Inhibition of human malignant neuroblastoma cell DNA synthesis by lipoxygenase metabolites of arachidonic acid.

In vivo studies have shown that inhibitors of cyclooxygenase metabolism of arachidonic acid may diminish growth and metastasis of certain tumors. Because cyclooxygenase inhibition may increase the production of lipoxygenase products of arachidonic acid metabolism, we have investigated the effect of two such products, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on tumor cell proliferation in vitro. When neuroblastoma cells (SK-N-SH) in culture were treated with 12-HETE for 18 hr, incorporation of [3H]thymidine was inhibited up to 64% at concentrations from 20 to 50 microM. Under the same conditions, 15-HETE resulted in inhibition of up to 46%, while arachidonic acid had no apparent effect. When evaluated in the presence of serum, 12-HETE at a concentration of 120 microM produced a 20.6 +/- 2.8% (S.E.) inhibition of the increase in total DNA content over 48 hr, while 15-HETE at this concentration produced a 16.5 +/- 5.3% inhibition. We conclude that 12-HETE, the product of platelet lipoxygenase, and 15-HETE, a product of neutrophil and lymphocyte lipoxygenases, can inhibit human neuroblastoma cell growth in vitro and may play a role in the effect of cyclooxygenase inhibitors on tumor growth in vivo.

Treatment of recurrent and refractory pediatric solid tumors with high-dose busulfan and cyclophosphamide followed by autologous bone marrow rescue.

PURPOSE: The purpose of this study was to determine the toxicities of and responses to high-dose busulfan and cyclophosphamide with autologous bone marrow transplant (ABMT) in patients with recurrent or refractory pediatric solid tumors. PATIENTS AND METHODS: We treated 18 patients (ages, 2 to 38 years; median, 14) who had tumors that were resistant to conventional chemotherapy and radiotherapy with busulfan 16 mg/kg and cyclophosphamide 200 mg/kg. Seventeen patients received bone marrow purged with 4-hydroperoxycyclophosphamide; one received unpurged marrow. RESULTS: Despite extensive prior treatment, including radiotherapy in 16 patients, toxicity generally was acceptable. For seven patients with measurable disease, there were three partial responses of 2, 10, and 20 months' duration, three patients with stable disease (SD), and one early, toxic death. Of the 11 patients with no measurable disease at the time of transplantation, one patient with osteosarcoma continues in remission at 57+ months and one third of the patients survived for at least 16 months. Mucositis was the predominant nonhematopoietic toxicity. CONCLUSION: Although the high-dose busulfan and cyclophosphamide combination showed modest activity, changes in the preparative regimen should be considered to improve the response rate in refractory tumors.

A Phase I trial of bryostatin-1 in children with refractory solid tumors: a Pediatric Oncology Group study.

Bryostatin-1, a macrocyclic lactone, appears to elicit a wide range of biological responses including modulation of protein kinase C (PKC). PKC, one of the major elements in the signal transduction pathway, is involved in the regulation of cell growth, differentiation, gene expression, and tumor promotion. Because of the potential for a unique mechanism of interaction with tumorgenesis, a Phase I trial of bryostatin-1 was performed in children with solid tumors to: (a) establish the dose-limiting toxicity (DLT) and maximum-tolerated dose (MTD); (b) establish the pharmacokinetic profile in children; and (c) document any evidence of antitumor activity. A 1-h infusion of bryostatin-1 in a PET formulation (60% polyethylene glycol 400, 30% ethanol, and 10% Tween 80) was administered weekly for 3 weeks to 22 children (age range, 2-21 years) with malignant solid tumors refractory to conventional therapy. Doses ranged from 20 to 57 microg/m2/ dose. Pharmacokinetics were performed in at least three patients per dose level. The first course was used to determine the DLT and MTD. Twenty-two patients on five dose levels were evaluable for toxicities. At the 57 microg/m2/dose level dose-limiting myalgia (grade 3) was observed in three patients; two of those patients also experienced photophobia or eye pain, and one experienced headache. Symptoms occurred in all patients within 24-72 h after the second dose of bryostatin-1 with resolution within 1 week of onset. Other observed toxicities (grades 1 and 2) included elevation in liver transaminases, thrombocytopenia, fever, and flu-like symptoms. The bryostatin-1 infusion was typically well tolerated. Although stable disease was noted in several patients, no complete or partial responses were observed. The recommended Phase II dose of bryostatin-1 administered as a 1-h infusion weekly for 3 of every 4 weeks to children with solid tumors is 44 microg/m2/dose. Myalgia, photophobia, or eye pain, as well as headache, were found to be dose limiting.

