Mark L. Gonzalgo

Hypermethylation of CpG Islands in Primary and Metastatic Human Prostate Cancer

Aberrant DNA methylation patterns may be the earliest somatic genome changes in prostate cancer. Using real-time methylation-specific PCR, we assessed the extent of hypermethylation at 16 CpG islands in DNA from seven prostate cancer cell lines (LNCaP, PC-3, DU-145, LAPC-4, CWR22Rv1, VCaP, and C42B), normal prostate epithelial cells, normal prostate stromal cells, 73 primary prostate cancers, 91 metastatic prostate cancers, and 25 noncancerous prostate tissues. We found that CpG islands at GSTP1, APC, RASSF1a, PTGS2, and MDR1 were hypermethylated in >85% of prostate cancers and cancer cell lines but not in normal prostate cells and tissues; CpG islands at EDNRB, ESR1, CDKN2a, and hMLH1 exhibited low to moderate rates of hypermethylation in prostate cancer tissues and cancer cell lines but were entirely unmethylated in normal tissues; and CpG islands at DAPK1, TIMP3, MGMT, CDKN2b, p14/ARF, and CDH1 were not abnormally hypermethylated in prostate cancers. Receiver operator characteristic curve analyses suggested that CpG island hypermethylation changes at GSTP1, APC, RASSF1a, PTGS2, and MDR1 in various combinations can distinguish primary prostate cancer from benign prostate tissues with sensitivities of 97.3–100% and specificities of 92–100%. Hypermethylation of the CpG island at EDNRB was correlated with the grade and stage of the primary prostate cancers. PTGS2 CpG island hypermethylation portended an increased risk of recurrence. Furthermore, CpG island hypermethylation patterns in prostate cancer metastases were very similar to the primary prostate cancers and tended to show greater differences between cases than between anatomical sites of metastasis.

Mutagenic and epigenetic effects of DNA methylation

Tumorigenesis begins with the disregulated growth of an abnormal cell that has acquired the ability to divide more rapidly than its normal counterparts (Nowell, P.C. (1976) Science, 194, 23-28 [1]). Alterations in global levels and regional changes in the patterns of DNA methylation are among the earliest and most frequent events known to occur in human cancers (Feinberg and Vogelstein (1983) Nature, 301, 89-92 ([2]); Gama-Sosa, M.A. et al. (1983) Nucleic Acids Res., 11, 6883-6894 ([3]); Jones, P.A. (1986) Cancer Res., 46, 461-466 [4]). These changes in methylation may impair the proper expression and/or function of cell-cycle regulatory genes and thus confer a selective growth advantage to affected cells. Developments in the field of cancer research over the past few years have led to an increased understanding of the role DNA methylation may play in tumorigenesis. Many of these studies have investigated two major mechanisms by which DNA methylation may lead to aberrant cell cycle control: (1) through the generation of transition mutations via deamination-driven events resulting in the inactivation of tumor suppressor genes, or (2) by altering levels of gene expression through epigenetic effects at CpG islands. The mechanisms by which the normal function of growth regulatory genes may become affected by the mutagenic and epigenetic properties of DNA methylation will be discussed in the framework of recent discoveries in the field.

Detection of methylated apoptosis-associated genes in urine sediments of bladder cancer patients.

