Background: Circulating tumor cells (CTCs) and cancer-associated macrophage-like (CAMLs) cells have been identified in the peripheral blood of patients with a variety of malignancies and are not present in patients without cancer. Identification of CTCs and cancer-associated cells in blood may provide a non-invasive technique for evaluating cancer progression, relapse, or treatment response. As proof of principle, isolation of CTCs and CAMLs in neuroblastoma patients was attempted.
Methods: In a pilot study, ten peripheral blood samples were collected from patients with high-risk neuroblastoma at different stages of disease. Size-exclusion, low-pressure filtration was used to isolate CTCs and CAMLs from the blood. Cells were stained with fluorescent antibodies (anti-human disialoganglioside GD2/anti-vimentin/anti-CD45/DAPI) to identify CTCs (GD2+, vimentin+, CD45-) and CAMLs (atypical nucleus and any combination of the 3 markers). Immunofluorescent microscopy was utilized for cell characterization and imaging.
Results: Ten patients with neuroblastoma were included in this study. Two samples (20%) were found to have clusters of GD2-positive cells, thus detecting neuroblastoma CTCs. The presence of CTCs was not reliably associated with any clinical or biological features in this small cohort. CAMLs were found from 25 to greater than 100 µm in size. CAMLs were identified in 9 of the samples (90%). On average, more CAML cells were identified in samples from patients with Stage 4 disease (ranging 1 to 26, mean 10 CAMLs) compared to Stage 3 (ranging 1 to 8, mean 3 CAMLs), although this observation was not statistically significant (P=0.27). MYCN amplification was not associated with the number of CTCs/CAMLs identified. CAMLs were not identified in 30 healthy controls. In this limited cohort, CAML detection in patients with HR neuroblastoma had sensitivity of 0.9 (95%CI 0.54-0.99) and specificity of 1 (95%CI 0.85-1).
Discussion: Although further work is necessary, identification of CTCs and CAMLs in the peripheral blood of neuroblastoma patients is technically feasible and may provide a non-invasive and economically viable method for cancer surveillance.