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.

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 differentiallyhypermethylatedin HNSCCindetectionof promoterhypermethylationin serum and salivary rinses associated with HNSCC.We did preliminary evaluation via quantitative methylation-specific PCR (Q-MSP) using a panel of 21genes 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 211patients 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.

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.

DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells ''the cancer methylome'' would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology.

No abstract

TMPRSS2-ETS gene fusions have been found recurrently in prostate carcinomas, but not in the presumed precursor lesion, high-grade prostatic intraepithelial neoplasia (HGPIN). However, HGPIN lesions may share chromosomal changes with prostate cancer. To determine the relative order of genetic events in prostate carcinogenesis, we have analyzed 34 prostate carcinomas, 19 paired HGPIN lesions, 14 benign prostate hyperplasias, and 11 morphologically normal prostatic tissues for TMPRSS2-ERG and TMPRSS2-ETV1 rearrangements and genomic imbalances. TMPRSS2 exon 1 was fused in-frame with ERG exon 4 in 17 of 34 (50%) prostate carcinomas and in 4 of 19 (21%) HGPIN lesions, but in none of controls. The findings were further validated by sequencing analysis and by the real-time polymerase chain reaction quantification of TMPRSS2-ERG fusion transcript and the ERG exons 5/6:exons 1/2 expression ratio. Chromosome copy number changes were detected by comparative genomic hybridization in 42% of clinically confined carcinomas and in none of the 16 HGPIN lesions analyzed. We demonstrate for the first time that the TMPRSS2-ERG fusion gene can be detected in a proportion of HGPIN lesions and that this molecular rearrangement is an early event that may precede chromosome-level alterations in prostate carcinogenesis.

The Androgen Receptor (AR) is the key-driving transcription factor in prostate cancer, tightly controlled by epigenetic regulation. To date, most epigenetic profiling has been performed in cell lines or limited tissue samples. Here, to comprehensively study the epigenetic landscape, we perform RNA-seq with ChIP-seq for AR and histone modification marks (H3K27ac, H3K4me3, H3K27me3) in 100 primary prostate carcinomas. Integrative molecular subtyping of the five data streams revealed three major subtypes of which two were clearly TMPRSS2-ERG dictated. Importantly, we identify a third subtype with low chromatin binding and activity of AR, but with high activity of FGF and WNT signaling. While positive for neuroendocrine-hallmark genes, these tumors were copy number-neutral with low mutational burden, significantly depleted for genes characteristic of poor-outcome associated luminal B-subtype. We present a unique resource on transcriptional and epigenetic control in prostate cancer, revealing tight control of gene regulation differentially dictated by AR over three subtypes.

No abstract