Comparative Analysis of Different Types of KRAS Mutation between Frozen and Paraffin Embeded Tissue Using the Pyrosequencing Method
Mariana P Macedo, Fernanda M Mello, Bianca CG Lisboa, Louise DB Andrade, Antonio Hugo Jose FM Campos, Dirce M Carraro, Fernando A Soares, Isabela W Cunha. Hospital A.C.Camargo, São Paulo, Brazil
Background: KRAS mutation is a negative predictor of response to treatment with Epidermal Growth Factor receptor inhibitors and is detected in ≈35% of Colorectal Carcinomas (CRC). KRAS mutation analysis is performed in routine practice in formalin fixed and paraffin embedded tissue (FFPET). Formalin fixation has been described to induce artifact mutations in selected genes, mostly due to error in DNA pairing during sequencing procedures. Some studies have compared KRAS status between FFPE and frozen tissue, but mostly regarding KRAS status (wild versus mutated). The objective of this study is to examine whether the detection of the most frequently described types of KRAS mutations found in CRC are reproducible between FFPET and frozen tissue using the pyrosequencing method.
Design: Twelve previously sequenced FFPET from CRC cases mutated for KRAS gene were selected based on the type of mutation detected and the availability of paired frozen tissue in Hospital A.C.Camargo Biobank. DNA from FFPET was extracted using the QIAmp DNA Extraction Kit; DNA from frozen tissue was extracted using an in house technique. PCR was performed with 100 ng of DNA, targeting the regions containing codons 12 and 13 using the KRAS PyroMarkTM Q24 kit (Qiagen). Pyrosequencing reaction was performed using the manufacturer's instructions (KRAS PyroMarkTM Q24 V2.0 (Qiagen).
Results: The twelve study cases consisted of seven tumors mutated in codon 12 and 5 in codon 13. Mutations in codon 12 were GGT>GAT (3 cases), GGT>GTT (3 cases), GGT>AGT (1 case). All mutations in codon 13 were GGC>GAC. The pyrosequencing using the DNA from the paired frozen tissue showed 100% of compatibility regarding the mutations detected in the FFPET.
Conclusions: KRAS mutation detection by pyrosequencing DNA from FFPET is highly reproducible with DNA from frozen tissue. This study shows no negative impact of formalin fixation in the detection of the most common types of KRAS mutation in CRC by pyrosequencing method.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 258, Wednesday Morning