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Using Plasma Transforming Growth Factor Beta-1 During Radiotherapy to Select Patients for Dose Escalation

From the Departments of Radiation Oncology, Medicine, and Community and Family Medicine, Duke University Medical Center, Durham, NC.

Address reprint requests to Mitchell S. Anscher, MD, Box 3085, Duke University Medical Center, Durham, NC 27710; email: anscher{at}radonc.duke.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: The ability to prescribe treatment based on relative risks for normal tissue injury has important implications for oncologists. In non–small-cell lung cancer, increasing the dose of radiation may improve local control and survival. Changes in plasma transforming growth factor beta (TGFß) levels during radiotherapy (RT) may identify patients at low risk for complications in whom higher doses of radiation could be safely delivered.

PATIENT AND METHODS: Patients with locally advanced or medically inoperable non–small-cell lung cancer received three-dimensional conformal RT to the primary tumor and radiographically involved nodes to a dose of 73.6 Gy (1.6 Gy twice daily). If the plasma TGFß level was normal after 73.6 Gy, additional twice daily RT was delivered to successively higher total doses. The maximum-tolerated dose was defined as the highest radiation dose at which one grade 4 (life-threatening) late toxicity and two grade 3 to 4 (severe life-threatening) late toxicities occurred.

RESULTS: Thirty-eight patients were enrolled. Median follow-up was 16 months. Twenty-four patients were not eligible for radiation dose escalation beyond 73.6 Gy because of persistently abnormal TGFß levels. Fourteen patients whose TGFß levels were normal after 73.6 Gy were escalated to 80 Gy (n = 8) and 86.4 Gy (n = 6). In the 86.4-Gy group, dose-limiting toxicity was reached because there were two (33%) grade 3 late toxicities.

CONCLUSION: It is feasible to use plasma TGFß levels to select patients for RT dose escalation for non–small-cell lung cancer. The maximum-tolerated dose using this approach is 86.4 Gy.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE ABILITY TO select patients for different treatments based on relative risks for normal tissue injury has important implications for oncologists. For certain malignancies, including non–small-cell lung cancer, the delivery of higher doses of radiation may result in both improved local control and survival.^1-5 However, the maximum-tolerated dose of radiation that can be safely delivered in the treatment of non–small-cell lung cancer is limited by the tolerance of the surrounding normal tissues.⁶ Furthermore, locally advanced malignancies, such as most cases of non–small-cell lung cancer, often require the irradiation of large volumes of normal tissue, making dose escalation difficult. Much emphasis has been placed on the role of physical factors (ie, volume of normal tissue radiated, and total dose and dose per fraction) in the development of risk estimates for normal tissue injury after radiotherapy (RT).^7,8 Although physical factors are important, none of these models consider molecular biologic events that may be responsible for the observed heterogeneity in normal tissue response between patients.

The underlying molecular mechanisms of radiation-induced lung injury have recently come under study. Exposure to ionizing radiation rapidly triggers a cascade of genetic and molecular events that ultimately lead to functional damage.^9,10 This is an active process involving a number of inflammatory and fibrogenic cytokines produced by macrophages, epithelial cells, pneumocytes, and fibroblasts.^11-13

Several gene products such as egr-1, NF-kß, c-jun, c-fos, and other transcriptional stimulatory elements have been shown to be induced by ionizing radiation.^14-21 These transcription factors activate genes for many cytokines and other gene products that may be related to the development of normal tissue injury. Several recent studies have shown that cytokines^10,12,22 and cell adhesion molecules^23,24 play an important role in radiation-induced pulmonary injury. Elevated levels of the cytokines interleukin 1 (IL-1), tumor necrosis factor alpha, platelet-derived growth factor, and transforming growth factor beta (TGFß) are consistently reported at various times after exposure of lung tissue to irradiation.^13,22,25-27 These, and other studies reviewed in Vujaskovic et al,²⁸ suggest that the initial proinflammatory cytokine response to radiation seems to be a general phenomenon followed by a cascade of molecular events continuing for weeks or months after the completion of RT. This response is part of a highly efficient, well-conserved, and predictable wound-healing pathway in which the cytokine TGFß has been found to play a crucial role.

