Poster Presentation Advances in Neuroblastoma Research Congress 2016

A flow cytometry backbone panel as a first step in detection of circulating tumor cells in neuroblastoma (#172)

Jalenka van Wijk 1 , Esther van Wezel 1 2 , Jan Koster 3 , Johan van Nes 3 , Lilly Zappeij-Kannegieter 1 , Rogier Versteeg 3 , Huib Caron 2 , c Ellen Schoot 1 , Godelieve Tytgat 2 4
  1. Department of Experimental Immunohematology, Sanquin Research, and Landsteiner Laboratory of the AMC, Amsterdam, the Netherlands
  2. Department of Pediatric Oncology, Emma Children’s Hospital, Academical Medical Center, Amsterdam, the Netherlands
  3. department of Oncogenomics, Academical Medical Center, University of Amsterdam, Amsterdam , The Netherlands
  4. Princess Máxima centre for Pediatric Oncology (PMC), Utrecht, the Netherlands

Introduction: Circulating tumor cells (CTCs) and disseminating tumor cells from neuroblastoma tumors are detected by immunocytology and RQ-PCR. However, these techniques are unable to characterize heterogeneity at the cellular level. Since neuroblastoma shows high heterogeneity between and within tumors we believe that subpopulations of CTC might exist. It is not clear if these subpopulations are detected with current techniques and they could have a difference in drug response or metastatic potential, therefore affecting prognosis.

 Aim: To develop a multicolour flow cytometry panel of antibodies consisting of a backbone panel identifying all neuroblastoma cells in blood and bone marrow, and additional heterogeneity markers to detect subpopulations. 

Methods: Possible backbone markers were identified by literature study and bioinformatical analysis on tumors, primary tumor cell lines (PTCs) and cell lines. Candidate markers were confirmed, in vitro on a panel of cell lines, with flow cytometry.

A peripheral blood dump channel was designed, consisting of antibodies directed against markers present on blood cells but absent on neuroblastoma cells. In combination with a neuroblastoma backbone panel, this dump channel can be used to distinguish malignant cells from the healthy blood background.

Results: We identified six new potential backbone markers for the detection of all neuroblastoma cells; EBAG9, ITGAV, RAP1A, CKAP4, CDH2 and IL6ST. 

Conclusion: The combination of markers commonly used in literature (CD45-, CD56+, CD81+ and CD9+) might not detect all neuroblastoma cells, since these markers were differentially expressed or even absent on cell lines and PTCs. A backbone panel with the new identified markers, in combination with a peripheral blood dump channel, is the first step in detection of distinct subpopulations in blood and bone marrow.