Clinical Utility of Circulating Tumour Cell Androgen Receptor Splice Variant-7 Status in Metastatic Castration-resistant Prostate Cancer.

Detection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumour cells (CTCs) is associated with worse outcome in metastatic castration-resistant prostate cancer (mCRPC). However, studies rarely report comparisons with CTC counts and biopsy AR-V7 protein expression. To determine the reproducibility of AdnaTest CTC AR-V7 testing, and associations with clinical characteristics, CellSearch CTC counts, tumour biopsy AR-V7 protein expression and overall survival (OS). CTC AR-V7 status was determined for 227 peripheral blood samples, from 181 mCRPC patients with CTC counts (202 samples; 136 patients) and matched mCRPC biopsies (65 samples; 58 patients). CTC AR-V7 status was associated with clinical characteristics, CTC counts, and tissue biopsy AR-V7 protein expression. The association of CTC AR-V7 status and other baseline variables with OS was determined. Of the samples, 35% were CTC+/AR-V7+. CTC+/AR-V7+ samples had higher CellSearch CTC counts (median CTC; interquartile range [IQR]: 60, 19-184 vs 9, 2-64; Mann-Whitney test p<0.001) and biopsy AR-V7 protein expression (median H-score, IQR: 100, 63-148 vs 15, 0-113; Mann-Whitney test p=0.004) than CTC+/AR-V7- samples. However, both CTC- (63%) and CTC+/AR-V7- (62%) patients had detectable AR-V7 protein in contemporaneous biopsies. After accounting for baseline characteristics, there was shorter OS in CTC+/AR-V7+ patients than in CTC- patients (hazard ratio [HR] 2.13; 95% confidence interval [CI] 1.23-3.71; p=0.02); surprisingly, there was no evidence that CTC+/AR-V7+ patients had worse OS than CTC+/AR-V7- patients (HR 1.26; 95% CI 0.73-2.17; p=0.4). A limitation of this study was the heterogeneity of treatment received. Studies reporting the prognostic relevance of CTC AR-V7 status must account for CTC counts. Discordant CTC AR-V7 results and AR-V7 protein expression in matched, same-patient biopsies are reported. Liquid biopsies that determine circulating tumour cell androgen receptor splice variant-7 status have the potential to impact treatment decisions in metastatic castration-resistant prostate cancer patients. Robust clinical qualification of these assays is required before their routine use.

Sharp A ，Welti JC ，Lambros MBK ，Dolling D ，Rodrigues DN ，Pope L ，Aversa C ，Figueiredo I ，Fraser J ，Ahmad Z ，Lu C ，Rescigno P ，Kolinsky M ，Bertan C ，Seed G ，Riisnaes R ，Miranda S ，Crespo M ，Pereira R ，Ferreira A ，Fowler G ，Ebbs B ，Flohr P ，Neeb A ，Bianchini D ，Petremolo A ，Sumanasuriya S ，Paschalis A ，Mateo J ，Tunariu N ，Yuan W ，Carreira S ，Plymate SR ，Luo J ，de Bono JS ... -  《-》

Associations between AR-V7 status in circulating tumour cells, circulating tumour cell count and survival in men with metastatic castration-resistant prostate cancer.

The interpretation of the presence of AR-V7 in circulating tumour cells (CTCs) in men with metastatic castration-resistant prostate cancer (mCRPC) remains to be elucidated. AR-V7 may hold promise as a predictive biomarker, but there may be prognostic impact of AR-V7 positivity as well. To investigate the clinical value of AR-V7, we determined whether AR-V7 detection in CTCs in patients with mCRPC is associated with CTC counts and survival. Between December 2011 and January 2019, three prospective clinical trials collected clinical data of patients with mCRPC, who progressed after docetaxel and/or enzalutamide or abiraterone. Baseline (and follow-up) blood samples were withdrawn determining CTC count and AR-V7 expression. The majority of patients started cabazitaxel as the next line of treatment after AR-V7 characterisation. A total of 127 samples were evaluable for the analysis of CTC count versus AR-V7 status. Although an association was observed between AR-V7 and CTC count in all patients with mCRPC (p = 0.017), no such association was found in the prognostic unfavourable subgroup of patients with ≥5 CTCs. After adjusting for clinical prognostic factors, AR-V7 expression in CTCs was not associated with overall survival (hazard ratio = 1.33, 95% confidence interval = 0.81-2.15, p = 0.25). We found that AR-V7 expression in CTCs had no additional prognostic value in patients with mCRPC, mostly treated with cabazitaxel. In patients with mCRPC with a predefined worse prognosis of a higher CTC count (≥5), a predictive biomarker is an important unmet medical need. Prospective trials should investigate whether AR-V7 detection in CTCs may guide treatment selection for these adverse prognosis patients.

Belderbos BPS ，Sieuwerts AM ，Hoop EO ，Mostert B ，Kraan J ，Hamberg P ，Van MN ，Beaufort CM ，Onstenk W ，van Soest RJ ，Martens J ，Sleijfer S ，de Wit R ，Mathijssen RHJ ，Lolkema MP ... -  《-》

AR-V7 in Peripheral Whole Blood of Patients with Castration-resistant Prostate Cancer: Association with Treatment-specific Outcome Under Abiraterone and Enzalutamide.

