Potential drugs for elimination of acute lymphatic leukemia cells from autologous bone marrow

Five drugs, selected because of minimal in vivo myelotoxicity, have been investigated for inhibition of the growth of three acute lymphatic leukemia-derived cell lines. Granulocyte-macrophage colony-forming units (GM-CFU) inhibition with these five drugs after 60-min incubation was first established. Drug concentrations giving up to 90% kill of GM-CFU were then used. Spirogermanium and L-asparaginase did not have an effect on any of the three cell lines under the culture conditions tested, while 4-hydroperoxycyclophosphamide (4-HC) and vincristine inhibited the growth of all three cell lines tested, and bleomycin inhibited the growth of two cell lines. In addition to 4-HC, bleomycin and vincristine should be considered as possible agents in eliminating leukemic cells from autologous marrow grafts. These drugs also deserve further investigation in clonal systems.     

Comparative regimens for the ex vivo chemopurification of B cell lymphoma-contaminated marrow

The success of autologous bone marrow transplantation for B cell lymphoma may depend on the efficacy of in vitro purification of patients' tumor cell-contaminated marrow. In this study, we tested the toxicity of seven different chemotherapeutic agents against two B cell lymphoma lines (LY-16 and SK-DHL-2) as compared to normal human bone marrow granulocyte-macrophage progenitor cells (CFU-GM). 4-Hydroperoxycyclophosphamide (4-HC), VP-16-213 (VP-16), nitrogen mustard, and vincristine showed a highly selective toxicity against cultured lymphoma cells; i.e., at doses sufficient to induce a 4-log clonogenic tumor cell reduction (4-HC 21 micrograms/ml, VP-16 50 micrograms/ml, nitrogen mustard and vincristine 5 micrograms/ml), 10.0 +/- 6.7, 3.0 +/- 3.2, 23.2 +/- 22.7 and 24.0 +/- 17.0% (mean +/- 1 SD), respectively, of normal bone marrow CFU-GM were preserved. The differential sensitivity of tumor cells and normal hematopoietic precursors was less prominent after exposure of cells to cis-diamminechloroplatinum II (cis-platinum); thus, at a drug dose of 100 micrograms/ml, all detectable lymphoma cells could be eradicated (i.e., greater than or equal to 4 log reduction) while a CFU-GM recovery of only 0.2 +/- 0.2% was observed. In contrast, adriamycin and bleomycin, at the highest tumoricidal concentrations tested (5 and 100 micrograms/ml, respectively) did not exhibit a selective toxicity toward lymphoma cell lines. In summary, our results suggest that nitrogen mustard and vincristine, as well as 4-HC and VP-16, may be useful agents for the ex vivo treatment of bone marrow grafts form B cell lymphoma patients.     

Autologous bone marrow transplantation in acute myelogenous leukemia: in vitro treatment with myeloid-specific monoclonal antibodies and drugs in combination

We report the results of a preclinical study comparing four different purging protocols using a promyelocytic human cell line HL-60 and myeloid leukemic progenitor cells (colony-forming unit-leukemic [CFU-L]) from acute myelogenous leukemia (AML) patients assayed in semisolid culture. We studied the antileukemic effect of (1) Single-cycle complement-mediated lysis by two different monoclonal antibodies (MoAbs) (M195 [CD33] and F23 [CD13] 40 micrograms/mL), reactive with distinct antigens found on early myeloid cells and monocytes, used alone and in combinations; (2) 4-Hydroperoxycyclophosphamide (4-HC) (80 mumol/L or 100 mumol/L) alone; or (3) combined with VP-16 (5 micrograms/mL) and (4) a cocktail of 1 through 3 as above (combined immunochemotherapy). More than 4 logs of HL-60 tumor cell elimination were observed after 1 hour of incubation with both MoAbs plus 4-HC + VP-16 while the single treatment (immunotherapy or chemotherapy) provided 1.5 and 3.5 logs of colony-forming inhibition, respectively. When the same protocols were tested on cryopreserved leukemic cells from eight patients with AML, we observed a mean value of CFU-L inhibition of 92.3% +/- 2.5% SD, 95.5% +/- 1.4% SD, and 99% +/- 0.8% SD after MoAbs and complement lysis, 4-HC, and 4-HC + VP-16 treatment, respectively. The combined treatment of MoAbs and 4-HC + VP-16 produced more than 3-log reduction of CFU-L colony formation. By comparison, the mean recovery of committed normal bone marrow progenitors after incubation with MoAbs and complement was 12% for CFU-granulocyte-macrophage (CFU-GM), 22.9% for burst-forming unit erythroid (BFU-E), and the recovery following 4-HC + VP-16 treatment was 4.4% for CFU-GM and 5.6% BFU-E. In subsequent experiments, highly purified CD34+ blast cells, enriched by positive selection, and stimulated in liquid culture by cytokines (interleukin-1 [IL-1], IL-3, and combination of both) or MO-conditioned medium (MoCM), demonstrated that immunochemotherapy spares hematopoietic colony-forming cells earlier than day 14 CFU-GM, in vitro.     

