Study Of Genetic And Immunological Markers For Radiotherapeutic Eiffcacy And Toxicities In Lung Cancer

ObjectivesThoracic radiation therapy (TRT) is one of the most effective modalities innon-small cell lung cancer (NSCLC) management. However, the prognosis andradiation-induced lung injury (RILI) vary from patients to patients after TRT. Itindicates that one-size can not fit all in TRT. So, Personalized TRT based ondifferent subtypes of NSCLC has been proposed. Furthermore, biomarker researchhas been considered as the most important way for personalized TRT. This studytested the single nucleotide polymorphisms (SNPs) in lung cancer-related genes andthe levels of autoantibodies against tumor-associated antigens (TAAs)/immunologicregulated molecules (IRMs), and explored any associations between thesebiomarkers and overall survival (OS) and/or radiation pneumonitis (RP) in NSCLCpatients treated with definitive TRT. The aim of this study was to identify usefulbiomarkers for personalized TRT.Methods1. Genetic study (done at the University of Michigan, USA)This study included newly pathologically diagnosed and clinical stage I-IIINSCLC patients treated with definitive TRT. Bioinformatic methods and moleculargenetic techniques were used to test some SNPs in the TGFβ1gene and11DNArepair genes (ATM、ERCC1、ERCC2、ERCC5、MGMT、NBN、TP53、XRCC1、XRCC2、XRCC4、XPC). A total of30SNPs from these12genes were selected asgenetic markers. Matrix-assisted laser desorption/ionization time-of-flight massspectrometer (MALDI-TOF) was utilized to genotypes these SNPs in NSCLCpatients. Kaplan-Meire method, Cox proportional hazards model, receiver operating characteristic (ROC) curve analysis and genetic statistical software package(SPSS19.0and STREE TREE) were used to analyze the association between SNPsand OS/RP.2. Immunologic study (done at Jilin Univerisity)This study included newly pathologically diagnosed and clinical stage I-IIINSCLC patients treated with definitive TRT. A total of7IRMs and TAAs wereselected as antigens in this study, including CD25，FOXP3，TGF-β1，p16，P53，OCT4II and OCT4III. The bioinformatics database and software were used toanalyze their structures and characters. Based on in silico mapping of MHCrestricted epitopes derived from these proteins, linear antigen fragments were thendesigned; the linear peptide antigens were synthezied with a chemical method and anin-house ELISA test was developed to detect circulating autoantibodies in NSCLCpatients. Kaplan-Meire method, Cox proportional hazards model, logistic regressionmodel, ROC curve analysis and genetic statistical software package (SPSS19.0andSTREE TREE) were used to analyze the association between circulatingautoantibodies to TAAs/IRMs and OS/RP.Results1. Genetic analysisOne hundred thirty-four patients who were enrolled in prospective trials withsurvival and lung toxicity data recorded prospectively were evaluated.8patientswere excluded with palliative dose (<60Gy).96patients (76%) underwentconcurrent chemo radiation therapy with the platinum-based regimen. The medianfollow-up time was45months, and the minimum follow-up time was12months.(1) Association between SNPs and OS of NSCLCIn the univariate analysis of clinical characteristics, there were statisticallysignificant associations between OS and sex, tumor volume, KPS or TRT dose. Indetail, patients of female, tumor volume≤118cc, KPS≥80and TRT dose≥70Gywould survive longer. Multivariate Cox analysis in30SNPs showed that5SNPs(ERCC2:rs238406, ERCC1:rs11615, ERCC1:rs3212948, XRCC4:rs9293329,XRCC4:rs2075685) were significantly associated with OS of NSCLC. In detail, patients with genotype of ERCC2:rs238406GT/TT、 ERCC1:rs11615TT、ERCC1:rs3212948CC、XRCC4:rs9293329GA/AA and XRCC4:rs2075685TTwould survive longer. Furthermore, combined analysis of unfavorable genotypes(UFGS) from these5SNPs showed significantly cumulative effects on OS, i.e., thedeath hazard was increased with the numbers of UFGS increasing (0-5). TRT dosesubgroup analysis showed that the effect of SNPs tested on OS was observed only inthe TRT dose <70Gy group (P=0.015) rather than in the TRT dose≥70Gygroup (P=0.112). Interactive analysis by STREE TREE showed that there wasinteractive effect between TRT dose and ERCC2:rs238406or ERCC1:rs11615onOS. ERCC2:rs238406and ERCC1:rs11615could serve as genetic markers for TRTdose <70Gy group and TRT dose≥70Gy group, respectively. ROC analysisshowed that the area under ROC curve (AUC) was less than0.7when clinical factorswere used only. However, the AUC would be more than0.8when clinical factorsand5SNPs were combined together.