Changes of Peripheral Blood Lymphocyte Subtypes in Patients with End Stage Cancer Administered Localized Radiotherapy and Bojungikki-Tang

Localized radiotherapy (RT) can cause immune dysfunction. Bojungikki-tang is known to restore immune function. We investigated the absolute counts and percentages of peripheral blood (PB) lymphocyte subtypes in end stage cancer patients before and after RT and after oral administration of Bojungikki-tang water extract (BJITE) and to evaluate the changes mediated by RT and BJITE. Absolute counts and percentages of lymphocyte and lymphocyte subsets were determined in whole blood using the TetraONE System (Beckman Coulter, USA). Flow cytometry results were compared before and after RT and after administration of BJITE. Absolute numbers of CD3+, CD4+, and CD8+ T cells and CD19+ B cells decreased significantly after RT (P < 0.05). Absolute numbers of CD3-CD56+ cells did not change in both groups. No significant differences were observed in the absolute counts of lymphocyte subtypes before and after administration of BJITE or vitamin group. When BJITE group was compared with vitamin group, absolute numbers of CD19+ B cells increased. RT-induced decrease in T cells and B cells in PB suggests that immune deterioration occurs after RT. Administration of BJITE might be effective in the restoration of number of B cells.


Introduction
Radiotherapy (RT) can be used to control symptoms of patients with end stage cancer. RT with palliative intent is administered in approximately one-half of cancer patients [1]. The purposes of palliative RT are to decrease symptoms, including pain, bleeding, and obstruction, and to improve the patient's quality of life [1,2]. However, there is an adverse effect of palliative RT. It has been demonstrated that localized radiation, even if administered to limited target volume, causes immune dysfunction [3][4][5].
Bojungikki-tang (Hochuekkito in Japanese and Buzhong-yi-qi-tang in Chinese) is a traditional herbal formula used in Korea, Japan, and China and it is composed of 10 species of medicinal plants. It has been traditionally used to improve severe weakness in Asian countries. Recent studies have demonstrated that Bojungikki-tang water extract (BJITE) has effect on restoring immune function [6] and inducing an increased protection against microbial agents [7]. It is useful not only for an enhancement of natural killer (NK) activity [8] but also for restoration of antitumor T cell response from stress-induced suppression [9]. Bojungikkitang is known to have protective effect of intestine and hematopoietic organs against radiation damage [10]. However, the immunological response of Bojungikki-tang in patients administrated RT has not yet been determined. The aims of the present study were to determine the absolute counts and percentages of peripheral blood (PB) lymphocyte subtypes in end stage cancer patients before and after RT and after oral administration of Bojungikki-tang using single-platform technology and to evaluate the changes mediated by RT and Bojungikki-tang.

Methods and Materials
2.1. Patients. Thirteen patients were enrolled in this single center, randomised controlled study. Patients meeting the following criteria were included: age 40 years or older; ECOG performance score 0-2; end stage cancer; selected for palliative RT. Exclusion criteria were as follows: patients who have been treated with operation, chemotherapy, or curative radiotherapy in the 2 months prior to randomisation; patients with severe hepatic or renal dysfunction (AST > 80 IU/L, ALT > 80 IU/L, BUN > 50 mg/dL, and creatinine > 3.4 mg/dL); subjects with a history or hypersensitivity to functional foods; women who are pregnant or nursing; patients who are of child bearing age and are not willing to use contraception; patients who have had major surgery in the 3 months prior to randomization; patients with neuropsychiatric disease; patients who have had cardiovascular or cerebrovascular disease during the previous 6 months; patients who have taken drug in the 3 months prior to randomization. All patients had to provide written informed consent before registration and the trial protocol was approved by the Institutional Review Board of Daegu Catholic University Medical Center, Korea.
Eligible patients were randomly assigned (1 : 1) to palliative RT with Bojungikki-tang (BJITE group) or palliative RT with vitamin (vitamin group). Table 1 shows the patients' clinical characteristics. All patients except one suffered from metastatic tumor.

Blood Collection.
Whole blood samples were collected one day before starting RT (Time 0), one day after completing RT (Time 1), and four weeks after oral administration of BJITE or vitamin (Time 2) in sterile EDTA vacutainers.

Percentages and Counts of Total Lymphocytes and Lymphocyte Subsets after Radiotherapy.
Using the single-platform technology, we initially compared both the percentages and absolute counts of lymphocyte subsets in all patients. Absolute counts and percentages of lymphocytes and lymphocyte subsets after RT in the BJITE group ( = 7) versus vitamin group ( = 6) are shown in Tables 2 and 3, respectively. There were no significant changes in total lymphocyte counts or percentages of lymphocytes during period of time in both groups. No significant differences were observed in the percentages of lymphocyte subtypes. However, absolute numbers of CD3+, CD4+, and CD8+ T cells and CD19+ B cells decreased significantly after RT ( < 0.05) (Figure 1). Absolute numbers of CD3-CD56+ cells did not change in both groups.

