Biochemical markers as predictors for pulmonary effects of radiation

Radiution Oncology Investigations 356-63 (1995)

Biochemical Markers as Predictors for Pulmononary Effects of Radiation

Sandra McDonald, M.D., Philip Rubin, M.D., Louis Constine, M.D., Jacky Williams, m . ~ . , Jacob Finkelstein, p h . ~ . , and Therese Smudzin, B.S.

Department of Radiation Oncology, University of Rochester Cancer Center (S.M., P . R., L. C., J . W., T.S.), and Department of PediatriclNeonatology, University of Rochester (J. F . ) ,

Rochester, New York

SUMMARY The delay in onset of clinidradiological expression of pulmonary radiation toxicity precludes modification of radiation factors during treatment. Early detection of normal lung damage by biochemical markers that would predict for pneumonitis and/or fibrosis could allow for timely intervention. This study was undertaken to analyze changes in blood levels of surfactant apoprotein, procollagen III, and transforming growth factor p (TGF-p) in patients undergoing pulmonary radiation therapy (RT) and to determine if a correlation exists between early changes of these biochemical markers and subsequent radiographic and clinical evidence of pneumonitis and/or fibrosis. Sixteen patients with primary lung cancer undergoing thoracic irradiation were enrolled in this study and blood samples were obtained pretreatment, weekly during treatment, and at follow-up. At the same time points, patients underwent physical examination, chest X-rays, and computerized tomographic (CT) scans. Surfactant apoprotein was determined by immunodot blot analysis, procollagen III levels were determined by radioimmunoassay (RIA), and TGF-P was determined by an enzyme-linked immunosorbent assay @LISA) kit. Radiographically, 9 of 16 patients entered developed moderate to severe evidence of acute radiation injury; 6 had mild or no changes. The mean increase in surfactant apoprotein level during treatment was different (P = 0.074) between the two groups: 6% in the moderate/severe group vs. 42% in the mild/no change group. The inverse correlation for surfactant apoprotein levels during treatment relative to radiographic changes at 2 months was statistically significant (R = -0.59, P < 0.05). Clinically, 5 of 16 developed pneumonitis. Lack of increase in surfactant apoprotein levels during treatment similarly corresponded to clinical pneumonitis. During treatment 50% (8 of 16) of the patients had procollagen III levels above the upper limit of normal. This group had increased radiographic evidence a t 4-6 months posttreatment of radiation late effects (fibrosis) compared to the group with normal procollagen levels during treatment. TGF-P values pretreatment were elevated for patients developing pneumonitis and moderate to severe radiological changes, but this did not have statistical significance. Analysis of preliminary data suggests that an increase in surfactant apoprotein levels during thoracic irradiation is associated with a decreased probability of clinical and radiographic evidence of radiation-induced lung injury. Further- more, an elevated procollagen III level during treatment may be useful in predicting the development of late radiographic evidence of more severe fibrosis than patients with normal values during treatment. The role of TGF-p as a predictor of fibrosis needs to be further investigated. These biochemical markers may prove useful as short-term endpoints to evaluate toxicity a t a time when treatment factors could be modified (e.g., volume or

Received original March 10, 1995; revised April 28, 1995; accepted April 28, 1995. Address correspondence/reprint requests to: Sandra McDonald, M.D., Department of Radiation Oncology, University of

Rochester Medical Center, Box 647,601 Elmwood Avenue, Rochester, NY 14642-8647. 0 1995 Wiley-Liss, lnc.

McDonald et al.: P d m m r y Effects of Radiution 5’7

dose) and particularly with the assessment of new modalities such as three-dimensional treatment planning. Radiat Oncol Invest 1995;3:5643. 0 1995 Wdey-Liss, Inc.