Recently, investigators have shown that changes in blood levels of certain cytokines may predict the risk of radiation-induced lung injury.^29,30 In a study of 73 patients receiving high-dose thoracic RT for lung cancer, Anscher et al²⁹ found that those patients whose plasma TGFß1 levels were normal at the completion of RT were at low risk for subsequent radiation-induced lung injury, whereas the risk of symptomatic lung damage was increased in the patients whose TGFß1 level remained elevated.²⁹ A subsequent analysis showed that these changes in plasma TGFß1 correlated with the risk of pulmonary injury independent of the volume of lung irradiated.³¹ Based on these preliminary data, we hypothesized that one could use serial plasma TGFß1 measurements to identify patients at low risk for normal tissue injury from conventional doses of radiation therapy and safely escalate the radiation dose in this subset of patients. Herein, we report the results of a prospective clinical trial designed to test this hypothesis in patients with non–small-cell lung cancer.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The goal of the study was to first determine the feasibility of using serial plasma TGFß1 measurements to select patients for RT dose escalation and then to determine the maximum-tolerated dose of radiation that could be given using this approach. This study began in November 1996, after receiving approval by the Duke University Medical Center institutional review board. All patients gave written informed consent.

Eligibility
Eligible patients had unresectable stage IIIA or IIIB, or medically inoperable stage I or II, non–small-cell lung cancer and an elevated plasma TGFß1 level (> 7.5 ng/mL [3 x 10^-7 mmol/L]) before treatment as determined by enzyme-linked immunosorbent assay.²⁹ In addition, patients were required to have a Karnofsky performance status 70, no prior thoracic RT, a life expectancy of at least 6 months, and a predicted forced expiratory volume in 1 second (FEV1) after RT of more than 800 mL (calculated using the formula: post-RT FEV1 = pre-RT FEV1 x [1 - {% total lung volume receiving > 35 Gy}]). Patients may have had prior chemotherapy, but chemotherapy concurrent with thoracic RT was prohibited. Patients were not stratified based on whether or not they had induction chemotherapy because we have not found this to be an independent risk factor for either symptomatic lung injury or change in regional lung function.^29,32

The eligibility criteria were modified in September 1998 to include patients regardless of their baseline TGFß1 level, based on a report from Anscher et al²⁹ in which it was found that plasma TGFß1 measurements could be used to identify patients at reduced risk of symptomatic radiation-induced lung injury irrespective of whether the pre-RT TGFß1 was elevated or normal. In February 1999, a final modification of the eligibility requirements was made permitting the enrollment of patients with metastatic or recurrent disease if, in the judgment of the attending radiation oncologist, aggressive treatment as prescribed in this protocol was warranted.

Pretreatment Evaluation
Pretreatment evaluation included a complete history and physical examination, chest x-ray, chest computed tomography (CT) scan, thoracic positron emission tomography (PET), bone scan, bronchoscopy, pulmonary function tests, and a complete blood count. Patients were staged according to the American Joint Committee on Cancer Manual for Staging of Cancer (ed 4).³³

RT
All patients were immobilized in a customized cradle (Alpha Cradle; Smithers Medical Products, Inc, Akron, OH) in the supine position with arms above the head throughout planning and treatment. A treatment planning CT scan was performed with the patient in their immobilization device. Contiguous 5-mm thick CT images were obtained from the level of the mandible through the liver. Gross tumor volumes (GTV), clinical target volumes (CTV), planning target volumes (PTV), and normal tissue contours were determined on all appropriate CT images and displayed using beams eye view (PLUNC; University of North Carolina, Chapel Hill, NC). Lung volumes did not include either the GTV or bronchi.

The GTV was defined as all known gross disease as determined by radiographic and clinical information. Gross tumor included the primary tumor and regional lymph nodes 1.5 cm or any lymph node with increased uptake on PET scan.

The CTV were the GTV plus volumes considered to contain microscopic/subclinical disease. The CTV1 included the primary tumor, ipsilateral hilum, and entire mediastinum from the thoracic inlet to 5 cm below the carina. For lower lobe tumors, the inferior and superior margins could be lowered to include the primary tumor and mediastinum. The contralateral hilum was not included unless involved with tumor. The CTV 2, 3, and 4 included the primary tumor plus any lymph nodes 1.5 cm and any PET scan–positive lymph nodes regardless of size.

RT consisted of 1.25 Gy bid to 45 Gy to PTV1 (CTV1 plus a 2-cm margin in all directions), with a concurrent boost of 0.35 Gy bid to 12.6 Gy to PTV2 (GTV plus 1.0-cm margin in all directions), thus providing 57.6 Gy at 1.6 Gy bid to PTV2. This was followed by 1.6 Gy bid to PTV3 (GTV plus 2.0-cm margin in all directions) to bring the total dose to 73.6 Gy. After 73.6 Gy, the plasma TGFß1 measurement was repeated. If the TGFß1 was both less than the pretreatment value and less than 7.5 ng/mL (3 x 10^-7 mmol/L), then treatment continued at 1.6 Gy bid to successively higher doses (initially 80 Gy, then 86.4 Gy) to PTV4 (same as PTV2). Otherwise, no further radiation was given. Once the decision was made to proceed to 80 Gy or 86.4 Gy, further TGFß1 measurements were not made until the first follow-up visit. The doses are not corrected for tissue inhomogeneity because our pilot data are based on uncorrected doses, and to change this policy in mid-study would have compromised the validity of the results. Elective nodal radiation to PTV1 was required even in medically inoperable stage I and II patients.

