Moshe Talpaz

A phase I trial of intravenously-administered recombinant tumor necrosis factor-alpha in cancer patients.

We report a phase I clinical investigation of 30-minute and four-hour intravenous (IV) infusions of recombinant tumor necrosis factor (rTNF)-alpha. Thirty-nine patients with disseminated cancer received escalating doses of rTNF-alpha for five consecutive days every 2 weeks for a total duration of 8 weeks. Dose escalations followed a modified Fibonacci scale with a minimum of four patients entered at each dose level: 5, 10, 25, 50, 75, 100, 150, 200, and 250 micrograms/m2/d. Toxicities consisted mainly of constitutional symptoms including fever, chills, headache, and fatigue, increasing in severity with dose escalation. No significant differences in dose-limiting toxicities were seen between the two rates of IV infusion. The maximum tolerated dose (MTD) was 200 micrograms/m2 with dose limiting toxicity being constitutional symptoms and hypotension. Hematologic changes included median decrease in both granulocyte and platelet counts of 38% and 41%, respectively (range, 16% to 85%), although clinically significant granulocytopenia and thrombocytopenia were not observed. Hematological parameters returned to baseline within 72 hours after rTNF-alpha was stopped. rTNF-alpha induced changes in lipid metabolism were manifested by median fasting triglyceride elevations above baseline (median, 103 micrograms/dL) of 157% (range, 16% to 389%) after five days of therapy with doses greater than 75 micrograms/m2, associated with a median increase in very-low-density lipoprotein (VLDL) of 80%. Serum rTNF peak levels exceeding 10 ng/mL were observed 30 minutes following rTNF-alpha infusions at MTD dose. Twelve of 34 patients had no change in their evaluable disease for a median duration of 18 weeks (range, 8 to 30 weeks), and 22 patients showed progressive disease. This study forms the framework for phase II trials of IV administered rTNF-alpha.

The human cellular abl gene product in the chronic myelogenous leukemia cell line K562 has an associated tyrosine protein kinase activity

Three antisera against the mouse v-abl gene product were used to identify two potential human c-abl gene products in the chronic myelogenous leukemia cell line K562. Two antipeptide sera were generated in rabbits using the predicted amino acid sequence of the mouse v-abl gene product. One antiserum was made against a polypeptide overlapping the in vivo tyrosine phosphorylation site of murine P120gag-abl and what is believed to be a homologous tyrosine phosphorylation site of the predicted normal human c-abl gene product (v-abl 263-280). The second antipeptide serum, abl 389-403, was generated against a predicted hydrophilic peptide of the v-abl gene product. Immunoprecipitation from K562 cells metabolically labeled with [32P]orthophosphate by a mouse tumor regressor and abl 389-403 antipeptide sera detected two proteins of 190,000 and 240,000 Da. Both proteins were labeled primarily at serine and, to a much lesser extent, at tyrosine residues. Immune complex kinase assays using conditions that allow the tyrosine phosphorylation of P120gag-abl showed that in vitro phosphorylation of P190 and P240 occurs primarily at tyrosine residues. The detection of these enzymatically active human c-abl gene products is a rare observation which may be in part attributed to the c-abl gene translocation from chromosomes 9 to 22 occurring in the vast majority of chronic myelogenous leukemia patients.

Clinical correlates of elevated serum levels of interleukin-6 in patients with untreated Hodgkin's disease

BACKGROUND Interleukin-6 (IL-6) is a potent immunomodulatory cytokine that may have pathogenetic significance in several malignancies. In addition, high IL-6 levels have been associated with a poor prognosis in multiple myeloma, nonHodgkins lymphoma, ovarian cancer, and renal cancer, as well in advanced Hodgkins lymphoma. In this study, we analyzed IL-6 levels in newly diagnosed Hodgkins disease and determined clinical correlates of elevated levels. PATIENTS AND METHODS Using a sensitive enzyme-linked immunosorbent assay (lower limit of sensitivity = 0.35 pg/mL) we measured IL-6 levels in sera from 33 healthy controls and 65 untreated patients with Hodgkins disease. RESULTS Interleukin-6 levels in the Hodgkins patients (median 2.7 pg/mL; range < 0.35 to 38.4 pg/mL) were significantly higher than in the controls (median < 0.35 pg/mL; range < 0.35 to 1.87 pg/mL; P < 0.0001). Interleukin-6 levels were also higher in males (P = 0.03) and in patients with bulky disease (P = 0.026) or advanced Ann Arbor stage (P = 0.017). In addition, serum levels of IL-6 also showed direct linear correlations with the erythrocyte sedimentation rate (r = 0.64, P = 0.0007), platelet count (r = 0.53, P < 0.0001), leukocyte count (r = 0.36, P = 0.003), and beta (2)-microglobulin level (r = 0.4, P = 0.0012); and an inverse linear correlation with serum albumin level (r = -0.43, P = 0.0003). In the 10 patients tested who had elevated serum IL-6 levels pretherapy and who achieved complete remission, serum IL-6 values decreased at the time of remission to the range found in healthy controls. CONCLUSIONS Our observations suggest that, in patients with Hodgkins disease, serum levels of IL-6 are frequently elevated at diagnosis, normalize during remission, and are associated with specific disease characteristics including several adverse prognostic features.

