Med Oncol (2015) 32:405 DOI 10.1007/s12032-014-0405-x
ORIGINAL PAPER
Protective effect of ulinastatin in patients with non-small cell lung cancer after radiation therapy: a randomized, placebo-controlled study Pengtao Bao • Weiguo Zhao • Yun Li Yu Liu • Yi Zhou • Changting Liu
•
Received: 21 November 2014 / Accepted: 24 November 2014 / Published online: 12 December 2014 Ó Springer Science+Business Media New York 2014
Abstract Radiation-induced lung injury (RILI) is a frequent, sometimes life-threatening complication of radiation therapy for the treatment of lung cancer. The anti-inflammatory role of ulinastatin has been well documented, and the potential application of ulinastatin in management of acute lung injury has been suggested in multiple animal studies. In this article, we described a double-blind, randomized, placebo-controlled study in patients with nonsmall cell lung cancer. A total of 120 patients were randomized into two groups: the trial group was treated with ulinastatin for 3 days prior to and for the first 7 days of radiation therapy and the control group was treated with placebo for 10 days following the same schedule. The results from follow-up studies showed that the incidence and grade of RILI were significantly lower in the trial group than in the control group. Reduction in pulmonary function from baseline was significantly smaller in the trial group than that in the control group. Production of serum TGF-b1, TNF-a and IL-6 decreased significantly in the trial group promptly following radiation therapy. However, no difference in survival or tumour response rate was found between the two groups. The results indicated that ulinastatin exerted a protective effect on radiation-induced lung injury. Treatment with ulinastatin could be an effective management
Pengtao Bao and Weiguo Zhao have contributed equally to this work. P. Bao C. Liu (&) Department of Nanlou Respiratory Pulmonology, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, People’s Republic of China e-mail: [email protected] P. Bao W. Zhao Y. Li Y. Liu Y. Zhou Department of Respiratory Medicine, The 309th Hospital of PLA, Beijing 100093, People’s Republic of China
strategy and greatly improve the clinical efficacy of radiation therapy for patients with lung cancer. Keywords Ulinastatin Non-small cell lung cancer Radiation-induced lung injury Inflammatory cytokines Abbreviations NSCLC Non-small cell lung cancer RILI Radiation-induced lung injury BALF Bronchoalveolar lavage fluid UTI Urinary trypsin inhibitor PF Pulmonary function IMRT Intensity-modulated radiotherapy FVC Forced vital capacity FEV1 Forced expiratory volume at 1s DLCO Diffusion capacity for carbon monoxide ELISA Enzyme-linked immunosorbent assay
Background Radiation-induced lung injury (RILI), also known as pulmonary radiation injury, is a frequent complication of pulmonary radiation therapy administered for the treatment of lung cancer [1–3]. Radiation-induced tissue damage to lung is usually unavoidable, followed by an acute phase of alveolitis and/or pneumonitis, and a late/chronic stage of pulmonary fibrosis [4–6]. A large proportion of patients with lung cancer receive radiation therapy, and *5–20 % of these patients develop symptomatic lung injury, and pulmonary function in 50–90 % of patients may decline [4, 7]. The clinical presentation of RILI is extremely variable and usually develops within 6 months of radiation therapy,
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characterised by cough, dyspnoea and fever [6]. If the lung injury affects a large volume of lung, it can be life-threatening [8]. Current treatment options for RILI remain limited, and many of them are used only for symptomatic relief [4, 9]. For example, the conventional glucocorticosteroid therapy suppresses the symptoms of RILI temporarily but results in increased relapse [10]. Therefore, the development of effective intervention for RILI is critical to the prognosis of patients treated with radiation therapy. After radiation therapy, acute inflammation and tissue repair system engage in the immediate repair machinery and restoration of normal lung function [11]. Previous research indicates that the pathogenesis of RILI is characterised by a sequence of biological changes, including early radiation pneumonitis, increased capillary permeability, interstitial oedema, alveolar capillary congestion, infiltration of inflammatory cells in the alveolar space and radiation fibrosis that results from distorted matrix deposition and mesenchymal cell proliferation [9, 11]. This process is dynamic and involves a number of proinflammatory cytokines, including IL-1, IL-6, TGF-b1 and TNFa [12, 13]. TGF-b1 is a pro-fibrotic cytokine and produced by a variety of cell types, including macrophages, epithelial cells and fibroblasts. Several recent studies have demonstrated the radiation-induced expression of TGF-b1 in lung cells, and the rise of TGF-b1 level in plasma and bronchoalveolar lavage fluid (BALF) directly reflects the severity of radiation-induced pulmonary injury [14–16]. Retrospective studies have indicated IL-6 as a biological predictor for the development of pulmonary injury after thoracic irradiation, and high pre-existing level of IL-6 is associated with an increased risk of radiation pneumonitis [17]. However, inhibition of IL-6 alone fails to ameliorate lung injury following radiation [18]. As an important proinflammatory cytokine and a principal mediator of cellular immune response, TNF-a functions to modulate the fibroblast proliferation and extracellular matrix protein synthesis in the subsequent fibrotic phase [19]. These studies have indicated the proinflammatory cytokines as master switch cytokines in the pathogenesis of radiation pneumonitis and inflammatory disorders. When activated after radiation, they promote a chain of molecular and cellular events that result in RILI. Therefore, modulating the levels of these cytokines by therapeutic agents may reflect an effective intervention for RILI. Ulinastatin, also known as urinary trypsin inhibitor or UTI, is a synthetic glycoprotein that can also be purified from human urine. Ulinastatin is derived from pre-/intera-trypsin inhibitors induced by neutrophil elastase during inflammation [20, 21]. As a Kunitz-type protease inhibitor containing two active functional domains, ulinastatin can simultaneously inhibit a wide variety of hydrolytic enzymes and inflammatory proteases [22]. Currently,
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ulinastatin has been used to treat acute pancreatitis, septic shock, severe burn injury and other inflammatory diseases [22, 23]. Application of ulinastatin in management of acute lung injury has been a research focus in recent years, and promising results have been obtained in multiple animal studies [24–27]. The mechanism of action has been attributed to the anti-inflammatory activity of ulinastatin [28]. In our previously conducted study, we demonstrated a strong correlation between expression of inflammatory cytokines and ulinastatin treatment in rats [29]. Furthermore, both pre- and post-treatment with ulinastatin significantly attenuated the grade of radiationinduced lung injury in rats, and a better therapeutic outcome was achieved through pre-treatment with a high dose of ulinastatin [29]. Despite all these exciting findings, the therapeutic effect of ulinastatin in prevention of RILI has not yet been tested in clinical practice. In this study, we demonstrated, for the first time, the efficacy of ulinastatin in amelioration of RILI in patients with lung cancer undergoing radiation therapy. Our results support the use of ulinastatin as a combinational therapeutic agent to improve the outcomes of radiation therapy for lung cancer.
