Tuesday, March 20

8:00 Breakfast on your own or Breakfast Workshop 
(Sponsorship Available)

Real-Time Methylation-Specific PCR

8:50 PCR Bias in Methylation Independent PCR (MIP) Amplifications
Tomasz K. Wojdacz, M.Sc., Institute of Human Genetics, University of Aarhus, Denmark
Many applications in methylation studies utilize methylation independent primers (MIP-primers) designed to amplify bisulfite modified sequences regardless of their methylation status. Guidelines for MIP primer design advise avoidance of CpG dinucleotides in primer sequences or replacement of Cs within CpGs by a mismatched base. We have shown that a limited number of CpGs in the primer sequences may be necessary, otherwise PCR bias towards the unmethylated template may lead to underestimation of the degree of methylation. Moreover, by manipulating the annealing temperature of the PCR amplification, we were able to control the efficiency of MIP primer binding to the methylated template and reverse PCR bias in favour of the methylated allele. Recently, the combination of the above strategy and High Resolution Melting technology (Methylation Sensitive High Resolution Melting, MS-HRM) allowed us to develop a panel of locus specific methylation assays capable of the detection of 0.1% methylated template in unmethylated background. Furthermore, we showed that MS-HRM can be designed to estimate the methylation level in a screened material. The overall parameters of MS-HRM make it the method of choice, readily applicable to both research and diagnostic settings.

9:10 CpG Island Methylator Phenotype (CIMP) in Colorectal Cancer Determined by Quantitative PCR (MethyLight) 
Shuji Ogino, M.D., Ph.D., Assistant Professor, Pathology, Harvard Medical School, Department of Pathology, Brigham and Women's Hospital, Department of Medical Oncology, Dana-Farber Cancer Institute
CpG island methylator phenotype (CIMP) has been established as a distinct phenotype in colorectal cancer. To discriminate biologically significant methylation from insignificant (low level) methylation, quantitative assays are necessary. Sodium bisulfite modification of genomic DNA and subsequent real-time TaqMan PCR (MethyLight) are robust with good precision, and useful for paraffin-embedded tumor tissue. Quantitative PCR on carefully-selected CpG islands enables us to precisely diagnose CIMP status and should be utilized to assess effects of CIMP on clinical outcomes - patient survival and treatment efficacy. 

9:40 Clinical and Molecular Responses in Lung Cancer Patients Receiving Chromatin Remodeling Agents 
David S. Schrump, M.D., Head & Principal Investigator, Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute
Our published studies have demonstrated that 5 aza 2’ deoxycytidine (DAC) and Depsipeptide FK228 (DP) simultaneously de-repress cancer-testis and tumor suppressor genes, and mediate growth arrest and apoptosis in cultured lung cancer cells, but not normal bronchial epithelia. Presently, limited information is available regarding modulation of gene expression in primary lung cancers exposed to these chromatin remodeling agents. During the past several years, more than 80 patients with primary thoracic malignancies have received DAC, DP, or sequential DAC/DP infusions on protocols initiated in the Thoracic Oncology Section, Surgery Branch, NCI. Clinical toxicities, and response to therapy have been assessed by CTCAE and RECIST criteria, respectively. Plasma DAC and DP levels have been evaluated by LC-MS and HPLC techniques. Quantitative RT-PCR, methylation-specific-PCR, immunohistochemistry, and ELISA techniques have been used to assess a variety of molecular endpoints in pre-and post-treatment tumor biopsies and sera. Microarray techniques have been used to comprehensively examine gene expression profiles in laser-captured tumor cells from pre- and post treatment biopsies from 21 individuals receiving DAC, DP, or sequential DAC/DP infusions. Results of these arrays have been compared to data derived from analysis of laser-captured tumor cells and adjacent, histologically normal bronchial epithelia from 20 patients undergoing definitive lung cancer resections. Whereas no objective clinical responses have been observed, several patients have exhibited prolonged stabilization of disease following DAC, DP, or sequential DAC/DP infusions. Plasma DAC and DP concentrations have approximated threshold levels for gene induction and apoptosis in cultured lung cancer cells. Approximately 30% of patients receiving DAC or DP infusions have exhibited enhanced expression of NY-ESO-1, p16, p21, or acetylated core histones in tumor biopsies. Long-oligo array analyses have revealed complex, heterogeneous responses to DAC, DP, and DAC/DP in lung cancer cells, with an apparent shift of gene expression profiles toward those observed in histologically normal bronchial epithelia. Collectively, these data confirm that DNA demethylating agents and HDAC inhibitors can modulate gene expression in primary lung carcinomas. These preliminary observations support further evaluation of chromatin remodeling agents alone or in combination with treatment regimens, which either specifically target induced gene products, or modulate survival signaling in lung cancer cells. 

10:10 Coffee Break, Poster and Exhibit Viewing 

11:00 Application of Quantitative PCR Methodology to Gene Promoter Hypermethylation Analysis in Colon Cancers
Yuriko Mori, M.D., Ph.D., Assistant Professor of Medicine, Division of Gastroenterology, Department of Medicine, Johns Hopkins University
Assessment of gene promoter methylation status is a growing research field in cancer genetics. We employed quantitative PCR methodology for the rapid, sensitive, and quantitative detection of gene promoter hypermethylation in human cancers. This approach enabled us to identify several novel genes undergoing promoter hypermethylation in human primary colon cancers.

