Supplemental Figure 1 and Figure 2 Supplemental Tables 1-5

Abstract Purpose: To determine a safe and biologically active dose of quizartinib (AC220), a potent and selective class III receptor tyrosine kinase (RTK) FLT3 inhibitor, in combination with salvage chemotherapy in children with relapsed acute leukemia. Experimental Design: Quizartinib was administered orally to children with relapsed AML or MLL-rearranged ALL following 5 days of high-dose cytarabine and etoposide (AE). A 3+3 dose escalation design was used to identify a safe and biologically active dose. Plasma inhibitory assay (PIA) testing was performed weekly to determine biologic activity. Results: Toxicities were consistent with intensive relapsed leukemia regimens. One of 6 patients experienced a dose-limiting toxicity (DLT) at 40 mg/m2/day (elevated lipase) and 1 of 9 had a DLT (hyperbilirubinemia) at the highest tested dose of 60 mg/m2/day. Of 17 response evaluable patients, 2 had complete response (CR), 1 complete response without platelet recovery (CRp), 1 complete response with incomplete neutrophil and platelet recovery (CRi), 10 stable disease (SD), and 3 progressive disease (PD). Of 7 FLT3-ITD patients, 1 achieved CR, 1 CRp, 1 Cri, and 4 SD. FLT3-ITD patients, but not FLT3 wild-type (WT) patients, had significantly lower blast counts post-quizartinib. FLT3 phosphorylation was completely inhibited in all patients. Conclusions: Quizartinib plus intensive chemotherapy is well tolerated at 60 mg/m2/day with near complete inhibition of FLT3 phosphorylation in all patients. The favorable toxicity profile, pharmacodynamic activity, and encouraging response rates warrant further testing of quizartinib in children with FLT3-ITD AML. Clin Cancer Res; 22(16); 4014–22. ©2016 AACR.

Abstract Introduction Remission induction rates after a second or greater relapse is a critical endpoint in phase II trials of childhood acute lymphoblastic leukemia (ALL). A robust benchmark is crucial for identification of novel multi-agent regimens worthy of further study. The Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL) consortium previously reported the response rates of children with multiply relapsed and refractory (R/R) ALL treated between 1995 and 2004, which provided a benchmark for clinical trials. To define more recent treatment patterns and test the robustness of this benchmark, we performed a retrospective cohort review of children with R/R ALL who experienced second or greater treatment failure at TACL consortium sites between 2005 and 2013. Patients and Methods Eligible patients were identified at participating TACL institutions. This cohort was comprised of patients with medullary R/R B-cell precursor ALL who experienced at least 2 treatment failures or relapsed after hematopoietic stem cell transplant. Patient demographic data and details of the initial and R/R disease characteristics were abstracted from medical records and entered into a central database. This study was approved by the IRB of each participating institution. Treatment failure was defined by the presence or re-emergence of circulating blasts, M2/M3 BM, or extramedullary (EM) disease despite therapy. Complete remission (CR) was defined as M1 marrow, no EM disease and evidence of peripheral count recovery. For the purpose of statistical analysis, patients who met these criteria without platelet recovery (CRp) or normal blood count recovery (CRi) were