3%. When ARMS was used, 6 more patients were defined as mutation positive, with the ORR of the 22 patients at 72.7%. For patients who provided plasma, 5 mutation positive patients were detected only by ARMS, with the ORR at 80%. Generally, our result was consistent with that of OPTIMAL and IPASS research, SB202190 mouse both using tumor tissue for EGFR

mutation analysis [5, 9]. The ORR for mutation positive patients in OPTIMAL using direct sequencing was 83%, higher than that of IPASS using ARMS strategy (71.2%). Interestingly, such difference also occurred in our study using pleural fluid samples (81.3% Vs 72.7%). The results implied that, more sensitive methods such as ADx-ARMS may find more positive patients, but for them, mutative cells may represent a minority of the whole tumor, which may influence the final clinical outcome of TKIs. The explanation is consistent with the work of Qing Zhou et al. which found that the relative

MEK phosphorylation EGFR mutation abundance could predict benefit from EGFR-TKIs treatment for advanced NSCLC [19]. Our data emphasized that, for mutation positive results, the predictive effect of body fluid was no less than that of tumor tissue. As considered for the two problems mentioned above, our research agreed with former reports that more sensitive method such as ARMS would be one of the feasible solutions [14, 20]. Compared with direct sequencing, ADx-ARMS assay found 18.8% (6/32) and 27.8% (5/18) more patients to be mutation positive for pleural fluid and plasma, respectively. Direct sequencing is currently the routine method used to detect EGFR mutations. The merits of this method are readily available and economic, but the procedure is complicated and time-consuming. Meanwhile, the sensitivity of sequencing is about 30%, which tends to cause false negative result [21]. Given the poor sensitivity of DNA sequencing, many patients and physicians opt to start TKIs treatment even if the sequencing results were Ribonucleotide reductase negative for EGFR mutation. If the tumor does not contain

activating mutations on EGFR, treatment with TKIs will most likely be ineffective. In our study, 11 former negative patients (6 pleural fluids, 5 plasmas) defined by sequencing were proved to be positive at last, and the clinical outcome for them was quite satisfactory. If the treatment plan was made according to the result of direct sequencing, those patients may lose the chance of TKIs therapy. Besides, by using ARMS, we also found 7 samples which harbouring double mutations (2 patients with 19 del and L858R, 1 with L858R and L861Q or S768I, 4 with 19 del and T790M). The clinical evaluations for the former 3 patients were all PR. This result was consistent with the study of Zhang et al. [22] which showed that patients with double activating mutations involving both exons 19 and 21 tend to respond well to TKIs and the sensitivity to TKIs was enhanced compared with either single mutant. As demonstrated by Qing Zhou et al.