Inhibition of differentiation and proliferation of colony-stimulating factor-induced clonal growth of normal human marrow cells in vitro by retinoic acid.

Necessary for growth and differentiation in many normal tissues and capable of inducing differentiation in human promyelocytic cell lines, retinoids were the subject of this study. Specifically, effects of 13-cis-retinoic acid and 13-trans-retinoic acid on the growth of normal human bone marrow cells in soft-agar system were studied. Both short-term incubation and continuous exposure to retinoic acid caused a decreased number of granulocyte colonies and an increased cluster-to-colony ratio. This effect was concentration-dependent. Examination of specimens stained with Wright-Giemsa or nitro blue tetrazolium stains showed a progressive increase in the percentage of immature granulocytic precursors with increasing concentrations of retinoic acid. No effect of retinoic acid was seen on a number of human tumor cell lines. Retinoic acid blocked both differentiation and proliferation and appeared to do so by specific, noncytotoxic mechanisms in normal human bone marrow cells.

Deletion of 3(p14p23) in secondary erythroleukemia arising in long-term survivors of small cell lung cancer.

Cytogenetic studies were done on the leukemia cells of two patients with small cell lung cancer (SCLC) who developed erythroleukemia (acute nonlymphocytic leukemia, French-American-British M6) after combined modality chemotherapy and radiotherapy for their lung cancer. Surprisingly, both erythroleukemias exhibited the del(3)(p14p23) predominantly found in SCLC. In four other patients who had secondary erythroleukemias associated with other cancers, no deletions of 3p were found. These findings could be accounted for by one of three possible mechanisms: (a) an inherited recessive gene (anti-oncogene or tumor suppressor gene) in this region of 3p was uncovered by the combined modality therapy, (b) an inherited predisposition to damage of both chromosomes at 3p14 leads to SCLC and erythroleukemia after exposure to carcinogens and/or chemotherapy-radiotherapy, or (c) the finding of lineage specificity for the 3p deletion with the presence of the 3p deletion in SCLC and erythroleukemia suggests a common bone marrow precursor.

Effect of fibroblast, lymphoid, and myeloid interferons on human tumor colony formation in vitro.

A variety of solid and hematological human tumors and normal human bone marrow specimens were assayed for colony formation in a short-term soft-agar culture system. The effect of human fibroblast, lymphoid, and myeloid interferons on inhibition of colony formation was assessed. The effect of interferon on colony formation formed a continuum from complete inhibition to stimulation of growth. Of 40 evaluable tumor specimens, 18 showed at least a 70% inhibition of colony formation in the presence of interferon, at concentrations of 1000 units/ml or less. Four specimens (acute myelogenous leukemia, osteogenic sarcoma, neuroblastoma, ovarian carcinoma) showed at least 3-fold stimulation of colony formation with interferon. Two of 12 normal bone marrow specimens grown with colony-stimulating, factor-conditioned media showed greater than 70% colony inhibition with interferon. A dose-response relationship was seen in all tumor specimens tested. While fibroblast interferon was the most active in this system, all interferons showed the same magnitude and direction of activity. Continuous exposure and 1-hr incubation of tumor cells with interferon were identical in terms of colony inhibition. These data support the ability of this assay system to select tumors responsive in vitro to interferon, suggest the optimal species and concentration for inhibition or stimulation of growth, and support a direct role of interferon in the regulation of cell growth independent of other immunoregulatory actions of interferon. Such information may prove useful for predicting response in vivo to interferon in Phase II trials.

Direct cloning of human parathyroid hyperplasia cells in soft-agar culture.

