Mutations of 1p genes do not consistently abrogate tumor suppressor functions in 1p-intact neuroblastoma.

BACKGROUND: Deletion of 1p is associated with poor prognosis in neuroblastoma, however selected 1p-intact patients still experience poor outcomes. Since mutations of 1p genes may mimic the deleterious effects of chromosomal loss, we studied the incidence, spectrum and effects of mutational variants in 1p-intact neuroblastoma. METHODS: We characterized the 1p status of 325 neuroblastoma patients, and correlated the mutational status of 1p tumor suppressors and neuroblastoma candidate genes with survival outcomes among 100 1p-intact cases, then performed functional validation of selected novel variants of 1p36 genes identified from our patient cohort. RESULTS: Among patients with adverse disease characteristics, those who additionally had 1p deletion had significantly worse overall survival. Among 100 tumor-normal pairs sequenced, somatic mutations of 1p tumor suppressors KIF1Bß and CHD5 were most frequent (2%) after ALK and ATRX (8%), and BARD1 (3%). Mutations of neuroblastoma candidate genes were associated with other synchronous mutations and concurrent 11q deletion (P = 0.045). In total, 24 of 38 variants identified were novel and predicted to be deleterious or pathogenic. Functional validation identified novel KIF1Bß I1355M variant as a gain-of-function mutation with increased expression and tumor suppressive activity, correlating with indolent clinical behavior; another novel variant CHD5 E43Q was a loss-of-function mutation with decreased expression and increased long-term cell viability, corresponding with aggressive disease characteristics. CONCLUSIONS: Our study showed that chromosome 1 gene mutations occurred frequently in 1p-intact neuroblastoma, but may not consistently abrogate the function of bonafide 1p tumor suppressors. These findings may augment the evolving model of compounding contributions of 1p gene aberrations toward tumor suppressor inactivation in neuroblastoma.

Neuroblastoma patient-derived cultures are enriched for a mesenchymal gene signature and reflect individual drug response.

Ex vivo evaluation of personalized models can facilitate individualized treatment selection for patients, and advance the discovery of novel therapeutic options. However, for embryonal malignancies, representative primary cultures have been difficult to establish. We developed patient-derived cell cultures (PDCs) from chemo-naïve and post-treatment neuroblastoma tumors in a consistent and efficient manner, and characterized their in vitro growth dynamics, histomorphology, gene expression, and functional chemo-response. From 34 neuroblastoma tumors, 22 engrafted in vitro to generate 31 individual PDC lines, with higher engraftment seen with metastatic tumors. PDCs displayed characteristic immunohistochemical staining patterns of PHOX2B, TH, and GD2 synthase. Concordance of MYCN amplification, 1p and 11q deletion between PDCs and patient tumors was 83.3%, 72.7%, and 80.0% respectively. PDCs displayed a predominantly mesenchymal-type gene expression signature and showed upregulation of pro-angiogenic factors that were similarly enriched in culture medium and paired patient serum samples. When tested with standard-of-care cytotoxics at human Cmax -equivalent concentrations, MYCN-amplified and non-MYCN-amplified PDCs showed a differential response to cyclophosphamide and topotecan, which mirrored the corresponding patients' responses, and correlated with gene signatures of chemosensitivity. In this translational proof-of-concept study, early-phase neuroblastoma PDCs enriched for the mesenchymal cell subpopulation recapitulated the individual molecular and phenotypic profile of patient tumors, and highlighted their potential as a platform for individualized ex vivo drug-response testing.