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We investigated the correlations between the biological effects of interferon-alpha (IFN-alpha) and clinical responsiveness in low-grade non-Hodgkin's lymphomas (NHL). In this disease, 40-50% of cases respond to IFN-alpha therapy. Patients with nodular NHL were selected for a phase II trial in which they were treated daily with 9 x 10(6) U of IFN-alpha 2a. Binding experiments with [125I]IFN-alpha 2a showed the presence of IFN-alpha receptors on tumor B-cells isolated from lymph nodes before therapy in 9 out of 10 patients. Receptor levels were not related to the subsequent clinical responses. However, no specific binding was detected in one patient who turned out to be unresponsive to IFN-alpha treatment. Single injections of IFN-alpha 2a before beginning the therapeutic protocol resulted in down-regulation of IFN-alpha receptors without change in their affinity in peripheral blood leukocytes from only patients who subsequently responded to therapy (4/10). In 4/5 non-responders and one patient displaying a minor response, receptor numbers did not decrease but Kd values rose markedly in all six cases. These results indicate that lack of in vivo IFN-alpha receptor down-regulation and reduced receptor affinity, as detected before therapy, may be correlated with failure of IFN-alpha therapy in nodular NHL.

Interferon-alpha is currently under evaluation as an antineoplastic agent in several types of tumour. Despite its clear in vitro effects, the effectiveness of interferon in vivo is limited. To assess whether this discrepancy reflects pharmacokinetic limitations, the authors analysed interferon distribution in 2 osteosarcoma patients by scintigraphy using 123I-interferon-alpha-2a. Numerical analysis of the scintigraphic records demonstrated that the main organs of elimination were the kidneys, when the calculation was made on the basis of surface area. On the other hand, the apparent total uptake by liver (whose projection surface--i.e. the area exposed to the lens--is greater) was higher, reaching about 25 to 30% of the injected dose. The projection surface of the tumour was able to take up radiolabelled interferon in both cases, resulting in a 4-fold increase in the external radiation count compared with the equivalent region of the contralateral limb (although it is not possible to determine whether the label is present on the tumour itself or on the surrounding inflammatory cells). Thus, interferon-alpha seems able to reach at least the immediate neighbourhood of osteosarcoma mass.

Phytohemagglutinin-induced interferon-gamma (IFN-gamma) was reported to act on mouse leukemic L1210 R cells resistant to IFN-alpha and -beta. Results reported here show that these cells are also sensitive to various preparations of murine IFN-gamma derived from different sources and purified to different degrees and that lymphokines present in the preparations are not involved in the antiviral effect of IFN. In addition, IFN-gamma preparation increases concanavalin A binding to L1210 S and L1210 R cells indicating that the sensitivity of L1210 R cells to IFN-gamma is not limited to its antiviral effect.

Fifty-four patients with hairy cell leukemia were treated for 7 months with three types of subcutaneously injected interferon: recombinant interferon alfa-2a from Hoffmann-La Roche (3 X 10(6) U daily); recombinant interferon alfa-2b from Schering (2 X 10(6) U/m2, three times per week); and partially purified human leukocyte interferon alpha from the Finnish Red Cross (3 X 10(6) U daily). After 4 months, in 27 patients hairy cells decreased dramatically (P less than 0.01) while platelets (P less than 0.05), monocytes (P less than 0.01), polymorphonuclear leukocytes (P less than 0.01) and hemoglobin (P less than 0.01) increased (in this order) as treatment progressed. Morphometric parameters were used to estimate the relative volume of hairy and myeloid cells present in bone marrow. Biopsies were performed at the beginning of the study and in the second, fourth, and seventh months thereafter. Bone marrow disorders were slower to improve as compared with blood. Six severe cases of hairy cell leukemia were responsive to treatment, but required special monitoring during the first 2 months. All three interferons studied are efficient. Interferon receptors, oncogene (mRNA levels) expression, and the relationship between PDGF and fibrosis in hairy cell leukemia are under investigation.

Alveolar II pulmonary tumor cells (A549 cells) maintained in continuous tridimensional organotypic culture were used to evaluate the eventual potentiation effect of mixtures of recombinant human interferon-alpha 2 and -gamma on growth inhibition of the tumor nodules. A continuous 45 day treatment (interferon renewed three times a week) with 10, 10(2) or 10(3) U/ml of IFN-alpha 2 or -gamma combined with a fixed high dose (10(3) U/ml) of either IFN-alpha 2 or -gamma resulted in an additive or synergistic growth inhibition according to the doses used. There was a close dose-effect relation, the percentage of inhibition increasing proportionally to the variable IFN doses added to the fixed high dose; moreover, the growth inhibition effect occurred earlier with the mixtures than with IFNs used separately. Furthermore, the growth inhibition observed with 2000 U/ml of the mixture (1000 U/ml of each IFN) was greater than that induced by 2000 U/ml of IFN-alpha 2 or -gamma used alone. A 35-day treatment with IFN-alpha 2 1000 U/ml plus IFN-gamma 1000 U/ml led to a complete growth inhibition and necrosis of the nodules. These data demonstrate that IFN-alpha 2 and -gamma cooperate to potentiate the IFN antiproliferative activity.

