Detection of Mutant RAS Subpopulations in Colorectal Cancer Patients

Acquired resistance to molecularly targeted therapies is a major Various detection techniques for RAS mutations are available, but obstacle blocking effective treatment of cancer patients. This is particularly true for therapies that target the epidermal growth factor receptor (EGFR) [1,2]. Acquired resistance to molecularly targeted therapies may occur de novo (meaning as a consequence of the selective pressure of treatment), or may be due to outgrowth of preexisting tumor subpopulations that are resistant to treatment [3]. Clinical studies have shown that undetected populations of tumor cells carrying KRAS mutations lead to therapeutic resistance and relapse in colorectal cancer (CRC) patients [4,5]. Consequently, the ability to assess the impact of minor mutant subpopulations on therapeutic resistance has important implications regarding the development of effective strategies for personalized cancer treatment. Detecting and analyzing the major and minor mutant subpopulations of cells carrying specific oncogene mutations in a patient's tumor, therefore, are crucial. The presence of some RASmutations, especially KRAS codon 12 mutations, in CRC patients is associated with poor prognosis and predicts lack of response to therapies that target the EGFR (e.g.,cetuximab, and panitumumab) [4]. Therefore, detecting KRAS mutations is important for CRC patients undergoing anti-EGFR therapy. Mutations in KRAS occur most frequently on exon 2 (codons 12 and 13) and to a lesser extent on exon 3 (codon 61). The multicenter RASCAL study provided evidence that there were different prognoses for CRC patients harboring different KRASmutations in their tumors. KRAS codon 12 mutations, especially G12V, is associated with poor prognosis, whereas patients with KRAS codon 13 mutations respond better to the therapies [6]. Accurate detection of RASmutations that occur at high and low frequency is critical for identifying the best strategies for intervention. An ideal assay for detection of RASmutations: 1) should have the ability to determine RAS mutationswith high levels of specificity and sensitivity, 2) be able to determine different levels of RASmutations in colorectal tumors quantitatively, 3) should also be able to detect minor mutant subpopulations of RASmutations; and 4) should employ a multiplex approach, in order to detect several RASmutations from small amount of DNA in a short turnaround time.