Comparative analysis of the effect of different radiotherapy regimes on lymphocyte and its subpopulations in breast cancer patients

Brief Research Article

Abstract

Purpose

The aim of this study was to determine whether different radiotherapy (RT) fractionation schemes induce disparate effects on lymphocyte and its subsets in breast cancer patients.

Methods

60 female patients diagnosed with breast cancer were recruited in this study after receiving modified radical mastectomy and were randomly divided into two groups. One group received irradiation at a standard dose of 50 Gy in 25 fractions and the other at a dose of 40.3 Gy in 13 fractions. Both total lymphocyte count and its composition were recorded at three timepoints: right before the radiation treatment (T0), immediately after the last fraction of radiotherapy (T1) and 6 months after irradiation therapy ended (T2).

Results

Both groups experienced temporal lymphopenia after finishing local radiation (T1) (13F T0 vs. T1 1570.6 ± 243.9 vs. 940.6 ± 141.8, **p < 0.01; 25F T0 vs. T1 1620.5 ± 280.2 vs. 948.5 ± 274.6, **p < 0.01), while the lymphocyte count recovered at follow-up time (T2), and the cell count in the hypofractionation group (13F) was higher than the standard fraction group (25F) (13F vs. 25F 1725.6 ± 225.6 vs. 1657.5 ± 242.4, *p < 0.05). With respect to the composition of lymphocyte, we found T cell, B cell, and NK cell reacted differently to different radiotherapy protocols.

Conclusions

Different RT protocols impose different impacts on immunity, leading us to further explore the optimal radiotherapy regimes to synergy with immunotherapy.

Keywords

Breast cancer Radiotherapy Peripheral lymphocyte Immune-oncology 

Introduction

Breast cancer is the most common cancer in females around the whole world [1, 2]. Adjuvant radiotherapy plays a significant role in the treatment of breast cancer; it can improve both local control and overall survival, as well as reduce the risk of recurrence and death [3, 4]. With respect to breast cancer irradiation protocols, apart from the standard 50 Gy/25F regime, there are also several hypofractionated schedules with increased dose per fraction and shorter treatment duration. The data of a big clinical trial show no significant difference between conventional and hypofractionated radiation schedules in terms of the risk of local recurrence and overall survival after 10 years of treatment in breast cancer patients prescribed post-surgery radiotherapy [5].

Radiotherapy has historically been regarded as immunosuppressive [6, 7], whereas the observation of “abscopal effect” elicited scientists’ reconsideration on its positive immunological effects [8, 9]. The term “abscopal” was defined by Mole in 1953; it referred to tumor regression out of radiation field while within the same organism [10]. On the contrary to inhibit the immune function, accumulating evidence has shown that radiotherapy can boost anti-tumor immunity. Briefly, its immunostimulatory response may include the followings: promote the cross presentation of tumor-derived antigens to lymphocytes by dentritic cells [11], enhance the ability of lymphocytes to migrate to and infiltrate the tumors [12], and stimulate lymphocyte’s ability to bind to and kill cancer cells [13]. Herein, we can see the pivotal role of lymphocyte in the anti-tumor immunity generated by radiation therapy. In animal experiments, the key role of lymphocyte has been verified, for example: to achieve the same tumor control, immunocompetent mice need 30 Gy, while T-cell-deficient mice need 60 Gy [14]. Lymphocyte count can partly reflect the function of immune system, and predict the outcome of cancer, e.g., Iris Kuss [15] reported that in patients diagnosed with squamous cell carcinoma of head and neck, those with active disease had significantly lower CD3+ and CD4+ T-cell counts than those with no evidence of disease. Zhang Ru Yang [16] found that in prostate cancer patients who received carbon ion radiotherapy (CIR), the CD4/CD8 ratio and CD19+ B-cell counts were higher in patients with a complete response or partial response to CIR than classified in the stable disease group.

Much has been learnt about the changes of lymphocyte subsets after radiotherapy of one specific fractionated schedule, yet little has been investigated to compare the influence of different RT fraction model. Thus, in this study, we enrolled women diagnosed with breast cancer and randomly classified them into two cohorts: one cohort received irradiation of 50 Gy/25F regime and another received 40.3 Gy/13F regime. We recorded every patient’s total lymphocyte count and subpopulation composition right before RT (T0), immediately after the last fraction (T1) and 6 months after the end of radiotherapy (T2) by flow cytometry, and then, comparative analysis was made between the two groups.

