Shortened time to recovery from chemotherapy induced neutropenia in pediatric patients with high dose combined cytokines.

Granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) are cytokines which have been extensively administered as monotherapy to patients with a variety of hematopoietic disorders at dosages of 5 mcg/kg/day. Because their spectrum of activity is both singular and simultaneously overlapping, we postulated that combined therapy would be more advantageous than monotherapy. Since 1992 we have carried out a study of G-CSF and GM-CSF as monotherapy or in combination in pediatric patients with solid tumors following chemotherapy induced nadirs of 0-800 WBC/mm3. When combined, the cytokines were given twice per day at 2.5 or 5.0 mcg/kg. For the monotherapy groups, either cytokine at 5 mcg/kg or 10 mcg/kg was given once daily. The mean time to recovery from neutropenia nadir ranged from 6.6-8.2 days in patients receiving a total of 10 mcg/kg/day compared to 10.4-10.6 days in patients treated with 5 mcg/kg/day. Side effects were ephemeral eosinophilia. The dosage of 10 mcg/kg/day appears to be a better dosage for pediatric patients with a slight advantage in the combined twice a day schedule (6.6 days).

Single and double autotransplants for relapsing/refractory Hodgkin's disease: results of two consecutive trials.

To evaluate a strategy of one cycle of dose-intensive chemotherapy for patients with Hodgkin's disease in sensitive relapse and two cycles for those with refractory disease, 122 patients received dose-intensive chemotherapy followed by autotransplant in two consecutive studies. Patients with refractory disease were offered a second transplant with different conditioning in the absence of progression or excessive toxicity. CR was present after treatment in 46% while 16% died in the peritransplant period. Of 41 patients with primary refractory disease and 42 with refractory relapse, 24 and 21 respectively received a second cycle. Of these 45 refractory patients, 12 were in CR and 11 in PR after the first cycle and 10 of these 11 in PR achieved a durable CR with the second transplant. The CR rate is 37% in patients with refractory relapse and 19% in those with primary refractory disease. At a median follow-up of 4 years, median survival is 45 months. Progression-free survival of the refractory patients who could receive a second cycle was similar to that of patients with sensitive disease. A sequential transplant strategy is feasible. A subgroup of patients with refractory disease can achieve long-term survival after sequential BMT.

Sporadic acute lymphocytic leukemia arising in a patient with neurofibromatosis and xanthogranulomatosis.

We present a case report of a child who developed acute lymphoblastic leukemia, neurofibromatosis, optic glioma, and xanthogranulomatosis. This complex is unusual, not previously described, and appears to be a coincidence of different diseases. The importance of this case is that it may offer a clue to the genetic base of neurofibrosis syndromes including leukemic associations.

Hemin toxicity in a human epithelioid sarcoma cell line.

The major cytotoxic component of hemin was identified as metal free protoporphyrin IX in an epithelioid sarcoma cell line (VA-ES-BJ) and a glioblastoma cell line (U-373 MG) by exposing the cell lines to the iron chelator deferoxamine, tin-protoporphyrin IX, and protoporphyrin IX. The contribution of lipid peroxidation and free radical generation to toxicity was examined using DL-buthionine-[S,R]-sulfoximine (BSO), and 21-aminosteroid (lazaroid, U74500A). Hemin caused significantly greater toxicity in VA-ES-BJ than in U-373 MG. While exogenous PpIX was more toxic than hemin in both cell lines, this toxicity was not due to iron depletion following intracellular heme formation since ferric citrate did not reverse PpIX toxicity. Pre-treatment with BSO enhanced hemin toxicity in the VA-ES-BJ cell line but not in U-373 MG, suggesting different modes of toxicity in the two cell lines. Exposure to lazaroid protected only VA-ES-BJ from protoporphyrin-induced toxicity implicating a specific sensitivity to lipid peroxidation and/or free radical generation by this cell line. These characteristics of the VA-ES-BJ cell line distinguish it from the glioblastoma and emphasize its utility for exploring cytotoxic effects of hemin and its precursors.

D-CECaT as preoperative chemotherapy for unresectable neuroblastoma in children over one year of age.

The role of intensive pre- and postoperative chemotherapy in unresectable nonmetastatic neuroblastoma is still controversial. A preoperative regimen that included deferoxamine, cyclophosphamide, etoposide, carboplatin and thiotepa (D-CECaT) was evaluated in 10 children over one year of age at diagnosis, and this was followed by surgery and postoperative chemotherapy. After four courses of D-CECaT, the response rate was 9/10 with 3 complete responses, 6 partial responses and 1 minor response. Severe but transitory myelosuppression was the major toxic effect. Complete remission by combined D-CECaT chemotherapy and surgery was obtained in 9/10 patients, while 1 case achieved complete remission only with postoperative chemotherapy. All children are disease-free with a median follow-up of 30.5 months (range: 1+ to 50+). This intensive treatment was effective in both standard- and high-risk unresectable NB. However, whether a less intensive approach and fewer courses can also give similar results in standard-risk cases warrants further study.

