Comparison of FFPE DNA Extraction Methods Using a Molecular Pathology Assay
Sertac Kip, Jennifer Winters, Kandelaria Rumilla, Benjamin Kipp. Mayo Clinic, Rochester, MN
Background: With the implementation of recent FDA approved companion diagnostic tests, some laboratories are required to validate additional xylene deparaffinizing extraction methods. Our laboratory has the Roche cobas® DNA Sample Preparation Kit (cobas) and the Qiagen QIAamp® DSP DNA FFPE Tissue Kit (DSP), in addition to the Qiagen QIAcube DNA extraction method. Since many cancer patients have limited specimen and require multiple tumor marker tests, our aim was to cross validate these techniques for molecular pathology testing.
Design: We analyzed 11 FFPE tumor samples that were originally ordered for clinical KRAS testing. Using the same amount of macrodissected tissue, DNA was extracted using the 3 methodologies and tested using the Qiagen therascreen® KRAS RGQ PCR Kit. This KRAS assay consists of a control reaction and seven mutation reactions. The crossing threshold (CT) value of the control reaction provides a metric for of the amount of amplifiable DNA, which is then compared to the CT values of the mutations reactions to determine mutation status. The CT value of the control reaction was compared between extraction methods for each of the 11 cases. There were 6 KRAS positive cases and their CT mutation values were compared between extraction methods. Final KRAS mutation status was also compared.
Results: The QIAcube method's control reaction's CT values ranged from 23.75 to 27.65; the Cobas method's values ranged from 21.54 to 26.46; and the DSP method's values ranged from 23.16 to 27.71. Of these 33 separate analyses, 32 had an acceptable CT values (21.92 to 32.00). One cobas extracted sample had too much DNA and had to be diluted. The %CV between extraction methods (control reaction's CT values) ranged from 0.3% to 6.2%. For the 6 positive cases, the %CV between extraction methods (mutation reaction's CT values) ranged from 1.9% to 8.2%. The final mutation results of the 11 cases were concordant for all three methodologies.
Conclusions: The results of this study indicate that the DNA extracted by any of these methods is acceptable for use with the therascreen® KRAS RGQ PCR Kit. For specimens with only enough tissue for one DNA extraction, cross validating extraction methods is necessary for testing additional markers. However, for some FDA approved assays, the different extraction method may be considered “off label” use based on current regulatory guidelines. However, the flexibility of using quality DNA from different extraction methods would likely reduce the need for multiple DNA extractions, reduce cost, and reduce the amount of tissue needed to perform these important clinical tests.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 275, Wednesday Morning