Randomized phase II trial of BCDT [carmustine (BCNU), cisplatin, dacarbazine (DTIC) and tamoxifen] with or without interferon alpha (IFN-alpha) and interleukin (IL-2) in patients with metastatic melanoma.

The purpose of this study was to evaluate in a randomized phase II trial the efficacy and toxicity of combination biochemotherapy compared with chemotherapy alone in patients with metastatic melanoma. Sixty-five patients with metastatic melanoma (ECOG performance status 0 or 1) were randomized to receive intravenous BCNU 100 mg m(-2) (day 1, alternate courses), cisplatin 25 mg m(-2) (days 1-3), DTIC 220 mg m(-2) (days 1-3) and oral tamoxifen 40 mg (BCDT regimen) with (n = 35) or without (n = 30) subcutaneous interleukin 2 (IL-2) 18 x 10(6) iu t.d.s. (day - 2), 9 x 10(6) iu b.d. (day - 1 and 0) and interferon 2 alpha (IFN-alpha) 9 MU (days 1-3). Evidence for immune activation was determined by flow cytometric analysis of peripheral blood lymphocytes. Treatment was repeated every 4 weeks up to six courses depending on response. The overall response rate of BCDT with IL-2/IFN-alpha was 23% [95% confidence interval (CI) 10-40%] with one complete response (CR) and seven partial responses (PR), and for BCDT alone 27% (95% CI 12-46%) with eight PRs; the median durations of response were 2.8 months and 2.5 months respectively. Sites of response were similar in both groups. There was no difference between the two groups in progression-free survival or overall survival (median survival 5 months for BCDT with IL-2/IFNalpha and 5.5 months for BCDT alone). Although 3 days of subcutaneous IL-2 resulted in significant lymphopenia, evidence of immune activation was indicated by a significant rise in the percentage of CD56- (NK cells) and CD3/HLA-DR-positive (activated T cells) subsets, without any change in the percentage of CD4 or CD4 T-cell subsets. Toxicity assessment revealed a significantly higher incidence of severe thrombocytopenia in patients treated with combination chemotherapy than with chemotherapy alone (37% vs 13%, P = 0.03) and a higher incidence of grade 3/4 flu-like symptoms (20% vs 10%) and fatigue (26% vs 13%). The addition of subcutaneous IL-2 and IFNalpha to BCDT chemotherapy in a randomized phase II trial resulted in immune activation but did not improve response rates in patients with metastatic melanoma, and indeed may increase some treatment-related toxicity.

An analysis of the normal ranges of lymphocyte subpopulations in children aged 5-13 years.

UNLABELLED: Peripheral blood lymphocytes subsets were examined in 233 healthy school children aged 4.9-13.7 years at the time of examination. Lymphocyte subsets were characterized according to the following cluster of differentiation (CD) numbers: CD2, CD4, CD8 and CD19 and quantified according to both their absolute number and as a percentage of total lymphocytes. For the purpose of analysis, the LMS (lambda, mu, sigma) method was utilized, with a 3%-97% confidence interval. Smoothing of the generated curves, was by multiple regression analysis, using the least squares method. The results of these analyses indicate distinct trends as a child ages, both in absolute numbers and in the percentage of each cell type. CONCLUSION: We characterized lymphocyte subsets in children aged 4.8-13.7 years. These data should prove of considerable value to pediatricians dealing with patients with known or suspected immunological problems, and ought to be used in place of the commonly used, but inappropriate, adult lymphocyte subset ranges.

Increased soluble interleukin-2 receptor concentration in plasma predicts a decreased cellular response to IL-2.

Interleukin 2 (IL-2) immunotherapy has met with limited success in the treatment of renal cell carcinoma (RCC) and malignant melanoma (MM). However, non-responders still account for up to 80% of those patients receiving IL-2. A high concentration of soluble IL-2 receptor (sIL-2R) is commonly found in the blood of such patients. We investigated the possibility that high sIL-2R concentration pretreatment may interfere with the bioavailability of IL-2. The mean concentration of sIL-2R in plasma from patients with MM, RCC and head and neck cancer was 3378 U ml-1, 8778 U ml-1 and 764 U ml-1 respectively, compared with 1315 U ml-1 in plasma from healthy volunteers. Inclusion of plasma from patients with RCC and MM patient plasma in cytotoxic T-lymphocyte leukaemic (CTLL) cell/IL-2 assays inhibited the ability of CTLL cells to respond to IL-2, and an inverse correlation was found between the concentration of sIL-2R and the growth response of CTLL cell to IL-2 (r = -0.86, P = 0.003). Plasma with soluble IL-2R concentrations greater than 3000 U ml-1 produced a reduction in cell growth of more than 50% when included in CTLL IL-2 assays. The addition of increasing concentrations of IL-2 to cultures containing suppressive plasma failed to restore CTLL cell growth response to normal. Failure to saturate sIL-2R by exogenous IL-2 addition therefore suggests that another factor, initially present at a concentration similar to the sIL-2R concentration, is responsible for the observed effect. Determination of the suppressive effect of patient plasma as presented here may allow more effective IL-2 dosing schedules.

The phenotypic changes in tumour infiltrating lymphocytes and tumour cells following intra-arterial infusion of interleukin-2 in patients with squamous cell carcinoma.

