Epigenetic Therapy for Thoracic Malignanceis

超过100例胸部恶性肿瘤患者接受了一系列临床方案治疗，检查了DNA去甲基化药物（Decitabine； DAC）和HDAC抑制剂（如罗米地辛（DP））单独或与其他研究药物联合输注后的毒性和临床反应。总的来说，这些试验没有证明客观的临床退化，尽管在大约10%的患者中观察到疾病的长期稳定（4 - 12个月）。在接受DAC输注的所有患者中，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。大约50%接受DP输注的患者表现出瘤内H3Ac和p21水平升高。此外，一些患者在DP输注后的肿瘤活检中显示NY-ESO-1和MAGE-A3的表达增强（120）。对接受DAC、DP或顺序DAC/DP输注的患者治疗前和治疗后活检中激光捕获的肿瘤细胞进行微阵列分析，揭示了从肺癌基因特征到正常呼吸上皮中观察到的基因特征的转变。这些早期试验为使用表观遗传方案结合免疫干预治疗胸部恶性肿瘤提供了概念证明。由于CT-X抗原似乎在多能性肿瘤细胞中优先表达，因此可以想象，自体表观遗传修饰的肿瘤细胞可能是独特的、个性化的疫苗，可以诱导对癌症干细胞的免疫反应。关于可靠建立原代细胞系的困难限制了我们在临床中正式评估这一问题的能力，因此这些方案被关闭。为了避免这些问题，我们研究了从K562红白血病和H1299肺癌细胞中产生的疫苗，这些细胞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，是否可以诱导对CT-X抗原的广泛免疫，这些抗原可能通过基因诱导方案在胸部恶性肿瘤中上调。在一项II期试验中，20名患者在完全切除恶性肿瘤后，接受了6次每月免疫接种，使用活辐照的表达GM-CSF （K562- gm）的K562细胞，并联合口服环磷酰胺和塞来昔布（抑制免疫抑制Tregs）作为辅助治疗。节律化疗耐受性良好，未观察到与疫苗相关的毒性。虽然一些患者在接种疫苗后对纯化的CT-X抗原表现出增加的血清学反应性，但没有人符合免疫反应标准。一些患者在接受治疗时异常地长时间无病间隔，然后在退出研究后不久迅速复发。这些发现提高了疫苗诱导短暂的抗肿瘤免疫的可能性，而对纯化的CT-X抗原的血清学反应分析并未检测到这种免疫；或者，患者可能从节律化疗中获益。一篇关于对碳水化合物抗原反应的论文正在接受同行评审，我们正在为另一篇论文完成对疫苗和自体肿瘤免疫反应的额外分析。在最近的研究中，我们评估了H1299冻融裂解液的免疫原性。这个NSCLC系是在NCI-NCI建立的，由于X染色体的扩增，相对于K562-GM，它表现出更广泛和更高水平的CT-X基因表达。在一项正在进行的试验中，20名接受常规治疗但复发风险高的胸部肿瘤患者被随机分配接受H1299溶出物与Iscomatix辅助治疗，伴或不伴节拍器环磷酰胺/塞来昔布。在6个月内每月接种疫苗，一个月后进行治疗评估。由于临床中心PDS的问题，该首次人体疫苗试验自2015年5月以来一直处于FDA临床搁置状态，这些问题与Iscomatrix没有直接关系，但仍然阻止了我们使用该小包装产品的当前批次。没有疫苗相关的不良事件。盲法分析显示，14名可评估治疗反应的患者中有8名（57%）对几种CT-X和内源性逆转录病毒抗原的血清转化或现有滴度显着增加。关于表观遗传治疗实体癌的一个主要问题是肿瘤的异质性和FNA标本反映癌细胞中靶基因调节的可靠性，以及药物治疗对肿瘤微环境的影响。鉴于最近的观察结果，DNA去甲基化剂和HDAC抑制剂通过抑制肿瘤微环境中的MDSC来增强对免疫检查点抑制剂的反应，后一个问题尤其相关（127）。最近，我们推测，如果患者的转移是克隆的，那么接受双侧肺转移切除术的患者可能是评估新的表观遗传方案的理想候选者。令人惊讶的是，关于这个问题的信息很少。因此，从12例患者切除的65例三切肺转移瘤中提取RNA和DNA。qRT-PCR技术用于评估广泛的CT-X和T-S基因的表达水平，以及编码人类恶性肿瘤中异常表达的表观遗传修饰因子的基因。利用Pyrosequencing技术定量分析了LINE1、NBL2和D4Z4重复序列的DNA甲基化水平和差异调控基因的启动子甲基化状态。该分析表明，肺转移表现出组织学相关和患者特异性的全球DNA去甲基化谱。即使在具有相似肿瘤组织学的个体中，也观察到显著的患者间基因表达异质性。无论切除时间（同步/异时）或解剖位置如何，来自同一患者的转移灶的表观遗传特征似乎一致。这些研究为最近批准的一项I期剂量递增研究提供了理论依据，该研究将口服DAC/THU作为辅助治疗，用于接受标准护理的双侧肺转移切除术的患者。患者将接受最初的半胸转移切除术，随后接受6周口服DAC/THU治疗，如果没有禁忌症，随后进行对侧转移切除术。耶鲁大学的转化免疫肿瘤学实验室将对PD终点进行全面的、最先进的转化分析，包括多重定量免疫结构、多参数细胞计数（CyTOF）和下一代测序。其他研究包括免疫亚群、循环肿瘤细胞和循环肿瘤DNA甲基化状态的分析将在NIH临床中心进行。此外，一项评估Nivolumab单独或联合口服DAC/THU作为NSCLC二线治疗的II期试验将在不久的将来开放。本方案将与。克利夫兰诊所的Yogen Saunthararajah和Vamsidhar Velcheti，以及上述全面的PD分析，是目前正在审查的UO-1资助（D. Schrump，校内PI； Yogen Saunthararajah，校外PI）的一部分。

More than 100 patients with thoracic malignancies have been treated on a series of clinical protocols examining toxicities and clinical responses following infusions of DNA demethylating agents (Decitabine; DAC), and HDAC inhibitors, such as romidepsin (DP) alone or in combination with other investigational agents. Collectively these trials demonstrated no objective clinical regressions, although prolonged stabilization of disease (4 - 12months) was observed in approximately 10% of patients. Nearly one quarter of all patients receiving DAC infusions 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. Approximately 50% of patients receiving DP infusions exhibited increased intratumoral levels of H3Ac and p21. In addition, several patients exhibited enhanced expression of NY-ESO-1 and MAGE-A3 in tumor biopsies following DP infusions (120). Micro-array analysis of laser captured tumor cells from pre