www.oncotarget.

com Oncotarget, 2018, Vol. 9, (No. 50), pp: 29403-29413

Research Paper
Circulating tumor cells mirror bone metastatic phenotype in
prostate cancer
Andreas Josefsson1, Karin Larsson1, Marianne Månsson1, Jens Björkman3, Eva
Rohlova3,4,5,6, Daniel Åhs1, Helena Brisby2, Jan-Erik Damber1 and Karin Welén1
1
Sahlgrenska Cancer Center, Department of Urology, Institute of Clinical Sciences, Sahlgrenska Academy, University of
Gothenburg, Gothenburg, Sweden
2
Department of Orthopaedics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg,
Sweden
3
TATAA Biocenter AB, Gothenburg, Sweden
4
Department of Anthropology and Human Genetics, Faculty of Science, Charles University, Prague, Czech Republic
5
Laboratory of Gene Expression, Institute of Biotechnology CAS, BIOCEV, Vestec, Czech Republic
6
Centre for Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic
Correspondence to: Karin Welén, email: [email protected]
Keywords: liquid biopsies; circulating tumor cells; skeletal metastases of prostate cancer
Received: April 19, 2018     Accepted: May 17, 2018     Published: June 29, 2018
Copyright: Josefsson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License
3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and
source are credited.

ABSTRACT

Circulating tumor cells (CTCs) are promising biomarkers in prostate cancer (PC)
because they derive from primary tumor and metastatic tissues. In this study, we
used quantitative real-time PCR (qPCR) to compare the expression profiles of 41
PC-related genes between paired CTC and spinal column metastasis samples from
22 PC patients that underwent surgery for spinal cord compression. We observed
good concordance between the gene expression profiles in the CTC and metastasis
samples in most of the PC patients. Expression of nine genes (AGR2, AKR1C3, AR,
CDH1, FOLH1, HER2, KRT19, MDK, and SPINK1) showed a significant correlation
between the CTC and metastasis samples. Hierarchical clustering analysis showed
a similar grouping of PC patients based on the expression of these nine genes in
both CTC and metastasis samples. Our findings demonstrate that CTCs mirror gene
expression patterns in tissue metastasis samples from PC patients. Although low
detection frequency of certain genes is a limitation in CTCs, our results indicate the
potential for CTC phenotyping as a tool to improve individualized therapy in metastatic
prostate cancer.

INTRODUCTION used for prognostic evaluation of disease progression and

therapeutic response. but analysis of circulating tumor
Prostate cancer (PC) is the most commonly cells (CTCs), cell-free DNA, cell-free RNAs or miRNAs,
diagnosed cancer and the sixth leading cause of cancer- exosomes and thrombocytes in liquid biopsies can provide
related death among men worldwide. Bone metastasis broader molecular details of the cancer phenotype, which
is the leading cause of morbidity and mortality in could be used for personalized therapy with specific
patients with PC and is the preferred target for therapy. targeting drugs [1].
Because metastatic biopsies are often difficult to obtain, CTCs can be used as tumor biomarkers because a
using blood samples would be preferred for biological high number of CTCs strongly correlate with metastatic
characterization of PC. Classical blood biomarkers are disease and poor prognosis in metastatic PC in both the

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 Table 1: Patient characteristics
At diagnosis At surgery Time from
diagnosis to
surgery (years)
Median age year (range)
   hormone naive (n=5) 68 (64-83) 68 (64-83) 0
   GnRH naive (n=1) 82 87 5.8
   GnRH initated (n=1) 69 70 0.1
  CRPC (n=15) 70 (57-83) 75 (59-88) 4.5 (1.0-14.8)
Median PSA ng/ml (range)
   hormone naive (n=5) 731 (111-1200) 731 (111-1200)
   GnRH naive (n=1) 3.12 93
   GnRH initated (n=1) 1200 x
  CRPC (n=15) 33.5 (5-334) 81.4 (5.8-276)
Gleason score (n) GS 6-7 GS 8-10 GS X
   hormone naive (n=5) 0 1 4
   GnRH naive (n=1) 1 0 0
   GnRH initated (n=1) 0 1 0
  CRPC (n=15) *
7 6 2
Therapy before surgery None TAB **
TAB+Docetaxel Docetaxel Enzalutamide
  CRPC (n=15) ***
6 3 2 3 1

