RELAPSE IN INDOLENT NHL BY VIRAL MARKING

该项目的目标是确定有助于 低度恶性非霍奇金淋巴瘤(NHL)初治后复发 化疗。以前的报告显示，低级别的患者 接受自体骨髓移植(ABMT)的NHL 自体移植物在体外净化后缺乏可被聚合酶链式反应检测的淋巴瘤细胞 与那些长期无病生存的患者相比， 接受的骨髓经聚合酶链式反应检测遗传标记仍为阳性 死于淋巴瘤。为了正式测试淋巴瘤细胞的存在 在后一组患者中，自体移植会导致复发， 我们建议用基因标记骨髓和外周血细胞 安全修饰的逆转录病毒(已被批准用于人类 FDA和NIH)对一组IV期低级别B细胞患者的治疗 NHL正在接受ABMT。遵循常规剂量的细胞还原剂 化疗和强化准备治疗前使用 环磷酰胺，VP-16，TBI，骨髓采集和 残留的淋巴瘤细胞将被阳性选择耗尽 造血祖细胞，利用CD34+细胞的免疫吸附 亲和柱(CellPro Ceprate扇区)，负面选择 CD10+、CD19+和CD20+淋巴瘤细胞。净化后的骨髓细胞将 与复制缺陷的无辅助病毒库存孵化 逆转录病毒，然后在患者接受了 强化放疗和化疗。存在或不存在 体外净化前后骨髓中的淋巴瘤细胞将 通过重排bcl-2或JH的PCR扩增来确定 免疫球蛋白基因序列。评卷的效率和稳定性 携带逆转录病毒基因的造血干细胞和淋巴瘤细胞将 通过单细胞分选，在异种骨上培养来评估 骨髓基质微环境，并对培养结果进行分析 用聚合酶链式反应检测逆转录病毒和bc1-2基因序列的存在。这个 淋巴瘤细胞残留在自体骨髓移植中的作用 体外净化后的临床结果将通过以下方式评估 检测术后复发患者的淋巴瘤细胞 ABMT检测逆转录病毒标记基因的存在。 我们还将使用动物模型来描述淋巴瘤细胞的特征 导致复发与其免疫表型、基因和 生长特性。这将通过移植非霍奇金淋巴瘤的细胞来完成 患者在被诊断为SCID小鼠时，一种模型 惰性淋巴瘤的中级非霍奇金淋巴瘤细胞和非霍奇金淋巴瘤细胞 复发的患者已经被发现有增殖现象。我们会 描述生长在体内的细胞的表型和基因 并将它们与临床上出现的淋巴瘤细胞进行比较 旧病复发。原代骨髓和外周血祖细胞，可能 复发患者体内含有非常低水平的淋巴瘤细胞 用逆转录病毒进行基因标记，然后移植到 免疫缺陷小鼠确定遗传标记是否稳定 在体内淋巴瘤的生长过程中。通过这种方式，中国的贡献 可以确定ABMT后的体外排泄到临床结果，以及 这些研究的结果随后可以应用于开发 对初次治疗后复发的患者更有效的治疗。

The goal of this project is to identify the factors that contribute to relapse of low grade non-Hodgkin's lymphoma (NHL) following initial chemotherapy. Previous reports have shown that patients with low grade NHL undergoing autologous bone marrow transplantation (ABMT) whose autografts lacked lymphoma cells detectable by PCR after ex vivo purging had an improved long-term disease-free survival compared to patients who received bone marrow that remained positive by PCR for genetic markers of lymphoma. In order to formally test if the presence of lymphoma cells in the autograft contributes to relapse in the latter group of patients, we propose to genetically mark marrow and peripheral blood cells with a safety-modified retrovirus (that has been approved for human use by the FDA and the NIH) in a group of patients with stage IV low grade B-cell NHL undergoing ABMT. Following conventional dose cytoreductive chemotherapy and before intensive preparative therapy with cyclophosphamide, VP-16 and TBI, bone marrow will be harvested and residual lymphoma cells will be depleted by positive selection of hematopoietic progenitor cells, using immunoadsorption of CD34+ cells to an affinity column (CellPro Ceprate Sector), and negative selection of CD10+, CD19+ and CD20+ lymphoma cells. the purged bone marrow cells will be incubated with a helper-virus free stock of replication-defective retrovirus and then re-infused to patients after they have received the intensive radiotherapy and chemotherapy. The presence or absence of lymphoma cells in the bone marrow before and after ex vivo purging will be determined by PCR amplification of rearranged bcl-2 or Jh immunoglobulin gene sequences. The efficiency and stability of marking hematopoietic stem cells and lymphoma cells with retroviral genes will be assessed by single cell sorting, culturing on a heterologous bone marrow stromal microenvironment, and subsequently analyzing the cultures for the presence of the retroviral and bcl-2 sequences by PCR. The contribution of lymphoma cells remaining in the bone marrow autograft following ex vivo purging to clinical outcome will be assessed by assaying lymphoma cells from patients whose disease relapsed following ABMT for the presence of the retroviral marker genes. We will also use an animal model to characterize the lymphoma cells that lead to relapse with respect to their immunophenotype, genotype, and growth properties. this will be done by transplanting cells from NHL patients at the time of diagnosis into SCID mice, a model in which intermediate grade NHL cells and NHL cells from indolent lymphoma patients at relapse have already been found to proliferate. We will characterize the phenotype and genotype of the cells which grow in the SCID mice and compare them to the lymphoma cells that appear at clinical relapse. Primary marrow and peripheral blood progenitor cells which may contain very low levels of lymphoma cells from relapsed patients will be genetically marked with the retrovirus, and then transplanted into immunodeficient mice to determine whether the genetic marker is stable during the growth of lymphoma in vivo. In this way, the contribution of ex vivo purging to clinical outcome following ABMT can be determined, and the results of these studies can then be applied to the development of more effective therapy for patients who relapse after initial therapy.