Cytotoxic antibody in normal human serums reactive with tumor cells from acute lymphocytic leukemia.

Serums showing complement-dependent cytotoxic reactions to acute lymphocytic leukemia cells were detected in three normal unimmunized subjects. These serums were reactive with tumor cells from 514 (514 tested) acute lymphocytic leukemia patients, and three (12 tested) patients with acute myelocytic leukemia; they did not react with tumor cells from patients with acute monocytic leukemia (two tested), with chronic lymphocytic leukemia (two tested) or with leukolymphosarcoma (two tested); nor did they react with normal lymphocytes from 52 different donors. These reactive serums appear to recognize antigens primarily associated with acute lymphocytic leukemia.

Developments with targeted enzymes in cancer therapy.

Cancer therapy based on the delivery of enzymes to tumour sites has advanced in several directions since antibody-directed enzyme/prodrug therapy was first described. It has been shown that methoxypolyethylene glycol (MPEG) can be used to deliver enzyme to a variety of solid tumours. MPEG-enzyme conjugates show reduced immunogenicity and may allow repeated treatment with enzymes of bacterial origin. Enzyme delivery to tumours by polymers can be used to convert a low toxicity prodrug to a potent cytotoxic agent. An example of such a prodrug is CB1954, which can be activated by a human enzyme in the presence of a cosubstrate. Tumour-located enzymes can also be used in conjunction with a combination of antimetabolites and rescue agents. The rescue agent protects normal tissue but is degraded at cancer sites by the enzyme, thus deprotecting the tumour and allowing prolonged antimetabolite action.

Isokinetic gradient sedimentation of normal human bone marrow cells: a simple method for isolation of proliferative granulocytic and erythroid elements.

Proliferative granulocytes and erythroid precursors were isolated from cell suspensions of human normal bone marrow by sedimentation on an isokinetic gradient of continuous low-density Ficoll. A fivefold enrichment with a 70% recovery of the proliferative granulocyte cohort and of early erythroid elements was achieved, as determined by differential morphology and tritiated thymidine incorporation. Viability and in vitro proliferative capacity following gradient centrifugation remained intact. This method of cell separation, based on differences in cell diameter, affords a simple and rapid means for the purification of specific cell populations from heterologous human normal bone marrow.

Chemotherapy of leukemia in mice, rats, and humans relating time of humoral stimulation, tumor growth, and clinical response.

Studies were conducted in the leukemic mouse and rat to test the hypothesis that enhanced effects of drugs given in sequence relate to a predictable increase in tumor growth and sensitivity to cycle-active agents. The rationale is based on a) evidence that, following drug-induced aplasia, resultant bone marrow proliferation in vivo corresponds temporally with induced humoral stimulatory activity, and on b) models that demonstrate increased cytotoxicity of beta-cytosine arabinoside (Ara-C) to myeloblasts cultured in humoral stimulatory activity (HSA). CD2F1 mice bearing L-1210 leukemia received a course of 60 mg Ara-C/kg every 8th hour (q.8 h) three times on day 0 and on another day in sequence (0,1 through 0,7). The longest survival (250% of controls) was in animals whose second course began on day 3, the time of peak HSA as measured by DNA synthesis induced in L-1210 cells in culture. LBN rats bearing acute myelocytic leukemia (AML) were treated with 100 mg Ara-C/kg q.8 h six times beginning on day 0 and on other days in sequence (0,1 through 0,12). The longest survival was in those treated on day 0,6 (760% of controls), the time of peak serum stimulation, and tumor labeling index (LI). Thirty-seven patients with AML received a single course of therapy with 45 mg Ara-C/kg by a 72-hour infusion and 1.0 mg daunorubicin/kg every day three times. On day 8, the time of peak HSA and tumor LI, Ara-C was again infused. Complete remission was achieved in 56% (65% of all patients less than 60 yr old) with a single cycle of therapy. Median duration of chemotherapy-free remission was 10 months. Of 11 relapsing patients, 8 achieved a second remission with the same regimen. These studies demonstrated that the amount of proliferation of residual tumor and thereby sensitivity to cycle-active drugs given in sequence relates to the initial drug effect on tumor proliferation and the induction of humoral stimulation.

