Mohammad O. Hoque

A quantitative Promoter methylation profile of prostate cancer

Purpose: Promoter hypermethylation is an alternative pathway for gene silencing in neoplastic cells and a promising cancer detection marker. Although quantitative methylation-specific PCR (QMSP) of the GSTP1 promoter has demonstrated near perfect specificity for cancer detection in prostate biopsies, we postulated that identification and characterization of additional methylation markers might further improve its high (80–90%) sensitivity. Experimental Design: We surveyed nine gene promoters (GSTP1, MGMT, p14/ARF, p16/CDKN2A, RASSF1A, APC, TIMP3, S100A2, and CRBP1) by QMSP in tissue DNA from 118 prostate carcinomas, 38 paired high-grade prostatic intraepithelial neoplasias (HGPIN), and 30 benign prostatic hyperplasias (BPH). The methylation levels were calculated and were correlated with clinical and pathologic indicators. Results: Only the methylation frequencies of GSTP1 and APC were significantly higher in prostate carcinoma compared with BPH (P < 0.001). Methylation levels of GSTP1, APC, RASSF1A, and CRBP1, differed significantly between prostate carcinoma and HGPIN, and/or HGPIN or BPH (P < 0.0001).With QMSP and empirically defined cutoff values, the combined use of GSTP1 and APC demonstrated a theoretical sensitivity of 98.3% for prostate carcinoma, with 100% specificity. Methylation levels were found to correlate with tumor grade (GSTP1 and APC) and stage (GSTP1, RASSF1A, and APC). Conclusions: Our data demonstrate the existence of a progressive increase of promoter methylation levels of several cancer-related genes in prostate carcinogenesis, providing additional markers to augment molecular detection of prostate carcinoma. Because methylation levels of GSTP1, APC, and RASSF1A are associated with advanced grade and stage, QMSP might augment the pathologic indicators currently used to predict tumor aggressiveness.

Quantitative Methylation-Specific Polymerase Chain Reaction Gene Patterns in Urine Sediment Distinguish Prostate Cancer Patients From Control Subjects

PURPOSE Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. PATIENTS AND METHODS We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14(ARF), MGMT, GSTP1, RARbeta2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. RESULTS Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARbeta2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. CONCLUSION Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.

Detection of aberrant methylation of four genes in plasma DNA for the detection of breast cancer.

PURPOSE Novel approaches to breast cancer screening are necessary, especially in the developing world where mammography is not feasible. In this study, we explored the hypothesis that blood-based biomarkers have potential for biomarkers for breast cancer. PATIENTS AND METHODS We first determined the frequency of aberrant methylation of four candidate genes (APC, GSTP1, Rassf1A, and RARbeta2) in primary breast cancer tissues from West African women with predominantly advanced cancers. We used a high-throughput DNA methylation assay (quantitative methylation-specific polymerase chain reaction) to examine plasma from 93 women with breast cancer and 76 controls for the presence of four methylated genes. Samples were randomly divided evenly into training and validation data sets. Cutoff values for gene positivity of the plasma-based assay and the gene panel were determined by receiver operating characteristic curves in the training data set and subsequently evaluated as a screening tool in the validation data set. RESULTS Methylation of at least one gene resulted in a sensitivity of 62% and a specificity of 87%. Moreover, the assay successfully detected 33% (eight of 24) of early-stage tumors. CONCLUSION These data suggest that epigenetic markers in plasma may be of interest for detection of breast cancer. Identification of additional breast cancer specific methylated genes with higher prevalence in early stage cancers would improve this approach.

Quantitative detection of promoter hypermethylation of multiple genes in the tumor, urine, and serum DNA of patients with renal cancer.

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of human cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the urine and serum samples of renal cancer patients. We examined the tumor and the matched urine and serum DNA for aberrant methylation of nine gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-β2, and ARF) from 17 patients with primary kidney cancer by quantitative fluorogenic real-time PCR. An additional 9 urine samples (total, 26) and 1 serum sample (total, 18) also were tested from renal cancer patients. Urine from 91 patients without genitourinary cancer and serum from 30 age-matched noncancer individuals were used as controls. Promoter hypermethylation of at least two of the genes studied was detected in 16 (94%) of 17 primary tumors. Aberrant methylation in urine and serum DNA generally was accompanied by methylation in the matched tumor samples. Urine samples from 91 control subjects without evidence of genitourinary cancer revealed no methylation of the MGMT, GSTP1, p16, and ARF genes, whereas methylation of RAR-β2, RASSF1A, CDH1, APC, and TIMP3 was detected at low levels in a few control subjects. Overall, 23 (88%) of 26 urine samples and 12 (67%) of 18 serum samples from cancer patients were methylation positive for at least one of the genes tested. By combination of urine or serum analysis of renal cancer patients, hypermethylation was detected in 16 of 17 patients (94% sensitivity) with high specificity. Our findings suggest that promoter hypermethylation in urine or serum can be detected in the majority of renal cancer patients. This noninvasive high-throughput approach needs to be evaluated in large studies to assess its value in the early detection and surveillance of renal cancer.

