Detection of circulating and disseminated neuroblastoma tumour cells using the Imagestream flow cytometer for use as predictive and pharmacodynamic biomarkers

Abstract

Background: Neuroblastoma (NB) is the commonest extracranial childhood solid tumour. Circulating tumour cells (CTCs) are a useful source of predictive and prognostic biomarkers in cancer and may serve as pharmacodynamic (PD) biomarkers for new treatments. Our research group is interested in developing MDM2/p53 inhibitors as a novel therapy NB are usually p53 wild-type even at relapse making them suitable for treatment with MDM2/p53 inhibitors. Objectives: 1) To detect CTCs from blood and disseminated tumour cells (DTC) from bone marrow (BM) from NB patients using the Imagestream flow cytometer (ISx) 2) To use these cells for predictive and PD biomarker studies for novel targeted therapies. Methods: 40 NB patients, (32-high, 6 -low and 2 intermediate risk) 24 from diagnosis, 14 from relapse including 2 diagnosis and relapse were recruited. 35 paired blood and BM samples were analysed for CTCs and DTCs and 5 unpaired blood samples for CTCs using the ISx to detect GD2+ve and CD45-ve NB cells. 4 patient samples were treated exvivo for 24 hours with 10µM Nutlin-3 and analysed for p53 and p21 expression before and after treatment. Results: CTCs were detected in 26/40 blood samples (range, 1-264/ml at diagnosis, 1- 39/ml at relapse) and DTCs in 25/35 BM (range, 57-169,635/ml at diagnosis, 112- 15688/ml at relapse) samples. Higher numbers of CTCs (but not DTCs) were found in patients with high risk NB who achieved a BM complete response after first line induction therapy versus those that did not ( n=23, p=0.006). Ex-vivo Nutlin-3 exposure in a patient with >200 CTCs/ml and >6000 DTCs/ml at diagnosis, led to increased p21 and p53 expression in GD2+ve/CD45-ve CTCs and DTCs compared to DMSO controls and GD2-ve/CD45+ve blood and BM cells. Conclusions: This the first study to show that DTCs and CTCs are detectable in NB patient samples at diagnosis and relapse using the ISx. Increased p53 and p21 expression in CTCs/DTCs following MDM2 inhibitor treatment is a useful PD tumour biomarker for use in early phase clinical trials. Enumeration of CTCs at diagnosis in high risk NB patients with BM infiltration may be a useful predictive biomarker of BM response.