Neuroblastomas with a high mitosis-karyorrhexis index (High-MKI) are often associated with MYCN amplification, MYCN protein overexpression and adverse clinical outcome. However, the prognostic effect of MYC-family protein expression on these neuroblastomas is less understood, especially when MYCN is not amplified. To address this, MYCN and MYC protein expression in High-MKI cases (120 MYCN amplified and 121 non-MYCN amplified) was examined by immunohistochemistry. The majority (101) of MYCN-amplified High-MKI tumors were MYCN(+), leaving one MYC(+), 2 both(+), and 16 both(−)/(+/−), whereas non-MYCN-amplified cases appeared heterogeneous, including 7 MYCN(+), 36 MYC(+), 3 both(+), and 75 both(−)/(+/−) tumors. These MYC-family proteins(+), or MYC-family driven tumors, were most likely to have prominent nucleolar (PN) formation (indicative of augmented rRNA synthesis). High-MKI neuroblastoma patients showed a poor survival irrespective of MYCN amplification. However, patients with MYC-family driven High-MKI neuroblastomas had significantly lower survival than those with non-MYC-family driven tumors. MYCN(+), MYC-family protein(+), PN(+), and clinical stage independently predicted poor survival. Specific inhibition of hyperactive rRNA synthesis and protein translation was shown to be an effective way to suppress MYC/MYCN protein expression and neuroblastoma growth. Together, MYC-family protein overexpression and PN formation should be included in new neuroblastoma risk stratification and considered for potential therapeutic targets.

Purpose: MYCN oncogene amplification is an independent predictor of poor prognosis in neuroblastoma. CX-5461 is a small molecular inhibitor that prevents initiation of ribosomal RNA (rRNA) synthesis by RNA Pol I, down-regulating MYCN/MYC proteins. We hypothesize that neuroblastoma tumor growth can be suppressed by CX-5461. Methods: MYCN-amplified (KELLY, IMR5) and non-amplified (SY5Y, SKNAS) neuroblastoma cells were treated with CX-5461. MYCN/MYC expression after 24-48 hours was determined by Western blot. Orthotopic neuroblastoma tumors created in mice using KELLY cells were treated with CX-5461-loaded silk films implanted locally. Tumor growth was monitored using ultrasound. Histologic evaluation of tumors was performed. Results: IC 50 for KELLY, IMR5, SY5Y, and SKNAS cells to CX-5461 was 0.75µM, 0.02µM, 0.8µM, and 1.7µM, respectively. CX-5461 down-regulated MYCN and MYC proteins at 0.25-1.0µM on Western blot analysis. CX-5461-loaded silk film released 23.7±3µg of the drug in 24 hours and 48.2±3.9µg at 120 hours. KELLY tumors treated with CX-5461-loaded film reached 800mm 3 after 7.8±1.4 days, while those treated with control film reached the same size on 5.1±0.6 days (p=0.03). CX-5461-treated tumors showed collapse of nucleolar hypertrophy and MYCN protein downregulation. Conclusion: We demonstrated that local delivery of CX-5461 via sustained release platform can suppress orthotopic neuroblastoma tumor growth, especially those with MYCN/MYC overexpression.

High-risk neuroblastoma requires surgical resection and multi-drug chemotherapy. This study aimed to develop an extended release, implantable and degradable delivery system for etoposide, commonly used for neuroblastoma treatment. Different concentrations of silk, a biodegradable, non-toxic, non-immunogenic material were employed to prepare etoposide-loaded wafer formulations. Secondary structure of silk in the formulations was characterized using Fourier Transform Infrared (FTIR) spectroscopy and optimized based on the crystalline structure. Accelerated in vitro degradation studies under different conditions such as acidic, alkaline, oxidizing mediums and high temperature, were performed. The integrity of the silk wafer structure was maintained unless exposed to 0. 1N NaOH for 24 hours. In vitro release of etoposide was performed in PBS (phosphate buffered saline) at 37°C. Silk coated 6% wafers released the drug up to 45 days, while uncoated wafers released the drug for 30 days. Cytotoxicity study was performed on KELLY cells to evaluate the etoposide cytotoxicity (LC50) and the long-term efficacy of the etoposide wafer formulations. The results showed that etoposide killed 50% of the cells at 1 μg/mL concentration and the wafer formulations demonstrated significant cytotoxicity up to 22 days when compared to untreated cells. Using an orthotopic neuroblastoma mouse model, intratumoral implantation of the coated 6%, uncoated 6%, or uncoated 3% silk wafers were all effective at decreasing tumor growth. Histological examination revealed tumor cell necrosis adjacent to the drug-loaded silk wafer.

Immunotherapy targeting GD2 is a primary treatment for patients with high-risk neuroblastoma. Dinutuximab is a monoclonal antibody with great clinical promise but is limited by side effects such as severe pain. Local delivery has emerged as a potential mechanism to deliver higher doses of therapeutics into the tumor bed, while limiting systemic toxicity. We aim to deliver dinutuximab locally in a lyophilized silk fibroin foam for the treatment of an orthotopic neuroblastoma mouse model. Dinutuximabloaded silk fibroin foams were fabricated through lyophilization. In vitro release profile and bioactivity of the release through complement-dependent cytotoxicity were characterized. MYCN-amplified neuroblastoma cells (KELLY) were injected into the left gland of mice to generate an orthotopic neuroblastoma model. Once the tumor volume reached 100 mm 3 , dinutuximab-, human IgG-, or buffer-loaded foams were implanted into the tumor and growth was monitored using high-resolution ultrasound. Post-resection histology was performed on tumors. Dinutuximab-loaded silk fibroin foams exhibited a burst release, with slow release thereafter in vitro with maintenance of bioactivity. The dinutuximab-loaded foam significantly inhibited xenograft tumor growth compared to IgG-and buffer-loaded foams. Histological analysis revealed the presence of dinutuximab within the tumor and neutrophils and macrophages infiltrating into dinutuximab-loaded silk foam. Tumors treated with local dinutuximab had decreased MYCN expression on histology compared to control or IgG-treated tumors. Silk fibroin foams offer a mechanism for local release of dinutuximab within the neuroblastoma tumor. This local delivery achieved a significant decrease in tumor growth rate in a mouse orthotopic tumor model. K E Y W O R D SCh14.18, dinutuximab, immunotherapy, local delivery, neuroblastoma, silk fibroin

Injecting vincristine-loaded sustained release silk gel at 8 separate locations halved the diffusion distance and doubled the time for the tumor to reach the growth inflexion point.