Multi-omics profiling of neuroblastoma identifies immunological biomarkers associated with higher risk of relapse: Results from the MICCHADO study

BACKGROUND: Neuroblastoma (NB) risk stratification relies on clinical and molecular features, though few molecular markers inform therapeutic interventions. Leveraging multi-omics approaches, we sought to identify biomarkers associated with relapse. AIMSTo characterize molecular and immunological features associated with relapse inhigh-risk NB (HR-NB), (MYCN-amplified or metastatic >12 months), and correlate findingswith clinical outcomes to identify relapse mechanisms and therapeutic opportunities. METHODS: For 197 NB patients (145 HR-NB, 52 low/intermediate-risk) in the MICCHADOprogram (NCT03496402, a 600-pediatric patients pancancer cohort), tumor and sequentialblood samples were analyzed using paired tumor/germline whole exome sequencing(WES), bulk RNA sequencing, sequential plasma cell-free DNA (cfDNA) analysis, and bloodflow cytometry by FACS. We assessed DNA alterations, transcriptomic subtypes,stemness, immune infiltration, and blood biomarkers. RESULTS: In HR-NB higher cfDNA and ctDNA levels were observed at diagnosis compared to non-HR. In addition to previously known genetic features (including MYCN, DDX1 and NBAS amplification, copy number alterations as well as recurrent SNVs), sequential cfDNA analyses identified novel alterations at relapse, including ALK, RAS-MAPK or CDKN2A/B. Transcriptomic profiling revealed a higher stemness index in HR-NB and in patients who experienced relapse, suggesting that a higher stemness index at diagnosis may predict relapse risk. Immune deconvolution of RNAseq from diagnostic samples revealed that patients who experienced relapse had higher levels of CD4+ memory T-cells, but lower natural killer (NK)Cells. Peripheral blood flow cytometry revealed, as expected, more T-cells in younger patients and a significant decrease in CD3+, CD4+, CD8+ T-cells, and NK cells from diagnosis to treatment and relapse. CONCLUSIONS: Multi-omics profiling of NB identified novel immune biomarkersassociated with relapse. A higher stemness index suggests more undifferentiated, relapse-prone tumors. Immune profiling revealed T-cell and NK cell changes, highlighting the need for integrated molecular and immune profiling in targeted therapies. Keywords: Neuroblastoma, Relapse, Biomarkers, Multi-Omics, Immune Profiling