Molecular-guided therapy predictions reveal drug resistance phenotypes and treatment alternatives in malignant peripheral nerve sheath tumors.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are rare highly aggressive sarcomas that affect 8-13% of people with neurofibromatosis type 1. The prognosis for patients with MPNST is very poor. Despite TOP2A overexpression in these tumors, doxorubicin resistance is common, and the mechanisms of chemotherapy resistance in MPNST are poorly understood. Molecular-guided therapy prediction is an emerging strategy for treatment refractory sarcomas that involves identification of therapy response and resistance mechanisms in individual tumors. Here, we report the results from a personalized, molecular-guided therapy analysis of MPNST samples. METHODS: Established molecular-guided therapy prediction software algorithms were used to analyze published microarray data from human MPNST samples and cell lines, with benign neurofibroma tissue controls. MPNST and benign neurofibroma-derived cell lines were used for confirmatory in vitro experimentation using quantitative real-time PCR and growth inhibition assays. Microarray data was analyzed using Affymetrix expression console MAS 5.0 method. Significance was calculated with Welch's t-test with non-corrected p-value < 0.05 and validated using permutation testing across samples. Paired Student's t-tests were used to compare relative EC50 values from independent growth inhibition experiments. RESULTS: Molecular guided therapy predictions highlight substantial variability amongst human MPNST samples in expression of drug target and drug resistance pathways, as well as some similarities amongst samples, including common up-regulation of DNA repair mechanisms. In a subset of MPNSTs, high expression of ABCC1 is observed, serving as a predicted contra-indication for doxorubicin and related therapeutics in these patients. These microarray-based results are confirmed with quantitative, real-time PCR and immunofluorescence. The functional effect of drug efflux in MPNST-derived cells is confirmed using in vitro growth inhibition assays. Alternative therapeutics supported by the molecular-guided therapy predictions are reported and tested in MPNST-derived cells. CONCLUSIONS: These results confirm the substantial molecular heterogeneity of MPNSTs and validate molecular-guided therapy predictions in vitro. The observed molecular heterogeneity in MPNSTs influences therapy prediction. Also, mechanisms involving drug transport and DNA damage repair are primary mediators of MPNST chemotherapy resistance. Together, these findings support the utility of individualized therapy in MPNST as in other sarcomas, and provide initial proof-of concept that individualized therapy prediction can be accomplished.

Genetic and functional studies of the intervertebral disc: a novel murine intervertebral disc model.

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.

Targeting receptor-activator of nuclear kappaB ligand in aneurysmal bone cysts: verification of target and therapeutic response.

Aneurysmal bone cyst (ABC) is a benign tumor of bone presenting as a cystic, expansile lesion in both the axial and appendicular skeleton. Axial lesions demand special consideration, because treatment-related morbidity can be devastating. In similar lesions, such as giant cell tumor of bone (GCTB), the receptor-activator of nuclear kappaB ligand (RANKL)-receptor-activator of nuclear kappaB (RANK) signaling axis is essential to tumor progression. Although ABC and GCTB are distinct entities, they both contain abundant multinucleated giant cells and are osteolytic characteristically. We hypothesize that ABCs express both RANKL and RANK similarly in a cell-type specific manner, and that targeted RANKL therapy will mitigate ABC tumor progression. Cellular expression of RANKL and RANK was determined in freshly harvested ABC samples using laser confocal microscopy. A consistent cell-type-specific pattern was observed: fibroblastlike stromal cells expressed RANKL strongly whereas monocyte/macrophage precursor and multinucleated giant cells expressed RANK. Relative RANKL expression was determined by quantitative real-time polymerase chain reaction in ABC and GCTB tissue samples; no difference in relative expression was observed (P > 0.05). In addition, we review the case of a 5-year-old boy with a large, aggressive sacral ABC. After 3 months of targeted RANKL inhibition with denosumab, magnetic resonance imaging demonstrated tumor shrinkage, bone reconstitution, and healing of a pathologic fracture. Ambulation, and bowel and bladder function were restored at 6 months. Denosumab treatment was well tolerated. Post hoc analysis demonstrated strong RANKL expression in the pretreatment tumor sample. These findings demonstrate that RANKL-RANK signal activation is essential to ABC tumor progression. RANKL-targeted therapy may be an effective alternative to surgery in select ABC presentations.