The Prognostic Significance Of Neutrophil Extracellular Traps And T Cell Receptor Diversity In Patients With Cancer

·Part ? The significance of neutrophil extracellular traps in tumor progression and prognosis evaluation·Chapter ? A high-risk luminal A dominant breast cancer subtype with high G-CSF secretion and neutrophil infiltrationBackground:The process that a transformed cell developed to an overt tumor depended on the cross-talk between the parenchymal cells and its immune microenvironment.Breast cancer is considered a collection of heterogeneous disease with different treatment schemes and clinical outcomes,rather than a single disease affecting the same organ.Although advances in molecular subtyping have been achieved in recent years,most traditional classification methods target individual genes independently of one another and primarily concentrated on proliferative markers.The contribution of other biological processes and infiltrating immune patterns have been ignored in breast cancer subtype classification.Aim:The study aimed to identify a new prognosis-related molecular classification of breast cancer based on the gene sets of tumor associated biological processes and immune cell subtypes.And we tried to explore the significance of the neutrophil extracellular traps(NETs)in the new subtype.Materials and methods:The study performed gene set variant analysis(GSVA)based on biological processes genesets(Hallmark genesets)to explore prognosis-related molecular classification using 985 breast invasive ductal/lobular carcinoma RNAseq samples in the TCGA database.And the infiltrating immune cells pattern of the new molecular subtype was described by 26 immune cell subtypes using GSVA algorithm.To verify the significance of NETs in breast cancer progression,we modified a "cancer cell migration and NETs detection system".Results:The samples were divided into three clusters and were categorized as "Hallmark-tsne" subtypes.There is a C3 subtype with poor prognosis in Hallmark-tsne subtypes.C3 is a high-risk luminal A dominant breast cancer subtype which displayed increased mobility,cancer stem cell-like phenotypes,a higher expression of hormone/luminal-related genes and immune dysfunction.With regard to immune dysfunction,the mobility-increased C3 subtype exhibited high granulocyte colony stimulating factor(G-CSF)expression accompanied with neutrophil aggregation.G-CSF secretion cancer cells stimulate neutrophils to form NETs,which increase the migration ability of cancer cells.This finding shed light on one potential explanation for why C3 subtype correlates with poor prognosis.Conclusions:The connection between biological processes and immune pattern of heterogenous breast cancer must be understood to improve clinical treatments.And the pro-tumor immune microenvironment induced by NETs is favorable to tumor progression.·Chapter ? Cooperation between neutrophil extracellular traps and coagulation systems conspire the survival of circulating tumor cells in renal cell carcinoma patientsBackground:Most cancer patients die from the metastasis or recurrence after the overt metastasis spreaded.However,the mechanisms underlying the intravascular survival of circulating tumor cells(CTCs)have not been completely understood.Elevated plasma C-reactive protein(CRP)concentration and thrombocytosis are correlated and both predict a poor prognosis in many cancer types.Neutrophils are important components of pro-tumor associated imflammatory cells,and NETs,a specific activiation status of neutrophils,can trigger the coagulation system.Although the morbidity of renal cancer ranked 16th among 35 common cancers in 2018,the 5-year survival rate of patients with distantly metastatic renal cell carcinoma(RCC)is only 8%-12%.Additionaly,RCC is strongly asccociated with inflammation and frequently accompanied with deep vein tumor thrombus.Aim:To explore how the inflammation and hypercoagulability bridged by NETs,and whether NETs shelter CTCs survival.Materials and methods:There are 106 treatment-naive patients with renal masses(9 benign renal masses and 97 RCC)enrolled in the study.Viable CTC counts were measured by oHS V1-hTERT-GFP method;treatment-naive clinical characteristics and hematological parameters were collected from patient medical records.Twenty-one peripheral blood leukocytes samples were performed next generation RNA sequencing(RNAseq),and immunophenoscore were calculated using gene set enrichment associated algorithms.RNAseq based NETs score of tumors in clear cell renal cell carcinoma(ccRCC)patients from TCGA database was used to evaluate the prognosis.Results:Patients with elevated CRP and fibrinogen(FIB)concentration had higher CTC counts than patients with normal levels of these indexes.Each pair of the three variables(CTC count,CRP and FIB concentration)was positively correlated.Combined with RNAseq data of peripheral blood,leukocytes in RCC patients with more viable CTCs were in a state similar to the spontaneous activation state of low density neutrophils(LDNs)or G-CSF primed leukocytes,namely,a NETs formation state.Additionally,RNAseq based-NETs score of peripheral blood was positively correlated with the plasma FIB concentration.and both of these values were increased in patients with elevated CRP.Moreover,immunofluorescence staining showed that NET frameworks were decorated with NET-derived tissue factor(TF),and entangled with viable renal cancer cells.Finally,NETs score of tumors are independent prognostic factors for ccRCC patients in TCGA database.Conclusions:NETs generated from peripheral neutrophils triggered coagulation cascade by their attached TF.Both the DNA scaffold sprouted and fibrin network activated by NETs anchor and shield CTCs from attack.