Comprehensive Determination of Prostate Tumor ETS Gene Status in Clinical Samples Using the CLIA Decipher Assay

Abstract

ETS family gene fusions are common in prostate cancer and molecularly define a tumor subset. ERG is the most commonly rearranged, leading to its overexpression, followed by ETV1, ETV4, and ETV5, and these alterations are generally mutually exclusive. We validated the Decipher prostate cancer assay to detect ETS alterations in a Clinical Laboratory Improvement Amendments-accredited laboratory. Benchmarking against ERG immunohistochemistry and ETV1/4/5 RNA in situ hybridization, we examined the accuracy, precision, and reproducibility of gene expression ETS models using formalin-fixed, paraffin-embedded samples. The m-ERG model achieved an area under curve of 95%, with 93% sensitivity and 98% specificity to predict ERG immunohistochemistry status. The m-ETV1, -ETV4, and -ETV5 models achieved areas under curve of 98%, 88%, and 99%, respectively. The models had 100% robustness for ETS status, and scores were highly correlated across sample replicates. Models predicted 41.5% of a prospective radical prostatectomy cohort (n = 4036) to be ERG⁺, 6.3% ETV1⁺, 1% ETV4⁺, and 0.4% ETV5⁺. Of prostate tumor biopsy samples (n = 509), 41.2% were ERG⁺, 8.6% ETV1⁺, 0.4% ETV4⁺, and none ETV5⁺. Higher Decipher risk status tumors were more likely to be ETS⁺ (ERG or ETV1/4/5) in the radical prostatectomy and the biopsy cohorts (P < 0.05). These results support the utility of microarray-based ETS status prediction models for molecular classification of prostate tumors.

Supplemental Figure S1

m-ERG (A) and ETV1 (B) model scores across 11 patients, each with 10 replicate samples, showing reproducibility and precision of models. The red dotted lines indicate the threshold of each model for calling ETS positivity.

Supplemental Figure S2

Examination of m-ERG and ETV1 gene expression models in prospective Decipher GRID RP specimens. A and B: Beeswarm plots of m-ERG and m-ETV1 model scores in the GRID RP cohort. The red dotted lines indicate the threshold of each model for calling ETS positivity. C–F: Heatmaps of ERG, ETV1, ETV4, ETV5 PSRs across GRID RP cohort showing the feasibility of predicting fusion using the Human Exon 1.0 ST arrays. GRID, Genomics Resource Information Database; PSR, probe selection region; RP, radical prostatectomy.

Figure 1

Examples of ERG IHC and ETV1/4/5 RISH results across the Johns Hopkins retrospective radical prostatectomy cohort. All depicted cases are concordant with m-ERG and ETV1/4/5 model calls by gene expression microarray. Original magnification, ×200. IHC, immunohistochemistry; RISH, RNA in situ hybridization.

Figure 2

Validation of the m-ERG and ETV1/4/5 gene expression models by comparison with IHC and RISH. A–D: Beeswarm plots of ERG, ETV1, ETV4, and ETV5 models in JHMI RP cohort showing model scores are highly concordant with IHC and RISH calls. The red dotted lines indicate the threshold of each model for calling ETS positivity. E and F: Density plots of m-ERG and ETV1 models in a subset of the JHMI RP cohort, prospective Decipher GRID RP, and biopsy cohorts and LNCaP cells with ETV1 fusion, showing distribution of models are highly similar across different data sets. ERG⁻, blue background; ERG⁺, mauve background. n = 358 JHMI RP cohorts (E and F); n = 4036 prospective Decipher GRID RP cohorts (E and F); n = 509 biopsy cohorts (E and F). GRID, Genomics Resource Information Database; IHC, immunohistochemistry; JHMI, Johns Hopkins Medical Institute; RISH, RNA in situ hybridization; RP, radical prostatectomy.

Figure 3

Examination of m-ERG and ETV1/4/5 gene expression models in prospective Decipher GRID RP and BX specimens. A: In GRID RP and BX data, ERG and ETV1/4/5 status are highly, although not entirely, mutually exclusive. B: Among cases in the RP cohort with high Decipher risk score compared with low Decipher risk score, a higher proportion of cases are m-ERG⁺. Among cases in the BX cohort with high Decipher risk score compared with low Decipher risk score, a high proportion of cases are ETV1/4/5⁺ (ETV⁺). BX, biopsy; GRID, Genomics Resource Information Database; RP, radical prostatectomy.