T6 monoclonal antibody reacts with blasts from cases of common antigen acute lymphocytic leukemia.

The expression of the T6 antigen on malignant lymphoid cells has been considered strong evidence in support of T-cell lineage and thymic stage of differentiation of the neoplastic cells. Thus, the authors have used the T6 monoclonal antibody for the last three years in the immunophenotyping of blasts from 60 consecutive cases of acute lymphocytic leukemia (ALL) and 8 cases of T-cell lymphoma. Blasts from 12 of 46 (26%) cases of common type ALL, 4 of 7 (57%) cases of T-cell ALL, 2 of 3 (66%) cases of lymphoblastic lymphoma, and 1 of 5 (20%) cases of peripheral (postthymic) T-cell lymphoma were positive for the T6 antigen. The authors conclude that the expression of T6 antigen on malignant lymphoid cells may not always indicate T-cell lineage.

Determination of the cytoprotective agent WR-2721 (Amifostine, Ethyol) and its metabolites in human blood using monobromobimane fluorescent labeling and high-performance liquid chromatography.

PURPOSE: WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] is a chemoprotective agent that is currently in pediatric clinical trials. It is a prodrug that is dephosphorylated by alkaline phosphatase to the active free thiol form, WR-1065 [S-2-(3-aminopropylamino)ethanethiol]. It is likely that adequate and sustained cellular levels of the drug are necessary for optimum cytoprotection. To date, a method to measure both plasma and cellular levels of WR-2721 and its metabolites in clinical samples has not been available. METHODS: In the study reported here the monobromobimane (mBBr) fluorescent labeling method was used to measure these levels when drug was added in vitro to blood samples from normal volunteers. In addition, we present pharmacokinetic data from a pediatric patient receiving WR-2721 (825 mg/m2 x 2). RESULTS: The results can be summarized as follows: (1) WR-2721 was detected in the patient's plasma with a half-life of about 10 min; (2) the WR-1065 concentration in the blood cellular fraction was similar to that of plasma; (3) both WR-1065 and WR-SS-low molecular weight (WR-SS-LMW) metabolites disappeared from plasma and the cellular fraction by 3.6 h after WR-2721 infusion; (4) a large proportion of WR-1065 was oxidized in plasma to WR-SS protein and WR-SS-LMW; (5) a large proportion of WR-1065 in the cellular fraction was oxidized to WR-SS-protein; (6) the WR-SS-LMW concentration in the cellular fraction was low; and (7) saturation of plasma and cellular protein binding sites was possible. CONCLUSIONS: The pharmacokinetic data that were generated with this technique could guide clinical trials using WR-2721.

WR-2721 (amifostine) infusion in patients with Ewing's sarcoma receiving ifosfamide and cyclophosphamide with mesna: drug and thiol levels in plasma and blood cells, a Pediatric Oncology Group study.

PURPOSE: Previous WR-2721 human pharmacokinetic studies were limited to plasma levels in patients receiving platinum-based compounds, and none includes the effects of WR-2721 on endogenous thiols. In the present study (Pediatric Oncology Group study no. 9457), we measured the levels of WR-2721, its active metabolites, as well as cysteine and glutathione in whole blood, plasma, and blood cells in patients receiving high-dose alkylating agents with mesna. METHODS: WR-2721 was administered (15 min intravenous infusion of 825 mg/m(2) per dose x2) to five patients with metastatic Ewing's sarcoma receiving ifosfamide and cyclophosphamide with mesna. Intracellular and extracellular blood thiols were labeled with monobromobimane (mBBr) at the time of collection, and the low molecular weight (LMW) thiols were subsequently separated by HPLC and detected by fluorescence. RESULTS: The active metabolite of the drug, WR-1065, peaked at 100 microM in plasma and blood cells at the end of WR-2721 infusion and decayed with a rapid initial half-life. Detectable levels of WR-1065 and its LMW disulfides were present in plasma and blood cells at approximately 1 h after the WR-2721 infusion. By the end of the first WR-2721 infusion (prior to mesna infusion), the mean cysteine level more than doubled and the mean Cys-SS-LMW (cystine and the mixed LMW disulfides) level decreased by approximately 50% in both plasma and blood cells. In four of five patients, reduced glutathione levels in blood cells increased by the end of the first WR-2721 infusions, the average increment being approximately 36%. CONCLUSIONS: (1) WR-1065 is rapidly formed from WR-2721 and equilibrates between plasma and blood cells; (2) WR-1065 decays in plasma and blood cells with similar rapid initial half-lives of approximately 16 min; (3) WR-2721 treatment increases cysteine in plasma and blood cells, an effect similar to that of mesna; (4) WR-2721 treatment appears to increase glutathione levels in blood cells; (5) Mesna does not have a substantial effect on the fate of WR-2721 in patients.