Purpose: There is increasing evidence for a fundamental role for epigenetic silencing of apoptotic pathways in cancer. Changes in DNA methylation can be detected with a high degree of sensitivity, so we used the MethyLight assay to determine how methylation patterns of apoptosis-associated genes change during bladder carcinogenesis and whether DNA methylation could be detected in urine sediments. Experimental Design: We analyzed the methylation status of the 5′ regions of 12 apoptosis-associated genes (ARF, FADD, TNFRSF21, BAX, LITAF, DAPK, TMS-1, BCL2, RASSF1A, TERT, TNFRSF25, and EDNRB) in 18 bladder cancer cell lines, 127 bladder cancer samples, and 37 samples of adjacent normal bladder mucosa using the quantitative MethyLight assay. We also analyzed the methylation status in urine sediments of 20 cancer-free volunteers and 37 bladder cancer patients. Results: The 5′ regions of DAPK, BCL2, TERT, RASSFIA, and TNFRSF25 showed significant increases in methylation levels when compared with nonmalignant adjacent tissue (P ≤ 0.01). Methylation levels of BCL2 were significantly associated with tumor staging and grading (P ≤ 0.01), whereas methylation levels of RASSF1A and ARF were only associated with tumor stage (P ≤ 0.04), and TERT methylation and EDNRB methylation were predictors of tumor grade (P ≤ 0.02). To investigate clinical usefulness for noninvasive bladder cancer detection, we further analyzed the methylation status of the markers in urine samples of patients with bladder cancer. Methylation of DAPK, BCL2, and TERT in urine sediment DNA from bladder cancer patients was detected in the majority of samples (78%), whereas they were unmethylated in the urine sediment DNA from age-matched cancer-free individuals. Conclusions: Our results indicate that methylation of the 5′ region of apoptosis-associated genes is a common finding in patients with bladder carcinoma. The ability to detect methylation not only in bladder tissue, but also in urine sediments, suggests that methylation markers are promising tools for noninvasive detection of bladder cancers. Our results also indicate that some methylation markers, such as those in regions of RASSF1A and TNFRSF25, might be of limited use for detection because they are also methylated in normal bladder tissues.

GSTP1 CpG island hypermethylation as a molecular biomarker for prostate cancer.

Somatic hypermethylation of CpG island sequences at GSTP1, the gene encoding the π‐class glutathione S‐transferase, appears to be characteristic of human prostatic carcinogenesis. To consider the potential utility of this epigenetic alteration as a biomarker for prostate cancer, we present here a comprehensive review of the literature describing somatic GSTP1 changes in DNA from prostate cells and tissues. GSTP1 CpG island hypermethylation has been detected in prostate cancer DNA using a variety of assay techniques, including (i) Southern blot analysis (SB), after treatment with 5‐mC‐sensitive restriction endonucleases, (ii) the polymerase chain reaction, following treatment with 5‐mC‐sensitive restriction endonucleases (RE‐PCR), (iii) bisulfite genomic sequencing (BGS), and (iv) bisulfite modification followed by the polymerase chain reaction, using primers selective for target sequences containing 5‐mC (MSP). In the majority of the case series so far reported, GSTP1 CpG island hypermethylation was present in DNA from at least 90% of prostate cancer cases. When analyses have been carefully conducted, GSTP1 CpG island hypermethylation has not been found in DNA from normal prostate tissues, or from benign prostatic hyperplasia (BPH) tissues, though GSTP1 CpG island hypermethylation changes have been detected in DNA from candidate prostate cancer precursor lesions proliferative inflammatory atrophy (PIA) and prostatic intraepithelial neoplasia (PIN). Using PCR methods, GSTP1 CpG island hypermethylation has also been detected in urine, ejaculate, and plasma from men with prostate cancer. GSTP1 CpG island hypermethylation, a somatic epigenetic alteration, appears poised to serve as a molecular biomarker useful for prostate cancer screening, detection, and diagnosis.

Roles of cell division and gene transcription in the methylation of CpG islands.

ABSTRACT De novo methylation of CpG islands within the promoters of eukaryotic genes is often associated with their transcriptional repression, yet the methylation of CpG islands located downstream of promoters does not block transcription. We investigated the kinetics of mRNA induction, demethylation, and remethylation of the p16promoter and second-exon CpG islands in T24 cells after 5-aza-2′-deoxycytidine (5-Aza-CdR) treatment to explore the relationship between CpG island methylation and gene transcription. The rates of remethylation of both CpG islands were associated with time but not with the rate of cell division, and remethylation of thep16 exon 2 CpG island occurred at a higher rate than that of the p16 promoter. We also examined the relationship between the remethylation of coding sequence CpG islands and gene transcription. The kinetics of remethylation of the p16exon 2, PAX-6 exon 5, c-ABL exon 11, andMYF-3 exon 3 loci were examined following 5-Aza-CdR treatment because these genes contain exonic CpG islands which are hypermethylated in T24 cells. Remethylation occurred most rapidly in the p16, PAX-6, and c-ABL genes, shown to be transcribed prior to drug treatment. These regions also exhibited higher levels of remethylation in single-cell clones and subclones derived from 5-Aza-CdR-treated T24 cells. Our data suggest that de novo methylation is not restricted to the S phase of the cell cycle and that transcription through CpG islands does not inhibit their remethylation.

Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging

Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long‐term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV‐Fluc) and passaged repeatedly for 3–8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843±28) to passage 20 (250±10) to passage 40 (44±3) to passage 60 (3±1 RLU/μg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5azacytidine treatment led to higher levels of firefly luciferase mRNA (RT‐PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5‐azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene‐based imaging of stem cell transplantation.

Clinical and Pathologic Outcome After Radical Prostatectomy for Prostate Cancer Patients With a Preoperative Gleason Sum of 8 to 10

Men with a biopsy Gleason sum of 8 to 10 are considered high‐risk. The current study sought to identify whether there was a subset of men with high biopsy Gleason sums who would have a good pathologic and biochemical outcome with surgical monotherapy. To increase the generalizability of the findings, data were used from patients treated at 2 very different practice settings: a tertiary care referral center (Johns Hopkins Hospital) and multiple equal‐access medical centers (Shared Equal Access Regional Cancer Hospital [SEARCH] Database).

Impact of surgical technique (open vs laparoscopic vs robotic-assisted) on pathological and biochemical outcomes following radical prostatectomy: an analysis using propensity score matching

Study Type – Therapy (case series)

Tertiary Gleason Patterns and Biochemical Recurrence After Prostatectomy: Proposal for a Modified Gleason Scoring System

PURPOSE We investigated the relationship between the tertiary Gleason component in radical prostatectomy specimens and biochemical recurrence in what is to our knowledge the largest single institution cohort to date. MATERIALS AND METHODS We evaluated data on 3,230 men who underwent radical prostatectomy at our institution from 2000 to 2005. Tertiary Gleason component was defined as Gleason grade pattern 4 or greater for Gleason score 6 and Gleason grade pattern 5 for Gleason score 7 or 8. RESULTS Biochemical recurrence curves for cancer with tertiary Gleason component were intermediate between those of cancer without a tertiary Gleason component in the same Gleason score category and cancer in the next higher Gleason score category. The only exception was that Gleason score 4 + 3 = 7 with a tertiary Gleason component behaved like Gleason score 8. The tertiary Gleason component independently predicted recurrence when factoring in radical prostatectomy Gleason score, radical prostatectomy stage and prostate specific antigen (HR 1.45, p = 0.029). Furthermore, the magnitude of the tertiary Gleason component effect on recurrence did not differ by Gleason score category (p = 0.593). CONCLUSIONS Although the tertiary Gleason component is frequently included in pathology reports, it is routinely omitted in other situations, such as predictive nomograms, research studies and patient counseling. The current study adds to a growing body of evidence highlighting the importance of the tertiary Gleason component in radical prostatectomy specimens. Accordingly consideration should be given to a modified radical prostatectomy Gleason scoring system that incorporates tertiary Gleason component in intuitive fashion, including Gleason score 6, 6.5 (Gleason score 6 with tertiary Gleason component), 7 (Gleason score 3 + 4 = 7), 7.25 (Gleason score 3 + 4 = 7 with tertiary Gleason component), 7.5 (Gleason score 4 + 3), 8 (Gleason score 4 + 3 with tertiary Gleason component or Gleason score 8), 8.5 (Gleason score 8 with tertiary Gleason component), 9 (Gleason score 4 + 5 or 5 + 4) and 10.

Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction.

The ability to detect methylation changes associated with oncogenic transformation is of critical importance in understanding how DNA methylation may contribute to tumorigenesis. We have developed a simple and reproducible fingerprinting method called methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) to screen for DNA methylation changes. This technique relies on digesting genomic DNA with methylation-sensitive and -insensitive restriction enzymes (e.g., HpaII and MspI) prior to AP-PCR amplification. Matched normal and tumor DNAs were compared to identify differential methylation. After the PCR products were resolved on high-resolution polyacrylamide gels, regions of genomic DNA that showed hypo- and hypermethylation associated with tumors were detected. These fragments were then isolated, cloned, and sequenced. Novel CpG islands were found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.