Relapsed Neuroblastoma Research Articles

Abstract 500: Bortezomib, a proteasome inhibitor, synergizes with DFMO to inhibit neuroblastoma cell proliferation via the reversal of the LIN28/Let-7 axis

Abstract Background: The most common extracranial solid pediatric tumor, Neuroblastoma (NB) is responsible for 8-10% of all cancer in children and 15% of pediatric cancer deaths. Due to a high rate of tumor relapse, less than 40% of patients with high-risk disease achieve long-term survival. High incidence of metastatic disease and relapse in NB, as well as cellular heterogeneity of tumor, suggests that high-risk NB is likely due to inherent drug resistance of cancer stem cells (CSCs) that survive treatment in order to regenerate differentiated tumor. One pathway associated with targeting CSCs is the LIN28/Let-7 pathway due to the inductive role of LIN28 in stem cell growth and metabolism. LIN28 is characteristically overexpressed in many malignancies, including NB. Overexpression of LIN28 correlates with poor outcome in NB, therefore drugs that target LIN28/Let-7 axis could be beneficial in treating NB patients by targeting CSCs and preventing relapse of disease. This study proposes the drug combination therapy of Difluoromethylornithine (DFMO)-an inhibitor of ornithine decarboxylase and polyamine biosynthesis-coupled with bortezomib-a proteasome inhibitor-as a new therapeutic upstream target of LIN28/Let-7 pathway and glycolytic metabolism. Methods: NB cell lines BE(2)-C and SMS-KCNR were used. Cell viability was measured using Calcein AM fluorescent assay at DFMO doses 0.15 - 40 mM, alone and in combination with bortezomib at doses 0.15 - 80 nM. Isobologram cell viability analyses with DFMO in combination with bortezomib were generated using Calcein AM fluorescent assay results. Western blot analysis was used to measure LIN28, MYCN, SOCS3 and NFKB. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with Cell Titer GLO luminescent cell viability assay. Neurosphere assays were used to evaluate sphere formation after DFMO treatment. Xenograft studies were performed with SMS-KCNR cells implanted in the flank of mice and treated with vehicle, 2% DFMO in drinking water, bortezomib 0.5mg/kg IP twice a week, or combination of DFMO and bortezomib. Results: Cell proliferation assays and isobologram experiments suggest synergistic cytotoxicity of the drug combination in NB cell lines BE(2)-C and SMS-KCNR. Further, western blot analysis and ATP per cell experiments demonstrate the inhibition of overexpressed proteins in the LIN28/Let-7 pathway and inhibition of ATP/cell activity in NB cells. DFMO inhibits neurosphere formation in NB. Xenograft studies show decrease in tumor size with combination treatment. Conclusion: This study indicates that bortezomib synergizes with DFMO in inhibiting NB cell proliferation and neurosphere formation both in vitro and in vivo. Given the current lack of effective treatments and the high incidence of relapse and metastatic disease for patients, this drug combination offers a potential new therapeutic treatment for children with NB. Citation Format: Maria Rich, Ping Zhao, Abhinav Nagulapally, Jeffrey Bond, Giselle Sholler. Bortezomib, a proteasome inhibitor, synergizes with DFMO to inhibit neuroblastoma cell proliferation via the reversal of the LIN28/Let-7 axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 500. doi:10.1158/1538-7445.AM2015-500

