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10547Background: Radiation and chemotherapy exposure are associated with the development of second malignant neoplasms (SMN) in neuroblastoma survivors. However, whether the incidence of SMN has ch...

Background Radiolabeled metaiodobenzylguanidine (MIBG) is sensitive and specific for detecting neuroblastoma. The extent of MIBG-avid disease is assessed using Curie scores. Although Curie scoring is prognostic in patients with high-risk neuroblastoma, there is no standardized method to assess the response of specific sites of disease over time. The goal of this study was to develop approaches for Curie scoring to facilitate the calculation of scores and comparison of specific sites on serial scans. Procedure We designed three semiautomated methods for determining Curie scores, each with increasing degrees of computer assistance. Method A was based on visual assessment and tallying of MIBG-avid lesions. For method B, scores were tabulated from a schematic that associated anatomic regions to MIBG-positive lesions. For method C, an anatomic mesh was used to mark MIBG-positive lesions with automatic assignment and tallying of scores. Five imaging physicians experienced in MIBG interpretation scored 38 scans using each method, and the feasibility and utility of the methods were assessed using surveys. Results There was good reliability between methods and observers. The user-interface methods required 57 to 110 seconds longer than the visual method. Imaging physicians indicated that it was useful that methods B and C enabled tracking of lesions. Imaging physicians preferred method B to method C because of its efficiency. Conclusions We demonstrate the feasibility of semiautomated approaches for Curie score calculation. Although more time was needed for strategies B and C, the ability to track and document individual MIBG-positive lesions over time is a strength of these methods.

Mass spectrometry (MS) is a powerful method for identifying proteins, and modern mass spectrometers are capable of remarkable speed, resolution and sensitivity. A single tandem mass spectrometry experiment can now lead to the identification and quantitation of thousands of proteins down to sub-femtomolar concentrations. Tandem mass spectrometry experiments generally involve extraction of proteins from cells, biofluid, or tissue followed by digestion of proteins to peptides, separation of peptides on an HPLC, and direct injection into a mass spectrometer (LC-MS/MS). The mass spectrometer measures the mass of each peptide ion (MS1) and selected ions are fragmented (MS/MS or MS2). Mass and fragmentation spectra of each peptide are compared against predicted peptide fragmentation spectra from the known proteome by database search engines (reviewed in Aebersold and Mann 2003). LC-MS/MS instruments also record peptide ion intensities, offering the potential for direct measurement of peptide concentration and thereby protein abundance. However, the extent of ionization of peptides by electrospray ionization is dependent on peptide sequence and modification, elution conditions, complexity of the sample, and other factors. As a result, the absolute intensities of ions derived from nonidentical peptides cannot provide accurate or direct quantitation. Approaches such as peptide ion chromatogram extraction and spectral counting have been developed to obtain relative quantitation of protein abundance (Ono et al. 2006; Fischer et al. 2006; Tang et al. 2006; Paoletti et al. 2006; Listgarten and Emili 2005; Wiener et al. 2004; Wang, Wu, Zeng, et al. 2006). Collectively termed “label-free” quantitation, these approaches require extensive analysis of reference samples and/or significant data redundancy, often requiring many hours of mass spectrometry time per sample. Although highly promising, label-free approaches remain impractical for users lacking access to dedicated mass spectrometry instrumentation and advanced informatic approaches. Stable isotope labeling provides an attractive alternative to label-free approaches. Stable isotopes are sufficiently stable to be non-radioactive. They have equal numbers of protons as their parental element but they differ in mass by the difference in the number of neutrons. Carbon, hydrogen, oxygen, nitrogen and sulfur have two or more isotopes with measurable abundance in Nature. For example, carbon is found as the predominant “light” isotope 12C

Recent proteomics studies suggest high abundance and a much wider role for lysine acetylation (K-Ac) in cellular functions. Nevertheless, cross influence between K-Ac and other post-translational modifications (PTMs) has not been carefully examined. Here, we used a variety of bioinformatics tools to analyze several available K-Ac datasets. Using gene ontology databases, we demonstrate that K-Ac sites are found in all cellular compartments. KEGG analysis indicates that the K-Ac sites are found on proteins responsible for a diverse and wide array of vital cellular functions. Domain structure prediction shows that K-Ac sites are found throughout a wide variety of protein domains, including those in heat shock proteins and those involved in cell cycle functions and DNA repair. Secondary structure prediction proves that K-Ac sites are preferentially found in ordered structures such as alpha helices and beta sheets. Finally, by mutating K-Ac sites in silico and predicting the effect on nearby phosphorylation sites, we demonstrate that the majority of lysine acetylation sites have the potential to impact protein phosphorylation, methylation, and ubiquitination status. Our work validates earlier smaller-scale studies on the acetylome and demonstrates the importance of PTM crosstalk for regulation of cellular function.

