| Objectives:1.85%NSCLC patients are at their advanced stages when diagnosed,and their OS is only 8-10 months after standard chemotherapy.Recently years have witnessed the revolution of NSCLC-treatment thanks to the discovery of driver mutations especially EGFR and the successful application of corresponding targeted drugs.The basis of realizing accurate therapy of lung cancer is the detection of molecular target,while it’s challenging to detect the mutation status of small tissue and cfDNA,a molecular detecting method of high sensitivity and specificity is seriously needed in clinical practice.2.During the process of EGFR-TKIs treatment,acquired resistance would happen in at least 60%patients due to the emergence of T790M mutation,so it is of great importance for treatment assessment and prognostic prediction to dynamically monitoring the EGFR mutation statues in order to find the resistant mutation and adjusting the regime as soon as possible.Meanwhile,the detection of EGFR T790M mutation has become a problem under the circumstance that the tumor tissue is unavailable after the resistance to first-generation EGFR-TKIs.Would the third-generation EGFR-TKIs a right choice?Therefore,the dynamic monitoring of cfDNA from peripheral blood requires a molecular detection method of high sensitivity and specificity.3.Pre-clinical trial showed that BRAF inhibitor Dabrafenib achieved good efficacy in treating adenocarcinoma lung patients and some other clinical trials have being processing,nevertheless,the mutation condition and the correlation between BRAF mutation and patients’ clinicopathological features are not well understood.Meanwhile,BRAF mutation detection in cfDNA of NSCLC is rarely reported thus it is of great significance to establish a stable,effective and sensitive detection platform,which would be of sound value to the identification of BRAF incidence,the correlation between BRAF mutation and patients’clinicopathological data and the subsequent usage of BRAF inhibitors.Methods:1.Paraffin-embedded tissue and clinicopathological features of 144 treatment-naive adenocarcinoma patients were collected from the Affiliated Drum Tower Hospital of Medical School of Nanjing University from November 2010 to November 2015.CastPCR was applied to detect EGFR mutations(exon 19,del2235-2249、de12236-2250);exon 20 T790M;exon21(L858)in the FFPE tissues,in order to explore the feasibility of applying CastPCR to clinical practice,the mutation rate of EGFR and the correlation between EGFR mutation and patients clinicopathological features were also studied.2.We utilized the same tissues described above and detected the BRAF mutation(exonll G469A,exonl5 D594G,V600E)rate using CastPCR and studied the correlation between BRAF mutation and patients’ clinicopathological features.3.103 matched peripheral blood samples were collected before patients’ treatment,and CastPCR was used to detect the EGFR and BRAF mutation rate in the plasma cfDNA,and the consistence calculated.Results:In the FFPE tissue of treatment-naive 144 adenocarcinoma lung patients,51.4%(74/144)cases harbored EGFR mutations,those carrying sensitizing mutations(exonl9 and/or exon21 L858R)accounted for 40.3%(58/144),and those carrying T790M accounted for 14.6%(21/144).Besides,6.9%(10/144)harbored double mutations,they were 3 19Del + T790M cases,5 19Del + L858R cases and 2 T790M+L858R cases.There are significant differences of the EGFR mutation rates between different genders and smoking status(P<0.05),while no differences in EGFR mutation rates were found between different ages,metastatic conditions,differentiation,clinical stages,tumor sizes and regional lymph metastatic(P≥0.05).2.In the FFPE tissue of 144 adenocarcinoma lung patients,8.3%(12/144)cases were found carrying BRAF mutations,16.7%(2/12)carried V600E,83.3%(10/12)carried non-V600E mutations.75.0%(9/12)cases harbored D594G and 8.3%(1/12)carried G469A.No differences in BRAF mutation rates were found between different genders,smoking status,ages,metastatic conditions,differentiation,clinical stages,tumor sizes and regional lymph metastatic(P≥0.05).3.The detection of EGFR in plasma cfDNA:sensitivity 56.4%(31/55),specificity 94.2%(49/52),concordance 74.8%(80/107).Specifically,for sensitizing mutations,sensitivity 57.5%(23/40),specificity 95.5%(64/67),concordance 81.3%(87/107).And for T790M mutation,sensitivity 45.0%(9/20),specificity 100.0%(87/87),concordance 89.7%(96/107).The detection of BRAF in plasma cfDNA:sensitivity 28.6%(2/7),specificity 93.0%(93/100),concordance 95.3%(102/107).Conclusions:CastPCR was successful in detecting EGFR and BRAF mutations in FFPE tissue of tumor and it had a high sensitivity and specificity,especially for the detection of EGFR T790M,and it has an advantage over the commonly used direct sequencing and ARMS.For patients whose tumor tissue is unavailable,CastPCR has satisfying specificity,consistency and certain sensitivity,which confers it the role of substitution to tumor tissue detection when the latter is not available.We applied CastPCR to the detecting of BRAF mutations in over 100 lung adenocarcinoma patients in their peripheral blood cfDNA for the first time,finding it’s high specificity and consistency,though its sensitivity was not high,we speculated that the following reasons might responsible:the heterogeneity of tumor,the low abundance of cell-free BRAF DNA and the reservation of tissue sample.More researches related to the optimization of detecting BRAF mutations in cfDNA of plasma are expected. |
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