Disseminated human malignant melanoma in congenitally immune-deficient (bg/nu/xid) mice.

Congenitally immune-deficient bg/nu/xid (BNX) mice are severely compromised in their ability to mount T-cell, B-cell, and lymphokine-activated killer (LAK) cell responses. Successful engraftment of BNX mice with human hematopoietic stem cells has been demonstrated recently. We have investigated the potential use of BNX mice for studies relating to the biology and immunotherapy of human malignant melanoma. The intravenous injection of fresh single-cell suspensions of human malignant melanomas into mice resulted in widely disseminated disease. Metastatic spread of human melanoma in BNX mice mimicked that observed in patients: eg, there were numerous tumor nodules identified in the subcutaneous tissues as well as in a variety of visceral organs, including spleen, kidneys, thyroid, adrenals, lungs, heart, and brain. BNX mouse lymph nodes were replaced consistently by human malignant melanoma cells. The presence of human tumor cells in these mice was confirmed by histologic analysis and microcytofluorometry analyses using human melanoma-specific monoclonal antibodies (MAbs). Moreover, human melanoma cells passaged in BNX mice remained lysable in vitro by specifically cytolytic, autologous human tumor-infiltrating lymphocytes (TILs). The capacity of fresh human malignant melanoma to disseminate widely in BNX mice may prove valuable not only for study of the biology of metastatic spread but also for studies of the immunotherapy of human melanoma using melanoma-specific MAbs and chemotherapeutic agents, as well as human TILs and LAK cells with or without retrovirus-mediated gene transfer modification.

Culture of human tumor infiltrating lymphocytes in hollow fiber bioreactors.

Human tumor infiltrating lymphocytes (TIL) from metastatic melanoma of six patients were grown using a new hollow fiber bioreactor system. After inoculating 0.35-10 X 10(8) TIL into the extra-fiber space (EFS), each Cellmax bioreactor was perfused with AIM-V medium, supplemented with rIL-2. The cells subsequently expanded 124-1170-fold to yield 1.5-5.4 X 10(10) TIL over a 14-32 day period. TIL were flushed from the EFS using 200 ml medium and possessed an average viability = 91%. The phenotype and the autologous tumor cell lytic capacity of these TIL were similar to those of TIL grown in the currently used gas-permeable culture bags. Tissue culture media use averaged 4.3 liters/10(10) TIL harvested. The TIL of one patient were re-expanded twice from cells remaining within the same bioreactor after harvest suggesting that one bioreactor cartridge could be used for repetitive, periodic studies. An estimated 80% decrease in technical time expended and in incubator space requirements were realized using this methodology. Cell culture on hollow fibers appears to be a useful method for producing large quantities of primary human lymphocytes for experimental, and perhaps, therapeutic needs.

Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients.

We have administered 1039 courses of high-dose interleukin-2 (IL-2) to 652 cancer patients. Five hundred ninety-six patients had metastatic cancer that either had failed standard effective therapies or had disease for which no standard effective therapy existed, and 56 patients were treated in the absence of evaluable disease in the adjuvant setting. IL-2 was administered either alone (155 patients) or in conjunction with activated immune cells such as lymphokine activated killer (LAK) cells (214 patients) or tumor infiltrating lymphocytes (TIL) (66 patients), with other cytokines such as alpha interferon (a-IFN)(128 patients) or tumor necrosis factor (TNF)(38 patients), with monoclonal antibodies (32 patients), or with the chemotherapeutic agent cyclophosphamide (19 patients). Initial results with the treatment of high-dose IL-2 alone or in conjunction with LAK cells have indicated that objective regressions of cancer can be achieved in 20% to 35% of patients with selected advanced metastatic cancers. Although most responses have been seen in patients with metastatic renal cell cancer, melanoma, colorectal cancer, and non-Hodgkin's lymphoma, many histologic types of cancer have not been treated in significant numbers. These regressions can be durable; of 18 patients achieving a complete response, ten have not experienced recurrence at intervals from 18 to 52 months. Although combinations of IL-2 with TNF do not appear to result in increased responses, there is a suggestion in our initial phase I studies that the combination of a-IFN and IL-2 is more effective than the administration of cytokine alone and this combination deserves further study. Similarly the adoptive transfer of TIL in conjunction with IL-2 also appears to be more effective than the use of IL-2 alone. The toxic side effects in patients treated with high-dose IL-2 are presented and include malaise, nausea and vomiting, hypotension, fluid retention, and organ dysfunction. Treatment-related deaths were seen in 1% of all treatment courses and in 1.5% of patients. These studies demonstrate that a purely immunologic manipulation can mediate the regression of advanced cancers in selected patients and may provide a base for the development of practical, effective biologic treatments for some cancer patients.

Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. A preliminary report.

Lymphocytes extracted from freshly resected melanomas can be expanded in vitro and can often mediate specific lysis of autologous tumor cells but not allogeneic tumor or autologous normal cells. We treated 20 patients with metastatic melanoma by means of adoptive transfer of these tumor-infiltrating lymphocytes and interleukin-2, after the patients had received a single intravenous dose of cyclophosphamide. Objective regression of the cancer was observed in 9 of 15 patients (60 percent) who had not previously been treated with interleukin-2 and in 2 of 5 patients (40 percent) in whom previous therapy with interleukin-2 had failed. Regression of cancer occurred in the lungs, liver, bone, skin, and subcutaneous sites and lasted from 2 to more than 13 months. Toxic effects of interleukin-2 occurred, although the treatment course was short (five days); these side effects were reversible. It appears that in patients with metastatic melanoma, this experimental treatment regimen can produce higher response rates than those achieved with interleukin-2 administered alone or with lymphokine-activated killer cells. It is too early to determine whether this new form of immunotherapy can improve survival, but further trials seem warranted.

Effect of split doses of radiation on mutation frequency in rodent cell lines.

Mutation frequencies in mouse cells and Chinese hamster cells were measured following single or split doses of UV light or X-rays. Split doses separated by a few to over 24 h induced no more ouabain- or 6-thioguanine-resistant mutants than did comparable single doses. These data lend no support to the possible existence of an inducible error-prone repair system in rodent cells.

Mutation induction by 5-fluorodeoxyuridine in synchronous Chinese hamster cells.

Chinese hamster ovary cells synchronized by mitotic detachment were treated with 5-fluorodeoxyuridine (FdUrd), 0.2 micrograms/ml, at various times in the cell cycle. FdUrd treatment in the early S period induced resistance to 6-thioguanine. Treatment of asynchronous cells with FdUrd caused little increase in 6-thioguanine resistance over the spontaneous frequency. Mitotic selection thus enhances the probability of a cell being in the portion of S period receptive to mutation. These observations suggest that FdUrd treatment leads to mutagenesis at the growing points of DNA replication.

Bromodeoxyuridine-induced mutations in synchronous Chinese hamster cells: temporal induction of 6-thioguanine and ouabain resistance during DNA replication.

Mutations were induced in synchronous Chinese hamster cells by bromodeoxyuridine (BUdR) incorporated into cells for one-hour periods in the cell cycle. There was a very pronounced temporal dependence during the first half of the DNA synthesis period for the induction of damage leading to 6-thioguanine (6TG) and ouabain resistance. No mutants above background were induced by exposure to BUdR in G1 and G2 cells, and very few mutants were induced in the latter part of the DNA synthesis period. The peak for the induction of 6TG resistance occurs at about two hr in the DNA synthesis period; one hour later there is a peak for the induction of ouabain resistance. Both peaks occur before the time of maximum incorporation of BUdR into DNA. These results suggest that the mutagenesis by BUdR is associated with at least two nuclear genes, which replicate at two hr and three hr in the DNA synthesis period.

Enhancement of postreplication repair in ultraviolet-light-irradiated Chinese hamster cells by irradiation in G2 or S-phase.

Postreplication repair in synchronous Chinese hamster cells was determined after split doses of ultraviolet (UV) radiation. Repair was enhanced by irradiation of cells in G2 or S-phase with a small dose of UV radiation at least 1.5 h before a three-fold larger dose of UV. There was significantly greater enhancement when the first dose was given in G2 than when it was given in the S-phase 0.5-1.5 h before the test dose. These data indicate that enhancement of postreplication repair does not require active DNA replication and qualitatively is independent of when in the cell cycle the cells are irradiated.

Mutagenic mechanism of 5-bromodeoxyuridine in Chinese hamster cells.

Resistance to 6-thioguanine was induced by 5-bromodeoxyuridine (BUdR) in synchronous Chinese hamster cells. The yield of mutant colonies was not proportional to the amount of BUdR incorporated into DNA; thus mutants were not due to mispairing of BUdR with guanine during replication. Few mutants were induced until BUdR concentrations exceeded that of the intracellular thymidine triphosphate pool and mutant yield was depressed by addition of thymidine to the medium. These data suggest that BUdR exerts an allosteric effect on the DNA synthesizing system which renders it more error prone.