EURURO Vol. 72 No. 2

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1. Introduction
Prostate cancer is the most common cancer diagnosed in
men, with nearly 410 000 diagnoses in Europe each year.
Approximately 20–25% will develop metastatic disease,
which inevitably progresses to lethal castration-resistant
prostate cancer (CRPC). CRPC is characterized by progres-
sive disease under maximal androgen blockade. Nonethe-
less, continued targeting of the androgen receptor (AR) has
demonstrated that this signalling pathway remains one of
the main drivers of progressive disease, even in the CRPC
setting . Besides taxane-based chemotherapy regimens,
next-generation androgen deprivation therapies, encom-
passing both the CYP17 inhibitor abiraterone acetate and
novel antiandrogens such as enzalutamide, have become
available. However, up to 20–40% of patients have resistant
disease at the start of these second-line AR therapies
Various AR perturbations, such as mutations ,
amplifications , and splice variants have
been associated with resistance to androgen deprivation
therapies. The emergence of mutations is affected by the
treatment history, as individual mutations have different
clinical consequences . Amplifications occur in 29–
45% of CRPC patients before starting a new antiandrogen
therapy and increase the expression of AR, which
is associated with resistance to next-generation androgen
deprivation therapies . Furthermore, AR splice variants
(ARVs) can act as constitutively active transcription factors,
bypassing the need for activating ligands and therefore
stimulating ligand-independent growth and progression of
the disease
Prostate cancer metastasises primarily to bone and
there are low success rates for obtaining adequate material
for profiling, even in the research setting . The
application of liquid biopsies, in the form of circulating
tumour cells (CTCs), circulating tumour DNA (ctDNA), or
exosomes, has potential for biomarker profiling without
access to metastatic tissue. Consequently, AR-V7 has
recently been linked to resistance to abiraterone acetate
and enzalutamide in multiple studies that applied various
forms of liquid biopsy
However, there is ongoing discussion about the discrim-
inatory value of detecting AR-V7 expression As other
AR perturbations have been associated with endocrine
treatment outcome, it is likely that a combination test at
both the DNA and RNA levels will improve patient
stratification. Previous work pioneered by Li and colleagues
demonstrated a connection between structural AR variation
and the generation of noncanonical transcripts . We
hypothesized that at least a subset of CRPC patients may
carry relevant intra-AR variations.
Therefore, we performed a pilot study in a selected
cohort of patients with CRPC involving thorough AR
profiling at both the DNA and RNA levels in liquid biopsies
(n = 34) from 30 patients. Our profiling combined muta-
tions, copy-number variations (CNVs), and sequencing of
the entire AR gene, including introns, in combination with
expression information from the full-length AR and seven
ARVs (AR45, AR-V1, AR-V2, AR-V3, AR-V5, AR-V7, and
AR-V9). The aim of the study was to investigate if a
comprehensive AR profile could provide additional infor-
mation to stratify patients beyond AR-V7 expression in the
context of endocrine treatment.
2. Patients and methods
The Supplementary material provides a detailed description of all the
methods. In brief, we collected blood samples from chemotherapy
pretreated and chemo-naı¨ve patients with CRPC in a non-interventional
clinical study. Ethical approval was obtained from the institutional
review and ethics board of GZA Sint-Augustinus. All patients provided a
written informed consent document. Blood collections included samples
for germline DNA extraction, CTC enumeration, CTC enrichment, and
extraction of cell-free DNA from plasma.
ARV expression levels were assessed by performing cDNA synthesis,
multiplex exon-junction–specific PCR (MASTR, Multiplicom NV), and
Illumina sequencing on RNA derived from CellSearch-enriched CTC
fractions. DNA-based library preparation was performed using a
ThruPLEX DNA-seq kit (Rubicon Genomics). Low-pass whole-genome
sequencing (1 50 bp) was performed for identification of copy-number
alterations. Targeted sequencing was performed using a SeqCap EZ
system (Roche Nimblegen) for detection of point mutations and intra-AR
structural variations (2 100 bp). Sequencing was conducted on a
Hiseq2500 instrument in rapid mode. Details on sequence data
processing and statistical analysis are available in the Supplementary
material. To identify intra-AR structural variations, we developed an in-
house structural variant–calling algorithm, svcaller, that is publicly
available .
3. Results
From October 2013 to June 2015, liquid biopsies (n = 34)
were collected from 30 patients with CRPC. Clinicopatho-
logic and radiologic data for the cohort are given in
Supplementary Table 1. The selected cohort encompasses
patients with poor prognosis, with 17/30 (56.7%) patients
having M1 disease at initial diagnosis. The goal was to
thoroughly investigate the AR molecular status in the
context of endocrine treatment .
Cell-free DNA (cfDNA) was successfully extracted from
33 plasma samples and sequencing libraries were con-
structed. The libraries were subjected to low-pass whole-
genome sequencing to determine the copy-number AR
status as well as genome-wide somatic CNVs (Supplemen-
tary Table 2, Supplementary Fig. 1). AR amplifications were
detected in 20/30 patients, with high-level amplifications in
11 patients.
Subsequently, targeted sequencing was performed via
in-solution hybridisation capture on the same sequencing
libraries used for low-pass whole-genome sequencing. The
target region contained baits complementary to 112 genes
(Supplementary Table 3), including all coding exons and
nonrepetitive intronic regions of AR (Supplementary Fig. 2).
The overall average coverage was 1169 (interquartile
range [IQR] 904.5 –2180 ; Supplementary Table 2). So-
matic mutations were detected in all profiled samples
(Supplementary Fig. 3, Supplementary Table 4). Genes
previously reported to be over-represented in CRPC
compared to primary prostate cancer , such as TP53,
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