From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models

Abstract

Background: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent.

Methods: Within the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models.

Results: Eighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as "very high priority", that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient's tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors.

Conclusions: PREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

Keywords: Neuroblastoma; Precision medicine; Preclinical models, next generation sequencing; Target therapy.

Fig. 1

Schematic representation of PREME. NB neuroblastoma, IHC immunohistochemistry, FCM flow cytometry, PDX Patient-Derived Xenograft, CDPX Cell Patient-Derived Xenograft, 3D patient-derived tumor-spheres

Fig. 2

Development, characterization, and therapeutic use of PDX/CPDX and 3D models. IHC immunohistochemistry, FCM flow cytometry, PDX Patient-Derived Xenograft, CDPX Cell Patient-Derived Xenograft, 3D patient-derived tumor-spheres, NB neuroblastoma, BM Bone Marrow derived from either iliac crests or tumor bone lesions, NSG NOD/SCID/IL2Rgammanull mice, nude athymic nude/nude mice, s.c. subcutaneous implant/injection of NB tumor fragments or NB cell suspension derived from tumor-infiltrated BM, orthotopic implantation/injection of NB tumor fragments or NB cell suspension derived from P2 generation mice, in the adrenal gland, WES whole exome sequencing

Fig. 3

Summary view of the potential therapeutically targetable somatic alterations. The data matrix shows the targetable pathogenic somatic variants detected in relapsed tumors (Pz). Mutated genes are reported by row, and samples are reported by column. On the top of the matrix, we reported the patient characteristics (gender, disease stage, MYCN amplification status and the age at diagnosis; Gt18 age at diagnosis ≥ 18mo, Lt18 age at diagnosis < 18mo). The matrix is split by rows based on the involved pathways (source: MyCancerGenome). The annotation track on the right reports the level of priority of the variant based on the pathogenicity and actionability (see Methods). TMB*: Tumor Mutational Burden. High TMB was considered if the patient had more than five variants per megabase of sequencing target regions

Fig. 4

Comparison of genetic variations and transcriptomic profiles among patient’s tumors and PDX or CPDX generations. For each analysis, tumors at first (P1) and second (P2) generations cluster together with the respective original patient tumors (Pz#). A–D Clustered heatmap of the correlation coefficients for SNVs and gene expression levels, respectively. B–E Hierarchical clustering using Euclidian distance and Ward’s method for SNVs and gene expression levels, respectively. C–F Principal Component Analysis and Scree Plot (bottom right) for SNVs and gene expression levels, respectively

Fig. 5

Tracking of potential therapeutically targetable somatic alterations among PDX or CPDX generations. A The figure shows the allele frequency of actionable pathogenic somatic variants detected in patient’s tumors and tracked in murine xenografts generations. B The figure shows the number of copies of actionable pathogenic somatic copy number variants detected in patient’s tumors and tracked in murine xenografts generations. Pz patient’s tumor, Pl first PDX generation, P2 second PDX generation, DEL Deletion; DUP Duplication, C Box plots showing the expression of gene levels of patient’s tumors and murine xenografts generations. The targetable gene showing an abnormal over-expression is reported in the red box