Marrow purging in autologous bone marrow transplantation for T-lineage acute lymphoblastic leukemia: efficacy of ex vivo treatment with immunotoxins and 4-hydroperoxycyclophosphamide against fresh leukemic marrow progenitor cells

A lymphoblast progenitor cell assay was used to evaluate the antileukemic efficacy of marrow-purging protocols that employed intact ricin immunotoxins (IT) and 4-hydroperoxycyclophosphamide (4-HC) against clonogenic primary T-lineage marrow blasts freshly obtained from 12 T-lineage acute lymphoblastic leukemia (ALL) patients. Residual T-lineage blast colonies were observed after treatment with 1 micrograms/mL T101 (anti-CD5)-Ricin (R) + G3.7 (anti-CD7)-R in eight of 12 cases and after 100 micrograms/mL 4-HC in six of nine cases. By comparison, a combination of IT and 4-HC proved very effective against T-lineage leukemic progenitor cells, and no residual blast colonies were observed in any of the eight cases studied. We conclude that future trials should consider combined treatment protocols such as IT + 4-HC for more effective purging of autologous marrow grafts.     

Preclinical studies of the combination of mafosfamide (Asta-Z 7654) and etoposide (VP-16-213) for purging leukemic autologous marrow

In the present study we evaluated the effect of etoposide (VP-16-213) compared to mafosfamide-cyclohexylamine (Asta-Z 7654) on normal granulocyte-macrophage colony-forming unit (GM-CFU) growth, T-cell response to mitogens, and a clonogenic promyelocytic cell line (HL-60). The incubation time (30 min vs 60 min) appeared to be a fundamental parameter. The GM-CFU recovery was 14.4% +/- 7.3% and 1.4% +/- 2.3%, respectively, at 50 micrograms/ml Asta-Z 7654, and 17.6% +/- 8.6% and 3.00% +/- 2.4%, respectively, at 50 micrograms/ml VP-16. ASTA-Z at 50 micrograms/ml was effective in inhibiting the T-cell response to phytohemagglutinin (98.7% +/- 1.2%), whereas VP-16 was not (2.3% +/- 1.7%). With the combined chemical agents ranging from 10 to 20 micrograms/ml for each drug, we obtained a better GM-CFU recovery (five to ten times) using a middle term liquid culture (21-day incubation) than with the standard colony assay (plated immediately after treatment). When using HL-60 cells as the target, the antileukemic activity of VP-16 was lower than of Asta-Z 7654. Both compounds, at 20 micrograms/ml, resulted in 3.3- and 2.3-log cell killing, respectively. On the other hand, lower doses of Asta-Z 7654 combined with VP-16 (ranging from 10 to 15 micrograms/ml each) induced greater than 4-log cell killing after 60 min incubation time. These data suggest that VP-16 could be combined with Asta-Z provided that the dose is reduced for both drugs (less than 20 micrograms/ml).     