Neuroblastoma (NB) is remarkable for its biologic heterogeneity and broad range of clinical behavior. Although survival rates are greater than 90% for patients with biologically favorable disease, outcomes for children with a high-risk clinical phenotype remains poor, with long-term survival still less than 40%. This issue of Journal of Clinical Oncology includes four studies that are focused on defining and treating high-risk NB. Modern molecular techniques have provided the tools to better define high-risk NB, and a modest improvement in outcome has been seen with dose intensification. However, each of the studies emphasizes the need for more effective therapeutic approaches. Efforts to identify variables that accurately predict outcome for patients with NB have been ongoing for more than 35 years. In the 1970s, the dramatic influence of stage and age on prognosis were reported. During this era, aggressive multimodality therapy was recommended for patients with skeletal metastases, whereas infants were treated more gently. In the 1980s, MYCN amplification emerged as a powerful marker of adverse prognosis in NB. The clinical significance of tumor histology, ploidy and other genetic aberrations, including 1p loss, 11q loss, and 17q gain were also demonstrated. Combinations of clinical and biologic prognostic variables are now routinely used for risk-group assignment and treatment stratification, although the criteria used to define risk vary greatly throughout the world. Because definitions of risk are not uniform, direct comparison of risk-based clinical trials conducted in different regions of the world has not been possible. To address this problem, an international task force recently established the International Neuroblastoma Risk Group (INRG) classification system, on the basis of the analysis of 13 prognostic variables in an 8,800 patient cohort. By defining homogenous pretreatment patient cohorts, the INRG classification system will greatly facilitate the development of international collaborative studies. To date, the strategy to improve outcome in high-risk patients has largely been focused on delivering increasingly intensive multimodality therapy. Matthay et al report the long-term results of a randomized Children’s Oncology Group (COG) trial comparing a more intensive arm of consolidation therapy (myeloablative therapy plus autologous bone marrow transplant [ABMT]) versus a less intensive arm (continued conventional-dose chemotherapy). All patients without evidence of disease after consolidation were then eligible for a second randomization to 13-cis-retinoic acid (cis-RA) versus no additional therapy. The initial results of the study, reported almost 10 years ago, demonstrated significantly better 3-year event-free survival (EFS) for the group randomly assigned to myeloablative therapy and ABMT and for patients randomly assigned to cis-RA. With additional followup, patients randomly assigned to the more intensive arm (myeloablative therapy and ABMT) continue to have significantly higher 5-year EFS. Similar results have been reported by European groups, although survival still remains poor. In the COG study, 30% SE 4% of the patients randomly assigned to the superior arm of therapy were event-free at 5 years. Although overall survival (OS) in the COG study was found to be significantly higher for each randomization at 5 years using a test of the log( log(.)) transformation, a significant advantage for OS was not observed in the European studies. Thus, the majority of the patients are not cured with intensive treatment strategies that include myeloablative therapy and stem-cell rescue. Furthermore, long-term follow-up studies have demonstrated that many survivors have serious late effects of therapy, including delays in growth and development, hearing loss, renal and cardiac dysfunction, learning problems, and treatment-related leukemia and second cancers. The study by Canete et al focuses on the outcome of infants younger than 12 months of age with high-risk disease treated on a International Society of Paediatric Oncology European Neuroblastoma (SIOPEN) clinical trial. In this cohort of 35 infants, 2-year EFS was 29% (SE 0.07) and OS was 30% (SE 0.08) after treatment with intensive multimodality therapy, including high-dose busulfan and melphalan with peripheral stem-cell support and cis-RA. Many of the tumors were resistant to chemotherapy, with 30% progressing or failing to respond to induction therapy. In the COG, infants with high-risk NB are treated on the same clinical trial as older patients with high-risk disease. Because of small numbers, international collaboration will likely be needed to determine if the clinical behavior of high-risk tumors diagnosed in infancy differs from high-risk tumors in older children. To ensure that patients with a lowor intermediate-risk clinical phenotype are spared toxic, dose-intensive, high-risk treatment regimens, accurate risk-group classification is critical. Historically, an age cutoff of 12 months has been used for risk stratification. However, recent analysis of a large series of 3,666 patients has demonstrated statistical evidence for increasing the age cutoff to 15 to 19 months. On the basis of these results, COG has modified eligibility criteria for its intermediate-risk clinical study to include toddlers, age 12 to 18 months, with favorable biology tumors. Similarly, an age cutoff of 18 months has been included in the INRG classification system. This new age cutoff will shift approximately 10% of patients previously classified as high risk to a lower-risk group. Newly designed JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 27 NUMBER 7 MARCH 1 2009