Parathyroid specimens removed from patients with clinical hyperparathyroidism were cultured in a two-layer soft-agar system. Four patients had parathyroid hyperplasia and one had a parathyroid adenoma. Colonies grew from single-cell suspensions of each specimen. Plating efficiency ranged from 0.001 to 0.05%. No colonies grew from normal bovine parathyroid specimens. Parathormone was detected in 0.9% NaCl solution incubated with the culture plates of three of the four human specimens tested. Parathormone levels determined by radioimmunoassay ranged from 10.4 < 100 ng/ml. Plates tested serially showed a progressive rise in parathormone levels with time and an increase in colony size and number. Microscopic evaluation of the cellular layer showed clusters of cells morphologically consistent with parathyroid origin. Colonies remained viable for approximately 3 weeks. These data confirm that malignancy of tissue in vivo is not necessary for colony formation in agar and that human parathyroid hyperplasia or adenoma cells produce and secrete parathormone in this system.

A phase I study of recombinant interleukin 2 plus recombinant beta-interferon.

Interleukin 2 (IL-2) therapies have antitumor activities against several neoplasms. In vitro these activities are enhanced by beta-interferon (IFN-beta). Therefore, we initiated a Phase I trial with a combination of IL-2 and IFN-beta three times weekly. The IFN-beta was administered i.v. Initially, the IL-2 was administered s.c. However, neutralizing antibody to the IL-2 developed in five patients, and the route of administration of the IL-2 was changed to i.v. Forty-seven patients were entered on the study. The maximum tolerated doses for the combination given i.v. were 5 x 10(6) units/m2 of IL-2 and 10 x 10(6) units/m2 of IFN-beta. Dose-limiting toxicities were profound fatigue/decreased performance status, anorexia/weight loss, depression, and arthralgias. Hypotension, exfoliative skin rash, thrombocytopenia, diarrhea, temperature greater than 40.6 degrees C, and peripheral edema were rarely dose limiting. Thirty-two patients were evaluable for response. After 4 weeks of treatment, 21 patients had stable disease, three patients had a minor response, and one patient had a partial response. Significant lymphokine-activated killer cell (LAK) activity was seen in seven patients (22%) and required 5 x 10(6) units/m2 of IL-2. Those who had progressive disease had significantly less LAK activity than those with either stable disease or a response. This therapy also induced more than 60 units/ml of endogenous gamma-interferon 4 h after the i.v. IL-2 administration. This study demonstrates that (a) intermittent i.v. bolus IL-2 therapy can generate LAK activity, (b) LAK activity may be associated with an antitumor response, (c) significant levels of gamma-interferon are induced by this therapy, and (d) IL-2 and IFN-beta given three times weekly i.v. is both tolerable and biologically active. The recommended Phase II dose is 5 x 10(6) units/m2 of IL-2 plus 6 x 10(6) units/m2 of IFN-beta.

Cell-mediated inhibition of tumor colony formation in agarose by resting and interleukin 2-stimulated human lymphocytes.

Human nonadherent peripheral blood mononuclear cells (PBMC) isolated from nonimmunized donors were preincubated for 18 h in medium alone or medium containing the lymphokine interleukin 2 and subsequently cocultured with tumor cells derived from malignant tumor cell lines or from fresh human tumors. The cell suspensions were subsequently inoculated into agarose; 14 days later, new tumor colony formation was determined. Although the different tumor cells displayed a wide range of sensitivity to the PBMC, in each instance, the number of colonies formed by the tumor cells exposed to the PBMC was consistently reduced relative to that of control cells. The inhibitory effect on the colony-forming cells was especially pronounced with PBMC preincubated with interleukin-2 and was dependent on the ratio of tumor cells to PBMC in the culture. This assay system provides an alternative to the standard 51Cr release assays in assessing the immunomodulatory effects of lymphokines and in quantitating the cytolytic or cytostatic activity of various effector cells against neoplastic stem cells from established cell lines and from heterogeneous cell preparations derived from fresh human tumors.

Pharmacokinetics of recombinant interleukin 2 in humans.