Dipyridamole enhancement of drug sensitivity in acute lymphoblastic leukemia cells

The effect of dipyridamole (DPM) on cell sensitivity to anticancer drugs was examined in acute lymphoblastic leukemia (ALL) cell lines. We established two ALL cell lines (KMO-90 and KMO-R) from bone marrow samples of a 12-year-old girl with ALL. The drug concentrations needed to reduce optical density to 50% of that of control cells (IC₅₀) showed that KMO-R was about twofold more resistant to doxorubicin (DOX), mitxantrone (MIT), vincristine (VCR), and etoposide (VP-16) than was KMO-90. Considering that both KMO-90 and KMO-R were established from a patient with ALL at the time of presentation and relapse, respectively, these two cell lines might be novel and useful models for research into the acquisition of drug resistance in ALL cells. Although cytotoxicity of DPM in KMO-90 was about 6% at 1 μg/ml, DPM enhanced cell sensitivity to DOX, MIT, VCR, and VP-16 at this concentration. Cytotoxicity of DPM in KMO-R was less than 5% at 1,5, and 10 μgg/ml. In KMO-R, DPM enhanced cell sensitivity to these four drugs in a dose-dependent manner. The plasma concentrations achieved by oral administration of DPM is about 1 μg/ml. At clinically achievable concentrations, DPM enhanced cell sensitivity to DOX, MIT, VCR, and VP-16 in both KMO-90 and KMO-R, thus showing DPM to be a useful agent for potentiating anticancer chemotherapy of hematopoietic malignancy.     

Ex vivo treatment of myeloma cells by 4-hydroperoxycyclophosphamide and VP-16-213

To assess the usefulness of 4-hydroperoxycyclophosphamide (4-HC) and VP-16-213 (VP-16) as a purging agent for myeloma cells in bone marrow (BM) ex vivo, myeloma cell lines (SK-RCS-1, RPMI-8226), lymphoma cell line (SK-DHL-2) and normal BM cells were treated at different concentrations of 4-HC or VP-16. Clonogenic tumor cells from all three cell lines could be reduced by more than 4 logs, when treated alone or as a mixture with irradiated normal BM cells at a 4-HC concentration of 60 microM. Under similar conditions, approximately 1% of the normal BM myeloid progenitor granulocyte-macrophage colony-forming cells survived. These observations support the use of 4-HC for purging myeloma cells for autologous BM transplantation.     

Sensitivity of fresh acute myeloid leukemia cells to etoposide: relationship with cell growth characteristics and DNA single-strand breaks.

In this study, we evaluated the individual in vitro sensitivity of fresh acute myeloid leukemia (AML) cells to VP-16, and attempted to correlate VP-16 cytotoxicity with AML cell growth characteristics and drug-induced DNA single-strand breaks (SSB). Primary (PE1) colony inhibition assays allowed us to characterize two distinct groups of AML: group I (patients 1 through 6), which displayed sensitivity to VP-16 similar to that of normal CFU-GM (IC90 of 20.52 +/- 2.44 micrograms/mL v 20.48 +/- 2.23 micrograms/mL after 1 hour drug exposure, respectively); and group II (patients 7 through 11), which was more sensitive to VP-16 (IC90 of 7.26 +/- 2.93 micrograms/mL, P = .004). Subsequently, groups I and II were termed normosensitive and hypersensitive, respectively. This objective VP-16 sensitivity classification, as determined by PE1, remained unaltered when assessed by secondary (PE2) colony inhibition assay (evaluating the self-renewal fraction of AML progenitors), or by cytofluorometric viability assay (evaluating the ultimately differentiated blast cell population). These findings would suggest that individual sensitivity to VP-16 of a particular cell population is maintained throughout CFU-AML differentiation. Finally, we report that sensitivity of AML cells to VP-16 did not correlate either with cell growth characteristics or with SSB generation. Indeed, AML cell sensitivity to VP-16 appeared more closely related to DNA repair kinetics after drug removal, ie, hypersensitivity being essentially characterized by a prolonged retention of SSB during the posttreatment period. Interestingly, the established HL-60 cell line, which presented greater sensitivity to VP-16 cytotoxicity than KG1, HEL, and K562, was also found to exhibit delayed DNA SSB repair kinetics, as compared with the other AML cell lines. These results suggest that hypersensitivity to VP-16 of some AML cells may be related to a deficient DNA-repair mechanism.     

