Refractory High-risk Neuroblastoma Research Articles

NANT 2012-01: Phase 1 study of DFMO and celecoxib with cyclophosphamide and topotecan for relapsed or refractory high-risk neuroblastoma.

10558Background: MYC drives polyamine expansion to support its oncogenic functions. Ornithine decarboxylase (Odc) is a direct MYC target that is rate-limiting for polyamine synthesis, and is itself...

Metronomic therapy has low toxicity and is as effective as current standard treatment for recurrent high-risk neuroblastoma

ABSTRACTThe metronomic therapy concept uses low doses of continuously applied chemotherapeutic, anti-angiogenetic, and immunomodulating drugs. Twenty patients with recurrent and 3 with refractory high-risk neuroblastoma were treated by the metronomic concept using celecoxib, cyclophosphamide, vinblastine, and etoposide for up to 24 months. The outcome was compared to 274 matched patients with a first recurrence from stage 4 neuroblastoma using the variables time from diagnosis to first recurrence, number of organs involved, and MYCN amplification. All were treated with dose-intensive conventional chemotherapy. The study patients experienced 1–3 recurrences and had 1–3 sites involved (osteomedullary, primary tumor, central nervous system, lymph nodes, liver, lungs) before the metronomic therapy started. Two patients in complete remission and three with active refractory disease following recurrence treatment were excluded from the outcome analysis. The curves for secondary event-free and overall survival demonstrated no significant differences. The toxicity was minimal except for ≥3 grade thrombocytopenia and leukopenia (all heavily pretreated). The treatment was realized in an outpatient setting. The metronomic approach is similarly effective as standard treatment in recurrent high-risk neuroblastoma, has low toxicity, and is applicable in an outpatient setting. A prospective study including propranolol as a fifth drug is underway.

Abstract 691: DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid pediatric tumor and is associated with MYCN amplification. MYCN is a regulator of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis. Inhibition of this pathway in MYCN-amplified NB tumors has been shown to be a target for treatment. Alpha-difluoromethylornithine (DFMO) inhibits ODC and is currently being used in a Phase II clinical trial for NB. BET inhibitors JQ1 and OTX-015 have been shown to be effective against MYCN-amplified cancers; it is hypothesized that they downregulate MYCN as well as cancer stem cell (CSC) signaling. We propose that inhibiting MYCN with a BET inhibitor, coupled with inhibition of ODC with DFMO, will result in enhanced inhibition of NB growth and tumor-initiating properties. Methods: Single and combination drug treatments were conducted on BE(2)-C, SMS-KCNR, CHLA90, and one patient-derived cell line. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability in 96-well plates. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. Drug combination studies were conducted in MYCN amplified tumors BE(2)-C and SMS-KCNR using ray designs to evaluate for synergism. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity and apoptosis in NB cells. Western blots measured protein levels of apoptosis markers (cleaved caspase 3 and cleaved PARP) and CSC markers (Nanog, Sox2, NF-kB, CXCR4, Lin28B, and MYCN). Neurosphere assays were used to evaluate tumor initiation via sphere formation. Results: Cell viability of MYCN amplified NB showed an IC50 range of 1.48-1.69 μM for JQ1 and 839.3 nM-2.36 μM for OTX-015; MYCN non-amplified NB showed an IC50 range of 4.22-11.58 μM for JQ1 and 2.99-11.03 uM for OTX-015. Combination treatment in MYCN NB had a synergistic effect, based on Loewe-additivity as the null hypothesis, for cell viability suppression by ray design experiments for BE(2)-C and SMS-KCNR. Western blots showed greater expression of apoptosis markers and decreased expression of CSC markers in combination relative to single drug treatments. A greater than 50% decrease in neurosphere formation with combination treatment in BE(2)-C, SMS-KCNR, and the patient cell line provides evidence for reduced CSC activity. IncuCyte imaging showed an increase in cell death with time post-treatment. Conclusion: The combination of DFMO with BET inhibitors has a synergistic effect in treating MYCN amplified NBs as shown by a decrease in cell viability. This combination targets CSC pathways and decreases tumor-initiating ability. Given the lack of effective treatment options for children with relapsed or refractory high-risk NB, this combination may be a promising novel therapy. Citation Format: Sarah DeCou, Ping Zhao, Tracey Avequin, Abhinav Nagulapally, Jeffrey Bond, Giselle Saulnier Sholler. DFMO synergizes with BET inhibitors targeting ODC and MYCN to impede neuroblastoma cell proliferation and tumor initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 691. doi:10.1158/1538-7445.AM2017-691

Abstract 3512: Dielectrophoretic capture and genetic analysis of individual disseminated solid tumor cells.