It has been demonstrated that androgen receptor splice variant 7 (AR-V7) expression in circulating tumor cells (CTCs) predicts poor treatment response in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone or enzalutamide. To develop a practical and robust liquid profiling approach for direct quantification of AR-V7 in peripheral whole blood without the need for CTC capture and to determine its potential for predicting treatment response in mCRPC patients. Whole blood samples from a prospective biorepository of 85 mCRPC patients before treatment initiation with abiraterone (n=56) or enzalutamide (n=29) were analyzed via droplet digital polymerase chain reaction. The association of AR-V7 status with prostate-specific antigen (PSA) response defined by PSA decline ≥50% and with PSA-progression-free survival (PSA-PFS), clinical PFS, and overall survival (OS) was assessed. High AR-V7 expression levels in whole blood were detectable in 18% (15/85) of patients. No patient with high AR-V7 expression achieved a PSA response, and AR-V7 status was an independent predictor of PSA response in multivariable logistic regression analysis (p=0.03). High AR-V7 expression was associated with shorter PSA-PFS (median 2.4 vs 3.7 mo; p<0.001), shorter clinical PFS (median 2.7 vs 5.5 mo; p<0.001), and shorter OS (median 4.0 vs. 13.9 mo; p<0.001). On multivariable Cox regression analysis, high AR-V7 expression remained an independent predictor of shorter PSA-PFS (hazard ratio [HR] 7.0, 95% confidence interval [CI] 2.3-20.7; p<0.001), shorter clinical PFS (HR 2.3, 95% CI 1.1-4.9; p=0.02), and shorter OS (HR 3.0, 95% CI 1.4-6.3; p=0.005). 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. 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.

Seitz AK ，Thoene S ，Bietenbeck A ，Nawroth R ，Tauber R ，Thalgott M ，Schmid S ，Secci R ，Retz M ，Gschwend JE ，Ruland J ，Winter C ，Heck MM ... -  《-》

Assessment of Total, PTEN(-), and AR-V7(+) Circulating Tumor Cell Count by Flow Cytometry in Patients with Metastatic Castration-Resistant Prostate Cancer Receiving Enzalutamide.

Metastatic castration-resistant prostate cancer (mCRPC) is a deadly disease. Enzalutamide is an oral second-generation anti-androgen that is active in mCRPC. Circulating tumor cells (CTC) count correlates with overall survival (OS) in mCRPC, whereas detection of the androgen-receptor splice variant 7 (AR-V7) in CTC predicts poor response to oral second-generation anti-androgens. Also, loss of PTEN (phosphatase and tensin homolog) in CTC is a biomarker of poor prognosis in mCRPC. In this translational study, we employed flow cytometry to assess total, PTEN-, and AR-V7+ CTC count per 7.5 mL of whole blood in a prospective cohort of patients with mCRPC receiving enzalutamide. CTCs were assessed in a total of 45 men with mCRPC at baseline and at 12 weeks. Overall, CTC, PTEN- CTC, and AR-V7+ CTC detection rate was high, at baseline, with 84.4%, 71.1%, and 51.1% of samples showing at least 1 cell/7.5-mL blood, respectively, and after 3 months, with 93.3%, 64.4%, and 77.7% of samples showing at least 1 cell/7.5-mL blood, respectively. Median radiographic progression-free survival (rPFS) and OS were 6 (95% confidence interval [CI], 5.6-9) and 14.3 (95% CI, 12.8-20.3) months, respectively. Median (interquartile range) total CTC count at baseline was 5 (3; 8), whereas median (interquartile range) PTEN- CTC count was 2 (0; 4) and median (interquartile range) AR-V7+ CTC count was 1 (0; 3). At baseline, ≥ 5 versus < 5 total CTC count was associated with worse rPFS (hazard ratio [HR], 2.35; 95% CI, 1.14-4.84; P= .021) and OS (HR, 3.08; 95% CI, 1.45-6.54; P = .003), whereas ≥ 2 versus < 2 PTEN- CTC count was associated with worse rPFS (HR, 3.96; 95% CI, 1.8-8.72; P= .001) and OS (HR, 2.36; 95% CI, 1.12-5; P= .025). Finally, ≥ 1 versus < 1 AR-V7+ CTC count was also associated with worse rPFS (HR, 5.05; 95% CI, 2.4-10.64; P< .001) and OS (HR, 2.25; 95% CI, 1.1-4.58; P= .026). Despite multiple limitations, including the small sample size, our preliminary study suggests that assessment of CTC via flow cytometry may provide potentially useful prognostic and predictive information in advanced prostate cancer. Further studies are warranted. Micro-Abstract: In this study, men with metastatic castration-resistant prostate cancer, scheduled to start enzalutamide, were assessed for circulating tumor cell count and molecular characterization (total, PTEN-, and AR-V7+ circulating tumor cell count) by the use of flow cytometry. We found that flow cytometry could be used to enumerate circulating tumor cells, but also to assess molecular biomarkers on their surface.

Di Lorenzo G ，Zappavigna S ，Crocetto F ，Giuliano M ，Ribera D ，Morra R ，Scafuri L ，Verde A ，Bruzzese D ，Iaccarino S ，Costabile F ，Onofrio L ，Viggiani M ，Palmieri A ，De Placido P ，Marretta AL ，Pietroluongo E ，Luce A ，Abate M ，Navaeiseddighi Z ，Caputo VF ，Celentano G ，Longo N ，Ferro M ，Morelli F ，Facchini G ，Caraglia M ，De Placido S ，Buonerba C ... -  《-》

Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cance

Therapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported. To characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay. AR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA. VTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

Liu HE ，Vuppalapaty M ，Hoerner CR ，Bergstrom CP ，Chiu M ，Lemaire C ，Che J ，Kaur A ，Dimmick A ，Liu S ，Metzner TJ ，Araya M ，Crouse S ，Sprenger-Haussels M ，Schlumpberger M ，Leppert JT ，Hauch S ，Sollier E ，Fan AC ... -  《BMC CANCER》