Key work radiation, pulmonary effects, biochemical markers

INTRODUCTION

Pulmonary complications resulting from anticancer therapy can range from acute fatal respiratory dis- tress to degrees of chronic pulmonary compromise, which may manifest years after the initial cancer therapy. The lung is thus a major dose-limiting organ in the delivery of optimal doses of radiation to eradicate malignant disease. This applies to irradiation of partial lung volumes for lung tumors and mediastinal masses due to Hodgkin’s disease and lymphomas, as well as whole lung volumes with total body irradiation (TBI) used for leukemia/ lymphoma and upper hemibody irradiation for metastatic cancers. The lung is unusual in its reaction following irradiation because both the initial pneumonitis-which has been considered an acute event-as well as the subsequent fibrosis are actu- ally both late effects. Radiographic abnormalities are commonly present even among patients who do not develop symptomatic pneumonitis. Patterns of radiographic changes associated with radiation injury have temporal courses, appearing weeks to years after radiotherapy (RT) corresponding to both pneumonitic and fibrotic phases. This delay between the damaging event and the detection of physiologic and morphologic abnormalities associated with pneumonitis has thus far precluded modification of radiation factors during treatment. In this era of multimodality therapy for cancer, the detection of biochemical markers for tissue damage in the subclinical phase, i.e., prior to the accumula- tion of significant injury, could allow for termina- tion of therapy or the institution of treatment strata- gems to prevent or attenuate later lesions.

For this investigation several possible biochemical markers were evaluated. Early ultrastructural changes of the type I1 pneumocyte have been detected in irradiated lungs of animals [ 1,2]. This cell is central to the biosynthesis and secretion of surfactant. Consequently, alteration in surfactant release was studied in mice and rabbits as a measure of predicting later fatal pneumonitis. Release of surfactant during the first week following radiation is quantifiable in bronchoalveolar lavage and was found to be a consistent early biochemical marker for later lethal radiation events in three murine strains and a rabbit model [3-51. However, bron-

choalveolar lavage is a time-consuming invasive procedure and as such not suitable for clinical situ- ations. A surfactant-associated protein component or apoprotein has been found to have a high degree of specificity for lung injury and the presence of this apoprotein in serum can be detected by sensitive radioimmunoassays (RIAs). It is hypothesized that the presence of this surfactant apoprotein in serum indicates damage to the capillary alveolar membrane followed by leakage into the blood [6] . In the rabbit model, serum surfactant apoprotein was demonstrated to be an accurate marker and predictor for later lethal radiation pneumonitis [7].

Fibroblast proliferation contributes to the increased lung collagen content in pulmonary fibrosis, and increases in procollagen 111, a biosynthetic precursor of collagen that is synthesized in fibroblasts, has been found in lungs of patients with idiopathic lung fibrosis [8]. Therefore, an assay for type I11 procollagen peptide may be useful in the early detection of pulmonary fibrosis as a complica- tion of RT.

Transforming growth factor beta (TGF-P) has been shown to stimulate collagen synthesis as well as proliferation of fibroblasts [9-121. There are data to suggest that plasma TGF-P may be a predictor for the later development of normal tissue damage. In a group of women treated with high dose chemotherapy and autologous bone marrow transplantation, the prechemotherapy plasma TGF-f! level was highly predictive for the later development of pulmonary and/or liver fibrosis following therapy [ 131. Changes in plasma TGF-f! levels during pulmonary RT have also been suggested as predictors for the risk of developing pneumonitis [ 141.

This study was undertaken to analyze changes in blood levels of surfactant apoprotein, procollagen 111, and TGF-P in patients undergoing thoracic irradiation and to determine if a correlation exists between early changes of these biochemical markers and subsequent radiographic and clinical evidence of pneumonitis and/or fibrosis.

Radiographic evidence of lung injury was evaluated using chest X-rays and more importantly serial computerized tomography (CT) of the chest. Because of its sensitivity to slight changes in lung densities, CT is presently favored for diagnostic detection of pulmonary damage in humans [ 15-17].

