B7-04: Detection of EGFR mutations in plasma by allele-specific real-time PCR in non-small cell lung cancer (NSCLC)

Abstract

Activating epidermal growth factor receptor (EGFR) mutations in NSCLC tumors are associated with rapid and sustained clinical response to EGFR tyrosine kinase inhibitors (TKIs). Due to tumor heterogeneity, identification of EGFR mutations using conventional DNA sequencing approaches can be problematic. Tumor-specific mutations such as KRAS have been detected in shed tumor DNA in patient plasma (Kimura, Ann N Y Acad Sci, 2004), and the ability to detect EGFR mutations in patient plasma would have potentially significant clinical utility. Here we describe the performance of an allele-specific real-time PCR system utilizing Scorpion primers (provided by DxS, UK) to detect EGFR mutations in tumor tissue and plasma. The sensitivity of this technique in terms of the ratio of mutant-to-wild-type genomic DNA and the minimum amount of DNA required for detection was determined using a dilution experiment. Genomic DNA from lung cancer cell lines containing either the exon 21 L858R point mutation or the exon 19 E746-750 deletion was mixed with wild-type genomic DNA at ratios ranging from 1:2 to 1:10,000. The presence of EGFR mutations was also determined in archival tumor material and/or plasma obtained from 39 consenting advanced stage NSCLC patients treated with EGFR-TKIs. In cell line experiments, mutant DNA was detectable utilizing the Scorpion technology at concentrations as low as 25 pg, and at ratios as little as 0.1% of the total pool of genomic DNA. Of the 39 NSCLC patients, only tissue was available for evaluation in seven patients; two were positive both with the Scorpion primers and direct sequencing while the rest were wild-type. For 21 patients where only plasma was available, 6 mutations were detected with Scorpion primers, none of which were detectable by direct sequencing. EGFR mutations were identified in both plasma and tissue of two patients who were complete responders to EGFR-TKI therapy, only one of which was detectable by direct sequencing. Two additional mutations were found in the tissue but not plasma of patients currently undergoing treatment. Neither of these mutations was detectable by direct sequencing. Allele-specific Scorpion technology is: 1) highly specific and sensitive for EGFR mutation analysis, 2) able to detect mutations not observable by direct sequencing in plasma and tissue, 3) capable of detecting mutations in shed tumor DNA in plasma and 4) may be suitable for monitoring response or detecting recurrence in advanced NSCLC patients.