Circulating tumor cells detection in neuroblastoma patients by EpCAM-independent enrichment and immunostaining-fluorescence in situ hybridization

Abstract

Detecting circulating tumor cells (CTCs) has proven valuable for evaluating the prognosis of cancer patients and for studying the mechanisms of treatment resistance. Owing to the lack of universal and specific tumor markers for neuroblastoma (NB), in this prospective study, we adopted an EpCAM-independent method to detect CTCs in the peripheral blood of NB patients. We used an EpCAM-independent assay to delete leukocytes and to enrich the CTCs. CTCs were identified by immunostaining of CD45, DAPI and DNA fluorescence in situ hybridization (FISH) of the centromere of chromosome 8 probe (CEP8). Cells that were DAPI+/CD45-/CEP8 ≥ 3 were considered CTCs. We collected peripheral blood from 28 NB patients as well as clinical and follow-up data. The number of CTCs among the different risk groups were significantly different ( p = .0208, Kruskal–Wallis test). Patients with metastasis had more CTCs than those without metastasis ( p < .0001, Mann–Whitney test). Patients with ≥3 CTCs per 4 ml of peripheral blood had an increased likelihood of having metastasis (sensitivity, 88.89%; specificity, 78.59%), and patients with ≥10 CTCs per 4 ml of peripheral blood had poorer overall survival. The EpCAM-independent assay along with immunostaining-FISH (i-FISH) described here can detect CTCs in patients with NB at a high sensitivity and may have clinical value for prognosis evaluation and diagnosing metastasis when imaging data are ambiguous.

The combination of CTCs and CEA can help guide the management of patients with SPNs suspected of being lung cancer

“…The detection of CTCs was performed as described previously by Liu et al and Ge et al [27,28] In briefly, CTC enrichment and identification was performed according to the instructions of the Cytelligen CTC Enrichment Kit and Human Tumor Cell Identification Kit (Cytelligen, San Diego, CA, USA). Blood samples were collected in 6-ml ACD-containing tubes from an antecubital vein in all patients before surgery.…”

PHOX2B expression in bone marrow and peripheral blood is associated with metastasis and prognosis in patients with neuroblastoma

“…Neuroblastoma patients with ≥ 3 CTCs per 4 ml of PB were showed with an increased likelihood of having metastasis with the sensitivity and specificity of 88.89% and 78.59%, respectively [27]. The univariate logistic regression results also showed that CTC and NSE levels were associated with metastasis [27].…”

Opportunities and challenges of circulating biomarkers in neuroblastoma

“…As with immunocytochemical analyses, RT-PCR-based studies have consistently identified NB-specific mRNA in peripheral blood at diagnosis in patients with metastatic disease and in a fraction of patients with localized and stage 4S disease (table 4). Diagnostic TH mRNA levels and CTC counts are typically higher in patients with more advanced metastatic disease [185,204,206,212] and in patients with high-risk disease [206,212], as observed in other solid malignancies [166]. However, NB-specific mRNA levels and CTC counts do not correlate with MYCN status [182,184,206,207,212].…”

“…High transcript levels of TH and other NB-specific genes at diagnosis have been associated with poor OS and/or EFS in patients with localized and metastatic disease, independent of risk status [187,193,194,197,203,206,208]. Similarly, high CTC counts (≥ 10 cells per 4 ml blood) have recently been shown to correlate with poor OS [212]. Among patients with high-risk disease, high expression of TH and PHOX2B mRNA at diagnosis has been shown to correlate with remarkably poorer outcome, thus identifying a subset of patients with ultra-high-risk disease who may benefit from novel treatment approaches [187].…”