Mutation detection of epidermal growth factor receptor and KRAS genes using the smart amplification process version 2 from formalin-fixed, paraffin-embedded lung cancer tissue

Abstract

Recent evidence indicates that the presence of epidermal growth factor receptor (EGFR) or KRAS mutations in non-small cell lung cancer (NSCLC) can predict the response of the tumor to gefinitib. However, it is difficult to detect these mutations using formalin-fixed, paraffin-embedded (FFPE) tissues because the fixation process and aging can damage the DNA. In this study, we describe our work in adapting the Smart Amplification Process version 2 (SmartAmp2) to detect EGFR or KRAS mutations in DNA extracted from FFPE tissues. We were able to detect these mutations in 37 (97%) of 38 FFPE lung cancer tissue samples within 60 minutes with the SmartAmp2 assay and to confirm the correlation between EGFR mutations in FFPE tissues and gefitinib responsiveness. All mutations had previously been confirmed in the 38 samples using DNA extracted from frozen tissues. Electrophoresis results indicated that PCR analysis was not reliable for DNA extracted from FFPE tissue when primers with a long amplicon (>300 bp) were used. This study confirms that the SmartAmp2 assay is suitable for use with DNA extracted from FFPE as well as frozen tissues.

Figure 1

A: Amplification by the SmartAmp2 assay requires five unique primers: turn-back primer (TP), folding primer (FP), boost primer (BP), and two outer primers (OP1 and OP2). The genomic sequence between and including the TP and FP primers is the target region to be amplified in the SmartAmp2 assay. B: SmartAmp2 assay PNA clamp. The PNA clamp competitive probe is designed for the wild-type allele sequence. The greater stability of the PNA probe in hybridization inhibits SmartAmp2 amplification and suppresses wild-type allele amplification. Amplification of the mutant allele is not inhibited by PNA regardless of the point mutation (KRAS) or deletion (EGFR exon 19).

Figure 2

A typical SmartAmp2 assay result. The graphs represent amplification curves of the L858R mutation detection in wild-type and mutant templates. If the amplification started within 40 minutes, it was defined as a positive reaction.

Figure 3

Electrophoresis results of samples 7, 23, and 33. The PCR products of DNA extracted from blood samples were used as a positive control. S, short; M, medium; L, long amplicon of each primer. For example, S lane of EGFR exon 19 represents the result of electrophoresis using the PCR product of the 192-bp amplicon of EGFR exon 19. The left lane is used as the 100-bp ladder marker.

Figure 4

Microscopic examination of sample number 31. A typical case of a tumor with severe fibrotic changes. Most of the tumor had undergone fibrotic changes, and true tumor cells were found in only a small portion of the tumor, as indicated by the arrows. In such cases, mutations were difficult to detect by direct sequencing.