ALBERT

Description: Introduction: Although recent studies have refined the classification of B-progenitor and T-lineage acute lymphoblastic leukemia into gene-expression based subgroups, a comprehensive integration of significantly mutated genes and pathways for each subgroup is needed to understand disease etiology. Methods: We studied 2789 children, adolescents and young adults (AYA) with newly diagnosed B-ALL (n=2,322 cases) or T-ALL (n=467) treated on Children's Oncology Group (n=1,872) and St. Jude Children's Research Hospital trials (n=917). The cohort comprised childhood NCI standard-risk (41.8%; age range 1-9.99 yrs, WBC ≤ 50,000/ml), childhood NCI high-risk (44.5%; age range ≥10 to 15.99 yrs) and AYA (9.9%; age range 16-30.7 yrs). Genomic analysis was performed on tumor and matched-remission samples using whole transcriptome sequencing (RNA-seq; tumor only; n=1,922), whole exome sequencing (n=1,659), whole genome sequencing (n=757), and single nucleotide polymorphism array (n=1,909). Results: For B-ALL, 2104 cases (90.6%) were classified into 26 subgroups based on RNA-seq gene expression data and aneuploidy or other gross chromosomal abnormalities (iAMP21, Down syndrome, dicentric), deregulation of known transcription factors by rearrangement or mutation (PAX5 P80R, IKZF1 N159Y), or activation of kinase alterations (Ph+, Ph-like). For T-ALL, cases were classified into 9 previously described subtypes based on dysregulation of transcription factor genes and gene expression. In 1,659 cases subject to exome sequencing (1259 B-ALL, 405 T-ALL) we identified 18,954 nonsynonymous single nucleotide variants (SNV) and 2,329 insertion-deletion mutations (indels) in 8,985 genes. Overall, 161 potential driver genes were identified by the mutation-significance detection tool MutSigCV or by presence of pathogenic variants in known cancer genes. Integration of sequence mutations and DNA copy number alteration data in B-ALL identified 7 recurrently mutated pathways: transcriptional regulation (40.6%), cell cycle and tumor suppression (38.0%), B-cell development (34.5%), epigenetic regulation (24.7%), Ras signaling (33.0%), JAK-STAT signaling (12.0%) and protein modification (ubiquitination or SUMOylation, 5.0%). The top 10 genes altered by deletion or mutation in B-ALL were CDKN2A/B (30.1%), ETV6 (27.0%), PAX5 (24.6%), CDKN1B (20.3%), IKZF1 (17.6%), KRAS (16.5%), NRAS (14.6%), BTG1 (7.5%) histone genes on chromosome 6 (6.9%) and FLT3 (6.1%), and for T-ALL, CDKN2A/B (74.7%), NOTCH1 (68.2%), FBXW7 (21.3%), PTEN (20.5%) and PHF6 (18.2%) (Figure 1A). We identified 17 putative novel driver genes involved in ubiquitination (UBE2D3, UBE2A, UHRF1, and USP1), SUMOylation (SAE1, UBE2I), transcriptional regulation (ZMYM2, HMGB1), immune function (B2M), migration (CXCR4), epigenetic regulation (DOT1L) and mitochondrial function (LETM1). We also observed variation in the frequency of genes and pathways altered across B-ALL subtypes (Figure 1B). Interestingly, alteration of SAE1 and UBA2, novel genes that form a heterodimeric complex important for SUMOylation, and UHRF1 were enriched in ETV6-RUNX1 cases. Deletions of LETM1, ZMYM2 and CHD4 were associated with near haploid and low hypodiploid cases. Deletion of histone genes on chromosome 6 and alterations of HDAC7 were enriched in Ph+ and Ph-like ALL. Mutations in the RNA-binding protein ZFP36L2 were observed in PAX5alt, DUX4 and MEF2D subgroups. Genomic subtypes were prognostic. ETV6-RUNX1, hyperdiploid, DUX4 and ZNF384 ALL were associated with good outcome (5-yr EFS 91.1%, 87.2%, 91.9% and 85.7%, respectively), ETV6-RUNX1-like, iAMP21, low hyperdiploid, PAX5 P80R and PAX5alt were associated with intermediate outcome (5-yr EFS 68.6%, 72.2%, 70.8%, 77.0% and 70.9%, respectively), whilst KMT2A, MEF2D, Ph-like CRLF2 and Ph-like other conferred a poor prognosis (55.5%, 67.1%, 51.5% and 62.1%, respectively). TCF3-HLF and near haploid had the worst outcome with 5-yr EFS rates of 27.3% and 47.2%, respectively. Conclusions: These findings provide a comprehensive landscape of genomic alterations in childhood ALL. The associations of mutations with ALL subtypes highlights the need for specific patterns of cooperating mutations in the development of leukemia, which may help identify vulnerabilities for therapy intervention. Disclosures Gastier-Foster: Bristol Myers Squibb (BMS): Other: Commercial Research; Incyte Corporation: Other: Commercial Research. Willman:to come: Patents & Royalties; to come: Membership on an entity's Board of Directors or advisory committees; to come: Research Funding. Raetz:Pfizer: Research Funding. Borowitz:Beckman Coulter: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Angiolillo:Servier Pharmaceuticals: Consultancy. Relling:Servier Pharmaceuticals: Research Funding. Hunger:Jazz: Honoraria; Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel.