Demyelinating and gliotic cerebellar lesions in Langerhans cell histiocytosis.

PURPOSE: To describe the involvement of the cerebellum by a gliotic and demyelinating process in Langerhans cell histiocytosis. METHODS: A retrospective analysis of all (N = 30) cases of Langerhans cell histiocytosis followed at our institution since 1975 yielded four patients with CT and/or MR evidence of cerebellar abnormalities. RESULTS: Four patients manifested strikingly similar findings of symmetric nonenhancing hypodensities in the dentate nuclei region of the cerebellum, which were hypointense on short-repetition-time/short-echo-time MR and hyperintense on long-repetition-time/long-echo-time MR. Biopsy in one patient yielded areas of demyelination, cell loss, and gliosis without histiocytic infiltration. CONCLUSION: Langerhans cell histiocytosis involves the cerebellum in a specific and poorly understood manner. Lesions on imaging may precede clinical findings by years. Lesions may occur in patients who have never experienced radiation therapy and may act as a marker for eventual central nervous system deterioration.

Intravenous immunoglobulin in the treatment of aplastic anemia.

Aplastic anemia is characterized by reduced production of mature erythrocytes, granulocytes, and platelets from marrow stem cells leading to peripheral blood pancytopenia. In many cases, it appears that there is an aberrant immune response suppressing stem cell differentiation and renewal, leading to bone marrow aplasia and the observed peripheral blood pancytopenia. This report describes a patient with aplastic anemia unresponsive to antithymocyte globulin and high-dose steroid therapy who did respond to intravenous immunoglobulin and now has normal peripheral blood counts.

Endothelial cell proliferation may be mediated via the production of endogenous lipoxygenase metabolites.

Endogenous regulators of endothelial cell proliferation have not been clearly defined. We investigated whether the cyclooxygenase and/or lipoxygenase metabolites are involved in this process, and report that lipoxygenase products can modulate endothelial cell growth. Nordihydroguaiaretic acid--a lipoxygenase inhibitor, inhibited endothelial cell proliferation as well as DNA synthesis. 5,8,11,14-Eicosatetraynoic acid--an inhibitor of both lipoxygenase and cyclooxygenase also inhibited endothelial cell DNA synthesis, while indomethacin--a selective cyclooxygenase inhibitor did not affect cell proliferation or DNA synthesis. While arachidonic acid stimulated DNA synthesis, this effect was completely abolished by nordihydroguaiaretic acid. These results demonstrate that products of the lipoxygenase pathway can affect endothelial cell proliferation.

Controversy in the management of optic nerve glioma.

The records of 16 patients with optic nerve glioma treated between 1961 and 1984 were reviewed. All patients except two had extension of tumor beyond the chiasm to the hypothalamus, adjacent brain and/or along the posterior optic tract. Eleven of 16 cases were biopsy-proven, two patients had craniotomy and visual inspection but no biopsy was performed, and in two cases the biopsy was not diagnostic. Fourteen patients received radiation therapy, usually consisting of 50 Gy in 5 weeks (range 40-56 Gy), one patient was treated surgically and one with chemotherapy. With a follow-up of 1 to 20 years, 7 of the 14 patients irradiated are alive, three patients are dead of disease at 3, 6 and 9 years post-treatment, three were lost to follow-up at 1, 8, and 8 years, and one is dead of intercurrent disease at 5.5 years. Overall vision was improved in five patients and stable in seven following treatment. In two patients, vision could not be evaluated because of young age at presentation. Four patients had recurrences. One was retreated with 30 Gy in 3 weeks and shows no evidence of disease at 20 years. The three other patients died of their disease. There is controversy over the best treatment for these patients. Based on these results and a review of the literature, the authors recommendation is to irradiate tumors with extension beyond the chiasm at the time of presentation rather than waiting for increasing symptoms because function that is lost may not always be recovered. Chemotherapy needs to be further investigated but holds promise, especially for the younger children.

Prognostic significance of mean cell volume in non-Hodgkin's lymphomas.

