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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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CTC AR nuclear expression in abiraterone- or enzalutamide-resistant CRPC patients. (A) AR intensity in individual CTC in samples from abiraterone- and enzalutamide-naïve patients (n=19) and patients who had progressed on abiraterone or enzalutamide but continued (n=10) or discontinued (n=14) treatment. The box plots show the AR nuclear expression in white blood cells in the same sample. Samples were sorted first on more or equal to five aCTCs and then by median AR expression (only patients with ≥1 aCTCs were included). (B) Percentage of aCTCs divided by classes of AR intensity expression. Only patients with ≥4 aCTCs were included. (C) Waterfall plot of the maximum percentage change in nuclear AR intensity of CTCs at baseline and at progression. Bars represent individual patients and grey colour correspond to response to treatment defined as a confirmed >50% decline in PSA in the absence of radiological progression. Increases were capped at 100%. (D) Scatter plot of nuclear AR intensity of individual cancer cells in formalin-fixed paraffin-embedded tissue CRPC tissue sections. Samples were sorted, first on previous exposure to abiraterone and second on the median intensity of AR. Median intensity±IQR is shown (n.c.–normalised counts).
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fig3: CTC AR nuclear expression in abiraterone- or enzalutamide-resistant CRPC patients. (A) AR intensity in individual CTC in samples from abiraterone- and enzalutamide-naïve patients (n=19) and patients who had progressed on abiraterone or enzalutamide but continued (n=10) or discontinued (n=14) treatment. The box plots show the AR nuclear expression in white blood cells in the same sample. Samples were sorted first on more or equal to five aCTCs and then by median AR expression (only patients with ≥1 aCTCs were included). (B) Percentage of aCTCs divided by classes of AR intensity expression. Only patients with ≥4 aCTCs were included. (C) Waterfall plot of the maximum percentage change in nuclear AR intensity of CTCs at baseline and at progression. Bars represent individual patients and grey colour correspond to response to treatment defined as a confirmed >50% decline in PSA in the absence of radiological progression. Increases were capped at 100%. (D) Scatter plot of nuclear AR intensity of individual cancer cells in formalin-fixed paraffin-embedded tissue CRPC tissue sections. Samples were sorted, first on previous exposure to abiraterone and second on the median intensity of AR. Median intensity±IQR is shown (n.c.–normalised counts).

Mentions: We then examined AR expression in aCTCs from CRPC patients according to prior exposure to abiraterone or enzalutamide therapy. We selected samples with at least four aCTCs and classified every aCTCs as AR-negative or AR-positive and then every one of the AR-positive aCTC into one of four quartiles (Figure 2D). We observed inter-patient and intra-patient heterogeneity in expression of AR in aCTCs (Figure 3A). Overall, we did not find significant changes in AR expression (median intensity and distribution of AR-positive classes) between aCTC from abiraterone and enzalutamide-naïve patients (n=11) and patients who had progressed on abiraterone or enzalutamide but continued (n=9) or discontinued (n=12) treatment (Figure 3B). Median nuclear AR intensity was 17.8 (IQR 13.9–50.6) in the abiraterone- and enzalutamide-naïve patients, 18.7 (IQR 8.0–35.1) in patients who had progressed on abiraterone or enzalutamide but still receiving treatment at the time of the blood draw, and 23.3 (IQR 10.9–35.8) in patients who had previously received abiraterone or enzalutamide and discontinued treatment owing to progression. Median percentage of AR-positive CTCs was 29% (IQR 10–71), 35% (5–54) and 44% (IQR 20–65), respectively. The difference was not significant (Supplementary Figures 4A and B).


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)

CTC AR nuclear expression in abiraterone- or enzalutamide-resistant CRPC patients. (A) AR intensity in individual CTC in samples from abiraterone- and enzalutamide-naïve patients (n=19) and patients who had progressed on abiraterone or enzalutamide but continued (n=10) or discontinued (n=14) treatment. The box plots show the AR nuclear expression in white blood cells in the same sample. Samples were sorted first on more or equal to five aCTCs and then by median AR expression (only patients with ≥1 aCTCs were included). (B) Percentage of aCTCs divided by classes of AR intensity expression. Only patients with ≥4 aCTCs were included. (C) Waterfall plot of the maximum percentage change in nuclear AR intensity of CTCs at baseline and at progression. Bars represent individual patients and grey colour correspond to response to treatment defined as a confirmed >50% decline in PSA in the absence of radiological progression. Increases were capped at 100%. (D) Scatter plot of nuclear AR intensity of individual cancer cells in formalin-fixed paraffin-embedded tissue CRPC tissue sections. Samples were sorted, first on previous exposure to abiraterone and second on the median intensity of AR. Median intensity±IQR is shown (n.c.–normalised counts).
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Related In: Results  -  Collection

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fig3: CTC AR nuclear expression in abiraterone- or enzalutamide-resistant CRPC patients. (A) AR intensity in individual CTC in samples from abiraterone- and enzalutamide-naïve patients (n=19) and patients who had progressed on abiraterone or enzalutamide but continued (n=10) or discontinued (n=14) treatment. The box plots show the AR nuclear expression in white blood cells in the same sample. Samples were sorted first on more or equal to five aCTCs and then by median AR expression (only patients with ≥1 aCTCs were included). (B) Percentage of aCTCs divided by classes of AR intensity expression. Only patients with ≥4 aCTCs were included. (C) Waterfall plot of the maximum percentage change in nuclear AR intensity of CTCs at baseline and at progression. Bars represent individual patients and grey colour correspond to response to treatment defined as a confirmed >50% decline in PSA in the absence of radiological progression. Increases were capped at 100%. (D) Scatter plot of nuclear AR intensity of individual cancer cells in formalin-fixed paraffin-embedded tissue CRPC tissue sections. Samples were sorted, first on previous exposure to abiraterone and second on the median intensity of AR. Median intensity±IQR is shown (n.c.–normalised counts).
Mentions: We then examined AR expression in aCTCs from CRPC patients according to prior exposure to abiraterone or enzalutamide therapy. We selected samples with at least four aCTCs and classified every aCTCs as AR-negative or AR-positive and then every one of the AR-positive aCTC into one of four quartiles (Figure 2D). We observed inter-patient and intra-patient heterogeneity in expression of AR in aCTCs (Figure 3A). Overall, we did not find significant changes in AR expression (median intensity and distribution of AR-positive classes) between aCTC from abiraterone and enzalutamide-naïve patients (n=11) and patients who had progressed on abiraterone or enzalutamide but continued (n=9) or discontinued (n=12) treatment (Figure 3B). Median nuclear AR intensity was 17.8 (IQR 13.9–50.6) in the abiraterone- and enzalutamide-naïve patients, 18.7 (IQR 8.0–35.1) in patients who had progressed on abiraterone or enzalutamide but still receiving treatment at the time of the blood draw, and 23.3 (IQR 10.9–35.8) in patients who had previously received abiraterone or enzalutamide and discontinued treatment owing to progression. Median percentage of AR-positive CTCs was 29% (IQR 10–71), 35% (5–54) and 44% (IQR 20–65), respectively. The difference was not significant (Supplementary Figures 4A and B).

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