A commercial real-time PCR kit provides greater sensitivity than direct sequencing to detect KRAS mutations: a morphology-based approach in colorectal carcinoma

Abstract

KRAS mutation testing has become a standard procedure in the management of patients with carcinomas. The most frequently used method for KRAS testing is direct sequencing of PCR products. The development of commercial real-time quantitative PCR kits offers a useful alternative since they are in theory much more sensitive than direct sequencing and they avoid post- PCR handling. We present our experience as a reference center for the study of KRAS mutations, comparing direct sequencing and the use of a commercial real-time quantitative PCR kit, as well as determining the sensitivity of both procedures in clinical practice. The TheraScreen K-RAS Mutation Kit identified mutations in 75 (44%) of the 170 tumors. Three cases were tested positive using TheraScreen K-RAS Mutation Kit and negative by direct sequencing. We then compared the sensitivity of the kit and that of direct sequencing using 74 mutant tumors. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in 13.5% of the tumors and, in 84%, KRAS mutation was identified at a dilution of 5%. Sequencing was able to detect KRAS mutations when the mutant DNA represented 10% of the total DNA in 20/74 (27%) of the tumors. When the mutant DNA represented 30% of the total DNA, sequencing could detect mutations in 56/74 (76%).

Figure 1

The sensitivity of the TheraScreen K-RAS Mutation Kit in comparison with direct sequencing. Mixtures of DNA from KRAS mutant and wild-type FFPE tumors were used to compare the sensitivity of the two methods. A: For sample P08-274, the TheraScreen K-RAS Mutation Kit was able to detect the KRAS mutation G12V when the DNA from a mutant tumor represented 5% of the total DNA. The 1%ΔCt value is the cut-off level provided by the manufacturer to detect the presence of a G12V KRAS mutation and it is derived from cell lines and synthetic constructs. B: For the same sample, sequencing was unable to detect the KRAS mutation G12V when mutant DNA was present as <30% of the total DNA. Percentages indicate the proportion of DNA from a mutant tumor relative to DNA from a wild-type tumor.

Figure 2

Morphological parameters that might interfere in the sensitivity of the KRAS mutational study. The samples were evaluated to establish the percentage of relevant lymphocytes (>10% in a ×20 field), (A) versus no lymphocytes (B); and relevant extracellular mucin (>50% of the tumor) (C) versus no mucin (D).