Comparing Platforms for Messenger RNA Expression Profiling of Archival Formalin-Fixed, Paraffin-Embedded Tissues

Abstract

Archival formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a readily available but largely untapped resource for gene expression profiling-based biomarker discovery. Several technologies have been proposed to cope with the bias from RNA cross-linking and degradation associated with archival specimens to generate data comparable with RNA from fresh-frozen materials. Direct comparison studies of these RNA expression platforms remain rare. We compared two commercially available platforms for RNA expression profiling of archival FFPE specimens from clinical studies of prostate and ovarian cancer: the Affymetrix Human Gene 1.0ST Array following whole-transcriptome amplification using the NuGen WT-Ovation FFPE System V2, and the NanoString nCounter without amplification. For each assay, we profiled 7 prostate and 11 ovarian cancer specimens, with a block age of 4 to 21 years. Both platforms produced gene expression profiles with high sensitivity and reproducibility through technical repeats from FFPE materials. Sensitivity and reproducibility remained high across block age within each cohort. A strong concordance was shown for the transcript expression values for genes detected by both platforms. We showed the biological validity of specific gene signatures generated by both platforms for both cohorts. Our study supports the feasibility of gene expression profiling and large-scale signature validation on archival prostate and ovarian tumor specimens using commercial platforms. These approaches have the potential to aid precision medicine with biomarker discovery and validation.

Figure 1

A–D: Correlations between technical replicates as a function of block age for all platforms and cohorts. Pairs of technical replicates from the same individual are shown by the same symbol. For prostate specimens normal tissue pairs are shown in gray, tumor is shown in black. E–H: Percentage of the genes that are either above or below the intensity threshold in each sample in a technical replicate pair. Each pair of replicates is plotted with an individual curve. J–L: Scatterplots of correlations between gene expression values for each gene represented on a platform across technical replicates. I and K: Presented as smoothed scatters in which color depth represents the density of the points in the plot for better visibility.

Figure 2

A and B: Bubble chart showing the relationship between the correlations of the gene expression values on the NuGen + Affymetrix and NanoString platforms and the average expression levels of those genes. Each circle represents a gene and the size is proportional to the absolute value of the correlations. Red represents positive correlations and blue represents negative correlations. C and D: Density plots of the observed correlations between the gene expression values on the NuGen + Affymetrix and NanoString platforms (solid lines) and the correlations under the null hypothesis of no association between the expression values measured on the NuGen + Affymetrix and NanoString platforms obtained by permuting sample labels (dashed lines).

Figure 3

Genes from the signatures that are represented on each platform were projected onto two principal components and plotted. Colors represent the compared classes and distinct plotting characters represent the technical replicates from the same specimen. A: NuGen + Affymetrix profiles of tumor versus normal signature on prostate samples. Tumor samples are shown in orange and normal samples are shown in blue. B: NanoString profiles of tumor versus normal signature on prostate samples. Tumor samples are shown in orange and normal samples are shown in blue. C: NuGen + Affymetrix profiles of Gleason signature on prostate tumor samples. Gleason 6 samples are shown in blue, Gleason 7 samples are shown in green, and Gleason 8 samples are shown in orange. D: NanoString profiles of Gleason signature on prostate tumor samples. Gleason 6 samples are shown in blue, Gleason 8 samples are shown in orange. E: NuGen + Affymetrix profiles of histology signature on ovarian samples. Clear cell carcinoma samples are shown in blue and serous carcinoma samples are shown in orange. F: NanoString profiles of histology signature on ovarian samples. Clear cell carcinoma samples are shown in blue and serous carcinoma samples are shown in orange. PCA, principal components analysis.