Relapsed Neuroblastoma Research Articles

High-dose 131I-metaiodobenzylguanidine therapy in patients with high-risk neuroblastoma in Japan.

The aim of the study was to investigate the outcomes and prognostic factors of high-dose 131I-metaiodobenzylguanidine (131I-MIBG) therapy in patients with refractory or relapsed neuroblastoma (NBL) in Japan. We retrospectively analyzed 20 patients with refractory or relapsed high-risk NBL who underwent 131I-MIBG therapy with an administration dose ranging from 444 to 666MBq/kg at Kanazawa University Hospital, Japan, between September 2008 and September 2013. We focused on measurements regarding their initial responses, prognostic factors, survivals, and toxicities following 131I-MIBG therapy using our hospital data and questionnaires from the hospitals that these patients were initially referred from. Furthermore, we performed Kaplan-Meier survival analysis to evaluate event-free survival (EFS) and overall survival (OS). In 19 patients with complete follow-up data, the median age at first 131I-MIBG treatment was 7.9years (range 2.5-17.7years). Following 131I-MIBG therapy, 17 of the 19 patients underwent stem-cell transplantations, and their treatment response was either complete (CR) or partial (PR) in three and two cases, respectively. The EFS and OS rates at 1year following 131I-MIBG therapy were 42% and 58%, respectively, and those at 5years following 131I-MIBG therapy were 16% and 42%, respectively. Using the two-sample log-rank test, the OS time following 131I-MIBG therapy was significantly longer for < 3-year time interval between the initial diagnosis and 131I-MIBG therapy (p = 0.017), Curie score < 16 just before 131I-MIBG therapy (p = 0.002), without pain (p = 0.002), without both vanillylmandelic acid (VMA) and homovanillic acid (HVA) elevation (p = 0.037) at 131I-MIBG therapy, and with CR or PR following 131I-MIBG therapy (p = 0.015). Although severe hematological toxicities were identified in all 19 patients, severe nonhematological toxicity was not recorded in any patient, except for one patient with grade 3 anorexia and nausea. High-dose 131I-MIBG therapy in patients with refractory or relapsed high-risk NBL can provide a favorable prognosis without severe nonhematological toxicities. Better prognosis may be anticipated in patients with the initial good response, no pain at 131I-MIBG therapy, no VMA and HVA elevation at 131I-MIBG therapy, low Curie score (< 16) just before 131I-MIBG therapy, and short time interval (< 3years) between the initial diagnosis and 131I-MIBG therapy.

NCMP-03. AN UNUSUAL CASE OF NEUROBLASTOMA ASSOCIATED OMS: HIGH RISK DISEASE REQUIRING IMMUNOTHERAPY

Abstract INTRODUCTION Opsoclonus-myoclonus-ataxia syndrome (OMS) is a rare paraneoplastic syndrome (PNS) typically associated with low-risk pediatric neuroblastoma (NB). Overall survival is excellent but patients are at risk for long-term neurologic sequelae. As with many PNS, the etiology of NB-associated OMS is thought to be immune mediated and patients are managed with immunosuppressive therapy. The use of other immunotherapy such as checkpoint inhibitors for advanced stage cancers has been associated with sporadic reports of the development of PNS. We report a case of a child with NB-associated OMS who received anti-GD2 antibody immunotherapy for advanced relapsed NB without exacerbation of OMS. While the mechanism of action of anti-GD2 is unique, this case demonstrates that further study is required to understand which patients are at risk for PNS development/flare during therapy for advanced stage cancers. CASE: Our patient presented at 16 months of age with acute onset of tremors, irritability and ataxia and was diagnosed with OMS; at that time, evaluation revealed intermediate risk stage 4 neuroblastoma which was treated with 4 cycles of chemotherapy with complete response. His OMS was managed initially with high dose dexamethasone, followed by monthly IVIG, ACTH and rituximab with improvement in neurologic symptoms. The patient developed NB relapse (stage 4) 18 months after completion of initial chemotherapy and received chemotherapy and anti-GD2 antibody immunotherapy. Through this second course of therapy he continued to wean his immunosuppressive anti-OMS therapy without flare of his neurologic symptoms. DISCUSSION We present an unusual case of a child with NB-associated OMS whose OMS symptoms did not flare despite the use of immunotherapy to treat relapsed stage 4 disease. CONCLUSION While immunosuppression is used to treat OMS and other paraneoplastic syndromes, the use of immunotherapy to treat the underlying malignancy may be tolerated. Further study is needed.

