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Introduction and Aim Although cytogenetics and response-guided therapy have considerably improved prognostication of pediatric AML (pedAML) patients, still 30-40% of the good responders relapse. Further delineation of the transcriptome of AML subpopulations, e.g. leukemic stem cells (LSCs), might result in a better understanding of pedAML biology and provide novel biomarkers for diagnostics, risk stratification, follow-up and targeted therapy. Methods Fluorescence-activated cell sorting (FACS) was used to isolate CD34+CD38- and CD34+CD38+ cells from pedAML patients/healthy controls (cord blood (CB), normal bone marrow (NBM)), defined as LSC/hematopoietic stem cell (HSC) and leukemic blast (L-blast)/control blast (C-blast), respectively. Sorting multiple phenotypes, both BM and blood, yielded 42 LSC and 35 L-blast fractions. Gene expression profiles (GEP) of LSCs and L-blasts were identified by a Cancer vs Normal (CvN) approach, whereas paired analysis of LSC vs L-blast aimed to identify LSC-specific aberrations. Micro-array analysis (4 pedAML, 3 CB) was followed by targeted quantitative PCR (qPCR) validation of the highest differentially expressed genes (DEGs) in LSC (n=52), L-blast (n=42) and between LSC and L-blast (n=15) (25 pedAML, 11 CB/9 NBM). DEGs were functionally analysed by protein association (STRING) and by gene set enrichment analysis (GSEA-Cytoscape). An overview of the workflow is shown in Fig. 1A. Results LSC vs HSC micro-array analysis revealed 83 up- and 212 downregulated targets (Fig. 1B). qPCR confirmed 8 and 11 of the 52 tested targets to be highly significantly up- and downregulated, respectively, in LSC (n=42) compared to HSC (n=20) (P L-blasts showed 157 and 332 up- and downregulated DEGs (Fig. 1C), and 8/42 were confirmed as significantly upregulated by qPCR (L-blast=35 vs C-blast=19, P Gene sets enriched in LSCs vs L-blasts addressed inflammatory responses, adipogenesis, TNF signaling and response. Pathway analysis showed repression of cell cycle genes, consistent with LSC quiescence. qPCR validation of 15 out of the 117 upregulated targets (Fig. 1D), of which 5/15 had a neural link (PCDHB2, GPRIN3, SLC22A23, CDR1, RPGRIP1L), did not confirm significant dysregulated expression (P>.05). Interestingly, the set of DEGs between LSCs and L-blasts shared only few genes with those differentially expressed between HSC and C-blast (28/306; 4 up- and 24 downregulated). This low intersection (7.7%) is in strong contrast to the previously reported 34% in adult AML (Gal et al. 2006). Moreover, none of those genes were detected in adult AML (except for CD38 downregulation). Remarkably, 3/4 mutual upregulated genes (IGF2, GPRIN3, PROS1, CYTH4) are involved in neural crossroads and have no relation to stemness nor AML. Conclusion Combining FACS with CvN transcriptomic profiling, followed by qPCR validation, identified novel DEGs in pedAML subpopulations. The majority of the most significant upregulated targets in LSCs (n=8) and L-blasts (n=8) showed no previous link to pedAML. Identification of 11 novel downregulated targets, often described as TSGs in solid tumors, warrants further studies whether hypomethylating therapy could result into LSC eradication in pedAML. LSCs reflected low cell cycle activity and elevated inflammatory response, hypoxia, metabolic dysregulation and signaling compared to HSC. GEP of LSCs vs L-blasts revealed a distinct molecular landscape compared to adult AML, and suggested a possible link between distortion of neural and hematopoietic systems in leukemogenesis. Figure Disclosures No relevant conflicts of interest to declare.

Acute lymphoblastic leukemia (ALL), which is the most common cancer in children, shows extensive genetic intra-tumoral heterogeneity. This heterogeneity might be the underlying reason for an incomplete response to treatment and for the development of relapse. In order to envision the clinical implementation of a refined risk-category strategy based on ALL subclonal composition, it is essential to first generate a reference single-cell map and accumulate evidence on how the subclonal composition affects the response to treatment. For that, we performed large-scale and integrative single-cell genome and transcriptome profiling of pediatric samples at diagnosis, during drug treatment and in case of relapse. We used the 10x Genomics platform for single-cell RNA-sequencing analysis (around 4000 cells per sample) and the Tapestri Platform (Mission Bio) for targeted single-cell DNA-sequencing (around 5000 cells per sample) of the most mutated genomic regions in ALL. For the later, we developed a custom panel that covers 305 ALL mutational hotspots across 110 genes. We have determined a reference single-cell map of the cellular (based on the gene expression profile) and the clonal composition (based on the co-occurrence of mutations at each individual cell) for pediatric ALL at diagnosis (8 T-ALL and 10 B-ALL patients). We have also reconstructed the tumor phylogeny highlighting the order of mutational acquisition and the most likely pattern of evolution. Moreover, we have studied how T-ALL evolves during drug treatment at single-cell resolution in 4 patients, unraveling which are the most sensitive clones to the therapy, which are the most resistant ones and when relapse clones originated. Single-cell RNA-sequencing also provided information on how normal hematopoiesis recovers during chemotherapy treatment. The results show that ALL is typically composed by a major clone and 5-10 smaller clones that have different sensitivities to the therapy. We have been able to detect minor clones ( Disclosures Maertens: Gilead Sciences: Other: Grants, personal fees and non-financial support; Cidara: Other: Personal fees and non-financial support; Amplyx: Other: Personal fees and non-financial support; F2G: Other: Personal fees and non-financial support; Merck: Other: Personal fees and non-financial support; Pfizer: Other: Grant and personal fees; Astellas Pharma: Other: Personal fees and non-financial support.

