High-risk B-precursor Acute Lymphoblastic Leukemia Research Articles

CD22 Exon 12 Deletion as an Independent Predictor of Poor Treatment Outcomes in B-ALL.

We previously reported a splicing defect (CD22ΔE12) associated with the deletion of exon 12 of the inhibitory co-receptor CD22 (Siglec-2) in leukemia cells from patients with CD19+ B-precursor acute lymphoblastic leukemia (B-ALL). CD22ΔE12 causes a truncating frameshift mutation and yields a dysfunctional CD22 protein that lacks most of the cytoplasmic domain required for its inhibitory function, and it is associated with aggressive in vivo growth of human B-ALL cells in mouse xenograft models. Although CD22ΔE12 with selective reduction of CD22 exon 12 (CD22E12) levels was detected in a high percentage of newly diagnosed as well as relapsed B-ALL patients, its clinical significance remains unknown. We hypothesized that B-ALL patients with very low levels of wildtype CD22 would exhibit a more aggressive disease with a worse prognosis because the missing inhibitory function of the truncated CD22 molecules could not be adequately compensated by competing wildtype CD22. Here, we demonstrate that newly diagnosed B-ALL patients with very low levels of residual wildtype CD22 ("CD22E12low"), as measured by RNAseq-based CD22E12 mRNA levels, have significantly worse leukemia-free survival (LFS) as well as overall survival (OS) than other B-ALL patients. CD22E12low status was identified as a poor prognostic indicator in both univariate and multivariate Cox proportional hazards models. CD22E12low status at presentation shows clinical potential as a poor prognostic biomarker that may guide the early allocation of risk-adjusted, patient-tailored treatment regimens and refine risk classification in high-risk B-ALL.

Prognostic significance of minimal residual disease in high risk B-ALL: a report from Children's Oncology Group study AALL0232

AbstractMinimal residual disease (MRD) is highly prognostic in pediatric B-precursor acute lymphoblastic leukemia (B-ALL). In Children's Oncology Group high-risk B-ALL study AALL0232, we investigated MRD in subjects randomized in a 2 × 2 factorial design to receive either high-dose methotrexate (HD-MTX) or Capizzi methotrexate (C-MTX) during interim maintenance (IM) or prednisone or dexamethasone during induction. Subjects with end-induction MRD ≥0.1% or those with morphologic slow early response were nonrandomly assigned to receive a second IM and delayed intensification phase. MRD was measured by 6-color flow cytometry in 1 of 2 reference labs, with excellent agreement between the two. Subjects with end-induction MRD <0.01% had a 5-year event-free survival (EFS) of 87% ± 1% vs 74% ± 4% for those with MRD 0.01% to 0.1%; increasing MRD amounts was associated with progressively worse outcome. Subjects converting from MRD positive to negative by end consolidation had a relatively favorable 79% ± 5% 5-year disease-free survival vs 39% ± 7% for those with MRD ≥0.01%. Although HD-MTX was superior to C-MTX, MRD retained prognostic significance in both groups (86% ± 2% vs 58% ± 4% for MRD-negative vs positive C-MTX subjects; 88% ± 2% vs 68% ± 4% for HD-MTX subjects). Intensified therapy given to subjects with MRD >0.1% did not improve either 5-year EFS or overall survival (OS). However, these subjects showed an early relapse rate similar to that seen in MRD-negative ones, with EFS/OS curves for patients with 0.1% to 1% MRD crossing those with 0.01% to 0.1% MRD at 3 and 4 years, thus suggesting that the intensified therapy altered the disease course of MRD-positive subjects. Additional interventions targeted at the MRD-positive group may further improve outcome. This trial was registered at www.clinicaltrials.gov as #NCT00075725.

