Molecular detection of noninvasive and invasive bladder tumor tissues and exfoliated cells by aberrant promoter methylation of laminin-5 encoding genes

Laminin-5 (LN5) anchors epithelial cells to the underlying basement membrane, and it is encoded by three distinct genes: LAMA3, LAMB3, and LAMC2. To metastasize and grow, cancer cells must invade and destroy the basement membrane. Our previous work has shown that epigenetic inactivation is a major mechanism of silencing LN5 genes in lung cancers. We extended our methylation studies to resected bladder tumors (n = 128) and exfoliated cell samples (bladder washes and voided urine; n = 71) and correlated the data with clinicopathologic findings. Nonmalignant urothelium had uniform expression of LN5 genes and lacked methylation. The methylation frequencies for LN5 genes in tumors were 21-45%, and there was excellent concordance between methylation in tumors and corresponding exfoliated cells. Methylation of LAMA3 and LAMB3 and the methylation index were correlated significantly with several parameters of poor prognosis (tumor grade, growth pattern, muscle invasion, tumor stage, and ploidy pattern), whereas methylation of LAMC2 and methylation index were associated with shortened patient survival. Of particular interest, methylation frequencies of LAMA3 helped to distinguish invasive (72%) from noninvasive (12%) tumors. These results suggest that methylation of LN5 genes has potential clinical applications in bladder cancers.     

Aberrant promoter methylation profile of prostate cancers and its relationship to clinicopathological features.

PURPOSE: We investigated the aberrant methylation profile of prostate cancers and correlated the data with clinical findings. EXPERIMENTAL DESIGN: Gene promoter methylation was analyzed in 101 prostate cancer samples. In addition, we analyzed 32 nonmalignant prostate tissue samples, which included 25 with benign disease, benign prostatic hypertrophy, or prostatitis, and 7 normal tissues adjacent to cancer. The methylation status of 10 genes was determined. The methylation index (MI) was calculated as a reflection of the methylated fraction of the genes examined. RESULTS: Methylation percentages of the genes tested in prostate cancers were: RARbeta, 53%; RASSF1A, 53%; GSTP1, 36%; CDH13, 31%; APC, 27%; CDH1, 27%; FHIT, 15%; p16(INK4A), 3%; DAPK, 1%; and MGMT, 0%. Methylation percentages in nonmalignant tissues were much lower. For clinicopathological correlations, we divided the cancer cases into low (6 or less) or high (7 or more) Gleason score (GS) groups, and into low (8 ng/ml or less) or high (greater than 8 ng/ml) preoperative serum prostate-specific antigen (PSA) groups. Methylation of RASSF1A, GSTP1, RARbeta, and CDH13 genes was significantly more frequent in the high GS group than in the low GS group. Methylation of RASSF1A, CDH1, and GSTP1 genes was significantly more frequent in the high PSA group than in the low PSA group. The median MIs were significantly higher in the high GS and the high PSA groups. According to the Spearman rank-correlation test, there was significant correlation between MI and GS (coefficient = 0.43, P < 0.0001) and the preoperative serum PSA (coefficient = 0.37, P = 0.0003). CONCLUSIONS: Our results indicate that the methylation profile of prostate cancers correlates with clinicopathological features of poor prognosis.     

Inactivation of cyclin D2 gene in prostate cancers by aberrant promoter methylation.

