Comparative Plasma Proteomics In Patients With Non-Small Cell Lung Cancer For Potential Biomarkers Of Radiation-Induced Lung Toxicity

Purpose:To investigate the plasma dynamics of 5 cytokines biologically relevant to inflammation/fibrosis, including interleukin-1beta (IL-1?), IL-6, IL-8, tumor necrosis factor alpha (TNF-?) and transforming growth factor betal (TGF-?1) to ascertain their prognostic value in predicting radiation induced lung toxicity (RILT) individually and to determine whether a model combing significant cytokines and physical dosimetric parameters will improve predictive accuracy for RILT.Methods and Materials:Fifty-eight patients, receiving definitive conventionally fractionated radiation therapy (RT) for inoperable stage?-?lung cancer were prospectively evaluated. The minimum follow-up was 18 months. Circulating cytokine levels were measured prior to and at weeks 2 and 4 during RT. The human cytokine Luminex xMAP plasma assay kit (microsphere-based sandwich immunoassay) was used to measure the concentrations of IL-1a, IL-6, IL-8, and TNF-?. TGF-?1 was measured by enzyme-linked immunosorbent assay (ELISA).The primary endpoint was symptomatic RILT defined as grade 2 and higher radiation pneumonitis or symptomatic pulmonary fibrosis.Results:Of the 58 eligible patients,10 patients (17.2%) developed RILT. Lower pre-treatment levels of IL-8 levels were significantly correlated with the development of RILT while radiation-induced elevations of TGF-?1 were marginally correlated with RILT. Significant correlations were not found for any of the remaining three cytokines or any clinical or dosimetric parameters. A model of incorporating physical parameter and IL-8 yielded an improved predictive ability as compared to models using any of the variable alone.Conclusions:Combining inflammatory cytokines with mean lung dose may provide a more accurate model for RILT prediction. Future study with a larger number of cases and events is needed to validate such findings. 2. A Longitudinal Plasma Proteomic Analysis of Patients Receiving Radiation for Non Small Cell Lung Cancer (NSCLC) to Identify Potential Biomarkers for Radiation-Induced Lung ToxicityPurpose:To study whether radiation induces differential changes of plasma proteomics in NSCLC patients with and without grade?2 RILT (RILT2).Materials/Methods:58 patients with NSCLC received radiotherapy (RT) were eligible.20 patients,6 with RILT2 with tumor stage matched to 14 without RILT2, were enrolled for this analysis. Platelet-poor plasma was obtained pre-RT, at 2,4,6 weeks during-RT, and 1,3 months post-RT. The plasma proteomes were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, RP-HPLC and LC-ESI-MS/MS. Variance components models were used to identify the differential protein expression between patients with and without RILT2.Results:Over 100 proteins were identified and quantified. After excluding proteins for which there were not at least two subjects with data for at least two time points,76 proteins remained for this preliminary analysis. C4b-binding protein alpha chain, Complement C3 and Vitronectin had significantly higher expression levels in patients with RILT2 compared to patients without RILT2, based on both the data sets of RT start to 3 months post-RT (P<0.01) and RT start to the end of RT (P<0.01). The expression ratios of patients with RILT2 vs without RILT2 were 1.60,1.36,1.46, and 1.66,1.34,1.46, for the above 3 proteins, respectively.Conclusions:This proteomic approach identified new plasma protein markers for future studies on RILT prediction. 3. Baseline Plasma Proteomic Analysis to Identify Potential Biomarkers that Predict Radiation-Induced Lung Toxicity in Patients Receiving Radiation for Non-Small Cell Lung CancerPurpose:To identify new plasma proteomic markers before radiotherapy start to predict grade?2 Radiation-Induced Lung Toxicity (RILT2)Methods:58 patients with NSCLC received radiotherapy (RT) were eligible.48 patients with minimum follow-up of 1 year,9 with RILT2 with tumor stage matched to 39 without RILT2, were enrolled for this analysis. Platelet-poor plasma was obtained within 2 weeks before radiotherapy. The plasma proteomes were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, RP-HPLC and LC-ESI-MS/MS. Z-scores and Bonferroni-adjusted p-values for the two-sample mean comparison were used to identify the differential protein expression between patients with and without RILT2.Results:Over 200 proteins were identified and quantified. After excluding proteins that were not detected in at least 40% of the 48 patient samples, C4b-binding protein alpha chain and Vitronectin had significantly higher (p<0.001 and p=0.02) expression levels in patients with RILT2 compared to patients without RILT2. These two proteins were validated by Western Blot. Ingenuity Pathway Analysis revealed that they both play important roles in the inflammatory response and are associated with the known pathways of radiation-induced lung damage. Conclusions:This proteomic approach demonstrates new plasma protein biomarkers before treatment for future studies on RILT2 prediction.