Low Dose Total Body Irradiation Combined With Recombinant CD19-Ligand × Soluble TRAIL Fusion Protein is Highly Effective Against Radiation-resistant B-precursor Acute Lymphoblastic Leukemia in Mice

In high-risk remission B-precursor acute lymphoblastic leukemia (BPL) patients, relapse rates have remained high post-hematopoietic stem cell transplantation (HSCT) even after the use of very intensive total body irradiation (TBI)-based conditioning regimens, especially in patients with a high “minimal residual disease” (MRD) burden. New agents capable of killing radiation-resistant BPL cells and selectively augmenting their radiation sensitivity are therefore urgently needed. We report preclinical proof-of-principle that the potency of radiation therapy against BPL can be augmented by combining radiation with recombinant human CD19-Ligand×soluble TRAIL (“CD19L–sTRAIL”) fusion protein. CD19L–sTRAIL consistently killed radiation-resistant primary leukemia cells from BPL patients as well as BPL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. Low dose total body irradiation (TBI) combined with CD19L–sTRAIL was highly effective against (1) xenografted CD19+ radiochemotherapy-resistant human BPL in NOD/SCID (NS) mice challenged with an otherwise invariably fatal dose of xenograft cells derived from relapsed BPL patients as well as (2) radiation-resistant advanced stage CD19+ murine BPL with lymphomatous features in CD22ΔE12xBCR-ABL double transgenic mice. We hypothesize that the incorporation of CD19L–sTRAIL into the pre-transplant TBI regimens of patients with very high-risk BPL will improve their survival outcome after HSCT.

­Resource Utilization and Cost Analysis By Treatment Arm on the Children’s Oncology Group AALL0232 Phase 3 High-Risk B-Precursor Acute Lymphoblastic Leukemia Trial: A Report from the Children’s Oncology Group

NCI-funded cooperative oncology group trials collect extensive biologic and outcome data but typically do not collect resource utilization (RU) or cost data. RU measures reflect therapy associated morbidity while cost data enable estimates of the financial burden of therapy. The ChildrenÕs Oncology Group (COG) trial AALL0232 used a 2x2 factorial randomization between 28 days of prednisone (PRED) or 14 days of dexamethasone (DEX) in Induction and high-dose (HD-MTX) or escalating ÒCapizziÓ methotrexate plus asparaginase (C-MTX) in Interim Maintenance I. Children ³10 years old were non-randomly assigned to PRED after May 2008 due to increased osteonecrosis on DEX. HD-MTX had superior event-free survival, and for patients <10 years on HD-MTX, DEX was superior. To estimate RU and inpatient costs on AALL0232, data from the Pediatric Health Information System (PHIS), a national database of daily inpatient billing data from 44 US hospitals, was merged with AALL0232 data. We hypothesized that inpatient RU and costs could be accurately estimated for AALL0232 in a merged COG-PHIS dataset and that RU and inpatient costs would be higher in children receiving HD-MTX, which requires hospitalization for administration.COG provided an AALL0232 patient list that was probabilistically matched to PHIS patients by hospital, dates of diagnosis and birth, and sex. Patients were observed in PHIS data for 365 days post COG enrollment. Daily billing codes were used to tabulate RU and inpatient days. RU data include 30 variables from 4 major categories: laboratory, pharmacy, radiology, and procedures. Inpatient treatment costs were estimated by multiplying geographically adjusted charges by hospital-specific cost-to-charge ratios (RCC) and then adjusted to 2013 US dollars using the consumer price index.Of 3083 patients enrolled on AALL0232, 935 (30.3%) were identified in PHIS. Two hospitals did not submit RCC to PHIS, so 20 patients were excluded from the cost analysis. As expected, age at enrollment differed significantly by study arm in the merged cohort (p<0.0001), while sex, race and ethnicity did not. Inpatient days were higher in the HD-MTX arms (median 53 days; interquartile range [IQR] 35-77) versus C-MTX (median 39 days; IQR 21-64.5; p<0.0001) and did not differ by steroid arm. Overall inpatient RU was similar in the HD-MTX and C-MTX arms, except for complete blood counts and antiemetics (Table 1). Children in the DEX arms had higher numbers of inpatient days of total, broad Gram-positive and antipseudomonal antibiotics than children on PRED (Table 2). There were no other RU differences that were both clinically and statistically significant, including intensive care RU. Inpatient costs were higher in the HD-MTX cohort and did not differ by steroid arm (Table 3).These data demonstrate that inpatient RU and costs can be estimated in COG trials using external data sources and support the hypothesis that inpatient costs are higher in children receiving HD-MTX than C-MTX. However, the median cost difference between HD-MTX and C-MTX was modest ($31,286) and driven by length of hospital stay rather than increased RU. Children randomized to DEX had increased use of antibiotics, which may reflect an effect of DEX on infection risk. The merging of RU and cost data with COG clinical trial data enables treatment arm intensity and cost comparisons that provide important data on the medical and financial burdens of therapy. Work is ongoing to increase the percentage of trial patients with RU and cost data and to incorporate outpatient RU and cost data. Inclusion of RU and cost data in NCI-funded cooperative group trials should provide a more comprehensive assessment of randomized therapies and enable cost-effectiveness analyses.Table 1:RU days by MTX arm (N=935)ResourceHD-MTX n=495 median (IQR)C-MTX n=430 median (IQR)pBlood counts33 (54-232)30 (50-275)0.048Antiemetics25 (35-208)13 (29-227)<0.0001Table 2:RU days by steroid arm (N=935)ResourceDEX n=378 median (IQR)PRED n=557 median (IQR)pTotal antibiotics37 (63-392)28 (59-388)0.022Broad Gram-positives7 (14-87)4 (12-164)0.0041Antipseudomonals2trun -10 (33-98)16 (28-139)0.011Table 3:Cost estimates per patient by randomization (N=915)ArmnCost median (IQR)pHD-MTX487$119,923 (73,981-194,465)<0.0001C-MTX428$88,637 (48,240-175,280)DEX372$101,717 (58,703-179,687)0.44PRED543$106,048 (55,551-193,731) DisclosuresHunger:Bristol Myers Squibb: Consultancy.

