Children's Oncology Group AALL0232 Research Articles

Characterizing Rare Variants in Drug Metabolism Genes in Refractory Pediatric High-Risk Pre-B ALL

Abstract 879 Introduction:We are working to understand how rare genetic variation can influence incidence and outcomes of pediatric high-risk leukemia. Despite improvements in standard-risk outcomes, children with high-risk precursor-B acute lymphoblastic leukemia (HR-ALL) have not enjoyed similar success. Recent studies demonstrate that there are critical metabolic pathways involved in the transition from normal precursor-B (pre-B) cells to leukemic cells as well as involved in proper therapeutic response (Mullighan, Nature 2007; Kang, Blood 2011). The Rare Variant Hypothesis predicts that a population of affected individuals would harbor a diverse collection of functionally significant variants in the genes involved in these pathways. Thus, the manner in which these critical genes or pathways become disrupted may be quite variable, but the outcome is very similar. Therefore, we hypothesize that rare variants in critical metabolic pathways influence the incidence and outcome of pediatric HR-ALL. We established Children's Oncology Group (COG) protocol AALL10B2 to study this question from existing patient DNA samples. Methods:Using our pooled sequencing strategy (Druley, Nat Methods 2009) and novel computational analysis software, SPLINTER (Vallania, Genome Res 2010), we have sequenced 54 candidate genes associated with pediatric leukemia in three pools of genomic DNA. One pool from 96 germline DNA samples from patients enrolled on COG HR-ALL protocol P9906, a second pool from 96 matched P9906 leukemia DNA samples, and a third from 93 ethnically matched unaffected pediatric controls. We called an average of 3,987 variants per pool (range 3822–4209) and validated variants by individual custom genotyping. The correlation between aggregated individual minor allele frequencies and pooled sequencing variant calling was excellent for all three pools (R2 range = 0.90–0.93). Results:By directly mapping the variants from the three pools, we identified gene regions with variation in the patients, but not controls. We found two genes with regions of excess patient-specific germline genetic variation, cytochromes 1A1 and 3A5 (CYP1A1 and CYP3A5). The CYP3A isozymes are involved in the activation of epipodophyllotoxins, anthracyclines and cyclophosphamide along with the clearance of vincristine and glucocorticoids, and common variants in these genes have previously been correlated with an increased incidence of pediatric ALL (Joseph, Pediatr Blood Cancer 2004; Aydin-Sayitoglu, Am J Hematol 2006; Borst, Eur J Haematol 2011). A heat map of genome-wide expression array results for our patient population (our 96 pilot patients from the P9906 trial and an additional 250 enrolled in the AALL0232 trial) against the 54 genes in our survey identified a previously unappreciated subpopulation (of ∼10% of patients) that trended toward inferior relapse-free survival and demonstrated significant overexpression of 14 genes: (in alphabetical order): ATM, CDKN1A, CYP1A1, CYP3A5, IKZF1, MDM2, MLL, MTHFR, NAT2, NQO1, PAX5, PTPN11, TCF3, TPMT. These patients did not possess other high-risk chromosomal translocations. Validation in 250 matched germline and HR-ALL DNA samples from participants in the COG AALL0232 study is underway, along with functional characterization and validation of the identified variants. Conclusions:We have identified a potential new genomic signature for a subset of HR-ALL patients who appear predisposed to poor therapeutic outcomes. Our preliminary results support the hypothesis that pediatric HR-ALL incidence and outcomes are influenced by a pre-existing profile of germline genetic variation, which is reasonable given the fact that it is exceptionally mathematically unlikely that these children would acquire the entire cadre of deleterious genetic variants required for malignant transformation solely through somatic mutation. Such results are “clinically-actionable” now. Children demonstrating functionally significant variants in these genes or similar patterns of gene expression could be immediately referred for hematopoietic stem cell transplantation in first remission rather than being exposed to years of highly toxic chemotherapy that is unlikely to be successful anyway. Meanwhile, in vitro functional studies will facilitate the discovery of alternate therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Pharmacokinetic (PK) and pharmacodynamics (PD) properties of SC-PEG e. coli L-asparaginase (EZN-2285) in the treatment of patients with acute lymphoblastic leukemia (ALL): Results from Children's Oncology Group (COG) study AALL07P4.