*
Gleason score was obtained at diagnosis, not at surgery.
*
TAB = total androgen blockade, GnRH analogue plus bicalutamide.
**
All patients were treated with GnRH analogues.

hormone naïve (HN) and castration-resistant (CR) settings metastases was low [12]. In breast cancer, concordance of
[2, 3]. Furthermore, detection of specific gene expression gene expression profiles in CTCs and primary tumors was
in CTCs can provide reliable information regarding highly variable [13].
the prognosis and therapeutic resistance to treatments In the present study, we analyzed the gene expression
targeting androgen receptor signaling [3–6]. CTCs may profiles in paired samples of CTCs and metastatic tissue
thus reflect not only the tumor burden but also the biology from the spines from PC patients (Table 1). Our data show
of the disease. that to a large extent the gene expression profile of CTCs
In various cancers, similarities have been detected mirrors that in the paired metastatic tissue. However, all
in the genomic compositions of CTCs and tumor tissues selected genes did not perform equally well. Therefore,
[7, 8]. In PC, for example, shared genomic alterations careful selection of genes and analysis of a biomarker
have been identified in CTCs and tumor tissue samples panel are required to develop CTC-based liquid biopsy
[9]. Moreover, androgen receptor (AR) amplification is strategies for clinical applications in PC.
concordant between CTCs and tumor tissue biopsies from
CRPC patients [10]. It is unclear, however, whether CTCs RESULTS
accurately reflect the phenotype of metastatic tissue in PC.
Cho et al. showed that expression of a small number of Detection of gene expression in spinal metastasis
genes (present or absent) was concordant between single tissues and CTCs from PC patients
CTCs and micro-dissected PC cells from bone marrow
biopsies in 75% of cases [11]. In colorectal cancer, gene We excluded 15 of the 46 genes in the PC-panel
expression in the CTCs was more similar to that in liver from further analysis. These included EPCAM used for
metastases than the primary tumors, but the concordance normalizing gene expression values, the general endogenous
between the gene expression profiles of CTCs and control genes GAPDH and GUSB, as well as CD45 and CD44

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 Table 2: Prostate cancer panel; detected gene expression signals in CTC and metastatic tissue and their correlation
Gene Detected Detected Correlation P-value Detected Detected
signals in signals in coefficient, R signals in signals in
CTC metastases CTC metastases
AGR2 18 22 0.778 p<0.001 Genes not included in analysis due to
low detection frequency in CTC samples
AHR 3 22 -0.500 0.667
AKR1C3 17 22 0.704 p<0.01 CYP11A1 1 22
AKT2 11 22 -0.159 0.640 CYP17A1 1 14
ALDH 13 22 0.316 0.293 CYP19A1 1 22
AR 14 22 0.565 p<0.05 ESR1 0 22
ARV7 7 22 0.750 0.052 ESR2 0 13
AURKA 12 22 -0.130 0.688 MET 1 22
BCL2 10 22 -0.395 0.258 PTCH1 0 22
CDH1 14 22 0.575 p<0.05 Genes not included in analysis due to
frequent detection in CTC negative samples
CDH2 2 22 - -
DDR1 2 22 - - MYC 22
EGFR 4 22 -0.200 0.800 TP53 22

EMP2 11 22 0.359 0.278 ANXA2R 22

FOLH1 16 22 0.900 p<0.001 Control genes not included in analysis
HER2 11 22 0.712 p<0.05 GAPDH 22 22
KLK3 18 22 0.451 0.061 GUSB 22 22
KRT19 16 22 0.693 p<0.01 CD44 22 22
MDK 18 22 0.720 p<0.01 CD45 2 2 22
POU5F1 13 21 0.478 0.099
PSCA 10 22 0.588 0.074
RUNX2 5 22 0.872 0.054
SNAI1 5 22 0.300 0.624
SPINK1 11 22 0.724 p<0.05
SRD5A1 15 21 0.445 0.110
TACSTD2 16 22 0.379 0.147
TOP2A 14 21 0.263 0.363
TUBB3 6 22 -0.029 0.957
Twist1 7 22 0.179 0.702
UPA 7 22 -0.414 0.355
VEGFA 14 22 0.066 0.822

Analysis are performed with Spearman rank correlation. Genes with a correlation between CTC and metastases with a
p-value less than 0.1 are marked in bold text.
which mainly reflect the white blood cell contamination in signals from the white blood cells (data not shown). Further,
the CTC samples. MYC, TP53, and ANXA2R were excluded ESR1, ESR2, PTCH1, MET, CYP11A1, CYP17A1, and
due to their frequent detection in CTC-negative (EPCAM- CYP19A1 genes were excluded because they were rarely
negative) samples, which was interpreted as contaminating detected in the CTC samples (Table 2).