Thirteen patients with malignant tumors were entered into a phase I trial with recombinant DNA human alpha 2 interferon (IFN alpha 2). The patients were given I.M. escalating doses of IFN alpha 2 ranging from 1-10(6) to 200-10(6) IU with a 72 hours washout between injections. In the majority of the patients, subjective symptoms were noted: fever, headache, chills, nausea, myalgias. Asthenia, anorexia, drowsiness appeared after the highest doses and disappeared without any sequellae. Leucopenia and thrombopenia were seen in 11 out of 13 patients. Hepatocellular toxicity was observed in 9 cases. Cardiac and vascular functions were not impaired by IFN alpha 2. The pharmacokinetic studies showed a maximum serum concentration between 4 and 6 hours after injection and the peak value was directly proportional to the dose. No neutralizing INF alpha 2 serum factor was detected during the treatment. The peak value for serum beta 2 microglobulin occurred 48 hours after and the N.K. activity was variably modified by IFN alpha 2 injections. A major clinical response was observed in 1 case, a minor response in 3 cases and a stabilisation of the disease in 4 cases.

The induction of endogenous interferon (IFN) was studied in 28 cases of Argentine hemorrhagic fever (AHF), a severe systemic disease caused by Junin virus. Serum samples were taken daily during the acute period, both before and after administration of immune plasma. This form of treatment has been found to reduce mortality when given early in the course of AHF. High titers of circulating IFN were present in the serum samples taken before treatment. IFN titers drastically dropped after transfusion of immune plasma. The antiviral activity was stable at pH 2 and was completely neutralized only by antibodies against IFN-alpha. Thus, we concluded that circulating endogenous IFN in patients with AHF can be considered as typical IFN-alpha. Fever, chills, and backache were associated with the higher levels of IFN. An inverse correlation between days of evolution of the disease and IFN activity was also observed.

IFN-alpha induces tumor regression with a high percentage of complete remissions in hairy cell leukemia. We have recently reported that hairy cells express specific IFN-alpha receptors which are down-regulated upon therapy. We show here that the activity of 2-5 A synthetase, an IFN-induced enzyme, is 2 to 7-fold stimulated in hairy cells from responsive patients within 12-24 h after the first IFN dose. This in-vivo enzyme induction paralleled the kinetics of receptor down-regulation. In one patient who was unresponsive to IFN-alpha treatment neither expression of IFN-alpha receptor nor change in 2-5 A synthetase were expressed and modulated as in sensitive patients. In-vitro treatment of hairy cells with recombinant interferons showed that IFN-beta 1 was able to induce this enzyme to the same extent than IFN-alpha 2, whereas IFN-gamma was inactive despite the presence of specific IFN-gamma receptors. Our results indicate that IFN-alpha can exert its therapeutic effects by acting directly on the hairy cells through interaction with surface membrane receptors. Induction of 2-5 A synthetase can be one of the steps involved in this action. However, both mechanisms, as well as receptor down-regulation are not sufficient to explain the responsiveness of hairy cell leukemia to IFN-alpha.

Hairy cells are stimulated to DNA synthesis by low molecular weight B cell growth factor (LMW-BCGF) and this proliferative response is suppressed by interferon (IFN)-alpha, both in vitro and in vivo. The suggestion that the CD23 molecule (Fc epsilon II receptor) might be involved in the signalling pathway of LMW-BCGF prompted us to study the expression of this molecule on hairy cells and its modulation by IFN-alpha. By flow cytometry and direct binding experiments with anti CD23 monoclonal antibodies, the presence of the CD23 antigen was detected in 7 of 12 cases tested, on variable percentages of cells, ranging from low to medium expression. In vitro incubation of hairy cells with IFN-alpha, which elicits a suppression of the proliferative response of these cells to LMW-BCGF, induced a parallel significant reduction of CD23 expression in only three cases. Similarly, a transient in vivo decrease of CD23 expression, concommitant with an inhibition of the LMW-BCGF response, could be detected in only one of three patients injected with IFN-alpha. Soluble sCD23/IgE-binding factor (BF) was quantitated in the serum from six other patients with hyperleukocytic hairy cell leukemia (HCL) undergoing a clinical trial of IFN-alpha therapy. Before treatment, these patients presented higher concentrations of the cleaved soluble form of the CD23 molecule than normal controls. Within a few weeks of IFN-alpha administration, these levels markedly decreased, paralleling a diminution of blood leukemic cells. Of interest, no such diminution was noticed for another patient resistant to IFN-alpha therapy. These results show that the proliferative response of hairy cells to LMW-BCGF is not linked to the expression of the CD23 marker. Besides, when the latter molecule was present, its decrease following IFN-alpha treatment, which could be detected in some cases, was not necessarily required for the suppression of the LMW-BCGF response and is thus not mandatory for the therapeutic efficacy of IFN-alpha. Our results point out that quantitation of serum sCD23/IgE-BF, whether related to a process of autocrine proliferation or not, is a parameter of potential importance for therapy monitoring.

