Reentry of nondividing leukemic cells into a proliferative phase in acute childhood leukemia.

Reentry of small leukemic blast cells into a proliferative phase was demonstrated in a 3 yr old girl with untreated acute lymphoblastic leukemia. Since the proliferating leukemic cell compartment in this disease is not self-maintaining, continual entry of cells into this compartment is necessary to prevent depletion of proliferating cells. In order to identify the source of replacement cells, the rate of change of tritiated thymidine-labeled cells in the proliferating compartment was observed by means of serial bone marrow samples under two conditions. In the first study period only 10% of small leukemic blast cells were labeled, and in the second study period 72% of this population had become lebeled. During the first period the proliferating blast cells were rapidly replaced by unlabeled cells, while during the second period the replacement cells were coming largely from a labeled cell source. The only identifiable source of cells for maintenance of the proliferating population which was virtually unlabeled during the first period and largely labeled during the second period was the population of small leukemic blast cells. The finding that the small blast cells are only temporarily nonproliferative could account for effectiveness of therapy directed primarily against a dividing cell population. Persistence of some cells with longer resting times into remission could provide a focus for subsequent relapse.

Variation of proliferative activity in leukemic cell populations of patients with acute leukemia.

In 31 children with acute leukemia, the proliferative activity of the leukemic marrow cell population as measured by mitotic and labeling indexes varied widely from patient to patient and from one disease stage to another. Leukemic marrow had a small but statistically significant diurnal variation of proliferative activity. Changes in labeling indexes were directly related to changes in the proportion of large dividing blasts in the marrow. Generation times of dividing leukemic blast cells in 3 patients were similar at diagnosis and in relapse. Changes in proliferative activity of leukemic marrow can be explained by progressive accumulation of nondividing leukemic cells.

Drug effect in acute leukemia.

The in vivo therapeutic effect of vincristine, cytosine arabinoside, and corticosteroids on leukemic blast cells in the bone marrow was evaluated. 24 studies were done in 21 children with acute leukemia. 19 children had acute lymphoblastic leukemia, and two children had acute myeloblastic leukemia. Direct cytotoxicity or lysis of blast cells after drug administration was looked for by serial measurements of the volume of marrow buffy coat. Changes in proliferative capacity were evaluated by serial measurements of number of cells in mitosis and the per cent of cells in deoxyribonucleic acid (DNA) synthesis, as indicated by tritiated thymidine incorporation. Corticosteroid administration caused lysis of leukemic blast cells. Each drug affected the proliferative capacity of the leukemic cells by an action at a different part of the mitotic cycle. Corticosteroids suppressed the entry of cells into DNA synthesis. Vincristine arrested cells in mitosis. Cytosine arabinoside inhibited DNA synthesis. After the inhibitory effect of cytosine arabinoside, an increased number of cells began to synthesize DNA, a phenomenon indicating that partial synchronization of the mitotic cycle had been achieved in the leukemic cell population. The action of these drugs at different parts of the mitotic cycle might be important in designing treatment regimens where in two or more of these drugs are used.

Synchronization and recruitment in acute leukemia.

The in vivo effects of several chemotherapeutic agents on the mitotic cycle of leukemic blasts in the bone marrow were evaluated by serial measurements of cells in mitosis and in deoxyribonucleic acid (DNA) synthesis as indicated by ability to incorporate tritiated thymidine or tritiated deoxyuridine. 28 studies were done in 23 children and 1 adult. The changes in the marrow after a single injection of L-asparaginase, hydrocortisone, cyclophosphamide, cytosine arabinoside, methotrexate, and an exchange transfusion (62% of the total blood volume) were evaluated. L-asparaginase and hydrocortisone were found to arrest the entry of cells into the S period. Cyclophosphamide appeared to inhibit DNA synthesis, arrest cells in mitosis, and inhibit the entry of cells into the S period. Cytosine arabinoside, and methotrexate inhibited DNA synthesis. During the period of time the cells were inhibited in the S phase by these two drugs, cells continued to enter the S period. Thus partial synchronization was achieved after these two drugs. An exchange transfusion had no consistent effect on the mitotic cycle, but partial synchronization in the S period was seen in one patient. To take advantage of the ability of cystosine arabinoside, to synchronize leukemic cells in the S phase, a second cycle-dependent drug was given at the time the leukemic blasts were synchronized. The second cycle-dependent drugs evaluated were vincristine, methotrexate, and cytosine arabinoside given by intravenous drip over a 12 hr period. Recruitment was found after cytosine arabinoside alone, and after prior synchronization with cytosine arabinoside and then the administration of either of these drugs. The results of these studies indicate that a greater therapeutic advantage can be achieved by a second cycle-dependent drug after synchronization than after the second drug alone.

