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Rapamycin demonstrated broad-spectrum tumor growth inhibition activity against the in vivo panels of childhood tumors used in the Pediatric Preclinical Testing Program (PPTP). Here we have evaluated rapamycin combined with agents used frequently in the treatment of childhood malignancies. Rapamycin was tested in vitro against 23 cell lines alone or in combination with melphalan, cisplatin, vincristine, or dexamethasone (leukemic models only). In vivo, the impact of combining rapamycin with a cytotoxic agent was evaluated using two measures: 1) the therapeutic enhancement measure, and 2) a linear regression model for time-to-event to formally evaluate for sub- and supraadditivity for the combination compared to the agents used alone. Combining rapamycin with cytotoxic agents in vitro gave predominantly subadditive or additive effects, except for dexamethasone in leukemia models for which supra-additive activity was observed. In vivo testing demonstrated that therapeutic enhancement was common for rapamycin in combination with cyclophosphamide and occurred for 4 of 11 evaluable xenografts for the rapamycin and vincristine combination. The combinations of rapamycin with either cyclophosphamide or vincristine were significantly more effective than the respective standard agents used alone at their maximum tolerated doses (MTD) for most evaluable xenografts. The combination of rapamycin and cisplatin produced excessive toxicity requiring cisplatin dose reductions, and therapeutic enhancement was not observed for this combination. Addition of rapamycin to either cyclophosphamide or vincristine at their respective MTDs appears promising, as these combinations are relatively well tolerated and as many of the pediatric preclinical models evaluated demonstrated therapeutic enhancement for these combinations. Mol Cancer Ther; 9(1); 101–12

Supplementary Table 5 from Molecular Characterization of the Pediatric Preclinical Testing Panel

Purpose: Identifying novel therapeutic agents for the treatment of childhood cancers requires preclinical models that recapitulate the molecular characteristics of their respective clinical histotypes.Experimental Design and Results: Here, we have applied Affymetrix HG-U133Plus2 profiling to an expanded panel of models in the Pediatric Preclinical Testing Program. Profiling led to exclusion of two tumor lines that were of mouse origin and five osteosarcoma lines that did not cluster with human or xenograft osteosarcoma samples. We compared expression profiles of the remaining 87 models with profiles from 112 clinical samples representing the same histologies and show that model tumors cluster with the appropriate clinical histotype, once “immunosurveillance” genes (contributed by infiltrating immune cells in clinical samples) are eliminated from the analysis. Analysis of copy number alterations using the Affymetrix 100K single nucleotide polymorphism GeneChip showed that the models have similar copy number alterations to their clinical counterparts. Several consistent copy number changes not reported previously were found (e.g., gain at 22q11.21 that was observed in 5 of 7 glioblastoma samples, loss at 16q22.3 that was observed in 5 of 9 Ewing's sarcoma and 4 of 12 rhabdomyosarcoma models, and amplification of 21q22.3 that was observed in 5 of 7 osteosarcoma models). We then asked whether changes in copy number were reflected by coordinate changes in gene expression. We identified 493 copy number–altered genes that are nonrandom and appear to identify histotype-specific programs of genetic alterations.Conclusions: These data indicate that the preclinical models accurately recapitulate expression profiles and genetic alterations common to childhood cancer, supporting their value in drug development.

Supplementary Table 6 from Molecular Characterization of the Pediatric Preclinical Testing Panel

Supplementary Table 4 from Molecular Characterization of the Pediatric Preclinical Testing Panel

Supplementary Table 3 from Molecular Characterization of the Pediatric Preclinical Testing Panel

Supplementary Tables S1-S2 from Molecular Characterization of the Pediatric Preclinical Testing Panel

Supplementary Tables, 1-5, Figure 1 from Stage 2 Combination Testing of Rapamycin with Cytotoxic Agents by the Pediatric Preclinical Testing Program

Signaling through the type 1 insulin-like growth factor receptor (IGF-1R) occurs in many human cancers, including childhood sarcomas. As a consequence, targeting the IGF-1R has become a focus for cancer drug development. We examined the antitumor activity of CP-751,871, a human antibody that blocks IGF-1R ligand binding, alone and in combination with rapamycin against sarcoma cell lines in vitro and xenograft models in vivo. In Ewing sarcoma (EWS) cell lines, CP751,871 inhibited growth poorly (In vivo CP-751,871, rapamycin, or the combination were evaluated against EWS, osteosarcoma, and rhabdomyosarcoma xenografts. CP751871 induced significant growth inhibition [EFS(T/C) >2] in four models. Rapamycin induced significant growth inhibition [EFS(T/C) >2] in nine models. Although neither agent given alone caused tumor regressions, in combination, these agents had greater than additive activity against 5 of 13 xenografts and induced complete remissions in one model each of rhabdomyosarcoma and EWS, and in three of four osteosarcoma models. CP751,871 caused complete IGF-1R down-regulation, suppression of AKT phosphorylation, and dramatically suppressed tumor-derived vascular endothelial growth factor (VEGF) in some sarcoma xenografts. Rapamycin treatment did not markedly suppress VEGF in tumors and synergized only in tumor lines where VEGF was dramatically inhibited by CP751,871. These data suggest a model in which blockade of IGF-1R suppresses tumor-derived VEGF to a level where rapamycin can effectively suppress the response in vascular endothelial cells. [Cancer Res 2009;69(19):7662–71]

Supplementary Materials and Methods, Tables 1-3, Figures 1-2 from The Insulin-like Growth Factor-1 Receptor–Targeting Antibody, CP-751,871, Suppresses Tumor-Derived VEGF and Synergizes with Rapamycin in Models of Childhood Sarcoma