Abstract Background: The purpose of this study was to develop a novel approach for isolation and genetic analysis of individual circulating and disseminated tumor cells (CTC, DTC) from patient samples. This work is motivated by our desire to develop non-invasive molecular diagnostics and understand single tumor cell heterogeneity. Methods: Here we used the DEPArray microelectronics and microfluidics platform for the manipulation and capture of individual tumor cells. A suspension of live or fixed cells, prelabeled with fluorochrome-conjugated antibodies, was loaded into the DEPArray cartridge, and application of non-uniform electrical fields (dielectrophoresis) was employed to trap individual cells into movable micro-cages. Cell images were acquired via the incorporated fluorescence microscope and CCD camera, and cells were selected based on morphology and expression of tumor antigens. To determine sensitivity and specificity of the capture process, cell lines were spiked into normal donor white blood cells (WBC) over a 5-log range. Pre-enrichment by FACSAria was employed when the starting tumor cell concentration was below 1 in 1,000 WBC. The whole genome amplification (WGA) product of single cells was then subjected to targeted sequencing. To apply this approach to patient samples, we obtained blood and bone marrow samples from patients with refractory high-risk neuroblastoma, a cancer with a high rate and quantity of distant metastatic disease. Results: Single tumor cells were recovered from all spiking experiments, including spiked samples with a tumor cell concentration as low as 1 in 106 WBC. Recovery of EpCAM-negative single cells was accomplished with the neuroblastoma cell lines SY5Y, NB1643, and Kelly labeled with an antibody for the disialoganglioside GD2, the breast cancer cell line MCF-7 with a HER2 antibody, and for the lung cancer cell line H292 with EGFR labeling. Recovery of EpCAM-positive cells was achieved with the colon carcinoma cell line SW480. Captured single cells were WGA'd and specificity was verified using Sanger sequencing. For the 2 neuroblastoma spiking experiments for which sequencing has been completed, the known ALK mutation (F1174L) was detected in 100% of cells. The known KRAS mutation (G12V) was detected in 100% of single SW480 cells sequenced to date. No mutations were detected in isolated WBC. We next analyzed the post-Ficoll fraction of 12 patient bone marrow and 1 patient blood sample, with a median tumor cell concentration of 0.119% (range 0.003 - 80.0%). A minimum of 10 single tumor cells were captured per patient. Genome-wide analysis of DNA and RNA copy number and mutation status from these samples is ongoing. Conclusion: These data demonstrate the development of a novel workflow for the reliable capture and genetic analysis of rare CTC. Efforts are ongoing to join this approach with a more comprehensive genome-wide analysis of single cells from neuroblastoma and other cancers. Citation Format: Erica L. Carpenter, JulieAnn Rader, Kateryna Krytska, Jacob Ruden, Eric Rappaport, Yael Mosse, Francesca Fontana, Manuela Banzi, Gianni Medoro, Nicolo Manaresi, Peter O'Dwyer, John Maris. Dielectrophoretic capture and genetic analysis of individual disseminated solid tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2013-3512

Phase II Study of Oral Capsular 4-Hydroxyphenylretinamide (4-HPR/Fenretinide) in Pediatric Patients with Refractory or Recurrent Neuroblastoma: A Report from the Children's Oncology Group