Isocentric photon irradiation using energies 4 MV was required. PTV1 was generally treated using opposed anterior/posterior and posterior/anterior fields. PTV2 to PTV4 could be treated using any beam arrangement.

Normal Tissue Dose Restrictions
The following normal tissue dose-volume restrictions could not be exceeded: whole lung, 30 Gy to two thirds, 17.5 Gy to 100%; esophagus, 60 Gy to the full circumference; brachial plexus, 60 Gy to any portion; spinal cord, 45.6 Gy to any portion; heart, 60 Gy to one third, 50 Gy to two thirds, and 40 Gy to 100%; and liver, 35 Gy to one half, 30 Gy to 100%.

Quantification of Plasma TGFß1
The method used for the analysis of plasma samples for TGFß1 has been previously reported.²⁹ First, TGFß1 was isolated from plasma using a modification of the acid-ethanol procedure of Roberts et al.³⁴ Then, an enzyme-linked immunosorbent sandwich assay was used to quantify TGFß1. Monoclonal antibodies 12H5 (0.5 mg/mL), a nonneutralizing anti-TGFß1 IgG2b,K antibody, and 4A11 (2 mg/mL), a neutralizing anti-TGFß1 IgG1,K antibody, were used as the capture and probe antibody, respectively (Genentech, Inc, South San Francisco, CA). Horseradish peroxidase–conjugated rabbit antimouse IgG1 (1:3000 dilution) (Zymed, South San Francisco, CA) was used as the secondary antibody; 2,2'-azino-di[3-ethylbenzthiazoline 6-sulfonic acid] (Bio-Rad Laboratories, Hercules, CA) was the substrate.

The 96-well microtiter plates were read at 405 nm using an automatic plate reader (v-max; Molecular Devices, Menlo Park, CA). The amount of TGFß1 in the patient samples was determined by comparing the peroxidase activity in wells containing known amounts of purified TGFß1 (R & D Systems, Minneapolis, MN) with the activity in the wells containing the patient’s plasma; samples were run in triplicate. Because the plasma TGFß1 was activated during extraction, this procedure could not distinguish between the active and latent forms. Therefore, unless otherwise specified, the term TGFß1 levels refers to the total TGFß1. The limit of detectability of this assay is 1 ng/mL (4 x 10^-8 mmol/L). Levels above 7.5 ng/mL (3 x 10^-7 mmol/L) were considered to be elevated. This threshold is two SDs above the mean level for normal controls.²⁹

Follow-Up
Follow-up evaluation occurred at 1 month after completing RT, then every 3 months for 1 year, every 4 months for 1 year, then every 6 months. At each follow-up, patients underwent a history and physical examination, with particular attention paid toward possible treatment-related toxicity, chest x-ray, and TGFß measurement. Bronchoscopy, PET scan, and pulmonary function tests were repeated at 6 and 12 months.

Statistical Considerations
The primary end point is the maximum-tolerated dose of radiation that can be delivered using changes in plasma TGFß1 levels as outlined above to select patients for dose escalation. In our previous study of accelerated RT for non–small-cell lung cancer, the incidence of grade 3 to 5 late toxicities among patients receiving 73.6 Gy using an identical irradiation protocol was 27%.³⁵ Based on this experience, an incidence of grade 3 or grade 3 to 4 toxicity of no more than 33% was considered acceptable (no grade 5 toxicities were permitted). Our previous work had demonstrated that normalization of plasma TGFß1 at the end of RT, as defined herein, predicted freedom from the development of pneumonitis with a sensitivity of 90% and a specificity of 60%.²⁹ Thus, analogous to a phase 1 trial of a new cytotoxic drug,³⁶ a minimum of five patients (maximum of seven patients) were to be treated at each dose group. The maximum-tolerated dose is defined as the highest radiation dose at which the following levels of grade 3 to 5 toxicities (Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer criteria) occur after completion of radiation treatment: one patient with grade 4 (life-threatening) late toxicity and two patients with grade 3 to 4 (severe-life threatening) late toxicity. Any grade 5 (fatal) toxicity, acute or late, would result in closure of the study, as it would indicate that the maximum-tolerated dose has been exceeded. Thus, the stopping rules were designed such that the maximum-tolerated dose could be reached without first encountering problems at a higher dose because of concerns that dose-limiting late toxicity was likely to be both severe and irreversible. Because radiation-related toxicity may take many months to manifest itself clinically, all patients within a dose level were followed for at least 6 months before escalation to the next dose level was permitted. Only one toxicity (highest grade) is counted per patient. Toxicity monitoring continued beyond the initial 6-month follow-up period, and subsequent toxicity was also used to determine the maximum-tolerated dose. Acute toxicity was defined as that which occurred during or within 6 months of completion of RT. A complication was considered a late toxicity if it persisted or developed at or beyond 6 months after RT was completed. Survival was estimated using the Kaplan-Meier method.³⁷