PURPOSE: Loss or abnormal expression of Cyclin D2, a crucial cell cycle-regulatory gene, has been described in human cancers; however, data for prostate tumors are lacking. We investigated the epigenetic silencing of Cyclin D2 gene in prostate cancers and correlated the data with clinicopathological features. EXPERIMENTAL DESIGN: Cyclin D2 promoter methylation was analyzed in 101 prostate cancer samples by methylation-specific PCR. In addition, we analyzed 32 nonmalignant prostate tissue samples, which included 24 samples of benign disease, benign prostatic hypertrophy, or prostatitis and 7 normal tissues adjacent to cancer. The methylation status of Cyclin D2 was correlated with the methylation of nine other tumor suppressor genes published previously from our laboratory on the same set of samples (R. Maruyama et al., Clin. Cancer Res., 8: 514-519, 2002). The methylation index was determined as a reflection of the methylated fraction of the genes examined. RESULTS: The frequency of methylation of Cyclin D2 promoter was significantly higher in prostate cancers (32%) than in nonmalignant prostate tissues (6%; P = 0.004), and it was not age related. Aberrant methylation was present at insignificant levels in peripheral blood lymphocytes (8%). We also compared methylation of cyclin D2 with methylation of nine tumor suppressor genes [published previously from our laboratory (R. Maruyama et al., Clin. Cancer Res., 8: 514-519, 2002)] studied in the same set of samples. The concordances between methylation of Cyclin D2 and the methylation of RARbeta, GSTP1, CDH13, RASSF1A, and APC were statistically significant, whereas methylation of P16, DAPK, FHIT, and CDH1 were not significant. The differences in methylation index between malignant and nonmalignant tissues for all 10 genes were statistically significant (P < 0.0001). Among clinicopathological correlations, the high Gleason score group had significantly greater methylation frequency of Cyclin D2 (42%; P = 0.004). Although the high preoperative serum prostate-specific antigen (PSA) group did not have significantly greater methylation frequency, methylation of Cyclin D2 had higher mean PSA value. Also, the prostate cancers in the high Gleason score group had high mean values of PSA. CONCLUSIONS: Our results indicate that methylation of Cyclin D2 in prostate cancers correlates with clinicopathological features of poor prognosis. These findings are of biological and potential clinical importance.     

Sun exposure related methylation in malignant and non-malignant skin lesions

We investigated the aberrant promoter methylation status of 12 genes in skin lesions, both malignant (basal cell carcinomas (BCCs), n=68 and squamous cell carcinomas (SCCs), n=35) and non-malignant (tags, n=58) skin lesions and compared the results of lesions from sun exposed (SE) and sun protected (SP) regions. Methylation was studied using a methylation specific PCR (MSP) and methylation of CDH1 was also measured using a semi-quantitative fluorescence based real-time MSP method. The methylation index (MI) was calculated as the methylated fraction of the genes examined. In this report, we found high frequencies of methylation of several known or suspected tumor suppressor genes in tags and skin cancers. Among the 12 genes, for the cadherin genes CDH1 and CDH3 and for two of the laminin 5 encoding genes LAMA3 and LAMC2 methylation frequencies greater than 30% were noted in one or more specimen types. We investigated whether methylation was tumor related. Surprisingly, the differences in the methylation profile of genes among the three specimen types were modest, and the MI, indicators of overall methylation frequencies, was nearly identical. However, significant differences were noted in the frequencies of methylation among the three specimen types for the genes RASSF1A (P=0.002), CDH1 (P=0.007) and one or more of three CAD genes (P=0.02). Methylation was highly significantly related to sun exposure, and sun protected specimens had little or no methylation. As methylation of CDH1 was completely SE specific we analyzed all the skin samples using a semi-quantitative real-time PCR assay for the CDH1 gene. The concordance between standard MSP and real-time MSP for all the samples (n=161) was 75% (P<0.0001). While weak signals were detected in the SP samples by real time PCR, the differences between SE and SP specimens were 148 fold for tags and 390 fold for BCCs. These differences were highly significant (P<0.0001). These findings suggest that methylation commences in UV exposed skin at a relatively early age and occurs in skin prior to the onset of recognizable preneoplastic changes.     

