Childhood Cancer Deaths Research Articles

Abstract 146: The long noncoding RNA MALAT1 promotes hypoxia-driven angiogenesis by upregulating pro-angiogenic gene expression in neuroblastoma cells

Abstract Neuroblastoma is the most common solid tumour during early childhood, and accounts for 15% of all childhood cancer death. One of the key features of neuroblastoma is aggressive progression due to tumour-driven angiogenesis. However, the mechanism through which neuroblastoma cells drive angiogenesis is unclear. Here we showed that the long noncoding RNA MALAT1 was over-expressed in human neuroblastoma cells under hypoxic condition, the condition which triggers pro-angiogenic switch and angiogenesis. In vitro angiogenesis assays demonstrated that conditioned medium from neuroblastoma cells transfected with MALAT1 siRNAs, compared with conditioned medium from neuroblastoma cells transfected with control siRNAs, induced considerably less endothelial cell migration, invasion and vascular sprouting under hypoxic condition. Affymetrix microarray differential gene expression study showed that one of the genes most significantly down-regulated by MALAT1 siRNAs in human neuroblastoma cells under hypoxic condition, was fibroblast growth factor (FGF). RT-PCR and immunoblot analyses confirmed that MALAT1 siRNAs reduced FGF mRNA and protein expression, and in vitro angiogenesis assays demonstrated that forced over-expression of FGF in neuroblastoma cells blocked MALAT1 siRNA-mediated reduction in endothelial cell migration, invasion and vascular sprouting. Taken together, our data suggest that over-expression of MALAT1 in human neuroblastoma cells plays an important role hypoxia-driven angiogenesis by up-regulating FGF mRNA expression. Citation Format: Andrew E. Tee, Peiyan Liu, Jesper Maag, Renhua Song, Jinyan Li, Belamy B. Cheung, Michelle Haber, Murray D. Norris, Glenn M. Marshall, Marcel Dinger, Tao Liu. The long noncoding RNA MALAT1 promotes hypoxia-driven angiogenesis by upregulating pro-angiogenic gene expression in neuroblastoma cells. [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 146. doi:10.1158/1538-7445.AM2015-146

Abstract 1627: Phenotypic and functional evaluation of peripheral blood cell subsets in children at the completion of induction therapy for acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, accounting for more than one quarter of the new cases of pediatric cancer each year. Despite significant improvements in cure rates, recurrent ALL causes 10% of childhood cancer deaths and the need for new treatment strategies has not diminished. Although ALL has long been viewed as a poor target for immune therapy, it has recently been reported that the peripheral blood absolute lymphocyte count (ALC) at completion of induction chemotherapy is prognostic for outcome. This finding suggests that the immune environment during therapy may play a role in protecting against disease progression. It is currently unknown, however, whether ALC is simply a marker of bone marrow recovery or whether particular immune cell subsets at this early time-point confer a beneficial effect. To address this question, we have initiated a flow-cytometric study of 20 peripheral blood leukocyte populations, obtained at Day 29 from children undergoing therapy for precursor B cell ALL. The goal of this study is to correlate the size and responsiveness of the cell subsets with ALC in order to identify potential immune-mediators of the high ALC-associated favorable outcome. To date we have evaluated 8 patients for T cells (5 subsets), B cells (6 subsets), NK cells (3 subsets), NKT cells (2 subsets), dendritic cells (2 subsets), monocytes, and granulocytes. In addition to this extensive phenotypic characterization, all Day 29 blood samples have been stimulated with a panel of Toll-like receptor (TLR) ligands to evaluate functional responsiveness. Although we have not yet identified a single lymphocyte population that significantly correlates with ALC, clear trends, most notably with myeloid dendritic cells, have emerged that suggest that ALC may be indicative of specific immune activity. TLR-induced cytokine production is currently being quantified to determine whether higher ALC correlates with a specific pattern of response. While a large-scale prospective study to validate prognostic significance of ALC in children with high-risk ALL is underway, to our knowledge our ongoing study is the first to investigate the specific leukocyte composition of Day 29 peripheral blood for significant links with ALC. The identification of the mechanism(s) underlying ALC-associated outcomes may reveal novel strategies for enhanced immune-mediated control of ALL after chemotherapy. Citation Format: Nina Rolf, Amina Kariminia, Kinga K. Smolen, Caron Strahlendorf, Gregor S. Reid. Phenotypic and functional evaluation of peripheral blood cell subsets in children at the completion of induction therapy for acute lymphoblastic leukemia. [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 1627. doi:10.1158/1538-7445.AM2015-1627

Childhood cancer mortality in Japan, 1980-2013.

