EURURO Vol. 71 No. 5

777 844

1. Introduction
The growth of normal and malignant prostate tissue is
regulated by androgens through activation of the androgen
receptor (AR) in both epithelial and stromal cells, and
androgen deprivation therapy (ADT) is standard treatment
for patients with advanced prostate cancer (PC). However,
after an initial period of reduced symptoms and tumor
growth, relapse occurs and the PC becomes castration
resistant (CRPC). Several mechanisms behind CRPC have
been described, including AR amplification, AR mutations,
expression of constitutively active AR variants, and intra-
crine steroid synthesis, as well as AR bypass mechanisms
. It has been shown that several new drugs prolong
survival and increase quality of life for men with CRPC,
including novel AR antagonists, cytostatic drugs, radio-
isotopes, steroidogenesis inhibitors, immunotherapies, and
therapies targeting the tumor microenvironment Thus,
there is a need for biomarkers that can guide CRPC therapy
selection. Moreover, the fatal outcome for patients with
CRPC highlights the necessity for further therapeutic
developments, particularly for patients characterized by
low AR activity and for whom no targeting therapy
currently exists.
We previously identified heterogeneous gene expression
patterns of clinical relevance in metastatic CRPC samples,
and found that high levels of the constitutively active AR
variant 7 (AR-V7) were associated with particularly poor
prognosis . Antonarakis and co-workers showed that
detectable levels of AR-V7 in circulating tumors cells are
predictive of poor response to AR-targeted therapies . We
also found a heterogeneous expression pattern for the
steroidogenic enzyme AKR1C3 in clinical samples of CRPC
metastases , and the relevance of AKR1C3 as a predictive
marker for therapy response to the steroidogenesis inhibi-
tor abiraterone is currently under evaluation.
The aim of this study was to further characterize gene
expression in bone metastases from men with CRPC to
identify subgroups of relevance for therapy choice.
2. Patients and methods
2.1. Patients
Fresh-frozen bone metastasis samples were obtained from a series of
men with PC (n = 65) or other malignancies (n = 14) who underwent
surgery for metastatic spinal cord compression at Umea˚ University
Hospital between 2003 and 2013. The PC patient series has been
described before and the clinical characteristics are summarized
in Formalin-fixed, paraffin-embedded (FFPE) metastasis
samples were available for 41 of the 54 CRPC patients in and
matched diagnostic prostate biopsies were available in 29 cases,
obtained at a median of 37 mo (interquartile range [IQR] 16–79) before
the metastasis biopsy. The study also included 12 separate men who
were treated with radical prostatectomy at Umea˚ University Hospital
between 2005 and 2006; the median age for these men was 61 yr (IQR
57–67) and median prostate-specific antigen (PSA) was 11 ng/ml (IQR
5.3–18 ng/ml). Clinical local stage was T2 (n = 3) or T3 (n = 9) and
Gleason score (GS) was 7 (n = 10) or 8 (n = 2).
Tissue microarrays (TMAs) were previously constructed from
samples taken during transurethral resection of the prostate (TURP)
performed between 1975 and 1991 as previously described Gleason
score was reevaluated by one pathologist (L.E.) and TMAs were
constructed containing five to eight samples of tumor tissue and four
samples of nonmalignant tissue from each patient. For this study, TMAs
from 284 patients had tissue available for analysis (Supplementary
Table 1). The patients had not received cancer therapy before TURP and,
according to the therapy traditions in Sweden at that time, the majority
( n = 202) were managed via watchful waiting.
The study was approved by the local ethics review board of Umea˚
University (Dnr 03-185, 2010-240-32, and 02-283).
2.2. Tissue preparation
Bone metastasis samples were instantly fresh-frozen in liquid nitrogen
or placed in 4% buffered formalin. Fixed samples were decalcified in
formic acid before being embedded in paraffin. Fresh radical prostatec-
tomy specimens were received at the pathology department immedi-
ately after surgery and cut in 0.5-cm-thick slices before fixation. From
these slices, 20 samples were taken using a 0.5-cm skin punch and frozen
in liquid nitrogen within 30 min after surgery. The prostate slices were
formalin-fixed, embedded in paraffin, cut in 5 mm-thick sections, whole-
mounted, and stained with hematoxylin-eosin. Tissue sample composi-
tion (nonmalignant or malignant) was determined according to location
in the whole-mount sections.
2.3. RNA extraction
Representative areas of fresh-frozen bone metastasis samples and of
malignant and nonmalignant prostate tissue (obtained in pairs from the
same patient) were cryosectioned into extraction tubes and RNA was
isolated using the Trizol (Invitrogen, Stockholm, Sweden) or AllPrep
(Qiagen, Stockholm, Sweden) protocol. The percentage of tumor cells in
the samples was determined by examination of parallel sections stained
with hematoxylin-eosin, and varied between 50% and 90%. The RNA
concentrations were quantified by absorbance measurements using a
spectrophotometer (ND-1000; NanoDrop Technologies, Wilmington, DE,
USA). The RNA quality was analyzed on a 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA, USA) and verified to have a RNA integrity
number 6.
2.4. Whole-genome expression profiling
For each sample, 300 ng of total RNA was amplified using an Illumina
TotalPrep RNA amplification kit (Ambion, Austin, TX, USA) according to
the manufacturer’s protocol. A total of 750 ng of cRNA from each sample
was hybridized to HumanHT-12 v4 Expression BeadChips, including
more than 47 000 probes covering over 31 000 annotated genes,
Patient summary: We studied heterogeneities at a molecular level in bone metastasis
samples obtained from men with castration-resistant prostate cancer. We found differ-
ences of possible importance for therapy selection in individual patients.
# 2016 European Association of Urology. Published by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license ).
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