Improved detection of EGFR mutations in the tumor cells enriched from the malignant pleural effusion of non-small cell lung cancer patient.

Previous studies focus on developing high sensitive PCR-related technologies to detect EGFR gene mutation in malignant pleural effusion (MPE) of non-small cell lung cancer patient (NSCLC) instead of improving the quality of clinical samples themselves. We therefore hypothesized that the enrichment of tumor cells in MPE could improve the quality of MPE for the more accurate detection of EGFR gene mutation in the patients with NSCLC. MPE were collected from 28 patients with NSCLC. The tumor cells in MPE were firstly enriched by the depletion of leukocytes with bi-antibodies and identified by multiple flow cytometry. CastPCR was performed to detect the deletions in exon 19 (DelEGFR19) and point mutations in 20 (T790M, c.2369C>T) and 21 (L858R, c.2573T>G) of EGFR. All the detected mutations were confirmed their presence by Sanger sequencing. Before enrichment, the tumor cells in MPE were detected in 46.4% (13/28) of samples and the median value of the percentage of the tumor cells was only 0.64% including 3 (10.7%, 3/28) of samples more than 10% on it. After enrichment, the tumor cells were detected in all samples (100%) and the median value of the percentage of tumor cells was increased to 40.8% including only one (3.6%, 1/28) less than 10% on it. EGFR gene mutations were detected in 28.6% (8/28) and 42.9% (12/28) of the samples before and after enrichment, respectively. The sensitivity on the detection rate was relatively increased by 44.4%. Moreover, the mutations can be confirmed their presence by Sanger sequencing in 6 samples before enrichment (75%, 6/8) and 11 samples (91.7%, 11/12) after enrichment. It is valuable to improve the quality of the sample by the enrichment of the tumor cells from MPE for the following genetic analysis.