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
From 1996 to 1999, a total of 38 patients were enrolled onto the study. Of these 38 patients, the plasma TGFß concentration remained elevated after 73.6 Gy in 24 patients, at which point RT was stopped. In the remaining 14 patients, the plasma TGFß level was normal after 73.6 Gy, and additional radiation was administered. Of these 14 patients, eight received a total dose of 80 Gy, and six received a total dose of 86.4 Gy. The characteristics of these groups are listed in Table 1. The characteristics of all three groups are similar, except that a higher percentage of the group receiving 86.4 Gy also received induction chemotherapy.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Non–small-cell lung cancer is one of the most common and deadly forms of cancer in the United States. In 2000, approximately 164,000 new cases of lung cancer will be diagnosed, of which approximately 80% will be non–small-cell lung cancer.³⁸ Approximately one third will present with locally advanced nonmetastatic tumors. Chemotherapy plus thoracic radiation has become the standard treatment for patients with locally advanced non–small-cell lung cancer, but cure rates remain low.^39-44

Failure to control local disease remains a significant clinical problem.^1,41,45 Standard doses of RT (60 to 65 Gy) produce local control rates of 15% or less in this patient population.⁴⁵ The importance of local control in improving survival for non–small-cell lung cancer patients has been noted by several authors.^1-5,46 Delivering higher doses of radiation to the tumor may significantly improve both local control and survival.^1-5,46

The maximum safe dose of radiation that can be delivered in the treatment of locally advanced non–small-cell lung cancer is limited by the tolerance of the surrounding normal tissues.⁶ The currently accepted tolerance doses have been derived empirically, often with little supporting data.⁶ These tolerance doses are based on risk estimates for the human population as a whole and are often skewed by unusually sensitive individuals.

Recently, the underlying molecular biologic mechanisms behind the development of normal tissue injury after cancer therapy have come under study.^10,47-50 The fibrogenic cytokine TGFß1 has been found to play a crucial role in this process. Ionizing radiation leads to both increased local expression and activation of TGFß1 by proteolytic enzymes⁵¹ or free radicals,⁵² resulting in increased fibrosis formation in irradiated tissues.^{13,22,48,49,53} It has been demonstrated in a mouse model that by lowering the level of circulating TGFß1 before radiation exposure, the severity of radiation-induced fibrosis can be reduced.⁵⁴ These data suggest that clinically apparent radiation injury may be a predictable entity that could be modifiable by reducing TGFß1 expression.^47,54

Plasma TGFß1 levels have recently been used to try and identify patients at risk for the development of normal tissue injury after exposure to chemotherapy and/or RT.^28,29,55-59 In the setting of RT for lung cancer, changes in plasma TGFß1 levels during treatment have most accurately identified patients who will not develop symptomatic pulmonary toxicity. Specifically, those patients whose plasma TGFß1 concentration returns to normal by the end of RT are at low risk of developing subsequent symptomatic radiation-induced lung injury.²⁹ This may be the group of patients most suitable for studies to determine whether escalating doses of RT can be safely delivered in an attempt to improve local control and survival for patients with unresectable non–small-cell lung cancer.

To our knowledge, the present study is the first attempt to individualize cancer treatment based on a simple biologic assessment of an individual patient’s risk of normal tissue injury. The data presented herein suggest that it is possible to identify patients with non–small-cell lung cancer who are at low risk for the development of late radiation-induced injury with standard doses of thoracic radiation. Furthermore, it seems that it is possible to use the approach outlined above to identify patients in whom doses of thoracic radiation may be safely escalated. The maximum-tolerated dose of radiation that can be delivered to the thorax using this approach is 86.4 Gy (uncorrected for tissue inhomogeneity). This dose is 17% higher than that delivered to the group who were not dose escalated and 30% to 40% higher than standard doses of RT for non–small-cell lung cancer commonly reported in the literature.^{1,4,5,39-41,45,46,60-62} Dose increases of this magnitude may result in clinically meaningful improvements in local control.⁶³ The present study is too small to expect to be able to detect dose-dependent improvements in local control or survival. A much larger group of patients will need to be studied to determine the impact of such high doses of RT on local control and/or survival in non–small-cell lung cancer.

