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Despite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed ex vivo drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials. Significance: Patients with relapsed/refractory leukemias face limited treatment options. Systematic integration of precision medicine efforts can inform therapy. We report the feasibility of identifying targetable mutations in children with leukemia and describe correlative biology studies validating therapeutic hypotheses and novel mutations. See related commentary by Bornhauser and Bourquin, p. 1322. This article is highlighted in the In This Issue feature, p. 1307

Activating variants of the MAPK pathway have been found in some Langerhans cell histiocytosis (LCH) lesions. Inhibition of the MAPK pathway with trametinib (MEK inhibitor) has been shown to induce responses in LCH patients. Two adolescent males with LCH driven by BRAF p.N486_P490del have received trametinib for >1 year with no reactivation in one and partial response in another (including stable lung disease). A third male with neonatal LCH and MAP2K1p.K57_G61del had a complete response to trametinib with no active disease after 22 months. All patients continue on trametinib monotherapy with tolerable skin and creatine phosphokinase toxicity.

10003 Background: In B-acute lymphoblastic leukemia (B-ALL), CNS2 was associated with inferior 5-year (yr) event-free and overall survival (EFS/OS) in recent trials. Here, we report the impact of CNS2 in T-ALL on AALL0434 and AALL1231, recently completed consecutive randomized phase 3 trials for children and young adults with T-ALL and T Lymphoblastic Lymphoma. This report is limited to T-ALL. Both trials used augmented Berlin Frankfurt Münster regimens. AALL0434 compared Capizzi escalating methotrexate+pegaspargase (C-MTX) vs High Dose MTX (HDMTX) +/- six nelarabine (Nel) courses; outcomes improved with CMTX and Nel. CNS1/CNS2 patients, except those defined as low risk (LR) received 12Gy cranial radiation (CRT); CNS3 patients received 18Gy CRT. AALL1231 randomized patients to +/- bortezomib (Bort). AALL1231 changed the AALL0434 backbone, using dexamethasone instead of prednisone throughout. CRT was given only to patients with CNS3 disease (18Gy) and those defined as very high risk (VHR) (12Gy). CNS2 patients could not be classified as LR on AALL0434 or standard risk (SR) on AALL1231. CNS1/CNS2 patients received the same intrathecal therapy frequency on both studies. Methods: CNS status was assigned at diagnosis. CNS2 defined as: presence of < 5/ μL WBCs and cytospin positive for blasts or ≥ 5/μLWBCs with negative Steinherz Bleyer algorithm. Outcomes by CNS status were compared between AALL0434 and AALL1231. Results: From 2007-2014, AALL0434 enrolled 1562 evaluable T-ALL patients, including 1128 (72.8%) CNS1, 306 (19.7%) CNS2 and 116 (7.5%) CNS3. 90.8% received CRT, including 90.4% of CNS1 patients. 5yr EFS rates for CNS1, 2, and 3 were 85.2±1.3%, 83.1±2.6%, and 71.4±5.2% (p = 0.0007); OS rates were 90.4±1.1%, 89.2±2.1%, and 83.1±4.3% (p = 0.0438). There were no differences in 5yr disease free survival (DFS) between CNS1 and CNS2 treated with CMTX (89.7% vs. 92.9%, p = 0.17) or CMTX+Nel (91.8% vs. 89.9%; p = 0.62). AALL1231 accrued 614 evaluable T-ALL patients [CNS1 437 (71.1%), CNS2 134 (21.8%), CNS3 43 (7.0%)] from 2014 to early closure in 2017. Of these, only 12% were scheduled to receive CRT. 3yr EFS rates for CNS1, 2 and 3 were 84.1±2.1%, 84.6±3.8% and 78.6±7.9% (p = 0.50). 3yr OS was: CNS1 87.5±1.9%, CNS2 92.2±2.8%, CNS3 78.5±7.9% (p = 0.017) . 3yr EFS was not statistically distinct without Bort in CNS1, 2 or 3 (85.3±2.9%, 81.4±5.6%, 71.9±13.4%) (p = 0.10) or with Bort (82.9±3.0%, 88.3±4.9%, 83.3±9,4%; p = 0.43). Intermediate risk (IR) CNS1 and CNS2 patients received identical therapy and had similar 3yr EFS (88.8±2.8% vs 88.8±3.5%, p = 0.98). Conclusions: Unlike in B-ALL, EFS/OS was similar for CNS1 and CNS2 on AALL0434 (with CRT) and AALL1231 (without CRT). Further, IR CNS1 and CNS2 on AALL1231 had similar outcomes with identical therapy. Thus, CNS2 status is non-prognostic in T-ALL on these contemporary COG regimens. CNS3 patients have poor outcomes in T-ALL despite CRT and intensive chemotherapy, novel approaches are needed. Clinical trial information: NCT00408005, NCT02112916.