BackgroundThis study aimed to provide an updated analysis of childhood cancer mortality rates and long-term trends to 2013 to describe the current level of deaths and identify changes in recent decades.MethodsData on number of deaths from cancer in children aged under 15 years were derived from Vital Statistics in Japan and the World Health Organization (WHO) mortality database for comparison countries. Trends in mortality were examined by fitting a joinpoint regression model.ResultsFor all cancers combined, the mortality rate during 2010–2013 was 19.9 per 1,000,000 population for boys and 17.5 for girls in Japan. Mortality from all cancers combined decreased significantly from 1980 to 2003 for boys and from 1980 to 2001 for girls. Afterwards, the rates remained stable for both sexes. Mortality from leukemia declined over the entire study period by 4.6 % per year (p <0.05) in boys and 4.3 % per year (p <0.05) in girls. For central nervous system (CNS) tumors, a slight increase in mortality was observed for both sexes, with a statistically significant annual percent change (APC) of 0.5 % (p <0.05) for boys and 0.6 % (p <0.05) for girls.ConclusionsWe provided updated information on recent trends of childhood cancer death. The establishment of a nationwide, childhood cancer registry is required in Japan. Moreover, trends in cancer mortality should be monitored continuously.

IM-12 * RNA NANOPARTICLE VACCINES RE-PROGRAM HOST IMMUNITY IN FAVOR OF ENHANCED EFFECTOR RESPONSES AGAINST INTRACRANIAL MALIGNANCIES

BACKGROUND: Pediatric brain tumors are the number one cause of solid cancer death in children, and thus necessitate the development of novel targeted therapeutics. To enhance the feasibility of cancer immunotherapy, off-the-shelf vaccination strategies have been proposed, but have proven to be only weakly immunogenic. Consequently we have designed a novel, translatable nanoparticle (NP) vaccine that can be embedded with immunomodulatory modifications to generate robust anti-tumor responses against murine models for glioblastoma (GBM) and medulloblastoma (MB). OBJECTIVES: We sought to assess if RNA-NP vaccines would induce superior immune responses against GBM and MB by stimulating intracellular pathogen recognition receptors (PRRs) while simultaneously downregulating regulatory pathways. RESULTS: We screened commercially available and clinically translatable NP formulations and determined that the cationic liposome DOTAP was the most superior NP for delivery of RNA to antigen presenting cells (APCs) in vitro and in vivo. Afterwards, we verified that these particles preserve RNA stability over time, and induce in vivo gene expression. These RNA-NPs elicited potent T cell immunity, superior to peptide vaccines formulated in Complete Freund's Adjuvant (CFA) and induced anti-tumor efficacy in adoptive cellular therapy platforms in murine GBM and MB models. We showed dramatic upregulation of activation markers including CD80 and CD86 and demonstrated abrogation of regulatory markers such as programmed death-ligand1 (PD-L1) when RNA-NPs were administered in combination with anti-PD-L1 monoclonal antibodies. Finally, we demonstrated enhanced immune responses through incorporation of immunomodulatory RNAs encoding for pathogen associated molecular patterns (PAMPs) derived from hepatitis C. CONCLUSION: Since clinically translatable RNA-NP vaccines can deliver combinatorial therapies using a single delivery platform, they represent a novel platform for inducing potent nontoxic immunity against intracranial tumors that can be harnessed to provide a more effective and specific therapy critical in improving clinical outcomes for children affected by these malignancies.

The outcomes and treatment burden of childhood acute myeloid leukaemia in Australia, 1997-2008: A report from the Australian Paediatric Cancer Registry.

Childhood acute myeloid leukaemia (AML) requires intensive therapy and is associated with survival rates that are substantially inferior to many other childhood malignancies. We undertook a retrospective analysis of Australian Paediatric Cancer Registry data from 1997 to 2008 together with a single-centre audit during the same period assessing burden on service delivery at a tertiary children's hospital (Royal Children's Hospital, Brisbane). Although survival improved from 54.3% (1997-2002) to 69.2% (2003-2008), childhood AML caused a disproportionate number of childhood cancer deaths, accounting for 5.5% of all childhood cancer diagnoses yet 7.9% of all childhood cancer mortality. Furthermore, treatment was associated with significant toxicity requiring intensive use of local health resources. Novel therapeutic strategies aimed at improving survival and reducing toxicity are urgently required.

Mesenchymal stromal cell secretome up-regulates 47 kDa CXCR4 expression, and induce invasiveness in neuroblastoma cell lines.

Neuroblastoma accounts for 15% of childhood cancer deaths and presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Previous studies have shown that the Mesenchymal Stromal Cell (MSC) secretome, triggers metastases in several cancer types such as breast and prostate cancer, but the specific role of the MSC factors in neuroblastoma metastasis is unclear. To better understand the effect of MSC secretome on chemokine receptors in neuroblastoma, and its role in metastasis, we studied a panel of 20 neuroblastoma cell lines, and compared their invasive potential towards MSC-conditioned-RPMI (mRPMI) and their cytokine receptor expression profiles. Western blot analysis revealed the expression of multiple CXCR4 isoforms in neuroblastoma cells. Among the five major isoforms, the expression of the 47 kDa isoform showed significant correlation with high invasiveness. Pretreatment with mRPMI up-regulated the expression of the 47 kDa CXCR4 isoform and also increased MMP-9 secretion, expression of integrin α3 and integrin β1, and the invasive potential of the cell; while blocking CXCR4 either with AMD 3100, a CXCR4 antagonist, or with an anti-47 kDa CXCR4 neutralizing antibody decreased the secretion of MMP-9, the expression of integrin α3 and integrin β1, and the invasive potential of the cell. Pretreatment with mRPMI also protected the 47 kDa CXCR4 isoform from ubiquitination and subsequent degradation. Our data suggest a modulatory role of the MSC secretome on the expression of the 47 kDa CXCR4 isoform and invasion potential of the neuroblastoma cells to the bone marrow.