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 Table 3: Correlation between gene expression in CTCs and metastases in individual patients
Patient ID CTC/Mets pairs Correlation coefficient, R P-value
1 24 0.532 <0.05
2 29 0.591 <0.05
5 28 0.508 <0.05
6 28 0.793 <0.001
7 22 0.676 <0.01
9 18 0.531 <0.05
10 28 0.487 <0.05
12 20 0.677 <0.01
14 24 0.25 0.187
16 18 0.185 0.27
18 31 0.307 0.092
19 29 0.593 <0.001
21 30 0.53 <0.001
22 16 0.506 =0.05

Spearman correlations and bootstrap p-values for the gene expression profiles in CTCs and metastatic tissue in individual patients.

In the bone metastasis tissue, the 31 genes that were patient to get a broader picture of the metastasis than
analyzed were detected in all samples, except for POU5F1, obtaining just the expression levels of individual genes.
SRD5A1, and TOP2A (96%), CYP17A1 (67 %), and ESR2 In this analysis, we included the calculated values for
(58 %). In the CTCs, the detection ratio of these 31 genes low detection levels in CTCs (i.e., cases with no signal
was much lower. The detection frequency in the CTC for a specific gene despite sufficient CTC content).
samples ranged from 87 % (AGR2, AKR1C3, and KLK3) We analyzed 14 patients whose CTC samples showed
to 4 % (CYP11A1, CDH11, CYP17A1, and MET) (Table 2). gene expression values for more than 50% of the 31
Prostate cancer origin of one of the CRPC patients could not included genes. Among these, 11 patients showed good
be confirmed because the CTCs did not show any detectable correlation (correlation coefficient ≥ 0.5) between the
expression of either KLK3 or FOLH1. Hence, this sample gene expression profiles in CTC and metastasis samples.
was excluded from further analyses. Six patients showed bootstrap generated p-values below
0.05 while five patients showed p-values between 0.05
Identification of genes in CTC reflecting and 0.1 (Table 3). Three of the 14 patients showed
expression in bone metastases poor correlation (0.185, 0.250 and 0.307, respectively)
between gene expression in CTC and metastatic tissue
We assessed the potential of the included genes samples. Two of these three patients showed metastasis
in the CTC-based analysis to reflect gene expression in in the lungs (66%), whereas only one of the eleven
tissue metastases by estimating the correlation in signal patients (14 %) with a correlation coefficient > 0.5
intensity of the expression of individual genes in the showed lung metastasis.
CTC and metastasis samples of all patients. Nine of the
thirty-one included genes showed a statistically significant Analysis of patient grouping based on CTC and
correlation between the expression in CTC and the metastatic gene expression profiles
metastasis samples (Table 2). In addition, ARV7, POU5F1,
PSCA, RUNX2, and KLK3 showed moderate to a high We selected the subset of 14 genes that showed
degree of correlation (R>0.4 and p<0.100). good correlation between the CTC and metastatic tissue
samples (Table 2) for hierarchical clustering analysis
Gene expression profiles in the CTCs and to determine whether patient grouping using the gene
metastatic tissue samples of individual patients expression data from CTCs is similar to their grouping
when the data from metastatic tissue samples were used.
We compared the gene expression profiles between However, to optimize the clustering analysis, we only
matched CTC and metastatic tissue samples of each included patients with expression values for more than