Methods Patients and inclusion criteria Using SPSS statistics software (version 19.0; SPSS Inc.,) and according to the published primary end-point event rates [30], to have [80 % power (a = 0.05) to detect an approximate 30 % reduction of incidence in trial group, in addition to the likelihood of dropout rates of patients (5–10 %), we estimated the number of subjects required for the study. This study was a double-blind, randomized, placebo-controlled investigation aiming to study the protective effect of ulinastatin in NSCLC patients after radiation therapy. The study was a single-site investigation conducted at the 309th Hospital of PLA, Beijing, China. Patient inclusion criteria consisted of the following: (1) age 30–75 years; (2) life expectancy C6 months; (3) locally unresectable stage III cancer, proven by either histology or cytology; (4) basal pulmonary function (PF) tests allowing radiotherapy or the absence of severe interstitial lung disease (ratio of FEV1 on vital capacity C50 %, ratio of diffusion capacity for carbon monoxide on alveolar volume C50 %); (5) Karnofsky performance status (KPS) C70; (6) adequate marrow, renal and hepatic function; and (7) no history of other malignancy. A total of 120 patients were randomly allocated into two groups: trial group (ulinastatin) and control group (placebo) with 1:1 ratio. The arms of the study were balanced
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with respect to gender, age, disease stage, gross tumour volume, lung function, smoking history, histology, chemotherapy and percentage of irradiated lung volume. In addition, patients in both groups who experienced RILI accepted the same radical radiation therapy. The study protocol was fully explained, and written informed consent was obtained from each participant. All of the study processes were performed by specified physicians or nurses who were blinded and trained to use the same set of questionnaires and guidelines. The patients were blinded from the treatment. The study protocol was approved by the Ethical Committee for Human Research of the Local Care and Use Committee. 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.
CT (read by an independent committee of experts including pneumologists, radiologists and radiation oncologists), and PF tests (reduction in vital capacity and/or diffusion capacity for carbon monoxide on alveolar volume). RILI was defined as a grade C1. The incidence and grade of RILI were observed at 8 and 24 weeks after the commencement of IMRT.
Thoracic radiation and ulinastatin therapy
Measurement of circulating cytokines
The dosing schedule for patients in this study was based on our previously conducted study [29]. Patients in the trial group were treated with ulinastatin (Techpool, Guangdong, China) at a dose of 20,000 U/kg body weight/day for 3 days prior to and for the first 7 days during the intensitymodulated radiotherapy (IMRT). Patients in the control group were treated with placebo (sterile freeze-dried powder without ulinastatin) for 10 days following the same schedule. Radiation therapy consisted of once-daily treatment with 2 Gy to a total of 60–70 Gy. Target volumes were defined using the International Commission on Radiation Units and Measurements (ICRU)-50 reports [31]. Total mean lung dose, the percentage of irradiated lung volume exceeding 20 and 30 Gy, was calculated from lung dose–volume histograms (DVHs) based on the computed tomography-defined lung volumes. All patients were treated with similar concomitant medications during the observation period. If patients received concurrent or sequential chemotherapy, the chemotherapeutic regimens, including paclitaxel, gemcitabine and pemetrexed disodium, were all platinum-based.
Five millilitres of blood was collected in EDTA-coated tubes and centrifuged for 20 min at 1,0009g at the start of IMRT and at 4, 8, 16 and 24 weeks after the commencement of IMRT. Enzyme-linked immunosorbent assay (ELISA) was performed to analyse the circulating TGF-b1, IL-6 and TNF-a levels. All measurements were performed independently by investigators, and the values were averaged. For all assays, the intra-observer and inter-observer variation coefficient was \5 %.
Classification criteria of RILI The RILI was graded 0–4 according to the system of the Radiation Therapy Oncology Group/European Organisation for the Research and Treatment of Cancer [32]. RILI was scored based on the clinical symptoms, radiological abnormalities and loss of pulmonary function. This scoring included three subjective scales and two objective scales. Subjective scales included cough, dyspnoea and thoracic pain. Objective scales consisted of chest X-ray and thoracic
Evaluation of pulmonary function PF measurements, including forced expiratory volume at 1 s (FEV1), forced vital capacity (FVC), and diffusion capacity for carbon monoxide were performed using a spirometer (VIASYS, USA). All measurements were performed at baseline before the start of IMRT and at 8 and 24 weeks after the commencement of IMRT. The measured values were expressed as changes in percentages compared to the baseline.
Follow-up visits and tumour response For measurements of tumour response and overall survival, patients were followed until September 2013 or until death. All patients underwent follow-up office visits, which included a visit at 4, 8, 16 and 24 weeks after completing IMRT, and a visit once every 3 months for the rest duration of the follow-up period. At each follow-up visit, evaluations included a complete history, physical examination, treatment response evaluation, concomitant medications, blood routine, renal and hepatic function and a CT scan of the thorax. Treatment response was assessed according to World Health Organisation criteria and based on the CT scan 2 months after the completion of IMRT. A complete response was defined as the disappearance of all known disease for at least 4 weeks. A partial response required a reduction of at least 50 % in the size of the tumour for at least 4 weeks. Progressive disease was defined as an increase in the size of the tumour by 25 % or more. Stable disease was defined as no change or \50 % reduction in tumour size or an increase in tumour size \25 % [33].
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The presence of pneumonitis was determined by the clinical examination and thoracic CT scan. Any radiological changes were considered diffuse haze, and groundglass opacification indicated the appearance of pneumonitis. The presence of fibrosis was assessed by a thoracic CT scan 6 months after the completion of IMRT. Statistical analysis
Med Oncol (2015) 32:405 Table 1 Patient characteristics in two groups Parameters
Trial group (57)
Control group (55)
P value
Mean age (years)
56.37
58.40
0.154
Range age (years)
37–76
43–72
Gender (male/ female) Region (u/s)
35/22
32/23
0.847
38/19
36/19
0.892
Smoke
Generally, the clinical presentation of RILI usually developed within 24 weeks following the radiation therapy. The primary end-point was during or after the course of treatment at 4, 8, 16 and 24 weeks. All computations were carried out using an SPSS software package (version 19.0; SPSS Inc.,). Continuous data were expressed as the mean ± SD, and discrete data were given as counts and percentages. Comparisons of patient characteristics between two groups were made with the Pearson’s Chisquare test or Fisher’s exact test. The independent-samples t test or one-way ANOVA was used for the circulating cytokines analysis, the pulmonary function analysis and the reduction in pulmonary function from baseline. Incidence and grade of RILI, and objective tumour responses were analysed by Mann–Whitney’s rank test. The Kaplan–Meier method was used to estimate the overall survival. All tests were two-sided and adopted a 5 % significance level. No adjustments for multiple testing were made.