Detection of Promoter Hypermethylation of Multiple Genes in the Tumor and Bronchoalveolar Lavage of Patients with Lung Cancer

Purpose: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the bronchoalveolar lavage (BAL) samples of lung cancer patients. Experimental Design: We examined the tumor and the matched BAL DNA for aberrant methylation of eight gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-β2, and ARF) from 31 patients with primary lung tumors by quantitative fluorogenic real-time PCR. BAL from 10 age-matched noncancer patients was used as a control. Results: Promoter hypermethylation of at least one of the genes studied was detected in all 31 lung primary tumors; 27 (87%) CDH1, 17 (55%) APC, 14 (45%) RASSF1A, 12 (39%) MGMT, 7 (23%) p16, 3 (10%) GSTP1, 3 (10%) RAR-β2, and 0 (0%) ARF. Methylation was detected in CDH1 (48%), APC (29%), RASSF1A (29%), MGMT (58%), p16 (14%), GSTP1 (33%), RAR-β2 (0%), and ARF (0%) of BAL samples from matched methylation-positive primary tumors, and in every case, aberrant methylation in BAL DNA was accompanied by methylation in the matched tumor samples. BAL samples from 10 controls without evidence of cancer revealed no methylation of the MGMT, GSTP1, p16, ARF, or RAR-β2 genes whereas methylation of RASSF1, CDH1, and APC was detected at low levels. Overall, 21 (68%) of 31 BAL samples from cancer patients were positive for aberrant methylation. Conclusion: Our findings suggest that promoter hypermethylation in BAL can be detected in the majority of lung cancer patients. This approach needs to be evaluated in large early detection and surveillance studies of lung cancer.

Evaluation of Promoter Hypermethylation Detection in Body Fluids as a Screening/Diagnosis Tool for Head and Neck Squamous Cell Carcinoma

Purpose: To evaluate aberrant promoter hypermethylation of candidate tumor suppressor genes as a means to detect epigenetic alterations specific to solid tumors, including head and neck squamous cell carcinoma (HNSCC). Experimental Design: Using promoter regions identified via a candidate gene and discovery approach, we evaluated the ability of an expanded panel of CpG-rich promoters known to be differentially hypermethylated in HNSCC in detection of promoter hypermethylation in serum and salivary rinses associated with HNSCC. We did preliminary evaluation via quantitative methylation-specific PCR (Q-MSP) using a panel of 21 genes in a limited cohort of patients with HNSCC and normal controls. Using sensitivity and specificity for individual markers as criteria, we selected panels of eight and six genes, respectively, for use in salivary rinse and serum detection and tested these in an expanded cohort including up to 211 patients with HNSCC and 527 normal controls. Results: Marker panels in salivary rinses showed improved detection when compared with single markers, including a panel with 35% sensitivity and 90% specificity and a panel with 85% sensitivity and 30% specificity. A similar pattern was noted in serum panels, including a panel with 84.5% specificity with 50.0% sensitivity and a panel with sensitivity of 81.0% with specificity of 43.5%. We also noted that serum and salivary rinse compartments showed a differential pattern of methylation in normal subjects that influenced the utility of individual markers. Conclusions: Q-MSP detection of HNSCC in serum and salivary rinses using multiple targets offers improved performance when compared with single markers. Compartment-specific methylation in normal subjects affects the utility of Q-MSP detection strategies.

Involvement of aquaporins in colorectal carcinogenesis

Aquaporins (AQPs) are important in controlling water permeability. As AQP1 is known as a serum-responsive gene, we hypothesized that AQP expression may be involved in the development of human cancer. By reverse transcriptase–polymerase chain reaction analysis, expression of AQPs 1, 3, and 5 was found in seven colon and colorectal cancer cell lines. Western blot analysis confirmed their expression in four of these cell lines. In situ hybridization demonstrated that during colorectal carcinogenesis, the expression of AQPs 1 and 5 was induced in early-stage disease (early dysplasia) and maintained through the late stages of colon cancer development. Expression of AQPs 1 and 5 was maintained even in metastatic lesions in the liver. These findings demonstrate that the expression of several AQPs is found in tumor cells and is associated with an early stage of colorectal cancer development. These novel observations suggest that multiple AQP expression may be advantageous to tumorigenesis, which may lead to a better understanding of colorectal carcinogenesis.