This report summarizes the pharmacokinetics in humans of recombinant interleukin 2 (IL-2) given as an i.v. bolus, i.v. or i.p. infusion, and i.m. or s.c. injection. Immediately after an i.v. bolus the serum IL-2 level equals the dose divided by the plasma volume, in a typical human 650 units/ml for a dose of 10(6) units/m2. The level initially decreases with a half-life of 12.9 min, followed by a slower phase with a half-life of 85 min out to 4 h after the bolus. The median steady state level during an i.v. infusion of 10(6) units/m2 over 6 h is 41 units/ml. A clearance rate of approximately 120 ml/min is obtained from either the i.v. bolus or infusion data and is consistent with the renal filtration being the major route of clearance. Serum levels remain fairly constant for about 8 h after s.c. or i.m. injection but are approximately 2% of the level seen immediately after an i.v. bolus. The area under the time-concentration curve suggests that about 30% of the IL-2 activity is transported from the site of an i.m. injection to the blood. After i.p. infusion IL-2 is only slowly transported to the blood. The median serum IL-2 levels are 430-fold lower than levels in the i.p. fluid and decrease with a median half-life of 6.3 h.

Effectiveness and tolerability of low-dose cyclophosphamide and low-dose intravenous interleukin-2 in disseminated melanoma [corrected].

We studied the effects on melanoma of low-dose recombinant interleukin-2 (IL-2) preceded by low-dose cyclophosphamide (CYC). Twenty-seven outpatients, aged 25 to 75 years, were treated with IL-2, 3.6 million U/m2 intravenously (IV), daily for five days on 2 successive weeks beginning three days after 350 mg/m2 of IV CYC. This schedule was repeated at least twice more at 1-week intervals. Six of 24 patients (25%) who received more than one 2-week cycle of treatment had a remission, one complete and five partial, with minor responses in eight others (33.3%). Three patients with rapidly progressive disease, who received only one cycle, were excluded from the analysis of response. The responses comprised remissions of liver metastases in two patients, one of them complete, two complete and two partial regressions of subcutaneous metastases, partial remission of lymph node metastases, and a partial remission of lung nodules. The mean duration of response exceeded 5 months, with two patients treated for greater than 1 year. Toxicity was moderate and controllable and only two patients required hospitalization, both overnight. Lymphokine-activated killer (LAK) cell activation was induced in 17 of the 24 patients, including all six responders, while none of seven patients without LAK activation had a remission. This regimen appeared to be as effective in melanoma as those involving ex vivo activation of LAK cells, and was generally tolerable to patients in all age groups.

Renal cell carcinoma: treatment with recombinant interleukin-2 plus beta-interferon.

Preclinical data have demonstrated synergy between interleukin-2 (IL-2) and beta-interferon (IFN-beta) in stimulating natural-killer (NK) cell activity and in increasing expression of IL-2 receptors. Based on results of a phase I trial, a combination of IL-2 and IFN-beta was administered three times weekly by intravenous (IV) bolus injection with 5 x 10(6) Cetus U/m2 of IL-2 and 6 x 10(6) U/m2 of IFN-beta to 24 patients with advanced renal cell carcinoma (RCC). Of 22 assessable patients there were six (27%) objective responses including one complete remission (CR) and five partial responses (PRs). There were three minor responses (MRs), 11 stable disease (SD), and two progressive disease (PD). Two of the objective responses have continued for almost 2 years. Response sites include lymph nodes, lungs, and bone. Toxicities requiring dose reduction include arthralgia, weight loss, fatigue, decreased performance status, depression, and hypotension. Five of 10 patients who had a prior nephrectomy without local recurrence achieved an objective response as compared with only one of 12 without a prior nephrectomy or with a local recurrence (P = .04). Mean peak lymphokine-activated killer (LAK) cell activity of the objective responders was 88 lytic units (LU) as compared with 4 LU in the nonresponders (P = .01). Mean peak NK cell activity was 288 LU in the objective responders as compared with 100 LU in the nonresponders (P = .10).(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-gamma induced by administration of recombinant interleukin-2 to patients with cancer: kinetics, dose dependence, and correlation with physiological and therapeutic response.