Post (y/n)
47/10
44/11
0.811
Present (y/n)
22/35
23/32
0.847
Worker
14
9
0.698
Farmer
13
12
Office staff
23
25
Other
7
9
Profession
Stage (IIIA/IIIB)
33/24
31/24
0.511
Histology (SCC/ADC) KPS (%)
20/37
21/34
0.845
85.79 ± 8.93
84.91 ± 9.21
0.61
FVC (%)
92.38 ± 9.26
91.06 ± 9.38
0.460
FEV1 (%)
88.66 ± 7.72
86.70 ± 8.52
0.205 0.581
DLCO (%)
85.32 ± 9.79
84.21 ± 11.38
CTV (cm3)
219.36 ± 49.76
234.98 ± 54.01
0.114
V20 (%)
29.54 ± 4.96
29.38 ± 4.86
0.865
V30 (%)
26.42 ± 5.14
27.48 ± 4.55
0.249
MLD (Gy)
15.59 ± 3.02
14.60 ± 3.14
0.726
CHE (y/n)
38/19
37/18
0.553
Sequential
15
18
0.489
Concurrent
23
19
1–2
5
7
3–4 5–6
15 10
19 6
[6
8
5
7
9
CHE (T)
Results Patients’ demographics and characteristics Using a double-blind, randomized, placebo-controlled design, 120 consecutive patients (67 men and 53 women; median age 56.9 years; age range 37–76 years; 60 in trial group and 60 in control group) were enrolled between August 2009 and August 2011 in this prospective study at the Radiation Oncology Department of our hospital. All patients were Han Chinese. The characteristics of all the patients are listed in Table 1. Three patients in the trial group and 5 in the control group died from metastatic disease or heart disease within 24 weeks after receiving radiation therapy and were excluded from this study. Consequently, a total of 112 patients (57 in the trial group and 55 in the control group) completed the study.
CHE (C) 0.478
CHE (A) Paclitaxel Gemcitabine
14
13
Pemetrexed disodium
17
15
0.365
Incidence and grade of RILI
Region (U/S) region (urban/suburban resident), SCC squamous carcinoma, ADC adenocarcinoma, KPS Karnofsky performance status, FEV1 forced expiratory volume at 1 s, FVC forced vital capacity, DLCO diffusion capacity for carbon monoxide, GTV gross tumour volume, CHE chemotherapy, CHE (T) timing of chemotherapy, CHE (C) chemotherapy cycles, CHE (A)chemotherapy agents, V20 percentage of irradiated lung volume exceeding 20 Gy, V30 percentage of irradiated lung volume exceeding 30 Gy, MLD mean lung dose
No adverse toxicological effects of ulinastatin were observed. Some patients showed subjective signs of RILI, such as cough, dyspnoea and thoracic pain within 8 weeks
post-radiation therapy. As shown in Table 2, according to the grading system of the Radiation Therapy Oncology Group, 14/57 (24.6 %) patients in the trial group and 32/55
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Table 2 Grade of RILI and variations of pulmonary function at 8 and 24 weeks post-radiation therapy Parameters
Group
Post-radiation therapy 8 weeks
P value
Post-radiation therapy 24 weeks
Trial
7
9
Control
14
10
P value
Grade of RILIa Grade 1 (n) Grade 2 (n) Grade 3 (n)
Trial
5
Control
11
0.0007
8 14
Trial
2
5
Control
7
11
FVC (%)
Trial
-0.96 ± 0.94
Control
-2.95 ± 0.85
FEV1 (%)
Trial Control
0.0055
Pulmonary functionb
DLCO (%)
\0.001
-3.35 ± 1.39
\0.001
-3.12 ± 1.72 -3.48 ± 1.39
0.235
-2.27 ± 1.73 -5.49 ± 2.07
\0.001
Trial
-4.61 ± 1.46
0.562
-2.09 ± 1.53
\0.001
Control
-4.78 ± 1.69
-4.81 ± 1.55
-4.66 ± 2.07
Patients in trial group (ulinastatin) and control group (placebo) were examined at 8 and 24 weeks after commencement of radiation therapy for grade of RILI and pulmonary function FVC forced vital capacity, FEV1 forced expiratory volume at 1 s, DLCO diffusion capacity for carbon monoxide a
The values are shown as the number of patients with each grade (n)
b
Baseline values of FEV1, DLCO and FVC are set as 0 in trial and control groups. Percentage changes in FEV1, DLCO and FVC at 8 and 24 weeks are calculated from baseline
(58.2 %) patients in the control group experienced grade C1 RILI at 8 weeks. At 24 weeks, 22/57 (38.6 %) patients in the trial group and 35/55 (63.6 %) patients in the control group experienced grade C1 RILI. The incidence and grade of RILI in the trial group were significantly lower than those in the control group at both 8 and 24 weeks post-therapy (P = 0.0007 and P = 0.0055, respectively). No grade 4 RILI was found in either group. Variation of pulmonary function The pulmonary function of all patients was recorded as baseline before radiation therapy commenced. The results of FVC, FEV1 and DLCO evaluations showed no statistical significance between the trial and control groups as shown in Table 1 [P = 0.460 (FVC), 0.205 (FEV1), 0.581 (DLCO)]. After IMRT, the FVC, FEV1 and DLCO values all decreased in both groups compared with the baseline (Table 2). A significant difference of reduction in FVC was observed between the trial group and the control group at week 8 (P \ 0.001). However, the reductions in FEV1 and DLCO were not statistically different between the two groups (P = 0.235 and 0.562, respectively). At week 24, significantly smaller reductions in FVC, FEV1 and DLCO were observed from the trial group than those from the control group (P \ 0.001), suggesting that the ulinastatin treatment protected patients against a decrease in pulmonary function after radiation therapy.
Changes in circulating TGF-b1, IL-6 and TNF-a At the commencement of IMRT, serum concentrations of proinflammatory cytokines TGF-b1, IL-6 and TNF-a were measured as baseline. Measurements of these cytokine were recorded at 4, 8, 16 and 24 weeks post-radiation therapy. As shown in Fig. 1, the circulating levels of TFGb1, IL-6 and TNF-a greatly increased at 4 weeks following IMRT in both trial and control groups and gradually decreased to baseline at 24 weeks. In the trial group treated with ulinastatin, the levels of all three cytokines were significantly lower compared with the control group at 4, 8 and 16 weeks (P \ 0.001). The results supported the role of ulinastatin as an anti-inflammatory agent and were consistent with the previous studies. Tumour response and survival Patients were followed until 24 weeks after completing of IMRT or until death. One hundred and twelve patients completed the study. The overall tumour response rates (complete response and partial response) were 52.6 % (95 % CI 47–73 %) in the trial group and 56.3 % (95 % CI 46–77 %) in the control group (Table 3, P = 0.979). The median duration of follow-up was 21.1 months. The median survival was 20.9 (95 % CI 18.8–23.1) months in the trial group and 21.4 (95 % CI 19.2–23.6) months in the control group. Survival curves of each group are shown in
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Fig. 2 using the Kaplan–Meier method (P = 0.842). No differences in survival or tumour response rate were found between the two groups. The results suggested that ulinastatin had no anti-tumour effect on NSCLC.