BN rat myeloid leukemia transferred to the (LEW x BN)F1 rat.

The BN rat myelocytic leukemia was transferred to (LEW x BN)F1 rats. In the F1 host the growth, dissemination, and response of this leukemia to chemotherapy were predictable, stable through serial passage, and similar to this leukemia's behavior in the parent strain. Rats given 10(7) spleen cells iv from leukemic donors died in about 3 weeks if untreated or responded to cytosine arabinoside even after overt leukemia had developed. This animal leukemia is useful as a model for human acute myelocytic leukemia.

Effects of the differentiating agent hexamethylene bisacetamide on normal and myelodysplastic hematopoietic progenitors.

Hexamethylene bisacetamide (HMBA; NSC 95580) is a potent polar-planar differentiating agent of leukemia and solid tumor cell lines in vitro at clinically achievable concentrations. HMBA is currently being studied in patients with myelodysplastic syndrome. Previous phase I trials have demonstrated that HMBA produces hematologic toxicity in morphologically normal bone marrows of patients with solid tumors. Because of concern that HMBA may produce more severe myelotoxicity in patients with myelodysplastic syndrome since these patients have limited hematopoietic reserves, we studied the effects of HMBA on myelodysplastic and normal hematopoietic progenitors in vitro. HMBA concentrations that are optimal for differentiation in vitro (2 to 5 mmol/L) and HMBA concentrations that are being achieved in clinical trials (1 to 2 mmol/L) inhibited the growth of granulocyte-macrophage colony-forming units and erythroid burst-forming units from all 15 patients with myelodysplastic syndrome and all 4 normal subjects, HMBA did not induce proliferation of myelodysplastic or normal progenitors at any concentration; rather, it produced nearly identical inhibition of normal and myelodysplastic hematopoietic progenitors. HMBA also produced quantitatively similar inhibition of clonogenic leukemic growth of two myeloid leukemia cell lines. For a differentiating agent to be effective, it will likely have to either produce both differentiation and proliferation of abnormal hematopoietic progenitors or show selective inhibitory effects on abnormal as compared with normal progenitors. Although the mechanisms responsible for the antiproliferative effects of HMBA cannot be determined from this study, similar inhibitory effects of HMBA on normal and abnormal hematopoietic progenitors suggest that HMBA may be of limited utility in producing and sustaining elevations of peripheral blood cell counts in patients with myelodysplastic syndrome.

Growth response of residual leukemia after initial drug therapy.

Clinical trials with laboratory correlates were conducted in patients with acute leukemia to determine if relationships exist between drug dose and growth of surviving leukemia cells. The therapeutic design was based on findings in the leukemic rat that relate the initial dose of drug and tumor kill to the magnitude of residual tumor proliferation and sensitivity to a second drug. Patients with acute myelocytic leukemia received cytarabine, either 2 or 6 g/m2/72 h by continuous infusion. The presence and magnitude of change between initial and residual tumor after treatment, as measured by change in labeling indices, depended on the "priming" dose of drug. The amount of perturbation correlated with clinical response to cytarabine given at the time of induced proliferation. With results which parallel the rat data, the direct relationship of initial drug dose and proliferation of residual tumor is demonstrated in humans, and lends support to the design of our clinical trials of timed sequential therapy.

Development of a cell kinetic approach to curative therapy of acute myelocytic leukemia in remission using the cell cycle-specific drug 1-beta-D-arabinofuranosylcytosine in a rat model.

