A comparability study of 5 commercial KRAS tests

Abstract

BackgroundActivating mutations in the KRAS gene occur frequently in human tumors, including colorectal carcinomas; most mutations occur in codons 12 and 13. Mutations in KRAS have been associated with poor response to anti-epidermal growth factor receptor antibodies. Therefore, an accurate and readily available analysis of KRAS mutational status is needed. The aim of this study was to evaluate concordance between KRAS assays performed by 6 different laboratories.MethodsForty formalin-fixed paraffin-embedded colorectal cancer tumor samples were obtained. Sample sections were submitted for KRAS mutation analysis to 5 independent commercial laboratories (Agencourt, Gentris, Genzyme, HistoGeneX, and Invitek) and to the Amgen DNA Sequencing Laboratory for direct polymerase chain reaction sequencing. The assay used by Invitek is no longer commercially available and has been replaced by an alternative technique. Results from the commercial services were compared with those from Amgen direct sequencing by κ statistics.ResultsKRAS mutations were observed in codon 12 and/or 13 in 20 of 40 (50%) samples in Amgen direct sequencing assays. Results from HistoGeneX (κ = 0.95), Genzyme (κ = 0.94), and Agencourt (κ = 0.94) were in almost perfect agreement with these results, and the results from Gentris were in substantial agreement with the results from Amgen (κ = 0.75). The Invitek allele-specific assay demonstrated slight agreement (κ = 0.13).ConclusionsThis study provides data on the comparability of KRAS mutational analyses. The results suggest that most (but not all) commercial services provide analysis that is accurate and comparable with direct sequencing.

Massively Parallel Tumor Multigene Sequencing to Evaluate Response to Panitumumab in a Randomized Phase III Study of Metastatic Colorectal Cancer

“…However, in a separate comparability study, we found assessment of KRAS status using Roche 454 pyrosequencing to have a high level of agreement (k ¼ 0.94) with direct sequencing (20).…”

“…The multigene sequencing assays were extensively qualified to show the ability to confidently detect mutations at a level of 5% of sequence reads per amplicon using both cell line DNA and procured FFPE colorectal tumor samples. Because of the nature of this qualification, the demonstration of correlation between this sequencing methodology and bidirectional sequencing (20), the lack of commercially available validated assays for all of these mutations and the limiting amount of material available for these samples, confirmation of the mutation status using an additional method was not pursued.…”

Concordance of Predictive Markers for EGFR Inhibitors in Primary Tumors and Metastases in Colorectal Cancer: A Review

“…All four of them reported discordance in some cases. As for the reliability of the other techniques used in the studies reported in Table 1, a paper by Oliner et al [64], evaluating five different KRAS tests, showed that classic DNA sequencing as well as pyrosequencing and AS-PCR provide accurate KRAS analysis. The commercial ASO kit described in that paper was reported to be less accurate.…”

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

“…However, they are characterized by different sensitivity, turnaround time, and cost. [12][13][14][15] Besides the use of robust KRAS testing methods, the selection of the appropriate tissue to be tested is also critical. Particularly, evaluation of the tumor cellularity is essential to guide the interpretation of the molecular data.…”