[Screening for EGFR mutations in lung cancer by a novel real-time PCR with double-loop probe and Sanger DNA sequencing].

OBJECTIVE

To map the frequency and types of EGFR gene mutations present in lung cancer tissues. To evaluate the clinical applicability of a novel real-time double-loop probe PCR of which the ADx-EGFR kit is based, and to compare its performance with traditional Sanger DNA sequencing in the detection of somatic mutations of tumor genes.

METHODS

A total of 208 formalin-fixed paraffin-embedded (FFPE) tumor samples were tested. Genomic DNA of the tissue samples was extracted and purified, and subjected to both traditional PCR amplification, Sanger sequencing of EGFR gene in exon 18, 19, 20, 21, and ADx's EGFR mutation detection kit. The mutation rates for EGFR gene in exon 18, 19, 20, 21, as well as the frequency of each mutation detected by the two methods, were analyzed.

RESULTS

The traditional Sanger DNA sequencing technique was successfully performed in 196 out of 208 (94.2%) lung cancer samples, and 22 samples (11.2%) showed EGFR gene mutations. ADx-EGFR kit was successfully used in the lung cancers of all of the 208 cases (100.0%), and 40 samples (19.2%) showed mutations. In the lung cancer samples analyzed, mutations were mainly detected in the exon 19 and exon 21 L858R point mutation, i.e. 4.8% (10/208) and 11.6% (23/208) of total mutations, respectively, and the remaining mutations were rare.

CONCLUSIONS

The success rate of ADx-EGFR real-time PCR for formalin-fixed and paraffin-embedded tissues samples is significantly higher than that of Sanger sequencing (P < 0.01). There are significant differences between the two methods. ADx-EGFR real-time PCR shows a much higher successful detection rate and mutation rate of lung cancer tissues compared with that of Sanger sequencing. As a result, the real-time PCR with ADx-EGFR kit is proved to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is an effective and reliable tool for clinical screening of somatic gene mutations in tumors.