To determine the clinical significance of minimal residual disease (MRD) in patients with prognostically relevant subtypes of childhood acute lymphoblastic leukemia (ALL), we analyzed data from 488 patients treated in St Jude Total Therapy Study XV with treatment intensity based mainly on MRD levels measured during remission induction. MRD levels on day 19 predicted treatment outcome for patients with hyperdiploid >50 ALL, National Cancer Institute (NCI) standard-risk B-ALL or T-cell ALL, while MRD levels on day 46 were prognostic for patients with NCI standard-risk or high-risk B-ALL. Patients with t(12;21)/( ETV6-RUNX1 ) or hyperdiploidy >50 ALL had the best prognosis; those with a negative MRD on day 19 had a particularly low risk of relapse: 1.9% and 3.8%, respectively. Patients with NCI high-risk B-ALL or T-cell ALL had an inferior outcome; even with undetectable MRD on day 46, cumulative risk of relapse was 12.7% and 15.5%, respectively. Among patients with NCI standard-risk B-ALL, the outcome was intermediate overall but was poor if MRD was ⩾1% on day 19 or MRD was detectable at any level on day 46. Our results indicate that the clinical impact of MRD on treatment outcome in childhood ALL varies considerably according to leukemia subtype and time of measurement.

With improved contemporary therapy, we reassess long-term outcome in patients completing treatment for childhood acute lymphoblastic leukemia (ALL) to determine when cure can be declared with a high degree of confidence. In six successive clinical trials between 1984 and 2007, 1291 (84.5%) patients completed all therapies in continuous complete remission. The post-therapy cumulative risk of relapse or development of a second neoplasm and the event-free survival rate and overall survival were analyzed according to the presenting features and the three treatment periods defined by relative outcome. Over the three treatment periods, there has been progressive increase in the rate of event-free survival (65.2% vs 74.8% vs 85.1% (P<0.001)) and overall survival (76.5% vs 81.1% vs 91.7% (P<0.001)) at 10 years. The most important predictor of outcome after completion of therapy was the type of treatment. In the most recent treatment period, which omitted the use of prophylactic cranial irradiation, the post-treatment cumulative risk of relapse was 6.4%, death in remission 1.5% and development of a second neoplasm 2.3% at 10 years, with all relapses except one occurring within 4 years of therapy. None of the 106 patients with the t(9;22)/BCR-ABL1, t(1;19)/TCF3-PBX1 or t(4;11)/MLL-AFF1 had relapsed after 2 years from completion of therapy. These findings demonstrate that with contemporary effective therapy that excludes cranial irradiation, approximately 6% of children with ALL may relapse after completion of treatment, and those who remain in remission at 4 years post treatment may be considered cured (that is, less than 1% chance of relapse).

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL.

Prognostic features in acute megakaryoblastic leukemia in children without Down syndrome: a report from the AML02 multicenter trial and the Children’s Oncology Group Study POG 9421

ETV6-RUNX1 fusion is the most common genetic aberration in childhood acute lymphoblastic leukemia (ALL). To evaluate whether outcomes for this drug-sensitive leukemia are improved by contemporary risk-directed therapy, we studied clinical features, response and adverse events of 168 children with newly diagnosed ETV6-RUNX1-positive ALL on St Jude Total Therapy studies XIIIA (N=36), XIIIB (N=38) and XV (N=94). Results were compared with 494 ETV6-RUNX1-negative B-precursor ALL patients. ETV6-RUNX1 was associated with age 1–9 years, pre-treatment classification as low risk and lower levels of minimal residual disease (MRD) on day 19 of therapy (P<0.001). Event-free survival (EFS) or overall survival (OS) did not differ between patients with or without ETV6-RUNX1 in Total XIIIA or XIIIB. By contrast, in Total XV, patients with ETV6-RUNX1 had significantly better EFS (P=0.04; 5-year estimate, 96.8±2.4% versus 88.3±2.5%) and OS (P=0.04; 98.9±1.4% versus 93.7±1.8%) than those without ETV6-RUNX1. Within the ETV6-RUNX1 group, the only significant prognostic factor associated with higher OS was the treatment protocol Total XV (versus XIIIA or XIIIB) (P=0.01). Thus, the MRD-guided treatment schema including intensive asparaginase and high-dose methotrexate in the Total XV study produced significantly better outcomes than previous regimens and demonstrated that nearly all children with ETV6-RUNX1 ALL can be cured.

