Abstract A033 Early relapse detection: The power and potential of adrenergic and mesenchymal neuroblastoma specific mRNA-based minimal residual disease detection in liquid biopsies
Authors: tytgat Godelieve , van Zogchel Lieke , Gelineau Nina , Ellen van der Schoot C. , Fiocco Marta , Letunovska Aleksandra , Van Gestel-Fadaie Zeinab 
Published: September 2024

Abstract
Outcome in high-risk neuroblastoma is still poor, about 50% of patients, assumed to be in complete remission, will suffer from a relapse. More sensitive biomarkers are needed to identify the patients at risk for an event, or to detect this event early. Neuroblastoma specific mRNA panels, including adrenergic (ADR) and mesenchymal (MES) markers, detected in liquid biopsies, are promising biomarkers. We performed a prospective neuroblastoma mRNA qPCR validation study in 345 high-risk neuroblastoma patients, treated in NB2004 (GPOH) or NBL2009 (DCOG) trials. Method: Serial paired bone marrow (BM) and peripheral blood (PB) samples were collected at designated time points and tested with the ADR marker panel (PHOX2B, TH, DDC, CHRNA3, and GAP43) and MES panel (PRRX1, POSTN and FMO3) by RT-qPCR. Association with event-free survival (EFS) and overall survival (OS) was estimated using Kaplan-Meier’s methodology, and hazard ratios were estimated with multivariable Cox regression models. Results: High ADR mRNA levels in BM and PB at diagnosis correlated with 5-year EFS of 17.1% and OS of 26.8% (p &lt; .001). Post-induction BM response correlated with 5-years EFS of 26.6% vs 60.4% (p &lt;.0001) and OS of 43.8% vs 65.7% (p = .001) for RT-qPCRpos patients vs RT-qPCRneg patients respectively. Post-induction BM immunocytology (IC) did not correlate with outcome, however qPCR stratified ICneg BM samples, with 5-year EFS of 20.8 (6.8) vs. 55.8 (7.6) and 5-year OS of 37.6 (8.0) vs. 62.8 (7.4) for ICneg/qPCRpos samples and ICneg/qPCRneg, respectively. For Dutch patients, combining post-induction MIBG (SIOPEN) scores with qPCR stratified patients with SIOPEN score ≤3, with 5-year relapse free survival of 32% vs. 57%, for qPCRpos vs qPCRneg, respectively. MES mRNA detection at diagnosis was prognostic for EFS, because these were detected in samples with high ADR mRNA levels, which correlated with poor outcome. Most GD2-ICneg BM samples were MES qPCR only positive, suggesting that the MES-phenotype is GD2 negative. After 2 chemotherapy courses MES-mRNA identified plasticity in paired analysis with BM GD2-IC, demonstrating 5Y EFS for GD2-ICneg/MESpos of 15.0% vs 57% for GD2ICneg/MESneg, respectively. In all samples, MES-mRNA was detected in more patients with relapse than those who remained disease free (48% vs. 33%, respectively; p=0.002). In patients with relapse, MES mRNA-levels increased during therapy but were not detectable at relapse, potentially reverting back to an ADR-phenotype. Finally, the ADR panel detected relapses up to 140 days prior to standard of care imaging. Conclusion: mRNA detection by qPCR is efficient, inexpensive, standardized, and very sensitive to detect BM disease in high-risk neuroblastoma. This prospective validation study shows the power and potential for sensitive MRD detection of ADR mRNA markers, its superiority to BM IC and MIBG-based response evaluation. Finally, the detection of plasticity by the MES markers identifies the patients at risk of an event when the tumor cells are undetectable by the current techniques.
Citation Format: Godelieve tytgat, Lieke van Zogchel, Nina Gelineau, C. Ellen van der Schoot, Marta Fiocco, Aleksandra Letunovska, Zeinab Van Gestel-Fadaie. Early relapse detection: The power and potential of adrenergic and mesenchymal neuroblastoma specific mRNA-based minimal residual disease detection in liquid biopsies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A033.