The aim of our study was to analyze the factors contributing to heterogeneity of prognosis in patients with hyperdiploidy50 chromosomes (HD50), a group of B-cell precursor acute lymphoblastic leukemia with favorable outcome. The 541 HD50 patients registered prospectively in the 58951 European Organisation for Research and Treatment of Cancer (EORTC) Children's Leukemia Group (CLG) trial, identified by karyotype (446 patients) and by DNA index (DI) (490 patients), had a 6-year event-free survival (EFS) of 89.0% (standard error [SE] = 1.5%) and a 6-year overall survival (OS) of 95.9% (SE = 0.9%). The strongest prognostic factor was the modal number of chromosomes (MNC): the 6-year EFS of 51-53, 54-57, and 58-66 MNC groups were 80%, 89%, and 99%, respectively (P.0001). Ploidy assessed by DI was also a favorable factor: the higher the DI, the better the outcome. The 6-year EFS of the 3 subgroups of DI1.16/≥1.16-1.24/≥1.24 were 83%, 90%, and 95%, respectively (P = .009). All usual combinations of trisomies (chromosomes 4, 10, 17, 18) were significant favorable factors but had lower EFS when MNC was lower than 58. In multivariate analysis, MNC remained the strongest factor. Consequently, the best indicator for excellent outcome was ploidy assessed by karyotype because patients with 58-66 chromosomes stood every chance of being cured (OS of 100% at 6-year follow-up) with less-intensive therapy. This trial was registered at www.clinicaltrials.gov as #NCT00003728. Registered: http://www.eortc.org/, http://clinicaltrials.gov/show/NCT00003728.

Data on incidence, characteristics, and prognosis in stage I childhood anaplastic large cell lymphoma are scarce. Of 463 patients enrolled in the international ALCL99 trial, 36 (8%) had stage I disease and were treated with a prephase chemotherapy, followed by either 3 chemotherapy courses in case of initial complete resection (6 patients) or otherwise by 6 courses of chemotherapy (30 patients). Disease localization was to the peripheral lymph nodes in 26, soft tissue mass in 8, and solitary bone and bronchial disease in 1 patient each. Of the 6 patients with complete resection, none experienced relapse, whereas of the 30 remaining stage I patients, 9 (30%) relapsed, including in all cases a new site of disease involvement and including 3 of 5 anaplastic lymphoma kinase–negative patients. In summary, the failure rate for incompletely resected stage I disease was similar to that for patients with stage II and stage III/IV disease. Whether anaplastic lymphoma kinase negativity contributed to this moderate outcome has to be proven prospectively. This study was registered at www.clinicaltrials.gov as NCT00006455.

Background Cranial radiotherapy (CRT) is associated with early and late side effects. Intrathecal (IT) and systemic chemotherapy could successfully replace CRT in most protocols for standard risk ALL. However, in medium and high risk ALL patients (pts) its omission is still debatable. Aim We investigated the long-term outcome, the occurrence of second malignant neoplasms (SMN) and the incidence of late toxicities in pts randomized for receiving or not CRT in the EORTC 58832 study. Methods From 1983 to 1989, ALL children under 18 years (yrs) were included in EORTC Children Leukemia Group BFM-oriented studies, either 58831, for standard risk pts (Riehm-Langerman Risk Factor (RF) < 1.2), or 58832, for medium risk (RF 1.2-1.69) and high risk pts (RF ≥1.7). Pts with central nervous system (CNS) involvement at diagnosis were ineligible. The present report focusses on pts included in the 58832 trial (randomized for receiving or not prophylactic CRT). Prophylactic CNS therapy consisted of 4 high-dose methotrexate (HD-MTX) injections (2500 mg/m2) during consolidation and 7 IT MTX injections scheduled during the treatment period. Pts still in complete remission (CR) after the end of late intensification were randomized for receiving prophylactic CRT (standard arm) or not (experimental arm) before the start of continuation therapy. Dose of CRT was age dependent: 24 Gy (> 2 yrs), 20 Gy (1-2 yrs) and 16 Gy (< 1 yr). Endpoints were: disease-free survival (DFS) (event: relapse, death in CR), incidence of SMN, event-free survival (EFS) (event: relapse, death in CR, SMN), incidence of late toxicities, and overall survival (OS) from randomization. Results 788 pts were included in the 58831/58832 study. Among them, 189 were randomized in the 58832 study to receive CRT (n=93) or No CRT (n=96). A total of 6 pts did not meet eligibility criteria, 2 had an early relapse, 3 had an early protocol violation and 2 refused allocated treatment. Finally, 176 randomized pts were included in the analyses: 84 in the CRT group and 92 in the No CRT group. The median follow-up was 20 yrs (range 4-32 yrs). Omission of CRT did not increase the 25-yr incidence of isolated CNS relapse, any CNS relapse or non-CNS relapse (4.8 vs 6.5; 11.9 vs 8.7 and 16.7 vs 21.8 in the CRT vs No CRT arms, respectively). No relapses occurred after 10 yrs. The 25-yr DFS rates were similar in both arms: 70.2% with CRT and 67.4% without CRT; No CRT vs CRT hazard ratio (HR)=1.08, 95% CI (0.63, 1.83). CRT was associated with an increase of the 25-yr SMN incidence: 13.2% with CRT and 3.9% without CRT. In the CRT arm, 9 pts (10.7%) developed SMN: 2 acute myeloid leukemias (AML), 1 non-Hodgkin lymphoma, 1 thyroid carcinoma, 4 meningiomas and 1 malignant histiocytosis. One SMN (meningioma) occurred after a CNS combined relapse. Three pts developed second SMN (meningiomas): 1 after an AML and 2 after a first meningioma. In the No CRT arm, 3 pts (3.3%) had SMN: 1 pleomorphic xanthoastrocytoma, 1 melanoma and 1 adenocarcinoma of the ileum. One SMN occurred after a bone marrow (BM) relapse. The 25-yr EFS rates were similar in both arms: 60.3% with CRT and 63.2% without CRT, HR=0.90, 95% CI (0.55, 1.46). CRT was also associated with an increase of late CNS and endocrine toxicities. Five pts (19.2% of the pts with available data) developed leukoencephalopathy in the CRT arm, versus 2 pts (8.7%) in the No CRT arm. Noteworthy, 1 of those 2 pts received CRT for a BM relapse, while the other received total body irradiation for a CNS relapse. Stroke was observed in 2 pts (7.7%) who received CRT. In contrast, there was no clear increase of the incidence of cognitive disturbance after CRT: 33.3% in the CRT arm vs 25.0% in the No CRT arm. Regarding endocrine toxicities, GH deficiency, hypothyroidism and precocious puberty were more frequent in the CRT arm: 53.1% vs 29.6%, 27.8% vs 0% and 29.4% vs 0%, respectively. Finally, the 25-yr OS rates were similar in both arms: 78.5% with CRT and 78.1% without CRT, HR=1.00, 95% CI (0.53, 1.88). Conclusion In medium and high risk pts without CNS involvement at diagnosis and treated with HD-MTX in the EORTC trial 58832 (1983-1989), omission of CRT did not increase the risk of CNS or non-CNS relapse. On long-term evaluation, CRT was associated with a higher incidence of SMN, late CNS and endocrine toxicities. These long-term results indicate that prophylactic CRT can be safely omitted in childhood medium and high risk ALL pts receiving IT and systemic chemotherapy (including HD-MTX) as CNS prophylaxis. Table Table. Disclosures No relevant conflicts of interest to declare.