Abstract Abstract 3115 Poster Board III-52 Mutations in the RAS proto-oncogenes are frequent in acute myeloid leukemia (AML) and serve as prototypic Class I lesions, initiating key downstream hyper-proliferative signal transduction pathways. N-RAS mutations (N-RASmut) are common in AML, occurring in 10-20% of adult and pediatric AML patients; however their prognostic significance in both adults and children remains disputed. Due to a frequent association with a normal karyotype, delineating the impact of these mutations on outcome may enable appropriate risk-adapted therapeutic approaches. Here, we report on the incidence and prognostic significance of N-RASmut in a cohort of 825 pediatric AML patients treated on 2 recent co-operative group studies, CCG-2961 and COG AAML03P1. In total, of the 825 children with de novo AML who underwent N-RAS mutational analysis, 86 (10%) were positive. Gender, age, race, presence of hepatosplenomegaly and FAB subtype were comparable between patients with and without N-RASmut. There was no statistically significant difference between those with and without N-RASmut with respect to specific chromosome class. FLT3/Internal tandem duplications (FLT3/ITD) were less common in those with N-RASmut (2% vs. 9%, p=0.03). In contrast, nucleophosmin (NPM) mutations were more common in those with N-RASmut (13% vs. 5%, p=0.02). Overall, N-RASmut showed a significant correlation with low risk AML (CBF, CEBPαa, or NPM, p=0.04) and an inverse relationship with high risk disease (-5/5q- or -7, FLT3/ITD/high allelic ratio, p=0.007). Patients with N-RASmut had similar complete remission (CR) rates following two courses of induction chemotherapy compared with non-mutated patients (79% vs. 79%, p=0.92). Those in CR had a similar relapse rate regardless of the presence of N-RASmut (RR 39% vs. 36%, p=0.97). Five year event free survival (EFS) and overall survival (OS) from study entry were also comparable, however N-RASmut patients demonstrated a marked increase in overall treatment related mortality (TRM) (21% vs. 14%, p=0.03), which was maintained when high and low risk patients were excluded (22% vs. 11%, p=0.05). Evaluation of TRM in CCG-2961 compared with COG AAML03P1 demonstrated that increased TRM in N-RASmut was only seen in patients treated on CCG-2961 and TRM was primarily due to invasive fungal and gram negative infections. Interestingly, for patients treated on CCG-2961, who received interval compressed intensive induction therapy, this increase in TRM correlated with a decrease in both OS and disease free survival from CR. Remission specimens from patients with N-RASmut were negative for the mutation, demonstrating that an increased infectious risk could not be attributed to a host polymorphism. We found N-RASmut did not contribute to increased disease recurrence in pediatric AML. Our findings indicate that N-RAS mutations do not identify a high risk population, however, as such mutations lead to downstream activation of signal transduction pathways, they may identify a target for directed therapy. Disclosures No relevant conflicts of interest to declare.

Review on 11q23 rearrangements in childhood acute lymphoblastic leukemia, with data on clinics, and the genes involved.

Recent whole-genome sequencing efforts led to the identification of IDH1R132 mutations in acute myeloid leukemia (AML) patients. We studied the prevalence and clinical implications of IDH1 genomic alterations in pediatric and adult AML. Diagnostic DNA from 531 AML patients treated on Children's Oncology Group trial COG-AAML03P1 (N=257), and Southwest Oncology Group trials SWOG-9031, SWOG-9333 and SWOG-9500 (N=274), were tested for IDH1 mutations. Codon R132 mutations were absent in the pediatric cohort, but were found in 12 of 274 adult patients (4.4%, 95% CI 2.3–7.5). IDH1R132 mutations occurred most commonly in patients with normal karyotype, and those with FLT3/ITD and NPMc mutations. Patients with IDH1R132 mutations trended toward higher median diagnostic white blood cell counts (59.2 × 109 vs 29.1 × 109 per liter, P=0.19) than those without mutations, but the two groups did not differ significantly in age, bone marrow blast percentage, overall survival or relapse-free survival. Eleven patients (2.1%) harbored a novel V71I sequence alteration, which was found to be a germ-line polymorphism. IDH1 mutations were not detected in pediatric AML, and are uncommon in adult AML.

