|Year : 2015 | Volume
| Issue : 1 | Page : 1
Zoledronic acid induces an immune response through increased central memory and effector memory gamma/delta T cells in early and metastatic breast cancer patients
Erika P Hamilton1, Kathleen K Harnden1, Amy C Hobeika2, Jeffrey Peppercorn3, Michael A Morse3, H Kim Lyerly4, Kouros Owzar5, Gretchen Kimmick3, P Kelly Marcom3, Kimberly L Blackwell3
1 Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, NC, United State
2 Department of Surgery, Duke University Medical Center, Durham, NC, United State
3 Department of Medicine, Division of Medical Oncology; Department of Medicine, Duke Cancer Institute, Durham, NC, United State
4 Department of Surgery; Department of Medicine, Duke Cancer Institute; Department of Immunology, Duke University Medical Center, Durham, NC, United State
5 Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC, United State
|Date of Submission||06-Aug-2014|
|Date of Acceptance||03-May-2015|
|Date of Web Publication||26-Mar-2015|
Kimberly L Blackwell
Department of Medicine, Division of Medical Oncology; Department of Medicine, Duke Cancer Institute, Durham, NC
Source of Support: None, Conflict of Interest: None
Introduction: Zoledronic acid (ZA) in combination with endocrine therapy (ET) and ovarian ablation yielded a disease-free survival and overall survival advantage in women with estrogen receptor+ early stage breast cancer (EBC). Evidence from preclinical studies suggests that ZA increases gamma/delta T cells (GDT). Materials and Methods: We examined immune responses following ZA in 24 BC patients by collecting peripheral blood mononuclear cells at day 0, 1, 7, and 28. GDT population and cytokine responses were assayed using flow cytometry and multi-analyte profiling beads (Luminex), respectively, and relative changes from baseline were analyzed using the Wilcoxon signed-rank test. Results: In total, 18 (75%) patients had metastatic breast cancer (MBC), 6 (25%) had EBC, 18 (75%) received ET, 4 (17%) chemotherapy (C), and 2 (8%) no concurrent therapy. Following ZA, an increase in both effector (CD3+/Vdelta2+/CD45RA−/CD27−) (P = 0.0005) and central memory GDT (CD3+/Vdelta2+/CD45RA−/CD27+) (P = 0.006), as well as a decrease in naïve GDT (CD3+/Vdelta2+/CD45RA+/CD27+) (P = 0.003) were observed at day 7. Cytokines, including interleukin-1 (IL-1) receptor antagonist (P < 0.003), IL-12 (P < 0.0005), macrophage inflammatory protein 1 beta (P < 0.0005), interferon-gamma inducible protein 10 (P < 0.00002), and monokine induced by IFN-gamma (P < 0.00006), were increased at day 1 compared to baseline. Conclusion: In both EBC and MBC patients, ZA appears to induce a significant change in GDTs and cytokines associated with cell-mediated immunity offering a possible biologic mechanism for ZA's anticancer activity. Further studies are necessary to determine which BC patients might achieve clinically meaningful anticancer benefits from ZA.
Keywords: Breast cancer, gamma/delta T-cells, immune response, zoledronic acid
|How to cite this article:|
Hamilton EP, Harnden KK, Hobeika AC, Peppercorn J, Morse MA, Lyerly H K, Owzar K, Kimmick G, Marcom P K, Blackwell KL. Zoledronic acid induces an immune response through increased central memory and effector memory gamma/delta T cells in early and metastatic breast cancer patients. Carcinomics Clin Commun 2015;2:1
|How to cite this URL:|
Hamilton EP, Harnden KK, Hobeika AC, Peppercorn J, Morse MA, Lyerly H K, Owzar K, Kimmick G, Marcom P K, Blackwell KL. Zoledronic acid induces an immune response through increased central memory and effector memory gamma/delta T cells in early and metastatic breast cancer patients. Carcinomics Clin Commun [serial online] 2015 [cited 2019 Mar 20];2:1. Available from: http://www.carcinomics.org/text.asp?2015/2/1/1/153998
| Introduction|| |
Zoledronic acid (ZA) is widely used to treat hypercalcemia of malignancy and reduce skeletal-related events in patients with metastatic breast cancer (MBC). In a landmark clinical trial, ABCSG-12, addition of ZA to endocrine therapy (ET) and ovarian suppression resulted in a significant improvement in both disease-free and overall survival (OS).  Explanations for this clinical benefit include direct antitumor effects and modulation of the immune response by ZA;  but the mechanism is not well-understood. Preclinical data exist from multiple cancer cell lines, including those derived from prostate cancer and multiple myeloma, that ZA inhibits tumor adhesion, invasion, and proliferation , and has antiangiogenic  and pro-apoptotic effects. 
