Phase I study of recombinant beta-interferon given by four-hour infusion.

Twelve patients with advanced malignant disease were entered onto a Phase I study of escalating doses of beta-interferon serine given by 4-h i.v. infusion twice a wk. Three patients each were entered at starting doses of 0.01, 1, 10, and 30 million units (MU)/m2. Doses escalation within individual patients was allowed to a maximum dose of 400 MU/m2. Fever, chills, fatigue, and acral cyanosis were commonly seen and increased in frequency at higher doses. Myalgia, nausea, diarrhea, headache, and confusion were seen at lesser frequencies. Mild leukopenia, paresthesia, infusion site erythema, and hypotension were each seen in one patient. No conventional maximal tolerated dose could be defined, since several patients underwent escalation to the highest allowable dose and seemed to develop tolerance to acute toxicities. However, a maximal starting dose of 10 MU/m2 was identified, such that those begun at this level or below tolerated semiweekly dose escalation, while those begun at 30 MU/m2 could not tolerate continued therapy. Detectable serum interferon levels were noted during treatment at 10 and 30 MU/m2, the levels at which significant toxicity also first appeared. A maximal starting dose of 10 MU/m2, with gradual escalation as tolerance to side effects develops, is suggested if therapy with high-dose beta-interferon serine is given by 4-h infusion.

Human monoclonal antibodies directed against melanoma tumor-associated antigens.

To analyze the humoral immune response to melanoma, human-mouse hybridomas were generated by the fusions of regional lymph node lymphocytes of patients with the mouse myeloma cell line M5. Six stable hybridomas were cloned from six separate lymphocyte parents obtained from three patients. Ascites were obtained from nude mice after i.p. injection with cultured hybridoma cells. The monoclonal antibodies, four immunoglobulin Gs and two pentameric immunoglobulin Ms, were partially purified to remove mouse immunoglobulin and then conjugated to biotin for immunocytochemical and immunohistochemical studies. With the avidin:biotin:peroxidase complex method to detect and amplify binding by the biotin-conjugated human monoclonal antibodies, we found the six antibodies to be reactive against cytoplasmic determinants in five short-term melanoma cultures and formalin-fixed paraffin-embedded melanoma tumors from four patients. The antigenic target of the antibodies identified was not carcinoembryonic antigen. Two antibodies, 2-139-1 and 6-26-3, were studied in more detail. Each stained 25 of 25 specimens of melanomas. Little or no reactivity was detected against fixed sections of normal skin, which included tissues such as epidermis, dermis, monocytes, lymphocytes, and vascular endothelium. More striking was the absence of binding to melanocytes in the basal layer of the skin or to pigmented nevus cells. Both antibodies showed cross-reactivity against other tumors, in particular colonic and prostatic carcinomas. In the normal colon, reactivity was restricted to the surface of the columnar epithelium; no reactivity was detected against normal prostatic epithelium. Reactivity was also not observed against liver and lung. However, the epithelia of the renal tubules, pancreatic ducts, and salivary ducts were all reactive. These human monoclonal antibodies identify cytoplasmic melanoma-associated tumor antigens that appear different from the membrane antigens defined by serological approaches and by most mouse monoclonal antibodies.

Active specific immunotherapy for melanoma: phase I trial of allogeneic lysates and a novel adjuvant.

