Microsatellite Instability Is Associated with Hypermethylation of the hMLH1 Gene and Reduced Gene Expression in Mycosis Fungoides
2003; Elsevier BV; Volume: 121; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2003.12496.x
ISSN1523-1747
AutoresJulia Scarisbrick, Tracey J. Mitchell, Eduardo Calonje, Guy Orchard, R. Russell‐Jones, Sean Whittaker,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoFifty-one mycosis fungoides samples were analyzed for microsatellite instability (MSI) using the panel of markers recommended for hereditary nonpolyposis colorectal cancer kindred and a panel we designed for cutaneous T cell lymphoma in order to compare detection rates and determine if MSI is a genome-wide phenomenon. Samples demonstrating MSI were analyzed for abnormalities of the hMLH1 gene including loss of heterozygosity, mutations, and promoter hypermethylation. MSI was detected in 16% using the hereditary nonpolyposis colorectal cancer panel and 22% with the cutaneous T cell lymphoma panel. Overall, 27% dem-onstrated MSI and 73% had a stable phenotype. hMLH1 gene studies did not detect loss of heterozygosity or reveal any mutations. Promoter hypermethylation was detected in nine of 14 patients with MSI, however (64%). In addition hMLH1 and hMSH2 protein expression was studied using immunohistochemical techniques. Five of nine patients with MSI and hMLH1 promoter methylation showed abnormal hMLH1 protein expression with normal hMSH2 gene expression. All other patients tested demonstrated normal hMLH1 and hMSH2 protein expression. MSI was found to be more prevalent in tumor stage mycosis fungoides (47%) than early stage disease (20%) and was associated with an older age of onset of mycosis fungoides. MSI may be a consequence of hMLH1 promoter hypermethylation in mycosis fungoides patients and may prevent transcription in a subset of patients. This suggests that the development of a mutator phenotype may contribute to disease progression in mycosis fungoides. Fifty-one mycosis fungoides samples were analyzed for microsatellite instability (MSI) using the panel of markers recommended for hereditary nonpolyposis colorectal cancer kindred and a panel we designed for cutaneous T cell lymphoma in order to compare detection rates and determine if MSI is a genome-wide phenomenon. Samples demonstrating MSI were analyzed for abnormalities of the hMLH1 gene including loss of heterozygosity, mutations, and promoter hypermethylation. MSI was detected in 16% using the hereditary nonpolyposis colorectal cancer panel and 22% with the cutaneous T cell lymphoma panel. Overall, 27% dem-onstrated MSI and 73% had a stable phenotype. hMLH1 gene studies did not detect loss of heterozygosity or reveal any mutations. Promoter hypermethylation was detected in nine of 14 patients with MSI, however (64%). In addition hMLH1 and hMSH2 protein expression was studied using immunohistochemical techniques. Five of nine patients with MSI and hMLH1 promoter methylation showed abnormal hMLH1 protein expression with normal hMSH2 gene expression. All other patients tested demonstrated normal hMLH1 and hMSH2 protein expression. MSI was found to be more prevalent in tumor stage mycosis fungoides (47%) than early stage disease (20%) and was associated with an older age of onset of mycosis fungoides. MSI may be a consequence of hMLH1 promoter hypermethylation in mycosis fungoides patients and may prevent transcription in a subset of patients. This suggests that the development of a mutator phenotype may contribute to disease progression in mycosis fungoides. hereditary