Targeting the Epigenome for Lung Cancer Therapy

针对肺癌治疗的表观基因组

• 批准号: 8763700
• 负责人: DAVID SCHRUMP
• 金额: $ 54.47万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763700
• 关键词: Aberrant DNA Methylation; Acute Erythroblastic Leukemia; Adjuvant; Adoptive Transfer; Aftercare; Aliquot; Allogenic; Antigens; Apoptosis; Aspirate substance; Attenuated; Award; Back; Biological Assay; Biopsy; CTAG1 gene; Cancer Patient; Cancer Vaccines; Cell Cycle Progression; Cell Line; Cell Proliferation; Cells; Chest; Chromatin Structure; Chromosome Mapping; Clinical; Clinical Protocols; Continuous Infusion; Cyclophosphamide; DNA; Data; Decitabine; Depsipeptides; Derivation procedure; Disease; Dose; Drug Kinetics; Endogenous Retroviruses; Epithelium; Erythema; Evaluable Disease; Evaluation; Exanthema; Excision; Exhibits; Freezing; Funding; Gene Expression; Genes; Genomic Instability; Germ Cells; Goals; Granulocyte-Macrophage Colony-Stimulating Factor; Growth; H1299; Histologic; Histology; Histone Deacetylase Inhibitor; Histones; Immune response; Immunocompetent; Immunologics; Infusion procedures; Injection of therapeutic agent; Institutes; K-562; K562 Cells; Laboratories; Lasers; Liquid substance; Lung; MAGEA3 gene; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of esophagus; Malignant neoplasm of lung; Malignant neoplasm of testis; Mediastinal; Mediating; Memorial Sloan-Kettering Cancer Center; Mesothelioma; Metastatic to; Modeling; Modification; Molecular; Molecular Profiling; Mus; Non-Small-Cell Lung Carcinoma; Oncogenes; Oral; Patients; Pattern; Peritoneal Fluid; Pharmaceutical Preparations; Phase; Phase I Clinical Trials; Phase II Clinical Trials; Pleural; Polycomb; Positioning Attribute; Primary Neoplasm; Process; Proteins; Publishing; RNA; Recombinants; Regulatory T-Lymphocyte; Relative (related person); Repression; Resected; Series; Serological; Serum; Signal Transduction; Site; Source; Specimen; Staging; Structure of respiratory epithelium; T-Lymphocyte; Techniques; Thoracic Neoplasms; Thoracic Oncology; Thymoma; Time; Tissues; Tobacco; Toxic effect; Transport Process; Tumor Suppressor Genes; Tumor Tissue; Universities; Up-Regulation; Vaccination; Vaccine Adjuvant; Vaccines; X Chromosome; bench to bedside; bronchial epithelium; cancer cell; cancer immunotherapy; cancer therapy; celecoxib; cell bank; cell growth; chemotherapy; chromatin remodeling; cohort; cytokine; demethylation; epigenome; established cell line; flavopiridol; functional group; gene induction; genome-wide; immunogenic; immunogenicity; imprint; in vivo; neoplastic cell; novel; nucleic acid metabolism; pre-clinical; preclinical study; research study; response; senescence; therapeutic vaccine; treatment duration; tumor

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 as well 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.