Promoter Hypermethylation as an Independent Prognostic Factor for Relapse in Patients with Prostate Cancer Following Radical Prostatectomy

Purpose: To analyze the prognostic significance of six epigenetic biomarkers (APC, Cyclin D2, GSTP1, TIG1, Rassf1A, and RARβ2 promoter hypermethylation) in a homogeneous group of prostate cancer patients, following radical prostatectomy alone. Patients and Methods: Biomarker analyses were done retrospectively on tumors from 74 prostate cancer patients all with a Gleason score of 3 + 4 = 7 and minimum follow-up period of 7 years. Using quantitative methylation-specific PCR, we analyzed six gene promoters in primary prostate tumor tissues. Time to any progression was the primary end point, and development of metastatic disease and/or death from prostate cancer was a secondary point. The association of clinicopathologic and biomolecular risk factors to recurrence was done using the log-rank test and Cox proportional hazards model for multivariate analysis. To identify independent prognostic factors, a stepwise selection method was used. Results: At a median follow-up time of 9 years, 37 patients (50%) had evidence of recurrence: biochemical/prostate-specific antigen relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression (TTP), the significant factors were age > 60 [hazard ratio (HR), 0.4; 95% confidence interval (95% CI), 0.2-0.8; P = 0.01], hypermethylation of GSTP1 (HR, 0.23; 95% CI; 0.09-0.64; P = 0.004), and hypermethylation of APC (HR, 3.0; 95% CI, 1.42-6.32; P = 0.004). In another multivariate analysis, a profile of hypermethylation of APC and cyclin D2 hypermethylation was significant as well: if either any one was hypermethylated (HR, 1.84; 95% CI, 0.92-3.72; P = 0.09) or if both were hypermethylated (HR, 4.3; 95% CI, 1.52-12.33; P = 0.01). Conclusions: Methylation status of selected genes in the prostate cancer specimen may predict for time to recurrence in Gleason 3 + 4 = 7 patients undergoing prostatectomy. These results should be validated in a larger and unselected cohort.

Inverse Correlation between Cyclin A1 Hypermethylation and p53 Mutation in Head and Neck Cancer Identified by Reversal of Epigenetic Silencing

Aberrant promoter hypermethylation of tumor suppressor genes is proposed to be a common feature of primary cancer cells. We recently developed a pharmacological unmasking microarray approach to screen unknown tumor suppressor gene candidates epigenetically silenced in human cancers. In this study, we applied this method to identify such genes in head and neck squamous cell carcinoma (HNSCC). We identified 12 novel methylated genes in HNSCC cell lines, including PGP9.5, cyclin A1, G0S2, bone-morphogenetic protein 2A, MT1G, and neuromedin U, which showed frequent promoter hypermethylation in primary HNSCC (60%, 45%, 35%, 25%, 25%, and 20%, respectively). Moreover, we discovered that cyclin A1 methylation was inversely related to p53 mutational status in primary tumors (P = 0.015), and forced expression of cyclin A1 resulted in robust induction of wild-type p53 in HNSCC cell lines. Pharmacological unmasking followed by microarray analysis is a powerful tool to identify key methylated tumor suppressor genes and relevant pathways.

Quantitative RARβ2 Hypermethylation: A Promising Prostate Cancer Marker

Retinoic acid receptor β2 (RARβ2) is a tumor suppressor gene frequently hypermethylated in several human neoplasms. To further characterize this epigenetic alteration in prostate cancer progression, we examined tumor tissue from 118 patients with prostate carcinoma (PCa), 38 paired high-grade prostatic intraepithelial neoplasias (HGPIN), and non-neoplastic prostate tissue from 30 patients with benign prostate hyperplasia (BPH), using quantitative methylation-specific PCR. We found RARβ2 hypermethylation in 97.5% of PCa, 94.7% of HGPIN, and 23.3% of BPH. Methylation levels were significantly higher in PCa compared with HGPIN and BPH (P < 0.00001). By establishing an empiric cutoff value, we were able to discriminate between neoplastic and non-neoplastic tissue, with 94.9% sensitivity and 100% specificity. Moreover, RARβ2 methylation levels correlated with higher pathological stage (r = 0.30, P = 0.0009). This quantitative assay represents a novel and promising molecular marker that may augment current approaches for prostate cancer detection.Retinoic acid receptor beta2 (RARbeta2) is a tumor suppressor gene frequently hypermethylated in several human neoplasms. To further characterize this epigenetic alteration in prostate cancer progression, we examined tumor tissue from 118 patients with prostate carcinoma (PCa), 38 paired high-grade prostatic intraepithelial neoplasias (HGPIN), and non-neoplastic prostate tissue from 30 patients with benign prostate hyperplasia (BPH), using quantitative methylation-specific PCR. We found RARbeta2 hypermethylation in 97.5% of PCa, 94.7% of HGPIN, and 23.3% of BPH. Methylation levels were significantly higher in PCa compared with HGPIN and BPH (P < 0.00001). By establishing an empiric cutoff value, we were able to discriminate between neoplastic and non-neoplastic tissue, with 94.9% sensitivity and 100% specificity. Moreover, RARbeta2 methylation levels correlated with higher pathological stage (r = 0.30, P = 0.0009). This quantitative assay represents a novel and promising molecular marker that may augment current approaches for prostate cancer detection.