Abstract
Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five KRAS and EGFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.
Highlights
The growing significance of identifying EGFR and KRAS mutations in lung cancer using molecular diagnostic approaches underlines the emphasis on the use of personalized medical care by physicians to help design optimal therapeutic regimens (Lynch et al, 2004; Paez et al, 2004; Pao et al, 2004; Pao et al, 2005a; Pao et al, 2005b)
Concatenated KRAS and EGFR (CRE) (Co-amplification of KRAS and EGFR) exons is a costeffective multiplex-PCR based method followed by concatenation of the PCR product as a single fragment for direct sequencing (Figure 1)
CRE-based KRAS-EGFR concatenation from fresh frozen primary tumors and tumor-derived cell lines Following CRE-based multiplex PCR of KRAS exon 2 and EGFR exons 18-21 with overlapping PCR bands (Figure 2A, lane 6), concatenation of the PCR product was performed with OAD176 and OAD152 primers using genomic DNA extracted from NCI-H1975 cells, a non-small-cell lung adenocarcinoma cell line
Summary
The growing significance of identifying EGFR and KRAS mutations in lung cancer using molecular diagnostic approaches underlines the emphasis on the use of personalized medical care by physicians to help design optimal therapeutic regimens (Lynch et al, 2004; Paez et al, 2004; Pao et al, 2004; Pao et al, 2005a; Pao et al, 2005b). While no direct evidence exists as yet, these studies may have implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al, 2006). Such information is especially important for lung cancer patients at an advanced-stage, who are not candidates for surgical intervention—wherein biopsy specimens obtained through fine-needle aspiration (FNA) may represent the only opportunity to obtain tissue material for diagnosis and molecular diagnostic analysis.
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