The Detection Method Of EGFR/KRAS Gene Mutations In Pleural Effusions Of NSCLC Patients And The Clinical Implication

BackgroundRecently, the morbidity and the mortality of lung cancer trend to ascend straightly inour country. Roughly500,000new cases occur each year, the increasing rate is about5％per year, and75%～80%in Non-small Cell Lung Cancer (NSCLC). The5-year survivalrate of patients with NSCLC was less than15%, if the advanced patients with metastasis,the median survival would be4～5months and the one-year survival rate would be lessthan10%. As surgery, chemotherapy, and radiotherapy given to NSCLC is in the bottleneck,the molecular-targeting antineoplastic agent has gradually become an irreplaceableimportant method in the advanced NSCLC treatment. According to a lot of clinical trials,the advanced NSCLC patients’lifetime can be prolonged by the molecular-targetingantineoplastic agent, represented by epidermal growth factor receptor tyrosine kinaseinhibitor (EGFR-TKI), but there is a close relation between the effect of EGFR-TKI on themolecular-targeting therapy in NSCLC and EGFR/KRAS gene. The mutations of exonEGFR19/21and exon EGFR20T790M are respectively the indexes of the sensitivity andthe resistance of EGFR-TKI, and KARS mutation is the index of the resistance of it. Hence,the detection of EGFR/KRAS gene plays a very important role of directing the targetingtherapy before accepting TKI treatment. Now the samples now used for themolecular-targeting detecting EGFR/KARS in NSCLC are mainly from tumor tissues bysurgery. Nevertheless, the tumor tissues usually cannot be acquired clinically. Under thatsituation, the difficulty of detecting EGFR/KARS in tumor tissues, consequently, restrictsthe choice in the molecular-targeting therapy of NSCLC patients. So it is imperative inNSCLC clinical diagnosis and treatment to seek nice substitutes as tissue specimens and thedetecting methods, showing important potentials for future clinical applications.AimsThe study aimed at establishing a substitute of detecting EGFR/KRAS from non-tumor tissue and exploring the clinical significance of EGFR/KRAS mutation in pleural effusionthrough detecting EGFR/KARS gene in pleural effusions of NSCLC patients. It wouldconfirm the relations of EGFR/KARS mutations with the effects of the clinical targetingtherapy, and provide the experimental evidences for forecasting the effect of moleculartargeting treatment.MethodsThe study was approved by Ethics Committees and all patients subscribed theinformation consent forms.63pleural effusion samples from NSCLC (IV, UICC,2009)in-patients who met inclusion criteria in the Department of oncology, from March2011toMarch2012and64tissue specimens were collected, and24tissue specimens matched thepleural effusion. The DNA were extracted from the above samples with QIAGEN Fluidsand Tissues DNA Extracting Kit, and then EGFR/KRAS gene mutations in63pleuraleffusion samples and64tissue ones were detected by Pyrosequencing. The KARS genes ofrandomly selected30pleural effusion samples were detected by (Locked Nucleic Acidsprobes Polymerase Chain Reaction)LNA-PCR-Sanger Sequencing, compared withPyrosequencing to analyze the difference of the sensitivities of both.Chi-square analysis was adopted to contrast the consistency of the results in those twosequencing methods and that of the EGFR/KRAS mutations from those two different tissues.The relationships of EGFR/KRAS mutations with gender, age, smoking history andpathologic types were analyzed. Following, Building standard of the inclusion criteria andexclusion criteria, we analyzed the samples from the patients who accepted the targetingtherapy and observed the relationship between their EGFR/KRAS genes and the clinicaleffects of TKI.Results1. The detection of EGFR mutations in pleural effusion by PyrosequencingThe mutation of EGFR in63pleural effusion samples was30.16%(19/63) withPyrosequencing. They all belonged to the mutation of exon EGFR19/21, in which therewere10cases having EGFR19deletion mutation (52.63%) and9cases having exonEGFR21L858R mutation (47.37%).2. The detection of KRAS mutations in pleural effusion by Pyrosequencing andLNA-PCR Sanger sequencing The mutation of KARS in63pleural effusion