EURURO Vol. 72 No. 5

Introduction

Prostate cancer (PCa) is the most common cancer and the

third leading cause of cancer death among European men

[1]

. In metastatic PCa, progression from a hormone-

sensitive state to castration resistance under androgen

deprivation therapy marks the transition to the lethal

phenotype of the disease. New insights into tumor biology

have contributed to the development of novel therapeutic

agents that have revolutionized the treatment landscape for

metastatic castration-resistant PCa (mCRPC) over recent

years

[2]

. On the basis of the discovery that the androgen

receptor (AR) is still active in mCRPC and responsible for

disease progression

[3]

, a new generation of AR-directed

agents such as the androgen biosynthesis inhibitor abir-

aterone and the AR inhibitor enzalutamide have been

developed and have been shown to improve overall survival

(OS)

[4 – 7]

However, treatment resistance still poses a major

challenge in mCRPC. Approximately one-third of patients

show primary resistance to abiraterone and enzalutamide

treatment without any decline in serum prostate-specific

antigen (PSA) levels, and virtually all of the initial

responders develop secondary resistance over time

[4 – 8]

A main research focus has been on the presence of AR

splice variants as a cause of this resistance. AR splice variant

7 (AR-V7) is the most abundant splice variant. It lacks the

androgen-binding site and remains constitutively active as a

transcription factor, independent of androgen signaling

[9,10]

. The clinical importance of this finding was recently

demonstrated by showing that AR-V7 is associated with

resistance to abiraterone and enzalutamide in mCRPC

patients

[8,11,12]

. Using a circulating tumor cell (CTC)-

based assay to determine AR-V7 expression, these studies

required elaborate processing of blood samples and

detectable CTCs.

Alternative quantification of AR-V7 mRNA levels directly

in peripheral whole blood has been reported

[13 – 15]

. The

main advantage of this approach is that it detects AR-V7

expression in all blood compartments at one time, reflecting

the overall AR-V7 status in blood. Previous studies

suggested the presence of AR-V7 transcripts not only in

CTCs

[8,11]

but also in exosomes

[16]

and as cell-free RNA in

plasma

[17]

. Moreover, AR-V7 detection in whole blood is

independent of detectable CTCs and their isolation via

enrichment. Epithelial-based CTC detection methods, such

as the widely used AdnaTest ProstateCancer and CellSearch

system, target epithelial cell

–

surface proteins for CTC

enrichment. However, epithelial-mesenchymal transition

(EMT) in CTCs, which plays a pivotal role in metastasis

development

[18]

, causes downregulation of epithelial

proteins. Hence, these cells will be invisible to epithelial-

based CTC detection methods. Taken together, these points

suggest high potential for determining AR-V7 status in

peripheral whole blood. However, an association between

AR-V7 status inwhole blood and resistance to treatment with

abiraterone or enzalutamide has not been reported to date.

In the present study, we established and validated a

liquid profiling approach with direct, absolute quantifica-

tion of AR-V7 and AR full length (AR-FL) mRNA levels in

peripheral whole blood using droplet digital polymerase

chain reaction (ddPCR), and determined its ability to predict

treatment resistance in mCRPC patients scheduled for

abiraterone or enzalutamide therapy.

Patients and methods

2.1.

Patient cohort and healthy donors

The study cohort included 85 patients with mCRPC who were treated at

the Department of Urology, Klinikum rechts der Isar, Technical University

of Munich in Germany between 2011 and 2016. These patients had

progressive disease as de

ned according to Prostate Cancer Clinical Trials

Working Group (PCWG2) criteria

[19]

at inclusion, and were scheduled

for a new line of systemic treatment of either abiraterone (

= 56) or

enzalutamide (

= 29). All patients signed institutional review board

–

approved consent before participation and were enrolled according to a

prospective biorepository protocol.

Treatment response was determined according to the institutional

standard procedure, including PSA levels within 1 wk before and every

4 wk after treatment initiation, as well as imaging procedures (computed

tomography and bone scan) within 4 wk before and every 3 mo after

treatment initiation.

The main endpoint was PSA response, de

ned as a PSA level decline

of 50%, as a marker for treatment response versus resistance. Further

study endpoints included PSA progression-free survival (PSA-PFS)

according to PCWG2 criteria

[19]

, clinical progression-free survival

(PFS), and OS. Clinical progression was de

ned as worsening of disease-

related symptoms or new cancer-related complications, radiographic

progression according to Response Evaluation Criteria In Solid Tumors

[20]

, two or more new bone lesions on bone scan, or death, whichever

occurred

rst

[19] .

Results are reported in compliance with REMARK

guidelines

[21]

2.2.

Blood samples

Blood samples were collected in 2.7-ml PAXgene blood RNA tubes

(Qiagen, Hilden, Germany) within 1 wk before treatment initiation. Total

Conclusions:

Testing of AR-V7 mRNA levels in whole blood is a simple and promising

approach to predict poor treatment outcome in mCRPC patients receiving abiraterone or

enzalutamide.

Patient summary:

We established a method for determining AR-V7 status in whole blood.

This test predicted treatment resistance in patients with metastatic castration-resistant

prostate cancer undergoing treatment with abiraterone or enzalutamide. Prospective

validation is needed before application to clinical practice.

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