Intraperitoneal lymphokine-activated killer cell/interleukin-2 therapy in patients with intra-abdominal cancer: immunologic considerations.

Lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2) were administered by the ip route to patients with intra-abdominal malignancies. Pharmacokinetic studies of IL-2 revealed 10- to 100-fold higher concentrations of IL-2 in peritoneal fluid versus serum. Ip levels of IL-2 were maintained well above those required to generate and maintain LAK cells in vitro. LAK cell activity was detectable in the peritoneal fluid for the duration of each treatment cycle and did not disappear until IL-2 was discontinued. Detection of interferon-gamma (IFN-gamma) in the peritoneal fluid of all patients was consistent with production in situ by activated lymphocytes. In some patients, low but detectable levels of IFN-gamma were also found in the serum. In vivo activation of monocytes in the peritoneal fluid as measured by in vitro production of hydrogen peroxide was documented in the majority of patients. Neither interleukin-1 nor tumor necrosis factor-alpha was detected in the peritoneal fluid. We found no correlation between the presence or levels of IL-2, IFN-gamma, or LAK cell lytic activity in peritoneal fluid or serum and response or nonresponse to therapy.

Hemocytometer leukocyte adherence inhibition technique.

The hemocytometer leukocyte adherence inhibition technique was employed in a criss-cross experimental design with two cancer patients (melanoma and colon carcinoma) and the corresponding tumor extracts. These extracts had been repeatedly tested for specific reactivity and lyophilized before transport to the workshop. The patients' leukocytes were mixed singly with each extract in the presence of normal serum, and the adherences of the cells were determined in hemocytometer chambers. Actual cell counts (total cells before washing and adherent cells after washing) are given in detail for the first time. Blood samples and reaction mixtures were coded by an independent observer. Determination of mean % adherence (+/- S.E.) showed that the melanoma patient's leukocytes (original adherence 70.8 +/- 2.8) reached with the melanoma extract (40.7 +/- 2.9; p less than 0.001) but not significantly with the colon carcinoma extract (61.5 +/- 4.1; p greater than 0.05). Similarly, the colon carcinoma patient's leukocytes (original adherence 68.6 +/- 2.7) reacted with the colon carcinoma extract (43.2 +/- 2.3; p less than 0.001) but adherence was not inhibited by the melanoma extract (76.9 +/- 2.6). The cancer patients were thus correctly identified with regard to tumor type in a simple blind trial.

Lymphokine-activated killer cells: culture conditions for the generation of maximal in vitro cytotoxicity in cells from normal donors.

The current method for generating lymphokine-activated killer (LAK) cells for use in human clinical trials is both labor intensive and expensive. Therefore, we altered cell culture conditions to determine whether LAK cells with enhanced lytic activity could be generated. Culture of normal human peripheral blood leukocytes for 7 days generated LAK cells with 4-fold more lytic activity than culture for 3 days. Although cell viability over this 7-day period dropped from 94% on Day 3 to 73% by Day 7, the recovery of cells from culture increased from 61 to 106%. If cells were exposed to CO2, lytic activity was further enhanced by up to 30-fold. Culture at a density of 1 or 2.5 X 10(6) cells/ml caused no difference in cell viability, recovery, or LAK activity when cells were cultured for up to 4 days; however, when cells were cultured for longer times, an initial density of 1 X 10(6) cells/ml yielded maximal LAK activity. Several commercially available serum-free defined media as well as human serum albumin supported LAK cell activation comparable to serum-containing media over a 4-day culture period. One defined medium, AIM V, supported LAK cell activation over a 7-day period even when cells were cultured at a density twice as high (2 X 10(6) cells/ml) as cells cultured in serum-containing medium. The results demonstrate that simple manipulation of human LAK cell culture conditions generates cells with greatly enhanced lytic activity and that serum-containing medium may not be necessary for generating LAK cells under the current clinical protocols.

An evaluation of leukocyte adherence inhibition in the immunodiagnosis of colorectal cancer.

