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Androgen receptor expression in circulating tumour cells from castration-resistant prostate cancer patients treated with novel endocrine agents.

Crespo M, van Dalum G, Ferraldeschi R, Zafeiriou Z, Sideris S, Lorente D, Bianchini D, Rodrigues DN, Riisnaes R, Miranda S, Figueiredo I, Flohr P, Nowakowska K, de Bono JS, Terstappen LW, Attard G - Br. J. Cancer (2015)

Bottom Line: The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis.Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments.Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.

View Article: PubMed Central - PubMed

Affiliation: Section of Medicine, The Institute of Cancer Research, Sutton, Surrey, SM2 5NG, UK.

ABSTRACT

Background: Abiraterone and enzalutamide are novel endocrine treatments that abrogate androgen receptor (AR) signalling in castration-resistant prostate cancer (CRPC). Here, we developed a circulating tumour cells (CTCs)-based assay to evaluate AR expression in real-time in CRPC and investigated nuclear AR expression in CTCs in patients treated with enzalutamide and abiraterone.

Methods: CTCs were captured and characterised using the CellSearch system. An automated algorithm to identify CTCs and quantify AR expression was employed. The primary aim was to evaluate the association between CTC AR expression and prior treatment with abiraterone or enzalutamide.

Results: AR expression in CTCs was evaluated in 94 samples from 48 metastatic CRPC patients. We observed large intra-patient heterogeneity of AR expression in CTCs. Prior exposure to abiraterone or enzalutamide was not associated with a change in CTCs AR expression (median intensity and distribution of AR-positive classes). In support of this, we also confirmed maintained nuclear AR expression in tissue samples collected after progression on abiraterone. AR staining also identified additional AR-positive CD45-negative circulating cells that were CK-negative/weak and therefore missed using standard protocols. The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis.

Conclusions: We developed a non-invasive method to monitor AR nuclear expression in CTCs. Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments. Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.

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Related in: MedlinePlus

Assay development for the AR marker using the CellSearch system. (A) Mean AR intensity was determined by automated image analysis. Each frame was evaluated for CTC using a global CK-PE threshold (I). After classification (II), the mean AR-FITC intensity was determined inside the Cytoplasm (III) and Nucleus (IV). (B) Prostate cancer cell lines were spiked in healthy volunteer (HV) blood and detected on the CellSearch platform. Median nuclear AR intensity of individual cells±interquartile range (IQR) is plotted. AR nuclear intensity was determined using the automated algorithm. ****P<0.001, One-way ANOVA followed by Dunett's multiple comparison test. (C) Representative images of AR expression in five prostate cancer cell lines detected on the CellSearch platform. PC3 and DU145 cells were used as negative controls and displayed absence of AR expression. 22Rv1, LNCaP and VCaP were used as positive controls. (D) LNCaP cells were grown in RPMI supplemented with 10% FBS and treated with indicated concentrations of enzalutamide or vehicle (DMSO 0.2%) for 24 h before spiking in HV blood. Cells were then isolated and detected on the CellSearch platform. AR nuclear intensity was evaluated using the automated algorithm. Median±IQR is showed. ****P<0.001, One-way ANOVA followed by Dunett's multiple comparison test.
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fig1: Assay development for the AR marker using the CellSearch system. (A) Mean AR intensity was determined by automated image analysis. Each frame was evaluated for CTC using a global CK-PE threshold (I). After classification (II), the mean AR-FITC intensity was determined inside the Cytoplasm (III) and Nucleus (IV). (B) Prostate cancer cell lines were spiked in healthy volunteer (HV) blood and detected on the CellSearch platform. Median nuclear AR intensity of individual cells±interquartile range (IQR) is plotted. AR nuclear intensity was determined using the automated algorithm. ****P<0.001, One-way ANOVA followed by Dunett's multiple comparison test. (C) Representative images of AR expression in five prostate cancer cell lines detected on the CellSearch platform. PC3 and DU145 cells were used as negative controls and displayed absence of AR expression. 22Rv1, LNCaP and VCaP were used as positive controls. (D) LNCaP cells were grown in RPMI supplemented with 10% FBS and treated with indicated concentrations of enzalutamide or vehicle (DMSO 0.2%) for 24 h before spiking in HV blood. Cells were then isolated and detected on the CellSearch platform. AR nuclear intensity was evaluated using the automated algorithm. Median±IQR is showed. ****P<0.001, One-way ANOVA followed by Dunett's multiple comparison test.

Mentions: The exported image files from the CellTracks Analyzer II were reanalyzed using an automated algorithm developed in Matlab (Matworks, Natick, MA, USA) as previously described (Ligthart et al, 2011, 2013). This algorithm was modified to measure nuclear AR expression (Figure 1A). Briefly, this algorithm evaluates all CK-PE-positive events above a threshold determined per cartridge. An event becomes an automatically detected CTC (aCTC) when it meets the following attributes: size range between 75 and 500 pixels, CK-PE standard deviation above 50, a DAPI peak value of at least 170 arbitrary units (a.u.) and a CD45-APC maximum value of less than 60 a.u. If an event had a standard deviation for CK-PE below 50, it was classified CK-negative/weak. A cartridge-wide threshold was also determined for DAPI. The average AR expression inside both the nuclear mask and the cytokeratin-based mask was calculated. The AR-FITC was corrected by subtracting the surrounding background. Automated classification for whether a CTC was positive or negative for AR was based on the threshold set using AR-negative prostate cell lines (25 a.u.).


Androgen receptor expression in circulating tumour cells from castration-resistant prostate cancer patients treated with novel endocrine agents.

