CpG Hypermethylation of MDR1 Gene Contributes to the Pathogenesis and Progression of Human Prostate Cancer

Clinical Cancer Research

et al. 2006

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129

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Purpose: We hypothesized that combined methylation analysis of Wnt antagonist genes could serve as a panel of biomarkers for diagnosis, staging, and prognosis in renal cell carcinoma (RCC). Experimental Design: Samples (n = 62) of RCC and corresponding normal renal tissue (NRT) were analyzed using methylation-specific PCR for methylation of six Wnt antagonist genes (sFRP-1, sFRP-2, sFRP-4, sFRP-5, Wif-1, and Dkk-3). To increase the sensitivity/specificity of RCC detection, the methylation score (M score) for multigene methylation analysis was developed. Receiver operator characteristic curve analysis was used to determine the optimal sensitivity/specificity of the M score. In addition, the M score was compared with the clinicopathologic outcome.Thirty-three serum DNA samples were also used to investigate the methylation status of Wnt antagonist genes. Results: The methylation levels of all Wnt antagonists were significantly higher in RCC than in NRT. In multivariate regression analysis, the methylation level of sFRP-1was a significant independent predictor of RCC, whereas for sFRP-2 and sFRP-4 there was a trend toward significance as independent predictors. The M score of Wnt antagonist genes was significantly higher in RCC than in NRT. Overall, the M score had a sensitivity of 79.0% and a specificity of 75.8% (area under the curve, 0.808) as a diagnostic biomarker. In addition, the M score could significantly distinguish grade, pTcategory, M category, and overall survival of RCC patients.The M score was independent of age and gender in predicting overall survival by the Cox proportional hazards model. In RCC patients, 72.7% of the methylation-specific PCR results had identical methylation in samples of tumor and serum DNA. No serum DNA in normal controls showed aberrant methylation of the Wnt antagonist genes. In addition, the methylation status of Wnt antagonist genes in serum DNA was significantly correlated with tumor grade and stage. Conclusions: This is the first report showing that M score analysis of Wnt antagonist genes can serve as an excellent epigenetic biomarker panel for detection, staging, and prognosis of RCC using serum DNA.

Section: Methodsmentioning

confidence: 99%

Wnt Antagonist Family Genes as Biomarkers for Diagnosis, Staging, and Prognosis of Renal Cell Carcinoma Using Tumor and Serum DNA

Clinical Cancer Research

et al. 2006

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129

106

“…The annealing temperature and PCR cycles used for MSP and USP primers were 64jC and 32 cycles, respectively. The sequences of primers for GSTP1 and MDR1, as well as their PCR conditions, were described previously (15,16). In each assay, the absence of DNA template served as negative controls.…”

Section: Methodsmentioning

confidence: 99%

“…The obtained MSP and USP products were analyzed by electrophoresis in 3% agarose gels and stained with ethidium bromide. With ImageJ software (http://rsb.info.nih.gov/ij), relative methylation levels (%) were calculated (15,16,23) by using the area under the curve corresponding to each band (MSP and USP). For methylation analysis of APC, we used 5.3% methylation as a cutoff value, which was the average percentage of methylation in 69 benign prostatic hyperplasia samples.…”

Section: Methodsmentioning

confidence: 99%

“…Using these criteria, MSP positivity for APC was defined as those prostate cancers with a percentage of methylation of >5.3%, and negative methylation was <5.3%. The criteria for MSP positivity of GSTP1 and MDR1 were described previously (15,16).…”

Section: Methodsmentioning

confidence: 99%

“…We have previously shown that the methylation status of GSTP1 or MDR1 is a good biomarker for detecting prostate cancer and correlates with clinicopathologic features (15,16). We hypothesize that multigene methylation analysis can be a good diagnostic and staging biomarker prior to treatment.…”

mentioning

confidence: 93%

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Multigene Methylation Analysis for Detection and Staging of Prostate Cancer

Clinical Cancer Research

et al. 2005

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100

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:Totestourhypothesis,prostate cancer samples (170) andbenignprostatic 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%, and11.6%, respectively.There was a significant correlation of M score with high pTcategory (P <0.001), high Gleason sum (P <0.001), high preoperative PSA (P = 0.027),andadvancedpathologic features.Forallpatients,the Mscorehadasensitivityof75.9% and a specificity of 84.1% as a diagnostic biomarker using a cutoff value of1.0. In patients with low or borderline PSAlevels (<10.0 ng/mL), the Mscorewas significantlyhigherinprostate cancers thanin benignprostatichyperplasias (2.635 F 0.200 and 0.357 F 0.121, respectively). ROC curve analysis revealed that the Mscore hada sensitivity of 65.4% and a specificity of 94.2% when1.0 wasused as a cutoff value. Forallpatients, Mscore candistinguishorgan-confined (VpT 2 ) fromlocallyadvanced cancer (zpT 3 ) with a sensitivity of 72.1% and a specificity of 67.8%. Moreover, consideringpatients 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 ...

Smoking influences aberrant CpG hypermethylation of multiple genes in human prostate carcinoma

et al. 2005

Cancer

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Objective Report a double‐blind, placebo‐controlled study of rituximab in patients with anti–MAG demyelinating polyneuropathy (A‐MAG‐DP). Methods Twenty‐six patients were randomized to four weekly infusions of 375mg/m2 rituximab or placebo. Sample size was calculated to detect changes of ≥1 Inflammatory Neuropathy Course and Treatment (INCAT) leg disability scores at month 8. IgM levels, anti‐MAG titers, B cells, antigen‐presenting cells, and immunoregulatory T cells were monitored every 2 months. Results Thirteen A‐MAG‐DP patients were randomized to rituximab and 13 to placebo. Randomization was balanced for age, electrophysiology, disease duration, disability scores, and baseline B cells. After 8 months, by intention to treat, 4 of 13 rituximab‐treated patients improved by ≥1 INCAT score compared with 0 of 13 patients taking placebo (p = 0.096). Excluding one rituximab‐randomized patient who had normal INCAT score at entry, and thus could not improve, the results were significant (p = 0.036). The time to 10m walk was significantly reduced in the rituximab group (p = 0.042) (intention to treat). Clinically, walking improved in 7 of 13 rituximab‐treated patients. At month 8, IgM was reduced by 34% and anti‐MAG titers by 50%. CD25+CD4+Foxp3+ regulatory cells significantly increased by month 8. The most improved patients were those with high anti‐MAG titers and most severe sensory deficits at baseline. Interpretation Rituximab is the first drug that improves some patients with A‐MAG‐DP in a controlled study. The benefit may be exerted by reducing the putative pathogenic antibodies or by inducing immunoregulatory T cells. The results warrant confirmation with a larger trial. Ann Neurol 2009;65:286–293