Molecular evidence of bone marrow involvement in advanced case ot Tgammadelta lymphoma with secondary myelofibrosis.

We describe the case of a middle-aged man with long indolent course of generalized Tgammadelta lymphoma. The onset of secondary myelofibrosis made cytological monitoring of the bone marrow infiltrates impossible. As during progression of the disease splenectomy revealed typical histological features of a high-grade hepatosplenic Tgammadelta lymphoma, the low-grade bone infiltrate was considered a secondary lymphoma. The use of the polymerase chain reaction helped to detect a constant and identical monoclonal rearrangement pattern of the T-cell receptor gamma-chain gene in both bone marrow and splenic T-cell infiltrates. The notion of a secondary spread of malignant T-cells to the bone marrow was thereby confirmed despite striking cytological differences between bone marrow and splenic infiltrates. This is the first report of a diagnostic DNA-based molecular approach using fixed decalcified bone marrow. This method may provide a major tool when dealing with myelofibrosis, which normally hampers sampling of cytological specimens.

Gemcitabine and etoposide in small cell lung cancer: phase I and II trials.

Gemcitabine and etoposide have both shown single-agent activity against multiple tumor types in clinical trials, including small cell lung cancer, but have not been previously used together. Forty-four patients with small cell and non-small cell lung cancer or other tumor types were enrolled in a phase I dose-finding trial using this drug combination. Gemcitabine 1,000 mg/m2 was given intravenously on days 1, 8, and 15 of a 28-day cycle, and etoposide (dose escalated from 20 to 80 mg/m2) was given on days 8, 9, and 10. Leukopenia, thrombocytopenia, and anemia were the major toxicities noted. Objective responses were observed in five of 44 patients. The maximum tolerated dose of etoposide was determined to be 80 mg/m2. On the basis of these results, a phase II trial of gemcitabine and etoposide in patients with small cell lung cancer has been initiated. Twelve patients have been enrolled in this ongoing trial, and toxicity to date has been manageable.

Paclitaxel administered over 3 h followed by cisplatin in patients with advanced head and neck squamous cell carcinoma: a clinical phase I study.

We have performed a clinical phase I trial of a combination treatment with paclitaxel given as 3-hour infusion and cisplatin to determine the maximum tolerated dose and the dose-limiting toxicity in patients with recurrent or metastatic squamous cell carcinoma of the head and neck. Treatment was repeated every 21 days. Doses administered ranged from 135 mg/m2 paclitaxel/75 mg/m2 cisplatin to 250 mg/m2 paclitaxel/100 mg/m2 cisplatin. Twenty-four patients have been entered into this study. The maximum tolerated dose was determined to be 225-250 mg/m2 paclitaxel/100 mg/m2 cisplatin. The dose-limiting toxicity of this regimen was myelosuppression (granulocytopenia). Neurosensory and neuromotor toxicity was moderate. However, analyses of threshold electrotonus studies indicated subclinical neurotoxicity in most patients. One patient receiving 200 mg/m2 paclitaxel/100 mg/m2 cisplatin developed grade 3 motor-neurotoxicity. Orthostatic hypotension was observed in 8 patients receiving doses of 200 mg/m2 paclitaxel/100 mg/m2 cisplatin or higher. Objective responses were observed at paclitaxel 175 mg/m2/ cisplatin 100 mg/m2 (n = 5; complete response in 1 patient), paclitaxel 200 mg/ m2/cisplatin 100 mg/m2 (n = 3; partial response in 3 patients) and at paclitaxel 225 mg/m2/cisplatin 100 mg/m2 (n = 8; partial response in 1 patient). Eleven additional patients had stable disease. We conclude that paclitaxel administered as a 3-hour infusion followed by cisplatin is an active regimen in advanced head and neck cancer and that orthostatic hypotension may be a potentially significant clinical toxicity.

Preclinical activity of trans-indazolium[tetrachlorobisindazoleruthenate(III)] (NSC 666158; IndCR; KP 1019) against tumour colony-forming units and haematopoietic progenitor cells.

