Abstract
Background/Aim: Adjuvant radiotherapy (RT) damages multiple layers of skin, muscle, blood vessels and blood cells that are included within the RT area. Indirect, bystander systemic effects could also develop in cells not directly hit by radiation. Materials and Methods: Ninety-three female patients recovering from breast cancer surgery and 82 female healthy blood donors were analyzed. For identification of systemic adaptive and innate immune response, rapid and low-cost blood-based biomarkers were assayed. Results: Post-operated breast cancer patients had a decreased number of circulating adaptive immune response cells but increased number of circulating immunosuppressive myeloid subpopulations. RT decreased the number of T-cells and platelets without influencing the number of immunosuppressive myeloid subpopulations. Alterations in the number and phenotypes of T-cell subpopulations were associated with SNPs. Conclusion: The combination of RT and immunotherapy might provide optimal treatment for cancer patients.
Adjuvant radiotherapy (RT) is an essential treatment of breast cancer and various other solid tumours after surgery. The anti-neoplastic properties of RT are primarily related to DNA damage and cell death. Direct damage in multiple layers of skin, muscle, blood vessels and blood cells that are included within the RT area could occur. Indirect, bystander systemic effects could also develop in cells not directly hit by radiation. Direct and indirect RT processes could lead to alterations of host immune response and inflammation (1-4).
Monocytes, neutrophils, natural killer (NK) cells and platelets are innate immune response cells that interact with lymphocytes, the adaptive immune response cells (5). Such interactions could result in either the activation or suppression of host immune response (6). Dysregulated numbers and ratios of circulating monocytes to lymphocytes (MLR), neutrophils to lymphocytes (NLR) or platelet to lymphocytes (PLR) suggests poor survival of cancer patients (7).
CD8+ cells in the T-lymphocyte group could be divided into cells expressing high levels of CD8 (CD8bright) with α/β heterodimer receptors and cells expressing low levels of CD8 (CD8dim) with α/α homodimer receptors (8-10). Surface CD8 substantially contributes to CD8+ cell-mediated functions such as cytokine production and cytolytic activity (9). Increased number of CD8dim cells has been associated with immunosuppressed state (10) and cytotoxic T-lymphocyte (CTL) impairment in patients and healthy individuals (11-13).
Granzyme B (GZB), a cytolytic enzyme and perforin (PRF), a pore-forming protein, are the major mediators of granule-exocytosis activity (14). Variation in GZB expression in CD8dim subpopulations due to single nucleotide polymorphisms (SNPs) has been detected in healthy individuals (15). Over-expression of GZB in CD8+ cells is a putative biomarker of impaired immunity in systemic lupus erythematosus patients (16).
Blood flow to normal breast tissue is highly variable between subjects, approximated 22±13 ml/min (17, 18). With this flow rate, innate and adaptive immune response cells in 1,100 ml to 1,750 ml circulating blood could be influenced directly by adjuvant RT.
The patient immune response status plays an important role in the outcome of patients with cancer (19). The present study aimed to determine systemic immune status of patients after recovery from surgery for breast cancer and the influence of adjuvant RT. The role of certain SNPs was also investigated. Rapid and low-cost methods analysing circulating biomarkers concerning innate and adaptive immune response were applied.
Materials and Methods
Patients and controls. Ninety-three female breast cancer patients aged over 18 years and scheduled for adjuvant RT after surgery were invited to participate. If they accepted the invitation, peripheral blood was drawn twice. The first baseline sample was obtained after recovery from surgery and before adjuvant RT (R0). The second follow-up sample was obtained directly, after completion of adjuvant RT (R1). Eighty two anonymous female healthy blood donors with no history of cancer or use of any immunomodulation agents were included as controls. One peripheral blood sample was obtained from controls.
Informed consent was obtained from all participants. The study was conducted in accordance with the Declaration of Helsinki and the Ethical Board at Linköping approved this investigation.
Adjuvant RT. Adjuvant RT was delivered at the Department of Oncology, Ryhov hospital Sweden, using Varian True beam machines (Varian Lina 2100 CD, Paulo Alto, CA, USA). According to size of the remaining breast after recover from partial mastectomy, two parallel opposing tangential fields was used. They were prescribed to the 95 % isodose according to International Commission on Radiation Units (ICRU), a 3-dimensional treatment planning system (Oncentra masterplan v 4.3, Elekta AB, Stockholm, Sweden). The absorbed dose was 50 Gy in 25 fractions given as 2 Gy per fraction. This treatment required approximately 2 min per day, 5 days per week for a total of 5 weeks.
Haematology and flow cytometry analysis. The levels of circulating platelets, total white blood cells (WBCs) and their subpopulations were analysed from whole blood samples using System XE5000 (Sysmex Corporation, Kobe, Japan).
