Establishment and Validation of Real-Time Polymerase Chain Reaction Method for CDH1 Promoter Methylation
2002; Elsevier BV; Volume: 161; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)64218-6
ISSN1525-2191
AutoresKiyomi O. Toyooka, Shinichi Toyooka, Anirban Maitra, Qinghua Feng, Nancy Kiviat, Alice L. Smith, John D. Minna, Raheela Ashfaq, Adi F. Gazdar,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoAberrant methylation of the promoter region has emerged as the major mechanism for silencing tumor suppressor genes. However, for some genes, such as E-cadherin (CDH1), methylation and protein expression demonstrate considerable heterogeneity, making correlations difficult. We compared methylation and protein expression status of CDH1 in 56 primary breast carcinomas using semiquantitative assays. Aberrant CDH1 methylation was studied by methylation-specific polymerase chain reaction (MSP) and semiquantitative real-time MSP assays. The Cdh1 expression was investigated by immunostaining on archival formalin-fixed sections from 34 primary carcinomas and their accompanying normal epithelium and preinvasive and metastatic lesions. Membrane-specific Cdh1 expression in the neoplastic cells was quantified by image analysis using an automated cellular imaging system and a continuous score. Aberrant promoter methylation of the CDH1 was present in 24 of 56 (43%) breast carcinomas by MSP assay. There was excellent concordance between the standard MSP assay and the real-time assay (91%, P < 0.0001). The concordance between loss of Cdh1 expression and CDH1 methylation by standard MSP was 71% (P = 0.02). Furthermore, there was a strong correlation between the semiquantitative assays for methylation and protein expression (r = 0.47, P = 0.005). We conclude that promoter methylation of CDH1 significantly correlated with the Cdh1 expression level, demonstrating that epigenetic silencing is a valid pathway for silencing of tumor suppressor genes in primary breast carcinomas. Aberrant methylation of the promoter region has emerged as the major mechanism for silencing tumor suppressor genes. However, for some genes, such as E-cadherin (CDH1), methylation and protein expression demonstrate considerable heterogeneity, making correlations difficult. We compared methylation and protein expression status of CDH1 in 56 primary breast carcinomas using semiquantitative assays. Aberrant CDH1 methylation was studied by methylation-specific polymerase chain reaction (MSP) and semiquantitative real-time MSP assays. The Cdh1 expression was investigated by immunostaining on archival formalin-fixed sections from 34 primary carcinomas and their accompanying normal epithelium and preinvasive and metastatic lesions. Membrane-specific Cdh1 expression in the neoplastic cells was quantified by image analysis using an automated cellular imaging system and a continuous score. Aberrant promoter methylation of the CDH1 was present in 24 of 56 (43%) breast carcinomas by MSP assay. There was excellent concordance between the standard MSP assay and the real-time assay (91%, P < 0.0001). The concordance between loss of Cdh1 expression and CDH1 methylation by standard MSP was 71% (P = 0.02). Furthermore, there was a strong correlation between the semiquantitative assays for methylation and protein expression (r = 0.47, P = 0.005). We conclude that promoter methylation of CDH1 significantly correlated with the Cdh1 expression level, demonstrating that epigenetic silencing is a valid pathway for silencing of tumor suppressor genes in primary breast carcinomas. The cadherins are a family of transmembrane glycoproteins expressed on the surface of epithelial cells and mediates homophilic, Ca2+-dependent intercellular adhesion that is essential for the maintenance of normal tissue architecture.1Takeichi M Morphogenetic roles of classic cadherins.Curr Opin Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1251) Google Scholar Among the cadherin family membranes, reduced expression of Cdh1 and Cdh13 is common in a variety of human tumors2Mareel M Bracke M Van Roy F Cancer metastasis: negative regulation by an invasion-suppressor complex.Cancer Detect Prev. 1995; 19: 451-464PubMed Google Scholar, 3Berx G Van Roy F The E-cadherin/catenin complex: an important gatekeeper in breast cancer carcinogenesis and malignant progression.Breast Cancer Res. 2001; 3: 289-293Crossref PubMed Scopus (347) Google Scholar including breast carcinoma4Takeichi M Cadherin cell adhesion receptors as a morphogenetic regulator.Science. 