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Association between plasma angiotensin-converting enzyme level and radiation pneumonitis
www.elsevier.com/locate/issn/10434666 Cytokine 37 (2007) 71–75
Association between plasma angiotensin-converting enzyme level and radiation pneumonitis Lujun Zhao, Luhua Wang *, Wei Ji, Xiaozhen Wang, Xiangzhi Zhu, Qinfu Feng, Weizhi Yang, Weibo Yin Department of Radiation Oncology, Cancer Hospital (Institute), Peking Union Medical College, Chinese Academy of Medical Science, Beijing, China Received 15 November 2006; received in revised form 11 February 2007; accepted 19 February 2007
Abstract Angiotensin-converting enzyme (ACE) plays an important role in pulmonary fibrosis and may be involved in the development of radiation-induced lung damage. The objective of this study was to evaluate the predictive value of plasma ACE in radiation pneumonitis (RP). Patients with stage I–III lung cancer were treated with radiotherapy with or without chemotherapy. ACE levels were measured using enzyme-linked immunosorbent assay before radiotherapy (pre-RT) and when a median dose of 45 Gy (Range: 40–48 Gy) was reached (during-RT). The primary end point was Pgrade 2 RP. Statistic significances were evaluated with independent T-test and chi-square. Thirty-nine patients were enrolled in this study, among which 33.3% experienced Pgrade 2 RP. ACE levels, either preRT or during-RT, were significantly lower in the RP group than in the non-RP group (P = 0.02 and 0.03, respectively). Nine out of the 19 patients (47.4%) with pre-RT ACE levels 6462 ng/mL experienced RP, versus 3 of 19 (15.8%) patients with ACE levels >462 ng/mL (P = 0.04). This study suggested that plasma ACE as a predictive factor for radiation pneumonitis deserves further study. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Radiation pneumonitis; TGF-b1; ACE; IL-6; Cytokines
1. Introduction Trying to identify simple and effective parameters to predict radiation pneumonitis (RP) is one of the most important areas of lung cancer research, which should allow physicians to safely determine a treatment regimen for patients and deliver a radiation dose tailored to a patient’s individual normal tissue sensitivity profile, rather than to the average tolerance of the whole population. Recently, valuable findings have been made, such as the impact of dose-volume parameters related to radiationinduced lung injury [1–3] and the relationship between some relevant cytokines and radiation-induced lung injury [4–10]. Unfortunately, neither dose-volume parameters, nor cytokines evaluated so far is reliable enough to be
*
Corresponding author. Fax: +861087716559. E-mail address: [email protected] (L. Wang).
1043-4666/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.02.019
applied in clinical practice. The accuracy of dose-volume parameters to predict the onset of RP has been reported to be 52–81% [11]. Plasma transforming growth factorbeta1 (TGF-b1) level changes after radiation therapy (RT) are not optimal either, with a predictability of only 40%, and when combined with dose-volume parameters, only 43% of high-risk patients experienced radiation pneumonitis [4]. Therefore, more reliable methods to predict the onset of RP need to be found. The relationships between TGF-b1, interleukin 6 (IL-6) and RP have been studied extensively [7,9,10,12]. Changes in the level of angiotensin-converting enzyme (ACE) after administration of bleomycin [13] or irradiation [14] in experiments on lung tissue have also been reported. It was also found that genetic differences in intrinsic pulmonary endothelial enzyme activity were correlated very highly with pulmonary radioresponsiveness in animal experiments [15]. But to our knowledge, no clinical study has focused on the predictive value of ACE in RP.
72
L. Zhao et al. / Cytokine 37 (2007) 71–75
The purpose of this work was to further evaluate the effects of IL-6 and TGF-b1, and to explore the value of ACE levels in predicting RP. These cytokines were chosen as representatives of inflammation-related, fibrosis-related and endothelial-related cytokines, respectively.
and IL-6 levels were determined with kits from R&D Systems Inc. (Minneapolis, MN). ACE levels were determined with kits from Chemicon International Inc (Temecula, CA). 2.4. Toxicity evaluation
2. Methods and materials 2.1. Patient eligibility All patients enrolled in this study must meet the following eligibility criteria: (1) histopathologically confirmed stage I–III lung cancer (2) a Karnofsky performance status >60; (3) expected survival >6 months; and (4) FEV1 > 50% of predictive value. Exclusion criteria were: (1) stage IV patients; (2) previous thoracic irradiation; (3) pneumonectomy; or (4) severe cardio-pulmonary disease. All eligible patients were fully informed of the purpose and schedule of the study. An informed consent form was signed by each enrolled patient. All protocol procedures were conducted in accordance with the ethical standards on human research in our institute. 2.2. Treatment planning Three-dimensional conformal radiotherapy (3DCRT) or conventional RT was administered. Generally, three to five coplanar fields were used when conformal RT was applied. The percentage of the whole lung which received a dose more than 20 Gy (V20) was limited to less than 25% for those who received concurrent chemoradiotherapy, and 35% for those who received sequential chemoradiotherapy or radiotherapy alone. The prescribed dose was 60–75 Gy for NSCLC, and 50–60 Gy for SCLC, given with fraction dose 1.8–2.0 Gy, five fractions per week. Chemotherapy could be given before, during or/and after RT. When chemotherapy was given sequentially with RT, platinum-based regimens were usually selected; for three cycles before or after RT. When chemotherapy was given concurrently, carboplatin and paclitaxel were commonly used, with a carboplatin dose of AUC = 2, and a paclitaxel dose of 45 mg/m2, once a week, for 4–6 weeks. 2.3. Measurement of the cytokines Blood samples were collected with EDTA as anticoagulant from every patient within one week before RT (preRT) and within one week after radiation dose reached 40 Gy (during-RT). The blood samples were centrifuged 1000g for 15 min at 4 °C, within 2 h after collection. The plasma was stored at 70 °C. Plasma samples were thawed at room temperature for the measurement of cytokines. They were then centrifuged at 10,000g for 30 min before testing for TGF-b1 levels (this removes the platelet contamination from the plasma). The cytokines (IL-6, TGF-b1, and ACE), were measured with enzyme-linked immunosorbent assays (ELISA). TGF-b1