Trans-indazolium[tetrachlorobisindazoleruthenate(III)] (KP 1019) is a new heavy metal complex with promising activity against tumour cell lines and in animal models. We studied the antineoplastic effects of KP 1019 (final concentrations: 1, 10, 100 micrograms/ml) on in vitro proliferation of clonogenic cells from freshly explanted human tumours in a capillary soft agar cloning system, and compared the activity of KP 1019 with conventional antineoplastic agents. 53 of 75 specimens (71%) showed adequate growth in controls. KP 1019 inhibited tumour colony formation in a concentration-dependent manner in both short- (1 h) and long-term (21 d) exposure experiments. KP 1019 at 100 micrograms/ml with 1 h exposure was as active as bleomycin, cisplatin, doxorubicin, etoposide, 5-fluorouracil, methotrexate, mitomycin-C and vinblastine, with only paclitaxel more active than KP 1019 (P = 0.002). The antitumour activity of KP 1019 was more pronounced after long-term exposure, indicating the potential schedule dependency of KP 1019. Activity was observed against non-small cell lung, breast and renal cancer. We conclude that if appropriate plasma levels can be achieved in patients, KP 1019 may have significant clinical activity against a variety of different tumour types.

Clinical phase I and pharmacokinetic trial of the new titanium complex budotitane.

Budotitane [cis-diethoxybis(1-phenylbutane-1,3-dionato)titanium (IV)] is a novel inorganic metal complex. Preclinical studies in established screening models indicate considerable antitumor activity. We have performed a clinical Phase I and pharmacokinetic trial with budotitane administered as i.v. infusion twice weekly. The starting dose of 100 mg/m2 was derived from a prior single dose Phase I study. Eighteen patients with solid tumors refractory to all other known treatment modalities were entered. 17 patients had received prior chemotherapy. Dose levels ranged from 100 mg/m2 to 230 mg/m2, with a total of 122 budotitane infusions administered. Neither leuko- nor thrombocytopenia were observed. 2/5 pts at 180 mg/m2 and 2/4 pts at 230 mg/m2 developed a 3-fold increase of reticulocytes without signs of hemolysis or bleeding. Nonhematologic toxicity was moderate at doses of < or = 180 mg/m2. Fifteen patients reported loss of taste at the day of infusion. At 230 mg/m2, 2/4 pts developed WHO grade 3 cardiac arrhythmias with polytope premature ventricular beats and nonsustained ventricular tachycardia. A limited pharmacokinetic analysis was performed at dose levels 180 mg/m2 and 230 mg/m2. At 180 mg/m2, Cmax was 2.9 +/- 1.2 microg/ml, t1/2 78.7 +/- 24.4 h, Cltot 25.3 +/- 4.6 ml/min and AUC 203 +/- 71.5 h x microg/ml. At 230 mg/m2, Cmax was 2.2 +/- 0.8 microg/ml, t1/2 59.3 +/- 12.1 h, Cltot 44.9 +/- 23.6 ml/min and AUC was 183 +/- 90.4 h x microg/ml. No objective tumor response was observed. We conclude that the maximum tolerated dose of budotitane administered twice weekly is 230 mg/m2, the dose limiting toxicity is cardiac arrhythmia. Further evaluation of the nature of the cardiac toxicities observed is warranted. Using this schedule, 180 mg/m2 is a safe dose for subsequent clinical studies.

Efficacy of 5'-nor-anhydrovinblastine (vinorelbine), against freshly explanted clonogenic human tumor cells in vitro.