specimens was7.93%(5/63) byPyrosequencing.4cases were12codon mutation, GGT>GAT mutation, p.G12D c.35G>A,and the another was13codon mutation, GGC>GAC mutation, p.G13D c.38G>A.12codonmutation rate was80%(4/5). Moreover, by LNA-PCR sanger, only6cases held KARSmutation, the rate was20%(6/30), and4cases were12codon mutation,3/4ones wereGGT>GAT mutation, p.G12D c.35G>A, and the another was GGT>TGT mutation，p.G12Cc.34G>T. The other two cases were14codon mutation that was GTA>ATA mutation.12codon mutation rate was66.67%(4/6). The positive rate of KARS mutation beingsequenced by LNA-PCR sanger was higher than that by Pyrosequencing, but the differencewas no significant, P=0.093. In overall30cases being sequenced by LNA-PCR Sangersequencing and Pyrosequencing, KARS mutation were detected in3cases withPyrosequencing all that were12codon mutation，GGT>GATmutation，p.G12D c.35G>A，the rate was10.00%(3/33), which was no different being compared with the result ofLNA-PCR sanger sequencing(20%6/30), P=0.470.3. The comparison of EGFR/KARS mutations in tumor tissues and in pleural effusionEGFR mutation rate was34.38%(22/64)in64tissue specimens, Compared EGFRmutation rates in64tissue specimens and in63pleural effusion(30.16%19/63)were notsignificantly different, P=0.705. Matched24tissue and and pleural effusion specimens, EGFRmutation rate was37.50%(9/24)and33.33%(8/24). There was no significance, P=0.500.KARS mutation rate was4.69%(3/64)in64tissue specimens,2cases were12codonmutation,66.7%, and one was13codon mutation,33.3%. The difference of KARSmutation rates in64tissue specimens and in63pleural effusion(7.93%，5/63) ones were notsignificant, P=0.350. There was no significance of KARS in24cases whose tissuespecimens(12.5%,3/24) matched pleural effusion(8.33%2/24) ones, P=0.500.4. Clinical implication of EGFRResults of detection to tissue samples and pleural effusion ones show there wererelations of EGFR mutation rate to smoking history(tissue P=0.019, pleural effusionP=0.014), pathohistologic type(tissue P=0.018, pleural effusion P=0,020), it was easier tooccur in non-smokers and patients with adenocarcinoma. Under RECIST criteria,18patients accepting TKI targeting treatment were assessed, there were5cases with EGFRsensitive mutation (exon19deletion mutation and exon21L858R mutation), but no CR after TKI treatment, only5PR, and the remission rate RR (CR+PR) was100%; there were13cases with wild type EGFR, no CR, but5PR,3SD and5PD, the remission rate RR(CR+PR) was38.5%. Compared with wild type, patients with EGFR sensitive mutationwere more sensitive to TKI treatment, there was significant difference between each other,(P=0.029).5. Clinical implication of KARSThere was no relation between KARS mutation in pleural effusion andclinicopathological characteristics including gender, age, pathohistologic type andsmoking(P>0.05). Under RECIST criteria,18patients accepting TKI targeting treatmentwere assessed, there was one case with KARS mutation who reached PR, and the remissionrate RR (CR+PR) was100%; there were17cases with KARS wild type, no CR after TKItreatment, but9PR,3SD and5PD, the remission rate RR (CR+PR) was56.25%. Therewas no significant relationship between KARS mutation and TKI treatment (P=0.556). Theresult didn’t accord with foreign reports in which KARS mutation led TKI resistance. Itsuggested that there would be a regional difference in KARS mutation and TKI clinicaleffect, which would be approved further with bigger sample size.Conclusions1. If tissue specimens were unable to be acquired, malignant pleural effusion could bea wonderful substitute for EGFR/KARS detection in patients with NSCLC.2. Pyrosequencing could be treated as a way to detect EGFR mutation;LNA-PCR-sanger and Pyrosequencing were both used to detect KARS mutation inmalignant pleural effusion, the former was more sensitive.3. Compared with EGRF wild type, EGFR gene with exon19and21mutations weremore sensitive to TKI treatment.4. There were no relation between KARS and TKI, which differed from the resistancemutations, reported by references. It showed that there was a regional difference in therelativity of KARS mutation and TKI clinical effect, which needed to be studied furtherwith bigger sample size.