Because cladribine can increase cytarabine triphosphate levels, we tested a cladribine–cytarabine combination in the St Jude AML97, trial in which this combination was administered before standard chemotherapy to 96 children with acute myeloid leukemia (AML) or myelodysplastic syndrome. Patients received a 5-day course of cladribine (9 mg/m 2 per dose) and cytarabine either as daily 2-h infusions (500 mg/m 2 per dose) (arm A) or a continuous infusion (500 mg/m 2 per day) (arm B). Ara-CTP levels and inhibition of DNA synthesis increased from day 1 to day 2, but were not different between the two arms. In addition, the median blast percentages at day 15 did not differ between arms A and B, but patients treated in arm A had shorter intervals between the initiation of the first and second courses of therapy. Thus, although there were trends toward better complete remission rates and overall survival for patients treated in arm B, the reduced efficacy of arm A may have been partially compensated by more intense timing of therapy for that group. For all patients, 5-year event-free survival and overall survival estimates were 44.1±5.4 and 50.0±5.5%. Our results suggest that cladribine in combination with continuous-infusion cytarabine is effective therapy for childhood AML.

To evaluate the impact of contemporary therapy on the clinical outcome of children with pre-B acute lymphoblastic leukemia (ALL) and the t(1;19)/ TCF3/PBX1 , we analyzed 735 patients with B-cell precursor ALL treated in four successive protocols at St Jude Children's Research Hospital. The 41 patients with the t(1;19) had a comparable event-free survival to that of the 694 patients with other B-cell precursor ALL ( P =0.63; 84.2±7.1% (s.e.) vs 84.0±1.8% at 5 years). However, patients with the t(1;19) had a lower cumulative incidence of any hematological relapse ( P =0.06; 0 vs 8.3±1.2% at 5 years) but a significantly higher incidence of central nervous system (CNS) relapse ( P <0.001; 9.0±5.1% vs 1.0±0.4% at 5 years). In a multivariate analysis, the t(1;19) was an independent risk factor for isolated CNS relapse. These data suggest that with contemporary treatment, patients with the t(1;19) and TCF3/PBX1 fusion have a favorable overall outcome but increased risk of CNS relapse.

We analyzed the long-term outcome of 1011 patients treated in five successive clinical trials (Total Therapy Studies 11, 12, 13A, 13B, and 14) between 1984 and 1999. The event-free survival improved significantly (P=0.003) from the first two trials conducted in the 1980s to the three more recent trials conducted in the 1990s. Approximately 75% of patients treated in the 1980s and 80% in the 1990s were cured. Early intensive triple intrathecal therapy, together with more effective systemic therapy, including consolidation and reinduction treatment (Studies 13A and 13B) as well as dexamethasone (Study 13B), resulted in a very low rate of isolated central nervous system (CNS) relapse rate (<2%), despite the reduced use of cranial irradiation. Factors consistently associated with treatment outcome were age, leukocyte count, immunophenotype, DNA index, and minimal residual disease level after remission induction treatment. Owing to concerns about therapy-related secondary myeloid leukemia and brain tumors, in our current trials we reserve the use of etoposide for patients with refractory or relapsed leukemia undergoing hematopoietic stem cell transplantation, and cranial irradiation for those with CNS relapse. The next main challenge is to further increase cure rates while improving quality of life for all patients.

The genetic risk factors for etoposide-induced leukemia with MLL translocations remain largely unknown. To identify genetic risk factors for and novel characteristics of secondary leukemia, we profiled 116 204 single nucleotide polymorphisms (SNPs) in germline and paired leukemic cell DNA from 13 secondary leukemia/myelodysplasia cases and germline DNA from 13 matched and 156 unmatched controls, all with acute lymphoblastic leukemia treated with etoposide. We analyzed global gene expression from a partially overlapping cohort. No single locus was altered in most cases. We discovered 81 regions of loss of heterozygosity (LOH) in leukemic blasts and 309 SNPs whose allele frequencies differed in cases vs controls. Candidate genes were prioritized on the basis of genes whose SNPs or expression differentiated cases from controls or showed LOH or copy number change in germline vs paired blast DNA from the13 cases. Three biological pathways were altered: adhesion, Wnt signaling and regulation of actin. Validation experiments using a genome scan for etoposide-induced leukemogenic MLL chimeric fusions in 15 HapMap cell lines also implicated genes involved in adhesion, a process linked to de novo leukemogenesis. Independent clinical epidemiologic and in vitro genome-wide approaches converged to identify novel pathways that may contribute to therapy-induced leukemia.