included as CR. Univariate and multivariate logistic regression were utilized to evaluate the risk of re-induction failure. Predictors included in this preliminary analysis were NCI risk criteria at diagnosis, duration of the prior remission, the treatment attempt number, and the EM and BM status at the start of each therapy attempt. Results This report includes 214 patients. Fifty-six percent were male. At initial diagnosis, 32% were at least 10 years old, 26% had initial white blood cell (WBC) counts over 50,000/µL, and 39% were classified as high risk by the NCI risk criteria (Table 1). Therapy involved various combinations of agents and ranged between 2 and 10 attempts. The CR rate was 42% for third treatment attempt and 24% for fourth and subsequent treatment attempts (Table 2). Treatment failures were significantly associated with increased number of treatment attempts (p < 0.001), shorter duration of previous CR (p < 0.001) and NCI risk category at diagnosis (p = 0.018). Conclusion This preliminary analysis found similar CR rates in patients with third treatment failure compared to the 1st TACL retrospective study of the prior decade (42% vs. 44%, Ko et al, 2010) and an Austrian report with a small cohort of patients (Reismüller et al, 2013). Further analysis will be performed in comparison to the initial TACL retrospective study cohort once enrollment to this study has been completed (approximately 400 patients). A robust, contemporary historical control may serve as an alternative to a randomized control when outcome with past therapies in unacceptably poor. Table 1. Patient Characteristics at Initial Diagnosis of Patients with ALL who received at least two treatment attempt (n = 214 patients) Characteristic No of patients % Age, years < 1 (infants) 18 8 1-9 126 59 10 and over 70 33 WBC count/uL < 50K 128 60 50K and over 56 26 Unknown 30 14 NCI risk criteria at diagnosis Non-infants, standard risk 82 38 Non-infants, high risk 84 39 Non-infants, unknown 30 14 Infants 18 8 Sex Female 94 44 Male 120 56 CNS disease Yes 42 20 No 148 69 Unknown 24 11 Karyotype1 Normal 68 30 11q23 (MLL gene) rearranged 19 8 Hypodiploidy 7 3 Hyperdiploidy 26 12 iAMP21 2 1 t(12;21) 6 3 t(1;19) 7 3 t(9;22) 15 7 Other 46 21 Unknown 28 12 1 Karyotype is available for 214 unique patients; 2 entries were reported for 7 patients, and 4 entries were reported for 1 patient. Table 2. Achievement of CR/CRp/CRi After Treatment of R/R ALL by Preceding Remission Duration and Treatment Attempt Third treatment attempt Fourth through tenth treatment attempt Duration of preceding CR Response Total % Response Total % Not achieved (refractory) 24 63 38 20 86 23 < 18 months duration 28 78 36 11 49 22 18 to 36 months duration 9 15 60 3 5 60 ≥ 36 months duration 8 8 100 0 1 0 All patients combined 69 164 42 34 141 24 Disclosures Sun: Gateway for Cancer Researchy: Research Funding; Amgen: Research Funding. Wilkes:Healthcare Research and Quality: Research Funding; Alex's Lemonade Stand Foundation: Research Funding. Gaynon:Bristol Meyers Squibb: Membership on an entity's Board of Directors or advisory committees; Sigma Tau: Speakers Bureau; JAZZ: Speakers Bureau. Wayne:Medimmune: Honoraria, Other: travel support, Research Funding; NIH: Patents & Royalties; Kite Pharma: Honoraria, Other: travel support; Pfizer: Honoraria; Spectrum Pharmaceuticals: Honoraria, Other: travel support, Research Funding. Whitlock:Amgen: Honoraria.

