Abstract
Independently of tumour and treatment modulation, the host immune response status plays an important role in the clinical outcome of patients with cancer. The influence of single nucleotide polymorphisms (SNPs) and adjuvant radiotherapy (RT) on the systemic immune response status of patients with breast cancer was investigated. Materials and Methods: Eighty-six female patients recovering from breast cancer surgery were investigated. As a control cohort, 82 healthy female blood donors were used. Blood-based SNPs, plasma C-reactive protein (CRP), cytokines and chemokines were analyzed for this purpose. Results: Independently of tumour stage and hormone receptor status, dysregulation of plasma CRP, chemokine (C-C motif) ligand 4 (CCL4) and interleukin 2 (IL2), but not CCL5, CCL2, platelet-derived growth factor, IL6, IL10, IL12, interferon-gamma or tumour necrosis factor alpha were detected in the patients when compared to controls. The extent of alteration in plasma levels of CRP and IL2 patients was significantly associated with SNPs in CRP rs1800947 and IL2 rs6822844, respectively. These SNPs had no influence on the levels of corresponding plasma biomarkers in the healthy controls. Adjuvant RT reduced plasma CRP and CCL5 levels in patients with regards to CRP rs1800947CC, CCL5 rs2107538GG and CCL5 rs2280789AA sequences. Conclusion: Dysregulation of immune responses, as indicated by plasma levels of CRP, CCL4 and IL2 were found in patients with breast cancer despite the removal of the tumour mass. The benefit of adjuvant RT, as indicated by reduced plasma amounts of inflammatory protein CRP and chemokine CCL5 were based on the SNPs of the patients. Analyses of blood-based SNPs, plasma CRP, IL2 and CCL5 are low cost, rapid and can be carried out using general laboratory facilities while requiring only a peripheral blood sample. The possibility of using these blood-based biomarkers as an indicator of patient immune status for selection of individual patient treatment warrants further investigation.
Adjuvant radiotherapy (RT) is an essential treatment of breast cancer and various types of solid tumours after surgery. Independently of tumour and treatment modulation, the host immune response status plays an important role in the clinical outcome of patients with cancer (1). Recently, a combination of RT and immunotherapy was suggested (2-4).
The anti-neoplastic properties of RT are primarily related to induction of DNA damage and cell death. Bystander effects can also develop in cells not directly hit by RT (5).These processes can in turn lead to the alteration of host immune responses driven by intra- and intercellular communication via inflammatory proteins, chemokines and cytokines (6-9).
The intensity of host systemic inflammatory responses can be assessed by monitoring the level of plasma C-reactive protein (CRP) (10, 11). Following the recovery stage, the plasma CRP level returns to a normal value. Increased plasma CRP levels were associated with a poor clinical outcome in patients with cancer independently of tumour-node-metastasis (TNM) staging (12).
Cytokines are small proteins that regulate local intercellular and systemic immune responses. One of these, interleukin 2 (IL2), is a potent immunomodulatory cytokine and has been used as single therapy for cancer (13). Chemokines are chemotactic cytokines that mediate host inflammatory responses, leukocyte trafficking and T-cell or natural killer (NK) cell activities (14-16). Chemokine (C-C motif) ligand (CCL4) and CCL5 are highly expressed in breast tumour cells and various cells in the tumour microenvironment but are minimally expressed in normal breast epithelial cells (17). The administration of anti-inflammatory chemokines was found to protect multipotent hematopoietic cells from the cytotoxic effects of chemotherapy (16). The use of anti-inflammatory chemokines as immunotherapy in patients with cancer has been suggested (16, 18-20).
The levels of inflammatory proteins, chemokines and cytokines are suggested to be associated with individual genetic variations (6, 8, 9). Major contributors to genetic variations are single nucleotide polymorphisms (SNPs) found within DNA sequences (9, 21).
The present study aimed to determine systemic immune responses of patients after recovery from surgery for breast cancer, as well as the influence of adjuvant RT and SNPs. Low-cost and rapid blood-based biomarkers were selected for this purpose. CRP, chemokines and cytokines in association with SNPs in the corresponding genes were analysed.
Materials and Methods
Patients and controls. Eighty-six female patients with breast cancer aged over 18 years and scheduled for standard adjuvant RT after breast cancer surgery were invited to participate. If they accepted the invitation, peripheral blood was drawn twice. The first, baseline, sample was obtained after the patient recovered from cancer surgery and before the commencement of adjuvant RT (R0). The second, follow-up, blood sample was obtained directly after completion of adjuvant RT (R1).
Eighty-two female healthy blood donors with no history of cancer or using any immunomodulation agents were included as controls. One peripheral blood sample was obtained.