1991; 251: 1451-1455Crossref PubMed Scopus (2974) Google Scholar, 5Takeichi M Cadherins in cancer: implications for invasion and metastasis.Curr Opin Cell Biol. 1993; 5: 806-811Crossref PubMed Scopus (828) Google Scholar, 6Behrens J The role of cell adhesion molecules in cancer invasion and metastasis.Breast Cancer Res Treat. 1993; 24: 175-184Crossref PubMed Scopus (148) Google Scholar, 7Moll R Mitze M Frixen UH Birchmeier W Differential loss of E-cadherin expression in infiltrating ductal and lobular breast carcinomas.Am J Pathol. 1993; 143: 1731-1742PubMed Google Scholar, 8Christofori G Semb H The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene.Trends Biochem Sci. 1999; 24: 73-76Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar, 9Toyooka KO Toyooka S Virmani AK Sathyanarayana UG Euhus DM Gilcrease M Minna JD Gazdar AF Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas.Cancer Res. 2001; 61: 4556-4560PubMed Google Scholar and they are regarded as tumor suppressor genes. Inactivation of these genes are hypothesized to be an important step in the progression from tumor formation to invasion and metastasis.10Katagiri A Watanabe R Tomita Y E-cadherin expression in renal cell cancer and its significance in metastasis and survival.Br J Cancer. 1995; 71: 376-379Crossref PubMed Scopus (132) Google Scholar These genes and some other cadherin family members are located on the long arm of chromosome 16 (16q),11Kremmidiotis G Baker E Crawford J Eyre HJ Nahmias J Callen DF Localization of human cadherin genes to chromosome regions exhibiting cancer-related loss of heterozygosity.Genomics. 1998; 49: 467-471Crossref PubMed Scopus (73) Google Scholar a chromosomal location that is frequently deleted in breast and other types of carcinomas.12Lindblom A Rotstein S Skoog L Nordenskjold M Larsson C Deletions on chromosome 16 in primary familial breast carcinomas are associated with development of distant metastases.Cancer Res. 1993; 53: 3707-3711PubMed Google Scholar, 13Tsuda H Callen DF Fukutomi T Nakamura Y Hirohashi S Allele loss on chromosome 16q24.2-qter occurs frequently in breast cancers irrespectively of differences in phenotype and extent of spread.Cancer Res. 1994; 54: 513-517PubMed Google Scholar, 14Tsuda H Hirohashi S Identification of multiple breast cancers of multicentric origin by histological observations and distribution of allele loss on chromosome 16q.Cancer Res. 1995; 55: 3395-3398PubMed Google Scholar, 15Sato M Mori Y Sakurada A Fujimura S Horii A The H-cadherin (CDH13) gene is inactivated in human lung cancer.Hum Genet. 1998; 103: 96-101Crossref PubMed Scopus (140) Google Scholar, 16Girard L Zochbauer-Muller S Virmani AK Gazdar AF Minna JD Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering.Cancer Res. 2000; 60: 4894-4906PubMed Google ScholarOn the basis of Knudson's17Knudson AG Hereditary cancer, oncogenes, and antioncogenes.Cancer Res. 1985; 45: 1437-1443PubMed Google Scholar “two-hit” hypothesis, both alleles of tumor suppressor genes have to be inactivated for loss of expression. Allelic loss of one copy is often associated with methylation, mutation, or some other form of inactivation of the second allele. Aberrant methylation of the promoter regions of tumor suppressor genes seems to be the major mechanism of gene silencing in human tumors.18Baylin SB Herman JG Graff JR Vertino PM Issa JP Alterations in DNA methylation: a fundamental aspect of neoplasia.Adv Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar Aberrant methylation of the CDH1promoter region has been observed in cancers of the breast, liver, prostate, lung, and stomach.18Baylin SB Herman JG Graff JR Vertino PM Issa JP Alterations in DNA methylation: a fundamental aspect of neoplasia.Adv Cancer Res. 1998; 72: 141-196Crossref PubMed Google Scholar, 19Kanai Y Ushijima S Hui AM Ochiai A Tsuda H Sakamoto M Hirohashi S The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas.Int J Cancer. 