This study comprehensively evaluated the landscape of genetic and epigenetic events that predispose to synchronous bilateral Wilms tumor (BWT). We performed whole exome or whole genome sequencing, total-strand RNA-seq, and DNA methylation analysis using germline and/or tumor samples from 68 patients with BWT from St. Jude Children’s Research Hospital and the Children’s Oncology Group. We found that 25/61 (41%) of patients evaluated harbored pathogenic or likely pathogenic germline variants, with WT1 (14.8%), NYNRIN (6.6%), TRIM28 (5%) and the BRCA-related genes (5%) BRCA1, BRCA2, and PALB2 being most common. Germline WT1 variants were strongly associated with somatic paternal uniparental disomy encompassing the 11p15.5 and 11p13/WT1 loci and subsequent acquired pathogenic CTNNB1 variants. Somatic coding variants or genome-wide copy number alterations were almost never shared between paired synchronous BWT, suggesting that the acquisition of independent somatic variants leads to tumor formation in the context of germline or early embryonic, post-zygotic initiating events. In contrast, 11p15.5 status (loss of heterozygosity, loss or retention of imprinting) was shared among paired synchronous BWT in all but one case. The predominant molecular events for BWT predisposition include pathogenic germline variants or post-zygotic epigenetic hypermethylation at the 11p15.5 H19/ICR1 locus (loss of imprinting). This study demonstrates that post-zygotic somatic mosaicism for 11p15.5 hypermethylation/loss of imprinting is the single most common initiating molecular event predisposing to BWT. Evidence of somatic mosaicism for 11p15.5 loss of imprinting was detected in leukocytes of a cohort of BWT patients and long-term survivors, but not in unilateral Wilms tumor patients and long-term survivors or controls, further supporting the hypothesis that post-zygotic 11p15.5 alterations occurred in the mesoderm of patients who go on to develop BWT. Due to the preponderance of BWT patients with demonstrable germline or early embryonic tumor predisposition, BWT exhibits a unique biology when compared to unilateral Wilms tumor and therefore warrants continued refinement of its own treatment-relevant biomarkers which in turn may inform directed treatment strategies in the future.

Despite the advent of personalized medicine, it is still difficult to predict how a cancer develops over time at the level of the individual patient or even in cancer model systems which begs the question whether certain aspects of cancer can ever be predicted or if there is an inherent unpredictability in cancer, similar to other complex biological systems, We demonstrate by a combination of agent-based mathematical modelling and analysis of data from patient-derived xenograft systems from multiple cancer types that certain conditions may invoke chaotic fluctuations in the clonal landscape of cancer cells. Our findings indicate that under those conditions, the cancer genome behaves as a complex dynamic system, making its long-term evolution inherently unpredictable.

Aim: Despite aggressive multiagent protocols, patients with metastatic rhabdomyosarcoma (RMS) have poor prognosis. In a recent high-risk trial (ARST0431), 25% of patients failed within the first year, while on therapy and 80% had tumor progression within 24 months. However, the mechanisms for tumor resistance are essentially unknown. Here we explore the use of preclinical models to develop resistance to complex chemotherapy regimens used in ARST0431. Methods: A Single Mouse Testing (SMT) protocol was used to evaluate the sensitivity of 34 RMS xenograft models to one cycle of vincristine, actinomycin D, cyclophosphamide (VAC) treatment. Tumor response was determined by caliper measurement, and tumor regression and event-free survival (EFS) were used as endpoints for evaluation. Treated tumors at regrowth were transplanted into recipient mice, and the treatment was repeated until tumors progressed during the treatment period (i.e., became resistant). At transplant, tumor tissue was stored for biochemical and omics analysis. Results: The sensitivity to VAC of 34 RMS models was determined. EFS varied from 3 weeks to > 20 weeks. Tumor models were classified as having intrinsic resistance, intermediate sensitivity, or high sensitivity to VAC therapy. Resistance to VAC was developed in multiple models after 2-5 cycles of therapy; however, there were examples where sensitivity remained unchanged after 3 cycles of treatment. Conclusion: The SMT approach allows for in vivo assessment of drug sensitivity and development of drug resistance in a large number of RMS models. As such, it provides a platform for assessing in vivo drug resistance mechanisms at a “population” level, simulating conditions in vivo that lead to clinical resistance. These VAC-resistant models represent “high-risk” tumors that mimic a preclinical phase 2 population and will be valuable for identifying novel agents active against VAC-resistant disease.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies in a systematic manner remains challenging in part due to the lack of faithful preclinical in vitro models. We established ten short-term patient-derived WT cell lines and characterized these models using low-coverage whole genome sequencing, whole exome sequencing and RNA-sequencing, which demonstrated that these ex-vivo models faithfully recapitulate WT biology. We then performed targeted RNAi and CRISPR-Cas9 loss-of-function screens and identified the nuclear export genes (XPO1 and KPNB1) as strong vulnerabilities. We observed that these models are sensitive to nuclear export inhibition using the FDA approved therapeutic agent, selinexor (KPT-330). Selinexor treatment of FHWT suppressed TRIP13 expression, which was required for survival. We further identified in vitro and in vivo synergy between selinexor and doxorubicin, a chemotherapy used in high risk FHWT. Taken together, we identified XPO1 inhibition with selinexor as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

SummaryAccelerating cures for children with cancer remains an immediate challenge due to extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs) from high-risk childhood cancers, many refractory to current standard-of-care treatments. Here, we genomically characterize 261 PDX models from 29 unique pediatric cancer malignancies and demonstrate faithful recapitulation of histologies, subtypes, and refine our understanding of relapsed disease. Expression and mutational signatures are used to classify tumors forTP53andNF1inactivation, as well as impaired DNA repair. We anticipate that these data will serve as a resource for pediatric oncology drug development and guide rational clinical trial design for children with cancer.HighlightsMultiplatform genomic analysis defines landscape of 261 pediatric cancer patient derived xenograft (PDX) modelsPediatric patient derived xenografts faithfully recapitulate relapsed diseaseInferredTP53pathway inactivation correlates with pediatric cancer copy number burdenSomatic mutational signatures predict impaired DNA repair across multiple histologies