Description: Introduction: Bendamustine is an alkylator with anti-metabolite properties that shows incomplete cross resistance with other alkylators such as cyclophosphamide. The combination of cyclophosphamide, clofarabine and etoposide is often used in the treatment of children with relapsed leukemia, most of whom have significant prior exposure to cyclophosphamide. We evaluated the maximum tolerated dose (MTD) and safety profile of bendamustine when used in combination with clofarabine and etoposide in pediatric patients with relapsed or refractory hematologic malignancies. Methods: Patients are eligible if they are younger than 22 years-old, have relapsed or refractory hematologic malignancies following 2 or more prior regimens, and have adequate organ function. Using the rolling 6 design, participants received bendamustine at one of 3 dose levels (escalating doses of 30, 40, or 60mg/m2/day) on Days 1-5 in combination with clofarabine (40 mg/m2/day), etoposide (100 mg/m2/day), and dexamethasone (8 mg/m2/day) daily on Days 1-5. We obtained pharmacokinetic (PK) studies to assess for potential time-dependent changes in bendamustine clearance over the 5 day course in this combination regimen since most PK studies of bendamustine have been conducted in adult patients with dose schedules of 90-120 mg/m2/day for 2 days. Results: Sixteen patients (12 males and 4 females) with median age 11 years (range 4 to 17 years) were enrolled: 10 B-cell acute lymphoblastic leukemia (B-ALL), 1 early T-cell precursor (ETP) leukemia, 1 gamma delta T-cell ALL, 2 Hodgkin lymphoma, and 2 T-cell non-Hodgkin lymphoma. Six patients were treated on dose level 1, six on dose level 2, and four on dose level 3. One patient with hyperleukocytosis died from severe systemic inflammatory response syndrome (SIRS). Dose limiting toxicity was failure to recover peripheral blood counts on Day 42. The recommended dose of bendamustine in this combination is 30mg/m2 daily over 5 days. Ten responses were observed: 6 complete remissions (CR), 1 durable minimal residual disease (MRD)-negative CR without platelet recovery in the patient with ETP-ALL, and 3 partial remissions. Eight patients proceeded to transplant. Nine patients died (5 from progressive disease, 2 from transplant complications, 1 from SIRS and 1 from complications of subsequent salvage chemotherapy). Six patients are alive at a median follow up of 12 months (range 2 to 29 months). Conclusions: Bendamustine is well tolerated in combination with clofarabine and etoposide and shows efficacy in multiple relapsed and refractory hematologic malignancies. Dose reductions on the first day of therapy are warranted in patients at risk of tumor lysis syndrome to avoid severe systemic inflammatory response. Disclosures Bhojwani: Amgen: Other: Blinatumumab global pediatric advisory board 2015.