1997; 71: 355-359Crossref PubMed Scopus (242) Google Scholar, 20Graff JR Herman JG Lapidus RG Chopra H Xu R Jarrard DF Isaacs WB Pitha PM Davidson NE Baylin SB E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas.Cancer Res. 1995; 55: 5195-5199PubMed Google Scholar, 21Tamura G Yin J Wang S Fleisher AS Zou T Abraham JM Kong D Smolinski KN Wilson KT James SP Silverberg SG Nishizuka S Terashima M Motoyama T Meltzer SJ E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas.J Natl Cancer Inst. 2000; 92: 569-573Crossref PubMed Scopus (323) Google Scholar, 22Zochbauer-Muller S Fong KM Virmani AK Geradts J Gazdar AF Minna JD Aberrant promoter methylation of multiple genes in non-small cell lung cancers.Cancer Res. 2001; 61: 249-255PubMed Google Scholar, 23Toyooka S Toyooka OK Maruyama R Virmani AK Girard L Miyajima K Harada K Ariyoshi Y Takahashi T Sugio K Brambilla E Gilcrease M Minna JD Gazdar AF DNA methylation profiles of lung tumors.Mol Cancer Therapeutics. 2001; 1: 61-67PubMed Google Scholar Some genes such as CDH1 demonstrate considerable heterogeneity of methylation and protein expression.24Graff JR Gabrielson E Fujii H Baylin SB Herman JG Methylation patterns of the E-cadherin 5′ CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression.J Biol Chem. 2000; 275: 2727-2732Crossref PubMed Scopus (329) Google Scholar Cdh1 expression in primary human breast cancers reflects a heterogeneous and unstable pattern of promoter region methylation, which begins early before invasion.24Graff JR Gabrielson E Fujii H Baylin SB Herman JG Methylation patterns of the E-cadherin 5′ CpG island are unstable and reflect the dynamic, heterogeneous loss of E-cadherin expression during metastatic progression.J Biol Chem. 2000; 275: 2727-2732Crossref PubMed Scopus (329) Google Scholar Such plasticity renders correlations between methylation and expression difficult, especially when the standard nonquantitative methylation-specific polymerase chain reaction (MSP) assay is used.In an effort to overcome some of these problems, we developed a semiquantitative real-time MSP assay and correlated our findings with the standard MSP assay. We also correlated it with a semiquantitative assay for protein expression using an image analyzer and immunostained sections.Materials and MethodsClinical SamplesSurgically resected specimens from 42 primary breast carcinomas, 38 cases of infiltrating ductal carcinoma, and 4 cases of infiltrating lobular carcinoma and 17 corresponding nonmalignant breast tissues from these patients were obtained from the Tumor and Tissue Repository at the Hamon Center. DNAs of 14 biopsy samples (13 cases of infiltrating ductal carcinoma and 1 case of infiltrating lobular carcinoma) of breast carcinoma were obtained from Senegal, Africa. The pathologist selected areas of viable appearing tissues consisting primarily of tumor cells. In general, the tumor tissues contained ∼50% tumor cells, although the percentage varied considerably. Epithelial cells from buccal swabs were obtained from 10 healthy nonsmoking volunteers. Paraffin blocks and slides were available from 34 of the resection samples and these were used for immunostaining. Collection of all specimens was obtained in accordance with procedures approved by the Human Subjects Committees of the participating institutions.DNA Extraction and Bisulfite TreatmentGenomic DNA was obtained from primary carcinomas and nonmalignant cells by digestion with proteinase K (Life Technologies, Inc., Grand Island, NY) for 24 hours at 37°C, followed by extraction with phenol:chloroform (1:1).25Herrmann BG Frischauf AM Isolation of genomic DNA.Methods Enzymol. 