Production of tumor necrosis factor alpha and interferon gamma in interleukin-2-treated melanoma patients: correlation with clinical toxicity.

Interleukin-2 (IL-2)-based immunotherapy regimens are accompanied by dose-limiting toxicity consisting of fever, tachycardia, chills and capillary leak syndrome. We hypothesized that the toxicity was caused by the induction and release of endogenous cytokines such as tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). We measured the serum levels of TNF alpha and IFN gamma in IL-2-treated melanoma patients and attempted a correlation with clinical toxicity. A total of 23 patients received either 6 x 10(6) IU or 12 x 10(6) IU Cetus IL-2/m2 by i.v. bolus daily for 5 consecutive days on weeks 1, 3 and 5. Serum TNF alpha and IFN gamma levels were measured by enzyme-linked immunosorbent assay. Clinical toxicity was scored each day by objective measurements of hypotension, tachycardia, fever and chills/rigors. Clinical toxicity and IFN gamma levels correlated nicely, peaking on the 5th day of each treatment cycle. The kinetics and magnitude of TNF alpha production, however, were not predictable and did not correlate with either IFN gamma or toxicity. Some patients had modest increases in TNF alpha production while others had markedly increased levels during the second and third treatment weeks. Remarkably, these high levels persisted during nontreatment weeks and after completion of therapy. This clinical study demonstrates novel kinetics for immunoreactive TNF alpha in IL-2 cancer patients, which do not correlate well with toxicity.

Cyclophosphamide-induced alterations in human monocyte functions.

This investigation was designed to study the effects of relatively low doses of cyclophosphamide (CY) on monocyte function in patients with surgically resected melanoma. Monocytes taken from patients 3 days after receiving 300 mg, 150 mg, or 75 mg CY/m2 had decreased interleukin-1 (IL-1) production. Production of tumor necrosis factor (TNF)-like molecules by the same monocytes appeared to be enhanced following 300 mg/m2 CY but not after 150 or 75 mg/m2 CY. In vitro studies of the direct effects of CY metabolites (mafosfamide and 4-hydroperoxycyclophosphamide) on human monocytes showed only concomitant decreases in production of IL-1 and TNF-like molecules. This occurred at concentrations that did not obviously affect viability, although monocyte spreading was inhibited. No evidence was obtained for in vitro enhancement of TNF-production. We conclude that CY can affect monocyte function. In vivo it may have both direct effects leading to decreased TNF and IL-1 production and indirect effects through lymphocytic or haematopoietic systems that activate monocytes to enhanced TNF production. The effects are dose-dependent. These CY-induced changes could be responsible in part for some of the alterations in host immunity and tumor resistance that follows administration of the drug.

Analysis of mammary tumour cell metastasis and release of bound n-acetylneuraminic acid.

A tumour model consisting of the highly metastatic mammary 13762 parental line, the non-metastatic and 6-thioguanine-resistant (TgR) variant line, and two TgR revertant lines (TgRrev, TgRrevM) that were occasionally metastatic, were used to determine whether the release of N-acetylneuraminic acid (NANA) was related to tumour metastasis. For comparative purposes, the occasionally metastatic R3230AC and the nonmetastatic DMBA8 tumour lines were studied. The NANA was considered to be in bound form, because acid hydrolysis was required to release it for high-pressure liquid chromatographic analyses. Sera of animals bearing the 13762 and R3230AC tumours had high levels of bound NANA. No differences were found in serum NANA levels in animals bearing metastatic or non-metastatic R3230AC tumours. Low levels of bound NANA were found in animals bearing the other tumour lines regardless of whether metastasis occurred or not. The experiments in vitro substantiated the in vivo findings. The phenotypic expression of bound NANA shedding did not correlate well with metastatic potential of the mammary tumour line. Our analyses suggest that this phenotypic marker cannot be used as a reliable indicator of metastasis.

Increased metastatic ability and bone formation of a mammary adenocarcinoma in vivo after in vitro passaging.

On in vitro passaging of the rat mammary adenocarcinoma R3230AC cell line, phenotypic changes occurred that were expressed in vivo. The histology of this mammary adenocarcinoma changed to a fibrosarcoma and then to an osteosarcoma. The overall population of early passaged cells consisted of a mixture of predominantly epithelial-like cells and few fibroblast-like cells; however, later passaged cells consisted more of the latter type. Along with the changes in histology, the tumor line also became highly metastatic in animals after in vitro passaging. The etiology of these phenotypic changes was not determined. Cytogenetic studies revealed chromosome changes in cells of later passages. Cells of 90 or greater passages that produced bone tumors were found to have fewer chromosomes and a metacentric marker isochromosome. In this report a correlation is made between the aberrant change in histology, increased metastatic ability and the presence of a marker chromosome of the R3230AC tumor.

Circulating immune complexes in rats bearing 6-thioguanine-resistant variants of the 13762 mammary adenocarcinoma.

The relationship between immune complex (IC) formation and tumor cell metastatic potential was investigated in rats inoculated in the footpad with parental 13762 mammary adenocarcinoma cells or 6-thioguanine-resistant (TGR) variant cells. These cell lines are either highly metastatic (13762), nonmetastatic (TGR), or occasionally metastatic ( TGRrev , TGRrevM ). The 13762 TGR rat tumor model thus provides the opportunity to examine host immune responses to tumor cells of different phenotypes, but derived from the same parent tumor line. IC levels were low in 13762 tumor-bearing rats. In contrast, animals with TGR tumors had high levels of ICs in their sera, while animals bearing TGRrev and TGRrevM tumors had intermediate levels of ICs. In this rat tumor model system, IC formation is inversely related to the metastatic potential of the tumor lines.

Circulating immune complexes and immunoglobulin M-class rheumatoid factor in rats bearing mammary adenocarcinomas which vary in ability to metastasize.

In order to explore whether immune complex (IC) formation and immunoglobulin M-class rheumatoid factor (RF) synthesis are related to tumor progression, solid-phase enzyme immunoassays were used to test for ICs and RF in rats bearing three different syngeneic mammary adenocarcinomas. The mammary adenocarcinoma cell lines used produced either extensive metastasis (13762), metastasis in only a proportion of the animals given injections (R3230AC), or no metastasis (DMBA8). DMBA8 and 13762 tumor-bearing rats developed only low levels of circulating ICs. Of 18 animals bearing R3230AC tumors, four developed palpable lymph node metastasis (macrometastasis), while another five showed evidence of metastasis only upon histological examination (micrometastasis). R3230AC tumor-bearing animals which did not develop metastasis were found to have significantly higher IC levels than those rats with metastasis. Several sera from rats bearing R3230AC tumors were fractionated by molecular sieve chromatography. Most of the ICs in these sera were 7S to 19S in size. Significant RF synthesis occurred only in rats bearing R3230AC tumors and only during terminal tumor growth. These results show that IC formation and RF synthesis varies in animals bearing different mammary adenocarcinomas.