AbstractPurpose: To determine the response rate to oral capsular fenretinide in children with recurrent or biopsy proven refractory high-risk neuroblastoma.Experimental Design: Patients received 7 days of fenretinide: 2,475 mg/m2/d divided TID (<18 years) or 1,800 mg/m2/d divided BID (≥18 years) every 21 days for a maximum of 30 courses. Patients with stable or responding disease after course 30 could request additional compassionate courses. Best response by course 8 was evaluated in stratum 1 (measurable disease on CT/MRI ± bone marrow and/or MIBG avid sites) and stratum 2 (bone marrow and/or MIBG avid sites only).Results: Sixty-two eligible patients, median age 5 years (range 0.6–19.9), were treated in stratum 1 (n = 38) and stratum 2 (n = 24). One partial response (PR) was seen in stratum 2 (n = 24 evaluable). No responses were seen in stratum 1 (n = 35 evaluable). Prolonged stable disease (SD) was seen in 7 patients in stratum 1 and 6 patients in stratum 2 for 4 to 45+ (median 15) courses. Median time to progression was 40 days (range 17–506) for stratum 1 and 48 days (range 17–892) for stratum 2. Mean 4-HPR steady-state trough plasma concentrations were 7.25 μmol/L (coefficient of variation 40–56%) at day 7 course 1. Toxicities were mild and reversible.Conclusions: Although neither stratum met protocol criteria for efficacy, 1 PR + 13 prolonged SD occurred in 14/59 (24%) of evaluable patients. Low bioavailability may have limited fenretinide activity. Novel fenretinide formulations with improved bioavailability are currently in pediatric phase I studies. Clin Cancer Res; 17(21); 6858–66. ©2011 AACR.

177Lu-DOTATATE Molecular Radiotherapy for Childhood Neuroblastoma

This study tested the principle that (68)Ga-DOTATATE PET/CT may be used to select children with primary refractory or relapsed high-risk neuroblastoma for treatment with (177)Lu-DOTATATE and evaluated whether this is a viable therapeutic option for those children. Between 2008 and 2010, 8 children with relapsed or refractory high-risk neuroblastoma were studied with (68)Ga-DOTATATE PET/CT. The criterion of eligibility for (177)Lu-DOTATATE therapy was uptake on the diagnostic scan equal to or higher than that of the liver. Of the 8 children imaged, 6 had abnormally high uptake on the (68)Ga-DOTATATE PET/CT scan and proceeded to treatment. Patients received 2 or 3 administrations of (177)Lu-DOTATATE at a median interval of 9 wk and a median administered activity of 7.3 GBq. Of the 6 children treated, 5 had stable disease by the response evaluation criteria in solid tumors (RECIST). Of these 5 children, 2 had an initial metabolic response and reduction in the size of their lesions, and 1 patient had a persistent partial metabolic response and reduction in size of the lesions on CT, although the disease was stable by RECIST. One had progressive disease. Three children had grade 3 and 1 child had grade 4 thrombocytopenia. No significant renal toxicity has been seen. (68)Ga-DOTATATE can be used to image children with neuroblastoma and identify those suitable for molecular radiotherapy with (177)Lu-DOTATATE. We have shown, for what is to our knowledge the first time, that treatment with (177)Lu-DOTATATE is safe and feasible in children with relapsed or primary refractory high-risk neuroblastoma. We plan to evaluate this approach formally in a phase I-II clinical trial.

Phase I trial of lestaurtinib for children with refractory neuroblastoma: a new approaches to neuroblastoma therapy consortium study

TrkB acts as an oncogenic kinase in a subset of human neuroblastomas. Lestaurtinib, a multi-kinase inhibitor with potent activity against Trk kinases, has demonstrated activity in preclinical models of neuroblastoma. Patients with refractory high-risk neuroblastoma received lestaurtinib twice daily for 5days out of seven in 28-day cycles, starting at 70% of the adult recommended Phase 2 dose. Lestaurtinib dose was escalated using a 3+3 design. Pharmacokinetics and plasma phospho-TrkB inhibitory activity were evaluated in the first cycle. Forty-seven subjects were enrolled, and 10 dose levels explored starting at 25mg/M(2)/dose BID. Forty-six subjects were evaluable for response, and 42 subjects were fully evaluable for determination of dose escalation. Asymptomatic and reversible grade 3-4 transaminase elevation was dose limiting in 4 subjects. Reversible pancreatitis (grade 2) was observed in 3 subjects after prolonged treatment at higher dose levels. Other toxicities were mild and reversible. Pharmacokinetic analyses revealed rapid drug absorption, however inter-patient variability was large. Plasma inhibition of phospho-TrkB activity was observed 1h post-dosing at 85mg/M(2) with uniform inhibition at 120mg/M(2). There were two partial responses and nine subjects had prolonged stable disease at dose levels≥5, (median: 6cycles). A biologically effective and recommended phase 2 dose of 120mg/M(2)/dose BID was established. Lestaurtinib was well tolerated in patients with refractory neuroblastoma, and a dose level sufficient to inhibit TrkB activity was established. Safety and signs of activity at the higher dose levels warrant further evaluation in neuroblastoma.

