Sensitive liquid biopsy monitoring correlates with outcome in the prospective international GPOH-DCOG high-risk neuroblastoma RT-qPCR validation study

Background: Liquid biopsies offer less burdensome sensitive disease monitoring. Bone marrow (BM) metastases, common in various cancers including neuroblastoma, is associated with poor outcomes. In pediatric high-risk neuroblastoma most patients initially respond to treatment, but in the majority the disease recurs with only 40% long-term survivors, stressing the need for more sensitive detection of disseminated disease during therapy. Methods: To validate sensitive neuroblastoma mRNA RT-qPCR BM testing, we prospectively assessed serial BM samples from 345 international high‐risk neuroblastoma patients, treated in trials NB2004 (GPOH) or NBL2009 (DCOG), using PHOX2B, TH, DDC, CHRNA3, and GAP43 RT-qPCR mRNA markers and BM GD2-immunocytology. Association between BM-infiltration levels and event-free survival (EFS) and overall survival (OS) was estimated by using Cox regression models and Kaplan-Meier’s methodology. Results: BM infiltration >10% by RT-qPCR at diagnosis was prognostic for survival (adjusted hazard ratio (HR) 1.82 [95%CI 1.25‐2.63] and 2.04 [1.33‐3.14] for EFS and OS, respectively). Any post-induction RT-qPCR positivity correlated with poor EFS and OS, with a HR of 2.10 [1.27-3.49] and 1.76 [1.01-3.08] and 5-years EFS of 26.6% [standard error 5.2%] versus 60.4% [6.7] and OS of 43.8% [5.9] versus 65.7% [6.6] for RT-qPCR-positive patients versus RT-qPCR-negative patients. In contrast, post-induction immunocytology positivity was not associated with EFS or OS (HR 1.22 [0.68-2.19] and 1.26 [0.54-2.42]). Conclusion: This study validates the association of not clearing of BM metastases by sensitive RT-qPCR detection with very poor outcome. We therefore propose implementation of RT-qPCR for minimal residual disease testing in neuroblastoma to guide therapy.