Granulocyte-macrophage stimulating factor (GM-CSF) increases circulating dendritic cells but does not abrogate suppression of adaptive cellular immunity in patients with metastatic colorectal cancer receiving chemotherapy

Abstract

Background: Advanced cancer and chemotherapy are both associated with immune system suppression. We initiated a clinical trial in patients receiving chemotherapy for metastatic colorectal cancer to determine if administration of GM-CSF in this setting was immunostimulatory.

Methods: Between June, 2003 and January, 2007, 20 patients were enrolled in a clinical trial (NCT00257322) in which they received 500 ug GM-CSF daily for 4 days starting 24 hours after each chemotherapy cycle. There were no toxicities or adverse events reported. Blood was obtained before chemotherapy/GM-CSF administration and 24 hours following the final dose of GM-CSF and evaluated for circulating dendritic cells and adaptive immune cellular subsets by flow cytometry. Peripheral blood mononuclear cell (PBMC) expression of γ-interferon and T-bet transcription factor (Tbx21) by quantitative real-time PCR was performed as a measure of Th1 adaptive cellular immunity. Pre- and post-treatment (i.e., chemotherapy and GM-CSF) samples were evaluable for 16 patients, ranging from 1 to 5 cycles (median 3 cycles, 6 biologic sample time points). Dendritic cells were defined as lineage (-) and MHC class II high (+).

Results: 73% of patients had significant increases in circulating dendritic cells of ~3x for the overall group (5.8% to 13.6%, p = 0.02) and ~5x excluding non-responders (3.2% to 14.5%, p < 0.001). This effect was sustained over multiple cycles for approximately half of the responders, but tachyphylaxis over subsequent chemotherapy cycles was noted for the remainder. Treatment also led to a significant reduction in the proportion of circulating regulatory T-cells (Treg; p = 0.0042). PBMC Tbx21 levels declined by 75% following each chemotherapy cycle despite administration of GM-CSF (p = 0.02). PBMC γ-interferon expression, however was unchanged.

Conclusions: This clinical trial confirms the suppressive effects of chemotherapy on Th1 cellular immunity in patients with metastatic colorectal cancer but demonstrates that mid-cycle administration of GM-CSF can significantly increase the proportion of circulating dendritic cells. As the role of dendritic cells in anti-tumor immunity becomes better defined, GM-CSF administration may provide a non-toxic intervention to augment this arm of the immune system for cancer patients receiving cytotoxic therapy.

Trial registration: ClinicalTrials.gov: NCT00257322.

Figures

Figure 1

Panel A. Levels of γ-interferon mRNA following the administration of GM-CSF after chemotherapy. γ-interferon mRNA levels were measured in 16 patients pre-chemotherapy/GM-CSF administration and post-GM-CSF/chemotherapy administration by quantitative real time PCR of peripheral blood mononuclear cells. γ-interferon mRNA levels were normalized against the housekeeping gene β-actin mRNA. There was no statistically significant change between the two groups. Panel B. Levels of T-bet transcription factor mRNA following the administration of GM-CSF after chemotherapy. T-bet transcription factor mRNA levels were measured in 16 patients pre-chemotherapy/GM-CSF administration and post-GM-CSF/chemotherapy administration by quantitative real time PCR of peripheral blood mononuclear cells. T-bet transcription factor mRNA levels were normalized against the housekeeping gene β-actin mRNA. There was an approximate 1.7 fold decrease in T-bet transcription factor mRNA levels following the administration of GM-CSF after chemotherapy (p = 0.043). The statistical significance was determined by a Wilcoxin matched pairs test.

Figure 2

Panel A. Changes in lymphocyte subsets associated with chemotherapy and GM-CSF administration. Individual lymphocyte subsets were evaluated by flow cytometry before and after the administration of chemotherapy and GM-CSF. The subsets that were evaluated include: CD4+, CD8 +, CD4+CD25 high (Treg), CD56+ CD3+ (NK-T) populations. Values depicted represent average percentages of nucleated cells in whole blood samples (n = 23) with error bars depicting standard error values. Significant differences were evaluated with a Wilcoxon matched pairs test. The decrease in CD4 and CD8 T lymphocyte subsets was statistically significant at p = 0.0064 and p = 0.0012 respectively. The decrease in the Treg population was also significant at p = 0. 0042 while the decrease in NK-T cells approached statistical significance p = 0.0997. Panel B. Percent change in lymphocyte subsets across multiple cycles of chemotherapy & GM-CSF. In the first and subsequent cycles of Chemotherapy & GM-CSF the percent change in lymphocyte subsets that include: CD4+, CD8 +, CD4+CD25 high (Treg), CD56+ CD3+ (NK-T) populations, were determined. Percent change was calculated in the standard fashion [(pre- treatment value - post-treatment value)/pre-treatment value] × 100. None of the differences in lymphocyte subset changes between the first and subsequent cycles reached statistical significance by two-tailed unpaired t-test with Welch's correction. Panel C. Changes in monocytes and myeloid DCs associated with chemotherapy and GM-CSF administration. Individual cell populations were evaluated by flow cytometry before and after the administration of chemotherapy and GM-CSF. Monocyte populations were identified as being CD14+. Myeloid dendritic cells (DCs) were identified using a lineage cocktail (CD3, CD20, CD14, CD56) negative cells that had MHC II high expression. Values depicted represent average percentages of nucleated cells in whole blood samples (n = 23) with error bars depicting standard error values. Differences were evaluated with a Wilcoxon matched pairs test. Differences in monocytes and DCs were statistically significant p = 0.0214 and p = 0.0198 respectively. Panel D. Percent change in monocytes and dendritic cells across multiple cycles of chemotherapy and GM-CSF. In the first and subsequent cycles of Chemotherapy & GM-CSF the percent change in monocytes (CD14+ cells) and dendritic cells (DCs) [lineage cocktail (CD3, CD20, CD14, CD56) negative, MHC II high expression] were determined. Percent change was calculated in the standard fashion [(pre-treatment value - post-treatment value)/pre-treatment value] × 100. None of the differences in lymphocyte subset changes between the first and subsequent cycles reached statistical significance by two-tailed unpaired t-test with Welch's correction.