9543 Background: Asparaginase is a critical therapeutic agent in the treatment of childhood ALL, and pegaspargase (Oncospar, ONC) is now the preferred product in COG frontline ALL trials. EZN-2285 replaces the succinimidyl succinate linker in ONC with a succinimidyl carbamate linker creating a more stable molecule. AALL07P4 was designed to determine the PK comparability of EZN-2285 to ONC when given intravenously in newly diagnosed patients with NCI high-risk (HR) B-precursor ALL. Methods: 162 eligible patients were randomized to receive EZN-2285 at 2100 (LD, n=66) or 2500 (HD, n=42) IU/m2 vs. 2500 IU/m2 of ONC (n=51) in a 2:1 manner based on the prednisone/Capizzi methotrexate arm of COG AALL0232. All groups were similar demographically with the exception of number of patients over the age of 16 years were 6, 5, and 19 in the ONC, HD and LD, respectively. Results: PK, PD and targeted AEs (Gr 3-5) after the first doses of study drug are presented in the table below. Adverse events in induction were not significantly different between arms with the exception of a higher incidence of hyperglycemia (p=0.042). Conclusions: Based on Cmax , AUC0-25d , and asparaginase activity 25 days post drug, EZN-2285 2500 IU/m2 appears to have an improved PK/PD profile compared to pegaspargase 2500 IU/m2 and a comparable toxicity profile in children with HR ALL. [Table: see text]

A BCR-ABL1-Like Gene Expression Profile Confers a Poor Prognosis In Patients with High-Risk Acute Lymphoblastic Leukemia (HR-ALL): A Report From Children's Oncology Group (COG) AALL0232

