Abstract
Background: Radiotherapy for lung cancer may induce pneumonitis. However, histological effects of radiotherapy on normal lung tissue are unknown. Transbronchial biopsy (TBB) is safe and accurate in monitoring parenchymal lesions in lung-transplanted patients. The aim of this prospective study was to evaluate whether histological changes of the healthy lung parenchyma after radiotherapy are present on TBB biopsies. Patients and Methods: Twelve patients with lung cancer necessitating radiation therapy participated in the study. Serial TBBs were obtained from lung parenchyma contra-lateral to the tumor before, just after radiotherapy, and at six months post-irradiation. Evaluation of each specimen was based on the presence of congestion, inflammation, hemorrhage and fibrosis. Results: A significant increase of interstitial fibrosis (thickness) and congestion was observed between the point prior to radiotherapy and after completion of radiotherapy (p=0.047), as well as between the pre-radiotherapy point and at six months after radiotherapy (p=0.014). Six patients (50%) showed intra-alveolar fibroblastic growth after radiotherapy. No patient showed clinical or radiographic findings of radiation pneumonitis. Conclusion: Even in the absence of clinical or radiographic findings, the lung parenchyma contra-lateral to the tumor suffers early histological lesions after radiation therapy, as monitored by serial TBBs.
Irradiation of the thorax affects the lung parenchyma, leading to the clinical syndrome of radiation pneumonitis (1, 2). Pneumonitis is directly linked, amongst other factors, to the volume of the irradiated lung above a threshold of 20-30 Gy (3). Thoracic irradiation may also affect the supposedly non-irradiated lung parenchyma. Thus, irradiation-related bronchiolitis obliterans organizing pneumonia (4, 5), and chronic eosinophilic pneumonia (6) have been described in patients with breast cancer.
Although pro-inflammatory and pro-fibrogenic cytokines, such as interleukine-6 (IL-6), and transforming growth factor-beta1 (TGF-β1) respectively, have been evaluated in the bronchoalveolar lavage fluid (BALF) of patients irradiated for lung cancer (7), the pathogenesis of these conditions affecting lung parenchyma is not fully-elucidated, as histological studies from living humans do not exist. Furthermore, it is totally unknown whether monitoring changes in lung parenchyma may predict both the outcome of these changes and their progression to fibrosis in order to treat early patients.
Transbronchial biopsy (TBB) is a very useful tool for the diagnosis of various conditions affecting the lung parenchyma and remains the ‘gold standard’ for evaluation of pulmonary allograft for acute rejection and opportunistic infection after lung transplantation (8, 9). Although there is debate on whether routine performance of TBB, as opposed to TBB performed after clinical evaluation allows early detection of acute rejection of the transplanted lung by showing obliterative bronchiolotis (9), new data suggest the utility of surveillance TBB in transplant patients, as supported by the presence of lymphocytic bronchiolitis that determines the outcome when acute rejection is detected (10).
Therefore, the aim of the present study was to prospectively assess histological changes of the non-cancerous lung parenchyma after irradiation, using serial TBBs.
Patients and Methods
Patients. This prospective study, conducted during a two-year period (October 2007-December 2008), included 14 patients who underwent thoracic irradiation for lung cancer. Twelve patients were finally assessable. Ethical approval was obtained from the Internal Review Board of the University Hospital of Alexandroupolis (# 1950/2007). All 14 patients signed an informed consent form prior to study inclusion. Survival was calculated from the time of treatment initiation (chemotherapy) until death. Characteristics of the 12 assessed patients (all male) are shown in Table I. Patients with non-small cell lung cancer were classified according to the clinical TNM staging system (11). Eight patients were current smokers [range=40-110 packs per year (ppy), median=80 ppy]. All patients were treated with chemotherapy prior to radiation therapy. Treatment schedules consisted of cisplatin-vinorelbine (N=7) or cisplatin–etoposide (N=5). Radiotherapy was delivered to the affected lung and mediastinal nodes. All patients were treated with 18-MV photons and three-dimension conformal radiotherapy, which spares healthy tissue as previously described (12). Nine fractions of 3.5 Gy were applied, delivering a median biological (for α/β=4 Gy dose) of 48 Gy to the mediastinum and the tumor mass, followed by five additional fractions of 3.5 Gy that increased the total biological dose to >70 Gy to the gross tumor area. However, since the TBBs were taken from the distal lung parenchyma of the diagonally contralateral lobe, it is unlikely that biopsies were within the irradiated field.
Study design. Patients were subjected to four serial TBS of the contralateral lung. The rationale for the contralateral biopsy was to have as much healthy tissue as possible and as less exposure to radiation therapy as possible to assess whether this minimal exposure led to demonstrable histological changes. The first biopsy was obtained after completion of the patient's chemotherapeutic regimen and prior to the initiation of irradiation (pre-RT point), the second at the completion of the radiotherapy (time-point 0), the third six months later (time-point 1), and the fourth biopsy at 12 months. However, none of our patients survived beyond 10 months from the initiation of radiation therapy to complete the last part of the study. All patients underwent a chest X-ray at each time point, together with their classical computed tomographic tumor evaluation. In cases of clinical or radiological suspicion of pneumonitis, additional high-resolution tomography was mandatory.