Molecular pathogenesis of ALK-positive Anaplastic Large Cell Lymphoma (ALK+ ALCL) is not completely understood. Approximately 80% of ALK+ ALCL cases harbor the t(2;5)(p23;q35)-associated NPM1-ALK rearrangement, while several variant 2p23/ALK translocations involving at least nine partner genes have been identified in the remaining cases. The 5'ALK partners play a key role in the constitutive activation of the chimeric protein by mediating its oligomerization and its subcellular localization. Their role in ALK lymphomagenesis is debated, but experimental studies showed that the partners impact a range of biological activities of ALK chimeras, including proliferation, transformation and metastatic capacities. Comparative analysis of biological properties of ALK oncoproteins, however, is hampered by the relative low frequency of different variant ALK fusions. To gain more insight into the molecular pathogenesis of ALK+ ALCL, we have characterized ALK fusions in five cases recently diagnosed in our institution. All tumors showed a cytoplasmic expression of ALK indicating the presence of a variant t(2p23/ALK). Using 5'RACE PCR, low coverage full genome sequencing and FISH, all five ALK rearrangements were characterized. The identified partner genes included EEF1G (Eukaryotic translation elongation factor 1 gamma), a novel ALK partner located at 11q12.3 (one case), and the already known partners, RNF213/ALO17 (17q25) (one case) and ATIC (2q35) (3 cases). Notably, all five cases displayed a similar LSI ALK break-apart FISH pattern indicative of a reciprocal t(2p23/ALK) associated with copy number gain of the rearranged ALK gene. The LSI 3'ALK/red signal was duplicated on der(11)t(2;11)(p23;q12.3) in the case with the novel EEF1G-ALK fusion and amplified in cases with the RNF213-ALK (5-7 extra red signals) and ATIC-ALK (2-4 extra red signals) rearrangements. The ATIC-ALK cases presumably harbor the inv(2)(p23q35) resulting in the reciprocal ATIC-ALK rearrangement, and 1 or 2 copies of the derivative i(2)(q10) carrying two additional ATIC-ALK loci each, as previously reported1. Of note, the original case with RNF213-ALK was also featured by the ALK copy number gain2. FISH findings were confirmed by array CGH in two available cases. These data together with our previous observations1,2 provide strong evidence that ALCL driven by at least three variant ALKfusions (EEF1G-, RFN213- and ATIC-ALK), but not by the classic NPM1-ALK, requires an increased gene dosage of the rearranged ALK. To assess whether this need is caused by the weaker promoter of EEF1G, RFN213 and ATIC, compared to the NPM1 promoter, we determined at first the relative mRNA expression level of the four ALK partner genes. The study revealed a significantly lower expression of EEF1G, RNF213 and ATIC in nonmalignant lymph nodes when compared to NPM1. Therefore, we presume that lymphomas driven by the EEF1G-, RNF213- and ATIC-ALK fusions may compensate the relatively low expression of ALK by an increased gene dosage. In the next step, we will compare oncogenic potential of all four fusions in the murine hematopoietic IL-3 dependent Ba/F3 cell line. Cloning of the EEF1G-, RFN213-, ATIC- and NPM1-ALK expression vectors is ongoing. Interestingly, the three variant fusions predominantly occurred in adult patients (4/5 in age > 48 years). All patients were treated with chemotherapy, but three of them relapsed after 8 (EEF1G-ALK), 13 (RNF213-ALK) and 20 (ATIC-ALK) months. The child with EEF1G-ALK relapsed three times and died 73 months after diagnosis due to GVHD post stem cell transplantation. In summary, we observed a recurrent gain of the rearranged ALK gene in ALK+ ALCL driven by three variant fusions: EEF1G-ALK (novel), RNF213-ALK and ATIC-ALK. Our findings suggest that expression pattern and/or transforming capacities of these fusions might be impacted by biological features of the partner genes. We plan to validate our hypothesis in functional studies. Preliminary clinical data suggest a poor clinical outcome of patients with the three variant fusions. References Wlodarska, I., et al. Blood.92, 2688-95 (1998) Cools, J., et al. Genes Chromosomes Cancer. 34, 354-62 (2002) Disclosures No relevant conflicts of interest to declare.

