Abstract
Background/Aim: Radiation pneumonia is a main side-effect that has limited the clinical usage of radiotherapy in locally advanced esophageal carcinoma. T helper cells 17 (Th 17) and T regulatory cells (Tregs) play an important role in inflammatory diseases. The balance between Treg and Th17 cells is a key factor in the progression of many inflammatory and autoimmune diseases. Whether Tregs and Th17 cells are predictive factors of radiation pneumonia has not yet been reported. In this study, we investigated the relationships of Treg/Th17 cells and radiation pneumonia in patients with locally advanced esophageal cancer who received radiotherapy. Patients and Methods: One hundred and forty-eight patients with locally advanced esophageal cancer who received radical and palliative radiotherapy were enrolled. The levels of Th17 and Treg cells in the blood of patients were detected using flow cytometry at the time point of pre-radiotherapy, 1st, 2nd, 3rd, 4th, 5th and 6th week from the start of radiation and 4 weeks after completion of radiotherapy. Radiation pneumonia was evaluated according to Radiation Therapy Oncology Group's acute radiation pneumonia standards, with the endpoint being grade 2 or above radiation pneumonia. Results: There were 24 cases of radiation pneumonia in 148 cases of locally advanced esophageal cancer patients who underwent radiotherapy. Th17 cells increased and, in contrast, Treg cells decreased in the radiation pneumonia group. The change in the ratio of Th17/Treg was more pronounced and the difference was statistically significant from the 5th week after irradiation compared to patients with no radiation pneumonia (p<0.05). There was no significant difference in dosimetric parameters, including V5, V20, V30 and mean lung dose (MLD) and clinical factors, such as gender, age, smoking history, history of surgery and chemotherapy. Conclusion: The ratio of Th17/Treg cells may be an effective predictive factor of radiation pneumonia.
Radiation therapy is an effective treatment strategy for esophageal cancer (1-3). However, radiation pneumonia is a main side-effect that has limited the clinical usage of radiotherapy. Therefore, if radiation pneumonia can be predicted early enough, individualized dosage regimens that may improve radiation treatment outcome can be administered to patients.
Many studies show that some cytokines are closely related to radiation pneumonia, such as interleukin (IL)-6, IL-10, transforming growth factor (TGF)-β1 (4-7). The most important cytokine is TGF-β1, but Novakova-Jiresova et al. reported that there is no significant difference in the concentration of serum TGF-β1 between patients with and without radiation pneumonia (8-10). Therefore, TGF-β1 is not a reliable predictive factor for the risk of radiation pneumonia. Recent studies have found that T helper cells 17 (Th17) and T regulatory cells (Tregs) play an important role in the inflammatory diseases. The balance between Tregs and Th17 cells is a key factor in the progress of many inflammatory and autoimmune diseases (11-13). Whether Treg cells and Th17 cells are predictive factors for radiation pneumonia has not yet been reported. In this article, we studied the predictive effect of Th17 and Treg cells on radiation pneumonia in locally advanced esophageal cancer.
Patients and Methods
Patients. One hundred and forty-eight esophageal cancer patients treated with thoracic radiation at the Jiangsu University Affiliated People's Hospital between January 2010 and October 2014 were enrolled in this study. Inclusion criteria: (a) histological diagnosis of primary esophageal squamous cell carcinoma; (b) no previous thoracic radiotherapy; (c) dose-volume histogram (DVH) data available for analysis; (d) expected survival time >6 months; (e) Karnofsky performance score (KPS)>70.
Three dimensional radiotherapy planning. Radiotherapy was delivered through three-dimensional conformal radiotherapy using a linear accelerator (Siemens Oncor Berlin, Germany or Elekta, Stockholm, Sweden) with 6 MV or 10 MV X-Ray. All radiotherapy plans were designed using the Pinnacle 3 system. Simulation computed tomography (CT) scan was performed with the patients breathing freely and included the entire lung volume. The scan thickness was 3 mm. Gross tumor volume (GTV) was defined as the gross tumor defined by the simulation CT. The clinical target volume (CTV) included the GTV with 3.0-3.5 cm margin longitudinally and 1 cm axially. The planning target volume (PTV) was defined as CTV plus 0.5-1.0 cm margin in all directions. The prescribed dose was approximately 60-65 Gy, 1.8-2.0 Gy per fraction. Ninety-five percent volume of PTV received 100% of the prescribed dose. The spinal cord received a dose not more than 45 Gy and the mean dose received by the heart was not more than 30 Gy. No more than 30% of the combined lung volume was intended to receive more than 20 Gy (V20<30%).
