Search

Purpose Large cell neuroblastomas (LCN) are frequently seen in recurrent, high-risk neuroblastoma but are rare in primary tumors. LCN, characterized by large nuclei with prominent nucleoli, predict a poor prognosis. We hypothesize that LCN can be created with high-dose intra-tumoral chemotherapy and identified by a digital analysis system. Methods Orthotopic mouse xenografts were created using human neuroblastoma and treated with high-dose chemotherapy delivered locally via sustained-release silk platforms, inducing tumor remission. After recurrence, LCN populations were identified on H&E sections manually. Clusters of typical LCN and non-LCN cells were divided equally into training and test sets for digital analysis. Marker-controlled watershed segmentation was used to identify nuclei and characterize their features. Logistic regression was developed to distinguish LCN from non-LCN. Results Image analysis identified 15,000 nuclei and characterized 70 nuclear features. A 19-feature model provided AUC > 0.90 and 100% accuracy when > 30% nuclei/cluster were predicted as LCN. Overall accuracy was 87%. Conclusions We recreated LCN using high-dose chemotherapy and developed an automated method for defining LCN histologically. Features in the model provide insight into LCN nuclear phenotypic changes that may be related to increased activity. This model could be adapted to identify LCN in human tumors and correlated with clinical outcomes.

Introduction High-risk neuroblastoma is a deadly disease; poor prognosticators are MYCN-amplification and TERT-overexpression. We hypothesized that Gene Set Enrichment Analysis (GSEA) could identify pathways associated with MYCN-amplification and that inhibition of these pathways could decrease tumor growth. Methods We analyzed the Neuroblastoma-Kocak dataset (GSE45547, n = 649) and identified pathways associated with MYCN-amplification. Inhibitors were selected from upregulated gene sets for in vitro cytotoxicity testing using ST16-patient-derived primary neuroblastoma cells and in vivo testing using orthotopic ST16-patient-derived xenografts (PDX) in mice. Tumor volume was measured with ultrasound and tumor sections examined after H&E staining. Results GSEA identified significantly overexpressed gene sets in MYCN-amplified tumors including MYC targets, cell cycle mitotic genes, TERT associated genes, loss of RB1 gene sets, and E2Fs targets. Several genes were potential Bromodomain-containing protein 4 (Brd4) targets, making Brd4 inhibitors - JQ1, AZD5153 - and cyclin-dependent kinase (Brd4’s binding partner) inhibitors – dinaciclib - potential therapeutic agents. JQ1 and dinaciclib were synergistic in inducing cytotoxicity in vitro. Dinaciclib-AZD5153 in vivo decreased tumor size compared to control, and increased tumor lymphocyte infiltration and necrosis on histology. Conclusions GSEA is a powerful approach to identify upregulated genes and potential therapeutic targets. Dinaciclib-AZD5153 combination therapy can be effective against MYCN-amplified and TERT-overexpressing neuroblastoma tumors.

PURPOSE: Advanced stage neuroblastoma patients require multi-agent chemotherapy. Intra-tumoral implantation of vincristine-loaded silk gel utilizes local diffusion to decrease orthotopic neuroblastoma tumor growth in mice. We hypothesize that injecting vincristine-loaded silk gel into eight locations within the tumor, instead of only centrally, decreases the diffusion distance and improves tumor growth suppression. METHODS: Human neuroblastoma cells, KELLY, were injected into mouse adrenal glands to create orthotopic tumors. After the tumors reached 100mm(3) by ultrasound, silk gels loaded with 50μg vincristine were injected centrally or in eight areas throughout the tumor. Drug-release profile was measured in vitro. Endpoints were tumor size>1000mm(3) and histologic examination. RESULTS: Vincristine-loaded silk gels suppressed tumor growth up to an “inflection point” (458.7±234.4mm(3) for central, 514.3±165.8mm(3) for eight-point injection) before tumor growth accelerated >200mm(3) over 3 days. The time to inflection point was 6.6days for central; 13.3days for eight-point injection(p