1987; 152: 180-183Crossref PubMed Scopus (257) Google Scholar For bisulfite treatment, 1 μg of genomic DNA was denatured by NaOH and modified by sodium bisulfite, which converts all unmethylated cytosines to uracil while methylated cytosines remain unchanged.26Wang RY Gehrke CW Ehrlich M Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues.Nucleic Acids Res. 1980; 8: 4777-4790Crossref PubMed Scopus (195) Google Scholar The modified DNA was purified using a Wizard DNA purification kit (Promega, Madison, WI), treated with NaOH to desulfonate, precipitated with ethanol, and resuspended in water.Standard MSP AssayAberrant promoter methylation of CDH1 was determined by the method of MSP assay as reported by Herman and colleagues27Herman 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 (5203) Google Scholar using primers specific for CDH1-methylated and -unmethylated sequences.28Graff JR Herman JG Myohanen S Baylin SB Vertino PM Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation.J Biol Chem. 1997; 272: 22322-22329Crossref PubMed Scopus (285) Google Scholar The polymerase chain reaction (PCR) amplicon of the methylated form encompassed nucleotides 945 and 1122 in the sequence of GenBank accession no. L34545. DNA from the lung cancer cell line NCI-H187 was used as a positive control for methylated alleles. Water blanks were included with each assay. PCR products were visualized on 2% agarose gels stained with ethidium bromide. Results were confirmed by repeating bisulfite treatment and MSP assays for all samples.Semiquantitative Real-Time MSP AssaySodium bisulfite-treated genomic DNA was amplified by fluorescence-based real-time MSP by using TaqMan technology (Perkin Elmer Corp., Foster City, CA) as described previously.29Eads CA Danenberg KD Kawakami K Saltz LB Danenberg PV Laird PW CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression.Cancer Res. 1999; 59: 2302-2306PubMed Google Scholar, 30Jeronimo C Usadel H Henrique R Oliveira J Lopes C Nelson WG Sidransky D Quantitation of GSTP1 methylation in non-neoplastic prostatic tissue and organ-confined prostate adenocarcinoma.J Natl Cancer Inst. 2001; 93: 1747-1752Crossref PubMed Scopus (267) Google Scholar We performed the MSP with the Gene Amp 5700 Sequence Detection System (Perkin Elmer Corp.). In brief, oligonucleotide primers were designed to specifically amplify bisulfite-converted DNA within the promoter of the CDH1 gene, and a probe was designed to anneal specifically within the amplicon during extension. For the internal reference gene, MYOD1, the primers and probe were designed to avoid CpG nucleotides. Thus, amplification of MYOD1 occurs independent of its methylation status, whereas the amplification of CDH1 is proportional to the degree of cytosine methylation within the amplicon. The methylation ratio was defined as the ratio of the fluorescence emission intensity values for the CDH1 PCR products to those of the MYOD1 PCR products, multiplied by 1000. This ratio was used as a measure for the relative level of methylated CDH1 alleles in the particular sample. The sequences of the primers and probe used to amplify and detect methylated CDH1 were 5′-AATTTTAGGTTAGAGGGTTATCGCGT-3′ (forward primer), 6FAM-5′-CGCCCACCCGACCTCGCAT-3′-TAMRA (probe), and 5′-tccccaaaacgaaactaacgac-3′ (reverse primer). The amplicon encompassed nucleotides 842 and 911 in the sequence of GenBank accession no. L34545. The sequences of the primers and probe used to amplify and detect MYOD1 were 5′-CCAACTCCAAATCCCCTCTCTAT-3′ (forward primer), 6FAM-5′-TCCCTTCCTATTCCTAAATCCAACCTAAATACCTCC-3′-TAMRA (probe), and 5′-TGATTAATTTAGATTGGGTTTAGAGAAGGA-[primes]3 (reverse primer). Semi-quantitative real-time MSP assays were performed in a reaction volume of 25 μl by using components supplied in a TaqMan PCR Core Reagent Kit (Perkin-Elmer Corp.). Separate amplification assays were performed for CDH1 and MYOD1; each assay was performed in duplicate. The final reaction mixtures contained the forward and reverse primers at 600 nmol/L each; the probe at 200 nmol/L; 200 μmol/L each of deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate; 400 μmol/L deoxyuridinetriphosphate; 5.5 mmol/L MgCl2; 1× TaqMan Buffer A; 1 U of Amplitaq Gold DNA polymerase (Perkin Elmer Corp.); and 3 μl bisulfite-converted genomic DNA. PCR was performed under the following conditions: 95°C for 12 minutes, followed by 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. We used DNA