RELAPSE IN INDOLENT NHL BY VIRAL MARKING

通过病毒标记导致惰性 NHL 复发

• 批准号: 3205536
• 负责人: ALBERT B DEISSEROTH
• 金额: $ 10.75万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-15 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3205536
• 关键词: CD antigens; Retroviridae; SCID mouse; affinity chromatography; autologous transplantation; bone marrow purging; bone marrow transplantation; cancer risk; flow cytometry; genetic markers; hematopoietic stem cells; human subject; human therapy evaluation; immunoglobulin genes; neoplasm /cancer chemotherapy; neoplasm /cancer radiation therapy; neoplasm /cancer relapse /recurrence; neoplasm /cancer therapy; nonHodgkin's lymphoma; polymerase chain reaction; prognosis

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.

该项目的目的是确定有助于 低级非霍奇金淋巴瘤（NHL）的复发 化学疗法。 先前的报告表明，低级患者 NHL接受自体骨髓移植（ABMT）的NHL 自体移植缺乏在体内净化后通过PCR检测到的淋巴瘤细胞 与患者相比 收到遗传标记的PCR保持阳性的骨髓 淋巴瘤。 为了正式测试是否存在淋巴瘤细胞 在自体移植中有助于后一组患者复发， 我们建议用A基因标记骨髓和外周血细胞 安全改性逆转录病毒（已批准了人类使用 一组IV期低级B细胞患者的FDA和NIH） NHL经历了ABMT。 遵循常规剂量细胞还原性 化学疗法和强化准备疗法之前 环磷酰胺，VP-16和TBI，骨髓将被收获，并 残留的淋巴瘤细胞将通过阳性选择的耗尽 造血祖细胞，使用CD34+细胞的免疫吸附到 亲和力柱（Cellpro ceprate扇区），以及负面选择 CD10+，CD19+和CD20+淋巴瘤细胞。 清除的骨髓细胞将 与无辅助病毒的复制缺陷库存一起孵育 逆转录病毒，然后在患者收到患者后重新注册 密集放疗和化学疗法。 存在或不存在 在体内净化之前和之后，骨髓中的淋巴瘤细胞将 通过PCR扩增重新排列的Bcl-2或JH确定 免疫球蛋白基因序列。 标记的效率和稳定性 造血干细胞和带逆转录病毒基因的淋巴瘤细胞将 可以通过单细胞分类来评估，在异源骨上培养 骨髓基质微环境，然后分析培养物 通过PCR存在逆转录病毒和Bcl-2序列。 这 残留在骨髓自体内的淋巴瘤细胞的贡献 在体内清除至临床结果将由 从疾病复发的患者中分析淋巴瘤细胞 ABMT存在逆转录病毒标记基因的存在。 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。 这将通过从NHL移植细胞来完成 诊断为SCID小鼠时的患者，该模型在其中 中间级的NHL细胞和NHL细胞来自惰性淋巴瘤 已经发现复发的患者正在增殖。 我们将 表征生长在细胞中的表型和基因型 SCID小鼠并将其与出现在临床上的淋巴瘤细胞进行比较 复发。 原发性骨髓和外周血祖细胞可能 来自复发患者的淋巴瘤细胞的水平非常低 用逆转录病毒在遗传上标记，然后移植到 免疫缺陷小鼠以确定遗传标记是否稳定 在体内淋巴瘤生长期间。 这样， 可以确定ABMT后的体内清除至临床结果，并且可以确定 然后，这些研究的结果可以应用于 初次治疗后复发的患者更有效的治疗。