The timing of sequentially administered antineoplastic drugs is one determinant of toxicity and therapeutic benefit. We have conducted a series of studies with 1-beta-D-arabinofuranosylcytosine (ara-C) in the rat model (Lewis X brown Norway F1 hybrid rats bearing brown Norway myelocytic leukemia) for human acute myelocytic leukemia to examine the factors determining optimum timing of sequential administration of this cell cycle DNA synthesis phase-specific drug. Late-stage disease in this model is not curable with ara-C, but the maximum survival is achieved by rats given serial 2-day courses of ara-C 6 days apart. ara-C given in 2- or 4-day-interval sequences to rats with late-stage disease is more toxic and not more effective. However, Lewis X brown Norway F1 hybrid rats bearing brown Norway myelocytic leukemia in early complete remission are curable with ara-C given in optimum timed sequence. In these experiments, groups of rats in early complete remission were given a 2-day course of ara-C in every-8-hr s.c. injections, and then a second 2-day course was given after 2-, 4-, 6-, 8-, 10-, or 12-day intervals. The best cure rate of rats surviving toxicity was achieved when sequentially administered 2-day courses of ara-C were given at 2- to 4-day intervals to rats in early complete remission. In the minimal residual disease state, as in late-stage disease, 2- and 4-day-interval sequencing was the most toxic. No significant number of cures of minimal residual disease could be obtained by even the maximum tolerated dose of ara-C given in longer than 6-day-interval sequences or by various continuous or intermittent schedules. The fact that the Lewis X brown Norway F1 hybrid rats bearing brown Norway myelocytic leukemia, while relatively refractory to ara-C, are curable with this drug when used in optimum timed sequence in early remission is encouraging for similar clinical trials in humans and suggests some principles for the design of such trials.

Enhancement of drug cytotoxicity by recruitment of leukemic myeloblasts with humoral stimulation.

To demonstrate that the effect of 1-beta-D-arabinofuranosylcytosine can be increased when the leukemic cell growth fraction is augmented by induced humoral factors, bone marrow cells from 14 patients with acute myeloblastic leukemia were studied in vitro. Cells were cultured in either pooled stimulatory serum, obtained from leukemic patients undergoing chemotherapy, or autologous leukemic pre-treatment serum. After 2 days in culture, serum containing 1-beta-D-arabinofuranosylcytosine was added. Cells cultured in stimulatory serum proliferated markedly when compared with cells cultured in pretreatment serum, as measured by tritiated thymidine incorporation into an acid-insoluble precipitate, tritiated thymidine labeling indices, and viable tumor cell counts. The cytotoxic effect of 1-beta-D-arabinofuranosylcytosine was enhanced in those cells initially cultured in stimulatory serum relative to cells initially cultured in pretreatment serum. These studies are consistent with the hypothesis that induced humoral stimulation of acute myeloblastic leukemic cells in vitro recruits a greater tumor growth fraction and thereby increases the cytotoxicity of cycle-dependent drugs.

Influence of humoral regulators on proliferation and maturation of normal and leukemic cells.

Factors that influence the proliferation of marrow elements can be detected in sera. To determine the function and to compare the effect of these factors, cells were obtained from patients with normal and leukemic bone marrows. The effects of drug-induced stimulatory and inhibitory sera and leukemic pretreatment sera over time (0 to 6 days) on proliferation and granulocytic morphology of normal and leukemic bone marrow cells in culture were evaluated. Increased proliferation was associated with stimulatory sera, while inhibitory and leukemic pretreatment sera retarded proliferation of both normal and malignant cells. Exposure of normal proliferative cells to inhibitory or leukemic pretreatment sera yielded the greatest increase in mature granulocyte forms. In contrast, although the proliferative response of leukemic cells to these sera was similar to normal, maturation was minimal. These data suggest that leukemic pretreatment sera are similar to inhibitory sera and are not leukemogenic. Both retard proliferation of normal and leukemic bone marrow cells while enhancing maturation of normal cells. Leukemic myeloblasts, however, cannot be made to mature by these humoral regulators.

Drug induced host factors which stimulate growth of residual leukemia in Lewis x Brown Norway F1 (LEW-BN) rats.