Radiation pneumonitis was assessed according to RTOG/EORTC criteria of acute radiation pneumonitis, with some modification: grade 0—no change; grade 1—mild symptoms of dry cough or dyspnea on exertion; grade 2—persistent cough requiring narcotics, antitussive agents or/and dyspnea with minimal effort but not at rest; grade 3—severe cough unresponsive to narcotic antitussive agents or dyspnea at rest or/and intermittent oxygen or steroids required; grade 4—severe respiratory insufficiency and/or continuous oxygen or assisted ventilation; grade 5—severe pneumonitis caused death. Radiationinduced lung damage was diagnosed based on the development of clinical symptoms, without any evidence of tumor recurrence, or any other specific etiology. Any related symptoms should become at least one grade severer than the baseline level. For the diagnosis of RP, radiographic evidence was required but not sufficient. The endpoint was grade 2 and above RP during or after the course of RT. 2.5. Follow-up and statistics Patients were followed up weekly during RT, 3 and 6 months after completion of RT. The significance of difference in mean values was evaluated with independent T-test, and comparison of the incidence of RP was made using chisquare test. All the P-values presented are from two-side test. 3. Results 3.1. Patient characteristics From February 2004 to December 2004, 39 patients were enrolled in this study: 35 males and 4 females. Their ages ranged from 40 to 81 years, with a median age of 56 years. Thirty-two patients presented with NSCLC, 7 with SCLC. Six patients underwent a lobectomy, and three patients had an exploratory thoracotomy. Seven patients had chronic obstructive pulmonary disease, 3 with cardiovascular disease, and 3 with diabetes. No patients had any evidence of interstitial pulmonary fibrosis. Thirty-five patients were treated with 3DCRT, and the other 4 with 2D conventional radiotherapy, with a median dose of 60 Gy (range: 46–70 Gy). The V20 for patients who received 3DCRT ranged from 10% to 31%, with a median value of 23.6%. Thirty-four (87.2%) patients received chemotherapy. Twenty-one patients (53.8%) received chemotherapy prior to RT and 19 (48.7%) received chemotherapy concurrently with RT.
L. Zhao et al. / Cytokine 37 (2007) 71–75
a
3.2. Radiation pneumonitis
73 RP non-RP
800 700
ACE Levels (ng/ml)
The median follow-up time for surviving patients was 8.5 months (range: 3.7–20.2 months). Grade 2 or above RP was observed in 13 patients (33.3%) by the time of last follow-up. Twelve of them occurred within 3 months of RT; the others occurred after 3 months and within 6 months from the beginning of RT. Grade 3 or above RP was found in 7 patients, accounting for 17.9% of all patients, including 3 died of radiation pneumonitis.
600 500 400 300 200 100 0 Pre-RT
3.3. Cytokine levels and RP
b
Among the 39 patients, two patients refused to have their blood drawn at the time when a dose of 40 Gy was reached. One patient prior to RT and another one after 40 Gy being reached had insufficient volume of blood drawn for the detection of ACE. The median dose was 45 Gy (Range: 40–48 Gy) when the during-RT blood samples were collected. The median pre-RT TGF-b1, IL-6, or ACE levels were 6.1 ng/mL, 1.6 pg/mL, and 461.9 ng/mL, respectively; and the median during-RT levels were 3.3 ng/mL, 3.9 pg/mL, and 489.7 ng/mL, respectively. There’s no significant difference in TGF-b1, IL-6, or ACE levels in patients with chronic obstructive pulmonary disease, cardiovascular disease, or diabetes and those without such comorbidities (P > 0.05 for all the cytokines). There were no significant difference either in the cytokine levels in patients with NSCLC and SCLC (P > 0.05 for all the cytokines) or in patients with different stage of disease (P > 0.05 for all the cytokines). No significant differences were found in cytokine levels in patients who received and those who did not receive chemotherapy before RT (P > 0.05 for all the cytokines). Pre-RT and during-RT cytokine levels in patients with and without RP are illustrated in Table 1. There was no significant difference in TGF-b1 levels pre- or during-RT between the RP and non-RP groups. Similarly, no significant difference was seen between the two groups in IL-6 levels. With regard to ACE, either pre-RT or during-RT, the level was significantly lower in the RP group than in the non-RP group (Fig. 1a). The mean pre-RT values were 394.2 and 525.9 ng/mL, for non-RP and RP groups, Table 1 Cytokine levels in patients with radiation pneumonitis (RP) and that in patients without RP (non-RP) (Means ± standard deviation) Cytokine
RP
Non-RP
P
Pre-RT TGF-b1 (ng/ml) Post-RT TGF-b1 (ng/ml) Pre-RT IL-6 (pg/ml) Post-RT IL-6 (pg/ml) Pre-RT ACE (ng/ml) Post-RT ACE (ng/ml)
7.74 ± 9.94 4.91 ± 2.97 5.08 ± 7.33 21.75 ± 38.35 394.17 ± 97.47 375.52 ± 119.62
7.02 ± 6.34 3.66 ± 2.23 7.85 ± 21.42 4.93 ± 8.53 525.89 ± 176.62 516.81 ± 208.28
0.785 0.159 0.655 0.143 0.021 0.032
RT, radiation therapy; TGF-b1, transforming growth factor b1; IL-6, Interleukin 6; ACE, angiotensin-converting enzyme.