Abstract 1611: The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting

Abstract Background: Despite the success of chemotherapy in improving the overall survival rate of childhood cancer, a number of types of children's cancers still have dismal outcomes. Included here are high risk neuroblastomas, Diffuse Intrinsic Pontine Gliomas (DIPG), and infant leukemias with MLL translocations. New treatments for these aggressive childhood cancers are urgently needed. Evidence is emerging of the importance of alterations in chromatin modifier genes in pediatric cancers. In this regard, CBL0137 is a carbazole-based anti-cancer agent with a unique mechanism of action. It is an indirect inhibitor of the chromatin remodeling complex FACT (Facilitates Chromatin Transcription). Inhibition of FACT by CBL0137 modulates the activity of several transcription factors involved in cancer: NF-kB and HSF1 are suppressed, while p53 is activated (Science Transl Med, 2011). We have examined FACT expression in neuroblastoma, DIPG and MLL leukemia, as well as the efficacy of CBL0137 in preclinical models of these diseases. Methods: Expression of the FACT subunits, SSRP1 and SPT16, was examined in neuroblastoma, DIPG and MLL leukemia cells using RT-PCR and Western analysis. The clinical significance of SSRP1 and SPT16 was also analysed using expression array data on 650 primary untreated neuroblastomas. Colony-forming assays were used to study the effect of CBL0137, either alone or combined with chemotherapeutic drugs. Cohorts of neuroblastoma, DIPG and MLL leukemia xenografted mice, as well as neuroblastoma-prone TH-MYCN mice, were treated with CBL0137, alone or combined with chemotherapeutic drugs. Results: High levels of SSRP1 and SPT16 expression were observed in all three types of child cancer. In addition, in neuroblastoma, the two FACT subunits were associated with MYCN amplification, and were strongly predictive of poor outcome (p<0.0001). As a single agent, CBL0137 administered iv had remarkable in vivo anti-tumor activity against neuroblastoma, DIPG and MLL leukemia xenografted mice. CBL0137 also synergized strongly with a variety of chemotherapeutic agents, including cyclophosphamide. Most dramatic results were observed when CBL0137 was combined with cyclophosphamide/topotecan, a highly active therapy for relapsed neuroblastoma that is currently in clinical trial. Cylophosphamide/topotecan plus CBL0137 resulted in cure of 90% of tumor-bearing MYCN-transgenic mice. Conclusions: Targeting FACT offers a highly promising novel therapeutic approach for aggressive childhood cancers. The results for CBL0137 are as good or better than any chemotherapy regimens we have tested in our preclinical models, and a Phase I COG trial of this nongenotoxic agent in refractory pediatric cancer patients is currently being planned. Citation Format: Michelle Haber, Jayne Murray, Laura Gamble, Ashleigh Carnegie-Clark, Hannah Webber, Michelle Ruhle, Michelle J. Henderson, Shiloh Middlemass, Daniel Carter, Maria Tsoli, Anahid Ehteda, Sandy Simon, Andre Oberthuer, Matthias Fischer, Katerina Gurova, Catherine Burkhart, Andrei Purmal, Richard B. Lock, David Ziegler, Glenn M. Marshall, Andrei V. Gudkov, Murray D. Norris. The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2015-1611

What we learned from bench to bedside with the neuroblastoma targeting CD171-specific CAR

s of the 51st Workshop for Pediatric Research 51st Workshop for Pediatric Research Gottingen, Germany 16-17 April 2015 This supplement has not been sponsored. Meeting abstracts Meeting abstract Despite the therapeutic efficacy of chimeric antigen receptor (CAR) redirected T cell immunotherapy in leukemia and lymphoma patients, similar clinical responses in solid tumor patients is, to date, an unrealized objective. We developed a CAR specific for CD171, an antigen expressed in several solid tumors including neuroblastoma, the most common extracranial tumor in childhood with an overall survival of less than 50% in high-risk patients. Since CD171 is also expressed in healthy tissues, including cerebellum and kidney, we proved the safety of targeting CD171 with CAR T cells in a non-human primate study. Further, we showed that CAR extracellular spacer and cytoplasmic signaling domain variants can be combined to tune the magnitude of cytotoxic CD8 + T lymphocyte (CTL) activation for tumor cell cytolysis and cytokine secretion. CAR constructs displaying the highest in vitro activity unexpectedly displayed the lowest in vivo anti-tumor activity, whereas CARs tuned for moderate signaling potency mediated tumor eradication. Recursively triggering hyperactive CARs rendered CTLs highly susceptible to activation-induced cell death resulting from augmented FasL expression, indicating that activation-induced cell death may be a critical parameter for achieving clinical efficacy against solid tumors. Our preclinical results assisted the design of a clinical trial comparing two CARs with different cytoplasmic signaling domains in patients with primary refractory or relapsed neuroblastoma, which was launched October 2014 at the Seattle Children's Hospital.

Prolonged progression-free survival after consolidating second or later remissions of neuroblastoma with Anti-GD2 immunotherapy and isotretinoin: a prospective Phase II study

Relapse of high-risk neuroblastoma (HR-NB) is deemed invariably fatal yet increasing numbers of HR-NB patients achieve a second complete/very good partial remission (CR/VGPR), hence the urgency to find a successful consolidative therapy. Identifying efficacy in patients without assessable disease, however, is problematic. We report the first study providing outcome data for this group of patients with poor prognosis. To prevent another relapse, HR-NB patients in second or later CR/VGPR received the anti-GD2 murine antibody 3F8 plus granulocyte-macrophage colony-stimulating factor plus isotretinoin in a Phase II trial. Upon meeting the target aim for progression-free survival (PFS) in the initial cohort of 33 patients, the trial was amended to allow patients who developed human anti-mouse antibody (HAMA) to receive rituximab to ablate HAMA with or without low-dose maintenance chemotherapy until immunotherapy could resume. For the total of 101 study patients, 5-year PFS and overall survival (OS) rates were 33% ± 5% and 48% ± 5%, respectively. Among the 33 long-term progression-free survivors, 19 had MYCN amplification, 19 had previously received anti-GD2 immunotherapy plus isotretinoin (as first-line therapy), and 15 never received maintenance chemotherapy. In a multivariate analysis of prognostic factors, only absence of minimal residual disease in bone marrow after 2 cycles of immunotherapy and before initiation of isotretinoin or anti-HAMA therapy was significantly favorable for both PFS and OS. Therefore, long-term PFS is possible for HR-NB patients who achieve at least a second CR/VGPR and receive consolidation that includes anti-GD2 immunotherapy plus isotretinoin, even if the patients received these biological treatments before relapse. Results from this prospective study will aid in the development of future Phase II studies for this growing ultra high-risk patient population.