Antitumor synergism occurs with two drugs in sequence when the second drug is given at the time of maximal regrowth of residual leukemia and peak humoral stimulatory activity (HSA). To determine if this enhancement relates to a host derived HSA, studies were conducted in Lewis x brown Norway F1 rats bearing brown Norway myelocytic leukemia. A significant cure rate was observed in rats treated initially with 1-beta-D-arabinofuranosylcytosine and then given injections of 10(6) leukemia cells and treated with a second 2-day course of 1-beta-D-arabinofuranosylcytosine in every-8-h s.c. injections in the 6-day period after the initial drug. No effect on survival of the initial drug or of the second drug given at intervals after day 6 was noted. This result is consistent with the efficacy of treatment at the time of peak HSA and tumor growth. The direct effect of HSA on tumor sensitivity to 1-beta-D-arabinofuranosylcytosine was evaluated by 18-h incubations of leukemia and HSA, followed by bioassay. Increased survival and high cure rates were observed when compared with cultured cells in normal serum. These studies support the notion that host derived factors operative during drug induced aplasia stimulate tumor growth and thereby, if the drugs are properly timed, increase sensitivity to cycle active agents.

Changes in topoisomerase I levels and localization during myeloid maturation in vitro and in vivo.

Changes in topoisomerase I (topo I) levels and localization were examined during the course of granulocytic maturation in vitro and in vivo. Western blotting revealed that granulocytic maturation in DMSO-treated HL-60 human leukemia cells was accompanied by a 5-fold decrease in topo I polypeptide content. Consistent with this result, 3- to 5-fold higher concentrations of the topo I poison camptothecin were required to stabilize topo I-DNA adducts in DMSO-treated HL-60 cells compared to untreated cells. Northern blotting revealed that these changes occurred without any decrease in topo I message. Immunolocalization studies revealed that these quantitative changes were accompanied by redistribution of topo I away from the nucleoli, where it was prominently accumulated in untreated HL-60 cells, to a more uniform nuclear distribution in DMSO-treated cells. Similar changes occurred during granulocytic maturation in human marrow in vivo. Western blotting revealed that topo I levels in normal progranulocytes were 50% as high as those in HL-60 cells, levels in metamyelocytes were 35% as high as HL-60 cells, and levels in peripheral blood granulocytes were 5% as high as HL-60 cells. Two other polypeptides that are concentrated in nucleoli, poly(ADP-ribose) polymerase and B23/nucleophosmin, also decreased during the course of granulocytic maturation. These changes were accompanied by an alteration in topo I localization similar to that observed in HL-60 cells during the course of granulocytic maturation. Conversely, treatment of human lymphocytes with the mitogenic lectin concanavalin A resulted in a 3-fold increase in topo I polypeptide content concomitant with a prominent increase in the amount of nucleolar antigen. These observations not only provide a context for understanding the recent observation that topo I levels are higher in human leukemia specimens than in normal marrow but also raise the possibility that elevated topo I levels in other cells might reflect alterations in nucleolar structure and function.

Direct relationship of marrow cell growth and 1-beta-D-arabinofuranosylcytosine metabolism.

To define the relationship between perturbed cell growth and intracellular metabolism of 1-beta-D-arabinofuranosylcytosine (ara-C) in sensitive human cells, growth kinetic, and biochemical pharmacological determinants were examined in normal human bone marrow populations in vitro in normal serum and in the presence of drug-induced humoral stimulatory activity (HSA). Cells cultured in HSA demonstrated both increased proliferation and greater ara-C-related inhibition of DNA synthesis than did cells maintained in normal serum, as measured by [3H]thymidine incorporation into DNA and [3H]thymidine granulocyte precursor labeling index. Parallel measurements of [3H]ara-C incorporation into DNA demonstrated similar behavior in HSA-perturbed cells. When these cultured cells were exposed to 1 and 10 microM ara-C, intracellular formation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate over 3 hr and retention of this active form during 1 subsequent hr in drug-free medium were both increased in HSA-stimulated cells relative to cells cultured in normal serum. These studies demonstrate coupling of induced cell growth kinetics with enhanced intracellular metabolism of the S-phase-specific antimetabolite ara-C in normal human marrow cells. The close direct relationship between growth kinetic perturbation and augmentation of intracellular ara-C activation in this normal hematopoietic model provides a basis for comparison with leukemic cell populations, in which uncoupling of growth kinetics and pharmacokinetics may signify divergence from normal drug-sensitive cell behavior and, thus, resistance to ara-C cytotoxicity.