Materials and methods

Study collective

We enlisted a total of 60 female patients with unilateral invasive breast carcinoma diagnosed and treated in our hospital from years 2015 to 2016. They were confirmed by pathology and received modified radical operation and auxiliary lymph node dissection. All of them had the indication to receive adjuvant radiotherapy. Patients with a history of prior thoracic radiotherapy, distant metastasis, second tumor, and immune system diseases were excluded from the study. Written informed consent was obtained from each individual patient. All the patients received 4–6 cycles of chemotherapy (CT) after the breast surgery. The selections of chemotherapy regimens and cycles were according to the TNM grade, status of ER/PR, Her-2, and Ki-67. There was an interval of 3 weeks between the end of CT and the initiation of RT. Random number table was used to assign 60 patients into two groups evenly: one group received 50 Gy/25F RT regime and another received 40.3 Gy/13 regime. In detail, 60 consecutive numbers were randomly chosen from the random number table, and they represent the patients recruited into the trial. When the first patient was enrolled if the first random number was an even number, then this patient would be in 50 Gy/25F RT, otherwise in 40.3 Gy/13. Likewise, other patients were assigned into one of the two groups. All the patients received conventional two-dimensional RT using a linear accelerator with 6MV-X and 6 MeV-E (Siemens Primus linear accelerator). The radiation fields contained ipsilateral chest wall and supraclavicular region. Patients’ characteristics are shown in Table 1.
Table 1

Patient characteristics

Characteristics

N

%

Age (years)

 30–39

16

26.7

 40–49

24

40.0

 50–59

20

33.3

Radiotherapy regime

 50 Gy/25F

30

50.0

 40.3 Gy/13F

30

50.0

pTNM

 T1

15

25.0

 T2

38

63.3

 T3

7

11.7

 N1

11

18.3

 N2

34

36.7

 N3

15

25.0

 M0

60

100

Hormone receptor status

 Estrogen receptor (ER)

  +

50

83.3

  −

10

16.7

 Progesterone receptor (PR)

  +

42

70.0

  −

18

30.0

Her-2 status

 +

18

30.0

 −

42

70.0

Ki-67 index

 < 14%

10

16.7

 > 14%

50

83.3

Blood collection and flow cytometry

Venous blood (10 ml) was collected in heparinized tubes at three timepoints: T0, T1, and T2. Blood samples were tested within 6 h of acquisition in laboratory. To analyse the percentage and count of lymphocyte, a standard single-platform technique was used. 100 μL anticoagulated whole blood was mixed with 10 μL fluorochrome-conjugated monoclonal antibodies. The monoclonal antibodies used in this study were: anti-CD45-FITC, anti-CD4-PE, anti-CD8-ECD, anti-CD3-PC5, anti-CD56-PE, and anti-CD19-ECD. The selection of lymphocyte was based on the expression of bright CD45 and low side scatter signals. The identification of T-cell subsets was based on the expression of CD3, CD4, and CD8. Meanwhile, the identification of B cell and NK cell was based on the expression of CD19 and CD56,respectively. The flow cytometric analysis was performed on the FCM, Cytomics (Beckman Coulter).

Statistical analysis

In this study, all the statistical analyses were performed with SPSS 22.0 software, and statistical significance was considered when p < 0.05. If samples followed normal distribution, paired t test or two-sided t test was used. If not, Wilcoxon test was used instead.

Results

Patient characteristics

A total of 60 female patients with unilateral breast cancer treated in our hospital were enrolled in this study. Before receiving radiotherapy, all the patients have received modified radical mastectomy and chemotherapy. The median age was 48 years ranged from 34 to 57 years. The mean age of patients in the standard RT schedule group and hypofractionation group was 49 and 53 years, respectively (p > 0.05). Patients in both groups are all in good general physical condition (the ECOG performance status in both groups was 0 or 1), and they had no immune system diseases; moreover, there was no significant difference in the total lymphocyte number of the two groups before the treatment of radiotherapy. They all finished the RT schedule, and serious side effects caused by radiotherapy were not observed. More detailed characteristics are listed in Table 1.