Cell kinetics and chemotherapy in neuroblastoma.

The proliferative behavior of neuroblastoma cells in bone marrow was evaluated before and after perturbation by chemotherapy. The data presented indicated that the proliferating fraction of the tumor cells before therapy was small. In most of the patients, initial therapy with a non-cell-cycle specific agent increased the mitotic and labeling indices. If initial therapy was followed by a cell cycle-specific agent, variable kinetic responses were observed. Although the number of patients was small and the follow-up was short, there was a correlation between the clinical response and the kinetic changes induced by the chemotherapy. Evaluation of cell kinetic changes during the initial course of chemotherapy might identify early those patients who are unlikely to have a good clinical response. Similar studies in the evaluation of new drugs to aid in identifying agents that favorably change cell kinetics and thus therapy for children with neuroblastoma was urged.

Correlation of cell kinetic and clinical response to chemotherapy in disseminated neuroblastoma.

Mitotic and labeling indices of bone marrow tumor cells were determined from patients with disseminated neuroblastoma. Although pretreatment values were variable, the median indices indicated that only a small proportion of tumor cells were proliferating. The pretreatment indices could not be correlated with presenting clinical features or with the response to chemotherapy. Studies were repeated after 7 daily doses of cyclophosphamide (150 mg/sq m) and serially after Adriamycin (35 mg/sq m) given on Day 8. Changes in the mitotic and labeling indices during the first course of therapy could be directly correlated with the clinical response as evaluated after 4 months of induction chemotherapy. In all patients who attained a complete or partial remission, an increase in these indices was observed after 7 days of cyclophosphamide. If this increase was associated with an observed kinetic effect of Adriamycin in the G1, S, and G2 phases of the cell cycle, a complete remission was attained. If, following Adriamycin, kinetic evidence of an effect was not present in all three phases of the cell cycle, only partial remission was attained. No clinical response to therapy was observed in those patients in whom the mitotic index and labeling index did not increase after 7 days of cyclophosphamide.

A clinical perspective on cell markers in acute lymphocytic leukemia.

Two hundred consecutive new patients, with acute lymphocytic leukemia (ALL) have been studied with a battery of five cell marker assays to determine if a classification system with prognostic significance can be developed; 182 have been classified among four groups as follows: 33 T-cell, 3 B-cell, 126 common, and 20 undifferentiated ALLs. Patients with T-cell disease are likely to have unfavorable clinical prognostic features and a poor response to therapy. Rare patients with B-cell disease are closely related clinically to non-Hodgkin's lymphoma. Those with common ALL infrequently have unfavorable clinical features and have a superior outcome to that of T-cell patients. Children with undifferentiated markers seem to respond less well to treatment than do those with common ALL, yet may not be identifiable as poor risk by clinical features. What remains to be resolved with further observation is whether these marker patterns are more reliable indicators of prognosis than the usual clinical determinants predisposing to treatment failure (high white blood cell count, mediastinal mass, and central nervous system disease). At the present time, it appears that in the absence of poor-risk clinical prognostic features, patients with common ALL are more likely to have lasting remissions than those with erythrocyte-rosette-positive T-cell disease or those with ALL that is undifferentiated by markers.

Detection of small cell lung cancer bone marrow involvement by discontinuous gradient sedimentation.

