Abstract A021: MYCN_TT is a zebrafish model of early metastatic dissemination in high-risk neuroblastoma

Abstract

Metastatic disease remains the primary cause of death in neuroblastoma (NB) patients, yet little is known about the mechanisms that control it. NB is the most common extracranial solid tumor in children and is disproportionally responsible for 10% of childhood cancer deaths. Patients are designated as high-risk (HR) for having MYCN-amplification or being over 18-months-of-age with metastatic disease. HR-NB genome is dominated by segmental chromosomal aberrations (e.g., 1p-loss and 17q-gain). Metastasis is an early event in NB and half of NB patients present with metastatic disease at diagnosis. NB metastases are found in a wide range of locations, including bone marrow. The metastatic cascade is a complex process that often requires genetic adaptation. The zebrafish has emerged as a transformative in vivo model for neuroblastoma research with a high conservation of genetics and disease pathology to human (70% of protein coding genes are shared). The MYCN_TT zebrafish line is an aggressive model of HR-NB that metastases spontaneously and features high MYCN expression with brightly GFP-labeled tumor cells. The primary tumor of MYCN_TT zebrafish arises from interrenal gland, an analog to the adrenal gland. Metastatic dissemination occurs early in MYCN_TT tumor development, where circulating tumor cells can be detected as early as 4 weeks post fertilization (wpf) within the vasculature or heart. By 8 wpf the primary tumor in MYCN_TT zebrafish has expanded considerably, eroded the wall of the adjacent common cardinal vein and tumor cells are directly showered into circulation. These disseminated tumor cells (DTC; <6 cells) seed a wide range of organs and tissues (6 and 8 wpf): mid/hind kidney (bone marrow analog), spleen (lymph organ), gills, liver, and eye. Micro-metastases (>6 cells) occur frequently at 6.5 wpf and are located in all tissues where DTCs seed. The kidney marrow is the most common site of dissemination and metastatic outgrowth. At 6-months-of-age, only 10% of MYCN_TT zebrafish have an overt metastasis, whereas at 12-months-of-age approximately 50% of MYCN_TT zebrafish will carry at least 1 overt metastasis (kidney marrow, eye or spleen). We have performed whole genome sequencing (WGS) on paired MYCN_TT primary tumors and metastasis (tail, reference). We analyzed single nucleotide polymorphisms, copy number alterations (CNA) and structural variants (SV), and found that MYCN_TT tumors primarily evolve via CNAs. Although primary tumors and matched metastases shared many CNAs and SVs, metastases acquired additional genomic alterations, including amplification of the zebrafish mycn locus, demonstrating continued genetic evolution following dissemination. Together, these findings establish the MYCN_TT zebrafish as a clinically relevant model of human HR-NB that recapitulates early hematogenous dissemination and progressive metastatic evolution. This model provides a powerful platform for defining the mechanisms that drive metastatic progression and identifying therapeutic vulnerabilities in metastatic neuroblastoma.