100 90 80 70 60 50 40 30 20 10 0
During-RT
RP non-RP
<352
352-462
462-558
>558
Pre-RT ACE Level (ng/ml) Fig. 1. Plasma angiotensin-converting enzyme (ACE) levels and radiation pneumonitis (RP). (a) Pre-RT and during-RT ACE levels in patients with and without RP. P value was 0.021 for pre-RT ACE level and 0.032 for during-RT ACE level. (b) Quartile of pre-RT plasma ACE levels and the incidence of RP (P = 0.165). RP, patients with radiation pneumonitis; non-RP, patients without radiation pneumonitis.
respectively (P = 0.02). The mean during-RT values were 375.5 and 516.8 ng/mL, respectively (P = 0.03). Using the pre-RT median level of the cytokines as a cutoff, it was found that patients with lower ACE level had a significant higher risk of RP (P = 0.04), and patients with lower TGF-b1 level also had a relative higher risk of RP, but not statistically significant (P = 0.11), see Table 2. When the median pre-RT ACE level of 462 ng/mL was used as the threshold to predict the occurrence of RP, the sensitivity was 75.0% (9/12), the specificity was 61.5% (16/26), and the accuracy was 65.8% (25/38). When the whole group was divided into four subgroups based on the quartile value of pre-RT ACE level, an apparent trend could be found that patients with lower ACE level had higher risk of RP (P = 0.165). Fig. 1b illustrates the relationship between quartile of pre-RT ACE level and the occurrence of RP. 3.4. Cytokine dynamics and RP The relationship between the occurrence of RP and cytokine changes after delivery of 40 Gy is shown in Table 3. It was found that after 40 Gy of irradiation, the incidence of RP in patients with increased levels of TGF-b1 or IL-6 was a little higher, but not statistically significant
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L. Zhao et al. / Cytokine 37 (2007) 71–75
Table 2 Pre-RT cytokine levels and radiation pneumonitis (RP) Cytokine level
No.
RP (%)
Non-RP (%)
P
TGF-b1
6 6.10 ng/ml > 6.10 ng/ml
20 19
9 (45.0) 4 (21.0)
11(55.0) 15(79.0)
0.113
IL-6
6 1.60 pg/ml > 1.6 pg/ml
20 19
6 (30.0) 7 (36.8)
14(70.0) 12(63.2)
0.651
ACEa
6 461.90 ng/ml > 461.90 ng/ml
19 19
9 (47.4) 3 (15.8)
10(52.6) 16(84.2)
0.043
RP, patients with radiation pneumonitis; Non-RP, patietns without radiation pneumonitis; TGF-b1, transforming growth factor; b1, IL-6, Interleukin 6; ACE, angiotensin-converting enzyme. a One patient had not donated enough volume of blood for ACE measurement. The patient experienced RP after radiation therapy.
Table 3 Changes of cytokine levels and radiation pneumonitis Cytokine change
No.
RP (%)
Non-RP (%)
P
TGF-b1a
Decreased Increased
24 13
7 (29.2) 6 (46.2)
17(70.8) 7(53.8)
0.501
IL-6a
Decreased Increased
20 17
5 (25.0) 8 (47.1)
15(75.0) 9(52.9)
0.161
ACEa,b
Decreased Increased
15 20
6 (40.0) 6 (30.0)
9(60.0) 14(70.0)
0.537
RP, patients with radiation pneumonitis; Non-RP, patients without radiation pneumonitis; TGF-b1, transforming growth factor b1; IL-6, Interleukin 6; ACE, angiotensin-converting enzyme. a Two patients refused to have their blood drawn post-RT. b Two patients had not donated enough volume of blood for ACE measurement (one patient pre-RT and one post-RT), one of which experienced RP.
(P values were 0.50 and 0.16 for TGF-b1 and IL-6, respectively). No relationship could be found between the change of plasma ACE level and the incidence of RP. 4. Discussion In the present series, we found significantly lower ACE levels in the RP group than that in the non-RP group, both before RT and when radiation dose reached 40 Gy. No significant correlation between the risk of radiation pneumonitis and plasma TGF-b1 or IL-6 levels was found. Some clinical trials demonstrated that the incidence of RP was significantly correlated with plasma TGF-b1 or IL-6 levels. The incidence of RP was significantly higher when TGF-b1 levels increased after RT, or if it failed to normalize after radiotherapy, [4,8–10]. IL-6 levels in bronchoalveolar lavage fluid were found to be significantly higher in the irradiated lung than in the unirradiated lung [16]; and plasma IL-6 levels before and after RT were much higher in patients with RP than those without RP [7]. Though our data showed higher incidence of RP in patients with lower pre-RT TGF-b1 level, the difference was not statistically significant. Large number of patients are needed to further test the findings.
