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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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Detection of AR+ CK-weak CTCs. (A) CK-AR+CD45- nucleated CTC counts significantly correlated with traditional CK+ aCTC. Linear regression. (B) Number of CK-AR+CD45- events detected in male healthy volunteers (N=4), breast cancer (N=6), colon cancer (N=1) and CRPC patients (N=48). Median±IQR is shown. (C) AR amplification and copy number gain was detected by FISH on CK-AR+CD45- events confirming malignant origin of these cells. Internal controls of the FISH method can be seen on the sample 5025 (event 682) containing three leukocytes with one AR/chromosome X copy number per leukocyte. Last image shows an example of a traditional CK+ CTC with AR and X chromosome copy gain.
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fig4: Detection of AR+ CK-weak CTCs. (A) CK-AR+CD45- nucleated CTC counts significantly correlated with traditional CK+ aCTC. Linear regression. (B) Number of CK-AR+CD45- events detected in male healthy volunteers (N=4), breast cancer (N=6), colon cancer (N=1) and CRPC patients (N=48). Median±IQR is shown. (C) AR amplification and copy number gain was detected by FISH on CK-AR+CD45- events confirming malignant origin of these cells. Internal controls of the FISH method can be seen on the sample 5025 (event 682) containing three leukocytes with one AR/chromosome X copy number per leukocyte. Last image shows an example of a traditional CK+ CTC with AR and X chromosome copy gain.

Mentions: Automated analysis of epithelial cellular adhesion molecule-positive events captured on CellSearch with additional AR staining identified a sub-class of AR-positive, CD45-negative, intact, nucleated circulating cells that had negative or weak CK (CK-) expression. CK-AR+CD45- nucleated cells counts ranged from 0 to 286 (median 2; mean 21) in CRPC patients and significantly correlated with traditional CK-positive aCTC (R2 0.72; P<0.0001) (Figure 4A). More than four CK-AR+CD45- events were found in 18/48 (37.5%) CRPC patients and were not observed in male healthy volunteers or other tumour types (Figure 4B). We detected AR amplification or AR copy number/X chromosome gain in three of these patients supporting a malignant origin of these CK weak/negative circulating cells (Figure 4C). The total number of CK weak/negative cells was associated with overall survival in univariate Cox Regression analysis (continuous) (P=0.004) in CRPC patients.


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)

Detection of AR+ CK-weak CTCs. (A) CK-AR+CD45- nucleated CTC counts significantly correlated with traditional CK+ aCTC. Linear regression. (B) Number of CK-AR+CD45- events detected in male healthy volunteers (N=4), breast cancer (N=6), colon cancer (N=1) and CRPC patients (N=48). Median±IQR is shown. (C) AR amplification and copy number gain was detected by FISH on CK-AR+CD45- events confirming malignant origin of these cells. Internal controls of the FISH method can be seen on the sample 5025 (event 682) containing three leukocytes with one AR/chromosome X copy number per leukocyte. Last image shows an example of a traditional CK+ CTC with AR and X chromosome copy gain.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Detection of AR+ CK-weak CTCs. (A) CK-AR+CD45- nucleated CTC counts significantly correlated with traditional CK+ aCTC. Linear regression. (B) Number of CK-AR+CD45- events detected in male healthy volunteers (N=4), breast cancer (N=6), colon cancer (N=1) and CRPC patients (N=48). Median±IQR is shown. (C) AR amplification and copy number gain was detected by FISH on CK-AR+CD45- events confirming malignant origin of these cells. Internal controls of the FISH method can be seen on the sample 5025 (event 682) containing three leukocytes with one AR/chromosome X copy number per leukocyte. Last image shows an example of a traditional CK+ CTC with AR and X chromosome copy gain.
Mentions: Automated analysis of epithelial cellular adhesion molecule-positive events captured on CellSearch with additional AR staining identified a sub-class of AR-positive, CD45-negative, intact, nucleated circulating cells that had negative or weak CK (CK-) expression. CK-AR+CD45- nucleated cells counts ranged from 0 to 286 (median 2; mean 21) in CRPC patients and significantly correlated with traditional CK-positive aCTC (R2 0.72; P<0.0001) (Figure 4A). More than four CK-AR+CD45- events were found in 18/48 (37.5%) CRPC patients and were not observed in male healthy volunteers or other tumour types (Figure 4B). We detected AR amplification or AR copy number/X chromosome gain in three of these patients supporting a malignant origin of these CK weak/negative circulating cells (Figure 4C). The total number of CK weak/negative cells was associated with overall survival in univariate Cox Regression analysis (continuous) (P=0.004) in CRPC patients.

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