nonpolyposis colorectal cancer loss of heterozygosity microsatellite instability microsatellite stable polymerase chain reaction single stranded conformation polymorphism Mycosis fungoides is an indolent form of primary cutaneous T cell lymphoma (CTCL). Patients present with cutaneous patches and plaques that may progress to tumor stage disease over several years. In the early cutaneous stages of disease there is no significant impact on survival (Zackheim et al., 1999Zackheim H.S. Amin S. Kashani-Sabet M. McMillan A. Prognosis in cutaneous T-cell lymphoma by skin stage: Long-term survival in 489 patients.J Am Acad Dermatol. 1999; 40: 418-425Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). A minority of patients with mycosis fungoides progress to tumor stage disease, however, and systemic spread may occur. In these patients the survival rate is poor with a median survival of 1–2 y (Salhany et al., 1988Salhany K.E. Causar J.B. Greer J.P. Casey T.T. Fields J.P. Collins R.D. Transformation of cutaneous T-cell lymphoma to large cell lymphoma. A clinicopathologic and immunologic study.AM J Pathol. 1988; 132: 265-277PubMed Google Scholar; Cerroni et al., 1992Cerroni L. Rieger E. Hodi S. Kerl H. Clinicopathologic and immunologic features associated with transformation of mycosis fungoides to large-cell lymphoma.Am J Surg Pathol. 1992; 16: 543-552Crossref PubMed Scopus (149) Google Scholar; Vergler et al., 2000Vergler B. Muret A. Beylot-Barry M. et al.Transformation of mycosis fungoides: Clinicopathological and prognostic features of 45 cases.Blood. 2000; 95: 2212-2218PubMed Google Scholar). Abnormalities of several tumor suppressor genes have been identified in mycosis fungoides including P53, P15, and P16 genes (Lauritzen et al., 1995Lauritzen A.F. Vejlsgaard G.L. Hou-Jensen K. Ralfkier E. P53 protein expression in cutaneous T-cell lymphomas.Br J Dermatol. 1995; 133: 32-36Crossref PubMed Scopus (34) Google Scholar; McGregor et al., 1995McGregor J.M. Dublin E.A. Levison D.A. MacDonald D.M. Smith N.P. Whittaker S. P53 immunoreactivity is uncommon in primary cutaneous lymphoma.Br J Dermatol. 1995; 132: 353Crossref PubMed Scopus (30) Google Scholar; McGregor et al., 1999McGregor J.M. Crook T. Fraser-Andrews E.A. Rozycka M. Crossland S. Whittaker S.J. 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Villuendas R. et al.p16 gene alterations are frequent in lesions of mycosis fungoides.Am J Pathol. 2000; 156: 1565-1572Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar; Scarisbrick et al., 2002Scarisbrick J.J. Woolford A.J. Calonje E. Photiou A. Ferreira S. Russell-Jones R. Whittaker S.J. Frequent abnormalities of the P15 and P16 genes in primary cutaneous T-cell lymphoma.J Invest Dermatol. 2002; 118: 493-499Crossref PubMed Scopus (95) Google Scholar). In addition, numerous cytogenetic abnormalities have been described in Sézary syndrome, the leukemic form of CTCL, frequently involving deletions on chromosomes 1p, 10q, 17p, and 17q (Nowell et al., 1982Nowell P.C. Finan J.B. Vonderheid E.C. Clonal characteristics of cutaneous T-cell lymphomas: Cytogenetic evidence from blood, lymph nodes and skin.J Inv Dermatol. 1982; 78: 69-75Crossref PubMed Scopus (74) Google Scholar; Shapiro et al., 1987Shapiro P.E. Warburton D. Berger C.L. Edelson R.L. Clonal chromosomal abnormalities in cutaneous T-cell lymphoma.Cancer Genet Cytogenet. 1987; 28: 267-276Abstract Full Text PDF PubMed Scopus (32) Google Scholar; Berger et al., 1988Berger R. Baranger L. Bernheimm A. Valensi F. Flandrin G. Cytogenetics of T-cell malignant lymphoma: Report of 17 cases and review of the chromosomal breakpoints.Cancer Genet Cytogenet. 