Evaluating the repair of DNA derived from formalin-fixed paraffin-embedded tissues prior to genomic profiling by SNP–CGH analysis

Abstract

Pathology archives contain vast resources of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples. Owing to the methods of tissue fixation and storage, the integrity of DNA and RNA available from FFPE tissue is compromized, which means obtaining informative data regarding epigenetic, genomic, and expression alterations can be challenging. Here, we have investigated the utility of repairing damaged DNA derived from FFPE tumors prior to single-nucleotide polymorphism (SNP) arrays for whole-genome DNA copy number analysis. DNA was extracted from FFPE samples spanning five decades, involving tumor material obtained from surgical specimens and postmortems. Various aspects of the protocol were assessed, including the method of DNA extraction, the role of Quality Control quantitative PCR (qPCR) in predicting sample success, and the effect of DNA restoration on assay performance, data quality, and the prediction of copy number aberrations (CNAs). DNA that had undergone the repair process yielded higher SNP call rates, reduced log R ratio variance, and improved calling of CNAs compared with matched FFPE DNA not subjected to repair. Reproducible mapping of genomic break points and detection of focal CNAs representing highlevel gains and homozygous deletions (HD) were possible, even on autopsy material obtained in 1974. For example, DNA amplifications at the ERBB2 and EGFR gene loci and a HD mapping to 13q14.2 were validated using immunohistochemistry, in situ hybridization, and qPCR. The power of SNP arrays lies in the detection of allele-specific aberrations; however, this aspect of the analysis remains challenging, particularly in the distinction between loss of heterozygosity (LOH) and copy neutral LOH. In summary, attempting to repair DNA that is damaged during fixation and storage may be a useful pretreatment step for genomic studies of large archival FFPE cohorts with long-term follow-up or for understanding rare cancer types, where fresh frozen material is scarce.

High-Throughput Detection of Clinically Relevant Mutations in Archived Tumor Samples by Multiplexed PCR and Next-Generation Sequencing

“…Figure 2C shows that FFPE-associated deamination was a very significant source of false positives for some samples, and the ruler assay was strongly predictive for this effect (presumably due to reduced primer annealing). Strategies exist for repairing (26) or selecting against (27,28) deaminated DNA, and such preprocessing steps may yield improved sequencing accuracy for degraded samples. Indeed, pretreatment of FFPE DNA samples with UDG to remove uracil-containing deaminated DNA molecules resulted in dramatic reduction in false positives compared with those without UDG treatment (Fig.…”

Genome‐wide DNA methylation analysis of formalin‐fixed paraffin embedded colorectal cancer tissue

“…Although DNA from formalin fixed paraffin embedded (FFPE) tissue has been used with some genome‐wide microarray applications with varying success (Lips et al, ; Thompson et al, ; Jacobs et al, ; Oosting et al, ; Tuefferd et al, ; Alvarez et al, ; Hosein et al, ), its potential for use with the suite of technology platforms now available for genome‐wide DNA methylation analysis has been largely untested (Thirlwell et al, ; Jasmine et al, ). The fixation process and long term storage of tissue embedded in paraffin can lead to DNA damage due to fragmentation, nucleotide base lesions, modified bases, and cross linking (Iwamoto et al, ; Srinivasan et al, ) resulting in poor quality DNA not ideally suited for genome‐wide studies; yet, archiving of clinical samples by this method is common.…”

Association of the DYX1C1 Gene with Chinese Literacy in a Healthy Chinese Population

“…Although the integrity of DNA could vary due to sample storage and processing issues and such variation could lead to variance of log-R ratio as shown by pathology archives of clinical material in the form of formalin-fixed paraffin-embedded (FFPE) tissue samples [39] , we do not think the log-R association observed is due to sample quality issues due to our vigilance in sample storage using high quality collection kits (Oragene) that is well tested, and our prompt processing of the saliva samples as soon as they are collected by a research assistant dedicated to this project, and also in light of the fact that the association is only observed in latent class 2 and not in latent class 1 samples. From the value of log-R ratio we observed (lower than 0), this region of DYX1C1 appeared to be altered by deletion rather than by amplification.…”