Fig. 1 Measurement of circulating TGF-b1, IL-6 and TNF-a levels in trial and control groups. At the commencement of IMRT, serum concentrations of TGF-b1, IL-6 and TNF-a in every patient from the trial and control groups were measured and averaged as baseline; follow-up measurements were performed for both groups at 4, 8, 16 and 24 weeks after commencement of IMRT. Solid line represents the trial group and dashed line represents the control group. Vertical lines represent SD. *No statistical significance (P [ 0.05). Statistical significance (P \ 0.001)
Discussion RILI, a consequence of radiation therapy, is sometimes life-threatening. Finding drugs with the ability to protect normal tissues against radiation damage is very important and will yield great benefit to the cancer patients. TGF-b1, TNF-a and IL-6 are the key mediators of inflammation and tissue damage, thus participating in the pathogenesis of RILI [9, 34]. Previous research has demonstrated that ulinastatin serves as an anti-inflammatory agent through suppression of synthesis of major inflammation cytokines [22, 23]. In our previously conducted study in animal models, both pre- and post-treatment with ulinastatin inhibited the radiation pneumonitis. Furthermore, pretreatment with a high dose of ulinastatin was significantly more potent than post-treatment in suppressing radiationinduced inflammation, particularly during the acute inflammatory phase [29]. However, it is not yet known whether ulinastatin is capable of down-regulating expression of these inflammatory cytokines in patients undergoing radiation therapy. The current study is the first that reports the protective effect of ulinastatin on radiationinduced lung injury in patients with NSCLC. In this study, ulinastatin treatment significantly reduced the incidence and grade of RILI, improved pulmonary function in patients and decreased circulating levels of
Fig. 2 Effects of ulinastatin on survival of patients in trial and control groups. A total of 112 patients (57 in the trial group and 55 in the control group) were followed, and survival curves of each group are shown. M indicates months of survival
Table 3 Objective tumour response and disease status in two groups Group
N
CR (%)
PR (%)
SD (%)
PD (%)
P value
Trial
57
11 (19.3 %)
19 (33.3 %)
15 (26.3 %)
12 (21.1 %)
0.979
Control
55
12 (21.8 %)
19 (34.5 %)
13 (23.6 %)
11 (20 %)
CR complete response, PR partial response, SD stable disease, PD progressive disease
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TGF-b1, IL-6 and TNF-a especially at acute phase, which were consistent with our previous results in rats. These findings confirmed the therapeutic effect of ulinastatin on RILI in patients with NSCLC after radiation therapy. The underlying mechanism of this effect may be due to an enhanced suppression of TGF-b1, TNF-a and IL-6 production by ulinastatin. However, treatment with ulinastatin had no apparent effect on survival and tumour response, indicating that ulinastatin as an anti-inflammatory drug had no anti-tumour effect on NSCLC. Although the results of this study are exciting which confirmed our hypothesis about the protective effect of ulinastatin on RILI in patients based on the previous investigation in rats, we should clearly see its limitations. Because of the relatively small sample size, we used one dose of ulinastatin (20,000 U/kg body weight/day) and set the treatment course for 10 days (3 days prior to and the first 7 days during IMRT). Analysis of cytokine levels in patients indicated that the serum concentration of TGFb1, TNF-a and IL-6 reached a peak around 4 weeks following IMRT. Whether treatment with higher doses and/or longer duration of ulinastatin that is concurrent with the increase in inflammatory cytokine levels could yield the greater inhibition of RILI requires further investigation. Previous studies have suggested an anti-tumour role for ulinastatin. Ulinastatin inhibits tumour cell invasion and prevents metastasis through the suppression of tumour cell receptor-bound plasmin activity or cathepsin B activity [35–37]. However, in our study, the use of ulinastatin did not result in increased tumour response rate or long-term survival. Given that the patients participating in our study had all been diagnosed with Stage IIIA or IIIB NSCLC, ulinastatin may have very limited effect on advanced tumour. It may be useful to investigate whether ulinastatin could exert its anti-tumour function with adjuvant chemotherapy. Current treatment and molecular interventions for RILI are focused on targeting the key components involved in pathogenesis of RILI. For example, sivelestat, a small molecule inhibitor of neutrophils elastase, is used to prevent collagen synthesis and deposition in order to suppress pulmonary fibrosis [38]. Ulinastatin also possesses elastase inhibitory activity [21]. Therefore, whether ulinastatin functions through the signalling pathway that regulates neutrophil activation in addition to inflammation needs further attention. Another therapeutic agent for RILI is amifostine that reduces the oxidative damages caused by reactive oxygen species during radiation therapy [39, 40]. It will be useful to investigate whether combining ulinastatin and amifostine would provide more adequate protection and yield greater clinical benefit to cancer patients undergoing radiation therapy.
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Conclusions Results of this study confirmed our hypothesis about the protective effect of ulinastatin on RILI in NSCLC patients. Treatment with ulinastatin at the proper dose could be an effective management strategy for patients who undergo radiation therapy. The use of radioprotective ulinastatin during radiation therapy could allow for greater doses of radiation that may increase tumour response and long-term survival of cancer patients. Conflict of interest peting interests.
The authors declare that they have no com-
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associated with chemotherapy and thoracic radiotherapy for lung cancer. J Thorac Oncol. 2012;7(1):177–82. Kim JY, Kim YS, Kim YK, Park HJ, Kim SJ, Kang JH, et al. The TGF-beta1 dynamics during radiation therapy and its correlation to symptomatic radiation pneumonitis in lung cancer patients. Radiat Oncol. 2009;4:59. Yang HJ, Youn H, Seong KM, Yun YJ, Kim W, Kim YH, et al. Psoralidin, a dual inhibitor of COX-2 and 5-LOX, regulates ionizing radiation (IR)-induced pulmonary inflammation. Biochem Pharmacol. 2011;82(5):524–34. Ogata T, Yamazaki H, Teshima T, Kihara A, Suzumoto Y, Inoue T, et al. Early administration of IL-6RA does not prevent radiation-induced lung injury in mice. Radiat Oncol. 2010;5:26. Redlich CA, Gao X, Rockwell S, Kelley M, Elias JA. IL-11 enhances survival and decreases TNF production after radiationinduced thoracic injury. J Immunol. 1996;157(4):1705–10. Kanai T, Ishiwata T, Kobayashi T, Sato H, Takizawa M, Kawamura Y, et al. Ulinastatin, a urinary trypsin inhibitor, for the initial treatment of patients with Kawasaki disease: a retrospective study. Circulation. 2011;124(25):2822–8. Hiyama A, Takeda J, Kotake Y, Morisaki H, Fukushima K. A human urinary protease inhibitor (ulinastatin) inhibits neutrophil extracellular release of elastase during cardiopulmonary bypass. J Cardiothorac Vasc Anesth. 1997;11(5):580–4. Inoue K, Takano H. Urinary trypsin inhibitor as a therapeutic option for endotoxin-related inflammatory disorders. Expert Opin Investig Drugs. 2010;19(4):513–20. Chen CC, Wang SS, Lee FY. Action of antiproteases on the inflammatory response in acute pancreatitis. JOP. 2007;8(4 Suppl.):488–94. Fang Y, Xu P, Gu C, Wang Y, Fu XJ, Yu WR, et al. Ulinastatin improves pulmonary function in severe burn-induced acute lung injury by attenuating inflammatory response. J Trauma. 2011;71(5):1297–304. Song Z, Chen G, Lin G, Jia C, Cao J, Ao G. The ultra-early protective effect of ulinastatin on rabbit acute lung injury induced by paraquat. BMC Emerg Med. 2013;13(Suppl. 1):S7. Wang N, Liu X, Zheng X, Cao H, Wei G, Zhu Y, et al. Ulinastatin is a novel candidate drug for sepsis and secondary acute lung injury, evidence from an optimized CLP rat model. Int Immunopharmacol. 2013;17(3):799–807. Katoh H, Ishikawa H, Hasegawa M, Yoshida Y, Suzuki Y, Ohno T, et al. Protective effect of urinary trypsin inhibitor on the development of radiation-induced lung fibrosis in mice. J Radiat Res. 2010;51(3):325–32. Shigetomi H, Onogi A, Kajiwara H, Yoshida S, Furukawa N, Haruta S, et al. Anti-inflammatory actions of serine protease inhibitors containing the Kunitz domain. Inflamm Res. 2010;59(9):679–87.
