Comparative analysis of EGFR mutations in circulating tumor DNA and primary tumor tissues from lung cancer patients using BEAMing PCR

Abstract

In this study, we measured human epidermal growth factor receptor (EGFR) mutations in both tissue and circulating tumor DNA (ctDNA) by using beads, emulsions, amplifications and magnetic polymerase chain reaction (BEAMing PCR). Noninvasive mutation detection by assessing circulating tumor DNA (ctDNA) offers many advantages over tumor biopsy. One hundred non-small cell lung cancer (NSCLC) patients were enrolled, and both preoperative plasma samples and formalin-fixed and paraffin-embedded (FFPE) samples were collected for the study. The EGFR mutation status was determined by BEAMing PCR in ctDNA. Real-time quantitative PCR (qPCR) data were collected from our hospital database (EMR-qPCR, Electronic Medical Records) for comparative analysis. Additionally, qPCR was also performed on FFPE tissues using a Diatech EGFR qPCR kit. The concordance rates were 98.8%, 98.9% and 95.5% for exons 19, 20 and 21, respectively, when the BEAMing data were compared with the EMR-qPCR data. Additionally, when the BEAMing and Diatech qPCR data were compared, 90%, 100%, 96% and 98% of the genes were obtained for exons 19, 20, 21 (L858R) and 21 (L861Q), respectively. For both comparisons, Cohen's kappa agreement was significant. The advantage of BEAMing is its ability to identify mutated DNA sequences in cancer cells in the background of normal cell DNA contamination. This could be useful for disease monitoring and progression.

Keywords: BEAMing; EGFR; NSCLC; ctDNA; qPCR.

Fig. 1

Microemulsions were made as described in Materials and Methods. One microliter of the emulsion was added in 1 µl of oil on a microscope slide and photograph was taken. Here in this picture, there were several compartments with a single bead (White arrowheads). Rests contain dual or multiple beads (100X magnification).

Fig. 2

Concentration of ctDNA in 100 samples (ng/mL).

Fig. 3

Purity of the ctDNA (A260/A280).

Fig. 4

Sensitivity and specificity analysis of the BEAMing PCR. A decreasing percentage of EGFR mutant DNA was added to the background of the wild-type DNA. Genomic and subsequent BEAMing PCR was carried out. The samples were hybridized and run in a FACSArea III. The mutant (Q1) and wild-type (Q3) populations were separated and are indicated. If the aqueous compartments were all the same size, the expected Poisson distribution was obtained. However, because they are not uniform, some compartments are larger and contain more than one template. This raises the number of double template beads (Q2).