and post treatment biopsies from patients receiving DAC, DP or sequential DAC/DP infusions revealed a shift from a lung cancer gene signature to one observed in normal respiratory epithelia. These early trials provided proof of concept for the use of epigenetic regimens in combination with immunologic interventions for the treatment of thoracic malignancies. Because CT-X antigens appear to be preferentially expressed in pluripotent tumor cells, it is conceivable that autologous epigenetically modified tumor cells may be unique, personalized vaccines to induce immune responses to cancer stem cells. Difficulties regarding reliable establishment of primary cell lines limited our ability to formally evaluate this issue in the clinic, and these protocols were closed. In an attempt to circumvent these problems, we examined if vaccines produced from K562 erythroleukemia and H1299 lung cancer cells which exhibit high level CT-X gene expression without pharmacologic manipulation could induce broad immunity to CT-X antigens that potentially can be up-regulated in thoracic malignancies by gene induction regimens. In a phase II trial, 20 patients received six, monthly immunizations with live irradiated K562 cells constitutively expressing GM-CSF (K562-GM) in conjunction with metronomic oral cyclophosphamide and celecoxib (to inhibit immunosuppressive Tregs) as adjuvant therapy following complete resection of their malignancies. Metronomic chemotherapy was well tolerated, and no vaccine-related toxicities were observed. Whereas several patients exhibited increased serologic reactivity to purified CT-X antigens following their vaccinations, none met criteria for immunologic response. Several patients had uncharacteristically long disease free intervals while on therapy, and then developed rapid recurrence shortly after being removed from study. These findings raise the possibility that the vaccine induced transient antitumor immunity that was not detected by analysis of serologic responses to purified CT-X antigens; alternatively, the patients may have derived benefit from the metronomic chemotherapy. A manuscript pertaining to responses to carbohydrate antigens is under peer review, and we are completing additional analysis of immune responses to the vaccine and autologous tumor for another manuscript. In more recent efforts, we have evaluated the immunogenicity of a freeze thaw lysate of H1299. This NSCLC line was established at the NCI-NCI and exhibits broader and much higher levels of CT-X gene expression relative to K562-GM due to amplification of the X chromosome. In an ongoing trial, 20 thoracic oncology patients rendered NED by conventional therapy, yet at high risk for recurrence have been randomized to receive H1299 lysates with Iscomatix adjuvant with or without metronomic cyclophosphamide/celecoxib. Vaccines have been administered monthly for 6 months followed by treatment evaluation one month later. A variety of biologic and immunologic endpoints are being assessed in this First-in-Humans vaccine trial which has been on FDA Clinical Hold since May, 2015 due to problems in the Clinical Center PDS, which were not directly related to Iscomatrix, but nevertheless have prevented us from using