Dose Intensification of Methotrexate and Cytarabine During Intensified Continuation Chemotherapy for High-risk B-Precursor Acute Lymphoblastic Leukemia

To determine the efficacy and toxicity of higher dose versus standard dose intravenous methotrexate (MTX) and pulses of high-dose cytosine arabinoside with asparaginase versus standard dose cytosine arabinoside and teniposide during intensified continuation therapy for higher risk pediatric B-precursor acute lymphoblastic leukemia (ALL). From 1994 to 1999, the Pediatric Oncology Group conducted a randomized phase III clinical trial in higher risk pediatric B-precursor ALL. A total of 784 patients were randomized in a 2×2 factorial design to receive MTX 1 g/m versus 2.5 g/m and to cytosine arabinoside/teniposide versus high-dose cytosine arabinoside/asparaginase during intensified continuation therapy. Patients receiving standard dose MTX had a 5-year disease-free survival (DFS) of 71.8±2.4%; patients receiving higher dose MTX had a 5-year DFS of 71.7±2.4% (P=0.55). Outcomes on cytosine arabinoside/teniposide (DFS of 70.4±2.4) were similar to higher dose cytosine arabinoside/asparaginase (DFS of 73.1±2.3%) (P=0.41). Overall survival rates were not different between MTX doses or cytosine arabinoside/teniposide versus cytosine arabinoside/asparaginase. Increasing MTX dosing to 2.5 g/m did not improve outcomes in higher risk pediatric B-precursor ALL. Giving high-dose cytarabine and asparaginase pulses instead of standard dose cytarabine and teniposide produced nonsignificant differences in outcomes, allowing for teniposide to be removed from ALL therapy.

Constitutive Function of the Ikaros Transcription Factor in Primary Leukemia Cells from Pediatric Newly Diagnosed High-Risk and Relapsed B-precursor ALL Patients

We examined the constitutive function of the Ikaros (IK) transcription factor in blast cells from pediatric B-precursor acute lymphoblastic leukemia (BPL) patients using multiple assay platforms and bioinformatics tools. We found no evidence of diminished IK expression or function for primary cells from high-risk BPL patients including a Philadelphia chromosome (Ph)+ subset. Relapse clones as well as very aggressive in vivo clonogenic leukemic B-cell precursors isolated from spleens of xenografted NOD/SCID mice that developed overt leukemia after inoculation with primary leukemic cells of patients with BPL invariably and abundantly expressed intact IK protein. These results demonstrate that a lost or diminished IK function is not a characteristic feature of leukemic cells in Ph+ or Ph- high-risk BPL.