634. Translation of a Neural Stem Cell-Mediated Enzyme/Prodrug Gene Therapy for Metastatic Neuroblastoma

Neuroblastoma (NB), a neuroendocrine tumor, is the most common extracranial solid tumor of childhood. Forty-five percent of these patients are diagnosed with metastatic high-risk tumors, for which treatment options are limited both in anti-tumor efficacy and patient tolerance. We have previously shown that neural stem cells (NSCs), engineered to secrete a modified rabbit carboxylesterase (CE), can home to metastatic NB tumor foci, and convert the prodrug CPT-11 (Irinotecan; IRN) to the 1000-fold more potent anti-cancer agent SN-38, resulting in significant therapeutic efficacy. The goal of our current biodistribution, efficacy and safety/toxicity IND-enabling studies is to identify the optimal dose and schedule of intravenously administered clinically relevant NSCs secreting a modified human CE (hCE1m6), followed by human equivalent doses of irinotecan. This would ideally provide a tumor selective, more effective and less toxic treatment for children with high-risk NB.In vitro cytotoxicity studies demonstrated IC50 values for four human NB tumors were significantly more sensitive to CPT-11 in the presence of hCE1m6 (similar to treatment with SN-38) compared to IRN alone. The sensitivity of NB tumor cells to CPT-11 was enhanced ≈6000-fold by hCE1m6 for KCNR tumor, while the sensitivity of SKNAS cells was enhanced ≈1200-fold for SKNAS, and approximately 150 to 600-fold decrease in IC50s of IRN for CHLA-136 and CHLA-255 NB cells. In vivo qPCR and MRI studies demonstrate NSC clearance through normal circulation and organs (lung, liver, brain) in naive non-tumor bearing Es1e/SCID mice is 24h following intravenous adminstration. In subcutaneous models of human NB we have quantified NSC tumor distribution and performed pharmacokinetics studies demonstrating a significant increase in tumor specific conversion of IRN to SN-38 with the CE-NSCs. Therapeutic efficacy studies are ongoing. If successful, we plan to be in Phase I studies in pediatric patients with refractory or relapsed high-risk neuroblastoma. This would be a first-in-human use of NSC-mediated enzyme/prodrug therapy in metastatic cancer patients.

Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application.

BackgroundVirus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was little apparent expansion of these cells in patients. In that study, VSTs were gene-modified on day 19 of culture and we hypothesized that by this time, sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism, we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV), Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2).ResultsAd-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates, so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively, and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2, TNF-α, IFN-γ, MIP-1α, MIP-1β and other cytokines in vitro.ConclusionsWe developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype, they should expand and persist in vivo, simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR).Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-015-0049-1) contains supplementary material, which is available to authorized users.

Iodine-131 metaiodobenzylguanidine therapy for neuroblastoma: reports so far and future perspective.

Neuroblastoma, which derives from neural crest, is the most common extracranial solid cancer in childhood. The tumors express the norepinephrine (NE) transporters on their cell membrane and take in metaiodobenzylguanidine (MIBG) via a NE transporter. Since iodine-131 (I-131) MIBG therapy was firstly reported, many trails of MIBG therapy in patients with neuroblastoma were performed. Though monotherapy with a low dose of I-131 MIBG could achieve high-probability pain reduction, the objective response was poor. In contrast, more than 12 mCi/kg I-131 MIBG administrations with or without hematopoietic cell transplantation (HCT) obtain relatively good responses in patients with refractory or relapsed neuroblastoma. The combination therapy with I-131 MIBG and other modalities such as nonmyeloablative chemotherapy and myeloablative chemotherapy with HCT improved the therapeutic response in patients with refractory or relapsed neuroblastoma. In addition, I-131 MIBG therapy incorporated in the induction therapy was proved to be feasible in patients with newly diagnosed neuroblastoma. To expand more the use of MIBG therapy for neuroblastoma, further studies will be needed especially in the use at an earlier stage from diagnosis, in the use with other radionuclide formations of MIBG, and in combined use with other therapeutic agents.