Abstract 1692: Genomic changes in relapsed neuroblastoma

Abstract Neuroblastoma (NB) is an aggressive pediatric malignancy originating from the sympathetic nervous system. Despite recent improvements in multimodal treatment, survival for children diagnosed with high-risk NB, remains below 50% at 5 years from treatment. Although an initial response to treatment is seen in a majority of patients, a significant portion will subsequently relapse with lesions resistant to standard therapy and for NB patients with relapsed or refractory metastatic disease, survival is below 10%. Genetic risk factors previously identified in primary neuroblastoma include MYCN-amplification, 11q-deletion, 1p-deletion and 17q gain and activating ALK mutations. However, treatment resistant tumors will likely have novel genetic aberrations differing from those present at time of diagnosis. To investigate the genetic mechanisms linked to recurrent disease, alterations between five triplet samples of primary tumor, relapsed/refractory tumor and constitutional DNA was investigated through whole genome sequencing. Sequencing was performed using Illumina instrumentation for an average coverage of at least 60X and 30X for tumor samples and normal tissue respectively. Read trimming, mapping to hg19 and variant calling were performed using the CLC Genomics Workbench software while copy number profiles were prepared through the CANVAS software. Systematic filtering was performed using the Ingenuity variant analysis tool. Variants with allele frequency above 3% in common population based cohorts were discarded as well as excluding all synonymous variants or variants in non-coding regions except those affecting canonical splice sites. A median of 21 and 59 non-silent somatic variants were detected in diagnostic and in relapsed samples respectively, while the median number of segmental alterations were 31 in diagnostic samples and 35 in relapse samples. Genes with recurrent alterations that emerged in relapse samples includes CCND1, CDK5RAP1, POLR2A, LSAMP, C3 and CARNS1. One patient showed only one single alteration common between primary and relapse; a t(11;17) resulting 11q-deletion and 17q-gain. The paired tumors did both have CCND1-gain although with different breakpoints and 6p-gain but of different parental alleles. In addition, in one patient an ARID1B mutation was detected in primary tumor but lost in the relapsed sample where an ARID1A mutation had emerged, which could indicate parallel evolution. Gene set analysis of variants in relapsed samples showed no significant functional enrichment differing from primary tumors where cell projection, Rap1-signaling and neuron part showed enrichment. In this study we investigated the genomes of primary and relapsed NB for five patients. We show that mutation load is higher in relapsed NB and includes alterations of previously linked to NB such as ATRX, TERT and CCND1 but lacked alterations in ALK, RAS or other genes of the RAS/MAPK pathway. Citation Format: Susanne Fransson, Angela Martinez Monleon, Anna Djos, Susanne Reinsbach, Rose-Marie Sjoberg, Per Sikora, Per Kogner, Tommy Martinsson. Genomic changes in relapsed neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1692.

Abstract 3123: Targeting SHP2 and RAS MAPK pathway in neuroblastoma

Abstract Introduction: Neuroblastoma (NB), the most common pediatric extra-cranial solid tumor, recurs in &amp;gt;50% of patients who present with metastases. Strategies to treat relapsed NB, which is often fatal, include targeting relapse-specific signaling pathways. Recently, sequencing studies have revealed that 78% of mutations detected in relapse samples are predicted to activate the RAS-MAPK pathway, suggesting there may be opportunities to pharmacologically target this pathway to treat recurrent NB. Experimental Design: Our lab previously reported that the PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and pharmacologic inhibition of SHP2 inhibited glioblastoma growth in vivo. To determine whether inhibiting SHP2 in NB would impact tumor growth we treated a panel of NB cells with diverse genetic backgrounds, including mutations in NRAS, with different SHP2 inhibitors (SHP099 and II-B08) alone and in combination with additional drugs that target RAS-MAPK signaling. Results: In comparison to NB cell lines harboring endogenous NRAS mutations (NRASmt), cells with NRAS wild-type (NRASwt) were more sensitive to SHP2 inhibitors. In addition, NRASwt cells engineered to overexpress NRASmt were more resistant to SHP099 than cells with endogenous or overexpressed NRASwt, as demonstrated by higher levels of proliferation, increased survival, and diminished apoptosis. Furthermore, SHP099 effectively inhibited SHP2 in both NRASwt and NRASmt cells, but only failed to inactivate the downstream RAS effector ERK1/2 in NRASmt cells, suggesting that SHP099 treatment alone is ineffective in cells harboring NRAS mutations. To determine whether SHP099 resistance in NRASmt cells could be overcome with combination strategies, sensitive and resistant NB cells were treated with SHP2 inhibitors alone or in combination with other RAS-MAPK pathway inhibitors (vemurafenib, trametinib and ulixertinib). Interestingly, in comparison to SHP099 or II-B08 alone, all three combinations significantly reduced survival and IC50 values in both NRASwt and NRASmt cells. Using the Bliss independence model we determined all three combinations were synergistic. Conclusions: Our studies demonstrate that NRAS mutations are associated with resistance to SHP2 inhibitors, and that in certain tumors, based on the genetic status of RAS-MAPK-SHP2 signaling components, combinations of drugs targeting this pathway could be effective strategies for relapsed NB. Citation Format: Ivette Valencia-Sama, Teresa Adderley, Lynn Kee, Gabriella Christopher, Yoshihito Kano, Michael Ohh, Meredith Irwin. Targeting SHP2 and RAS MAPK pathway in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3123.