1988; 36: 123-130Abstract Full Text PDF PubMed Scopus (35) Google Scholar; Thangavelu et al., 1997Thangavelu M. Finn W.G. Yelavarthi K.K. et al.Recurring structural chromosomal abnormalities in peripheral blood lymphocytes of patients with mycosis fungoides/Sézary syndrome.Blood. 1997; 89: 3371-3377PubMed Google Scholar; Scarisbrick et al., 2001Scarisbrick J.J. Woolford A.J. Russell-Jones R. Whittaker S.J. Allelotyping in mycosis fungoides and Sézary syndrome.J Invest Dermatol. 2001; 117: 663-670Crossref PubMed Scopus (51) Google Scholar). No disease-specific genetic abnormalities have been identified in CTCL, however. Microsatellite instability (MSI) is a form of genetic instability that is highly characteristic of hereditary nonpolyposis colorectal cancer (HNPCC), a familial cancer syndrome with a strong predisposition to colorectal, gastric, and endometrial cancers. MSI in HNPCC tumors tends to occur at high levels (greater than 40%, defined as MSI-H) but lower levels of MSI have been identified in various sporadic internal malignancies (Cunningham et al., 1998Cunningham J.M. Christensen E.R. Tester D.J. Kim C.-Y. Roche P.C. Burgart L.J. Thibodeau S.N. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability.Cancer Res. 1998; 58: 3455-3460PubMed Google Scholar; Esteller et al., 1998Esteller M. Levine R. Baylin S.B. Ellenson L.H. Herman J.G. MLH1 promoter hypermethylation is associated with the microsatellite instability phenotype in sporadic endometrial carcinomas.Oncogene. 1998; 17: 2413-2417Crossref PubMed Scopus (430) Google Scholar; Leung et al., 1998Leung S.Y. Chan T.L. Chung L.P. et al.Microsatellite instability and mutation of DNA mismatch repair genes in gliomas.Am J Pathol. 1998; 153: 1181-1188Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar; Leung et al., 1999Leung S.Y. Yuen S.T. Chung L.P. Chu K.M. Chan A.S.Y. Ho J.C.I. hMLH1 promoter methylation and lack of hMLH1 expression in sporadic gastric carcinomas with high-frequency microsatellite instability.Cancer Res. 1999; 59: 159-164PubMed Google Scholar; Ghimenti et al., 1999Ghimenti C. Tannergard P. Wahlberg S. et al.Microsatellite instability and mismatch repair gene inactivation in sporadic pancreatic and colon tumours.Br J Cancer. 1999; 80: 11-16Crossref PubMed Scopus (56) Google Scholar; Halling et al., 1999Halling K.C. Harper J. Moskaluk C. et al.Origin of microsatellite instability in gastric cancer.Am J Path. 1999; 155: 205-211Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), cutaneous tumors, including basal cell carcinoma, squamous cell carcinoma, and melanoma (Peris et al., 1995Peris K. Keller G. Chimenti S. Amantea A. Kerl H. Hofler H. Microsatellite instability and loss of heterozygosity in melanoma.J Invest Dermatol. 1995; 105: 625-628Crossref PubMed Scopus (67) Google Scholar; Quinn et al., 1995Quinn A.G. Healy E. Rehman I. Sikkink S. Rees J.L. Microsatellite instability in human non-melanoma and melanoma skin cancer.J Invest Dermatol. 1995; 104: 309-312Crossref PubMed Scopus (109) Google Scholar), and in a variety of lymphomas and leukemias (Peng et al., 1996Peng H. Du Chen G.M. Singh N. Isaacson P.G. Pan L. Replication error phenotype and p53 gene mutation in lymphomas of mucosa-associated lymphoid tissue.Am J Pathol. 1996; 148: 643-648PubMed Google Scholar; Gartenhaus et al., 1996Gartenhaus R. Johns III, M.M. Wang P. Rai K. Sidransky D. Mutator phenotype in a subset of chronic lymphocytic leukemia.Blood. 