What we learned from bench to bedside with the neuroblastoma targeting CD171-specific CAR

s of the 51st Workshop for Pediatric Research 51st Workshop for Pediatric Research Gottingen, Germany 16-17 April 2015 This supplement has not been sponsored. Meeting abstracts Meeting abstract Despite the therapeutic efficacy of chimeric antigen receptor (CAR) redirected T cell immunotherapy in leukemia and lymphoma patients, similar clinical responses in solid tumor patients is, to date, an unrealized objective. We developed a CAR specific for CD171, an antigen expressed in several solid tumors including neuroblastoma, the most common extracranial tumor in childhood with an overall survival of less than 50% in high-risk patients. Since CD171 is also expressed in healthy tissues, including cerebellum and kidney, we proved the safety of targeting CD171 with CAR T cells in a non-human primate study. Further, we showed that CAR extracellular spacer and cytoplasmic signaling domain variants can be combined to tune the magnitude of cytotoxic CD8 + T lymphocyte (CTL) activation for tumor cell cytolysis and cytokine secretion. CAR constructs displaying the highest in vitro activity unexpectedly displayed the lowest in vivo anti-tumor activity, whereas CARs tuned for moderate signaling potency mediated tumor eradication. Recursively triggering hyperactive CARs rendered CTLs highly susceptible to activation-induced cell death resulting from augmented FasL expression, indicating that activation-induced cell death may be a critical parameter for achieving clinical efficacy against solid tumors. Our preclinical results assisted the design of a clinical trial comparing two CARs with different cytoplasmic signaling domains in patients with primary refractory or relapsed neuroblastoma, which was launched October 2014 at the Seattle Children's Hospital.

Abstract B41: Whole genome sequencing identifies tumor evolution events in relapse neuroblastoma

Abstract Treatment of primary neuroblastoma tumors tends to initially cause tumor regression, but relapse occurs in 50-60% of patients with high-risk tumors. These relapses are generally very aggressive and to date no curative regimen is known for these refractory tumors. We performed whole genome sequencing for six triplets of primary NB tumors, corresponding relapses and normal tissue from the same patient to determine if tumor evolution events on DNA level occurred. We first analyzed the number of mutations and structural variations affecting the coding sequence in the primary tumors and relapses. The relapses consistently show more aberrations than the primary tumors, although this varies between pairs. Some mutations are conserved from primary tumor to relapse, while others that are observed in the primary tumor are not present (at detectable levels) in the relapse. The majority of events however appear de novo in the relapses which indicates that clonal selection occurs and that secondary mutations take place in the formation of neuroblastoma relapses. We next focused on the mutations that are detected in the relapse tumor and not in the primary tumor. Analysis revealed several tumor evolution events in known oncogenes which have also been reported in primary neuroblastoma tumors in low frequency. Two relapses show strong enrichment for point mutations in ALK and PTPN11. Tree other tumors show de novo structural aberrations in NF1 (homozygous deletion), ALK (amplification) and BRAF (truncated protein). All events affect the RAS-MAPK signaling pathway which suggests involvement of this pathway in neuroblastoma tumor evolution. To further investigate the high frequency of events in this pathway in relapsed tumors we performed WGS on a panel of 25 neuroblastoma cell lines which were mainly derived from pretreated or relapsed neuroblastoma tumors. The frequency of genomic aberrations in NF1, NRAS, KRAS, BRAF, PTPN11 and ALK was much higher compared to primary tumor samples. Currently we are validating the effect of the tumor evolution events in non-malignant neuroblasts and neuroblastoma cell lines. The effect on RAS-MAPK pathway activity and phenotypic affects are studied. For the ALK mutation and the NF1 deletion we could indeed show activation of the RAS-MAPK pathway in vitro which is accompanied by an increase in colony forming capacity. Our findings show that tumor evolution events do occur in neuroblastoma mainly in genes which are also mutated in primary tumors. We found a high frequency of mutations that affect the RAS-MAPK pathway which suggests involvement of this pathway in NB tumor evolution. This warrants further validation of the RAS-MAPK pathway as a potential therapeutic target in relapsed refractory neuroblastoma. Citation Format: Thomas F. Eleveld, Linda Schild, Jan Koster, Danny Zwijnenburg, Marli E. Ebus, Peter Sluis van, Johannes H. Schulte, Huib N. Caron, Rogier Versteeg, Jan J. Molenaar. Whole genome sequencing identifies tumor evolution events in relapse neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B41.