Whole exome sequencing has facilitated the discovery of causal genetic variants associated with human diseases at deep coverage and low cost. In particular, the detection of somatic mutations from tumor/normal pairs has provided insights into the cancer genome. Although there is an abundance of publicly-available software for the detection of germline and somatic variants, concordance is generally limited among variant callers and alignment algorithms. Successful integration of variants detected by multiple methods requires in-depth knowledge of the software, access to high-performance computing resources, and advanced programming techniques. We present ExScalibur, a set of fully automated, highly scalable and modulated pipelines for whole exome data analysis. The suite integrates multiple alignment and variant calling algorithms for the accurate detection of germline and somatic mutations with close to 99% sensitivity and specificity. ExScalibur implements streamlined execution of analytical modules, real-time monitoring of pipeline progress, robust handling of errors and intuitive documentation that allows for increased reproducibility and sharing of results and workflows. It runs on local computers, high-performance computing clusters and cloud environments. In addition, we provide a data analysis report utility to facilitate visualization of the results that offers interactive exploration of quality control files, read alignment and variant calls, assisting downstream customization of potential disease-causing mutations. ExScalibur is open-source and is also available as a public image on Amazon cloud.

10019 Background: Exposures to radiation and chemotherapy are associated with increased risk of second malignant neoplasms (SMN) in neuroblastoma survivors. However, it remains unclear if modifications in risk-based treatment strategies during the past 25 years have changed SMN rates. Methods: The International Neuroblastoma Risk Group (INRG) Task Force created a database of neuroblastoma patients diagnosed from 1974-2013. SMN risk was measured by cumulative incidence, standardized incidence ratios (SIR) and absolute excess risk (AER) per 10,000 person-years relative to a matched United States population. Poisson regression compared rates of SMN between different groups. Results: Of the 16,520 patients in the INRG database, 9,261 enrolled on Children’s Oncology Group protocols had SMN data available. 79 (0.85%) patients developed SMN, including hematologic malignancies (n = 38), sarcomas (n = 19), carcinomas (n = 10), CNS tumors (n = 10), hepatoblastoma (n = 1), and nephroblastoma (n = 1). The incidence o...