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 10 of the included genes in their CTC samples, and we that displayed more than 50% detection frequency in
only included genes with positive expression signals in the CTC-derived data. Thus, we obtained a matrix of
more than 10 of these patients. This resulted in a matrix 8 genes and 13 patients (5 HN and 8 CR). Clustering
of 13 patients and 12 genes (ARV7 and PSCA genes of these patients based on the gene expression data
were excluded). Based on expression in metastatic tissue from metastatic tissue samples revealed two clusters,
samples, the patients were grouped into three clusters. of which one included all HN and two CR patients.
Concurrent analysis using CTC-derived data showed that Clustering analysis of the CTC-derived dataset from
the eleven of the thirteen patients (exceptions: patient these patients again grouped all HN samples, with two
numbers 14 and 21) grouped similarly into three clusters more CR patients clustered together with the HN patients
(Figure 1). (Figure 3). Notably, the two CR patients that grouped
Next, we included all genes that were detected with the HN cluster in the CTC-based analysis (18 and 6)
in more than 10 CTC samples and all patient samples displayed high expression (green signal) of both AR and
whose CTC analyses resulted in 10 or more signals to AKR1C3 genes, which was atypical for most HN samples
determine the performance of the PC Panel in grouping (highlighted in Figure 3).
patients based on CTC data without pre-analysis of
significantly correlating genes or patients. We obtained DISCUSSION
a matrix of 15 patients and 23 genes. Hierarchical
clustering of this dataset demonstrated that PC patient CTCs have been used for a long time as
grouping based on the analyses of gene expression in biomarkers for PC prognosis and therapy response.
CTCs was similar to that based on the metastatic dataset However, the potential of obtaining phenotypical
(Figure 2). information from these circulating cancer cells has not
been satisfactorily explored. In the present study, we
CTC analysis reflects patient treatment status analyzed the gene expression of CTCs to explore the
potential of gaining phenotypical information regarding
Next, we analyzed if the gene expression data from the metastatic disease.
the CTCs identified phenotypes that match the clinical The CTCs are intact cancer cells, and therefore
status of the patients. To address this, we classified the carry biologically relevant information regarding
patients based on their hormonal status at sampling, i.e., the disease in addition to their use as biomarkers
if they were resistant to treatment with GnRH analogues based on their enumeration. In the present study, we
[castration-resistant (CR), n=12] or were hormone naïve show a strong correlation between CTCs and bone
(HN; hormone naïve and GnRH naïve, n=6, Table 1). metastasis samples from the same patient regarding
For clustering analysis, we used nine genes that were the expression levels of several genes that are related
differentially expressed in the metastasis samples of to PC progression, metastasis, and therapy resistance.
these two categories. However, due to the low detection However, when analyzing the genes individually, it
frequency of certain genes in the CTC samples, the was obvious that the potential to reflect the expression
dataset was modified to include patients with genes level in metastases by CTC analysis differed among

Figure 1: Heat map illustrates hierarchical clustering using significantly correlating genes in PC patients based on their expression in
(A) metastatic tissue (B) and CTCs. Note: a, b and c represent groups of patient samples that cluster similarly in both metastases and CTC
analyses. Arrows indicate samples switching clusters between the two analyses.

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 genes. Our analysis showed that nine out of forty- factor that influences detection of gene expression from
one genes displayed a strong correlation between tumor cells alone. This is exemplified in the present study
their expression in CTCs and metastasis samples. by the poor correlation of urokinase-type plasminogen
Among these was ARV7 which expression in CTCs activator (UPA) and vascular endothelial growth factor
is related to abiraterone and enzalutamide resistance A (VEGFA) genes, which are both expressed by tumor
in several studies [4–6]. Previously, detection of any cells as well as the tumor stromal cells. Thus, care must
of the other eight genes in CTCs has not been related be taken in selecting genes that enable CTC analysis
to any clinically relevant characteristic, and is for the to provide useful information regarding the metastatic
first time presented to be found in CTCs in prostate tumor cells.
cancer patients. The MDK and AGR2 genes are related In the individual patients, the gene expression
to neuro-endocrine differentiation in CRPC [14, 15], profile of CTC samples correlated with the gene
which is a biological process that is associated with a expression profile of the corresponding metastatic
non-functional AR signaling axis and correlates with samples in most cases. However, we also encountered
poor prognosis. These tumors likely do not respond patients where the correlation was poor or absent. We
optimally to drugs that are targeted towards sustained are unable to reliably predict the reasons for these
AR signaling in CRPC. Similarly, although high differences between patients because the patient
expression of AR, ARV7, and AKR1C3 indicates that the material in this study was too small. However, the
tumors are dependent on activated AR-signaling their presence of lung metastasis in two out of three patients
overexpression may be a part of the activated resistance that showed poor concordance between gene expression
mechanisms that lower sensitivity to AR targeting drugs in the CTCs and bone metastatic tissue samples
[5, 16]. This was illustrated by our findings that show indicates that these CTC samples may also represent
higher expression of AR and AKR1C3 the CR patients lung metastases, which presumably represent a different
than the HN patients. phenotype than the bone metastases. This suggests
There are at least two plausible reasons for the that CTCs represent the whole metastatic disease, and
differences in expression of several genes between therefore, the gene panel should potentially represent
CTC and metastasis samples. First, these genes may be the biological characteristics of metastases in different
differentially regulated in a CTC than a metastatic tumor organs. This is supported by previous findings that
cell. Second, the CTC and metastasis samples may the single cell CTC mutational status may be highly
contain different amounts and types of contaminating heterogeneous [17].
cells. In the CTCs, leukocyte contamination affects Our study also showed that patient grouping
the detection levels of certain genes, whereas, in the based on gene expression analysis of CTC samples
metastatic tissue, tumor stroma is a major contaminating was similar to grouping based on the analysis of the