SUMMARY Accelerating cures for children with cancer remains an immediate challenge as a result of extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs)—many of which are refractory to current standard-of-care treatments—from high-risk childhood cancers. Here, we genomically characterize 261 PDX models from 37 unique pediatric cancers; demonstrate faithful recapitulation of histologies and subtypes; and refine our understanding of relapsed disease. In addition, we use expression signatures to classify tumors for TP53 and NF1 pathway inactivation. We anticipate that these data will serve as a resource for pediatric oncology drug development and will guide rational clinical trial design for children with cancer., Graphical Abstract, In Brief Rokita et. al provide an extensively annotated genomic dataset of somatic oncogenic regulation across 37 distinct pediatric malignancies. The 261 patient-derived xenograft models are available to the scientific community, and the genomic annotations will enable rational preclinical agent prioritization and acceleration of therapeutic targets for early-phase pediatric oncology clinical trials.

The lack of model systems has limited the preclinical discovery and testing of therapies for Wilms tumor (WT) patients who have poor outcomes. Herein, we establish 45 heterotopic WT patient-derived xenografts (WTPDX) in CB17 scid-/- mice that capture the biological heterogeneity of Wilms tumor (WT). Among these 45 total WTPDX, 6 from patients with diffuse anaplastic tumors, 9 from patients who experienced disease relapse, and 13 from patients with bilateral disease are included. Early passage WTPDX show evidence of clonal selection, clonal evolution and enrichment of blastemal gene expression. Favorable histology WTPDX are sensitive, whereas unfavorable histology WTPDX are resistant to conventional chemotherapy with vincristine, actinomycin-D, and doxorubicin given singly or in combination. This WTPDX library is a unique scientific resource that retains the spectrum of biological heterogeneity present in WT and provides an essential tool to test targeted therapies for WT patient groups with poor outcomes., The progress in pre-clinical drug discovery for Wilms tumor (WT) is limited by a lack of disease models. Here, the authors develop 45 heterotopic WT patient-derived xenografts including several anaplastic models that recapitulate the biological heterogeneity of WT, and propose this as a resource for evaluating future therapeutics for WT.

International audience; Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

Background Contained in-bag spleen morcellation is a conventional extraction technique for safe spleen removal during laparoscopic splenectomy. Existing data for the use of in-bag enzymatic splenic digestion as an alternative to morcellation are lacking. This proof-of-concept study sought to evaluate the effectiveness of single and combinatorial enzyme digestion of murine spleens. Materials and methods Murine spleens were digested with collagenase alone or with combinations of commercially available enzymes (collagenase, elastase, hyaluronidase, neutral protease) to determine their degradation effect. The primary end point was the percentage of mass reduction at 15 and 30 min. Results For collagenase alone (n = 15), the mean reduction in mass was 14 ± 10% (range: 2%-31%) at 15 min and 30 ± 25% (range: 7%-100%) at 30 min. Using combinatorial dissolution with collagenase, hyaluronidase, and elastase (n = 8), the mean reduction in mass was 27 ± 16% (range: 6%-42%) at 15 min and 48 ± 27% (range: 3%-100%) at 30 min. Injecting the enzyme solution into whole spleens (n = 9) yielded a mean reduction in mass of 22 ± 13% (range: 9%-42%) at 15 min and 55 ± 31% (range: 9%-100%) at 30 min; mean reduction was 9 ± 13% (range: 0%-39%) at 15 min and 23 ± 13% (range: 3%-53%) with no injection (n = 12). Conclusions We provide the first demonstration of successful enzymatic murine spleen digestion as an alternative method for in-bag spleen removal during laparoscopic splenectomy. However, the significant cost and quantities of commercial enzyme required for clinical application dampens the enthusiasm for this novel approach.

SummaryRhabdomyosarcoma is a soft-tissue sarcoma with molecular and cellular features of developing skeletal muscle. Rhabdomyosarcoma has two major histologic subtypes, embryonal and alveolar, each with distinct clinical, molecular, and genetic features. Genomic analysis shows that embryonal tumors have more structural and copy number variations than alveolar tumors. Mutations in the RAS/NF1 pathway are significantly associated with intermediate- and high-risk embryonal rhabdomyosarcomas (ERMS). In contrast, alveolar rhabdomyosarcomas (ARMS) have fewer genetic lesions overall and no known recurrently mutated cancer consensus genes. To identify therapeutics for ERMS, we developed and characterized orthotopic xenografts of tumors that were sequenced in our study. High-throughput screening of primary cultures derived from those xenografts identified oxidative stress as a pathway of therapeutic relevance for ERMS.