Camptothecin cytotoxic effects in vitro: dependency on exposure duration and dose.

A survey of in vitro cytotoxic effects of camptothecin in human epitheliod sarcoma, colon, breast and ovarian carcinomas, glioblastoma, and neuroblastoma (PNET) cell lines, was done. We chose the MTT assay to measure survival and observed that 24 h exposures to camptothecin caused consistently greater toxicity than 1 h exposures. The LD50 for camptothecin was in the 12.5-25 ng/ml range. There was a 10-fold range of growth rates measured by OD after 5 days exposure and varied expression of MDR1 in these cell lines--none of which could be correlated with tumor sensitivity to drug. The most sensitive cell lines were colon and glioblastoma, and the most resistance were ovarian, breast and epithelioid sarcoma.

Deferoxamine followed by cyclophosphamide, etoposide, carboplatin, thiotepa, induction regimen in advanced neuroblastoma: preliminary results. Italian Neuroblastoma Cooperative Group.

Based upon phase I and II studies of deferoxamine alone and in combination with cytotoxic agents cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CECaT), we initiated a single arm multicentre trial in 1992 for advanced neuroblastoma. 57 of 65 patients who entered the trial were evaluable. Following 4 courses of the D-CECaT, almost all the patients underwent surgery. Toxicity was moderate and mainly reversible myelosuppression. The post-surgically defined responses in stage 3 high risk, stage 4 moderate risk and stage 4 high risk patients included 24 complete responses, 26 partial responses, and 3 minor responses, and 4 patients had progressive disease. These patients are being followed to determine the impact of this programme on their overall survival.

Va-es-bj - an epithelioid sarcoma cell-line.

A tumor cell line was derived and established from a bone marrow aspirate of a 41-year-old male who presented with motor symptoms of cord compression and an epidural tumor with osseous, pulmonary and hepatic metastases. The epidural tumor was an epithelioid sarcoma staining positive for cytokeratin markers of epithelial differentiation AE-1 and AE-3. This cell line was composed of lowly refractile, multinucleated giant or mononuclear round or elongated cells with monopolar or bipolar short processes. When permitted to grow beyond confluence these cells clumped up and formed mounds progressing to free floating spheroids. The cells were characterized by chromosomal triploidy with marker chromosomes, rapid growth in nude mice and secretion of immunoreactive and biologically functional granulocyte macrophage colony stimulating factor. This epithelioid sarcoma cell line stained positive for AE-1, AE-3, vimentin, epithelial membrane antigen and was designated VA-ES-BJ.

Phase I-II trial of high dose Ara-C, carboplatinum, etoposide and steroids in patients with refractory or relapsed lymphomas.

Thirty-three patients, including 20 with non-Hodgkin's lymphomas (NHL) and 13 patients with Hodgkin's disease, were treated with a combination of high dose Ara-C 3 gm/m2 over 3 h, carboplatinum 300 mg/m2 over 15 min, etoposide 300-750 mg/m2 continuous infusion over 24 h and solumedrol 1250 mg. Probantheline was given prophylactically. The etoposide dose was escalated from 300 mg/m2 to 600 mg/m2 to 750 mg/m2. The median age was 44 years (range 28-63). Median Karnofsky performance status was 80 (range 60-100). Patients treated included: primary refractory six, first relapse 14, > first relapse seven, and resistant relapse six. Responses were seen in 11 patients (33%; 95% CI 17-49). Of note, no responses were seen in ten patients receiving < 750 mg/m2 etoposide vs. 11/23 patients receiving 750 mg/m2 etoposide (p = 0.013). Responses were seen in patients who were refractory to previous chemotherapy and occurred in all sites. Toxicity was tolerable with most patients not requiring hospitalization following chemotherapy. Two patients died on study: one of Guillain-Barre syndrome and the other of a sudden death. Severe mucositis was not observed. High dose etoposide based salvage chemotherapy offers a greater probability of response than lower dose etoposide. This regimen is well tolerated and can be administered with relatively brief hospitalization.

Incidence and treatment of tumor lysis syndrome in patients with acute leukemia.

Tumor lysis syndrome (TLS) is a complication associated with electrolyte abnormalities that is observed in patients with acute leukemia who are receiving intense doses of chemotherapy. Forty-one patients with acute leukemia were treated with high-dose combination chemotherapy and were evaluated for TLS. A grading system developed for the evaluation of these patients was applied. Grade I tumor lysis was observed in 22 patients, grade II TLS in 2 patients and grade III in 1 patient. All patients were treated with intravenous fluids, mannitol, allopurinol and in some patients, aluminum-based antacids. Treatment for TLS prior to intensive chemotherapy reduced morbidity and mortality associated with high-dose chemotherapy for acute leukemias.

