Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue

Abstract

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for clinical research. However, nucleic acids extracted from FFPE tissues are fragmented and chemically modified making them challenging to use in molecular studies. We analysed 23 fresh-frozen (FF), 35 FFPE and 38 paired FF/FFPE specimens, representing six different human tissue types (bladder, prostate and colon carcinoma; liver and colon normal tissue; reactive tonsil) in order to examine the potential use of FFPE samples in next-generation sequencing (NGS) based retrospective and prospective clinical studies. Two methods for DNA and three methods for RNA extraction from FFPE tissues were compared and were found to affect nucleic acid quantity and quality. DNA and RNA from selected FFPE and paired FF/FFPE specimens were used for exome and transcriptome analysis. Preparations of DNA Exome-Seq libraries was more challenging (29.5% success) than that of RNA-Seq libraries, presumably because of modifications to FFPE tissue-derived DNA. Libraries could still be prepared from RNA isolated from two-decade old FFPE tissues. Data were analysed using the CLC Bio Genomics Workbench and revealed systematic differences between FF and FFPE tissue-derived nucleic acid libraries. In spite of this, pairwise analysis of DNA Exome-Seq data showed concordance for 70-80% of variants in FF and FFPE samples stored for fewer than three years. RNA-Seq data showed high correlation of expression profiles in FF/FFPE pairs (Pearson Correlations of 0.90 +/- 0.05), irrespective of storage time (up to 244 months) and tissue type. A common set of 1,494 genes was identified with expression profiles that were significantly different between paired FF and FFPE samples irrespective of tissue type. Our results are promising and suggest that NGS can be used to study FFPE specimens in both prospective and retrospective archive-based studies in which FF specimens are not available.

Figure 1. Isolation of RNA from FF and FFPE tissues and generation of RNA-Seq libraries.

(A) Bioanalyzer profiles and yields of RNA isolated from FF tissue and from sections of the matching FFPE block of a normal tonsil (reactive) sample. RNA was isolated in triplicate (representative profile shown) from sections of the FFPE block using three different purification kits. Yields of RNA and purification kits are stated in the text above each profile. (B) Bioanalyzer profiles of RNA (left) isolated by ExpressArt FFPE RNAready (Amp Tec) from FFPE samples of normal tonsil (reactive) stored for different times as indicated above the profile together with the yields. Isolations were conducted in triplicate and a representative profile is shown. The resulting profiles of RNA-Seq libraries (right, with library ID top-right), with or without preceding fragmentation of the RNA as indicated in the middle section.

Figure 2. Single nucleotide variants detected in DNA-Exome-Seq data from the paired FF/FFPE samples.

The percentage of common (grey), exclusively FF (white) and exclusively FFPE (red) single nucleotide variants are shown as bars referring to the left axis. The average coverage is shown as stars for FF (white) and FFPE (red) on the right axis. Patient ID and number of years of storage are shown below.

Figure 3. Post mapping results from RNA-Seq of the paired FF/FFPE colon set.

(A) The fractions of reads mapping to the different targets. (B) Fractions of non-perfect matches among matches in RNA-Seq forward. (C) Fractions of reads mapping to total exon regions (total intron is the remaining fraction up to 100%). Data from FF and FFPE specimens are shown as white bars and red bars, respectively.

Figure 4. Effects of storage time on post-mapping results from the paired FF/FFPE samples.

Fractions of non-specifically mapped DNA-Exome-Seq (A) and RNA-Seq (B) reads; fractions of non-perfectly mapped DNA-Exome-Seq (C) and RNA-Seq (D) reads for FF (black) and FFPE (red) for each of the ten samples by number of years since sampling.

Figure 5. Effect of the FFPE process on the expression profiles.

(A) Correlation plots of log₂(1+RPKM) values for FF and FFPE colon samples stored for 13 years (top) and 2 years (bottom). (B) Pearson correlation coefficients of log₂(1+RPKM) values for FF vs. FFPE from the three paired FF/FFPE sample sets. (C) Intersection analyses of the results from paired statistical tests for significantly (FDR<0.05) affected genes comparing FF and FFPE samples within each sample set (divided into all affected, reduced and increased genes). (D) Plots of log-fold change values of the 1,494 genes affected in common, among all affected genes, for each pairwise combination of the three tissue sets with numbers of genes indicated for each quadrant.

Figure 6. MDS analysis of expression profiles of the colon trio-sets.

MDS analysis of the expression profiles of the six trio-sets (FF and FFPE samples from colon tumours with adjacent normal colon tissue) from the paired FF/FFPE colon set. CX: patient ID; T: tumour; N: normal; FF: fresh frozen; PE: FFPE.

Figure 7. MDS analysis and pairwise plots of the expression profiles from bladder specimens.

(A) MDS analysis of the ten FF bladder carcinoma samples and the three FF/FFPE pairs of Ta tumours based on the expression profiles of the genes included in the previously published 12-gene expression signature for progression in bladder carcinoma. Pro: progressing carcinoma; np: non-progressing carcinoma; FF: fresh frozen; PE: FFPE. (B) Pairwise plots of log₂(1+RPKM) values from the three FF/FFPE paired samples with Pearson correlation coefficients noted in the bottom-right corner.