Differential gene expression profile correlates with bortezomib responsiveness in steroid-resistant cGVHD. Homo sapiens

Nineteen patients with steroid-refractory cGVHD (chronic graft versus host disease) received weekly bortezomib from 2 – 12 months (1.6 mg/m2 q wk x 4 followed by one week of rest). Treatment was well tolerated with no > grade 3 adverse events. Six patients achieved CR or good PR (gPR). Average Rodnan scores decreased from 22.6 ± 12.8 to 5.9 ± 6.2 (p 2 treatments, including one patient with marked healing of suppurating lesions and significantly reduced chronic diarrhea in another. Thus, weekly bortezomib was safe and produced early improvements in some patients with longstanding refractory disease. CR or gPR correlated with a younger age and lower number of involved organs with a severity score of > 2. Based on gene expression array and pathway analyses, High Responders displayed an immune response profile that resembled that of hematopoietic stem cell-transplanted patients without cGVHD. By comparison, Low Responders showed markedly altered gene expressions in both cell-mediated immune response and proinflammatory pathways. Our preliminary findings suggest that bortezomib responsiveness may be predicated by distinct immunopathological manifestations among cGVHD patients. This trial was registered at www.clinicaltrials.gov as NCT01158105. Overall design: 54 total samples, 0 replicates, 8 healthy controls, 6 disease controls. Sample details follow. Eligible patients were 18 years or older with a confirmed clinical diagnosis of steroid-refractory cGVHD, defined as either failure to improve after 2 months or progression after 1 month of standard steroid-based therapy, and had no prior bortezomib treatment for cGVHD. Enrolled patients were required to be in remission from his/her primary malignancy for which HSCT was indicated, ECOG performance status 3 months, have not had myocardial infarction within 6 months prior to enrollment and not having received radiation therapy within 3 weeks, platelet count ≥ 50 x 10^9/L, absolute neutrophil count ≥ 1.0 x 10^9/L, total bilirubin ≤ 1.5x upper normal limit, creatinine clearance ≥ 30 mL/min and no peripheral neuropathy episodes (≥ Grade 2) within 14 days before enrollment. Patients with prior hypersensitivity to bortezomib, boron or mannitol were excluded from trial. Bortezomib was supplied in vials as open-label stock. Patients received Kytril premedication per institutional standards (1 mg intravenous/IV push) before start of VELCADE® (bortezomib; Millennium Pharmaceuticals) infusion (1.6 mg/m2). Bortezomib was administered on days 1, 8, 15, 22 of a 35-day cycle. Steroid dose that was in effect on Day 1 was maintained. All other agents given for GVHD treatment were withdrawn as tolerated. Patients with stable disease or better after two cycles continued for up to six cycles of bortezomib. Patients who continued to do well were eligible for up to six months of maintenance. Those patients continued on the same dose of bortezomib but on days 1 and 15 of a 28-day cycle. Followup after the final dose was for 6 months. Subjects were assessed for signs and symptoms of cGVHD on day 1 of each cycle by standardized criteria for cGVHD diagnosis, new clinical scoring system, and new guidelines for global assessment of cGVHD severity (NIH Consensus Development Project on Criteria for Clinical Trials in cGVHD). Safety was assessed according to adverse event incidents for 30 days following the last dose for up to 6 months. Adverse events were recorded throughout the trial and followed by investigator until resolution. Enrolled patients also completed a neurotoxicity-directed questionnaire on presence and intensity of neuropathic pain and/or peripheral neuropathy from the patient's perspective. Responses were reviewed to assist with evaluation of onset and intensity of peripheral neuropathy and other neurotoxicities that may require intervention or dose modification. No change from baseline was considered as stable disease (SD). >50% improvement in any specific organ score or overall performance was considered partial response (PR). Disappearance of symptoms of cGVHD was considered complete response (CR). Worsening of symptoms in specific organ or overall observed in two consecutive assessment time points was considered as progressive disease (PD). All human subject-related study protocols were reviewed and approved by the institutional Review Board for Human Protection, Baylor University Medical Center. All patients signed informed consent before being enrolled into study. This trial was registered at www.clinicaltrials.gov as NCT01158105.

