A Simple and Cost-Effective Method of DNA Extraction from Small Formalin-Fixed Paraffin-Embedded Tissue for Molecular Oncology Testing: A Practical Approach
Anthony N Snow, Aaron A Stence, Jonathan A Pruessner, Susan Forde, Shelly Edler, Aaron D Bossler, Deqin Ma. University of Iowa Hospitals and Clinics, Iowa City, IA
Background: Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncology testing. This is particularly challenging with tiny biopsies with limited tumor cells in a background of non-tumor cells. It is cost prohibitive for many laboratories to invest in laser capture microdissection. We compared our standard procedure of manually scraping tissue from unstained slides with a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides and optimized the DNA extraction protocol.
Design: FFPE tissue blocks were selected from previously tested cases from our laboratory. Areas with tumor ranging from 1-2 mm2 were marked by a pathologist. Sections were either scraped with a razor blade or deparaffinized and H&E stained for tumor cell capture using the Pinpoint solution. Tissue bound to the solution was microdissected, and genomic DNA was isolated according to the manufacturer's instructions for the Pinpoint Slide DNA Isolation or QIAamp DNA FFPE Tissue kit (Qiagen, Inc.).
Results: DNA yield was 10-fold higher using the QIAamp method compared with the Pinpoint method when following the manufacturer's recommendation of a 4 hour proteinase K (PK) digestion. Although longer incubation with PK increased the DNA yield from this method, it was still about 60 ng/mm2 less than with the QIAamp method. Mutations detected included BRAF V600E in melanoma by primer extension, a 15 base pair deletion and a SNP in the EGFR gene from a transbronchial lung biopsy using Sanger sequencing, G12D and G12C mutations in KRAS from colon biopsies by primer extension, and a multiplex PCR-based assay for microsatellite instability from a colon biopsy with clusters of signet ring cells in pools of mucin. DNA from both kits gave equivalent results on all molecular tests performed. When significantly less DNA (about 90% decrease on average) was used, the test performance was not affected in comparison with the original testing. Cost analysis revealed a savings of 24.4% with the new procedure.
Conclusions: Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of tissue from the slides easier. Blending the two kits increased yield and reduced cost. Valid test results were obtained across multiple molecular oncologic testing platforms with 90% less tissue and 70.9% to 93.6% less DNA. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 278, Wednesday Morning