Using circulating tumor cells to inform on prostate cancer biology and clinical utility

Abstract

Substantial advances in the molecular biology of prostate cancer have led to the approval of multiple new systemic agents to treat men with metastatic castration-resistant prostate cancer (mCRPC). These treatments encompass androgen receptor directed therapies, immunotherapies, bone targeting radiopharmaceuticals and cytotoxic chemotherapies. There is, however, great heterogeneity in the degree of patient benefit with these agents, thus fueling the need to develop predictive biomarkers that are able to rationally guide therapy. Circulating tumor cells (CTCs) have the potential to provide an assessment of tumor-specific biomarkers through a non-invasive, repeatable "liquid biopsy" of a patient's cancer at a given point in time. CTCs have been extensively studied in men with mCRPC, where CTC enumeration using the Cellsearch® method has been validated and FDA approved to be used in conjunction with other clinical parameters as a prognostic biomarker in metastatic prostate cancer. In addition to enumeration, more sophisticated molecular profiling of CTCs is now feasible and may provide more clinical utility as it may reflect tumor evolution within an individual particularly under the pressure of systemic therapies. Here, we review technologies used to detect and characterize CTCs, and the potential biological and clinical utility of CTC molecular profiling in men with metastatic prostate cancer.

Keywords: Androgen receptor; EpCAM; PSA; biomarker; castration resistant prostate cancer; liquid biopsy; microfluidic.

Figure 1

Schematic illustration of the three major steps of a CTC assay. Due to CTC rarity in peripheral blood, most capture techniques require CTC enrichment, followed by detection. (1) There are multiple approaches to enrich CTCs based on physical or biological properties that distinguish CTCs from other circulating cells. (2) After enrichment, CTCs can be detected by different techniques, including protein-based detection, nucleic acid-based and telomerase activity-based detection. (3) Successful molecular characterization of CTCs could provide a real-time assessment of cancer metastasis biology, tumor biomarkers such as mutations or epigenetic signatures or gene expression levels and avoid the necessity of repeated invasive biopsies.

Figure 2

Epithelial plasticity during prostate cancer dissemination. Reproduced from Bitting et al.. Due to genetic or epigenetic changes, normal prostate cells begin to grow un-controllably, a premalignant process known as prostate intraepithelial neoplasia (PIN). In response to signaling from the surrounding stroma, some of these cells undergo an epithelial–mesenchymal transition (EMT) and invade through the basement membrane. These invasive cells enter the bloodstream and may exist as epithelial circulating tumor cells (CTCs), mesenchymal CTCs, or CTCs with a dual phenotype. Upon exiting the vasculature, disseminated tumor cells (DTCs) may sit dormant or undergo apoptosis. Other DTCs undergo a mesenchymal–epithelial transition (MET) and grow as detectable macrometastases. In prostate cancer (PC), bone metastases are typical and are initially AR dependent, progressing through a range of AR mutations or splice variants and other oncogenic and tumor suppressor mutations. Visceral metastases are atypical, are variably AR dependent, and generally involve loss of an epithelial phenotype (EP) and are enriched for a neuroendocrine or anaplastic phenotype. EP is not clearly linked to the process of lymph node metastasis; instead, nodal metastases likely involve other forms of invasion or migration.

Figure 3

Co-expression of epithelial and mesenchymal proteins in CTCs from men with metastatic castration resistant prostate cancer (mCRPC). Reproduced from Armstrong et al.. All panels represent merged images derived from phase/DAPI, CD45/DAPI, CK/DAPI and either vimentin (Vim)/DAPI, N-cadherin (N-cad)/DAPI expression or O-cadherin (O-cad)/DAPI as indicated. Shown are examples of (A) a leukocyte with CD45 expression, (B) a CTC with no vimentin expression, (C) a CTC with vimentin expression, (D) a CTC with N-cadherin expression, (E) 3 CTCs, 2 with N-cadherin expression (arrowheads), (F) a CTC with O-cadherin expression and a nearby leukocyte and (G) an additional CTC with O-cadherin expression. Scale bars represent 20 nm and were added from an image taken at identical magnification and resolution. Control cells were assayed in parallel at the same time of CTC collection and analysis with each set of patient samples and are shown in the Supplementary material.