Vinorelbine (5'-nor-anhydrovinblastine) is a semisynthetic vinca alkaloid currently undergoing extensive clinical evaluation. We have studied the antitumor effect of vinorelbine (final concentrations: 8.4-1000.0 ng/ml) against freshly explanted clonogenic cells from 102 human tumors using a capillary soft agar cloning system and have compared the compound's activity with vinblastine, vincristine, vindesine, paclitaxel, docetaxel, and other clinically used anticancer agents. Four specimens were excluded from further analyses (3 bacterial or fungal contamination, 1 benign histology). Fifty-one of the remaining 98 (52%) specimens had adequate colony formation in control capillaries. Vinorelbine showed concentration-dependent antitumor activity against a variety of solid tumors. At clinically relevant concentrations (0.1 x peak plasma concentrations in humans) vinorelbine inhibited 21 of 49 specimens (43%) and was as active as vinblastine, vincristine, vindesine, bleomycin, doxorubicin, 5-fluorouracil, mitomycin-C, cisplatin, methotrexate, and etoposide. However, paclitaxel (71% inhibition, p = 0.006) and docetaxel (78% inhibition, p = 0.002) were significantly more active than vinorelbine. Moreover, vinorelbine showed antitumor activity against several tumor types and in particular against breast cancer but also in non-small cell lung cancer. We conclude that vinorelbine has a wide spectrum of in vitro activity against freshly explanted human tumors and that the clinical activity of this compound against breast cancer and non-small cell lung cancer is reflected in vitro.

Clinical and pharmacokinetic phase I trial of oral dimethylaminoetoposide (NK611) administered for 21 days every 35 days.

We have conducted a clinical and pharmacokinetic trial of the novel podophyllotoxin derivative NK611 administered orally for 21 consecutive days. The treatment was repeated every 35 days. Eighteen patients were included into the study, all of whom were eligible. Due to early progression of tumor disease in two patients, 16 patients were evaluable for toxicity [7 female, 9 male, median age 64 years (range: 44 to 73)]. Dose escalation steps were 5 mg/day [105 mg per cycle (pc)], 10 mg/day (210 mg pc), 12.5 mg/day (265 mg pc) and 15 mg/day (315 mg pc). A total of 37 courses was administered. Toxicity was evaluated using NCI-CTC criteria. Granulocytopenia was the main hematologic toxicity. Other hematologic toxicities were sporadic. Non-hematologic toxicities were mild and consisted of grade 1 nausea and grade 2 alopecia. Pharmacokinetic analyses were performed in six patients each treated with 10 mg/day and 12.5 mg per day, and in one patient treated with 15 mg/day. Using a two-compartment model, t1/2 alpha ranged from 0.47 to 1.54 h and t1/2 beta from 2.0-11.6 h. Mean values for Cmax and AUC were 1.47 +/- 0.331 microgram/ml and 13.67 +/- 3.81 micrograms/ml.h. No objective tumor responses were observed. However, one patient with metastatic breast cancer had stable disease for twelve months. We conclude that the Maximum Tolerated Dose of NK611 administered daily for 21 consecutive days is 12.5 mg/day. The Dose-Limiting Toxicity is granulocytopenia. The recommended dose for further clinical Phase II studies is 10 mg/day.

Clinical phase I study of paclitaxel followed by cisplatin in advanced head and neck squamous cell carcinoma.

We performed a clinical phase I trial of the combination of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) and cisplatin in patients with recurrent or metastatic squamous cell carcinoma of the head and neck, using a 3-hour infusion of paclitaxel followed by a 1-hour infusion of cisplatin. Treatment with this combination was repeated every 21 days. Patients who had received prior treatment with platinum-containing regimens were excluded. However, patients who had received two or fewer courses of radiochemotherapy not including platinum compounds were eligible. At present, 21 patients have been entered into this ongoing study. Doses ranged from paclitaxel 135 mg/m2 plus cisplatin 75 mg/m2 to paclitaxel 250 mg/m2 plus cisplatin 100 mg/m2. The maximum tolerated dose was reached at paclitaxel 250 mg/m2 and cisplatin 100 mg/m2. The dose-limiting toxicity of this regimen was myelosuppression (leukopenia, granulocytopenia). Clinically, neurosensory toxicity was moderate. However, preliminary analyses of threshold electrotonus studies indicate the presence of subclinical neurotoxicity in most patients. One patient receiving paclitaxel 200 mg/m2 plus cisplatin 100 mg/m2 developed grade 3 motor neurotoxicity. Profound orthostatic hypotension was observed in five patients receiving paclitaxel 200 mg/m2 plus cisplatin 100 mg/m2 or higher. Neurotoxicity was of delayed onset and slowly reversible, and its severity appeared to be dose related. Twelve patients are currently evaluable for response. Of these, three partial remissions were observed (6, 6+, and 3+ months). Five additional patients had stable disease. We conclude that the combination of paclitaxel administered as a 3-hour infusion followed by cisplatin is an active regimen in advanced head and neck cancer. In addition to myelosuppression, orthostatic hypotension may be a potentially significant clinical toxicity. Clinical phase II studies have been initiated, using a dose of paclitaxel 200 mg/m2 and cisplatin 100 mg/m2.