Aberrant promoter methylation of multiple genes in non-small cell lung cancers

Aberrant methylation of CpG islands acquired in tumor cells in promoter regions is one method for loss of gene function. We determined the frequency of aberrant promoter methylation (referred to as methylation) of the genes retinoic acid receptor beta-2 (RARbeta), tissue inhibitor of metalloproteinase 3 (TIMP-3), p16INK4a, O6-methylguanine-DNA-methyltransferase (MGMT), death-associated protein kinase (DAPK), E-cadherin (ECAD), p14ARF, and glutathione S-transferase P1 (GSTP1) in 107 resected primary non-small cell lung cancers (NSCLCs) and in 104 corresponding nonmalignant lung tissues by methylation-specific PCR. Methylation in the tumor samples was detected in 40% for RARbeta, 26% for TIMP-3, 25% for p16INK4a, 21% for MGMT, 19% for DAPK, 18% for ECAD, 8% for p14ARF, and 7% for GSTP1, whereas it was not seen in the vast majority of the corresponding nonmalignant tissues. Moreover, p16INK4a methylation was correlated with loss of p16INK4a expression by immunohistochemistry. A total of 82% of the NSCLCs had methylation of at least one of these genes; 37% of the NSCLCs had one gene methylated, 22% of the NSCLCs had two genes methylated, 13% of the NSCLCs had three genes methylated, 8% of the NSCLCs had four genes methylated, and 2% of the NSCLCs had five genes methylated. Methylation of these genes was correlated with some clinicopathological characteristics of the patients. In comparing the methylation patterns of tumors and nonmalignant lung tissues from the same patients, there were many discordancies where the genes methylated in nonmalignant tissues were not methylated in the corresponding tumors. This suggests that the methylation was occurring as a preneoplastic change. We conclude that these findings confirm in a large sample that methylation is a frequent event in NSCLC, can also occur in smoking-damaged nonmalignant lung tissues, and may be the most common mechanism to inactivate cancer-related genes in NSCLC.     

Aberrant promoter methylation profile of bladder cancer and its relationship to clinicopathological features.

We investigated the aberrant promoter methylation profile of bladder cancers and correlated the data with clinicopathological findings. The methylation status of 10 genes was determined in 98 surgically resected bladder cancers, and we calculated the median methylation index (MI), a reflection of the methylated fraction of the genes tested. Methylation frequencies of the genes tested in bladder cancers were 36% for CDH1, 35% for RASSF1A and APC, 29% for CDH13, 16% for FHIT, 15% for RAR beta, 11% for GSTP1, 7% for p16(INK4A), 4% for DAPK, and 2% for MGMT. Methylation of four of the individual genes (CDH1, RASSF1A, APC, and CDH13) and the MI were significantly correlated with several parameters of poor prognosis (tumor grade, growth pattern, muscle invasion, tumor stage, and ploidy pattern). Methylation of CDH1, FHIT, and a high MI were associated with shortened survival. CDH1 methylation positive status was independently associated with poor survival in multivariate analyses. Our results suggest that the methylation profile may be a potential new biomarker of risk prediction in bladder cancer.     

Methylation of multiple genes in prostate cancer and the relationship with clinicopathological features of disease

Promoter methylation plays an important role in the inactivation of tumor suppressor genes during tumorigenesis. We examined the methylation status of glutathione s-transferase Pi1 (GSTP1), retinoic acid receptor beta (RARB), CD44, E-cadherin (ECAD), RAS association domain family protein 1A (RASSF1A) and endothelin B receptor (EDNRB) genes in 81 prostate cancer and 42 benign prostatic hyperpasia specimens. Genomic DNA was isolated from archived formaldehyde-fixed and paraffin-embedded tissue blocks. Methylation-specific PCR (MSP) was carried out after bisulfite treatment of genomic DNA. Methylation frequencies in prostate cancer and benign prostatic hyperplasia were 72% and 5% for GSTP1, 40% and 0% for RARB, 72% and 38% for CD44, 61% and 14% for ECAD, 49% and 19% for RASSF1A and 72% and 62% for EDNRB, respectively. Methylation of GSTP1, RARB, CD44, ECAD and RASSF1A, but not of EDNRB was detected at a statistically higher frequency in prostate cancer than in the benign prostatic hypertrophy specimens. Methylation of RARB occurred more frequently in early onset (age <55 years) as compared to late onset disease (age >70 years) (odds ratio, 8.6; 95% CI, 1.4-51.4; P=0.02). Methylation of RARB also occurred more frequently in stage III as compared to stage II disease (odds ratio, 3.2; 95% CI, 1.1-8.8; P=0.03). A methylation index (MI) was calculated as the total number of genes methylated, excluding EDNRB. A trend toward higher MI was noted in stage III as compared to stage II disease, and in Gleason score 7 as compared to Gleason score 6 tumors. Our results suggest that the methylation of selected genes in prostate cancers correlates with clinicopathological features of poor prognosis.     