INTRODUCTION: Neuroblastoma is the most common pediatric extracranial solid malignancy with limited effective treatment. We have shown that sustained release single drugs delivered locally through a silk-based biomaterial are effective in decreasing orthotopic neuroblastoma xenograft growth. We further optimized this approach and hypothesized that increasing doses of local chemotherapy or delivering two chemotherapeutic agents simultaneously inhibit additional tumor growth. METHODS: MYCN-amplified (KELLY) and non-MYCN-amplified (SKNAS) neuroblastoma cells were treated with combinations of cisplatin, vincristine, doxorubicin, and etoposide to determine cytotoxicity and synergy. Drug-loaded silk material was created, and the amounts of drug released from the material over time were recorded. Murine orthotopic neuroblastoma xenografts were generated; tumors were implanted with single- or dual-agent chemotherapy-loaded silk. Ultrasound was used to monitor tumor growth, and tumor histology was evaluated. RESULTS: In vitro, vincristine/cisplatin combination was synergistic, significantly decreasing cell viability relative to other combinations. Both drugs loaded into silk could be released effectively for over two weeks. Locally-implanted vincristine/cisplatin silk induced increased tumor growth suppression compared to either agent alone in KELLY tumors (p50μg-500μg for vincristine-cisplatin, respectively. Tumor histology demonstrated tumor cell necrosis adjacent to drug-loaded silk material and presence of large cell neuroblastoma. CONCLUSIONS: Local delivery of sustained release chemotherapy can suppress tumor growth especially at high doses or with two synergistic drugs. Locally delivered dual therapy is a promising approach for future clinical testing.

Purpose Precise excision of neuroblastoma is challenging, especially when tumors adhere to vital structures. Indocyanine green (ICG), an FDA-approved dye with absorption peaking at 800 nm, can absorb the near IR laser energy and release heat in the dyed tissue. We hypothesize that by injecting ICG at tumor sites followed by precise laser application, tumor cell death can be selectively targeted. Methods Orthotopic neuroblastoma tumors were created in the adrenal gland of immunocompromised mice. Tumor, liver, kidney, and muscle tissues were chosen for ICG injection. Intervention variables included presence of tumor capsule, continuous vs. pulsed laser treatment and total energy delivered. Control groups included laser or ICG only. Tissues were stained with hematoxylin/eosin. Results Continuous wave laser generated excessive heat, causing damage in all tissues. When using pulsed laser treatment, liver, kidney, muscle, and intact tumor tissues showed no cell death when treated with laser alone or laser plus ICG. Tumor tissue with the capsule removed, however, showed cell death on histology. Conclusions Pulsed laser treatment combined with ICG causes targeted tumor cell death in neuroblastoma tumor without capsule. No cell death was observed when tumor capsule was present, when only laser was used, or when applied over non-tumor tissues.

Local drug delivery minimizes systemic toxicity while delivering high-dose chemotherapy for neuroblastoma patients. We hypothesized that varying burst and maintenance dosing of implanted silk platforms would improve survival.Platforms were loaded with vincristine 25μg, 50μg, 100μg, and 200μg varying burst (released 1-4days postimplantation) and maintenance (over the next 20days) dosing. Orthotopic tumors were created in mice using human neuroblastoma KELLY cells. Silk platforms were implanted into tumors when volumewas300mmDrug release ranged from burst dosing of 18.2 to 80.9μg, maintenance of 5.0 to 111.6μg, and cumulative of 23.3 to 177.4μg. Animals treated with 200μg platform died 9-13days postimplantation, corresponding to 128.1-141.2μg released (toxic dose). Animals received 30.2±3.4μgday-one survived longer than those that received 10.1±1.1μg (p=0.03), suggesting10.1μgday-one was insufficient. Tumors treated with 100μg or 50μg silk platform took longer to reach 1000 mmPlatform formulations can be manipulated to vary burst and maintenance dosing, summarized by an equation consisting of these variables.