Interferons (IFNs) initiate their effects by interacting with specific high-affinity cell surface receptors, but little is known about the physiology of IFN receptor interaction in vivo. Treatment of patients suffering from hairy cell leukemia (HCL) with human recombinant alpha IFN results in significant tumor regression, with clinical improvement in a high percentage of cases. To investigate a possible relevance of binding parameters as response markers, IFN receptor interaction on tumor cells responsive to IFN in vivo was studied. Binding of human alpha 2 IFN to circulating hairy cells was analyzed before and during IFN therapy in ten patients selected on the basis of high numbers of peripheral hairy cells. Binding experiments were carried out on Ficoll-Paque fractionated peripheral cell samples containing a majority of hairy cells. All patients reacted to recombinant alpha IFN treatment with a striking decrease in binding capacity within 12 hours after the first injection. As demonstrated by using a monoclonal antibody able to recognize alpha 2 IFN bound to its receptor, this decreased binding capacity was not due to blocking by circulating IFN but rather to a decrease in receptor number. This receptor "down-regulation" was partially reversible after the first IFN injection. However, upon prolonged IFN therapy, all patients displayed a stable state of decreased receptor expression. Down-regulation of IFN receptors can be regarded as a response marker to IFN treatment. This response marker, however, was not correlated with the clinical response within the first months of IFN therapy.

Eleven patients with metastasized breast cancer received 8 intramuscular injections of 6 x 10(6) units of human fibroblast interferon over a period of 40 days. The injections did not cause local irritation or inflammation. Fever occurred in only 1 of the 11 patients. Although several types of metastases were monitored, only skin nodules consistently (10 out of 11 patients) exhibited changes that were suggestive of a therapeutic effect of the treatment regimen: either a simple decrease in size of some nodules or central necrosis accompanied by an inflammatory reaction. NK-activity of peripheral blood leukocytes was significantly increased after administration of the first dose; the effect of subsequent injections was less clear. Receptors for estrogens and progestogens were increased in the tumor biopsies of 2 out of 2 and 5 out of 6 patients tested respectively.

Twenty-six patients with histologically proven metastatic malignant melanoma were included in a phase II trial of interferon alfa-2b (Intron A; Schering-Plough). Patients were given 10 X 10(6) IU/m2 of interferon alfa-2b subcutaneously three times a week until major intolerance or progression of disease. General signs of intolerance were seen in all patients; hematological toxicity with leukopenia (below 1,800/mm3) and/or thrombocytopenia (below 600/mm3) was seen in six patients and therapy was interrupted in one patient. Mild liver toxicity was seen in most patients after two weeks of treatment. These manifestations disappeared 1-2 weeks after treatment was discontinued. Twenty-four patients were evaluable for response. There were two complete responses; one skin and one lymph node going into remission for 12 and 12.5 months respectively. A partial response was observed in five cases lasting 1, 1.8, 2, 3 and 5 months respectively. These results indicate a potential role for interferon alfa-2b in treating patients with metastatic malignant melanoma, however, further trials are required to determine the optimum dose and schedule of administration and use of interferon alfa-2b in combination with cytotoxic drugs.

This report presents the results of a clinical trial on 53 patients with hairy cell leukaemia using low-dose alpha-interferon as therapy. Improvement of cytopenia and/or bone marrow hairy cell infiltration occurred in all but one patient. Often, blood and bone marrow improvements were dissociated and, after 7 or 13 months of therapy, complete remission was only observed in about 40% of patients. Recurrence of the disease was observed in some cases after cessation of therapy. A summary of the following results is presented: alpha-interferon receptor analysis, oncogene expression, study of the sensitivity of hairy cells to natural killer cells, and the effects of interferon on T-cell receptor gene rearrangement and on the function of T-cell clones. Results are also presented which show that myelofibrosis may be due to a release of platelet derived growth factor. The immunological findings and oncogene expression in 2 patients with a variant form of hairy cell leukaemia for which resistance to therapy was observed are also described. All the results show that interferon acts on hairy cells and are consistent with a direct effect by interferon in the treatment of hairy cell leukaemia.