Birinapant (TL32711), a Small Molecule Smac Mimetic, Induces Regressions in Childhood Acute Lymphoblastic Leukemia (ALL) Xenografts That Express TNFα and Synergizes with TNFα in Vitro – A Report From the Pediatric Preclinical Testing Program (PPTP)

AbstractAbstract 3565 Introduction:Birinapant is a small molecule mimetic of Smac that potently and specifically antagonizes multiple inhibitors of apoptosis proteins (IAPs). Birinapant rapidly degrades cIAPs and enables cytokines (TNFα, TRAIL) to activate the extrinsic apoptosis pathway, while it rapidly turns off the canonical NF-κB survival pathway, causing cancer cell death. Preclinical studies using adult cancer models have shown that birinapant causes tumor regressions as a single agent in selected models and that it has potent antitumor activity when combined with chemotherapies and death receptor ligands. Methods:Birinapant was evaluated against the 23 cell lines of the PPTP in vitro panel (including 1 AML and 5 ALL lines) using 96 hour exposure at concentrations from 1.0 nM to 3.0 μM, both as a single agent and in combination with TNFα (10 ng/mL) or TRAIL (10 ng/mL). Birinapant was tested against 2 PPTP solid tumor xenografts (rhabdomyosarcoma, Rh30; Ewing, CHLA-258), an anaplastic large cell lymphoma (ALCL) xenograft (Karpas-299), and 3 B-precursor ALL xenografts (ALL-2, ALL-17, & ALL-19) at a dose of 30 mg/kg administered by the intraperitoneal route using a Q3 day × 5 schedule. Gene expression data for the PPTP cell lines and xenografts was available using both Affymetrix U133 Plus 2.0 and Agilent SurePrint G3 arrays. Results:Birinapant demonstrated limited single agent activity (median relative IC50 (rIC50) > 3 μM), with only the AML cell line Kasumi-1 showing Relative In/Out% (Rel I/O%) values < 0% with rIC50 of 37 nM. Marked potentiation of birinapant was observed for a subset of cell lines with the addition of TNFα or TRAIL. The 5 ALL cell lines showed a median rIC50 value of 3.6 nM for birinapant in combination with TNFα, with Rel I/O% values between −50% and −100% (indicative of a profound cytotoxic effect). Four of 5 ALL cell lines showed little or no potentiation of birinapant effect with the addition of TRAIL. Among solid tumor cell lines, potentiation of birinapant effect was observed for selected rhabdomyosarcoma, rhabdoid tumor, Ewing sarcoma, and neuroblastoma cell lines with the addition of either TNFα or TRAIL.Birinapant was well tolerated in vivo. Birinapant induced significant differences in event-free survival (EFS) distribution compared to control in 3 of 3 (100%) of the B-precursor ALL xenografts, but in none of the solid tumor or ALCL xenografts. Objective responses were not observed for the solid tumor and ALCL xenografts, whereas for the ALL panel one xenograft (ALL-17) achieved a complete response (CR) and another (ALL-2) achieved a maintained CR, with treated animals remaining in remission at day 42, approximately 30 days after their last treatment with birinapant. Given the mechanism of action of Smac mimetics, TNFα expression was examined. TNFα expression was significantly higher for the PPTP ALL xenografts compared to the PPTP solid tumor xenografts and to 15 normal tissues. TNFα expression in ALL clinical specimens was examined using publicly available datasets, with the observation that its expression is significantly higher for high-risk B-precursor ALL compared to a set of normal tissues, but with a wide range of TNFα expression among ALL cases. Lymphotoxin A and B also show significantly elevated expression in ALL compared to normal tissues. Among the ALL xenografts tested with birinapant, the best responding xenograft (ALL-2) showed the highest TNFα expression. Karpas-299 also showed high TNFα expression, but the two solid tumor xenografts did not. Unlike the ALL cell lines for which exogenous TNFα potentiated birinapant in vitro activity, exogenous TNFα did not potentiate birinapant in vitro activity against Karpas-299. Conclusion:Birinapant showed little single agent in vitro activity against ALL cell lines, though it markedly potentiated the activity of exogenously added TNFα for these cell lines. In vivo, birinapant showed remission-inducing activity against 2 of 3 ALL xenografts, with one of these showing a maintained CR. TNFα is mechanistically associated with the activity of Smac mimetics, and the initial PPTP in vivo data for ALL xenografts are consistent with a relationship between TNFα expression and responsiveness to birinapant. The PPTP results suggest that birinapant may show high level activity against a subset of childhood ALL, and additional in vivo testing is ongoing to better identify predictive markers that can reliably select responsive cases. Disclosures:Chunduru:TetraLogic Pharmaceuticals: Employment, Equity Ownership. Graham:TetraLogic Pharmaceuticals: Employment, Equity Ownership. Geier:TetraLogic Pharmaceuticals: Honoraria. Houghton:TetraLogic Pharmaceuticals: Honoraria.