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ORIGINAL PAPER
Protective effect of ulinastatin in patients with non-small cell lung cancer after radiation therapy: a randomized, placebo-controlled study Pengtao Bao • Weiguo Zhao • Yun Li Yu Liu • Yi Zhou • Changting Liu
•
Received: 21 November 2014 / Accepted: 24 November 2014 / Published online: 12 December 2014 Ó Springer Science+Business Media New York 2014
Abstract Radiation-induced lung injury (RILI) is a frequent, sometimes life-threatening complication of radiation therapy for the treatment of lung cancer. The anti-inflammatory role of ulinastatin has been well documented, and the potential application of ulinastatin in management of acute lung injury has been suggested in multiple animal studies. In this article, we described a double-blind, randomized, placebo-controlled study in patients with nonsmall cell lung cancer. A total of 120 patients were randomized into two groups: the trial group was treated with ulinastatin for 3 days prior to and for the first 7 days of radiation therapy and the control group was treated with placebo for 10 days following the same schedule. The results from follow-up studies showed that the incidence and grade of RILI were significantly lower in the trial group than in the control group. Reduction in pulmonary function from baseline was significantly smaller in the trial group than that in the control group. Production of serum TGF-b1, TNF-a and IL-6 decreased significantly in the trial group promptly following radiation therapy. However, no difference in survival or tumour response rate was found between the two groups. The results indicated that ulinastatin exerted a protective effect on radiation-induced lung injury. Treatment with ulinastatin could be an effective management
Pengtao Bao and Weiguo Zhao have contributed equally to this work. P. Bao C. Liu (&) Department of Nanlou Respiratory Pulmonology, Chinese PLA General Hospital, 28 Fuxing Road, Beijing 100853, People’s Republic of China e-mail: [email protected] P. Bao W. Zhao Y. Li Y. Liu Y. Zhou Department of Respiratory Medicine, The 309th Hospital of PLA, Beijing 100093, People’s Republic of China
strategy and greatly improve the clinical efficacy of radiation therapy for patients with lung cancer. Keywords Ulinastatin Non-small cell lung cancer Radiation-induced lung injury Inflammatory cytokines Abbreviations NSCLC Non-small cell lung cancer RILI Radiation-induced lung injury BALF Bronchoalveolar lavage fluid UTI Urinary trypsin inhibitor PF Pulmonary function IMRT Intensity-modulated radiotherapy FVC Forced vital capacity FEV1 Forced expiratory volume at 1s DLCO Diffusion capacity for carbon monoxide ELISA Enzyme-linked immunosorbent assay
Background Radiation-induced lung injury (RILI), also known as pulmonary radiation injury, is a frequent complication of pulmonary radiation therapy administered for the treatment of lung cancer [1–3]. Radiation-induced tissue damage to lung is usually unavoidable, followed by an acute phase of alveolitis and/or pneumonitis, and a late/chronic stage of pulmonary fibrosis [4–6]. A large proportion of patients with lung cancer receive radiation therapy, and *5–20 % of these patients develop symptomatic lung injury, and pulmonary function in 50–90 % of patients may decline [4, 7]. The clinical presentation of RILI is extremely variable and usually develops within 6 months of radiation therapy,
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characterised by cough, dyspnoea and fever [6]. If the lung injury affects a large volume of lung, it can be life-threatening [8]. Current treatment options for RILI remain limited, and many of them are used only for symptomatic relief [4, 9]. For example, the conventional glucocorticosteroid therapy suppresses the symptoms of RILI temporarily but results in increased relapse [10]. Therefore, the development of effective intervention for RILI is critical to the prognosis of patients treated with radiation therapy. After radiation therapy, acute inflammation and tissue repair system engage in the immediate repair machinery and restoration of normal lung function [11]. Previous research indicates that the pathogenesis of RILI is characterised by a sequence of biological changes, including early radiation pneumonitis, increased capillary permeability, interstitial oedema, alveolar capillary congestion, infiltration of inflammatory cells in the alveolar space and radiation fibrosis that results from distorted matrix deposition and mesenchymal cell proliferation [9, 11]. This process is dynamic and involves a number of proinflammatory cytokines, including IL-1, IL-6, TGF-b1 and TNFa [12, 13]. TGF-b1 is a pro-fibrotic cytokine and produced by a variety of cell types, including macrophages, epithelial cells and fibroblasts. Several recent studies have demonstrated the radiation-induced expression of TGF-b1 in lung cells, and the rise of TGF-b1 level in plasma and bronchoalveolar lavage fluid (BALF) directly reflects the severity of radiation-induced pulmonary injury [14–16]. Retrospective studies have indicated IL-6 as a biological predictor for the development of pulmonary injury after thoracic irradiation, and high pre-existing level of IL-6 is associated with an increased risk of radiation pneumonitis [17]. However, inhibition of IL-6 alone fails to ameliorate lung injury following radiation [18]. As an important proinflammatory cytokine and a principal mediator of cellular immune response, TNF-a functions to modulate the fibroblast proliferation and extracellular matrix protein synthesis in the subsequent fibrotic phase [19]. These studies have indicated the proinflammatory cytokines as master switch cytokines in the pathogenesis of radiation pneumonitis and inflammatory disorders. When activated after radiation, they promote a chain of molecular and cellular events that result in RILI. Therefore, modulating the levels of these cytokines by therapeutic agents may reflect an effective intervention for RILI. Ulinastatin, also known as urinary trypsin inhibitor or UTI, is a synthetic glycoprotein that can also be purified from human urine. Ulinastatin is derived from pre-/intera-trypsin inhibitors induced by neutrophil elastase during inflammation [20, 21]. As a Kunitz-type protease inhibitor containing two active functional domains, ulinastatin can simultaneously inhibit a wide variety of hydrolytic enzymes and inflammatory proteases [22]. Currently,
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ulinastatin has been used to treat acute pancreatitis, septic shock, severe burn injury and other inflammatory diseases [22, 23]. Application of ulinastatin in management of acute lung injury has been a research focus in recent years, and promising results have been obtained in multiple animal studies [24–27]. The mechanism of action has been attributed to the anti-inflammatory activity of ulinastatin [28]. In our previously conducted study, we demonstrated a strong correlation between expression of inflammatory cytokines and ulinastatin treatment in rats [29]. Furthermore, both pre- and post-treatment with ulinastatin significantly attenuated the grade of radiationinduced lung injury in rats, and a better therapeutic outcome was achieved through pre-treatment with a high dose of ulinastatin [29]. Despite all these exciting findings, the therapeutic effect of ulinastatin in prevention of RILI has not yet been tested in clinical practice. In this study, we demonstrated, for the first time, the efficacy of ulinastatin in amelioration of RILI in patients with lung cancer undergoing radiation therapy. Our results support the use of ulinastatin as a combinational therapeutic agent to improve the outcomes of radiation therapy for lung cancer.