The leukocyte adherence inhibition technique was used to assess cell-mediated immunoreactivity and serum-blocking factors related to adenocarcinoma of the colon or rectum. In the group of 48 patients with confirmed tumors of this type, 36 of 38 had reactive leukocytes and 46 of 47 had serum-blocking factors. Patients whose tumors had been removed surgically, with no sign of recurrence, retained their leukocyte activity for up to 3.5 years in 6 of 6 cases, but only a small proportion (7 of 30) retained blocking factors. In 67 controls (who were patients with nonmalignant gastrointestinal disorders, patients with gastrointestinal tumors other than colorectal adenocarcinoma, patients with other cancers, or healthy volunteers), negative reactions were obtained, with diverticular disease the only prominent exception. The leukocyte adherence inhibition test appeared to be highly sensitive and specific. Application to the immunodiagnosis of colorectal cancer thus seems to be warranted.

Immunomodulatory properties and toxicity of interleukin 2 in patients with cancer.

We performed a phase Ia/Ib study of interleukin 2 (IL2) in patients with cancer. Single doses of IL2 from 10(3) units/m2 to 10(7) units/m2 were well tolerated but failed to induce significant immunological changes. Chronic IL2 treatment for 5 days out of 7 for 3 weeks was well tolerated at doses below 10(7) units/m2 and was accompanied by significant immunological changes. Following chronic treatment with intramuscular injections of IL2 at 1 x 10(6) units/m2, we observed augmentation of peripheral blood natural killer activity and induction of peripheral blood LAK activity. Induction of LAK activity was most evident when IL2 was included in the cytotoxicity assay. There was a marked increase in the number of peripheral blood mononuclear cells bearing the Leu-19 marker in association with the observed increases in natural killer and LAK activity. A small percentage of Leu-19+ cells coexpressed CD3. There was heterogeneous expression of the low affinity Fc receptor (CD16). In vivo induced Leu-19+ cells could be divided into two populations, dim and bright, based on the intensity of fluorescent staining with antibodies to Leu-19. The majority of Leu-19 bright cells were CD16- while the majority of Leu-19 dim cells were CD16+. In addition, the intensity of CD16 staining was higher for Leu-19 dim cells than for Leu-19 bright cells. Increases in the amounts of CD38 and CD8 antigens were also observed. Significant increases in serum levels of the soluble IL2 receptor were observed during treatment. One partial remission was noted in a woman with non-Hodgkin's lymphoma.

The determination of an immunologically active dose of interferon-gamma in patients with melanoma.

This study was undertaken to determine an immunologically active regimen for the administration of recombinant gamma-interferon (rIFN-gamma). The patient population included patients with completely resected melanoma, stage I (Clark's level IV or V) or stage II. All patients exhibited no evidence of disease (NED) at the time of the study. Patients received rIFN-gamma by intramuscular (IM) injection daily for 15 days at 0.0001 mg/m2, 0.001 mg/m2, 0.01 mg/m2, 0.1 mg/m2 (ten patients/group), or 0.25 mg/m2 (five patients). Interferon (IFN) was well tolerated, with non-dose-limiting constitutional symptoms occurring in the majority of patients at 0.1 mg/m2 and 0.25 mg/m2. All five patients receiving 0.25 mg/m2 developed elevated transaminase levels, which led to a discontinuation of therapy in one patient. Immunological activity was assessed by serial measurements of natural killer (NK) cell activity, hydrogen peroxide production by monocytes, and changes in expression of Fc receptors and human leukocyte class II antigen (HLA-DR) on monocytes. These changes were determined at baseline (X2), six to seven time points during rIFN-gamma therapy, and two times after the last dose of rIFN-gamma. No changes were observed at the two lowest doses. At the 0.01 mg/m2 dose, all parameters were elevated but not as consistently nor to the same levels as seen following administration of 0.1 mg/m2. At 0.25 mg/m2, H2O2 production was enhanced, but unlike at 0.1 mg/m2, it declined during the last few days of IFN therapy. Subcutaneous (SC) administration was compared with IM administration using the 0.1 mg/m2 dose. SC administration resulted in enhanced H2O2 production and Fc receptor expression by monocytes. More consistent elevations in peroxide generation and higher levels of Fc receptor expression were seen following SC administration. No significant difference was found between the two routes of administration. A comparison of two schedules, daily and three times weekly, suggested that monocyte activation may return to normal 72 hours after IFN administration. Of the doses tested, 0.1 mg/m2 administered daily appeared to be the most effective biological response modifier (BRM) regimen, and because of ease of administration, we favor the SC route.