Diamine oxidase as a plasma marker of rat intestinal mucosal injury and regeneration after administration of 1-beta-D-arabinofuranosylcytosine.

Diamine oxidase (DAO; EC 1.4.3.6) is an enzyme found in high activity in the mature upper villus cells of rat intestinal mucosa and only in very low activity in all other tissues except for the placenta in the pregnant rat. The present study was designed to investigate whether plasma and mucosal DAO could be used to monitor the timing and severity of injury and recovery of the intestinal mucosa after administration of the chemotherapeutic agent 1-beta-D-arabinofuranosylcytosine (ara-C). A dose of 0.3 g/kg s.c. every 8 hr for 6 doses was given to adult Lewis x Brown Norway rats. This resulted in death of the proliferating crypt cells, followed by regeneration of the mucosa from the surviving crypt cells, with recovery by Day 8. This mucosal damage and recovery was reflected by histological changes and a decrease in activity of mucosal disaccharidases and alkaline phosphatase. Both mucosal and plasma DAO levels also fell markedly to less than 10% of basal levels (N = 30, p less than 0.005) by Day 4 and recovered with a time course similar to the histological and biochemical changes indicative of injury and recovery. With increasing dosage and/or increasing duration of ara-C treatment, mucosal injury was progressive, with increasing loss of both plasma and mucosal DAO levels as compared to controls (N = 38, p less than 0.005). Plasma DAO levels in three patients with leukemia following ara-C chemotherapy decreased markedly to less than 30% of basal pretreatment levels (p less than 0.05) by Days 9 to 12, with a time course that was compatible with clinical intestinal mucosal injury. Our data document that plasma DAO levels reflect the mucosal injury and subsequent recovery after ara-C treatment in the rat and humans. Thus, plasma DAO may serve as a marker of the integrity of the intestinal mucosa after chemotherapy.

Correlation of proliferative and clonogenic tumor cells in multiple myeloma.

To expand on the findings from previous clinical trials that the growth of residual tumor is increased at a predictable time following initial drug administration, malignant plasma cells from bone marrows of patients with multiple myeloma (MM) were examined for changes in proliferation and clonogenicity induced in vivo by cyclophosphamide and in vitro by drug-induced humoral stimulatory activity. Peak plasma cell [3H]thymidine labeling index (LI) occurred predictably following drug and paralleled changes in agar colony formation by marrow cells obtained during therapy. Colony-forming capacity of pretreatment MM marrow populations was enhanced when those cells were cultured with humoral stimulatory activity, similar to the increased colony formation detected in Day 9 postcyclophosphamide marrows at the time of peak plasma cell LI. To further define a relationship between proliferative plasma cells and colony-forming tumor cells, MM marrows were fractionated by sedimentation on an isokinetic gradient. Enrichment of a proliferative tumor cell cohort was achieved, evidenced by [3H]thymidine LI. Colony-forming cells were also enriched by isokinetic gradient sedimentation, and agar colony formation by MM marrow cell fractions correlated with the kinetic characteristics of the isolated subpopulations. These studies of whole and fractionated human MM marrow cell populations suggest that the kinetically active cells which are induced to proliferate in vivo and in vitro are closely related to the clonogenic tumor cells which produce colonies in agar and which, like those cells measured by [3H]thymidine LI, respond to growth stimulation by drug-induced humoral stimulatory activity.

Bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by human NAD(P)H quinone oxidoreductase 2: a novel co-substrate-mediated antitumor prodrug therapy.