ACE is an ectoglycoprotein located mainly in the luminal surface of vascular endothelial cells [17]. It has been shown to be expressed in high levels selectively in lung tissue [18]. In severe interstitial fibrosis and in other restrictive syndromes such as adult respiratory distress syndrome, acute pulmonary edema, lung sepsis, or bronchogenic carcinoma, loss of endothelial cells and damage to the pulmonary vascular bed have been shown to disclose low serum ACE activity [17]. The correlation between lung damage induced by chemotherapy or radiotherapy and changes of ACE has also been reported. Villani et al. [13] investigated the mechanism of the decrease in diffusion capacity (DLCO) of lung tissue after usage of bleomycin-including chemotherapy. They found that pulmonary capillary blood volume showed a significant decline, and a significant correlation existed between the change of DLCO and the change of serum ACE (P < 0.02). Ward et al. [14] found that, 2 months after exposure to single doses (0–30 Gy) of 60Co gamma rays to the right hemithorax, rats developed a dose-dependent decrease in ACE activity in the right lung. This endothelial dysfunction was accompanied by an increase in hydroxyproline content in the irradiated lung. Further study demonstrated that pulmonary endothelial dysfunction induced by hemithorax irradiation represents a direct response of the endothelium to radiation injury and is not secondary to other phenomena such as shunting of function to the shielded lung [19]. Ward et al. [15] further found that low ACE activity before irradiation was associated with sensitivity to radiation fibrosis; C3H and CBA mice had as two times high ACE levels as C57BL mice had, and did not demonstrate pulmonary radiation fibrosis after a single dose 14 Gy of irradiation, while C57BL mice did exhibit substantial fibrosis at 52 weeks after irradiation. The detailed mechanism of ACE activity decreasing after RT was studied by Papapetropoulos et al. [20]. They found that after irradiation of cultured bovine pulmonary arterial endothelial cell monolayers, the number of viable endothelial cells decreased in a dose- and time-dependent manner. But ACE activity per surviving cell increased in a time- and dose-dependent manner, reaching a maximum fourfold increase at 96 h after 30 Gy. However, the ACE activity decreased per culture well. It was hypothesized that the increase in ACE activity per cell could not keep up with the decrease in the number of viable endothelial cells, leading to an overall decrease in ACE activity per culture well. One must note that the actual role of ACE in development of RP is far from known. It is unclear to us why some animal experiments showed that ACE inhibitors (ACEI) could significantly ameliorate radiation damage to lung tissue [21]. One clinical investigation failed to find any protective effect of ACEI on the onset of symptomatic radiation pneumonitis [22]. Nevertheless, our data demonstrate that ACE levels before RT in patients with RP was significantly lower than that in patients without RP, 394.2 and 525.9 ng/mL, respectively, (P = 0.02). When irradiated to 40–50 Gy, patients with RP still had significantly lower
L. Zhao et al. / Cytokine 37 (2007) 71–75
ACE levels than those without RP, being 375.5 and 516.8 ng/mL, respectively, (P = 0.03). This is consistent with the results of animal experiment conducted by Ward et al. [15]. Our study provides the first clinical data suggesting that plasma ACE level may have a predictive value for radiation pneumonitis, and it is worth further study. Acknowledgments This study is supported by the Beijing medicine development foundation, NO: 2002-101. We thank Dr. Rene Olivier Mirimanoff from University Hospital of Lausanne for his invaluable assistance during the preparation of this manuscript and helpful comments. References [1] Yorke ED, Jackson A, Rosenzweig KE, Braban L, Leibel SA, Ling CC. Correlation of dosimetric factors and radiation pneumonitis for non-small-cell lung cancer patients in a recently completed dose escalation study. Int J Radiat Oncol Biol Phys 2005;63:672–82. [2] Oetzel D, Schraube P, Hensley F, Sroka-Perez G, Menke M, Flentje M. Estimation of pneumonitis risk in three-dimensional treatment planning using dose-volume histogram analysis. Int J Radiat Oncol Biol Phys 1995;33:455–60. [3] Kwa SL, Lebesque JV, Theuws JC, et al. Radiation pneumonitis as a function of mean lung dose: an analysis of pooled data of 540 patients. Int J Radiat Oncol Biol Phys 1998;42:1–9. [4] Fu XL, Huang H, Bentel G, et al. Predicting the risk of symptomatic radiation-induced lung injury using both the physical and biologic parameters V30 and transforming growth factor b. Int J Radiat Oncol Biol Phys 2001;50:899–908. [5] Rubin P, Johnston CJ, Williams JP, McDonald S, Finkelstein JN. A perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis. Int J Radiat Ocol Biol Phys 1995;33:99–109. [6] Marks LB, Yu X, Vujaskovic Z, Small Jr W, Folz R, Anscher MS. Radiation-induced lung injury. Semin Radiat Oncol 2003;13:333–45. [7] Chen Y, Williams J, Ding I, et al. Radiation pneumonitis and early circulatory cytokine markers. Semin Radiat Oncol 2002;12(1 Suppl. 1):26–33. [8] Anscher MS, Murase T, Prescott DM, et al. Changes in plasma TGF beta levels during pulmonary radiotherapy as a predictor of the risk of developing radiation pneumonitis. Int J Radiat Oncol Biol Phys 1994;30:671–6.