Beta-tubulin and P-glycoprotein: major determinants of vincristine accumulation in B-CLL cells.

Vincristine (VCR) accumulation in chronic lymphatic leukemia of B-cell origin (B-CLL) has recently been shown not to be inversely correlated to P-glycoprotein (PGP) levels. Therefore, we studied, in addition to PGP expression and accumulation of VCR, the cellular beta-tubulin content in quiescent and rhIL-2 activated B-CLL cells. VCR mediates cytotoxicity by binding to tubulin. Constitutive beta-tubulin levels in B-CLL cells varied considerably. Upon activation with rhIL-2, beta-tubulin expression increased significantly. Therefore, tubulin levels could be correlated over a wide range to VCR accumulation. When the PGP-mediated drug efflux was blocked by verapamil (VRP), tubulin levels correlated linearly to VCR accumulation. All B-CLL cases expressed PGP at different levels. There was no linear correlation between PGP expression and VCR accumulation. A modulation factor m was defined as a quotient of VCR accumulation in the presence and absence of VRP to define the extent by which VRP inhibited a steady-state accumulation of VCR. The factor allowed discrimination between B-CLLs expressing low versus high PGP, irrespective of the levels of tubulin. However, PGP and beta-tubulin levels together were predictive for VCR accumulation in steady state. There was no uniform-accumulation defect for VCR in B-cell CLL because beta-tubulin and PGP were expressed independently. Non PGP-mediated VCR transport seems to play a minor role in B-cell CLL. Leukemia-associated varying of cytoskeletal organization in B-cell CLL might be one reason for the diverse cellular responses to receptor-mediated signals.

Characterization and mutual ligand-induced modulation of epidermal growth factor and interferon-alpha receptors on renal carcinoma cells in vitro.

We have determined the binding of epidermal growth factor (EGF) and interferon (IFN)-alpha to their specific receptors on four renal carcinoma cell lines. CaKi-2, a-498 and ACHN cell lines express high numbers, and CaKi-1 expresses low number of EGF receptors (EGFRs). On all four renal carcinoma cell lines, we have also detected specific IFN-alpha binding sites. EGF and IFN-alpha binding to their receptors caused modulation of the other ligand's receptor binding. Scatchard analyses of binding data showed that IFN-alpha treatment leads to an increase of EGFR number in three out of four cell lines and to a decrease of EGFR number in one out of four (CaKi-1). No significant changes in EGF binding affinities were detected. EGF induced a reduction in IFN-alpha receptor number in all four cell lines without significant changes in IFN-alpha binding affinities. We hypothesize that presence of EGF in the microenvironment of renal cancer cells may modulate the biological effects of IFN and consequently decrease its antiproliferative activity.

Activity of NK 611, a new epipodophyllotoxin derivative, against colony forming units from freshly explanted human tumours in vitro.

