Circulating Plasma MicroRNAs As Potential Markers To Identify EGFR Mutation Status And To Monitor Epidermal Growth Factor Receptor-tyrosine Kinase Inhibitor Treatment In Patients With Advanced Non-small Cell Lung Cancer

Introduction Lung cancer continues to represent the first leading cause of cancer-related mortality worldwide.Despite the rapid advances,the prognosis of lung cancer remains poor.Non-small cell lung cancer(NSCLC)accounts for approximately 80.0–85.0% of all lung cancers.The majority of NSCLC patients are diagnosed at advanced stages with poor prognosis.Traditional chemotherapy,targeted therapy,radiation as well as immunotherapy are mainstays for the lung cancer patients.Recently,molecular targeted therapy guided as the “driver oncogenes” has attained great success.To date,the epidermal growth factor receptor(EGFR)gene remains the most important oncogenic driver of NSCLC.EGFR mutation accounts for 44% of all driver genes for NSCLC.EGFR mutations are detected in 30%-40% of NSCLC from Asian patients and 10%-20% from Caucasian patients.EGFR exon 19 deletions(EGFR19DEL)and EGFR exon 21 L858R(EGFR L858R)point mutations are the two major classic sensitive mutations which account for 85%-90% in all EGFR mutations.The recommended samples as the guidelines are tumor tissues and cells identifying as the “gold standard”.While in the clinical work,tumor biopsy specimens can sometimes be difficult to obtain from certain patients,which promoted the study of“liquid biopsy”,especially the focus on the circulating tumor DNA(ct DNA).The sensitivity of ct DNA has been reported to be relative low.We attempted to identify a panel of convenient and economical markers as supplements to ct DNA.Micro RNAs exert a wide range of biological functions,including early tumorigenesis and tumor progression.Accumulating evidence has proven that circulating micro RNA signatures in human plasma or serum may serve as disease fingerprints and novel molecular markers for NSCLC with high stability,sensitivity and specificity.However,associations between circulating micro RNAs in plasma and EGFR mutation status and their application for monitoring EGFR-TKI treatment and disease progression have not been systematically studied.The limited number of very recent studies reported are exploratory studies with small samples with quite different results.In this study,we retrospectively analyzed the detection of EGFR mutation for the newly diagnosed NSCLC patients in real clinic world.Then,we aimed to identify a panel of circulating plasma micro RNAs that can distinguish between NSCLC patients with EGFR-sensitive mutations and EGFR wild type(EGFR WT)patients and to explore the potential of this micro RNA panel to monitor tumor responses to EGFR-TKI treatment.Section Ⅰ: Retrospective analysis of EGFR testing data in newly diagnosed NSCLC patients in real-world from our single center.Objective: To summarize the actual situation of EGFR mutational testing in real-world from our single center.Methods: Clinicopathological characteristics of 627 primarily diagnosed NSCLC patients hospitalized in our center from April 2013 till April 2016 were retrospectively analyzed.We especially focused on the EGFR mutational testing and the progression free survival(PFS)for the patients harboring sensitive EGFR mutations.Results: Among the 627 NSCLC patients,the median age was 58 years old,with 56.0%(315/627)of male gender,48.6%(305/627)of no smoking history,82.6%(518/627)of stage IV and 84.4%(529/627)of adenocarcinoma.75.4%(473/627)patients had EGFR mutational testing with the median diagnosis time of 12 days(5-27 days).The majority of the samples for EGFR testing were tumor tissues with the percentage of 63.8%(302/473),and the latter were plasma sample of 23.0%(109/473)and pleural effusion samples of5.5%(26/473).36 patients had both the tumor tissue and plasma samples for EGFR examination.Plasma sample sent for EGFR mutational testing increase by the year with the percentage of 40.7% in April 2015 till April2016.35.7%(169/473)patients harbored sensitive EGFR mutations,with 55.6% EGFR 19 DEL and 40.8% EGFR L858 R mutations.As compared of EGFR MU(+)patients with the EGFR WT groups,it was found that the age,gender,smoking history and pathological type were with significant difference between the two groups.133 patients had the first-line EGFR-TKI therapies with the objective response rate(ORR)of 73.7%,median PFS of10.0 months(95% CI 8.75-11.25).PFS of EGFR-TKI for EGFR 19 DEL patients was significantly better than that of the EGFR L858 R patients(11.0m vs 8.0m,HR=0.57,p=0.011).Conclusions: We had relatively high EGFR mutational testing for the primarily diagnosed NSCLC patients in our center.Percentage of plasma sample sent for EGFR mutational testing increased by the year.NSCLC patients harboring EGFR 19 DEL mutational genotype had better PFS than those of EGFR L858 R.Section Ⅱ: Microarray analysis of candidate plasma microRNAs for EGFR-sensitive mutations.Objective: Preliminarily screening out the candidate plasma miRNAs with significantly dysregulated expressed in the EGFR mutational groups compared with the wild type groups.Methods: Plasma samples of 3 EGFR exon 19 deletion,3 EGFR p.L858 R mutation,and 3 EGFR WT patients and 4 healthy controls(HCs)were selected for Agilent micro RNA microarray analysis to detect differences in the expression levels of circulating plasma micro RNAs between the above cohorts.Raw data were extracted using Agilent’s Feature Extraction software and normalized using Gene Spring software.Unpaired t-test was applied for the differential miRNA expression analysis.Hierarchical clustering was performed for the