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.
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