Methods Patients and inclusion criteria Using SPSS statistics software (version 19.0; SPSS Inc.,) and according to the published primary end-point event rates [30], to have [80 % power (a = 0.05) to detect an approximate 30 % reduction of incidence in trial group, in addition to the likelihood of dropout rates of patients (5–10 %), we estimated the number of subjects required for the study. This study was a double-blind, randomized, placebo-controlled investigation aiming to study the protective effect of ulinastatin in NSCLC patients after radiation therapy. The study was a single-site investigation conducted at the 309th Hospital of PLA, Beijing, China. Patient inclusion criteria consisted of the following: (1) age 30–75 years; (2) life expectancy C6 months; (3) locally unresectable stage III cancer, proven by either histology or cytology; (4) basal pulmonary function (PF) tests allowing radiotherapy or the absence of severe interstitial lung disease (ratio of FEV1 on vital capacity C50 %, ratio of diffusion capacity for carbon monoxide on alveolar volume C50 %); (5) Karnofsky performance status (KPS) C70; (6) adequate marrow, renal and hepatic function; and (7) no history of other malignancy. A total of 120 patients were randomly allocated into two groups: trial group (ulinastatin) and control group (placebo) with 1:1 ratio. The arms of the study were balanced
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with respect to gender, age, disease stage, gross tumour volume, lung function, smoking history, histology, chemotherapy and percentage of irradiated lung volume. In addition, patients in both groups who experienced RILI accepted the same radical radiation therapy. The study protocol was fully explained, and written informed consent was obtained from each participant. All of the study processes were performed by specified physicians or nurses who were blinded and trained to use the same set of questionnaires and guidelines. The patients were blinded from the treatment. The study protocol was approved by the Ethical Committee for Human Research of the Local Care and Use Committee. 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.
CT (read by an independent committee of experts including pneumologists, radiologists and radiation oncologists), and PF tests (reduction in vital capacity and/or diffusion capacity for carbon monoxide on alveolar volume). RILI was defined as a grade C1. The incidence and grade of RILI were observed at 8 and 24 weeks after the commencement of IMRT.
Thoracic radiation and ulinastatin therapy
Measurement of circulating cytokines
The dosing schedule for patients in this study was based on our previously conducted study [29]. Patients in the trial group were treated with ulinastatin (Techpool, Guangdong, China) at a dose of 20,000 U/kg body weight/day for 3 days prior to and for the first 7 days during the intensitymodulated radiotherapy (IMRT). Patients in the control group were treated with placebo (sterile freeze-dried powder without ulinastatin) for 10 days following the same schedule. Radiation therapy consisted of once-daily treatment with 2 Gy to a total of 60–70 Gy. Target volumes were defined using the International Commission on Radiation Units and Measurements (ICRU)-50 reports [31]. Total mean lung dose, the percentage of irradiated lung volume exceeding 20 and 30 Gy, was calculated from lung dose–volume histograms (DVHs) based on the computed tomography-defined lung volumes. All patients were treated with similar concomitant medications during the observation period. If patients received concurrent or sequential chemotherapy, the chemotherapeutic regimens, including paclitaxel, gemcitabine and pemetrexed disodium, were all platinum-based.
Five millilitres of blood was collected in EDTA-coated tubes and centrifuged for 20 min at 1,0009g at the start of IMRT and at 4, 8, 16 and 24 weeks after the commencement of IMRT. Enzyme-linked immunosorbent assay (ELISA) was performed to analyse the circulating TGF-b1, IL-6 and TNF-a levels. All measurements were performed independently by investigators, and the values were averaged. For all assays, the intra-observer and inter-observer variation coefficient was \5 %.
Classification criteria of RILI The RILI was graded 0–4 according to the system of the Radiation Therapy Oncology Group/European Organisation for the Research and Treatment of Cancer [32]. RILI was scored based on the clinical symptoms, radiological abnormalities and loss of pulmonary function. This scoring included three subjective scales and two objective scales. Subjective scales included cough, dyspnoea and thoracic pain. Objective scales consisted of chest X-ray and thoracic
Evaluation of pulmonary function PF measurements, including forced expiratory volume at 1 s (FEV1), forced vital capacity (FVC), and diffusion capacity for carbon monoxide were performed using a spirometer (VIASYS, USA). All measurements were performed at baseline before the start of IMRT and at 8 and 24 weeks after the commencement of IMRT. The measured values were expressed as changes in percentages compared to the baseline.
Follow-up visits and tumour response For measurements of tumour response and overall survival, patients were followed until September 2013 or until death. All patients underwent follow-up office visits, which included a visit at 4, 8, 16 and 24 weeks after completing IMRT, and a visit once every 3 months for the rest duration of the follow-up period. At each follow-up visit, evaluations included a complete history, physical examination, treatment response evaluation, concomitant medications, blood routine, renal and hepatic function and a CT scan of the thorax. Treatment response was assessed according to World Health Organisation criteria and based on the CT scan 2 months after the completion of IMRT. A complete response was defined as the disappearance of all known disease for at least 4 weeks. A partial response required a reduction of at least 50 % in the size of the tumour for at least 4 weeks. Progressive disease was defined as an increase in the size of the tumour by 25 % or more. Stable disease was defined as no change or \50 % reduction in tumour size or an increase in tumour size \25 % [33].