肝癌血浆游离DNA甲基化谱检测及临床意义

  目的  在外周血游离DNA中筛选肝癌特异的甲基化谱。  方法  收集55例肝癌和54例慢性肝病患者血浆标本,应用甲基化特异性PCR方法检测血浆游离DNA的12个抑癌基因甲基化状态。  结果  在肝癌组中,APC、p16、GSTP1、Cyclin D2、LHX1、TFPI2、DKK2、DKK3、SFRP2、14-3-3 sigma、ppENK、NPTX2基因的甲基化频率分别为78.18%、63.64%、58.18%、49.09%、49.09%、47.27%、40.00%、18.18%、16.36%、9.09%、7.27%和5.45%,而在慢性肝病组中,各基因的甲基化频率分别为27.78%、22.22%、7.41%、3.70%、16.67%、37.04%、37.04%、11.11%、20.37%、7.41%、7.41%和9.26%。APC、Cyclin D2、TFPI2、DKK3和GSTP1基因在肝癌组中的甲基化频率高于慢性肝病组（P < 0.01）。将它们组成5基因甲基化谱,肝癌组甲基化指数（中位值为0.6,IQR 0.4~0.8）显著高于慢性肝病组（中位值为0.2,IQR 0~0.2）。在肝癌组中,甲基化指数与患者年龄具有相关性,年龄大者甲基化指数较高,而甲基化指数与其它临床病理参数未见相关性。甲基化指数与肝癌患者的无瘤生存期及总体生存期均未见相关性。  结论  该甲基化谱可能成为肝癌辅助诊断的核酸标志物。      

Multigene methylation analysis for detection and staging of prostate cancer.

PURPOSE: Aberrant gene promoter methylation profiles have been well-studied in human prostate cancer. Therefore, we rationalize that multigene methylation analysis could be useful as a diagnostic biomarker. We hypothesize that a new method of multigene methylation analysis could be a good diagnostic and staging biomarker for prostate cancer. EXPERIMENTAL DESIGN: To test our hypothesis, prostate cancer samples (170) and benign prostatic hyperplasia samples (69) were examined by methylation-specific PCR for three genes: adenomatous polyposis coli (APC), glutathione S-transferase pi (GSTP1), and multidrug resistance 1 (MDR1). The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. We further investigated whether methylation score (M score) can be used as a diagnostic and staging biomarker for prostate cancer. The M score of each sample was calculated as the sum of the corresponding log hazard ratio coefficients derived from multivariate logistic regression analysis of methylation status of various genes for benign prostatic hyperplasia and prostate cancer. The optimal sensitivity and specificity of the M score for diagnosis and for staging of prostate cancer was determined by receiver-operator characteristic (ROC) curve analysis. A pairwise comparison was employed to test for significance using the area under the ROC curve analysis. For each clinicopathologic finding, the association with prostate-specific antigen (PSA) failure-free probability was determined using Kaplan-Meier curves and a log-rank test was used to determine significance. The relationship between M score and clinicopathologic findings was analyzed by either the Mann-Whitney U test, Kruskal-Wallis test, or the Spearman rank correlation test. RESULTS: The frequency of positive methylation-specific PCR bands for APC, GSTP1, and MDR1 genes in prostate cancer samples was 64.1%, 54.0%, and 55.3%, respectively. In benign prostatic hyperplasia samples, it was 8.7%, 5.8%, and 11.6%, respectively. There was a significant correlation of M score with high pT category (P < 0.001), high Gleason sum (P < 0.001), high preoperative PSA (P = 0.027), and advanced pathologic features. For all patients, the M score had a sensitivity of 75.9% and a specificity of 84.1% as a diagnostic biomarker using a cutoff value of 1.0. In patients with low or borderline PSA levels (<10.0 ng/mL), the M score was significantly higher in prostate cancers than in benign prostatic hyperplasias (2.635 +/- 0.200 and 0.357 +/- 0.121, respectively). ROC curve analysis revealed that the M score had a sensitivity of 65.4% and a specificity of 94.2% when 1.0 was used as a cutoff value. For all patients, M score can distinguish organ-confined (< or =pT(2)) from locally advanced cancer (> or =pT(3)) with a sensitivity of 72.1% and a specificity of 67.8%. Moreover, considering patients with PSA levels of <10 ng/mL, the M score has a sensitivity of 67.1% and a specificity of 85.7%. The ROC curve analysis showed a significant difference between M score and PSA (P = 0.010). CONCLUSIONS: This is the first report demonstrating that M score is a new method for multigene methylation analysis that can serve as a good diagnostic and staging biomarker for prostate cancer.     