Description: Introduction: Pegaspargase (Oncaspar, PEG-ASP) has replaced native E.coli asparaginase (L-ASP) in the first-line treatment for acute lymphoblastic leukemia (ALL) in many places because of its longer half-life and lower immunogenicity. Risk factors for allergic reactions to PEG-ASP remain unclear. Besides L-ASP, polyethylene glycol (PEG) is also a potential allergen, but the contribution of antibodies directed against PEG vs. L-ASP antigens has not been well characterized in front-line ALL trials. Herein, we assessed the usefulness of serum antibodies for diagnosing allergic reactions to PEG-ASP and to identify risk factors in a front-line ALL trial featuring intensive PEG-ASP treatment. Methods: Of 600 patients enrolled in St. Jude's Total XVI study, 599 were evaluable for reactions to asparaginase and received either low-risk (LR) or standard/high-risk (SHR) therapy based on their risk for relapse. All patients received PEG-ASP at 3000 U/m2 on day 3 of remission induction; those with minimal residual disease (MRD) ≥ 1% on day 15 of induction received an additional dose of PEG-ASP. After induction, patients were randomized to receive PEG-ASP at 2500 U/m2 or 3500 U/m2 once every other week starting from week 1 of continuation treatment (SHR arm) or reinduction 1 (LR arm) (Figure 1). Reactions were graded using CTCAE v3.0. Nine serum samples per patient were scheduled on induction days 1, 8 and 15, consolidation day 1, and day 1 of continuation weeks 7, 8, 9, 17, and 19. Serum samples (n=5369) were analyzed for anti-PEG-ASP IgG by ELISA; positive samples were tested separately for anti-PEG and anti-L-ASP. Patient- and treatment-related variables were identified as potential risk factors for reactions and anti-PEG-ASP by χ2 test or multiple linear regression. Results: Eighty-one (13.5%) patients developed grade 2-4 reactions to PEG-ASP; of these, 58 (72%) were grade 3/4. In univariate analysis, contrary to our prior front-line ALL trial using L-ASP (Liu, Leukemia 2012), there was no difference in the frequency of reactions between SHR (13.2%) and LR (13.8%) patients (P = 0.83). Patients with reactions received a median of only 3 doses of PEG-ASP before their reaction. The majority of reactions occurred upon the first few re-exposures to PEG-ASP following a 12- to 19-week hiatus after the induction doses, regardless of risk arm (P = 0.076): 72% of reactions among LR patients occurred during reinduction I, and 88% of reactions among SHR patients occurred during continuation weeks 1-6. Anti-PEG, not anti-L-ASP, was the predominant component of anti-PEG-ASP IgG (96% vs. 17%, P = 0.021), reflecting the antigenicity of PEG itself, as has been observed for other PEGylated drugs. Anti-L-ASP became a more common component of antibodies as therapy progressed (from 10% during induction to 36% at week 19 of continuation), whereas anti-PEG became a slightly less common component as therapy progressed (from 100% to 87%). Pre-treatment anti-PEG was present in 30 of 583 (5.1%) patients, more common in older patients (P = 2.1x10-5), and associated with a risk of subsequent reactions (P = 0.025), with 8 out of 30 developing reactions. Anti-PEG-ASP at all time points was positively associated (P ≤ 0.025 for days 1 & 15, consolidation day 1, continuation weeks 7, 8, 9, 17, & 19) with risk of a reaction during therapy except on induction day 8 (P = 0.24). For predicting future reactions, anti-PEG-ASP as early as consolidation day 1 had a sensitivity of 61% and a specificity of 90%, with an optimum receiver operating characteristic (ROC) curve at continuation week 7 (AUC = 0.85). In a multivariate analysis of clinical risk factors, only a lower number of intrathecal (IT) injections (P = 2.7x10-5) was predictive of reactions. Patients received 1 to 7 doses of IT therapy during induction: those who received more ITs (4-7 doses) had fewer reactions (7.6% of 301 patients) than those who received fewer (1-3 doses) ITs (20.2% of 248 patients); they were also less likely to have anti-PEG-ASP antibodies (P = 4.0x10-8). Conclusions: Serum anti-PEG-ASP has good specificity and sensitivity for clinical allergic reactions to PEG-ASP treatment. Patients who received fewer IT injections during induction had a higher risk of allergy to PEG-ASP, possibly due to increased immunosuppression from multiple ITs. Anti-PEG, commonly observed pre-treatment, is likely the major mediator of PEG-ASP reactions and may be predictive of future reactions. Figure 1. Figure 1. Disclosures Inaba: Shire: Research Funding. Relling:Shire Pharmaceuticals: Research Funding.