Marrow involvement by small cell lung cancer (SCLC) is detected in 10-23% of patients at initial diagnosis by marrow aspirate and biopsy techniques. To improve the detection and potentially monitor marrow involvement by SCLC we have attempted to concentrate malignant cells with clonogenic potential on a discontinuous density gradient (DDG). The bone marrow from 43 patients with SCLC (36 with histologically negative marrow aspirates and biopsies) were separated on a Ficoll-based DDG. Samples were also separated by conventional Ficoll-diatrizoate (FD) (density, 1.077) gradient sedimentation. The cellular interphase from three fractions (F X) corresponding to specific densities 1.050 (F X 1), 1.055 (F X 2), and 1.060 (F X 3) as well as cells separated by Ficoll-diatrizoate (F X FD) centrifugation were isolated and 2.5 X 10(5) cells from each fraction were cultured in 2 ml of 0.3% agar in McCoy's media with 10% fetal calf serum, 2.5 micrograms transferrin, 1 microgram insulin, and 1% penicillin-streptomycin. Colony growth was assessed after 14 days of culture at 37 degrees C and 6% CO2. Tumor colony growth was seen in eight of 36 (22%) patients with histologically negative marrows as well as in four of seven (57%) patients with known involvement. Mean colony growth per 2.5 X 10(5) cells for all 12 patients was 4.3 colonies for F X 1; 8.8 for F X 2; and 2.7 for F X 3. In contrast mean growth from the F X FD was 1.0 colonies. Cells with clonogenic potential could be demonstrated from F X 2 and F X 3 in seven of 12 and eight of 12 patients, respectively; in F X FD four of 12 patients had tumor growth. We conclude that separation of marrow samples by DDG concentrates malignant cells with clonogenic potential at least 8-fold compared to FD separation and that the sensitivity of the clonogenic assay in detecting marrow involvement by SCLC is enhanced by DDG sedimentation.

The importance of an isolated central nervous system relapse in children with acute lymphoblastic leukemia.

We assessed the influence of an initial isolated meningeal relapse on treatment outcome in 839 children with acute lymphoblastic leukemia (ALL) who were admitted to St Jude Children's Research Hospital (Memphis) from mid-1967 through mid-1979. The patients were entered in a series of five clinical trials (Total Therapy Studies V through IX), each designed to test one or more modifications of treatment for ALL. Two groups were compared: 699 children who received CNS prophylaxis (2,400-rad craniospinal irradiation or 2,400-rad cranial irradiation plus intrathecal methotrexate) v 56 who did not. Our results, obtained with a time-dependent covariate model and a matching technique, indicate a 2 to 3.5-fold increase in the risk of hematologic relapse or death among patients who experienced an isolated CNS relapse compared with similar patients (matched for leukocyte count and length of complete remission) who remained free of CNS involvement. Of the 107 children with an initial isolated CNS relapse, 89 (83%) have died or have had a subsequent relapse. There was no detectable difference in the rate of hematologic relapse or death after a CNS relapse between patients who had received preventive therapy and those who had not. We conclude that CNS prophylaxis is important both for the prevention of initial CNS leukemia and for reducing the risk of hematologic relapse or death subsequent to a CNS relapse.

Pharmacodynamics of prolonged oral etoposide in patients with advanced non-small-cell lung cancer.

