Background
Tumor infiltration by cells involved in the cellular immune response appears to correlate with prognosis for patients with colon cancer, suggesting that the immune system plays a critical role in patient outcome [
1‐
3]. Following surgical resection of a colonic tumor, improved overall survival in patients with early stage (I-III) disease correlates with the extent of immune infiltration of the primary tumor with dendritic cells and T-cells. The reduced relapse rate in these patients suggests that sensitization to tumor antigens and an associated viable cellular immune response may eradicate microscopic residual disease. While patients with stage IV (metastatic) colorectal cancer are generally considered incurable, it is theoretically possible that stimulation of anti-tumor aspects of the immune system might positively affect response to therapy and overall median survival [
4], analogous to the phenomenon noted for earlier stages of this disease. In addition, patients with advanced cancer are known to have tumor-related immune suppression and chemotherapy, typically administered in this setting, is also immunosuppressive [
5,
6]. However, the degree of immunosuppression of different chemotherapy regimens, and the precise effects of chemotherapy on specific lymphocyte subsets have not been rigorously defined. Still, for patients with colon cancer, reversing the innate immune suppression caused by any of the potential etiologies may be advantageous.
GM-CSF is frequently utilized in cancer patients to aid in hematopoietic recovery but it also has pleiotropic effects on components of the immune system, including being a requisite growth factor for myeloid dendritic cells (DCs) that play an important role in tumor antigen presentation and stimulation of immune responses [
7]. Our hypothesis was that GM-CSF would be beneficial for patients with advanced colorectal cancer by stimulating cellular immune responses, potentially including Th1 immune responses, specifically by activating dendritic cells and also might abrogate chemotherapy induced immunosuppression common in this patient population. We devised a phase II trial augmenting standard chemotherapy with mid-cycle GM-CSF and monitored effects on the circulating dendritic cell and other peripheral blood mononuclear cell (PBMC) populations along with evaluation of Th1 type immune responses. The principal endpoints of this study were biologic in order to provide a potential rationale for larger studies with survival endpoints including GM-CSF.
Materials & methods
Clinical trial design and conduct
Patients who were receiving chemotherapy for stage IV colorectal cancer were identified for this study. Enrollment for this study began in June, 2003 and ended January 2007, with 20 patients enrolled onto clinical trial NCT00257322. The study was approved by the University of California, Irvine Institutional Review Board and all patients signed written informed consent prior to participation. All research was conducted in compliance with the Helsinki Declaration for ethical research in humans. Patients received 500 μg GM-CSF daily for 4 days starting 24 hours after completion of each chemotherapy cycle. Initial patients received weekly 5-fluorouracil with leucovorin (n = 12). With changes in standard of care that occurred during the conduct of the study, a protocol modification allowed enrollment of patients receiving biweekly mFOLFOX6 (5FU, leucovorin, oxaliplatin) chemotherapy (n = 8). Blood was obtained before chemotherapy/GM-CSF administration and 24 hours following the final dose of GM-CSF.
Quantitative Real-time PCR (qRT-PCR)
Blood samples drawn from the patients were immediately processed for the isolation of PBMCs using Histopaque 1083 (Sigma-Aldrich). Trizol (Invitrogen) was added to the PBMC pellets and stored at 4°C for subsequent RNA extraction. cDNA preparation for each RNA sample was performed using the high capacity cDNA reverse transcription kit (Applied Biosystems). PBMC expression of γ-interferon, T-bet transcription factor (Tbx21), DC-LAMP, and Wnt 5a by quantitative real-time PCR was performed, the former two as measures of Th1 immunity. All experimental mRNA were normalized to the housekeeping gene β-actin mRNA.
Flow cytometry
Standard whole blood staining protocols were used to evaluate circulating leukocyte populations. Phlebotomy samples in EDTA anti-coagulated blood were collected and individual aliquots of 200 μl of blood were placed into #2059 tubes (BD Biosciences) and stained with the appropriate experimental or isotype control antibodies. Tubes were then incubated at room temperature (RT) for 60 minutes in the dark, with mixing every 15 minutes. RBCs were lysed with ACK lysis media for 15 minutes at RT and residual cells pelleted by centrifugation. Supernatant was discarded by aspiration, cells washed and the cell pellet was resuspended in either 500 μl of staining media (if flow cytometry analysis is to be immediate) or 50% volume of staining media and 50% volume freshly prepared 4% paraformaldehyde. In the latter case tubes were stored at 4°C in the dark for no more than 48 hours before flow cytometry analysis. Calabrite 3, a calibration bead set (BD Biosciences) were used for standardization of the conditions and compensation controls for the three-color flow cytometry analysis according to manufacturer's instructions. Additional fluorophore controls, CD3-FITC, CD8-PE, CD4-TC, were used to verify the uniformity of conditions for the analyses.