PD-L1 expression in metastatic neuroblastoma as an additional mechanism for limiting immune surveillance

Neuroblastomas (NB) are neuroectodermal tumors that account for 15% of all childhood cancer deaths. Different prognostic factors including age, stage and amplification of MYCN ( MYCN ampl ), are critical for defining NB risk and for guiding therapeutic choices 1 . In this context, although different strategies have been approached, the prognosis of high-risk neuroblastoma (NB) remains poor due the high frequency of relapse which not only occurs at the primary tumor site but frequently arises in the bone marrow. Thus, in vivo, neuroblastoma exploits different mechanisms to escape the immune surveillance 2 . These might include the so called “adaptive immune resistance” characterized by the de-novo expression of Programmed Death Ligands (PD-Ls) 3 induced by IFN-γ whose storm occurs after different immunotherapeutic approaches. In the present study we analyzed the constitutive and the inducible surface expression of PD-Ls in NB cells. We showed that PD-L1 is constitutively express by virtually all HLA class I pos NB cell lines, whereas PD-L2 is rarely detected. Moreover PD-L1 could be acquired/upregulated in both NB cell lines and NB engrafted nude/nude mice. Importantly, after IFN-γ stimulation, PD-L1 can be expressed by metastatic neuroblasts isolated from bone marrow aspirates of high-risk NB patients, characterized by different MYCN amplification status. Interesting - ly, in one case, metastatic neuroblasts were poorly responsive to IFN-γ stimulation, suggesting that responsiveness to IFN-γ might represent a further element of heterogeneity in metastatic neuroblasts. Finally, we documented the presence of PD-1-positive lymphocytes in NB-infiltrated bone marrow of patients. These cells are mainly represented by aβ T cells but also include small populations of gδ T cells and NK cells. Moreover, PD-1 pos T cells expressed higher levels of activation markers as com- pared to the negative counterpart. Overall our data show that a PD-L1-mediated immune resistance mechanism might occur in metastatic neuroblasts and provide a biological rationale for blocking the PD-1/PD-Ls axis in future combined immunotherapeutic approaches.

DNA Methylation Profiling of Pediatric AML Reveals That Hypomethylation of MN1 Is Characteristic of Inv(16) AML and a Driver of MN1 Overexpression

Primary refractory and relapsed pediatric acute myeloid leukemia (AML) still lead to a significant number of childhood cancer deaths, despite the current chemotherapeutic regimens. AML leukemogenesis is driven by collaborative genetic abnormalities that induce hematopoietic maturation arrest and cell proliferation. Particular AML-associated maturation inhibiting aberrations are known to target chromatin regulators, thus directly influencing the transcriptional program of leukemic cells. Therapies targeting epigenetic processes, e.g. with hypomethylation-inducing agents, are therefore becoming an attractive therapeutic strategy in adult AML. AML biology in children is not equivalent to that of adults, thus methylation patterns seen in adult AML cannot be extrapolated to pediatric AML. Therefore there is a need to unravel the mechanism behind changes in epigenetic processes as the result of AML-causing genetic abnormalities in order to develop new drugs for pediatric AML.We hypothesized that pediatric AML samples have distinct DNA-methylation patterns which may provide a rationale for treatment with demethylating agents in specific pediatric AML subtypes. Furthermore, these differences in methylation could be characteristic for AML subgroups and that particular methylation patterns drive the expression of specific genes which may play a key role in the tumorigenesis of these AML leukemias.We performed genome-wide CpG-island methylation profiling on a representative and molecularly characterized cohort of pediatric patients with de novo AML. Empirical Bayes Wilcoxon rank-sum test showed that AML patients carrying inv(16)(p13;q22) (n=9) have distinct DNA methylation patterns when compared to non-inv(16) AML patients (n=143) (consisting mainly of MLL-rearranged, t(8;21), t(15;17), t(8;16) AML and AML cases with a normal karyotype). The MN1 gene ranked as most significantly differentially methylated in inv(16) AML compared to non-inv(16) AML, with inv(16) AML cases having significantly (p=2x10-6) lower methylation levels compared to non-inv(16) AML cases. Hypomethylation of specific regions of the MN1-associated CpG-island was confirmed by methylation specific PCR and bisulfite sequencing. Subsequent gene expression (GEP) data on 294 pediatric AML patients showed that MN1 was 8 fold higher expressed in patients carrying inv(16) compared to all other patients (9.9, n=35 vs 6.9, n=259, p<0.001). Furthermore, integrating GEP and methylation array data showed that MN1 expression negatively correlated (ρs= 0.82, p=0.011) with methylation levels, which is in agreement with the biological assumption of methylation and gene expression.Since genes known to regulate DNA methylation have frequently been shown to be mutated in adult AML we determined whether a decreased expression of DNA methyltransferases, DNMT1, DNMT2, DNMT3A, DNMT3B, could be the cause of a hypometylated MN1 locus in inv(16) AML. Our findings show that only DNMT3B expression was significantly (p=8x10-15) lower in inv(16) cases compared to non-inv(16) cases. To test whether hypomethylation of the MN1 CpG-island and the overexpression of MN1 is the result of decreased DNMT1 expression, HL60 cells which express negligible levels of MN1 were treated with the DNMT1 inhibitor Decitabine. This showed that treatment of HL60 cells with Decitabine led to increase of MN1 transcript levels, however, not as high as those observed in patient samples. This suggests that DNMT1 activity may not be the only DNA methyltransferase influencing expression of MN1 in inv(16) patients. Interestingly, we observed a high (ρs= 0.42) correlation between MN1 methylation and DNMT3B expression, which suggests DNMT3B could be an important DNA methyltransferase involved in regulating MN1expression.Overall we show that pediatric AML patients carrying and inv(16) have a characteristic DNA methylation pattern compared to other AML patients carrying specific cytogenetic aberrations. Furthermore, our data suggest that hypomethylation of the MN1 gene is an underlying mechanism for high MN1 expression in inv(16)(p13;q22) patients possibly regulated by multiple DNA methyltransferases. DisclosuresNo relevant conflicts of interest to declare.

