O2143: Microfluidic Isolation, Characterization, and ex-vivo Expansion of Neuroblastoma Ctcs for Molecular Analysis and Risk Classification

Background: Neuroblastoma (NB) is the most common extracranial solid tumor in children. Most patients succumb to relapsed and progressive high-risk disease refractory to current therapies. Circulating tumor cells (CTCs) have been associated with metastasis, relapse, and therapy resistance. The mechanisms by which CTCs contribute to cancer progression has not been characterized in neuroblastoma. In this study we utilized a novel state of the art labyrinth device to isolate neuroblastoma CTCs from high-risk patients, and single cell sequencing for molecular analysis and risk classification.

Methods: Peripheral blood was collected from high-risk NB patients (n=3) and a healthy control. Blood samples were collected just prior to surgery and after four cycles of chemotherapy per standard Children’s Oncology Group (COG) treatment protocols. From the acquired blood samples CTCs were isolated using a novel labyrinth device and fixed with a 10x genomics fixation kit. 10x genomics fixed cells were processed for single cell RNA sequencing.

Results: Cell clusters containing CD90+, PHOX2B+, CD45-, CD235- were considered CTCs. We identified five NB CTC clusters (3, 14, 16, 19 and 23) that were not present in the control sample. Single cell sequencing data revealed that PHOX2B is a more consistent CTC screening marker than TRKB, TH and GD2 in this series. Analysis of differential gene expression revealed that molecular pathways related to proliferation, DNA repair, cell cycle progression, and angiogenesis were upregulated in NB CTC. These included the DNA repair genes Lig3, Lig1, PARP1 and POLQ. TGFB/SMAD pathway- SMAD1, 2, 4, 5 and 9 were also highly expressed in NB CTCs.

Conclusion: These results suggest that CTCs can be isolated and analyzed from peripheral blood samples of NB patients and used for diagnosis and molecular characterization. Such data has important mechanistic and precision medicine implications.