Solid Cancer In Children Research Articles

Recommendations for the use of nuclear medicine imaging in patients with neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid cancer in children. Despite intensive multimodality treatment, long-term survival of patients with high-risk NB, which comprises more than half of all cases, remains poor. Nuclear medicine is key in diagnosis, staging, response assessment and long-term follow-up of NB. The emergence of novel tracers and the increasing complexity of studies require updated guidelines for nuclear medicine imaging in NB. Standardising diagnostic techniques are essential for improving study comparability and ensuring test quality. This article aims to provide a comprehensive review of NB radionuclide diagnostic imaging, including its characteristics, accuracy, advantages, and limitations. It offers practical recommendations to multidisciplinary teams responsible for treating patients with NB. This review summarises the opinions of leading experts from the Neuroblastoma Spanish Group within the Spanish Society of Paediatric Haematology and Oncology (SEHOP) and the Spanish Society of Nuclear Medicine and Molecular Imaging (SEMNIM).

EPID-07. INCIDENCE OF PAEDIATRIC BRAIN TUMORS IN AN ITALIAN REGIONAL COHORT: AN EPIDEMIOLOGICAL STUDY FROM 2012 TO 2023

Abstract BACKGROUND Central nervous system (CNS) tumors are the most common solid cancer in children, second only to leukaemia. METHODS We conducted a retrospective epidemiological study of primary CNS tumors diagnosed in children up to 16 years of age between 2012 and 2023, in order to estimate the incidence in a regional cohort of 1,2 million inhabitants (Northeastern Italy). Only primitive lesions were enrolled, relapses were excluded. RESULTS Over a period of twelve years, 100 new diagnoses were made (44% Female, 56% Male) with a mean age of 8,92 years (range 0-16 years) at time of onset. In terms of location, we recorded an equal number of supratentorial and subtentorial diagnoses (50% vs 50%). A time-series analysis was performed to verify possible variations in the incidence rate of brain tumors over years, and no significant change was described (R2 0.301, p=0.06). However, after 2017 a significant increase was noted (R2 0.725, p=0.015), as the incidence of new diagnosis quite doubled, from 3.16 in 2018 (95%CI 3.15-3.17) to 5.59 in 2023 (95%CI 5.57-5,61). Data were therefore divided in two groups: 2012-2017 (group 1), and 2018-2023 (group 2). There were no differences in terms of gender or age. The number of low-grade (LG) and high-grade (HG) diagnosis were compared: HG tumors, including high grade glioma, ependymoma and medulloblastoma, were significantly higher in the group 2 than group 1 (27/50, 54% vs 6/43, 14%, p<0.001). CONCLUSIONS In childhood, CNS tumors incidence has been fairly stable in the last twelve years in our regional cohort, but a recent concern of an increased rate of high-grade diseases emerged according to our data. In case of a confirmation of this trend, further analysis will be required in order to understand the reason.

JS04.5.A Enhancing the activity of anti-CD47 antibody therapy with radiotherapy in preclinical models of medulloblastoma

Abstract Background Brain cancers are the most common solid cancer in children and the leading cause of cancer-related deaths in children. Medulloblastoma is the most common paediatric brain tumour. Treatment for medulloblastoma involves surgery, craniospinal irradiation (CSI) and chemotherapy. These therapies are extremely damaging to the developing brain and have not changed in decades, resulting in stagnation in the survival outcomes for children with medulloblastoma, and poor quality of life for children who survive their treatment. Immunotherapy has become a focus of novel treatment development. While there are multiple clinical trials aiming to increase immune recognition of medulloblastoma, none have been successful to date. Anti-CD47 is an immune-modulating therapeutic antibody which blocks the anti-phagocytic signal, CD47, expressed by brain cancer cells. Anti-CD47 has shown promising preliminary efficacy in brain cancer models. Material and Methods Using a small animal radiotherapy platform, we have developed a preclinical CSI protocol which mimics clinical radiotherapy. Using an orthotopic xenograft model of medulloblastoma, mice were treated with either anti-CD47 antibody therapy, CSI, or the combination of both anti-CD47 and CSI. Results CSI was found to deplete adaptive immune cells in the brain, while myeloid cells remained the dominant populations. Anti-CD47 antibody therapy was ineffective as a single agent against a patient derived xenograft model of Group 3 medulloblastoma, and CSI as a monotherapy resulted in temporary tumour regression. We found that the combination of anti-CD47 with CSI resulted in marked and persistent tumour regression. Conclusion This preclinical work has shown promising efficacy of anti-CD47 in combination with CSI, which we are currently testing in additional models. Our work is currently employing a range of techniques such as high dimensional flow cytometry and single cell sequencing to elucidate the mechanisms by which radiotherapy enhances the anti-tumour activity of myeloid cells. This work will enable the rational design and translation of optimal combination therapies for medulloblastoma clinical trials.