The administration of recombinant interleukin-2 as an i.v. bolus at dose levels of from 1 to 30 MIU/m2 to patients with cancer induces easily measurable serum interferon-gamma levels of 1 to 500 U/ml. After a lag of 1 h, interferon-gamma rises to a maximum at 4 h and then slowly decreases. The peak values are poorly correlated with the dose of interleukin-2, and thus must be also be dependent on other factors. Successive administration of interleukin-2 typically increases the peak level of interferon-gamma fourfold, but does not diminish the lag period. Peak levels of interferon-gamma are also increased by concurrent administration of interferon-beta with interleukin-2. Continuous i.v. infusion of 1.5 to 20 MIU/m2 of interleukin-2/day results in interferon-gamma levels of 1 to 7 U/ml. Hypotension, which is characteristically associated with interleukin-2 administration, is correlated with interferon-gamma levels in only some patients. There was no apparent correlation between tumor regression and serum interferon-gamma levels.

Therapy of acquired immune deficiency syndrome with recombinant interleukin-2.

Recombinant human interleukin-2 (rIL-2) was administered to 87 patients with the acquired immune deficiency syndrome (AIDS) to test the hypothesis that this lymphokine would correct the underlying qualitative and quantitative deficiency in cellular immunity. Patients were divided into two groups by the presence or absence of Kaposi's sarcoma and subjects within each of these groups received intravenous rIL-2 three times weekly for eight weeks. Subjects received one of several doses which ranged from 1,000 to 2,000,000 units per square meter body surface area. Toxicity at high doses consisted of flu-like symptoms and hypotension at highest doses. Partial objective tumor regression was observed in three patients with Kaposi's sarcoma. Seventeen patients had progression of disease (new opportunistic infection or increase in Kaposi's sarcoma) during therapy. No improvement in immunologic status was observed. This study does not suggest a role for single-agent rIL-2 therapy of established AIDS but its use in less symptomatic persons or in conjunction with antiretroviral agents such as azidothymidine should be investigated.

Skeletal manifestations in cutaneous T-cell lymphomas.

The clinical course of three patients with cutaneous T-cell lymphoma (CTCL) in whom skeletal disease developed is presented and the literature on skeletal involvement in these disorders is reviewed. Three separate types of skeletal manifestations occurred: (1) osteolytic lesions, (2) osteoblastic lesions, and (3) diffuse osteoporosis. Hypercalcemia was present in two cases. Tumor cells from two patients in short-term culture secreted osteoclast-activating factor(s). Both of these patients had pathologic evidence of osteoclast activation in bone sections. Thus, the tumor cells in certain patients with CTCL may derive from a monoclonal proliferation of a T-cell subset capable of producing humoral bone-resorbing factor(s) similar to those demonstrated in cultures of mitogen- and antigen-activated normal lymphocytes. Since skeletal lesions are unusual, it would follow that other T-cell subsets account for pathologic cell proliferation in most patients with CTCL.

Obesity in families of extremely obese women.

In order to assess the prevalence of obesity in families of extremely obese individuals, we conducted a mail survey of a national obesity organization. Thirty-nine percent (N=981) of the questionnaires were completed and returned. Respondents were excluded from further analysis if they were adopted, male, their gender could not be determined, provided incomplete information about their parents or their own height and weight, or were less than 22 or greater than 63 years of age. The analyses included 729 probands and their families. Both the prevalence and the extent of obesity were high in the families members. The average family members' body mass index (BMI=kg/m2) was 30, and 78% of the families studied had at least one other obese (BMI>30 kg/m2) first-degree relative (parent, sibling, or child). Although obesity was common in the families, survey respondents were generally the heaviest members of their families, having an average BMI of 47 kg/m2. Correlations among first-degree relatives were similar to those found for average weight groups, suggesting that obesity and BMI are similarly influenced by family genetic factors in this extremely obese population.

Interleukin 2-activated human killer cells are derived from phenotypically heterogeneous precursors.