Recombinant leukocyte A interferon therapy for advanced hairy cell leukemia. Therapeutic and immunologic results.

Hairy cell leukemia is a lymphoproliferative disorder characterized clinically by cytopenias. Standard therapy following variable periods of disease stability consists of splenectomy that often restores normal hematologic parameters for periods ranging from weeks to years. Fifteen patients (five without prior splenectomy or chemotherapy) were treated with 3 X 10(6) units per day of recombinant leukocyte A interferon and 14 of 15 patients completed eight weeks of therapy and were evaluated for response. There was one complete and 12 partial responses for an overall response rate of 93 percent. All of these patients' conditions have remained in complete or partial remissions and they continue to receive interferon with a median follow-up of six months. Coincident with the normalization of peripheral blood counts was a return of natural killer activity and normalization of immunologic surface markers as determined by monoclonal antibodies. This study confirms and extends earlier observations with natural alpha-interferon and indicates that recombinant leukocyte A interferon in low daily doses is also very effective treatment for hairy cell leukemia. In fact, it may be the best single modality of therapy for inducing both hematologic and immunologic recovery of these patients and deserves consideration as initial therapy.

Evaluation of the immunogenicity of freeze-dried skin allografts in humans: cell-mediated response.

PATIENTS RECEIVING freeze-dried skin (FDS) allografts were evaluated for cell-mediated response by a lymphocytotoxicity test. Eleven patients received single or multiple FDS allografts from a donor typed for human leukocyte antigen (HLA). Heparinized blood samples were drawn before the procedure and at 2, 4, and 8 weeks after grafting. Mononuclear cells were isolated by Ficoll-Hypaque gradients. Cell-mediated lymphocytotoxicity tests were conducted, with mononuclear cells from the FDS allograft recipients used as effector cells. Phytohemagglutinin-stimulated blast target cells of the same HLA type as that of the skin donor were labeled with chromium 51. Lympholysis was evaluated by measuring the amount of 51Cr release after 18 hours' incubation with target/effector cell ratios of 1:100, 1:50, and 1:25. Negative control wells contained target cells alone. Positive control wells contained target cells and effector cells sensitized in vitro against the target cells. Viability of effector cells was tested by trypan-blud dye exclusion and response to phytohemagglutinin. None of the experimental blood samples showed 51Cr release significantly greater than shown by the negative controls. Our findings of no cell-mediated response and the findings of a previous study showing no production of anti-HLA antibody in response to FDS allografts indicate that allogeneic FDS is an immunologically safe material for use in periodontal surgical procedures.

Immunological monitoring of clinical trials using biological response modifiers.

Phase I clinical trials of Biological Response Modifiers must be designed to provide information not only regarding toxicities, pharmacokinetics and anti-tumor response, but also on their many immunomodulatory properties. If BRMs are to be used rationally, then detailed standardized data must be obtained in such a way as to enable meaningful conclusions to be drawn from the monitoring assays. Assay selection, timing for monitoring and methods for data interpretation are important considerations in the design of such trials.

Assessment of biological responses: what to measure and when.