Abstract IA24: Targeting MYCN-driven neuroblastoma using novel therapeutic approaches

Abstract Despite improvements in survival rates for childhood cancer over recent decades, a number of malignancies remain refractory to standard chemotherapy. Amongst these is neuroblastoma, the commonest solid tumour of young children. Children diagnosed with high-risk neuroblastoma still have dismal survival rates, hence alternative therapies are urgently required. The MYCN oncogene is the single most powerful biologic prognostic factor identified in childhood neuroblastoma. Children whose tumours display MYCN amplification have particularly poor prognosis, and the possibility of delivering highly effective therapies targeting the MYCN pathway is an area of research with enormous potential. In this regard, we have used a variety of approaches, to identify and validate key genes associated with MYCN, to use existing or novel therapeutics in new combinations to develop promising treatment strategies targeting these genes, and finally, to develop novel inhibitors of key MYCN-associated genes through cell-based screening of chemical small molecule libraries. One of the best characterised MYCN downstream target genes is the ornithine decarboxylase (ODC1) gene, which is role-limiting for polyamine biosynthesis. We have previously shown that ODC1 is a therapeutic target for neuroblastoma (Hogarty et al 2008, Cancer Res 68 (23): 9735-9745), and that in a range of pre-clinical models, targeting multiple steps in the polyamine pathway enhances therapeutic efficacy. Based on these findings, an international Phase I trial for refractory neuroblastoma, coordinated by the NANT consortium is now active, using ODC1 inhibition by high dose DFMO and SAT1 induction by celecoxib. We have since shown that MYCN regulates not only ODC1 but every gene in the polyamine pathway, in order to drive cellular proliferation, highlighting the importance of this pathway as a therapeutic target in neuroblastoma. In seeking to identify molecular targets that interact with or regulate MYCN/myc, are relevant to neuroblastoma biology and which have clear potential for molecular targeting in the clinical setting, we have recently focused on FACT (Facilitates Chromatin Transcription), a chromatin remodelling complex which has been shown to associate with myc. High levels of the genes encoding each of the two FACT subunits are strongly predictive of poor neuroblastoma outcome. Chromatin-trapping of the FACT complex by Curaxins, a recently described class of non-genotoxic compounds which simultaneous inhibit NFkB and active p53, is associated with strong anti-tumour activity (Gasparian et al 2011, Sci Transl Med 3:95ra74. DOI:10.1126). We have studied the effects of the lead curaxin compound in clinical trials, CBL0137, in two independent mouse models of neuroblastoma. As a single agent, CBL0137, administered either by gavage or iv had remarkable anti-tumour activity in both MYCN transgenic mice and also in nude mice xenografted with human neuroblastoma BE(2)-C cells. Most dramatic results were observed when iv CBL0137 was combined with the cyclophosphamide/topotecan, commonly used to treat relapsed neuroblastoma. This treatment resulted in cure of 90% of tumour-bearing MYCN-transgenic mice and a doubling of the lifespan of nude mice xenografted with BE(2)-C human neuroblastoma cells. These results are the most impressive we have ever observed for any therapeutic protocols in these models, and a Phase I trial of CBL0137 in refractory pediatric cancer patients is currently being planned. We have previously shown that the Multidrug Resistance-associated Proteins MRP1 and MRP4 are regulated by the MYCN oncogene and that high levels of these genes are strongly predictive of poor outcome in neuroblastoma (Henderson et al 2011, J Natl Cancer Inst 103 (16): 1236-1251). We have employed cell based screening of chemical small molecule libraries to successfully identify inhibitors of these molecular targets. More recently, we have used chemical library screening to identify a novel molecule, M606, which dramatically reduces MYCN protein levels and activity in human MYCN-amplified neuroblastoma cells, as well as c-myc protein levels in myc-overexpressing tumour cells. M606 inhibits growth of human neuroblastoma cells in a dose-dependent manner. M606 causes HIF1a protein accumulation and nuclear translocation, but this effect is independent of MYCN/MYC down-regulation. M606 is structurally related to a family of prolyl hydroxylase inhibitors, and inhibits prolyl hydroxylase activity. In common with some, but not all propyl hydroxylase inhibitors, for example, desferrioxamine (DFO), M606 appears to have iron chelating activity, and the effects of M606 on MYCN/Myc and HIF1a levels are entirely reversible by addition of iron. However, M606 is far more potent and less toxic than DFO, and inhibits the progression of tumours in MYCN transgenic mice. M606 thus appears to represent a novel molecule for targeting the MYCN oncogene in neuroblastoma. Citation Format: Michelle Haber. Targeting MYCN-driven neuroblastoma using novel therapeutic approaches. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA24.

