# Distinct Clinical Significance of Minimal Residual Disease Detected by 7NB-mRNAs Expression in Bone Marrow at Different Time Points of High-Risk Neuroblastoma Patients

## TL;DR

This study shows that measuring specific mRNAs in bone marrow at certain treatment stages can predict relapse in high-risk neuroblastoma patients.

## Contribution

The study identifies EOH and EOC as optimal time points for evaluating BM-MRD using a 7NB-mRNA ddPCR assay in high-risk neuroblastoma.

## Key findings

- BM-MRD at EOH and EOC was significantly associated with relapse in high-risk neuroblastoma patients.
- Higher BM-MRD levels (≥ 3.5) at EOH and EOC correlated with worse 3-year event-free survival.
- The 7NB-mRNA ddPCR assay provides a reliable method for MRD detection at specific treatment stages.

## Abstract

More than half of patients with high-risk neuroblastoma (NB) relapse, likely driven by chemoresistant minimal residual disease in the bone marrow (BM-MRD). Although several quantitative PCR (qPCR) and droplet digital PCR (ddPCR) assays measuring different but overlapping sets of NB-associated mRNA (NB-mRNAs) have demonstrated a significant prognostic value at various time points, the optimal combination of MRD markers (a set of NB-mRNAs) and evaluation timing remain uncertain. Here, we evaluated BM-MRD with a 7NB-mRNAs ddPCR assay quantifying CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs across clinically relevant treatment stages. We analyzed 89 bone marrow samples obtained from mostly overlapping 30 high-risk NB patients at four time points: diagnosis (Dx), end of induction (EOI), end of high-dose chemotherapy (EOH), and end of consolidation (EOC). At EOH and EOC time points, BM-MRD was significantly associated with subsequent relapse. Moreover, patients with higher BM-MRD levels (7NB-mRNAs ≥ 3.5) had inferior 3-year event-free survival. These findings suggest that EOH and EOC are clinically informative evaluation time points for BM-MRD detected by 7NB-mRNAs expression.

Patients with high-risk neuroblastoma (NB) continue to have long-term survival rates below 60%, with relapse occurring in more than half of patients, likely driven by chemoresistant minimal residual disease in the bone marrow (BM-MRD). Although several quantitative PCR (qPCR) and droplet digital PCR (ddPCR) assays measuring different but overlapping sets of NB-associated mRNAs (NB-mRNAs) have shown a significant prognostic value at various time points, the optimal combination of MRD markers (a set of NB-mRNAs) and evaluation timing remains unclear. In the present study, 89 bone marrow samples were collected from mostly overlapping 30 high-risk NB patients at four time points: diagnosis (Dx), end of induction (EOI), end of high-dose chemotherapy (EOH), and end of consolidation (EOC). BM-MRD was assessed with a 7NB-mRNAs ddPCR assay quantifying CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs. BM-MRD at EOH and EOC time points was significantly associated with relapse. Moreover, patients with higher BM-MRD levels at EOH (7NB-mRNAs ≥ 3.5) and EOC (7NB-mRNAs ≥ 3.5) time points had significantly inferior 3-year event-free survival (EOH, p = 0.003; EOC, p = 0.033). These results indicate that EOH and EOC are clinically informative evaluation time points for BM-MRD detected by 7NB-mRNA expression.