During the past several years, increasing attention has been devoted to the use of biological response modifiers (BRMs) for the treatment of cancer. Phase I trials of BRMs must be designed to provide information not only regarding toxic effects, pharmacokinetics, and antitumor activities, but also on the many immunomodulatory effects. Biological monitoring must be carefully planned to enable meaningful conclusions to be drawn as to the optimal dose, schedule, and route of administration. Important considerations include selection of assays for the various biological effects, timing of testing following BRM administration, and methods for data interpretations.

The cellular mechanism of leukocyte adherence inhibition.

Human blood leukocytes from three subjects who had been contact sensitized to dinitrochlorobenzene were used in direct and indirect leukocyte-adherence-inhibition (LAI) reactions in an attempt to elucidate the cellular mechanism of reactivity. The leukocytes were separated and purified by standard procedures. In direct LAI, only T cells or populations containing T cells gave positive reactions (significantly reduced adherence) with the antigen. Supernatants from suitable leukocyte-antigen mixtures contained a soluble leukocyte-adherence-inhibition-factor (LAIF) that reduced the adherence of normal leukocytes. Only T cells or populations containing T cells were active in LAIF production; B cells, granulocytes, and monocytes were inactive. The cellular requirement for the action of preformed LAIF was not restricted: all major types of blood leukocytes were susceptible to its effect.

Response of warts in epidermodysplasia verruciformis to treatment with systemic and intralesional alpha interferon.

The susceptibility of human papillomavirus infection to polyclonal human leukocyte interferon (IFN-alpha) has been evaluated in patients with epidermodysplasia verruciformis (EV), a disease with extensive chronic papillomavirus-induced warts. In a double-blind, placebo-controlled study with intralesional IFN-alpha, four of five IFN-alpha-treated warts regressed; none of the placebo-treated warts responded (p = 0.024). Three patients with EV were treated with systemic IFN-alpha for 4 weeks in an open study, achieving partial regression of warts in all three. In a double-blind, placebo-controlled study, warts in two children with EV regressed with systemic IFN-alpha while two who received placebo showed no improvement. The lesions recurred following cessation of therapy. At the completion of therapy with IFN-alpha, histologic normalization was accompanied by a 95% decrease in the number of viral antigen-containing cells in the warts (p less than 0.001). We conclude that warts in EV respond to systemic and intralesional IFN-alpha.

Deficient natural killer cell (NK) activity and macrophage functioning in schizophrenic patients.

The cytotoxic potential of mononuclear blood cells from 27 chronic schizophrenic patients was evaluated using quantitative measures of natural killer cell activity and macrophage inhibition of tumor cell growth. 44% of the patients had some evidence of deficient in vitro mononuclear cell function which was not correlated with a qualitative or quantitative alteration in the histologic appearance of these peripheral blood cells.

Augmentation of E-rosette formation by lymphocytes of cancer patients stimulated in vitro with tumor extracts.

The ability of tumour extracts to augment E-rosette formation by cancer patients' lymphocytes was demonstrated for colorectal carcinoma, breast carcinoma and melanoma. Positive augmentation reactions were obtained with 14 to 20 patients tested with extracts related to their own tumour types. Some lack of specificity was suggested by 5 positive reactions in the same patients tested with unrelated extracts. No false positives were found in normal subjects. The technique is simple, rapid and appears to depend on tumour-associated antigens in the extracts. Simultaneous leucocyte adherence inhibition tests on split samples of blood had a high degree of sensitivity and specificity, confirming the potential reactivity of the leucocytes and extracts used.

Leukocyte adherence inhibition and specific immunoreactivity in malignant melanoma.