Abstract 2874: De novo acquisition of RD3 loss with intensive multi-modal therapy harmonize neuroblastoma relapse

Abstract The majority of high-risk neuroblastoma that initially responds to therapy will ultimately relapse. Acquired genetic/molecular rearrangement contributes to tumor relapse and currently, no salvage treatment regimens are known to be curative. Our studies identified the loss of retinal-degeneration-protein-3 (RD3) in progressive disease (PD) and its association with poor clinical outcomes. Herein we investigated the acquisition of RD3-loss after current standard of care (SOC, intensive multimodal therapy) in neuroblastoma. Acquired transcriptional (RNA in situ hybridization, QPCR) and translational (IHC on a custom archived cell micro array, immunoblotting) loss of RD3 with SOC was investigated with bed-to-bench approach utilizing a panel of 15 stage-4 patient derived cell-lines with known therapy status, diagnosis [DX] vs. PD after SOC). Further, RD3-loss in progressive disease, its direct role in the pathogenesis disease progression (cell migration, invasion, tumorosphere formation and metastatic potential) was investigated using mouse model of progressive neuroblastoma and in experimental in vitro and ex vivo settings coupled with gene manipulation strategies. In addition, RD3 status in response to therapy were also affirmed by in silico analysis of independent studies on bed-to-bench and experimental models. Results demonstrated RD3-loss (transcriptional/translational) acquisition with SOC in human PD. Experimental in vitro, in vivo, ex vivo studies affirmed RD3 loss in PD and, further forced RD3-rearrangements in these models defined its functional role in PD pathogenesis. Data-mining experimental studies with salvage therapeutic agents affirmed the acquisition of RD3-loss in resistant cells and in residual tumors. For the first time, these results demonstrate the de novo acquisition of RD3 loss in PD after intensive multi-modal therapy. Defining the criticality of RD3-loss in PD and the defined role of RD3 loss in PD pathogenesis, benefit of targeted reinforcement could lead to improved salvage therapy for high-risk neuroblastoma. Citation Format: Dinesh Babu Somasundaram, Karthikeyan Subramanian, Sheeja Aravindan, Zhongxin Yu, Mohan Natarajan, Terence S. Herman, Natarajan Aravindan. De novo acquisition of RD3 loss with intensive multi-modal therapy harmonize neuroblastoma relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2874.

Abstract 2160: Microfragmented human fat tissue is a natural scaffold for drug delivery: potential application in cancer chemotherapy

Abstract Localization of chemotherapy at the tumor site can improve therapeutic efficacy and reduce systemic toxicity. In previous studies we have shown that mesenchymal stromal cells (MSCs) isolated from bone marrow or fat tissue, can be loaded with the anti-cancer drug Paclitaxel (PTX) and kill cancer cells when localized nearby. We here investigated the capacity of human micro-fragmented adipose tissue (MFAT), used as a natural container of MSCs, to delivery PTX with the idea to improve local drug concentration and to prolong the therapeutic activity. Surprisingly, we found that both fresh but also devitalized MFAT (DMFAT) (by freezing/thawing procedure) were very effective biomaterials able to deliver and release significant amount of PTX, killing several human cancer cell lines in vitro with an impressive long lasting activity. In an orthotopic mice model of Neuroblastoma (NB) transplant, we found that DMFAT loaded with PTX prevents or delay NB relapse when placed in the surgical area of tumor resection, without any collateral toxicity. We concluded that MFAT, but also DMFAT, may work as natural biomaterials that can be used to localize and release anti-cancer molecules at the tumor site. In addition, the procedure to easily prepare DMFAT did not alter its natural scaffold structure. This suggest that such biomaterial may have a prominent role in the near future in many oncological applications to delivery anti-cancer drugs, helping to fight cancer in human. Citation Format: Giulio Alessandri, Valentina Coccè, Fabio Pastorino, Rita Paroni, Michele Deicas, Francesco Restelli, Carlo Tremolada, Angiola Berenzi, Eugenio Parati, Anna Teresa Brini, Giampietro Bondiolotti, Augusto Pessina, Mirco Ponzoni. Microfragmented human fat tissue is a natural scaffold for drug delivery: potential application in cancer chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2160.