A Phase I trial of active specific immunotherapy for melanoma was performed to measure the toxicity and immunological effects of the therapy. A mixture of mechanical lysates (homogenates) of two melanoma cell lines was injected together with a novel adjuvant, DETOX, into 22 patients. Several types of cell-mediated and humoral immunity to melanoma-associated antigens were measured serially. In the 17 patients with measurable disease, the sizes of lesions were also noted serially. At least six patients per group were injected s.c. with either 100, 200, or 400 antigenic units (approximately 10, 20, and 40 million tumor cell-equivalents) of the lysates mixed with 0.25 ml of DETOX s.c. on weeks 1, 2, 3, 4, and 6. Three patients at each dose level also received 300 mg/m2 of cyclophosphamide i.v. 4 days before the start of immunization. Evidence for successful immunization was obtained in 13 of the 22 patients. An increase in the frequency of peripheral blood cytolytic lymphocyte precursors reacting against melanoma cells occurred in 12 patients, as measured by a limiting dilution assay involving in vitro re-exposure to irradiated melanoma cells for 9-10 days. Eight of the 12 patients had received cyclophosphamide. By cold-target competition assays, these cytolytic lymphocytes appeared to be atypical T-cells, which recognized melanoma-associated antigens on several allogeneic lines without apparent major histocompatibility complex restriction. An increase in antibody titers against melanoma-associated antigens, measured by enzyme immunoassay, was found in five of 22 patients, and a change in delayed hypersensitivity against the melanoma lysate, in three patients. Responses were found at all three dosage levels of lysate, without an obvious dose optimum. No toxicity except minor local soreness was noted. Therefore, no maximum tolerable dose was defined. Five of 17 patients with measurable lesions had a remission of their melanoma, two complete and three partial, with three additional minor responses. A patient whose complete remission lasted 5.5 months, has no evidence of disease 22+ months after entry onto the study, with the aid of surgical resection of small s.c. recurrences on two separate occasions. Sites of regression included s.c. nodules, lymph nodes, and pulmonary nodules, with no responses in liver, adrenal gland, or bone. The patients who had an increase in cytolytic lymphocyte precursors comprised all eight with a clinical remission (five major, three minor). In contrast, none of the seven patients lacking an increase in cytotoxic lymphocytes had a clinical response.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between cytotoxic and suppressor activities of human pulmonary alveolar macrophages.

We have reported previously that pulmonary alveolar macrophages (PAMs) from individuals with lung cancer and active chronic pulmonary diseases were cytotoxic to tumor cells in vitro, whereas PAMs from normal individuals or patients with acute pulmonary disorders were noncytotoxic. In the present study, we evaluated 20 PAM preparations for both suppressor and cytotoxic functions to determine if PAMs could function as suppressor cells and, if so, whether a correlation between the two functions exists. Cytotoxicity was assessed in a 60-hr cytotoxicity assay against [3H]proline-prelabeled human melanoma target cells. More than 20% cytotoxicity was considered to be significant. Suppressor activity was measured by determining whether admixing PAMs at various ratios with autologous or allogeneic mononuclear cells could suppress concanavalin A-induced blastogenesis by T-lymphocytes. At least 50% suppression was considered to be significant. Of the 20 specimens evaluated, 13 were cytotoxic and 5 of these exhibited suppressor activity. None of the 7 noncytotoxic PAM preparations had suppressor activity. Suppression was nonspecific and not HLA restricted, since autologous and allogeneic mononuclear cells were inhibited to a similar extent. Suppression was probably not due to prostaglandin production by the PAMs since assays were performed under optimal conditions and required extremely high concentrations of prostaglandins. A significant correlation between suppressor and cytotoxic activity was found. Suppression was observed only with PAM specimens that were also highly cytotoxic to tumors, but not all cytotoxic PAM specimens were suppressive. Whether these actions reflect different levels of activation of PAMs or are the properties of different macrophage subsets remains to be clarified.

Cytotoxic activity of human pulmonary alveolar macrophages.

The functions of human pulmonary alveolar macrophages (PAMs) have been relatively little studied compared with those of their circulating counterparts, blood monocytes. This study examined the ability of human PAMs to kill primary human tumor cell cultures and control normal fibroblasts in vitro. PAMs were derived by bronchial lavage from patients with lung cancer of various histological types and stages, patients with acute or chronic noncancerous pulmonary disorders, and subjects with a presumed illness who proved to be normal. After extensive washing, the PAMs were cocultured with [3H]proline-labeled tumor cells, principally lung cancers and melanomas, at various effector:target ratios for 60 hr. Cytotoxicity was measured by comparing radioactivity associated with the remaining adherent tumor cells cultured in the presence or absence of PAMs. Twenty-eight of 42 preparations of PAMs from 42 individuals were cytotoxic to one or more short-term primary tumor cultures. All 28 specimens from patients with lung cancer or chronic pulmonary disease were cytotoxic; all of the 14 PAM preparations lacking cytotoxicity were from individuals with acute pulmonary disorders or who were proved free of pulmonary disease. PAMs were cytotoxic even at effector:target ratios of 2.5:1 or 1.25:1. Fibroblasts were unaffected at any ratio. Sarcoidosis patients in remission had noncytotoxic PAMs, whereas the disease in relapse was characterized by cytotoxic PAMs. Serial study of 2 patients confirmed a loss of reactivity during remission. Smoking did not correlate with the presence or absence of spontaneous cytotoxicity and did not influence the degree of cytotoxicity in "reactors." Partially purified alpha-interferon enhanced the killing of cytotoxic PAMs in 10 of 21 instances but did not induce cytotoxicity in 9 tests on nonreactive PAMs. We conclude that human PAMs from patients with lung cancer or chronic pulmonary diseases, including active sarcoidosis, were cytotoxic to several recently explanted tumor cell cultures. PAMs from acute pulmonary dysfunctions and those from patients with inactive sarcoidosis were not spontaneously cytotoxic.

