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e14547 Background: Glioblastoma (GBM) is the most aggressive brain malignancy with a heterogeneity molecular profile and accounts for no more than 3-5% of Central Nervous System tumors in children. Despite its rarity, pediatric GBM pertain different molecular genetics, outcome and effectiveness to therapies and remains an equally lethal tumor in children. Identification of genetic alterations in GBM of childhood and adolescence is important to refine molecular classification, define prognosis and therapeutic strategies. We aimed to detect and investigate molecular changes with potential prognostic marker and therapeutic target in GBM, using next generation sequencing (NGS) strategy. Methods: We selected 41 GBM samples from patients treated at Pediatric Oncology Institute/GRAACC. NGS was performed to identified genetic alterations in tumor samples using Oncomine Childhood Cancer Research Assay (OCCRA) panel, specific genetic panel for childhood and adolescence neoplasms. Results: We selected 41 GBM samples and identified 33 mutated genes. The most commonly detected genetic alterations were involving TP53, PDGFRA, PIK3CA, NF1, MYC, MET and genes with histone-related functions, including H3F3A and ATRX. Mutations in H3F3A K28M, ATRX and TP53 were most recurrent. ATRX alterations, either nonsense substitution or frameshift deletion, were exclusively found in patients with H3F3A K28M mutations. TP53 loss-of-function was exclusive in 9 of 41 (22%) tumors. In 7 of 9 cases (78%) harboring both H3F3A and TP53 mutations, patients died due to disease progression . In these patients, H3F3A-TP53 mutation is suggested to be unfavorable prognostic factor. Copy number alterations were found in 30% of GBM cases and PDGFRA-amplifications were the most frequent. PDGFRA-amplification was exclusively found in patients with H3F3A K28M mutations and 75% of these patients did not survived longer than two years. Conclusions: Molecular profiling based on NGS genetic panel, specific for pediatric tumors, can provide information about potential prognostic biomarkers for GBM of childhood and adolescence. Thus, wider understanding about GBM biologic heterogeneity may lead to personalized therapeutic strategies for pediatric patients.

10525Background: In the phase 1 portion of KEYNOTE-051 (NCT02332668), the 2-mg/kg-Q3W dose of pembro was identified as the pediatric recommended phase 2 dose. We provide an update on the safety and...

10525 Background: In phase 1 of the KEYNOTE-051 study (NCT02332668), the 2 mg/kg Q3W approved adult dose of pembro was also determined to be the pediatric recommended phase 2 dose. In phase 2, presented herein, we further evaluated this dose in pediatric pts with advanced cancer. Methods: Children aged 6 mo to < 18 y with advanced melanoma or a PD-L1+ advanced, relapsed or refractory solid tumor or lymphoma, measurable disease per RECIST v1.1, and performance score ≥50 using Lansky Play or Karnofsky scales received pembro 2 mg/kg Q3W for 35 cycles or until confirmed disease progression per immune-related RECIST by investigator review, intolerable toxicity, or pt/investigator decision to discontinue. Tumor imaging was performed every 8 wk for the first 6 mo, then every 12 wk thereafter. AEs were graded by NCI CTCAE v4.0. Key efficacy endpoints were ORR, disease control rate (DCR), and PFS per RECIST v1.1 by investigator and OS. Results: Of 369 pts prescreened, 364 were evaluable for PD-L1 expression; of these, 121 (33.2%) were PD-L1+. 66 pts were enrolled; median follow-up was 2.5 mo (range, 0.2-18). As of the data cutoff (Nov 7, 2016), 23 (34.8%) pts were still on treatment. Median age was 13 y (range, 1-17), 77.3% had metastatic disease, and 34.8% had ≥3 prior lines of therapy for recurrent/metastatic disease. Primary diagnoses were non-CNS solid tumors (n = 45), CNS tumors (n = 16), and lymphoma (n = 5). 5 (7.6%) pts had grade 3-4 treatment-related AEs (TRAEs), most commonly neutropenia (n = 2). No treatment-related deaths occurred; 1 pt discontinued for a TRAE (grade 3 AST increased). 1 pt each with Hodgkin lymphoma, adrenocortical carcinoma, mesothelioma, and glioblastoma had partial response for an ORR of 6.1% (95% CI, 1.7-14.8); 7 (10.6%) pts had stable disease for a DCR of 16.7% (95% CI, 8.6-27.9). Median PFS and OS were 1.8 mo and 9.2 mo, respectively; 12-mo PFS was 10.2% and OS was 40.5%. Potential effects of pembro on the developing immune system (eg, T and B cells, vaccinated antibodies) will also be presented. Conclusions: Pembro showed low toxicity and warrants further study to determine activity in select pediatric tumors. Enrollment in KEYNOTE-051 is ongoing. Clinical trial information: NCT02332668.

