Determination of ERG(+), EZH2, NKX3.1, and SPINK‐1 subtypes to evaluate their association with clonal origin and disease progression in multifocal prostate cancer

Abstract

cause death. The criteria to establish PCa prognosis include PSA level, Gleason score, and clinical TNM stage, but these criteria still do not establish the prognosis with su cient certainty (2).Many studies have established different classi cations of PCa in molecular subtypes that can contribute to determining the prognosis, among them ETS(+) subtypes, characterized by chromosomal fusions, which cause the overexpression of ETS transcription factors. The most prevalent ETS fusion in PCa is TMPRSS2-ERG, which leads to ERG overexpression (3-5). ERG overexpression leads to an increase in the expression of EZH2, which acts as a histone methyltransferase enzyme, as well as to a decrease in the expression of the tumor suppressor gene NKX3.1, which is an androgen-regulated prostate-speci c homeobox gene and a transcription factor. NKX3.1 also binds to TMPRSS2 upstream sequences, and in TMPRSS2-ERG cases, it negatively regulates ERG expression and constitutes a negative feedback control since ERG in turn directly represses NKX3.1 (6,7). EZH2 and NKX3.1 had also been described as PCa subtypes. Another subtype reported in PCa is characterized by SPINK-1 overexpression, which is speci cally overexpressed in a subset of ETS(-) and is associated with another subtype, the SPOP subtype, characterized by a mutation in this gene (3). SPINK-1 protein is a trypsin inhibitor, which can function as an autocrine growth factor (8,9), and SPOP is an E3 ubiquitin ligase substrate-binding subunit of the proteasome complex that mediates the ubiquitination of target proteins, leading more frequently to their proteasomal degradation (10).The prognostic relevance of PCa molecular subtypes remains controversial (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). The presence of multiple foci -with the possibility of different subtypes-in the same patient with PCa could contribute to the di culty of predicting the clinical behavior of a speci c PCa subtype (21-23). It is not well known whether these foci have a monoclonal origin that expands through the prostate, giving rise to new foci, and share some molecular characteristics by origin, which begins to acquire changes in subclonal events; or whether each focus appears independently, so they have a multiclonal origin and they are expected to be very heterogeneous at the molecular level (22,24,25).To help elucidate the origin of heterogeneity in PCa and its association with disease progression, ERG(+), SPOP, EZH2, SPINK-1, and NKX3.1 subtypes were analyzed in 83 samples from prostatic foci and regional metastasis to lymph nodes (LN) from 20 PCa patients with poor prognosis. We also established molecular patterns in samples and patients, combining the presence of all subtypes analyzed. Methods SamplesSamples from formalin-xed para n-embedded (FFPE) tissues obtained from radical prostatectomy (RP) of 20 PCa patients with poor prognosis (regional metastases and/or biochemical recurrence (BCR)) and 5-years of follow-up were included. Cases were selected from the project "Exploration of potential predictor b...