Noninvasive monitoring of drug biodistribution and metabolism: studies with intraarterial Pt-195m-cisplatin in humans.

We have performed a comparative evaluation of systemic (i.v.) and intraarterial (i.a.) cisplatin by using a trace dose of the radiolabeled form of this drug [( 195mPt]cisplatin) to monitor the drug's biodistribution by dynamic scintigraphic imaging. We have analyzed the drug's metabolism using a compartmental model both following i.a. and i.v. administration in patients with gliomas. Significantly larger amounts of radioactivity (up to 10 times higher than in the uninvolved brain) were measured in tumors following i.a. administration, whereas the differential localization following i.v. drug administration was, at best, only twofold that of the uninvolved brain. On the other hand, no significant differences could be detected in the pharmacokinetics of either free cisplatin or platinated proteins in blood. The washout slope in tumors following i.a. administration may be an indicator of the higher local concentration of free cisplatin; no such washout could be observed in tumors following i.v. administration. The present noninvasive methods may help document the amount and the rate of (active) drug deposition at the desired target site. They may also assist in monitoring, prospectively and/or, on line, the probable effect of chemotherapy in an individual patient. In turn it may lead to novel methods for optimizing chemotherapeutic effectiveness at specific tumor-bearing sites and in defined treatment protocols.

Recombinant alfa interferon in renal cell carcinoma: a randomized trial of two routes of administration.

Ninety-seven patients with recurrent or metastatic renal cell carcinoma were randomized to receive recombinant interferon (IFN) alfa-2b (Intron A; Schering-Plough, Kenilworth, NJ) by either the subcutaneous (SC) or intravenous (IV) route. The SC dosage was 2 X 10(6) IU/m2 three times weekly, and the IV dose 30 X 10(6) IU/m2 for five consecutive days every 3 weeks. Dose escalation to a maximum of 10 X 10(6) IU/m2 SC and 50 X 10(6) IU/m2 IV was allowed for patients with minimal or absent toxicity. Five of 51 of the SC-treated patients (10%) and three of 46 of the IV-treated patients (7%) had a partial response (PR) or complete response (CR). Patients with prior nephrectomy, no prior treatment, and lack of bone metastases were most likely to respond, and a retrospective analysis of this subgroup revealed a 23% response rate. Achievement of response took from 3 weeks to 11 months, while response duration lasted from 3 to 31+ months. All responders had prior nephrectomy; six of eight had no prior chemotherapy or hormonal therapy; five had lung metastases, and none had bone metastases. Regardless of route, almost all patients developed a flu-like syndrome; however, grade 3 or greater toxicity was much more common for IV-treated patients. This trial demonstrates modest, but definite antitumor activity for recombinant interferon in advanced renal cell carcinoma. SC administration with lower dose and toxicity is as effective as treatment administered IV.

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.

Active-specific immunotherapy for melanoma.

Twenty-five patients with metastatic melanoma were treated with a therapeutic vaccine ("theraccine") consisting of allogeneic melanoma lysates and a novel adjuvant, DETOX (Ribi ImmunoChem Research, Inc, Hamilton, MT). Each patient received 200 antigenic units (20 x 10(6) tumor cell equivalents) subcutaneously on weeks 1, 2, 3, 4, and 6. Clinical responses included one complete remission, three partial remissions, and a long-term (17-month) stability. Two other patients had mixed responses, with partial remissions of numerous subcutaneous nodules. Sites of responsive disease included primarily the skin, but ileal, breast, and a liver metastasis also responded. Removal of residual lesions in patients with partial remissions, whose other lesions had disappeared during treatment, led to long disease-free survivals. The median duration of remission was 17 months, with four of the five responders alive for at least 24 months after treatment. An increase in precursors of cytolytic T cells (CTLs) correlated with clinical outcome, when complete, partial, and mixed responses and long-term stability were considered. The CTLs recognized melanoma-associated antigens on many cell lines, but not other types of tumor or normal lymphocytes. Skin-test reactivity to melanoma antigens and serum antibodies against the melanoma cells was unrelated to clinical response. Toxicity was minimal, restricted largely to minor soreness at the site of injection. Only five patients, four of whom were treated with repeated courses, developed severe granulomas. These results confirm that active-specific immunization with allogeneic lysates of melanoma administered with the adjuvant DETOX can induce immunity to melanoma, and can induce regressions of disease in a proportion of patients with metastatic disease with little toxicity.

