| Objective:EGFR mutation status is the important biomarker of EGFR-TKI response and prognosis in NSCLCs. At present, there's no unified method to detect EGFR mutation. This article aims to explore a more accurate, sensitive and convenient method for clinical use in China by comparing direct sequencing and amplification refractory mutation system (ARMS) and to investigate the different prediction of EGFR-TKI response by the two methods.Method:The NSCLC patients of Zhongshan Hospital in need of EGFR mutation detection were included from 2008 to March,2011. Surgery resection and other biopsy tumor tissue of these patients were submitted to mutational analysis by direct sequencing method and ARMS method. Exon 18,19,20 and 21 of EGFR were examined. In addition, patients who received EGFR-TKI were followed up to compare the different TKI response in different EGFR mutation status by the two detection methods.Results:There are 364 patients included.344 patients were detected by direct sequencing,188 patients detected by ARMS method and 168 patients were detected by both methods. In the patients who were detected by both methods, There were 25 cases detected the same EGFR mutation and 3 cases different mutation by both methods, while 107 cases were detected EGFR wild-type by both methods.29 cases were detected EGFR mutation only by ARMS method, otherwise 4 cases only by sequencing method, so the two detection methods are to some extent coincident (K=0.51, P<0.01).Integrating detection results by two methods, there are 61 cases identified as mutated EGFR. The sensitivity of direct sequencing is 52.5%, whereas the sensitivity of ARMS is 93.4%(P<0.001). In the 29 cases who can only detected EGFR mutation by ARMS, when we review the sequencing results we found that there were 10 cases identified as no mutation because of very low mutation peak. The other 19 cases got the same results when we repeat detection by both methods. In addition, direct sequencing methods detected 2 rare mutations which are not included in ARMS kit. The mutation detection rate of ARMS is significantly higher than that of direct sequencing(33.9% vs 19.0%, P 0.002).In the 88 small specimen detected by both methods, the mutation detection rate of ARMS is significantly higher than that of direct sequencing (42.0% vs 19.3%, P=0.001), while in 80 big specimen, there's no difference (25% vs 18.8%, P=0.339)。There are 107 patients have received EGFR-TKI in second plus line regularly who detected EGFR mutation by sequencing and /or ARMS. Female, nonsmoker and patients whose histopathology type is adenocarcinoma have better TKI response.There are 103 patients detected EGFR mutation by sequencing methods and received EGFR-TKI in the second line plus regularly. The objective response rate(ORR) and disease control rate(DCR) of EGFR mutated-type patients by sequencing are respectively 69.1% and 94.5%, which are superior than EGFR wild-type patients.respectively 22.9%(P<0.001) and 35.4%(P<0.001).The progression-free survival(PFS) of EGFR mutated-type patients by sequencing is 16.3months(95%CI:12.8～19.8), which is significantly longer than EGFR wild-type patients 6.3 (95%CI:3.7～8.9)(P<0.001). In the same way, the ORR and DCR of EGFR mutated-type patients by ARMS are respectively 78.3% and 95.7%, which are superior than EGFR wild-type patients,8.3%(P<0.001) and 8.3%(P<0.001) respectively. The PFS of EGFR mutated-type patients by ARMS is 17.5 months (95%CI:11.9～23.1), which is significantly longer than EGFR wild-type patients 1.7 months (95%CI:0.8～2.6) (P<0.001). In the patients who were detected by both methods, we find that the ORR and DCR of EGFR wild-type patients by sequencing method is 25% and 28.1%, while by ARMS method is 8.3%(P=0.162)和8.3%(P=0.093). The PFS of patients who were detected EGFR mutated-type by ARMS but EGFR wild-type by sequencing is 11.2 months (95%CI:7.2～15.1), which is shorter than patients who are detected EGFR mutated-type by both methods 21 months (95%CI:17.3～24.7), but longer than patients who are detected EGFR wild-type by both methods 1.7months (95%CI:0.8～2.6) (P<0.001).To quantify the mutation cell rate of tumor tissue by calculating the difference of mutation Ct and control Ct, it is found that the Ct difference has a rising trend with the TKI response changing from CR, PR, SD to PD. In addition, there is no significant difference in the median PFS of patiets with deletion mutation of 19 exon and L858R point mutation in 21 exon.Conclusion:ARMS method is more sensitive than direct sequencing and can elevate the mutation detection rate, especially superior in small biopsy specimen. Moreover, ARMS method is also more simple and convenient and can be widely applied in clinical detection in place of direct sequencing. Direct sequencing can detect rare mutation not included by ARMS kit, so integrating the two methods can make detection more repliable. No matter sequencing or ARMS method we use to detect, EGFR mutated-type patients have better ORR and DCR and longer PFS than EGFR wild-type patients. ARMS method can predict the TKI response better than sequencing method. There is association between mutated cell rate and TKI response. The more mutated cell rate is, the longer PFS is, and it needs more investigation. In our study, there's no response difference between patients who have deletion mutation in 19 exon and those who have point mutation in 21 exon...
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