19名激素难治性慢性移植物抗宿主病（chronic graft versus host disease, cGVHD）患者接受了为期2至12个月的每周一次硼替佐米（bortezomib）治疗：剂量为1.6 mg/m²，每周给药1次，连用4周后休息1周。该治疗耐受性良好，未出现≥3级不良事件。6名患者达到完全缓解（complete response, CR）或良好部分缓解（good partial response, gPR）。平均Rodnan评分从22.6±12.8降至5.9±6.2（原文此处p值及后续标注缺失），治疗2个周期后，1名患者的化脓性病变明显愈合，另1名患者的慢性腹泻显著改善。综上，每周一次硼替佐米治疗安全，可使部分长期难治性cGVHD患者出现早期病情改善。CR或gPR与患者年龄更轻、受累器官数量更少以及严重评分>2相关。
通过基因表达芯片与通路分析发现，高应答者的免疫应答谱与未发生cGVHD的造血干细胞移植（hematopoietic stem cell transplantation, HSCT）患者相似；相比之下，低应答者的细胞介导免疫应答及促炎通路上的基因表达均发生显著改变。本研究的初步结果提示，硼替佐米的治疗应答可能由cGVHD患者不同的免疫病理表现所决定。
本试验在www.clinicaltrials.gov注册，编号为NCT01158105。

### 总体设计
共纳入54份样本，无重复样本，其中包含8名健康对照者、6名疾病对照者，样本细节如下。

#### 入组标准
符合入组条件的患者需满足：年龄≥18岁，经临床确诊为激素难治性cGVHD（定义为接受标准糖皮质激素治疗2个月后病情未改善，或治疗1个月后病情进展），且此前未因cGVHD接受过硼替佐米治疗；因造血干细胞移植指征罹患的原发恶性肿瘤已达到缓解；东部肿瘤协作组（Eastern Cooperative Oncology Group, ECOG）体能状态在入组前3个月内符合要求（原文此处具体评分标注缺失）；入组前6个月内未发生心肌梗死，入组前3周内未接受放射治疗；血小板计数≥50×10^9/L，中性粒细胞绝对计数≥1.0×10^9/L，总胆红素≤1.5倍正常上限，肌酐清除率≥30 mL/min；入组前14天内未出现≥2级周围神经病发作。

#### 排除标准
对硼替佐米、硼或甘露醇存在既往超敏反应的患者被排除本试验。

#### 治疗方案
硼替佐米以开放标签制剂的瓶剂形式供应。患者在VELCADE®（硼替佐米；千禧制药（Millennium Pharmaceuticals））输注前，按照机构标准接受Kytril预处理（1 mg静脉推注），给药剂量为1.6 mg/m²。硼替佐米在35天周期的第1、8、15、22天给药。维持患者在治疗第1天所用的糖皮质激素剂量。其他用于GVHD治疗的药物在可耐受的前提下停用。
完成2个周期治疗后病情稳定或更佳的患者，可继续接受最多6个周期的硼替佐米治疗；病情持续良好的患者可接受最多6个月的维持治疗，此时维持给药剂量不变，但调整为在28天周期的第1、15天给药。最后一剂给药后，对患者进行为期6个月的随访。

#### 疗效与安全性评估
每周期第1天，采用cGVHD标准化诊断标准、全新临床评分系统以及cGVHD严重程度全球评估新指南（美国国立卫生研究院cGVHD临床试验标准共识开发项目），对患者的cGVHD体征与症状进行评估。
安全性评估针对最后一剂给药后30天内发生的不良事件，随访时长最长为6个月。整个试验期间均需记录不良事件，并由研究者跟进至不良事件缓解。
入组患者还需完成一份针对神经毒性的问卷，从患者自身视角评估神经性疼痛和/或周围神经病的存在与强度，以协助评估可能需要干预或调整剂量的周围神经病及其他神经毒性的发生与严重程度。

#### 疗效判定标准
与基线状态无变化视为病情稳定（stable disease, SD）；任一特定器官评分或整体体能状态改善≥50%视为部分缓解（partial response, PR）；cGVHD症状完全消失视为完全缓解（complete response, CR）；连续两个评估时间点出现特定器官或整体症状恶化视为进展性疾病（progressive disease, PD）。

#### 伦理审查与知情同意
所有涉及人类受试者的研究方案均经贝勒大学医学中心人类保护机构审查委员会审查批准。所有患者在入组研究前均签署了知情同意书。

本试验在www.clinicaltrials.gov注册，编号为NCT01158105。