Construction Of A Neoadjuvant Chemoradiotherapy Responsiveness Prediction Model For Locally Advanced Rectal Cancer And Study Of The Mechanisms Associated With Its Resistance To Radiotherapy

Objective:The purpose of this study was to analyze the mutation characteristics of locally advanced rectal cancer(LARC)patients with different sensitivities to neoadjuvant chemoradiotherapy(nCRT)using whole exome sequencing(WES)and investigate the molecular mechanisms influencing radiotherapy resistance through basic experiments and transcriptome sequencing.Methods:In this retrospective study,a total of 54 LARC patients were included.Tumor tissue biopsy specimens and fresh peripheral blood samples were collected before nCRT.DNA was extracted from both tumor tissues and peripheral blood for WES analysis.Based on the tumor regression grade(TRG),patients were divided into responsive and resistant groups.Bioinformatics methods were employed to analyze genomic mutations,gene site mutations,copy number variations(CNV),tumor mutation burden(TMB),chromosomal ploidy,and microsatellite instability(MSI)between the responsive and resistant groups.KRASG12V and KRASA146T mutations,identified from the gene mutation differences between the two groups,were selected to establish overexpression cell lines.The mechanisms underlying radiotherapy resistance were explored through RT-qPCR,western blot,and transcriptome sequencing.Results:WES analysis revealed no significant differences in genomic mutations,CNV,TMB,chromosome ploidy,and MSI between the responsive and resistant groups.However,specific gene mutations,such as KRASG12V and KRASA146T,were observed in the resistant group but not in the responsive group.After radiation treatment of KRASG12V,KRASA146T,and KRAS wild-type(KRASWT)cells,Transcriptome sequencing of the irradiated KRASG12V,KRASA146T,and KRASWT cells revealed significant down-regulation of some pathways,such as interferon signaling,DNA damage and so on.Western blot showed significantly decreased expression levels of γ-H2AX and significantly increased expression levels of Ku80 in KRASG12V and KRASA146T mutated cells compared to KRASWT cells.Conclusion:This study indicates that KRASG12V and KRASA146T mutations are potential genetic mutation sites associated with resistance to nCRT in LARC.These mutations may affect DNA damage repair by regulating non-homologous end joining,thereby mitigating radiation-induced DNA damage.Additionally,KRASG12V and KRASA146T mutations may downregulate immune-related pathways,such as interferon signaling,further enhancing radiotherapy resistance.Objective:Many biomarkers have predictive value for therapeutic effect and prognosis in tumor patients.However,the role of neutrophil-lymphocyte ratio(NLR)in locally advanced rectal cancer(LARC)patients treated with neoadjuvant chemoradiotherapy(nCRT)has not been studied.This study aimed to explore the predictive value of the NLR before nCRT(pre-NLR)for therapeutic effect and prognosis of LARC patients treated with nCRT.Methods:A total of 326 LARC patients undergoing nCRT with total mesorectal excision(TME)were enrolled.Preoperative clinical features and postoperative pathological characteristics were collected.Logistic regression analysis was conducted to determine the correlation between pre-NLR and tumor regression grade(TRG).Cox regression analysis was performed,and a Cox-based nomogram was developed to predict overall survival(OS).We also assessed the predictive performance of the nomogram with calibration curve,receiver operating characteristic(ROC)curve,and decision curve analysis(DCA).Results:Among 326 patients,the median pre-NLR was 2.16(interquartile range,IQR:1.69-2.71).In the logistic regression analysis,only the pre-NLR was statistically significant(odds ratio,OR=0.62,95%CI:0.47-0.80,P<0.001)for TRG in LARC patients treated with nCRT.In the Cox multivariate regression analysis,we found that pre-NLR,nCRT with surgery interval,ypTNM stage,TRG,vascular invasion,adjuvant chemotherapy,and carbohydrate antigen 19-9 before nCRT were predictors of OS.The predictive nomogram was developed to predict 3-year and 5-year OS with an area under the ROC curve(AUC)of 0.8343 and 0.8625,respectively.Good statistical performance on internal validation was shown by calibration curve and DCA.Conclusion:This study demonstrated that a lower pre-NLR was associated with a greater rate of TRG in LARC patients treated with nCRT.Furthermore,pre-NLR was correlated with OS in LARC patients treated with nCRT,Meanwhile,we successfully constructed a nomogram based on available clinicopathologic factors for prediction of 3-year and 5-year OS in LARC patients treated with nCRT.