Abstract 743We have previously identified a subset of National Cancer Institute (NCI)-HR B-cell precursor (BCP) ALL patients with a gene expression profile similar to that of BCR-ABL1 ALL (BCR-ABL1-like ALL (Mullighan, N Engl J Med 2009; den Boer, Lancet Oncology 2009; Harvey, Blood, 2010, and unpublished data) and poor outcome on the COG P9906 trial, which was limited to a selected subset of HR BCP ALL patients. These cases are BCR-ABL1-negative but commonly have deletion or mutation of IKZF1. Up to half of these cases harbor rearrangements, deletions and/or mutations activating cytokine receptors and tyrosine kinase signaling (e.g. CRLF2 and activating JAK1/2 mutations), although the kinase-activating mutations in many cases remain unknown. In this analysis, we have assessed the prognostic significance of this BCR-ABL1-like signature in an unselected cohort of BCR-ABL1 negative BCP ALL patients consecutively enrolled on COG AALL0232. This phase 3 trial utilized a 2×2 factorial design comparing dexamethasone (DEX) versus prednisone (PRED) during induction, and high dose methotrexate (HD-MTX) versus Capizzi methotrexate (C-MTX) during interim maintenance 1 (IM-1). We recently reported improved event free survival (EFS) for patients receiving HD-MTX versus C-MTX (Larsen, J Clin Oncol 29: 6s, 2011) and for DEX versus PRED among patients <10 years old randomized to HD MTX (Winick, J Clin Oncol 29: 586s, 2011). We used two algorithms, Recognition of Outliers by Sampling Ends (ROSE) and Predictive Analysis of Microarrays (PAM), to define 66 of 565 (ROSE) and 81 of 572 (PAM) patients as BCR-ABL1-like.Event-free survival (EFS) for BCR-ABL1-like cases was inferior to that of non-BCR-ABL1-like cases, irrespective of the clustering algorithm used to identify them, with 5-yr EFS rates of 63.1±7.2% vs. 84.9±2.0% (p<0.0001) for ROSE clustering and 62.6±6.9% vs. 85.8±2.0% (p<0.0001) for PAM. These differences were maintained regardless of randomized treatment arm.We next examined variables that contributed to outcome in patients who displayed the BCR-ABL1-like signature, identified either by ROSE or PAM. Older (≥ 10 years) BCR-ABL1-like patients were significantly more likely to have an initial white blood count greater than 100,000/ul (ROSE: p< 0.001, PAM: p< 0.001). Interestingly, older females with the BCR-ABL1-like signature had superior EFS compared to males (4-yr EFS for ROSE: 73. ±9.8% vs. 43.0 ±10.3%, p=0.02; 4-yr EFS for PAM: 69. ±10.2% vs. 43. ±9.4%, p=0.04). In a multivariate COX regression analysis of the entire cohort that included identification of BCR/ABL1-like by PAM (HR 1.88, p=0.011), the other significant predictors of poor outcome were the presence of minimal residual disease (MRD) ≥ 0.01% in the bone marrow as measured by flow cytometric methods on day 29 (HR 3.09, p < 0.0001) and the presence of hypodiploidy (HR 3.14, p=0.027). In a COX model including identification of BCR/ABL1-like by ROSE (HR 1.65, p=0.053), other significant factors were day 29 MRD positivity (HR 3.26, p<0.0001), age ≥ 10 years (HR 1.61, p=0.047), presenting white blood cell count > 100,000/ul at diagnosis (HR 1.62, p=0.047), and hypodiploidy (HR 3.0, p=0.034).In summary, the BCR-ABL1-like gene expression profile identified a subset of unselected BCP ALL patients using two different clustering algorithms that was strongly associated with a high rate of treatment failure, even with the best available therapy recently identified in COG AALL0232. The prognostic significance of these gene signatures was also independent of other known risk factors. Ongoing work to determine the genetic and biochemical landscape that contribute to this phenotype will hopefully yield new approaches to treatment for these BCR-ABL1-like patients in order to improve outcome. Disclosures:Borowitz:BD Biosciences: Research Funding. Wood:BD Biosciences: Research Funding. Hunger:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speaker's children own stock in BMS.

Improved Post-Induction Chemotherapy Does Not Abrogate Prognostic Significance of Minimal Residual Disease (MRD) for Children and Young Adults with High Risk Acute Lymphoblastic Leukemia (ALL). A Report From Children's Oncology Group (COG) Study AALL0232