A phase II study of recombinant alpha interferon in patients with recurrent or metastatic breast cancer.

Thirty-three patients with advanced breast cancer were treated with a recombinant alpha interferon (rIFN-alpha 2). All patients were ambulatory (performance status greater than or equal to 50 Karnofsky scale) and almost all had received previous chemotherapy. Large intravenous dosages of 30 to 50 X 10(6) IU/m2 were given for five consecutive days every two to three weeks to 22 patients and smaller subcutaneous dosage of 2 X 10(6) IU/m2 three times a week to 11 patients. No complete or partial responses were seen. Two patients had stable disease and the remainder progressed. Flu-like syndromes were seen in all patients. Nausea, vomiting, and anorexia were frequent. Hypotension and confusion were noted in six and five patients, respectively. Life-threatening leukopenia was noted in two patients receiving intravenous dosage and thrombocytopenia was noted in one; no sepsis or bleeding complications were noted. In this study, a highly purified and biologically active rIFN-alpha 2 was not associated with activity in previously treated women with metastatic breast cancer.

Human monoclonal antibodies that distinguish cutaneous malignant melanomas from benign nevi in fixed tissue sections.

Human monoclonal antibodies were generated by fusing a nonsecretory variant of murine myeloma cells with lymphocytes obtained from the lymph nodes of patients with metastatic cutaneous malignant melanoma. Two human IgG monoclonal antibodies, designated 2-139-1 and 6-26-3, were extensively studied for their patterns of binding to cells in 64 specimens of formalin-fixed, paraffin-embedded tissue sections. These comprised: 23 cutaneous and 2 ocular melanomas; 4 specimens of lentigo maligna; 27 benign nevi; 2 basal and 2 squamous cell neoplasms of the skin; and 4 specimens of normal skin. A direct avidin-biotin-immunoperoxidase staining method was used. Under these conditions, the antibodies reacted with variable intensity to all 18 primary cutaneous malignant melanomas, 5 metastatic cutaneous melanomas, and both ocular melanomas. Antibody 2-139-1 reacted with 1 of 4 specimens and 6-26-3 with 3 of 4 specimens of lentigo maligna. Two of 5 dysplastic nevi reacted with both antibodies, each with a smaller proportion of cells than with melanomas. There was no reactivity with the 22 other nevi representing a spectrum of histologic types or with normal melanocytes. Basal cell and squamous cell carcinomas of the skin also were not stained. These human monoclonal antibodies appear to be useful in distinguishing malignant melanomas from benign nevi, with the exception of dysplastic nevi, and from basal and squamous cancers of the skin in routinely prepared tissue sections. They may also help to identify the cytoplasmic antigens that are immunogenic in humans.

Transient focal neurologic deficits complicating interleukin-2 therapy.

We report 2 patients with transient, focal, repetitive, stereotyped episodes of neurologic deficits during treatment with interleukin-2. One patient had recurrent monocular blindness, while the 2nd had recurrent homonymous quadrantanopia. We suggest that these attacks are provoked by endothelial cell activation induced by interleukin-2.

Reduction of fatal graft-versus-host disease by 3H-thymidine suicide of donor cells cultured with host cells.

The effect of the tritiated thymidine (3H-TdR) suicide technique on the ability of donor cells to induce fatal graft-versus-host disease (GVHD) was studied. C57BL/6 (H-2b) spleen cells were stimulated in vitro with irradiated BALB/c (H-2d) Moloney lymphoma cells in mixed culture and 3H-TdR of high-specific activity added to eliminate proliferating cells. The ability of such cells to induce fatal GVHD was assayed by injecting them i.v. into adult BALB/c mice immunosuppressed with cyclophosphamide (180 mg/kg). These cells induced fatal GVHD in fewer mice (52 per cent) than did C57BL/6 cells cultures with BALB/C lymphoma cells but without 3H-TdR (87%) and C57BL/L cells cultured with irradiated C57BL/6 cells with (95 per cent) or without 3H-TdR (86 per cent). Thus, the 3H-TdR suicide technique greatly diminished the ability of cells to induce lethal GVHD.