ET-04 * MEBENDAZOLE IS EFFICACIOUS IN DIVERSE MEDULLOBLASTOMA TUMOR MODELS AND INHIBITS TUMOR ANGIOGENESIS

Medulloblastoma is the leading cause of cancer death in children. Surgery, radiotherapy and chemotherapy regimens are the current standard for treatment. While effective in most patients, those have long-term neurological sequelae in survivors, and a significant fraction of patients still succumb to the disease. In this study, we found that mebendazole (MBZ), an FDA-approved antiparasitic, demonstrated significant anti-tumor efficacy in etiologically distinct medulloblastoma mouse models. MBZ significantly improved the survival of mice with orthotopic xenograft tumors derived from the SHH group and group 3 medulloblastomas and was also highly efficacious against a PTCH1-mutant medulloblastoma with acquired resistance to the SMO inhibitor vismodgib. Analysis of the vasculature in rodent tumors revealed that MBZ selectively inhibited tumor angiogenesis but not the normal brain vasculature, and inhibited the kinase activity of VEGFR2 in vitro and in vivo. This study demonstrates that MBZ could be a highly promising therapeutic for medulloblastoma with anti- angiogenesis activity.

Abstract B39: IGF-IR regulation of osteoclastogenesis in neuroblastoma bone metastasis

Abstract Neuroblastoma is a tumor that is responsible for 15% of cancer deaths in children and has a mortality rate greater than 90% percent upon metastasis to bone. Due to the high mortality rate and lack of effective treatment options, our laboratory is investigating potential new therapeutic targets in neuroblastoma bone metastases. Since, insulin-like growth factor I (IGF-I) production in the bone promotes neuroblastoma growth and survival, our hypothesis is that inhibiting IGF-I receptor (IGF-IR) will prevent metastasis of this tumor to bone. Furthermore, neuroblastoma with increased IGF-IR expression induces osteoclasts to differentiate, with the percentage of differentiated osteoclasts in vitro increasing to 30% in the presence of conditioned medium from high IGF-IR expressing cells. The addition of a monoclonal, neutralizing antibody to the IGF-IR decreases the percentage of mature osteoclasts formed by over 50%. We are testing the hypothesis that IGF-IR activation in neuroblastoma alters the levels of RANK, RANKL and OPG, proteins involved in osteoclast differentiation. This alteration in RANK, RANKL, and OPG may cause osteoclast differentiation and osteolysis. Neuroblastoma cells with high IGF-IR protein levels express low levels of the decoy receptor OPG, while some lines also express high levels of RANKL. Therefore, we are currently testing the effect of IGF-IR inhibition on RANKL and OPG levels and subsequent effect on osteolysis. Citation Format: Chris A. Rivera-Pintado, Yufei Shan, Cynthia M. van Golen. IGF-IR regulation of osteoclastogenesis in neuroblastoma bone metastasis. [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 B39.

