Carmen Jerónimo

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.

Mitochondrial mutations in early stage prostate cancer and bodily fluids

We recently demonstrated the existence of specific patterns of somatic mitochondrial DNA (mtDNA) mutations in several cancers. Here we sought to identify the presence of mtDNA mutations in prostate cancer and their paired PIN lesions. The D-loop region, 16S rRNA, and the NADH subunits of complex I were sequenced to identify mtDNA mutations in 16 matched PIN lesions and primary prostate cancers. Twenty mtDNA mutations were detected in the tumor tissue of three patients. Identical mutations were also identified in the PIN lesion from one patient. This patient with multiple point mutations also harbored a high frequency of microsatellite instability (MSI-H) in nuclear mononucleotide repeat markers. Remarkably, identical mutations were also detected in all (3/3) matched urine and plasma samples obtained from these patients. Although mitochondrial mutations are less common in prostate adenocarcinoma, they occur early in cancer progression and they can be detected in bodily fluids of early stage disease patients. The identification of MtDNA mutations may complement other early detection approaches for 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.

P-Cadherin Overexpression Is an Indicator of Clinical Outcome in Invasive Breast Carcinomas and Is Associated with CDH3 Promoter Hypomethylation

Purpose: P-cadherin overexpression has been reported in breast carcinomas, where it was associated with proliferative high-grade histological tumors. This study aimed to analyze P-cadherin expression in invasive breast cancer and to correlate it with tumor markers, pathologic features, and patient survival. Another purpose was to evaluate the P-cadherin promoter methylation pattern as the molecular mechanism underlying this gene regulation. Experimental Design: Using a series of invasive breast carcinomas, P-cadherin expression was evaluated and correlated with histologic grade, estrogen receptor, MIB-1, and p53 and c-erbB-2 expression. In order to assess whether P-cadherin expression was associated with changes in CDH3 promoter methylation, we studied the methylation status of a gene 5′-flanking region in these same carcinomas. This analysis was also done for normal tissue and for a breast cancer cell line treated with a demethylating agent. Results: P-cadherin expression showed a strong correlation with high histologic grade, increased proliferation, c-erbB-2 and p53 expression, lack of estrogen receptor, and poor patient survival. This overexpression can be regulated by gene promoter methylation because the 5-Aza-2′-deoxycytidine treatment of MCF-7/AZ cells increased P-cadherin mRNA and protein levels. Additionally, we found that 71% of P-cadherin-negative cases showed promoter methylation, whereas 65% of positive ones were unmethylated (P = 0.005). The normal P-cadherin-negative breast epithelial cells showed consistent CDH3 promoter methylation. Conclusions: P-cadherin expression was strongly associated with tumor aggressiveness, being a good indicator of clinical outcome. Moreover, the aberrant expression of P-cadherin in breast cancer might be regulated by gene promoter hypomethylation.

Epigenetics in Prostate Cancer: Biologic and Clinical Relevance

CONTEXT Prostate cancer (PCa) is one of the most common human malignancies and arises through genetic and epigenetic alterations. Epigenetic modifications include DNA methylation, histone modifications, and microRNAs (miRNA) and produce heritable changes in gene expression without altering the DNA coding sequence. OBJECTIVE To review progress in the understanding of PCa epigenetics and to focus upon translational applications of this knowledge. EVIDENCE ACQUISITION PubMed was searched for publications regarding PCa and DNA methylation, histone modifications, and miRNAs. Reports were selected based on the detail of analysis, mechanistic support of data, novelty, and potential clinical applications. EVIDENCE SYNTHESIS Aberrant DNA methylation (hypo- and hypermethylation) is the best-characterized alteration in PCa and leads to genomic instability and inappropriate gene expression. Global and locus-specific changes in chromatin remodeling are implicated in PCa, with evidence suggesting a causative dysfunction of histone-modifying enzymes. MicroRNA deregulation also contributes to prostate carcinogenesis, including interference with androgen receptor signaling and apoptosis. There are important connections between common genetic alterations (eg, E twenty-six fusion genes) and the altered epigenetic landscape. Owing to the ubiquitous nature of epigenetic alterations, they provide potential biomarkers for PCa detection, diagnosis, assessment of prognosis, and post-treatment surveillance. CONCLUSIONS Altered epigenetic gene regulation is involved in the genesis and progression of PCa. Epigenetic alterations may provide valuable tools for the management of PCa patients and be targeted by pharmacologic compounds that reverse their nature. The potential for epigenetic changes in PCa requires further exploration and validation to enable translation to the clinic.