Cell volumes of neoplastic lymphocytes collected from lymph nodes of 53 patients with non-Hodgkin's lymphoma were compared to lymphocytes from 18 patients with reactive hyperplasia. The mean cell volume (MCV) and the modal volume (MV) of neoplastic lymphocytes were larger than the MCV and MV of lymphocytes from reactive hyperplasia. The cell volumes of neoplastic lymphocytes from patients with non-Hodgkin's lymphoma were more heterogeneous within and among cases than observed in volumes from lymphocytes of patients with reactive hyperplasia. The cell volumes of neoplastic lymphocytes corresponded to subgroups within the Rappaport Classification and the Working Formulation. Cell volumes of neoplastic cells from low-grade lymphomas were smaller than intermediate grade lymphomas which in turn were smaller than high-grade lymphomas. When cases of NHL were placed into three subtypes based on the MCV, large cell lymphomas had a significantly shorter survival then small and intermediate cell lymphomas at 12 months. However, a stepwise multiple regression analysis failed to demonstrate any independent value of cell volume in the prediction of survival.

Significance of aberrant immunophenotypes in childhood acute lymphoid leukemia.

Leukemic cells from 51 pediatric patients (younger than 18 years) diagnosed with acute lymphoid leukemia by standard morphologic and cytochemical methods were subjected to flow cytometric studies using a panel of monoclonal antibodies against T-cell (CD1, 2, 3, 4, 5, 7, 8), B-cell (CD10, 19, 20, 21), myeloid (CD13, 14, 15, 33), and HLA-DR antigens. Cases of "conventional" acute lymphoid leukemia (leukemic cells with a normal configuration of B-cell or T-cell differentiation antigens) were observed in 26 of 51 (51%) cases, whereas cases of "aberrant" acute lymphoid leukemia (cells with abnormal patterns of B-cell or T-cell antigens or with concomitant myeloid antigens) were noticed in 25 (49%) cases. Myeloid antigen-positive acute lymphoid leukemia was observed in the leukemic cells of eight (16%) individuals. No significant differences were observed between conventional and aberrant ALL in the distribution of sex, age, leukocyte count, hemoglobin concentration, platelet count, blast count, French-American-British (FAB) type, lymphadenopathy, organomegaly, rate or duration of remission, or survival. When only myeloid antigen-positive cases were compared with myeloid antigen negative-cases, no significant correlations were observed except for duration of first remission (myeloid antigen positive, 26+ +/- 22 months; myeloid antigen negative, 40+ +/- 18 months; P less than 0.001), and duration of survival (myeloid antigen positive, 27+ +/- 24 months; myeloid antigen negative, 62+ +/- 17 months; P = 0.001). These data suggest that pediatric patients with ALL blasts possessing myeloid antigens may represent a high-risk group for length of remission and survival.

Blood thiols following amifostine and mesna infusions, a pediatric oncology group study.

The Pediatric Oncology Group study for metastatic Ewing's sarcoma used amifostine and mesna with the alkylating agents. To determine the fate of combined drug thiols, we measured thiol levels in plasma, red blood cells (RBC), and peripheral blood mononuclear cells (PBMC) of four patients. We also conducted analogous measurements on two patients who received mesna alone and a volunteer's blood following in vitro treatment. Thiols were labeled with monobromobimane, separated on high-pressure liquid chromatography, and detected by fluorescence. Incubation of a volunteer's blood with mesna, WR-1065, or both revealed that cellular uptake of total reducible drug was approximately 10% of plasma level for mesna but approximately 60% for WR-1065. Cellular drugs were mainly the thiol form, whereas half of the plasma drugs were disulfides. Combined incubation with both thiols did not change the extent or form of uptake. WR-1065 and mesna prevented glutathione depletion by 4-hydroperoxycyclophosphamide. Results from patients were similar. WR-1065 and mesna appeared in the cells by the end of the drug infusions, although WR-1065 uptake was more efficient than mesna. The concentration-time profiles of mesna in RBC paralleled those in plasma. Amifostine administration during mesna infusion caused transient increase in mesna levels. Both agents increased blood cysteine and decreased total reducible cysteine. Mesna alone and mesna plus amifostine prevented cellular glutathione depletion. In conclusion, mesna is imported by RBC and PBMC, but less efficiently than WR-1065. When present at equal levels, these thiols do not influence each other's uptake. Adequate dosing of either drug is necessary for protecting the cells from toxic effects of alkylating agents.