Elimination of drug-resistant myeloma tumor cell lines by monoclonal anti-P-glycoprotein antibody and rabbit complement

The effectiveness of ex vivo chemotherapy with drugs, such as vincristine, etoposide, and Adriamycin (doxorubicin, Adria Labs, Columbus, OH) for elimination of residual tumor cells from human bone marrow grafts could be undermined by the presence of multidrug- resistant tumor cells in the bone marrow. Therefore, to supplement chemoseparation, we investigated whether MRK-16, a monoclonal antibody (MoAb) to the surface moiety of multidrug resistance-associated P- glycoprotein antigen, can eliminate drug-resistant tumor cells in the presence of rabbit complement (RC). Two doxorubicin (DOX)-resistant human myeloma tumor cell line, 8226/DOX40 (resistant to 4 x 10(-7) mol/L DOX) and 8226/DOX6 (6 x 10(-8) mol/L DOX) with high and low amounts of cell surface P-glycoprotein, respectively, and the drug- sensitive parent cell line 8226/S were used as tumor models in this study. Using the limiting dilution assay, we have shown that three cycles of treatment with 25 micrograms/mL of MRK-16 MoAb and a 1:4 final dilution of RC eliminated 2.90 +/- 0.10 logs of 8226/DOX40 cells and 1.94 +/- 0.18 logs of 8226/DOX6 cells. One and two cycles of treatment were less effective, eliminating 0.47 +/- 0.40 and 1.94 +/- 0.36 logs of 8226/DOX40 and 0.12 +/- 0.20 and 1.63 +/- 0.58 logs of 8226/DOX6 cells, respectively. The 8226/S cell growth was unaffected by one to three cycles of treatment. The cell kill was not impaired when the antibody plus complement treatment was carried out on a mixture of 8226/DOX40 or 8226/DOX6 cells with a ninefold excess of irradiated bone marrow mononuclear cells (MNCs). The three cycles of treatment with antibody plus complement did not adversely affect granulocyte- macrophage colony-forming unit (GM-CFU) survival in hematologically normal marrows (92.5% to 104% survival) or in myeloma patient marrows (85% to 100%). These results show that it is possible to eliminate drug- resistant myeloma tumor cell lines from the admixed human bone marrow by treatment with MRK-16 MoAb plus RC. This method could prove to be effective for elimination of other drug-resistant tumor cell lines including those of leukemia and solid tumors, and will be further useful for supplementing chemopurging, and immunopurging of bone marrow with other antitumor cell antibodies.     

Use of etoposide in combination with cyclosporin for purging multidrug-resistant leukemic cells from bone marrow in a mouse model.

Cyclosporin (CsA) is a potent modulator of multidrug resistance (MDR) and has been combined with etoposide (VP-16) to purge MDR leukemic cells from human bone marrow (BM) in vitro. We studied the feasibility of this approach in an in vivo model for autologous BM transplantation using the murine leukemia cell line P388 and its MDR variant P388/ADR. Colony-forming assays with 2-h drug exposure revealed a tumor selectivity of VP-16 for P388 cells compared to normal murine marrow granulocyte-macrophage colony-forming units (CFU-GM), whereas P388/ADR cells were resistant to VP-16. Simultaneous incubation with CsA restored sensitivity in these cells. Almost 4 logs of cell kill were achieved by treating P388/ADR cells with 60 microM VP-16 plus 2.5 microM CsA (combination A) or 40 microM VP-16 plus 10 microM CsA (combination B), whereas there was a 2.5-log reduction of CFU-GM at these doses. Even though the myelotoxicity of VP-16 was increased by the addition of CsA, this effect was nonspecific as shown by a similar chemosensitization in sensitive P388 as well as in P388/VP 2.5 cells, an atypical MDR variant lacking P-glycoprotein. In vivo experiments addressed the ability of BM treated with VP-16 and CsA to rescue lethally irradiated mice and to purge leukemic cells. In total, 1/14 lethally irradiated mice died due to sepsis within 10 days after receiving 15 x 10(6) BM cells treated ex vivo with combination A in contrast to 1/4 for combination B. All 16 surviving animals demonstrated long-term engraftment. When simulated remission marrow contaminated with 0.1% P388/ADR was purged with VP-16 (60 microM) or CsA (2.5 microM) alone, all mice died from leukemia before day 16 after transplantation (median 14.3 and 12.2 days). In contrast, nine of ten animals receiving similar marrow purged with combination A survived > 60 days without any evidence of disease (p < 0.01). We conclude that combining VP-16 and CsA was effective in purging MDR leukemia cells from transplanted BM in this murine model.     