(2) Association between SNPs and radiation pneumonitisIn the univariate analysis of clinical characteristics, there were statisticallysignificant associations between RP and mean lung dose (MLD) and/or the lungvolume receiving more than20Gy (V20). In detail, patients of MLD≤16.3Gy orV20≤26%would be less likely to develop RP after TRT. Multivariate Cox analysisshowed that2out of30SNPs, XRCC4:rs1478486and XRCC4:rs2075685weresignificantly associated with RP. In detail, patients with genotypes ofXRCC4:rs1478486CC or XRCC4:rs2075685could have higher risk of RP after TRT.Furthermore, combined analysis of unfavorable genotypes (UFGS) from these2SNPs showed a significantly cumulative effect on RP, i.e. an increase in the RPhazard with the numbers of UFGS increasing (0-2). Interactive analysis showed thatthere was an interactive effect between lung dosimetric metrics (MLD/V20) andXRCC4:rs1478486CC or XRCC4:rs2075685on RP. ROC analysis showed that theAUC was0.66when MLD/V20was used only. However, the AUC was more than0.76when MLD/V20and2SNPs were combined together. 2. Immunologic analysisEighty-eight patients who were enrolled in prospective trials with survival andlung toxicity data recorded prospectively were evaluated,10of whom were excludeddue to palliative dose (<60Gy), and61(78%) underwent concurrent chemoradiation therapy with the platinum-based regimen. The median follow-up time was10.3months, and the minimum follow-up time was6months.(1) Association between circulating antibodies to TAAs/IRMs and OS of NSCLCIn the univariate analysis of clinical characteristics, there were statisticallysignificant associations between OS and age or chemotherapy. In detail, patientsaged≤60, who received systemic chemotherapy, could survive longer. MultivariateCox analysis showed that none of circulating autoantibodies to7TAAs/IRMs wassignificantly associated with OS of NSCLC (P>0.05)(2) Association between TAAs/IRMs and radiation pneumonitisIn the univariate analysis of clinical characteristics, there was no clinicalvariable significantly associated with RP. Multivariate logistic analysis of antibodiesto7TAAs/IRMs showed that anti-CD25IgG and anti-OCT4II IgG weresignificantly associated with RP. In detail, patients with a low level of anti-CD25IgG or a high level of anti-OCT4II IgG could have a higher risk of RP after TRT.ROC analysis showed that the AUC was0.59when MLD/V20was used only.However, the AUC would be0.77when MLD/V20and2TAAs/IRMs werecombined together.Conclusion(1) Genetic variants in DNA repair genes are significantly associated with survivalof NSCLC patients treated with definitive TRT. Some SNPs from the DNA repairgenes can serve as biomarkers in predicting survival of NSCLC.(2) Higher dose TRT (≥70Gy) may improve survival in NSCLC patients withunfavorable genotypes of DNA repair genes. However, NSCLC patients carryingfavorable genotypes of DNA repair genes may not benifit from high dose TRT.(3) Genetic variants in DNA repair genes are significantly associated with radiationpneumonitis after TRT. Some SNPs from the DNA repair genes can serve asbiomarkers for prediction of radiation pneumonitis. (4) Circulating autoantibodies against CD25and OCT4II are significantlyassociated with radiation pneumonitis after TRT and can serve as biomarkers forprediction of radiation pneumonitis.(5) A novel genetic and immunological method can be set up based on present workto screen biomarkers for cancer.