58

Table 1. RTOG Acute Radiation Morbidity Scoring Criteria for Lung

McDonald et al.: Pzrlmonury Eflects of Radiation

Grade

No change Mild symptoms of dry cough Persistent cough requiring Severe cough unresponsive Severe respiratory or dyspnea on exertion narcotic antitussive to narcotic antitussive insufficienc ylcontinuous

agentsldyspnea with agents or dyspnea at oxygen or assisted minimal effort but not at restklinical or radiological ventilation rest evidence of acute

pneumonitisloxygen or steroids may be required

RTOGIEORTC Lute Radiation Morbidity Scoring Scheme None Asymptomatic or mild Moderate symptomatic Severe symptomatic fibrosis Severe respiratory

symptoms (dry cough); fibrosis or pneumonitis or pneumonitis; dense insufficienc ylcontinuous slight radiographic (severe cough); low grade radiographic changes oxygen or assisted appearances fever; patchy radiographic ventilation

appearances

Table 2. Patient Assessments*

Post-RT

During End Week Week Week Month Month Procedure Pre-RT RT RT 1 2 3 1 3+Q3

H & P X X X X X Chest X-ray X X X X X Chest CT X X X X X Biochemical markers: surfactant X X X X X X X X

apoprotein, procollagen III, TGF-P

*H & P = history and physical; Q3 = every 3 months thereafter

MATERIALS AND METHODS

Patient Eligibility Patients undergoing irradiation for malignant disease, in which normal lung would be included in the treatment field, were eligible. This included patients with lung cancer, or mediastinal masses due to Hodgkin’s disease and lymphomas, patients undergoing TBI for leukemia, and upper hemibody irradiation for metastatic cancers. All patients had a complete medical evaluation including hemogram, WBC, and platelet count and were eligible if no significant abnormalities were found. Blood param- eters did not apply to TBI patients. Internal review board-approved informed written consent was obtained.

Patient Characteristics Sixteen patients with primary lung cancer who underwent thoracic irradiation were evaluated. Me- dian age was 67 years; 15 patients were male and 1 patient was female. Median follow-up was 6 months. Nine patients have died of the disease and one patient has died of treatment complications.

Treatment Description The average RT dose for these patients was 5,495 cGy with a range of 4,000 cGy in 250 cGy fractions to 6,950 cGy in 120 cGy bid. Treatment time was 4-6 weeks. Radiation portals encompassed the partial or total lung volumes, as required for therapy of the particular malignancy. Fractionation and doses were as required for the specific disease.

Clinical and Radiographic Evaluation History and physical examination, emphasizing the respiratory system, were performed. Clincal evidence for pulmonary injury was evaluated and graded according to RTOG acute morbidity scoring criteria (Table 1). Chest X-rays and chest CT were performed at intervals indicated in Table 2. Changes observed in irradiated normal lung were graded using a modified scoring system based on publications by Mah et al. [16], Arriagada et al. [ 181, and Maasilta et al. [ 191. The following radiographic findings (chest X-ray and CT based) were scored (maximum score): extent of loss or air content (5), degree of opacification (4), anatomic extent of involvement (6), other associated changes

McDonald et al.: Pulmmry Ef fea of Radktim 59

standard curve constructed under identical condi- tions. The normal range for this test was determined (by Behring) using 451 healthy men and women. By calculating the 5th and 95th percentiles a normal range of 0.3-0.8 U/ml was established.

such as air bronchogram, pleural thickening, or ef- fusion (6). The scores from each finding were added giving a maximum score of 21. A radiographic score of 10 or greater out of a maximum score of 21 was determined to be moderate to severe and would fall into the late morbidity scoring system of grade 2 and higher (Table l ) , while a radiographic score of less than 10 was considered unaf- fected to mild corresponding to the late morbidity score of grade 0 or 1.