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The presence of pneumonitis was determined by the clinical examination and thoracic CT scan. Any radiological changes were considered diffuse haze, and groundglass opacification indicated the appearance of pneumonitis. The presence of fibrosis was assessed by a thoracic CT scan 6 months after the completion of IMRT. Statistical analysis
Med Oncol (2015) 32:405 Table 1 Patient characteristics in two groups Parameters
Trial group (57)
Control group (55)
P value
Mean age (years)
56.37
58.40
0.154
Range age (years)
37–76
43–72
Gender (male/ female) Region (u/s)
35/22
32/23
0.847
38/19
36/19
0.892
Smoke
Generally, the clinical presentation of RILI usually developed within 24 weeks following the radiation therapy. The primary end-point was during or after the course of treatment at 4, 8, 16 and 24 weeks. All computations were carried out using an SPSS software package (version 19.0; SPSS Inc.,). Continuous data were expressed as the mean ± SD, and discrete data were given as counts and percentages. Comparisons of patient characteristics between two groups were made with the Pearson’s Chisquare test or Fisher’s exact test. The independent-samples t test or one-way ANOVA was used for the circulating cytokines analysis, the pulmonary function analysis and the reduction in pulmonary function from baseline. Incidence and grade of RILI, and objective tumour responses were analysed by Mann–Whitney’s rank test. The Kaplan–Meier method was used to estimate the overall survival. All tests were two-sided and adopted a 5 % significance level. No adjustments for multiple testing were made.
Post (y/n)
47/10
44/11
0.811
Present (y/n)
22/35
23/32
0.847
Worker
14
9
0.698
Farmer
13
12
Office staff
23
25
Other
7
9
Profession
Stage (IIIA/IIIB)
33/24
31/24
0.511
Histology (SCC/ADC) KPS (%)
20/37
21/34
0.845
85.79 ± 8.93
84.91 ± 9.21
0.61
FVC (%)
92.38 ± 9.26
91.06 ± 9.38
0.460
FEV1 (%)
88.66 ± 7.72
86.70 ± 8.52
0.205 0.581
DLCO (%)
85.32 ± 9.79
84.21 ± 11.38
CTV (cm3)
219.36 ± 49.76
234.98 ± 54.01
0.114
V20 (%)
29.54 ± 4.96
29.38 ± 4.86
0.865
V30 (%)
26.42 ± 5.14
27.48 ± 4.55
0.249
MLD (Gy)
15.59 ± 3.02
14.60 ± 3.14
0.726
CHE (y/n)
38/19
37/18
0.553
Sequential
15
18
0.489
Concurrent
23
19
1–2
5
7
3–4 5–6
15 10
19 6
[6
8
5
7
9
CHE (T)
Results Patients’ demographics and characteristics Using a double-blind, randomized, placebo-controlled design, 120 consecutive patients (67 men and 53 women; median age 56.9 years; age range 37–76 years; 60 in trial group and 60 in control group) were enrolled between August 2009 and August 2011 in this prospective study at the Radiation Oncology Department of our hospital. All patients were Han Chinese. The characteristics of all the patients are listed in Table 1. Three patients in the trial group and 5 in the control group died from metastatic disease or heart disease within 24 weeks after receiving radiation therapy and were excluded from this study. Consequently, a total of 112 patients (57 in the trial group and 55 in the control group) completed the study.
CHE (C) 0.478
CHE (A) Paclitaxel Gemcitabine
14
13
Pemetrexed disodium
17
15
0.365
Incidence and grade of RILI
Region (U/S) region (urban/suburban resident), SCC squamous carcinoma, ADC adenocarcinoma, KPS Karnofsky performance status, FEV1 forced expiratory volume at 1 s, FVC forced vital capacity, DLCO diffusion capacity for carbon monoxide, GTV gross tumour volume, CHE chemotherapy, CHE (T) timing of chemotherapy, CHE (C) chemotherapy cycles, CHE (A)chemotherapy agents, V20 percentage of irradiated lung volume exceeding 20 Gy, V30 percentage of irradiated lung volume exceeding 30 Gy, MLD mean lung dose
No adverse toxicological effects of ulinastatin were observed. Some patients showed subjective signs of RILI, such as cough, dyspnoea and thoracic pain within 8 weeks
post-radiation therapy. As shown in Table 2, according to the grading system of the Radiation Therapy Oncology Group, 14/57 (24.6 %) patients in the trial group and 32/55
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Table 2 Grade of RILI and variations of pulmonary function at 8 and 24 weeks post-radiation therapy Parameters
Group
Post-radiation therapy 8 weeks
P value
Post-radiation therapy 24 weeks
Trial
7
9
Control
14
10
P value
Grade of RILIa Grade 1 (n) Grade 2 (n) Grade 3 (n)
Trial
5
Control
11
0.0007
8 14
Trial
2
5
Control
7
11
FVC (%)
Trial
-0.96 ± 0.94
Control
-2.95 ± 0.85
FEV1 (%)
Trial Control
0.0055
Pulmonary functionb
DLCO (%)
\0.001
-3.35 ± 1.39
\0.001
-3.12 ± 1.72 -3.48 ± 1.39
0.235
-2.27 ± 1.73 -5.49 ± 2.07
\0.001
Trial
-4.61 ± 1.46
0.562
-2.09 ± 1.53
\0.001
Control
-4.78 ± 1.69
-4.81 ± 1.55
-4.66 ± 2.07
Patients in trial group (ulinastatin) and control group (placebo) were examined at 8 and 24 weeks after commencement of radiation therapy for grade of RILI and pulmonary function FVC forced vital capacity, FEV1 forced expiratory volume at 1 s, DLCO diffusion capacity for carbon monoxide a
The values are shown as the number of patients with each grade (n)
b
Baseline values of FEV1, DLCO and FVC are set as 0 in trial and control groups. Percentage changes in FEV1, DLCO and FVC at 8 and 24 weeks are calculated from baseline
(58.2 %) patients in the control group experienced grade C1 RILI at 8 weeks. At 24 weeks, 22/57 (38.6 %) patients in the trial group and 35/55 (63.6 %) patients in the control group experienced grade C1 RILI. The incidence and grade of RILI in the trial group were significantly lower than those in the control group at both 8 and 24 weeks post-therapy (P = 0.0007 and P = 0.0055, respectively). No grade 4 RILI was found in either group. Variation of pulmonary function The pulmonary function of all patients was recorded as baseline before radiation therapy commenced. The results of FVC, FEV1 and DLCO evaluations showed no statistical significance between the trial and control groups as shown in Table 1 [P = 0.460 (FVC), 0.205 (FEV1), 0.581 (DLCO)]. After IMRT, the FVC, FEV1 and DLCO values all decreased in both groups compared with the baseline (Table 2). A significant difference of reduction in FVC was observed between the trial group and the control group at week 8 (P \ 0.001). However, the reductions in FEV1 and DLCO were not statistically different between the two groups (P = 0.235 and 0.562, respectively). At week 24, significantly smaller reductions in FVC, FEV1 and DLCO were observed from the trial group than those from the control group (P \ 0.001), suggesting that the ulinastatin treatment protected patients against a decrease in pulmonary function after radiation therapy.
