Epigenetic Therapy for Thoracic Malignanceis

超过100名胸部恶性肿瘤患者已经接受了一系列临床方案的治疗，这些方案检查了DNA去甲基化剂（地西他滨; 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。总体而言，这些试验表明没有客观的临床消退，尽管在约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，近四分之一的患者显示肿瘤组织中p16、法师-3或NY-ESO-1的表达增加。在几名接受DAC治疗超过6个月的患者中观察到对NY-ESO-1的血清学应答。大约50%接受DP输注的患者表现出肿瘤内H3 Ac和p21水平升高。此外，一些患者在DP输注后肿瘤活检中表现出NY-ESO-1和MAGE-A3的表达增强。对接受DAC、DP或连续DAC/DP输注的患者治疗前和治疗后活检的激光捕获肿瘤细胞的微阵列分析显示，从肺癌基因特征转变为在正常呼吸道上皮中观察到的基因特征。这些早期试验为表观遗传方案联合免疫干预治疗胸部恶性肿瘤提供了概念证据。由于CT-X抗原似乎优先在多能肿瘤细胞中表达，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疫苗，以诱导对癌症干细胞的免疫应答。关于可靠建立原代细胞系的困难限制了我们在临床上正式评价该问题的能力，这些方案被关闭。在试图规避这些问题，我们已经检查了，如果从同种异体癌细胞产生的疫苗可以诱导广泛的免疫CT-X抗原，可能会上调胸部恶性肿瘤的基因诱导方案。在最近的II期试验中，我们评估了H1299肺癌细胞冻融裂解物的免疫原性。该NSCLC细胞系在NCI建立，由于X染色体扩增，相对于K562-GM，该细胞系表现出许多相关CT-X基因表达的高水平表达。在一项正在进行的首次人体试验中，21例通过常规治疗导致NED的胸部恶性肿瘤患者被随机分配接受来自H1299肺癌细胞的冻融裂解物，该细胞表现出高水平的CTA表达，Iscomatrix通过皮内注射q month x 6 +/-口服节拍循环/细胞。主要终点是第6次接种后1个月对纯化肿瘤抗原的血清学应答。所有患者在接种疫苗后均表现出持续72-96小时的局部和全身炎症反应。无治疗相关毒性。14例患者（67%）完成了所有6次疫苗接种; 7例患者因疾病复发提前退出研究。8名患者（57%）对NY-ESO-1表现出血清学应答。观察到针对GAGE 7、XAGE和MAGE-C2的额外应答。疫苗治疗还降低了TcB百分比（p=0.0067）和TcB上的PD-1表达（p=0.0023），以及CD 14+单核细胞（p=0.0089）、经典单核细胞（p=0.0159）和中间单核细胞（p= 0.0031）上的PD-L1表达。Cy/cel不增加免疫应答或增强疫苗诱导的外周免疫亚群的改变。H1299裂解物疫苗诱导对CTA的免疫应答，并以可能增强抗肿瘤免疫的方式调节外周免疫亚群。这些发现支持在胸部肿瘤患者中评价该裂解物与免疫检查点抑制剂的组合。这项试验的结果已被选为美国外科医师学会年度临床大会的口头报告，与这些发现有关的手稿将在不久的将来提交出版。关于在癌症免疫治疗方案中使用DNA去甲基化剂的主要限制涉及短半衰期（5分钟）和由于胞苷脱氨酶（CDA）引起的差的生物分布，所述胞苷脱氨酶（CDA）在整个身体中以高水平存在。最近在非人灵长类动物中的研究和在镰状细胞病患者中的临床试验已经证明，四氢尿苷（THU）的口服制剂-一种先前已经静脉内施用给数百名癌症患者而没有毒性的化合物可以显著增加口服地西他滨的Cmax（50 nM）和T1/2（4小时）。这些发现为评价口服DAC/THU和免疫检查点抑制剂Nivolumab作为NSCLC患者二线治疗的II期试验以及口服DAC/THU和派姆单抗作为表现出高水平PDL 1表达的不可手术NSCLC患者一线治疗的I/II期剂量递增研究提供了依据。将在耶鲁大学的转化免疫肿瘤学实验室进行PD终点的综合最先进转化分析，包括多重定量IHC、多参数质谱细胞术（CyTOF）和下一代测序。将在NIH临床中心进行其他研究，包括免疫亚群、循环肿瘤细胞和循环肿瘤DNA甲基化状态分析。

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. 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 have examined if vaccines produced from allogeneic cancer cells can induce broad immunity to CT-X antigens that potentially can be up-regulated in thoracic malignancies by gene induction regimens. In a recent phase II trial, we have evaluated the immunogenicity of a freeze thaw lysate of H1299 lung cancer cells. This NSCLC line was established at the NCI and exhibits high level expression of numerous relevant CT-X gene expression relative to K562-GM due to amplification of the X chromosome. In an ongoing first-in- humans trial, 21 patients with thoracic malignancies rendered NED by conventional therapy were randomized to receive freeze-thaw lysates from H1299 lung cancer cells exhibiting high-level CTA expression with Iscomatrix via intradermal injection q month x 6 +/- oral metronomic cy/cel. The primary endpoint was serologic response to purified tumor antigens one month after the 6th vaccination. All patients exhibited local and systemic inflammatory responses lasting 72-96 hours following vaccinations. There were no treatment related toxicities. 14 patients (67%) completed all six vaccinations; 7 patients were removed from study early due to disease recurrence. 8 patients (57%) exhibited serologic responses to NY-ESO-1. Additional responses were observed against GAGE7, XAGE and MAGE-C2. Vaccine therapy also decreased percent Tregs (p=0.0067) and PD-1 expression on Tregs (p=0.0023), as well as PD-L1 expression on CD14+ monocytes (p=0.0089), classical monocytes (p=0.0159), and intermediate monocytes (p= 0.0031). Cy/cel did not increase immune responses or enhance vaccine-induced alterations in peripheral immune subsets.H1299 lysate vaccines induce immune responses to CTAs, and modulate peripheral immune subsets in a manner that may enhance antitumor immunity. These findings support evaluation of this lysate in combination with immune checkpoint inhibitors in thoracic oncology patients. Results of this trial have been selected for oral presentation at the American College of Surgeons Annual Clinical Congress, and a manuscript pertaining to these findings will be submitted for publication in the near future. A major limitation regarding the use of DNA demethylating agents in cancer immunotherapy regimens pertains to the short half-life (5min) and poor biodistribution due to cytidine deminase (CDA) which is present at high levels throughout the body. Recent studies in non-human primates and a clinical trial in patients with sickle cell disease, have demonstrated that an oral formulation of tetrahydrouridine (THU)- a compound that has been previously administered intravenously to hundreds of cancer patients with no toxicities can markedly increase Cmax (50nM)and T1/2 ( 4 hours) of oral Decitabine. These findings have provided the rationale for a phase II trial evaluation oral DAC/THU and the immune checkpoint inhibitor Nivolumab as second line therapy for patients with NSCLC, as well as a phase I/II dose escalation study of oral DAC/THU and pembrolizumab as first line therapy for patients with inoperable NSCLC exhibiting high level PDL1 expression. 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.