Biochemical Marker Assays '

Su$actant Apoprotein The serum concentration of surfactant-associated protein was determined by dot blot analysis. Briefly, samples were applied to nitrocellulose membrane gel-blot paper (Schleicher & Schuell, Keene, NH) and blocked with BLOTTO [20]. Pri- mary antibody, mouse antisurfactant apoprotein IgA (Cappel-Organon Teknika Corp., Durham, NC), was applied, incubated, and washed. Second- ary antibody, biotinated goat anit-mouse, was applied, incubated, and washed. Vectastain (Vector Laboratories, Burlingame, CA) was applied and followed by the enhanced chemiluminescence (ECL) Western detection system (Amersham Life Science, Arlington Heights, IL). The nitrocellulose was then exposed to Kodak Scientific Imaging Film (Kodak, Rochester, NY). Film was developed and read densitometrically . Blood samples were collected pretreatment, during treatment, and posttreatment as described. Analysis of all collected samples was performed at one time. Since the pretreatment baseline value varied for each individual (both for the patients and normal controls), each patient was his or her own pretreatment control. Therefore, values given are relative changes compared to baseline.

procoll4lgen III Serum procollagen 111 concentrations were determined by RIA-gnost P III P Kit (Behring Behring- werke, Marburg, Germany). In vitro determination of human procollagen 111 peptide in human serum (or plasma) was made using a two-stage sandwich assay. A complex of solid phase anti-P 111 P antibodies (monoclonal, mouse), procollagen 111 peptide in the sample, and 1251-labeled anti-P I11 P antibodies (monoclonal, mouse) is formed during this process. At the end of the reaction the free tracer is removed by decanting (or aspiration) and subsequent washing. The amount of tracer specifi- cally bound to the coated test tubes is measured with a gamma scintillation counter. Quantification of the unknowns is carried out by reading off a

TGF-f3 TGF-P, concentrations in patient plasma were determined by enzyme-linked immunosorbent assay (ELISA) using a quantification kit (Genzyme Diag- nostics, Cambridge, MA). As suggested in the kit instructions, our laboratory, using 9 healthy sub- jects, established its own expected normal values of 17.88 ng/ml in plasma (95% confidence limits 14.24, 21.52). Samples and standard are activated by acidification with HC1 and then neutralization with NaOH. These are then added to test wells allowing any TGF-f3 I to be bound by antibodies on the microtiter plate. The wells are washed and a direct-labeled HRT-conjugated polyclonal antibody to TGF-f3, is added. This subsequently binds to the captured TGF-PI . After washing, a substrate solu- tion is added producing a color in the presence of peroxidase. Once the color reaction is stopped, the absorbance of each well at 450 nm is determined. The intensity of color is directly proportionate to the amount of TGF-f3, present in the well. Quanti- fication of the test sera is carried out by reading off a standard curve.

Statistical Methods Significance of correlation for the surfactant/ procollagen levels was determined by the t-test for correlation. Percent changes in the marker levels for each patient were determined by dividing the raw values during and posttreatment by the pretreatment value. Blood samples during treatment were not taken at consistent time points for all patients; therefore, changes in each patient's values during treatment were averaged. The mean values for each treatment group were then calculated. Significance of the difference of mean values for different groups of patients was determined by a two-sample t-test.

RESULTS Of the 16 patients entered into the study, 5 developed clinical pneumonitis (grade 3) at 1 4 months posttreatment; in 4 of the cases this was transient and responsive to steroids. One patient developed fatal (grade 5) pneumonitis at 1 month. Radio- graphically, 10 patients had moderate to severe changes, defined as a radiographic score of 10 or greater out of a maximum score of 21, 3 patients

60 M c D m U et al.: Pdmonury Egects of Radiation

c, c -40 z I? -60 aJ

Ei Surfactant Level Radiographic Score

-

I I 1 1 _. . ..- ...

n=6

I I 1 Fig. 1. errors. The pretreatment radiographic score is zero.