Elevated Plasma Thrombopoietic Activity in Patients With Metastatic Cancer-Related Thrombocytosis

BACKGROUND AND PURPOSE High platelet counts are occasionally seen in patients suffering from progressive malignant disorders. While granulocyte colony-stimulating factor (G-CSF) has been implicated in paraneoplastic leukemoid reactions, the stimulus for thrombocytosis is unknown. Our purpose in this study was to determine if plasma from cancer patients with thrombocytosis contains a factor or factors with thrombopoietic activity. METHODS We tested the effects of plasma obtained from 5 individuals with advanced tumors and high platelet counts and from 4 patients with advanced cancer and normal platelet counts on megakaryocytic differentiation of two megakaryoblastic cell lines (Dami and HEL). Differentiation was evaluated by assessing the expression of the platelet-specific cell-surface antigens CD41 (HUPL-mI) and glycoprotein IIb-IIIa using an immunocytochemical staining score. In addition, plasma samples from 7 of the 9 patients and from 5 additional cancer patients with thrombocytosis were assayed for the levels of interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and IL-1 beta protein using an enzyme-linked immunosorbent assay (ELISA). RESULTS Expression of platelet-specific cell-surface antigen was increased in HEL cells after exposure to plasma from all 5 of the cancer patients with thrombocytosis, and in Dami cells after exposure to plasma from 4 of the 5. Similar, but less significant, results were found when these cells were incubated with control combinations of recombinant GM-CSF plus IL-6 or of IL-3 plus IL-6. Platelet-specific cell-surface-antigen expression was not increased in HEL or Dami cells after exposure to the plasma from the 4 cancer patients with normal platelet counts or to normal control plasma. ELISA revealed elevated levels of IL-6 in the plasma from 4 patients with thrombocytosis (38, 40, 63, and 99 pg/mL). In addition, GM-CSF concentration was high in 3 of these 4 patients (33, 47, and 127 pg/mL), and the G-CSF level was elevated in 1 (543 pg/mL). IL-1 beta and IL-3 levels were undetectable. CONCLUSIONS Our data suggest that the thrombocytosis observed in individuals with advanced malignant disease is mediated by a humoral mechanism. Levels of IL-6, GM-CSF, and G-CSF are elevated in some of these patients, but the plasma concentrations are generally lower than those required for in vitro induction of megakaryocytic differentiation. Plasma from patients with paraneoplastic thrombocytosis may therefore contain thrombopoietins that have not yet been identified, and which might have clinical usefulness.

Molecular characteristics of chronic myelogenous leukemia in blast crisis

We have studied the expression of c-abl and c-myc in leukemic cells of patients in all clinical phases of chronic myelogenous leukemia. We demonstrate that an aberrant 8-Kb c-abl related transcript is present in the RNA of the leukemic cell from all patients with Ph+ CML and that the loss of both normal chromosome #9 is associated with the loss of the normal c-abl related transcripts. This represents direct evidence that the normal c-abl related transcripts derive from the normal c-abl gene locus on the normal chromosome #9, while the aberrant c-abl related transcript in Ph+ CML derives from the hybrid bcr-abl gene formed as a result of the t(9;22). We further demonstrate that trisomy 8 in some instances is associated with enhanced expression of the c-myc oncogene.

Immune restoration and/or augmentation of local graft versus host reaction by traditional chinese medicinal herbs

The in vitro restorative effect of aqueous extracts from two traditional Chinese medicinal herbs were studied in 19 cancer patients and in 15 normal healthy donors. Using the local graft versus host (GVH) reaction as a test assay for T‐cell function, the extract from astragalus membranaceus (10 μg/ml) induced a restored reaction in nine of ten patients with an increase in local GVH reaction from 18.2 ± 15.8 mm3 to 112.9 ± 94.2 mm3 (P < 0.01). The extract from ligustrum lucidum, likewise effected an immune restoration in nine of 13 cancer patients with an increase in local GVH reaction from 32.3 ± 36.1 mm3 to 118 ± 104.9 mm3 (P < 0.01). This degree of immune restoration appears to be complete as it equals the local GVH reaction observed among untreated mononuclear cells from normal healthy donors (82.8 ± 41.1 mm3, P > 0.1). These results suggest that both extracts of the traditional Chinese medicinal herbs contain potent immune stimulants which may provide the rational basis for their therapeutic use as biological response modifiers. Cancer 52:70‐73, 1983.

Recombinant interferon-alpha therapy of Philadelphia chromosome-negative myeloproliferative disorders with thrombocytosis.