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[9] Anscher MS, Kong FM, Marks LB, Bentel GC, Jirtle RL. Changes in plasma transforming growth factor beta during radiotherapy and the risk of symptomatic radiation-induced pneumonitis. Int J Radiat Oncol Biol Phys 1997;37:253–8. [10] Anscher MS, Kong FM, Andrews K, et al. Plasma transforming growth factor beta1 as a predictor of radiation pneumonitis. Int J Radiat Oncol Biol Phys 1998;41:1029–35. [11] Rodrigues G, Lock M, D’Souza D, Yu E, Van Dyk J. Prediction of radiation pneumonitis by dose-volume histogram parameters in lung cancer—a systematic review. Radiother Oncol 2004;71:127–38. [12] Kong F-M, Haken RT, Eisbruch A, Lawrence TS. Non-small cell lung cancer therapy-related pulmonary toxicity: an update on radiation pneumonitis and fibrosis. Semin Oncol 2004;32(S3):S42–54. [13] Villani F, Bacchella L, Bulgheroni A, et al. Study of functional and biochemical indicators of subclinical lung damage in bleomycintreated patients. Respir Med 1992;86:327–33. [14] Ward WF, Kim YT, Molteni A, Ts’ao C, Hinz JM. Pentoxifylline does not spare acute radiation reactions in rat lung and skin. Radiat Res 1992;129:107–11. [15] Ward WF, Sharplin J, Franko AJ, Hinz JM. Radiation-induced pulmonary endothelial dysfunction and hydroxyproline accumulation in four strains of mice. Radiat Res 1989;120:113–20. [16] Barthelemy-Brichant N, Bosquee L, Cataldo D, et al. Increased IL-6 and TGF-b1 concentrations in bronchoalveolar lavage fluid associated with thoracic radiotherapy. Int J Radiat Oncol Biol Phys 2004;58:758–67. [17] Beneteau-Burnat B, Baudin B. Angiotensin-converting enzyme: clinical applications and laboratory investigations on serum and other biological fluids. Crit Rev Clin Lab Sci 1991;28:337–56. [18] Danilov SM, Muzykantov VR, Martynov AV, et al. Lung is the target organ for a monoclonal antibody to angiotensin-converting enzyme. Lab Invest 1991;64:118–24. [19] Ward WF, Molteni A, Ts’Ao CH, Solliday NH. Pulmonary endothelial dysfunction induced by unilateral as compared to bilateral thoracic irradiation in rats. Radiat Res 1987;111:101–6. [20] Papapetropoulos A, Burch SE, Topouzis S, Catravas JD. Radiationinduty in cultured bovine pulmonary arterial endothelial cell monolayers. Toxicol Appl Pharmacol 1993;120:96–105. [21] Molteni A, Moulder JE, Cohen EF, et al. Control of radiationinduced pneumopathy and lung fibrosis by angiotensin-converting enzyme inhibitors and an angiotensin II type 1 receptor blocker. Int J Radiat Biol 2000;76:523–32. [22] Wang LW, Fu XL, Clough R, et al. Can angiotensin-converting enzyme inhibitors protect against symptomatic radiation pneumonitis?. Radiat Res 2000;153:405–10.
Association between plasma angiotensin-converting enzyme level and radiation pneumonitis Lujun Zhao, Luhua Wang *, Wei Ji, Xiaozhen Wang, Xiangzhi Zhu, Qinfu Feng, Weizhi Yang, Weibo Yin Department of Radiation Oncology, Cancer Hospital (Institute), Peking Union Medical College, Chinese Academy of Medical Science, Beijing, China Received 15 November 2006; received in revised form 11 February 2007; accepted 19 February 2007
Abstract Angiotensin-converting enzyme (ACE) plays an important role in pulmonary fibrosis and may be involved in the development of radiation-induced lung damage. The objective of this study was to evaluate the predictive value of plasma ACE in radiation pneumonitis (RP). Patients with stage I–III lung cancer were treated with radiotherapy with or without chemotherapy. ACE levels were measured using enzyme-linked immunosorbent assay before radiotherapy (pre-RT) and when a median dose of 45 Gy (Range: 40–48 Gy) was reached (during-RT). The primary end point was Pgrade 2 RP. Statistic significances were evaluated with independent T-test and chi-square. Thirty-nine patients were enrolled in this study, among which 33.3% experienced Pgrade 2 RP. ACE levels, either preRT or during-RT, were significantly lower in the RP group than in the non-RP group (P = 0.02 and 0.03, respectively). Nine out of the 19 patients (47.4%) with pre-RT ACE levels 6462 ng/mL experienced RP, versus 3 of 19 (15.8%) patients with ACE levels >462 ng/mL (P = 0.04). This study suggested that plasma ACE as a predictive factor for radiation pneumonitis deserves further study. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Radiation pneumonitis; TGF-b1; ACE; IL-6; Cytokines
1. Introduction Trying to identify simple and effective parameters to predict radiation pneumonitis (RP) is one of the most important areas of lung cancer research, which should allow physicians to safely determine a treatment regimen for patients and deliver a radiation dose tailored to a patient’s individual normal tissue sensitivity profile, rather than to the average tolerance of the whole population. Recently, valuable findings have been made, such as the impact of dose-volume parameters related to radiationinduced lung injury [1–3] and the relationship between some relevant cytokines and radiation-induced lung injury [4–10]. Unfortunately, neither dose-volume parameters, nor cytokines evaluated so far is reliable enough to be
*
Corresponding author. Fax: +861087716559. E-mail address: [email protected] (L. Wang).
1043-4666/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.02.019
applied in clinical practice. The accuracy of dose-volume parameters to predict the onset of RP has been reported to be 52–81% [11]. Plasma transforming growth factorbeta1 (TGF-b1) level changes after radiation therapy (RT) are not optimal either, with a predictability of only 40%, and when combined with dose-volume parameters, only 43% of high-risk patients experienced radiation pneumonitis [4]. Therefore, more reliable methods to predict the onset of RP need to be found. The relationships between TGF-b1, interleukin 6 (IL-6) and RP have been studied extensively [7,9,10,12]. Changes in the level of angiotensin-converting enzyme (ACE) after administration of bleomycin [13] or irradiation [14] in experiments on lung tissue have also been reported. It was also found that genetic differences in intrinsic pulmonary endothelial enzyme activity were correlated very highly with pulmonary radioresponsiveness in animal experiments [15]. But to our knowledge, no clinical study has focused on the predictive value of ACE in RP.