Informed consent was obtained from all participants. The study was conducted in accordance with the Declaration of Helsinki and the Ethical Board at Linköping University approved this investigation, Dnr 2010/331-31.
Adjuvant RT. Adjuvant RT was delivered at the Department of Oncology, Ryhov Hospital, Sweden using Varian True Beam machines (Varian, Palo Alto, CA, USA). The patients received standard treatment with a total of 50 Gy to the operated breast in 25 fractions, 1 fraction per day administered 5 times per week for a total of 5 weeks.
Plasma CRP analysis. The level of high-sensitive CRP in the plasma of the controls and patients at R0 and R1 were analysed using Siemens Adevia 1800 (Siemens Healthcare, Erlangen, Germany) with reagents and protocols from this company.
The characteristics of 86 female patients with breast cancer.
Plasma cytokine and chemokine analysis. Based on previous investigations, plasma IL2, IL6, IL10, IL12, interferon-gamma (IFNγ) or tumour necrosis factor alpha (TNFα), CCL2, CCL4, CCL5 and platelet-derived growth factor (PDGF)-bb were selected as candidate blood-based biomarkers (1, 12, 21, 22). Customised fluorochrome kits for multiplex fluorochrome technique (Luminex xMAPTM Technology, Austin, TX, USA) were purchased from Bio-Rad Laboratories (Bio-Rad Laboratories, Hercules, CA, USA). The levels of plasma cytokines and chemokines were analysed according to the Bio-Rad Laboratories protocol (www.Bio-Rad.com/bio-Plex/x-Plex).
DNA and SNP analysis. High-molecular weight DNA was extracted from peripheral blood samples using Qiagen Bio Robot M48 with MagAttract DNA Blood M48 kits (Qiagen, Hilden, Germany). For SNP analysis, all genotypes fulfilling the Hardy–Weinberg equilibrium (chi-squared test, p<0.05) were used. SNPs in genes associated with CRP, IL2, CCL4 and CCL5 expression that showed a tendency for association (p<0.1) between levels in controls vs. patient at R0 or between patient levels at R0 vs. R1 were investigated.
The selected SNPs were CRP rs1800947, IL2 rs6822844, CCL4 rs1719153, CCL5 rs2107538 and CCL5 rs2280789. All of these sequences were tested and passed two hits in the dbSNP database and were HapMap-validated with an Illumina design ability score according to the manufacturer's protocol (23). The genotyping of the SNPs was performed by Illumina Golden Gate Genotyping assay at the SNP & SEQ Technology Platform, Uppsala University, Sweden (http://www.genotyping.se).
Statistics. Student's t-tests were used for comparing the plasma levels of proteins, chemokines and cytokines between the control and patient levels at R0. Paired Student t-tests were used for comparisons between patients before (R0) and after (R1) adjuvant radiotherapy.
Plasma level (Mean±SD) of C-reactive protein (CRP), cytokines and chemokines in healthy blood donors (Controls) and patients with breast cancer before (R0) and after (R1) adjuvant radiotherapy.
Influence of single nucleotide polymorphisms (SNP) on plasma levels (Mean±SD) of C-reactive protein (CRP), interleukin 2 (IL2), chemokine (C-C motif) ligand 4 (CCL4) and CCL5 among controls.
The influence of SNPs on the levels of plasma biomarkers within the group of controls and patients at R0 or R1, were analysed. The influence of pathological conditions and SNPs on plasma biomarkers at R0 were analysed by comparisons between the controls and the patient with the SNP genotype using Student t-tests. The influence of adjuvant RT and SNPs on plasma biomarkers were analysed by comparisons between patient levels at R0 and R1 using paired Student t-tests.
All statistical analyses were two-sided and the threshold for significance was set at p≤0.05.
Results
Characteristics of the patients and controls. A total of 86 female patients with breast cancer with a median age of 67 (range=41-86) years were prospectively included after recovery from surgery (Table I). The patients were heterogeneous regarding TNM stage and hormone-receptor expression in the tumour. The control cohort comprised 82 female healthy blood donors with a median age of 56 (range=41-70) years.
The median age of the patients was higher than that of the controls but this did not reach statistical significance (data not shown).
Plasma CRP, cytokine and chemokine levels in controls and patients before and after adjuvant RT. After recovery from the removal of their breast cancer mass, the patients had significantly higher plasma levels of CRP (p=0.001) and CCL4 (p=0.004) but a lower level of IL2 (p=0.003) than the controls (Table II). Despite similar levels of CCL5 in the controls and the patient at R0, adjuvant RT significantly reduced the plasma CCL5 level in patients (R0 vs. R1: p=0.01).