Previous work has demonstrated that ZA increases the number of gamma-delta T cells (GDTs); however, these studies were not in a BC population and were not in vivo. Clezardin et al. has proposed that the antitumor effect of ZA may be mediated through the mevalonate pathway.  ZA is a potent inhibitor of farnesyl pyrophosphate synthase, resulting in mevalonate pathway blockade and inducing intracellular accumulation of the isopentyl pyrophosphate (IPP).  In ZA treated mice with BC xenografts, GDTs infiltrated and inhibited growth of tumors that produced high IPP levels, but not those expressing low levels.  They suggest that ZA was able to induce recruitment of GDTs to tumors via accumulation of IPP, representing a potential mechanism of immune activation against cancer cells. ,,
The purpose of this study was to further characterize the immunomodulatory effects of ZA in BC patients in an attempt to explain its potential anticancer effect in women with BC. While the anticancer effect has been observed in early BC patients, we sought to examine this in both early and MBC patients. It has been clearly established that the immune system plays a role in BC control and it is hypothesized that the quality of this immune response and the interplay between the immune system and the tumor microenvironment will impact antitumor immunity. ,, We specifically hypothesized that we would observe increases in cytokine levels involved with T cell maturation and differentiation with a subsequent decrease in naïve GDTs and increase in mature memory GDTs in response to clinical ZA administration.
| Materials and Methods|| |
The procedures were followed in accordance with the ethical standards of the Institutional Review Board and with the Helsinki Declaration of 1975, as revised in 2000. All patients signed informed consent approved by the Institutional Review Board.
This was a translational science study involving patients with BC recruited from our institution's BC clinic. Patients who were >18 years of age with any stage of BC who were prescribed ZA (Zometa® , Novartis Corporation, Basel, Switzerland) by their treating physician and who had not yet received their first dose were eligible for participation. Patients with autoimmune disease or those receiving concurrent immunosuppression (>5 mg prednisone daily) or experimental immunotherapy were excluded.
Peripheral blood including whole cells and serum was collected and banked at 4 set time- points: Day 0 (within 7 days prior to ZA initiation), day 1 (between 20 and 48 h post-ZA administration), day 7 (between 6 and 8 days post-ZA administration, and day 28 (between 26 and 32 days post-ZA administration). In addition to the collection of peripheral blood, patient symptoms after the first administration of ZA were captured by the clinical BC team to allow us to explore the relationship between symptomatic complaints and immune modulation. Specific attention was focused on the capture of any flu-like symptoms such as fever, malaise, myalgia, nausea, and headache using Common Terminology Criteria for Adverse Events v3.0 (CTCAE).
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Ficoll-Hypaque™ Plus (Amersham Pharmacia Biotech AB, Upsala, Sweden) and cryopreserved for subsequent analysis. For donor serum isolation, whole blood was allowed to clot followed by centrifugation at 1800 g for 10 min. Serum was then collected and cryopreserved.
