RELAPSE IN INDOLENT NHL BY VIRAL MARKING

该项目的目标是确定有助于 低级别非霍奇金淋巴瘤（NHL）的复发， 化疗 以前的报道表明，低级别的患者 接受自体骨髓移植（ABMT）的NHL， 自体移植物在离体净化后缺乏通过PCR可检测到的淋巴瘤细胞 长期无病生存率比那些 接受的骨髓通过PCR检测遗传标记仍呈阳性 淋巴瘤 为了正式测试是否存在淋巴瘤细胞 在自体移植物中导致后一组患者的复发， 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（已被批准用于人类使用的 FDA和NIH）在一组IV期低级别B细胞 接受ABMT的NHL。 常规剂量细胞减灭后 化疗和强化准备治疗前， 环磷酰胺、VP-16和TBI，将收获骨髓， 残留的淋巴瘤细胞将通过阳性选择被耗尽， 造血祖细胞，使用CD 34+细胞的免疫吸附， 亲和柱（CellPro Ceprate Sector），和阴性选择 CD 10+、CD 19+和CD 20+淋巴瘤细胞。 净化的骨髓细胞将 与无辅助病毒的复制缺陷型 逆转录病毒，然后重新注入病人后，他们接受了 强化放疗和化疗。 的存在或不存在 在离体净化之前和之后骨髓中的淋巴瘤细胞将 通过PCR扩增重排的bcl-2或Jh 免疫球蛋白基因序列。 打标的效率和稳定性 具有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、在异源骨上培养 骨髓基质微环境，随后分析培养物 通过PCR检测逆转录病毒和bcl-2序列的存在。 的 自体骨髓移植物中残留淋巴瘤细胞的贡献 将通过以下方法评估离体清除后的临床结局 分析来自疾病复发患者的淋巴瘤细胞， ABMT检测是否存在逆转录病毒标记基因。 我们还将使用动物模型来表征淋巴瘤细胞， 导致其免疫表型、基因型和 生长特性 这将通过移植NHL的细胞来完成， 将诊断时的患者移植到SCID小鼠中， 中度NHL细胞和惰性淋巴瘤NHL细胞 已经发现复发的患者会增殖。 我们将 表征在细胞中生长的细胞的表型和基因型。 SCID小鼠，并将其与临床上出现的淋巴瘤细胞进行比较。 复发 原代骨髓和外周血祖细胞， 含有非常低水平的淋巴瘤细胞的复发患者将 用逆转录病毒进行基因标记，然后移植到 免疫缺陷小鼠，以确定遗传标记是否稳定 在体内淋巴瘤的生长过程中。 这样， 可以确定ABMT后的离体净化至临床结果，并且 这些研究的结果可以应用于开发 对初次治疗后复发的患者更有效。

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.