Franclim R. Ribeiro

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 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.

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.

Epigenetic Heterogeneity of High-Grade Prostatic Intraepithelial Neoplasia: Clues for Clonal Progression in Prostate Carcinogenesis

High-grade prostatic intraepithelial neoplasia (PIN) is the most likely precursor of prostate adenocarcinoma, but the frequency and timing of epigenetic changes found in prostate carcinogenesis has not been extensively documented. Thus, the promoters of three genes (APC, GSTP1, and RARβ2) involved in prostate carcinogenesis were tested by quantitative methylation-specific PCR in tissue DNA from 30 prostate carcinomas, 128 high-grade PIN lesions, and 30 normal prostate tissue samples dissected from 30 radical prostatectomy specimens using laser capture microdissection. The percentage of methylated alleles (PMA) was calculated for each gene, and hierarchical cluster analysis was used to define the degree of similarity of epigenetic alterations among the various samples. We found that PMA values of APC and RARβ2 were higher than those of GSTP1 in all three types of tissue samples and median PMA values for all three genes were higher in prostate cancer. By cluster analysis, 26 of 30 prostate carcinomas and 82 of 128 high-grade PIN lesions were grouped in the “high methylation” branch, whereas 24 of 30 normal prostate tissue samples were allocated in the “low methylation” branch. Although high-grade PIN lesions are epigenetically more similar to prostate carcinoma than to normal prostate tissue, paired prostate carcinoma and high-grade PIN lesions did not always segregate together. We concluded that APC and RARβ2 hypermethylation is frequent in normal prostate tissue and the progressive enrichment in cells carrying methylated alleles observed in high-grade PIN and prostate carcinoma is consistent with clonal progression. Because GSTP1 promoter methylation is mainly observed in prostate carcinoma and some high-grade PIN lesions, it represents an important marker for the transition of in situ to invasive neoplasia. (Mol Cancer Res 2006;4(1):1–8)

8q Gain Is an Independent Predictor of Poor Survival in Diagnostic Needle Biopsies from Prostate Cancer Suspects

Purpose: The main procedure to confirm a suspected diagnosis of prostate cancer is histologic analysis of ultrasound-guided sextant prostate biopsies. As it is difficult to reliably assess tumor stage and grade in such minute samples, the clinical significance of some tumor foci remains unclear. Genetic markers that could augment pretreatment prognostic information would improve the clinical management of the disease. Experimental Design: We have analyzed by comparative genomic hybridization a consecutive series of prostate needle biopsies obtained prospectively from 100 prostate cancer suspects. For 25 of these patients, a second independent biopsy core was analyzed to assess possible tumor heterogeneity. Additionally, a three-color fluorescent in situ hybridization assay was done in paraffin-embedded biopsy cores to validate the comparative genomic hybridization findings and to confirm their prognostic value. Results: Sixty-one of 100 biopsy samples had morphologic evidence of prostate cancer and 41 (67%) of these displayed genomic copy number changes as opposed to none of the morphologically normal biopsies. The presence of losses, amplifications, and the total number of genomic imbalances were significantly associated with poorly differentiated tumors. Kaplan-Meier curves with log-rank test showed that patients whose tumors displayed 8q gains had a significantly worse survival even when tumor grade was taken into account (P = 0.008). Restricting the analysis to cases with Gleason score 7, the most troublesome category in terms of prognostic information, gains at 8q were still significantly associated with poor survival (P = 0.011), something that was confirmed by fluorescent in situ hybridization in an independent series of biopsies with much longer follow-up time (P = 0.023). Conclusions: We show that whole genomic information can be obtained from minute needle biopsies of prostate cancer suspects and that genetic data can provide additional prognostic information before a therapeutic decision is taken.

FLI1 is a novel ETS transcription factor involved in gene fusions in prostate cancer.

To characterize the pattern of ETS rearrangements and to uncover novel ETS fusion genes, we analyzed 200 prostate carcinomas (PCa) with TaqMan low‐density arrays (TLDAs), followed by selective analyses with fluorescence in situ hybridization (FISH), RT‐PCR, and sequencing. Besides confirming the recurrent presence of ERG, ETV1, ETV4, and ETV5 rearrangements, we here report FLI1 as the fifth ETS transcription factor involved in fusion genes in prostate cancer. Outlier expression of the FLI1 gene was detected by TLDAs in one PCa that showed relative overexpression of FLI1 exons 4:5 as compared with FLI1 exons 2:3. A structural rearrangement was found using FISH probes flanking the FLI1 gene and RT‐PCR and sequencing analyses showed fusion of SLC45A3 exon 1 with FLI1 exon 3. Interestingly, we found four cases with two different ETS rearrangements in the index tumor, thus revealing intratumor genetic heterogeneity. Correlation analysis with clinico‐pathological data showed association of ERG rearrangements with locally advanced disease (pT3, P = 0.007) and MYC overexpression (P = 0.001), and association of ETV1 rearrangements with PTEN downregulation (P = 0.015). We report that FLI1 is a novel ETS transcription factor involved in gene fusions in prostate cancer and that intratumor genetic heterogeneity of ETS rearrangements can occasionally be found in index primary tumors.