In vitro evaluation of combination drug purging for autologous bone marrow transplantation

Using an in vitro model, we studied whether combining 4-hydroperoxycyclophosphamide (4HC) with other drugs could improve its effectiveness as an ex vivo purging agent for autologous bone marrow transplantation. 4HC was incubated simultaneously with vincristine and etoposide, and sequentially with methylprednisolone, in various combinations. Compared to 4HC alone, all drug combinations increased the kill of the leukemia cell lines K562 and CEM without increasing the kill of granulocyte-macrophage colony-forming units (CFU-GM). The combination of 4HC, vincristine and methylprednisolone was the most active, and this drug combination was also the only combination which showed improved selective cytotoxicity (compared to 4HC alone) toward REH cells. This combination inhibited at least 8 logs of clonogenic leukemia cells from all three cell lines at doses which spared 1% of CFU-GM. This was an increase of 1.7 to 6.6 logs of clonogenic leukemia cell kill over 4HC alone. This drug combination displayed similar differential activity between fresh clonogenic leukemia cells and CFU-GM cultured from the bone marrows of seven patients about to undergo autologous bone marrow transplantation for acute lymphocytic leukemia.     

Effect of mafosfamide (ASTA-Z-7654) on the clonogenic cells in precursor-B acute lymphoblastic leukaemia: significance for ex vivo purging of bone marrow for autologous transplantation.

Lymphoblasts from 11 patients with acute lymphoblastic leukaemia (ALL) of precursor-B type were exposed to the cyclophosphamide derivative mafosfamide (ASTA-Z-7654), and examined for growth inhibition using an in vitro colony assay. Leukaemic clonogenic cells were significantly more resistant to this cytotoxic drug (mean IC50 29.2 micrograms/ml, IC90 64.8 micrograms/ml) compared with myeloid progenitors from seven normal bone marrow samples (mean IC50 9.0, IC90 19.9) (p less than 0.0001). This effect was most pronounced in the four previously treated cases examined (mean IC90 84.4 micrograms/ml). The implications of these findings for bone marrow purging with ASTA-Z in patients with ALL for autologous marrow transplantation are discussed.     

Stromal cells regulate survival of B-lineage leukemic cells during chemotherapy

Approximately 20% of B-lineage acute lymphoblastic leukemias are not cured by traditional chemotherapy. The possibility was examined that residual leukemic cells that potentially contribute to relapse are harbored in association with fibroblastic stromal cells in the bone marrow. Modulation of cytarabine (Ara-C) and etoposide (VP-16) efficacy by bone marrow stromal cells in vitro was investigated. Stromal cell coculture was shown to sustain the proliferation of B-lineage leukemic cells and to reduce leukemic cell apoptosis when exposed to Ara-C or VP-16. Direct contact with stromal cells was essential for the protection of leukemic cells during chemotherapy, whereas soluble factors had negligible effect. Specifically, signaling mediated through interaction with the stromal cell adhesion molecule VCAM-1 was required to maintain the maximum viability of leukemic cells during Ara-C and VP-16 exposure. In contrast, the interaction of leukemic cells with fibronectin did not confer significant resistance to either chemotherapeutic agent. These observations suggest a role for the bone marrow microenvironment in modulating the response of B-lineage leukemic cells to Ara-C or VP-16, and they indicate specific molecular interactions that may be important in determining the sensitivity of leukemic cells to treatment. (Blood. 2000;96:1926-1932)     

In vitro cytotoxicity of VP-16-213 and nitrogen mustard: agonistic on tumor cells but not on normal human bone marrow progenitors

