High quality copy number and genotype data from FFPE samples using Molecular Inversion Probe (MIP) microarrays.

Yuker Wang,Zhigang C Wang,Ralf Krahe,Melissa Bondy,Andrea L Richardson,Axel Walther,Musaffe Tuna,Ronald Sapolsky,Martin Moorhead,Aysegul Sahin,Robert Warren,Malek Faham,Gordon B Mills,Victoria Eh Carlton,Joe W Gray,Patricia A Thompson,George Karlin-Neumann,Paul T Spellman,Li Zhang

doi:10.1186/1755-8794-2-8

Abstract

BackgroundA major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. As FFPE protocols may vary widely between labs and samples may be stored for decades at room temperature, an ideal FFPE CN technology should work on diverse sample sets. Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from cell line and frozen tumor DNA. Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess degraded FFPE DNA. We assessed CN with a MIP panel of 50,000 markers in 93 FFPE tumor samples from 7 diverse collections. For 38 FFPE samples from three collections we were also able to asses CN in matched fresh frozen tumor tissue.ResultsUsing an input of 37 ng genomic DNA, we generated high quality CN data with MIP technology in 88% of FFPE samples from seven diverse collections. When matched fresh frozen tissue was available, the performance of FFPE DNA was comparable to that of DNA obtained from matched frozen tumor (genotype concordance averaged 99.9%), with only a modest loss in performance in FFPE.ConclusionMIP technology can be used to generate high quality CN and genotype data in FFPE as well as fresh frozen samples.

Highlights

A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE)
While in previous studies with fresh frozen and cell line DNA, we found that the reference set selection had little impact on final data quality, we noted that for FFPE samples reference set selection markedly affected data quality
We have previously described the performance of the Molecular Inversion Probe (MIP) technology using receiver operating characteristic (ROC) curves with true positive/false positive analysis in samples with known CN changes

Summary

Introduction

A major challenge facing DNA copy number (CN) studies of tumors is that most banked samples with extensive clinical follow-up information are Formalin-Fixed Paraffin Embedded (FFPE). DNA from FFPE samples generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking during FFPE fixation and processing. Many genomic assays fail to produce high quality CN and genotype data from FFPE samples [4,5,6,7,8,9,10], restricting the application of these promising whole genome scanning technologies to the limited number of fresh frozen samples. The chemical damage and modifications that FFPE DNA may suffer from can inhibit the enzyme-dependent chemistries necessary for a number of approaches [11] This damage is reflected in the high rate of sequencing artifacts and genotyping failures seen with FFPE extracted DNA [13,14]. When genotypes are measured, FFPE samples have substantially lower call rates (suggesting a loss of performance) and genotype discordances between FFPE and fresh frozen samples raise troubling questions about data reliability [5,6,7,8,9,10]

Results

Discussion

Conclusion