Effects of BJITE on Total Lymphocytes and Lymphocyte
Subsets. Absolute counts and percentages of lymphocytes and lymphocyte subsets after administration of BJITE or vitamin are shown in Tables 2 and 3, respectively. After administration of BJITE, there were no differences in total lymphocyte number and all lymphocyte subsets compared with baseline data. No differences were found, before and after administration of BJITE or vitamin group, in the CD3+, CD4+, and CD8+ T cells as well in of CD56+ cells. However, there were significant differences in CD19+ B cell counts and percentages of CD19 in vitamin group. In vitamin group, the percentages and absolute counts of CD19 cells did not increase compared with baseline status and remained the decreased status.

Discussion
Bojungikki-tang is known to restore immune functions and to improve anti-tumor activity [11][12][13]. It is usually indicated for patients with general weakness and anemia. Palliative RT relieves clinical symptoms in advanced cancer patients but it can also cause immunological changes. In the present study, we have investigated the percentages and absolute counts of PB lymphocytes and lymphocyte subtypes in patients with end stage cancer administered localized radiotherapy and Bojungikki-tang. In our study, total lymphocyte in counts and percentages of lymphocytes did not change after palliative RT in both groups. This is contrast to the results of the earlier study of radiation-induced lymphocytopenia [3][4][5]14]. This is probably due to differences of radiation dose and interval.
Absolute counts of T cells and B cells declined after localized RT. Absolute counts of NK cells were not affected by RT. Local radiation at therapeutic doses always triggered some activation of the innate and adaptive immune system Evidence-Based Complementary and Alternative Medicine 5 [15]. Results of present study correspond well with those of the earlier study, which reported that naive T cells and B cells are highly radiosensitive [16,17]. No significant differences were observed in the percentages of lymphocyte subtypes in all patients. These findings differ from the results of the earlier study, which reported that, unlike absolute numbers, the percentages of all analyzed lymphocyte subsets of cervical cancer patients significantly elevated after RT [18]. Safwat et al. [14] reported that localized RT is associated with a significant increase in the percentage of CD4+ T cells and a significant reduction of the absolute number of lymphocyte subsets in patients with non-Hodgkin's lymphoma. This result is similar to our result except for the increase of CD4+ T cell percentages.
Absolute counts of lymphocyte subpopulations in PB have been traditionally measured by dual-platform technologies, which were standard, widely used methodologies. These methods couple percentages of positive cell subsets determined by flow cytometry with the absolute lymphocyte count obtained by automated hematology analyzers. However, they may be responsible for substantial differences in absolute lymphocyte counts determined by different analyzers [19]. Recently, single-platform technologies were developed and are performed entirely on flow cytometry. These methods have significantly improved the assay precision and accuracy and agreement of results between laboratories [19,20]. Using the single-platform flow cytometry, it was possible to independently analyze the percentages and absolute counts of lymphocyte subsets. In this study, absolute counts of lymphocyte subpopulations decreased significantly after RT, although the percentages of lymphocyte subsets did not change. The values of percentages did not consider total WBC count, which might be changed in cancer patients, particularly after administration of RT. Therefore, it may be useful to determine absolute counts, not percentages, of lymphocyte subsets for exactly reflecting immune status of patients.
Absolute counts or percentages of lymphocytes from BJITE or vitamin group showed no significant differences before and after administration of BJITE or vitamin. Another study also reported that the total number of circulating lymphocytes; CD3+, CD4+, and CD8+ T cells; and CD20+ B cells remained unchanged [21].
Interestingly, CD19+ B cells increased significantly after administration of BJITE. These findings are in contrast to the results of the earlier study, which reported that BJITE is remarkably effective in the restoration of number of T cells and NK cells [8]. Bojungikki-tang is known to have beneficial effects on anti-tumor activity [9] or NK cell activity [21]. In this study, measurement of NK cell activity was not performed. The administration of Bojungikki-tang polysaccharide fraction was associated with elevated expression levels of CD19/CD40 specific for pre-B cells [22].

Conclusions
In conclusion, RT-induced decrease in helper T cells, cytotoxic T cells, and B cells in PB suggests that immune deterioration occurs after RT. Administration of BJITE might be effective in the restoration of number of B cells.