Abstract B43: Role of activated SHP2 in high-risk neuroblastoma pathogenesis

Abstract Neuroblastoma, an embryonic tumor of peripheral sympathetic nervous system (PSNS), accounts for 10% of all childhood cancer deaths. We recently developed a robust zebrafish model of neuroblastoma and demonstrated that activated ALK synergizes with MYCN by inhibiting a developmentally-timed apoptotic response that is otherwise induced by MYCN. Genomic studies show that PTPN11 (encoding SHP2) is the second most frequently mutated genes in high-risk neuroblastoma. We have now used our zebrafish model to ask whether mutationally activated SHP2 functions as an oncogene in neuroblastoma. We developed transgenic lines in which ptpn11E69K is overexpressed in the PSNS under control of the dopamine-beta-hydroxylase promoter. Interestingly, ptpn11E69Ksynergized with MYCN to accelerate the onset of neuroblastoma in the interrenal gland, the zebrafish analogue of the adrenal medulla. Tumors began to appear at 15 weeks in transgenic fish overexpressing MYCN alone, whereas latency was shortened to 7 weeks and the penetrance was increased more than four-fold in transgenic fish co-expressing MYCN and ptpn11E69K. Co-expression of ptpn11E69K and MYCN also induced ganglioneuroma in the sympathetic ganglia, an outcome that has not been observed in transgenic fish overexpressing MYCN and activated ALK. These results suggest that activated SHP2 can collaborate with MYCN in neuroblastoma tumorigenesis via mechanisms different from those of activated ALK. Thus, zebrafish appears to provide a robust model system for functional genomic analysis and for the investigation of the mechanisms and pathways underlying mutations identified in ongoing tumor resequencing studies. Citation Format: (Jane) shizhen Zhu, Benjamin G. Neel, A. Thomas Look. Role of activated SHP2 in high-risk neuroblastoma pathogenesis. [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 B43.

Curcumin induces apoptosis in human neuroblastoma cells via inhibition of AKT and Foxo3a nuclear translocation

Neuroblastoma (NB) is one of the most frequent extracranial solid tumors in children. It accounts for 8–10% of all childhood cancer deaths, and there is a need for development of new drugs for its treatment. Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), has been shown to exert anti-tumor activity on NB, but the specific mechanism by which curcumin inhibits cancer cells proliferation remains unclear. In the present study, we investigated the anti-proliferative effect of curcumin in human LAN5 NB cells. Curcumin treatment causes a rapid increase in reactive oxygen species and a decrease in the mitochondrial membrane potential—events leading to apoptosis activation. Furthermore, curcumin induces decrease in haet shock protein (Hsp)60 and hexokinase II mitochondrial protein levels and increase in the pro-apoptotic protein, bcl-2 associated death promoter (BAD). Moreover, we demonstrate that curcumin modulates anti-tumor activity through modulation of phosphatase and tensin homolog deleted on chromosome 10 and consequential inhibition of the survival Akt cell-signaling pathway. Inhibition of Akt causes its translocation into the cytoplasm and import of Foxo3a into the nucleus where it activates the expression of p27, Bim, and Fas-L pro-apoptotic genes. Together, these results take evidence for considering curcumin as a potential therapeutic agent for patients with NB.

Abstract 4380: The miR-15 family members are therapeutic candidates to treat chemoresistant neuroblastomas

Abstract Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for approximately 15% of cancer deaths in children. Neuroblastomas are very heterogeneous, with prognosis ranging from spontaneous regression to aggressive clinical course, and often become refractory to all current forms of treatment. While low and intermediate-risk patients have good prognosis, high-risk patients have poor prognosis and need intense chemotherapy. Despite the aggressive treatment, 60-70% of patients will die, mainly due to resistance to therapy and metastasis. Owing to the multiple different mechanisms that confer NBL resistance to therapy, targeting single components of a pathway may not suffice to restore chemosensitivity. In this respect, it is desirable to find molecules that can regulate multiple cellular processes or different component of the same pathways, thereby overcoming MDR and improving the therapeutic response. Our strategy is aimed to use microRNAs (miRNAs) to target the oncogenic properties of MDR neuroblastomas and render them vulnerable to treatment. The mRNA expression of genes related to multidrug resistance was analyzed in multiple neuroblastoma data sets. The 3′UTR of the genes differentially expressed in the highest-risk neuroblastoma group (Stage 4, MYCN amplified) was scanned with miRNA-binding sites prediction algorithms (TargetScan, PicTar, miRANDA). MicroRNAs predicted to regulate the expression of 2 or more overexpressed genes were selected for functional validation. Thirty-six miRNAs mimetics were transfected in two different chemoresistant neuroblastoma cell lines and effects on cell proliferation were evaluated. We have found a substantial number of genes implicated in the DNA damage response deregulated in high-risk NBL. MicroRNA-binding prediction algorithms revealed that most of them could potentially be regulated by microRNAs. Functional analysis in chemoresistant NBL cell lines identified several microRNAs that regulate proliferation, the best of which were the miR-15 family members. The overexpression of miR-497 reduces de proliferation of chemoresistant NBL cell lines and induces apoptotic cell death only in MYCN-amplified cell lines. Moreover, conditional overexpression of miR-497 in neuroblastoma xenografts led to a reduction in tumor growth. CONCLUSIONS: A substantial number of genes implicated in the MDR phenotype are deregulated in high-risk NBL and can be simultaneously targeted with miRNAs, providing a new therapeutic approach for chemoresistant neuroblastomas. This work is supported by Instituto de Salud Carlos III (CP11/00052, RD12/0036/0016 and PI11/00740) cofinanced by the European Regional Development Fund (ERDF) and Asociación Española Contra el Cáncer-Junta de Barcelona. Citation Format: Aroa Soriano, Laia Paris-Coderch, Luz Jubierre, Ana Almazán-Moga, Carla Molist, Josep Roma, Soledad Gallego, José Sánchez de Toledo, Miguel F. Segura. The miR-15 family members are therapeutic candidates to treat chemoresistant neuroblastomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4380. doi:10.1158/1538-7445.AM2014-4380