NK 611 is a new semisynthetic analogue of etoposide, which presumably also acts through inhibition of topoisomerase II, and has been found to be more potent against several cancer cell lines in vitro than etoposide. The objectives of our study were to determine the activity of NK 611 against freshly explanted clonogenic cells from human tumours and compare this agent with etoposide and other clinically useful agents. After exposure for 1 h in 45 evaluable tumour specimens, NK 611 showed clear concentration-dependent antitumour activity. At 51 microM, 49% of specimens were markedly inhibited. Using a long-term (21-28 day) exposure at 6.8 microM, 58% of 50 evaluable specimens were profoundly inhibited. At equimolar concentrations, NK 611 was as active as etoposide. Across all tumour types studied, NK 611 was as active as vinblastine, bleomycin, doxorubicin, 5-fluorouracil, mitomycin-C and cisplatin. Our results showed cross resistance to etoposide in the majority of specimens. Activity of NK 611 was greater with long-term exposure than with short-term exposure indicating schedule dependency. We conclude that NK 611 has a wide spectrum of in vitro antitumour activity. Since preliminary clinical information suggests that this drug is well tolerated at high doses, further development of this agent in Phase II trials with multiple dosing schedules is warranted.

Interleukin 1 modulates growth of human renal carcinoma cells in vitro.

We have investigated the influence of interleukin 1 (IL-1) on growth of human renal carcinoma cells in vitro. Using a capillary soft-agar cloning system, 18% of freshly explanted renal carcinomas were stimulated to grow by IL-1 and 4% were inhibited. Subsequent experiments with established renal cancer cell lines demonstrated that two out of four cell lines (Caki-2, A-498) were sensitive to IL-1. [3H]Thymidine incorporation as well as monolayer growth was enhanced in Caki-2 cells in the presence of high (10%) and low (1%) serum concentrations. Although clonogenic growth of A-498 cells was stimulated by IL-1, overall [3H]thymidine incorporation and monolayer proliferation were decreased. Using radioligand experiments, 250 cell-surface receptors of high affinity (KD 4.5 x 10(-11) M) and 2500 receptors of low affinity (KD 1.3 x 10(-9) M) were detected on A-498 cells. IL-1 binding was reduced under the influence of IL-1. Competition experiments with inhibiting antibodies against IL-1 receptor type I and type II revealed that signal transduction was performed via type I receptors. After cross-linking to IL-1, receptor type I was immunoprecipitated using anti-IL-1 antibodies. We hypothesise that, since IL-1 modulates in vitro growth of a subgroup of human renal cancer cells, interference with its mechanism of action may be of potential value in order to modulate tumour proliferation.

Effects of vinorelbine on epidermal growth factor-receptor binding of human breast cancer cell lines in vitro.

Epidermal Growth Factor (EGF) is a mitogenic peptide that binds to surface membrane receptors (EGFR) of breast cancer cells. After binding, secondary transmitter molecules are activated by tyrosine phosphorylation of the intracellular receptor domaine. The activity of the EGF/EGFR system can be modulated by a variety of chemically unrelated compounds including cytostatic agents. The purpose of our present study was to determine the effects of vinorelbine, a novel semisynthetic vinca alkaloid on EGF receptor binding on human breast cancer cells. We have found that MDA-231 and MDA-468 cells bind substantially more [125I]-EGF after preincubation with vinorelbine. This effect was concentration- and time-dependent reaching a maximum at 100 ng/ml and 24 h incubation. Subsequent experiments showed an increase in the rate of EGF binding as well as maximal binding capacity. Scatchard analysis of binding experiments under equilibrium conditions indicated that this was mainly due to an increase in the number of apparent EGF binding sites. Modulation of EGF receptor binding by vinorelbine was not detectable when isolated membranes were used indicating that intact cytoplasmatic mechanisms are required for the upregulation of EGF receptors.

Investigation of the comparative effects of 2-chlorodeoxyadenosine on tumor colony forming units in vitro.