INTRODUCTION Despite improved response rates following the use of novel therapeutics, including proteasome inhibitors (PI) and immunomodulatory drugs, the majority of MM patients develop chemoresistance and relapse. Thus, it is essential to identify novel drugs that overcome resistance and promote remission. Our previous work detailed the synergistic activity of combined treatment with selective inhibitor of nuclear export (SINE) selinexor and PI carfilzomib (CFZ) in MM (Rosebeck et al, Blood 2013:122(21):279). In this study, we sought to characterize the underlying mechanism of selinexor, a novel drug being tested in multiple phase I/II clinical trials of advanced-stage cancers. METHODS Plasma cells (PC) were purified from MM patient bone marrow (BM) aspirates using EasySep (STEMCELL Tech). RPMI 8226, 8226/Dox40, and MM1S cells were cultured in RPMI1640/10% FBS. RNA was isolated with RNeasy minicolumns (Qiagen) from control or MM1S cells treated for 6 hrs with selinexor. Gene expression profiling was done on Illumina HumanHT12 microarray and InfernoRDN. Differentially expressed genes were input into Ingenuity Pathway Analysis software (Qiagen). Fisher’s exact test was used to calculate p-values for significant pathway enrichment. TF profiling plate array was from Signosis. RESULTS Selinexor treatment inhibited key MM pathway regulators including transforming growth factor beta 1 (TGFB1; p=1.65E-14), which is involved in paracrine BM signaling and bone formation, and the epidermal growth factor (EGF) receptor HER2 (p=4.84E-08), which deregulates EGF signaling and promotes survival. SINE also affected integrin-associated signaling (p=2.42E-03), which controls MM/BM stromal cell adhesion and contributes to chemoresistance. We profiled SINE-dependent changes in TF activation and found increased DNA binding of cAMP response element-binding protein (CREB), which represses IL-6 production, an essential MM cell survival cytokine, and retinoid X receptor (RXR), which is associated with MM cell death. We also found impaired DNA binding of heat shock factors (HSF), which sensitizes MM cells to PI, Kruppel-like factor 4 (KLF4) and multidrug resistance (MDR) promoter-enhancing factors 1 and 2 (MEF-1/2), which are associated with chemoresistance in MM, nuclear respiratory factor 1 (NRF-1), which mediates a recovery pathway to compensate for reduced proteasome function, and paxillin 2 (Pax2), a transcriptional repressor of p53 and enhancer of Wilms tumor protein (WT1). In addition, TF associated with poor prognosis MM, including the pre-B cell homeobox (PBX) oncogene, soluble mucin 1 (SMUC), and WT1, were inhibited. Next, we tested the ability of selinexor to overcome drug resistance in MM cells. 8226/Dox40 are insensitive to the effects of CFZ via upregulation of p-glycoprotein, however selinexor effectively killed these cells. Importantly, we demonstrate efficacy of SINE on PC from patients refractory to and/or relapsing (R/R) on PI-containing regimens. Additionally, we will present preliminary responses from CFZ-refractory patients enrolled in our newly-opened multi-site phase I clinical trial conducted in the MM Research Consortium combining selinexor and CFZ with dexamethasone in R/R MM (NCT02199665). CONCLUSIONS Our results suggest selinexor-dependent inhibition of pathways that promote intrinsic MM cell survival and contribute to chemoresistance in the BM milieu, which may be mediated by suppression of essential growth factors and genes involved in MM cell/BM stroma adhesion. We also found inhibition of TF that may influence MDR/PI resistance either directly (KLF4, MEF-1/2) or indirectly (HSF, NRF-1). Finally, we demonstrate the ability of SINE to overcome MDR in MM cells and PC from patients with R/R disease. Our studies provide evidence of the pleiotropic effects of SINE and underscore the potential of selinexor to be an effective therapeutic for advanced-stage cancers, including MM. Disclosures Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics: Employment. Jakubowiak:Bristol Myers-Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background & Objectives: Relapse of Ewing sarcoma (ES) can occur months or years after initial remission, and salvage therapy for relapsed disease is usually ineffective. There is great need to develop biomarkers that can predict which patients are at risk for relapse, so that therapy and post-therapy evaluation can be adjusted accordingly. In the current study we sought to identify prognostic gene expression signatures in patients diagnosed with ES. Methods: Specimens were obtained from the Children's Oncology Group (COG) Biorepository. All samples were prospectively acquired from patients treated on clinical trials INT-0154 and AEWS0031. Criteria for inclusion included confirmation of localized ES, registration on a clinical trial and availability of frozen tumor. Total RNA was processed for whole genome expression profiling using Affymetrix GeneChip Human Exon 1.0 ST arrays. Affymetrix Power Tools (APT) were used to generate normalized gene-level signal intensity estimates from raw CEL file data. Differentially expressed genes with false discovery rate (FDR) of < 0.2 and absolute fold-change > 1.3 were considered to be significantly associated with outcome (survivor vs. non-survivor, relapse vs. no-relapse). Differentially expressed genes were used to create prognostic gene signatures. An independent group of ES samples from the Euro-Ewing tumor Biorepository in Muenster, Germany was similarly analyzed as a validation cohort. Results: Frozen tumor tissue was available from 254 COG patients. Following RNA and sample QC, Affymetrix CEL files were successfully generated from 56 unique patients with localized disease for whom outcome data were available. Analysis of processed gene expression data led to exclusion of 10 cases from further analysis due to issues of batch effect and outlier status. The demographic, event-free (EFS), and overall (OS) characteristics of the remaining 46 cases were shown to be representative of the INT-0154 and AEWS0031 studies as a whole. Unsupervised clustering of transcript-level data failed to demonstrate segregation of the 46 tumors into groups based on relapse or survival status. Supervised analysis of survivors vs. non-survivors identified a small number of differentially expressed genes and several statistically significant gene signatures, but none of these candidate classifiers could be internally validated. Interestingly, gene set enrichment analysis (GSEA) reproducibly revealed that integrin and chemokine receptor pathways were significantly over-represented as discriminators of EFS and OS (FDR Conclusions: Robust prognostic gene signatures that pass internal and external cross-validation were not identified in this study. These findings demonstrate the profound degree of molecular heterogeneity in ES and suggest that the heterogeneous nature of these tumors is likely to preclude identification of prognostic signatures that will be clinically useful for all cases. Whether prognostic gene signatures will be useful for differentiating among subsets of ES cases is unknown and will require larger cohort analyses. Nevertheless, these studies support recent laboratory-based discoveries implicating cell adhesion and chemokine receptor signaling in ES aggression. Further investigation of these pathways in the context of tumor cell:tumor stroma interactions is warranted. Citation Format: Samuel L. Volchenboum, Jorge Andrade, Donald A. Barkauskas, Mark Krailo, Richard Sposto, Andreas Ranft, Jenny Potratz, Uta Dirksen, Timothy J. Triche, Elizabeth Lawlor. Do prognostic gene signatures exist in Ewing sarcoma? A report from the Children's Oncology Group. [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 A72.

Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron-transferring flavoprotein, and is a member of the acyl-CoA dehydrogenase (ACD) enzyme family. Human IVD crystal structure with a bound substrate analogue shows the guanidino group of Arg387, a conserved residue among other members of the ACD enzyme family, juxtaposed to a phosphate oxygen of the 4'-phosphopantothiene moiety of the substrate analogue. Site-directed mutagenesis was used to investigate the role of Arg387 in substrate binding and enzyme function. Replacing this residue with Lys, Ala, Gln, or Glu resulted in stable proteins. Spectrophotometric substrate binding assays indicated that the Arg387Lys mutant was able to form the charge-transfer complex intermediate with similar efficiency to wild type, while the rest of the mutants were significantly less able to properly form this intermediate. However, the Km of the isovaleryl-CoA for the Arg387Lys mutant was 20.3 compared to 1.5 microM for the wild type. The Km for the rest of the mutants were 75.6, 195, and 550 microM, respectively. The catalytic efficiency per mole of FAD was 20.3, 3.3, 2.0, and 0.34 for the mutants, respectively, compared to 260 microM(-1) x min(-1) for the wild type. These results substantiate the important role of Arg387 in anchoring the substrate, and are consistent with the hypothesis that residues distant from the active site are important for stabilizing the enzyme:substrate/product complex, and could play an important role in the mechanism of the enzyme-catalyzed reaction.

9583 Background: Clinical information, “-omic” datasets, and tissue samples are becoming more difficult to harmonize and manage for advanced data mining. We believe that clinical research data can be centralized and provide direct access to sample availability and associated data from a variety of information stores. Methods: We obtained a standardized set of anonymized patient data from the International Neuroblastoma Risk Group. The cohort consists of more than 11,000 children diagnosed worldwide between 1974 and 2002. The data consist of 34 metrics, such as age at diagnosis, stage of tumor, and other clinical and biological markers. We instantiated the dataset into a Postgres database, and using the Django web framework, created a data model for rapid development of tools and views and built a front-end interface for generating complex queries. To test the feasibility of accessing information on disparate and geographically distinct data samples, we have a formal agreement with the Children's Oncology Group Tumor Bank at The Research Institute at Nationwide Children's Hospital. Based on query results, we consume the Tumor Bank tissue inventory data through a web-facing application programming interface. The end-user is presented only with the number of patients who match their query search terms and for whom tissue samples are available. Results: We have completed our initial implementation and have agreements for collaboration with other international consortium groups. We have created a paradigm for statisticians to securely update and add data, and a verification system checks for internal validity and provides a report of the transaction. Our system can initiate queries and accept results in a variety of standards-compliant formats, and will be available in demonstration form by May 2012. Conclusions: Querying patient data while interrogating external sources allows researchers to observe which ancillary data and samples are available and to quickly download data or request any samples. While designed around a neuroblastoma dataset, our system can be applied to a variety of clinical scenarios and will be made available through an open-source license.

