Table 4.
Summary of studies reporting direct and indirect detection of CTCs in peripheral blood of patients with NB. CTC, circulating tumour cell; EFS, event-free survival; FISH, fluorescence in situ hybridization; ICC, immunocytochemistry; MRD, minimal residual disease; NB, neuroblastoma; ND, not determined; RT-(q)PCR, reverse transcription (quantitative real-time) PCR; INSS, International Neuroblastoma Staging System; OS, overall survival; PFS, progression-free survival.
| detection method(s) | marker(s) | sensitivity (tumour cell n/non-tumour cell n) | main observation(s) in peripheral blood | study |
|---|---|---|---|---|
| ICC | NCAM, unknown NB-specific antigen | ND | CTCs detected in patients with metastatic disease at diagnosis and relapse. | Lanino et al. [168] |
| ICC | GD2, Thy-1, unknown NB-specific antigen | 2/105 | CTCs detected in patients with metastatic disease at diagnosis and during therapy. The presence of CTCs in patients during therapy associated with disease relapse. |
Moss & Sanders [170] |
| ICC | GD2, Thy-1, unknown NB-specific antigen | ND | CTCs detected in patients with metastatic disease at diagnosis. CTCs detected during therapy in one patient. |
Sanders et al. [171] |
| ICC; RT-PCR | ICC: NCAM, GD2, Thy-1; RT-PCR: UCH-L1 | ICC: ND; RT-PCR: 1/107 | CTCs/NB-mRNA detected in patients with localized and metastatic disease, and in patients with no evidence of disease. RT-PCR more sensitive than ICC, though analytical sensitivity of ICC not determined. |
Mattano et al. [172] |
| ICC | NCAM, GD2, Thy-1 | ND | CTCs detected in patients with metastatic disease at diagnosis and during therapy. Around half of ICC-positive blood samples and several negative samples showed clonogenic growth of NB cells in vitro. |
Moss et al. [169] |
| RT-PCR | TH | 1–10/106 |
TH mRNA detected in patients with metastatic disease at diagnosis, with some patients undetectable 6–8 weeks into therapy. TH mRNA detected in patients with relapsed disease and patients in remission, one of whom later relapsed. |
Burchill et al. [176] |
| RT-PCR | TH | ND |
TH mRNA detected in patients with metastatic disease at diagnosis, with some patients undetectable 6–8 weeks into therapy. TH mRNA detected in patients with relapsed disease and patients in remission, some of whom later relapsed. Some patients in remission and with no detectable TH mRNA eventually relapsed. |
Burchill et al. [177] |
| RT-PCR | TH | 1/105–106 | TH mRNA detected in patients with localized and metastatic disease, including INSS stage 4S patients. | Miyajima et al. [178] |
| RT-PCR | TH | ND |
TH mRNA detected in patients with localized and metastatic disease, and in patients in remission. Significantly higher TH mRNA levels in samples taken before complete remission. |
Miyajima et al. [179] |
| RT-PCR | TH | ND | TH mRNA detected in a patient with metastatic disease and undetectable by conventional histology. | Kuroda et al. [180] |
| RT-PCR | TH, UCH-L1 | ND | NB-mRNA detected in patients of unknown tumour stage, some of whom tested negative following treatment. | Gao et al. [175] |
| RT-PCR | GAGE, TH | GAGE: 1/106; TH: ND | GAGE mRNA, but not TH mRNA, detected in some patients with metastatic disease at diagnosis. | Cheung & Cheung [190] |
| RT-PCR | TH, UCH-L1 |
TH: 10−4 µg RNA UCH-L1: 10−6 µg RNA |
NB-mRNA detected at diagnosis in patients aged 1 year or older with metastatic disease. RT-PCR detection of UCH-L1 more sensitive than that for TH. | Yanagisawa et al. [173] |
| ICC | GD2 | 1/106 | CTCs detected in patients with metastatic disease and INSS stage 4S disease at diagnosis. | Faulkner et al. [209] |
| RT-PCR | TH | 1 cell/2 ml blood | TH mRNA detected in patients with metastatic disease at diagnosis. | Burchill et al. [191] |
| RT-PCR | TH | ND | TH mRNA detected in patients with metastatic disease at diagnosis. | Kuroda et al. [181] |
| RT-PCR | TH | 1/106 | Detection of TH mRNA in patients older than 1 year with metastatic disease associated with poorer OS and EFS. Detection of TH mRNA in patients with no evidence of disease associated with increased risk of relapse and death. |
Burchill et al. [194] |
| ICC; RT-PCR | ICC: GD2; RT-PCR: TH, CHGA | GD2: 1/106; TH: 1/103; CHGA: 1/106 | TH RT-PCR less sensitive than CHGA RT-PCR and GD2 ICC. | Pagani et al. [195] |
| RT-qPCR | TH | 70 transcripts/1 ml blood | At diagnosis, TH mRNA levels significantly higher in patients with metastatic/INSS stage 4S disease than limited disease. Some patients positive for TH mRNA before, during and 24 h after surgery. |
Träger et al. [185] |
| RT-qPCR | TH | 1/106 | TH mRNA detected in patients with localized and metastatic diagnosis, and in a patient after seven months of chemotherapy. | Lambooy et al. [196] |
| RT-qPCR | TH | 1/106 | TH mRNA detected at diagnosis, during chemotherapy and at relapse in patients aged 1 year or older with metastatic disease. High frequency of tumour cell contamination in autologous PBSC harvests; high TH mRNA levels associated with poorer survival. | Tchirkov et al. [197] |
| ICC; RT-PCR | ICC: GD2; RT-PCR: TH, UCH-L1 | GD2: 1/106 TH: 1/105 UCH-L1: 1/106 |