Type I (alpha, beta) and type II (gamma) murine interferons are able to potentiate each other with respect to the inhibition of encephalomyocarditis (EMC) virus and of herpes simplex virus type 1 (HSV-1) multiplication in a murine cell line (DBT). Examination of two double-stranded RNA-dependent enzymes in DBT cells, the 2-5A synthetase and the 67,000 MW protein phosphokinase indicates that mixed interferon preparations act synergistically at least with respect to an increase in the activity of the former enzyme. The results obtained with gamma interferons of different origin and of different specific activity suggest that interferon itself, rather than the lymphokines present in the interferon preparations, is responsible for the synergistic effect.

Twenty-four patients with histologically proven metastatic malignant melanoma were included in a Phase II trial of human DNA recombinant interferon (rDNA IFN alpha 2). They were given 10 X 10(6) IU of IFN alpha 2 subcutaneously three times a week until progression of disease or major intolerance developed. Twenty-two patients were evaluable for toxicity and response. General manifestations of intolerance were seen in all the patients. Hematologic toxicity was seen in six patients and therapy had to be interrupted in one patient. Mild liver toxicity was seen in most patients after 2 weeks of treatment. These manifestations disappeared within 2 weeks after treatment was discontinued. A partial response was seen in four cases lasting 2, 4, 4, and 5 months, respectively. There were two complete responses (one skin, one lymph node metastasis) lasting 20 and 6 weeks, respectively. These results indicate a potential role for rDNA IFN alpha 2 in treating patients with metastatic malignant melanoma. However, further trials are required to determine the optimal dose and schedule of administration and modalities of combination.

Seventeen patients with hairy cell leukemia (HCL) were treated with low doses of recombinant alpha interferon (IFN) for over 4 months. Marked improvement was observed in peripheral blood and bone marrow in 15 of 17 patients. Comparison of pretreatment values and hemograms obtained after 4 months of treatment showed a marked decrease in circulating hairy cells (P less than .01), a decrease in the number of lymphocytes (P less than .01), a rise in the number of platelets (P less than .05), granulocytes (P less than .05), and monocytes (P less than .01), and a rise in the hemoglobin level (P less than .01). Transient reduction in the number of granulocytes was noted during the first month. Correction of thrombocytopenia often appeared within 2 months and usually preceded improvement of anemia, monocytopenia, and neutropenia. Bone marrow biopsy specimens were taken before treatment and 2, 4, and 7 months after its initiation. The volumes occupied by hairy cells, cells of the myeloid lines, and adipocytes were studied by stereological analysis of semithin sections. Decrease in the volume occupied by hairy cells was seen after 4 months of treatment (P less than .01), and the volume continued to decrease at the seventh month (P less than .05). Hairy cells were no longer detected on bone marrow biopsies of 4 of 17 patients by the fourth month and in 3 of 8 additional patients by the seventh month. A rise in the volume occupied by normal myeloid cells was visible by the second month of treatment (P less than .01). Nevertheless, the volume occupied by granulocytes remained lower than in the normal controls (P less than .01). After an initial increase during the first 2 months of treatment (P less than .01), the overall cellularity remained unchanged at 4 months and decreased significantly (P less than .05) at 7 months. Except for biopsies at 2 months, mean cellularity was below that of control biopsies (P less than .01).

Using a radioimmunoassay for human leukocyte interferon (IFN alpha), pharmacokinetic studies were carried out in twelve cancer patients given sequential intramuscular injections of Hu IFN alpha 2. Even though individual monitoring of serum IFN titers emphasized, for a given dose, marked quantitative variations of the observed maximum concentrations, their mean values were found to be dose-dependent (358 +/- 167 U/ml at 30.10(6) U and 1044 +/- 599 U/ml at 100.10(6) U doses). Comparison with bioassay results showed that IFN activities measured in sera were of the same order of magnitude as those calculated from radioimmunoassay standard curves. Data obtained from this series on observed peak time, half-life value and serum concentrations were consistent with those reported by the other groups using recombinant leukocyte interferon in clinical trial. Therefore, radioimmunoassay is an useful method for routinely assaying IFN alpha used either as antitumour or antivirus agent because of its high sensitivity (4 U/ml) and its simplicity.

Binding of 125I-labelled human recombinant DNA interferons (IFNs) alpha-2, beta and gamma was compared on various human lymphoid cells and embryonic fibroblasts. While binding constants were within an order of magnitude for all three interferons (10(-10) to 10(-9) M), no competition was observed between IFN-gamma on the one hand and IFN-alpha 2 and IFN-beta on the other. However, consistent with previous reports, IFN-alpha 2 and IFN-beta competed for presumably common receptors. Depending on the cell type, binding sites for IFN-gamma were expressed in different numbers compared to those for IFN-alpha 2 and IFN-beta. These direct comparative binding studies support the hypothesis that the receptor system for IFN-gamma is unrelated to the IFN-alpha/beta system.