Background: Haemophilia A (HA), the most common inherited bleeding disorder, is well suited for gene therapy because a modest increase in the plasma factor VIII (FVIII) levels to ≥1% of normal levels will substantially ameliorate the bleeding diathesis and improve quality of life. Earlier gene transfer strategies for FVIII replacement approaches using plasmid electroporation, retroviral vector, or adenoviral vector failed to achieve persistent phenotypic correction of bleeding. We have recently shown that a single peripheral vein administration of adeno-associated viral (AAV) vectors expressing the FIX transgene results in stable long-term expression of transgenic FIX at therapeutic levels without long term toxicity in patients with severe haemophilia B (ClinicalTrials.gov:NCT00979238). However, the use of AAV vectors for HA gene therapy has been limited by inefficient expression of transgenic FVIII and the large size of the FVIII cDNA. To overcome these obstacles, we developed two AAV-FVIII expression cassettes containing a small synthetic liver specific promoter (HLP) driving the expression of codon optimized FVIII variants. These vectors mediated therapeutic expression of FVIII in murine and non-human primate models (McIntosh et al 2013). The first of these constructs, AAV-HLP-hFVIII-SQ, encoding a B-domain deleted FVIII variant, was recently shown (Rangarajan et al, 2017) to mediate sustained (>1 year) normalisation of factor VIII activity in six of seven participants following a single intravenous infusion of AAV serotype 5 pseudotyped vector. However, high vector doses (6x1013 vector genomes/kg [vg/kg]) were required for efficacy, possibly because this product was manufactured using the insect cell/baculovirus system. In this report we describe the preliminary results of our on-going Phase I/II clinical trial (GO-8) evaluating the second FVIII cassette (AAV-HLP-hFVIII-V3), which contains a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain that are highly conserved through evolution. In murine studies, AAV-HLP-hFVIII-V3 mediated expression of FVIII at 3-fold higher levels when compared to AAV-HLP-hFVIII-SQ. Methods: The safety and efficacy of a single intravenous infusion of AAV8-HLP-hFVIII-V3, pseudotyped with AAV serotype 8 capsid was assessed in three adult men with severe hemophilia A (FVIII activity levels ≤1% of normal) in the context of an Investigator led, Phase I/II, open-label, non-randomized, dose-escalation trial (ClinicalTrials.gov: NCT03001830GO-8). The first subject received a dose of 6x1011vg/kg and the subsequent two patients each received a dose of 2x1012vg/kg. AAV8-HLP-hFVIII-V3 was manufactured in mammalian HEK 293T cells. The subjects have been followed up for 13-47 weeks after vector administration. Results: Peripheral vein administration of AAV8-HLP-hFVIII-V3 was well tolerated in all patients with no infusion-related reactions. Transgenic FVIII was detectable within two weeks and was more than 5 IU/dl by 6 weeks of gene transfer in all three subjects. Factor VIII activity (one stage clotting assay) levels have remained stable at 7±1IU/dl in patient 1 over a period of 47 weeks. The second participant is 20 weeks following administration of 2x1012 vg/kg of AAV8-HLP-hFVIII-V3 and has steady-state FVIII activity of 6±2IU/dl. In the third subject, who was also treated at a dose of 2x1012 vg/kg, the steady state FVIII activity is almost 10 times higher at 69±7 IU/dl. Elevation of serum alanine aminotransferase was observed in patients 1 and 3 at between weeks 4-6 after gene transfer, reaching peak levels that were 1.5 X upper limit of the normal range. Both patients were treated with corticosteroids within 48 hours of the onset of transaminitis with no loss of transgene expression. No participant has developed a FVIII inhibitor. Conclusion: Our preliminary results from the ongoing Phase I/II study demonstrate FVIII activity levels >5% in all three subjects with normalization of FVIII:C levels in one patient. These levels are sufficient to reduce/prevent spontaneous hemorrhage and have been achieved using relatively lower doses of AAV8-HLP-FVIII-V3 than reported previously with a related FVIII expression cassette. No Grade III (CTCAE v4.03) or greater adverse events have been observed over a period of 47 weeks after administration of AAV8-HLP-hFVIII-V3. Disclosures Nathwani: Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tuddenham:Freeline: Consultancy; BioMarin: Consultancy, Patents & Royalties. Chowdary:Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy. McIntosh:Freeline: Consultancy.

Background: We have previously shown that a single intravenous administration of a self-complementary adeno-associated virus (scAAV) vector containing a codon-optimised factor IX gene, under control of a synthetic liver specific promoter and pseudotyped with serotype 8 capsid, (scAAV2/8-LP1-hFIXco) resulted in a dose-dependent increase in plasma FIX levels in all 10 enrolled severe hemophilia B (HB) patients (ClinicalTrials.gov:NCT00979238; Nathwani et al 2011). FIX activity was stably maintained for at least 3 years (Nathwani et al 2014) but concerns over FIX expression declining over time remain. This is because AAV-mediated transgene expression is mediated mainly by episomally retained viral genomes, which may be lost with natural hepatocyte turn-over. The only vector-associated adverse event was an asymptomatic rise in liver enzymes associated with a decline in FIX levels, occurring within 3 months of gene transfer in two-thirds of the patients treated at a dose of 2x1012 vector genomes(vg)/kg. Liver enzymes normalized with corticosteroids without complete loss of transgene expression. There was no long-lasting toxicity over a period of 3 years but further follow-up is required. The vector preparation used contained an excess of empty capsids, which lacked a full-length viral genome, and are therefore, non-functional but capable of provoking an immune response against transduced hepatocytes. Therefore, a new clinical preparation of scAAV2/8-LP1-hFIXco was manufactured from which most of the empty particles were removed by caesium chloride density centrifugation in the hope that this would reduce the risk of hepatotoxicity. We report on the evaluation of this new vector preparation in severe HB patients and provide an update on up to 8 years follow-up of our original cohort of patients. Methods: Ten subjects were recruited in 2010-2012 to the initial dose-escalation/extension study arm, which entailed a single intravenous infusion of scAAV2/8-LP1-hFIXco (full: empty capsid ratio ~1:10) at a dose of either 2x1011vg/kg, 6x1011vg/kg or 2x1012vg/kg. Two severe HB patients (FIX Results: Transgenic FIX activity levels have remained stable in all 10 subjects treated in the initial dose escalation/extension arm over a median follow-up of 6.7±1.0 years with mean levels in the three dose cohorts at the time of reporting of 1.9±0.6, 2.3±0.3 and 5.1±1.4 IU/l respectively. Over this period, annual FIX concentrate usage has dropped by 66% and annual bleed rate has declined by 82% when compared to pre-gene therapy levels. No late toxicity was observed. Neutralising antibodies to FIX were not detected in any patient but anti-AAV8 capsid-specific antibody levels persisted at high titres in 9 of 10 patients. In patients treated with the new preparation of scAAV2/8-LP1-hFIXco (median follow up = 2.1±1.4 years), mean FIX activity in the 2x1012vg/kg dose cohort was 2.6±0.7 IU/l. This is lower than observed previously at this dose level, but the difference is not statistically significant. Mean steady state FIX levels in the 5x1012vg/kg cohort were 17±5 IU/l. FIX antigen to activity ratio was 1:1. Elevation of serum alanine aminotransferase was observed in 3 of 4 patients treated with the new vector preparation, requiring treatment with corticosteroids. Conclusion: This is the first report to demonstrate stable therapeutic expression of FIX in patients with severe HB over a period of 8 years following systemic administration of scAAV2/8-LP1-hFIXco without late toxicities. We show for the first time that reducing the capsid load by removing empty AAV capsids does not appear to reduce the incidence of hepatotoxicity in patients with severe HB suggesting that other factors are involved in the aetiology of this process. Disclosures Nathwani: Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Chowdary:Freeline: Consultancy; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria. McIntosh:Freeline: Consultancy. Recht:Biogen: Research Funding; Shire: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees.