Background: T-cell acute lymphoblastic leukemia is characterized by clonal and mutual exclusive chromosomal rearrangements that recurrently activate TAL1, LMO2, TLX1, NKX2-1, TLX3, HOXA or MEF2C oncogenes. Most of these translocations or chromosomal rearrangements occur as erroneous D-J or V-DJ rearrangement attempts of T-cell receptor beta (TCRB) or TCR alpha/delta (TCRAD) genes, mostly positioning oncogenes under the transcriptional control of TCR enhancer elements. Alternatively, oncogenes can also be activated as consequence of BCL11B chromosomal rearrangements. Although many oncogenes are known in T-ALL, the driving oncogenic lesion in particular T-ALL cases remains unknown. Aims: In this study, we aimed to clone reciprocal breakpoint sequences to elucidate cellular mechanisms that lead to recurrent BCL11B -TLX3 chromosomal translocations. Moreover, we want to identify oncogene candidates in various T-ALL patient samples with BCL11B-, TCRB- or TCRAD-translocations for which the candidate oncogene so far has not been identified. Methods: We used Targeted Locus Amplification procedure, a recently developed method that relies on the crosslinking of DNA in live cells, DNA digestion and re-ligation to allow formation of circular DNA ligation fragments and inverted polymerase chain reaction amplification from specific view-point loci. Amplified DNA fragments are sequenced by next generation sequencing, allowing sequence identification in a region covering 2MB around selected regions of interest, including TLX3, TLX1, TAL1, LMO2, BCL11B, TCRAD (TRAJ61), TCRB (TCRBC2) Results: TLA was successfully performed on 10 T-ALL patients having FISH validated TAL1 translocations (2 patients), LMO2 translocations (3 patients), TLX3 translocations (3 patients), TLX1 translocations (2 patients) or an inversion targeting NKX2.1 (1 patient). Analysis of both TAL1 translocated cases revealed a TAL1-TCRAD genomic fusion due to a classical t(1;14)(q32;q11) in 1 patient, but surprisingly reveal a TAL1-TCF7 genomic fusion due to a t(1;5)(q32;q31.1) chromosomal translocation in the second patient. For the LMO2 translocated cases, two patients showed classical LMO2-TCRAD (t(11;14)(p13;q11)) or LMO2-TCRB (t(7;11)(q35;p13) translocations, whereas the third patient presented with an unusual LMO2-BCL11B genomic fusion due to a t(11;14)(p13;q32). Two out of 3 TLX3-translocated patients had classical t(5;14)(q35;q32) translocations, whereas the TLX3 gene in the third patient was rearranged to the calcyphosine-like gene (CAPSL, which flanks the IL7Ra gene) on chromosomal 5p13.2 due to a t(5;5)( p13.2;q35) or an inv(5). One patient had an inversion on chromosome 14, i.e. inv(14)(q11;q13), that brings the NKX2.1 oncogene under the transcriptional control of the TCRAD enhancer. Finally, one TLX1-rearranged patient had a classical TLX1-TCRAD translocation, whereas the other presented with a chromosomal inversion involving the chromosomal band 10q24 (which included TLX1), but also revealed a novel translocation involving the centromere protein P gene (CENPP) on chromosome 9q22.31 with the TCRAD locus. Summary/Conclusions: Targeted Locus Amplification identification of chromosomal rearrangements and genomic breakpoint sequences reveals novel complex translocations in 3 out of 10 T-ALL cases analyzed thus far, indicating higher complexity of chromosomal translocations of known T-ALL oncogenes as thus far anticipated. It further proved a useful tool to identify novel translocation partners from various loci such as the TCR or BCL11B genes that are recurrently involved in these chromosomal rearrangements in T-ALL. Cloning of molecular translocation breakpoints of diagnostic T-ALL patient samples may further provide excellent minimal residual disease markers for disease monitoring during the course of treatment. Disclosures Splinter: Cergentis BV: Employment. van Min:Cergentis BV: Employment.

Background In childhood BCP-ALL, the presence of t(12;21)/ETV6 -RUNX1 defines one of the most prevalent oncogenic subgroup and is usually associated with a favorable outcome. Nevertheless, an excellent prognosis has not been reported by all collaborative groups, suggesting that the outcome of ETV6 -RUNX1 patients (pts) could be influenced by the treatment. To address this issue, the long-term outcome of ETV6 -RUNX1 pts was investigated into the EORTC 58881 and 58951 studies, with particular attention to the effect of the randomized treatments. Methods The 58881 study (1989-1998) used a BFM backbone without cranial irradiation, and aimed to compare E-Coli (Medac®) Asparaginase (A'ase) with Erwinia A'ase and to assess the value of 6-Mercaptopurine (6-MP) i.v. (1 g/m²/month) when added to classic maintenance. The subsequent 58951 study (1999-2008) used the best arm of the trial 58881, i.e. E-Coli A'ase and classic maintenance. The aims of this study were to compare dexamethasone (Dexa) (6 mg/m²/day) with prednisone (Pred) (60 mg/m²/day) during induction and maintenance; to evaluate increased number of A'ase administrations (24 vs 12) for non-very high risk pts; and to assess the value of vincristine/corticosteroid pulses during maintenance for average risk pts. Detection of ETV6 -RUNX1 by FISH and/or RT-PCR was centralized. Pts less than 1 year (yr) or with t(9;22)/BCR-ABL were excluded from the analysis. Results An ETV6 -RUNX1 was evidenced in 104/363 (28%) and 380/1493 (27%) newly diagnosed BCP-ALL pts enrolled in the 58881 and 58951 trial respectively. A majority of ETV6-RUNX1 pts were below the age of 10 yrs (93.3% in 58881 and 91.3% in 58951). The median follow-up was 11.8 yrs for the 58881 and 6.7 yrs for the 58951. In both studies, the 10-yr event-free-survival (EFS) rate was significantly higher for ETV6 -RUNX1 pts than for all BCP-ALL pts (82.5% vs 74.9% in 58881 and 