the current lot of this vialed product. There were no vaccine related adverse events. Blinded analysis demonstrated sero-conversion or significant increases in existing titers to several CT-X and endogenous retroviral antigens in 8 of 14 patients (57%) evaluable for treatment response. A major concern regarding epigenetic therapy for solid cancers pertains to tumor heterogeneity and reliability of FNA specimens to reflect target gene modulation in cancer cells, as well as effects of drug treatment on the tumor microenvironment. This latter issue is particularly relevant given recent observations that DNA demethylating agents and HDAC inhibitors enhance responses to immune checkpoint inhibitors by inhibiting MDSC within the tumor microenvironment (127). Recently we speculated that patients undergoing bilateral pulmonary metastasectomy procedures might be ideal candidates for evaluation of novel epigenetic regimens provided their metastases are clonal. Surprisingly little information was available regarding this issue. As such, RNA and DNA were extracted from 65 trisected pulmonary metastases resected from 12 patients. qRT-PCR techniques were used to evaluate expression levels of a broad panel of CT-X and T-S genes as well as genes encoding epigenetic modifiers aberrantly expressed in human malignancies. Pyrosequencing techniques were used to quantitate DNA methylation levels in LINE1, NBL2, and D4Z4 repetitive sequences and promoter methylation status of differentially regulated genes. This analysis demonstrated that pulmonary metastases exhibit histology-related and patient-specific global DNA demethylation profiles. Significant inter-patient heterogeneity of gene expression was observed even among individuals with similar tumor histologies. Epigenetic signatures appeared consistent among metastases from the same patient, irrespective of time of resection (synchronous/metachronous) or anatomic location. These studies provided the rationale for a recently approved phase I dose escalation study of oral DAC/THU administered as adjuvant therapy for patients undergoing standard of care bilateral pulmonary metastasectomy procedures. Patients will undergo initial hemithoracic metastasectomy, and thereafter receive six weeks of oral DAC/THU therapy followed by contralateral metastasectomy if no contraindications. Comprehensive state of the art translational analyses of PD endpoints including multiplex quantitative IHC, multiparametric mass cytometry (CyTOF) and next-gen sequencing will be conducted in the Translational Imuno-Oncology Laboratory at Yale University. Additional studies including analysis of immune subsets, circulating tumor cells and methylation status of circulating tumor DNA will be performed at the NIH Clinical Center. Additionally, a phase II trial evaluating Nivolumab alone or in combination with oral DAC/THU as second line therapy for NSCLC will open in the near future. This protocol, which will be conducted in collaboration with Drs. Yogen Saunthararajah and Vamsidhar Velcheti at the Cleveland Clinic, and incorporate comprehensive PD analyses described above, are part of a UO-1 grant (D. Schrump, intramural PI; Yogen Saunthararajah, extramural PI) presently under review.