Novel RNA Hybridization Method for the In Situ Detection of ETV1, ETV4, and ETV5 Gene Fusions in Prostate Cancer

Abstract

The genetic basis of 50–60% of prostate cancer is attributable to rearrangements in ETS (ERG, ETV1, ETV4, and ETV5), BRAF and RAF1 genes and overexpression of SPINK1. The development and validation of reliable detection methods are warranted to classify various molecular subtypes of prostate cancer for diagnostic and prognostic purposes. ETS gene rearrangements are typically detected by fluorescence in situ hybridization (FISH) and reverse transcription PCR methods. Recently, monoclonal antibodies against ERG have been developed that detect the truncated ERG protein in immunohistochemical assays where staining levels are strongly correlated with ERG rearrangement status by FISH. However, specific antibodies for ETV1, ETV4 and ETV5 are unavailable, challenging their clinical use. We developed a novel RNA in situ hybridization based assay for the in situ detection of ETV1, ETV4, and ETV5 in formalin fixed paraffin embedded tissues from prostate needle biopsies, prostatectomy, and metastatic prostate cancer specimens using RNA probes. Further, with combined RNA in situ hybridization and immunohistochemistry we identified a rare subset of prostate cancer with dual ETS gene rearrangements in collisions of independent tumor foci. The high specificity and sensitivity of RNA in situ hybridization provides an alternate method enabling bright field in situ detection of ETS gene aberrations in routine clinically available prostate cancer specimens.

Prostate Cancer SubtyPINg BiomarKers and Outcome: Is Clarity EmERGing?

“…If ERG rearrangements occurred at any point in PCa development and progression, one would expect to observe with some frequency small pockets of nascent ERG + cancer arising within large ERG − tumor foci, which has never been reported. In contrast, we and others have published examples of apparent ERG + /ERG − , ERG + /ETV1 + and ERG + /SPOP mut cases which represent “collisions” of genetically distinct foci on whole section evaluation(4, 8, 11, 12). …”

Characterization of 1577 Primary Prostate Cancers Reveals Novel Biological and Clinicopathologic Insights into Molecular Subtypes

“…We also identified a total of 3% of cases with m-ERG + /m-ETS + or m-ERG + /m-SPINK1 + profiles. In our experience, ERG , non- ERG ETS and SPINK1 subtype defining alterations are nearly always mutually exclusive, and observed co-occurrence is most likely due to either misclassification (given the lack of gold standard training data for non-ERG classifiers) or profiling of collisions between genetically distinct tumor clones (which may appear morphologically indistinguishable), although exceptionally rare examples of focal SPINK1 expression in otherwise ERG + tumor have been reported[7,37,38]. As shown by multivariate analysis (Table S8), conflict cases identified herein show similar clinicopathological associations as m-ERG + PCa, consistent with an enrichment of m-ERG + tumors in these conflict cases Thus, studies are ongoing to generate gold standard data for these non-ERG based classifiers.…”

“…Approximately 10% of PCa, which are nearly exclusively negative for ERG or other ETS gene fusions (ETS − ), harbor marked SPINK1 over-expression, consistent with a unique molecular subtype (SPINK1 + )[3,5]. Although we and others have validated antibodies (against ERG and SPINK1) and fluorescence in situ hybridization (FISH) or RNA in situ hybridization (RISH) assays (against ETV1 , ETV4 , and ETV5 )[4,6,7], routine comprehensive subtyping remains challenging and is cost prohibitive given the lack of current clinical indications.…”

Somatic molecular subtyping of prostate tumors from HOXB13 G84E carriers

“…ETV1 (NM_004956), ETV4 (NM_001986.2) and ETV5 (NM_004454.2) probes, validated in a recent study, were utilized [38]. Probes for PPIB (NM_000942.4) were used as positive control.…”