Reversion of p-glycoprotein mediated multidrug-resistance to vincristine and adriamycin by psc-833, a cyclosporine derivative in human neuroblastoma cell-lines.

We evaluated the effect of PSC-833 a cyclosporine derivative for its MDR1 reversing activity on vincristine and adriamycin resistance in human neuroblastoma cell lines. The cell lines have high, intermittent and low MDR1 expression. Resistance to vincristine and adriamycin was inverse to the degree of MDR1 expression. Resistance could be lowered with longer exposures to drug in all three cell lines. PSC-833 was able to reverse resistance in the SK-N-FI cells to a greater degree than in the two cell lines with lower expression of MDR1. These data suggest that MDR1 may play a protective role against vincristine and adriamycin in human neuroblastoma cells, and PSC-833 can reverse this protection. The duration of exposure to drug alters the response and the effect of PSC-833 on reversal of resistance. Where MDR1 is minimally expressed, PSC-833 is ineffective. Overall activity of vincristine and its modulation by PSC-833 were more consistent than that of adriamycin. Multiple drug resistance mechanisms are complex and vary among different cell lines when challenged with MDR1 drugs and reversing agents.

delta-Aminolevulinic acid effects on neuronal and glial tumor cell lines.

Acute intermittent porphyria (AIP) or precursor syndrome is a well described neuropathic clinical entity with incompletely known etiology. The most prominent biological abnormalities associated with this syndrome are elevations in serum and hepatic delta-aminolevulinic acid (ALA) and porphobilinogen (PBG). We determined the impact of ALA and PBG on human neuroblastoma and glioblastoma tumor cell survival as measured by the MTT assay. ALA proved to be cytotoxic in neuroblastoma cells, while PBG lacked cytotoxic effects. This cytotoxic effect of ALA could be enhanced by deferoxamine and diminished by heme, presumably through modulation of ALA synthesis. In conclusion, ALA excess may prove to be associated with the development of neuropathy in AIP.

Peripheral blood stem cells in children with solid tumors. Part II. Immunocytologic detection of tumor cells in bone marrow and peripheral blood stem cell harvests.

Simultaneous comparative assessment of tumor contamination in bone marrow aspirates and peripheral blood stem cell collections using immunocytochemical techniques was done in 6 children with neuroectodermal tumors. In 3 neuroblastoma patients tumor cells were detected in 5 of 16 marrow samples but not in peripheral blood stem cells. Clonogenic tumor cell reinfusion in the autologous support setting may be avoided in neuroblastoma patients by using peripheral blood stem cells.

Peripheral blood stem cells in children with solid tumors. Part I. Feasibility and application.

A method for collecting peripheral blood mononuclear cells following mobilizing chemotherapy in pediatric patients is described. The critical elements of the method included temporary heparinization of the patient to reduce citrate overload, and limiting extracorporeal circulation to 15% of the patient's blood volume using packed red blood cells and albumin. A median of 0.9 x 10(8) mononuclear cells/kg per collection were harvested in 40 collections from eight patients with only one episode of fever and chills. Peripheral blood stem cells were reinfused into six of these patients with refractory/recurrent pediatric tumors after intensive chemotherapy. Bone marrow reconstitution followed with a mean of 30 days (19-38) for absolute neutrophils and 48 days (32-275+) for platelets. Previous chemotherapy did not appear to affect peripheral blood stem cell efficacy in reconstituting chemotherapy-ablated bone marrow.

Cerebral ventricular fluid distribution of subcutaneous granulocyte-macrophage colony stimulating factor.

After treating a child suffering from disseminated primitive neuroectodermal tumor and hydrocephalus with bilateral ventriculostomies, we administered intravenous high dose thiotepa followed by a single subcutaneous dose of GM-CSF 24 hours later. The appearance and clearance of GM-CSF were measured from both ventricles, one of which was surrounded by tumor. Peak levels of GM-CSF were recorded simultaneously in both ventricles 11 hours after injection. Complete clearance from injection required 15 hours and 31 hours for the tumor-free right ventricle and the tumor-involved left ventricular wall, respectively. Tumor response was ephemeral and limited to ventricular fluid WBC, protein and LDH decreases. Microglia were detected; however, there was no evidence of anti-tumor activity in biopsied tumor tissue. Tumored regions of the brain may have perturbation of GM-CSF distribution and clearance which may contribute to the lack of microglial activity.

A saturation threshold for taxol cytotoxicity in human glial and neuroblastoma cells.

