Abstract 451: Transferrin Receptor identifies a distinct pool of circulating tumor cells from metastatic prostate cancer patients with unique molecular profiles enriched in AR variants
Authors: Gjyrezi Ada , Galletti Giuseppe , Zhang Jiaren , Bareja Rohan , Halima Ahmed , Worroll Daniel , Nanus David , Tagawa Scott , Beltran Himisha , Giannakakou Paraskevi 
Published: August 2019

Abstract
Androgen receptor (AR) drives prostate cancer (PC) progression, even in castrate-conditions due to expression of AR variants (AR-V), which are constitutively active in the nucleus. Castration-resistant prostate cancer (CRPC) is currently lethal, due to the development of drug resistance. The molecular basis of clinical drug resistance remains poorly elucidated, due to tumor heterogeneity and limited availability of serial tumor sampling as disease progresses. Circulating tumor cells (CTCs), isolated from patient peripheral blood, have emerged as a reliable, easy to obtain source of tumor cells which can be and used to interrogate the molecular disease evolution. To date, EpCAM-based CTC enrichment is commonly used but has important shortcomings as EpCAM is often lost during EMT, thus, missing the more aggressive mesenchymal CTCs. On the other hand, Transferrin Receptor (TfR) which is overexpressed in cancer cells, is a cell surface protein required for iron uptake and is critical for several biological processes such as cell proliferation and metabolism. Mining large gene expression databases in PC we observed that TfR expression increased with disease stage, from adenocarcinoma (TCGA) to CRPC (SU2C) to the more aggressive neuroendocrine PC (NEPC), while EpCAM expression remained unchanged. We also found that TfR expression was increased in EMT cell models, while EpCAM expression was lost, suggesting that TfR may perform well in CTC isolation across the EMT spectrum. We then tested TfR- and EpCAM-based CTC enrichment in a cohort of 31 CRPC patients, where we observed a significantly higher number of TfR+ CTCs (median, 304) than EpCAM+-CTCs (median, 21) (p&lt;0.01). RNA-sequencing of TfR+ CTCs in comparison to EpCAM+ CTCs, followed by gene-set enrichment analyses revealed that the TfR+ subpopulation was significantly enriched in prostate specific oncogenic pathways including EMT, suggesting distinct molecular profiles between the two subsets.
To investigate the clinical relevance of the two CTC subsets, we quantitated AR-FL and splice variants AR-V7 and AR-v567es expression by digital droplet PCR (ddPCR) in pools of TfR+-CTCs vs EpCAM+-CTCs. Our results from 16 CRPC patients showed that while expression of AR-FL was similar in the two CTC subsets, both AR variants were enriched in TfR+-CTCs. To better map the tumor heterogeneity of AR-V expression we isolated 102 single TfR+- and 63 single EpCAM+-CTCs from 3 CRPC patients and quantified AR-V expression by ddPCR. The single CTC analysis revealed that AR-V7 and AR-v567es were expressed in 21% and 18% of TfR+-CTCs, respectively, vs 0% in EpCAM+-CTCs. These data identify TfR as a promising marker for CTC identification across the EMT gradient, and provide a unique insight into tumor cell heterogeneity, which was not appreciated before, and which may have significant clinical impact.
[A. Gjyrezi and G. Galletti contributed equally to this work.]
Citation Format: Ada Gjyrezi, Giuseppe Galletti, Jiaren Zhang, Rohan Bareja, Ahmed Halima, Daniel Worroll, David Nanus, Scott Tagawa, Himisha Beltran, Paraskevi Giannakakou. Transferrin Receptor identifies a distinct pool of circulating tumor cells from metastatic prostate cancer patients with unique molecular profiles enriched in AR variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 451.