GM-CSF with biochemotherapy (cisplatin, DTIC, tamoxifen, IL-2 and interferon-alpha): a phase I trial in melanoma.

BACKGROUND: Ineffective tumour antigen processing is recognised as an important cause of failure of immunotherapy in melanoma. GM-CSF may augment the cytotoxic lymphocyte response by activating antigen-presenting cells. This study evaluates a schedule combining GM-CSF with biochemotherapy. PATIENTS AND METHODS: Nineteen patients with advanced malignant melanoma received cisplatin (25 mg/m2 days 1-3). dacarbazine (220 mg/m2 days 1-3), interleukin-2 (9 MIU/m2/24 h) and interferon-alpha2b (5 MIU/m2) both days 6-10 and days 17-21, and tamoxifen 40 mg/day continuously. Subcutaneous GM-CSF was given in escalating doses to three cohorts: 1) 450 microg/m2 days 4-5 and 15-16; 2) as 1) plus 225 microg/m2 days 6-10 and 17-21; 3) 450 microg/m2 days 4-10 and 15-21. Each cycle was 28 days. RESULTS: Constitutional side effects were the major non-haematological toxicity and lymphopaenia the main haematological toxicity. Six patients responded (32%, 95% confidence interval: 13%-57%), two patients had complete remission. There was an apparent trend for increasing responses with increasing GM-CSF dose; zero of six responses in cohort 1, two of seven in cohort 2 and three of six in cohort 3 (P = 0.016). Median overall survival was 6.2 months. Increasing GM-CSF doses significantly increased serum concentrations of neopterin and TNF-alpha. CONCLUSIONS: The combination of GM-CSF with biochemotherapy is feasible and there appears to be a dose-response relationship with GM-CSF in terms of host immunological response, and possibly clinical efficacy.

Reduced IL-4 and interferon-gamma (IFN-gamma) expression by CD4 T cells in patients with chronic lymphocytic leukaemia.

CD7 co-expression by CD4 T cells has been reported to be higher in the Th1 compared with the Th2 functional subset. Clinical immunodeficiency and immune dysregulation are more prevalent in the advanced stages of B cell chronic lymphocytic leukaemia (B-CLL). To analyse this further 25 patients with B-CLL and 11 healthy subjects were examined for cell surface CD7 and intracellular IFN-gamma and IL-4 expression in the peripheral blood CD4+ T helper cell population. Significantly decreased CD7, IFN-gamma and IL-4 expression was observed in the patients with B-CLL (P < 0.001). While CD7 negativity and IL-4 expression were more frequent in the later stages of the disease, this did not attain statistical significance. These results suggest a possible explanation for the reduced cellular and humoral immunity in B-CLL.

A simple flow cytometry assay using dihydrorhodamine for the measurement of the neutrophil respiratory burst in whole blood: comparison with the quantitative nitrobluetetrazolium test.

The neutrophil respiratory burst is essential for the host's ability to kill ingested microorganisms. Several flow cytometric assays have recently been developed to measure this process. These assays are largely unvalidated. In this study a whole blood flow cytometry assay using dihydrorhodamine 123 (DHR) as a substrate was compared with the quantitative nitrobluetetrazolium (NBT) test, an accepted measure of the earliest events in the respiratory burst. Because whole blood is used, the new assay is quicker and simpler than existing flow cytometry assays. Specimens as small as 0.1 ml can be used which makes the assay ideal for use in neonates and young children. There was a high degree of correlation between the DHR assay and the quantitative NBT test (r(s) = 0.76, P < 0.01). It is concluded that the whole blood DHR assay is an accurate and sensitive measure of the respiratory burst.

Substance P binding to peripheral blood mononuclear leukocytes in atopic dermatitis.

Substance P has various immunomodulatory effects, including in vitro modification of lymphocyte proliferation and cytokine release. Elevated levels of substance P and increased staining of substance P-positive nerve fibres have been reported in atopic dermatitis patients. We examined fluoresceinated substance P binding to a range of lymphocyte subsets and compared the results in atopic dermatitis, non-atopic psoriasis patients and normal controls. Fluoresceinated substance P and phycoerythrin-labelled monoclonal antibodies to CD3, CD4, CD8, CD57, CD19 and CD14 were incubated in duplicate with Ficoll-Hypaque separated peripheral blood mononuclear leukocytes. With flow cytometry the fluoresceinated substance P-positive cells were identifiable as a peak of positively fluorescent cells, and the percentages of positive cells were measured. We have demonstrated binding of fluoresceinated substance P to all subsets examined, with significantly less binding to atopic dermatitis CD3-, CD8- and CD57-positive cells. This may affect cytokine release and hence be important in the pathogenesis of atopic dermatitis.

A simple method for culturing myeloma cells from human bone marrow aspirates and peripheral blood in vitro.

A double layer agar technique has been developed to grow myeloma colonies (MY-CFUc) from human bone marrow aspirates and peripheral blood. Heavily irradiated HL60 cells (5 x 10(5)/plate) are added to an agar underlay in growth medium containing 0.5% agar. Mononuclear cells from the test bone marrow or blood are overlayered in either 0.2 ml HL60-conditioned medium (HL60-CM) or in 0.5 ml growth medium containing 0.23% agar, and the cultures are incubated at 37 degrees C in an atmosphere of 5% CO2, 10% O2 and 85% N2. Colonies (greater than 50 cells) form between 2 and 3 weeks. Using this method 60/68 samples of bone marrow and 7/12 samples of blood from 54 patients have produced colonies in soft agar and in liquid on an agar underlay. The cells which form these colonies are of two distinct sizes, the larger cells being plasmacytoid and the smaller lymphoid. The two cell types are usually, but not always, present in separate colonies. Both plasmacytoid and lymphoid cells carry the isotype of the respective patient's myeloma protein and the plasma cell marker (HAN PC1). This technique has enabled us to culture myeloma cells from patients with as few as 2% plasma cells in the bone marrow but it does not permit the growth of normal B, T or granulocyte-macrophage colonies (GM-CFUc). The drug sensitivity of myeloma cells (MY-CFUc) compared with normal haemopoietic cells (GM-CFUc) can be measured using dose-response curves in individual patients. Furthermore, this method can detect resistant subpopulations within a given myeloma sample.

Double staining immunofluorescence procedure for enumeration of cells containing cytoplasmic immunoglobulin in human bone marrow: 40 selected cases.

A double staining immunofluorescence method was developed that allowed the reliable differential counting of cells containing cytoplasmic immunoglobulin in human bone marrow. These cells were stained with a polyspecific antihuman immunoglobulin serum conjugated with rhodamine. In separate preparations subpopulations of cells containing each heavy chain class and light chain type were identified by prior staining with isotype specific fluorescein conjugated antihuman immunoglobulin sera. A standardised counting procedure was adopted to indicate the degree of plasma cell infiltration and to establish the percentage of cells containing each type of immunoglobulin. A series of 122 patients requiring haematological investigation of the bone marrow for various disorders but without evidence of paraproteinaemia was tested to provide results for a nonmyelomatous reference group. These results were compared with those from 40 patients with paraproteins of various classes in their serum. The procedure described here clearly differentiated the two groups. When combined with serum paraprotein assays and conventional haematological cytology of bone marrow these counts provided useful additional information in cases of diagnostic difficulty and in the assessment of individual patients before, during, and after treatment.