Abstract 1711: Detection of circulating tumor cells for use as prognostic, predictive and pharmacodynamic biomarkers in neuroblastoma

Abstract

Abstract Introduction: Neuroblastoma (NB) is the commonest extracranial childhood solid tumour. New treatments and a better understanding of drug resistance are needed to improve survival. Circulating tumour cells (CTCs) may provide a source of tumour cells for genetic biomarkers, give insights into tumour load and serve as pharmacodynamic (PD) biomarkers for new treatments. In clinical diagnostics, slide based FISH is used to detect MYCN amplification in neuroblastoma tumours. As it is not possible to conduct slide based FISH on an entire blood sample due to the infrequency of CTCs in peripheral blood (1 in 106 leucocytes), an alternative method FISH-IS (in suspension) was used in the present study to detect MYCN gene amplification using the Imagestream imaging flow cytometer (ISx). Aims: 1) To detect CTCs from blood and disseminated tumour cells (DTC) from bone marrow (BM) from NB patients using the (ISx) 2) To use these cells for genetic and PD biomarker studies for novel targeted therapies. Methods: A panel of NB cell lines (n=6) cloned into neuronal (N) and substrate-adherent (S) types were used to determine expression of NB cell surface markers (GD2 and NCAM) using the ISx. Western blotting and fluorescence activated cell sorting (FACS) were used to validate NCAM and GD2 expression respectively. 25 paired NB patient blood and BM samples were analysed for CTCs and DTCs, 4 unpaired blood samples were analysed for CTCs and one unpaired BM sample was for DTCs using the ISx to detect GD2 +ve and CD45-ve cells. Results: Using the ISx 80-90% of N-type cells immunostained positive for GD2 compared to 0.8 -10% of S-type cells confirmed by FACS. CTCs were detected in 21/29 patient samples and DTCs in 19/26 cases. The numbers of CTCs ranged from 10-522/ml blood and DTCs from 57-35,515/ml of bone marrow. After validating FISH-IS protocol on ISx using NGP and SHSY5Y cell lines, 4 NB patient DTC samples were analysed for MYCN amplification. 2/4 cases showed MYCN gene amplification consistent with results obtained on the primary tumour. Conclusion: This the first study to show that DTCs and CTCs are detectable in NB patient samples using the ISx. Future work will involve further genetic characterisation and development of PD biomarkers for Phase I clinical trials. Citation Format: Swathi Merugu, Deborah Tweddle. Detection of circulating tumor cells for use as prognostic, predictive and pharmacodynamic biomarkers in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1711. doi:10.1158/1538-7445.AM2017-1711