Statistical Methods
Expression of mRNA by real-time PCR was compared, pre- and post-treatment, with a Wilcoxin matched pairs signed rank test. Changes in lymphocyte subsets pre- and post-treatment were compared similarly. Differences across cycles of chemotherapy/GM-CSF administration were analyzed for statistical significance by a two-tailed unpaired t-test with Welch's correction.
Discussion
GM-CSF is utilized primarily for its activity in increasing granulocyte production following administration of bone marrow suppressive cytotoxic chemotherapy. However, largely because of its role in stimulating monocytic precursors, it has long been recognized that it possesses immunostimulatory activity [
7]. Our utilization of GM-CSF in this trial was to attempt to abrogate the immunosuppression induced by chemotherapy [
5] and by the cancer itself [
6]. In this population of patients receiving chemotherapy for metastatic colorectal cancer, GM-CSF was administered without significant toxicity.
GM-CSF did not augment cellular, Th1, immune functioning, as measured by the methodologies utilized in this study. The reduction in T-bet is postulated to be secondary to chemotherapy. The lack of concomitant reduction of γ-interferon may be due to GM-CSF-dependent stimulation of dendritic cells, which can also produce this cytokine. Together with the decreases in the CD4+, CD8+, CD14+ and CD56 =/CD3+ populations, the decrease in T-bet suggests that there was not selective sparing of Th1 biased lymphocytes by the cytotoxic chemotherapy to account for the stable γ-interferon mRNA expression in PBMCs. As there was no control arm of patients receiving chemotherapy without GM-CSF on this study, we cannot quantitate the absolute reduction in PBMC γ-interferon production which might be seen following chemotherapy alone. With respect to T-cell subset changes, approximately 2/3 (66%) of subjects had a significant decrease in CD4+ lymphocytes and approximately 80% of subjects had a significant decrease in CD8+ lymphocytes with each cycle.
One of the most significant findings from this clinical trial was the increase in the number of circulating myeloid DCs (Lineage-, MHC high). These increased in approximately 75% of subjects across all cycles. In subsequent cycles of chemotherapy and GM-CSF, approximately 50% of the subjects experienced increased DC numbers over the initial cycle. Dendritic cells are potent antigen presenting cells and play a pivotal role in the induction of immune responses. Dendritic cells have been directly implicated in anti-tumor immune responses [
12‐
15]. The increase in circulating DCs, likely a consequence of GM-CSF administration, may contribute to the anti-tumor effects of chemotherapy administered in the adjuvant or metastatic setting.
Another important observation was the reduction in regulatory T-cells (Treg) following chemotherapy/GM-CSF. Approximately 90% of subjects had a significant (> 20%) decrease in Treg lymphocytes with the first cycle. Tregs can suppress the activity of cytotoxic T cells through direct cell-to-cell contact or via the release of cytokines such as transforming growth factor beta. Depletion of intratumoral Tregs has been shown to enhance antitumor immunity and promote tumor rejection in mouse models [
16]. In patients with colon cancer, increased numbers of Treg cells has been associated with a poor clinical prognosis [
17]. This decrease in circulating Treg cells argues against a significant increase in myeloid derived suppressor cells, implicated in the induction of Tregs [
18], as a result of the administration of GM-CSF; an effect that has been inconsistently reported in other settings[
19‐
22]. Thus, the reduction in Tregs seen in our study, and the concomitant loss of immune system suppression that these cells induce, may be prognostically favorable.
As noted previously, this study did not contain a chemotherapy only control arm. Therefore, the effects of chemotherapy cannot be distinguished specifically from the effects of GM-CSF. Still, in patients with metastatic colorectal cancer receiving chemotherapy and GM-CSF, cellular immune responses are inhibited and the impairment in Th1 type immune parameters are not abrogated by administration of GM-CSF. However, administration of GM-CSF along with chemotherapy does lead to an increase in circulating DCs and a reduction in inhibitory Tregs. Together, this may provide a mechanism for augmentation of innate immune responses and may provide benefit to colorectal cancer patients receiving chemotherapy.
Conclusions
This phase II clinical trial confirms that chemotherapy in patients with metastatic colorectal cancer suppresses Th1 cellular immunity but demonstrates that mid-cycle administration of GM-CSF can significantly increase the proportion of circulating dendritic cells and reduce the proportion of circulating Treg cells. Th1 activity, as measured by γ-interferon production, was not affected. These finding suggest that GM-CSF can abrogate important aspects of the immune suppression seen in patients with advanced colorectal cancer and may provide a non-toxic intervention to augment anti-tumor immunity for cancer patients receiving cytotoxic chemotherapy.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MM, NO, MP and KP conducted immunologic assays including PCR and flow cytometry. MM assisting in maintaining clinical trial related data according to IRB guidelines. ELN and RFH conceived this study, provided oversight for all aspects of the laboratory investigation, were involved in obtaining informed consent and providing treatment for patients and directed the overall project. All authors were involved in manuscript preparation.