90.8% vs 82.7% in 58951), and was similar to the 10-yr EFS of hyperdiploid pts. Noteworthy, very few EFS events were observed during treatment period or after 6 yrs from diagnosis. The main prognostic factors of the ETV6-RUNX1 subgroup in both studies were the white blood cell count (WBC) and the response to prephase. As shown in the table below, the analysis of the relationship between treatment modalities and outcome revealed that the in vivo drug sensitivity of ETV6-RUNX1 ALL was distinct from that of other BCP-ALL. In this subgroup, the benefit of a more potent A'ase (58881) or of intensified A'ase administrations (58951) was less pronounced as compared to other pts, and 6-MP i.v. during maintenance was particularly deleterious. Moreover, the overall benefit of vincristine/corticosteroid pulses was not observed in ETV6-RUNX1 average risk group pts, who already had an outstanding outcome. By contrast, the use of Dexa in place of Pred significantly improved the 10-yr EFS of ETV6-RUNX1 pts (95.0% vs 87.2%, hazard ratio (HR)=0.44, 95% CI 0.20-0.96) whereas no difference was observed in the remaining population (HR=1.01, 95% CI 0.77-1.33) (test for interaction: p=0.04). Table.5888158951All (n=363)ETV6-RUNX1 (n=104)Hyperdiploid (n=102)Others1 (n=155)All (n=1493)ETV6-RUNX1 (n=380)Hyperdiploid (n=484)Others1 (n=619)10-yr EFS ratesAll pts74.9%82.5%83.3%65.2%82.7%90.8%88.4%73.2%Pred ² (n=745)81.8%87.2%88.8%73.0%Dexa ² (n=748)83.6%95.0%88.0%73.5%Medac A'ase 2,3 (n=320)76.6%83.2%88.6%65.7%Other A'ase 2,3 (n=43)62.8%78.6%50.0%60.0%10-yr disease-free-survival ratesShort A'ase 4 (n=607)83.5%91.2%90.1%72.4%Long A'ase 4 (n=622)87.0%94.8%88.2%80.7%No 6-MP iv 2,5 (n=96)85.4%100%90.9%72.5%6-MP iv 2,5 (n=94)72.3%70.6%78.6%68.8%No Pulse 5,6 (n=148)87.5%96.1%88.5%71.1%Pulse 5,6 (n=153)82.8%95.1%91.3%82.3%1Others, MLL rearrangements excluded 2All risk groups 3Pts randomized or not for A'ase 4Non-very high risk pts 5Pts who started maintenance 6Average risk pts Conclusions Within the EORTC 58881 and 58951 trials, the use of Dexa rather than Pred allowed to further improved the long-term outcome for ETV6-RUNX1 pts. Our data also show that this excellent outcome can be jeopardized by slight changes in therapy, such as the addition of 6-MP i.v. to classic maintenance. Together, these results stress the importance of analyzing homogeneous oncogenetic subgroups when comparing different therapeutic schemes, to unmask specific drug effects that could be hidden when analyzing the whole group of patients. Disclosures Bertrand: ERYTECH Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Background: Contemporary treatment schedules for pediatric acute myeloid leukemia (AML) have improved 5 year-overall survival rates up to 70% . These schedules usually consist of 4 to 5 intensive courses mainly based on cytarabine (AraC) and anthracyclines, with allo-HSCT upfront in a subset of patients . Long-term side effects such as late cardiotoxicity caused by high cumulative anthracycline dosages are a major concern. Therefore, the DCOG/BSPHO collaboration adapted the NOPHO 2004 protocol to DB-AML01 to reduce cumulative anthracyclines from 420-450 mg/m² to 300-330 mg/m² and to omit HSCT in CR1. Material: The treatment consisted of 5 chemotherapy courses: first induction course AIET (AraC conventional dose and Idarubicin (Ida) 36 mg/m2); second induction course AM (AraC conventional dose and Mitoxantrone (Mitox) 30 mg/m2) when blasts at day 15 < 5% or FLA-DNX (high dose AraC and Daunoxome (DNX) 180 mg/m2) when blasts > 5% at day 15 or t(8;21) AML; 3 consolidation courses (high dose AraC with Etoposide as course 3 and 5 and in between high dose AraC alone). Cumulative dose of AraC is 43,4 gr/m2 and of anthracyclines 300 mg/m² for patients receiving AIET+AM or 330 mg/m² for patients receiving AIET/FLA-DNX. Results: From 2010 to 2014 a total of 113 children with AML, median age 6.0 yrs (range 0-16), gender 56 males, were included. Second induction course was AM in n=75 and FLA-DNX in n= 33. Death in induction occurred in 4 patients, CR rate was 102/113 (90.3%) and 7 patients were refractory after induction. The later events were death in CR (n=2), relapse in CR (n=40), and secondary malignancies (n=0). HSCT was given to 3 patients in CR1 (physician's decision). Median follow up time is 2.6 years (range (range 1-5). Probability of EFS at 2 years was 60% (95% CI 49-69%) and 2-yr OS was 76 (95% CI 66-83%)77.9%. Especially inv 16 (n=10) and t(8;21) AML (n=16) had an excellent outcome (inv 16:2-yr EFS: 70% (95% CI 32-89%) and 2-yr OS 91% (95% CI 51-99%) resp t(8;21):2-yr EFS: 87% (95% CI 57-97%) and OS: 94% (95% CI 63-99%)), EFS nor OS in other genetic subgroups such as FLT3-ITD and MLL rearrangements were significantly different from the total group. Children with an older age (> 10 years) (n=34) or high WBC count (>50.109/l) (n=41) had the poorest 2-yr EFS: 51 (95% CI 33-67%) resp EFS: 40% (95% CI 24-56%), whereas OS was not significantly worse. For the patients with a relapse, 32 achieved a second CR and 4 a partial remission (90%) . In second CR 34 patients received an allo-HSCT. Until now 26 of the 40 patients are still in second CR. Conclusion: The DB-AML01 outcome data suggest that the choice of high cumulative doses of AraC with moderate cumulative doses of anthracyclines up to 300-330 mgr/m² results in a favorable overall survival for particularly those children with t(8;21), inv (16) AML, young age or lower WBC counts. Children with relapsed AML could be rescued with second line treatment and allo-HSCT. These results underscore the relevance of identification of subgroups of pediatric AML patients, based on age, WBC count and t(8;21) or inv 16, curable with "lower risk" treatment arms without allo-HSCT in CR1. Disclosures Kaspers: Janssen-Cilag: Research Funding.