Bronchoscopy and biopsies. TBBs were performed during fiberoptic bronchoscopy under local anesthesia only. In every case, TBBs were taken from the distal lung parenchyma of the lobe diagonally contralateral to the tumor. All biopsies were fixed in 10% formalin solution and processed routinely to paraffin wax. They were subsequently cut serially into 3 μm sections, stained with hematoxylin and eosin and evaluated with an optical microscope.
Tissue specimens were qualitatively evaluated for the following patterns: acute lung injury, chronic cellular infiltrates, fibrosis, and cellular or noncellular elements filling the alveoli. A scoring system (13) of four parameters, namely alveolar congestion, hemorrhage, leukocyte infiltration in airspace or the vessel wall, and thickness of the alveolar wall, was used as a semi-quantitative way of evaluating changes of lung parenchyma. The parameters were scored on a scale of 0-4, as previously described (13): a score of 0 represented normal lung; 1, mild, <25% specimen involvement; 2, moderate, 25-50% specimen involvement; 3, severe, 50-75% specimen involvement; and 4, very severe, >75% specimen involvement. An overall score was obtained based on the sum of all the scores.
Statistical analysis. Statistics were performed using StatView software. Student's t-test was used to compare differences between groups. The differences were considered significant if p-values were less than 0.05.
Patients' characteristics (n=12).
Results
Patients. Patients' overall survival (Table I) ranged from nine to 16 months (median=14.5 months); all deaths were cancer-related after disease progression. No patient exhibited clinical or radiographic findings of radiation-induced lung injury.
Histopathological evaluation. The number of tissue fragments ranged from two to six per biopsy (median=4), with size ranging from 0.1 cm to 0.6 cm (median=0.3 cm). Twelve cases were considered adequate, containing at least two fragments of alveolated parenchyma at all three time-points.
Semi-quantitative evaluation (Table II): Between the pre-RT point and point 0 (p=0.048), as well as between the pre-RT point and time-point 1 (p=0.014) (Figure 1), a significant increase of the total score was observed [especially interstitial fibrosis (thickness) and congestion] (Figure 2), but not between time-points 0 and 1 (p=0.15) (Table II). Hemorrhage was not found in the biopsies studied, but hemosiderin-laden macrophages were present in eleven cases (91.6%), eight of them post-irradiation (Table III). Leucocytes were present in the biopsies of two patients at time-point 1 (Figure 2B).
Score (mean±SD) of histopathological findings according to time-point of biopsy relative to time of radiotherapy (RT).
Qualitative evaluation (Table III): Hyaline membranes, indicative of acute lung injury, were present in two cases (16.6%), at the pre-RT point. No other findings of acute lung injury, such as fibrin or type II cell hyperplasia, were encountered. Two serial biopsies of the same patient and the specimen of another patient (time-point 1) showed the presence of eosinophils after irradiation. Lymphocytes were present in biopsies of two patients (time point 0 and 1) (Figure 2D). Intra-alveolar fibroblastic tissue (Figure 2A, C and D), either mature or immature, was found in four patients (33.3%) at time-point 0; of these, two patients had the same findings at time-point 1. Two additional patients (16.6%) exhibited intra-alveolar fibroblastic growth at time-point 1. Overall, six patients (50%) exhibited intra-alveolar fibroblastic growth after radiotherapy.
Discussion
Our study shows that radiotherapy for various types of lung cancer causes microscopic lesions, even in the absence of corresponding clinical manifestations of the non-cancerous lung, as monitored by serial TBBs. Septal fibrosis and alveolar congestion were the main findings at the time of completion of the radiotherapy and at six months post-irradiation, showing statistically significant differences from the pre-irradiation specimen.
In our study, six patients (50%) exhibited early features of organizing pneumonia, such as intra-alveolar fibroblastic growth (Table III), occurring in the contralateral lung, without evidence of clinical/radiographic syndrome. Histological findings of our patients were consistent with the biopsies of patients with breast cancer suffering from bronchiolitis obliterans organizing pneumonia (4, 5). Organizing pneumonia, occurring in the early post-irradiation phase in the contralateral lung, is characterized by fever, non-productive cough, mild dyspnea, and migratory alveolar opacities on chest radiography and computed tomography (4, 5). We have not encountered any case of clinical or radiographic manifestation in our series, possibly because our study was prospective, enrolling few patients (1, 2). It has been suggested that the increase of lymphocytes found in BALF of patients with breast cancer following unilateral irradiation mainly consisted of activated cluster of differentiation 4+ cells (CD4+) migrating from the irradiated lung to the contralateral one (14, 15).