Microfragmented human fat tissue is a natural scaffold for drug delivery: Potential application in cancer chemotherapy

Localization of chemotherapy at the tumor site can improve therapeutic efficacy and reduce systemic toxicity. In previous studies we have shown that mesenchymal stromal cells (MSCs) isolated from bone marrow or adipose tissue can be loaded with the anti-cancer drug Paclitaxel (PTX) and kill cancer cells when localized nearby. We here investigated the capacity of human micro-fragmented adipose tissue (MFAT), used as a natural scaffold of MSCs, to deliver PTX with the idea to improve local drug concentration and to prolong the therapeutic activity. Surprisingly, we found that both fresh but also devitalized MFAT (DMFAT) (by freezing/thawing procedure) were able to deliver and release significant amount of PTX, killing several human cancer cell lines in vitro with a long lasting activity. In an orthotopic mice model of Neuroblastoma (NB) transplant, DMFAT loaded with PTX prevents or delays NB relapse when placed in the surgical area of tumor resection, without any collateral toxicity. We concluded that MFAT, but also DMFAT, may represent very innovative natural biomaterials able to localize and release anti-cancer molecules at the tumor site, helping to fight cancer in human.

RAS-MAPK Pathway-Driven Tumor Progression Is Associated with Loss of CIC and Other Genomic Aberrations in Neuroblastoma.

Mutations affecting the RAS-MAPK pathway frequently occur in relapsed neuroblastoma tumors, which suggests that activation of this pathway is associated with a more aggressive phenotype. To explore this hypothesis, we generated several model systems to define a neuroblastoma RAS-MAPK pathway signature. Activation of this pathway in primary tumors indeed correlated with poor survival and was associated with known activating mutations in ALK and other RAS-MAPK pathway genes. Integrative analysis showed that mutations in PHOX2B, CIC, and DMD were also associated with an activated RAS-MAPK pathway. Mutation of PHOX2B and deletion of CIC in neuroblastoma cell lines induced activation of the RAS-MAPK pathway. This activation was independent of phosphorylated ERK in CIC knockout systems. Furthermore, deletion of CIC caused a significant increase in tumor growth in vivo These results show that the RAS-MAPK pathway is involved in tumor progression and establish CIC as a powerful tumor suppressor that functions downstream of this pathway in neuroblastoma.Significance: This work identifies CIC as a powerful tumor suppressor affecting the RAS-MAPK pathway in neuroblastoma and reinforces the importance of mutation-driven activation of this pathway in cancer. Cancer Res; 78(21); 6297-307. ©2018 AACR.

Abstract B42: The properties of ex vivo expanded γδ T cells provide for the rational use of combination therapies

Abstract Cell-based immunotherapies have relied mainly on the use of αβ T cells or dendritic cells, and technologies to expand and engineer these cells have resulted in potent antitumor products. Although the use of adaptive immunocompetent cells has advanced more rapidly than innate immune cells, such as natural killer or γδ T cells, these alternative cellular products can provide equivalent antitumor activity. However, the methods to expand clinically relevant innate cell products are not as advanced as those for other immunocompetent cells, such as αβ T cells. We have developed a safe and efficient method for expanding, storing, and genetically engineering γδ T cells, and have focused on the use of these ex vivo expanded cells to treat pediatric neuroblastoma. γδ T cells are an attractive candidate for cell-based products because their antitumor activity is directly cytolytic to tumor cells, in part, due to their receptors NKG2D and CD16. NKG2D facilitates the innate recognition of stress-induced ligands such as MICA/B and UL-16 binding proteins (ULBP), and CD16 enables mechanisms of antibody-dependent cellular cytotoxicity (ADCC). We have developed a serum-free expansion protocol using apheresis products from healthy donors and from neuroblastoma patients. γδ T cells from all starting products can be expanded to greater than 70% of the culture within 12-14 days. These γδ T cells are cytotoxic against K562 cells, which has become our standard for which to compare the various expansion products, and 5 human derived neuroblastoma cell lines. We have tested several freezing media and developed a method using serum-free conditions between days 12-14 of expansion, resulting in equivalent cytotoxicity by cells pre- and post-freezing while maintaining greater than 70% viability post-thaw. We also show that our expansion protocol results in γδ T cells that are greater than 90% positive for surface expression of the receptors NKG2D and CD16. Therefore, we are using these cells in combination treatments that include standard chemotherapy regimens for relapsed neuroblastoma patients, i.e., temozolomide (TMZ), and the monoclonal antibody dinutuximab. We have shown that stress antigens such as MICA/B and ULBP2/5/6 are upregulated in vitro on IMR5 cells for 6 hours after a 1-hour exposure to TMZ. Further, combining patient-expanded γδ T cells that express CD16 with a GD2-specific antibody, dinutuximab, induces 30% increased neuroblastoma cell death compared to γδ T cells alone. To test the in vivo effectiveness of 12-day expanded, frozen, and thawed cells, we injected NSG male and female mice with 5 x 106 IMR5 cells subcutaneously. After a palpable 125-mm3 tumor was established, we began treatment with various combinations of γδ T cells, dinutuximab, and TMZ. These ongoing studies show that γδ T cells alone do not provide an antitumor benefit, but tumor regression is achieved when incorporating γδ T cells into dinutuximab and TMZ treatment regimens. This advantage is only observed at lower TMZ doses, whereas at higher TMZ doses, &amp;gt;60 mg/kg, chemotherapy dominates the antitumor effect. Overall, these studies show that a safer, serum-free expanded γδ T cell product can be produced, which can be stored frozen without affecting the cytotoxic properties. The expression of NKG2D and CD16 on the expanded cells allows for the development of combination therapies using cytotoxic chemotherapy agents and antibody-based treatments. The combination of γδ T cells, TMZ, and dinutuximab can provide a rational advancement for treating pediatric neuroblastoma patients, and we hypothesize that this combination therapy is preferred over high-dose chemotherapy regimens because it can reduce treatment-related toxicities and provide a multifaceted immunotherapeutic approach to neuroblastoma treatment. Citation Format: Jaquelyn T. Zoine, Kathryn S. Sutton, Kristopher A. Knight, Kelly C. Goldsmith, Christopher B. Doering, H. Trent Spencer. The properties of ex vivo expanded γδ T cells provide for the rational use of combination therapies [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B42.