Comparison of Diffusion and Perfusion Techniques in Differentiating High Versus Low Grade Pediatric Brain Tumors

Background/Objective: Brain tumors are the most common solid cancer in children and cause significant mortality and morbidity. We compare the effectiveness of different parameters in predicting tumor grade between dynamic contrast enhancement (DCE), intravoxel incoherent motion (IVIM), dynamic susceptibility contrast (DSC) perfusion and diffusion weighted imaging (DWI). 
 
 Methods: A retrospective blinded review of pediatric brain tumors with DCE, IVIM, DWI, and DSC was performed. Parametric maps were registered to T2 weighted images. Volumetric regions of interest (ROI) were manually segmented from solid tumor components for each patient by a neuroradiologist (CH), neuroradiology fellow (BG), and medical student (EC). Resulting mean values for parameters from DCE (Ktrans, Kep, Ve, Vp,), IVIM (D, D*, f), DSC (rCBV) and DWI (ADC) were compared using Student’s t-test for high- and low-grade tumor groups based on WHO grading from pathology. For significant parameters, receiver operating characteristic (ROC) analysis with area under curve (AUC) was performed. 
 
 Results: 20 subjects were included with 9 low grade and 11 high grade tumors. Significant differences between low versus high grade were demonstrated for D (10−3 mm2/s) (1.4±0.4 vs 0.9±0.2, p=0.01), f (0.04±0.02 vs 0.07±0.02, p=0.02), ADC (10−3 mm2/s) (1.4±0.4 vs 0.9±0.3, p=0.009) and rCBV (2.2±0.9 vs 4.7±2.1, p=0.003). No significant difference was found for D* or any DCE parameter. AUC from ROC was similar for all significant parameters [D (0.81, p=0.003); f (0.80, p=0.003); ADC (0.83, p=0.001); rCBV (0.83, p=0.0005)]. 
 
 Conclusion: D and f parameters from IVIM can significantly differentiate high versus low grade pediatric brain tumors similar to ADC and rCBV. Conversely, no DCE parameter was significant. 
 
 Scientific Implications: The results will assist the selection of MRI sequences that best predict tumor grade, as well as guide tumor biopsy for the most aggressive tumor portions. Further study of these techniques may correlate with molecular profiling and predict outcome.

Abstract 5871: Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma

Abstract Background: Neuroblastoma (NB) is the most common extra-cranial solid cancer in children. Although multimodal therapies with differentiating agents and immunotherapy with anti-GD2 antibody and GM-CSF have shown promising results, it remains deadly in approximately 50% of patients with high-risk disease. Chimeric antigen receptor T-cell therapies (CAR-T) have been found to be effective in treating refractory and relapsed leukemia and lymphoma, and two of them have been recently approved by the FDA. However, current CARs frequently lose efficacy due to T cell exhaustion and CARs against solid tumor antigens often lack enough specificity due to a low incidence of somatic mutations resulting in a paucity of tumor neoantigens. There have not been effective CAR-T therapies against other solid cancers to date although many clinical trials are underway. We previously identified 2 cell surface cancer-associated antigens, GPC2 (Glypican-2) and CD276 (B7-H3), both highly expressed in NB tumor cells but expressed at low or undetectable levels in normal organs. Here we attempt to develop a high throughput way of identifying optimal CART cell binders that show activation and expansion in the presence of GPC2 and CD276 but lack of exhaustion. Methods: Binders targeting GPC2 or CD276 were cloned into CAR lentiviral constructs and then were separately transduced into T cells to develop CAR-T cells. Then we analyzed cytotoxicity of these CART cells individually. To identify the most effective GPC2 or CD276-specific targeting CAR-T cells, we utilized a combined proteomics and transcriptomics method for every single CAR-T cell to quantify RNA and protein at the same. All CAR-T cells were pooled and co-cultured with CD276/GPC2-expressing NB cancer cells (target cells) for 24 hr. Co-cultured CAR-T cells were examined for their activation, exhaustion, cytotoxicity state and distinguished different cell types by staining with CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) antibodies, and then molecularly barcoded using 10X Genomics platform for single cell RNA-sequencing (scRNA-seq). Results: We attempted to test the efficacy of CAR-T cells using 14 established and novel binders against GPC2 or CD276. According to cytotoxicity assay of these 14 CART cells, we found CT3 was the most effective GPC2 targeting CART cells but majority of CD276 CART cells showed high cytolytic activity in vitro. Conclusions and Future Directions: Using the CITE-seq and RNAseq analyses, we can identify which of the CARs are enriched and have an activated T cell signature, but lack exhaustion markers. Top candidate binders for each antigen are currently being developed into “AND” or “OR” CARs that are being tested in in-vitro and in-vivo models of NB. Thus, we have developed a high throughput method to identify high affinity functional binders against tumor cell surface antigens and provide a novel immunogenomics methods of CARs optimization for development of highly effective immunotherapies against NB and other cancers. Citation Format: Meijie Tian, Adam Tai-Chi Cheuk, Yong Song, Sivasish Sindiri, Nan Li, Christopher M. Dower, Mitchell Ho, Bradley St Croix, Javed Khan. Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5871.