Background Quisinostat (JNJ-26481585) is a second-generation pyrimidyl-hydroxamic acid histone deacetylase (HDAC) inhibitor with high cellular potency towards Class I and II HDACs. Quisinostat was selected for clinical development as it showed prolonged pharmacodynamic effects in vivo and demonstrated improved single agent antitumoral efficacy compared to other analogs. Procedures Quisinostat was tested against the PPTP in vitro panel at concentrations ranging from 1.0 nM to 10 μM and was tested against the PPTP in vivo panels at a dose of 5 mg/kg (solid tumors) or 2.5 mg/kg (ALL models) administered intraperitoneally daily × 21. Results In vitro quisinostat demonstrated potent cytotoxic activity, with T/C% values approaching 0% for all of the cell lines at the highest concentration tested. The median relative IC50 value for the PPTP cell lines was 2.2 nM (range

Purpose: Pediatric adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy. Conventional chemotherapeutic agents have shown limited utility and are largely ineffective in treating children with advanced ACC. The lack of cell lines and animal models of pediatric ACC has hampered the development of new therapies. Here we report the establishment of the first pediatric ACC xenograft model and the characterization of its sensitivity to selected chemotherapeutic agents. Experimental Design: A tumor from an 11-year-old boy with previously untreated ACC was established as a subcutaneous xenograft in immunocompromised CB17 scid−/− mice. The patient harbored a germline TP53 G245C mutation, and the primary tumor showed loss of heterozygosity with retention of the mutated TP53 allele. Histopathology, DNA fingerprinting, gene expression profiling, and biochemical analyses of the xenograft were conducted and compared with the primary tumor and normal adrenal cortex. The second endpoint was to assess the preliminary antitumor activity of selected chemotherapeutic agents. Results: The xenograft maintained the histopathologic and molecular features of the primary tumor. Screening the xenograft for drug responsiveness showed that cisplatin had a potent antitumor effect. However, etoposide, doxorubicin, and a panel of other common cancer drugs had little or no antitumor activity, with the exception of topotecan, which was found to significantly inhibit tumor growth. Consistent with these preclinical findings, topotecan as a single agent in a child with relapsed ACC resulted in disease stabilization. Conclusion: Our study established a novel TP53-associated pediatric ACC xenograft and identified topotecan as a potentially effective agent for treating children with this disease. Clin Cancer Res; 19(7); 1740–7. ©2013 AACR.

Background The DNA methylating agent temozolomide was developed primarily for treatment of glioblastoma. However, preclinical data have suggested a broader application for treatment of childhood cancer. Temozolomide was tested against the PPTP solid tumor and ALL models.

Background Nuclear factor-κB (NF-κB) has been implicated in tumor cell proliferation and survival, and tumor angiogenesis. We sought to evaluate the effects of curcumin, an inhibitor of NF-κB, on a xenograft model on disseminated neuroblastoma.

We explored adeno-associated viral vector (AAV)–mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 1013 vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 1011 vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.

LAMP1 oversialylation results in excessive lysosomal exocytosis, promoting tumor invasion and drug resistance., Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf−⁄− mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance.

Background MLN4924 is an investigational first-in-class small molecule inhibitor of NEDD8-activating enzyme (NAE). NAE is an essential component of the NEDD8 conjugation pathway, controlling the activity of a subset of ubiquitin-proteasome system (UPS) E3 ligases, multiprotein complexes that transfer ubiquitin molecules to substrate proteins. Procedures MLN4924 was tested against the PPTP in vitro panel using 96-hour exposure time at concentrations ranging from 1.0 nM to 10 µM. It was tested in vivo at a dose of 100 mg/kg [66 mg/kg for the acute lymphoblastic leukemia (ALL) xenografts] administered orally twice daily × 5 days. Treatment duration was 3 weeks. Results The median relative IC50 for MLN4924 against the PPTP cell lines was 143 nM, (range: 15–678 nM) with that for the Ewing panel being significantly lower (31 nM). MLN4924 induced significant differences in EFS distribution compared to control in 20 of 34 (59%) evaluable solid tumor xenografts. MLN4924 induced intermediate activity (EFS T/C values >2) in 9 of the 33 evaluable xenografts (27%), including 4 of 4 glioblastoma xenografts, 2 of 3 Wilm's tumor xenografts, 2 of 5 rhabdomyosarcoma xenografts, and 1 of 4 neuroblastoma xenografts. For the ALL panel, 5 of 8 evaluable xenografts showed intermediate activity for the EFS T/C measure. MLN4924 did not induce objective responses in the PPTP solid tumor or ALL panels. Conclusions MLN4924 showed potent activity in vitro and in vivo showed tumor growth inhibitory activity against a subset of the PPTP solid tumor and ALL xenografts. Pediatr Blood Cancer 2012;59:246–253. © 2011 Wiley Periodicals, Inc.

