Targeting the Epigenome for Lung Cancer Therapy

在过去的十年中，一系列临床方案被启动，以确定接受DNA去甲基化药物和HDAC抑制剂的胸部肿瘤患者的毒性和临床反应。在一项I期试验中，35名患者在35天周期的第1至第3天通过持续输注接受了不断增加的DAC剂量。虽然没有观察到客观的反应，但两名肺癌患者表现出较长时间的疾病稳定（1年）。近四分之一的患者肿瘤组织中p16、MAGE-3或NY-ESO-1的表达增加。在几个接受DAC治疗超过6个月的患者中观察到NY-ESO-1的血清学反应。在随后的II期试验中，19名肺癌患者在21天周期的第1天和第7天以4小时输注的MTD方式接受DP治疗。在DP治疗后，约50%的患者瘤内H3Ac和p21水平升高。此外，一些患者在DP输注后的肿瘤活检中表现出NY-ESO-1和MAGE-A3的表达增强。在最近的一项I期试验中，31例胸部肿瘤患者（包括20例肺癌，4例食管癌和3例MPM患者）接受了顺序DAC/DP输注。DAC按MTD （75mg/m2）连续输注72小时，从35天治疗周期的第1天开始。DP在DAC后立即输注4小时，第10天；在顺序队列中，DP的剂量增加到先前确定的MTD （18mg/m2）。中位稳态DAC和DP水平分别为13.4 ng/ml（范围：4.7-78 ng/ml; 5-80nM）和323 ng/ml（范围：136-923 ng/ml）。虽然没有观察到客观的肿瘤消退，但一些患者表现出持续4-12个月的疾病稳定。采用微阵列技术对8例肺癌患者治疗前和治疗后活检中激光捕获的肿瘤细胞中扩增的RNA中的基因表达谱进行了全面检测；这些阵列的结果与激光捕获的肿瘤细胞和邻近组织学正常的支气管上皮的分析数据进行了比较，这些数据来自20例肺癌确诊切除术患者。DAC/DP暴露后，肺癌细胞中大约1230个基因被显著抑制，而500个基因被上调。这些发现与其他阵列数据一致，表明在模拟临床环境暴露的条件下，DAC/DP处理的培养肺癌细胞中1002个基因被抑制，383个基因被上调。相对于邻近的正常肺上皮，DAC/DP上调的基因在肺癌中被抑制，而DAC/DP抑制的基因在肺癌中比正常肺上皮上调。DAC/DP在体内调节的富集功能群包括细胞信号转导、分子转运、核酸代谢、细胞生长和增殖、肿瘤等。在一项正在进行的I期剂量递增试验中，24例胸部恶性肿瘤患者（包括7例肺癌、4例食管癌、3例MPM和2例胸腺瘤患者）在临床前实验中使用的暴露条件下连续接受了4h DP/72h黄酮吡醇输注。虽然没有观察到客观反应，但7例患者表现出疾病稳定，持续时间为4至12个月。尽管这些令人鼓舞的结果，该试验将很快结束，由于不良应计。该试验的药代动力学和分子终点分析尚未进行。进行了一系列实验室实验，以检查使用表观遗传修饰的肿瘤细胞用于癌症疫苗的潜在可行性。简而言之，对不同组织学的胸部恶性肿瘤患者的组织/液体进行原代培养。组织来源包括胸膜和腹膜液、通过内窥镜技术进行的胸膜和纵隔活检、ct引导的fna和切除的原始标本。38例患者中有10例（26%）建立了细胞系，其中14例NSCLC中有3例，2例SCLC中有1例，6例食管癌中有2例，6例间皮瘤中有4例。从肿瘤消化物的初始培养/抽吸到细胞系（包括冷冻的备用和准备用于药物操作的1x107细胞）的平均时间约为2-3个月。其中一些癌细胞系用DAC （1-3mcM × 6天）治疗，有或没有DP （10-25ng/ml × 6-24小时）。qRT-PCR分析显示，未经处理的细胞系中CT-X基因表达异质；在药物治疗后，基因诱导的模式和程度在这些细胞系中变化很大。使用异体PBL表达NY-ESO-1和MAGE-A3重组TCRs的细胞因子释放试验证实，hla限制了DAC或顺序DAC/DP治疗的肿瘤靶点的识别。在这些暴露条件下，DP并没有一致地增强dac介导的基因诱导或CTL识别。上述研究为评估表观遗传修饰肿瘤细胞作为原发性胸部恶性肿瘤患者的辅助疫苗，以及通过标准治疗导致NED的胸外恶性肿瘤转移到胸部的患者提供了临床前依据。疫苗将与Iscomatrix一起施用，Iscomatrix是一种专利佐剂，已被证明可引起对纯化CTA疫苗的有效免疫反应；此外，口服塞来昔布将用于抑制Tregs的活性，Tregs已被证明可以减弱癌症患者对治疗性疫苗的免疫反应。迄今为止，已有25名患者参与了这些试验。尽管临床前研究证明了这种方法的可行性，但从原发肿瘤中可靠地提取细胞系一直是一个重大挑战，部分原因是切除的恶性肿瘤的组织学。在其他研究中，我们已经确定了几种已建立的细胞系，包括K562红白血病细胞和H1299肺癌细胞，它们在没有药理学操作的情况下表现出高水平的CT-X基因表达，我们试图对其作为佐剂疫苗进行评估。到目前为止，已有15名患者参加了试验，试验涉及给药K562细胞组成性表达GM-CSF (K562- gm)，联合口服节律环磷酰胺和塞来昔布来抑制Treg活性。这些细胞是从约翰霍普金斯大学的一个主细胞库中扩增出来的，使用的资金来自之前的“从实验室到床边奖”。每隔28天x 6天皮下注射疫苗，一个月后评估免疫反应。标准分期研究在基线、第四次疫苗接种前和治疗评估期间进行。几乎所有患者都能耐受节律化疗，并且没有疫苗相关的毒性。所有可评估患者的血清将在MSKCC的路德维希癌症研究所使用能够检测超过30种cta反应的elisa进行全面评估。8名患者在接种部位出现红斑和皮疹，提示对K562-GM注射的某些成分有反应。

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.