Key Points Children treated with blinatumomab for B-ALL with MRD had few side effects and proceeded to hematopoietic cell transplant without delay. Blinatumomab given prior to transplant reduces MRD and results in favorable leukemia-free survival, toxicity, and overall survival.

Erwinia chrysanthemi-derived asparaginase is increasingly integral to acute lymphoblastic leukemia therapy. In our series, 16% of patients developed symptomatic hyperammonemia following Erwinia administration with symptoms including refractory nausea, vomiting, profound fatigue, malaise, and coma. This series of patients receiving Erwinia indicates higher than expected incidence of hyperammonemia, correlation between ammonia and asparaginase levels and therapeutic asparaginase activity levels despite dose reduction. The series provides evidence for investigation into which patients require intervention to prevent toxicity, which patients may have ammonia levels used as an asparaginase activity surrogate and which patients may achieve equivalent efficacy with abridged dosing.

Introduction: Despite progress in the treatment of acute myeloid leukemia (AML) over the past few decades, long-term survival of children, adolescents and young adults remains poor. Most treatment failure is secondary to the development of chemotherapy resistance resulting in relapsed or refractory disease. Epigenetic changes in leukemia cells, such as DNA methylation and/or histone acetylation, can lead to alterations in gene expression thereby causing resistance to chemotherapeutic agents. These epigenetic changes are potentially reversible with agents that demethylate DNA and/or inhibit histone deacetylation which ultimately leads to restoration of normal gene expression and chemosensitivity. We are reporting an ongoing phase I trial of decitabine and vorinostat, two epigenetic modifying agents, followed by chemotherapy in children, adolescents and young adults with relapsed or refractory AML. Methods: T2016-003 is a phase I study that was developed and activated in the TACL consortium in July 2017 for patients 1 to 25 years of age with relapsed or refractory AML. The study followed a standard 3 + 3 phase I cohort escalation design. Three dose levels of decitabine were investigated: dose level 1 (7.5 mg/m2); dose level 2 (10 mg/m2); and dose level 3 (15 mg/m2). Patients received decitabine (at assigned dose level) and vorinostat (age 18 years, 200 mg twice daily) days 1 to 5 followed by fludarabine (30 mg/m2, days 6 to 10), cytarabine (2000 mg/m2, days 6 to 10) and granulocyte colony stimulating factor (G-CSF) (5 μ/kg/dose, days 5 until neutrophil recovery) (FLAG). Intrathecal therapy was given up to 72 hours prior to the initial doses of decitabine/vorinostat, with additional intrathecal therapy for central nervous system disease. Patients could receive up to two cycles of therapy. End of course evaluation by bone marrow minimal residual disease (MRD) testing using flow cytometry was performed between days 35 and 42 or upon adequate blood count recovery. Results: Twenty-five patients have enrolled to date with 24 evaluable for dose limiting toxicity (DLT) and response. Median age was 7 (range, 1 to 20) years. Fourteen patients (58.3%) had relapsed after a prior bone marrow transplant and 7 (29.2%) had refractory disease at time of enrollment. There were no dose limiting toxicities at dose level 1 (n=3, 7.5 mg/m2) or 2 (n=3, 10 mg/m2) of decitabine. However, at dose level 3 (n=12, 15 mg/m2), 3 subjects reported Grade 4 invasive fungal infection (Aspergillus terreus, Candida parapsilosis and Rhizomucor pusillus) leading to de-escalation of decitabine to dose level 2. The study continued dose expansion at dose level 2 without DLT, enrolling 7 additional patients. Twenty-two subjects have completed the study and are evaluable for response. Responses were noted at all dose levels. Eight subjects (36.4%) achieved a complete response (CR) or complete response with incomplete hematologic recovery (CRi). Five of the 8 subjects who achieved a CR/CRi were also negative for MRD. Eight subjects had known epigenetic lesions (i.e. KMT2Ar, CEBPα, IDH2) with 6 (75%) achieving a CR, 4 of whom (66.7%) were also MRD negative. Conclusion: Overall, we identified decitabine (10 mg/m2) in combination with vorinostat and followed by FLAG chemotherapy to be well-tolerated and an effective regimen in pediatric patients with relapsed AML, particularly in subjects with known epigenetic lesions. The study continues to enroll at dose level 2 of decitabine for a total accrual goal of 33 subjects. Correlative analysis of peripheral blood and bone marrow investigating methylation, histone acetylation and gene expression is ongoing. Disclosures Burke: Amgen, Inc.: Consultancy, Speakers Bureau.