Phase I trial of lestaurtinib for children with refractory neuroblastoma (NB): A New Approach to Neuroblastoma Therapy (NANT) Consortium study.

9532 Background: TrkB acts as an oncogenic kinase in a subset of human NB. Lestaurtinib, a multi-kinase inhibitor with potent activity against Trk kinases, has demonstrated anti-tumor activity in preclinical models of human NB. Methods: We performed a Phase I trial of Lestaurtinib in patients with recurrent or refractory high-risk neuroblastoma starting at 80% of the adult recommended Phase II dose. Subjects received lestaurtinib twice daily for five days on, two days off continuous dosing. Lestaurtinib dose was escalated using a 3+3 design. Pharmacokinetics and plasma phospho-TrkB inhibitory activity were evaluated in the first cycle defined as 28 days. Response data were obtained after the first and then every other cycle. Results: Forty-seven patients were enrolled, and 10 dose levels explored starting at 25 mg/m2/dose BID, with 42 patients evaluable for dose escalation and 47 patients evaluable for response. Asymptomatic and reversible gr 3-4 transaminase elevation was dose limiting in 4 patients. Pancreatitis (gr 2) was observed in 3 patients after prolonged treatment (≥5 cycles) at dose levels ≥5 and considered possibly related to drug. Symptoms resolved with discontinuation or dose reduction. Other toxicities were mild and reversible. Pharmacokinetic analyses indicate that drug is rapidly absorbed in children, however systemic exposure (Cmax and AUC on d1, Ctrough on d5 and d26) did not consistently increase with increasing dose, and inter-patient variability was large. Plasma inhibition of phospho-TrkB activity was observed 1 hour post-dosing at 85 mg/m2 with uniform inhibition at 120 mg/m2. There were two objective responses and 10 patients had prolonged stable disease at dose levels ≥5, (median: 12 cycles) before disease progression (pending review). A biologically effective and recommended phase II dose of 120 mg/m2/dose BID was established. Conclusions: Lestaurtinib was well tolerated in this heavily pre-treated patient group with refractory NB, and a dose level required to provide enough free drug to inhibit TrkB activity was established. Safety and signs of activity at the higher dose levels warrants further evaluation of this drug or other Trk-directed therapies in NB. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Cephalon Cephalon Cephalon

Temozolomide phase II study in children with relapsing refractory high-risk neuroblastoma

9012 Background: To determine the response rate (RR) of neuroblastoma (NB) in children to temozolomide (TMZ), and evaluate the duration of response and tolerance of the drug in this patient population. Methods: A multicenter, phase II evaluation of an oral, daily schedule of TMZ (200 mg/m2 on 5 consecutive days and repeated every 28 days) was undertaken in children with a refractory or relapsed high-risk NB (metastatic or localized with Myc-N amplification). Evidence of activity was defined by radiologic or MIBG scan evidence of sustained reduction in lesion size or activity whenever it occurs. Methodology included a two-step study using Fleming’s method with a first step of 15 patients and a second of 10 additional patients if 2 to 4 responses had been observed in the first cohort. All data were centrally reviewed by a panel. Results: Among 34 registered patients over a 14 month period in 14 centres, twenty five are finally evaluable and received 94 cycles of chemotherapy. Disease status was metastatic NB (n=23) either refractory (n=9) or in relapse (n=14). Grade ¾ thrombocytopenia was the most frequent toxic event (16% of the cycles). Myelosuppression resulted in significant treatment delays and dose reductions (24% and 21% of cycles respectively). Out of 25 patients, response (CR, VGPR or PR) was observed in 5 (RR=20 ± 8%) with a median duration of 6 months. Furthermore a mixed response or an objective effect was observed in respectively 2 and 3 additional patients. Conclusions: Temozolomide is effective in heavily pretreated patients with NB, and deserves further evaluation in combination with another drug No significant financial relationships to disclose.