2-CdA is a deaminase-resistant purine analogue which has shown clinical activity against various hematological tumors, and is currently undergoing clinical phase II trials. The objectives of our study were to determine the activity of 2-CdA against freshly explanted clonogenic cells from non-hematological human tumors and compare this agent with other clinically useful anticancer agents. We also compared short-term (1 hour) and long-term (21-28 days) exposures. For short-term exposure (1-hour), final concentrations were 0.57, 5.7, 57, and 114 ng/ml. Inhibition of tumor specimens was concentration-dependent: 0.57 ng/ml: 1/51 (2%), 5.7 ng/ml: 4/52 (7%), 57 ng/ml: 11/52 (21%), 114 ng/ml: 27/50 (54%). At concentrations > or = 57 ng/ml, 2-CdA was as active as cisplatin, doxorubicin, 5-fluorouracil, mitomycin-C, vinblastine, and etoposide. For long-term exposures (21-28 days), final concentration of 2-CdA were 0.57, 5.7, and 57 ng/ml. At 0.57 ng/ml, 2-CdA was active in 4/54 (7%) specimens [5.7 ng/ml: 13/54 (24%), 57 ng/ml: 40/54 (74%)]. A head-to-head comparison with short-term exposures demonstrated greater activity if the drug exposure time was extended. Using the strategy for testing other standard agents (in vitro dose of 1/10th achievable peak plasma concentration), one would predict clinical response rates for single agent bolus or short-term administration of 2-CdA to be in the neighborhood of 7%. Longer durations of infusion or multiple doses might increase the response rate to about 24%. If higher peak plasma concentration could be achieved, dose-dependent increases in clinical responses might be achievable. We conclude that 2-CdA is active against clonogenic cells from freshly explanted non-hematological human tumor specimens at high concentrations.

Chemomodulation of drugs involved in multidrug resistance in chronic lymphatic leukemia of the B-cell type.

Reduced drug accumulation may be one reason for intrinsic drug resistance in chronic lymphatic leukemia of the B-cell type (B-CLL). Immunophenotyped leukemic human B-cells from 38 cases of B-CLL were characterized for P-glycoprotein (PGP) content. In all, 30 cases of B-CLL were additionally analyzed for further parameters: accumulation of daunorubicin (DNR, n = 20) and rhodamine 123 (Rh123, n = 30) in both the presence and the absence of verapamil (VRP). Also, 16 cases of B-CLL were analyzed for vincristine (VCR) accumulation with or without VRP. Concerning the relative expression of PGP, these 16 cases of B-CLL were representative for the whole group of 30 cases. Spontaneous accumulation of Rh123 and VCR varied over a wide range: accumulation of Rh123, by a factor of 11.8; accumulation of VCR, by a factor of 9.7; and accumulation of DNR, by a factor of 3.6. VRP modulated the accumulation of RH123 in 16/30 cases (53%), that of VCR in 69% of cases, and that of DNR in 11% of cases. The maximal VRP-mediated increases in accumulation amounted to factors of 1.3 for DNR, 2.3 for Rh123, and 7.8 for VCR. Spontaneous drug accumulation did not correlate with the extent of chemomodulation. The amount of PGP in B-CLL cells (all cases studied were PGP-positive) did not correlate with drug accumulation or with the extent of VRP-mediated chemomodulation. Thus, high expression of PGP is only partially responsible for defective drug accumulation in B-CLL. Only the degree of chemomodulation by VRP is predictive for the extent of the PGP-related functional drug accumulation defect. Thus, in B-CLL, PGP-independent drug accumulation defects seem to be as important as those mediated by PGP. The extent of this drug accumulation defect varies for the different drugs in the following order VCR > Rh123 > DNR. The relevance of PGP-mediated and -independent drug accumulation defects in vivo may depend to a large extent on the drug being used and on the individual cell type. Both types of defect in drug accumulation are of high importance when regimens include VCR a drug commonly used in second-line chemotherapy of B-CLL. Both defects in drug accumulation may be responsible for intrinsic VCR resistance in B-CLL.

Effects of the microtubule-disturbing agents docetaxel (Taxotere), vinblastine and vincristine on epidermal growth factor-receptor binding of human breast cancer cell lines in vitro.