Abstract 80: Activated SHP2 collaborates with MYCN in neuroblastoma tumorigenesis

Abstract Neuroblastoma, an embryonic tumor of peripheral sympathetic nervous system (PSNS), accounts for 10% of all childhood cancer deaths. We recently developed a robust zebrafish model of neuroblastoma and demonstrated that activated ALK synergizes with MYCN by inhibiting a developmentally-timed apoptotic response that is otherwise induced by MYCN. Genomic studies show that PTPN11 (encoding SHP2) is the second most frequently mutated genes in high-risk neuroblastoma. We have now used our zebrafish model to ask whether mutationally activated SHP2 functions as an oncogene in neuroblastoma. We developed a transgenic line in which ptpn11E69K is overexpressed in the PSNS under control of the dopamine-beta-hydroxylase promoter. Interestingly, ptpn11E69K synergized with MYCN to accelerate the onset of neuroblastoma in the interrenal gland (IRG), the zebrafish analogue of the adrenal medulla. Tumors began to appear at 15 weeks in transgenic fish overexpressing MYCN alone, whereas latency was shortened to 7 weeks and the penetrance was increased more than four-fold in transgenic fish coexpressing MYCN and ptpn11E69K. Coexpression of ptpn11E69K and MYCN also induced ganglioneuroma in the sympathetic ganglia, an outcome that has not been observed in transgenic fish overexpressing MYCN and activated ALK. In addition, coexpression of ptpn11E69K and MYCN induced adrenal neuroblastic hyperplasia in the IRG of all the transgenic fish at 5 weeks of age, a phenotype that has only been observed in about 50% of transgenic fish overexpressing activated ALK and MYCN or MYCN alone. Thus, these results suggest that activated SHP2 can collaborate with MYCN in neuroblastoma tumorigenesis via mechanisms different from those of activated ALK. Thus, zebrafish appears to provide a robust model system for functional genomic analysis and for the investigation of the mechanisms and pathways underlying genetic alterations identified in integrative genomic studies. Citation Format: Xiaoling Zhang, Guang Yang, A.Thomas Look, Benjamin G. Neel, Shizhen (Jane) Zhu. Activated SHP2 collaborates with MYCN in neuroblastoma tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 80. doi:10.1158/1538-7445.AM2014-80

Abstract 41: 47 kda CXCR4: A marker for highly invasive neuroblastoma

Abstract Neuroblastoma; a pediatric cancer of the sympathetic nervous system, which accounts for 15% of childhood cancer deaths, presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Although studies have shown that the CXCL12 chemokine receptor CXCR4 mediates the process of metastasis in many cancer types, its role in the bone marrow metastasis of neuroblastoma is unclear. In an effort to better understand the role of the CXCR4- CXCL12 axis in bone marrow metastasis of neuroblsatoma, we screened 20 neuroblastoma cell lines for their invasive potential, and expression of CXCR4, and sought to identify a correlation between tumor invasiveness and CXCR4 expression. Western blot analyses of cell lysates revealed the presence of multiple CXCR4 isoforms with 3 prominent species at about 35 kda, 47 kda and 62 kda. Cell lines expressing the 47 kda isoform showed higher secretion of MMP9 in gelatin zymography and higher migration and invasion towards mesenchymal stem cells (MSCs) and MSC-conditioned-RPMI, in transwell migration/invasion studies. Far western blot analysis with immuno-precipitated CXCR4, showed that the 47kda protein bound with CXCL12 at a higher rate. Blocking CXCR4 with AMD3100 or a neutralizing antibody against the 47 Kda isoform decreased the secretion of MMP9, the expression of integrin α3 and integrin β1 and the invasive potential of the cell. These data suggest that the 47 kda CXCR4 plays a prominent role in the bone marrow metastasis of neuroblastoma and could possibly be used as a diagnostic marker for highly invasive neuroblastoma and the development of minimal residual disease. Citation Format: Vipin Shankar, Lei Qi, Kentaro Kihira, Yoshihiro Komada, Hiroki Hori. 47 kda CXCR4: A marker for highly invasive neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 41. doi:10.1158/1538-7445.AM2014-41

Abstract 118: The cytoplasmic domain of ICAM-2 interacts with α-actinin to confer a non-metastatic phenotype in neuroblastoma cells

