Investigation On The Predictor Of Efficacy For EGFR-TKIs And Platinum-based Chemotherapy In Advanced Non-small Cell Lung Cancer And Their Drug Resistance Mechanisms

Objective:(1)To determine these biomarkers from the inflammatory parameters of complete blood counts for predicting prognosis in EGFR-mutant NSCLC patients treated with EGFR-TKIs.(2)Systematic analysis of non-small cell lung cancer related single nucleotide variation,Indel and other types of gene mutations from genomics level to explore mutations and their downstream targets related to the therapeutic efficacy and prognosis of EGFR-TKIs.(3)To investigate the value of kinesin KIF4 A in predicting the therapeutic efficacy of platinum therapeutics after EGFR-TKIs acquired resistance and its molecular mechanism.Methods:(1)We retrospectively investigated 127 cases of stage IIIB/IV NSCLC patients with activating EGFR mutations treated with EGFR-TKIs.The receiver operating characteristic(ROC)curves were utilized to determine the optimal cut-off for the immunologic markers in complete blood counts as prognostic factors for EGFR-TKIs therapy.Univariate and multivariate analysis were used to identify prognostic factors for progression-free survival(PFS)and overall survival(OS)of EGFR-mutant NSCLC patients treated with EGFR-TKIs.(2)The drug-resistant plasma samples from 14 NSCLC patients with EGFR mutations targeted by EGFR-TKIs were collected.Extraction of circulating tumor DNA(ctDNA)for next-generation targeted capture sequencing.The target genes were 143 tumor-associated genes,such as TP53,EGFR,KRAS,ERBB2,PIK3 CA and so on.Combined with bioinformatics methods to identify mutations and enriched signal pathways of tumor-driven genes associated with EGFR-TKIs resistance and tumor immunity.Comparison of gene mutation between two groups of advanced NSCLC patients with significantly different progression-free survival(PFS≤14 months;PFS>14 months).(3)The expression of kinesin KIF4 A in lung cancer tissues,which from EGFR-TKIs acquired resistance advanced NSCLC patients,and normal tissues was detected by immunohistochemistry.The relationship between the expression of KIF4 A and the efficacy and prognosis of platinum-based chemotherapy was analyzed.The expression of KIF4 A and LRP in A549 cells and A549/DDP cells was detected by RT-PCR and Western Blot.Exogenous KIF4 A was overexpressed in A549 cells by cell transfection technique or knockdown of endogenous KIF4 A expression in A549/DDP cells by RNAi technique.The effects of cisplatin with different concentration gradients on the growth and proliferation of two cells were detected by MTT assay.Immunoprecipitation was used to detect the interaction between intracellular protein KIF4 A and LRP.The different domains of KIF4 A and LRP were constructed.Identification of the domain of kinesin KIF4 A interacting with LRP protein by protein co-immunoprecipitation assay.The co-localization of KIF4 A and LRP in the cells was observed by immunofluorescence staining.The small interfering RNA(siRNA)was used to knock down the endogenous KIF4 A of A549/DDP cells,and the immunofluorescence staining was used to detect whether KIF4 A could regulate the cytoplasmic distribution of LRP,verified by RNAi rescue experiment at last.Results:(1)(1)According to the receiver operating characteristic curve and the Youden index,the optimal cut-off values of lymphocyte-to-monocyte ratio(LMR),neutrophil and lymphocyte ratio(NLR)were 3.37 and 2.90 respectively.WBC,HB,PLT,PLR,RDW,MPV and other indicators in the complete blood counts were not suitable as potential markers for prognostic analysis(P>0.05).(2)There were no significant difference between the age,smoking status,pathological type,clinical stage,ECOG score,EGFR-TKIs drug type and EGFR mutation type in LMR and NLR different groups.Neutrophil count decreased(P=0.002),lymphocyte count increased(P<0.001),monocyte count decreased(P<0.001)were significantly related with high LMR and low NLR levels.(3)ECOG score(P<0.001),LMR(P=0.007),NLR(P<0.001)were significantly associated with the PFS of patients and were risk factors for PFS.The lower the ECOG score,high LMR(>3.37)and low NLR(≤2.90)were significantly correlated with long-term PFS.ECOG score(HR 1.909,95% CI: 1.283-2.840,P=0.001),NLR(HR 1.049,95% CI: 1.009-1.090,P=0.015)were independent prognostic factors for PFS.