JNJ-26854165 was originally developed as an activator of p53 capable of inducing apoptosis in cancer cell lines. In vitro, JNJ-26854165 demonstrated cytotoxic activity. The ALL cell line panel had a significantly lower median IC(50) (0.85 µM) than the remaining cell lines. In vivo JNJ-26854165 induced significant differences in EFS distribution compared to control in 18 of 37 solid tumors and in 5 of 7 of the evaluable ALL xenografts. Objective responses were observed in 4 of 37 solid tumor xenografts, and 2 of 7 ALL xenografts achieved PR or CR. Responses were noted in xenografts with both mutant and wild-type p53.

Background High-grade glioblastomas have immature, leaky tumor blood vessels that impede the efficacy of adjuvant therapy. We assessed the ability of human interferon (hIFN)-β delivered locally via gene transfer to effect vascular stabilization in an orthotopic model of glioblastoma xenograft resection. Methods Xenografts were established by injecting 3 grade IV glioblastoma cell lines (GBM6-luc, MT330-luc, and SJG2-luc) into the cerebral cortex of nude rats. Tumors underwent subtotal resection, and then had gel foam containing an adeno-associated virus vector encoding either hIFN-β or green fluorescence protein (control) placed in the resection cavity. The primary endpoint was stabilization of tumor vasculature, as evidenced by CD34, α-SMA, and CA IX staining. Overall survival was a secondary endpoint. Results hIFN-β treatment altered the tumor vasculature of GBM6-luc and SJG2-luc xenografts, decreasing the density of endothelial cells, stabilizing vessels with pericytes, and decreasing tumor hypoxia. The mean survival for rats with these neoplasms was not improved, however. In rats with MT330-luc xenografts, hIFN-β resulted in tumor regression with a 6-month survival of 55% (INF-β group) and 9% (control group). Conclusion The use of AAV hIFN-β in our orthotopic model of glioblastoma resection stabilized tumor vasculature and improved survival in rats with MT330 xenografts.

LCL161, a SMAC mimetic, was tested against the PPTP in vitro panel (1.0 nM to 10.0 µM) and the PPTP in vivo panels (30 or 75 mg/kg [solid tumors] or 100 mg/kg [ALL]) administered orally twice in a week. LCL161 showed a median relative IC50 value of >10 µM, being more potent against several leukemia and lymphoma lines. In vivo LCL161 induced significant differences in EFS distribution in approximately one-third of solid tumor xenografts (osteosarcoma and glioblastoma), but not in ALL xenografts. No objective tumor responses were observed. In vivo LCL161 demonstrated limited single agent activity against the pediatric preclinical models studied. Pediatr Blood Cancer 2012; 58: 636–639. © 2011 Wiley Periodicals, Inc.

Background IMC-A12, a fully human antibody that blocks ligand binding to the Type 1 insulin-like growth factor receptor, and rapamycin, a selective inhibitor of mTORC1 signaling, have both demonstrated significant antitumor activity against PPTP solid tumor models. Here we have evaluated antitumor activity of each agent individually and in combination against nine tumor models. Procedures IMC-A12 was administered twice weekly and rapamycin was administered daily for 5 days per week for a planned 4 weeks. The impact of combining IMC-A12 with rapamycin was evaluated using two measures: (1) the “therapeutic enhancement” measure, and (2) a linear regression model for time-to-event to formally evaluate for sub- and supra-additivity for the combination compared to the agents used alone. Results Two osteosarcomas, and one Ewing sarcoma of the nine xenografts tested showed therapeutic enhancement. The combination effect was most dramatic for EW-5 for which PD2 responses of short duration were observed for both single agents and a prolonged PR response was observed for the combination. Both OS-2 and OS-9 showed significantly longer times to progression with the combination compared to either of the single agents, although objective response criteria were not met. Conclusions The combination of IMC-A12 with rapamycin was well tolerated, and induced tumor responses that were superior to either single agent alone in several models. These studies confirm reports using other antibodies that inhibit IGF-1 receptor-mediated signaling that indicate enhanced therapeutic effect for this combination, and extend the range of histotypes to encompass additional tumors expressing IGF-1R where this approach may be effective. Pediatr Blood Cancer 2012; 58: 729–735. © 2011 Wiley Periodicals, Inc.

Background The centromere kinesin motor protein CENP-E plays a crucial role in mitosis, and is an appealing molecular target in cancer. GSK923295A is an allosteric inhibitor of CENP-E that is undergoing clinical evaluation.

Background Genz-644282 is a novel non-camptothecin topoisomerase I poison that is in clinical development. Procedures Genz-644282 was tested against the PPTP in vitro panel (0.1 nM to 1 µM), and in vivo using three times per week × 2 schedule repeated at day 21 at its maximum tolerated dose (MTD) of 4 mg/kg. Subsequently Genz-644282 was tested at 4, 3, 2, and 1 mg/kg in 3 models to assess the dose–response relationship. mRNA gene signatures predictive for Genz-644282 response in vitro were applied to select 15 tumor models that were evaluated prospectively. Results In vitro, Genz-644282 demonstrated potent cytotoxic activity with a median IC50 of 1.2 nM (range 0.2–21.9 nM). In vivo, Genz-644282 at its MTD (4 mg/kg) induced maintained complete responses (MCR) in 6/6 evaluable solid tumor models. At 2 mg/kg Genz-644282 induced CR or MCR in 3/3 tumor models relatively insensitive to topotecan, but there were no objective responses at 1 mg/kg. Further testing at 2 mg/kg showed that Genz-644282 induced objective regressions in 7 of 17 (41%) models. There was a significant correlation between predictive response scores based on Affymetrix U133Plus2 baseline tumor expression profiles and the observed in vivo responses to Genz-644282. Conclusions Genz-644282 was highly active within a narrow dose range (2–4 mg/kg), typical of other topoisomerase I poisons. As with other topoisomerase I poisons, how accurately these data will translate to clinical activity will depend upon the drug exposures that can be achieved in children treated with this agent. Pediatr Blood Cancer 2012; 58: 200–209. © 2011 Wiley Periodicals, Inc.