Targeting JAK1/2 and mTOR in murine xenograft models of Ph-like acute lymphoblastic leukemia

AbstractCRLF2 rearrangements, JAK1/2 point mutations, and JAK2 fusion genes have been identified in Philadelphia chromosome (Ph)–like acute lymphoblastic leukemia (ALL), a recently described subtype of pediatric high-risk B-precursor ALL (B-ALL) which exhibits a gene expression profile similar to Ph-positive ALL and has a poor prognosis. Hyperactive JAK/STAT and PI3K/mammalian target of rapamycin (mTOR) signaling is common in this high-risk subset. We, therefore, investigated the efficacy of the JAK inhibitor ruxolitinib and the mTOR inhibitor rapamycin in xenograft models of 8 pediatric B-ALL cases with and without CRLF2 and JAK genomic lesions. Ruxolitinib treatment yielded significantly lower peripheral blast counts compared with vehicle (P < .05) in 6 of 8 human leukemia xenografts and lower splenic blast counts (P < .05) in 8 of 8 samples. Enhanced responses to ruxolitinib were observed in samples harboring JAK-activating lesions and higher levels of STAT5 phosphorylation. Rapamycin controlled leukemia burden in all 8 B-ALL samples. Survival analysis of 2 representative B-ALL xenografts demonstrated prolonged survival with rapamycin treatment compared with vehicle (P < .01). These data demonstrate preclinical in vivo efficacy of ruxolitinib and rapamycin in this high-risk B-ALL subtype, for which novel treatments are urgently needed, and highlight the therapeutic potential of targeted kinase inhibition in Ph-like ALL.

Outcome in adolescent and young adult (AYA) patients compared with younger patients treated for high-risk B-precursor acute lymphoblastic leukemia (HR-ALL): A report from the Children’s Oncology Group study AALL0232.

CRA9508 Background: Historically, AYA patients (&gt;16yrs of age) with HR-ALL have an inferior outcome compared to HR-ALL patients 1-15 yrs old, due to increased rates of both relapse and toxicity. Methods: AALL0232 was a phase III randomized trial for patients 1-30 years of age with newly diagnosed B-precursor HR-ALL that utilized a 2 x 2 factorial design with an augmented intensity Berlin-Frankfurt-Münster (BFM) backbone. Patients were randomized to receive dexamethasone versus prednisone during Induction and high dose methotrexate versus escalating Capizzi methotrexate during Interim Maintenance I. A total of 2,574 eligible, evaluable, non-Down Syndrome, non-very high risk patients were randomized between January 2004 and January 2011. AYA patients comprised 20% of this group (n=501; 16-21 yrs: 466 and 22-30 yrs: 35). Results: The 5-yr event-free survival (EFS) and overall survival (OS) rates were 68.0% and 79.8% for AYA patients compared to 80.9% and 88.4% for younger patients (p&lt;0.0001). The 5-yr cumulative incidence of relapse was 21.3% for AYA patients and 13.4% for younger patients (p=0.0018), largely due to higher rate of marrow relapse (15.2% vs. 9.0%; p&lt;0.0007) and not CNS relapse (5.2% vs. 3.7%; p=0.5776). In addition, fewer AYA patients achieved remission, defined as &lt;5% marrow blasts at end induction (97.2% vs. 98.8%; p=0.0134). There was no significant difference in induction mortality between AYA and younger patients, 2.4% vs. 1.8% (p=0.36). However post-induction remission deaths were significantly higher in AYA compared with younger patients, 5.5% vs. 2.1% at 5 years (p&lt;0.0001). Conclusions: This is the largest cohort of AYA ALL patients presented to date and confirms high cure rates for AYA patients treated on pediatric ALL trials. AYA HR-ALL patients had lower EFS and OS compared to younger patients, primarily attributable to both higher rates of marrow relapses and remission deaths. Effective strategies to improve the outcome of AYA patients with HR-ALL should be aimed at both furthering leukemia eradication and lowering toxicity of therapy for this patient population.

Outcome in adolescent and young adult (AYA) patients compared with younger patients treated for high-risk B-precursor acute lymphoblastic leukemia (HR-ALL): A report from the Children’s Oncology Group study AALL0232.

