Expression profiling of formalin-fixed paraffin-embedded primary breast tumors using cancer-specific and whole genome gene panels on the DASL® platform

Abstract

Background: The cDNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel v1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.

Methods: Bland-Altman plots and Spearman correlations evaluated intra/inter-panel agreement of normalized expression values. Unequal-variance t-statistics tested for differences in expression levels between HER2 + and HER2 - tumors. Regulatory network analysis was performed using Metacore (GeneGo Inc., St. Joseph, MI).

Results: Technical replicate correlations ranged between 0.815-0.956 and 0.986-0.997 for the 1.5K and 24K panels, respectively. Inter-panel correlations of expression values for the common 498 genes across the two panels ranged between 0.485-0.573. Inter-panel correlations of expression values of 17 probes with base-pair sequence matches between the 1.5K and 24K panels ranged between 0.652-0.899. In both panels, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) was the most differentially expressed gene between the HER2 + and HER2 - tumors and seven additional genes had p-values < 0.05 and log2 -fold changes > |0.5| in expression between HER2 + and HER2 - tumors: topoisomerase II alpha (TOP2A), cyclin a2 (CCNA2), v-fos fbj murine osteosarcoma viral oncogene homolog (FOS), wingless-type mmtv integration site family, member 5a (WNT5A), growth factor receptor-bound protein 7 (GRB7), cell division cycle 2 (CDC2), and baculoviral iap repeat-containing protein 5 (BIRC5). The top 52 discriminating probes from the 24K panel are enriched with genes belonging to the regulatory networks centered around v-myc avian myelocytomatosis viral oncogene homolog (MYC), tumor protein p53 (TP53), and estrogen receptor α (ESR1). Network analysis with a two-step extension also showed that the eight discriminating genes common to the 1.5K and 24K panels are functionally linked together through MYC, TP53, and ESR1.

Conclusions: The relative RNA abundance obtained from two highly differing density gene panels are correlated with eight common genes differentiating HER2 + and HER2 - breast tumors. Network analyses demonstrated biological consistency between the 1.5K and 24K gene panels.

Figure 1

Bland-Altman plots displaying intra-panel agreement of technical and extract replicates. a. Technical-replicate agreement of normalized expression for the 1.5K panel. b. Technical-replicate agreement of normalized expression for the 24K panel. c. Extract-replicate agreement of normalized expression for the 1.5K. d. Extract-replicate agreement of normalized expression for the 24K panel. The vertical axis denotes the difference in expression values between the technical replicates and the horizontal axis denotes the average expression for each of the 498 genes in common. A local regression line is superimposed on figures a-d. e. Summarized standard deviations of the differences for each of the eight samples with technical replicates and eight samples with extract replicates.

Figure 2

Bland-Altman plots displaying inter-panel agreement of common probes. a. Inter-panel agreement for the 17 sequence-matched probes for a single representative sample, with a local regression line superimposed. b. A local regression line representing each of the 20 samples. c. Inter-panel agreement for the 498 gene symbols in common across the 1.5K and 24K panels for a single sample, with a local regression line superimposed. d. A local regression line for each of the 20 samples.

Figure 3

ERBB2 gene expression. a. ERBB2 (HER2) expression for the three probes on the 1.5K panel. b. ERBB2 (HER2) expression for the three probes on the 24K panel. From left to right the three probes are ordered from the 5-prime to 3-prime end of ERBB2. The ERBB2 probe positions for the 1.5K panel are: 607 for GI.4758297.S.1789-5prime; 1192 for GI.4758297.S.1787-middle; and 4106 for GI.4758297.S.1786-3prime. The ERBB2 probe positions for the 24K panel are: 47 for ILMN_1717902-5prime; 370 for ILMN1728761-middle; and 4390 for ILMN_2352131-3prime. Internal to each probe the samples are sorted by HER2 IHC 0-3+ values. Open circles represent tumors with HerceptTest IHC scores of 0-1+ and closed triangles represent tumors with HerceptTest IHC scores of 2-3+.

Figure 4

Fold change agreement of genes differentiating HER2+ versus HER2- tumors. a. Agreement of fold change estimates for the 1.5K and 24K panels for a range of noise thresholds. b. Agreement of fold change using a noise threshold of |0.20|, i.e., considering only genes that have a log2-fold change large than |0.20| in at least one of the panels.

Figure 5

MetaCore network analyses. The top 47 genes that matched to the probes that were differentially expressed between HER2+ and HER2- samples (p-values < 0.01 and log2-fold change > |1.0|) in the 24K panel are enriched with genes (marked by red cycles) belonging to two distinct regulatory networks. a. The first network includes genes (marked by red cycles) functionally connected to MYC (c-Myc). b. The second network is enriched by genes (marked by red cycles) centered around TP53 (p53) and ESR1. c. The 8 genes differentially expressed in both the 24K and 1.5K panels (marked by red cycles) are all connected in a network that includes TP53 (p53), ESR1, and MYC (c-Myc) d. Symbol legend. Abbreviations for gene names presented in Figure 5 are defined in Additional File 1, Tables S1-S3.