72
L. Zhao et al. / Cytokine 37 (2007) 71–75
The purpose of this work was to further evaluate the effects of IL-6 and TGF-b1, and to explore the value of ACE levels in predicting RP. These cytokines were chosen as representatives of inflammation-related, fibrosis-related and endothelial-related cytokines, respectively.
and IL-6 levels were determined with kits from R&D Systems Inc. (Minneapolis, MN). ACE levels were determined with kits from Chemicon International Inc (Temecula, CA). 2.4. Toxicity evaluation
2. Methods and materials 2.1. Patient eligibility All patients enrolled in this study must meet the following eligibility criteria: (1) histopathologically confirmed stage I–III lung cancer (2) a Karnofsky performance status >60; (3) expected survival >6 months; and (4) FEV1 > 50% of predictive value. Exclusion criteria were: (1) stage IV patients; (2) previous thoracic irradiation; (3) pneumonectomy; or (4) severe cardio-pulmonary disease. All eligible patients were fully informed of the purpose and schedule of the study. An informed consent form was signed by each enrolled patient. All protocol procedures were conducted in accordance with the ethical standards on human research in our institute. 2.2. Treatment planning Three-dimensional conformal radiotherapy (3DCRT) or conventional RT was administered. Generally, three to five coplanar fields were used when conformal RT was applied. The percentage of the whole lung which received a dose more than 20 Gy (V20) was limited to less than 25% for those who received concurrent chemoradiotherapy, and 35% for those who received sequential chemoradiotherapy or radiotherapy alone. The prescribed dose was 60–75 Gy for NSCLC, and 50–60 Gy for SCLC, given with fraction dose 1.8–2.0 Gy, five fractions per week. Chemotherapy could be given before, during or/and after RT. When chemotherapy was given sequentially with RT, platinum-based regimens were usually selected; for three cycles before or after RT. When chemotherapy was given concurrently, carboplatin and paclitaxel were commonly used, with a carboplatin dose of AUC = 2, and a paclitaxel dose of 45 mg/m2, once a week, for 4–6 weeks. 2.3. Measurement of the cytokines Blood samples were collected with EDTA as anticoagulant from every patient within one week before RT (preRT) and within one week after radiation dose reached 40 Gy (during-RT). The blood samples were centrifuged 1000g for 15 min at 4 °C, within 2 h after collection. The plasma was stored at 70 °C. Plasma samples were thawed at room temperature for the measurement of cytokines. They were then centrifuged at 10,000g for 30 min before testing for TGF-b1 levels (this removes the platelet contamination from the plasma). The cytokines (IL-6, TGF-b1, and ACE), were measured with enzyme-linked immunosorbent assays (ELISA). TGF-b1
Radiation pneumonitis was assessed according to RTOG/EORTC criteria of acute radiation pneumonitis, with some modification: grade 0—no change; grade 1—mild symptoms of dry cough or dyspnea on exertion; grade 2—persistent cough requiring narcotics, antitussive agents or/and dyspnea with minimal effort but not at rest; grade 3—severe cough unresponsive to narcotic antitussive agents or dyspnea at rest or/and intermittent oxygen or steroids required; grade 4—severe respiratory insufficiency and/or continuous oxygen or assisted ventilation; grade 5—severe pneumonitis caused death. Radiationinduced lung damage was diagnosed based on the development of clinical symptoms, without any evidence of tumor recurrence, or any other specific etiology. Any related symptoms should become at least one grade severer than the baseline level. For the diagnosis of RP, radiographic evidence was required but not sufficient. The endpoint was grade 2 and above RP during or after the course of RT. 2.5. Follow-up and statistics Patients were followed up weekly during RT, 3 and 6 months after completion of RT. The significance of difference in mean values was evaluated with independent T-test, and comparison of the incidence of RP was made using chisquare test. All the P-values presented are from two-side test. 3. Results 3.1. Patient characteristics From February 2004 to December 2004, 39 patients were enrolled in this study: 35 males and 4 females. Their ages ranged from 40 to 81 years, with a median age of 56 years. Thirty-two patients presented with NSCLC, 7 with SCLC. Six patients underwent a lobectomy, and three patients had an exploratory thoracotomy. Seven patients had chronic obstructive pulmonary disease, 3 with cardiovascular disease, and 3 with diabetes. No patients had any evidence of interstitial pulmonary fibrosis. Thirty-five patients were treated with 3DCRT, and the other 4 with 2D conventional radiotherapy, with a median dose of 60 Gy (range: 46–70 Gy). The V20 for patients who received 3DCRT ranged from 10% to 31%, with a median value of 23.6%. Thirty-four (87.2%) patients received chemotherapy. Twenty-one patients (53.8%) received chemotherapy prior to RT and 19 (48.7%) received chemotherapy concurrently with RT.
L. Zhao et al. / Cytokine 37 (2007) 71–75
a
3.2. Radiation pneumonitis
73 RP non-RP
800 700
ACE Levels (ng/ml)
The median follow-up time for surviving patients was 8.5 months (range: 3.7–20.2 months). Grade 2 or above RP was observed in 13 patients (33.3%) by the time of last follow-up. Twelve of them occurred within 3 months of RT; the others occurred after 3 months and within 6 months from the beginning of RT. Grade 3 or above RP was found in 7 patients, accounting for 17.9% of all patients, including 3 died of radiation pneumonitis.