Epidermal growth factor (EGF) is a mitogenic peptide that binds to surface membrane receptors (EGFR) of breast cancer cells. After binding, secondary transmitter molecules are activated by tyrosine phosphorylation of the intracellular receptor domaine. The activity of the EGF/EGFR system can be modulated by a variety of chemically unrelated compounds including cytostatic agents. The purpose of our present study was to determine the effects of mitotic inhibitors on EGF receptor binding on human breast cancer cells. We found that MDA-231 and MDA-468 cells bind substantially more [125I]EGF after preincubation with docetaxel, vinblastine and vincristine. This effect was concentration- and time-dependent, reaching a maximum at 3000 ng/ml and 48 h incubation for docetaxel, and 100 ng/ml and 48 h incubation for vinca alcaloids. Subsequent experiments showed an increase in the rate of EGF binding as well as maximal binding capacity. Scatchard analysis of binding experiments under equilibrium conditions indicated that this was due to an increase in the number of apparent EGF binding sites. Modulation of EGF receptor binding by docetaxel, vinblastine, and vincristine was not detectable when isolated membranes were used, indicating that intact cytoplasmatic mechanisms are required for the upregulation of EGF receptors.

Removal of breast cancer cells from bone marrow by in vitro purging with ether lipids and cryopreservation.

Four cell lines from human breast cancer (HTB 19, HTB 133, IZB B, and MCF 7M) were investigated for in vitro growth before and after incubation with the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) for 4 h and at increasing concentrations (25, 50, 75, and 100 micrograms/ml), as well as with and without cryopreservation. When measured with the human tumor cloning assay in agar or methyl-cellulose the surviving fraction showed an inverse correlation with the concentrations of ET-18-OCH3. After a 4-h exposure with 75 micrograms/ml ET-18-OCH3 at a cell density of 2 x 10(5)/ml the number of colonies of HTB 19 decreased from 75 +/- 10/10(3) cells (100%) to 1 +/- 0/10(3) cells (1%), and after subsequent cryopreservation no remaining colonies were found. In order to simulate the situation in autologous bone marrow transplantation we contaminated normal human bone marrow cells with malignant HTB 19 breast cancer cells at a ratio of 100:1. After incubation with 75 micrograms/ml ET-18-OCH3 for 4 h and subsequent cryopreservation there was a considerable reduction of HTB 19 colonies whereas the majority of normal human hematopoietic progenitors recovered. We conclude that ET-18-OCH3, in combination with cryopreservation, can remove breast cancer cells from bone marrow by up to 1 log and may be useful for purging bone marrow for autologous bone marrow transplantation not only in acute leukemia and non-Hodgkin's lymphoma but also in breast cancer.

Phase I clinical and pharmacokinetic trial of dextran conjugated doxorubicin (AD-70, DOX-OXD).

Coupling of anthracyclines to high-molecular-weight carriers may alter drug disposition and improve antitumor effects. We have performed a clinical phase I trial of doxorubicin coupled to dextran (70000 m.w.). The drug was administered as single dose i.v. every 21-28 days. Thirteen patients have received a total of 24 courses (median 2; range 1-3). At the starting dose of 40 mg/m2 doxorubicin equivalent (DOXeq), WHO grade IV thrombocytopenia was noted in 2/2 patients. WHO grade IV hepatotoxicity and WHO grade III cardiotoxicity were noted in a patient with preexisting heart disease. Five patients were treated with 12.5 mg/m2 DOXeq. Maximal toxicity at this dose level was WHO grade III thrombocytopenia and local phlebitis (WHO grade II) in 1/5 patients, elevation of alkaline phosphatase (WHO grade III) and WHO grade III vomiting in another patient. Subsequently, five patients received 20 mg/m2 DOXeq. Hepatotoxicity was noted in 5/5 patients (1 x WHO grade IV, 1 x WHO grade III). Thrombocytopenia was noted in 3/5 patients (1 x WHO grade IV, 2 x WHO grade III). At 12.5 mg/m2 DOXeq, a patient diagnosed with a malignant fibrous histiocytoma had stable disease for 4 months. Pharmacokinetic analyses of total and free doxorubicin were performed in plasma and urine. The maximum peak plasma concentration (ppc) for total DOX was 12.3 micrograms/ml at 40 mg/m2 DOXeq. The area under the plasma concentration time curve (AUC) ranged from 28.83-80.21 micrograms/ml*h with dose-dependent elimination half lives (t1/2 alpha: 0.02-0.87 h; t1/2 beta: 2.69-11.58 h; t1/2 gamma: 41.44-136.58 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Preclinical activity of taxotere (RP 56976, NSC 628503) against freshly explanted clonogenic human tumour cells: comparison with taxol and conventional antineoplastic agents.