Abstract 1902: Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma

Abstract The purpose of this study was to determine the effect of the natural product Harmine in human neuroblastoma (NB). NB is an early childhood malignancy arising from the developing peripheral nervous system. In up to 25% of cases MYCN gene amplification is correlated to high tumor stage and poor patient prognosis. High stage NB relapses frequently in spite of multimodal therapy and is then virtually untreatable. There is a clear need for specific, novel therapeutics. Harmine is a tricyclic β-carboline alkaloid from the harmal plant with cytostatic and cytotoxic effects on tumor cells. It is capable of blocking the activity of mitogen activate protein kinase (MAPK) and the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK) family proteins. These kinases inhibit apoptosis and encourage proliferation. Four human NB cell lines were used to study the effects of Harmine treatment: SK-N-BE(2)-C and KELLY (MYCN amplified) as well as SK-N-AS and SK-N-FI (MYCN single copy). Molecular interaction models of Harmine bound to DYRK family kinases were generated by computational docking using x-ray structures. The anti-cancer properties of Harmine were analyzed by real-time cell viability assays, in a dose-dependent manner, over a 72 hour period. The IC50 values were 169.9, 170.8, and 791.7 μM for SK-N-BE(2)-C, KELLY, and SK-N-FI, respectively, after 72 hours. Apoptosis assays for caspase activation, PARP cleavage, and annexin V induction, were performed using Western blot and flow cytometry. NB cell line exposure to 100 μM Harmine resulted in caspase-3/7 and caspase 9 activation, PARP cleavage, and annexin V-positive stained cells as early as 24 hours after treatment, indicating apoptosis induction. These results led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that Harmine induces NB cell death through a cellular mechanism that involves DYRK family kinases and triggers caspase-mediated apoptosis. Citation Format: Katie L. Uhl, Chad R. Schultz, Dirk Geerts, Andre S. Bachmann. Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1902.

Too many targets, not enough patients: rethinking neuroblastoma clinical trials.

Neuroblastoma is a rare solid tumour of infancy and early childhood with a disproportionate contribution to paediatric cancer mortality and morbidity. Combination chemotherapy, radiation therapy and immunotherapy remains the standard approach to treat high-risk disease, with few recurrent, actionable genetic aberrations identified at diagnosis. However, recent studies indicate that actionable aberrations are far more common in relapsed neuroblastoma, possibly as a result of clonal expansion. In addition, although the major validated disease driver, MYCN, is not currently directly targetable, multiple promising approaches to target MYCN indirectly are in development. We propose that clinical trial design needs to be rethought in order to meet the challenge of providing rigorous, evidence-based assessment of these new approaches within a fairly small patient population and that experimental therapies need to be assessed at diagnosis in very-high-risk patients rather than in relapsed and refractory patients.