600 500 400 300 200 100 0 Pre-RT
3.3. Cytokine levels and RP
b
Among the 39 patients, two patients refused to have their blood drawn at the time when a dose of 40 Gy was reached. One patient prior to RT and another one after 40 Gy being reached had insufficient volume of blood drawn for the detection of ACE. The median dose was 45 Gy (Range: 40–48 Gy) when the during-RT blood samples were collected. The median pre-RT TGF-b1, IL-6, or ACE levels were 6.1 ng/mL, 1.6 pg/mL, and 461.9 ng/mL, respectively; and the median during-RT levels were 3.3 ng/mL, 3.9 pg/mL, and 489.7 ng/mL, respectively. There’s no significant difference in TGF-b1, IL-6, or ACE levels in patients with chronic obstructive pulmonary disease, cardiovascular disease, or diabetes and those without such comorbidities (P > 0.05 for all the cytokines). There were no significant difference either in the cytokine levels in patients with NSCLC and SCLC (P > 0.05 for all the cytokines) or in patients with different stage of disease (P > 0.05 for all the cytokines). No significant differences were found in cytokine levels in patients who received and those who did not receive chemotherapy before RT (P > 0.05 for all the cytokines). Pre-RT and during-RT cytokine levels in patients with and without RP are illustrated in Table 1. There was no significant difference in TGF-b1 levels pre- or during-RT between the RP and non-RP groups. Similarly, no significant difference was seen between the two groups in IL-6 levels. With regard to ACE, either pre-RT or during-RT, the level was significantly lower in the RP group than in the non-RP group (Fig. 1a). The mean pre-RT values were 394.2 and 525.9 ng/mL, for non-RP and RP groups, Table 1 Cytokine levels in patients with radiation pneumonitis (RP) and that in patients without RP (non-RP) (Means ± standard deviation) Cytokine
RP
Non-RP
P
Pre-RT TGF-b1 (ng/ml) Post-RT TGF-b1 (ng/ml) Pre-RT IL-6 (pg/ml) Post-RT IL-6 (pg/ml) Pre-RT ACE (ng/ml) Post-RT ACE (ng/ml)
7.74 ± 9.94 4.91 ± 2.97 5.08 ± 7.33 21.75 ± 38.35 394.17 ± 97.47 375.52 ± 119.62
7.02 ± 6.34 3.66 ± 2.23 7.85 ± 21.42 4.93 ± 8.53 525.89 ± 176.62 516.81 ± 208.28
0.785 0.159 0.655 0.143 0.021 0.032
RT, radiation therapy; TGF-b1, transforming growth factor b1; IL-6, Interleukin 6; ACE, angiotensin-converting enzyme.
100 90 80 70 60 50 40 30 20 10 0
During-RT
RP non-RP
<352
352-462
462-558
>558
Pre-RT ACE Level (ng/ml) Fig. 1. Plasma angiotensin-converting enzyme (ACE) levels and radiation pneumonitis (RP). (a) Pre-RT and during-RT ACE levels in patients with and without RP. P value was 0.021 for pre-RT ACE level and 0.032 for during-RT ACE level. (b) Quartile of pre-RT plasma ACE levels and the incidence of RP (P = 0.165). RP, patients with radiation pneumonitis; non-RP, patients without radiation pneumonitis.
respectively (P = 0.02). The mean during-RT values were 375.5 and 516.8 ng/mL, respectively (P = 0.03). Using the pre-RT median level of the cytokines as a cutoff, it was found that patients with lower ACE level had a significant higher risk of RP (P = 0.04), and patients with lower TGF-b1 level also had a relative higher risk of RP, but not statistically significant (P = 0.11), see Table 2. When the median pre-RT ACE level of 462 ng/mL was used as the threshold to predict the occurrence of RP, the sensitivity was 75.0% (9/12), the specificity was 61.5% (16/26), and the accuracy was 65.8% (25/38). When the whole group was divided into four subgroups based on the quartile value of pre-RT ACE level, an apparent trend could be found that patients with lower ACE level had higher risk of RP (P = 0.165). Fig. 1b illustrates the relationship between quartile of pre-RT ACE level and the occurrence of RP. 3.4. Cytokine dynamics and RP The relationship between the occurrence of RP and cytokine changes after delivery of 40 Gy is shown in Table 3. It was found that after 40 Gy of irradiation, the incidence of RP in patients with increased levels of TGF-b1 or IL-6 was a little higher, but not statistically significant
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L. Zhao et al. / Cytokine 37 (2007) 71–75
Table 2 Pre-RT cytokine levels and radiation pneumonitis (RP) Cytokine level
No.
RP (%)
Non-RP (%)
P
TGF-b1
6 6.10 ng/ml > 6.10 ng/ml
20 19
9 (45.0) 4 (21.0)
11(55.0) 15(79.0)
0.113
IL-6
6 1.60 pg/ml > 1.6 pg/ml
20 19
6 (30.0) 7 (36.8)
14(70.0) 12(63.2)
0.651
ACEa
6 461.90 ng/ml > 461.90 ng/ml
19 19
9 (47.4) 3 (15.8)
10(52.6) 16(84.2)
0.043
RP, patients with radiation pneumonitis; Non-RP, patietns without radiation pneumonitis; TGF-b1, transforming growth factor; b1, IL-6, Interleukin 6; ACE, angiotensin-converting enzyme. a One patient had not donated enough volume of blood for ACE measurement. The patient experienced RP after radiation therapy.
Table 3 Changes of cytokine levels and radiation pneumonitis Cytokine change
No.
RP (%)
Non-RP (%)
P
TGF-b1a
Decreased Increased
24 13
7 (29.2) 6 (46.2)
17(70.8) 7(53.8)
0.501
IL-6a
Decreased Increased
20 17
5 (25.0) 8 (47.1)
15(75.0) 9(52.9)
0.161
ACEa,b
Decreased Increased
15 20
6 (40.0) 6 (30.0)
9(60.0) 14(70.0)
0.537
RP, patients with radiation pneumonitis; Non-RP, patients without radiation pneumonitis; TGF-b1, transforming growth factor b1; IL-6, Interleukin 6; ACE, angiotensin-converting enzyme. a Two patients refused to have their blood drawn post-RT. b Two patients had not donated enough volume of blood for ACE measurement (one patient pre-RT and one post-RT), one of which experienced RP.
(P values were 0.50 and 0.16 for TGF-b1 and IL-6, respectively). No relationship could be found between the change of plasma ACE level and the incidence of RP. 4. Discussion In the present series, we found significantly lower ACE levels in the RP group than that in the non-RP group, both before RT and when radiation dose reached 40 Gy. No significant correlation between the risk of radiation pneumonitis and plasma TGF-b1 or IL-6 levels was found. Some clinical trials demonstrated that the incidence of RP was significantly correlated with plasma TGF-b1 or IL-6 levels. The incidence of RP was significantly higher when TGF-b1 levels increased after RT, or if it failed to normalize after radiotherapy, [4,8–10]. IL-6 levels in bronchoalveolar lavage fluid were found to be significantly higher in the irradiated lung than in the unirradiated lung [16]; and plasma IL-6 levels before and after RT were much higher in patients with RP than those without RP [7]. Though our data showed higher incidence of RP in patients with lower pre-RT TGF-b1 level, the difference was not statistically significant. Large number of patients are needed to further test the findings.