A novel prodrug activation system, endogenous in human tumor cells, is described. A latent enzyme-prodrug system is switched on by a simple synthetic, small molecule co-substrate. This ternary system is inactive if any one of the components is absent. CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is an antitumor prodrug that is activated in certain rat tumors via its 4-hydroxylamine derivative to a potent bifunctional alkylating agent. However, human tumor cells are resistant to CB 1954 because they are unable to catalyze this bioactivation efficiently. A human enzyme has been discovered that can activate CB 1954, and it has been shown to be commonly present in human tumor cells. The enzyme is NQO2 [NAD(P)H quinone oxidoreductase 2], but its activity is normally latent, and a nonbiogenic co-substrate such as NRH [nicotinamide riboside (reduced)] is required for enzymatic activity. There is a very large (100-3000-fold) increase in CB 1954 cytotoxicity toward either NQO2-transfected rodent or nontransfected human tumor cell lines in the presence of NRH. Other reduced pyridinium compounds can also act as co-substrates for NQO2. Thus, the simplest quaternary salt of nicotinamide, 1-methyl-3-carboxamidopyridinium iodide, was a co-substrate for NQO2 when reduced to the corresponding 1,4-dihydropyridine derivative. Increased chain length and/or alkyl load at the 1-position of the dihydropyridine ring improved specific activity, and compounds more active than NRH were found. However, little activity was seen with either the 1-benzyl or 1-(2-phenylethyl) derivatives. A negatively charged substituent at the 3-position of the reduced pyridine ring also negated the ability of these compounds to act as cosubstrates for NQO2. In particular, 1-carbamoylmethyl-3-carbamoyl-1,4dihydropyridine was shown to be a co-substrate for NQO2 with greater stability than NRH, with the ability to enter cells and potentiate the cytotoxicity of CB 1954. Furthermore, this agent is synthetically accessible and suitable for further pharmaceutical development. NQO2 activity appears to be related to expression of NQO1 (DT-diaphorase), an enzyme that is known to have a favorable distribution toward certain human cancers. NQO2 is a novel target for prodrug therapy and has a unique activation mechanism that relies on a synthetic co-substrate to activate an apparently latent enzyme. Our findings may reopen the use of CB 1954 for the direct therapy of human malignant disease.

Topoisomerase II levels during granulocytic maturation in vitro and in vivo.

Western blotting, indirect immunolocalization, flow cytometry, and a functional assay for drug-induced strand breakage were utilized to examine topoisomerase (topo) II levels during granulocytic maturation in HL-60 human progranulocytic leukemia cells and in samples of normal human marrow. Indirect immunofluorescence revealed that the intensity of the signal for topo II in unsynchronized log phase HL-60 cells varied widely. Indirect immunolabeling combined with propidium iodide staining and two-parameter flow cytometry revealed that topo II levels increased an average of 2-fold as cells progressed from G1 to G2/M. When HL-60 cells were induced to mature toward granulocytes, topo II levels progressively decreased and became undetectable by functional assays, by indirect immunoperoxidase staining, and by Western blotting with an antibody which identified Mr 170,000 and Mr 180,000 forms of topo II. Similar changes were detected during normal granulocytic maturation in human marrow in vivo. Western blotting revealed that levels of the Mr 170,000 (proliferation-associated) isoform of topo II were highest in marrow fractions enriched in progranulocytes and myelocytes, intermediate in unfractionated marrow from normal volunteers, and undetectable in mature granulocytes. The Mr 180,000 topo II polypeptide was also diminished or absent from mature granulocytes. In further experiments, marrow samples from normal volunteers were subjected to flow cytometry after labeling of topo II and various cell surface markers. Levels of the Mr 170,000 topo II polypeptide in CD34-positive cells (multipotent and committed progenitors from several hematopoietic lineages) were indistinguishable from levels observed in the HL-60 leukemia cell line. These results suggest that topo II levels in highly proliferative normal human myeloid cells in vivo approach levels found in corresponding neoplastic cell lines in vitro. Conversely, as the same cells mature into granulocytes in vivo or in vitro, levels of both molecular weight forms of topo II diminish. These results provide a framework for the further investigation of topo II levels and drug sensitivity in human leukemia.

Phase I and pharmacodynamic study of taxol in refractory acute leukemias.