Score (mean±SD) of histopathological findings according to time-point of biopsy: a significant increase of total score between before radiation therapy (Before RT) and just after RT (After RT) (p=0.048), as well as between before radiation therapy and six months after RT (At 6 months) (p=0.014).
We also found alveolar congestion, with inflammatory cells and fibrosis (Tables II and III). Furthermore, by serial TBBs, those histological features gradually increased over time (Table II). Pathological events of the irradiated lung successively consist of an acute exudative phase with secondary infiltration of the interstitium by mononuclear and other inflammatory cells, with finally the development of fibrosis (chronic phase) (1, 15). Findings such as hyaline membranes, marked cytological atypia within hyperplastic pneumocytes, and prominent vascular changes are also present (1, 5, 15). However, these histological features of the contralateral non-irradiated lung have never been monitored prospectively to our knowledge.
Septum fibrosis was seen in our patients very early, raising questions regarding the underlying mechanism of this manifestation. Studies on DNA damage induced by irradiation of rat lung showed the induction of chromosomal aberrations even in non-irradiated pulmonary regions, possibly due to the action of superoxide radicals, activated lymphocytes, and chromosomal-damaging factors produced in the irradiated lung and transported to the non-irradiated region via diffusion or blood circulation (16). Similarly, alveolar macrophages of both lungs exhibited functional abnormalities after unilateral irradiation in another experimental model (17).
A: Transbronchial biopsy (TBB) at six months post-radiotherapy (RT) with severe septal thickening and intra-alveolar fibroblastic growth (×200). B: TBB at six months post-RT with severe septal thickening and leucocytes both in and outside the lumen of a vessel (×200). C: TBB at completion of RT with intra-alveolar fibroblastic growth (×200). D: TBB at six months post-irradiation with moderate septal thickening, intralveolar fibroblastic growth and lymphocytic infiltration (×200).
Qualitative evaluation of histopathological findings.
Another possible explanation for the early fibrotic lesions observed in some of our patients might be previous treatment by chemotherapy. In particular, hyaline membranes, found in the alveoli of two patients, may be attributed to chemotherapy, as it is known to cause lung injury (2, 18). Despite the classical diffused alveolar damage caused by many chemotherapeutic agents resulting in non-cardiogenic edema (18), the risk of developing pneumonitis is increased after thoracic irradiation when a cytostatic agent is applied, together with other factors such as old age, low performance status, low baseline pulmonary function and PaO2 (19). Finally, the process of acute radiation pneumonitis is probably tightly linked to fibrosis, involving complex molecular mechanisms (20). Yet, to design such a study in patients with advanced-stage lung carcinoma and good performance status is not ethical since external beam irradiation as single treatment is not indicated. Therefore, all our patients had previously undergone chemotherapy, after which the baseline biopsies were considered for the study.
In our study, eosinophils were found in the biopsies of two patients after irradiation (Table III). Radiotherapy for breast cancer is known to cause a slightly elevated number of eosinophils in BALF (21), or even the syndrome of radiation-induced chronic eosinophilic pneumonia, characterized by dyspnea, cough, alveolar or infiltrative pulmonary opacities, and marked alveolar and peripheral eosinophilia (6). Mild alveolar eosinophilia may also accompany radiation-induced organizing pneumonia (4, 5).
Limitations of our study are the small number of patients enrolled, and the short follow-up period. Indeed, patients with breast cancer had a median follow-up period of 15 months after diagnosis of radiation-induced lung injury (5). Because such a long follow-up period is unlikely to be achieved in patients with lung cancer, due to poor survival in those with advanced-stage disease, a minimum of 12 months is necessary to fully-assess those patients likely to present late lung injury (22). Although we designed this study accordingly, we were unable to achieve this goal due to our patients' rapidly deteriorating condition and death. This is probably why we did not observe changes in the chronic or late fibrotic phase comparing to the breast cancer series. Yet, as far as we are aware of, our study is the first reporting results prospectively.
In conclusion, our study shows that in patients with lung carcinoma undergoing radiation therapy, healthy lung parenchyma presents early histological changes, even in the absence of any clinical or radiographic finding, and these lesions can be monitored by serial TBB. The value of such surveillance of histological alterations in predicting the evolution of post-radiotherapy lung damage, and therefore in identifying patients at risk of developing a clinical syndrome, requires further larger prospective studies.
Clinical Relevance
This prospective study of histological biopsies taken by transbronchial way shows that, early lesions of the supposed “healthy” lung parenchyma are present in patients with lung carcinoma after radiation therapy.
Footnotes
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Conflicts of Interest
All Authors state no conflict to disclose
This study was presented in part at the European Congress of Interventional Pulmonology, Marseille 2011, and at the European Respiratory Society Congress, Amsterdam 2011.
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Funding Source
This research was supported with a grant from “Fondation Lancardis”, Martigny, Switzerland.
- Received January 28, 2014.
- Revision received March 30, 2014.
- Accepted April 1, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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