Description: Key Points Adherence rates were significantly lower in African Americans (87%) and Asian Americans (90%), as compared with non-Hispanic whites (95%). Adherence to 6MP at

Description: Background: Mercaptopurine (MP) and asparaginase (ASP) are critical components in the treatment of acute lymphoblastic leukemia (ALL). Dose-limiting toxicities of the two drugs are common, resulting in therapy interruption, which has been associated with inferior treatment outcome in some studies. However, the interaction between these drugs has not been clearly identified. Merryman et al (Pediatr Blood Cancer 2012) reported that in DFCI ALL 05-01, patients had lower blood counts and more dosage reductions of MP during consolidation therapy (with concomitant ASP treatment) than during continuation therapy (identical treatment without concomitant ASP). Among groups of homogeneously treated patients with ALL, variability in ASP exposure due to inactivating antibodies can affect ASP pharmacodynamics: we have reported that ASP antibodies were associated with lower plasma ASP activity and higher dexamethasone (DEX) clearance, leading to a lower risk of osteonecrosis and a higher risk of CNS relapse (Liu, Leukemia 2012; Kawedia, Blood 2012). Here we studied the possible effect of ASP antibodies on MP tolerability in St. Jude Children's Research Hospital Total XV, a clinical trial featured intensive ASP treatment. Methods: A total of 390 children with ALL treated on St. Jude Total XV protocol were evaluable. TPMT genotype was used to guide starting doses of MP. During maintenance treatment, planned MP doses were higher on the low-risk arm (LR; n = 202) than on the standard/high-risk arm (SHR; n = 188). MP dose intensity was estimated as (prescribed dose)/(protocol dose) for weeks 1-146 (boys) or 1-120 (girls) for patients on the LR and SHR arms of maintenance therapy. Native E.coli-ASP (Elspar) was administered intramuscularly at 10000 U/m2 thrice weekly for 6 or 9 doses during remission induction. During maintenance therapy, patients on the LR arm received ASP only during reinductions I (weeks 7-9) and II (weeks 17-19), whereas those on the SHR arm received 19 weekly doses at 25000 U/m2 during weeks 1-19. Patients were tested for serum anti-Elspar antibodies at days 5, 19, 34 of remission induction, day 1 of reinduction I and day 1 of reinduction II, and were grouped based on whether they were ever positive for antibodies at any time during therapy or not. The area under the antibody concentration-time curve (AUC) for the entire period up to week 19 was also estimated in 360 patients. Result: Overall MP dose intensity was higher in those with vs without ASP antibodies in patients on the LR (median 83 vs 75%, P = 0.003) and SHR arms (median 86 vs 76%, P = 3.3 × 10-5; Figure 1A), and MP dose intensity was correlated with ASP antibody AUC in patients on both treatment arms (LR, P = 7.7 × 10-3 and SHR, P = 2.4 × 10-4; Figure 1B). In a multivariate model including age, sex, risk arm, ancestry, TPMT status, NUDT15 genotype and ASP antibody status, TPMT genotype was the strongest determinant of MP dose intensity (-17% in heterozygotes, P = 1.9 × 10-8), followed by ASP antibody positivity (+8.9% dose intensity in those with antibodies, P = 5.8 × 10-6). The model also confirmed previously identified associations of higher MP dose intensity with higher African ancestry (Bhatia et al. Blood 2014) (P = 1.8 × 10-4) and lower Asian ancestry (P = 0.05) (Yang et al. J Clin Oncol 2015). Conclusion: Interindividual differences in ASP systemic exposure, as reflected by ASP antibodies, had a strong impact on MP tolerance, especially in patients on the SHR arm who received intensive ASP therapy. We have previously shown that patients who are positive for ASP antibodies not only have lower exposure to ASP but also to dexamethasone (Kawedia, Blood 2012; Liu, Leukemia 2012). These data further emphasize the capacity for variation in ASP exposure to impact yet another critical component of ALL therapy. Figure 1 Asparaginase antibodies associated with higher mercaptopurine tolerance in patients on the low-risk (n = 202) and standard/high-risk (n = 188) arms. P values were estimated using the (A) Mann-Whitney U test and (B) linear regression model. DI, dose intensity; MP, mercaptopurine; ASP, asparaginase; NEG, anti-asparaginase antibody negative; POS, anti-asparaginase antibody positive. Figure 1. Asparaginase antibodies associated with higher mercaptopurine tolerance in patients on the low-risk (n = 202) and standard/high-risk (n = 188) arms. / P values were estimated using the (A) Mann-Whitney U test and (B) linear regression model. DI, dose intensity; MP, mercaptopurine; ASP, asparaginase; NEG, anti-asparaginase antibody negative; POS, anti-asparaginase antibody positive. Disclosures Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Relling:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping.