from NCI-H187 cells in which CDH1 is methylated (positive control), DNA from NCI-H1395 cells in which CDH1 is not methylated (negative control), and two wells that contained water instead of DNA (control for PCR specificity). We used serial dilutions of the positive control DNA to create a standard curve (1 to 1000 ng).Immunohistochemistry and Automated Cellular ImagingImmunostaining was performed at room temperature and performed on the DAKO Autostainer (DAKO, Carpinteria, CA). Reagents were used as supplied in the Envision Plus Detection Kit (DAKO). DAKO Target Retrieval Solution, pH 6.0, was used. Optimum primary antibody dilutions were predetermined using known positive control tissues. A known positive control section was included in each run to assure proper staining. Staining was performed using a DAKO Autostainer.Evaluation of immunostaining was performed using a recently developed system of image analysis, the Automated Cellular Imaging System (ACIS) (ChromaVision Medical Systems, Inc., San Juan, CA)31Wang S Saboorian MH Frenkel EP Haley BB Siddiqui MT Gokaslan S Wians Jr, FH Hynan L Ashfaq R Assessment of HER-2/neu status in breast cancer. Automated cellular imaging system (ACIS)-assisted quantitation of immunohistochemical assay achieves high accuracy in comparison with fluorescence in situ hybridization assay as the standard.Am J Clin Pathol. 2001; 116: 495-503Crossref PubMed Scopus (127) Google Scholar according to the manufacturer's instructions. The ACIS system consists of an automated robotic bright-field microscope module, a computer, and a Windows NT-based software interface. The robotic microscope module scans the immunohistochemically stained slides, and a computer monitor displays the digitized tissue images. The ACIS system, as described on the manufacturer's website (www.chromavision.com), is able to distinguish cell membrane staining from cytoplasmic staining, using so-called color-space transformation proprietary technology. Only tumor cells were selected for analysis by image analysis. An average score for 10 selected areas most positive for immunostaining was calculated for each sample, and a continuous score generated for the entire sample set.Data AnalysisStatistical differences between groups were examined using Fisher's exact tests. The quantitative ratios of different groups were compared using the Mann-Whitney U nonparametric test. Correlation value was analyzed by the Pearson correlation test. All statistical tests were two-sided. For all tests, probability values of <0.05 were regarded as statistically significant.ResultsStandard MSP AssayMSP assay for CDH1 was performed in 56 infiltrating breast carcinomas (51 ductal and 5 lobular) and 17 corresponding nonmalignant breast tissues and 10 samples of buccal mucosa. The representative MSP assay examples were illustrated in Figure 1. In breast carcinoma samples, which consisted of mixtures of tumor cells and nonmalignant cells, either only the unmethylated band was present or both methylated and unmethylated bands were present. The presence of unmethylated CDH1 promoter sequences in all of the tissues analyzed reflected the presence of contaminating nonmalignant cells and confirmed the integrity of the DNA in these samples.Fifty-six breast cancer specimens were analyzed by standard nonquantitative MSP assay. Of these 24 (43%) were positive. The various intensity of the bands demonstrated heterogeneity, and 12 (21%) of the tumors gave a relatively weak (+) band and 12 (21%) gave a strong (++) band. Of interest, all 5 infiltrating lobular carcinomas (100%) and 19 of 51 (37%) of infiltrating ductal carcinomas were methylated (P = 0.02). Seventeen corresponding adjacent nonmalignant tissues were available and of these two, 12% gave weak bands. There was no methylation detected in DNAs from the buccal mucosa (n = 10) of healthy nonsmoking volunteers. Examples of the bands are presented in Figure 1.Real-Time MSP AssayAll of the 56 available breast cancer specimens were analyzed by semiquantitative real-time PCR. No fluorescent signal could be detected in 14 (25%) samples. Of the 42 samples that gave signals, the