This study raises several questions. It is possible that higher doses may have been achievable if more limited volumes were irradiated. We cannot address this question directly because the volumes irradiated were similar in all groups. The fact that the irradiated volumes were similar, as was the pretreatment pulmonary function, supports the validity of using plasma TGFß1 levels to select patients for radiation dose escalation.

Others have taken a different approach, choosing to stratify patients into dose groups according to the volume of lung to be irradiated.⁶⁴ Recent evidence suggests that combining both the volume of irradiated lung and plasma TGFß1 measurements may permit a better estimation of lung injury risk than using either method alone.³¹ The recent report that plasma IL-6 levels may also be predictive of radiation-induced lung injury³⁰ suggests that it may soon be possible to generate a sophisticated normal tissue injury risk profile for an individual patient using a combination of physical and biologic factors. Because circulating IL-6 and TGFß1 levels may reflect different components of the pathologic process (ie, inflammation and fibrosis, respectively), it soon may also be possible to monitor the effectiveness of interventions directed against these phenomena.

The effect of induction chemotherapy on the tolerance of the lung to RT cannot be directly addressed by this study. There is a suggestion that the more frequent use of chemotherapy in the later years of the study may have limited the radiation dose that could be delivered. This issue will become even more important if the trend towards concurrent chemoradiotherapy for non–small-cell lung cancer continues. The fact that a greater proportion of patients in the 86.4-Gy group received induction chemotherapy does not invalidate the findings of this study. The biologic processes related to normal tissue injury are not specific to RT, and it has been shown that TGFß1 is also an important component in injury from antineoplastic drugs,^55,65,66 as well as other diseases associated with excessive fibrosis.^67,68 Thus, changes in circulating TGFß1 may simply reflect a patient’s risk of injury from cancer therapy, which can vary depending on the presence or absence of other comorbidities.

The fact that only 14 of 38 patients had a normal TGFß1 after 73.6 Gy is not surprising. Many tumors, including non–small-cell lung cancer, are associated with increased expression of TGFß1, which is reflected in the plasma,^69-73 possibly as a result of altered bioavailability of this cytokine.⁷⁴ A persistently elevated plasma TGFß1 after treatment suggests that the treatment being given is not eradicating the tumor,⁷⁵ and thus, more of the same treatment may not only be ineffective but also may predispose the patient to toxicity from the treatment. This issue requires further study.

Interestingly, two of the three late grade 3 toxicities in the two dose-escalated groups were esophageal. Thus, significant late pulmonary injury was not common. The esophageal toxicity may have been related to the twice-daily treatment schedule used in this study, as well as to the elective irradiation of the mediastinal nodes. Some authors have advocated the elimination of elective nodal irradiation, which might both reduce toxicity and permit further dose escalation.⁷⁶ The validity of this approach requires further study.

One criticism of this study relates to the potential inaccuracies introduced by not correcting the radiation dose for lung inhomogeneities. When the study was implemented, it was the practice in our institution not to perform such corrections. In addition, the historical data from which estimates of anticipated toxicity were generated consisted of patients in whom dose inhomogeneity corrections were not made. To change this practice in mid-study could have invalidated the results because the data obtained in patients whose doses were defined differently would not be directly comparable. It is estimated that the doses delivered in this study are actually 6% to 8% higher than reported if homogeneity corrections are made (data not shown). Future studies will include corrections for tissue inhomogeneity from the inception.

In summary, it seems to be feasible to use biologically based criteria, in this case TGFß1, to determine an individual patient’s normal tissue injury risk from cancer therapy. Furthermore, it seems to be possible to use this risk assessment to select patients for RT dose escalation for locally advanced or medically inoperable non–small-cell lung cancer. The maximum-tolerated dose of RT deliverable using this approach is 86.4 Gy uncorrected for tissue inhomogeneities. Continued research in this area is warranted to determine whether this approach to treatment will lead to improved local control and/or survival for patients with non–small-cell lung cancer.

	ACKNOWLEDGMENTS

 
Supported by grant no. 1R21CA83721-01 from the National Institutes of Health, Bethesda, MD.

We thank our colleagues in the Department of Radiation Oncology at the University of North Carolina at Chapel Hill for PLUNC treatment planning software.

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Submitted February 9, 2001; accepted June 13, 2001.

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