在过去的十年中，启动了一系列临床方案，以确定接受DNA脱甲基剂和HDAC抑制剂的胸部肿瘤患者的毒性和临床反应。在I期试验中，在35天周期的第1至第3天连续输注通过连续输注进行了35例DAC的不断升级。尽管未观察到客观反应，但两名肺癌患者表现出长时间的疾病稳定（> 1年）。在所有患者中，将近四分之一的患者表现出肿瘤组织中P16，MAGE-3或NY-ESO-1的表达增加。在接受DAC的几名患者中观察到了对NY-ESO-1的血清学反应超过六个月。在随后的II期试验中，在21天周期的第1天和第7天，在MTD中用DP治疗19例肺癌患者。在DP治疗后，大约50％的患者增加了肿瘤内H3AC和P21的水平。此外，在DP输注后，几名患者在肿瘤活检中表现出增强的NY-ESO-1和MAGE-A3的表达。在最近的I期试验中，有31例胸腔肿瘤患者（包括20例肺癌，4例食管癌和3个MPM患者）接受了顺序的DAC/DP输注。 DAC以MTD（75mg/m2）的形式施用，作为在35天治疗周期的第1天开始连续的72H输注。 DP在DAC后立即作为4H输注，并在第10天进行。在顺序组中，DP的剂量增加到了先前建立的MTD（18mg/m2）。中位稳态DAC和DP水平为13.4 ng/ml（范围：4.7-78 ng/ml; 5-80nm）和323 ng/ml（范围：136-923 ng/ml）。尽管未观察到客观肿瘤回归，但几名患者表现出持续4-12个月的疾病稳定。微阵列技术用于全面检查来自八名八名肺癌患者的前后治疗前和治疗后活检中从激光捕获的肿瘤细胞扩增的RNA中的基因表达谱；将这些阵列的结果与来自激光捕获的肿瘤细胞分析以及来自20例明确肺癌切除术的20名患者的组织学正常支气管上皮的数据进行了比较。在DAC/DP暴露后，大约1230个基因被显着抑制，而500个基因在肺癌细胞中上调。这些发现与其他阵列数据一致，这些阵列数据表明在培养的肺癌细胞中，在用DAC/DP处理的培养的肺癌细胞中，在模仿临床环境中暴露的培养肺癌细胞中的383个基因上调。与正常的肺癌相比，与正常的肺癌相比，与相邻的名义肺上皮相对于相邻的名义肺上皮抑制了由DAC/DP上调的基因在肺癌中受到抑制，而被DAC/DP抑制的基因在肺癌中上调。由DAC/DP在体内调节的富集官能团包括细胞信号传导，分子转运，核酸代谢，细胞生长和增殖以及癌症。在正在进行的I期剂量升级试验中，24例胸腔恶性肿瘤患者，包括7例肺癌，4例食管癌，3个MPM和2例胸腺瘤患者，已在暴露条件下接收了4H DP/72H黄叶肽输注，与我们的临时性实验中使用的情况相对应。尽管尚未观察到客观反应，但七名患者表现出持续4到12个月的疾病稳定。尽管有这些令人鼓舞的结果，但由于应计差，该试验很快就会结束。尚未进行该试验的药代动力学和分子终点的分析。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进行癌症疫苗的潜在可行性。简而言之，对各种组织学的胸腔恶性肿瘤患者的组织/液体进行了处理以进行原发性培养。组织的来源包括胸膜和腹膜液，胸膜和纵隔活检通过内窥镜技术，CT引导的FNA和切除的主要标本。从38例患者中的10名（26％）中建立了细胞系，包括14个NSCLC中的3例，2个SCLC中的1例，6种食管癌中的2例和6名间皮瘤患者中的4例。从肿瘤消化的初始培养/抽吸到细胞系的平均时间，包括冷冻等分试样的备份和> 1x107细胞进行药物操纵的细胞，约为2-3个月。这些癌细胞系中有几个用DAC（1-3mcm x 6天）处理，有或没有DP（10-25ng/ml x 6-24H）。 QRT-PCR分析显示未处理细胞系中的异质CT-X基因表达。在药物治疗后，在这些系中，基因诱导的模式和大小差异很大。使用同种异体PBL表达重组TCR的NY-ESO-1和MAGE-A3的细胞因子释放测定证实了对用DAC或顺序DAC/DAC治疗的肿瘤靶标的HLA限制性识别。在这些暴露条件下，DP并未始终增强DAC介导的基因诱导或CTL识别。上述研究提供了临床前的理由，用于评估原发性胸腔恶性肿瘤患者的表观遗传改性肿瘤细胞作为辅助疫苗，以及对胸部胸腔恶性肿瘤转移的患者，这些患者是通过标准治疗对胸部进行的。该疫苗应用Iscomatrix施用，Iscomatrix是一种专有辅助物，已显示出对纯化的CTA疫苗的有效免疫反应；此外，口服塞来昔布将用于抑制已显示可减弱癌症患者治疗疫苗的免疫反应的Treg的活性。迄今为止，已有25名患者接受了这些试验。尽管临床前研究证明了这种方法的可行性，但原发性肿瘤细胞系的可靠推导是一个重大挑战，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红血球血症和H1299肺癌细胞，这些细胞表现出高水平的CT-X基因表达，而无需药理操作，我们试图将其评估为辅助疫苗。迄今为止，已有15名患者被纳入涉及施用K562细胞的试验，这些K562细胞与口服分类环磷酰胺和CelecoxiB结合使用，以构成表达GM-CSF（K562-GM），以抑制Treg活性。这些细胞从约翰·霍普金斯大学的大型细胞银行扩展，使用了以前的基准奖中的资金。疫苗是皮下施用的Q 28天X 6，然后在一个月后评估免疫反应。标准分期研究是在基线，第四次疫苗和治疗评估期间获得的。几乎所有患者都耐受性化疗，并且没有与疫苗相关的毒性。所有可评估患者的血清将在MSKCC的路德维希癌症研究所进行全面评估，使用能够检测到30多个CTA的反应的ELISA。八名患者在其疫苗接种部位出现了红斑和皮疹，这表明对K562-gm注射的某些组成部分反应。