When cultured with native or recombinant human interleukin 2 (IL 2), human peripheral blood non-adherent mononuclear cells (NAMNC) acquire the ability to lyse both NK-sensitive and NK-resistant tumor target cells. The development of these IL 2-activated killer (IAK) cells, also known as LAK, is observed in the absence of exogenous antigen or mitogen. This study describes the ability of various subpopulations of human peripheral blood NAMNC with defined surface phenotype to generate the IAK activity. Human NAMNC were separated into various subpopulations on the basis of the ability to bind monoclonal antibodies, activated with IL 2, and were examined for the cytolytic effect on various tumor target cells. Although CD16+ (Leu-11+) NK cells from NAMNC could become IAK cells when cultured with IL 2, removal of these cells from NAMNC had no effect on the latter's ability to generate the IAK effect. When CD16- NAMNC were separated into CD2+ E rosette-forming T cells (ERFC) and CD2- non-T (non-ERFC) subpopulations, both subpopulations generated the IAK activity. The ability of monoclonal antibody-defined subpopulations of T and non-T cells to generate IAK cells was then examined. Both CD4+ and CD8+ subsets isolated by either positive or negative selection generated the IAK activity. Similarly, CD20+ (B1+) B cells and CD20- non-T (null) cells developed into IAK cells when cultured with IL 2. In contrast, Leu-7+ T cells failed to generate the IAK activity. CD4+ and CD8+ subsets were additionally separated into narrower subpopulations by using monoclonal antibodies anti-Leu-8 and 9.3 respectively, and were examined for their ability to generate IAK cells. Precursors of IAK cells were derived from each of the four: CD4+, Leu-8+ (inducer), CD4+, Leu-8- (helper/amplifier), CD8+, 9.3+ (cytolytic), and CD8+, 9.3- (suppressor) subpopulations of T cells. Thus, the IAK activity appears to be derived from phenotypically heterogeneous and otherwise functionally diverse human lymphoid cells and is not confined to any single subpopulation.

The human tumor colony-forming chemosensitivity assay: a biological and clinical review.

Over forty papers describing correlations between in vitro human tumor sensitivity to a variety of chemotherapeutic agents and the in vivo response of patients to those agents have been published since the publication in 1978 by Salmon and Hamburger of their results of a human tumor colony-forming chemosensitivity assay (CFCA). The true positive rate in over 1600 correlations is 71% and the true negative rate is 94%. The biological elements of the assay, its developmental history, its place in the spectrum of in vitro chemosensitivity assays, and its theoretical and practical limitations are discussed. The scope, design, and limitations of key clinical trials are presented and an analysis of the potential errors of statistical interpretation of the trials as well as the results of the trials is given.

Evidence for tumor reduction in refractory or relapsed B-CLL patients with infusional interleukin-2.

Recombinant interleukin-2 (rIL-2) is a biologic response modifier that is capable of enhancing or restoring the cytolytic capacity of large granular lymphocytes (LGL). We utilized this biologic response modifier in the treatment of B-chronic lymphocytic leukemia (B-CLL), a disease frequently characterized by deficient or absent natural killer activity. B-CLL (n = 12) patients previously refractory to chemotherapy or with progressive disease post cessation of chemotherapy were eligible. rIL-2 was given as i.v. infusion (2 x 10(6) units/m2) over 2 h 5 times per week for 3 weeks as induction. Responding patients were placed on maintenance therapy. Although there were no complete or partial responses (by ECOG criteria) there was clear evidence of tumor reduction. Seven of 10 evaluable patients had a reduction of the peripheral blood B cell clone, 3 had node reduction and 2 had reduction in their splenomegaly. All patients experienced mild to moderate toxicity and 1 patient died while on induction therapy. Three B-CLL patients following induction rIL-2 treatment were placed back on chemotherapy because of progressive disease. Interestingly, these 2 B-CLL patients achieved extremely rapid and complete responses to chemotherapy which had previously been ineffective. These data suggest a possible role for rIL-2 in treatment of B-CLL.