Human NHL Research Articles

A novel antifolate 10-propargyl-10-deazaaminopterin (PDX) displays synergistic effects with gemcitabine in non-Hodgkin's lymphoma models in vitro and in vivo

6596 Background: PDX is a rationally designed antifolate aimed at improving membrane transport and polyglutamylation in tumor cells. Previous studies have shown that PDX has better activity and a better hematological toxicity profile than MTX against human NHL. PDX as single agent is currently in phase I and II clinical trials for patients with NHL. Methods: The combination of PDX (2nM) and gemcitabine (20nM) was tested in vitro in 4 NHL cell lines. Cells were exposed to one drug for 24h, then exposed to the same or other drug for additional 24h or exposed to both drugs for 24h, then cultured in RPMI alone for 24h. Efficacy was measured at 48h by Alamar Blue and Trypan Blue assays. The same combination was evaluated in vivo in a model of human NHL in irradiated NOD/SCID mice. Mice were inoculated with 10x106 SKI-DLCL cells via a sc. route. When tumors approached 300 mm3, mice were divided into 4 groups of 5 and treated twice weekly for 4 doses i.p. with normal saline (control), MTD of PDX (60 mg/kg) and gemcitabine (60 mg/kg) or a combination of the drugs, each given at 30 mg/kg. Results: PDX displayed synergistic effects with gemcitabine in vitro in all cell lines, with strongest synergy observed in cells first treated with PDX and subsequently exposed to gemcitabine. This combination produced 10.9±4.1% greater effects in SKI-DLCL, 13.5±5.4% in HT, 25.5±7.2 % in DOHH2, and 28.4±8.7 % in RL compared to the additive effects of both drugs given as monotherapy. Animals in the control group had rapid POD and were sacrificed. PDX or gemcitabine treated animals initially responded to treatment but by day 44 regrew tumors to 739.0% and 317.7% of their initial tumor volumes, respectively. Animals in the PDX+gemcitabine arm had significant reduction (63.1%) of their initial tumor volume, with 3 animals in CR, 1 in PR and 1 with POD. Conclusions: The potential of the PDX-gemcitabine combination is very promissing. Future experiments are planned in order to: 1) determine the optimal schedule of these drugs, and 2) compare the eficacy of the optimal combination to the “standard” antifolate-pyrimidine analog combination (methotrexate-Ara-C). No significant financial relationships to disclose.

A novel antifolate 10-propargyl-10-deazaaminopterin (PDX) displays synergistic effects with gemcitabine in non-Hodgkin's lymphoma models in vitro and in vivo

6596 Background: PDX is a rationally designed antifolate aimed at improving membrane transport and polyglutamylation in tumor cells. Previous studies have shown that PDX has better activity and a better hematological toxicity profile than MTX against human NHL. PDX as single agent is currently in phase I and II clinical trials for patients with NHL. Methods: The combination of PDX (2nM) and gemcitabine (20nM) was tested in vitro in 4 NHL cell lines. Cells were exposed to one drug for 24h, then exposed to the same or other drug for additional 24h or exposed to both drugs for 24h, then cultured in RPMI alone for 24h. Efficacy was measured at 48h by Alamar Blue and Trypan Blue assays. The same combination was evaluated in vivo in a model of human NHL in irradiated NOD/SCID mice. Mice were inoculated with 10x106 SKI-DLCL cells via a sc. route. When tumors approached 300 mm3, mice were divided into 4 groups of 5 and treated twice weekly for 4 doses i.p. with normal saline (control), MTD of PDX (60 mg/kg) and gemcitabine (60 mg/kg) or a combination of the drugs, each given at 30 mg/kg. Results: PDX displayed synergistic effects with gemcitabine in vitro in all cell lines, with strongest synergy observed in cells first treated with PDX and subsequently exposed to gemcitabine. This combination produced 10.9±4.1% greater effects in SKI-DLCL, 13.5±5.4% in HT, 25.5±7.2 % in DOHH2, and 28.4±8.7 % in RL compared to the additive effects of both drugs given as monotherapy. Animals in the control group had rapid POD and were sacrificed. PDX or gemcitabine treated animals initially responded to treatment but by day 44 regrew tumors to 739.0% and 317.7% of their initial tumor volumes, respectively. Animals in the PDX+gemcitabine arm had significant reduction (63.1%) of their initial tumor volume, with 3 animals in CR, 1 in PR and 1 with POD. Conclusions: The potential of the PDX-gemcitabine combination is very promissing. Future experiments are planned in order to: 1) determine the optimal schedule of these drugs, and 2) compare the eficacy of the optimal combination to the “standard” antifolate-pyrimidine analog combination (methotrexate-Ara-C). No significant financial relationships to disclose.