(4)ECOG score(P=0.002),LMR(P=0.034),NLR(P<0.001)were significantly associated with the OS of patients and were risk factors for OS.The lower the ECOG score,high LMR(>3.37)and low NLR(≤2.90)were significantly correlated with long-term OS.ECOG score(1.976,95% CI: 1.209-3.232,P=0.007),NLR(HR 1.043,95% CI: 0.997-1.092,P=0.038)were independent prognostic factors for PFS.(2)(1)A total of 2830 SNVs and 925 Indels on 143 tumor-associated genes in the targeted capture list were detected in 14 samples.In the SNVs,there were mainly 720 intron regions and 1912 exon regions mutations.Exon mutation types include 473 synonymous mutations,1370 missense mutations.(2)Plasma ctDNA concentrations were 2.731±5.544ng/μl and 0.446±0.178ng/μl in short-term PFS group and long-term PFS,respectively,with significant difference between the two groups(P=0.040).The numbers of non-synonymous mutations in short-term PFS group were more than long-term PFS.(3)C>T is the mainly base substitution type in 14 cases of targeted capture sequencing samples.(4)KEGG pathway enrichment analysis revealed that 25 genes including MTOR,JAK1,AKT3,RAF1 and FGFR3 are somatic mutation genes closely related to EGFR-TKIs resistance.(5)Kaplan-Meier survival analysis showed that the median PFS of the CTNNB1,BRAF,CSF1 R and PIK3 CA wild-type groups was longer than that of the gene mutation group(P=0.040,P=0.024,P=0.005,P<0.001).(3)(1)The positive rates of KIF4 A protein expression in EGFR-TKIs acquired resistance non-small cell lung cancer tissues and normal lung tissues were 60.00% and 26.67%,respectively,with significant difference between the two groups(P=0.038).The PFS of platinum therapy,which after EGFR-TKIs acquired resistance in KIF4 A positive and negative patients were 6 months and 9 months,respectively,with a significant difference between the two groups(P=0.003).The OS of KIF4 A positive and negative patients were 16.5 months and 22 months respectively,with a significant difference between the two groups(P=0.007).(2)The mRNA and protein expressionn of KIF4 A and LRP in A549 cells was significantly higher than A549/DDP cells.(3)Afer overexpressing GFP-KIF4 A in A549,the cell were less sensitive to cisplatin.(4)Co-immunoprecipitation experiments showed that both KIF4A-C230 and KIF4A-C896 binding to LRP similar to KIF4A-FL.On the contrast,the N-terminal of KIF4A(N351 and N1018)can not binding to LRP.GFP-KIF4 A was immunoprecipitated by LRP-WT and LRP-N447,while the LRP-C446 failed to bind to GFP-KIF4 A.(5)KIF4A and LRP partially co-localized in the microtubules in the cytoplasm.In control siRNA-transfected cells,LRP was dispersed throughout the cytoplasm,whereas a large number of LRP aggregated in the perinuclear region in KIF4A-depleted cells.LRP is mainly concentrated in the perinuclear region in KIF4 A knockout A549 cells.LRP is normally distributed in the cytoplasm in cells expressing GFP-KIFA-FL but lacking endogenous KIF4 A.In contrast,LRP clustered in the perinuclear region in cells depleted KIF4 A but expressing truncated KIF4 A mutants N351,N1018,C896 or C230.Conclusion:(1)Inflammatory and immune indicators NLR and LMR were two of the important biomarkers,which reflect the therapeutic efficacy of EGFR-TKIs in patients with advanced EGFR-mutated NSCLC.high LMR and low NLR were good prognostic factors in EGFR-mutant NSCLC patients receiving EGFR-TKIs treatment.Low NLR is an independent prognostic biomarker for long-term PFS and OS.Moreover,the NLR measurement has better prognostic value than LMR.(2)(1)The ctDNA concentration,gene mutation number and mutation frequency of NSCLC patients were significantly correlated with the PFS of EGFR-TKIs therapy.(2)Mutations in CTNNB1,BRAF,CSF1 R,and PIK3 CA genes may indicate poor prognosis of EGFR-TKIs targeted therapy in NSCLC patients.(3)The positive rate of KIF4 A protein expression(1)in non-small cell lung cancer tissues was significantly higher than that in normal lung tissues.Patients with negative KIF4 A expression had a significantly longer PFS and OS than those with positive KIF4 A expression.Kinesin KIF4 A promotes drug resistance of(2)platinum-based drugs in non-small cell lung cancer by binding to LRP in the cytoplasm and transporting LRP.The tail domain of KIF4A(cargo binding domain)interact(3)s with the N-terminus of LRP.The distribution of LRP in the cytoplasm requires the full-length function of KIF4 A,and the kinestin KIF4 A is one of the transporters in the cytoplasm of LRP.