Dynamic changes of lymphocyte count from T0 (before RT) to T2 (6 months after the end of RT)

As shown in Fig. 1, similar dynamic changes were seen in terms of total lymphocyte count from T0 to T2 in the two cohorts. Before the initiation of radiotherapy, there were no significant differences between two groups (13F vs. 25F 1570.6 ± 243.9 vs. 1620.5 ± 280.2, p > 0.05). After the last fraction of RT, lymphocyte count dropped significantly (13F T0 vs. T1 1570.6 ± 243.9 vs. 940.6 ± 141.8, **p < 0.01; 25F T0 vs. T1: 1620.5 ± 280.2 vs. 948.5 ± 274.6, **p < 0.01), and no difference was observed between two cohorts (T1 13F vs. 25F: 940.6 ± 141.8 vs. 948.5 ± 274.6, p > 0.05). After 6 months, lymphocyte number recovered to the baseline level (T0) in both groups, and the cell count in the hypofractionation group (13F) was higher than the standard fraction group (25F) (T2 13F vs. 25F 1725.6 ± 225.6 vs. 1657.5 ± 242.4, *p < 0.05).
Fig. 1

Total lymphocyte number (cells/μl) in the two groups at T0, T1, and T2. After the last fraction of radiotherapy (T1), lymphocyte count in both groups dropped significantly, and recovered to baseline 6 months after the end of radiotherapy. At T2, patients in the hypofractionated RT group had higher lymphocyte count than patients in standard RT group. t test, *p < 0.05, **p < 0.01

Alteration of lymphocyte subpopulation from T0 to T2 in two groups

Figure 2a reveals the alteration of the percentage of CD3+ T lymphocyte from T0 to T2. No significant difference was found during our observation in both groups. From Fig. 2b, c, we found that CD3+/CD4+ T cell and CD3+/CD8+ T cell reacted differently to RT. In the hypofractionation group (13F), the percentage of CD3+/CD4+ T cell kept elevated from T0 to T2 (T0 vs. T1 41.5 ± 6.9 vs.43.7 ± 7.1, *p < 0.05, T0 vs. T2 41.5 ± 6.9 vs.44.5 ± 9.2, *p < 0.05). Whereas in the standard fractionation group, the percentage of CD3+/CD4+ T cell increased after the last fraction of radiotherapy and then dropped to the pre-treatment level after 6 months of RT (T0 vs. T1 41.9 ± 6.3 vs. 46.6 ± 8.4 *p < 0.05, T0 vs. T2:41.9 ± 6.3 vs. 43.1 ± 6.2 p>0.05) (Fig. 2b). For the percentage of CD3+/CD8+ T cell, the two groups also had disparate changes. The percentage showed no significant difference in hypofractionation cohort (13F) during the duration of observation (T0 vs. T1 30.5 ± 4.4 vs. 29.1 ± 5.6 p>0.05, T0 vs. T2 30.5 ± 4.4 vs. 31.7 ± 6.2 p>0.05); however, in the other cohort (25F), the percentage kept unchanged from T0 to T1 and then dropped significantly 6 months later (T0 vs. T1 34.1 ± 6.1 vs. 35.0 ± 6.3 p>0.05, T0 vs. T2 34.1 ± 6.1 vs.30.4 ± 6.1 *p < 0.05) (Fig. 2c).
Fig. 2

Percentage of lymphocyte subpopulations before RT after the last fraction of RT and 6 months after RT in two groups breast cancer patients receiving different radiotherapy regimes: a CD3+ T lymphocyte; b CD3+ CD4+ T lymphocyte; c CD3+ CD8+ T lymphocyte; d CD3–CD19+ B cell; e CD3–CD56+ NK cell. Wilcoxon test *p < 0.05

In terms of CD3−/CD19+ B cell, the percentage of cell dropped when the radiotherapy was finished in both groups (13F T0 vs. T1 6.7 ± 4.2 vs. 5.1 ± 3.8 *p < 0.05; 25F: T0 vs. T1 5.1 ± 2.9 vs. 4.0 ± 2.4 *p < 0.05). Then, after 6 months of recovery, the value returned to the baseline in hypofractionation group (T0 vs. T2 6.7 ± 4.2 vs.6.2 ± 4.1 p>0.05), whereas in the standard group, the percentage was still lower than the pre-RT value (T0 vs. T2 5.1 ± 2.9 vs.3.9 ± 2.2 *p < 0.05) (Fig. 2d).