Background: Growing evidence indicates that aberrant DNA hypermethylation is associated with leukemogenesis, drug resistance, and relapse. It has been shown that pre-treating leukemia cells with AZA or decitabine could partially reverse the aberrant DNA methylation, restore the expression of tumor suppressor genes, and sensitize cells to chemotherapeutic agents.

Abstract Introduction Children with relapsed T-ALL and T-LL have a dismal prognosis, with survival of less than 25% (Goldberg et al, J Clin Oncol 2003; Reismuller et al, Brit J Haematol 2009). Fewer than a third of children with relapsed T-ALL/LL attain a second remission with standard reinduction therapies (Raetz et al, J Clin Oncol 2008). Nelarabine (NEL) is a purine nucleoside analogue prodrug of AraG, which is resistant to cleavage by endogenous purine nucleoside phosphorylase and cytotoxic to T-lymphoblasts at micromolar concentrations. NEL has substantial single-agent activity in first and multiply relapsed T-ALL (Berg et al, J Clin Oncol 2005). In an effort to improve reinduction rates for children with T-ALL and T-lymphoblastic lymphoma (T-LL) in first relapse, we evaluated the safety and preliminary efficacy of NEL in combination with cyclophosphamide (CPM) and etoposide (ETOP) in this setting. Methods T2008-002: A Phase I trial of NECTAR (Nelarabine, Etoposide and Cyclophosphamide in T-ALL Relapse), is an investigator-initiated collaboration between the Therapeutic Advances in Childhood Leukemia & Lymphoma consortium (TACL), the Pediatric Oncology Experimental Therapeutics Investigators' Consortium (POETIC), and the Innovative Therapies for Children with Cancer consortium (ITCC). Eligible patients had first relapse or initial induction failure of T-ALL or T-LL, no CNS-3, no prior stem cell transplantation, and adequate performance status and organ function. The studyÕs primary objective was to establish the recommended phase 2 doses (RP2D) of NEL and CPM in combination with a fixed dose of ETOP. Subjects received a single course of NEL (325-650 mg/m2) and CPM (330-440 mg/m2) at 1 of 3 assigned dose levels in combination with a fixed ETOP dose (100 mg/m2), each for 5 consecutive days. Intrathecal therapy was administered no less than 7 days prior to or 21 days following the start of a course. Responding patients without dose-limiting toxicity (DLT) were eligible for a second course. The primary study endpoint was the occurrence of dose-limiting toxicity (DLT) during course 1. Secondary endpoints included rates of CR2, complete remission without platelet recovery (CRp), or partial response (PR), and minimal residual disease (MRD) levels at the end of each course. Results Of 19 children enrolled on T2008-002, 6 were enrolled at Dose Level (DL) 1 (NEL 325 mg/m2, CPM 330 mg/m2), 7 at DL2 (NEL 650 mg/m2, CPM 330 mg/m2), and 6 at DL3 (NEL 650 mg/m2, CPM 440 mg/m2). Two potential DLTs led to subject removal during course 1, but were later reversed upon committee review and those subjects replaced. 17 patients were evaluable for DLT and response. Confirmed DLTs occurred in 2 patients: 1 at DL2 (grade 2 (Gr2) motor neuropathy and Gr3 sensory neuropathy) and 1 at DL3 (Gr3 sensory neuropathy). Other ³ Gr3 non-hematologic adverse events are listed in the Table. Of 9 T-ALL patients evaluable for response, there were 2 CRs,1 CRp and 1 CR in the bone marrow/PR in an extramedullary site, with responses at all dose levels, for a response rate of 44% in the T-ALL cohort; at the RP2D, 2/4 evaluable T-ALL patients had a response. Of 8 T-LL patients evaluable for response, there were 2 CRs (1 each at DL1 and DL2), for an overall T-LL response rate of 25%. Eight patients received a second course of NECTAR; 9 subsequently underwent HSCT. MRD levels were available for 2 responding T-ALL patients, with 0.027% and 0.07% blasts after Course 1 and 0.07% and <0.001% blasts after Course 2.TableGrade ³3 Non-Hematologic Adverse Events Occurring in >5% of SubjectsToxicity (CTC3.0)Grade ³ 3 Anorexia3 (16%)Aspartate aminotransferase increased2 (11%)Diarrhoea NOS2 (11%)Febrile neutropenia6 (32%)Hyperkalemia2 (11%)Hypoalbuminaemia3 (16%)Hypocalcaemia4 (21%)Hypokalemia8 (42%)Hypotension NOS2 (11%)Nausea3 (16%)Pleural effusion3 (16%)Vomiting NOS2 (11%)Pain-Other2 (11%) Conclusions The recommended phase 2 doses (RP2D) for NECTAR are NEL 650 mg/m2, CPM 440 mg/m2 and ETOP 100 mg/m2, each given daily for 5 days. The activity and toxicity seen in this phase I dose escalation cohort compare favorably with established reinduction regimens in relapsed T-ALL/LL. An ongoing cohort expansion at the RP2D will better define the activity of NECTAR in first relapse of T-ALL and T-LL. Acknowledgments: Glaxo-Smith-Kline; Higgins Family Foundation; Women's Auxiliary Millennium Chair in Haematology/Oncology Disclosures Whitlock: Glaxo-Smith-Kline: Research Funding. Off Label Use: Nelarabine, cyclophosphamide and etoposide for relapsed T-ALL/T-LL are off-label drug uses.. Zwaan:GSK: Research Funding. Gore:GSK: Research Funding.