Abstract A13: Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid cancer in children. Although multimodal therapies with differentiating agents and immunotherapy with anti-GD2 antibody and GM-CSF have shown promising results, it remains deadly in approximately 50% of patients with high-risk disease. Chimeric antigen receptor T-cell therapies (CAR-T) have been found to be effective in treating refractory and relapsed leukemia and lymphoma, and two of them have been recently approved by the FDA. However, current CARs frequently lose efficacy due to T-cell exhaustion, and CARs against solid tumor antigens often lack enough specificity due to a low incidence of somatic mutations resulting in a paucity of tumor neoantigens. There have not been effective CAR-T therapies against other solid cancers to date, although many clinical trials are under way. Therefore, we attempted to develop a high-throughput way of identifying optimal CART cell binders that show activation and expansion in the presence of targets but lack of exhaustion. We previously identified two cell surface cancer-associated antigens, GPC2 (Glypican-2) and CD276 (B7-H3), both highly expressed in NB tumor cells but expressed at low or undetectable levels in normal organs. 14 established binders as well as novel binders targeting these two antigens were cloned into CAR lentiviral constructs and then were separately transduced into T cells to develop 14 CAR-T cells using a 2nd-generation design. All 14 CAR-T cells were pooled and cocultured with CD276/GPC2-expressing NB cancer cells (target cells) for 24 hr. To identify the effective GPC2 or CD276-specific targeting CAR-T cells, we utilized a combined proteomics and transcriptomics method for every single CAR-T cell to quantify RNA and protein at the same. Cocultured CAR-T cells were examined for their activation, exhaustion, cytotoxicity state and distinguished different cell types by staining with CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) antibodies, and then molecularly barcoded using 10X Genomics platform for single-cell RNA-sequencing (scRNA-seq). The data are currently being analyzed and will be presented. Using this method, we will be able to identify which of the CARs are enriched and have an activated T-cell signature, and lack exhaustion marks as determined by the CITE-seq and RNAseq analyses. Finally, top candidate binders for each antigen will be developed into “AND” or “OR” CARs and will be tested in in vitro and in vivo models of NB. Thus, we will develop a high-throughput way to identify high-affinity functional binders against tumor cell surface antigens. This study also will provide novel immunogenomics methods of CARs optimization for development of highly effective immunotherapies against NB and other cancers. Citation Format: Meijie Tian, Adam Tai-Chi Cheuk, Jeetendra Kumar, Young K. Song, Sivasish Sindiri, Nan Li, Christopher M. Dower, Mitchell Ho, Brad St. Croix, Javed Khan. Immunogenomic approaches to optimize immunotherapeutic targeting of neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A13.

Simvastatin Induces Apoptosis in Medulloblastoma Brain Tumor Cells via Mevalonate Cascade Prenylation Substrates.

