RESUMEN

OBJECTIVES: To use a non-biased assay for circulating tumour cells (CTCs) in patients with prostate cancer (PCa) in order to identify non-traditional CTC phenotypes potentially excluded by conventional detection methods that are reliant on antigen- and/or size-based enrichment. PATIENTS AND METHODS: A total of 41 patients with metastatic castration-resistant PCa (mCRPC) and 20 healthy volunteers were analysed on the Epic CTC platform, via high-throughput imaging of DAPI expression and CD45/cytokeratin (CK) immunofluorescence (IF) on all circulating nucleated cells plated on glass slides. To confirm the PCa origin of CTCs, IF was used for androgen receptor (AR) expression and fluorescence in situ hybridization was used for PTEN and ERG assessment. RESULTS: Traditional CTCs (CD45- /CK+ /morphologically distinct) were identified in all patients with mCRPC and we also identified CTC clusters and non-traditional CTCs in patients with mCRPC, including CK- and apoptotic CTCs. Small CTCs (≤white blood cell size) were identified in 98% of patients with mCRPC. Total, traditional and non-traditional CTCs were significantly increased in patients who were deceased vs alive after 18 months; however, only non-traditional CTCs were associated with overall survival. Traditional and total CTC counts according to the Epic platform in the mCRPC cohort were also significantly correlated with CTC counts according to the CellSearch system. CONCLUSIONS: Heterogeneous non-traditional CTC populations are frequent in mCRPC and may provide additional prognostic or predictive information.

Asunto(s)

Metástasis de la Neoplasia/patología , Células Neoplásicas Circulantes/patología , Neoplasias de la Próstata Resistentes a la Castración/sangre , Neoplasias de la Próstata Resistentes a la Castración/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Progresión de la Enfermedad , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia/genética , Fosfohidrolasa PTEN/sangre , Fosfohidrolasa PTEN/genética , Fenotipo , Antígeno Prostático Específico/sangre , Antígeno Prostático Específico/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Receptores Androgénicos/genética

RESUMEN

BACKGROUND: PTEN gene loss occurs frequently in castration-resistant prostate cancer (CRPC) and may drive progression through activation of the PI3K/AKT pathway. Here, we developed a novel CTC-based assay to determine PTEN status and examined the correlation between PTEN status in CTCs and matched tumour tissue samples. METHODS: PTEN gene status in CTCs was evaluated on an enrichment-free platform (Epic Sciences) by fluorescence in situ hybridisation (FISH). PTEN status in archival and fresh tumour tissue was evaluated by FISH and immunohistochemistry. RESULTS: Peripheral blood was collected from 76 patients. Matched archival and fresh cancer tissue was available for 48 patients. PTEN gene status detected in CTCs was concordant with PTEN status in matched fresh tissues and archival tissue in 32 of 38 patients (84%) and 24 of 39 patients (62%), respectively. CTC counts were prognostic (continuous, P=0.001). PTEN loss in CTCs associated with worse survival in univariate analysis (HR 2.05; 95% CI 1.17-3.62; P=0.01) and with high lactate dehydrogenase (LDH) in metastatic CRPC patients. CONCLUSIONS: Our results illustrate the potential use of CTCs as a non-invasive, real-time liquid biopsy to determine PTEN gene status. The prognostic and predictive value of PTEN in CTCs warrants investigation in CRPC clinical trials of PI3K/AKT-targeted therapies.

Asunto(s)

Células Neoplásicas Circulantes/patología , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Anciano , Progresión de la Enfermedad , Humanos , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , L-Lactato Deshidrogenasa/genética , Masculino , Fosfatidilinositol 3-Quinasas/genética , Pronóstico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo

RESUMEN

The Epic Platform was developed for the unbiased detection and molecular characterization of circulating tumour cells (CTCs). Here, we report assay performance data, including accuracy, linearity, specificity and intra/inter-assay precision of CTC enumeration in healthy donor (HD) blood samples spiked with varying concentrations of cancer cell line controls (CLCs). Additionally, we demonstrate clinical feasibility for CTC detection in a small cohort of metastatic castrate-resistant prostate cancer (mCRPC) patients. The Epic Platform demonstrated accuracy, linearity and sensitivity for the enumeration of all CLC concentrations tested. Furthermore, we established the precision between multiple operators and slide staining batches and assay specificity showing zero CTCs detected in 18 healthy donor samples. In a clinical feasibility study, at least one traditional CTC/mL (CK+, CD45-, and intact nuclei) was detected in 89 % of 44 mCRPC samples, whereas 100 % of samples had CTCs enumerated if additional CTC subpopulations (CK-/CD45- and CK+ apoptotic CTCs) were included in the analysis. In addition to presenting Epic Platform's performance with respect to CTC enumeration, we provide examples of its integrated downstream capabilities, including protein biomarker expression and downstream genomic analyses at single cell resolution.

RESUMEN

Retrospective analysis of patient tumour samples is a cornerstone of clinical research. CTC biomarker characterization offers a non-invasive method to analyse patient samples. However, current CTC technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. We sought to investigate CTC recovery from frozen, archived patient PBMC pellets. Matched samples from both mCRPC patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed "fresh" via Epic CTC Platform or from "frozen" PBMC pellets. Samples were analysed for CTC enumeration and biomarker characterization via immunofluorescent (IF) biomarkers, fluorescence in-situ hybridization (FISH) and CTC morphology. In the frozen patient PMBC samples, the median CTC recovery was 18%, compared to the freshly processed blood. However, abundance and localization of cytokeratin (CK) and androgen receptor (AR) protein, as measured by IF, were largely concordant between the fresh and frozen CTCs. Furthermore, a FISH analysis of PTEN loss showed high concordance in fresh vs. frozen. The observed data indicate that CTC biomarker characterization from frozen archival samples is feasible and representative of prospectively collected samples.