Ganetespib, an Hsp90 inhibitor, was tested against the PPTP in vitro cell line panel and selected xenografts in vivo, including JAK2- and BRAF-mutated models. Ganetespib demonstrated potent in vitro cytotoxic activity (median rIC50 8.8 nM, range 4.4-27.1 nM). In vivo, ganetespib induced significant differences in EFS distribution for 4 of 11 xenografts. Intermediate activity (EFS T/C > 2) was noted only for the MV4;11 xenograft, and there were no objective responses. Administered as single agents, Hsp90 inhibitors examined by the PPTP have shown limited evidence for a therapeutic window against both solid tumor and leukemia pediatric preclinical models.

Background Lenalidomide, a novel immunomodulatory agent, is reported to modulate stem cell differentiation, and have direct antiproliferative activity as well as inhibit inflammation and hyperalgesia. On the basis of this varied pharmacological profile, lenalidomide is under investigation as a treatment for a range of oncologic indications. Procedures Lenalidomide was evaluated against the PPTP in vitro panel using 96-hr exposure at concentrations ranging from 1 nM to 10 µM. It was tested against the PPTP in vivo panels at a dose of 30 mg/kg administered orally (PO) once daily for a planned for 6 weeks. Results In vitro activity was not observed at concentrations up to 10 µM. Lenalidomide was well tolerated, and induced significant differences in EFS distribution compared to control in 7 of 37 (18.9%) of the evaluable solid tumor xenografts and in 0 of 8 (0%) of the evaluable ALL xenografts. The best response in the solid tumor panel was PD2 [progressive disease with growth delay (EFS T/C > 1.5)], observed in 4 of 37 (10.8%) solid tumor xenografts. A single ALL xenograft showed a PD2 response. Conclusions Direct antiproliferative effects of lenalidomide were not observed in vitro. In vivo lenalidomide demonstrated low activity against tumors in immune-deficient mice. Our results suggest that lenalidomide's utility in the pediatric clinical setting may depend upon its ability to induce antitumor activity through effects on host immune and stromal cells rather than through direct effects on tumor cells. Pediatr Blood Cancer 2011; 57: 606–611. © 2011 Wiley-Liss, Inc.

Background PG11047 is a novel conformationally restricted analog of the natural polyamine, spermine that lowers cellular endogenous polyamine levels and competitively inhibits natural polyamine functions leading to cancer cell growth inhibition. The activity of PG11047 was evaluated against the PPTP's in vitro and in vivo panels. Procedures PG11047 was evaluated against the PPTP in vitro panel using 96 hr exposure at concentrations ranging from 10 nM to 100 µM. It was tested against the PPTP in vivo panels at a dose of 100 mg/kg administered by the intraperitoneal route weekly for 6 weeks. Results In vitro PG11047 demonstrated a concentration–response pattern consistent with cytostatic activity. The median EC50 for PG11047 was 71 nM. Cell lines of the Ewing sarcoma panel had a lower median EC50 value compared to the remaining cell lines in the panel, while cell lines of the neuroblastoma panel had a higher median EC50 value. In vivo PG11047 induced significant differences in EFS distribution compared to control in 5 of 32 (15.6%) of the evaluable solid tumor xenografts and in 0 of 7 (0%) of the evaluable ALL xenografts. The single case of tumor regression occurred in an ependymoma xenograft. Conclusions Further pediatric development of PG11047 will require better defining a target population and identifying combinations for which there is a tumor-selective cytotoxic effect. The regression observed for an ependymoma xenograft and the exquisite sensitivity of some Ewing sarcoma cell lines to the antiproliferative effects of PG11047 provide leads for further preclinical investigations. Pediatr Blood Cancer 2011; 57: 268–274. © 2011 Wiley-Liss, Inc.

Background AZD8055 is a small molecule ATP-competitive inhibitor of the serine/threonine kinase mTOR that regulates cap-dependent translation through the mTORC1 complex and Akt activation through the mTORC2 complex. Procedures AZD8055 was tested against the PPTP in vitro panel at concentrations ranging from 1.0 nM to 10 µM and against the PPTP in vivo panels at a dose of 20 mg/kg administered orally daily × 7 for 4 weeks. Results In vitro the median relative IC50 for AZD8055 against the PPTP cell lines was 24.7 nM. Relative I/O values >0% (consistent with a cytostatic effect) were observed in 8 cell lines and 15 cell lines showed Relative I/O values ranging from −4.7 to −92.2% (consistent with varying degrees of cytotoxic activity). In vivo AZD8055 induced significant differences in EFS distribution compared to controls in 23 of 36 (64%) evaluable solid tumor xenografts, and 1 of 6 evaluable ALL xenografts. Intermediate activity for the time to event activity measure (EFS T/C >2) was observed in 5 of 32 (16%) solid tumor xenografts evaluable. The best response was stable disease. PD2 (progressive disease with growth delay) was observed in 20 of 36 (55.6%) evaluable solid tumor xenografts. AZD8055 significantly inhibited 4E-BP1, S6, and Akt phosphorylation following day 1 and day 4 dosing, but suppression of mTORC1 or mTORC2 signaling did not predict tumor sensitivity. Conclusions AZD8055 demonstrated broad activity in vitro, but at the dose and schedule studied demonstrated limited activity in vivo against the PPTP solid tumor and ALL panels. Pediatr Blood Cancer 2012; 58: 191–199. © 2011 Wiley Periodicals, Inc.