Crespo M, van Dalum G, Ferraldeschi R, Zafeiriou Z, Sideris S, Lorente D, Bianchini D, Rodrigues DN, Riisnaes R, Miranda S, Figueiredo I, Flohr P, Nowakowska K, de Bono JS, Terstappen LW, Attard G - Br. J. Cancer (2015)

Assay development for the AR marker using the CellSearch system. (A) Mean AR intensity was determined by automated image analysis. Each frame was evaluated for CTC using a global CK-PE threshold (I). After classification (II), the mean AR-FITC intensity was determined inside the Cytoplasm (III) and Nucleus (IV). (B) Prostate cancer cell lines were spiked in healthy volunteer (HV) blood and detected on the CellSearch platform. Median nuclear AR intensity of individual cells±interquartile range (IQR) is plotted. AR nuclear intensity was determined using the automated algorithm. ****P<0.001, One-way ANOVA followed by Dunett's multiple comparison test. (C) Representative images of AR expression in five prostate cancer cell lines detected on the CellSearch platform. PC3 and DU145 cells were used as negative controls and displayed absence of AR expression. 22Rv1, LNCaP and VCaP were used as positive controls. (D) LNCaP cells were grown in RPMI supplemented with 10% FBS and treated with indicated concentrations of enzalutamide or vehicle (DMSO 0.2%) for 24 h before spiking in HV blood. Cells were then isolated and detected on the CellSearch platform. AR nuclear intensity was evaluated using the automated algorithm. Median±IQR is showed. ****P<0.001, One-way ANOVA followed by Dunett's multiple comparison test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4385957&req=5

fig1: Assay development for the AR marker using the CellSearch system. (A) Mean AR intensity was determined by automated image analysis. Each frame was evaluated for CTC using a global CK-PE threshold (I). After classification (II), the mean AR-FITC intensity was determined inside the Cytoplasm (III) and Nucleus (IV). (B) Prostate cancer cell lines were spiked in healthy volunteer (HV) blood and detected on the CellSearch platform. Median nuclear AR intensity of individual cells±interquartile range (IQR) is plotted. AR nuclear intensity was determined using the automated algorithm. ****P<0.001, One-way ANOVA followed by Dunett's multiple comparison test. (C) Representative images of AR expression in five prostate cancer cell lines detected on the CellSearch platform. PC3 and DU145 cells were used as negative controls and displayed absence of AR expression. 22Rv1, LNCaP and VCaP were used as positive controls. (D) LNCaP cells were grown in RPMI supplemented with 10% FBS and treated with indicated concentrations of enzalutamide or vehicle (DMSO 0.2%) for 24 h before spiking in HV blood. Cells were then isolated and detected on the CellSearch platform. AR nuclear intensity was evaluated using the automated algorithm. Median±IQR is showed. ****P<0.001, One-way ANOVA followed by Dunett's multiple comparison test.
Mentions: The exported image files from the CellTracks Analyzer II were reanalyzed using an automated algorithm developed in Matlab (Matworks, Natick, MA, USA) as previously described (Ligthart et al, 2011, 2013). This algorithm was modified to measure nuclear AR expression (Figure 1A). Briefly, this algorithm evaluates all CK-PE-positive events above a threshold determined per cartridge. An event becomes an automatically detected CTC (aCTC) when it meets the following attributes: size range between 75 and 500 pixels, CK-PE standard deviation above 50, a DAPI peak value of at least 170 arbitrary units (a.u.) and a CD45-APC maximum value of less than 60 a.u. If an event had a standard deviation for CK-PE below 50, it was classified CK-negative/weak. A cartridge-wide threshold was also determined for DAPI. The average AR expression inside both the nuclear mask and the cytokeratin-based mask was calculated. The AR-FITC was corrected by subtracting the surrounding background. Automated classification for whether a CTC was positive or negative for AR was based on the threshold set using AR-negative prostate cell lines (25 a.u.).

Bottom Line: The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis.Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments.Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.

View Article: PubMed Central - PubMed

Affiliation: Section of Medicine, The Institute of Cancer Research, Sutton, Surrey, SM2 5NG, UK.

ABSTRACT

Background: Abiraterone and enzalutamide are novel endocrine treatments that abrogate androgen receptor (AR) signalling in castration-resistant prostate cancer (CRPC). Here, we developed a circulating tumour cells (CTCs)-based assay to evaluate AR expression in real-time in CRPC and investigated nuclear AR expression in CTCs in patients treated with enzalutamide and abiraterone.

Methods: CTCs were captured and characterised using the CellSearch system. An automated algorithm to identify CTCs and quantify AR expression was employed. The primary aim was to evaluate the association between CTC AR expression and prior treatment with abiraterone or enzalutamide.

Results: AR expression in CTCs was evaluated in 94 samples from 48 metastatic CRPC patients. We observed large intra-patient heterogeneity of AR expression in CTCs. Prior exposure to abiraterone or enzalutamide was not associated with a change in CTCs AR expression (median intensity and distribution of AR-positive classes). In support of this, we also confirmed maintained nuclear AR expression in tissue samples collected after progression on abiraterone. AR staining also identified additional AR-positive CD45-negative circulating cells that were CK-negative/weak and therefore missed using standard protocols. The number of these events correlated with traditional CTCs and was associated with worse outcome on univariate analysis.

Conclusions: We developed a non-invasive method to monitor AR nuclear expression in CTCs. Our studies confirm nuclear AR expression in CRPC patients progressing on novel endocrine treatments. Owing to the significant heterogeneity of AR expression in CTCs, studies in larger cohorts of patients are required to identify associations with outcome.

Show MeSH
Related in: MedlinePlus