Granulocytic sarcoma (GS) is a rare manifestation of myeloid proliferation, characterized by formation of a mass comprised of immature cells of myeloid origin. Orbital granulocytic sarcoma is rarer still, with only a small fraction of GS patients having orbital involvement. Given the rarity of orbital GS, no unified therapy plan has been identified, as large prospective trials are not feasible, but it is widely accepted that patients with GS ought to be treated with systemic intensive chemotherapy consistent with standard of care regimens for acute myelogenous leukemia (AML) or chronic myelogenous leukemia (CML). Development of a treatment plan for GS in poor responders involves a systemic leukemia plan as novel therapeutics have not been investigated for treatment GS per se, but used more widely for AML. GS is most commonly associated with AML and thus will be addressed in that context in this review. Patients with GS associated with CML should receive CML-specific therapy. When conventional and traditional cytotoxic GS/AML chemotherapy regimens are insufficient, patients often require a combination of novel therapeutics, stem cell transplantation (SCT), and radiation. Much of the recent advancement in AML therapy, as well as in AML translational research, has been in targeting molecular facets of the disease and enabling more specificity with treatment. The aim of treating patients for whom conventional treatment was unsuccessful with personalized therapy has not yet been realized, but many of the novel therapeutics reviewed below have demonstrated promise and are cause for optimism. In our center, when a GS/AML patient is refractory to frontline therapy, we rely on novel chemotherapy therapeutic options as outlined below.

We report a 14 year-old female with Giant Cell Tumor of Bone, successfully treated with denosumab, who developed critical hypercalcemia after completion of therapy. Five months after her last denosumab treatment, serum calcium rose to 16.5 mg/dL (normal 8.7–10.8 mg/dL), nearly double her prior level of 8.4 mg/dL while receiving denosumab. She required emergent intervention to treat her hypercalcemia, which was attributed to rebound osteoclast activity and osteopetrotic bone. Denosumab is widely used in adults and increasingly in pediatric oncology populations and our experience demonstrates the need for close monitoring for electrolyte derangements following discontinuation. Pediatr Blood Cancer 2015;62:1078–1080. © 2015 Wiley Periodicals, Inc.

Despite the remarkable pace of characterizing the genomics of pediatric acute leukemias, the integration of real-time sequencing results into clinical practice has lagged. With increased availability of molecularly-targeted therapies, the promise of matching genetic lesions in patients' leukemia cells to treatment has not yet been fully realized. We established the first pediatric leukemia clinical genomics consortium in the United States, known as the Leukemia Precision-based Therapy (LEAP) Consortium, which includes 13 major pediatric cancer institutions. We hypothesized that it is feasible to identify and match, in real-time, actionable alterations with a targeted therapy for pediatric patients with relapsed, refractory or high-risk leukemias or myelodysplastic syndrome (MDS). Using a combination of a DNA-based next-generation sequencing panel and RNA-based gene fusion testing, followed by data review by our multidisciplinary molecular tumor board, we are conducting a clinical trial to test this hypothesis. To date, we have enrolled and reviewed data from 143 patients stratified by disease status: Cohort 1, patients with relapsed or refractory leukemias (n=93), and Cohort 2, patients with de novo high-risk leukemias or MDS (n=50). A matched targeted therapy (MTT) recommendation has been made for 72% (n=103) of patients, tiered based on the level of evidence linking the mutation to potential activity of targeted therapy in the context of each patient's disease (Tier 1: 11%, Tier 2: 4%, Tier 3: 41%, Tier 4: 6%, Tier 5: 10%). Of the 44 patients in Cohort 1 with clinical follow-up data, 5 (11%) had alterations in therapy made based upon sequencing results and MTT recommendation. These include the use of the MEK inhibitor trametinib for RAS mutant acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (n=2), dasatinib for B-ALL and T-ALL with NUP214-ABL1 translocations (n=2) and ponatinib for B-ALL with an ABL1 T315I mutation (n=1). In addition, this genomic data refined diagnosis and supported additional germline assessment in a subset of patients. In parallel to the genomic sequencing, we are conducting high-throughput drug sensitivity (HTS) assays to study in vitro anti-leukemia effects of a panel of up to 120 targeted inhibitors in the context of leukemia-associated genetic alterations. Of the initial 106 accrued patients, 40 (38%) had adequate amounts of blood or bone marrow for in vitro drug testing. All samples were tested in the inhibitor panel assay, and 65% of the samples yielded interpretable results. Inhibitor screening data was compiled for all 12 patients with genetic alterations resulting in Tier 1, 2 or 3 MTT recommendations. This subset of patients had leukemias with two distinct molecular profiles: 1) oncogenic RAS signaling pathway mutations or 2) oncogenic tyrosine kinase alterations. Our molecular tumor board recommended trametinib for the first group of patients and tyrosine kinase inhibitors (TKIs), specific to the mutation, for the second group of patients. In vitro HTS data analysis demonstrated dose-response sensitivity of leukemia cells with RAS pathway mutations to trametinib, many of which had half-maximal inhibitory concentration (IC50) less than 50 nM. Similarly, leukemia cells from patients with FLT3, ABL1 or KIT mutations generally showed dose-responses to relevant TKIs with low IC50s. Overall, HTS data were concordant with MTT recommendations informed by the sequencing results. This first in the US multi-institutional prospective leukemia genomics trial brings state-of-the-art clinical genetic testing, assessment of prognostic biomarkers, selection of patients for germline testing, and targeted therapeutic treatment recommendations to children and young adults with high-risk, relapsed or refractory leukemias or MDS. Our collaboration provides a unique opportunity to perform sophisticated patient-specific in vitro drug testing assays to impact MTT discovery efforts, which we plan to validate in vivo using patient-derived xenograft models where feasible. We believe that a model like the LEAP Consortium has the potential to transform precision medicine approaches for children with high-risk leukemias and to inform future genomics-guided therapeutic trials and drug discovery efforts. Disclosures Tasian: Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding. Burke:JAZZ: Speakers Bureau; Shire: Speakers Bureau; AMGEN: Speakers Bureau. Tyner:AstraZeneca: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Janssen: Research Funding; Array: Research Funding; Constellation: Research Funding; Gilead: Research Funding; Aptose: Research Funding; Incyte: Research Funding; Genentech: Research Funding.