Background: Approximately 3540 children are diagnosed with leukemia in the United States yearly (Bhatia and Robison, Oncology of Infancy and Childhood, 2008). Cooperative group trials have increased survival, particularly for acute lymphoblastic leukemia (ALL), but successful treatment of recurrent leukemia remains an unmet medical need. Resistance pathways and epigenetic alterations suggest a role for histone deacetylase (HDAC) inhibitors in children with leukemia (Burke and Bhatla, Frontiers in Pediatrics, 2014). Panobinostat is an orally administered pan-deacetylase inhibitor with activity against HDACs at concentrations in the nanomolar range (Atadja, Cancer Letters, 2009), and for which there is pre-clinical evidence of activity in pediatric leukemia (Stubbs, et al., ASH 2010). Panobinostat shows promise in a variety of adult hematologic malignancies (Khot et al., Expert Opinion on Investigational Drugs, 2013). We undertook a phase I trial of panobinostat in children with recurrent hematologic malignancies, and herein report the safety and pharmacokinetics (PK) from enrolled children with leukemia. Methods: T2009-012 is a first-in-child study coordinated by Therapeutic Advances in Childhood Leukemia and Lymphoma (TACL). Children with relapsed or refractory leukemia between the ages of 1 and 21 years were enrolled to a multi-center, single agent trial of panobinostat dosed once per day three days per week for four successive weeks. Dose escalation was a standard 3+3 design with three dose levels planned. Subjects underwent lumbar puncture with prophylactic chemotherapy at treatment start and after a 28 day course. Blood was sampled pre-dose, at 0.5, 1, 6, 24 and 28-48 hours following the first dose. PK was obtained from blood on patients concurrently with optional specimens obtained from cerebrospinal fluid (CSF) on Day 29. Subjects who received fewer than 11 of the 12 planned doses and did not experience a dose limiting toxicity (DLT) were considered not evaluable for DLT, but were included in the summary of toxicities. Serial ECGs were monitored. Results: Seventeen subjects were enrolled with a diagnosis of acute leukemia, 10 with ALL and 7 with acute myelogenous leukemia (AML). Five were enrolled at dose level 1, 24 mg/m2/dose, 6 at dose level 2, 30 mg/m2/dose, and 6 at dose level 3, 34 mg/m2/dose. There have been no DLTs. Nine subjects are evaluable for DLT and 4 subjects were taken off study early due to increasing blast count. No subjects required removal from protocol therapy for QTc prolongation. One subject with infant ALL was removed early for progressive Aspergillus infection, 1 subject only received 10 doses owing to electrolyte abnormalities, and 2 subjects had nausea and vomiting after administration of 4 doses and did not continue. Grade 3/4 adverse events occurring in more than 20% of subjects included anemia in 82%, diarrhea in 24%, febrile neutropenia in 65%, hypokalemia in 41%, and hypophosphatemia in 24%. Concentration-time profiles were obtained from 9 subjects ages 16 months to 14 years in the 3 dose levels. Mean ± SE of PK for all subjects were Cmax 28.8 ± 6.1 ng/mL, Tmax 2.0 ± 0.8 hours, and T1/2 12.8 ± 3.0 hours. Two toddlers had the highest dose-normalized AUC0-inf and lowest oral clearance. Apparent oral clearance proportionally increased with increase in BSA. To date, 4 CSF specimens have been evaluated and found to have panobinostat below the lower limit of the quantification of 0.1 ng/mL, despite appreciable levels in the plasma. Two subjects on dose level two began a second cycle of therapy; one completed a second cycle for MLL rearranged leukemia and one discontinued study participation in the second cycle to undergo hematopoietic stem cell transplant for secondary AML after achieving a CRp in the first cycle. Conclusions: Panobinostat was tolerated these heavily pre-treated patients without unanticipated toxicities. PK in larger children and adults appears similar but PK in smaller children needs to be further explored. Penetration of panobinostat into the CSF was negligible. Two of 17 patients were able to receive a second cycle of therapy, but 4 had to be withdrawn early because of rapid increase in blast counts. Future trials will explore combination therapy in children with refractory hematologic malignancies, particularly those known to be driven by epigenetic mechanisms, in order to better control risk of rapid progression and improve efficacy through synergy. Disclosures Off Label Use: panobinostat for leukemia. Manley:Seattle Genetics, Inc.: Employment, Equity Ownership. Thomson:Epizyme, Inc: Employment.

To address the therapeutic challenges in childhood relapsed ALL, a phase 1 study combining a survivin mRNA antagonist, EZN-3042, with reinduction chemotherapy was developed for pediatric patients with second or greater bone marrow relapses of B-lymphoblastic leukemia. EZN-3042 was administered as a single agent on days −5 and −2 and then in combination with a 4-drug reinduction platform on days 8, 15, 22, and 29. Toxicity and the biological activity of EZN-3042 were assessed. Six patients were enrolled at dose level 1 (EZN-3042 2.5 mg/kg/dose). Two dose-limiting toxicities were observed: 1 patient developed a grade 3 &ggr;-glutamyl transferase elevation and another patient developed a grade 3 gastrointestinal bleeding. Downmodulation of survivin mRNA and protein were assessed after single-agent dosing and decreased expression was observed in 2 of 5 patients with sufficient material for analysis. Although some biological activity was observed, the combination of EZN-3042 with intensive reinduction chemotherapy was not tolerated at a dose that led to consistent downregulation of survivin expression. The trial was terminated following the completion of dose level 1, after further clinical development of this agent was halted.

