P-0195 - Clinical validation of a novel multiplex kit for all RAS mutations in colorectal cancer: Results of RASKET (RAS KEy Testing) prospective multicenter...
|Date||28 June 2014|
|Event||World GI 2014|
|Citation||Annals of Oncology (2014) 25 (suppl_2): ii14-ii104. 10.1093/annonc/mdu165|
T. Kudo1, K. Muro2, H. Taniguchi2, K. Akagi3, T. Nishina4, T. Kajiwara4, T. Denda5, S. Hironaka5, W. Okamoto6, T. Yoshino7
It is increasingly important to diagnose all RAS statuses in KRAS and NRAS among patients with advanced colorectal cancer (CRC) and to administer anti-epidermal growth factor receptor antibodies. Although direct sequencing (DS) with manual microdissection (MMD) is a widely used approach, a quality controlled diagnostic kit that simultaneously and rapidly detects RAS mutations is needed. In this study, we evaluated a novel multiplex kit using a Luminex (xMAP) assay to detect 48 mutations reported in the PRIME study (NEJM 2013;369:1023-34), or those with a >0.1% incidence in the COSMIC database in all RAS of exon (ex) 2 (codon 12/13), ex 3 (codon 59/61), and ex 4 (codon 117/146) and with a 1–5% sensitivity in a single reaction using 50–100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissue without MMD.
Samples were taken from patients with histologically confirmed colorectal adenocarcinoma and sufficient archived FFPE; all patients provided written informed consent. The primary endpoint was the concordance rate of all RAS mutations between the results of the kit and the two reference methods (DS with MMD and TheraScreen KRAS Mutation Kit). We defined a result as being KRAS ex 2 mutant positive if a mutation was found in DS with MMD or TheraScreen KRAS Mutation Kit. The secondary endpoints included the concordance of minor RAS mutations, other than KRAS ex 2, for the KRAS ex 2 wild-type (WT) population and the concordance of each genotype for the overall population. Separate data sets were generated using the kit, DS with MMD and TheraScreen KRAS Mutation Kit at the central testing laboratories.
309 samples from six institutes were registered. For the reference methods, 307 FFPE were analyzable and 140 RAS mutations (46%) were detected, with 111 KRAS ex 2 and 29 minor RAS mutations. The kit detected 143 RAS mutations (47%) with 114 KRAS ex 2 and 29 minor RAS mutations. Each assay in the kit was performed using 50-100 ng of template DNA, with an overall hands-on time of 4.5 h per each 96-specimen-set. The concordance rate between the two methods was 97% (95% CI, 94 to 98%). Minor RAS mutations were detected in 15% of the KRAS ex 2 WT population (n = 191), and the concordance rate was 98% (95% CI, 96 to 100%). The concordance of each genotype for the overall population was 100% (95% CI, 97 to 100%).
The clinical validity of the multiplex kit was demonstrated. The kit allows for the rapid and exact detection of all RAS mutations in ex 2, ex 3, and ex 4 from an FFPE of CRC (Study ID: UMIN000011784).