TPS10079 Background: Large cooperative group studies have shown the efficacy of risk-adapted treatment for Ewing sarcoma (ES). However, validation and local adaptation by national cooperative group...

e21034 Background: Over 80% of patients with Wilms tumor (WT) can be cured from their disease, most of them with minimal late effects; nonetheless, patients with advance stage tumor, unfavorable hi...

10532 Background: Preclinical models show that a daily antiangiogenic regimen at low-dose may be effective against chemotherapy-resistant tumors. In patients (pts) with high grade, operable osteosa...

10533 Background: Osteosarcoma (OS) is a class of cancer originating from the bone, affecting mainly children and young adults. In a previous work we observed that the overexpression of MAPK7 gene was significantly associated to tumor progression, a poor response to treatment, and worse overall survival, suggesting that MAPK7 could play an important role in osteosarcoma tumorigenesis. This present study investigated if any genomic structural alteration could be related to MAPK7 overexpression in OS samples. Methods: The MAPK7 gene was fully sequenced in 33 OS samples (15 prechemotherapy,15 post-chemotherapy and 3 OS cell lines) using the 3500 Genetic Analyser (Applied Biosystem) and 15 custom primers pairs that amplified overlapping genomic fragments. Results: From the 30 patient samples analyzed, 16 could not be completely sequenced. Samples containing these deleted regions were significantly correlated to clinical and pathologic features of patients with OS. No deletions were observed in patients with the favorable outcome (non-metastatic at diagnosis, >90% degree of necrosis in the surgical specimen and with complete remission at end of treatment). There was no correlation found between deletions and overexpression in pre-chemotherapy, even in patients that after chemotherapy still overexpressed MAPK7. Conclusions: Progress related to improved survival of patients with OS is at a plateau, and the intensification of therapy or incorporation of new therapeutic agents has not been successful, especially in patients metastatic at diagnosis. To our knowledge, no molecular marker for OS that could be related to its development, evolution or progression has been described yet. Based on our findings, we observed that patients who have no genomic deletion of MAPK7 gene respond better to treatment and have a favorable outcome. Studies available to date suggest that a single targeted therapy may not provide clinically significant anti-tumor effects, therefore we believe that specific inhibitors of the MAPK/ERK pathway are emerging as a relevant addition to multimodal therapies.

10535 Background: Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. Glutathione S-transferase (GST) and Cytochrome P-450 (CYP) family genes are involved in almost all anticancer drugs metabolism. The polymorphisms in these genes are associated with anticancer drugs resistance and toxicity events. This study aims to investigate the genotype frequencies of GSTM1, GSTT1, GSTM3, CYP1A2, CYP2C9 and CYP3A5 genes in OS patients and the influence of these polymorphisms in their clinical outcome. Methods: We investigated the peripheral blood DNA of 70 OS patients (25 metastatic and 45 nonmetastatic at diagnosis), following the GLATO - Latin American Group of Osteosarcoma Treatment - 2006. GSTM1 and GSTT1 deletion polymorphisms were examined through a multiplex-PCR and the GSTM3 polymorphism of three base pair-deletion using PCR-RFLP method. CYP1A2, CYP2C9 and CYP3A5 single nucleotide polymorphisms (SNPs) were investigated through real time PCR using TaqMan probe. Results: We found that GSTM1 null genotype was correlated with relapse occurrence (p=0,031) in patients that received high doses of chemotherapy. The CYP1A2*F allele was associated with lung metastasis (p=0,032), lung relapse (p=0,018) and high grade of ototoxicity (p=0,039). The CYP3A5*3 allele was associated with high grade of hepatotoxicity (p=0,010). The presence of at least one GSTM3*B allele was associated with better overall survival (p=0,045). The presence of CYP3A5*3 homozygous genotype was associated with better overall survival (p=0,043) in metastatic patients at diagnosis. The genotype GSTM3*B/ GSTM1 present, GSTM3*B/ GSTT1 present and GSTM3*B/CYP3A5*3 in metastatic patients at diagnosis were associated with better survival (p=0,048; p=0,004; p=0,012, respectively). Conclusions: The findings of this study suggest that GST and CYP polymorphisms may have a role in treatment response and may be an important marker in the future to personalized therapy in OS. Furthermore, CYP1A2*F allele is correlated with risk of lung metastasis and GSTM3*B homozygous genotype in combination with GSTM1 present, GSTT1 present and CYP3A5*3 were associated with better survival.