PURPOSE: This study was undertaken to investigate the pharmacodynamic relationship between etoposide drug levels on 21-day oral treatment courses and hematologic toxicities in patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Thirty-two patients with stage IIIB or IV NSCLC were treated with oral etoposide 50 mg/m2/d for 21 consecutive days in combination with cisplatin 100 mg/m2 on day 1. Treatment was repeated every 28 days for up to six courses. Patients had not received previous chemotherapy and had a performance status of 0 to 2. Patients were monitored weekly while on treatment for compliance with oral etoposide and toxicity, including complete blood cell counts, and a blood sample before the daily etoposide dose (drug trough levels). Etoposide concentrations were measured in the plasma by high-performance liquid chromatography (HPLC). RESULTS: Three patients achieved a complete response (CR) and 10 patients a partial response for an objective response rate of 41% (95% confidence interval, 24% to 58%). The median survival was 4 months (range, 1 to 23). Neutropenia was dose-limiting, and two patients died of neutropenic sepsis. Pharmacodynamic correlations for drug concentrations and hematologic toxicities were available for 27 patients and a total of 76 treatment courses, and correlations were significant for graded hematologic toxicity and nadir counts of leukocytes, neutrophils, hemoglobin, and platelets. The grade of infection (77 courses) was also related to drug levels. Using data from 27 initial courses, a pharmacodynamic model was developed to estimate the nadir leukocyte or neutrophil count (WBCn, ANCn) based on the pretreatment count (WBCp, ANCp) and the etoposide concentration (Ec) as follows: WBCn = 0.35 (1 + WBCp x e-1.12 x Ec) and ANCn = 0.32 (1 + ANCp x e-2.47 x Ec). CONCLUSION: Etoposide concentrations are related to the resulting hematologic toxicities. It is possible to predict nadir counts in the first course by a pharmacodynamic model. The above equations need to be validated prospectively and may be useful in future studies of prolonged oral etoposide.

Phase I study of docetaxel with concomitant thoracic radiation therapy.

PURPOSE: The taxanes have demonstrated activity as radiation sensitizers in preclinical studies. This study was designed to determine the maximum-tolerated dose (MTD), optimal schedule, and toxicities of docetaxel in combination with concomitant standard chest radiotherapy. PATIENTS AND METHODS: Twenty-nine patients with advanced non-small-cell lung or esophageal cancer enrolled in this phase I study to evaluate escalating docetaxel doses at three schedules. Docetaxel was administered as two 21-day cycles at doses of 40, 60, and 75 mg/m2 per cycle. Docetaxel administration schedules were as follows: schedule A, once every 3 weeks; schedule B, 2 of 3 weeks; or schedule C, weekly. Six weeks of concomitant standard chest radiotherapy in 1.8- to 2.0-Gy daily fractions was delivered to 60 Gy total. RESULTS: Dose-limiting esophagitis and neutropenia were encountered with schedules A and B at docetaxel doses of 60 mg/m2 per cycle. The docetaxel MTD for schedules A and B was 40 mg/m2 per cycle. Dose-limiting esophagitis was also observed with schedule C; however, there was no neutropenia. For schedule C, we identified the MTD as 60 mg/m2 per cycle (20 mg/m2/wk). Other toxicities encountered included thrombocytopenia, hypersensitivity reaction, and pulmonary infiltrates (fatal in two patients). Late toxicity of esophageal stricture occurred in five patients. CONCLUSION: Esophagitis and neutropenia are the dose-limiting toxicities of docetaxel administered with concomitant chest radiotherapy. Weekly administration of docetaxel allows for the highest total docetaxel dose during chest radiotherapy. We identified the recommended phase II docetaxel dose as 20 mg/m2 administered weekly with concomitant chest radiotherapy for 6 weeks.

Paclitaxel/carboplatin plus ifosfamide in non-small cell lung cancer.

Chemotherapy has a positive role in managing patients with stage IV non-small cell lung cancer. Randomized studies and meta-analyses comparing chemotherapy with best supportive care have confirmed a significant prolongation of survival for chemotherapy-treated patients. In recent years, several new active agents have been identified. These include the taxanes paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) and docetaxel, the antimetabolite gemcitabine, the mitotic spindle inhibitor vinorelbine, and (potentially) the topoisomerase I inhibitors. Combinations of either vinorelbine or paclitaxel with cisplatin have resulted in a significant increase in median survival times compared with both cisplatin and vindesine and cisplatin and etoposide. Paclitaxel combined with either carboplatin or ifosfamide also has been promising. Since even the best current combination regimens result in median survival times of less than 1 year, the identification of more active drugs and combinations remains a high priority. One possible strategy to identify more active regimens may be the combination of three rather than two active agents into a combination regimen. Although the three-drug regimens used in the 1980s appeared to be no more active than two-drug combinations, the advent of additional active compounds with novel mechanisms of action allows this question to be readdressed. Based on this background, we have initiated a phase I/II study of carboplatin and paclitaxel with escalating doses of ifosfamide. The study design and dosing schedule are discussed in this report.