1996; 87: 38-41PubMed Google Scholar; Hatta et al, 1998; Zhu et al, 1999). In addition, MSI may be a poor prognostic marker in adult T cell leukemia (Hayami et al., 1999Hayami Y. Komatsu H. Iida S. et al.Microsatellite instability as a potential marker for poor prognosis in adult T cell leukemia/lymphoma.Leuk Lymphoma. 1999; 32: 345-349PubMed Google Scholar). Furthermore, mice deficient in the mismatch repair gene hMSH2 demonstrate MSI and have an increased propensity to develop lymphoma (Lowsky et al., 1997Lowsky R. DeCoteau J.F. Reitmair A.H. et al.Defects of the mismatch repair gene MSH2 are implicated in the development of murine and human lymphoblastic lymphomas and are associated with the aberrant expression of rhombotin-2 and Tal-1.Blood. 1997; 89: 2276-2282Crossref PubMed Google Scholar). We recently detected MSI in 12 out of 44 patients (27%) with mycosis fungoides (Scarisbrick et al., 2000Scarisbrick J.J. Woolford A.J. Russell-Jones R. Whittaker S.J. Loss of heterozygosity on 10q and microsatellite instability in primary cutaneous T-cell lymphoma is found in advanced disease and may be associated with homozygous deletion of PTEN.Blood. 2000; 95: 2937-2942PubMed Google Scholar). This study screened for MSI using eight dinucleotide markers on a specific region at 10q22–26. This prompted us to test whether MSI was a genome-wide phenomenon by studying rates of MSI at different chromosomal loci. The difference in genetic instability may be reflected not only by the chromosomal location of markers, however, but also by the types of markers used. Dinucleotide repeats are inherently less stable than mononucleotide repeats and are more sensitive at detecting the MSI-L phenotype (MSI at fewer than 40% of markers) whereas mononucleotide markers are more specific for detecting MSI-H tumors (Dietmaier et al., 1997Dietmaier W. Wallinger S. Bocker T. Kullman F. Fishel R. Ruschoff J. Diagnostic microsatellite instability: Definition and correlation with mismatch repair expression.Cancer Res. 1997; 57: 4749-4756PubMed Google Scholar; Jass et al., 1999Jass J.R. Biden K.G. Cummings M.C. et al.Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways.J Clin Pathol. 1999; 52: 455-460Crossref PubMed Scopus (222) Google Scholar). A comparison of MSI rates in different tumors is difficult because most studies employ different numbers, types, and loci of microsatellite markers. In order to further explore the role of MSI in mycosis fungoides we analyzed 51 samples using a panel of microsatellite markers recommended at a National Cancer Institute Workshop on Microsatellite Instability for detection of MSI in HNPCC families (Boland et al., 1998Boland C.R. Thibodeau S.N. Hamilton S.R. 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. 1998; 58: 5248-5257PubMed Google Scholar). This HNPCC panel includes two mononucleotide repeats (BAT25 (4q12), a 25-repeat thymidine tract within exon 16 of the c-kit oncogene, which is a reliable marker for identifying MSI-H; and BAT26 (2p21–22), a 26-repeat adenine tract within intron 5 of the hMSH2 gene, which is often inactivated in HNPCC kindreds) and three dinucleotide repeats (D17S250 (17q11.2–12), which is close to the locus for the tumor suppressor gene BRCA-1; D2S123 (2p16), which is telomeric to mismatch repair genes hMSH-2 and hMSH-6; and D5S346 (5q21–22) close to the locus for the adenomatosis polyposis coli tumor suppressor gene). In order to establish whether this HNPCC panel of markers has a high sensitivity for detecting MSI in CTCL we compared this panel with a second panel, which also incorporates BAT25 as it is a reliable marker of the MSI-H phenotype and