The possible presence of tumor cells in remission bone marrow (BM) is one of the major problems for the success of autologous BM transplantation (ABMT), because the reinfusion of viable malignant cells may result in relapse. In this study we attempted the purging of the malignant cells by the use of VP-16-213 (VP-16) and nitrogen mustard (NM) either alone or in combination. Four cell lines from various hematological malignancies were utilized: SK-DHL-2 was established from a B-cell diffuse histiocytic lymphoma; RAJI was from an Epstein-Barr virus (EBV)-infected B-cell lymphoma cell line; K-562 were from a chronic myelogenous leukemia (CML) blastic crisis; and HL-60, derived from a human promyelocytic leukemia, were used in exponential growth phase. Four logs of tumor cell-elimination were observed after 1-h incubation of RAJI cells with 25 micrograms/ml of VP-16. K-562 and SK-DHL-2 cells showed a greater than 4 logs reduction after 1-h exposure to 75 micrograms/ml of VP-16, and HL-60 cell line growth was inhibited by 3.2 logs. Under the same conditions (i.e., the treatment with 75 micrograms/ml), we observed a mean recovery of 2.7% of BM granulocyte-macrophage colonies (granulocyte-macrophage colony-forming units, CFU-GM), 3.2% of erythroid (erythroid burst-forming units, BFU-E), and 2.5% of pluripotent (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM) progenitors, respectively. More than 3 logs reduction of leukemia and lymphoma cell lines were reached following 1-h treatment with 1 micrograms/ml of NM. After exposure to the same concentration of the drug we obtained 2.5% CFU-GM, 1.2% BFU-E, and 2% CFU-GEMM recovery. A drug mixture containing constant doses of VP-16 (10 and 20 micrograms/ml) and NM (1 micrograms/ml) reduced HL-60 and SK-DHL-2 cell growth to undetectable levels (i.e., 4 and 5 logs elimination) in the presence of an excess of irradiated BM cells, whereas it did not further affect the recovery of the BM precursors as compared to the single drugs used alone. These results suggest that the combination of these two drugs at the selected dose level could provide a better therapeutic index (i.e., higher tumor cell killing coupled with no additional cytotoxic effect on normal BM cells) than the same chemotherapeutic agent used alone and that this mixture may be useful for the "ex vivo" treatment of BM grafts.     

Modulation of etoposide (VP-16) cytotoxicity by verapamil or cyclosporine in multidrug-resistant human leukemic cell lines and normal bone marrow

We studied the effects of two modulators of multidrug resistance (MDR), cyclosporine and verapamil, on the cytotoxicity of etoposide (VP-16) in normal human bone marrow; two human leukemia cell lines, K562 and CEM; their MDR variants, K562/DOX and CEM/VLB; and mixtures of normal marrow and leukemic cells. VP-16 was selectivity toxic to the parental leukemic cells, with IC-50 values of 2 microM for CEM cells, 1.5 microM for K562 cells, and 12 microM for normal marrow CFU-GM. This selectivity was lost in the MDR variant leukemia cells, with IC-50s of 20 microM in K562/DOX and 8 microMs in CEM/VLB. Cyclosporine, 6 microMs, and verapamil, 20 microM, alone were nontoxic to bone marrow CFU-GM, and did not significantly increase the toxicity of VP-16 to normal marrow cells or to the two drug-sensitive leukemic cell lines. However, cyclosporine specifically enhanced the cytotoxicity of VP-16 in the MDR leukemia cells, reducing the IC-50 to the same level as the parental sensitive cells. Verapamil was considerably less effective. In a mixing experiment that included K562/DOX cells and normal bone marrow, cyclosporine increased the toxicity of VP-16 to the resistant leukemic cells by nearly 20-fold. Because the cytotoxic effect of cyclosporine is additive for resistant tumor cells, its combination with VP-16 may be useful in the purging of contaminating tumor cells prior to autologous bone marrow transplantation.     