Abstract 1440Improved Post-Induction Chemotherapy Does Not Abrogate Prognostic Significance of Minimal Residual Disease (MRD) for Children and Young Adults with High Risk Acute Lymphoblastic Leukemia (ALL). A Report from Children's Oncology Group (COG) Study AALL0232.Minimal residual disease is one of the strongest prognostic factors in pediatric ALL. COG AALL0232 was a phase 3 randomized trial for patients 1–30 years old with newly diagnosed NCI HR B precursor ALL that used a 2×2 factorial study design comparing dexamethasone (DEX) versus(vs.) prednisone(PRED) during induction, and high dose methotrexate (HD-MTX) vs. Capizzi methotrexate (C-MTX) during interim maintenance 1(IM-1). We previously reported improved event-free survival (EFS) for patients receiving HD-MTX vs. C-MTX (J Clin Oncol 29: 6s, 2011) and for DEX vs. PRED among patients <10 years old randomized to HD-MTX(J Clin Oncol 29: 586s,2011). MRD was measured by 6 color flow cytometry in two central labs (MJB and BLW) to a level of sensitivity of 0.01% at end induction. Patients with >=0.1% MRD at end induction, as well as patients with morphologic slow early response or specific adverse genetic features received intensified therapy including IM-2 and a second delayed intensification, and then had MRD determined at end consolidation, (about 13 weeks post diagnosis). End induction MRD > =0.01% was highly predictive of inferior outcome, though patients with 0.1–1% MRD who received intensive therapy had very low rates of early relapse and a much higher rate of late relapse. [Display omitted] 5 year EFS for end-induction MRD positive (>=0.01%) patients was 63±5% vs. 86±2% for MRD negative patients. However, patients who were MRD positive at end induction who became negative by end consolidation had improved 5y EFS of 79±9%(n=136) compared to 52±14% for those who remained MRD positive(n=52) (p=.0012). Both end induction MRD positive and negative patients benefitted from HD-MTX vs. C-MTX, though the effect was small and did not reach statistical significance for MRD positive patients. By contrast, end-induction MRD was highly predictive of outcome for patients receiving either HD-MTX or C-MTX.5 y EFS as a function of MRD status and IM regimen.End induction MRDCapizziHDMTXP value<.01%84 ± 3%88 ± 2%.04>.01%59 ± 6%67 ± 7%.12P value<.0001<.0001End induction MRD negative patients <10y receiving DEX had better outcome than those getting PRED (5 y EFS 92±3% vs. 87±4% P=.027) while MRD positive patients or those>10y showed no difference. However, DEX patients <10y if anything had a slightly higher rate of end induction MRD positivity than those given PRED (22% vs. 17%, p=.073). In multivariate analysis, end consolidation MRD was the most powerful prognostic factor for the small subset of patients in whom this was assessed. Excluding this, end induction MRD was the most significant variable; age, white blood cell count, day 15 marrow morphology and HD-MTX vs. C-MTX were also significant. We conclude that MRD remains the most powerful prognostic factor even in the context of improved therapy. Additionally, for those patients who were MRD positive at end induction, achieving MRD negative status by end consolidation improved outcome significantly. The higher frequency of MRD in younger patients receiving DEX calls into question the validity of using end induction MRD as a surrogate for outcome when testing novel interventions during induction therapy. Disclosures:Borowitz:BD Biosciences: Research Funding. Wood:BD Biosciences: Research Funding.

Cytogenetic and early-response characteristics of adolescents and young adults with acute lymphoblastic leukemia (ALL): A Children's Oncology Group (COG) study.

9509 Background: Outcomes foradolescents and young adults (AYA) with ALL are inferior to those for younger children, raising the question of whether there are differences in the underlying biology of the disease or host. Methods: Blast cytogenetic features, clinical and early disease response characteristics for two cohorts of patients (ages 1-15 vs. 16-30 years) enrolled on the current COG AALL0232 therapeutic trial for NCI high risk B-precursor ALL were compared. All patients were required to have blast cytogenetic studies for prognostically significant chromosome translocations and ploidy as well as measurements of marrow minimal residual disease (MRD) by flow cytometry at end-induction in COG Reference Laboratories. The AALL0232 trial includes a standard 4-drug induction with a randomization to dexamethasone (10 mg/m2/day) for 14 days or prednisone (60 mg/m2/day) for 28 days. These analyses are confined to patients randomized to receive prednisone during induction therapy, as the results for the steroid randomization remain blinded. Results: 1379 eligible/evaluable patients were analyzed; 1113 patients ages 1-15 and 266 patients ages 16-30 years (AYA). AYA ALL was characterized by a male predominance (69% vs. 54%, p<0.0001) and a lower initial median white blood cell count (9 vs. 33 × 109/L, p<0.0001). Fewer AYA patients had the ETV6-RUNX1 fusion (3.7% vs. 14.4%, p<0.0001); however, simultaneous trisomies of chromosomes 4, 10, 17 were more common in AYA ALL (16.0% vs. 10.2%, p=0.02). There were no significant differences in the presence of very high risk features including the BCR-ABL1 fusion, MLL translocations, or hypodiploidy among the two age cohorts. End-induction MRD was negative (<0.01%) in 59% of AYA patients compared to 74% of younger patients (p<0.0001) and only 67% of AYA vs. 80% of younger patients (p=0.0015) achieved an M1 marrow (< 5% blasts) on day 15 of induction. Conclusions: AYA patients with ALL have similar very high risk cytogenetic profiles when compared to younger patients, but display slower initial responses to induction therapy, suggesting that additional biologic features in AYA ALL have yet to be discovered. No significant financial relationships to disclose.

