To date, nearly 120 patients with thoracic malignancies (mostly lung cancers) have received deoxyazacytidine (DAC) Depsipeptide (DP), or sequential DAC/DP infusions on protocols initiated in the Thoracic Oncology Section. 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. Micro-array 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 histones H3 and H4 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. A manuscript summarizing the DAC/DP clinical trial is in preparation. Whereas coordinate activation of BORIS variant, stem cell, and CT-X genes in cancer cells suggests either direct or functional interactions of their respective gene products during germ cell development, the frequency and clinical relevance of activation of these genes during pulmonary carcinogenesis have not been examined in a comprehensive manner. Experiments have been initiated to comprehensively examine BORIS variant- relative to stem cell, and CT-X gene expression profiles using RNA and DNA from lung cancer specimens in the Ludwig Cancer Institute/CUMC lung cancer bank. Clinical-pathologic data including tumor histology and stage, treatment regimens, and outcome for the respective patients, all of whom have undergone surgery at New York-Presbyterian Hospital, have been entered into a database at CUMC. Because the DNA and RNA have been derived from whole tumors, not laser captured cancer cells, techniques are being validated to assess mRNA copy numbers for BORIS variant, stem cell, and CT-X genes relative to thyroid transcription factor 1 (TTF1), which is expressed in pulmonary adenocarcinomas, but not in normal lung, or inflammatory cells. Data from these array experiments will be merged with the CUMC clinical database. Results of this analysis should establish the frequency of, relationships between, and prognostic significance of BORIS variant, stem cell, and CT-X gene activation in pulmonary adenocarcinomas. One potential strategy to overcome limitations of immunotherapy for pulmonary malignancies related to low level, heterogeneous intratumoral CT-X antigen expression, and the lack of recombinant polyvalent vaccines, involves the use of epigenetically-modified autologous tumor cells to immunize lung cancer patients against a variety of CT-X antigens that potentially can be up-regulated in their respective primary cancers by systemic gene induction regimens. To date, no such efforts have been reported. To examine the potential feasibility of this strategy, tissues/fluids from patients with thoracic tumors of various histologies were processed for primary culture. Sources of tumor cells included peritoneal fluid, endoscopic pleural or mediastinal biopsies, as well as CT-guided FNAs. To date, 15 cell lines have been established from thirty-seven patients. Several of these lines have been exposed to DAC+/-DP under exposure conditions exceeding those achievable in clinical settings. These experiments revealed heterogeneous, robust CT-X gene induction;following drug treatment, primary HLA-A201 cancer lines were recognized by allogeneic PBL expressing HLA-A201 TCR for NY-ESO-1 and MAGE-A3. These studies provided the preclinical rationale for a protocol evaluating this use of epigenetically modified autologous tumor cell vaccines as a means to broadly immunize thoracic oncology patients against multiple CT-X antigens following complete resection of their malignancies. The data were presented in oral format at the recent AATS meeting, and a full manuscript is nearing completion for publication
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