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, accounting for 15% of all childhood cancer deaths. Despite recent improvements in outcome for children with early stage disease, the majority of children with high-risk neuroblastoma survive less than 5 years. Nearly all newly diagnosed patients achieve apparent remission, but patients with high-risk disease relapse and die from metastatic progression within 2-5 years. Our long-range goal is to develop therapies that prevent metastatic relapse. We recently made the novel observation that intercellular adhesion molecule-2 (ICAM-2) prevents development of disseminated tumors in a murine model of metastatic neuroblastoma. Interestingly, ICAM-2 suppressed metastatic but not tumorigenic potential in preclinical models, supporting a novel mechanism of regulating metastatic disease. Aside from this observation, little is known about the function of ICAM-2 in tumor cells. Importantly, and consistent with data generated using neuroblastoma cell lines, we also showed that primary human neuroblastoma cells expressing ICAM-2 are those recognized clinically to have limited metastatic potential. Because the actin cytoskeletal network is a primary regulator of cell motility and metastatic potential, we hypothesized that ICAM-2 affected the phenotype of neuroblastoma cells by interacting with the actin cytoskeletal linker protein α-actinin. We used in silico modeling to examine the likelihood that the cytoplasmic domain of ICAM-2 binds directly to α-actinin. We then used site-directed mutagenesis to express variants of ICAM-2 with mutated α-actinin binding domains, and compared the impact of ICAM-2 wild type (WT) and each variant on neuroblastoma cell motility. We used dual immunofluorescence and co-immunoprecipitation approaches to demonstrate co-localization of ICAM-2 WT and α-actinin in situ; and we evaluated ICAM-2 WT and variants for their ability to produce disseminated tumors in in vivo models. In vitro and in vivo characteristics of cells expressing ICAM-2 variants with modified α-actinin binding domains differed from cells expressing ICAM-2 WT and also from cells that express no detectable ICAM-2. Unlike the WT protein, ICAM-2 variants did not completely suppress development of disseminated neuroblastoma tumors in vivo. We concluded that the interaction of ICAM-2 with α-actinin was critical to conferring the ICAM-2-mediated non-metastatic phenotype in NB cells. This work was supported by the University of Alabama at Birmingham-Comprehensive Cancer Center-New Faculty Development Award (P30 CA013148). Citation Format: Joseph M. Feduska, Stephen G. Aller, Stuart Cramer, Robert C.A.M. van Waardenburg, Karina J. Yoon. The cytoplasmic domain of ICAM-2 interacts with α-actinin to confer a non-metastatic phenotype in neuroblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 118. doi:10.1158/1538-7445.AM2014-118

Abstract LB-201: MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically

Abstract Disease recurrence following multi-modal therapy is the single most adverse event in medulloblastoma (MB). Currently &amp;gt;90% of relapsing patients die, accounting for ∼10% of childhood cancer deaths. MB is heterogeneous at diagnosis, comprising four molecular subgroups with distinct clinicopathological and molecular features and outcomes. The relevance of these features at relapse is unknown, making characterisation, modelling and targeted therapy of relapse biology essential to improve outcomes. However, relapsed MBs are not routinely biopsied in clinical practice. We undertook a first comprehensive investigation of the molecular, clinical and pathological features of 29 relapsed MBs and paired tumour samples taken at diagnosis, including the assessment of features with established significance at diagnosis (e.g. chromosome 17 and TP53 pathway status, MYC family (MYC, MYCN) gene amplification, polyploidy, CTNNB1 mutation and molecular subgroup status). Molecular subgroup was concordant at diagnosis and relapse, however evidence of alteration of all other features examined was found in relapsed tumours, with the majority of changes (30/44) representing acquired high-risk events. Most notably, MYC family gene amplifications and TP53 pathway defects commonly emerged in combination at relapse following conventional multimodal treatment (P=0.02, 7/22, 32%) and predicted rapid progression to death (P=0.016). These observations suggested aberrant activation of MYC family genes synergizes with TP53 inactivation in the genesis of biologically aggressive MB. To investigate any such relationship, we examined Trp53 status in our transgenic mouse model of spontaneously-arising MYCN-driven MB (GTML; Glt1-tTA/TRE-MYCN-Luc). Somatic Trp53 mutations were found in 83% of tumors (n=10/12). Direct modelling of this interaction in GTML/Trp53KI/KI mice dramatically enhanced MB formation with 100% penetrance (43/43, median survival 47 days) in GTML/Trp53KI/KI versus 6% (3/50) in GTML; P&amp;lt;0.0001), faithfully mimicked clinicopathological characteristics of TP53-MYC family gene-associated relapsed human tumors, and validated the essential role of TP53 in potentiating the growth of MYCN-driven MB. Finally, therapeutic inhibition of Aurora-A kinase using MLN8237 in these tumours, and in derived neurospheres in vitro, promoted degradation of MYCN, reduced tumor growth and prolonged survival. In summary, while subgroup status remains stable, MBs display altered molecular, pathological and clinical features at relapse, and the emergence of combined TP53-MYC family gene defects is common following conventional therapy. Their association with rapid demise, coupled with their biological validation as driving and therapeutically exploitable events in a novel mouse MB model, strongly support further investigation and routine biopsy of relapse disease to drive future individualised therapeutic strategies. Citation Format: Rebecca M. Hill, Sanne Kuijper, Janet Lindsey, Ed C. Schwalbe, Karen Barker, Jessica Boult, Daniel Williamson, Zai Ahmad, Albert Hallsworth, Sarra Ryan, Evon Poon, Simon Robinson, Ruth Ruddle, Florence Raynaud, Louise Howell, Colin Kwok, Abhijit Joshi, Sarah Nicholson, Stephen Crosier, Stephen Wharton, Tom Jacques, Keith Robson, Antony Michalski, Darren Hargrave, Barry Pizer, Simon Bailey, Fredrik J. Swartling, Kevin Petrie, William A. Weiss, Louis Chesler, Steve Clifford. MYC and TP53 defects interact at medulloblastoma relapse to define rapidly progressive disease and can be targeted therapeutically. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-201. doi:10.1158/1538-7445.AM2014-LB-201