Medulloblastoma is a common pediatric brain tumor and one of the main types of solid cancers in children below the age of 10. Recently, cholesterol-lowering “statin” drugs have been highlighted for their possible anti-cancer effects. Clinically, statins are reported to have promising potential for consideration as an adjuvant therapy in different types of cancers. However, the anti-cancer effects of statins in medulloblastoma brain tumor cells are not currently well-defined. Here, we investigated the cell death mechanisms by which simvastatin mediates its effects on different human medulloblastoma cell lines. Simvastatin is a lipophilic drug that inhibits HMG-CoA reductase and has pleotropic effects. Inhibition of HMG-CoA reductase prevents the formation of essential downstream intermediates in the mevalonate cascade, such as farnesyl pyrophosphate (FPP) and gernaylgerany parophosphate (GGPP). These intermediates are involved in the activation pathway of small Rho GTPase proteins in different cell types. We observed that simvastatin significantly induces dose-dependent apoptosis in three different medulloblastoma brain tumor cell lines (Daoy, D283, and D341 cells). Our investigation shows that simvastatin-induced cell death is regulated via prenylation intermediates of the cholesterol metabolism pathway. Our results indicate that the induction of different caspases (caspase 3, 7, 8, and 9) depends on the nature of the medulloblastoma cell line. Western blot analysis shows that simvastatin leads to changes in the expression of regulator proteins involved in apoptosis, such as Bax, Bcl-2, and Bcl-xl. Taken together, our data suggests the potential application of a novel non-classical adjuvant therapy for medulloblastoma, through the regulation of protein prenylation intermediates that occurs via inhibition of the mevalonate pathway.

Therapy-related Acute Myeloid Leukemia After the Treatment of Primary Solid Cancer in Children: A Single-center Experience.

Therapy-related acute myeloid leukemia (t-AML) has a dismal prognosis and is one of the most frequent second malignant neoplasms which could be encountered by pediatric oncologists. Between October 2000 and September 2016, 16 patients who had primary solid tumors were diagnosed with t-AML at the Seoul National University Children's Hospital. The median patient age at the time of diagnosis of their primary solid tumors was 9.6 years (range, 0.1 to 15.4 y), and that of t-AML was 14.0 years (range, 4.7 to 23.9 y). The median latency period from the end of the primary tumor treatment to the initial diagnosis of t-AML was 29 months (range, 6 to 130 mo). Twelve patients achieved complete remission. Of them, only 7 patients underwent hematopoietic stem cell transplantation (HSCT). The 3-year overall survival (OS) rates and event-free survival rates were 33.7±12.2% and 26.9±11.5% respectively. The patients who underwent HSCT showed favorable 5-year OS rates (57.1±18.7%), whereas the 5-year OS rates of those who did not undergo HSCT was 0%. This study demonstrates that an achievement of complete remission and a subsequent HSCT can be the optimal solution for the treatment of t-AML, and this strategy showed acceptable outcomes.

Radiomics in paediatric neuro-oncology: A multicentre study on MRI texture analysis.

Brain tumours are the most common solid cancers in children in the UK and are the most common cause of cancer deaths in this age group. Despite current advances in MRI, non-invasive diagnosis of paediatric brain tumours has yet to find its way into routine clinical practice. Radiomics, the high-throughput extraction and analysis of quantitative image features (e.g. texture), offers potential solutions for tumour characterization and decision support. In the search for diagnostic oncological markers, the primary aim of this work was to study the application of MRI texture analysis (TA) for the classification of paediatric brain tumours. A multicentre study was carried out, within a supervised classification framework, on clinical MR images, and a support vector machine (SVM) was trained with 3D textural attributes obtained from conventional MRI. To determine the cross-centre transferability of TA, an assessment of how SVM performs on unseen datasets was carried out through rigorous pairwise testing. The study also investigated the nature of features that are most likely to train classifiers that can generalize well with the data. Finally, the issue of class imbalance, which arises due to some tumour types being more common than others, was explored. For each of the tests carried out through pairwise testing, the optimal area under the receiver operating characteristic curve ranged between 76% and 86%, suggesting that the model was able to capture transferable tumour information. Feature selection results suggest that similar aspects of tumour texture are enhanced by MR images obtained at different hospitals. Our results also suggest that the availability of equally represented classes has enabled SVM to better characterize the data points. The findings of the study presented here support the use of 3D TA on conventional MR images to aid diagnostic classification of paediatric brain tumours.