Combined modality therapy in non-small cell lung and esophageal cancer: a phase I dose-escalation study of docetaxel with concurrent radiotherapy.

The effective treatment of non-small cell lung cancer requires both locoregional and systemic therapy. Outcome in intermediate-stage malignancies of the chest may be improved by combining chemotherapy and radiotherapy. Several combined-modality trials involving the platinum compounds, vinorelbine, gemcitabine, and the taxanes have been recently conducted. In one such phase I dose-escalation study of 29 patients with non-small cell lung cancer or esophageal cancer, the concurrent administration of docetaxel and radiotherapy at a dose of 2 Gy/d for 6 weeks was shown to be feasible. Objective tumor responses were observed, and further studies of docetaxel in combination with radiotherapy are indicated.

Ifosfamide-based three-drug combination regimens in non-small cell lung cancer.

The role of chemotherapy in the treatment of non-small cell lung cancer (NSCLC) has expanded in recent years, resulting in increased median survival and higher 5-year survival rates in some studies. Few patients with NSCLC can be cured, however, and the median survival time is still modest. Research strategies are aimed at identifying new active single agents, testing their combination in non-cisplatin- and non-carboplatin-containing regimens, and their incorporation into regimens containing more than two drugs, in efforts to improve response and survival and/or reduce toxicity. Several trials of triplet chemotherapy combinations for the treatment of NSCLC have been conducted at the University of Chicago. The three-drug regimen of vinorelbine, paclitaxel, and ifosfamide with granulocyte colony-stimulating factor was evaluated in a phase I trial. Doses of all three drugs were reduced from their standard doses so that toxicity would be manageable, although toxicity was still high. The low response rate (<20%) at the recommended phase II doses led to early closure of this trial. The University of Chicago is also assessing the three-drug combination of carboplatin, paclitaxel, and ifosfamide in patients with stage IIIB and IV NSCLC. The carboplatin and paclitaxel doses are being maintained within their active single-agent range, with the goal of identifying the maximum tolerated dose of ifosfamide when added to this combination. Additional end points include response rates, survival time, and dose-limiting toxicities. This study is ongoing.

Clinical studies of docetaxel (Taxotere) and concomitant chest therapy.

The administration of concomitant chemoradiotherapy has been shown to increase local and regional control of non-small cell lung cancer. Docetaxel (Taxotere; Rhône-Poulenc Rorer, Antony, France) has single-agent activity in non-small cell lung cancer, as well as radiation-enhancing potential in preclinical studies. Therefore, its investigation with concomitant radiotherapy in the clinical setting is justified. Since the clinical interactions of docetaxel and concomitant chest radiotherapy have not been previously described, we initiated a phase I study with the goal of determining the maximum tolerated dose of docetaxel and the optimal schedule for its administration during a standard course of radiation therapy to the chest, in addition to defining the dose-limiting toxicities of this regimen. This report describes the design and preliminary results of this study.

Factors leading to delay in the diagnosis and affecting survival of children with head and neck rhabdomyosarcoma.

Rhabdomyosarcoma of the head and neck often presents with vague symptoms which mimic other disease conditions. These factors lead to undue delay in the establishment of the correct diagnosis and the delivery of acceptable therapy, including surgery, radiation therapy, and chemotherapy. There is, however, evidence of improved results of treatment of these tumors since the addition of multiple drug chemotherapy to surgery and radiotherapy.

Assessment of lumiaggregometry for research and clinical laboratories.

Platelet aggregometry is often used to help diagnose storage pool disease (SPD-reduced amounts of granule nucleotides) and release defects (abnormal release of granule nucleotides). The general assumption that normal aggregation patterns are sufficient to rule out the diagnosis of one of these disorders has been invalidated by the recent publication of two papers describing patients with clinical bleeding, prolonged bleeding times and normal aggregation patterns in spite of defective release. The lumiaggregometer provides a tool for measuring platelet release and aggregation simultaneously. This paper presents a standardized, reproducible method for the use of the lumiaggregometer based on a "standard curve". Data obtained during the development of the procedure are presented including normal ranges of release at different concentrations of agonists, release measured in intrinsic disorders as well as in patients on aspirin, and values for release relative to varying platelet counts. A monoclonal antibody (anti-p24/CD9; MAb7) which activates platelets similarly to thrombin and may be a useful reagent for distinguishing SPD and release defects is also introduced.