·Chapter ? Neutrophil extracellular traps associated gene expression signature identifies prognosis in pan-cancerBackground:Tumors are wounds that do not heal,inducing an uncontrolled systemic inflammation in host.Neutrophils in the tumor microenviroment tried but failed to heal the wound,especially the outburst of NETs stimulate the inflammation and coagulation cascade which built a comfortable environment for cancer progression.Infiltrating NETs participate death induced by pathology processes associated with tumor in multifacets,involving the tumor invasion and metastasis,pro-angiogenesis in tumor bed,cancer-asccociated thrombosis formation,organ dysfunction and so on.Although the parenchyma cells in cancer originated from different lineage and exhibited heterogeneity due to randomly accumulated mutations,a mesenchymal microenvironment with immune suppressive status is essential for the development of cancer.Aim:Based on the theory that pro-tumor microenvironment is universal necessary for cancer progression,the study aimed to train a simple pan-cancer prognosis prediction model with NETs associated genes.Materials and methods:Pan-cancer samples,8739 primary tumor tissue RNA sequencing data of 32 solid cancer types from TCGA database,were sampled by computer-generated random numbers,so that 70%of them was assigned to the training datasets.The remaining 30%samples were imported as testing datasets.Additionally,the glioma samples from CGGA database,brest cancer samples from METABRIC database and lung cancers from GEO database were also enrolled as testing datasets.The optimized NETs associated gene sets were imported as feature values to train the prognosis models with Cox proportion hazard regression and random forest for survival algorithme.To elucidate the biological mechanism that NETs-associated prognosis models,the relationship among NETs,angiogenesis and cell proliferation was quantified with the z-score calculated by their corresponding gene signatures.Results:NETs score calculated with the Cox models and random forest models,which named as "NETs score(Cox)" and "NETs score(RF)”respectively.For the pan-cancer in traning and testing datasets,pantients in group with higher NETs score(Cox)or NETs score(RF)were suffered worse disease specific survival(DSS),overall survival(OS)and progression free survival(PFI)than that in group with lower scores.Univariate Cox hazard analysis indicates that NETs score(RF)is a significant risk factor for prognosis in the 30 kinds of solid tuomors(except DLBC and KICH)in TCGA pan-cancer training datasets.Time dependent ROC curves and DCA curves are unanimous suggesting NETs score(RF)is more precisely to predict pan-cancer prognosis,but NETs score(Cox)is more stable in predicting long-term prognosis.Additionally,each pair of the three variables,the z-score of NETs,angiogenesis and cell cycle,showed a positive correlation.That's the evidence of data,why NETs-associated model is feasible to predict prognosis,from that NETs can promote cancer progression by promoting angiogenesis and cell proliferation.Conclusions:NETs scores calculated based on the Cox model or random forest for survival are powerful to predict pancancer prognosis·Part ? Characteristics,dynamic changes and prognositic significance of peripheral T cell receptor repertoire profiling in patients with different tumor burdenBackground:Cancer is a complex disease with immunogenic phenotype.Renal cell carcinoma(RCC)is considered a strongly immunogenic cancer due to spontaneous or cytokine-induced tumour regression.The immune system co-evolves with tumour cells,but the two are mutually dysregulated.Although tumour induced T cells were mobilized with the stimulation from immunogenic burden released by tumour,the cytotoxicity of those T cells was blocked by tumour-derived PDL1.T cells play an important role in adaptive immnity and cancer survillance,while their dysfunction is involved in carcinogenesis and tumour progression.To some extent,T cell receptor diversity is an important index to mirror the function of T cells.Aim:This study aimed to explore how the baseline and dynamic clear cell RCC(ccRCC)tumour burden affects the T cell repertoire and whether the baseline T cell receptor ? chain(TCRB)diversity predicts prognosis.Materials and methods:Fifty patients with renal massed were enrolled in this study.Among them,38 treatment-naive ccRCC and 2 benign patients with different clinical stages(tumor burdens)were collected the paired pre-operative and post-operative peripheral blood samples,and the samples were performed TCR repertoire sequencing(TCR-seq).The group was used to analyse the effect of surgery(the alteration of the tumour burden)on the immune status,hereafter referred to as "surgery-associated cohort".And 28 patients from the surgery-associated cohort were performed the bulk peripheral leukocytes transcriptome sequencing.Another group,including 18 advanced ccRCC patients,named "prognosis-asscociated metastastic cohort",were used to predict prognosis with their baseline TCR-seq data.To validate the results,peripheral blood samples from a stage ? ccRCC with inferior vena cava tumour embolus was perform flow cytometry to analyse the changes in exhausted T cells;and an advanced ccRCC patient was subjected to single RNA and TCR sequencing(10x Genomics).Results:Integrated the data of TCR-seq and transcriptome sequencing from surgery-associated cohort,it suggested that patients with low TCRB diversity are in severe inflammatory response and in relatively hypercoagulable state;while high TCRB diversity indicates a homeostatically balanced immune system armed with plentiful naive T cells,namely the immune surveillance relatively functioned.The pre-operative TCRB diversity was significantly higher in stage ?(low tumour burden)ccRCC patients than in stage ?(high tumour burden)patients.For the stage ? ccRCC patients,a higher baseline TCRB diversity indicates a longer overall survival.After the major tumour burden of advanced patients removed by cytoreductive nephrectomy(CN),the TCRB diversity was restored,the proportion of exhausted T cell decreased and naive T cells were mobilized.Additionally,the counts of emerging TCRB clonotypes with low concentration increased and the concentration of overwhelming persistent TCRB clonotypes shrinked after the immunogenic burden reduced by CN in advanced patients.Conclusions:The circulating TCRB repertoire could reflect the immune status and predict prognosis.And to some extent,CN reduces the burden of the immune system in advanced patients,which might provide a good opportunity for immunotherapy.