Assessment of clonal relationships in ipsilateral and bilateral multiple breast carcinomas by comparative genomic hybridisation and hierarchical clustering analysis

The issue of whether multiple, ipsilateral or bilateral, breast carcinomas represent multiple primary tumours or dissemination of a single carcinomatous process has been difficult to resolve, especially for individual patients. We have addressed the problem by comparative genomic hybridisation analysis of 26 tumours from 12 breast cancer patients with multiple ipsilateral and/or bilateral carcinoma lesions. Genomic imbalances were detected in 25 of the 26 (96%) tumours. Using the genomic imbalances detected in these 26 lesions as well as those previously found by us in an independent series of 35 unifocal breast carcinomas, we compared a probabilistic model for likelihood of independence with unsupervised hierarchical clustering methodologies to determine the clonal relatedness of multiple tumours in breast cancer patients. We conclude that CGH analysis of multiple breast carcinomas followed by unsupervised hierarchical clustering of the genomic imbalances is more reliable than previous criteria to determine the tumours’ clonal relationship in individual patients, that most ipsilateral breast carcinomas arise through intramammary spreading of a single breast cancer, and that most patients with bilateral breast carcinomas have two different diseases.

Statistical dissection of genetic pathways involved in prostate carcinogenesis.

Molecular markers that could stratify prostate cancer patients according to risk of disease progression would allow a significant improvement in the management of this clinically heterogeneous disease. In the present study, we analyzed the genetic profile of a consecutive series of 51 clinically confined prostate carcinomas and 27 benign prostatic hyperplasias using comparative genomic hybridization (CGH). We then added our findings to the existing literature data in order to perform a meta‐analysis on a total of 294 prostate cancers with detailed CGH and clinicopathological information, using multivariate statistical methods that included principal component, hierarchical clustering, time of occurrence, and regression analyses. Whereas several genomic imbalances were shared by organ‐confined, locally invasive, and metastatic prostate cancers, 6q and 10q losses and 7q and 8q gains were significantly more frequent in patients with extra‐prostatic disease. Regression analysis indicated that 8q gain and 13q loss were the best predictors of locally invasive disease, whereas 8q gain and 6q and 10q losses were associated with metastatic disease. We propose a genetic pathway of prostate carcinogenesis with two distinct initiating events, namely, 8p and 13q losses. These primary imbalances are then preferentially followed by 8q gain and 6q, 16q, and 18q losses, which in turn are followed by a set of late events that make recurrent and metastatic prostate cancers genetically more complex. We conclude that significant differences exist in the genetic profile of organ‐confined, locally invasive, and advanced prostate cancer and that genetic features may carry prognostic information independently of Gleason grade.

Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

BackgroundAberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors.MethodsA panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters.ResultsSignificant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively.ConclusionThe major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

Hypermethylation of Cyclin D2 is associated with loss of mRNA expression and tumor development in prostate cancer

D-type cyclins play a pivotal role in cell cycle regulation and their abnormal expression was associated with several human malignancies. To assess Cyclin D2 promoter methylation status and expression levels in prostate tissues, quantitative methylation-specific PCR and quantitative reverse transcription PCR assays were performed in a large series of prostate carcinomas, high-grade prostatic intraepithelial neoplasias (HGPIN), benign prostate hyperplasias (BPH), normal prostate tissue (NPT) samples, and prostate cancer (PCa) cell lines (before and after demethylating treatment). Methylation levels were correlated with mRNA expression levels and key clinicopathologic parameters. Cyclin D2 promoter methylation was found in 117/118 PCa, 38/38 HGPIN, 24/30 BPH, 11/11 NPT, and 4/4 cell lines. Methylation levels were significantly higher in PCa compared with HGPIN, NPT, and BPH (P<0.0001), correlating with tumor stage and Gleason score (r=0.29, P=0.0014; and r=0.32, P=0.0005, respectively). Conversely, Cyclin D2 mRNA levels were significantly lower in PCa (P<0.01) and a significant inverse correlation between Cyclin D2 methylation and expression levels was found in prostatic tissues (r=−0.61, P<0.000001). Demethylating treatment induced a substantial increase in Cyclin D2 mRNA in LNCaP cells whereas decreased levels were observed in DU-145 and PC-3 cells. We concluded that Cyclin D2 promoter methylation downregulates gene transcription and occurs with high frequency at low levels in normal, hyperplastic, and preneoplastic prostate tissues. Conversely, high Cyclin D2 methylation levels characterize invasive prostatic carcinoma, correlating with clinicopathologic features of tumor aggressiveness.