The influence of SNPs and adjuvant RT on plasma CRP, IL2, CCL4 and CCL5 levels in the controls and the patients before and after adjuvant RT. No statistically significant influence of the studied SNPs on the corresponding CRP, IL2, CCL4 and CCL5 levels were found in the controls (Table III). SNP variations in CRP rs1800947 and IL2 rs6822844 were significantly associated with levels of CRP (p=0.05) and IL2 (p=0.01) of patients at R0. An impact of adjuvant RT and SNP sequence on the levels of CRP (p=0.05) and CCL5 (p=0.007) were detected in the patients at R1 (Table IV).
Influence of single nucleotide polymorphisms (SNP) on plasma levels (Mean±SD) of C-reactive protein (CRP), interleukin 2 (IL2), chemokine (C-C motif) ligand 4 (CCL4) and CCL5 among patients with breast cancer before (R0) and after (R1) adjuvant radiotherapy.
In those with the same SNP genotype (Table V), higher levels of CRP in the patients at R0 when compared to the controls were associated with CRP rs1800947 CC (p=0.003). Lower levels of plasma IL2 in these patients were associated with IL2 rs6822844AC (Controls vs. patients at R0: p=0.007). Combination effects of adjuvant RT and SNP on levels of plasma CCL5 were detected when comparing levels in patients at R0 with those at R1 based on CCL5 rs2107538 GG (p=0.04) and CCL5 rs2280789 AA (p=0.05).
Discussion
Despite recovering from surgery, significantly elevated levels of CRP and CCL4, as well as down-regulation of immune modulator cytokine IL2 were detected in patients with breast cancer. It has been reported that CCL2 and CCL5 are highly expressed in breast cancer cells or cells found in the tumour microenvironment (17). Similar levels of plasma CCL2 and CCL5 in our controls and patients before adjuvant RT suggested that the majority of tumour cells were successfully removed by surgery. SNPs in CRP and IL2 gene were associated with plasma levels of CRP and IL2 in patients but not in the controls. Since all visible tumour mass were removed, SNPs might influence systemic inflammation and immunosuppressive condition among patients with breast cancer (24, 25).
Significance of differences in plasma levels of C-reactive protein (CRP), interleukin 2 (IL2), chemokine (C-C motif) ligand 4 (CCL4) and CCL5 according to genotype when comparing controls and patients before (R0) and after (R1) adjuvant radiotherapy.
The majority of cells receiving adjuvant RT in our investigation were expected to be normal cells. RT-mediated cell death and its bystander effects can enhance host immune responses that contribute to the clinical outcome of patients (5, 15). An effect of adjuvant RT in our study was only documented for CRP and CCL5. The down-regulation of systemic inflammatory biomarkers CRP and CCL5 by adjuvant RT suggests a benefit from this treatment, especially in patients with CRP rs1800947 CC genotype and CCL5 rs2107538GG or CCL5 rs2280789 AA genotype (26).
Genetic variations based on SNP sequences had no influence on the plasma levels of these immune response biomarkers under healthy conditions. The lack of association between the levels of CCL4 and CCL4 rs1719153 might be due to the limited number of patients. Alternatively, the selected SNPs might only marginally influence CCL4 production in the systemic circulation.
The plasma levels of IL2, CRP and CCL5 have been suggested to be associated with the clinical outcome of patients with cancer (12, 13, 19, 27). Blood-based analyses of SNPs and plasma CRP, IL2 and CCL5 are low cost, rapid and can be undertaken using routine laboratory facilities while requiring only a peripheral blood sample. The possibility of using these blood-based biomarkers as indicators of immune status for selection of individual patient treatment warrants further investigation (28).
Acknowledgements
The Authors would like to thank the patients and healthy blood donors who participated in this study, Tomas Axelsson for SNPs analysis and the staff of Ryhov Hospital, Jönköping for practical support.
This investigation was partially supported by the Jönköping Clinical Cancer Research Foundation (Grant 110426-1), Futurum (Grant 144631), FORSS (Grant 567001), the Thai Office of Science and Technology in Brussels as well as the Ministry of Science and Technology. The funders had no role in study design, data collection and preparation of this manuscript or the decision to publish this investigation.
Footnotes
Authors' Contributions
NL, LER, SL and FL designed the study and analysed the results. TL and BÅA carried out the blood-based biomarkers experiments and data. DO, MS and MN responsible for included the patients, adjuvant RT and statistical analysis. All authors wrote, read and approved this manuscript.
Conflicts of Interest
The Authors declare that they have no conflicts of interest to disclose in regard to this study.
- Received January 21, 2019.
- Revision received February 11, 2019.
- Accepted February 14, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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