CRA9508 The full, final text of this abstract will be available at abstract.asco.org at 12:01 AM (EDT) on Saturday, June 2, 2012, and in the Annual Meeting Proceedings online supplement to the June 20, 2012, issue of Journal of Clinical Oncology. Onsite at the Meeting, this abstract will be printed in the Saturday edition of ASCO Daily News.

Augmented therapy improves outcome for pediatric high risk acute lymphocytic leukemia: Results of Children's Oncology Group trial P9906

The augmented BFM regimen improves outcome for children with NCI high acute lymphoblastic leukemia (ALL). Patient age, sex, and presenting white blood cell count (WBC) can be used to identify a subset of approximately 12% of children with B-precursor ALL that had a 5-year continuous complete remission (CCR) rate of only about 50% on earlier Pediatric Oncology Group (POG) trials. Children's Oncology Group trial P9906 evaluated a modified augmented BFM regimen in 267 patients with particularly high risk B-precursor ALL. Minimal residual disease (MRD) was assessed in blood at day 8 and in marrow at day 29 of induction and correlated with outcome. The 5-year CCR probability for patients in P9906 was significantly better than that observed for similar patients on POG trials 8602/9006 (62.2 ± 3.7% vs. 50.6 ± 2.4%; P = 0.0007) but similar to POG 9406 (63.5 ± 2.4%; P = 0.81). Interim analysis showed poor central nervous system (CNS) control, especially in patients with initial WBC ≥ 100,000/microliter. Day 29 marrow MRD positive (≥ 0.01%) vs. negative patients had 5 year CCR rates of 37.1 ± 7.4% vs. 72.6 ± 4.3%; day 8 blood MRD positive vs. negative patients had 5 year CCR rates of 57.1 ± 4.6% vs.83.6 ± 6.3%. End induction marrow MRD predicted marrow but not CNS relapse. In multivariate analysis, day 29 MRD > 0.01%, initial WBC ≥ 100,000/µl, male gender, and day 8 blood MRD > 0.01% were significant prognostic factors. Augmented BFM therapy improved outcome for children with higher risk ALL. Day 8 blood and day 29 marrow MRD were strong prognostic factors in these patients.

Identification of novel cluster groups in pediatric high-risk B-precursor acute lymphoblastic leukemia with gene expression profiling: correlation with genome-wide DNA copy number alterations, clinical characteristics, and outcome

AbstractTo resolve the genetic heterogeneity within pediatric high-risk B-precursor acute lymphoblastic leukemia (ALL), a clinically defined poor-risk group with few known recurring cytogenetic abnormalities, we performed gene expression profiling in a cohort of 207 uniformly treated children with high-risk ALL. Expression profiles were correlated with genome-wide DNA copy number abnormalities and clinical and outcome features. Unsupervised clustering of gene expression profiling data revealed 8 unique cluster groups within these high-risk ALL patients, 2 of which were associated with known chromosomal translocations (t(1;19)(TCF3-PBX1) or MLL), and 6 of which lacked any previously known cytogenetic lesion. One unique cluster was characterized by high expression of distinct outlier genes AGAP1, CCNJ, CHST2/7, CLEC12A/B, and PTPRM; ERG DNA deletions; and 4-year relapse-free survival of 94.7% ± 5.1%, compared with 63.5% ± 3.7% for the cohort (P = .01). A second cluster, characterized by high expression of BMPR1B, CRLF2, GPR110, and MUC4; frequent deletion of EBF1, IKZF1, RAG1-2, and IL3RA-CSF2RA; JAK mutations and CRLF2 rearrangements (P < .0001); and Hispanic ethnicity (P < .001) had a very poor 4-year relapse-free survival (21.0% ± 9.5%; P < .001). These studies reveal striking clinical and genetic heterogeneity in high-risk ALL and point to novel genes that may serve as new targets for diagnosis, risk classification, and therapy.