ACE is an ectoglycoprotein located mainly in the luminal surface of vascular endothelial cells [17]. It has been shown to be expressed in high levels selectively in lung tissue [18]. In severe interstitial fibrosis and in other restrictive syndromes such as adult respiratory distress syndrome, acute pulmonary edema, lung sepsis, or bronchogenic carcinoma, loss of endothelial cells and damage to the pulmonary vascular bed have been shown to disclose low serum ACE activity [17]. The correlation between lung damage induced by chemotherapy or radiotherapy and changes of ACE has also been reported. Villani et al. [13] investigated the mechanism of the decrease in diffusion capacity (DLCO) of lung tissue after usage of bleomycin-including chemotherapy. They found that pulmonary capillary blood volume showed a significant decline, and a significant correlation existed between the change of DLCO and the change of serum ACE (P < 0.02). Ward et al. [14] found that, 2 months after exposure to single doses (0–30 Gy) of 60Co gamma rays to the right hemithorax, rats developed a dose-dependent decrease in ACE activity in the right lung. This endothelial dysfunction was accompanied by an increase in hydroxyproline content in the irradiated lung. Further study demonstrated that pulmonary endothelial dysfunction induced by hemithorax irradiation represents a direct response of the endothelium to radiation injury and is not secondary to other phenomena such as shunting of function to the shielded lung [19]. Ward et al. [15] further found that low ACE activity before irradiation was associated with sensitivity to radiation fibrosis; C3H and CBA mice had as two times high ACE levels as C57BL mice had, and did not demonstrate pulmonary radiation fibrosis after a single dose 14 Gy of irradiation, while C57BL mice did exhibit substantial fibrosis at 52 weeks after irradiation. The detailed mechanism of ACE activity decreasing after RT was studied by Papapetropoulos et al. [20]. They found that after irradiation of cultured bovine pulmonary arterial endothelial cell monolayers, the number of viable endothelial cells decreased in a dose- and time-dependent manner. But ACE activity per surviving cell increased in a time- and dose-dependent manner, reaching a maximum fourfold increase at 96 h after 30 Gy. However, the ACE activity decreased per culture well. It was hypothesized that the increase in ACE activity per cell could not keep up with the decrease in the number of viable endothelial cells, leading to an overall decrease in ACE activity per culture well. One must note that the actual role of ACE in development of RP is far from known. It is unclear to us why some animal experiments showed that ACE inhibitors (ACEI) could significantly ameliorate radiation damage to lung tissue [21]. One clinical investigation failed to find any protective effect of ACEI on the onset of symptomatic radiation pneumonitis [22]. Nevertheless, our data demonstrate that ACE levels before RT in patients with RP was significantly lower than that in patients without RP, 394.2 and 525.9 ng/mL, respectively, (P = 0.02). When irradiated to 40–50 Gy, patients with RP still had significantly lower
L. Zhao et al. / Cytokine 37 (2007) 71–75
ACE levels than those without RP, being 375.5 and 516.8 ng/mL, respectively, (P = 0.03). This is consistent with the results of animal experiment conducted by Ward et al. [15]. Our study provides the first clinical data suggesting that plasma ACE level may have a predictive value for radiation pneumonitis, and it is worth further study. Acknowledgments This study is supported by the Beijing medicine development foundation, NO: 2002-101. We thank Dr. Rene Olivier Mirimanoff from University Hospital of Lausanne for his invaluable assistance during the preparation of this manuscript and helpful comments. References [1] Yorke ED, Jackson A, Rosenzweig KE, Braban L, Leibel SA, Ling CC. Correlation of dosimetric factors and radiation pneumonitis for non-small-cell lung cancer patients in a recently completed dose escalation study. Int J Radiat Oncol Biol Phys 2005;63:672–82. [2] Oetzel D, Schraube P, Hensley F, Sroka-Perez G, Menke M, Flentje M. Estimation of pneumonitis risk in three-dimensional treatment planning using dose-volume histogram analysis. Int J Radiat Oncol Biol Phys 1995;33:455–60. [3] Kwa SL, Lebesque JV, Theuws JC, et al. Radiation pneumonitis as a function of mean lung dose: an analysis of pooled data of 540 patients. Int J Radiat Oncol Biol Phys 1998;42:1–9. [4] Fu XL, Huang H, Bentel G, et al. Predicting the risk of symptomatic radiation-induced lung injury using both the physical and biologic parameters V30 and transforming growth factor b. Int J Radiat Oncol Biol Phys 2001;50:899–908. [5] Rubin P, Johnston CJ, Williams JP, McDonald S, Finkelstein JN. A perpetual cascade of cytokines postirradiation leads to pulmonary fibrosis. Int J Radiat Ocol Biol Phys 1995;33:99–109. [6] Marks LB, Yu X, Vujaskovic Z, Small Jr W, Folz R, Anscher MS. Radiation-induced lung injury. Semin Radiat Oncol 2003;13:333–45. [7] Chen Y, Williams J, Ding I, et al. Radiation pneumonitis and early circulatory cytokine markers. Semin Radiat Oncol 2002;12(1 Suppl. 1):26–33. [8] Anscher MS, Murase T, Prescott DM, et al. Changes in plasma TGF beta levels during pulmonary radiotherapy as a predictor of the risk of developing radiation pneumonitis. Int J Radiat Oncol Biol Phys 1994;30:671–6.
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