5' CpG island methylation of the FHIT gene is correlated with loss of gene expression in lung and breast cancer

Allele loss and loss of expression of fragile histidine triad (FHIT), a putative tumor suppressor gene located in chromosome region 3p14.2, are frequent in several types of cancers. Tumor-acquired methylation of promoter region CpG islands is one method for silencing tumor suppressor genes. We investigated 5' CpG island methylation of the FHIT gene in 107 primary non-small cell lung cancer (NSCLC) samples and corresponding nonmalignant lung tissues, 39 primary breast carcinomas, as well as in 49 lung and 22 breast cancer cell lines by a methylation-specific PCR assay. In addition, we analyzed brushes from the bronchial epithelium of 35 heavy smokers without cancer. FHIT methylation was detected in 37% of primary NSCLCs, 31% of primary breast cancers, and 65% of lung and 86% of breast cancer cell lines. The frequency of methylation in small cell and NSCLC cell lines were identical. Methylation was found in 9% of the corresponding nonmalignant lung tissues and in 17% of bronchial brushes from heavy cigarette smokers. FHIT methylation was significantly correlated with loss of FHIT mRNA expression by Northern blot analysis in lung cancer cell lines and with loss of Fhit expression in NSCLC and breast tumors by immunostaining. We conclude that methylation of FHIT is a frequent event in NSCLC and breast cancers and is an important mechanism for loss of expression of this gene. Methylation of FHIT commences during lung cancer pathogenesis and may represent a marker for risk assessment.     

胰腺组织p15、p16抑癌基因CpG岛甲基化研究

Objective： To detect the aberrant methylation patterns in the CpG islands of p16 and p15 tumor suppressor genes, and to analyze its correlation with pancreatic carcinogenesis and with clinicopathological characteristics of patients with pancreatic cancer （PC）. Methods： The methylation-specific polymerase chain reaction （MSP） method was used to monitor methylation patterns in the CpG islands of p15 and p16 genes from 29 cases of PC and 3 cases of chronic pancreatitis （CP） paraffin-embedded tissue, as well as 2 cases of normal liver tissues and 12 cases of normal blood samples. Results： p15 and p16 genes were detected to show unmethylation patterns and no amplification using methylation-specific primers in control group. The aberrant methylation rates of p16 in carcinoma tissue and adjacent noncarcinoma tissue were 37.9% （11 of 29 cases） and 34.5% （10 of 29 cases） respectively. Of the 11 aberrant methylated samples, 5 showed complete methylation and 6 hemimethylation. The methylation rates of p15 gene in carcinoma tissue and adjacent noncarcinoma tissue were 27.5% （8/29） and 24.4% （7/29） respectively. Of the 8 aberrant methylated samples, 3 showed complete methylation and 5 hemimethylation. In 6 PC samples, aberrant methylation in CpG islands of both p15 and p16 genes existed simultaneously. The aberrant methylation patterns in CpG islands of p15 and p16 genes had no close correlation with the clinicopathological characteristics （age, sex, smoking, volume of primary tumor, differentiation, clinical stage and histological classification） of the patients with PC （P〉0.05）. Conclusion： The aberrant methylation in CpG islands of p15 and p16 genes could be regarded as an early molecular event in PC and had no close correlation with the clinicopathological characteristics of the patients with PC.     

Frequent epigenetic inactivation of p53 target genes in seminomatous and nonseminomatous germ cell tumors

