Exploratory Study Of Potential Predictive Biomarkers In Digestive System Tumors Treated With Immune Checkpoint Inhibitors

Background.The development of immune checkpoint inhibitors(ICIs)-based immunotherapy has created a new concept of anti-tumor therapy and has shown durable survival advantage in pan-cancer patients.Digestive system cancers,among the malignancies with a high incidence and mortality,also benefit a lot from ICIs.Unfortunately,only a fraction of patients can response to ICIs,while the majority of unselected patients cannot respond to them.Moreover,specific immune-related adverse events and hyperprogressive diseases limit those application.Thus,it is imperative to identify valid biomarkers in optimizing patient selection for maximizing the clinical benefits and minimizing the risk of toxicity.Both T cell receptors(TCR)and cytokines are important components in the antitumor immune cycle,which directly affect the response to ICIs.The correlation between these two markers and digestive system tumors treated with ICIs is worth researching.This study aimed to explore the predictive role of peripheral blood TCR repertoire and cytokines in digestive system cancer patients treated with ICIs.Methods.This study was comprised of two parts.The first part aimed to explore the predictive value of the circulating TCR repertoire for clinical outcomes in digestive system cancer patients who received ICIs.A cohort of patients with digestive system tumors such as esophageal cancer,gastric cancer,hepatocellular cancer and colorectal cancer was included.All patients received anti-programmed death receptor-1(PD-1)antibody monotherapy or anti-PD-1combination therapy.Peripheral blood samples prior to starting treatment and when the first clinical response evaluated according to the Response Evaluation Criteria in Solid Tumors were collected.Peripheral blood samples were also obtained from some healthy controls.Tissue samples before anti-PD-1 therapy were collected for PD-L1 test.The TCR repertoire was evaluated by sequencing of complementarity-determining region 3(CDR3)in the TRB gene.The Shannon index was used to measure the diversity of the TCR repertoire,and Morisita's overlap index was used to determine TCR repertoire similarities between preand post-treated samples.Multivariable logistic regression was used to determine whether variables were associated with disease control.Cox proportional hazard regression was used to assess survival outcomes comparing different predictors using the same variables in the logistic regression model.The second part included two cohorts treated with ICIs.Peripheral blood samples before treatment were collected for the test of 59 cytokines using a multiplex immunoassay kit.The discovery cohort aimed to explore the correlation between cytokines and clinical benefits.The prognostic value of peripheral cytokine levels was evaluated by the C-index of the receiver operating characteristic,integrated discrimination improvement,and net reclassification improvement indexes by using the R3.6.2.Subsequently,the candidate peripheral biomarkers were tested in the validation cohort.Moreover,we analyzed tumor infiltrating lymphocytes using multiplex immunohistochemistry in a subset of tissue samples from the combined cohort,aiming to determine the association between peripheral cytokines and local immune microenvironment.Results.The first part enrolled 137 patients,and the median age was 54.0 years old(range 21-70 years old).76 patients received anti-PD-1 monotherapy and 61 patients received anti-PD-1 combination therapy.Overall,patients showed significantly lower TCR diversity compared to healthy controls(P<0.001),while the distribution of TCR diversity was similar across tumor types.A negative correlation was found between TCR diversity and age(r=-0.306,P=0.0003).In the anti-PD-1 monotherapy cohort,patients with higher baseline TCR diversity exhibited a significantly higher disease control rate(DCR)(77.8% vs.47.2%;HR,3.92;95%CI,1.14-13.48;P=0.030)and a longer progression-free survival(PFS)(median: 6.47 months vs.2.77 months;HR,2.10;95% CI,1.16-3.79;P=0.014)and overall survival(OS)(median: NA vs.8.97 months;HR,3.53;95% CI,1.49-8.38;P=0.004)than those with lower diversity.Moreover,patients with a higher TCR repertoire similarity still showed a superior PFS and OS even in the cohort with lower baseline diversity.However,neither biomarker showed predictive value in the anti-PD-1 combination therapy cohort.Interestingly,the combination of TCR diversity and PD-L1 expression can facilitate patient stratification in the pooled cohort.In the second part,21 esophageal squamous cell cancer(ESCC)patients treated with anti-PD-1 antibody were enrolled in discovery cohort and 61 pancancer patients treated with anti-PD-1/PD-L1 antibody were enrolled in validation cohort.In the discovery cohort,multiplex immunoassay profiling data revealed that both soluble PD-L1 and C-C motif chemokine 5(CCL5/RANTES)showed rising trends across the three subgroups PD,SD and PR.Further investigation demonstrated the predictive and prognostic value of the pre-treatment levels of PD-L1,CCL5/RANTES,and their combinatorial signature the “2-cytokine signature”.As expected,the signature-high patients displayed a remarkably increased DCR and prolonged survival versus that of the lower subgroup.More importantly,the relevance between the three signatures and the efficiency of immunotherapy was confirmed in the pan-cancer validation cohort.Notably,the significant association between the “2-cytokine signature” and longer survival was validated.Further quantitative analyses of the tumor microenvironment composition suggested a link between the “2-cytokine signature” and NK cell infiltration.Conclusions.1.Both peripheral blood TCR repertoire and cytokine levels can be used as predictive biomarkers for the clinical outcome of digestive system tumors treated with ICIs monotherapy.2.TCR diversity is hopeful to develop as a supplementary indicator for PD-L1 testing.3.The combination of cytokines can receive a significant improvement in the accuracy of prediction of clinical benefits.4.Biomarkers for anti-PD-1 combination therapy require further study.