The molecular genetics of Philadelphia chromosome-positive leukemias.
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Acute Disease↗
Biomedical subjects
J U Gutterman
Publications and source records attributed to J U Gutterman.
Effect of differentiation-inducing agents on oncogene expression in a chronic myelogenous leukemia cell line.
K562 is a Philadelphia (Ph) chromosome-positive chronic myelogenous leukemia (CML) blast crisis cell line representing a pluripotent precursor cell. At the molecular level, K562 cells express high levels of the aberrant bcr-abl product, p210bcr-abl, believed to be critical to the pathogenesis of CML. The authors demonstrate that exposure of K562 cells to hemin causes a state of partial, reversible erythroid maturation, accompanied by a marked decrease in p210bcr-abl. The change in bcr-abl expression may be mediated at the translational level since steady state amounts and enzymatic activity of the bcr-abl protein are reduced whereas bcr-abl mRNA levels are unaltered. The decrease in p210bcr-abl phosphokinase enzymatic activity can be detected within 2 hours after addition of hemin to the culture media, indicating that changes in expression of this oncogene probably occur before or concurrent with differentiation. No change in bcr-abl protein occurred in a CML cell line (KBM-5) which did not undergo differentiation after exposure to hemin, consistent with a direct relationship between altered p210bcr-abl expression and hemin-induced erythroid differentiation. Importantly, the marked diminution in bcr-abl protein was not associated with a disruption in K562 growth rates, indicating that the proliferative capacity of these cells may be independent of the bcr-abl product. In contrast to hemin, cytosine arabinoside (Ara-C) caused terminal erythroid differentiation of K562 cells, characterized by irreversible hemoglobin accumulation and cytostasis; and no change in bcr-abl protein expression was observed. The distinct effects of Ara-C and hemin could reflect the existence of pleiotropic differentiation pathways. Both Ara-C and hemin-exposed cells showed a decrease in c-myc and c-myb transcripts, suggesting that altered levels of these proto-oncogenes may be associated with erythroid maturation, regardless of the rate of cell division. K562 cells provide a useful model for analyzing the interaction between oncogene expression and CML cell growth and differentiation.
Cell Differentiation↗
Multiple restriction fragment length polymorphisms of the human epidermal growth factor receptor gene.
We have examined the epidermal growth factor (EGF) receptor gene for structural alterations in fresh human tumors. DNA samples from 92 patients with solid tumors (lung cancer, 37; breast cancer, 24; head and neck cancer, 17; other tumors, 14) were analyzed and compared with those from 22 leukemia patients and 14 individuals without malignant neoplasms. When DNA samples were digested with HindIII restriction endonuclease, Southern blot analysis demonstrated 3 distinct polymorphic bands (9.8, 11, and 12 kilobases) after hybridization to the HER-A64-1 probe and another 2 distinct polymorphic bands (4.9 and 5.2 kilobases) after hybridization to the HER-A64-3 probe. Pedigree analysis of 43 members of a single family and comparative analysis of tumor and normal DNA samples from the same patients demonstrated that the variations in fragment size observed were due to 2 independent restriction fragment length polymorphisms in the region of the EGF receptor gene. Amplification of the EGF receptor gene was detected in 3 cases of breast cancer, but not in other tumors studied. We conclude that the human EGF receptor gene has multiple restriction fragment length polymorphisms and that in fresh human tumor samples rearrangement and amplification of the gene occur infrequently, if ever, within the region encompassed by the 2 complementary DNA probes used.
Alleles↗
Effect of recombinant granulocyte/macrophage colony-stimulating factor on human monocyte activity in vitro and following intravenous administration.
