A Molecular Touch-Prep Method for Preserving Tumor DNA from Fresh Clinical Specimens
JA Lefferts, ML Petras, A Suriawinata, GJ Tsongalis. Dartmouth-Hitchcock Medical Center, Dartmouth Medical School, Norris Cotton Cancer Center, Lebanon, NH
Background: Molecular diagnostic laboratories are facing an increasing demand for detecting mutations in tumor samples archived in formalin-fixed, paraffin-embedded (FFPE) tissue blocks. This testing has been driven in part by studies that led the FDA to update anti-EGFR antibody drug labels to mention their lack of benefit in colorectal cancer patients with tumors harboring KRAS codon 12 or 13 mutations. Due to the prevalence of FFPE tumor specimens, DNA isolated from this source is most easily obtained. However, processing of FFPE tissue results in partial fragmentation of DNA and carry-over of PCR inhibitors during extraction protocols. As a potential remedy to this situation, we evaluated the use of Whatman FTA paper cards for collection of fresh colorectal tumor samples before tissue fixation and for isolation of DNA for use in two different KRAS mutation assays.
Design: Eleven colon tumor samples were collected by making a cut into the fresh tumor and applying Whatman FTA paper to the cut surface. The samples were allowed to dry completely at room temperature and held for DNA extraction based on Whatman FTA protocols. KRAS mutation analysis was performed using seven independent custom TaqMan PCR assays (Applied Biosystems). Samples in which KRAS mutations were detected were also subjected to pyrosequencing analysis of KRAS. DNA from matched FFPE tissue blocks from these tumors were also collected for use in the TaqMan analysis.
Results: Of the eleven colon tumors sampled, TaqMan PCR analysis revealed that six were positive for mutations via the Whatman FTA paper preparations. The samples found to have mutations were confirmed by pyrosequencing and by performing the TaqMan PCR testing using DNA isolated from corresponding FFPE tumor specimens. In addition to the six specimens found to have mutations, there were two low-level/equivocal mutations identified via the FTA method which were not identified by pyrosequencing or with the TaqMan assay in DNA isolated from FFPE tissue.
Conclusions: Whatman FTA paper cards provide an intriguing alternative to FFPE tissue for collection of colorectal tumor samples for isolation of DNA and have many advantages including immediate specimen availability, faster DNA isolation protocol, decreased need for hazardous chemicals and potentially higher quality DNA, while still allowing for long-term storage of tumor specimens at room temperature before DNA isolation.
Monday, March 22, 2010 1:00 PM
Poster Session II # 242, Monday Afternoon