Cryopreserved PBMC from pre-ZA and days 1, 7, and 28 following ZA were stained for 30 min at room temperature in a total volume of 25 μl 1% BSA/phosphate buffered saline (PBS) containing cocktails fluorochrome-conjugated antibodies. Individual antibody cocktails consisted of the following: For GDT analysis, CD3-FITC (BD, San Jose, CA), Vd2-PE (BD), CD45RA-PerCP (Abcam), CD27-APC (BD Pharmingen, La Jolla, CA); for dendritic cell analysis, CD14-FITC (BD), CD86, 83, or 80-PE (Abcam), HLA-DR-PerCP (BD), CD11c-APC (BD); for monocyte analysis, CD14-FITC (BD), CD163-PE (BD Pharmingen), CD80-PerCP (BD Pharmingen), CD86 (BD Pharmingen) or 16-APC (Abcam). Cells were washed using 1% BSA/PBS and analyzed by multiparameter flow cytometry. Approximately, 100,000 gated events were collected. Flow cytometry acquisition was performed on a BD FACSCalibur and analyzed using Cellquest software (BD).
Serum 0.5 mL aliquots, which had not been previously thawed, were packed in dry ice and sent to the laboratory. Cytokines, chemokines and growth factors were quantified in previously frozen serum samples using multiplex Luminex xMap™ technology. Cytokines of interest were quantified using a manufactured human Cytokine 30-Plex Panel from Invitrogen (Carlsbad, CA) (endothelial growth factor [EGF], Eotaxin, fibroblast growth factor, granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage CSF, hepatocyte growth factor, interferon-a (IFN-a), IFN-g, interleukin-1 receptor antagonist (IL-1RA), IL-1 a, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, interferon-gamma inducible protein 10 (IP-10), MCP-1, monokine induced by IFN-gamma (MIG), macrophage inflammatory protein 1 alpha (MIP-1a), MIP 1b, RANTES, tumor necrosis factor - a, and vascular EGF). Assays were performed on a BioPlex system (BioRad) in our Human Vaccine Institute Immune Reconstitution and Biomarker Facility according to manufacturer's protocols.
For days 1, 7, and 28, the change from baseline for each measurement was quantified using a log-ratio. For a given day, the marginal hypothesis of change from baseline was tested using the Wilcoxon signed-rank test.  The Hodges-Lehmann estimator was used to quantify and estimate the effect size.  Marginal unadjusted P values were produced. All analyses were carried out using the R statistical environment. 
| Results|| |
Twenty-four patients, 75% (18) with MBC and 25% (6) with early stage BC (EBC), were enrolled between October 2009 and March 2011 [patient demographics, [Table 1]]. Concurrent treatments included ET in 18 patients (75%) and chemotherapy in 4 (17%) while the remaining 2 (8%) received no other therapy. The ZA was well-tolerated with expected typical adverse events: Flu-like syndrome symptoms such as fever, myalgia, and arthralgia within 48 h of ZA administration. These symptoms occurred in 13 of 24 (54%) patients. However, there were no serious adverse events including osteonecrosis of the jaw. There was no observed correlation between flu-like syndrome symptoms and changes in cytokine levels or GDT subsets.
Interleukin-1 receptor antagonist (P < 0.003), IL-12 (P < 0.0005), MIP-1b (P < 0.0005), IP-10 (P < 0.00002) and MIG (P < 0.00006), were observed to increase at day 1 post-ZA administration [Table 2] and [Figure 1]. By day 28, these cytokine levels (IL-1RA, IL-12, MIP-1b, and MIG) appeared to return to baseline with the exception of IP-10 which remained elevated compared to baseline at all time points (day 1, 7, and 28 post-ZA) [Table 2] and [Figure 1]. In this small sample, no significant differences were observed between patients with EBC versus MBC, ET versus chemotherapy, and pre- versus post-menopausal patients.
|Figure 1: Interleukin - 1 (IL - 12), IL - 1 receptor antagonist, interferon - gamma inducible protein 10, monokine induced by IFN - gamma, and macrophage inflammatory protein 1 beta increase 24 h post-zoledronic acid (ZA) administration cytokine response to ZA|
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|Table 2: Immune cytokines are primarily effected after 24 h of ZA, and the majority are not maintained at day 7 or 28|
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Gamma/delta T-cell population subsets
Following ZA administration, a transient decrease in total GDT (CD3+/Vdelta2+) at day 1 was observed (P = 0.00006) that persisted at days 7 and 28 [Table 3]. In addition to a decrease in total GDTs, an increase in the mature memory cells and a decrease in the naïve cells were observed [Table 3] and [Figure 2]. At day 7, an increase in both effector (CD3+/Vdelta2+/CD45RA-/CD27−) (P = 0.0005) and central memory GDT (CD3+/Vdelta2+/CD45RA−/CD27+) (P = 0.006) was observed, with a decrease in naïve GDT (CD3+/Vdelta2+/CD45RA+/CD27+) (P = 0.003) [Table 3] and [Figure 2]. In this limited patient sample, power was inadequate to detect differences between patients with EBC versus MBC, ET versus chemotherapy, and pre- versus post-menopausal.