Detection of CTCs by GD2 ICC at diagnosis in patients with localized disease correlated with unfavourable outcome. No correlation of TH mRNA status with outcome in patients with metastatic disease. GD2 ICC showed greatest accuracy and UCH-L1 RT-PCR showed poor accuracy. | Corrias et al. [174] |
| RT-PCR | GALGT | 1/106 | GALGT mRNA detected in patients with metastatic disease at diagnosis and associated with higher rates of relapse and death. | Cheung et al. [208] |
| RT-PCR | GAGE | ND | GAGE mRNA detected in high proportion of healthy subjects; thus, GAGE mRNA not specific for NB. | Oltra et al. [210] |
| RT-PCR | DDC, TH | 1/106 | DDC and TH mRNA detected at similar frequencies in patients with metastatic disease at diagnosis. | Bozzi et al. [198] |
| RT-PCR | DCX, TH | 1/105 |
DCX and TH mRNA detected at similar frequencies in patients with high-risk disease at diagnosis. High correlation between DCX and TH mRNA levels in positive samples. |
Oltra et al. [199] |
| RT-qPCR | TH | ND |
TH mRNA detected in patients with localized and metastatic disease at diagnosis. Detection of TH mRNA after therapy associated with poorer prognosis. |
Parareda et al. [183] |
| RT-PCR | TH | ND | TH mRNA detected in patients with metastatic disease at diagnosis, and all deaths in mRNA-positive patients related to systemic metastases. No association of TH mRNA positivity with MYCN status. | Kuroda et al. [184] |
| RT-PCR | TH, GALGT, ELAVL4 | TH: 1/106; GALGT: 1/104; ELAVL4: 1/106 | Persistence of high ELAVL4 mRNA levels during therapy has prognostic value in patients with metastatic disease. GALGT RT-PCR less sensitive than that for TH or ELAVL4. |
Swerts et al. [200] |
| RT-qPCR | TH | 1/106 | For CTC detection, blood should be collected into PAXgene or EDTA tubes and stored for 48 h or less. Implementation of standardized protocols increased sensitivity of TH RT-PCR from 58% to 90%. |
Viprey et al. [201] |
| RT-PCR | TH | ND |
TH mRNA detected in patients with metastatic disease during treatment, but not in patients with stage 3 disease. No association of TH mRNA with MYCN status. All mRNA-positive patients died of disseminated disease. |
Kuroda et al. [211] |
| RT-qPCR | TH, DDC, GD2S | ND | High NB-mRNA levels associated with poor prognosis in patients with various disease stages. RT-qPCR for GD2S less specific than that for TH and DDC. |
Träger et al. [192] |
| RT-qPCR | TH, DDC, DBH, PHOX2B |
TH: 1/106; DDC: 1/106–107; DBH: 1/107 PHOX2B: 1/106–107 |
Panel of RT-qPCR markers more sensitive than PHOX2B alone in detecting MRD. | Stutterheim et al. [202] |
| RT-qPCR | TH, DCX | ND | TH and/or DCX mRNA detected in some patients with localized disease and associated with poorer EFS but not OS. | Yáñez et al. [203] |
| RT-qPCR | TH, GALGT, DDC, DCX, ELAVL4, STX, PHOX2B | ND | All markers except PHOX2B expressed in healthy donors. TH mRNA levels significantly lower in patients with localized versus metastatic disease. Weak correlation between detection of different markers in patients with localized disease, but perfect agreement between TH mRNA and GALGT mRNA in patients with metastatic disease. | Corrias et al. [204] |
| RT-qPCR | CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B, TH | ND | Sensitivity of MRD detection is greater with multiple RT-qPCR markers. | Hartomo et al. [205] |
| RT-qPCR | TH, PHOX2B, DCX | ND | High levels of TH, PHOX2B or DCX mRNA at diagnosis in patients with metastatic disease strongly associated with poorer EFS and OS. High levels of TH and PHOX2B at diagnosis identify patients with ultra-high-risk disease who may benefit from novel treatment approaches. | Viprey et al. [187] |
| RT-qPCR | TH | ND | Higher TH mRNA levels at diagnosis in high-risk patients and patients with metastatic disease, but no association with MYCN status. Expression of TH mRNA at diagnosis correlated with poorer 5 year PFS, both independent of risk stage and also when assessed in high-risk patients. No significant correlations with EFS were made after induction therapy. | Lee et al. [206] |
| RT-qPCR | CHGA, DCX, DDC, PHOX2B, TH | ND | NB-mRNA detectable in patients with relapsed/refractory disease. Lower RT-qPCR ΔCT values correlated with poorer PFS, independent of disease stage or MYCN status. |
Marachelian et al. [193] |
| RT-qPCR | TH, PHOX2B, DCX | ND | Levels of PHOX2B and DCX mRNAs, but not TH mRNA, in the highest tertile were associated with shorter EFS in a cohort of high-risk patients with metastatic disease. Correlation of mRNA and EFS was independent of MYCN status. Prognostic power of PHOX2B mRNA lost when combined with TH mRNA. | Corrias et al. [207] |
| FISH | CD45, CEP8, DAPI | ND | CTCs identified by depletion of non-tumour cells with magnetic beads coated with anti-CD45 antibodies, followed by analysis of non-bound cells with CEP8 probe and DAPI. CTC = CD45-negative, DAPI-positive, CEP8 ≥ 3 spots. Significantly more CTCs identified in high-risk patients and patients with metastatic disease. Patients with greater than or equal to 3 CTCs per 4 ml blood had greater likelihood of having metastases, and patients with greater than or equal to 10 per 4 ml blood had poorer OS. No correlation of CTC count with MYCN status. Higher CTC counts in patients with partial response to therapy or progressive disease relative to patients with complete response. | Liu et al. [212] |