Molecular profiling of ETS and non-ETS aberrations in prostate cancer patients from northern India

Abstract

Background: Molecular stratification of prostate cancer (PCa) based on genetic aberrations including ETS or RAF gene-rearrangements, PTEN deletion, and SPINK1 over-expression show clear prognostic and diagnostic utility. Gene rearrangements involving ETS transcription factors are frequent pathogenetic somatic events observed in PCa. Incidence of ETS rearrangements in Caucasian PCa patients has been reported, however, occurrence in Indian population is largely unknown. The aim of this study was to determine the prevalence of the ETS and RAF kinase gene rearrangements, SPINK1 over-expression, and PTEN deletion in this cohort.

Methods: In this multi-center study, formalin-fixed paraffin embedded (FFPE) PCa specimens (n = 121) were procured from four major medical institutions in India. The tissues were sectioned and molecular profiling was done using immunohistochemistry (IHC), RNA in situ hybridization (RNA-ISH) and/or fluorescence in situ hybridization (FISH).

Results: ERG over-expression was detected in 48.9% (46/94) PCa specimens by IHC, which was confirmed in a subset of cases by FISH. Among other ETS family members, while ETV1 transcript was detected in one case by RNA-ISH, no alteration in ETV4 was observed. SPINK1 over-expression was observed in 12.5% (12/96) and PTEN deletion in 21.52% (17/79) of the total PCa cases. Interestingly, PTEN deletion was found in 30% of the ERG-positive cases (P = 0.017) but in only one case with SPINK1 over-expression (P = 0.67). BRAF and RAF1 gene rearrangements were detected in ∼1% and ∼4.5% of the PCa cases, respectively.

Conclusions: This is the first report on comprehensive molecular profiling of the major spectrum of the causal aberrations in Indian men with PCa. Our findings suggest that ETS gene rearrangement and SPINK1 over-expression patterns in North Indian population largely resembled those observed in Caucasian population but differed from Japanese and Chinese PCa patients. The molecular profiling data presented in this study could help in clinical decision-making for the pursuit of surgery, diagnosis, and in selection of therapeutic intervention.

Keywords: PTEN; RAF kinase; SPINK1; TMPRSS2-ERG; genetic rearrangements.

Figure 1

IHC staining and FISH for ERG rearrangement and RNA‐ISH for ETV1. Panel A, whole section view of a prostate carcinoma tissue (Gleason score 3 + 3) showing ERG expression by IHC staining (panel on left). Right panel showing IHC staining for ERG over‐expression (×20 magnification) with the corresponding H&E stained sections (top). Bottom panel shows FISH for ERG genetic rearrangement using ERG break‐apart probes. Yellow arrow shows rearranged ERG, and co‐localizing red‐green signal depicts intact ERG locus. Panel B shows ETV1 expression by RNA‐ISH with a maximum intensity score 4 (×20 magnification).

Figure 2

IHC staining for SPINK1. Panel A and B show a prostatic adenocarcinoma with SPINK1 positive immunostaining with the corresponding H&E stained section; Gleason score 3 + 4 (×20 magnification). Panel C and D show a needle core biopsy which demonstrates SPINK1 positive immunostaining with the corresponding H&E stained section; Gleason score 3 + 3 (×4 and ×10 magnification in inset).

Figure 3

FISH for PTEN aberrations. Panel A, schematic diagram showing the genomic organization of PTEN gene on Chr10 q23.3 and chromosome 10 control probe (q11.12). Green and red bars indicate the chromosome 10 control probe and PTEN BAC clones, respectively. FISH images of the PCa specimens, PCA‐87 shows normal PTEN copy number with two red and two green signals corresponding to PTEN and Chr10 control probe, respectively. PCA‐74 shows heterozygous deletion of PTEN showing loss of one red signal, but two green centromere signals. PCA‐77 depicts homozygous deletion of PTEN (loss of red signals), but two green centromere signals. PCA‐78 shows aneuploidy with 3‐4 copies of chromosome 10 and PTEN. Panel B, percent distribution of the PTEN aberrations. Panel C shows ERG genetic rearrangement using ERG break‐apart probes and homozygous deletion of PTEN in the same patient (PCA‐61). Likewise, PCA‐70 shows ERG genetic rearrangement and normal PTEN status.

Figure 4

FISH for RAF genetic rearrangements. Panel A, schematic diagram showing the genomic organization of BRAF gene on chr7q34. Green and red bars indicate the 5' and 3' BAC clones, respectively. FISH images of the PCa specimens (PCA‐73 & 25) show BRAF amplification (left) and BRAF rearrangement (middle) in PCA‐77 (right). Panel B, schematic diagram showing the genomic organization of the RAF1 gene on chr3p25. Green and red bars indicate 5' and 3' BAC clones, respectively. FISH images of the PCa specimens (PCA‐40) show RAF1 rearrangement with 5' deletion (green signal missing), 3' deletion (red signaling missing), and 5' deletion with 3 copies of normal RAF1 in the top panel. Likewise, PCa specimens PCA‐23, PCA‐16, and PCA‐56 show RAF rearrangement, amplification, and 3' deletion, respectively (bottom panel).