9584 Background: Neuroblastoma is the most common extracranial solid tumor found in children. The most common marker prognostic of poor outcome is amplification of the MYCN oncogene, yet the biological programs by which MYCN affects its aggressive phenotype are largely unknown. In order to identify biological pathways affected by MYCN amplification, we performed global analysis of genes and proteins in the Tet 21/N neuroblastoma cell line. Methods: MYCN expression in Tet21/N cells is regulated by tetracycline. Gene arrays were performed on MYCN-high and MYCN-low Tet21/N cells and SH-EP parental cells using Affymetrix GeneChip Human Exon 1.0 ST. For proteomic analysis, the cells were labeled with stable isotopes for relative quantitation, enriched for phosphoproteins to enhance detection of signaling proteins, and analyzed by GeLC-MS/MS analysis. Integrative pathway analysis was performed on the genomic and proteomic datasets using DAVE and GeneGo. Results: Integrating genomic and proteomic data in MYCN-high and MYCN-low expressing cells identified 88 proteins and 300 genes that change in abundance. We compare these results with previous studies and find both novel and previously identified genes and proteins. Integrating proteomic and genomic data identified over 50 gene products that are present in both analyses and are altered in abundance in response to modulating MYCN expression. The majority of these have discordant abundance changes, with protein levels more frequently altered with no change in gene expression. We have validated changes in protein levels using Western blots including NPM1 and MATR3. We present interaction networks and pathways that correlate with MYCN expression. Conclusions: We showed a significant discordance between gene and protein expression, underscoring the need for integrative genomic and proteomic analysis to describe complex systems. Our analysis identified previously reported and novel genes and proteins and networks regulated by MYCN expression that may provide new insights into the biology of MYCN-amplified tumors.

e20008 Background: Neuroblastoma is the most common solid tumor found in children and is difficult to treat given its genetic and clinical heterogeneity. The tyrosine kinase receptor, TrkB, is often co-expressed with the MYCN oncogene in high-risk tumors, whereas the TrkA receptor is most often found expressed in low-risk, non-MYCN amplified samples. There are differences in the gene expression profiles of TrkB- and TrkA-over-expressing cell lines, but they do not explain the phenotypic variation. We hypothesize that differences in protein translation and post-translational modifications have profound downstream effects on cellular signaling and disease phenotype. Methods: We performed quantitative proteomics using stable isotope labeling, phosphopeptide enrichment, and tandem mass spectrometry on parental SY5Y neuroblastoma cells and cell lines stably transfected with either TrkA or TrkB. Receptors were activated with NGF or BDNF and activation was inhibited with CEP-701 (Lestaurtinib), a selective tyrosine kinase inhibitor, currently in clinical trials. Samples were separated by gel electrophoresis, digested with trypsin, and applied to the mass spectrometer for protein identification. Results: We have performed quantitative phosphoproteomic analysis and compared protein expression levels and patterns in TrkA overexpressing, TrkB overexpressing, and parental SY5Y cells. The TrkA and TrkB receptors were activated with NGF and BDNF ligands, respectively, as evidenced by increased phosphorylation of ERK and AKT, with inhibition by CEP-701. Changes in protein abundance and pathway activation following both ligand binding and inhibition are being determined by quantitative phosphoproteomic analysis, and TrkA-specific and TrkB-specific differences will be presented. Conclusions: As current genomic techniques may underestimate the differences in protein expression, proteomic profiling holds great promise for describing how post-translational modifications such as phosphorylation can affect tumor phenotype. Our work may reveal key elements of TRK signaling pathways important in neuroblastoma tumorigenesis, and may lead to the identification of novel targets for therapy development.