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1.
Introduction
In prostate cancer the only predictive biomarkers of
resistance to therapy are the androgen receptor (AR) splice
variants and DNA-repair deficiency, relative to hormonal
therapy (HT) and poly(adenosine diphosphate-ribose)
polymerase inhibitors, respectively
[1,2]. The clinical
condition of patients and radiographic or symptomatic
progression are still the key parameters for therapeutic
intervention
[3], whereas prostate-specific antigen (PSA) is
a poor predictor of clinical response to HT or chemothera-
py. Castration-resistant prostate cancer (CRPC) is managed
by taxane-based chemotherapy (docetaxel, cabazitaxel),
anti-AR therapies (abiraterone acetate, enzalutamide),
immunotherapies (sipuleucel-T6), and radium-223. Un-
fortunately, a validated biomarker for predicting the
outcome of second-line HT in CRPC is still elusive
[4]. Antonarakis et al
[1]found in circulating tumor cells
(CTCs) that one splicing variant, androgen receptor splice
variant 7 (AR-V7), is associated with resistance to
enzalutamide and abiraterone. AR-splice variants are
truncated receptor isoforms lacking the C-terminal li-
gand-binding domain (LBD) that is a key regulator region of
the full-length AR (AR-FL). The LBD is responsible for
androgen-dependent receptor activity and the target of
flutamide, bicalutamide, and enzalutamide
[5,6] .There-
fore, LBD deletion results in loss of the antiandrogen
binding site and constitutive activation of AR-V7
[6]. AR-V7
is the hallmark of biological disease progression, and its
detection can be of strategic importance for treatment
management. To implement this test into clinical practice,
it should be highly sensitive, specific and easy to perform,
and cost effective. AR-V7 may be detected in tumor tissue
[7], CTCs
[1,8] ,or inmessenger RNA (mRNA) extracted from
whole blood
[9]. Unfortunately, these methods have
substantial limitations: a biopsy to assess the molecular
tumor evolution and heterogeneity is invasive and not
always feasible, and the cost and complexity of isolating
CTCs and the low sensitivity of mRNA extracted fromwhole
blood are relevant drawbacks. Even though it has been
demonstrated in vitro and in a few studies in patients that
AR-V7 is a biomarker of resistance, its role needs to be
confirmed. The present study aimed at confirming the role
of AR-V7 to predict resistance to HT and developing a new
methodological approach based on digital droplet poly-
merase chain reaction (ddPCR) to assess this marker
reliably. Plasma-derived exosomal RNA was used as the
source material, and the study provided new data to
address the correlation between exosomal AR-V7 and
therapy resistance, given that the translation of available
data on CTCs to exosomes is not obvious.
2.
Materials and methods
2.1.
Extraction of VCaP AR-V7 RNA and its detection by digital
droplet polymerase chain reaction
VCaP cells (ATCC CRL-2876) were used to set up the ddPCR
method because they are known carriers of AR-V7. RNA was
extracted from VCaP using the RNeasy Mini Kit (Qiagen,
Valencia, CA, USA), transcribed into complementary DNA
(cDNA) and amplified using the Duplex One-Step RT-ddPCR
Kit (Bio-Rad, Hercules, CA, USA). Primers and probes for both
AR-FL and AR-V7 were designed in our laboratory by Primer3
software (ThermoFisher Scientific, Waltham, MA, USA). FAM/
HEX labeling and primer synthesis were done by Bio-Rad:
AR-FL forward primer: 5
0
-CATCAAGGAACTCGATCGT-3
0
; AR-
FL reverse primer: 5
0
-GAACTGATGCAGCTCTCTC-3
0
; AR-FL
probe: 5
0
-ACATCCTGCTCAAGACGCTCCT-3
0
; AR-V7 forward
primer: 5
0
-CTGTGCGCCAGCAGAAAT-3
0
; AR-V7 reverse prim-
er: 5
0
-TCAGGGTCTGGTCATTTTGA-3
0
; AR-V7 probe: 5
0
-
TGTCCATCTTGTCGTCTTCG-3
0
.
PCR reactions were assembled into individual wells
according to the following protocol: 1 ng RNA template
(5
m
l), 1
m
l 20X AR-V7 primer/probe assay (FAM), 1
m
l 20X
AR-FL primer/probe assay (HEX), 5
m
l 1X ddPCR Super Mix,
2
m
l RT 20 U/
m
l, 1
m
l 300 mM DTT, 5
m
l DNase/RNase-free
water (total volume: 20
m
l). Droplet generation oil (70
m
l)
was added, and the eight-well cartridge was placed into the
droplet generator; 40
m
l of the droplet solution was then
transferred into a 96-well PCR plate. The following
conditions were used for the reverse transcriptase PCR
reaction: 50
8
C 60 min, 95
8
C 10 min, 95
8
C 30 s and
55
8
C 60 s (40 cycles), 98
8
C 10 min, 4
8
C hold. The
droplet reader was used for fluorescence signal quantifica-
tion. The QuantaSoft software (Bio-Rad) measures the
number of positive versus negative droplets for both
fluorophores (FAM/HEX); their ratio is then fitted to a
Poisson distribution to determine the copy number of the
target molecule, as copies per milliliter (copies/ml), in the
input reaction.
To evaluate the sensitivity of the ddPCR AR-V7 assay,
cDNA obtained from the RNA of VCaP cells was diluted in
water at 250, 100, 50, 25, 15, 10, 5, 1, 0.5, and 0.1 ng/
m
l. The
number of copies/ml was measured by ddPCR.
2.2.
Patient selection
A total of 36 participants with metastatic CRPC treated
with enzalutamide or abiraterone as per approved label
were enrolled. Patients were required to have histologi-
cally confirmed prostate adenocarcinoma, progressive
disease despite castration levels of serum testosterone
(
<
500 ng/l) while on stable androgen-deprivation thera-
py, and documented metastases, confirmed by computed
tomography or technetium-99 bone scans. Patients must
have had at least three increasing serum PSA values taken
at least 2 wk before the last value of at least 2.0 ng/ml,
consistent with the Prostate Cancer Working Group-2
guidelines. Prior taxane-based chemotherapy was permit-
ted. The analysis of AR-V7 in primary tumors was not
performed because this is not part of clinical practice. The
study was approved by the Ethics Committee of Pisa
University Hospital and conducted in accordance with the
principles of the Declaration of Helsinki. All patients gave
their signed informed consent before blood collection and
data analysis.
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