Cell surface vimentin-positive circulating tumor cell-based relapse prediction in a long-term longitudinal study of postremission neuroblastoma patients

Abstract

Neuroblastoma (NB) is a deadly childhood disease that carries a 50% chance of relapse for anyone in remission and similar level of 5-year survival. We investigated the value of our proprietary approach-cell surface vimentin (CSV) positive circulating tumor cells (CTC) to monitor treatment response and predict relapse in NB patients under remission in a Phase II long-term preventative clinical trial. We longitudinally analyzed peripheral blood samples from 93 patients for 27 cycles (~25 months) and discovered that the presence of CSV+ CTCs in the first two sequential samples (baseline, cycle 4 [month 3-4]) was a significant indicator of earlier relapse. We observed strong correlation between relapse-free survival (RFS) and lack of CSV+ CTCs in first 4 cycles of therapy (95%). There was sensitivity reaching 100% in predicting RFS in patients who had neither CSV+ CTCs nor MycN amplification. Of note, the low number of CSV+ CTCs seems equivalent to low tumor load because the prevention therapy difluoromethylornithine yields faster reduction of relapse risk when none or only 1-2 CSV+ CTCs (every 6 mL) are present in the blood samples compared to >3 CSV+ CTCs. To the best of our knowledge, this is the first study that directly observes CTCs in under remission NB patients for relapse prediction and the first to gather sequential CSV+ CTC data in any study in a long-term longitudinal manner.

Trial registration: ClinicalTrials.gov NCT01586260 NCT02395666.

Keywords: circulating tumor cell; difluoromethylornithine; liquid biopsies; neuroblastoma; relapse.

Conflict of interest statement

Competing Interests

The authors declare that they have no competing interests.

© 2020 UICC.

Figures

Figure 1

Cell line staining and CSV validation. a Neuroblastoma cell line CHP-134 stained for the neuronal marker CD56 (ThermoFisher Scientific, USA), and CSV (MD Anderson, USA) and nucleus (DRAQ5). b Flow cytometry analysis for CSV is shown in panel b. 50,000 events were captured in duplicate.

Figure 2

Study outline and early relapse. Panel a shows the study outline containing 27 cycles (28 days per cycle) of DFMO therapy. Blood samples were collected at 3–4 cycle intervals as shown by the arrows indicating the most frequent time points at which samples were received. In each cycle, treatments were given once a week for the first 3 weeks followed by rest for 1 week prior to initiation of next cycle. Panels b, c, and d illustrate the number of patients who relapsed within 25 months, respective to the number CTCs detected at baseline (b) based on the consented patients whose samples were available at baseline (n=54), S1 (c) based on the consented patients whose samples were available at S1 (n=34), or those who maintained the indicated CTC levels through first 2 samples (d) based on the patients whose samples were available at either baseline or S1 (n=60), respectively.

Figure 3

Relapse-free survival prediction based on CTC and MycN amplification status. Panels a and b show the relapse-free survival difference between patients with or without detectable CTCs at baseline (a) or between patients who were or were not CTC from baseline through first 4 months (b). Pairwise comparison between different groups was conducted. Significant difference in relapse-free survival was observed between CTC-&MycNAmp- and CTC+&MycNAmp- groups (p = 0.041) without adjusting for multiple testing correction (c).

Figure 4

Treatment monitoring in pool of patients. Each panel represents the number of patients that relapsed or not. Panels differ based on number of detected CTCs in each pool as indicated in the title of each graph. a Number of patients with 0 CTC who did or did not relapse at the indicated cycles. b Number of patients with 1 to 2 CTCs who did or did not relapse at the indicated cycles. c Number of patients with 3 to 4 CTCs who did or did not relapse at the indicated cycles. d Number of patients with ≥5 CTCs who did or did not relapse at the indicated cycles. B = Baseline. A Fisher exact test was performed on the B + S1 populations, p = 0.0278

Figure 5

Overall CTC profiles of six individual patients. Three patients who maintained relapse-free survival are shown in panels a, b, and c; three relapsed patients are shown in panels c, d, and e. Graph depicts CTC count per 6ml of blood at treatment cycle (total 27 cycles; 25 months). Arrow and red number indicates days after sample collection at indicated cycle when the patient relapsed.