Surfactant apoprotein level vs. low radiographic score. Error bars represent standard

Ei Surfactant Level Radiographic Score

c c,

-40 L aJ a

-60 PRE-FIT During-RT 1 Month 2 Month 4 - 6 E Y t h n=10 n=10 n=6 n=4

Fig. 2. errors.

Surfactant apoprotein level vs. high radiographic score. Error bars represent standard

had mild changes (a radiographic score of less than lo), and 3 had no changes.

For all 16 patients, overall surfactant apoprotein levels increased an average of 20% compared to baseline during RT. Figure 1 illustrates the changes in the surfactant apoprotein level relative to the radiographic score of normal lung injury for patients with mild to no evidence of lung injury. For these patients , surfactant apoprotein level during treatment increased 42% above pretreatment baseline, remained high at 19% above baseline at 1 month posttreatment, and decreased during the follow-up period.

Figure 2 illustrates the average percentage change in surfactant apoprotein levels for patients with moderate to severe radiographic changes. In this group, the level did not increase significantly (6%, n = 10) during treatment compared to baseline, was not elevated at 1 month ( -5%, n = 6), and remained stable during the follow-up period. The radiographic score was high in this group, no- tably at 2 months (17.6), consistent with the time period for pneumonitis. The mean increase in surfactant levels during radiation for the two groups was 42% vs. 6% (P = 0.074). Surfactant apoprotein levels during radiation were inversely associ-

McDonaM et al.: Pdmtmary Eflectv of Radiation 61

70 QU Normal

w W c 40 ID

Pneumonitis NO Pneumonitis

s s

n=3 T i 9 30 LL

h (3 20 I-

10

0

N T c 0

k a V " E E L

-40 Pre-RT During 1 month 2 month 6 month Post Treatment

Fig. 3. none. Error bars represent standard errors.

Surfactant apoprotein levels: pneumonitis vs.

20 R A 7 16 0 G

P H

2 12

A a

: 4

S

R E

0

Increase in Procollagen Normal Procollagen Levels

T

n=8 n.8 n-4 n=6 n=4 n=5 1 month 2 month 4-6month

Time Post Treatment

Fig. 4. represent standard errors.

Procollagen 111 vs. radiographic score. Error bars

ated with radiographic changes at 2 months (R = -0.59, P < 0.05, n = 10, H,: R = 0, H,: R z 0).

The changes over pretreatment baseline in surfactant apoprotein levels in patients who developed clinical pneumonitis compared to those who did not are illustrated in Figure 3. Surfactant apoprotein levels in patients who developed pneumonitis rose only slightly to 6% (n = 5) over baseline during treatment. Patients without clinical pneumonitis had a rise in surfactant level to 28% (n = 11) over baseline, indicating that clinical pneumonitis and surfactant apoprotein levels were inversely come- lated.

The radiographic scores for patients with normal procollagen III (0.3-0.8 U/ml) during treatment were compared with the scores of patients who had elevated procollagen level (Fig. 4). Pa- tients with an increase in procollagen during treat-

Fig. 5. TGF-P levels in patients with and without pneumonitis. Error bars represent standard errors. Wk = weeks; mon = month@).

ment had a higher radiographic score at 4-6 months (time period for fibrosis) than patients with no increase, although this difference is not significant.

TGF-P Levels of TGF-P were determined on these patients. For patients with no pneumonitis, the mean pretreatment value of TGF-P was 24.44 ng/ml (n = 1 1 ) . This mean value was slightly higher at 26.53 (n = 5) n g / d for patients who developed pneumonitis and there was a large rise in TGF-P to a mean of 51.5 ng/ml at 6 months (n = 2) post- irradiation in this group, which was not seen in patients who did not develop pneumonitis (n = 3) (Fig. 5). The patient who developed fatal pneumonitis had a pretreatment value of 26.60 ng/ml. Of the 10 patients who developed radiological evidence of moderate to severe damage, the mean pretreatment values were 25.80 and 24.20 ng/ml for those with mild or no damage.