Phenotypes of ex vivo, peripheral WBCs were analyzed using Becton Dickinson FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) within three hours. All monoclonal antibodies were purchased from BD Biosciences. These cells were directly stained for surface marker expression of CD3, CD4, CD8, CD11, CD13, CD14, CD33 and CD56 or intracellularly stained for GZB and PRF expression, according to BD's protocol. Phenotypes and the real numbers of circulating blood lymphocyte, monocyte and neutrophil subpopulations were analyzed after gating according to their location.
SNP analysis. High-molecular weight DNA was extracted from ex vivo peripheral blood samples using QIAGEN Bio Robot M48 with MagAttract DNA Blood M48 kits (Qiagen, Valencia, CA, USA). The quantity and quality of DNA was determined by the Pico-green method using Quant-iT™ Pico Green™ dsDNA Assay Kit (ThermoFischer Scientific, MA, USA). The Pico Green fluorescence intensity was determined by Hidex Sense Microplate reader (Hidex Oy, Turku, Finland).
Characteristics of 93 female breast cancer patients.
Based on our previous investigation, the following SNPs in GZB rs8192917, PRF rs10999426 and PRF rs3758562 were analyzed (15). These SNP sequences were HapMap-validated with Illumina design ability score according to the manufacturer's protocol (20). The genotyping of SNPs was performed at the SNP & SEQ Technology Platform, Uppsala University, Sweden (www.genotyping.se).
Statistical analysis. Mann-Whitney U-test was used for comparisons of immune response parameters between controls and patients before adjuvant RT, patient R0. To examine the influence of adjuvant radiotherapy, Wilcoxon test was used for comparisons between the patients paired blood sample, before adjuvant RT (Patient R0) and after adjuvant RT (Patient R1), respectively. All comparisons were two-sided and p≤0.05 were considered statistically significant.
Results
Characteristics of patients and controls. A total of 93 female breast cancer patients were prospectively included after recovering from partial mastectomy. Their median age was 65 years (range=41-86 years). They were heterogeneous regarding TNM stage, histology and hormone receptor expression in the tumour (Table I).
Eighty-two female healthy blood donors, median age of 56 years (range=41-70 years), were included as controls. The median age of patients was higher than the controls, but this did not reach statistical significance.
The phenotypes and the numbers of circulating innate and adaptive immune response cells in controls and patients. After recovery from removal of their breast cancer mass, the patients had lower number of circulating lymphocytes compared to controls (p=0.0014) but similar number of monocytes or neutrophils (Table II). As a consequence, the monocyte to lymphocyte ratio (MLR), neutrophils to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) were statistically significantly increased in the patients R0 (Table II).
Circulating innate and adaptive immune response parameters in controls and patients before (R0) or after (R1) adjuvant radiotherapy.
Alterations in the phenotypes of lymphocytes (Figure 1), monocytes and neutrophils (Figure 2) were detected in the patients compared to controls. The number of CD3+, CD4+ and CD56+ cells in lymphocyte population were significantly lower in patients R0 compared to controls (Table II and Figure 1). Number of CD13+CD56+ cells in monocyte population or neutrophil population were significantly higher (p=0.0001) in patients R0 compared to controls (Table II and Figure 2). Decreased ratio of CD4+ to CD8+ cells (CD4/CD8 ratio) was also detected in the patients R0 (p=0.001).
Adjuvant RT significantly decreased real numbers of all investigated adaptive and innate immune response subpopulations, except eosinophils (Table II). Despite decreasing real numbers of cells, increased MLR, NLR and PLR were observed in patients R1 compared to R0 (p=0.0001). At lymphocytes gate, lowest levels of CD3+, CD56+, CD4+, CD8+, CD8dim and CD8bright cells were also detected in the patients R1 compared to patients R0 (Table II). A higher CD4/CD8 ratio was also detected after adjuvant RT (p≤0.0001). The adjuvant RT had no influence on circulating numbers of CD13+CD56+ expressing cell in monocyte or neutrophil gate.
Number of circulating CD4+ and CD8+ cells expressing GZB and PRF in relation to SNPs and adjuvant RT. Lower numbers of GZB and PRF expressing in CD4+ cells were detected in patient R0 compared to controls (Table III). The PRF rs375862 AG+GG sequence correlated to increasing real numbers of PRF+ cells in CD8+ dim population of patients compared to controls (p=0.046).