The cytotoxic effects of taxol at concentrations of 0.001-1.0 microgram/ml were determined in two human glioblastoma multiforme, two neuroblastoma and two primitive neuroectodermal tumor cell lines. The neuroectodermal cell lines were established from previously treated patients, while the glioblastomas were from untreated patients. At exposure durations of 1, 4 and 24 h there was an inverse taxol concentration-survival relationship for all six cell lines as measured by the MTT method. Significant differences in sensitivity to taxol among these cell lines were observed; the most resistant cell line SK-N-FI is characterized by very high levels of MDR1 expression and the most sensitive SK-N-AS by very low levels. An additional level of complexity concerns a saturation threshold for taxol-induced cytotoxic effects which when reached precludes additional effects of prolonged or additional exposure. Tumors of the brain and peripheral nervous system appear to be sensitive to taxol. However, dosage necessary to maximize cytocidal effects in tumors requires knowledge of at least the range of each tumors constitutive sensitivity to taxol and a way to optimize drug delivery.

Propantheline prevention of mucositis from etoposide.

Mucosal toxicity is dose limiting for etoposide. This may be related to the direct effects of etoposide on the mucosa. Twelve patients receiving etoposide 1800 mg/m2 as part of a myeloablative pre-transplant regimen were randomized to receive propantheline 30 mg or placebo orally every 6 h for six doses. Mucositis was less frequent (2 of 6 vs 5 of 6) and less severe (p = 0.05) in the propantheline arm. There were no differences in tumor response or survival between the two groups. Propantheline is an anticholinergic that causes xerostomia by decreasing salivation. Propantheline may reduce the salivary excretion of etoposide and could reduce its toxic effects on the mucosa. Propantheline is effective in reducing the incidence and severity of mucositis in patients receiving high-dose etoposide.

D-CECaT: a breakthrough for patients with neuroblastoma.

In view of the high relapse rate following chemotherapy for patients with advanced neuroblastoma (NB) and primitive neuroectodermal tumors (PNET), we designed a novel chemotherapy program which incorporated the iron chelator deferoxamine. The purpose of the deferoxamine was to sensitize the cells to standard chemotherapy. The D-CECaT regimen contained (in mg/m2): deferoxamine 4500 during days 1-5; cyclophosphamide 600 mg over days 6 and 7; etoposide 300 mg over days 7 and 8; carboplatin 100 mg over days 7 and 8; and thiotepa 30 mg over days 6-8. Between October 1989 and May 1992 we entered 23 advanced NB and two PNET patients. Sepsis occurred in four courses, nausea and vomiting in 30 courses, and 50 courses required blood and platelets. Responses observed in previously untreated patients with stage III NB: six out of six CR (17+ to 41+ months), with stage IV NB, nine out of 11 CR (14+ to 28+ months), two out of 11 VGPR (22+ months), with stage IV PNET two out of two CR (1+ to 35+ months). With previously treated and failed stage IV NG, two out of six VGPR for 19+ and 20 months, and four out of six PR 1, 8, 9 and 11 months. Median survival for 19 new patients was 22+ months (6 to 41+ months; two patients in CR died at 7 months during adjuvant autologous marrow transplant). In conclusion, D-CECaT is an effective initial cytoreductive regimen for advanced stage NB/PNET patients. Additional patients and studies are required to determine its use as an alternative to autologous bone marrow transplantation.

Sequential addition of deferoxamine and hemin inhibits glioma tumor-cell growth.

The toxicity of deferoxamine, a potent iron chelator may be antagonized by hemin a potential iron source. Using the MTT assay we explored the effects of different concentrations and schedules of deferoxamine and hemin or deferoxamine and iron free tin protoporphyrin (SnPP) in two neuroblastoma (VA-N-BR, SK-N-AS), and two glioblastoma multiforme (VA-MG-SL, and U-373 MG) cell lines. In these cell lines, survival after exposure to 10 mug/ml deferoxamine for three days ranged from 28% to 59%. Incubation with hemin alone, had variable effects on growth depending on the cell line. Concomitant exposure to equimolar concentrations of deferoxamine and hemin resulted in the reversal of deferoxamine induced toxicity. Surprisingly, in the glioblastoma multiforme cell lines sequential exposure to deferoxamine and then hemin resulted in additional toxic effects rather than abrogation of deferoxamine toxicity. Sequential exposure of all cell lines to deferoxamine, hemin, and the chemotherapeutic agent thiotepa resulted in enhanced toxicity over any drug used alone. Exposure of the cells to deferoxamine and SnPP or deferoxamine and FeCl3 did not result in increased toxicity. These results implicate iron as the toxic element but indicate that the iron is only toxic when presented to the cell bound to protoporphyrin, such as found in hemin.