Long-term disease stabilization in a patient with relapsed neuroblastoma after allogeneic hematopoietic stem cell transplantation. Clinical case and literature review

Neuroblastoma (NB) is a malignant pediatric tumor originating from undifferentiated neural crest cells. About 20 % of high-risk group patients are high-risk group patients are refractory to first line therapy and about 50 % of initial responders later develop a relapse. There is no common tactics for salvage therapy. In spite of a c relatively high response rate observed for most second- and third-line therapy regimens used, the long-term event-free survival is still as low as 5 %, which implies a need for consolidation, e.g. by allogeneic hematopoietic stem cell transplantation (allo-HSCT) from haploidentical donor. We present a case report of a patient with systemic NB relapse achieving a long-term disease stabilization after a haplo-HSCT and post-transplant therapy.

Vincristine resistance in relapsed neuroblastoma can be efficiently overcome by Smac mimetic LCL161 treatment

PurposeIn spite of good initial therapy response neuroblastomas often spread to distant organs or relapse after periods of remission. Dysregulation of apoptosis, a hallmark of cancer, is often effected by elevated levels of antiapoptotic signals leading to resistance against chemotherapeutic drugs. Inhibitors of apoptosis proteins (IAPs) are crucial cellular apoptosis regulators. Targeting IAPs with Smac mimetics has been demonstrated as a promising strategy for treatment of neuroblastoma and other tumors. MethodsIn paired neuroblastoma cell lines, obtained from the same patient at time of diagnosis (CHLA-15) and postchemotherapy during progressive disease (CHLA-20), expression of crucial IAPs was determined. Furthermore, effects of vincristine on viability, cytotoxicity, apoptosis induction and caspase-3/7 activation were determined. ResultsCellular IAP-1 (cIAP-1) and X-linked IAP (XIAP) expression was increased in cell line CHLA-20. Moreover, biological effects of vincristine were significantly lower in these cells. Treatment of cells with Smac mimetic LCL161 increased the effects of vincristine in CHLA-15 cells and more importantly was able to overcome vincristine resistance in CHLA-20 cells. ConclusionsThese findings demonstrate the potential of Smac mimetics for the development of novel therapeutic approaches for the treatment of relapsed/resistant neuroblastoma.

Characterisation of the p53 pathway in cell lines established from TH-MYCN transgenic mouse tumours.

Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation(IR) were determined in 6adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or exvivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting invivo toxicity studies of MDM2 inhibitors.

Haploidentical Stem Cell Transplantation for Refractory/Relapsed Neuroblastoma

Pediatric patients with refractory or relapsed metastatic neuroblastoma (NBL) have a poor prognosis despite autologous stem cell transplantation (SCT). Allogeneic SCT from a haploidentical donor has a remarkable alloreactive effect in patients with leukemia; thus, we evaluated this approach in children with very high-risk NBL. We analyzed data from 2 prospective phase I/II trials. A total of 26 patients with refractory (n = 5), metastatic relapsed (n = 20), or locally relapsed MYCN-positive (n = 1) NBL received a median of 17 × 106/kg T/B cell-depleted CD34+ stem cells with 68 × 103/kg residual T cells and 107 × 106/kg natural killer cells. The conditioning regimen comprised melphalan, fludarabine, thiotepa, OKT3, and a short course of mycophenolate mofetil post-transplantation. Engraftment occurred in 96% of the patients. Event-free survival and overall survival at 5 years were 19% and 23%, respectively. No transplantation-related mortality was observed, and the single death was due to progression/subsequent relapse. The median duration of follow-up was 8.1 years. Patients in complete remission before SCT had a significantly better prognosis than those with residual tumor load (P < .01). All patients with progressive disease before SCT relapsed within 1 year. Grade II and grade III acute graft-versus-host disease (GVHD) occurred in 31% and 12% of the patients, respectively. Chronic limited and extensive GVHD occurred in 28% and 10%, respectively. Our data indicate that haploidentical SCT is a feasible treatment option that can induce long-term remission in some patients with NBL with tolerable side effects, and may enable the development of further post-transplantation therapeutic strategies based on the donor-derived immune system.

TrkAIII signals endoplasmic reticulum stress to the mitochondria in neuroblastoma cells, resulting in glycolytic metabolic adaptation.

