During the past decade, a series of clinical protocols were initiated to determine toxicities and clinical responses in thoracic oncology patients receiving DNA demethylating agents, and HDAC inhibitors. In a phase I trial, 35 patients received escalating doses of DAC administered via continuous infusion on days 1 through 3 of a 35 day cycle. Although no objective responses were observed, two lung cancer patients exhibited prolonged stabilization of disease (>1 yr). Nearly one quarter of all patients exhibited increased expression of p16, MAGE-3, or NY-ESO-1 in tumor tissues. Serologic responses to NY-ESO-1 were observed in several patients receiving DAC for more than six months. In a subsequent phase II trial, 19 lung cancer patients were treated with DP at the MTD administered as a 4h infusion on days 1 and 7 of a 21 day cycle. Intratumoral levels of H3Ac and p21 were increased in approximately 50% of patients following DP therapy. In addition, several patients exhibited enhanced expression of NY-ESO-1 and MAGE-A3 in tumor biopsies following DP infusions. In a more recent phase I trial, 31 patients with thoracic neoplasms (including 20 lung cancer, 4 esophageal cancer and 3 MPM patients) received sequential DAC/DP infusions. DAC was administered at the MTD (75mg/m2) as a continuous 72h infusion commencing on day 1 of a 35 day treatment cycle. DP was administered as a 4h infusion immediately following DAC, and on day 10;the dose of DP was increased in sequential cohorts to the previously established MTD (18mg/m2). Median steady state DAC and DP levels were 13.4 ng/ml (range: 4.7-78 ng/ml;5-80nM), and 323 ng/ml (range: 136-923 ng/ml), respectively. Whereas no objective tumor regressions were observed, several patients exhibited stabilization of disease lasting 4-12 months. Micro-array techniques were used to comprehensively examine gene expression profiles in RNA amplified from laser-captured tumor cells from pre- and post treatment biopsies from eight lung cancer patients;results of these arrays were 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. Approximately 1230 genes were significantly repressed, whereas 500 genes were up-regulated in lung cancer cells following DAC/DP exposure. These findings were consistent with additional array data demonstrating repression of 1002 genes and up-regulation of 383 genes in cultured lung cancer cells treated with DAC/DP under conditions mimicking exposures in clinical settings. Genes that were up-regulated by DAC/DP were repressed in lung cancers relative to adjacent nomal lung epithelia, whereas genes that were repressed by DAC/DP were up-regulated in lung cancers compared to normal lung epithelia. Enriched functional groups modulated by DAC/DP in vivo included cell signaling, molecular transport, nucleic acid metabolism, cell growth and proliferation, and cancer.In an ongoing phase I dose-escalation trial, 24 patients with thoracic malignancies including 7 lung cancer, 4 esophageal cancer, 3 MPM, and 2 thymoma patients, have received sequential 4h DP/72h Flavopiridol infusions under exposure conditions corresponding to those used in our preclinical experiments. Whereas no objective responses have been observed, seven patients have exhibited disease stabilization lasting from 4 to >12 months. Despite these encouraging results, this trial will close soon due to poor accrual. Analyses of pharmacokinetics and molecular endpoints for this trial have not been performed as yet.A series of laboratory experiments were conducted to examine the potential feasibility of using epigenetically modified tumor cells for cancer vaccines. Briefly, tissues/fluids from patients with thoracic malignancies of various histologies were processed for primary culture. Sources of tissue included pleural and peritoneal fluid, pleural aswell as mediastinal biopsies via endoscopic techniques, CT-guided FNAs, and resected primary specimens. Cell lines were established from 10 of 38 patients (26%) including 3 of 14 NSCLC, 1 of 2 SCLC, 2 of 6 esophageal cancer, and 4 of 6 mesothelioma patients. Average time from initial culture of tumor digests/aspirate to cell lines, including frozen aliquot back-up and >1x107 cells ready for drug manipulation, was approximately 2-3 months. Several of these cancer lines were treated with DAC (1-3mcM x 6 days) with or without DP (10-25ng/ml x 6-24h). qRT-PCR analysis revealed heterogeneous CT-X gene expression in untreated cell lines;patterns and magnitude of gene induction varied considerably in these lines following drug treatment. Cytokine release assays using allogeneic PBL expressing recombinant TCRs for NY-ESO-1 and MAGE-A3 confirmed HLA-restricted recognition of tumor targets treated with DAC or sequential DAC/DP. DP did not consistently enhance DAC-mediated gene induction or CTL recognition under these exposure conditions.The aforementioned studies provided the preclinical rationale for evaluation of epigenetically-modified tumor cells as adjuvant vaccines in patients with primary thoracic malignancies, as well as patients with extra-thoracic malignancies metastatic to the chest, who are rendered NED by standard therapy. The vaccines are to be administered with Iscomatrix, a proprietary adjuvant that has been shown to elicit potent immunologic responses to purified CTA vaccines;additionally, oral celecoxib will be used to inhibit activity of Tregs that have been shown to attenuate immune responses to therapeutic vaccines in cancer patients. To date, 25 patients have been accrued to these trials. Despite preclinical studies demonstrating feasibility of this approach, reliable derivation of cell lines from primary tumors has been a significant challenge, in part due the histologies of the resected malignancies. In additional studies, we have identified several established cell lines including K562 erythroleukemia, and H1299 lung cancer cells that exhibit high level CT-X gene expression without pharmacologic manipulation, which we have sought to evaluate as adjuvant vaccines. To date, 15 patients have been accrued to trials involving administration of K562 cells constitutively expressing GM-CSF (K562-GM) in conjunction with oral metronomic cyclophosphamide and celecoxib to inhibit Treg activity. These cells were expanded from a master cell bank at Johns Hopkins University using funds from a previous Bench-to-Bedside Award. The vaccines are administered subcutaneously q 28days x 6, followed by evaluation of immune response one month later. Standard staging studies are obtained at baseline, prior to the fourth vaccine, and during treatment evaluation. Virtually all patients have tolerated metronomic chemotherapy, and there have been no vaccine-related toxicities. Sera from all evaluable patients will be comprehensively assessed at the Ludwig Cancer Institute at MSKCC using ELISAs capable of detecting responses to more than 30 CTAs. Eight patients have developed erythema and rash at their vaccination sites, suggesting response to some component of the K562-GM injections.
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