Figure 2: Heat map illustrates hierarchical clustering using significant genes without prior selection based on their expression in (A)
metastatic tissue and (B) and CTCs. Note: a, b and c represent groups of patient samples that cluster similarly in analyses of both metastases
and CTCs. Arrows indicate samples that switch clusters between the two analyses.

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 metastatic tissue. Moreover, patients were grouped CTC profiling is sensitivity as a result of the limited
according to the hormonal status based on the gene amount of material, the usefulness of the method is
expression profiles of both CTC and metastasis dependent on the amount of CTCs that are available for
samples. This shows that although all independent detection. Thus, patients with a non-metastatic disease or
genes may not display a good correlation between the those responding to therapy are less suitable for this type
CTC and metastasis samples, a combination of genes of analysis. The present study includes only patients with
may represent clinically relevant information regarding metastatic disease, but despite their severe condition,
the tumor phenotype. we observed significant variations in the amount of
Although the analysis of CTCs from liquid CTCs isolated from individual patients. This affected
biopsies has many advantages, the technology requires the detection frequency of certain genes depending on
further optimization to overcome the limitations. We their relative expression levels and limited their use in
encountered two technical issues in the present study. the analyses.
The first relates to the contamination by leukocytes Another challenge we encountered in our study was
during CTC isolation, which limits the expression panel about interpreting the absent gene expression signals from
to genes that are not expressed in leukocytes. This samples with low CTC content. It was obvious from our
represents a major obstacle that needs improvement results that some genes were less expressed than others
in CTC isolation methodologies to develop reliable despite comparable CTC content. Thus, an absent signal in
treatment prediction analysis for immune-related CTC-derived expression data cannot simply be interpreted
therapies such as programmed death-ligand 1 (PD-L1) as a low expression signal because it may be a result of
antibody-based immunotherapy. The second technical a too low CTC content to allow detection of the specific
limitation was the reliance on EPCAM and HER2 gene. In the present study, we developed a strategy to
antibodies for the isolation of CTCs. This limits the identify the threshold of CTC content that would enable
detection of CTC populations with low expression of detection of each gene individually and avoid false low
these antigens [18] or those masking these antigens detection values. If a gene was not detected in a sample
by macromolecules [19]. Hence, CTCs that were not despite sufficient CTC content, it was assigned an
isolated by our methodology may represent other sub- expression value lower than the lowest detected for that
populations of CTC, and their genetic profile may also gene. If the CTC content was below the threshold and the
be critical for evaluating the status of PC metastasis. This gene expression could not be detected, that absent signal
problem may be overcome by novel isolation methods was excluded from further analysis. In the future, to enable
that are not based on epithelial antigens. Therefore, it expression profiling of CTCs for clinical applications,
is plausible that we may overcome the insufficient there is a need to increase the detection sensitivity and
sensitivity to detect expression of certain genes in the develop robust methods to handle absent gene expression
CTC samples by utilizing other isolation methods. signals as a result of limited CTC content.
De Bono et al. showed that the amount of CTCs In conclusion, our study demonstrates the potential
reflects the progression and treatment response of PC of CTCs to mirror the gene expression profile of PC
[20]. Since one of the technical issues encountered with bone metastases in individual patients. The study also

Figure 3: Heat map illustrates the hierarchical clustering using the limited gene subset discriminating HN and CR metastases, based on
their expression in (A) metastases and (B) CTCs. Note: ‘A: a’ represents a cluster that includes all HN samples in the metastases analyses;
‘B: a’ represents a similar cluster in the CTCs. Arrows indicate samples that cluster differently in the CTC-based analysis; white box
highlights the AR and AKR1C3 signals in two CR samples that change clusters.