Alternative TrkAIII splicing characterises advanced stage metastatic disease and post-therapeutic relapse in neuroblastoma (NB), and in NB models TrkAIII exhibits oncogenic activity. In this study, we report a novel role for TrkAIII in signaling ER stress to the mitochondria in SH-SY5Y NB cells that results in glycolytic metabolic adaptation. The ER stress-inducing agents DTT, A23187 and thapsigargin activated the ER stress-response in control pcDNA SH-SY5Y and TrkAIII expressing SH-SY5Y cells and in TrkAIII SH-SY5Y cells increased TrkAIII targeting to mitochondria and internalisation into inner-mitochondrial membranes. Within inner-mitochondrial membranes, TrkAIII was subjected to Omi/HtrA2-dependent cleavage to tyrosine phosphorylated 45–48kDa carboxyl terminal active fragments, localised predominantly in tyrosine kinase-domain mitochondrial matrix orientation. This stress-induced activation of mitochondrial TrkAIII was associated with increased ROS production, prevented by the ROS scavenger Resveratrol and underpinned by changes in Ca2+ movement, implicating ROS/Ca2+ interplay in overcoming the mitochondrial TrkAIII activation threshold. Stress-induced, cleavage-activation of mitochondrial TrkAIII resulted in mitochondrial PDHK1 tyrosine phosphorylation, leading to glycolytic metabolic adaptation. This novel mitochondrial role for TrkAIII provides a potential self-perpetuating, drug reversible way through which tumour microenvironmental stress may maintain the metastasis promoting “Warburg effect” in TrkAIII expressing NBs.

Complications of Emerging Oncology Therapies Requiring Treatment in the Pediatric Intensive Care Unit

The development of immunotherapies has resulted in significant improvements in survival for children with cancer and congenital blood disorders. The purpose of this review is to guide the recognition and treatment of significant side of effects three increasingly used immunotherapies—T cell-mediated therapies, immune checkpoint inhibitors, and anti-GD2 antibodies. Cytokine release syndrome (CRS) is a cytokine-induced systemic inflammatory state secondary to chimeric antigen receptor T cells (CAR–T cells), a therapy used for relapsed and refractory leukemia. CRS can range from fever and mild flu-like symptoms to cardiopulmonary collapse and neurologic sequelae including encephalitis, seizures, and cerebral edema. Immune check point inhibitors work to redirect the T cell response towards cancer cells and have shown a survival advantage for patients with advanced melanoma. These agents also activate an immune response which can mimic autoimmune disease and cause organ failure and neurologic complications like Guillain–Barre and posterior reversible encephalopathy. Children with high-risk and chemotherapy-resistant neuroblastoma have seen improved survival with the use of anti-GD2, an immunotherapy directed at the highly abundant and conserved GD2 antigen expressed on neuroblastoma cells. Severe pain is the consistently reported side effect of anti-GD2, occurring in an estimated 50% of patients and often requiring narcotic and sedative agents for the duration of therapy. Immunotherapy has become critical to the treatment of previously irremediable diseases like relapsed leukemia and refractory neuroblastoma. The survival advantage offered by these therapies requires an understanding of diagnosis and treatment of their life-threatening toxicities.

Elevated TIMP-1 expression is associated with a prometastatic phenotype, disease relapse, and poor survival in neuroblastoma.

Approximately two-thirds of patients with neuroblastoma are found to have metastatic disease at time of diagnosis with frequent skeletal, lymph node, central nervous system, and liver involvement. Using a serial in vivo splenic injection model, we have isolated an aggressive subclone (BE(2)-C/LM2) from MYCN-amplified neuroblastomas that demonstrate an enhanced propensity to develop metastatic liver lesions. BE(2)-C/LM2 subclone cells demonstrate increased adherent, soft agar colony and tumorsphere growth in vitro. Components of the tumor microenvironment regulate cancer progression, via networks of cytokines and growth factors. Cytokine array analysis identified increased TIMP-1 in the plasma of mice injected with BE(2)-C/LM2 subclone cells, leading us to hypothesize that TIMP-1 may play a role in our observed prometastatic phenotype. Immunoblotting and ELISA demonstrated enhanced endogenous TIMP-1 expression in our isolated neuroblastoma subclone. Silencing endogenous TIMP-1 successfully blocked in vitro proliferation, soft agar colony formation and tumorsphere formation by BE(2)-C/LM2 cells. Stable RNA interference of endogenous TIMP-1 failed to reverse the prometastatic phenotype of our BE(2)-C/LM2 subclone in our liver metastasis model, suggesting that endogenous TIMP-1 levels may not be an essential component of this in vivo behavior. Notably, tissue microarray analysis and Kaplan-Meier by gene expression demonstrates that elevated TIMP-1 expression is correlated with increased disease relapse and mortality in patients with neuroblastoma. Taken together, our study identifies TIMP-1 as a novel soluble factor that is associated with a prometastatic phenotype in our in vivo model and adverse outcomes in patients with neuroblastoma.