With respect to CD3−/CD56+ NK cell, the change tendency in the two groups kept in line. We found no significant difference in the percentage from T0 to T2 (13F T0 vs. T1/T2 18.6 ± 4.9 vs.20.4 ± 4.7/19.7 ± 4.7 p>0.05; 25F T0 vs. T1/T2 17.3 ± 5.3 vs. 15.4 ± 5.4/16.1 ± 5.1 p>0.05) (Fig. 2e).

Discussion

Radiotherapy has been applied to treat malignant tumors for over 100 years; its ability to directly kill tumor cells has been accepted widely. While RT has traditionally been considered as immunosuppressive and acted only as a local modality, a large amount of evidence indicates that these perspectives are not comprehensive and precise. Accumulating data has proved that ionizing radiation could elicit systemic anti-cancer immune response [17, 18, 19], and the “abscopal effect” was exactly a good demonstration. It has been learnt the immunostimulatory properties of RT may be achieved by modifying both tumor cells and immune compartments [20]. Radiation can cause several types of cell deaths, one of which is called immunogenic cell death [ICD]. In detail, ICD turns tumor cells into in situ vaccines, making them be more sensitive to immune-mediated eradication [21, 22]. The characteristics of ICD embody the following: 1. release of tumor antigens; 2. translocation of calreticulin (CRT) to the tumor cell membrane; 3. ATP release; and 4. release of high-morbidity group protein B1 (HMGB1), uric acid, and heat shock proteins (HSPs) [23, 24, 25]. Translocation of CRT plays an incredible role in the process of ICD, because it acts as an “eat me” signal to macrophages and dendritic cells (DCs), thus facilitating the tumor antigen uptake and presentation by DCs, then results in the cross priming of cytotoxic T lymphocytes (CTLs) [20]. The release of ATP and HMGB-1 also contributes to the above process. When sublethal dose of radiation is given, the phenotype of tumor cells will alter via immunogenic modulation (IM). Molecules changed by non-lethal irradiation include major histocompatibility complex 1 (MHC-1), adhesion molecules, death receptors, and tumor-associated antigens. Up-regulation of these molecules is believed to lead to enhanced tumor killing mediated by CTLs [18, 26]. Apart from influencing tumor cells, RT also affects immune microenvironment, including tumor-specific CD8+ T-cell generation, DCs maturation, and antibody production by B cell [20, 27]. Current studies have shown that “abscopal effect” was mediated by anti-tumor T lymphocyte [28]. As the mechanisms of RT-generated systemic anti-cancer response to be elucidated gradually, on one side, we should recognize that the response is harnessed by the cooperation of proper tumor alteration and appropriate host immune system; on the other side, the significance of the immunity of patients must be highlighted, since a normal immune system is the radical premise for the generation of RT’ systemic anti-cancer response.

Since lymphocyte plays an indispensable role in the immune system, so our study seeks to compare the influence imposed on lymphocyte by different RT regimes. With regard to total lymphocyte number, similar change trend was seen in both groups, and they all experienced temporal lymphopenia. The result was in accordance with earlier studies, indicating that lymphocyte was radiosensitive [2]. Some studies found that lower lymphocyte count was a predictor of poorer prognosis in cancer patients [29]; for lowered peripheral lymphocyte, number could predispose patients to tumor recurrence or a second malignancy. Fortunately, the RT-induced lymphocyte suppression was temporal; at the follow-up time (T2), the cell count recovered to pre-RT level. Nevertheless, our study did not document the consecutive lymphocyte number change; there was a 6-month interval between T1 and T2. Previous studies neither reached a consensus about when lymphocyte began to recover after receiving radiation. We discussed the systemic anti-tumor effect generated by radiotherapy above; however, RT alone rarely elicited effective anti-tumor immunity. Perhaps, the temporal diminution of lymphocyte is one of the reasons. Thus, oncologists wondered what should do to counteract the disadvantage of lymphopenia and augment the anti-tumor response induced by RT. Recently, they have focused their attention on integrating immunotherapy with radiotherapy to achieve this purpose. Selected as “Breakthrough of the Year 2013” by the editors of Science Magazine [30], the immune checkpoint inhibitor was one type of immunotherapy, and it could block the co-inhibitory molecules expressed on T cells; herein, unleash brakes on T cells and generates durable anti-cancer effect. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) were the widely studied checkpoints. Expressed on the surface of T cell, CTLA-4 could bind to B7 family ligand on antigen-presenting cell with greater affinity than CD28 (a co-stimulatory molecule), hampering the activation of anti-tumor T cell [31]. PD-1 was recognized as another brake on T lymphocyte when combined with PD-L1 ligand expressed by tumor cells,and it could inhibit the proliferation of lymphocyte and the release of cytokines [32]. Multiple preclinical models have demonstrated the synergistic relationship between radiotherapy and checkpoint inhibitors. Demaria found in a mouse model of breast cancer that radiation could turn a tumor unresponsive to anti-CTLA4 mAb into a sensitive one by converting the irradiated tumor to an immunogenic hub [33]. Dewan showed in mouse model that anti-CTLA4 mAb monotherapy could delay the tumor growth in primary site, while a complete regression in primary site was achieved with the combination of RT and mAb. Furthermore, the incorporation of RT and anti-CTLA4 mAb hindered tumor growth in a secondary site [34]. Furthermore, several clinical reports confirmed the efficacy of this integrative modality. For instance, a melanoma patient underwent tumor progression while using ipilimumab (an anti-CTLA4 mAb approved by FDA); surprisingly, tumor response exhibited after local radiation to the metastatic site and concurrent treatment of ipilimumab [35]. Given that no data of randomized clinical studies proving the synergic action between RT and anti-CTLA4 antibodies were available, several prospective trials are ongoing in melanoma patients.