Flow cytometry analysis. Peripheral blood samples from the patients with locally advanced esophageal cancer were collected at the time point of pre-radiotherapy, 1st, 2nd, 3rd, 4th, 5th and 6th week from the start of radiation and 4 weeks after radiotherapy had been completed. Samples were then centrifuged at 1,000 rpm for 5 min, with the supernatant being discarded and added into the lymphocyte separation medium. Peripheral blood mononuclear cells were separated by density gradient centrifugation and seeded into 6-well plates at a concentration of 2×106/ml. CD4+ IL-17+ cells (Th17) and CD4+ CD25+Foxp3 cells (Tregs) were evaluated by flow cytometry analysis.
Follow-up and evaluation of radiation pneumonia. Early radiation pneumonia usually occurs 1 to 6 months after radiation, whereas late lung fibrosis usually occurs 6-24 months after radiation. Since we were interested in the prediction of early radiation pneumonia, a 4-month period as cut-off for diagnosis was used.
Patients were evaluated by radiation oncologists every two weeks during radiation and once a month until four months after radiotherapy. A chest CT scan was performed at each follow-up evaluation after completion of radiotherapy. Radiation pneumonia was scored according to the Radiation Therapy Oncology Group's acute radiation morbidity scoring criteria for pneumonia and the diagnosis of radiation pneumonia was based on the clinical symptoms and the corresponding radiographic changes in the radiation field.
Ethics statement. The study was approved by the Jiangsu University Affiliated People's Hospital ethics committee. All patients gave their consent for their information to be stored in the hospital database and subsequently used for research. All of the participants provided their written consent to participate in this study. Further, at the time of patient follow-up, in order to document the process, the presence of a qualified physician, a staff member of the Hospital ethics committee and a staff member of the medical records Department were required at the same time.
Statistical analysis. Data are represented as mean±standard deviation. Statistical analysis was performed using Pearson's χ2 test in the frequency tables or using one-way analysis of variance (ANOVA) in the comparison of the cell number of Th17 cells, Tregs and the ratio of Th17/Tregs between the two groups. In ANOVA, when a significant difference between groups was apparent, multiple comparisons of means were performed using the Dunnett test. All statistical assessments were two-sided and evaluated at the 0.05 level of significance. All statistical analyses were performed using SPSS 19.0 software (SPSS, Chicago, IL, USA).
Results
The incidence of radiation pneumonia. One hundred and forty-eight patients were included in this study with a median follow-up of 13 months (range=6-24 months). There were 24 patients (16.2%) with grade 2 or above radiation pneumonia.
The relationship between clinical factors and radiation pneumonia. There was no significant relationship between clinical factors and radiation pneumonia. The clinical factors, including sex, age, smoking history, chemotherapy and thoracic surgery, are shown in Table I.
The relationship between DVH parameters and radiation pneumonia. The lung dosimetric factors V5, V30 and mean lung dose (MLD) were calculated in patients with or without radiation pneumonia at grade 2 or above. The results are presented as mean±standard deviation. The results showed all dosimetric parameters were slightly higher in patients with than in patients without radiation pneumonia (Table II); however, there was a significant difference between the two groups (p>0.05).
The relationship between Th17 cells, Treg and the ratio of Th17/Tregs and radiation pneumonia. Before radiotherapy, Th17 cells (the proportion of CD4+ T-cells) in the radiation pneumonia group and non-radiation pneumonia group were 2.59±1.11% and 2.51±1.21%, respectively, whereas Treg cells were 0.72±4.19% and 4.13±1.02%. There was no significant difference between the two groups. After the start of radiotherapy, Th17 cell numbers gradually increased; however, the Treg cell numbers decreased, so the Th17/Treg ratio further increased, and the changes in the radiation pneumonia group were more obvious. Four weeks after completion of radiotherapy, Th17 and Treg cells were more pronounced and there was a significant difference between the radiation pneumonia and non-radiation pneumonia groups (Figure 1A and B). The Th17/Treg ratio increased significantly in the radiation pneumonia group compared with the non-radiation pneumonia group at the fifth week after the start of radiotherapy (Figure 1C).
Relationship between clinical factors and radiation pneumonia.