|Figure 2: Memory gamma/delta T cells increase at day 7 in response to zoledronic acid (ZA) response to ZA|
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|Table 3: GDTs are affected at day 7 post - ZA but do not persist at day 28|
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| Discussion|| |
Gamma/delta T cell lymphocytes produce proinflammatory cytokines which mediate key cytotoxic effector functions. An excellent candidate for cancer immunotherapy, GDT lymphocytes have been shown to selectively kill malignant cells while showing minimal activity towards nonmalignant cells.  In this study, we demonstrated that ZA induces a significant change in GDTs and cytokines associated with an anticancer immune response in both early stage and MBC patients.
We demonstrated that ZA treatment induced several markers of tumor immunity including IL-12, IP-10, and MIG. These cytokines, which are involved in T cell activation and recruitment, were observed to increase on day 1 in response to ZA administration. Increases in cytokine levels, like those seen in this study, have been implicated in direct antitumor immunity [Table 4]. IL-12 induces IFN-gamma production and is involved in T cell activation. Specifically, IL-12 is implicated in the differentiation of naive T cells into mature T cells, as well as the proliferation of mature T cells. , Thus, the increase in IL-12 at day 1 is concordant with the increase in memory GDT subsets (including effector memory and central memory GDT) later observed at day 7, and could be responsible for the memory GDT proliferation seen in this study.
Similarly, IP-10 was observed to increase in response to ZA infusion. IP-10 has been implicated in T cell activation,  T cell recruitment to inflammatory/tumor sites, , and plays a significant role in T-cell synthesis. , IP-10 was uniformly (17/17 patients) increased at day 1 compared to baseline. It is possible that the transient decline in total GDT at day 1 could be associated with IP-10 mediated recruitment of T-cells to inflammatory sites, including areas of metastatic tumor. Further work is needed to determine if this effect is seen more frequently among patients with known malignancy compared to patients at low or minimal risk of disease, such as patients in the adjuvant setting or those receiving ZA for osteoporosis. Based on the putative role of IP-10 in T-cell activation,  our data may also suggest that the subsequent increase seen in the mature memory subsets of GDTs was IP-10 mediated.
Zoledronic acid appears to induce a highly significant change in immune effector cells in both early stage and MBC patients receiving ET or chemotherapy. A decrease in the total GDTs was seen at day 7 and persisted at day 28. The etiology and significance of this change is unclear, but this could reflect infiltration of cells into sites of inflammation or metastases as has been described with increases of IP-10 and MIG, both of which serve as a chemoattractants. In addition to recruiting GDT to inflammatory sites, IP- 10 and MIG have been linked with T-cell mediated tumor eradication.  Finally, a change in GDT population subsets was observed at day 7 with a decrease in naïve GDTs and an increase in both effector memory and central memory GDTs at day 7. Increases in memory GDTs, as seen in response to ZA in this study, serve as a possible mechanism of ZA's observed antitumor effect.
We did not observe a correlation between flu-like symptoms post-ZA administration and changes in cytokine levels or GDTs. Interestingly, there have been recent publications suggesting that the presence of fever within 48 h of ZA administration correlates with a higher proportion and absolute number of GDTs in patients given ZA for osteoporosis.  We may not have seen this in our study due to concomitant medication administration, such as steroids and antihistamines, which are often given with chemotherapy infusions. In addition, it has been suggested that younger patients are more likely to have flu-like symptoms postinfusion, possibly due to a more robust immune system.  However, the patients in our study likely have unmeasured changes in the ability to mount an entirely normal immune response due to their underlying malignancy and recent anticancer therapies. These inherent differences in our study population, in comparison to the osteoporosis population, may account for differences in the observed acute phase reactants. It is unclear how concomitant chemotherapy impacts the immune response, although it does not appear to have prevented changes in cytokines or T-cell populations.