In the above, we have discussed the synergistic anti-tumor effect of combining RT and checkpoint inhibitors together; however, it is still controversial to determine the optimal RT protocols in the combinatorial treatment. Appropriate radiotherapy dose is significant to generate anti-tumor response, because too low irradiation is unable to activate anti-tumor immunity and excessive irradiation can destroy the immunity. Dörthe Schaue showed in mice model bearing B16-OVA melanoma, tumor growth was delayed when local single-dose radiation between 7.5 and 15 Gy was given, while a single-dose irradiation of 5 Gy had little immunostimulatory effect [36]. Between radiation dose of 7.5 and 10 Gy, anti-OVA T-cell response increased, together with the decrease of regulatory T-cell percentage in spleen. Nevertheless, after single dose of 15 Gy, both effector cell and Treg increased. Notably, regulatory T cell was regarded to inhibit anti-cancer immunity. From radiobiological point of view, single-dose RT and multi-fractions RT would have different impacts on tumor cell and immune system, thus leads to diverse treatment outcome. Tsai demonstrated that fractionated 2 Gy×5 radiotherapy resulted in up-regulation of some gene expression, e.g., IFN-related genes; however, this effect could not be seen after single dose of 10 Gy was given [37]. Lugade discovered that fractionated radiotherapy was superior to single-dose radiation in the aspects of activating CD8+ T cell and triggering abscopal effect [27]. Dewan MZ tested the ability of disparate radiation schemes to synergy with anti-CTLA4 mAb. Mouse models bearing breast carcinoma and colon carcinoma were used in that study. Mice were randomly divided into eight groups: receiving no RT or three different RT protocol, respectively (20 Gy×1, 8 Gy×3, 6 Gy×5) combining with CTLA-4 antibody or not. Primary tumor control and abscopal effect were achieved only in the groups of integrating mAb and fractionated RT (8 Gy×3, 6 Gy×5) [34]. From the studies discussed above, perhaps, multi-fractionated irradiation would be more adequate than single-dose irradiation to synergy with immunotherapy. Moreover, the choice of fractionated RT enabled the normal tissue to repair during and after radiotherapy. In animal model, hyofractionated RT was found to be superior to conventionally fractionated RT [38], but very limited clinical trial results were available to bolster this view.

Conclusions

From the studies showed above, it is convincing that radiotherapy is immunostimulatory and has promising potential to synergy well with immunotherapy, particularly with the checkpoint inhibitors. Further studies are needed to determine the optimal irradiation dose and fractionation regime in the combinatorial treatment. Perhaps, such high-dose commonly used in the radical radiotherapy is not needed; lower dose that can just expose the immunogenicity of tumor cells will be better. Which one is more optimal to combine with immunotherapy, hypofractionated radiotherapy or standard fractionated radiotherapy? Unfortunately, we have no confirming answer. More clinical studies are urgently needed to unravel the significant question.

Notes

Compliance with ethical standards

Conflict of interest

All authors have stated that they have no conflicts of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.

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Copyright information

© Federación de Sociedades Españolas de Oncología (FESEO) 2018

Authors and Affiliations

  1. 1.Cancer Research CenterQilu Hospital of Shandong UniversityJinanChina

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