Taxol, a novel antimicrotubule agent that enhances tubulin polymerization and microtubule stability, was administered to adults with refractory leukemias as a 24-h i.v. infusion in a Phase I study. The primary objectives were to determine the maximum tolerated dose of taxol administered on this schedule to patients with acute leukemias and describe the nonhematological toxicities which became dose limiting. The starting dose, 200 mg/m2, was based on the maximum tolerated dose in solid tumor trials, in which myelosuppression precluded dose escalation. Seventeen patients received 28 evaluable courses at 200, 250, 315, and 390 mg/m2. Severe mucositis limited further dose escalation. Other nonhematological effects included peripheral neuropathy, alopecia, myalgias, arthralgias, nausea, vomiting, diarrhea, and an acute pulmonary reaction that was presumptively due to taxol's Cremophor vehicle. Mean peak taxol plasma concentrations at all dose levels were in the range of concentrations that were previously demonstrated to induce microtubule bundles, a morphological effect associated with cytotoxicity, in leukemia cells in vitro. Pretreatment blasts from 12 patients were incubated with taxol ex vivo. Taxol-induced microtubule bundles were apparent in the blasts of eight patients who also had cytoreduction of tumor, and sensitivity to bundle formation was related to the magnitude of antitumor activity. In contrast, taxol did not induce microtubule bundles ex vivo in the blasts of the other four total nonresponders. Based on this study, the maximum tolerated doses and recommended Phase II doses for taxol, limited by nonhematological toxicity and administered as a 24-h i.v. infusion to patients with refractory leukemias, are 390 and 315 mg/m2. Phase II trials at these myelosuppressive doses are required to determine taxol's activity in the treatment of leukemias. In addition, further evaluation of microtubule bundle formation ex vivo in Phase II studies is necessary to determine the ultimate utility of this assay in assessing tumor sensitivity to taxol.

ZD2767, an improved system for antibody-directed enzyme prodrug therapy that results in tumor regressions in colorectal tumor xenografts.

ZD2767 represents an improved version of antibody-directed enzyme prodrug therapy. It consists of a conjugate of the F(ab')2 A5B7 antibody fragment and carboxypeptidase G2 (CPG2) and a prodrug, 4-[N,N-bis(2-iodoethyl)amino]phenoxycarbonyl L-glutamic acid. The IC50 of the prodrug against LoVo colorectal tumor cells was 47 microM, and cleavage by CPG2 released the potent bis-iodo phenol mustard drug (IC50 = 0.34 microM). The drug killed both proliferating and quiescent LoVo cells. Administration of the ZD2767 conjugate to nude mice bearing LoVo colorectal xenografts resulted in approximately 1% of injected ZD2767 conjugate localizing/g of tumor after 72 h, and blood and normal tissue levels of the conjugate were 10-50-fold lower. A single round of therapy involving the administration of the prodrug 72 h after the conjugate to athymic mice bearing established LoVo xenografts resulted in approximately 50% of the tumors undergoing complete regressions, tumor growth delays greater than 30 days, and little toxicity (as judged by body-weight loss). Similar studies using a control antibody-CPG2 conjugate that does not bind to LoVo tumor cells resulted in a growth delay of less than 5 days, confirming the tumor specificity of this approach. These studies demonstrate the potential of ZD2767 for the treatment of colorectal cancer.

Effective chemotherapy of acute myelocytic leukemia occurring after alkylating agent or radiation therapy for prior malignancy.

Eleven consecutive patients with acute myelocytic leukemia occurring as a second malignancy were treated with high-dose, timed, sequential chemotherapy. Eight of the patients were felt to have "secondary" acute leukemia because they had received an alkylating agent or radiation therapy. The other three patients were considered controls. Despite a median age of 65, four of the eight secondary leukemia patients achieved complete remission with this regimen. One of the three control patients also achieved complete remission. This remission rate and duration are comparable to what was achieved with this treatment of "primary" acute myelocytic leukemia during the same period of time. These results suggest that patients with leukemia occurring after an alkylating agent or radiation therapy are not at especially high risk if treated aggressively.