Background Reduced intensity conditioning (RIC) regimens for HSCT in hematologic malignancies are successful in reducing transplant related mortality (TRM) and making HSCT available for a select group of high risk patients. Despite HSCT and its associated graft-versus-leukemia (GVL) effect, the risk of relapse remains high with RIC regimens. Clofarabine is a second generation purine analogue with significant anti-leukemic activity in pediatric patients. We postulated that a non-myeloablative (NMA) preparative regimen with clofarabine and 2 Gy TBI may lead to sustained engraftment with low TRM in high-risk children with hematologic malignancies undergoing related and unrelated HSCT. Patient and Methods The primary aim of the Phase I study was to establish the maximum feasible dose (MFD) of clofarabine in conjunction with 2 Gy TBI that leads to durable engraftment with acceptable toxicity in a high-risk pediatric population. Patients between 1-21 years of age with leukemia and undergoing HSCT with significant co-morbidities or any condition that will make them at high risk of TRM with a myeloablative regimen were eligible. Patients had to be in CR or CRp (no morphologic disease, ANC >0.75 x 109/L but without platelet recovery) status prior to HSCT with a PS score of >50 with no active infection, a creatinine clearance of >70 ml/min/1.73m2 and liver enzymes Results Seventeen pediatric patients with a median age of 9 years (range 1-18) enrolled on the study between 7/2010 and 5/2013 and 16 have completed 100-day post-HSCT evaluations. Ten patients had ALL (CR2 =5; CR3 =4 and CR4=1) while 7 had AML (PIF=1; CR1= 1; CR2=3 and secondary AML=3); 9 received UD and 8 received RD grafts. All patients were heavily pre-treated and the indication for the NMA approach was: prior HSCT (n=6), prior liver transplant (n=2), trisomy 21 (n=2) or significant comorbidities (n=7). There was no observed TRM or DLT in the first cohort (clofarabine 40 mg/m2/dose x 5 days) in both the UD and RD strata. The dose was escalated to 52 mg/m2/dose in the next cohorts. All patients tolerated the maximum dose well and no DLT was reported. Significant non-hematologic toxicities (CTC ≥ grade 3) observed were- transient transaminitis (ALT increase in 30% and AST increase in 17%); anorexia (35%) and skin reactions in 16%. PBSC was the stem cell source for all patients, except 3 who had received BM from a RD. Median cell dose was 6.8 x 106 CD34+/kg (range: 2.2-11.5 x 106/kg). All patients achieved neutrophil and platelet engraftment at a median of 19 days (range: 9-25) and 11 days (range: 3-29), respectively. CD3 chimerism analysis at day+30 post-transplant showed donor engraftment in all patients; 15 were full donor (>95%) and 2 showed mixed chimerism (53% and 81% donor). Ten (58%) patients developed acute GVHD, 8 (47%) had ≤ Grade II and 2 (11%) had Grade III acute GVHD. Seven (41%) patients relapsed (4/8 RD and 3/9 in UD arms) at a median time of +106 days (range: 88-190) post-HSCT and all of them died due to disease. Ten patients (58%) are alive and disease free; six have completed 1 year evaluations and 4 are still in follow-up. Conclusion The MFD of clofarabine in combination with 2 Gy TBI was determined to be 52 mg/m2. This regimen is well tolerated and leads to brisk and sustained donor engraftment in a pediatric population that is at high risk of TRM. Disclosures: Thompson: Sanofi Aventis: Speakers Bureau.

