Exploratory Study Of Potential Predictive Biomarkers In Gastrointestinal Cancer Treated With Molecular Targeted Therapy And Immune Checkpoint Inhibitors

Background.Due to the breakthrough in immunotherapy and molecular targeted therapy,we will truly enter a new era in oncology from the era of traditional cytotoxic drugs.A number of molecular-targeted agents and immunotherapy agents have been approved for gastrointestinal cancer,which really prolong the life of patients and improve the quality of life.To make best use of these compounds has become a research hotspot.Dynamic monitoring of gene mutations and tumor microenvironment to identify potential prognostic and predictive biomarkers is an important means to achieve precise individual therapy.The development of liquid biopsy technology provides a research basis for the precision treatment.Extracellular vesicles(EVs)and peripheral immunological factors are both important components of liquid biopsy.However,there are few studies on EVs and immunological factors for gastrointestinal cancer,which is worth further study.This study aimed to explore the predictive role of extracellular vesicle DNA(EV DNA)and peripheral blood immunological factors in molecular targeted therapy and immunotherapy for patients with gastrointestinal tumors.Methods.This study is divided into two parts.The first part is to explore the significance of EV DNA in prognosis and targeted therapy resistance for patients with advanced colorectal cancer.Patients with metastatic colorectal cancer who received chemotherapy combined with molecular-targeted agents were included.Peripheral blood samples were collected during treatment,and serum EVs was extracted.The marker proteins of EVs were identified by western blotting,morphological characteristics of EVs were observed by electron microscopy,and the diameter distribution of EVs was analyzed by nano-particle tracking analysis(NTA).EV DNA was centrifuged for PCR amplification of targeted gene fragments and high-throughput sequencing of PCR products,aiming to investigate the predictive signifisance of EV DNA in prognosis and treatment resistance for colorectal cancer patients.Thus,it is verified that EVs,abundant in blood,can be used to predict tumor prognosis and monitor drug resistance.The second part explores the predictive value of peripheral immunological factors in patients with advanced gastrointestinal tumors treated with immune checkpoint inhibitors(ICIs).A total of two cohorts were included.In the discovery cohort of patients with gastrointestinal cancer treated with anti-PD-1 /PD-L1 antibody,peripheral blood samples were collected at baseline before treatment and 59 immune factors were detected to explore their association with clinical benefit.The validation cohort was used to validate candidate biomarkers in other gastrointestinal cancer patients.Results.In the first part,81 patients were enrolled,and EV DNA mutations were dynamically monitored,and it was found that patients with TP53 and KRAS comutation in EV DNA before treatment had worse survival than those with wildtype,KRAS mutation or TP53 mutation alone.In addition,patients with TP53/KRAS co-mutation after treatment had a worse prognosis than those without co-mutation(OS 7.98 m vs 16.6 m,P = 0.001),and patients with TP53/KRAS co-mutation after treatment were more likely to develop metastasis of new lesions(57.14% vs.13.73%,P = 0.0019).TP53/KRAS co-mutations is a factor of poor prognosis in advanced colorectal cancer was further varified in the validation cohort of 982 patients with advanced CRC in the MSKCC database.The effect of TP53/KRAS co-mutated EVs promoting metastasis was preliminarily observed in zebrafish in vivo.EV DNA detection can detect secondary resistant mutations of cetuximab,mainly KRAS.By dynamic monitoring of drug-resistant gene mutations in EV DNA,54% of patients could detect an increase in the abundance of drug-resistant gene mutations in advance and predict tumor progression.The second part included 82 patients in the discovery cohort and 30 patients in the validation cohort.By analyzing the expression of 59 peripheral blood immune factors in plasma samples from patients with digestive system tumors who received anti-PD-1 /PD-L1 monotherapy,it was found that patients with high expression of "immune checkpoint signature"(14 immune checkpoint molecules including PD1 and PD-L1,etc)and "T cell migration signature"(two chemokines CXCL10/IP10 and CCL5/RANTES)had higher ORR and longer PFS and OS than those with low expression in the exploration cohort.The ORR was significantly higher in validation cohort(66.7% vs.13.3%,P<0.01),and the high expression of "immune checkpoint characteristics" was associated with OS improvement.In the exploration and validation cohort,the expression of three immune checkpoint molecules,PD-L1,TIM3 and CD28,was up-regulated in the response group.The AUC values of the characteristic set of these three immune checkpoint molecules were 0.705 and 0.847,respectively.The high expression of "immune checkpoint signature" in plasma can be used as a novel biomarker to predict the efficacy and prognosis of anti-PD-1/PD-L1 immunomonotherapy in patients with gastrointestinal tumors.Conclusions.1.EVs,as a non-invasive liquid biopsy,has potential significance for the prognosis of colorectal cancer or drug-resistance monitoring;2.TP53/KRAS co-mutation is a poor prognostic factor for advanced colorectal cancer;3.The peripheral blood immunological factors can predict the efficacy of immunotherapy for gastrointestinal cancer.