The purpose of these studies was to examine the antitumor properties of blood monocytes from patients undergoing a phase I trial with recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF). Peripheral blood monocytes from 7 patients receiving various doses of rGM-CSF by continuous infusion were isolated prior to therapy and at various times during the 2-wk infusion. Monocytes/cubic centimeter of blood increased in a dose-dependent fashion in patients receiving rGM-CSF. However, activation of monocyte-mediated tumorilytic properties was seen in only 1 of 7 patients. rGM-CSF administration also did not stimulate interleukin-1 or tumor necrosis factor production by blood monocytes. The failure to detect monocyte-mediated tumoricidal activation was not secondary to an inherent "defect" in the patients' monocytes because in vitro incubation with lipopolysaccharide alone or human recombinant gamma-interferon plus muramyl dipeptide resulted in monocyte-mediated cytotoxicity and in the secretion of interleukin-1 and tumor necrosis factor. rGM-CSF in vitro also did not stimulate the tumoricidal function of normal monocytes or the secretion of interleukin-1 or tumor necrosis factor. We concluded that systemic administration of rGM-CSF did not result in routine activation of monocyte-mediated cytotoxicity but did result in a dose-dependent rise in the number of circulating monocytes. Because these monocytes could be activated in vitro, we propose that, in an adjuvant therapy setting, rGM-CSF be combined with other activating agents to increase the pool of potential killer cells.
Cells, Cultured↗
Interferon-induced changes in pharmacokinetics and tumor uptake of 111In-labeled antimelanoma antibody 96.5 in melanoma patients.
The type I interferons [both partially purified human leukocyte interferon (HuIFN-alpha) and recombinant alpha interferon] and the type II interferons have been shown to increase the expression of tumor-associated antigens in vitro. To determine whether HuIFN-alpha could increase tumor acquisition of the antimelanoma antibody 96.5 in vivo, five patients with metastatic malignant melanoma were treated with HuIFN-alpha at a dose of 3 X 10(6) units daily by im administration. Twenty-four hours after the first dose of HuIFN-alpha, 1 mg of antibody 96.5 labeled with 5 mCi of 111In was coadministered with 19 mg of unlabeled 96.5. Five patients matched for metastatic site and lesion size who had not received HuIFN-alpha were also given a dose of 5 mCi of radiolabeled 96.5 at the same total antibody dose (20 mg). In patients treated with HuIFN-alpha, there was a statistically significant increase in the plasma half-life of the 111In label (39.7 +/- 3.3 hr) compared to the untreated control group (29.8 +/- 3.2 hr). In addition, there was an increase in the apparent volume of distribution of the antibody in the HuIFN-alpha group (5.56 +/- 0.67 L) compared to controls (3.15 +/- 0.5 L) suggesting both an increased immediate extravascular distribution of radiolabeled antibody and a decrease in the subsequent rate of clearance of antibody from plasma. These two phenomena result in a 28% decrease in the area under the concentration curve in the HuIFN-alpha-treated group compared to controls. Computer analysis of whole-body scans from patients showed a threefold increase in radiolabeled antibody distributed to tumor relative to blood pool but no change in organ:blood ratios for liver, spleen, bone, or kidney compared to controls. This pilot study suggests that treatment of patients with HuIFN-alpha results in an improved distribution of radiolabeled antibody to tumor target without a concomitant increase of label in normal nontarget tissues. In addition, this change in whole-body distribution of antibody is manifested by changes in the pharmacokinetic parameters measured for monoclonal antibody.
Antibodies, Monoclonal↗
Synergy of tumor necrosis factor and interleukin 2 in the activation of human cytotoxic lymphocytes: effect of tumor necrosis factor alpha and interleukin 2 in the generation of human lymphokine-activated killer cell cytotoxicity.