The effects of tumor necrosis factor-alpha on early human hematopoietic progenitor cells treated with 4-hydroperoxycyclophosphamide

We have previously reported that 20 hours' preincubation of human bone marrow cells with interleukin-1 beta (IL-1) can protect early progenitor cells from 4-hydroperoxycyclophosphamide (4-HC) cytotoxicity. Since tumor necrosis factor-alpha (TNF alpha) shares many of the biologic properties of IL-1, we have compared the protective effects of TNF alpha with IL-1 against 4-HC. Incubation of human bone marrow mononuclear cells or an enriched progenitor population for 20 hours with either TNF alpha or IL-1 resulted in the survival of an increased number of single- and mixed-lineage colonies, including replatable blast cell colonies, while only rare colonies were seen in the control group. Antibodies to TNF alpha completely abolished the protection observed with IL-1, while antibodies to IL-1 alpha and IL-1 beta decreased but did not abolish the protection seen with TNF alpha. Combinations of low doses of TNF alpha and IL-1 showed synergy in their protective effects. Furthermore, no protection was observed by IL-1, IL- 1 bone-marrow-conditioned medium (IL-1-BMCM), or TNF alpha for HL-60, K562, KG1, KG1a, and DU.528 leukemic-cell lines or primary acute myelogenous leukemic (AML) blast cells from the lethal effects of 4-HC. In the case of HL-60 and KG1a cell lines, TNF alpha preincubation resulted in increased cytotoxicity. Furthermore, preincubation of a mixture of AML cells and normal bone-marrow cells with IL-1 + TNF alpha before 4-HC resulted in the protection of normal but not leukemic progenitors. These results suggest that TNF alpha is necessary for the protection of normal, early, human hematopoietic progenitors from 4-HC, while IL-1 is not mandatory but will synergize with TNF alpha to offer increased protection. In addition, no protection from 4-HC is observed by TNF alpha, IL-1, or IL-1-BMCM for primary leukemic blast cells or leukemic cell lines.     

Density-gradient separation of autologous bone marrow grafts before ex vivo purging with 4-hydroperoxycyclophosphamide

We found in previous studies that the cytotoxicity of 4-hydroperoxycyclophosphamide (4-HC) toward normal and malignant cells in autologous bone marrow grafts was modulated by the variable erythrocyte content of the incubation mixture. This affected both the kinetics of engraftment and the probability of leukemic relapse. To decrease this variability in 4-HC cytotoxicity, we initiated a trial of density-gradient separation of the autografts to remove erythrocytes and other mature blood cells before the 4-HC treatment. A series of 64 consecutive patients was studied. Light-density (sp. gr. much less than 1.077 g/ml) cells were isolated in a 2-step procedure by Ficoll-diatrizoate separation of previously isolated buffy-coat cells. Overall cell recovery after buffy-coat and gradient separations was 28.9 +/- 11.7% ( +/- SD). These cells were then treated with 60 micrograms/ml of 4-HC. The erythrocyte content of the incubation mixtures averaged 0.8%, and did not predict percent CFU-GM survival after the 4-HC incubation. The variability in CFU-GM survival after the purge was less than for previous patients who received buffy-coat cells (with erythrocyte contents ranging from 4-22%) treated with 100 micrograms/ml of 4-HC, indicating that a more uniform purge was achieved. Although the selected dose of 4-HC (60 micrograms/ml) yielded a lower CFU-GM survival, the kinetics of engraftment did not significantly differ between the recipients of light-density or buffy-coat cells. No patient required use of a 'backup' graft because of engraftment failure.     

Utilization of a colony assay to assess the variables influencing elimination of leukemic cells from human bone marrow with monoclonal antibodies and complement