The leukocyte adherence inhibition (LAI) test has been used to assess specific anti-tumour immunoreactivity in 80 patients with malignant melanoma, 21 of whom had apparently been successfully treated by surgery, and 44 control subjects. Reaction with melanoma extracts in vitro enabled the activity of blood leukocytes to be detected by inhibition of their adherence to glass, while serum was tested for factors which modified this inhibition. Of the patients with tumours (ranging from primary melanoma in situ to advanced disseminated disease), 22/24 had active leukocytes and 50/58 has serum blocking factor; two of the sera, from patients with regressing tumours were unblocking. After surgery with no clinical recurrence, leukocytes continued to be active except when tested several years after operation. Blocking factor rapidly disappeared in 16/20 patients tested, and in several patients examined serially the serum became unblocking. In three cases, persistence of serum blocking was followed by clinical diagnosis of metastases. Leukocyte activity was nerver detected in control subjects (0/10), many of whom had other kinds of tumours or skin lesions. Blocking activity in serum was found in only 3/38 controls with no history of melanoma (1 had a fibrosing cellular blue naevus and 2 had liver disease). Thus the LAI test correlated well with clinical and pathological findings, and shows great promise for the reliable, rapid and specific immunodiagnosis of malignant melanoma.

Effects of recombinant interferon-alpha on immune function in cancer patients.

Patients with a variety of malignancies were treated in phase I clinical trials of recombinant leukocyte A interferon (IFL-rA) schedules consisting of either twice-daily doses for 28 days (48 patients) or three times weekly for 28 days (86 patients). Extensive monitoring of several immune functions was done on these patients, with rigorously standardized assays and determination of inherent variability of function for each individual. The assays performed were natural killer (NK) cell cytotoxicity, monocyte cytostatic activity, lymphoproliferative responses to concanavalin A (con A) and mixed leukocyte culture, and an analysis of changes in leukocyte populations as determined using a panel of monoclonal antibodies and flow cytometry. No appreciable increase in NK activity was observed on any of the treatment regimens, and an unexpected observation was the depression of activity in a substantial proportion (30%) of patients. In contrast, monocyte function, as measured in an assay of growth inhibition of tumor target cells, was found to be elevated in 70% of the patients. Lymphoproliferative responses were depressed in most patients in response to both con A and the mixed leukocyte culture. Cell surface marker studies revealed an increase in the percentage of OKT10 positive cells in the majority of patients studied and a transient increase in cells reacting with the antibody MO2. The data have been analyzed in terms of their relationship to dose and schedule of administration, and the depression of NK activity was shown to be greatest at the highest doses and more frequent schedule of administration.

Serum soluble IL-2 receptor as a tumor marker in patients with hairy cell leukemia.

Activated T cells synthesize and express a cell membrane-bound receptor for interleukin-2 (IL-2) and have recently been shown to secrete a soluble form of the same receptor. Hairy cell leukemia is a chronic disorder caused by expansion of a clonal population of an unusual mononuclear cell of B cell origin. These cells have previously been shown to express an IL-2 receptor on the cell membrane. The sera of 26 patients with hairy cell leukemia were examined for the presence of a soluble IL-2 receptor before and during therapy with either recombinant interferon alpha-2a or 2'-deoxycoformycin. Before therapy, all patients had markedly elevated levels of this soluble IL-2 receptor ranging from five to 60 times the highest level observed in normal control sera. In individual patients changes in the level during therapy correlated well with clinical assessments of tumor response; levels fell to near the normal range in patients responding to therapy. Patients not responding to interferon alpha had no significant change in the soluble IL-2 receptor level. These results suggest that hairy cells secrete a soluble IL-2 receptor and that serial measurements of the level of this receptor in the serum can be used as a noninvasive means to assess disease response to therapy.

Specific immunodiagnosis of hepatocellular carcinoma by leucocyte adherence inhibition.

The leucocyte adherence inhibition test provides a rapid, reliable, and specific technique for the immunodiagnosis of primary hepatocellular carcinoma (malignant hepatoma). The patient's blood leucocytes are tested in vitro for cell-mediated immunity against tumour-associated antigens and the serum is tested for blocking factor which interferes with the immunological reaction. Specific reactivity of both leucocytes and serum was consistently detected in patients with malignant hepatoma, and negative reactions were obtained in other liver diseases including secondary tumours of the liver. The test has provided positive evidence for the presence of hepatoma when more conventional methods gave doubtful or negative results. A positive result preceded the clinical appearance of tumour by up to three years in two patients.