Background Germline mutations in the N-terminal of CCAAT/enhancer binding protein α (CEBPA) are a feature of autosomal dominant AML. Despite the strong penetrance of these mutations, the age of disease onset varies considerably and is usually precipitated by acquiring a C-terminal mutation. Although biallelic CEBPA-mutations in sporadic AML are associated with favorable clinical outcomes, little is known about long-term survival or the secondary molecular events linked with familial cases. Aims We sought to establish the long term clinical outcomes in familial CEBPA-mutated AML and to examine the patterns of secondary mutations associated with leukemic transformation. Methods and Results Disease specific and follow up information was collected in 16 affected patients, from 7 pedigrees, published between 2004 and 2011. In 94% of patients (n=15), at least 3 years follow up was achieved. All pedigrees had a germline N-terminal CEBPA mutation and 17 of 18 documented disease episodes had an acquired C-terminal mutation. The age at AML diagnosis varied from 2-39 years (median 24.5 yrs) with a single asymptomatic carrier detected (now 23 yrs). With the exception of 1 patient diagnosed in 1963, all cases received combination chemotherapy at diagnosis. Additional consolidation comprised autologous stem cell transplantation (SCT, n=3) and allogeneic SCT in 1 patient failing to achieve CR post induction therapy. Ten patients had at least 1 further disease episode, the first recurrence presenting after a median of 2.1 years (range 0.5-14 yrs), 5 continued in CR and 1 patient was lost to follow up. In 3 out of 4 patients, where CEBPA was screened at recurrence, the acquired C-terminal mutations differed from diagnosis, signifying new episodes of AML. The median overall survival (OS) for the entire cohort was 20 years (1.1-46 yrs, n=16) and 17.3 years for patients with multiple disease episodes, reflecting durable responses to second line therapies. To identify potential co-operating mutations in CEBPA pedigrees, whole exome sequencing (WES) was performed in 7 tumor samples from 5 patients across 3 pedigrees, all with the germline mutation p.P23fs (Figure 1). All tumor DNA samples were sequenced with matched remission or skin DNA as a germline control. The number of acquired mutations in familial tumors was similar to sporadic disease, with 10-22 (median=14) non-synonymous tier 1 mutations per sample. In addition to the acquired C-terminal CEBPA mutation, these included established AML loci such as EZH2, TET2, WT1, GATA2, NRAS, CSF3R and the recently identified cohesin complex gene, SMC3. Of note, FLT3-ITD, NPM1 and DNMT3A mutations were absent in all tumors. A minimum of 2 established mutations were identified in each tumor and, at present, we can only speculate on which additional mutations are ‘driver' or ‘passenger' events. Reflecting findings in sporadic AML, biallelic CEBPA and GATA2 mutations co-occurred in both siblings from Pedigree 1 and were subsequently identified by Sanger sequencing in the child III.2 (Figure 1). All 3 patients continue in long term remission following chemotherapy. We were able to trace the clonal evolution in patient I.2 (Pedigree 3) by WES of 3 consecutive tumor samples which arose over a 17 year period. At diagnosis (Dx) the patient received induction and consolidation chemotherapy and remained disease free for 14 years. The second disease episode (R1) was treated with chemotherapy followed by autologous SCT and the third presentation (R2) was chemo-refractory. Tumor DNA from R2 was clonally related to Dx, sharing 7 identical mutations, including the original C-terminal CEBPA deletion. In contrast, R1 appeared molecularly distinct from Dx and R2, most likely representing a new clone which was subsequently eradicated with treatment. Conclusion This is the first report of long term clinical outcomes in familial CEBPA-mutated AML. Although many patients experienced disease recurrence, our extended follow up showed that OS remained favorable despite multiple episodes of disease. Assessment of C-terminal CEBPA mutations provided a unique insight into the recurrence of AML, with some patients appearing to develop completely new leukemic episodes. Although the penetrance of germline mutations is high, healthy carriers and late onset disease are noted, emphasizing the need for clinical vigilance and screening of all related potential SCT donors. Disclosures: No relevant conflicts of interest to declare.

Abstract 134 Background: Asparaginase (ASP) is an essential component in combination chemotherapy for childhood ALL and NHL, as indicated by several randomized trials. However, the optimal number of ASP administrations is still unknown. We conducted a randomized phase III trial comparing conventional E.coli ASP regimen (short-ASP, 12 doses) with prolonged E.coli ASP therapy (long-ASP, 24 doses). Methods: The European Organization for Research and Treatment of Cancer Children's Leukemia Group (EORTC-CLG) phase III 58951 trial was open to de novo ALL or NHL patients (pts) < 18 y. This BFM-based study addressed 2 main randomized questions. The first evaluated the value of dexamethasone (DEX, 6mg/m2/d) vs prednisolone (PRED, 60mg/m2/d) in induction for all pts. In the second question all non-very high risk (VHR) pts were randomized for either short- or long-ASP. All patients had to receive 8×10000 U/m2 in induction. In the short-ASP arm pts had to receive 4×10000 U/m2 in late intensification; pts in the long-ASP arm had to receive 8×5000 U/m2E.coli ASP injections in consolidation and 8 (4×10000 U/m2 + 4×5000U/m2) in late intensification. Patients with grade ≥2 allergy to E.coli ASP had to be switched to equivalent doses of Erwinia or PEG ASP. Central randomization was stratified by the 1st randomized arm, risk group (VLR, AR1, AR2) and center. The primary endpoint of the study was disease-free survival (DFS), secondary endpoints were overall survival (OS) and toxicity. Intention-to-treat analysis was performed. Results: Between December 1998 and August 2008, 2038 patients were randomized for the 1st question and 1552 pts, ALL (n=1481) and NHL (n=71), were randomly assigned to receive long-ASP (n=775) or short-ASP (n=777). At a median follow-up of 7 years there were 97 vs 112 events in the long- vs short-ASP group (see table). The 8-year DFS rate was 87.0% in the long-ASP and 84.2% in short-ASP group (hazard ratio (HR) = 0.87, 95% CI 0.66–1.14, 2-sided logrank p=0.30). The 8-year OS rate was comparable in both treatment arms: 92.6% in the long-ASP group and 91.3% in the short-ASP group (HR = 0.89, 95% CI 0.61–1.29, 2-sided log rank p=0.53). Similar treatment differences were observed in each risk group, in randomized arm (PRED vs DEX), and B- and T-lineage ALL pts. The incidence of grade 3–4 infection was higher in the long- vs short-ASP group during consolidation (25.2% vs 14.5%) and late intensification (22.6% vs 15.9%). This difference was more pronounced in pts who were randomly assigned to DEX (see table). In the long- vs short-ASP group grade 2–4 allergy to ASP was 22.5% vs 0.3% in consolidation and 10.3% vs 21.5% in late intensification. During the whole treatment period, the incidence of grade 2–4 allergy was 30.5% in the long-ASP arm and 21.7% in the short-ASP arm. In the long- vs short-ASP arm approximately 67% vs 95% pts received at least the total number of E.coli or equivalent ASP administrations as planned according to the treatment arm. Conclusion: At long follow-up (median= 7 yrs) prolonged E.coli asparaginase therapy in consolidation and late intensification for VLR and AR pts did not improve significantly the outcome. Intensive ASP treatment did increase infection rate in consolidation and late intensification and resulted in more grade 2–4 allergic reactions. In the future, we aim to improve outcome rates by the use of PEG ASP and monitoring of asparaginase activity and antibody formation. Disclosures: No relevant conflicts of interest to declare.

