Abstract 1550: Multi-biomarker subtyping of circulating tumor cells using sequential fluorescence quenching
Authors: Adams Daniel L. , Tsai Susan , Tang Cha-Mei , Stefansson Steingrimur 
Published: July 2016

Abstract
Background: Circulating tumor cells (CTCs) are rare but clinically valuable indicators of cancer status. However their clinical utility is limited to 2-3 positive fluorescent markers and 1 negative fluorescent marker. By contrast, tissue biopsies allow for numerous subtyping markers, yielding information about the tumor's biology and predicted treatment response. If CTC analysis is to be more useful, it must move beyond 2-3 identification markers. We describe a simple and inexpensive method to capture and identify CTCs using traditional fluorescence biomarkers with repeated restating of 9 unrelated fluorescent antibodies. In this study, we sought to subtype CTCs with the epithelial to mesenchymal-like phenotype (EMTCTCs) from pancreatic cancer samples first identified using a conventional CTC marker panel: Cytokeratin (CK), EpCAM, and CD45. We further subtyped these EMTCTCs with an immunosuppression therapy panel (PD-L1, CXCR4, PD-1) and then with a mesenchymal marker panel (CD14, CD34, Vimentin) to better interrogate the EMT phenotype. Alternative predictive and prognostic biomarkers may also be utilized. The order of staining does not affect the result. Our data demonstrate the ability to sequentially analyze, subtype and track 9 distinct cancer markers on each individual cell.
Methods: We developed a method of fluorescence quenching using cell lines: A2058, MB231, MCF7, HUVEC, and LnCAP. The technique consists of the steps: quench, underivatize, amine strip and restain (“QUASR”). Cells isolated on CellSieveTM microfilters were stained using the CTC marker panel, after which the QUASR protocol was applied and the cells were stained with a second mesenchymal marker panel. After imaging, QUASR was repeated for a third immunotherapy marker panel, and cells imaged a third time. QUASR was then used on 12 pancreatic cancer patient blood samples previously identified with EMTCTCs provided by Medical College of Wisconsin.
Results: No degradation was observed in cell surface, or intracellular markers for the 3 rounds of QUASR. There were 764 EMTCTCs identified as CK+/CD45- in the cancer patient samples, while EpCAM was positive in only 2% of these EMTCTCs. Post quenching, most EMTCTCs had additional mesenchymal phenotypes, i.e. 97% of cells were vimentin+ and 11% were CD34+. Expression of the immunotherapy markers were highly heterogeneous between patients, ranging from 0% to 100% positivity for PD-L1 and from 0% to 90% positivity for CXCR4. None of the CTCs were PD-1+ nor CD14+.
Conclusions: Our data demonstrates that sequential multi-panel restaining of clinically applicable cancer biomarkers can provide a greater amount and broader variety of information from patient blood samples. The ability to analyze CTCs beyond simple enumeration will greatly enhance the clinical utility of blood based biopsies, as patient samples can now be screened for multiple prognostic and predictive biomarkers.
Citation Format: Daniel L. Adams, Susan Tsai, Cha-Mei Tang, Steingrimur Stefansson. Multi-biomarker subtyping of circulating tumor cells using sequential fluorescence quenching. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1550.