10518Background: Despite the remarkable pace in characterizing the genomics of acute leukemias, integration of sequencing into clinical practice is lagging. With increased availability of targeted ...

// Nathan P. Gossai 1, * , Jordan A. Naumann 1, * , Nan-Sheng Li 4 , Edward A. Zamora 1 , David J. Gordon 3 , Joseph A. Piccirilli 4, 5 , Peter M. Gordon 1, 2 1 Department of Pediatrics, Division of Pediatric Hematology and Oncology, University of Minnesota, Minneapolis, MN, USA 2 University of Minnesota Masonic Cancer Center, Minneapolis, MN, USA 3 Department of Pediatrics, Division of Pediatric Hematology/Oncology, University of Iowa, Iowa City, IA, USA 4 Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA 5 Department of Chemistry, University of Chicago, Chicago, IL, USA * These authors have contributed equally to this work Correspondence to: Peter M. Gordon, email: gord0047@umn.edu Keywords: gold nanoparticles, drug delivery, leukemia, molecularly targeted therapy, anti-sense Received: February 08, 2016 Accepted: May 01, 2016 Published: May 18, 2016 ABSTRACT We describe a customizable approach to cancer therapy in which a gold nanoparticle (Au-NP) delivers a drug that is selectively activated within the cancer cell by the presence of an mRNA unique to the cancer cell. Fundamental to this approach is the observation that the amount of drug released from the Au-NP is proportional to both the presence and abundance of the cancer cell specific mRNA in a cell. As proof-of-principle, we demonstrate both the efficient delivery and selective release of the multi-kinase inhibitor dasatinib from Au-NPs in leukemia cells with resulting efficacy in vitro and in vivo . Furthermore, these Au-NPs reduce toxicity against hematopoietic stem cells and T-cells. This approach has the potential to improve the therapeutic efficacy of a drug and minimize toxicity while being highly customizable with respect to both the cancer cell specific mRNAs targeted and drugs activated.

In patients with relapsed ALL, minimal residual disease (MRD) identified prior to allogeneic hematopoietic cell transplantation (HCT) is a strong predictor of relapse. We report our experience using a combination of reduced-dosing clofarabine, CY and etoposide as a 'bridge' to HCT in eight patients with high risk or relapsed ALL and pre-HCT MRD. All patients had detectable MRD (>0.01%, flow cytometry) at the start of therapy with all eight achieving MRD reduction following one cycle. The regimen was well tolerated with seven grade 3/4 toxicities occurring among four of the eight patients. Five patients (62.5%) are alive, one died from relapse (12.5%) and two from transplant-related mortality (25%). The combination of reduced-dose clofarabine, CY and etoposide as bridging therapy appears to be well tolerated in patients with relapsed ALL and is effective in reducing pre-HCT MRD.