Abstract Abstract 3605 Background: AC220 is a novel class III receptor tyrosine kinase (RTK) inhibitor that is potent and highly selective for mutant and wild type (WT) FLT3 and other class III RTK's including KIT, CSF1R, RET and PDGFR. In childhood acute myeloid leukemia (AML), ∼18% of children have FLT3 internal tandem duplication mutations (FLT3-ITD), and ∼10% high WT FLT3 expression. FLT3-ITD is associated with poor prognosis. In childhood acute lymphoblastic leukemia (ALL), the highest levels of FLT3 mRNA expression occur in cases of infants (80%) and childhood ALL with MLL rearrangements (MLL-r) (5%), both conferring poor prognosis.1,2 Study Design: TACL 2009–004is a first-in-children study using AC220 in combination with cytarabine and etoposide. Children > 1 month and < 21 years of age with relapsed/refractory AML or MLL-rearranged ALL are eligible. A standard 3+3 dose escalation design is utilized. The three doses tested (25, 40 and 60 mg/m2/day) are significantly lower than those tested in adults. Dose escalation past 60 mg/m2 occurs only if adequate biologic activity as determined by a plasma inhibitory assay (PIA) is not achieved. Intravenous (IV) cytarabine (1 gm/m2/dose every 12 hours) and IV etoposide (150 mg/m2/dose daily) are given over 5 days. AC220 is administered once daily as an oral solution on days 7–28. Patients can receive up to 2 courses of therapy. PIA testing is performed at trough time points weekly during exposure to AC220 to determine biologic activity. Results: To date, 13 patients (pts) were enrolled and 12 are evaluable for toxicity and response. One pt died from infectious complications (not drug-related) after a single dose of AC220 and was replaced. Median age at study entry was 10.2 years (range 11 mo – 20 yrs), average number of prior regimens was 2.8 (range 1–5), and 5 pts had prior stem cell transplant. Nine pts had relapsed AML, 2 had relapsed MLL-r ALL, and 1 had secondary AML. Of patients with AML, 4 had FLT3-ITD mutations and one had a D835 mutation. Toxicities were consistent with intensive relapsed leukemia regimens. Across all dose levels, non-hematologic toxicities ≥ grade 3 attributed to AC220 included vomiting (n=1), elevated transaminases (n=1), anorexia (n=2), and infection (n=3). One pt experienced a dose-limiting toxicity (DLT) on dose level 2 (40 mg/m2/day) of recurrent grade 3 elevated lipase. Dose level 2 was expanded to 6 pts without additional DLTs. Of 3 pts treated at 60 mg/m2/day, there have been no DLTs. Near total (>99%) inhibition of FLT3 phosphorylation by PIA is seen in every patient across all dose levels. Of 12 pts evaluable for response to date, 1 patient achieved a complete response (CR), 3 achieved complete response with incomplete neutrophil and platelet recovery (CRi), 5 had stable disease (SD), and 3 had progressive disease (PD). Responses in the 4 FLT3-ITD pts include 1 CR, 2 CRi and 1 SD. The FLT3-ITD patient with SD had reduction in marrow blasts without peripheral blood count recovery. An additional 6 pts will be enrolled at 60 mg/m2/day to complete safety evaluation and confirm biologic activity. Conclusions: AC220 plus intensive chemotherapy is well tolerated at doses up to 60 mg/m2/day with near complete inhibition of FLT3 phosphorylation in all pts tested to date. Response rates to date in pre-treated children with relapsed FLT3-ITD AML are encouraging. Disclosures: Off Label Use: AC220 in relapsed/refractory pediatric acute leukemia. Gammon:Ambit Biosciences: Employment.

Therapy of relapsed pediatric acute lymphoblastic leukemia (ALL) is hampered by low remission rates and high toxicity, especially in second and subsequent relapses. Our phase 1 study, T2005-003, showed that the combination of bortezomib with vincristine, dexamethasone, pegylated asparaginase, and doxorubicin had acceptable toxicity. We report the phase 2 expansion of this combination in patients with relapsed ALL who failed 2-3 previous regimens. Twenty-two patients with relapsed ALL were treated with bortezomib combined with this regimen; their ages ranged from 1 to 22 years, and they had either B-precursor ALL (n = 20) or T-cell ALL (n = 2). Grade 3 peripheral neuropathy developed in 2 (9%) patients. After 3 patients died from bacterial infections, treatment with vancomycin, levofloxacin, and voriconazole prophylaxis resulted in no further infectious mortality in the last 6 patients. Fourteen patients achieved complete remission (CR), and 2 achieved CR without platelet recovery, for an overall 73% response rate, meeting predefined criteria allowing for early closure. B-precursor patients faired best, with 16 of 20 (80%) CR + CR without platelet recovery, whereas the 2 patients with T-cell ALL did not respond. Thus, this combination of bortezomib with chemotherapy is active in B-precursor ALL, and prophylactic antibiotics may be useful in reducing mortality. Bortezomib merits further evaluation in combination therapy in pediatric B-precursor ALL. This study is registered at http://www.clinicaltrials.gov as NCT00440726.

