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1.
Introduction
Widespread screening combined with the rise in the aging
population has led to increased diagnosis of prostate cancer
(PCa), but the majority of men diagnosed are at low risk
[14_TD$DIFF]
for
disease progression
[1,2]. Rather than undergoing invasive
procedures, many men are opting for active surveillance,
whereby treatment is delayed until signs of overt progres-
sion
[2,3]. It would be advantageous to better understand
the risk of disease progression at the individual patient level
and to identify molecular features predictive of response for
specific interventions, which is the premise of precision
cancer prevention
[4].
Because of the critical dependence of PCa on the androgen
receptor (AR)
[5,6], many interventions for PCa target AR
signaling or androgen biosynthesis
[7] .Among these are
agents that target 5
a
-reductase, which catalyzes the
conversion of testosterone to dihydrotestosterone (DHT)
[8] .Although 5
a
-reductase inhibitors (5-ARIs), including
finasteride and dutasteride, have been evaluated in pro-
spective clinical trials for both primary prevention
[9–12]and secondary prevention for patients on active surveillance
[13,14] ,their benefits in these settings remain controversial.
We performed co-clinical analyses to evaluate the
phenotypic and molecular consequences of finasteride
treatment in a genetically engineered mouse model
(GEMM) that is analogous to low-risk PCa in humans
[15,16]. Using cross-species computational analyses to
compare finasteride treatment of GEMMs with retrospec-
tive cohorts of 5-ARI–treated patients, we show that
reduced expression of
NKX3.1
is a predictor of response
to 5-ARIs. We propose that expression of
NKX3.1
be
evaluated as a means of stratifying men on active
surveillance as candidates for intervention with 5-ARIs.
2.
Patients and methods
2.1.
Preclinical analyses
Experiments using animals were performed according to protocols
approved by the Institutional Animal Care and Use Committee at
Columbia University Medical Center.
Nkx3.1
wild-type (
Nkx3.1
+/+
[1_TD$DIFF]
) and
germline homozygous mutant (
Nkx3.1
/
) mice, and
Pten
germline
heterozygous mutant mice (
Pten
+/
) have been described previously
[16,17]. Finasteride (Kemprotec, Carnforth, UK) was dissolved in ethanol
at a concentration of 20 mg/ml and diluted using sterile phosphate-
buffered saline to a working stock of 1 mg/ml. Cohorts of mice were
randomly assigned to treatment with finasteride or vehicle. At sacrifice,
prostate tissues were fixed in 10% formalin and paraffin-embedded or
snap-frozen in liquid nitrogen. Histopathological grading was performed
according to the classification of Park et al
[18]. Immunohistochemical
staining was done as previously described
[19]; images were captured
using an Olympus VS120 whole-slide scanning microscope. Levels of
steroids in serum were determined by extraction with hexane/
dichloromethane (3:2 v/v) followed by purification using a XEVO TQS
tandem mass spectrometer (Waters, Milford, MA, USA) with a detection
limit of 10 pg/ml. Quantitative real-time polymerase chain reaction
(PCR) was carried out on RNA prepared using TRIzol reagent (Life
Technologies, Carlsbad, CA, USA) with a QuantiTect SYBR Green PCR kit
(Qiagen, Hilden, Germany)
[19]. RNA sequencing was performed using a
MagMAX-96 total RNA isolation kit (Life Technologies) as previously
described
[19]. Raw counts for RNA sequencing (RNAseq) data were
normalized and the variance was stabilized using the DESeq2 package
(Bioconductor) in R-studio 0.99.902, R v3.3.0.
2.2.
Patient cohorts
Patient specimens were obtained following protocols approved by the
institutional review board of Weill Cornell Medicine (WCM) and Fred
Hutchinson Cancer Research Center (FHCRC). Two independent retro-
spective patient cohorts were used for training and testing/validation
( Table 1 ). The WCM cohort (
n
= 9) included patients with clinically
localized PCa who had been receiving finasteride or dutasteride before
prostatectomy. The FHCRC cohort (
n
= 15) included samples collected as
part of the multicenter ARI40010 study
[20]from patients who had
received dutasteride for 4 mo before prostatectomy. Immunohistochem-
istry was performed on paraffin-embedded tissues with a rabbit
polyclonal NKX3.1 antibody (Biocare Medical, Pacheco, CA, USA) using
a Leica Bond III automated stainer (Leica Biosystems, Wetzlar, Germany),
and quantified using HALO software (Indica Labs, Corrales, NM, USA).
2.3.
Statistical analysis
Independent groups were compared using a two-tailed two-sample
Welch
t
test assuming that variances between the populations were not
equal. When two phenotypes were compared (
[16_TD$DIFF]
i.e., finasteride- vs
vehicle-treated samples), the Welch
t
test was applied to estimate the
difference in RNAseq counts (
[16_TD$DIFF]
i.e., differential expression) between these
phenotypes for each gene. Differential expression signatures were thus
defined as the list of genes ranked by
t
values from a two-tailed two-
sample
t
test comparing the finasteride- and vehicle-treated samples.
For comparison with human gene signatures, mouse genes were mapped
histopathological and expression profiling analyses. Cross-species computational anal-
ysis comparing finasteride-treated mice with two independent 5-ARI–treated patient
cohorts showed that reduced
NKX3.1
expression is predictive of response to 5-ARI. A
limitation of the study is that these retrospective human cohorts have relatively few
patients with limited clinical outcome data. Future prospective clinical trials are needed
to validate whether stratifying patients on the basis of
NKX3.1
expression improves the
benefit of 5-ARIs during active surveillance.
Conclusions:
This co-clinical study implicates
NKX3.1
status as a predictor of response to 5-
ARIs, and suggests that molecular features, including
NKX3.1
expression, may help to
identify PCa patients most likely to benefit from 5-ARIs during active surveillance.
Patient summary:
The aim of precision cancer prevention is to tailor interventions on the
basis of individualized patient characteristics. We propose that patients with low
NKX3.1
expression are optimal candidates for intervention with 5
a
-reductase inhibitors as an
adjunct to active surveillance.
#
2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.
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