Both the brisk cytokine increase at day 1 and the observed proliferation in memory GDTs by day 7 post-ZA could offer an important biologic mechanism for the improvement in OS seen when ZA was added to ET and ovarian suppression in ABCSG-12.  Although, a subsequent trial, AZURE, did not show an anticancer benefit for all BC patients, subset analyses demonstrated lower risk of disease recurrence among women who had been menopausal for at least 5 years  suggesting that ZA's effect may be most clinically significant in a low estrogen environment. The clinical heterogeneity in patient characteristics, tumor biology, and treatment may explain the differences between these trials and should be further evaluated in a larger study stratified by age, disease setting, and treatment.
Our patient population not only included EBC patients such as those seen in ABCSG-12 and AZURE, but also included a high percentage of patients with MBC. In the current literature, evidence for ZA anticancer activity has only been observed in early BC patients (ABCSG-12 and AZURE), and this cannot be generally applied to patients with metastatic disease. Indeed, one small series by Santini et al. demonstrated that ZA activates effector subsets of GDTs in endocrine-responsive early BC patients with osteopenia.  However, the immune response changes in patients with metastatic disease are not well-described in the literature. One study by Meraviglia et al. described effector maturation of GDTs in advanced breast patients in response to ZA with low-dose IL-2 administration.  However, in contrast to Meraviglia et al., our study demonstrates a response to ZA alone, as it would be given in the clinical setting and not in combination with other immune modulating compounds like IL-2. In addition, our study is unique in the inclusion of both early BC and MBC patients, and we performed all cytokine analyses in a single batch on the same platform. This gives further evidence that the immune system in these metastatic patients is competent enough to respond similarly to ZA as the immune systems of early BC patients. It is unknown how these findings would translate to clinical benefit and further studies are needed.
While our study is limited by sample size and heterogeneity of patients, ZA had a significant impact on T cells and cytokines, which may mediate the impact of ZA on nonskeletal clinical outcomes. GDT lymphocytes are optimal targets for cancer immunotherapy, given their selectivity for killing malignant cells, and our study results indicate that ZA may potentiate this response in early stage and MBC patients. This is a possible mechanism for ZA's anticancer activity, and the ability of ZA to induce in vivo expansion of GDT lymphocytes should be explored further. The results of this study can help guide the design of future studies evaluating the immune response to bisphosphonates.
Further studies are needed to better characterize the immune response to ZA and to correlate this response with clinical outcomes. Evaluation of immune response and changes in GDT lymphocytes among larger subsets of pre- and postmenopausal women and among patients on chemotherapy versus ET may help explain the differences in ZA benefit seen in adjuvant clinical trials. Finally, optimal use of ZA, including dosing and frequency, could be guided by better understanding all of the physiologic effects induced by this agent.
| Acknowledgments|| |
We would like to thank Karrie Comatas and Amanda Summers for technical assistance with immune assays.
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| Authors|| |
Erika P. Hamilton: Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, NC
Kathleen K. Harnden: Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, NC
Amy C. Hobeika: Department of Surgery, Duke University Medical Center, Durham, NC
Jeffrey Peppercorn: Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, Duke Cancer Institute, Durham, NC
Michael A. Morse: Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, Duke Cancer Institute, Durham, NC
H. Kim Lyerly: Department of Surgery, Duke University Medical Center, Duke Cancer Institute, Department of Immunology, Duke University Medical Center, Durham, NC
Kouros Owzar: Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC
Gretchen Kimmick: Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, Duke Cancer Institute, Durham, NC
P. Kelly Marcom: Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, Duke Cancer Institute, Durham, NC
Kimberly L. Blackwell: Department of Medicine, Division of Medical Oncology, Duke University Medical Center, Durham, Duke Cancer Institute, Durham, NC.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]