Preparation of purified lymphoma cells suitable for therapy.

Very few tumoral Ags have yet been isolated in NHL B cells. It is nevertheless possible to use whole tumor cells as a source of tumor Ags. We describe the purification of large numbers of human NHL B cells directly from lymph node or spleen biopsies, and different preparations allowing their use in a clinical setting. The purification procedure consists of the negative selection of tumor B cells: cells to be eliminated are opsonized by CD2 Abs, and then coupled to magnetic beads for separation by the Isolex 300 magnetic separator. The mean yield of the purification was 74% for CD19+ cells, with a mean purity of 87%, dependent on the initial fraction of tumor cells in the biopsy. Using this procedure, a large number of purified tumor cells can be recovered from a biopsy in sterile conditions. We also describe treatments of B cells that can enhance their uptake by APCs, a critical step in anti-tumor immunotherapy strategies. Cells were opsonized by rituximab, or induced in apoptosis by irradiation, or necrosis by heating. Cell lysates were directly prepared from purified tumor cells. These procedures were reproducible on every lymphoma cell, and treated cells were phagocytosed by APCs. The methodology described here allows the evaluation of the immunological potential of apoptotic, necrotic, opsonized lymphoma cells, or their lysates, in a clinical setting.

Endostatin induces tumor stabilization after chemo- or ANTI-CD20 therapy of high-grade non-hodgkin's lymphoma (Nhl)

Both chemotherapy and chimeric anti-CD20 monoclonal antibodies are effective agents against B-cell NHL. However, patients achieving remission are at risk of relapse. To evaluate the effect of the anti-angiogenic drug endostatin alone and after the administration of cyclophosphamide (CTX) or anti-CD20 rituximab antibody, we have generated a new model of human NHL by transplanting ip Namalwa cells in NOD/SCID mice. First, we evaluated the more effective treatment schedule of the different drugs. When administered alone, CTX (3 courses of 75 mg/Kg ip) rituximab (3 courses of 25 mg/Kg ip) and endostatin (5 courses of 50 μg sc) delayed tumor growth, and CTX was the more effective in controlling bulky disease. When given after chemo- or immuno-therapy, endostatin effectively induced tumor stabilization. In fact, when mice given CTX or rituximab on day 3, 5 and 7 after transplantation were randomized to receive endostatin or PBS on day 15 to 19, in endostatin-treated mice tumor growth was prevented as long as the drug was administered. Furthermore, endostatin administered on day 25- to 29 after tumor re-growth was still able to induce significant tumor regression, whereas CTX and rituximab were not effective. Sequential administration of chemotherapy and endostatin seems promising for bulky NHL, and the less toxic sequential administration of rituximab and endostatin is promising for limited disease.

Suitability of Epstein-Barr virus-based episomal vectors for expression of cytokine genes in human lymphoma cells.

Plasmids carrying the Epstein-Barr virus (EBV) latent gene EBNA1 and the EBV latent origin of replication (oriP) stay in transfected human cells as autonomously replicating extrachromosomal genetic units. They thus might represent a suitable tool for cytokine gene introduction into human tumor cells with the prospect of therapeutic antitumor vaccination. The aim of this study was to analyze whether such plasmids permit stable and efficient expression of cytokine genes in human non-Hodgkin lymphoma cells. We tested physical stability and expression levels of plasmids carrying EBNA1 and oriP for episomal maintenance, immunoglobulin light chain enhancer elements for augmentation of expression, and cytokine or marker genes after introduction into human NHL cell lines in vitro and in vivo after inoculation into nude mice. Data obtained with these EBV-based vectors were compared with another plasmid, not carrying EBNA1 and oriP. cDNAs coding for GM-CSF, IL6, TNF alpha, the chloramphenicolacetyltransferase (CAT) and the beta-galactosidase (lacZ) gene were transfected into the EBV-positive Burkitt's lymphoma cell line BL60 and the EBV-negative B cell lymphoma cell line BJA-B. EBV-derived vectors permitted a high, host cell independent transfection efficiency and high and host cell independent levels of expression. After removal of the selection pressure (hygromycin B) cytokine expression could be detected for several weeks in vitro and in vivo but, however, declined continuously. These experiments suggest that episomal BC-derived vectors represent an effective tool for cytokine gene transfer in human lymphoma cells.