The role of HLA-DP as transplantation antigen in contributing to GvHD is a matter of current debate. HLA-DP, is encoded centromeric to DR-DQ and its alleles are in weak linkage disequilibrium with the rest of the MHC; thus a certain number of HLA matched pairs could be actually DP incompatible to study a possible correlation between HLA-DP matching and GvHD, 24 HLA identical BMT/donor-recipient sibling pairs (serologically tested for HLA Class I and DR antigens) were tested for DQ and DP genes using restriction fragment length polymorphism (RFLP) analysis.DNA extracts were digested according to a standard procedure with two different restriction enzymes (HIND III and MSP I) and hybridised with DQ (alpha and beta) and DP (alpha and beta) specific probes. Highly stringent hybridization and washing conditions were used to prevent cross-hybridizations.Twenty four out of 24 pairs proved to be DQ and DP identical. GvHD developed in 16 out of 24 (66.6%) recipients.These data suggest that DNa analysis of DQ-DP regions, with the probes and enzymes used, does not give predictive informations for GvHD in HLA genotypically identical pairs.
Fifty patients with recurrent Hodgkin's disease have been treated with high-dose therapy followed by autologous bone marrow transplantation. Forty-one patients had extranodal sites of relapse and 31 patients had constitutional symptoms. Two patients had been treated with mechlorethamine, vincristine, procarbazine, and prednisone (MOPP), lomustine, vinblastine, procarbazine, and prednisone (CcVPP), and radiation; 16 patients with MOPP, doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD), radiation, and lomustine, etoposide, and prednisone (CEP); 20 patients with alternating MOPP/ABVD, and 12 patients with alternating MOPP/ABVD followed by CEP and radiation. Eighteen patients had progressive disease during alternating MOPP/ABVD protocol alone or during conventional salvage therapy; 32 patients had had a complete remission with first-line therapy but later relapsed, 25 of them having received conventional salvage therapy; 12 achieved no response or progression ("resistant-relapse" patients); and 13 responded partially or completely ("sensitive-relapse" patients). Complete remission occurred in 24 patients (48%) with a median duration of 24 months and 16 patients (32%) achieved partial response with a median duration of 9 months, for an overall response rate of 80%. Ten patients failed to respond and died in progressive disease 1 to 10 months (median, 6 months) after transplantation. Toxicity was significant including infections (20%), liver enzymes and alkaline phosphatase elevations (100%), and carmustine lung toxicity (7%). There were two treatment-related deaths; one patient died of Pseudomonas aeruginosa septicemia and another patient died of cerebral hemorrhage. These results validate the procedure of high-dose therapy followed by autologous bone marrow transplantation in inducing remission in these advanced, highly-treated patients. Clearly, the question of whether high-dose therapy and transplantation will eventually supersede new conventional salvage therapies will be addressed after controlled clinical studies.
Great excitement surrounded the initial discovery of the Philadelphia chromosome translocation and the molecular rearrangement of the abl protooncogene which is generated by this translocation in chronic myelogenous leukemia (CML). In this review, we will discuss how molecular analysis of the biological abnormalities generated in the CML cells by this molecular rearrangement can be applied to new directions in therapy for this disease.
Insulin-like growth factor-I (IGF-I), by itself, cannot sustain the growth of BALB/c 3T3 cells, but requires the cooperation of other growth factors, such as platelet-derived growth factor or epidermal growth factor. In 3T3 cells constitutively overexpressing the human IGF-I receptor, called p6 cells, IGF-I by itself is fully mitogenic. We show here that p6 cells are also stimulated to enter DNA synthesis by the sole addition of basic fibroblast growth factor (bFGF), which, by itself, is incapable of stimulating parental 3T3 cells. Although bFGF does not bind directly to the IGF-I receptor, it induces its autophosphorylation. Stimulation of p6 cells by bFGF is not inhibited by an antibody to the IGF-I receptor that inhibits IGF-I-mediated DNA synthesis, and IGF-I is not detectable in the medium of bFGF-treated p6 cells. Stimulation cannot be explained by an increased number of FGF receptors, because p6 cells actually have slightly fewer FGF receptors than parental BALB/c 3T3 cells. Basic FGF also stimulates DNA synthesis in 3T3 cells overexpressing a mutant IGF-I receptor that does not autophosphorylate in response to IGF-I and has lost its mitogenic potential. Although we were unable to demonstrate directly that bFGF causes transphosphorylation of the IGF-I receptor, we conclude that in cells overexpressing the IGF-I receptor, bFGF can stimulate DNA synthesis either by an unknown mechanism or through transphosphorylation of the IGF-I receptor.
Marrow cells were exposed to the LNL6 or G1N safety-modified variants of the N2 retrovirus, which contain the G418 bacterial resistance gene neo. The frequency of acquisition of the G418 resistance phenotype following exposure to LNL6 or G1N was compared among hematopoietic progenitor cells from the marrow of patients with chronic phase chronic myelogenous leukemia (CML), blast crisis CML, or from nonleukemic individuals. Under the conditions of our experiments, the myeloid committed progenitor cells from 3 of 6 nonleukemic individuals, 9 of 18 chronic-phase CML patients, and 2 of 4 blast crisis CML patients acquired resistance to at least 1 mg/ml G418 following incubation with cell-free supernatants from the PA317 LNL6 or PA317 G1N producer cell lines. Ten of the 32 colonies growing up in 0.8 mg/ml G418 from chronic-phase marrow exposed to LNL6 were shown to contain the neo gene by polymerase chain reaction (PCR) assay of DNA. These results were consistent with estimates of the transduction frequency based on acquisition of resistance to G418 as the number of colonies growing under G418 selection was always greater at 0.8 mg/ml G418 than at higher concentrations of G418 (1.0–1.4 mg/ml). The average transduction frequency at each G418 concentration (1.0, 1.2, and 1.4 mg/ml) in cells from blast crisis CML cells ranged from 2 to 14%, as measured by acquisition of G418 resistance. Chronic-phase CML showed slightly lower average frequencies of transduction (0.6–2.8% of the colonies are G418 resistant). The average transduction frequency of cells from nonleukemic marrow was as high as that seen from the marrow of chronic-phase CML individuals. These results have implications for retroviral marking strategies which are designed to follow the purging of blastic leukemia cells from the marrow of CML patients used for autologous bone marrow transplantation. Retroviral-mediated gene transfer using a marker gene (NeoR) is being used by a number of investigators to ask clinically important questions. One critical question is: When a leukemia patient who has received purged autologous bone marrow relapses, do the leukemic cells come from the administered marrow or do they arise endogenously in the patient's body? Etkin et al. demonstrate that cells from the marrow of patients with advanced stages of chronic myelogenous leukemia can be marked efficiently and, therefore, the question of relapse origin can be studied.