Changes in circulating TGF-b1, IL-6 and TNF-a At the commencement of IMRT, serum concentrations of proinflammatory cytokines TGF-b1, IL-6 and TNF-a were measured as baseline. Measurements of these cytokine were recorded at 4, 8, 16 and 24 weeks post-radiation therapy. As shown in Fig. 1, the circulating levels of TFGb1, IL-6 and TNF-a greatly increased at 4 weeks following IMRT in both trial and control groups and gradually decreased to baseline at 24 weeks. In the trial group treated with ulinastatin, the levels of all three cytokines were significantly lower compared with the control group at 4, 8 and 16 weeks (P \ 0.001). The results supported the role of ulinastatin as an anti-inflammatory agent and were consistent with the previous studies. Tumour response and survival Patients were followed until 24 weeks after completing of IMRT or until death. One hundred and twelve patients completed the study. The overall tumour response rates (complete response and partial response) were 52.6 % (95 % CI 47–73 %) in the trial group and 56.3 % (95 % CI 46–77 %) in the control group (Table 3, P = 0.979). The median duration of follow-up was 21.1 months. The median survival was 20.9 (95 % CI 18.8–23.1) months in the trial group and 21.4 (95 % CI 19.2–23.6) months in the control group. Survival curves of each group are shown in
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Fig. 2 using the Kaplan–Meier method (P = 0.842). No differences in survival or tumour response rate were found between the two groups. The results suggested that ulinastatin had no anti-tumour effect on NSCLC.
Fig. 1 Measurement of circulating TGF-b1, IL-6 and TNF-a levels in trial and control groups. At the commencement of IMRT, serum concentrations of TGF-b1, IL-6 and TNF-a in every patient from the trial and control groups were measured and averaged as baseline; follow-up measurements were performed for both groups at 4, 8, 16 and 24 weeks after commencement of IMRT. Solid line represents the trial group and dashed line represents the control group. Vertical lines represent SD. *No statistical significance (P [ 0.05). Statistical significance (P \ 0.001)
Discussion RILI, a consequence of radiation therapy, is sometimes life-threatening. Finding drugs with the ability to protect normal tissues against radiation damage is very important and will yield great benefit to the cancer patients. TGF-b1, TNF-a and IL-6 are the key mediators of inflammation and tissue damage, thus participating in the pathogenesis of RILI [9, 34]. Previous research has demonstrated that ulinastatin serves as an anti-inflammatory agent through suppression of synthesis of major inflammation cytokines [22, 23]. In our previously conducted study in animal models, both pre- and post-treatment with ulinastatin inhibited the radiation pneumonitis. Furthermore, pretreatment with a high dose of ulinastatin was significantly more potent than post-treatment in suppressing radiationinduced inflammation, particularly during the acute inflammatory phase [29]. However, it is not yet known whether ulinastatin is capable of down-regulating expression of these inflammatory cytokines in patients undergoing radiation therapy. The current study is the first that reports the protective effect of ulinastatin on radiationinduced lung injury in patients with NSCLC. In this study, ulinastatin treatment significantly reduced the incidence and grade of RILI, improved pulmonary function in patients and decreased circulating levels of
Fig. 2 Effects of ulinastatin on survival of patients in trial and control groups. A total of 112 patients (57 in the trial group and 55 in the control group) were followed, and survival curves of each group are shown. M indicates months of survival
Table 3 Objective tumour response and disease status in two groups Group
N
CR (%)
PR (%)
SD (%)
PD (%)
P value
Trial
57
11 (19.3 %)
19 (33.3 %)
15 (26.3 %)
12 (21.1 %)
0.979
Control
55
12 (21.8 %)
19 (34.5 %)
13 (23.6 %)
11 (20 %)
CR complete response, PR partial response, SD stable disease, PD progressive disease
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TGF-b1, IL-6 and TNF-a especially at acute phase, which were consistent with our previous results in rats. These findings confirmed the therapeutic effect of ulinastatin on RILI in patients with NSCLC after radiation therapy. The underlying mechanism of this effect may be due to an enhanced suppression of TGF-b1, TNF-a and IL-6 production by ulinastatin. However, treatment with ulinastatin had no apparent effect on survival and tumour response, indicating that ulinastatin as an anti-inflammatory drug had no anti-tumour effect on NSCLC. Although the results of this study are exciting which confirmed our hypothesis about the protective effect of ulinastatin on RILI in patients based on the previous investigation in rats, we should clearly see its limitations. Because of the relatively small sample size, we used one dose of ulinastatin (20,000 U/kg body weight/day) and set the treatment course for 10 days (3 days prior to and the first 7 days during IMRT). Analysis of cytokine levels in patients indicated that the serum concentration of TGFb1, TNF-a and IL-6 reached a peak around 4 weeks following IMRT. Whether treatment with higher doses and/or longer duration of ulinastatin that is concurrent with the increase in inflammatory cytokine levels could yield the greater inhibition of RILI requires further investigation. Previous studies have suggested an anti-tumour role for ulinastatin. Ulinastatin inhibits tumour cell invasion and prevents metastasis through the suppression of tumour cell receptor-bound plasmin activity or cathepsin B activity [35–37]. However, in our study, the use of ulinastatin did not result in increased tumour response rate or long-term survival. Given that the patients participating in our study had all been diagnosed with Stage IIIA or IIIB NSCLC, ulinastatin may have very limited effect on advanced tumour. It may be useful to investigate whether ulinastatin could exert its anti-tumour function with adjuvant chemotherapy. Current treatment and molecular interventions for RILI are focused on targeting the key components involved in pathogenesis of RILI. For example, sivelestat, a small molecule inhibitor of neutrophils elastase, is used to prevent collagen synthesis and deposition in order to suppress pulmonary fibrosis [38]. Ulinastatin also possesses elastase inhibitory activity [21]. Therefore, whether ulinastatin functions through the signalling pathway that regulates neutrophil activation in addition to inflammation needs further attention. Another therapeutic agent for RILI is amifostine that reduces the oxidative damages caused by reactive oxygen species during radiation therapy [39, 40]. It will be useful to investigate whether combining ulinastatin and amifostine would provide more adequate protection and yield greater clinical benefit to cancer patients undergoing radiation therapy.
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Conclusions Results of this study confirmed our hypothesis about the protective effect of ulinastatin on RILI in NSCLC patients. Treatment with ulinastatin at the proper dose could be an effective management strategy for patients who undergo radiation therapy. The use of radioprotective ulinastatin during radiation therapy could allow for greater doses of radiation that may increase tumour response and long-term survival of cancer patients. Conflict of interest peting interests.
The authors declare that they have no com-
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