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.

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.

Three epigenetic biomarkers, GDF15, TMEFF2 and VIM, accurately predict bladder cancer from DNA-based analyses of urine samples.

Purpose: To identify a panel of epigenetic biomarkers for accurate bladder cancer (BlCa) detection in urine sediments. Experimental Design: Gene expression microarray analysis of BlCa cell lines treated with 5-aza-2′-deoxycytidine and trichostatin A as well as 26 tissue samples was used to identify a list of novel methylation candidates for BlCa. Methylation levels of candidate genes were quantified in 4 BlCa cell lines, 50 BlCa tissues, 20 normal bladder mucosas (NBM), and urine sediments from 51 BlCa patients and 20 healthy donors, 19 renal cancer patients, and 20 prostate cancer patients. Receiver operator characteristic curve analysis was used to assess the diagnostic performance of the gene panel. Results: GDF15, HSPA2, TMEFF2, and VIM were identified as epigenetic biomarkers for BlCa. The methylation levels were significantly higher in BlCa tissues than in NBM (P < 0.001) and the cancer specificity was retained in urine sediments (P < 0.001). A methylation panel comprising GDF15, TMEFF2, and VIM correctly identified BlCa tissues with 100% sensitivity and specificity. In urine samples, the panel achieved a sensitivity of 94% and specificity of 100% and an area under the curve of 0.975. The gene panel could discriminate BlCa from both healthy individuals and renal or prostate cancer patients (sensitivity, 94%; specificity, 90%). Conclusions: By using a genome-wide approach, we have identified a biomarker panel that allows for early and accurate noninvasive detection of BlCa using urine samples. Clin Cancer Res; 16(23); 5842–51. ©2010 AACR.

High Promoter Methylation Levels of APC Predict Poor Prognosis in Sextant Biopsies from Prostate Cancer Patients

Purpose: Prostate cancer is a highly prevalent malignancy and constitutes a major cause of cancer-related morbidity and mortality. Owing to the limitations of current clinical, serologic, and pathologic parameters in predicting disease progression, we sought to investigate the prognostic value of promoter methylation of a small panel of genes by quantitative methylation-specific PCR (QMSP) in prostate biopsies. Experimental Design: Promoter methylation levels of APC, CCND2, GSTP1, RARB2, and RASSF1A were determined by QMSP in a prospective series of 83 prostate cancer patients submitted to sextant biopsy. Clinicopathologic data [age, serum prostate-specific antigen (PSA), stage, and Gleason score] and time to progression and/or death from prostate cancer were correlated with methylation findings. Log-rank test and Cox regression model were used to identify which epigenetic markers were independent predictors of prognosis. Results: At a median follow-up time of 45 months, 15 (18%) patients died from prostate cancer, and 37 (45%) patients had recurrent disease. In univariate analysis, stage and hypermethylation of APC were significantly associated with worse disease–specific survival, whereas stage, Gleason score, high diagnostic serum PSA levels, and hypermethylation of APC, GSTP1, and RASSF1A were significantly associated with poor disease-free survival. However, in the final multivariate analysis, only clinical stage and high methylation of APC were significantly and independently associated with unfavorable prognosis, i.e., decreased disease-free and disease-specific survival. Conclusions: High-level APC promoter methylation is an independent predictor of poor prognosis in prostate biopsy samples and might provide relevant prognostic information for patient management.