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