We have previously used a chromium-release assay to demonstrate that the cocktail of monoclonal antibodies BA-1, BA-2, BA-3, and complement can effectively lyse human leukemic cells in the presence of excess bone marrow. Using a leukemic cell colony assay, we have reinvestigated the variables influencing lysis of human leukemic cells (KM-3, HPB- NULL, NALM-6) in bone marrow using BA-1, BA-2, BA-3, and complement. Specific variables addressed included the concentration of excess bone marrow cells, the number of treatments, the presence or absence of DNase during the treatment, the combination of antibodies, and the sensitivity of different leukemic cell lines to lysis. Using the colony assay, the BA-1,2,3 cocktail was shown to be more effective than any single antibody or combination of two antibodies. We also determined that the concentration of excess bone marrow cells and number of treatments had a direct bearing on leukemic cell lysis. Although two cycles of treatment were significantly superior to one cycle, three cycles were not significantly superior to two cycles. Inclusion of DNase (10 micrograms/mL) was a critical adjunct that eliminated clumping and facilitated plating cells in the colony assay. Finally, we could show that striking differences existed in the sensitivity of the leukemic cell lines to lysis with the BA-1,2,3 cocktail and complement. NALM-6 cells were the most sensitive (approximately four logs of kill), and KM-3 cells were the most resistant (less than two logs of kill). Our results strongly support the utility of sensitive leukemic cell colony assays in the analysis of marrow treatment variables in autologous bone marrow transplantation.     

Autologous bone marrow transplantation for childhood acute lymphoblastic leukemia: a novel combined approach consisting of ex vivo marrow purging, modulation of multi-drug resistance, induction of autograft vs leukemia effect, and post-transplant immuno- and chemotherapy (PTIC)

In an attempt to reduce the high relapse rate associated with ABMT, five children with high-risk first CR and 19 in second or subsequent CR lacking matched family allogeneic donors underwent ABMT with chemopurged bone marrow utilizing verapamil (VPL), vincristine, and VP-16. Patients were conditioned with TBI, VPL bolus and infusion with VP-16 and cyclophosphamide. The first cohort of patients (n = 4) received only cyclosporin A (CsA). The second cohort (n = 7) received CsA and alpha interferon (total = 11 with post-transplant immunotherapy alone.) The third cohort (n = 13) received CsA and six alternating cycles of alphaIFN and chemotherapy and six additional cycles of chemotherapy (vincristine, VP-16, Ara-C, prednisone) followed by G-CSF (post-transplant immune chemotherapy (PTIC)). The 2-year DFS is 42+/-10% (90% confidence interval (CI) is 26.5-58.5%) and 2-year overall survival is 54+/-10% (90% CI is 37.5-70.5%). Furthermore, patients receiving PTIC (n = 13) vs immunotherapy alone (CsA+/-aIFN) (n = 11) had a substantially better 2 year DFS and OS: 69+/-13% vs 13+/-12% and 85+/-10% vs 25+/-15% (P = 0.008 and P = 0.06, respectively). These results suggest that the use of ABMT with chemopurging, combined with PTIC is well tolerated and may be an alternative new approach in the treatment of a subset of children with high-risk first CR or > or = second CR ALL who lack closely matched family-related allogeneic donors.     

Cytostatic drug testing in human leukemias by means of multiparametric flow cytometry

Summary Human bone marrow cells from 20 patients as well as the permanent human B-cell lines RPMI 1788, Raji, Daudi, T-cell lines Molt, CEM, Jurkat and the promyelocytic line HL 60 were assayed by means of a newly developed in vitro flow cytometric cytostatic drug assay. The cells were exposed to cytosine-arabinoside, L-asparaginase, daunorubicin, prednisone or vincristine. Surviving cells were stained after an incubation period of 2 to 7 days with esterase and pH-indicator dye ADB (1,4-diacetoxy-2,3-dicyanobenzene), dead cells with DNA-dye PI (propidium iodide). Dose-response curves were established using percent surviving cells. It was possible to evaluate bone marrow samples from 16 out of 20 patients. Seven samples were leukemic (acute myeloid leukemia (AML) n=6, Non-Hodgkin's Lymphoma (NHL) n=1). Nine samples were from patients either in complete remission or with benign diseases. Daunorubicin and cytosine-arabinoside were cytotoxic in both groups, whereas vincristine was effective mainly in the leukemic group (p<0.05). There was significant heterogeneity in the reactivity of AML-marrow cells from different patients to different drugs. The cell lines exhibited different patterns of sensitivity. Vincristine arrested cells in G2/M-phase, cytosine-arabinoside caused an increase of cells in the S-phase. This work was supported in part by grant (Ne 310/1-1) from the Deutsche Forschungsgemeinschaft