Neuropeptide Y receptor Y5 as an inducible pro-survival factor in neuroblastoma: implications for tumor chemoresistance.

Neuroblastoma (NB) is a pediatric tumor of neural crest origin with heterogeneous phenotypes. While low stage tumors carry a favorable prognosis, over 50% of high risk NB relapses after treatment with a fatal outcome. Thus, developing therapies targeting refractory NB remains an unsolved clinical problem. Brain-derived neurotrophic factor (BDNF) and its TrkB receptor are known to protect NB cells from chemotherapy-induced cell death, while neuropeptide Y (NPY), acting via its Y2 receptor (Y2R), is an autocrine proliferative and angiogenic factor crucial for maintaining NB tumor growth. Here, we show that in NB cells, BDNF stimulates the synthesis of NPY and induces expression of another one of its receptors, Y5R. In human NB tissues, the expression of NPY and Y5R positively correlated with the expression of BDNF and TrkB. Functionally, BDNF triggered Y5R internalization in NB cells, while Y5R antagonist inhibited BDNF-induced p44/42-MAPK activation and its pro-survival activity. These observations suggested TrkB-Y5R transactivation that resulted in cross-talk between their signaling pathways. Additionally, NPY and Y5R were up-regulated in a BDNF-independent manner in NB cells under pro-apoptotic conditions, such as serum deprivation and chemotherapy, as well as in cell lines and tissues derived from post-treatment NB tumors. Blocking Y5R in chemoresistant NB cells rich in this receptor sensitized them to chemotherapy-induced apoptosis and inhibited their growth in vivo by augmenting cell death. In summary, the NPY/Y5R axis is an inducible survival pathway activated in NB by BDNF or cellular stress. Upon such activation, Y5R augments the pro-survival effect of BDNF via its interactions with TrkB receptor and exerts an additional BDNF-independent anti-apoptotic effect, both of which contribute to NB chemoresistance. Therefore, the NPY/Y5R pathway may become a novel therapeutic target for patients with refractory NB, thus far an incurable form of this disease.

Likelihood of Bone Recurrence in Prior Sites of Metastasis in Patients With High-Risk Neuroblastoma

Despite recent improvements in outcomes, 40% of children with high-risk neuroblastoma will experience relapse, facing a guarded prognosis for long-term cure. Whether recurrences are at new sites or sites of original disease may guide decision making during initial therapy. Eligible patients were retrospectively identified from institutional databases at first metastatic relapse of high-risk neuroblastoma. Included patients had disease involving metaiodobenzylguanidine (MIBG)-avid metastatic sites at diagnosis and first relapse, achieved a complete or partial response with no more than one residual MIBG-avid site before first relapse, and received no total body irradiation or therapy with (131)I-MIBG before first relapse. Anatomically defined metastatic sites were tracked from diagnosis through first relapse to determine tendency of disease to recur at previously involved versus uninvolved sites and to assess whether this pattern was influenced by site irradiation. Of 159 MIBG-avid metastatic sites identified among 43 patients at first relapse, 131 (82.4%) overlapped anatomically with the set of 525 sites present at diagnosis. This distribution was similar for bone sites, but patterns of relapse were more varied for the smaller subset of soft tissue metastases. Among all metastatic sites at diagnosis in our subsequently relapsed patient cohort, only 3 of 19 irradiated sites (15.8%) recurred as compared with 128 of 506 (25.3%) unirradiated sites. Metastatic bone relapse in neuroblastoma usually occurs at anatomic sites of previous disease. Metastatic sites identified at diagnosis that did not receive radiation during frontline therapy appeared to have a higher risk of involvement at first relapse relative to previously irradiated metastatic sites. These observations support the current paradigm of irradiating metastases that persist after induction chemotherapy in high-risk patients. Furthermore, they raise the hypothesis that metastatic sites appearing to clear with induction chemotherapy may also benefit from radiotherapeutic treatment modalities (external beam radiation or (131)I-MIBG).

Emergence of new ALK mutations at relapse of neuroblastoma.

Emergence of new <i>ALK</i> mutations at relapse of neuroblastoma.