Abstract A051: Characterization of IDO1 and TDO2 in pediatric central nervous system tumors

Abstract Purpose: Indoleamine 2,3 dioxygenase 1 (IDO1) and tryptophan dioxygenase (TDO2) are tryptophan catabolic enzymes that have been implicated in facilitating immunosuppression in cancer. With respect to the central nervous system (CNS), both enzymes have been correlated with decreased overall survival in adult patients with glioma. Based on these observations, we questioned whether pediatric CNS tumors, which constitute the most common form of solid cancer in children, also possess expression and activity for these immunosuppressive mediators. Experimental Design: Purified RNA from pediatric patient tumor tissue was obtained from the Children's Brain Tumor Tissue Consortium (Philadelphia, PA) and utilized for qRT-PCR analysis of IDO1, TDO2, CD3e, IFNγ, and TGFβ1. Primary cell lines were propagated from fresh tumor tissue acquired by Dr. Rintaro Hashizume, Ph.D. (Northwestern University) and cultured for 48 hours alone or with a combination of 100 ng/mL IFNγ and 10, 50, 250 or 1000 nM L1MT, D1MT and BGB-5777; a novel pharmaceutical-grade IDO1 inhibitor (Beigene). Cell culture supernatants were collected at 48 hours for detection of tryptophan (Trp) and kynurenine (Kyn) levels by high pressure liquid chromatography. The cells were collected confirmed for IDO1 protein expression via western blot. Results: RT-PCR defined two populations of pediatric high grade glioma (HGG) based on high (>3 fold expression compared to average) versus low (<3 fold expression compared to average) IDO1-expressing tumors. In contrast, low grade glioma (LGG) and diffuse intrinsic pontine glioma (DIPG) universally expressed low IDO1 levels when compared to HGG. All patient tumors expressed TDO2 mRNA. All tumors were infiltrated by T cells, based on the ubiquitous presence of CD3ϵ mRNA. Primary human pediatric cell lines comprised of HGG and DIPG inducibly expressed IDO1 mRNA and protein after treatment IFNγ (P < 0.001). Unexpectedly, IFNγ treatment also led to increased TDO2 mRNA expression in DIPG cells. To determine whether these enzymes were functionally active, BGB-5777, in addition to historical IDO1 pathway modulators, L1-MT and D1-MT, were co-incubated with the pediatric cell lines plus IFNγ. At 250 nM, BGB-5777 significantly decreased the Kyn/Trp ratio in both HGG and DIPG cell lines (p<0.001), while treatment with L1-MT and D1-MT concentrations had no impact. Conclusion: These data show for the first time that, IDO1 is expressed in pediatric tumors and the novel blood brain barrier-penetrating IDO1 inhibitor, BGB-5777, is a highly potent inhibitor of enzymatic activity in this setting. Given that IDO1 inhibitors are already in multiple clinical trials for adults with brain cancer, these data suggest that BGB-5777 may be a valuable tool for inhibiting IDO1 in the setting of malignant pediatric brain cancer. Citation Format: Kristen Lauing, Rishi Lulla, Lijie Zhai, Rintaro Hashizume, Jason Fangusaro, Derek A. Wainwright. Characterization of IDO1 and TDO2 in pediatric central nervous system tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A051.

In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma.

Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects. We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ(9)-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells. Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts. Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a nonpsychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

Evaluation of the Histo-Epidemiological Profile of Solid Childhood Cancers in Togo.

Cancer is a subject of continuing concern, more common in adults than in children, but often with a poor outcome in the latter. Our study set itself the objective to describe the epidemiological and histological aspects of solid cancers in children in Togo. This descriptive, cross-sectional study focused on cases of solid cancers in children diagnosed from 2010 to 2014 (5 years) at the pathology laboratory of the Tokoin teaching hospital. Data were collected from the records of that laboratory. We collected 66 cases of childhood cancer representing 5% of all solid cancers. The annual incidence was 13.2 cases. The sex ratio (M/F) was 1.4; mean age was of 7.2±1.6 years. The age group most affected was that of 5-9 years (40.9%). Four histological groups of solid childhood cancers were listed: lymphoma (n=34 cases; 51.5%), embryonic cancer (n=17 cases; 25.8%), sarcomas (n=13 cases; 19.7%) and carcinoma (n=2 cases; 3%). The most common histological types were Burkitt lymphoma (36.4%), nephroblastoma (10.6%) and retinoblastoma (10.6%). This study shows that solid cancers in children are relatively frequent in Togo with a male predominance. They are still largely dominated by Burkitt lymphoma, followed by retinoblastoma and nephroblastoma.