Human lymphocytes can respond to interleukin 2 (IL-2) under serum-free conditions with generation of major histocompatibility locus-unrestricted oncolytic activity. This function has been named lymphokine activated killing (LAK). Although IL-2 is sufficient for the development of LAK, this function can be regulated positively by the addition of tumor necrosis factor alpha or beta (TNF-alpha or -beta). The cytotoxic synergy observed with TNF enables production of optimal LAK function at a 10-fold lower IL-2 concentration. Neither TNF-alpha nor -beta is able to induce LAK function in the absence of IL-2. Using TNF-alpha as a model, we demonstrate that (a) the cytotoxic synergy occurs with both fresh human tumors and cell lines; (b) the degree of IL-2/TNF-alpha synergy, for most peripheral blood lymphocyte donors, is dependent upon the IL-2 concentration used for activation with the most striking synergy observed at lower IL-2 doses; (c) synergy is specific for TNF-alpha and can be abrogated by neutralizing antibody against this cytokine; (d) addition of high-dose neutralizing antibody to IL-2 alone-stimulated peripheral blood lymphocytes can reduce the cytotoxicity capacity of these effectors suggesting an immunoregulatory role for endogenous TNF-alpha; and (e) TNF-alpha addition to IL-2-stimulated peripheral blood lymphocytes does not increase proliferation or cell recovery but does result in enhanced IL-2 receptor expression. Collectively, our results suggest that TNF-alpha (and -beta) have immunopotentiating roles in the amplification of non-major histocompatibility locus-restricted lymphocyte effector function.
Cell Line↗
Amplified and overexpressed epidermal growth factor receptor gene in uncultured primary human breast carcinoma.
We analyzed the epidermal growth factor receptor gene using a complementary DNA probe of the epidermal growth factor receptor gene in 21 uncultured primary breast carcinomas and found that the gene was amplified in three of these tumors. We further demonstrated by immunohistochemistry using a monoclonal antibody to the epidermal growth factor receptor that the receptor protein product of this gene was overexpressed and displayed elevated kinase activity. Our data indicate that one of the molecular mechanisms for overexpression of epidermal growth factor receptor in human breast cancer is epidermal growth factor receptor gene amplification without rearrangement in a subset of tumors.
Adult↗
Recombinant interferon gamma in hairy cell leukemia, multiple myeloma, and Waldenstrom's macroglobulinemia.
Fifteen patients with multiple myeloma, five with hairy cell leukemia, and five with Waldenstrom's macroglobulinemia were treated with recombinant interferon gamma (rINF-gamma) to determine the antitumor activity of this agent. The rIFN-gamma was administered by daily intramuscular injection at doses ranging from 0.125 to 0.5 mg/m2. No responses were observed in patients with multiple myeloma, although in one patient the disease has remained stable for over 16 months. Minimal improvement in some hematologic indexes were observed in three of five patients with hairy cell leukemia. One partial remission and one minor response were documented in two of the five patients with Waldenstrom's macroglobulinemia. In five patients, an increase in normal serum immunoglobulins was observed. These results suggest that there is only minimal activity of rIFN-gamma as a single agent in neoplasms of B-cell origin.
Adult↗
Preclinical and clinical effects of the hematopoietic colony stimulating factors.
Hematopoietic colony stimulating factors are available in purified recombinant form and have used for assessment of hematopoietic activities in mice, monkeys and in phase I clinical trials with humans. This report reviews the preclinical and clinical studies involving these factors.
Animals↗
Philadelphia-negative chronic myelogenous leukemia with breakpoint cluster region rearrangement: molecular analysis, clinical characteristics, and response to therapy.
We have detected rearrangement in the breakpoint cluster region (bcr) on chromosome 22 in cells derived from seven chronic myelogenous leukemia (CML) patients who had no cytogenetic evidence of a chromosome abnormality. These Philadelphia (Ph)-negative, bcr rearrangement-positive CML patients had clinical features and laboratory parameters that bore a strong resemblance to those of Ph-positive CML; all patients have shown a favorable response to hydroxyurea, busulphan, or alpha interferon (IFN-alpha) therapy. In one patient, because of the deletion of distal 3' sequences, detection of bcr rearrangement required a large probe that recognized proximal 5' sequences. Cells obtained from five patients were studied by Northern blotting and showed an aberrant 8 kilobase (kb) mRNA indistinguishable from the bcr-abl transcript that is felt to be a pathogenetic factor in Ph-positive CML. In three patients with a normal karyotype, bcr rearrangement was detected at the time of hematologic remission, and represented the only evidence for persistent malignancy. Our results suggest that: (1) the presence of bcr rearrangement in CML is associated with clinical features of Ph-positive disease, even in the absence of the Ph chromosome; (2) deletions occur within bcr and necessitate the use of probes covering both 5' and 3' DNA segments for accurate diagnosis; (3) molecular analysis may provide a useful approach to the follow-up of leukemia therapy in some patients; and (4) these patients respond to hydroxyurea, busulphan, and IFN-alpha therapy.
