The Study Of Genetic Expression Characteristic Of The First Generation Of EGFR-TKI Responders And Prescription Regularity And Mechanism Of TCM Treatment

ObjectiveWe retrospectively included long-term responders who defined as a specific subset of EGFR-mutant NSCLC patients treated with the first generation EGFR-TKI and experienced≥3 years response to treatment.By analyzing their pre-therapeutic tissue specimens using Whole Exome Sequencing,we accessed their clinical and genetic characteristic and tried to explore the mechanism of EGFR-TKI long-term response.To summarize the prescription regularity of 1^st-gen EGFR-TKI long-term responder and to explore the effective ingredients and molecular mechanism of the core formula on treatment on EGFR-mutated NSCLC patients via data mining and network pharmacology.MethodsThe 1^stgen EGFR-TKI long-term responders who were admitted to the first affiliated hospital of Guangzhou University of Traditional Chinese Medicine,the first hospital affiliated with Guangzhou Medical University,the oncology hospital affiliated with Zhongshan University,and Pearl River Hospital of Southern Medical University were required to sequenced their pathological tissue specimens by Whole Exome Sequencing which included mutation type,base type,mutation signaling pathway,tumor mutation burden.Herbal prescriptions of 1^stgen EGFR-TKI long-term responders（LTR）in the first affiliated hospital of Guangzhou University of Traditional Chinese Medicine were collected to analyze by the ancient and modern medical case platform for descriptive analysis and correlation analysis,cluster analysis,complex network analysis.Using network pharmacology,the network of Formulas-ingredients-targets and the network of core protein was constructed while The intersection targets were inputted to perform GO and KEGG pathway enrichment analysis to explore the mechanism of Chinese traditional medicine on treatment of NSCLC and Chinese traditional medicine on delaying EGFR-TKI resistance.ResultsThere were 7 women and 4 men among 11 participants.The median PFS of them was52 months and the average onset age of them was 66 years old.6/11 patients harbored EGFR exon19 deletion while 5/11 patients harbored EGFR L858R mutation.10/11 patients were at stage IV disease while only 1/11 patients was at stage III disease.Overall gene mutation Rate ranked top 30 in 11 LTRs are EGFR,ESRRA,TP53,DSPP,MUC2,MUC4,NCOR2,PABPC1,UBXN11,CTNNB1,DDX27,ELF2,HDAC2,MAML2,NCOA3,NEFH,PPL,PHPN2,SPEN,SRRT,TDRD9,ZNF479,ZNF729,ZNF93,ZP4,ABCB10,ABCC6,ABT1,ACOT2,ACRC.In term of mutation signaling pathway,Epstein-Barr virus infection carcinogenic pathway was the unique pathway in patients whose PFS more that 5 years while cell cycle and PI3K/Akt pathway were the unique signaling pathway in those with PFS less than 5 years.Thyroid hormone signaling pathway and Notch signaling pathway were shared pathway in the two groups of patients.Missense was the main mutation type of 1^st-gen LTR accounting for 88.2%and there was no difference with the remaining 8mutation types.C>T base replacement was the main types of base replacement which accounted for 56.3%;followed by T>C base substitutions accounting for 18%;the remaining four types of base substitutions accounted for little difference.The median TMB of LTR was0.38/MB.High-frequency drugs for treating NSCLC patients included Codonopsis pilosula,Radix Ranunculi Ternati,Thunberg Fritillary Bulb,Gecko,Tuber Fleeceflower,Glycyrrhiza uralensis,Wild jujube seed,Codonopsis lanceolata,Semen Armeniacae Amarum,Panax notoginseng.The occurrence frequency of sweet,bitter and spicy drugs was higher in the total drugs.The drugs with tonifying lung and spleen and drugs with removing blood stasis and dispersing knot are the most commonly used medicine pair in the treatment of NSCLC patients.Based on network pharmacology,the core formulas of 1^st-gen EGFR-TKI LTR were made up of Codonopsis pilosula,Radix Ranunculi Ternati,Thunberg Fritillary Bulb,Gecko,Tuber Fleeceflower.A total of 3518 targets found in 35 active ingredients of the core formulas.1482 targets were found in 15 active ingredients of Codonopsis codonopsis,911 targets were found in 10active ingredients of Radix Ranunculi Ternati,702 targets were found in 8 active ingredients of Fritillaria thunbergii,401 targets were found in 4 active ingredients of Tuber Fleeceflower,32 targets were found in only one ingrident of Gecko.5,280 targets of NSCLC were retrieved from the Genecard database and the OMIM database.521 concurrent targets were screened out in the core formulas and NSCLC by R programming language.521 concurrent targets were inputted into String database to acquire protein-to-protein interaction network of which interaction degree ranked top 10 were IL6（33）、JUN（30）、IL1B（29）、MAPK1（27）、EGF（24）、MMP9（24）、CXCL8（24）、IL10（23）、ICAM1（23）、PTGS2（23）.GO bubble showed that the main biological function of the key targets involved cell signaling,protein phosphate,hormone secretion and regulation.KEGG pathway enrichment analysis showed enrichment pathways of the key targets including Fluid shear stress and atherosclerosis,Kaposi sarcoma-associated herpesvirus infection,AGE-RAGE signaling pathway in diabetic complications,IL-17signaling pathway,TNF signaling pathway,Hepatitis B,Chagas disease,Human cytomegalovirus infection,Prostate cancer,Hepatitis B.ConclusionGenetic test results indicated that EGFR gene mutation was predictable biomarker for EGFR-TKI efficacy.TP53 gene mutation was the only common mutation of lung cancer in1^st-gen EGFR-TKI LTRs.Lower mutation rate of TP53 means a better prognosis for EGFR-mutated NSCLC patients.Co-mutation rate is thought as a factor to predict survival benefit and efficacy of EGFR-TKI.HDAC2 co-mutation may be a newly founded mechanism of EGFR-TKI resistance.The mutation of HDAC2 leads to the inactivation of HDAC2 and the apoptosis of tumor cells,which maybe the potential mechanism of long-term survival benefit.TMB is negatively correlated with the efficacy of EGFR-TKI for which low TMB indicated a longer-term survival benefit of EGFR.The herbal prescription analysis of 1^st-gen EGFR-TKI LTRs suggested that drugs with complementary pulmonary and spleen and drugs with removing blood stasis and dispersing knot are the most commonly used medicine pair in the treatment of NSCLC patients.The core formulas of 1^st-gen EGFR-TKI LTRs were made up of Codonopsis pilosula,Radix Ranunculi Ternati,Thunberg Fritillary Bulb,Gecko,Tuber Fleeceflower.Result of network pharmacology analysis indicated that beta-sitosterol,Luteolin,Emodin-1-O-β-D-glucopyranoside,palmitic acid,Peimisine were the mainly active ingredients in the core formulas and the key target were IL6,JUN,IL1B,MAPK1,EGF,MMP9.The signal pathway involving Cell proliferation and apoptosis,inflammatory response,tumor angiogenesis which reflected the characteristics of anti-tumor by multi-ingredients and multi-targets interaction of herbal prescription.Our study revealed the mechanism of traditional Chinese medicine on the treatment of NSCLC patients and on delaying resistance of EGFR-TKI.