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 points out the importance of careful selection of genes kit (Qiagen Hannover GmbH, Germany) as previously
to accommodate the technical and biological aspects that described [3]. Briefly, the patient blood samples were
limit the interpretation of expression of different genes. collected immediately before surgery in AdnaCollect tubes
and refrigerated at 4°C. CTC isolation was performed
MATERIALS AND METHODS within 24 hours by capturing them on EPCAM- and
HER2 antibody-conjugated magnetic beads. The mRNAs
Patients from lysed CTCs were isolated using oligodT-conjugated
magnetic beads and transcribed into cDNA.
We recruited twenty-five patients undergoing
surgery for spinal cord compression symptoms related to Design of the PC gene expression panel
metastatic PC, between 2013 and 2016, at the Department of
Orthopedics, Sahlgrenska University Hospital, Gothenburg. As shown in Table 2, the gene expression panel,
The study protocol was approved by the ethics committee in which is referred to as PC-panel, was composed of 46
Gothenburg, Sweden (# 936-12 and # 455-11). We excluded genes, of which five were control genes, and 41 were
two patients that had other cancer diagnoses in addition to PC PC-related genes. The genes were selected based on their
and one patient that was CTC-negative. Clinical information role in PC progression, metastasis, steroid synthesis and
of the included 22 patients is shown in Table 1. The hormone signaling, stemness and neuroendocrine differentiation
naïve patients (n=5) were diagnosed with metastatic PC at as reported in the literature. All the included genes were
the time of surgery and did not receive any hormonal therapy expected to be highly expressed in PC cells and negligible
before surgery. The patient annotated as GnRH naïve in this expression in the leukocytes (contaminating white blood
study received bicalutamide three months before surgery, cells in the CTC samples).
but responded to GnRH therapy initiated after surgery.
One patient annotated as GnRH initiated in this study was Gene expression profiling
diagnosed with metastatic PC and was treated with GnRH We pre-amplified 2 μl of cDNA samples from
antagonists for one month before surgery, after which he metastases and CTC samples (including beads from CTC
responded well to GnRH agonists. The CRPC patients (n=15) collection) using the TATAA PreAmp Primer Mix and
relapsed with metastasis in the spine after GnRH therapy TATAA PreAmp GrandMaster® Mix (Cat. No. #TA05,
alone, or after second or third line therapy (Table 1). TATAA Biocenter) in a T100 BioRad thermocycler. We
also pre-amplified non-template control and human gDNA
RNA preparation and cDNA synthesis from (0.5 ng/μl, TATAA Biocenter) samples. The preamplified
metastatic tissue samples were centrifuged to pellet the magnetic beads, and
a fraction of the supernatant was diluted 10X in a separate
Metastatic tissue that was removed during surgery tube. We performed qPCR analyses of the diluted samples
was immersed in RNAlater (Ambion) and frozen at (45 assays) using the ValidPrime™ assay kit (TATAA
-80°C. Total RNA was prepared from 40-100 mg tissue Biocenter) with specific primers designed for this study as
(dependent on the content of bone) using the RNeasy shown in Supplementary Table 1. This assay is now available
Plus Universal Mini kit (Qiagen) according to the as part of the GrandPerformance CTC Assay Panel at the
manufacturer’s instructions. Briefly, the tissue was TATAA Biocenter. The qPCR analysis was performed using
homogenized in the Qiazol Lysis Reagent in a Tissue Lyser the TATAA Probe GrandMaster® Mix Low ROX (TATAA
II homogenizer at 25 Hz for5 min twice, and the resulting Biocenter) and GE 96.96 Dynamic Array™ Sample & Assay
homogenate was treated with gDNA eliminator solution Loading Reagent Kit (P/N 85000802-R, Fluidigm). We also
(Qiagen), extracted with chloroform. After centrifugation, included preamplified no template control (preAmp NTC)
the aqueous phase was mixed with 70% ethanol and and no template control (NTC) for the qPCR, which was
directly loaded onto the RNeasy Mini spin column. After performed on the BioMark system (Fluidigm) using the
washing, the total RNA was eluted from the column in 30 96.96 Dynamic Array™ IFC (Integrated Fluidic Circuit). All
μl RNase-free water, and its concentration and purity were the samples (including the NTCs and gDNA) were analyzed
measured in a NanoDrop (Thermo Scientific, Waltham, in duplicates. The assays we use were designed with ISO
MA, USA). Forty nanograms of the RNA from metastatic 17025 accredited methods (TATAA Biocenter) and validated
tissue samples were converted to cDNA using the TATAA in compliance with the MIQE guidelines [21], which is
GrandScript cDNA Synthesis Kit (Cat. No. #A103a, considered sufficient for research and most diagnostic usage.
TATAA Biocenter, Gothenburg, Sweden).
Preprocessing, normalization, and interpretation
CTC isolation and cDNA synthesis of gene expression data