Taxotere (TER) and taxol (TA) are new antitumour agents currently undergoing clinical evaluation. We studied the antineoplastic effects of these agents (final concentrations: 4.0, 0.4, 0.04 mumol/l) on the in vitro proliferation of clonogenic cells from freshly explanted human tumours using a capillary soft agar cloning system. We also compared the activity of these new compounds to conventional antineoplastic agents (bleomycin, cisplatin, dacarbazine, doxorubicin, etoposide, 5-fluorouracil, vinblastine, interferon-alpha 2). Using a 21-28-day continuous drug exposure, 54/81 specimens (67%) were evaluable for comparisons, and using a 1-h drug exposure followed by 21-28 days incubation, 50/80 specimens (63%) were similarly evaluable. With both schedules, TA and TER showed concentration-related antitumour activity. At 0.4 mumol/l, median colony survival was 0.61 x control (range 0.09-0.96) for TA and 0.51 x control (0.15-0.81) for TER in the 1-h incubation (P = 0.0002). Median colony formation was also reduced significantly more by TER as compared to TA in the long-term incubation schedule. Statistical analysis indicated that TER but not TA was significantly more active than cisplatin (P = 0.02), doxorubicin (P = 0.01), 5-fluorouracil (P = 0.01) and interferon-alpha 2 (P = 0.01). We conclude that TER and TA are more active against in vitro tumour colony formation from freshly explanted human tumours. TER appears to be slightly more active than taxol and promises to be active against tumours resistant to conventional antineoplastics.

Differential cytotoxicity of an ether lipid on lymphoma and bone marrow cells and its role in purging malignant lymphoid cells from remission bone marrow contaminated with tumor cells.

Four human clonogenic malignant lymphoid cell lines (CEM, Su-DHL-4, Li-A, and Raji) as well as normal human bone marrow stem cell progenitor cells were investigated for clonal in vitro growth before and after incubation with the ether lipid ET-18-OCH3 for various times (1, 4, and 18 h) and at increasing concentrations of the drug (25, 50, 75, and 100 micrograms/ml). The clonal growth of the malignant lymphoid cell lines was inversely correlated with concentrations and times of drug incubation. The antineoplastic effect of ET-18-OCH3 was further amplified by subsequent cryopreservation. In a situation of 4-h exposure to less than or equal to 50 micrograms/ml ET-18-OCH3 and subsequent cryopreservation, in which greater than 50% of the normal human bone marrow progenitor cells survived, 1-3 logs of the malignant lymphoblastoid cells were killed, indicating a potential value of this drug for bone marrow purging in lymphoid malignancy. In order to simulate the situation of autologous bone marrow transplantation (ABMT) in complete remission of the disease, we contaminated normal human bone marrow cells with malignant CEM or Su-DHL-4 lymphoid cells at a ratio of 100:1. Results show that 4 h of incubation with 75 micrograms/ml ET-18-OCH3 and subsequent cryopreservation can eliminate 2-3 logs of clonogenic cells of the malignant lymphoblastoid cell lines under conditions that allow recovery of greater than 50% of the normal human hematopoietic progenitors.