Prognostic value of 18F-DOPA PET/CT at the time of recurrence in patients affected by neuroblastoma

The aim of this study was to investigate the relationship between (123)I-metaiodobenzylguanidine (MIBG) scan semi-quantification and a new (18)F-DOPA positron emission tomography (PET)/CT score in patients with suspected or documented neuroblastoma (NB) relapse and to assess the association between these two parameters and progression-free survival (PFS)/overall survival (OS). We analysed 24 NB patients who had undergone (123)I-MIBG and (18)F-DOPA PET/CT scans at the time of suspected relapse, after applying a proper scoring system for each scan. In time-to-event analyses, the score distributions were regarded as continuous and were categorized in tertiles and medians. We used Kaplan-Meier curves and Cox proportional hazard models for PFS and OS in order to estimate the independent prognostic impact of (123)I-MIBG and (18)F-DOPA PET/CT scans. The (123)I-MIBG and (18)F-DOPA scores were highly and positively correlated (Spearman's rho = 0.8, p < 0.001). Over a median follow-up of 14 months (range 6-82), 12 cases of disease progression and 6 deaths occurred. Multivariate Cox models showed a higher risk of disease progression [hazard ratio (HR) 17.0, 95% confidence interval (CI) 2.7-109] in NB patients with (123)I-MIBG score > 3 (3rd tertile) and an even higher risk (HR:37.2, 95% CI 2.4-574) in those with (18)F-DOPA whole-body metabolic burden (WBMB) >7.5 (median), after adjustment for all main clinical/pathological factors considered. Kaplan-Meier analyses showed a significant association with OS (log-rank p = 0.01 and p = 0.03 for (123)I-MIBG and (18)F-DOPA WBMB, respectively). Our results confirm the good agreement between (18)F-DOPA PET/CT and (123)I-MIBG scan in patients affected by NB relapse. In time-to-event analyses, (123)I-MIBG scan and (18)F-DOPA PET/CT scores were independently and significantly associated with disease progression.

Late effects in children treated with intensive multimodal therapy for high-risk neuroblastoma: High incidence of endocrine and growth problems

Due to the poor prognosis of high-risk (HR) neuroblastoma (NBL), scant data exist on late effects after treatment. Recently, protocols utilizing intense multimodal treatment have resulted in improved long-term survival. The objective of this study was to determine the prevalence of late effects in survivors of HR NBL. A retrospective review of clinical data for serial patients completing treatment between September 1994 and October 2007 and surviving for at least 1 year was performed. Therapy included aggressive chemotherapy, surgery, radiation and single or tandem SCT. Oncology follow-up was standard; clinical criteria were utilized for referrals to endocrinology and other services. Fifty-one eligible patients were identified. Median follow-up was 6.1 years (range 1.0-15.2). Height was significantly impacted (ΔZ-score -1.91 in those treated with TBI and -0.77 in those without). Pre-diabetes or diabetes, hypothyroidism and ovarian insufficiency were observed in 50, 59 and 75% of at-risk survivors, respectively. Hearing loss and dental issues were common. Nine patients had relapse of NBL; seven died of progressive disease. As there is a high prevalence of late effects in long-term survivors of HR NBL, close monitoring and further studies after treatment are indicated, and in particular after more modern, non-TBI regimens.

Inhibition of Select HSV Receptors in Human Neuroblastoma Cell Lines Demonstrate the Pathway for Oncolytic Herpes Simplex Viral Entry

Neuroblastoma, a tumor of neural crest origin, is the most common extracranial solid tumor of children and is responsible for over 15% of all pediatric cancer deaths. Refractory or relapsed neuroblastoma continues to carry a dismal prognosis emphasizing the need for more effective and novel therapies. We have previously shown that an oncolytic herpes simplex virus (oHSV) was cytolytic against neuroblastoma cell lines in vitro however the mechanism responsible for viral entry into the cells remains unexamined.

Anti-tumor activity of the beta-adrenergic receptor antagonist propranolol in neuroblastoma

Neuroblastoma (NB) is a pediatric tumor of the sympathetic nervous system, which is often associated with elevated catecholamines. More than half of patients with metastatic NB relapse and survival is extremely poor with current therapies. In a high-throughput screen of FDA-approved drugs we identified anti-NB activity for the nonselective β-adrenergic receptor antagonist propranolol hydrochloride. Propranolol inhibited growth of a panel of fifteen NB cell lines irrespective of MYCN status, and treatment induced apoptosis and decreased proliferation. Activity was dependent on inhibition of the β2, and not β1, adrenergic receptor, and treatment resulted in activation of p53 and p73 signaling in vitro. The majority of NB cell lines and primary tumors express β2 adrenergic receptor and higher mRNA levels correlate with improved patient survival, but expression levels did not correlate with in vitro sensitivity to propranolol. Furthermore, propranolol is synergistic with the topoisomerase I inhibitor SN-38 and propranolol inhibits growth of NB xenografts in vivo at doses similar to those used to treat infants with hemangiomas and hypertension. Taken together, our results suggest that propranolol has activity against NB and thus should be considered in combination treatments for patients with relapsed and refractory NB.