Telomere maintenance is pivotal for high-risk neuroblastoma

Neuroblastoma, a malignant tumor of the sympathetic nervous system, is the most common extracranial solid cancer in children. The clinical courses of the disease are remarkably heterogeneous: While...

JARID1B Expression Plays a Critical Role in Chemoresistance and Stem Cell-Like Phenotype of Neuroblastoma Cells.

Neuroblastoma (NB) is a common neural crest-derived extracranial solid cancer in children. Among all childhood cancers, NB causes devastating loss of young lives as it accounts for 15% of childhood cancer mortality. Neuroblastoma, especially high-risk stage 4 NB with MYCN amplification has limited treatment options and associated with poor prognosis. This necessitates the need for novel effective therapeutic strategy. JARID1B, also known as KDM5B, is a histone lysine demethylase, identified as an oncogene in many cancer types. Clinical data obtained from freely-accessible databases show a negative correlation between JARID1B expression and survival rates. Here, we demonstrated for the first time the role of JARID1B in the enhancement of stem cell-like activities and drug resistance in NB cells. We showed that JARID1B may be overexpressed in either MYCN amplification (SK-N-BE(2)) or MYCN-non-amplified (SK-N-SH and SK-N-FI) cell lines. JARID1B expression was found enriched in tumor spheres of SK-N-BE(2) and SK-N-DZ. Moreover, SK-N-BE(2) spheroids were more resistant to chemotherapeutics as compared to parental cells. In addition, we demonstrated that JARID1B-silenced cells acquired a decreased propensity for tumor invasion and tumorsphere formation, but increased sensitivity to cisplatin treatment. Mechanistically, reduced JARID1B expression led to the downregulation of Notch/Jagged signaling. Collectively, we provided evidence that JARID1B via modulation of stemness-related signaling is a putative novel therapeutic target for treating malignant NB.

ED-23 * ESTIMATING THE IMPACT OF CHILDHOOD CANCER IN THE UNITED STATES: YEARS OF LIFE LIVED WITH DISEASE AND YEARS OF POTENTIAL LIFE LOST, 2009

BACKGROUND: Cancer is one of the leading causes of death among children in the US, and central nervous system (CNS) tumors are the most common type of solid cancer in children <20 years. Incidence rates and overall survival are commonly reported cancer statistics, but they may fail to capture the full impact of childhood cancers. We describe the Years of Potential Life Lost (YPLL) and Years of Life Lived with Disease (YLLD) in children <20 years of age in the US to better understand the public health, economic and personal impact of childhood cancer. METHODS: We examined mortality data due to neoplasm in 2009 among children <20 years old in both the National Vital Statistics System (NVSS) and the Surveillance, Epidemiology, and End Results (SEER) datasets. Histology-specific YPLL and YLLD of CNS tumors, leukemias and lymphomas were measured using SEER data. RESULTS: There were 2,233 deaths and 153,390.4 YPLL due to neoplasm in 2009. CNS tumors were the largest cause of YPLL (31%). Among specific histologies examined, the greatest mean YPLL (mYPLL) was due to atypical teratoid/rhabdoid tumor (ATRT) (78 mYPLL) and high grade glioma (71 mYPLL). The histologies with the highest mean YLLD (mYLLD) were primitive neuroectodermal tumor (4.59 mYLLD), medulloblastoma (3.17 mYLLD) and acute lymphoblastic leukemia (3.09 mYLLD); ATRT had the lowest (0.63 mYLLD). CONCLUSIONS: CNS tumors are the second most common malignancy in children but have the highest cost in YPLL. This study offers the first description of YPLL in childhood tumors defined by histology and proposes a new measure of cancer impact, YLLD. YPLL and YLLD complement the traditional indicators of mortality and help place CNS tumors in the context of other childhood malignancies.