Longer Time to the Start of Continuation Therapy Is Associated with Improved Survival In High Risk Pediatric Acute Lymphoblastic Leukemia (ALL): A Report From the Children's Oncology Group (COG)

AbstractAbstract 3223 Background:Delays in treatment are common in pediatric ALL and may occur more often in high risk ALL (HR-ALL). Delays during maintenance therapy have adverse impact on survival, but the significance of delays prior to maintenance is unknown (Schmiegelow, et al, American Journal of Pediatric Hematology Oncology 1990 and Dibenedetto et al, Pediatric Hematology Oncology 1994). We studied the effect of delays in therapy prior to maintenance by analyzing data from two HR-ALL clinical trials, CCG1961 and POG9906. The objective was to determine the association between time to maintenance (TTM) from starting induction therapy and event-free survival (EFS) in children with high-risk ALL. Methods:CCG1961 enrolled 2078 patients from September 1996 to May 2002 with B- and T-lineage ALL with high-risk features, defined as those aged 1–9 years with initial white blood cell count (WBC) ≥ 50,000/μl, any patient aged ≥ 10 years and ≤ 21 years, and those with central nervous system (CNS) leukemia or overt testicular leukemia. Rapid early responders (RERs), those with < 25% blasts in the bone marrow on day 7 of therapy, were randomized to receive standard or augmented BFM therapy with either a single interim maintenance and delayed intensification phase (SDI) or two interim maintenance and delayed intensification (DDI) courses as outlined in Table 1. Slow early responders (SERs) were randomized to augmented BFM with DDI with either doxorubicin or idarubicin and cyclophosphamide in the DI courses. POG9906 enrolled 276 patients from March 2000 to April 2003 with high-risk B-precursor ALL, defined as all patients with CNS disease, testicular disease, MLL rearrangement, age ≥ 16 years, or WBC ≥ 100K. In addition, it included selected patients aged 12–15 with high-risk disease based on the Shuster criteria for age and initial WBC. Patients with favorable cytogenetic features (ETV6-RUNX1 fusion or trisomy of chromosomes 4 and 10) were excluded unless they had CNS or testicular leukemia. Patients were treated with augmented BFM with DDI. Patients who made it to start of maintenance are included in this report. TTM was calculated as time from start induction until start of maintenance therapy. Expected TTM for those with B-lineage ALL is shown in Table 1. TTM was dichotomized by plotting the Akaike information criterion (AIC) versus all possible cutoff values of TTM and identifying the cut-point which best discriminated between groups who did and did not fail. The identified threshold was then examined as a predictor of EFS using the Kaplan Meier method and log rank test. The outcomes were 5-year EFS for CCG1961 and 4-year EFS for POG9906. Results:Among the B-lineage patients, longer TTM was significantly associated with improved EFS in CCG1961 Std BFM DI and SER Aug BFM Doxo arms, as well as on POG 9906. For example, in Std BFM DI, 5-year EFS was 83% in those with longer TTM and 71.9% in those with shorter TTM (P=.036). Among the T lineage cohort enrolled on CCG1961, similar results were seen with longer TTM being significantly associated with improved EFS in Std BFM DDI and SER Aug BFM Ida, and a trend toward improved EFS with longer TTM in each of the other arms. Conclusions:These data suggest that longer TTM may be associated with superior EFS in pediatric high-risk ALL. Multiple factors may contribute to this association. For instance, pharmacogenetic differences resulting in more treatment toxicity and delays may also be associated with more potent anti-leukemic effects, or patients with infectious complications may have an endogenous immune response that also has an anti-leukemia effect. Further research is needed to validate the association between TTM and EFS and to characterize the timing and causes of these delays in protocol therapy. Disclosures:No relevant conflicts of interest to declare.

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.

Gene expression classifiers for relapse-free survival and minimal residual disease improve risk classification and outcome prediction in pediatric B-precursor acute lymphoblastic leukemia

AbstractTo determine whether gene expression profiling could improve outcome prediction in children with acute lymphoblastic leukemia (ALL) at high risk for relapse, we profiled pretreatment leukemic cells in 207 uniformly treated children with high-risk B-precursor ALL. A 38-gene expression classifier predictive of relapse-free survival (RFS) could distinguish 2 groups with differing relapse risks: low (4-year RFS, 81%, n = 109) versus high (4-year RFS, 50%, n = 98; P < .001). In multivariate analysis, the gene expression classifier (P = .001) and flow cytometric measures of minimal residual disease (MRD; P = .001) each provided independent prognostic information. Together, they could be used to classify children with high-risk ALL into low- (87% RFS), intermediate- (62% RFS), or high- (29% RFS) risk groups (P < .001). A 21-gene expression classifier predictive of end-induction MRD effectively substituted for flow MRD, yielding a combined classifier that could distinguish these 3 risk groups at diagnosis (P < .001). These classifiers were further validated on an independent high-risk ALL cohort (P = .006) and retainedindependent prognostic significance (P < .001) in the presence of other recently described poor prognostic factors (IKAROS/IKZF1 deletions, JAK mutations, and kinase expression signatures). Thus, gene expression classifiers improve ALL risk classification and allow prospective identification of children who respond or fail current treatment regimens. These trials were registered at http://clinicaltrials.gov under NCT00005603.