This study evaluates the morphological and phenotypic changes that occur in squamous cell carcinoma of the head and neck when local infusions of interleukin-2 (IL-2) are given. Twelve patients were treated with a range of doses of IL-2 (3 x 10(3) to 3 x 10(6) international units/day) by continuous intra-arterial infusion for 10 days. Biopsies of the tumour were taken pre- and 48 h post-therapy, snap-frozen, cut, and examined histologically and immunocytochemically. Local infusions of IL-2 increase the numbers of antigen-presenting Langerhans cells (CD1a-positive) and infiltrating lymphocytes, predominantly of the CD3 and CD4 (T-helper) phenotypes. Locally infused IL-2 results in the expression of MHC (major histocompatibility complex) class II antigens on the surface of the tumour cells, capillary and post-capillary endothelial cells, and peri-tumoural macrophages. Intratumoural NK (natural killer) cells and CD8-positive (T-cytotoxic) infiltrating lymphocytes were not increased by this therapy and CD25 (IL-2 receptor) was only increased in those patients treated at the lower dose levels. The system of intra-arterial cytokine infusion into head and neck tumours developed in this study is a useful model to examine the biological effects of cytokines, since in vivo they are mainly produced and act locally. Furthermore, the infused tumours are easily accessible to biopsy. The results from studies such as this may influence the design of tumour-targeted cytokine gene therapy programmes.

Immunological parameters in peripheral blood of patients with renal cell carcinoma before and after nephrectomy.

OBJECTIVE: To determine the effects of nephrectomy on the immune response of patients with renal cell carcinoma (RCC). PATIENTS AND METHODS: Five patients with RCC were monitored before and over a period of up to 3 months after nephrectomy. The aspects measured were the phenotypic expression of markers on circulating lymphocytes, circulating concentrations of cytokines, markers of inflammatory and immune responses, and natural killer (NK) cell and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells (PBMC). The suppressive activity of patients' plasma on NK activity and ability to generate interleukin-2 (IL-2) induced LAK cells in PBMC of normal volunteers was also investigated. RESULTS: The results indicated that high CD4/8 ratios were present pre-nephrectomy with evidence of inflammatory responses and immune activation in some patients, particularly those with metastatic disease. CONCLUSION: The effect of nephrectomy was to decrease the inflammatory response and increase immune activation. Various defects in NK cell activity and LAK cell generation were demonstrated pre-surgery which slowly improved once the primary tumour had been removed and it is suggested that such defects could have contributed to tumour growth and development due to an ineffective immune response.

Immune changes in peripheral blood resulting from locally directed interleukin-2 therapy in squamous cell carcinoma of the head and neck.

Interleukin-2 (IL-2) was administered locally by constant intra-arterial infusion in four escalating doses from 3 x 10(4)-3 x 10(7) IU/day to 12 patients with squamous cell carcinoma of the head and neck (SCCHN) in a phase I trial. Lymphocyte phenotypic markers and serum cytokine concentrations were measured over the course of treatment. Serum IL-1-alpha, -beta and IL-6 were not induced at any dose level. Tumour necrosis factor (TNF)-alpha was induced in the 2 patients who showed a clinical response (at the lowest dose) as well as in 4/10 of the non-responders. In addition TNF-beta was induced in 3/10 and IFN-gamma in 5/10 non-responders. Soluble IL-2 receptor concentrations were increased at the two higher doses. The highest dose of IL-2 produced a lymphocytosis after day 5 until the end of administration reflected by a general rise in lymphocyte phenotypic markers. CD25, CD3/HLA-DR and CD56 showed an additional upregulation not accounted for by the lymphocytosis with a suggestion of a bell-shaped dose-response curve for CD25 and CD3/HLA-DR. Administration of IL-2 in this manner has been shown to be well tolerated and has some anti-tumour activity at low doses, with little toxicity.

Plasma neopterin as an adjunct to C-reactive protein in assessment of infection.

C-reactive protein (CRP) concentrations are increased in plasma in people with inflammatory conditions and bacterial infections. Plasma neopterin concentrations are increased in people with bacterial septicemias, viral infections, and graft vs host disease. Plasma concentrations of CRP and neopterin were measured daily in 21 bone-marrow transplant (BMT) patients, 64 patients in intensive-care units (ICU), and 12 patients with squamous cell carcinoma of the head and neck (HN). In the BMT patients, plasma neopterin measurements in addition to CRP measurements allowed infectious episodes to be distinguished from graft vs host disease. In the ICU patients, increased concentrations of CRP were not specific for infection and the additional plasma neopterin measurements did not improve this specificity. In all three patient groups, the derivation of a neopterin/CRP ratio was of no clinical use. These three groups of patients showed patterns of CRP and neopterin concentrations characteristic of their underlying diseases, the BMT patients with the immunological activation of graft vs host disease showed predominantly increased concentrations of plasma neopterin, ICU patients with infectious and inflammatory conditions had increased concentrations of both CRP and neopterin in plasma, and the HN group with localized inflammation showed increased plasma concentrations of CRP without increases in neopterin.

Combined detection of phenotype and Y chromosome by immunoenzyme labelling and in situ hybridisation on peripheral lymphocytes.

A novel staining method for simultaneously determining the immunophenotype and sex of peripheral lymphocytes is described. Cell surface markers on lymphocytes are identified using specific monoclonal antibodies located by a peroxidase anti-peroxidase staining technique (PAP). Lymphocytes are subsequently hybridised with a biotinylated Y chromosome-specific sequence probe and this is located by avidin-biotin-alkaline phosphatase staining. Following the two staining steps the preparation is examined and in the same field lymphocytes show brown peroxidase staining of cell surface markers and red staining of the Y chromosome. The application of this combined staining technique provides a convenient method for studying the development of different cell lineages following sex-mismatched bone-marrow transplantation and for the identification of chimeric situations. The method has been shown to be sensitive and reproducible.