classification of the differentially expressed miRNAs.Results: Compared to healthy controls,76 dysregulated micro RNAs were found in the plasma of all 9 NSCLC patients.6 significantly upregulated miRNAs and 4downregulated miRNAs were found in the EGFR MU(+)patients compared with the EGFR WT cohorts.14 upregulated miRNAs and 7 downregulated miRNAs were found in the EGFR 19 DEL patients compared with the EGFR WT cohorts.2 upregulated miRNAs and 3 downregulated miRNAs were found in the EGFR L858 R patients compared with the EGFR WT cohorts.22 differentially expressed miRNAs were found between the EGFR 19 DEL and EGFR L858 R patients.Conclusions: Mi RNAs with significantly different expression could be found among the NSCLC and HCs,which supplied the candidate miRNAs for the further validation study.Section Ⅲ: Identification of reference genes for quantitative real-time reverse-transcription polymerase chain reaction(q RT-PCR)testing for circulating miRNA analysis in plasma.Objective: To identify the suitable reference genes for q RT-PCR testing for candidate circulating miRNA analysis in plasma.Methods: Plasma samples of 8 HCs,8 patients with benign pulmonary diseases,8adenocarcinoma lung cancer(ACs),4 squamous cell lung cancer(SCC),4small cell lung cancer patients were collected.12 candidate reference genes were tested with q RT-PCR analysis.Stability for the candidate genes was firstly analyzed with the scatter plot and thereafter analyzed by statistical algorithms of GeNorm,Normfinder and Bestkeeper.Results: From the scatter plot,5SrRNA,18 SrRNA and RNU43 were found to be most stable.With the Bestkeeper analysis,5Sr RNA and 18 Sr RNA were found to be with the lowest CP value of 1.806 and 1.821 separately.With the Normfinder analysis,5Sr RNA and RNU43 were found to be with the lowest stability value of 0.303 and 0.631 separately.As to the Ge Norm,combine with the M value and Vn/n+1 paired variation analysis,5Sr RNA combined with RNU38 B as panel was recommended.Conclusions: The 5SrRNA was to be used as an endogenous control to normalize the raw quantification cycle(Ct)values in the latter study for the miRNA q RT-PCR analysis.Section Ⅳ: q RT-PCR validation of candidate plasma micro RNAs significantly differently expressed in EGFR MU(+)patients compared with EGFR WT patients.Objective: To discover and validate the candidate circulating plasma microRNAs significantly differently expressed in the EGFR MU(+)patients compared with the EGFR WT patients with the q RT-PCR analysis.Methods: In total,20 NSCLC patients(12 EGFR 19 DEL,8 EGFR L858R)and 8 HCs were selected for the 10 preliminary candidate miRNAs using q RT-PCR in a pre-experiment.Thereafter,the validation was performed in the enlarging samples of 153 patients with 64 EGFR 19 DEL,36 EGFR L858 R,53 EGFR WT.Unpaired t-test or one-way analysis of variance(ANOVA)was applied for the differential miRNA expression analysis.Chi-square test was used to analyze the qualitative data.Receiving Operating Characteristics Curve(ROC)and Logistic Regression analysis were performed to determine the sensitivity and specificity of the miRNAs as diagnostic markers.Results: miR-107,miR-122,miR-125a-5p and miR-195 were determined as the candidate miRNAs in the pre-experiment.Thereafter,miR-125a-5p was screened out for exhibiting no statistically significant differences between the cohorts.Mi R-107 yielded the sensitivity of 64.7%,specificity of 76.6%at cutoff of 0.097 and AUC of 0.72 in discriminating EGFR 19 DEL and EGFR WT,as well as sensitivity of 64.2%,specificity of 80.6% at cutoff of0.153 and AUC of 0.77 in discriminating EGFR L858 R and EGFR WT.Mi R-122 yielded the sensitivity of 73.6%,specificity of 63.9% at cutoff of0.124 and AUC of 0.75 in discriminating EGFR L858 R and EGFR WT.Mi R-195 yielded the sensitivity of 71.8%,specificity of 69.1% at cutoff of0.876 and AUC of 0.75 in discriminating EGFR 19 DEL and EGFR WT.Panel of miR-107 combined with miR-122 or miR-195 deserves better diagnostic value than each single marker.The multivariate analysis results revealed that miR-107 and smoking status were important diagnostic predictors of EGFR-sensitive mutations.Conclusions: miR-107,miR-122 and miR-195 had the potential to be biomarkers for discriminating the EGFR 19 DEL or L858 R mutational genotypes with the WT genotype.Section Ⅴ: Dynamic changes of miRNAs in monitoring EGFR-TKI treatment.Objective: To elucidate the fluctuations in circulating microRNAs with the course of EGFR-TKI treatment in the EGFR 19 DEL NSCLC patients.Methods: Plasma of 36 NSCLC patients harboring EGFR 19 DEL was collected before and during EGFR-TKI treatment(12 patients also had the plasma collected at the time of disease progressed).Expression of circulating miR-107 and miR-195 were tested with the q RT-PCR.Line chart was performed preliminarily to analyze the dynamic changes of miRNAs.Unpaired t test or one-way ANOVA was applied for the differential miRNA expression analysis.Results: miR-107 as well as miR-195 in patients with a complete or partial response(CR/PR)to EGFR-TKI appeared to decrease more sharply in comparison to that of patients with stable disease(SD)with statistical significance.No significant difference was detected between patients with a PFS>8.0 months compared with those of PFS≤8.0 months for EGFR-TKI treatment.No matter the response of the tumor,miR-107 and miR-195 expression levels at the time of PD exhibit a tendency to return to the levels before EGFR-TKI treatment in the majority of patients.Conclusions: Circulating plasma miRNAs exhibited dynamic changes with the course of EGFR-TKI treatment which rendered them the potential in monitoring EGFR-TKIs.