Bioinformatic description of immunotherapy targets for pediatric T-cell leukemia and the impact of normal gene sets used for comparison.

Pediatric lymphoid leukemia has the highest cure rate of all pediatric malignancies, yet due to its prevalence, still accounts for the majority of childhood cancer deaths and requires long-term highly toxic therapy. The ability to target B-cell ALL with immunoglobulin-like binders, whether anti-CD22 antibody or anti-CD19 CAR-Ts, has impacted treatment options for some patients. The development of new ways to target B-cell antigens continues at rapid pace. T-cell ALL accounts for up to 20% of childhood leukemia but has yet to see a set of high-value immunotherapeutic targets identified. To find new targets for T-ALL immunotherapy, we employed a bioinformatic comparison to broad normal tissue arrays, hematopoietic stem cells (HSC), and mature lymphocytes, then filtered the results for transcripts encoding plasma membrane proteins. T-ALL bears a core T-cell signature and transcripts encoding TCR/CD3 components and canonical markers of T-cell development predominate, especially when comparison was made to normal tissue or HSC. However, when comparison to mature lymphocytes was also undertaken, we identified two antigens that may drive, or be associated with leukemogenesis; TALLA-1 and hedgehog interacting protein. In addition, TCR subfamilies, CD1, activation and adhesion markers, membrane-organizing molecules, and receptors linked to metabolism and inflammation were also identified. Of these, only CD52, CD37, and CD98 are currently being targeted clinically. This work provides a set of targets to be considered for future development of immunotherapies for T-ALL.

Ultrastructural features of neuroblastic tumours in relation to morphological, and molecular findings; a retrospective review study

BackgroundNeuroblastoma is the most common solid tumour of infancy and is responsible for 15% of childhood cancer deaths. Presence of amplified MYCN in neuroblastoma is associated with poor prognosis and rapid tumour progression. The aim of this study was to examine and compare the ultrastructural features of high-risk MYCN amplified neuroblastomas, with lower-risk non-MYCN amplified tumours.MethodsThis was a retrospective study evaluating archival diagnostic tissue samples, in which Fluorescence in-situ hybridisation (FISH) had been used at diagnosis to establish MYCN status. 22 (11 MYCN amplified tumours and 11 non-MYCN amplified) tumours of similar light microscopic appearance (poorly differentiated neuroblastoma) were then selected for ultrastructural examination.ResultsThere is a relationship between ultrastructural features in neuroblastoma and MYCN status, although with marked overlap between groups. MYCN amplified tumours generally exhibited a ‘less differentiated’ ultrastructural phenotype, with significantly smaller neurotubules (NT) in the cell body (p < 0.002). Non-MYCN amplified tumours show increased features of neuronal differentiation, with fewer neurosecretory granules (NSG) and NT in the cytoplasm.ConclusionsMYCN amplification is associated with a less differentiated ultrastructural phenotype, and lack of MYCN amplification with relative ultrastructural neuronal differentiation.

Towards optimal clinical and epidemiological registration of haematological malignancies: Guidelines for recording progressions, transformations and multiple diagnoses

Haematological malignancies (HM) represent over 6% of the total cancer incidence in Europe and affect all ages, ranging between 45% of all cancers in children and 7% in the elderly. Thirty per cent of childhood cancer deaths are due to HM, 8% in the elderly. Their registration presents specific challenges, mainly because HM may transform or progress in the course of the disease into other types of HM. In the context of cancer registration decisions have to be made about classifying subsequent notifications on the same patient as the same tumour (progression), a transformation or a new tumour registration. Allocation of incidence date and method of diagnosis must also be standardised.We developed European Network of Cancer Registries (ENCR) recommendations providing specific advice for cancer registries to use haematology and molecular laboratories as data sources, conserve the original date of incidence in case of change of diagnosis, make provision for recording both the original as well as transformed tumour and to apply precise rules for recording and counting multiple diagnoses. A reference table advising on codes which reflect a potential transformation or a new tumour is included.This work will help to improve comparability of data produced by population-based cancer registries, which are indispensable for aetiological research, health care planning and clinical research, an increasing important area with the application of targeted therapies.