IN-VITRO MAINTENANCE AND RETROVIRAL TRANSDUCTION OF HUMAN MYELOMA CELLS IN LONG-TERM MARROW CULTURES

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplan t is attributable to contamination of the autograft with myeloma cells . A requirement in these studies is ex vivo genetic marking of maligna nt cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of clono genic myeloma cells in marrow aspirates from 14 patients with MM. To e ffect gene transfer we utilized a long-term marrow culture (LTMC) syst em previously shown to facilitate gene transfer into a spectrum of hem atopoietic progenitor and stem cells. Transduction of cells in LTMC wa s performed by multiple supernatant exposure. At LTMC initiation and a fter 21 days of culture malignant cells were assessed by morphology, f low cytometry, and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoi etic progenitors as determined by the number of colonies positive for proviral DNA by PCR, G418 resistance, and X-gal staining was also with in the expected range; 65%, 44% and 23%, respectively. Thus, there was no evidence that prior chemotherapy exposure or malignant cell contam ination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 2 1-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 1 2 informative patients (50%). PCR using allele-specific primers when a vailable confirmed the specificity of IgVH rearrangements for the myel oma clone. In 2 of the 14 patients, expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for tran sfer of reporter genes (neo' and LacZ) into the myeloma clone: morphol ogically abnormal G418-resistant colonies demonstrated intense stainin g for beta-galactosidase, and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patie nt, individual colonies positive for beta-galactosidase bore a cytogen etic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primer s was positive. Our results demonstrate the maintenance of myeloma cel ls in vitro for up to 21 days in LTMC. They further illustrate that th ese cells can be genetically marked using transduction protocols curre ntly being tested in clinical trials of hematopoietic cell gene transf er.