Discussion
Radiation-induced lung injury is a common side-effect in the radiotherapy of thoracic cancers (14-16), which reduces patients' quality of life and overall survival. Therefore, it would be advantageous to limit the clinical use of higher irradiation doses in the radiation course. Radiation pneumonia is affected by many factors, including physical factors (dose, irradiation area, V5, V20, V30, MLD) and clinical factors (age, gender, pulmonary function, smoking history, chemotherapy) (17-19). Many studies have shown that dosimetric parameters are closely related to radiation pneumonia, especially MLD (20, 21), which is significantly correlated to the occurrence of radiation pneumonia. Present existing data shows that when the dosimetric parameters are at the levels V10>34%, V20>25%, V30%>18%, MLD>15 Gy, the incidence of radiation pneumonia will significantly increase. Among these parameters, V20 is thought of as the most important that, when controlled to less than 25%, will reduce the incidence of radiation pneumonia. In our study, the dosimetric parameters V5, V20, V30, MLD were 60.4±5.7, 26.2±4.8, 16.4±4.5, 15.8±4.1, respectively, which is consistent with the results reported in the literature. However, there is no significant difference between the two groups in our study perhaps due to the small sample size.
Radiation pneumonia always occurs within one to three months after completion of radiotherapy, so some researches have focused on monitoring the expression of certain cytokines in the course of radiotherapy to predict the occurrence of radiation pneumonia. Among them, some cytokines could predict radiation pneumonia, such as tumor necrosis factor (TNF)-α, IL-6, IL-10, TGF-beta 1 and angiotensin-converting enzyme (ACE). Th17 and Treg cells play an important role in the inflammatory diseases (11-13), such as allergic rhinitis, bronchial asthma, rheumatoid arthritis and inflammation of the bowel. In these diseases, Th17 cells increase and Treg cells decrease; therefore, as radiation pneumonia is also an inflammation-related disease, we wished to examine whether Th17 and Treg cells are related to radiation pneumonia. In our earlier study, we established an animal model of radiation pneumonia. The results showed that unilateral lung irradiated with 20 Gy X-ray could successfully induce radiation pneumonia and Th17 cells in the blood were significantly increased. On the contrary, however, Treg cells were decreased (22). To further investigate whether Th17 and Treg cells could predict the occurrence of radiation pneumonia in patients with thoracic radiation, we collected blood samples from locally advanced esophageal cancer patients in the course of radiotherapy. This study shows that there is no significant difference in Th17 and Treg cells between the radiation pneumonia group and non-radiation pneumonia group before radiation. After the start of radiotherapy, Th17 cells increased and Treg cells decreased gradually. Until completion of radiotherapy, the difference between the two groups of Th17 and Treg cells (% of CD4+ cells) was statistically significant. Therefore, we can hypothesize that Th17/Treg cells are involved in the development of radiation pneumonia. For earlier prediction of radiation pneumonia occurrence, we analyzed the changes of the ratio of Th17/Treg, with the results showing that the ratio of Th17/Treg significantly increased at the 5th week after the start of radiotherapy in the radiation pneumonia group than in the non-radiation pneumonia group. Therefore, the ratio of Th17/Treg could predict the occurrence of radiation pneumonia earlier than Th17 cells or Tregs separately.
Relationship between DVH parameters and radiation pneumonia.
In summary, the ratio of Th17/Treg cells is likely to be a predictor for radiation pneumonia; however, clinical trials with larger samples are needed for further studies. The role of Th17/Treg cells in the incidence of radiation pneumonia and the molecular mechanisms are not yet clear, questions that will be answered with further in vivo and in vitro experiments.
Relationship between Th17 cells, Treg cells, ratio of Th17/Treg and radiation pneumonia in patients with locally advanced esophageal carcinoma. Peripheral blood samples from the patients with locally advanced esophageal cancer were collected at the time point of pre-radiotherapy, 1th, 2th, 3th, 4th, 5th, 6th week from the radiation started and 4 weeks after radiotherapy was completed and, then, separated by density gradient centrifugation and seeded in 6-well plates at a concentration of 2×106/ml. CD4+ IL-17+ cells (Th17) and CD4+ CD25+Foxp3 cells (Tregs) were evaluated by flow cytometry analysis. A: Th17 and radiation pneumonia; B: Treg and radiation pneumonia; C: Ratio of Th17/Treg and radiation pneumonia. **p<0.01 vs. non-radiation pneumonia group.
Acknowledgements
This work was funded by Zhenjiang Science and Technology Development Program, No. SH2011030. We express our heartfelt thanks to all patients who participated in the study.
Footnotes
↵* These Authors contributed equally to this study.
- Received May 2, 2017.
- Revision received May 25, 2017.
- Accepted May 26, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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