MultiDimensional ClinOmics for Precision Therapy of Children and Adolescent Young Adults with Relapsed and Refractory Cancer: A Report from the Center for Cancer Research.

PURPOSE: We undertook a multidimensional clinical genomics study of children and adolescent young adults with relapsed and refractory cancers to determine the feasibility of genome-guided precision therapy. EXPERIMENTAL DESIGN: Patients with non-central nervous system solid tumors underwent a combination of whole exome sequencing (WES), whole transcriptome sequencing (WTS), and high-density single-nucleotide polymorphism array analysis of the tumor, with WES of matched germline DNA. Clinically actionable alterations were identified as a reportable germline mutation, a diagnosis change, or a somatic event (including a single nucleotide variant, an indel, an amplification, a deletion, or a fusion gene), which could be targeted with drugs in existing clinical trials or with FDA-approved drugs. RESULTS: Fifty-nine patients in 20 diagnostic categories were enrolled from 2010 to 2014. Ages ranged from 7 months to 25 years old. Seventy-three percent of the patients had prior chemotherapy, and the tumors from these patients with relapsed or refractory cancers had a higher mutational burden than that reported in the literature. Thirty patients (51% of total) had clinically actionable mutations, of which 24 (41%) had a mutation that was currently targetable in a clinical trial setting, 4 patients (7%) had a change in diagnosis, and 7 patients (12%) had a reportable germline mutation. CONCLUSIONS: We found a remarkably high number of clinically actionable mutations in 51% of the patients, and 12% with significant germline mutations. We demonstrated the clinical feasibility of next-generation sequencing in a diverse population of relapsed and refractory pediatric solid tumors. Clin Cancer Res; 22(15); 3810-20. ©2016 AACR.

Massively parallel sequencing reveals an accumulation of de novo mutations and an activating mutation of LPAR1 in a patient with metastatic neuroblastoma.

Neuroblastoma is one of the most genomically heterogeneous childhood malignances studied to date, and the molecular events that occur during the course of the disease are not fully understood. Genomic studies in neuroblastoma have showed only a few recurrent mutations and a low somatic mutation burden. However, none of these studies has examined the mutations arising during the course of disease, nor have they systemically examined the expression of mutant genes. Here we performed genomic analyses on tumors taken during a 3.5 years disease course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal primary tumor taken at surgical resection, and a liver metastasis at autopsy). Whole genome sequencing of the index liver metastasis identified 44 non-synonymous somatic mutations in 42 genes (0.85 mutation/MB) and a large hemizygous deletion in the ATRX gene which has been recently reported in neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the primary tumor and bone marrow biopsy, while the other 30 were unique to the index tumor, indicating accumulation of de novo mutations during therapy. Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1) had high level of expression of the mutant alleles, suggesting potential oncogenic driver roles of these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1 was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant demonstrated a significantly increased motility through elevated Rho signaling, but had no effect on growth. Therefore, this study highlights the need for multiple biopsies and sequencing during progression of a cancer and combinatorial DNA and RNA sequencing approach for systematic identification of expressed driver mutations.

Synthetic lethal screen identifies NF-κB as a target for combination therapy with topotecan for patients with neuroblastoma.

BACKGROUND: Despite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma. METHODS: We took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies. RESULTS: We found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib) significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments. CONCLUSIONS: Synthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma.

Screening a panel of drugs with diverse mechanisms of action yields potential therapeutic agents against neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite current aggressive therapy, the survival rate for high risk NB remains less than 40%. To identify novel effective chemo-agents against NB, we screened a panel of 96 drugs against two NB cell lines, SK-N-AS and SH-SY5Y. We found 30 compounds that were active against NB cell lines at < or =10 microM concentration. More interestingly, 17 compounds are active at < or =1 microM concentration, and they act through a wide spectrum of diverse mechanisms such as mitotic inhibition, topoisomerase inhibition, targeting various biological pathways, and unknown mechanisms. The majority of these active compounds also induced caspase 3/7 by more than 2-fold. Of these 17 active compounds against NB cell lines at sub-micromolar concentration, eleven compounds are not currently used to treat NB. Among them, nine are FDA approved compounds, and three agents are undergoing clinical trials for various malignancies. Furthermore, we identified four agents active against these NB cell lines that have not yet been tested in the clinical setting. Finally we demonstrated that Cucurbitacin I inhibits neuroblastoma cell growth through inhibition of STAT3 pathway. These drugs thus represent potential novel therapeutic agents for patients with NB, and further validation studies are needed to translate them to the clinic.

New Technologies for Diagnosing Pediatric Tumors Expert Opinion on Medical Diagnostics.

BACKGROUND: The completion of Human Genome Project (HGP) has paved the way for novel, more detailed and accurate molecular diagnostic classification of cancer. With the information from the HGP, cancers can be categorized not only on the morphology or limited immunohistological markers, but according to their "molecular fingerprints" such as gene expression profiles. Technologies detecting these signatures have been developed to simultaneously measure multiple genes or proteins in one assay with high sensitivity and specificity. OBJECTIVE: To evaluate potential innovative novel methods of diagnosis and prognosis in pediatric cancers. METHODS: We selected a variety of promising new diagnostic technologies utilizing molecular signatures which harness the results from HGP including DNA microarray, bead-based detection system, multiplexed RT-PCR, MesoScale Discovery (MSD), and isotope-coded affinity tag (ICAT), as well as their applications in biomarker discovery for pediatric tumors. Label-free detection technologies and the obstacles for taking these new diagnostic technologies from the bench to the bedside are also discussed. CONCLUSION: The use of molecular signatures is gaining acceptance in clinical practice. However, technical challenges need to be addressed before incorporating these new technologies into current diagnostic and prognostic schema.