Abstract
The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes.
Highlights
Molecular therapy targeting transmembrane receptor tyrosine kinases with a variety of tyrosine kinase inhibitors has become part of the standard treatment for many patients with common forms of cancer
Seven of 201 cases analyzed for BRAF gave no amplifiable products due to excessive DNA degradation with both Sanger sequencing and ASLNAqPCR
We considered a result true positive (TP), false positive (FP), true negative (TN) or false negative (FN) as follows
Summary
Molecular therapy targeting transmembrane receptor tyrosine kinases with a variety of tyrosine kinase inhibitors has become part of the standard treatment for many patients with common forms of cancer. Evidence of both tyrosine kinase activation and lack of activating mutations in the tyrosine kinase downstream effectors is expected as a general precondition for successful patient treatment [1]. Among transmembrane tyrosine kinases the EGF receptor (EGFR) is one of the main therapeutic targets since it is active in both colorectal (CRC) and non-small cell lung cancers (NSCLC). Novel BRAF inhibitor molecules like vemurafenib are proving highly effective to treat patients with BRAF mutated tumors, like melanoma [15]
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