Background PR-104 is rapidly hydrolyzed to PR-104A in vivo, which is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and amine (PR-104M) to produce DNA interstrand cross-links. PR-104 activation can occur via hypoxia-dependent reductases and also independently of hypoxia by aldo-keto reductase (AKR) 1C3. Procedures PR-104A was tested against the PPTP in vitro panel (10 nM to 100 µM), and PR-104 in vivo using a weekly × 6 schedule at its maximum tolerated dose (MTD) of 550 mg/kg. Subsequently PR-104 was tested at 270 and 110 mg/kg. Pharmacokinetics for PR-104 and its metabolites were determined, as were levels of AKR1C3 RNA and protein in xenografts. Results In vitro, the leukemia models were most sensitive to PR-104A. In vivo, PR-104 induced objective responses at its MTD in 21/34 solid tumor models and maintained complete responses against 7/7 acute lymphoblastic leukemia (ALL) models. At 270 mg/kg and lower dose levels, PR-104 did not induce solid tumor regressions, suggesting a steep dose–response relationship. Pharmacokinetic analysis suggests higher systemic exposures to PR-104A and its metabolites in mice compared to those achievable in patients. Levels of AKR1C3 protein did not correlate with tumor responsiveness. Conclusions As monotherapy, PR-104 demonstrated a high level of activity against both solid tumor and ALL models at its MTD, but the activity was almost completely lost at half the MTD dose for solid tumors. Pharmacokinetic data at the PR-104 MTD from human trials suggest that PR-104 metabolites may not reach the plasma exposures in children that were associated with high-level preclinical activity. Pediatr Blood Cancer 2011; 57: 443–453. © 2010 Wiley-Liss, Inc.

Background BMS-754807 is a small molecule ATP-competitive inhibitor of the type-1 insulin-like growth factor receptor currently in phase 1 clinical trials. Procedures BMS-754807 was tested against the Pediatric Preclinical Testing Program (PPTP) in vitro panel at concentrations ranging from 1.0 nM to 10 µM and was tested against the PPTP in vivo panels at a dose of 25 mg/kg administered orally BID for 6 days, repeated for 6 weeks. Results In vitro BMS-754807 showed a median EC50 value of 0.62 µM against the PPTP cell lines. The median EC50 for the four Ewing sarcoma cell lines was less than that for the remaining PPTP cell lines (0.19 µM vs. 0.78 µM, P = 0.0470). In vivo BMS-754807 induced significant differences in EFS distribution compared to controls in 18 of 32 evaluable solid tumor xenografts (56%) tested, but in none of the ALL xenografts studied. Criteria for intermediate activity for the time to event activity measure (EFS T/C > 2) were met in 7 of 27 solid tumor xenografts evaluable for this measure. The best response was PD2 (progressive disease with growth delay), which was observed in 18 of 32 solid tumor xenografts. PD2 responses were most commonly observed in the rhabdomyosarcoma, neuroblastoma, osteosarcoma, Ewing sarcoma, and Wilms tumor panels. Conclusions BMS-754807 activity in vitro is consistent with a specific IGF-1R effect that has half-maximal response in the 0.1 µM range and that is observed in a minority of the PPTP cell lines. In vivo intermediate activity was most commonly observed in the neuroblastoma and rhabdomyosarcoma panels. Pediatr Blood Cancer 2011;56:595–603. © 2010 Wiley-Liss, Inc.

Background The National Cancer Institute (NCI) has established the Pediatric Preclinical Testing Program (PPTP) for testing drugs against in vitro and in vivo childhood cancer models to aid in the prioritization of drugs considered for early phase pediatric clinical trials. Procedures In vitro cytotoxicity testing employs a semi-automated fluorescence-based digital imaging cytotoxicity assay (DIMSCAN) that has a 4-log dynamic range of detection. Curve fitting of the fractional survival data of the cell lines in response to various concentrations of the agents was used to calculate relative IC50, absolute IC50, and Ymin values. The panel of 23 pediatric cancer cell lines included leukemia (n = 6), lymphoma (n = 2), rhabdomyosarcoma (n = 4), brain tumors (n = 3), Ewing family of tumors (EFT, n = 4), and neuroblastoma (n = 4). The doubling times obtained using DIMSCAN were incorporated into data analyses to estimate the relationship between input cell numbers and final cell number. Results We report in vitro activity data for three drugs (vincristine, melphalan, and etoposide) that are commonly used for pediatric cancer and for the mTOR inhibitor rapamycin, an agent that is currently under preclinical investigation for cancer. To date, the PPTP has completed in vitro testing of 39 investigational and approved agents for single drug activity and two investigational agents in combination with various “standard” chemotherapy drugs. Conclusions This robust in vitro cytotoxicity testing system for pediatric cancers will enable comparisons to response data for novel agents obtained from xenograft studies and from clinical trials. Pediatr Blood Cancer 2011;56:239–249. © 2010 Wiley-Liss, Inc.

Background GSK690693 is a small molecule ATP-competitive inhibitor of the pro-survival kinase Akt. Since Akt regulates multiple downstream targets including transcription factors, glycogen synthase 3, the pro-apoptotic protein Bad, as well as MDM2 and mTORC1, it was tested against the in vitro and in vivo panels of the Pediatric Preclinical Testing Program (PPTP). Procedures GSK690693 was tested in vitro at concentrations from 1 nM to 10 µM, and against the in vivo panel of xenografts at a dose of 30 mg/kg daily × 5 for 6 consecutive weeks. Three measures of in vivo antitumor activity were used: (1) an objective response measure modeled after the clinical setting; (2) a treated to control (T/C) tumor volume measure; and (3) a time to event measure based on the median event-free survival (EFS) of treated and control animals for each xenograft. Results GSK690693 inhibited cell growth in vitro with IC50 values between 6.5 nM and >10 µM. In vivo, GSK690693 significantly increased EFS in 11 of 34 (32%) solid tumor xenografts, most notably in all 6 osteosarcoma models, but not in any of the 8 ALL xenografts tested. No objective responses were observed and only one solid tumor met EFS T/C criteria for intermediate activity. Conclusions GSK690693 demonstrated broad activity in vitro, however our results against both the solid tumor and ALL PPTP in vivo panels demonstrate that, as single agent at the dose and schedule used, GSK690693 has only modest antitumor activity. Pediatr Blood Cancer. 2010;55:1329–1337. © 2010 Wiley-Liss, Inc.