PURPOSE The clinical course of patients with myeloproliferative disorders and excessive thrombocytosis may be complicated by serious hemorrhagic or thrombotic events. We have previously reported that interferon-alpha can control severe refractory thrombocytosis in patients with advanced chronic myelogenous leukemia. Therefore, we treated a group of thrombocythemic patients with Ph-negative myeloproliferative disorders, including polycythemia vera and essential thrombocythemia, with recombinant interferon-alpha (rIFN-alpha 2a). PATIENTS AND METHODS Eight patients with profound elevations in platelet counts received a median induction dose of 5.4 X 10(6) U/day (range, 5.0 to 10.0 X 10(6) U/day) of rIFN-alpha 2a administered intramuscularly or subcutaneously. RESULTS We observed a significant decline in platelet counts from a median baseline value of 1,929 X 10(9)/L (range, 960 to 2,960 X 10(9)/L) to a median posttreatment value of 431 X 10(9)/L (range, 71 to 1,150 X 10(9)/L) (p less than 0.01). Concomitantly, white blood cell counts declined from a median baseline value of 20.8 X 10(9)/L (range, 10.5 to 40.8 X 10(9)/L) to a median posttreatment value of 6.1 X 10(9)/L (range, 2.9 to 29.0 X 10(9)/L) (p less than 0.02). Correction of thrombocytosis was rapid, with a median of only eight days from the start of therapy to the achievement of a platelet count less than 1,000 X 10(9)/L. Six of eight patients have shown an ongoing response with a median follow-up period of 11 months (range, one to 30 months). There have been no bleeding or thrombotic events during the study. Side effects of rIFN-alpha 2a therapy consisted of fever and flu-like symptoms, with tachyphylaxis developing after one to two weeks of therapy. CONCLUSION Our observations suggest that alpha interferon may be a promising therapeutic agent for myeloproliferative disorders characterized by thrombocytosis.

Molecular analysis of chromosome 22 breakpoints in adult Philadelphia‐positive acute lymphoblastic leukaemia

Summary. The Philadelphia (Ph) translocation. t(9;22)(q34:q11). is found in the majority of patients with chronic myelogenous leukaemia (CML) as well as in approximately 20% of adult acute lymphoblastic leukaemia (ALL) patients. The chromosome 22 breakpoint in CML has been localized within a restricted 5.8 kb segment of DNA known as the breakpoint cluster region (bcr). To investigate the chromosome 22 breakpoint in ALL. we analysed five adult Ph‐positive ALL patients for bcr rearrangement. Rearrangement was detected within bcr in two patients. However, in one patient the break occurred 5’to the first exon of bcr and in two patients the bcr region was not involved. We conclude that the identical cytogenetic marker, t(9;22), may yield a different genomic configuration in ALL and CML.

Cytokines : interleukins and their receptors

Preface R. Kurzrock, M. Talpaz. Receptors for Hematopoietic Regulatory Cytokines: Overview of Structure and Function U. Gullberg, et al. Control of Thrombocytopoiesis: Current State of the Art R. Hoffman, M.W. Long. Interleukin-I and its Inhibitors: Implications for Disease Biology and Therapy Z. Estrov, et al. Interleukin- 2: Its Rationale and Role in the Treatment of Patients with Cancer J.T. Rubin. Interleukin-2 and its Receptor A. Lindemann, R. Mertelsmann. Structure and Function of Interleukin 4 and its Receptors R.K. Puri. Interleukin-5: Overview K. Takatsu. Interleukin-t: a Comprehensive Review M. Lotz. Interleukin-7 and Lymphopoiesis: Biological and Clinical Implications P.M. Appasamy. A Novel Leukocyte Chemotactic and Activating Cytokine, Interleukin 8 (IL-8) N. Mukaida, et al. Interleukin 9: Structural Characteristics and Biologic Properties J.-C. Renauld. Interleukin 10 (IL-10) D. Benjamin. Interleukin (IL)-11 and its Receptor: Biology and Potential Clinical Applications in Thrombocytopenic States Yu-Chung Yang. Interleukin-12: a Pivotal Regulator of Cell-Mediated Immunity M.K. Gately, M.J. Brunda. Interleukin-13: Characterization and Biologic Properties A.N.J. McKenzie, G. Zurawski. Index.

Studies of natural killer cell activity and antibody-dependent cell-mediated cytotoxicity among patients with acute leukemia in complete remission

SummaryLow natural killer (NK) cell activity against the K-562 leukemia cell line was observed in patients with acute leukemia in the early stages of remission, i.e., 2–4 months (11.3%±7.95% specific target cell lysis). This parameter was found to be normal among leukemia patients after a longer time in remission (19.53%±7.55%) when compared with healthy donors (18.46%±12.98%). A similar pattern of activity was observed in studies of antibody-dependent cell-mediated cytolysis (ADCC) to the CEM lymphoid tumor cell line in the same group (37.58%±12.4% vs. 51%±6.79% specific target cell lysis).ADCC to chicken red blood cells (CRBC) and to human red blood cells (HRBC) was not significantly different from that for healthy controls at either duration of remission.Nine patients relapsed over a follow-up period of 9 months. They were found to have slightly lower NK activity (14.4%±9.3%) and ADCC to CEM (41.4%±8.5%) than the patients who remained in remission (17.1%±6.8%; 48.7%±9.7%, respectively).These data indicate a lymphocyte deficit which may persist for some time after remission has been induced, and which may be due to the effect of leukemic cell burden or the effect of aggressive chemotherapy.