10534 Background: Osteosarcoma (OS) is the most common malignant bone tumor in adolescents. A feature of the OS is its high risk of lung metastasis. Unlike other sarcomas, OS does not have genetic markers. We suggest that abnormalities in the expression levels of genes involved in important cell signaling pathways, as MAPKs pathway, may contribute to OS progression and aggressiveness. In a recent study, we found an overexpression of MAPK7 gene in prechemotherapy specimens when compared to postchemotherapy specimens and when compared with normal bone. This previous study suggests that MAPK7 overexpression may be contributing to the worse response to treatment and therefore a poor overall survival. To understand the relationship between the OS genesis process and the MAPK pathway, we investigated the expression of genes involved in the regulation of MAPK pathway (MAP2K5, MAP2K6, MAP3K1, MAP3K3, MAP4K3 and DUSP1). Methods: Total RNA was extracted from 68 tumor samples obtained from 28 patients (28 prechemotherapy specimens, 28 postchemotherapy specimens and 12 metastasis specimens) and five normal bone tissues. cDNA was synthesized and expression of the six genes were determined by quantitative real-time PCR. Clinical variables were correlated with gene expression. Results: MAP2K6 and MAP4K3 had a similar gene expression profile. Both genes were overexpressed in postchemotherapy specimens and were strongly associated with important clinical parameters as presence of metastasis (P=0.0159 and P=0.0014, respectively), shorter overall survival (P=0.0040 and P=0.0040, respectively) and disease progression (P=0.0142 and P=0.0109, respectively). DUSP1 overexpression in postchemotherapy specimens was related with metastasis at diagnosis (P=0.0004), worse response to treatment (P=0.0009) and disease recurrence (P=0.0039). MAP2K5, MAP3K1 e MAP3K3 showed no significant correlations in the present study. Conclusions: The overexpression of MAP2K6, MAP4K3 and DUSP1 in postchemotherapy specimens presented a significant association with poor prognosis. The current study suggests that these genes could play an important role in OS tumorigenesis and may be in the future new therapeutic targets in OS treatment.

9528 Background: To identify prognostic factors in the OS patients enrolled in the BOTG studies III, IV and V. Methods: A total of 604 patients younger than 30 years of age with high grade OS (meta...

10032 Background: Based on results from leukemia maintenance therapy, anti-angiogenic approaches have been indicated as alternatives for solid tumors. In this rationale, metronomic therapy (MT) eme...

e21507 Background: This study represents a prospective assessment of angiogenesis genes mRNA expression in tumors and blood from patients treated with pre- and post-operative Brazilian osteosarcoma protocol (GCBTO 2006) that introduce metronomic chemotherapy (anti-angiogenic) in order to try to increase survival of osteosarcoma patients. Methods: Tumor samples from 27 patients were analyzed before and after chemotherapy to determine VEGFA, VEGFR1, VEGFR2, PDGFC, SDF1 and TSP1 genes expression profile by Quantitative Real Time PCR. Also, blood samples of these patients were investigated pre- and post-chemotherapy and at the end of high-dose chemotherapy trying to evaluate potential for proangiogenic factors and antiangiogenic factor (TSP1) which could be used to monitor treatment activity. Results: Of all six genes studied pre- and post- chemotherapy, in tumor samples, only SDF1 and VEGFR2 were underexpressed. SDF1 gene has the lowest expression at all. In tumor samples, TSP1 and VEGFA expression was higher than SDF1, VEGFR2 and PDGFC expression in biopsies and surgeries (P=0.001). VEGFR1 expression was higher than VEGFR2 expression (P=0.001). PDGFC and VEGFR1 overexpression were associated with necrosis grade I and II (Huvos score) (P=0.005). VEGFA and TSP1 were overexpressed in 96% and 92% of surgery samples, respectively. In blood samples from biopsy, surgery and end of treatment there were no statistically significant changes in the marker genes expression. Conclusions: The study suggests an association between PDGFC and VEGFR1 overexpression and lower grade necrosis. TSP1 and VEGFA were the most expressed genes in all tumor samples but TSP1 was lower than VEGFA in biopsies and VEGFA was lower than TSP1 in surgery (P=0.001). Although VEGFR2 is the primary receptor of VEGF, VEGFR1 was the most expressed VEGF receptor. No significant financial relationships to disclose.