Abstract 3000: Defining the subclonal landscape of high-risk neuroblastoma

Abstract We recently showed that relapsed neuroblastoma (NB) tumors harbor an increased somatic mutational burden, enriched for ALK or RAS-MAPK activating lesions, nominating targeted therapeutic strategies for a condition that is largely incurable. We also showed that these mutations can be present in subclonal tumor cell populations at diagnosis, suggesting that they may contribute to acquired chemoradiotherapy resistance. We hypothesize that subclonal mutations in NB oncogenes are common at diagnosis, are biomarkers for treatment failure, and can be targeted in conjunction with standard chemotherapy to improve patient survival. Here, we focus on the first aspect of this broad hypothesis by presenting the subclonal landscape of diagnostic, high-risk neuroblastomas. A meta-analysis of our previously published primary/relapse (N=23), whole exome (N=232) and whole genome (N=159) diagnostic data, and recent sequencing study of 78 diagnostic and 67 relapse cases were used to inform design of a custom amplicon panel for ultra-deep sequencing. The 28-gene panel covers 57 mutations in frequently mutated genes. Here, 250 high-risk primary tumors were sequenced to an average depth of 50,000X. More subclonal mutations were called (mean=36) compared to clonal alterations (mean=3), demonstrating the complex subclonal architecture in these tumors. We validated five clonal ALK mutations we originally discovered and identified 15 additional pathogenic ALK mutations with mutant allele frequencies (MAFs) ranging from 0.03-23%. Known ALK activating mutations were found at codons 1170, 1174, 1196, 1245, 1275 in 15 of 38 diagnostic primary tumors. This 39.5% frequency is much higher than the 14% frequency defined by us using Sanger or other sequencing methods. Improvement of subclonal mutation detection of actionable, relapse-specific driver mutations demonstrates the clinical utility of utilizing this sequencing methodology at diagnosis to enable timely improvement of outcomes for children with high-risk refractory NB. Citation Format: Jo Lynne Harenza, Maura A. Diamond, Derek A. Oldridge, Olivia Padovan-Merher, Pichai Raman, Yael P. Mosse, John M. Maris. Defining the subclonal landscape of high-risk neuroblastoma [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 3000. doi:10.1158/1538-7445.AM2017-3000

Abstract 5823: Modulation of the Hippo pathway protein YAP as a mechanism of resistance to MEK inhibition in MAPK hyperactivated neuroblastoma

Abstract Background: Current therapies for high-risk neuroblastoma are highly toxic and ineffective, as a majority of patients will face therapy-resistant relapses. Our recent whole genome sequencing analysis revealed that a majority of relapsed tumors had mutations causing RAS-MAPK pathway activation. Recent studies have implicated the Hippo pathway transcriptional coactivator protein YAP in relapsed neuroblastoma as well as resistance to MEK inhibition in other cancer models. Based on these findings, we hypothesized that YAP activity may lead to acquired resistance to MEK inhibition, and that dual inhibition of these two pathways may have synergistic therapeutic efficacy. Methods: We selected the cell line NLF from a panel of NB cell lines due to its high YAP expression and NF1 loss. NLF cells were treated with MEK inhibitor trametinib (20 nM) over a 72-hour period and whole cell lysates and nuclear lysates were collected for immunoblot. Effects of YAP knockdown via siRNA transfection with and without trametinib co-treatment on survival were assessed by CellTiter-Glo and RT-CES. Cell signaling analyses of tumor lysates from mouse SKNAS (NRAS Q61K) xenografts treated with vehicle, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg for YAP, ERK, and apoptosis pathways were performed via immunoblot. Results: Immunoblots of 72-hour time course treatments of trametinib in NLF showed decreased pYAP, the cytosolic inactive form of the YAP protein, indicating increased YAP capable of translocating to the nucleus, which was confirmed by analyses of nuclear extracts. siRNA-mediated knockdown of YAP alone in NLF cells had a modest effect on survival, while combinatorial inhibition of MEK and YAP signaling caused a significant reduction in cellular proliferation and survival. Immunoblotting of trametinib-treated SKNAS xenografts indicated decreased pYAP and pERK in response to increasing trametinib dose, while total YAP levels remained constant. Conclusion: Our results demonstrate that prolonged trametinib exposure causes a marked nuclear enrichment of unphosphorylated YAP and co-inhibition of MEK and YAP in neuroblastoma models effectively reduces proliferation and survival. We are currently investigating the effects of MEK inhibition on the expression of YAP target genes and on cellular proliferation upon modulation of YAP protein expression. Note: This abstract was not presented at the meeting. Citation Format: Grace E. Coggins, Lori S. Hart, Jimmy Elias, John M. Maris. Modulation of the Hippo pathway protein YAP as a mechanism of resistance to MEK inhibition in MAPK hyperactivated neuroblastoma [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 5823. doi:10.1158/1538-7445.AM2017-5823