Carney complex: report of three cases.

The authors report three patients with features of the Carney complex: cardiac myxomas (n = 2), multicentric (n = 1) or recurrent (n = 1); pigmented skin lesions (n = 2); cutaneous myxomas (n = 2); melanotic schwannoma (n = 2); and sonographic evidence of multicentric, large cell, calcifying Sertoli cell tumors of the testes (n = 1). The initial component of the complex was cutaneous myxoma (n = 1), cardiac myxoma (n = 1), or psammomatous melanotic schwannoma (n = 1). Two patients died of metastatic schwannoma.

Suppression of lymphocyte proliferation by human tumor cells grown in short term cultures.

We studied the effect of human tumor cells grown in short term cultures on the proliferative response fo normal human peripheral blood mononuclear cells (PBMC) to mitogens and alloantigens. In 6 or 7 short term cancer cell lines studied, the addition of 10(3) cells to mitogen- or alloantigen-stimulated cultures of 10(5) PBMC caused from 20 to 60% suppression of 3H thymidine incorporation. Cells from short term cultures established from biopsies of normal skin did not suppress at these concentrations. The suppression was not due to a change in kinetics, or to a decrease in viability or recovery of either the tumor cells or PBMC in the cultures. Prior treatment of tumor cells with mitomycin C abrogated the suppression, while prior X-irradiation with 1,000, 2,000 or 3,000 rads had no effect. One of the tumor cells caused suppression when separated from the PBMC by a cell-impermeable membrane while another line required cell-to-cell contact for suppression.

Antitumor effects of doxorubicin against a virally-induced rat osteosarcoma with minimal immunosuppression.

Intratibial inoculation of a Moloney strain of Murine Sarcoma Virus (MSV-M) in neonatal Wistar-Lewis rats produced osteosarcoma in 96% of animals and resulted in a median survival of 20 days. Intraperitoneal (i.p.) administration of doxorubicin (adriamycin) (1-2 mg/kg/d, on day 10-12) resulted in reduced tumor growth and prolonged median survival to 95+ and 64 days, respectively. Higher dose doxorubicin (3-4 mg/kg/d, on day 10-12) caused early lethal toxicity. Autopsy data revealed a characteristic sarcomatous tumor producing osteoid. Gross pulmonary nodules appeared in 30% of both treated and untreated animals. Microscopic evaluation of lung tissue revealed anaplastic tumors without osteoid in as many as 90% of rats. Hepatosplenomegaly was usually present but microscopic sections of the spleen did not reveal tumor. Long bone metastases were increased in frequency in those animals receiving doxorubicin. Cell mediated immunity (CMI) to osteosarcoma cells by peripheral blood lymphocytes of tumor-bearing animals was detectable between days 21-48. This was bimodal with an early peak at day 21 (CMI = 56%) and a late peak at day 39 (CMI = 48%). CMI in rats given 1 mg/kg/d x 3d of doxorubicin was similar, with peak cytotoxicity (CMI = 61%) on day 26. Two mg/kg/d x 3d of doxorubicin did not significantly suppress either the early response (CMI = 50% on day 22) or the second peak (CMI = 38% and 50% on day 40 and 46, respectively). Thus, doxorubicin was effective in decreasing the growth of an MSV-M induced osteosarcoma and prolonging survival in the rat while usually failing to suppress CMI against rat osteosarcoma cells.

Phase II study of low dose cyclophosphamide and intravenous interleukin-2 in metastatic renal cancer.

Thirteen patients with metastatic renal cancer were treated in a phase II trial with interleukin-2, 21.6 million IU/m2 intravenously daily for five days on two consecutive weeks, starting 3 days after the administration of low dose cyclophosphamide 350 mg/m2 intravenously. Treatment cycles were repeated every 21 days. No responses were seen (95% Confidence Interval: 0-22%). The most common toxicities were fever, fatigue, hypotension, nausea/emesis, and myalgia/arthralgia. There were 11 episodes of Grade III toxicity including Grade III hypotension in 7 patients. Because of the significant toxicity and the lack of observed response, the study was discontinued. Cyclophosphamide and interleukin-2 at the dose and schedule used in this study has considerable toxicity and is unlikely to improve on response rates previously seen with other IL-2 based regimens in metastatic renal cancer.