To correlate leukemic cell karyotype with immunophenotype, we studied 364 children with acute lymphoblastic leukemia (ALL). A prognostically favorable cytogenetic feature, hyperdiploidy greater than 50 chromosomes, was found in 33% of cases classified as common ALL antigen positive (CALLA+) early pre-B (common) ALL, in contrast to 18% of pre-B cases (P = .012), 5% of T cell cases (P less than .001), and none of the B cell cases (P less than .001) or cases of CALLA negative (CALLA-) early pre-B ALL (P = .002). The frequency of translocations, an adverse cytogenetic feature, was significantly lower in CALLA+ early pre-B ALL cases (35%) than in B cell (100%; P less than .0001), pre-B (59%; P less than .001), or CALLA- early pre-B (62%; P = .016) cases. Thus, patterns of chromosomal change differ widely among the major immunophenotypic groups of ALL and may account for reported differences in responsiveness to treatment.

Secreted protein, acidic and rich in cysteine (SPARC), is a matricellular glycoprotein with growth-inhibitory and antiangio-genic functions. Although SPARC has been implicated as a tumor suppressor in humans, its function in normal or malignant hematopoiesis has not previously been studied. We found that the leukemic cells of AML patients with MLL gene rearrangements express low to undetectable amounts of SPARC whereas normal hematopoietic progenitors and most AML patients express this gene. SPARC RNA and protein levels were also low or undetectable in AML cell lines with MLL translocations. Consistent with its tumor suppressive effects in various solid tumor models, exogenous SPARC protein selectively reduced the growth of cell lines with MLL rearrangements by inhibiting cell cycle progression from G1 to Sphase. The lack of SPARC expression in MLL-rearranged cell lines was associated with dense promoter methylation. However, we found no evidence of methylation-based silencing of SPARC in primary patient samples. Our results suggest that low or absent SPARC expression is a consistent feature of AML cells with MLL rearrangements and that SPARC may function as a tumor suppressor in this subset of patients. A potential role of exogenous SPARC in the therapy of MLL-rearranged AML warrants further investigation.

6549 Background: Recurring balanced chromosome aberrations involving band 11q23 have been detected in both de novo AL/MDS and therapy-related AL/MDS (t-AL/MDS), but de novo 11q23 AL/MDS has seldom been compared directly with 11q23 t-AL/MDS. Methods: We compared pretreatment characteristics and overall survival (OS) of 128 adult patients (pts) with de novo 11q23 AL (n=126) or MDS (n=2) enrolled onto CALGB study 8461 with those of 131 adults with 11q23 t-AL (n=118) or t-MDS (n=13) studied at the IWBCAt-AL/MDS. Results: In analyses restricted to acute myeloid leukemia (AML) and MDS, pts with de novo disease had higher platelet counts (P=.008), and % marrow (P<177>.0001) and blood blasts (P=.0008) than pts with t-AML/MDS, whereas other pretreatment characteristics were similar. The 1- and 3-year OS rates were 43% and 20% for de novo AML/MDS pts, but only 25% and 8%, respectively, for t-AML/MDS pts (P<.001, see Table). In pts receiving stem cell transplantation (SCT), de novo AML/MDS pts (n=30) had 1- and 3-year OS rates of 80% and 37% compared with, respectively, 43% and 20% for t-AML/MDS pts (n=20, P<.01). The difference in OS between de novo AML/MDS and t-AML/MDS was mainly due to the significantly better OS of pts with de novo AML/MDS harboring t(9;11)(p22;q23) or t(11;19)(q23;p13.3). There were no significant differences in pretreatment characteristics or OS between pts with de novo (n=31) and therapy-related (n=11) acute lymphoblastic leukemia (ALL) with 11q23 abnormalities, the majority of whom had t(4;11)(q21;q23). Conclusions: OS of pts with de novo AML/MDS was superior to OS of t-AML/MDS pts, primarily because of more favorable outcomes of de novo AML/MDS with t(9;11)(p22;q23) and t(11;19)(q23;p13.3). Although SCT improved OS of both de novo and therapy-related pts, the outcome for both types of 11q23 AML/MDS remains poor, highlighting the need for novel therapeutic approaches. Author Disclosure OS of de novo and t-AL/MDS with balanced 11q23 abnormalities [*11q23 breakpoint partner] Type of AL/MDS All 11q23 AML/MDS cases 9p22* 6q27* 19p13.1* 19p13.3* Other 11q23 AML/MDS pts with SCT ALL 4q21* De novo #pts 1-yr 3-yr 97 43% 20% 42 48% 31% 17 47% 12% 11 27% 18% 7 57% 14% 20 35% 15% 30 80% 37% 27 41% 22% Therapy-related #pts 1-yr 3-yr 120 25% 8% 65 19% 6% 8 38% 13% 15 53% 18% 14 26% 26% 18 29% 0% 20 43% 20% 11 46% 46% P value <.001 <.001 .78 .69 .04 .13 <.01 .32 No significant financial relationships to disclose.