Abstract 428: Phenotyping pancreatic cancer CTCs as biomarkers for RX-3117 clinical trials
Authors: Eisen Andrew , Kim DJ , Yang Jie , Wu Weiguo , Davis Darren W. 
Published: August 2019

Abstract
Background:
RX-3117 is an oral, small molecule nucleoside analogue pro-drug being used in combination with nab-paclitaxel in a Ph2 clinical trial for pancreatic cancer. RX-3117 can be transported intracellularly by SLC29A1 (hENT1) and is converted to an active agent by the cancer-enriched enzyme, uridine-cytidine kinase 2 (UCK2) for incorporation into RNA and DNA. This leads to cancer cell apoptosis. We developed an approach to enumerate and phenotype CTCs from pancreatic cancer subjects by quantitative immunofluorescence (QIF) to assess whether CTC numbers, phenotypic features and/or early apoptotic responses to therapy predict or presage clinical response.
Methods:
QIF staining parameters were developed with cancer cell lines sensitive and resistant to RX-3117 utilizing monoclonal antibodies to hENT1 and UCK2. The staining methods were then applied to CTCs isolated from 10 ml anticoagulated blood by dielectrophoretic (DEP) properties that differentiate cancer cells and normal PBMCs using the ApoStream device (ApoCell, TX). In addition to the drug-related markers, CTCs were stained for a panel of either Epithelial or Mesenchymal markers, CD45 and nuclei (DAPI). Phenotyped CTCs were binned into 6 categories defined by whether they were EPI+/-, EMT+/- or CD45+/-. The percentage of cells in each category also positive for hENT1 or UCK2 was determined as was the mean fluorescence intensity of the marker+ sub-population. In addition, assessment of hENT1 and UCK2 expression was performed by qRT-PCR utilizing mRNA isolated from isolated CTCs, the buffy coat from spun blood and in plasma.
Results:
In a pilot study, blood was obtained from five subjects with Stage IV pancreatic cancer on various regimens. CTCs were isolated, enumerated and phenotyped. CTC numbers ranged from 100 – 20,000 per ml. The percentages of UCK2+ cells were higher in EPI+/EMT+ subsets than EPI-/EMT- subsets. The percentages of hENT1+ cells were generally low in all the subsets. hENT1 and UCK2 transcripts were observed at acceptable levels in the cell populations, and borderline, but consistent levels in plasma samples. CEACAM5 transcripts were observed only in one CTC and PBMC fraction from one subject. After normalization vs. housekeeping transcripts, a greater than 2-fold increase of UCK2 was observed in three CTC samples compared to PBMC samples. A greater than 2-fold increase of hENT1 was observed in two CTC samples.
Conclusion:
CTCs from patients with pancreatic cancer can be obtained in numbers suitable for multiparameter phenotyping to identify features that might serve as selective, predictive or prognostic biomarkers in clinical trials. Multiparameter phenotyping of several relevant markers is being performed by quantitative immunofluorescence on CTCs isolated from pancreatic cancer patients in an on-going Ph2 clinical trial of RX-3117 in combination with nab-paclitaxel.
Citation Format: Andrew Eisen, DJ Kim, Jie Yang, Weiguo Wu, Darren W. Davis. Phenotyping pancreatic cancer CTCs as biomarkers for RX-3117 clinical trials [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 428.