10547 Background: Chemotherapy (CT) with ifosfamide, carboplatin, and etoposide (ICE) is effective in refractory solid tumors. The aim of this trial was to determine the efficacy and safety of ICE as front line therapy for patients (pts) with newly-diagnosed EFT. Methods: Induction with 2 courses of ICE (ifosfamide 3g/m2/d ×3, carboplatin 450mg/m2 ×1, etoposide 150mg/m2/d ×3) plus 2 of VDC (vincristine 1,5mg/m2/d ×1, doxorubicin 60mg/m2/d ×1, cyclophosphamide 1,5g/m2/d ×1) was followed by surgery and/or radiotherapy. After induction, pts were allocated to low (LR) or high-risk (HR) according to LDH (1,5×NL), site (pelvic/non-pelvic) and resection (complete/unresectable) or metastatic (mets). Postoperative CT included 10 alternating courses of IE and VDC for LR and 12 alternating courses of ICE and VDC for HR. Toxicity was assessed following 523, 648, 268 and 114 courses of ICE, VDC, IE and VC, respectively; 35 site visits were carried out for data quality control. Results: From 10/2003 to 10/2008, 150 of 159 enrolled pts were evaluable; median age 13y (0.2–29.3y). 44 pts were classified as LR and 101 as HR, 56 mts. Toxicity was mainly grade III-IV neutropenia, thrombocytopenia and febrile neutropenia, and was significantly higher following ICE. The 3y EFS and OS were 65% and 27% for localized pts and 80% and 43% for mets pts. 3y EFS for lung and non-lung mets were 40% and 19% (P=0.013). Multivariate analysis showed that mets disease was the only independent prognostic factor (P=0.006) for the entire group. However, when mts pts were analyzed separately, high LDH (P=0.01) and extra-lung site (P=0.016) had significant negative impact on survival. Conclusions: ICE regimen is safe and despite a high proportion of pts with advanced disease the survival are similar to larger series. Elevated LDH and the presence of extra-lung mets were significant negative prognostic factors in mets pts. Randomized trials to identify pts who may benefit from more intensive CT are warranted. No significant financial relationships to disclose.

9046 Background: Febrile neutropenia (FN) after chemotherapy requires prompt introduction of empiric antimicrobial therapy. It has been demonstrated that FN patients do not represent a homogeneous group, and the stratification of infectious risk leads to changes in therapy. Our objectives were to evaluated the efficacy of the antimicrobial regimens used according to the FN institutional protocol and to identify risk factors related to invasive infection in children and adolescents during episodes of FN. Methods: We conducted a prospective study in the Pediatric Oncology Institute (IOP-GRAACC-UNIFESP/SP-Brazil) from March/2003 to June/2004. We analyzed 448 FN episodes in 200 patients, they were classified in high and low risk of invasive infection according to the underling disease and comorbities. Low risk patients (solid tumors, ALL and lymphomas in remission, without comorbities) were treated with oral gatifloxacin as outpatient; whereas high risk (AML, solid tumors, Lymphomas and ALL with comorbities or activity) were treated with cefepime as inpatient. Invasive infection was considered when a culture obtained from a sterile site resulted positive for bacterial and/or fungus or there were clinical and laboratory findings that strongly suggest sepsis. Results: Of 204 (45.5%) episodes treated with gatifloxacin, 72.1% obtained success without modification. Of 206 (46.0%) episodes treated with cefepime, 34.5% obtained success without modification, 38 (8.5%) patients were treated with distinct antibiotic regimen. Invasive infection was observed in 133 episodes (29.6%); 16 (3.6%) deaths occurred, all of them in the high risk group and the overall success was 84.3%. In the logistic regression analysis remained as independent risk factors: age ≤3 ys (OR: 2.3), temperature ≥38.5ºC (OR:1.6), central venous catheter (OR:1.7), underlying active disease (OR:1.6), mucositis (OR:2.4) and diarrhea (OR:2.4). Conclusions: Applying a institutional protocol, it is possible to predict the infectious risk and treat the patients individually .The observed overall success (84.3%), confirms that the therapy based in the protocol was adequate. There was no toxicity associated with gatifloxacin in this study, and no deaths in the low risk group. No significant financial relationships to disclose.