c-kit has not been implicated in either HNPCC or CTCL. As in the HNPCC panel the other markers were chosen because they are close to putative tumor suppressor genes previously found to be abnormal in CTCL (BAT34R (17p13.1), a mononucleotide repeat in the eleventh exon of P53, and three dinucleotide repeats, D10S215 (10q), D1S201 (1p), IFNA (9p)). Germline mutations in the hMLH1 and hMSH2 genes have been identified in HNPCC kindred and may correlate with absent protein expression (Thibodeau et al., 1998Thibodeau S.N. French A.J. Cunningham J.M. et al.Microsatellite instability in colo-rectal cancer: Different mutator phenotypes and the principal involvement of hMLH1.Cancer Res. 1998; 58: 1713-1718PubMed Google Scholar; Thibodeau et al., 1998Thibodeau S.N. French A.J. Cunningham J.M. et al.Microsatellite instability in colo-rectal cancer: Different mutator phenotypes and the principal involvement of hMLH1.Cancer Res. 1998; 58: 1713-1718PubMed Google Scholar; Dietmaier et al., 1997Dietmaier W. Wallinger S. Bocker T. Kullman F. Fishel R. Ruschoff J. Diagnostic microsatellite instability: Definition and correlation with mismatch repair expression.Cancer Res. 1997; 57: 4749-4756PubMed Google Scholar). hMLH1 gene abnormalities have also been detected in some sporadic malignancies demonstrating MSI including lymphoma (Herman et al., 1998Herman J.G. Uma A. Polyak K. et al.Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma.Proc Natl Acad Sci. 1998; 95: 6870-6875Crossref PubMed Scopus (1693) Google Scholar; Cunningham et al., 1998Cunningham J.M. Christensen E.R. Tester D.J. Kim C.-Y. Roche P.C. Burgart L.J. Thibodeau S.N. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability.Cancer Res. 1998; 58: 3455-3460PubMed Google Scholar; Leung et al., 1999Leung S.Y. Yuen S.T. Chung L.P. Chu K.M. Chan A.S.Y. Ho J.C.I. hMLH1 promoter methylation and lack of hMLH1 expression in sporadic gastric carcinomas with high-frequency microsatellite instability.Cancer Res. 1999; 59: 159-164PubMed Google Scholar). In order to determine if the hMLH1 gene is abnormal in patients with mycosis fungoides demonstrating MSI, all samples were analyzed for allelic loss in the region of the hMLH1 gene, for mutations using polymerase chain reaction single stranded conformation polymorphism (PCR-SSCP) analysis, and for aberrant promoter methylation using methylation-specific PCR. In addition, hMLH1 and hMSH2 protein expression was studied in samples demonstrating MSI using immunohistochemical techniques, which have been found to be a reliable screening method for hMLH1 and hMSH2 protein expression in HNPCC-related skin tumors (Mathiak et al., 2002Mathiak M. Rutten A. Mangold E. et al.Loss of DNA mismatch repair proteins in skin tumors from patients with Muir–Torre syndrome and MSH2 or MLH1 germline mutations: Establishment of immunohistochemical analysis as a screening test.Am J Surg Pathol. 2002; 26: 338-343Crossref PubMed Scopus (138) Google Scholar). Fifty-one archival specimens of tumor and normal DNA from individual patients were selected. All tumor samples were chosen because either Southern blot or PCR-SSCP-based T cell receptor gene analysis had previously shown a dominant tumor population (Whittaker et al., 1991Whittaker S.J. Smith N.P. Russell-Jones R. Luzzatto L. Analysis of β, γ and δ T-cell receptor genes in mycosis fungoides and Sézary syndrome.Cancer. 1991; 68: 1572-1582Crossref PubMed Scopus (87) Google Scholar; Whittaker, 1997Whittaker S.J. T-cell receptor gene analysis in cutaneous T-cell lymphomas.Clin Exp Dermatol. 