values ranged from 0.1 to 24.1 (median, 2.7; mean, 5.5).Correlation between Standard and Real-Time MSP AssaysWe correlated the results of the two MSP assays (Figure 2). The mean value (by real-time MSP assay) of the 32 samples scored negative by the standard MSP assay was 0.4, the mean value of the 12 samples scored as weak positive was 5.3, and the mean value of the 12 samples scored as strong positive was 12.8. The mean values of the three groups were significantly different (Figure 2).Figure 2The relationship between quantitative ratio by semiquantitative real-time MSP assay and standard MSP assay. MSP results were classified into three groups based on the intensity of MSP product: negative (−, white circle), relatively weak positive (+, gray circle); and relatively strong positive (++, dark gray circle). Dotted lines indicate the mean of each group. n = sample number. Values of 0.01 really represent completely negative samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Of the 32 samples scored negative by standard MSP assay, 14 samples lacked a fluorescent signal and were scored negative by real-time MSP assay. Of the remaining 18 samples, the mean value was 0.7, and the mean value for the whole group was 0.4. Based on our results we selected a value of 1.0 as the cutoff between methylation-negative and -positive samples. Using this value, the concordance between the two assays was 91%.Loss of Cdh1 Expression by ImmunostainingImmunostaining for Cdh1 was performed on a subset of 34 primary breast carcinoma samples and their adjacent nonmalignant tissues and lymph node metastases, when available. Although all of these samples were examined microscopically, only the primary tumors were scored by image analysis.Immunostaining was limited to epithelial cells (normal, premalignant, malignant). Stromal cells, including lymphocytes and blood vessels, were completely negative. Of the 34 samples, the majority of the samples (20, 59%) demonstrated heterogeneity of staining in the primary tumor, and 12 (35%) were homogeneously negative, including all 4 (100%) of infiltrating lobular carcinomas. Only two samples (6%) demonstrated strong staining throughout the tumor. The corresponding in situ components and lymph node metastases also showed heterogeneity. The heterogeneity of staining indicated the need for a quantitative scoring system based on both the staining intensity and the percentage of positive cells. In general, the normal ducts and lobules demonstrated intense uniform staining when the tumor stained homogeneously, but showed focal loss of staining when the tumor demonstrated heterogeneity. Ducts in the vicinity of negatively stained tumors usually also demonstrated a negative pattern. Of interest, foci of hyperplasia and apocrine metaplasia stained less intensely and less uniformly than the corresponding normal ducts. Examples of these staining patterns are demonstrated in Figure 3.Figure 3Immunostaining of Cdh1 protein in breast carcinomas and adjacent tissues. A: Normal ducts showing uniform strong positive Cdh1 immunostaining. B: Normal duct showing strong immunostaining, whereas duct with apocrine metaplasia (arrow) shows decreased staining. C: Normal ducts showing loss of expression except for one focus of cells (arrow). D: Homogeneously strong positive staining in ductal carcinoma in situ. E: Clusters of metastatic ductal carcinoma cells in afferent lymphatics of an axillary lymph node showing heterogeneous expression (arrow). F: Completely Cdh1-negative lobular carcinoma. Normal ducts (arrows) surrounded by malignant cells also show loss of expression. CDH1 methylation status by standard MSP assay was negative in the corresponding tumors of the samples illustrated in A, B, and D and positive in samples illustrated in C, E, and F. Numbers below figures indicate sample identification number. Original magnifications: ×200 (A, F); ×400 (B, C); ×600 (E); ×800 (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Using the ACIS image analysis system, Cdh1 immunostaining was scored as a continuous variable with values within the range 0 to 5 Cdh1 staining index units. The range of the 34 test samples was 0 (12 samples) to 2.7, and the mean value of all samples was 0.86. We correlated the protein expression values with degree of methylation as determined by real-time PCR (Figure 4). There was a inverse correlation between these two sets of values, with a r value of 0.47 and a P value of 0.005. However, four breast cancers were completely negative for both methylation and protein expression.Figure 4The correlation of Cdh1 staining index (CSI) and by semiquantitative real-time MSP assay (quantitative ratio).