Adolescent↗
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.
Adult↗
Characterization of human recombinant tumor necrosis factor-alpha antiproliferative effects on human cells in culture.
TNF is a macrophage-derived polypeptide known to cause hemorrhagic necrosis of transplanted tumors and is cytotoxic or cytostatic to a variety of malignant human cells in vitro. However, little is known about its mechanism of antiproliferative activity. Human cervical carcinoma (ME-180) cells in log-phase were treated with various doses of rTNF (from Genentech, Inc., Sp. Act. 5.7 x 10(7) U/mg) for 72 hours. Fifty percent inhibition of cell growth was obtained at a dose of 1000 U/ml (ID50). An identical ID50 was obtained for confluent cells. Maximal antiproliferative effects were observed only with 72 hours continuous exposure to rTNF. Exposure of cells to an ID50 dose for 12 hours or less resulted in no antiproliferative effect; similar to results obtained with murine TNF. The incorporation of [3H] uridine and [3H] thymidine was reduced by 25 and 40% respectively by 24 hours after TNF treatment. [3H] uridine incorporation rebounded to 170% of controls level 72 hours after TNF addition. There was a transient 40% decrease in [3H] leucine incorporation at 48 hours which recovered to control value by 72 hours. These studies show that TNF can suppress both DNA and RNA synthesis while protein synthesis is only transiently affected. Considering its short plasma half-life (approximately 20 min) in vivo, these studies also suggest that for optimal antiproliferative effect rTNF should be administered in a fashion which provides sustained drug levels.
2',5'-Oligoadenylate Synthetase↗
The role of interferons in the treatment of hematologic malignancies.
The antiviral and antigrowth activity of interferon (IFN) makes this agent a promising approach to cancer therapy. Three tumors that are unusually sensitive to IFN-a are chronic myelogenous leukemia (CML) in the benign phase, hairy-cell leukemia, and the T-cell lymphomas associated with mycosis fungoides. Studies using partially purified IFN-a or IFN-A2a (Roferon-A) therapy have shown that most patients with benign-phase CML achieve hematologic remission. Furthermore, in some patients, long-term therapy results in hematologic remission and complete suppression of the Philadelphia chromosome. Early intervention, within six months after diagnosis, increases the response rate among CML patients treated with IFN-A2a. IFN-a, IFN-A2a (Roferon-A) and IFN-A2b (Intron A) are remarkably active against hairy-cell leukemia, with similar results achieved in clinical studies. The question of antibody formation in trials of recombinant IFN-a products will be discussed in this paper. Neutralizing antibody development and antibody positivity as determined by enzyme-linked immunosorbent assay (ELISA) have been reported with both IFN-A2a and IFN-A2b (Intron A), but the rate reported with IFN-A2b was significantly lower. However, possible differences in the sensitivity of the assays have been mentioned. The effects of differing dosing regimens also were noted. Additional trials conducted by clinical investigators using identical methods will help answer questions arising from these discrepancies and determine the full value of interferon in cancer therapy.
Humans↗
Phase I study of recombinant human granulocyte-macrophage colony-stimulating factor in patients with bone marrow failure and malignancy.
Twenty-five patients with malignancy and/or bone marrow failure were treated with recombinant human granulocyte macrophage colony-stimulating factor (rh GM-CSF) (specific activity 5 x 10(7) units/mg, purity greater than 95%) given by continuous intravenous infusion at various fixed dose levels (15-500 micrograms/m2/day), as part of a phase I study. Treatment was associated with marked increases in white blood cell counts (2-70 fold), consisting mainly of neutrophilic granulocytes. Significant increases were also observed in eosinophils, monocytes and lymphocytes. In addition six patients had multilineage responses characterized by increases in platelet counts (greater than 2 fold and greater than 100,000/mm3) and reticulocyte counts (greater than 2 fold). Three of these 6 patients did not require red cell or platelet transfusion for 17 to 37 weeks. Treatment also resulted in an increase in bone marrow cellularity and myeloid: erythroid cell ratio. The common side effects were constitutional symptoms and bone pain. These findings demonstrate that rh GM-CSF is well tolerated when given by continuous IV infusion and is a potent stimulator of hematopoiesis in vivo.