Abstract LB-68: MYCN-induced TRPM7 expression and channel activity occurs through a mechanism that involves ornithine decarboxylase

Abstract Neuroblastoma (NB) is an extra-cranial solid cancer in children. MYCN is a transcription factor that regulates the expression of genes that are involved in cell proliferation, growth, cell migration, etc. MYCN gene amplification is a prognostic indicator of poor outcome in NB. Recent studies have shown that intracellular free calcium regulates multiple steps involved in cell migration. Although significant advances have been made in understanding the role of calcium during migration, little has been achieved towards understanding its impact on the malignant progression of NB. Our results showed that MYCN gene amplification or over-expression of MYCN in NB cells increased TRPM7 expression, increased peak TRPM7 current, induced constitutive TRPM7 channel activity, enhanced calcium signaling, and promoted cell migration, implicating TRPM7 as a potential regulator of calcium signals that promote NB cell migration and invasion. Recent results suggest that MYCN may regulate TRPM7 expression through a process that involves ornithine decarboxylase (ODC), a rate limiting enzyme in the biosynthesis of polyamines, and a direct transcriptional target of MYCN. Treatment of NB cells with α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, or transfection of ODC specific siRNA effectively decreased TRPM7 expression. This effect was attenuated by supplementing the culture media with spermidine, confirming that ODC may function downstream of MYCN to regulate TRPM7 expression. Interestingly, western blot analysis of NB cell lysates showed that MYCN induced the cleavage of the TRPM7 kinase domain, a post-translational modification that has been shown to increase TRPM7 currents and induce constitutive TRPM7 channel activity. Additionally, DFMO inhibited TRPM7 cleavage, an effect that was reversed by spermidine supplementation. Taken together, these results suggest that MYCN may regulate TRPM7 expression and channel activity through a mechanism that involves ODC-mediated polyamine biosynthesis. Citation Format: Dana-Lynn T. Koomoa, Ingo Lange. MYCN-induced TRPM7 expression and channel activity occurs through a mechanism that involves ornithine decarboxylase. [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-68. doi:10.1158/1538-7445.AM2014-LB-68

MycN promotes TRPM7 expression and cell migration in neuroblastoma through a process that involves polyamines

Neuroblastoma is an extra-cranial solid cancer in children. MYCN gene amplification is a prognostic indicator of poor outcome in neuroblastoma. Recent studies have shown that the multiple steps involved in cell migration are dependent on the availability of intracellular calcium (Ca2+). Although significant advances have been made in understanding the role of Ca2+ during migration, little has been achieved towards understanding its impact on the progression of diseases such as cancer. Interestingly, previous studies showed that cancer cell migration is regulated by TRPM7, a calcium-permeable ion channel. The objective of the current study was to elucidate the mechanism by which MycN promotes NB cell migration and the mechanism regulating TRPM7 expression. The results showed that MycN increased TRPM7 expression, induced TRPM7 channel activity, increased intracellular Ca2+ signaling, and promoted cell migration in NB cells. The results also showed that inhibition or down-regulation of ornithine decarboxylase (ODC) inhibited TRPM7 expression, a process that was reversed by spermidine. Overall, this study provides evidence that MycN promotes TRPM7 expression and cell migration through a mechanism that involves ODC synthesis of polyamines.

Abstract 3781: MYCN-induced TRPM7 mediates calcium influx and promotes neuroblastoma cell migration.

Abstract Neuroblastoma (NB) is the most common extra-cranial solid cancer in children. One of the genetic characteristics of advanced stage NB is MYCN gene amplification, a prognostic indicator of poor outcome. MYCN is a transcription factor from the myc proto-oncogene family that regulates fundamental processes by regulating the expression of genes that are involved in cell proliferation, growth, cell migration, etc. One of the hallmarks of the malignant progression of primary tumors is uncontrolled growth and spread of tumor cells, with the consequence of metastatic cells entering the blood stream or lymphatic system and nesting into secondary tissues and organs. Metastatic tumors affect the function of cells and/or organs at the secondary site, and compromise the health and survival of cancer patients. Recent studies have shown that the multiple steps involved in cell migration and invasion (such as lamellapodia formation, focal adhesion turnover, and cytoskeletal dynamics) are dependent on the availability of free intracellular calcium. Although significant advances have been made in understanding the role of calcium during migration and invasion itself, little has been achieved towards understanding its impact on the progression of diseases such as cancer where cell motility is critical for the spread of tumor cells. To address this deficiency, the objective of the current study was to elucidate the mechanism by which calcium signaling regulates NB migration and invasion. Our initial results showed that tetracycline-inducible over-expression of MYCN in NB cells increased TRPM7 expression, enhanced calcium signaling, and promoted cell migration, compared to control NB cells. In addition, increasing extracellular calcium augmented these processes. These results implicate TRPM7 as a potential regulator of calcium-dependent NB cell migration and invasion. In order to confirm the role of TRPM7 in these processes, we used pharmacological inhibitors of TRPM7 and TRPM7-specific siRNA to modulate TRPM7 activity in NB cells, and performed live-cell calcium measurements, electrophysiological patch-clamp recordings, and cell migration assays. The results showed that TRPM7 plays a role in regulating intracellular calcium signaling and NB cell migration. Overall, the results from this study suggest that MYCN over-expression and minute changes in extracellular calcium can promote NB cell migration and invasion through a process that involves TRPM7. Furthermore, this study identifies a new biomarker for NB (TRPM7) and novel target for the development of more effective chemotherapeutic drugs for the treatment of advanced stage NB. Citation Format: Ingo Lange, Dana-Lynn T. Koomoa. MYCN-induced TRPM7 mediates calcium influx and promotes neuroblastoma cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3781. doi:10.1158/1538-7445.AM2013-3781