9055 Background: Ifosfamide (IFO) is very effective and widely used in pediatric oncology protocols. Patients are usually admitted to receive IFO infusions due to potential urological and neurological complications. The current study was conducted to assess the feasibility of outpatient IFO infusions in children and adolescents with cancer, the compliance of the patients and their parents, and the acute toxicities in this setting. Methods: All patients with newly diagnosed solid tumors included in protocols with at least three cycles of IFO/mesna were eligible for this study. IFO was administered I.V. over one hour. Doses varied according to the underlying malignancy and protocol from 2.5–3.0g/m2/day × 3 days to 2.7g/m2/day × 5 days. Three doses of mesna were administered intravenously in the outpatient clinic at 75% of the IFO dose divided on hours 0, 3 and 6 and a fourth dose of mesna was given as oral tablets at home 12 hours after IFO. Hydration was 2L/m2 I.V. over 5 hours in the hospital, followed by another 1L/m2 P.O. at home. Kidney function was assessed before, during, and after treatment through glomerular filtration rate; sodium, magnesium, calcium, uric acid renal excretions; urinary phosphate threshold and retinol-binding protein. Results: From November 2003 to August 2005, 30 patients received a total of 160 IFO cycles. Compliance was 100%. The incidence of grades 3 and 4 toxicities in all IFO cycles was: gastrointestinal 15%, leucopenia 92%, neutropenia 88%, thrombocytopenia 29%, hepatotoxicity 3%, and genitourinary 7%. No patient had gross hematuria, neurotoxicity or had to be admitted for chemotherapy. One third of the patients developed subclinical tubular nephropathy. Conclusions: Ambulatory infusion of IFO is safe and toxicities are comparable to the expected with inpatient administration. No significant financial relationships to disclose.

9536 Background: To evaluate whether clinical signs or symptoms of congestive heart failure, serial assessment of systolic and diastolic cardiac function by low dose dobutamine stress echo (LDSE) and serum cardiac troponin T (cTnT) can predict doxorubicin (DOXO) cardiotoxicity. Methods: Twenty five patients with osteosarcoma enrolled in the Brazilian osteosarcoma treatment group study 2000, from january 2000 to may 2004, were studied with LDSE (>5μg/kg/min) before chemotherapy, 160 mg/m2 DOXO and after 160 mg/m2 DOXO. cTnT were measured before and during DOXO infusion. Cardiotoxicity was defined as shortening fraction (SF) less than 30% assessed by rest echo 1 to 6 months off chemotherapy. Group A comprised those without cardiotoxicity (16 patients, 10 male, 14.3 ± 4.7 years) whereas group B included those with a SF < 30% (9 patients, 6 male, 15.4 ± 3 years). Elevated serum cTnT was defined as seric levels above 0.01ng/ml. Results: Patients were submitted to a mean 3.4 LDSE and a mean of 32.5 serum cTnT. One patient (group B) presented clinical manifestation of cardiotoxicity. There was no statistical difference of elevated serum cTnT between the group B and group A (87.5% vs 46.2%; p=0,06). Left ventricular dimensions by M- MODE and transmitral Doppler inflow diastolic parameters were not significantly different between the two groups. Resting SF showed comparable values in both groups until cumulative doses of DOXO reached 160mg/m2, then the resting SF in group B was significant lower than group A (27% ± 2 and 34.1% ± 2, p2 (SF 36.1%±3,4 and ΔSF 2.1±2.3 vs. SF 45.2%±4.9 and ΔSF 9.4±3; p< 0.01 and p < 0.01) as well as at a DOXO dose > 160mg/m2 (30.3%±3 and 3.1±1.9 vs. 40.8%±5.9 and 7.2±4.2;p No significant financial relationships to disclose.

8544 Background: MRD is an important prognostic factor for children with ALL and it provides crucial information on the response to treatment and risk of relapse. However, the frequency which MRD c...