Mutations in the RAS Signaling, B-Cell Development, TP53/RB1, and JAK Signaling Pathways Are Common in High Risk B-Precursor Childhood Acute Lymphoblastic Leukemia (ALL): A Report From the Children's Oncology Group (COG) High-Risk (HR) ALL TARGET Project.

Abstract 85▪▪This icon denotes an abstract that is clinically relevant.Despite the overall favorable treatment outcome for childhood ALL, about 20% of patients still experience relapse. Moreover, many ALL cases lack sentinel chromosomal alterations, and the genetic factors contributing to leukemogenesis in these cases are poorly understood. Genome-wide profiling of DNA copy number alterations (CNA) coupled with focused candidate gene resequencing has identified novel genetic alterations that contribute to leukemogenesis and are associated with treatment outcome in ALL. However, large-scale analysis of somatic sequence mutations in ALL has not yet been performed. As part of the COG HR-ALL TARGET (Therapeutically Applicable Research to Generate Effective Targets) Project, 125 genes (selected based on recurrent CNAs, gene expression profiles and known cancer genes) were sequenced in 187 HR B-precursor ALL patients enrolled in COG P9906. The entire coding region and UTRs of each gene were sequenced and more than 98% of the targeted nucleotides have high quality sequence coverage. We found that somatic mutations are frequent in genes that encode for proteins involved in signal transduction, B-cell development and p53/RB signaling. A notable finding was the presence of somatic mutations resulting in constitutive activation of RAS signaling in at least 39% of the cohort. Seventy-three cases have at least one mutation in NRAS (30), KRAS (28), PTPN11 (9), FLT3 (7) and NF1 (6), including 7 patients with multiple mutations (KRAS and NRAS (3), FLT3 and NF1 (1), PTPN11 and FLT3 (1), PTPN11 and FLT3 (1), PTPN11 and NRAS (1), PTPN11 and KRAS (1)). There was no association between RAS pathway mutations and event-free survival or cumulative incidence of relapse in this HR patient cohort. Notably, RAS pathway mutations were uncommon in ALL cases with TCF3-PBX1 (1/22 cases) or MLL translocations (2/18 cases), but occurred frequently in cases lacking known sentinel cytogenetic lesions (68/145 cases, 47%, p<0.0001). Sequence mutations that are known or predicted to impair normal B-cell development were observed in at least 14% of the cohort (PAX5 (21), IKZF1 (7)), with confirmatory germline sequencing underway to clarify whether lymphoblast sequence alterations present in other B-cell development genes including BLK, ETV6, IKZF3, TCF3, RAG1, and BCL11A are somatic or germline. Sequence mutations disrupting TP53/RB1 signaling ((TP53 (10), RB1 (4), CDKN2A (4)) occurred in 10% of cases. Activating sequence mutations in JAK family members (JAK2 (16) and JAK1 (3)) were present in 10% of the cases. Of the 103 cases (55%) with at least one sequence mutation in these four (RAS signaling, B-cell development, p53/RB, and JAK) pathways, 30 have somatic mutations in multiple pathways. When both CNAs and sequence mutations are considered, 94% of the cases have lesions in at least one of the pathways and 31% of the cases have somatic alterations in all three of the B-cell development, RAS, and p53/RB1 signaling pathways, suggesting that aberrations in multiple pathways may be central to development of HR-ALL. Our results contrast sharply with known genetic alterations in T-cell ALL, where RAS pathway alterations are relatively uncommon (<10% of cases). PTEN somatic mutations and deletions occur in approximately 25% of cases of T-cell ALL, but we found no PTEN alterations in this cohort, suggesting differential activation of RAS and PI-3K signaling pathways in T-cell and HR B-precursor ALL. Validation of putative deleterious mutations in other sequenced genes (e.g., TBL1XR1, BLK, and CREBBP) is ongoing. These results from the TARGET COG HR-ALL Pilot Project confirm and extend prior knowledge of genetics of this subtype of ALL and point the way to new potential therapeutic strategies for this patient population. Disclosures:No relevant conflicts of interest to declare.