Abstract Abstract 251 Literature review and TACL experience support an expected less than 40% complete remission rate with variety of regimens in patients with ALL in second and subsequent relapse (Ko, J Clin Oncol 2010; 28: 648–654). We had shown that bortezomib might be safely combined with vincristine, dexamethasone, pegylated asparaginase, and doxorubicin (VXLD) in the phase I portion of our study (Messinger, Pediatr Blood Cancer 2010;55:254–9). We now report the phase II expansion of that study. ALL patients who relapsed or were refractory after 2 or 3 regimens were treated with bortezomib 1.3 mg/m2/dose on days 1, 4, 8 and 11, combined with VXLD. Patients between ages 1 to 21 years old, with more than 25% bone marrow blasts, were eligible. One patient from the phase I cohort with these criteria was included in the phase II extension. In the phase II extension 22 patients were treated with this combination and all are included in analyses. All patients had relapsed or failed at least 2 prior regimens. Overall 14 achieved complete remission (CR; M1 marrow with ANC and platelet recovery and no extramedullary disease or circulating blasts) and 2 achieved CRp (CR with no platelet recovery) for total 73% remission rate (Table). This level of response exceeded the predefined criteria, allowing for early termination of the study. Three patients (14%) died from bacterial infections and two patients (9%) had no response (Table). One patient (4.5%) was not evaluable for response due to protocol violation, when additional therapy was administered before CR was confirmed with peripheral blood count recovery. B-Lineage ALL patients fared best, with 16/20 achieving CR + CRp (overall response rate 80%), whereas the two patients with T-cell ALL did not respond. Similarly, B-Lineage ALL had superior bone marrow response (M1 marrow): B-Lineage = 17/20 (85%) vs. T-cell = 0/2 (0%). Severe grade 3 or more peripheral neuropathy (PN) was seen in 2 (9%) patients, (one had prior vincristine PN). One patient has developed mucor invasive sinus and orbital infection, requiring halting therapy after day 14 but achieved CRp. After the 3 (14%) septic deaths, the use of vancomycin, levofloxacin and voriconazole or posaconazole prophylaxis in the last 6 patients has prevented further infectious mortality.ResponseAllBTn22202CR14 (64%)14 (70%)0CRp2 (9%)2 (10%)0Total Response16 (73%)16 (80%)0Deaths3 (14%)3 (15%)0SD/PD2 (9%)02 (100%)N/E1 (4.5%)1 (5%)0 In conclusion, the regimen of bortezomib + VXLD is exceptionally effective in multiple relapsed B-Lineage ALL with the highest response rate for any multiply relapsed ALL trial reported thus far. The use of prophylactic antibiotics may be effective in reducing mortality. Bortezomib with VXLD should be further evaluated in randomized fashion on frontline relapse and high-risk pediatric B-Lineage ALL clinical studies. Disclosures: Messinger: Genzyme: Consultancy. Off Label Use: Bortezomib (Velcade®) is approved for multiple myeloma and mantle cell lymphoma both B cell malignancies. We are describing use in relapsed B cell Acute Lymphoblastic Leukemia which is off label.”

Background. Current event-free survival (EFS) rates for children with newly diagnosed acute myeloid leukemia (AML) approach 50-60%. We hypothesize that further improvements in survival are unlikely to be achieved with traditional approaches such as dose intensive chemotherapy or hematopoietic stem cell transplants, since these therapies have been rigorously explored in clinical trials. This report highlights efforts to assess the response rates and survival outcomes after first or greater relapse in children with AML. Procedure. We performed a retrospective cohort review of pediatric patients with relapsed and refractory AML (rAML) previously treated at TACL institutions between the years of 1995 and 2004. Data regarding disease characteristics at diagnosis and relapse, treatment response, and survival was collected on 99 patients and 164 medullary relapses or treatment failures. Results. The complete response (CR) rate following the second therapeutic attempt was 56 +/- 5%. CR rates following a third treatment attempt was 25 +/- 8% while 17 +/- 7% achieved CR following the fourth through sixth treatments. The 5-year disease-free survival in patients achieving CR following a second therapeutic attempt was 43 +/- 7%. The 5-year EFS and overall survival (OS) rates for all patients receiving a second treatment attempt was 24 +/- 5% and 29 +/- 5%, respectively. Conclusions. This CR rate following a second therapeutic attempt and OS rate in patients with rAML is consistent with the literature. There are limited published data of CR rates for subsequent relapses. Our data can serve as a historical benchmark to compare outcomes of future therapeutic trials in rAML against traditional chemotherapy regimens. Pediatr Blood Cancer. (C) 2010;55:421-429. 2010 Wiley-Liss, Inc.

Outcomes remain poor for children after relapse of acute lymphoblastic leukemia (ALL), especially after early marrow relapse. Bortezomib is a proteasome inhibitor with in vitro synergy with corticosteroids and clinical activity in human lymphoid malignancies.

PurposeDespite improvements in treatment, approximately 20% of patients with acute lymphoblastic leukemia (ALL) experience relapse and do poorly. The Therapeutic Advances in Childhood Leukemia (TACL) Consortium was assembled to assess novel drugs for children with resistant leukemia. We hypothesize that novel agents and combinations that fail to improve baseline complete remission rates in comparable populations are unlikely to contribute to better outcomes and should be abandoned. We sought to define response rates and disease-free survival (DFS) rates in patients treated at TACL institutions, which could serve as a comparator for future studies.Patients and MethodsWe performed a retrospective cohort review of patients with relapsed and refractory ALL previously treated at TACL institutions between the years of 1995 and 2004. Data regarding initial and relapsed disease characteristics, disease response, and survival were collected and compared with those of published reports.ResultsComplete remission (CR) rates (mean +/- SE) were 83% +/- 4% for early first marrow relapse, 93% +/- 3% for late first marrow relapse, 44% +/- 5% for second marrow relapse, and 27% +/- 6% for third marrow relapse. Five-year DFS rates in CR2 and CR3 were 27% +/- 4% and 15% +/- 7% respectively.ConclusionWe generally confirm a 40% CR rate for second and subsequent relapse, but our remission rate for early first relapse seems better than that reported in the literature (83% v approximately 70%). Our data may allow useful modeling of an expected remission rate for any population of patients who experience relapse.