Preclinical Evaluation of Engineered Oncolytic Herpes Simplex Virus for the Treatment of Neuroblastoma

Despite intensive research efforts and therapeutic advances over the last few decades, the pediatric neural crest tumor, neuroblastoma, continues to be responsible for over 15% of pediatric cancer deaths. Novel therapeutic options are needed for this tumor. Recently, investigators have shown that mice with syngeneic murine gliomas treated with an engineered, neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ134.5 gene, enabling replication in tumor cells but precluding infection of normal neural cells. We hypothesized that M002 would also be effective in the neural crest tumor, neuroblastoma. We showed that M002 infected, replicated, and decreased survival in neuroblastoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly decreased tumor growth, and that this effect was augmented with the addition of ionizing radiation. Importantly, survival could be increased by subsequent doses of radiation without re-dosing of the virus. Finally, these studies showed that the primary entry protein for oHSV, CD111 was expressed by numerous neuroblastoma cell lines and was also present in human neuroblastoma specimens. We concluded that M002 effectively targeted neuroblastoma and that this oHSV may have potential for use in children with unresponsive or relapsed neuroblastoma.

Phase II study of temozolomide in combination with topotecan (TOTEM) in relapsed or refractory neuroblastoma: A European Innovative Therapies for Children with Cancer-SIOP-European Neuroblastoma study

PurposeTo assess objective response rate (ORR) after two cycles of temozolomide in combination with topotecan (TOTEM) in children with refractory or relapsed neuroblastoma. Patients and MethodsThis multicenter, non-randomised, phase II study included children with neuroblastoma according to a two-stage Simon design. Eligibility criteria included relapsed or refractory, measurable or metaiodobenzylguanidine (mIBG) evaluable disease, no more than two lines of prior treatment. Temozolomide was administered orally at 150mg/m2 followed by topotecan at 0.75mg/m2 intravenously for five consecutive days every 28days. Tumour response was assessed every two cycles according to International Neuroblastoma Response Criteria (INRC), and reviewed independently. ResultsThirty-eight patients were enroled and treated in 15 European centres with a median age of 5.4years. Partial tumour response after two cycles was observed in 7 out of 38 evaluable patients [ORR 18%, 95% confidence interval (CI) 8–34%]. The best ORR whatever the time of evaluation was 24% (95% CI, 11–40%) with a median response duration of 8.5months. Tumour control rate (complete response (CR)+partial response (PR)+mixed response (MR)+stable disease (SD)) was 68% (95% CI, 63–90%). The 12-months Progression-Free and Overall Survival were 42% and 58% respectively. Among 213 treatment cycles (median 4, range 1–12 per patient) the most common treatment-related toxicities were haematologic. Grade 3/4 neutropenia occurred in 62% of courses in 89% of patients, grade 3/4 thrombocytopenia in 47% of courses in 71% of patients; three patients (8%) had febrile neutropenia. ConclusionTemozolomide–Topotecan combination results in very encouraging ORR and tumour control in children with heavily pretreated recurrent and refractory neuroblastoma with favourable toxicity profile.

Treatment with topotecan plus cyclophosphamide in children with first relapse of neuroblastoma

Reports of responses and toxicities of salvage therapies for relapsed neuroblastoma are rare and often confounded by effects of additional treatments. Our objective was to describe the outcomes and toxicities for a topotecan and cyclophosphamide (TOPO/CTX) regimen for first relapse or progression of high-risk neuroblastoma. We retrospectively reviewed charts of relapsed or refractory neuroblastoma patients treated between 1999 and 2009 with our standard-of-care outpatient TOPO/CTX (0.75 and 250 mg/m(2) /day × 5 days q3-4 weeks). Twenty-seven patients received 343 cycles of TOPO/CTX (median 10 cycles per patient, range 1-32). Most patients (N = 25) had undergone autologous stem cell transplantation. Seventeen (63%) patients had an objective response (CR + PR + MR). The 3-year progression-free survival (PFS) after relapse was 11 ± 6% and 3-year overall survival (OS) after relapse was 33 ± 9%. The median PFS was 1.2 years and the median OS was 2.3 years. Five patients are alive with follow-up of 3.1-5.5 years. Shorter time from diagnosis to relapse (6-18 months) was associated with shorter OS. The majority of patients experienced chemotherapy delays, transfusions, and febrile neutropenia, including eight bacterial infections. The mean number of hospitalized days was less than one per cycle. TOPO/CTX was well tolerated and resulted in response rates and PFS similar to those reported for patients treated on COG 9462. Our study provides additional toxicity, historical endpoints, and time-to-progression data against which new agents and combination therapies using TOPO/CTX as a backbone can be measured.