A38
Evaluation of techniques for the analysis of KRAS mutation status in formalin fixed tissue
Brendan O'Sullivan1, Sarah Whelton2, Susanna Akiki2, Susan Sharpe1, Nayneeta Deshmukh1, Rahul Hejmadi1, Jenny Bell1, Philippe Taniere1
1University Hospital Birmingham NHS Foundation Trust, Birmingham, UK, 2Birmingham Women's Hospital, Birmingham, UK
Background
K-RAS mutation analysis for codons 12 and 13, is required in bowel adenocarcinoma before treatment with anti EGFR1 drugs, since the presence of a mutation is predictive of lack of response. Analysis is performed in formalin fixed paraffin embedded tissue.
Method
We have analysed 40 cases for KRAS mutation status by 3 different methods: direct DNA sequencing, Pyrosequencing and Real time PCR (DxS Ltd). All cases were subject to microdissection on slides. Two DNA extraction protocols were compared: in house Tris EDTA/NP40 and Qiagens FFPET tissue kit. Validation of the presence of mutation was obtained when at least one technique produced reproducible results according to interpretation guidelines.
Results
33/40 (83%) cases showed concordance with all 3 techniques. Of the remaining 7 cases: (1) Direct sequencing failed to amplify DNA in 2 cases and in 5 failed to show the mutation, (2) Pyrosequencing failed to amplify DNA in one case; gave an unreproducible result in one case; and failed to show mutation in one case, (3) Real time PCR failed to produce interpretable results in one case.
Qiagens FFPET tissue kit produced extracts which, when tested, elicited greater average peak heights in pyrosequencing. Furthermore, control Ct values from the Real time analysis were inferior when using the in house DNA extraction method.
Conclusion
Results demonstrate the benefits of access to different techniques and confirm previous data suggesting that direct DNA sequencing may be of compromising sensitivity when more sensitive kit based tests are available.
We recommend using the Qiagens FFPET tissue kit.