The outcome of relapsed ALL remains very poor, and many re-induction regimens are highly toxic. We report here the Phase I component of a Phase I/II study of bortezomib added to 4-drug induction with vincristine, dexamethasone, pegylated asparaginase, and doxorubicin in children with relapsed ALL. A total of 10 patients were enrolled, 5 in 1 st marrow relapse (2 early and 3 late relapses) and 5 in 2 nd or subsequent relapse. Four patients were enrolled at dose level 1 (1 mg/m 2 ) to obtain 3 evaluable patients. One patient was inevaluable for toxicity because of an error in dexamethasone doses and was removed after 8 days. No dose-limiting toxicity (DLT) was observed. Six patients were enrolled at dose level 2 (1.3 mg/m 2 ), the standard single agent dose. One patient had a DLT (hypophosphatemia and rhabdomyolysis) after1 dose of bortezomib, and that patient died from diffuse zygomyces infection by day 17. Five additional patients were enrolled with no further DLT’s. As planned, no further dose escalation was pursued. The regimen had predictable toxicity related to the chemotherapy drugs. Only 2 patients had mild peripheral neuropathy (grades 1 and 2). Seven of the10 patients (70%) achieved a CR, and 1/10 had bone marrow CR with persistent central nervous system (CNS) leukemia despite weekly intrathecal (IT) methotrexate, for a total of 80% bone marrow CR rate. In conclusion: the combination of bortezomib with 4 standard drugs is highly active with acceptable toxicity in heavily pretreated relapsed pediatric ALL patients. We are expanding the MTD cohort for a phase II estimate, and intend to report updated results at the ASH meeting.

10042 Background: Despite significant progress in childhood AML, with 5-year event free survival ∼50%, relapse remains common. Survival after relapse is no better than 20–30% despite fair success in obtaining second and subsequent complete remissions (CR2, CR3). There is limited data on CR rates for large cohorts of pediatric rAML patients. Methods: The TACL Consortium (www.tacl.us) was formed to evaluate novel drug combinations in patients with relapsed leukemia. To estimate baseline CR2 and CR3 rates in AML, we reviewed all patients treated for rAML between 1995 and 2004 at 8 TACL institutions, examining disease features at diagnosis, therapy, and response. Results: Fifty-three percent of 100 rAML patients were male; 36% were ≥10 years at diagnosis, and 27% had presenting WBC counts ≥50,000/uL. Eighty-nine percent attained CR1 with initial treatment. The majority (97/100) received traditional cytarabine-based AML regimens as initial therapy as well as in first (67/100) relapse, and a significant proportion (16/46) in second relapse. Patients underwent 1 to 5 therapeutic attempts for refractory or relapsed AML. Fifty-seven percent (56/99) of patients achieved CR after a first relapse, 31% (11/35) after a second relapse, and 25% (5/20) after a third relapse. Response to first re-induction was independent of gender, presenting age ≥10 years, or presenting WBC ≥50,000/uL. Patients with CR1 >12 months were more likely to attain CR2 than those with initial CR1<12 months (83% vs 51%, p=0.005). CR rates for initial re-induction attempts depended on whether or not a remission had been initially achieved (63% vs 33%, p=.016). Conclusions: Duration of prior remission is an important predictor of response in childhood rAML. Drug combinations with response rates no better than those reported above are unlikely to contribute to cure. Our findings emphasize the need for novel drug combinations with higher response rates. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Therapeutic Advances in Childhood Leukemia Consortium

Introduction: Event-free survival (EFS) for children with ALL is approximately 80%. Despite substantial success in achieving second and subsequent remissions, survival of patients with relapsed ALL (rALL) remains dismal. We propose that progress depends on identification of novel drug combinations with more activity in rALL than those commonly employed. The literature suggests a 40% CR rate for second and subsequent relapse (Br. J Haematol 2005; 131:579). However, CR rates depend on the number of prior therapeutic attempts and duration of any prior response, complicating identification of promising regimens. Limited patient numbers and a large number of potential candidate regimens discourage randomized trials. We surveyed local experience with rALL to establish a robust benchmark for evaluation of novel drug combinations.