Increased Incidence of Osteonecrosis (ON) with a Dexamethasone (DEX) Induction for High Risk Acute Lymphoblastic Leukemia (HR-ALL): A Report from the Children's Oncology Group (COG).

Treatment with DEX, rather than prednisone (PRED), improves outcome for children with standard risk ALL. However, DEX exposure is strongly associated with the development of therapy-related ON, particularly in adolescents. Previous COG HR-ALL studies have shown a lower ON risk for patients receiving 1 vs. 2 delayed intensification (DI) phases, and for DI using discontinuous DEX (days 1–7 & 15–21) vs. continuous DEX (days 1–21), suggesting a strategy for giving the drug with acceptable toxicity. The HRALL study COG AALL0232 utilizes a modified augmented BFM backbone that compares in a 2x2 randomized design:induction DEX (10 mg/M2/day x14 days) vs PRED (60 mg/M2/day x28 days), andinterim maintenance (IM) escalating-dose “Capizzi” methotrexate vs. high-dose (HD) MTX.Induction rapid early responders (RER) receive single DI while slow responders receive double DI; all patients receive monthly 5-day DEX pulses during maintenance. To limit ON risk in adolescents, in the initial study design children ≥ 13y received discontinuous DEX during single or double DI; those <13y received continuous DEX. In 10/2006 the study was amended due to an unexpectedly high ON incidence in patients 10–12y receiving continuous DEX (28% @ 18 months) compared with historical controls given discontinuous DEX (3.4%). Subsequently all patients ≥ 10y have received discontinuous DEX; it is too early to assess the impact of this change. A comprehensive interim ON analysis was completed 4/2008 (reported as 24-month cumulative incidences). Overall ON incidence is 10.4% (110/1647), and is higher for those age ≥ 10 vs. <10y (15.2 vs 2.6%, p<0.0001, RHR=6.38); 99/110 cases of ON occurred in the older cohort. Among all patients, ON incidence is higher in DEX vs. PRED regimens (11.6 vs 8.7%, p=0.014, RHR 1.64); rates are similar for Capizzi MTX vs. HD-MTX regimens (10.4 vs 9.8%). Among patients ≥ 13y, incidence is higher in DEX vs. PRED regimens (18.9 vs 9.9%, p=0.02, RHR 1.97). There is no difference between regimens for children <10y. Among randomized RER patients ≥ 10y, incidence is higher in DEX vs. PRED regimens (17.2 vs 12.6%, p=0.006, RHR=1.79). For historical comparison, ON incidences by age cohort for RER patients on AALL0232 regimen PC (PRED + Capizzi MTX) vs. CCG-1961 regimen D (double DI with discontinuous DEX) were: <10y 4.1±3.4 vs. 2.0±1.4%, 10–12y 21.9±10.6 vs. 7.1±2.8%, and ≥ 13y 7.1±10.1 vs. 6.6±3.8%. To address these findings, the study was amended 6/2008. Patients ≥ 10y will be non-randomly assigned to induction PRED; the induction steroid randomization will continue for younger patients. Patients of all ages will receive discontinuous DEX during DI and PRED pulses during maintenance. Of note, compared with CCG-1961 the AALL0232 augmented BFM backbone was non-randomly modified in several ways that may affect ON incidence, including the use of pegaspargase during induction, a higher 15 mg intrathecal methotrexate dose for those age ≥ 9y, and monthly DEX instead of PRED maintenance pulses. Heightened awareness among caregivers may also have led to increased recognition and reporting of this toxicity. Using CTCAE v3.0 criteria, clinical ON severity among the 110 patients is: 3% grade 1, 60% grade 2, 35% grade 3, and 2% grade 4. Data regarding surgical intervention are being collected. These findings will directly influence the design of future trials in an effort to lessen the incidence and burden of this toxicity.