The CTCs were isolated from blood samples and The raw data (averaged Cq-values) was controlled
detected using the AdnaTest ProstateCancerSelect/Detect and corrected for genomic DNA contamination using the

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 GenEx software (MultiD Analyses AB) with implemented was applied to the number of available complete pairs of
functions for the ValidPrime™ concept [22]. The averaged matched samples for each patient. Hierarchical clustering
Cq values corrected with more than one cycle (Cq) were was performed using normalized and mean centered data
considered compromised due to large-scale genomic DNA with Spearman Rank Correlation and Average linkage in
contamination and removed from the analysis. a MultiExperiment Viewer (MeV, Dana-Farber Cancer
EPCAM expression was considered as a measure of Institute, US). All statistical tests were two-sided and p-values
epithelial cell (i.e. CTC) content. The expression of other < 0.05 were considered statistically significant.
genes was normalized to EPCAM expression to eliminate
the contamination from normal bone tissue or white blood Abbreviations
cells in metastatic tissue and CTC samples, respectively.
When expression was not detected for some genes in CTC, Circulating tumor cells; CR, Castration
the CTC samples, the results were not automatically resistant; HN, Hormone naïve; PC, Prostate cancer; Cq,
interpreted as displaying low expression. Instead, we Cycles of qPCR reaction; qPCR, quantitative real-time
individually identified a cut-off level of CTC content polymerase chain reaction; CRPC, Castration-resistant
(based on EPCAM expression) so that the gene expression prostate cancer; GnRH, Gonadotropin-releasing hormone;
levels could be reliably detected in CTC samples, and AR, androgen receptor; RNA, ribonucleic acid; DNA,
eliminate false low CTC expression values. If the CTC deoxyribonucleic acid; cDNA, Cyclic-DNA.
content was now high enough for the gene to be reliably
detected, the low expression value was used instead of the Author contributions
absent signal. On the other hand, if the CTC content was
lower than the cut-off detection level that was required Andreas Josefsson contributed to project planning,
to detect the expression of a particular gene, the absent ethical approvals, logistics, the inclusion of patients,
signal was excluded from further analyses. Therefore, if a analysis, preparation of tables and manuscript writing.
specific gene was not detected in a specific CTC sample, Karin Welen contributed to study initiation, project
the Cq(EPCAM) value for that CTC sample was compared planning, ethical approvals, logistics of sample handling,
to the Cq(EPCAM) values in other CTC samples where isolation of circulating tumor cells and mRNA from
the specific gene was detected. We interpreted the absent metastasis samples, statistical analysis, preparation of
signal as a valid detection value if the Cq(EPCAM) value tables and figures, and manuscript writing.
in the specific sample was at least 2Cq values lower (i.e. Jan-Erik Damber contributed to study initiation,
four times higher EPCAM expression) than the highest writing ethical approvals, project planning, interpretation
Cq(EPCAM) value at which the specific gene could of results and manuscript writing.
be detected in all the CTC samples analyzed. In such a Helena Brisby was responsible for the inclusion
scenario, the expression of that specific gene was assigned of patients and collaboration at the orthopedic center,
a delta Cq value, which was one Cq value higher than the interpretation of some results and finalizing the manuscript.
highest Cq value detected for that specific gene. If the Karin Larsson was responsible for handling the
Cq(EPCAM) value in the CTC sample was higher than patient samples, logistics, isolation of circulating tumor
the cut-off Cq(EPCAM) value, i.e., displaying a lower cells and extraction of the mRNA from the metastases, and
EPCAM expression, the absent signal was regarded as finalizing the manuscript.
absent and excluded from further analyses. Marianne Månsson performed the majority of the
statistical analysis and interpreted the results.
Statistics Jens Björkman was involved in optimizing the
experimental set-up, interpretation of results, writing the
Spearman Rank Correlation was used to compare the methods section, and finalizing the manuscript.
gene expression levels in CTC and metastasis samples from Eva Rohlova performed the technical work at the
individual patients using the IBM SPSS Statistics 22.0.0.0 TATAA Biocenter, including optimization of assays
software. Since expression levels of the genes included and validation of the pre-amplification steps. She also
in the panel may partly be dependent on each other within participated in writing the methods section, interpretation
individual patients, the p-values were derived using the of results and finalization of the manuscript.
bootstrapping method as follows: Two patients were sampled Daniel Åhs was involved in patient inclusion,
randomly and the correlation between the CTC expression logistics, the collection of patient data and data evaluation,
values from one patient and the metastasis expression values and finalization of the manuscript.
from the second patient were calculated. This was repeated
100000 times to achieve the empirical distribution function ACKNOWLEDGMENTS
for the correlation between independent individuals. The
p-value was then derived by comparing the observed The authors acknowledge Anita Fae for help with
correlations within patients to this distribution. The procedure CTC isolation, Mikael Kubista and Robert Sjöback