Description: Introduction: Treatment for acute lymphoblastic leukemia (ALL) with asparaginase (ASP) and glucocorticoids, such as dexamethasone (DEX), can cause changes in serum triglycerides (TGs). Glucocorticoids increase the activity of lipoprotein lipase (LPL), the enzyme responsible for removing TGs from the plasma, and increase production of TG-rich particles, resulting in the mobilization and redistribution of body fat. In contrast, ASP decreases the activity of LPL. Therefore, when glucocorticoids and ASP are given together, it is likely that TG-rich particles are rapidly formed, but insufficiently cleared, resulting in elevated serum TGs. The ASP formulation, e.g., native E. coli ASP (Elspar, L-ASP) vs. PEGylated ASP (Oncaspar; PEG-ASP), may influence the magnitude of TG changes. Our aim was to compare changes in TGs, the incidence of hypertriglyceridemia (hyperTG), and associated toxicities in two front-line pediatric St. Jude ALL trials that used different formulations of ASP: Total XV (L-ASP) and Total XVI (PEG-ASP). Methods: Patients on TXV (n=392) and TXVI (n=583) were assigned to the low-risk (LR) or standard/high risk (SHR) therapeutic arms. Serum TGs were measured as research biomarkers at four time-points: consolidation day 15 ("baseline" was 〉 35 days after the last dose of ASP during remission induction and 〉 21 days after the last dose of glucocorticoid); day 1 of week 7, 8, and either week 12 (TXV) or week 17 (TXVI) of continuation. Patients received DEX for 5 days/week during weeks 1, 4, and 14, for 8 days at week 7, and for 7 days at week 9 of continuation. In TXV, SHR patients received L-ASP weekly during the observed course of continuation (25,000 U/m2/dose); LR patients received L-ASP thrice weekly during continuation weeks 7-9 (10,000 U/m2/dose). In TXVI, patients were randomized to receive PEG-ASP 2,500 or 3,500 U/m2/dose during continuation: SHR weeks 1, 3, 5, 7, 9, 11, 13, and 15; LR weeks 7 and 9. HyperTG was defined as TG 〉 1000 mg/dL (dotted horizontal line in Figure 1). Clinically relevant toxicities in TXV, including thrombosis (≥ grade 3), osteonecrosis (≥ grade 2) and pancreatitis (≥ grade 3) were graded according to CTCAE v2.0; in TXVI CTCAE v3.0 was used, with identical grades except for pancreatitis (≥ grade 2). Results: Similar trends in TGs were observed in both protocols for LR patients: there was a decrease in TGs from baseline to week 7, when DEX but not ASP had been given (TXV: p= 2 x 10-8, TXVI: p〈 2 x 10-16); TGs increased from week 7 to 8 after both DEX and ASP (TXV: p=3 x 10-14, TXVI: p〈 2 x 10-16); and the week 12/17 TG did not differ from baseline (〉 16 days after last dose of DEX and ASP, TXV: p= 0.7, TXVI: p= 0.1). Among LR patients, the only difference between protocols was that the week 12/17 TG was higher on TXVI (with PEG-ASP) than TXV (with L-ASP) (p= 0.04). In contrast, among SHR patients, TGs were elevated at all time points relative to baseline (e.g. week 7 compared to baseline, TXV: p= 1 x 10-5, TXVI: p 〈 2 x 10-16); TGs increased from week 7 to 8 after both DEX and ASP (TXV: p= 6 x 10-16, TXVI: p 〈 2 x 10-16); and the week 12/17 TG remained higher than baseline (TXV: p= 9 x 10-9, TXVI: p 〈 2 x 10-16) at 7-16 days after last dose of DEX or ASP. Among SHR patients, TGs were higher in TXVI vs. TXV after baseline: at week 7 (p= 8 x 10-7), week 8 (p= 6 x 10-4), and at the week 12/17 TG (p= 8 x 10-4), suggesting a greater effect of PEG-ASP vs. L-ASP in increasing TGs. More patients developed hyperTG on TXVI vs TXV: 10% (59/583) vs. 5% (20/392), respectively (p= 0.007). In both protocols, only patients on SHR therapy developed hyperTG, suggesting an association with ASP. Patients with hyperTG were more likely to be diagnosed with thrombosis and osteonecrosis. However, this association was only significant after adjustment for age and risk group in TXVI: 17% of patients with vs. 4% without hyperTG developed post-induction thrombosis (p= 0.009); 39% of patients with vs. 11% without hyperTG developed osteonecrosis (p= 0.006). In TXV, associations of hyperTG with thrombosis (p= 0.2) and osteonecrosis (p= 0.08) were not significant after adjustment for age and risk group. There was no association between hyperTG and pancreatitis in either protocol. Conclusions: These data support a greater association of PEG-ASP than L-ASP with elevation of serum TGs, and a greater effect of ASP than DEX. Whether serum hyperTG contributes to toxicities such as thrombosis or osteonecrosis, or is a biomarker of greater drug exposure or sensitivity, is unclear. Figure. Figure. Disclosures Inaba: Shire: Research Funding. Relling:Shire Pharmaceuticals: Research Funding.