1997; 21: 81-87Crossref Scopus (16) Google Scholar). These samples consisted of 51 mycosis fungoides patients with different cutaneous stages of disease (stage T1 n=15, stage T2 n=15, stage T3 n=17, and stage T4 n=4). Only four patients with T4 stage disease were selected for this study as some patients with erythrodermic disease did not have a dominant clone in the skin and others had no nonclonal DNA for use as control. MSI was detected by amplifying microsatellite markers using a PCR-based methodology and comparing the size of microsatellite repeats in tumor and normal DNA. MSI was defined as the presence of novel bands in the tumor DNA compared to the normal control. Samples showing MSI were scored as noninformative for LOH in accordance with standard criteria (Boland et al., 1998Boland C.R. Thibodeau S.N. Hamilton S.R. 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. 1998; 58: 5248-5257PubMed Google Scholar). Samples were analyzed for MSI using the HNPCC and CTCL panels of microsatellite markers listed in Table I. The sequences of oligonucleotide primers for the microsatellite markers were obtained from the Human Genethon Linkage Map (Gyapay et al., 1994Gyapay G. Morissette J. Vignal A. et al.The 1993–4 Genethon Human Genetic Linkage Map.Nat Genet. 1994; 7: 246-249Crossref PubMed Scopus (1978) Google Scholar) (D5S346, D2S123, D17S250, D10S215, D1S211); IFNA primer sequences were used as previously published (Heyman et al., 1996Heyman M. Rasool O. Brandter L. et al.Prognostic importance of p15 and p16 gene inactivation in childhood acute lymphocytic leukaemia.J Clin Oncol. 1996; 14: 1512-1520PubMed Google Scholar); and sequences for the mononucleotide repeats BAT34C, BAT25, and BAT26 primers were as previously described (Zhou et al., 1997Zhou X.P. Hoang J.M. Cottu P. Thomas G. Hamelin R. Allelic profiles of mononucleotide repeat microsatellites in control individuals and in colorectal tumours with and without replication errors.Oncogene. 1997; 15: 1713-1718Crossref PubMed Scopus (118) Google Scholar).Table IThe presence of MSI at microsatellite markers at different cytogenetic loci in 14 patients with mycosis fungoidesHNPCC panelCTCL panelCutaneous stage of mycosis fungoidesD17S250, 17q11–12D2S123, 2p16D5S346, 5q21–22BAT26, 2p21–22BAT25, 4q12D10S215, 10q24D1S201, 1p32IFNA, 9p21BAT34R, 17p13.11T1MSIMSI––MSIMSIMSI–MSI2T1MSI––––––––3T1––––––MSI––4T2–––––MSI–––5T2–MSIMSIMSI––MSIMSI–6T2–MSI–––––––7T3––––MSIMSIMSI––8T3––MSI–MSIMSI–––9T3––MSI––––––10T3–MSI–––––MSI–11T3––––––MSI––12T3––––––MSI––13T3–––––MSI–––14T3–––––––MSI–D17S250, D2S123, D5S346, BAT26, and BAT25 comprise the HNPCC panel and D10S215, D1S201, IFNA, BAT24R, and BAT25 comprise the CTCL panel.MSI, microsatellite instability; –, normal. Open table in a new tab D17S250, D2S123, D5S346, BAT26, and BAT25 comprise the HNPCC panel and D10S215, D1S201, IFNA, BAT24R, and BAT25 comprise the CTCL panel.MSI, microsatellite instability; –, normal. Samples demonstrating MSI were also analyzed for LOH at the loci for hMLH1 (3p21) using D3S1611, which is an intragenic dinucleotide marker. PCR was performed in a reaction mixture of 20 μL, including 20 pmol of each oligonucleotide primer, 0.2 mM dATP, 0.2 mM dGTP, 0.2 mM dTTP Amersham-Pharmacia Biotechnology, (Amersham Biosciences, Little Calfont, Buckinghamshire, UK) 0.03 mM dCTP, 0.1 μCi [α33P]-dCTP (3000 Ci per mmol), 50 ng of sample DNA, 1×PCR buffer containing 1.5 mM MgCl2, 1.25 units Taq polymerase (Amersham-Pharmacia Biotechnology), and 1.25 units Taq antibody (Clontech Palo Alto, CA). Twenty-five to 30 cycles of PCR were performed in a DNA Perkin Elmer 