www.oncotarget.com 29411 Oncotarget

 (TATAA Biocenter) for help with setting up the CTC 7. Heitzer E, Auer M, Gasch C, Pichler M, Ulz P, Hoffmann
analyses, and the staff at the orthopedic surgery unit of EM, Lax S, Waldispuehl-Geigl J, Mauermann O, Lackner C,
the Sahlgrenska University Hospital for their valued Höfler G, Eisner F, Sill H, et al. Complex tumor genomes
assistance with blood and tissue sampling. The authors inferred from single circulating tumor cells by array-CGH
also acknowledge the funding support for this project and next-generation sequencing. Cancer Res. 2013; 73:2965–
from the Swedish Cancer Society, the Swedish Society 75. https://doi.org/10.1158/0008-5472.CAN-12-4140.
of Medicine, the Movember Foundation, BioCARE, the 8. Maheswaran S, Sequist LV, Nagrath S, Ulkus L,
Göteborg Medical Society, the Swedish PC Federation, Brannigan B, Collura CV, Inserra E, Diederichs S, Iafrate
the County Council for West Sweden (ALF project), the AJ, Bell DW, Digumarthy S, Muzikansky A, Irimia D,
Swedish Society for Strategic Research, and the Research et al. Detection of mutations in EGFR in circulating
Foundations of P. Falk, M. Ågren, U. and K-E. Winberg. lung-cancer cells. N Engl J Med. 2008; 359:366–77.
https://doi.org/10.1056/NEJMoa0800668.
9. Jiang R, Lu YT, Ho H, Li B, Chen JF, Lin M, Li F, Wu K, Wu H,
CONFLICTS OF INTEREST
Lichterman J, Wan H, Lu CL, OuYang W, et al. A comparison
of isolated circulating tumor cells and tissue biopsies using
J. Björkman and E. Rohlova are employed by the
whole-genome sequencing in prostate cancer. Oncotarget.
TATAA Biocenter AB; J. Björkman is a stock owner in
2015; 6:44781–93. https://doi.org/10.18632/oncotarget.6330.
TATAA Biocenter AB. All the other authors declare that
there are no potential conflicts of interest. 10. Podolak J, Eilers K, Newby T, Slottke R, Tucker E, Olson
SB, Lue HW, Youngren J, Aggarwal R, Small EJ, Graff
JN, Alumkal JJ, Beer TM, Thomas GV. Androgen receptor
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