Adjuvant RT decreased the real numbers of lymphocytes expressing GZB and PRF in CD4+ cells and CD8+ cells. Based on the GZB rs8192917 sequences, the number of GZB expressing CD4+, CD8bright and CD8dim cells was lower in patients R1 than patients R0 (Table III). The influence of adjuvant RT on PRF expression in CD4+, CD8bright and CD8dim differed. Decreased real numbers of CD8dim cells expressing PRF after adjuvant RT were associated with the SNPs in PRF rs10999426 and PRF rs375862 (p<0.0001) of these patients. The influence of adjuvant RT and SNPs on PRF expression in circulated CD8bright cells were not detected in these patients.
Circulating CD13+CD56+ cells in monocyte (M) and neutrophil (N) populations. Flow cytometry dot plot results presented ex vivo fresh blood cells of one control (Control) and one breast patient before (R0) and after (R1) adjuvant radiotherapy, respectively.
Discussion
Real numbers and distribution of innate and adaptive immune response subpopulations play an important role in cancer patients (7). Elevated levels of circulating innate and adaptive immune response cells in patients indicate systemic inflammation and poor survival (7). Decreased numbers of circulating helper T-cells, NK cells and increased number of immunosuppressive monocytes and neutrophils in cancer patients has been documented (21, 22). After the patient's recovery from removal of visible tumour, our investigation suggests that a general immunosuppression persists.
Circulating perforin (PRF+) and granzyme B (GZB+) expressing CD4+, CD8dim and CD8bright cells populations. Flow cytometry dot plot results represented ex vivo fresh blood cells of one control (Control) and one breast cancer patient before (R0) and after (R1) adjuvant radiotherapy, respectively.
The impact of adjuvant RT on human body is more complex than in in vitro or in vivo models. Blood vessels and blood flow under the treatment area will be affected during adjuvant RT. Given the general blood flow rate, immune response cells in more than 1000 ml blood are expected to be directly in contact with radiation (17, 18).
Decreasing platelet numbers in blood circulation after adjuvant RT might be a consequence of platelet activation and consumption due to blood vessel destruction. Adjuvant RT did not influence the real number of circulating CD13+CD56+ monocytes and neutrophils. The impaired immune response in patients R1 might associate to decreased numbers of lymphocytes but stable number of immunosuppressive CD13+CD56+ monocytes and neutrophils (21, 22) .
In healthy individuals, the CD4/CD8 ratio declines with age indicating immunological changes due to the ageing process or viral infection (23, 24). The decreasing CD4/CD8 ratio is correlated to increased mortality in the elderly (23). Increased circulating CD8dim cells are associated with peripheral exhaustion or impairment of effective CTL (12, 13).
SNPs in GZB and PRF genes influence numbers of GZB and PRF expressing cells in blood circulation (15). SNPs in GZB and PRF genes seem to have a higher impact on the numbers of GZB and PFR expressing cells in the CD4+ population than in the CD8+population. Adjuvant RT and GZB rs8192917 influenced the number of circulating GZB expressing CD4+, CD8bright and CD8dim cell populations. The impact of adjuvant RT and SNPs in the PRF gene on the number of cells expressing PRF seem to be pronounced in CD4+ and CD8dim cells but not in the CD8bright population. Thus, decreasing numbers of GZB expressing CD8dim cells, stable numbers of GZB expressing CD8bright and increasing CD4/CD8 ratio might be the beneficial effects of RT on adaptive immunological functions (9, 25).
SNP genotypes and real numbers of circulating granzyme B and perforin expressing cells in controls and patients before (R0) or after (R1) adjuvant radiotherapy.
Conclusion
A state of immunosuppression persists in breast cancer patients after recovery from removal of visible tumor by surgery. This suppression is manifested by a decreasing number of circulating adaptive immune response, CD4+ cells and increased numbers of immunosuppressive, CD13+CD56+ myeloid cells. Alteration in this adaptive immune response was associated with genotype variations in GZB and PRF genes. Adjuvant RT decreased the real numbers of platelet and adaptive immune response subpopulations without influencing the number of immunosuppressive cells in myelocytes. Our investigation suggests that the combination of RT and immunotherapy may provide an optimized treatment for these patients. The use of these rapid and low-cost blood-based biomarkers for the identification of patient's immune status and the influence of RT needs further investigation.
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 throughout this investigation. Our investigation was 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, Ministry of Science and Technology. The funders had no influence in the study design, data collection, analysis, preparation or decision to publish this investigation.
Footnotes
↵* These Authors contributed equally to this work.
Authors' Contributions
NL, LER, SL and FL designed the studies. TL, LS, DO and B-ÅA included the blood samples and carried out the experiments. All Authors analyzed data, wrote, read and approved the final manuscript.
Conflicts of Interest
The Authors declare that they have no conflicts of interest to disclose in regard to this study.
- Received July 10, 2019.
- Revision received July 22, 2019.
- Accepted July 23, 2019.
- Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
