Stem cell transplantation in multiple myeloma : Clinical aspects and experimental gene transfer studies

Sammanfattning: STEM CELL TRANSPLANTATION IN MULTIPLE MYELOMA Clinical aspects and experimental gene transfer studies Thesis by Bo Björkstrand, M.D.. Department of Medicine, Karolinska Institutet at Huddinge University Hospital, S-141 86 Huddinge, SwedenMultiple myeloma (MM) is a hematological malignant disease. considered essentially incurable despite its sensitivityto cytotoxic chemotherapeutic drugs and radiation. High-dose chemo/radiotherapy with allogeneic- or autologoushematopoietic stem cell transplantation (tx) can induce a disease-free state and may prolong survival and offer animproved quality of life to MM patients. The aim of this thesis was to improve the results of high-dose treatmentwith hematopoietic stem cell tx in MM. 523 patients reported to the Myeloma Registry of the European Group for Blood and Marrow Transplantation(EBMT) were subjected to retrospective studies: 334 patients who underwent autologous stem cell transplantation(ASCT) were analyzed with respect to parameters of prognostic importance for the outcome after tx. 189 patientswho umderwent allogeneic bone marrow transplantation (BMT) with an HLA-matched sibling donor were comparedto an equal number of patients treated with ASCT after case-matching for gender and number of lines of previouslyadministered treatment, and were evaluated with respect to the outcome after tx. A program utilizing two sequentialcycles of high-dose treatment and autografting was applied on 23 patients at Huddinge Hospital. and was prospectivelyevaluated with respect to feasibility and efficacy, and selected patients in complete remission (CR) were analyzedfor minimal residual disease (MRD) using tbe PCR-based immunoglobulin-gene "fingerprinting" method. In theexperimental studies, the effect of including basic fibroblast growth factor (bFGF) in the prestimulation medium inaddition to the three-cytokine combination of stem cell factor, interleukin-3 and interleukin-6 on the rate of retroviral-mediated transfer (RMGT) of the neomycin resistance (neoR) gene in human hematopoietic cells in vitro wasassessed, and the feasibility of RMGT in antibody-separated human MM cells in vitro was studied. Finally, aclinical protocol for gene marking of CD34+ cells in the autograft, with the aim to detect a possible graft-origin ofrecurring MM cells at relapse after ASCT, was prepared and activated. For the 334 ASCT-patients analyzed, the median overall survival (OS) after tx was 38 months and actuarial OS at90 months was 25%. Factors significantly predictive for a better OS or progression-free survival (PFS) followingASCT were tx as part of front-line treatment before disease progression, responsiveness to chemotherapy, malegender, post-tx alpha-interferon maintenance treatment, a low beta-2-microglobuln value at diagnosis, and to achieveCR after tx, and tbe first four of these parameters were also independently predictive for a bettcr outcome in amultivariate analysis. In addition, for patients in first chemotherapy-responsive phase of disease, stage I-II atdiagnosis and treatment in a double-ASCT program were associated with a signficantly better outcome. In thecomparison between BMT and ASCT, OS was significantly better after ASCT (median OS 34 months, vs 18 forBMT), which was evident during the first 3 years post-tx, but later the survival curves merged and long-term OSwas similar. The survival difference was only observed in males, while in females the survival was similar.Tramsplamt-related mortality (TRM) was significantly higher for BMT, which was the primary reason for the pooreroutcome, even tbough relapse rate was higher after ASCT. For the double-ASCT program, the CR-rate was 69%,actuarial OS at 49 months was 85%, amd 61% of the patients were in continuous CR. There were two MM-unrelateddeaths. The original clonal band could initially not be detected by PCR in any of the six patients amalyzed, but later aband appeared in two patients who subsequently relapsed from CR. RMGT into humam hematopoietic cells wassignificantly enhanced by the addition of bFGF to the three-factor medium. The transduction rate in terms ofG418-resistmt CFU-GM was estimated to 38% and 27% with and without bFGF, respectively, and the optimalconcentration of bFGF was in the range between 20 and 2tl0 ng/ml. Tbe feasibility of RMGT in humam MM cellswas demonstrated, with G418-resistance ranging between 1.5% and 50% in liquid cultures. The presence of theneoR-gene in the transduced cytohne-stimulated hematopoietic cells as well as in the transduced humam MM cellswas proven by PCR-analysis. In conclusion, the outcome after ASCT in MM patients is influenced by several factors of prognostic importance.Survival is significantly better after ASCT as compared to BMT, but the relapse rate is lower after BMT, and themain reason for tbe difference in survival is tbe higher rate of TRM associated with BMT. A program with twosequential ASCT-procedures is feasible, and em induce long-term CR at a substantial rate, where MRD in someCR-patients could not be detected by PCR. A foreign selectable marker gene can be introduced by RMGT into MMcells in vitro, and this forms the basis of a clinical study with the intention to mark cells in the autograft prior toreinfusion, which may enable the determination of the origin of clonal MM cells at relapse following ASCT.Keywords: multiple myeloma, bone marrow transplantation, allogeneic, autologous, prognostic factors, hemato-poietic stem eells, gene therapy, gene transfer, retrovirus, basic fibroblast growth factor