Detection of Mutant RAS Subpopulations in Colorectal Cancer Patients.

Acquired resistance to molecularly targeted therapies is a major 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 RAS mutations, 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 KRAS mutations 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 RAS mutations that occur at high and low frequency is critical for identifying the best strategies for intervention. An ideal assay for detection of RAS mutations: 1) should have the ability to determine RAS mutations with high levels of specificity and sensitivity, 2) be able to determine different levels of RAS mutations in colorectal tumors quantitatively, 3) should also be able to detect minor mutant subpopulations of RAS mutations; and 4) should employ a multiplex approach, in order to detect several RAS mutations from small amount of DNA in a short turnaround time.

Various detection techniques for RAS mutations are available, but screening of KRAS mutations for anti-EGFR therapy in CRC patients is done mostly by direct sequencing (DS) or real time PCR assays, which may be expensive and time consuming. In this issue of EBioMedicine, Dr. Takayuki Yoshino and colleagues report on the utility of the MEBGEN RASKET KIT (RASKET), a multiplex assay using PCR-reverse sequence specific oligonucleotide with xMAP technology. This assay can detect 48 RAS (KRAS and NRAS) mutations in exons 2, 3 and 4, with a short turnaround time of 4.5 h for 96 specimens [7]. Moreover, the RASKET results had high concordance with those obtained using DS and the TheraScreen KRAS RGQ Polymerase chain reaction kit. The RASKET can detect mutant fractions of around 1–5% which is similar to TheraScreen KRAS mutation kit and is higher than DS. There are other sensitive assays for KRAS detection such as the Allele Specific Competitive Blocker Polymerase Chain Reaction (ACB-PCR) that can detect KRAS mutations fractions as low as 0.001% (1 mutant out of 100,000 wild-type) [8]. The sensitivity of an assay will impact whether a colorectal tumor is KRAS mutant or wild-type [9]. With ACB-PCR, KRAS codon 12 mutations are detected in 100% of colon tumors and also in some normal colonic mucosa [8]. The levels of different RAS mutations that are “pathological” have not yet been rigorously established for CRC patients [8]. Nevertheless, it has been demonstrated that the KRAS mutant subclones known to cause resistance to anti-EGFR therapy are quite prevalent and frequently occur at low levels (levels below the detection limit by DS [8]). There is evidence that a significant number of CRC patients characterized as KRAS wild-type fail to respond to EGFR therapy [9]. This could be due in part to the insensitive techniques that clinicians use for KRAS mutation detection or due to the presence of other undetected mutations in different genes in the RAF–MEK–ERK kinase pathway. RASKET, similar to DS and the TheraScreen kit, does not provide quantitation of the detected RAS mutations. Therefore, these methodologies may not be sufficient for establishing whether particular levels of mutation are associated with clinical response. RAS mutations are frequently present as small subpopulations within colon tumors, which are remarkably heterogeneous and may often be polyclonal in origin [10]. Therefore, it seems likely that both sensitive and quantitative methodologies to characterize RAS mutations will be required to advance personalized treatment of colon cancer.

The RASKET kit has its limitations: it does not measure the RAS mutations quantitatively and also there are other more sensitive assays available to measure RAS mutations. Nonetheless, the RASKET multiplex assay detects exons 2, 3 and 4 KRAS and NRAS mutations from a small amount of DNA (50–100 ng) that is extracted from formalin fixed paraffin embedded tissues in a short turnaround time of 4.5 h.