11:30 Panel Discussion with Morning Speakers

12:00 Luncheon Solutions Showcases
(Sponsorship Available)

Developing Epigenetically-Effective Drugs

2:05 The Landscape of DNA Methylase Inhibitors: From Single Nucleosides to Modified Oligodeoxynucleotides 
Victor E. Marquez, Ph.D., Chief, Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute-Frederick, National Institutes of Health
Aberrant methylation of cytosine residues in the promoter region of genes has been shown to result in gene silencing associated with cancer progression. Some specific inhibitors of cytosine methyltransferases are cytidine analogues, such as 5-azacytidine and 5-aza-2’-deoxycytidine. Recently, another cytidine analogue [zebularine or [2(1H)pyrimidinone riboside] has shown activity in reactivating several tumor suppressor genes. Because of its chemical stability and oral activity in mice, zebularine was considered for clinical trials; however, an unexpected toxicity in primates has temporarily halted its progress. In view of the tortuous metabolic pathway of zebularine, prior to DNA incorporation, efforts to either circumvent its inefficient activation and to bypass first liver passage (a suspected cause of toxicity) are being explored with synthetic oligodeoxynucleotides (ODNs) containing 2(1H)pyrimidinone in place of cytosine. The crystal structure of bacterial M.HhaI methylase in complex with zebularine-modifed ODNs shows that the nucleotide is rotated out of the double helix. In forming a tight complex with the enzyme, the rotating motion of the nucleotide is more critical than the nature of the nucleobase itself. Moreover, strong inhibition of DNA methylation can be achieved with ODNs containing abasic sites. While exploring the activity of a 13-mer ODN containing a conformationally locked abasic pseudosugar at the recognition site of HhaI methylase, we were able to capture the DNA-protein complex at the mid-point of the flipping trajectory. These ODNs are some of the most potent inhibitors of DNA methylase known to date. Our approaches to optimize modified nucleosides and ODNs as potential clinical candidates will be highlighted. 

2:35 Epigenetic Therapy Combinations: A Rational Next Step
Andrew Allen, M.D., MRCP, Ph.D., Chief Medical Officer and Executive Vice President, Pharmion Corporation
Epigenetic regulation of gene expression involves several modalities of DNA and histone modification operating in tandem. Drugs are now available in the clinic to address both DNA methylation and histone acetylation, and combining these agents may offer an effective approach to cancer therapy. Data exploring such combinations are now available in hematologic malignancies, particularly in acute myeloid leukemia, and these will be discussed.

3:05 Spotlight Presentation (Sponsorship Available)

3:20 Refreshment Break 
(Last Chance for Poster and Exhibit Viewing)

3:45 Histone Deacetylase as a Therapeutic Target: Development of Vorinostat
Victoria M. Richon, Ph.D., Senior Director, Department of Cancer Research, Merck & Co., Inc.
Histone deacetylase (HDAC) inhibitors represent a promising new class of anti-tumor agents. Vorinostat (suberoylanilide hydroxamic acid, SAHA) is a nanomolar inhibitor of HDAC activity and is currently undergoing evaluation in multiple Phase 1 and 2 clinical trials. HDACs are enzymes that catalyze the removal of the acetyl modification from the lysine residues of proteins, including the core nucleosomal histones. Together with histone acetyltransferases (HATs), HDACs regulate the level of protein acetylation. Alterations in both HAT and HDAC activity occur in cancer. HAT activity has been found to be disrupted by translocation, amplification, over-expression, or mutation in a variety of cancers, including those of hematological or epithelial origin. HDACs have been found to be over-expressed or associated with oncogenic transcription factors. Vorinostat induces growth arrest, differentiation or apoptosis in a wide variety of transformed cells. The anti-proliferative effects of vorinostat are believed to be due to drug-induced accumulation of acetylated proteins, including the core nucleosomal histones and other proteins (e.g., bcl6, alpha-tubulin and Hsp90). Phase 1 trials have been conducted for both intravenous and oral formulations of vorinostat. Results from these trials show that vorinostat can inhibit its target enzyme, HDAC, in peripheral mononuclear cells, at doses that are well-tolerated. Anti-tumor activity has been seen in patients with both hematological and solid tumors. 

4:15 Success and Challenges of Developing HDAC Inhibitors as Anti-Cancer Agents
Wenlin Shao, Ph.D., Research Investigator, Oncology Drug Discovery, Novartis Institutes for BioMedical Research
HDAC inhibitors have demonstrated anti-tumor efficacy in the treatment of both solid and hematological malignancies. However, the mechanisms underlying such effects of HDACi remain to be elucidated. We have taken both candidate-based and genomic approaches to decipher the molecular events that mediate the HDACi anti-cancer effect. An increased understanding at the molecular level will assist stratification of tumor types that could benefit from HDACi therapy.

4:45 The Power of HDACs
Donald F. Corcoran, MBA, President & Chief Executive Officer, MethylGene Inc. 
HDACs are a hot, new field in cancer and are being studied in other indications such as neurodegenerative disease, inflammation and antifungals. MethylGene has taken advantage of novel chemistry to develop a set of selective molecules targeted against cancer as well as several non-oncology indications. There are several HDAC pan-inhibitors in clinical development; however, because of the isotype-specificity of MethylGenes MGCD0103, treatment with this compound may result in an improved therapeutic window. MGCD0103 is currently in multiple Phase II clinical trials, including Hodgkins and B-cell lymphomas and has also launched a program to develop molecules against histone methyltransferases.

5:15 Close of Conference