9700 thermal cycler. Each cycle consisted of denaturing at 94°C for 20 s, annealing at 55–65°C (according to the Tm for each oligonucleotide primer pair) for 20 s, and extension at 72°C for 50 s. PCR cycles were limited to 25 to ensure analysis was semiquantitative in order to eliminate potential false negative results due to increased amplification of contaminating normal DNA. A negative control reaction containing no DNA was examined for each PCR assay. PCR products were diluted 2-fold with stop solution (95% formamide, 20 mM ethylenediamine tetraacetic acid (EDTA), 0.05% xylene cyanol FF, and 0.05% bromophenol blue) and denatured for 10 min at 95°C before being loaded onto denaturing 6% polyacrylamide (Gibco BRL Gaithersburg, MD) gels containing 7 M urea (United States Biochemicals) and electrophoresed using an S2 sequencing gel apparatus (Gibco BRL, Swampscott, MA). The gel was dried on 3 mm Whatman paper and exposed to X-ray film (Genetic Research Instrumentation, Essex, UK) with an intensifying screen (Appligene Oncor, Graffenstaden, France) at room temperature for 48–72 h. Radiographs were examined for novel bands in tumor DNA, compared to normal DNA from individual patients, by two independent observers without knowledge of histologic tumor type or clinical details. MSI was defined as the presence of additional smaller or larger novel bands in tumor DNA compared to the corresponding normal DNA. All samples showing MSI were subjected to repeat amplification and analysis for confirmation. MSI at one of five loci was scored as MSI-L; MSI at two or more of five loci (>40%) was considered to be representative of a highly unstable phenotype (MSI-H). Samples that failed to demonstrate MSI were termed microsatellite stable (MSS). All samples where two distinct allelic bands were present in the normal DNA, in the absence of novel bands (MSI) in the tumor DNA, were considered to be informative. Samples that produced a single allelic band in normal DNA or failed to amplify for a given microsatellite marker were scored as noninformative. LOH was scored as positive when a clear reduction in signal intensity was detected in one of the alleles of the tumor DNA compared to the same allele in the paired normal DNA. In order to quantify signal intensities we also analyzed our samples using a scanning densitometer with Image Quant softwear (Amersham Pharmacia); a reduction in signal intensity of more than 50% in one of the alleles in the tumor DNA compared to the paired normal DNA was defined as LOH. Mutational analyses using PCR-SSCP were performed on the four exons (2, 9, 15, and 16) of the hMLH1 gene, in which more than two mutations had been previously reported (Han et al., 1995Han H.J. Maruyama M. Baba S. Park J.G. Nakamura Y. Genomic structure of human mismatch repair gene, hMLH1, and its mutation analysis in patients with hereditary non-polyposis colorectal cancer (HNPCC).Hum Mol Genet. 1995; 4: 237-242Crossref PubMed Scopus (167) Google Scholar; Liu et al., 1996Liu B. Parsons R. Papadopoulos N. et al.Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients.Nat Med. 1996; 2: 169-174Crossref PubMed Scopus (854) Google Scholar; Wijnen et al., 1996Wijnen J. Khan P.M. Vasen H. et al.Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15–16.Am J Hum Genet. 1996; 58: 300-307PubMed Google Scholar; Hangaishi et al., 1997Hangaishi A. Ogawa S. Mitani K. Hosoya N. Chiba S. Yazaki Y. Hirai H. Mutations and loss of expression of a mismatch repair gene, hMLH1, in leukemia and lymphoma cell lines.Blood. 