Optimizing DNA Extraction from Archival Prostate Biopsy Samples.

PURPOSE: In this paper, we compare the performance of three distinct nucleic acid extraction protocols in a large population-based prostate cancer cohort, aiming to optimize DNA yield from limited tumor samples in prostate needle biopsies.

MATERIAL AND

METHODS

We utilized two commercially available extraction kits and identified the minimum digitally measured tumor area yielding sufficient DNA for sequencing panels (defined as >50 ng of total DNA). Representative FFPE prostate cancer biopsies were processed from 643 patients recruited through population-based U.S. cancer registries as part of the multi-institutional RESPOND study. Scanned whole slide images were used to annotate and measure the tumor region prior to manual macrodissection and DNA isolation using the extraction kits.

RESULTS

The QIAamp® FFPE Advanced® kit resulted in the highest median DNA yield compared to combined RNA/DNA or DNA extraction using the AllPrep® kit (4.3 vs 2.1 or 2.1 ng per mm2 total tumor, p=0.0004 and p<0.0001, respectively). DNA yield significantly correlated with total tumor area for both kits. Using a tumor area cutoff of 2 mm2 for the Advanced® kit, or 5 mm2 for the AllPrep® DNA/RNA kit, with macrodissection across ten 4 μm tissue sections, 86% to 94% of samples generated more than 50 ng of total DNA. Both kits resulted in comparable panel-based sequencing quality metrics, including percentage duplicated reads and median insert length. For DNA quantity and median target coverage at sequencing, the Advanced® kit outperformed the AllPrep® kit, but it does not allow simultaneous RNA extraction.

CONCLUSIONS

For a DNA yield of at least 50 ng, we suggest a cutoff of 2 mm2 of tumor area with ten 4 μm macrodissected tissue sections for a prostate cancer needle core biopsy to be considered sufficient for DNA extraction.