Comorbidity factors were also investigated. Patients with chronic obstructive pulmonary disease (COPD) prior to RT had a rise of 34% in surfactant apoprotein level during treatment and an average radiographic score of 1 1 .3 at 2 months posttreatment, whereas patients without COPD had a slight rise (7%) in surfactant and developed evidence of severe injury (mean radiographic score of 17.6). This is consistent with the above data. All patients were cigarette smokers, although 8 had quit a minimum of 6 months prior to treatment. There was no difference in radiation effects between those who quit and those who continued to smoke during RT. Total radiation doses varied, but lower total doses were administered using larger fractions. Six of the 10 patients with a moderate or severe

62

radiographic score had >5,400 cGy and 4 had <5,400 cGy.

McDonald et al.: Pdmonury Eflects of Radiation

DISCUSSION The use of biochemical markers to predict for damaging late radiation effects in lung would provide a valuable tool in patient care. The markers analyzed in this study were selected for the relative ease with which the appropriate samples could be obtained from patients and their ability to reflect pulmonary damage based on our preclinical experience with these in studies evaluating radiation pulmonary effects [ 1-14]. Increase in surfactant apoprotein levels during treatment correlated with a low likeli- hood of clinical and radiographic evidence of radiation-induced lung injury; conversely, if surfactant apoprotein levels remained stable compared to baseline, lung injury was apparent. The finding of an inverse correlation between surfactant apoprotein levels during treatment and the clinical radiographic changes during the pneumonitic phase of lung injury appears contrary to the findings in our animal model where surfactant increase predicted for lethal pulmonary effects [7]. Confounding factors in the clinical model, which may explain this, include the use of fractionated radiation vs. single dose in the animal model. We have shown in our animal model that surfactant release is dose depen- dent; therefore, in the clinical setting, where the radiation dose is small, subsequent fractions of radiation are given in the presence of released surfactant, which may itself offer a protective effect. Following completion of treatment, there was a de- crease in surfactant apoprotein levels that could be due to type I1 cell depletion. Support for the protective role of surfactant is found in the group where surfactant release was not obvious, but late effect lung damage was observed.

Another difference between the clinical setting as opposed to the animal model is partial vs. whole lung irradiation. Abscopal effects must be considered: radiation induces immediate release of cytokines within the radiation field, followed by later releases from outside the irradiated area [21,22]. This interplay of messages between both irradiated and unirradiated cells is intimately related to development of pneumonitis and fibrosis and the roles of various cytokines and growth factors are unknown at the present time.

A correlation exists for an elevation of procollagen 111 during treatment and evidence of fibrosis at 4-6 months posttreatment. This correlation with later evidence of pulmonary injury may be useful in predicting the development of late radiographic ev-

idence of more severe fibrosis in patients with high values during treatment.

Other researchers [ 13,141 have presented preliminary data showing that elevated TGF-p levels in the pretreatment stage may be predictive for late radiation effects. In our study the patients who developed pneumonitis did have increased mean TGF-p values prior to treatment, but this was not statistically different from either the healthy nor- mals or the patients who did not develop pneumonitis. Before the conclusion of a predictive effect can be made, larger numbers of patients need to be evaluated. Symptoms of acute radiation pneumonitis usually only become evident 1-3 months following completion of thoracic irradiation, but occa- sionally they can occur as late as 6 months after irradiation. In general, the early onset of symptoms implies a more serious and more protracted clinical course. In any event, it is usually too late to modify the radiation factors and current management of established toxicity is symptomatic. These biochemical markers may allow one to adjust radiation or possibly be used therapeutically in individuals predicted to be at higher risk of toxicity. The prom- ising results in this study will focus our future re- search efforts to utilize these and other markers for early detection and monitoring of patients undergoing pulmonary irradiation.

ACKNOWLEDGMENTS This work was supported by grants from the NCI CA11051-22, CA27791, and CA-91-04.

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Biochemical markers as predictors for pulmonary effects of radiation