Abstract 133 Background: T-cell acute lymphoblastic leukemia (ALL) accounts for 15% of ALL cases in children and has been associated with a higher risk for central nervous system (CNS) relapse and a worse prognosis. In EORTC trials 58831 and 2, standard risk (SR) patients (pts) were not irradiated but received intermediate dose methotrexate (MTX) courses; for medium and high risk pts, high dose (HD) MTX was added to the treatment regimen and the administration of cranial radiotherapy (RT) was randomised. The omission of RT didn't result in an increase of CNS or systemic relapse and consequently, CNS-directed chemotherapy was substituted for RT in all following trials. The long-term outcome of T-ALL pts in the subsequent phase III trials (58881 and 58951) are presented here. Methods: The BFM backbone for ALL treatment was applied to all EORTC-CLG trials since 1983. As CNS treatment in study 58881, SR pts received 4 HD MTX courses (5 g/m2) in interval therapy and 10 IT MTX injections during the intensive treatment phases. Pts with CNS-3 status at diagnosis received 2 additional IT injections during induction, 2 during consolidation and 6 HD MTX courses + IT during maintenance. T-ALL pts with poor prephase response (PPR) at day 8 or who didn't achieve complete remission (CR) after induction were included in the very high risk (VHR) group. VHR CNS-directed chemotherapy included 10 IT MTX injections, 6 IT triple and 10 HD MTX courses during intensive treatment phases, followed by 4 IT MTX injections during maintenance (the latter for CNS-3 pts only). In the 58951 trial, all T-ALL pts had an intensified induction. The CNS-directed therapy of all average risk T-ALL pts was intensified to 11 HD MTX courses, 1 IT with MTX and 15 triple IT. MRD ≥ 1% at the end of induction was added as VHR criterium. All non-transplanted VHR pts received 1 IT MTX injection, 19 IT triple and 9 HD MTX courses. Several randomized questions were addressed in both trials of which most relevant for T-ALL pts: in study 58881 the comparison E.coli asparaginase (ASP) Medac versus (vs) “other ASP” (= Erwinia ASP or E.coli ASP Bayer); in trial 58951 1) the comparison dexamethasone (DEX 6 mg/m2/d) vs prednisolone (PRED 60 mg/m2/d) in induction and 2) conventional vs prolonged E.coli ASP for non-VHR pts. Results: 303 and 296 T-ALL pts were included in trials 58881 and 58951 resp, representing 14.5% and 15.2% of all pts. Outcome results and type of events for the entire 58881 and 58951 cohorts and according to several subgroups are presented in the table. The 8-year isolated and overall CNS relapse incidences were 6.8% and 10.9% in study 58881, 5.3% and 8.5% in study 58951. The 8-year EFS, DFS and OS improved remarkably in study 58951. In the latter trial, outcome improvement was particularly seen in pts with initial WBC100x10E9/L and for pts with PPR treated in the DEX arm (HR (99%CI): 1.52 (0.63;3.64) and 1.47 (0.64;3.35)). Prolonged ASP treatment did not improve outcome of the whole T-ALL 58951 cohort. Conclusion: Prophylactic and therapeutic RT can safely be omitted from frontline treatment of children with T-ALL. Adequate ASP therapy, intensified induction treatment and CNS directed therapy can result in a significant improvement of the outcome of at least 2/3rd of T-ALL pts, particularly those with initial WBC Disclosures: No relevant conflicts of interest to declare.

Abstract 2615 Background: The first EORTC-CLG AML pilot study (58872) demonstrated the efficacy of Mitoxantrone (MTZ), substituted for daunorubicin, in the treatment of childhood AML. The subsequent trial (58921) aimed to compare MTZ and Idarubicin (IDA), an anthracycline with a favorable pharmacokinetic profile (such as good CSF penetration) and suggested to be more efficacious than daunorubicin in adult AML trials performed in the last two decades. Methods: Between March 1993 and December 2002, 227 eligible patients (pts) =70×10E9/L was abandoned from November 1994 on. Randomization was done centrally. The primary endpoint was EFS; secondary endpoints were OS, CR rate after induction/1st intensification, DFS and toxicity. Intention-to-treat analysis was used. Results: A total of 112 and 115 eligible pts were randomly assigned to receive IDA and MTZ, respectively. The rate of CR after two courses was 79.5% (IDA) vs 85.2% (MTZ). At an overall median follow-up of 9.9 y (range 0.25–16 y), there were 65 vs 59 events in the IDA vs MTZ group: failure to achieve CR (23 vs 17), relapse (35 vs 40), and death without relapse (7 vs 2). The 5-year EFS rate was 42.0% (SE 4.7%) in the IDA group and 48.4% (SE 4.7%) in the MTZ group (hazard ratio (HR) = 1.20, 95% CI 0.84–1.71, 2-sided log rank p=0.29). The 5-year OS rate was comparable in both treatment arms: 59.8% (SE 4.6%) in the IDA group and 57.5% (SE 4.7%) in the MTZ group (HR = 1.03, 95% CI 0.70–1.54, 2-sided log rank p=0.87). In CR patients with (N=46/187) or without (N=141/187) an HLA-identical sibling donor, the 5-year DFS rate from CR was 65.1% (SE 7.1%) and 51.5% (SE 4.2%) respectively (HR=0.65, 95% CI 0.37–1.11, 2-sided log rank p=0.11) and the 5-year OS rate 78.0% (SE 6.1%) and 60.7% (SE 4.1%) respectively (HR=0.53, 95% CI 0.28–1.01, 2-sided log rank p=0.048). This advantage for patients with a donor remained important regarding both DFS (HR=0.60, p=0.07) and OS (HR=0.49, p=0.03), even after adjustment for WBC count at diagnosis, age, cytogenetic features and randomized arm. The interval between start of induction and start of 1st intensification was similar in both arms (median 5.2 weeks). Grade 3–4 infection following the induction course was 37.5% (IDA) vs 25.4% (MTZ); incidence of fever grade 3–4 was 25% (IDA) vs 22.8% (MTZ). In this trial, the cumulative anthracycline dosage (conversion factor 5) was 380 mg/m2. Acute and late-onset cardiotoxicity was comparable in both treatment arms. Conclusion: There was no significant difference in efficacy and in toxicity between the two randomized treatment groups, IDA versus MTZ, although grade 3–4 infection rate following the induction course was slightly higher in the IDA arm. Patients who reached CR and who had a HLA compatible sibling donor had a longer DFS and OS than pts without a donor. Disclosures: No relevant conflicts of interest to declare.