Bone Marrow↗
Recent advances in the therapy of chronic myelogenous leukemia.
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Combined Modality Therapy↗
Chronic myelogenous leukemia--past, present, and future.
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Humans↗
Aberrant expression of the K-ras proto-oncogene in B-cell malignancies.
The proto-oncogenes have the capacity for conversion to an oncogene that is capable of inducing or maintaining the transformed state when they are overly expressed or altered by mutation or rearrangement. To study the possible involvement of these genes in the neoplastic transformation of B-cell malignancies, we have analyzed their expression in 18 fresh samples obtained from the peripheral blood of patients with a variety of B-cell malignancies. A high-molecular weight c-k-ras transcript (5.2 kb) was detected in all samples including normal lymphocytes. In contrast, a low-molecular weight c-k-ras transcript (1.2 kb) was not detected in our control normal lymphocytes, but was found in 9 fresh samples. The 1.2 kb c-k-ras transcript was expressed 2-3-fold higher than the 5.2 kb c-k-ras transcript. C-h-ras (2.9 kb, 1.4 kb) was expressed at a low level in 4 and 13 samples, but not in normal lymphocytes. C-myc (2.3 kb) and c-raf (3.8 kb) were expressed in all samples including normal lymphocytes without significant variation in the level of expression. C-fos (2.2 kb) was expressed in 15 samples and normal lymphocytes with a wide range in the level of expression. C-myb (3.7 kb) and c-fes (2.7 kb) were expressed at a low level in 6 and 10 samples, respectively, including normal lymphocytes. C-sis (4.0 kb) was not detected in any sample including normal lymphocytes.
DNA↗
Differential in vitro effects of recombinant alpha-interferon and recombinant gamma-interferon alone or in combination on the expression of melanoma-associated surface antigens.
The effect of individual in vitro concentrations of recombinant alpha-interferon species A (rIFN-alpha-A), recombinant gamma-interferon (rIFN gamma), and combinations of both interferons on the expression of melanoma-associated surface antigens p97 and the high molecular weight proteoglycan (HWM-MAA-240K) as detected by monoclonal antibodies (MoAb) 96.5 and ZME 018 respectively, was examined. rIFN-alpha-A at optimal concentrations of 50 and 500 U/ml caused significant (p less than 0.05 and p less than 0.01, respectively) increases in expression of p97 on the melanoma cell line Hs 294t following a 48-h incubation. No change in 96.5 binding was noted with rIFN-alpha-A concentrations up to 1000 U/ml after a 24-h incubation. Likewise, rIFN gamma at an optimal concentration of 500 U/ml enhanced p97 expression slightly at 24 h (p = 0.053) and more significantly by 48 h at concentrations of 50, 500, and 1,000 U/ml (p less than 0.01, p less than 0.05, and p less than 0.025, respectively). In contrast, neither rIFN gamma nor rIFN-alpha-A had an effect on modulation of the HMW-MAA as detected by ZME 018, irrespective of dose or incubation time. Optimal and suboptimal concentrations of rIFN-alpha-A plus rIFN gamma in combination did not significantly enhance expression of either antigen to a greater extent than that observed with optimal concentrations of each IFN alone, despite the fact that the combination had a synergistic anti-proliferative effect. These data demonstrate that the effects of each interferon (IFN) on the expression of melanoma-associated antigens (MAA) are heterogeneous and depend on IFN type and concentration, exposure time, and characteristics of the antigen studied. Moreover, the data regarding combinations of rIFN-alpha-A and rIFN gamma suggest that mechanisms responsible for IFN effects on MAA modulation are independent from its effects on proliferation. These findings may have clinical relevance with respect to trials using combinations of alpha- and/or gamma-IFN plus MoAb in vivo.
Antigens, Neoplasm↗