1997; 89: 1740-1747Crossref PubMed Google Scholar). The sequences of the exon specific primers are as follows: exon 2 (nt 117–207) 5′-AATATGTACATTAGAGTAGTTG-3′, 5′-CAGAGAAAGG-TCCTGACTC-3′; exon 9 (nt 678–790) 5′-CAAAAGCTTCAGAATCTC-3′, 5′-CTGTGGGTGTTTCCTGTGAGTGG-3′; exon 15 (nt 1668–1731) 5′-CCCATTTGTCCCAACTGG-3′, 5′-CGGTCAGTTGAAATGTCAG-3′; exon 16 (nt 1732–1896) 5′-CATTTGGATGCTCCGTTAAAAGC-3′, 5′-CACCCGGCTGGAAATTTTATTTG-3′. PCR were performed using standard methodology with Amplitaq Gold (Applied Biosystems, Foster City, CA) in a 20 μL volume using approximately 100 ng genomic DNA as a template. Amplicons specific for exons 2, 9, and 16 were amplified in 1×PCR buffer (PCR Buffer II, Applied Biosystems) containing 1.5 mM MgCl2; the amplicons specific for exon 16 were amplified in 1×PCR buffer containing 3.0 mM MgCl2. Each PCR included 46.25 Bq radiolabeled 5′-[α33P]-dCTP. Reaction conditions were the same for all primers: 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. On the final cycle the extension time was increased to 8 min. Three microliters of the amplified DNA was diluted with 8 μL of 95% formamide, 1 mM EDTA, 0.25% bromophenol blue, and 0.25% xylene cyanol. Samples were denatured for 5 min at 95°C and snap frozen in liquid nitrogen. Seven microliters of each denatured sample were loaded onto a native acrylamide gel with or without 5% glycerol. The gels consisted of 0.5×SequaGel MD (National Diagnostics USA, Atlanta, GA) and 0.6 × TBE. Electrophoresis conditions were 6–8 W for 15–17 h. Gels were dried and autoradiography was performed at –70°C. Samples were analyzed for methylation of CpG-rich islands within the hMLH1 gene promoter using methylation-specific PCR with DNA chemically modified by sodium bisulfite (Herman et al., 1996Herman J. Graff J. Myohanen S. et al.Methylation-specific PCR. A novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci. 1996; 93: 9821-9826Crossref PubMed Scopus (5225) Google Scholar). Bisulfite modification normally changes cytosine into uracil; however, this reaction is inhibited if DNA is methylated. In methylation-specific PCR the modified DNA is subjected to separate PCR amplification using primers designed to anneal to methylated bisulfite-modified DNA and primers for unmethylated bisulfite-modified DNA within a given gene (Herman et al., 1996Herman J. Graff J. Myohanen S. et al.Methylation-specific PCR. A novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci. 1996; 93: 9821-9826Crossref PubMed Scopus (5225) Google Scholar). Amplification with the primers for methylated DNA confirms the gene is hypermethylated, whereas amplification with the unmethylated primers should occur in all cases as tumor cells are always mixed with some reactive cells and ensures that the DNA bisulfite modification has been successful (Baur et al., 1999Baur A.S. Shaw P. Burri N. Delacketaz F. Basman F.T. Caubert P. Frequent Methylation silencing of p15 and p16 in B-cell and T-cell lymphomas.Blood. 1999; 94: 1773-1781Crossref PubMed Google Scholar). The LS180 cell line (CAMR, Salsbury, Wiltshire, UK) is hypermethylated at the hMLH1 gene promoter and DNA extracted from these cell lines was used as positive controls for the methylated primer reaction. For each patient sample 5 μg genomic DNA was subjected to bisulfite modification. DNA was first denatured in 0.2 mol per L NaOH in a volume of 50 μL for 20 min at 37°C. After the addition of 30 μL 10 mM hydroquinone and 520 μL 3 M sodium bisulfite (pH 5-6) samples were incubated overnight for 16 h at 55°C. The bisulfite-treated DNA was then puri
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