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionCDH1 is a potent invasion/tumor suppressor of breast cancer, and partial or complete loss of expression has been found to correlate with poor prognosis in breast cancer patients.32Asgeirsson KS Jo JG Tryggvadottir L Olafsdottir K Sigurgeirsdottir JR Ingvarsson S Ogmundsddottir HM Altered expression of E-cadherin in breast cancer. Patterns, mechanisms and clinical significance.Eur J Cancer. 2000; 36: 1098-1106Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Cdh1 is inactivated in a high percentage of breast cancers, especially lobular carcinomas. The CDH1 gene is located on human chromosome 16q22.1, a region frequently demonstrating allelic loss in sporadic breast cancer,33Cheng CW Wu PE Yu JC Huang CS Yue CT Wu CW Shen CY Mechanisms of inactivation of E-cadherin in breast carcinoma: modification of the two-hit hypothesis of tumor suppressor gene.Oncogene. 2001; 20: 3814-3823Crossref PubMed Scopus (195) Google Scholar thus providing a mechanism of inactivation of the second allele. Because methylation of CDH1 is a dynamic, plastic process affected by a variety of local conditions,20Graff JR Herman JG Lapidus RG Chopra H Xu R Jarrard DF Isaacs WB Pitha PM Davidson NE Baylin SB E-cadherin expression is silenced by DNA hypermethylation in human breast and prostate carcinomas.Cancer Res. 1995; 55: 5195-5199PubMed Google Scholar down-regulation of its protein product is frequently heterogeneous, complicating interpretation of expression and its correlation with clinicopathological parameters. Variations in the overall level of methylation may be the result of 1) a variable degree of methylation in the promoter sequence of the gene; 2) mixtures of methylated and unmethylated tumor cells; and 3) varying percentages of nontumor and tumor cell mixtures. In addition, protein expression may be down-regulated by mechanisms other than methylation.There was an excellent correlation between the nonquantitative standard MSP assay and the semiquantitative real-time PCR assay. The wide range of positive values by the real-time PCR assay reflected the known heterogeneity of expression, a finding that was confirmed by immunostaining. There was a significant inverse relationship between real-time PCR and image analysis results. Although methylation seemed to be the major mechanism of gene silencing, other mechanisms including gene mutations (especially in lobular carcinomas)33Cheng CW Wu PE Yu JC Huang CS Yue CT Wu CW Shen CY Mechanisms of inactivation of E-cadherin in breast carcinoma: modification of the two-hit hypothesis of tumor suppressor gene.Oncogene. 2001; 20: 3814-3823Crossref PubMed Scopus (195) Google Scholar, 34Berx G Cleton-Jansen AM Strumane K de Leeuw WJ Nollet F van Roy F Cornelisse C E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain.Oncogene. 1996; 13: 1919-1925PubMed Google Scholar, 35Droufakou S Deshmane V Roylance R Hanby A Tomlinson I Hart IR Multiple ways of silencing E-cadherin gene expression in lobular carcinoma of the breast.Int J Cancer. 2001; 92: 404-408Crossref PubMed Scopus (190) Google Scholar, 36Pierceall WE Woodard AS Morrow JS Rimm D Fearon ER Frequent alterations in E-cadherin and alpha- and beta-catenin expression in human breast cancer cell lines.Oncogene. 1995; 11: 1319-1326PubMed Google Scholar, 37Hiraguri S Godfrey T Nakamura H Graff J Collins C Shayesteh L Doggett N Johnson K Wheelock M Herman J Baylin S Pinkel D Gray J Mechanisms of inactivation of E-cadherin in breast cancer cell lines.Cancer Res. 1998;
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