Kinetic studies of cells in childhood leukemias.

A review of cell kinetic studies in acute childhood leukemia with a comparison of leukemic transformation of non-Hodgkin's lymphoma is presented in this paper. Leukemic cell populations have a longer cell cycle than their normal cell counterparts. The cell populations are comprised of proliferating and resting fractions and are capable of self-maintaining growth. Growth regulation is determined primarily by the size of the proliferating cell population or growth fraction. The growth fraction can vary as to site of disease, the clinical phase, following chemotherapeutic perturbation, and most importantly is related to the specific tumor cell type. Within a specific type of leukemia there is considerable variability of proliferative activity at time of diagnosis, but this variability bears no relationship to the subsequent clinical course. Those leukemias, such as the E rosette-positive form of lymphocytic leukemia characterized by rapid tumor growth and large tumor bulk, are also associated with tumor cell populations having larger growth fractions than standard lymphocytic leukemia. There is evidence for growth regulation of leukemic cell populations on systemic, regional, and, perhaps most importantly of all, intrinsic cell levels. It is this area of growth regulation for these tumor cell populations which currently needs the greatest research attention.

A phase I study of STEALTH cisplatin (SPI-77) and vinorelbine in patients with advanced non small-cell lung cancer.

STEALTH cisplatin (SPI-77) is a liposomal formulation of cisplatin that has activity in animal models of non small-cell lung cancer (NSCLC). Vinorelbine has documented clinical activity in NSCLC. The purpose of this study was to determine the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of SPI-77 when administered in combination with a fixed dose of vinorelbine to patients with stage IIIB or IV NSCLC refractory to or recurrent following previous chemotherapy. SPI-77 was given on day 1 in combination with vinorelbine at a fixed dose of 25 mg/m2 on days 1 and 8 of a 3-week treatment cycle. Dose escalation of SPI-77 progressed as follows: 20, 40, 80, 100, 120, and 140 mg/m2. Twenty patients were entered (11 men and nine women; median age, 63 years). Sixty-four complete cycles of therapy were administered, and 19 of 20 patients completed at least 1 cycle of combination chemotherapy. Neutropenia was dose limiting at a SPI-77 dose of 140 mg/m2. Neuropathy and nephrotoxicity were minimal and not dose related. A partial response was observed in three of 17 patients eligible for a response evaluation and response duration ranged from 6 weeks to 5 months. In conclusion, treatment with combination SPI-77 and vinorelbine was well tolerated, and our recommended phase II dose is 120 mg/m2 of SPI-77 in combination with vinorelbine at 25 mg/m2. Activity was observed in this patient population, and additional phase II testing of this regimen in a less extensively pretreated cohort of patients with NSCLC is indicated.

Ethical complications of clinical therapeutic research on children.

Usual discussion by ethicists and physicians of the ethical implications of research on children with catastrophic disease, and the guidelines established by the Federal Government for this research, rest on applying general moral principles to problems. Whatever the merits of this approach for establishing policy, it does not adequately reflect the life situation of patients and the complexity of a single regimen which is simultaneously both therapeutic and research oriented. Also, the issues become more complex when there is disagreement among the parents about a course of treatment. It is our contention that such cases are properly resolved by considering the degree of paternalism to be exercised by the clinician-researcher.

KIE: Much of the discussion of ethical issues in therapeutic research on children is based on the application of general moral principles to categories of problems. The authors believe that this approach does not adequately address the complexity of a treatment regimen for catastrophically ill children which is both therapeutic and research oriented. Using a case study to illustrate the potential complications of such a situation, they argue that medical paternalism is justified in therapeutic research on children even if it diminishes the patient's autonomy, on the grounds that the best interests of the patient must take precedence over preservation of complete autonomy.