Hypermethylation of tumor-suppressor genes has been implicated in the pathogenesis of human cancers. This study was designed to examine the methylation profiles of a selected group of p53 target genes (APAF-1, CASP-8, DAPK-1, IGFBP-3) and to correlate the findings with the histopathological characterization of testicular germ cell tumors (TGCT). Promoter methylation status was analysed by highly sensitive real-time methylation-specific PCR in 46 primary TGCTs (26 seminomas and 20 nonseminomas) and 15 normal testicular tissue samples. APAF-1 methylation was detected in all of the seminomatous and nonseminomatous TGCTs as well as in 60% of normal testicular tissue. Methylation of DAPK-1 was frequent in seminomas (50%) and nonseminomas (20%), but not in normal testicular tissue (6%). The degree of DAPK-1 methylation correlated with the clinical stage of the disease (P=0.05) and was useful in differentiating seminomatous from nonseminomatous, and malignant from nonmalignant testicular tissue (P=0.04 and 0.02, respectively). The APAF-1 methylation index achieved a highly significant differentiation between seminomatous or nonseminomatous tissue and nonmalignant testicular tissue (P=0.0001). In testicular tumorigenesis, promoter methylation of specific p53 target genes occurs at early stage but to varying degrees. Methylation also occurs in normal testicular tissue, which is in contrast to findings in other urogenital malignancies. Further studies will be necessary to determine whether the methylation level may be used as marker for risk estimation, especially in clinical stage I disease.     

Colorectal Cancers with both p16 and p14 Methylation Show Invasive Characteristics

Recent studies indicated that p16 and p14 inactivation owing to promoter methylation was important for colorectal tumorigenesis. In this study, we examined the methylation status of these genes in 86 primary colorectal cancers using methylation-specific PCR (MSP) and correlated the results with the clinicopathological features of the patients. Aberrant promoter methylation of p16 and p14 genes was detected in 43 of 86 (50%) and 25 of 86 (29%) colorectal cancers, respectively. Next, we examined the correlation of methylation status with the clinicopathological features. We found a significant difference in maximal tumor size ( P =0.022) when patients with both p16 and p14 methylation were compared to other patients. On the other hand, there was no significant difference in other factors, such as the extent of tumor and Dukes stage. These results suggested that colorectal cancer with both p16 and p14 methylation has the same invasiveness at a smaller size compared to that of the cancer with neither p16 nor p14 methylation. Inactivation of both p16 and p14 genes may result in a malignant change in colorectal cancer cells, leading to advanced cancers with a smaller size than those with p16 or p14 activity.     

RUNX3 promoter region is specifically methylated in poorly-differentiated colorectal cancer

BACKGROUND, MATERIALS AND METHODS: It has recently become clear that RUNX3 expression is frequently silenced by aberrant methylation in gastric cancers. In this study, we investigated the methylation status of the RUNX3 gene in 92 resected primary colorectal cancers using methylation-specific PCR (MSP) and correlated the results with the clinicopathological features of affected patients. RESULTS: Aberrant promoter methylation of the RUNX3 gene was detected in 31 out of 92 (34%) colorectal cancers. A significant difference in histology (p = 0.028) was also found on comparing the RUNX3 methylation of poorly-differentiated colorectal cancers to that of other differentiated ones. CONCLUSION: RUNX3 aberrant methylation might play an important role in colorectal cancers, especially in poorly-differentiated colorectal cancers.     

In silico mining identifies IGFBP3 as a novel target of methylation in prostate cancer

Promoter hypermethylation is central in deregulating gene expression in cancer. Identification of novel methylation targets in specific cancers provides a basis for their use as biomarkers of disease occurrence and progression. We developed an in silico strategy to globally identify potential targets of promoter hypermethylation in prostate cancer by screening for 5' CpG islands in 631 genes that were reported as downregulated in prostate cancer. A virtual archive of 338 potential targets of methylation was produced. One candidate, IGFBP3, was selected for investigation, along with glutathione-S-transferase pi (GSTP1), a well-known methylation target in prostate cancer. Methylation of IGFBP3 was detected by quantitative methylation-specific PCR in 49/79 primary prostate adenocarcinoma and 7/14 adjacent preinvasive high-grade prostatic intraepithelial neoplasia, but in only 5/37 benign prostatic hyperplasia (P < 0.0001) and in 0/39 histologically normal adjacent prostate tissue, which implies that methylation of IGFBP3 may be involved in the early stages of prostate cancer development. Hypermethylation of IGFBP3 was only detected in samples that also demonstrated methylation of GSTP1 and was also correlated with Gleason score > or =7 (P=0.01), indicating that it has potential as a prognostic marker. In addition, pharmacological demethylation induced strong expression of IGFBP3 in LNCaP prostate cancer cells. Our concept of a methylation candidate gene bank was successful in identifying a novel target of frequent hypermethylation in early-stage prostate cancer. Evaluation of further relevant genes could contribute towards a methylation signature of this disease.