JLK1486, a Bis 8-Hydroxyquinoline-Substituted Benzylamine, Displays Cytostatic Effects in Experimental Gliomas through MyT1 and STAT1 Activation and, to a Lesser Extent, PPARγ Activation

Gliomas account for 5% to 7% of all solid cancers in adults and up to 30% of solid cancers in children; glioblastomas are the most malignant type of glioma and often have dismal prognoses. The alkylating agent temozolomide provides the greatest chemotherapeutic benefits currently available; however, glioblastoma patients cannot be cured. Novel drugs that efficiently combat glioblastomas are therefore of great interest. We report here that JLK1486, an 8-hydroxyquinoline-substituted benzylamine, could represent a novel chemical scaffold to reach this goal. Indeed, JLK1486 mediated anticancer activity in vivo (through intravenous as well as oral routes of administrations) in an orthotopic xenograft model and displayed efficiency similar to that of temozolomide. The therapeutic benefits of JLK1486 seem to relate to its ability to activate various transcription factors (including Myt1, STAT1, and peroxisome proliferator-activated receptor γ) in glioma cells. These transcription factors are implicated in the control of glioma cell proliferation, and the resultant global effect of their activation by JLK1486 was cytostatic, not cytotoxic. Thus, the current study opens the door for the development of novel compounds to combat glioblastoma using 8-hydroxyquinoline benzylamine analogs.

Abstract 5259: Application of HT-RNAi-based functional genomics in neuroblastoma

Abstract Neuroblastoma is the most common extra-cranial solid cancer in children. About 70% of patients with neuroblastoma have metastatic disease at the time of diagnosis. Children with favorable disease characteristics and younger than one year of age (stages I and II) have a higher likelihood of disease-free survival. However, older children with advanced disease (stages III and IV) have only a 30% chance of survival even with aggressive therapy. Additionally, amplification of the MYC-N gene has been associated with stage III and IV tumors, thus establishing it as a poor prognostic marker. Current treatment primarily consists of combinations of surgery, radiation, and chemotherapy. Newer regimens also include the topoisomerase I inhibitor topotecan, which has been found to be effective against recurrent disease. In order to identify “Achilles heel” targets that modulate growth of neuroblastoma, we applied a functional genomics approach using High Throughput RNA interference (HT-RNAi). Furthermore, by treatment of cells with low dose topotecan, we hope to identify genes the can improve the response to this current therapy. We first identified optimal siRNA transfection conditions for the four neuroblastoma cell lines; MYC-N amplified, IMR-5 and IMR-32 and MYC-N non-amplified, SK-N-AS and NB-EB. Since these cell lines can be difficult to transfect with siRNA, several cationic lipids were tested at varying lipid:siRNA ratios to obtain the optimal reagent and ratio for each cell line. Next, we determined the optimal low dose (IC10-30) of topotecan for each cell line. Following these optimizations, HT-RNAi assays were conducted on the four neuroblastoma cell lines using a kinase siRNA library targeting 572 kinases and a siRNA library targeting 532 apoptosis associated genes treated with vehicle or low dose topotecan. These HT-RNAi screens identified genes whose silencing decreased neuroblastoma cell growth and genes whose silencing improved topotecan response. We are currently in the process of validating these results. After validation, we hope to develop these molecular targets into innovative therapeutics for children affected with this disease. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5259.