Abstract 565FN2 Hyperdiploidy >50 chromosomes (HD>50) has long been recognized as favorable group in childhood B-ALL but there is still debate on the factors contributing to heterogeneity of prognosis observed within this entity. Better outcome has been reported for patients (pts) presenting DNA index (DI) >1.16 (Blood 1985;10:213), ≥56 chromosomes (Leukemia 1996;10:213), triple trisomies (TT) +4,+10,+17, double trisomies +4,+10 (Leukemia 2005;19:734) and trisomy 18 (Blood 2003;102:2756) but there is no consensus between the reports and these factors are differently applied in current protocols. We studied these factors in the pts with HD>50 enrolled in the ALL 58951 trial, BFM related. HD>50 were detected by cytogenetics (karyotype/FISH) and/or flow cytometry (DI). In order to analyze the outcome of HD>50 itself, pts with recurrent unfavorable translocations t(9;22), 11q23/MLL+, with t(1;19), t(12;21) or Down Syndrome were excluded, as well as near-triploidies/duplication of hypodiploidies 30–39 chromosomes. Pts were stratified into 4 risk groups (VLR/AR1/AR2/VHR) according to DI, Modal Number of Chromosomes (MNC), WBC, CNS/gonadal involvement, presence of VHR features (unfavorable translocations, poor response to prephase, residual disease (MRD) at the end of induction >10-2). VLR was defined as: DI>1.16 or MNC>50, WBC Overall Results: Out of 1651 B-lineage pts registered in the 58951 study over a 10-year period (1998-2008), a total of 541 pts had HD>50. Median age was 3 years and median WBC was 5.6×109/L. After prephase, 3% (N=17) were poor responders; initial risk group distribution into VLR/AR1/AR2/VHR was 45%/47%/5%/3%. After induction, 540 (99.8%) reached complete remission, 455 of whom had an MRD evaluation: MRD10-3 (N=26;6%) or MRD≥10-2 (N=13;2%). At median follow-up of 6 years, 48 pts (9%) relapsed, 22 pts (4%) died and 6 (1%) died without relapse. The 6-yr EFS was 89% (SE=1.5%). MNC was assessed in 446 pts (82%) with successful karyotypes, MNC ranged from 51 to 66 chromosomes and peaked at 55–56; 87 pts had 51 to 53 chromosomes (HD51-53), 258 pts had 54 to 57 (HD54-57) and 101 pts had ≥58 chromosomes (HD≥58). In these 3 groups, VLR regimen was given to 16%, 50% and 64% respectively. Structural abnormalities were detected in 46% of pts and associated with all MNCs. DI, assessed in 460 pts was Prognostic Factors: The only significant prognostic factors for EFS were MNC, DI, TT, DT and MRD. EFS was clearly improved (p10-2 and 2 pts: MRD≥10-3) and only 1 pt (MNC=58) relapsed. No specific profile of chromosome gains was identified in HD ≥58 since all chromosomes contributed to tri/tetrasomies, except chromosome 1. Likewise, the higher the DI the better the outcome (p=0.01): 6-yr EFS was 83% (DI TT were detected in 168 out of 468 pts, and DT in 242 out of 466 pts. There was no overlap with HD ≥58 since TT and DT were distributed from 52 to 66 chromosomes. One third of TT and of DT had MNC≥58. TT and DT were also of prognostic importance for outcome: the 6-yr EFS rate was 96% (TT) vs 86% (non-TT) (p=0.005) and 94% (DT) vs 84% (non-DT) (p=0.003). A hierarchical variable based on presence of HD≥58, TT or DT showed that HD ≥58 (N=101; 6-yr EFS: 99%) group had a better outcome than TT without HD≥58 (N=115; 6-yr EFS: 93%) and DT without HD≥58 and without +17 (N=55; 6-yr EFS: 84%) groups (p=0.04). We can infer from our results that the good outcome observed for all of the TT and DT was partially due to their association with HD '58. Consequently, the best indicator for excellent outcome was a high MNC (≥58 chromosomes). From our 58951 trial, we can assume that among children with B-ALL and HD>50, those with ≥58 chromosomes, stand every chance of being cured. Our results stress the necessity of karyotype for identifying them since this is the only way to assess MNC. They can also be detected (less accurately) by DI (DI ≥1.24). Therefore, both MNC and DI should be used for stratifying pts in the very low risk groups. Disclosures: No relevant conflicts of interest to declare.

Background: An overview of trials dating back to the ‘70s and ‘80s has shown that addition of vincristine (VCR)+prednisone/prednisolone pulses to continuation therapy of childhood ALL, i.e. 6-mercaptopurine (6-MP) + methotrexate (MTX), improved disease-free survival (DFS) (Lancet, 1996). However, a recent randomized intergroup trial (IGT) has shown that, when given to intermediate risk (age=6 yrs or WBC>20×109/L) patients (pts) treated according to the BFM protocol, VCR+dexamethasone (DEX) pulses in continuation therapy failed to improve DFS and overall survival (Lancet, 2007). Methods: In 12/1998, EORTC CLG started the randomized phase III 58951 trial addressing 3 questions: R1) the value of DEX vs PRED in induction; R2) the value of prolonged (24 injections) vs conventional (12 injections) duration of L-asparaginase (ASPA) courses during consolidation and late intensification; R3) for average risk (AR) pts only: the value of 6 (VCR+corticosteroid) pulses every 10 weeks during continuation therapy. The corticoid of the pulses was that assigned to the patient at R1: DEX (6 mg/sm/d) or prednisolone (PRED) (60 mg/sm/d) during 7 days. AR pts were defined by being neither low risk (hyperploidy > 50 or DNI>1.16, and WBC Results: Between 6.1999 and 11.2004, 411 pts, ALL (N=384) and NHL (N=27), were randomized for the pulse question. In the Pulses group, 101 vs 101 pts were randomized for PRED vs DEX. At a median follow-up of 6.3 years, there were 19 vs 34 DFS events for the Pulses vs No Pulses comparison: BM (10 vs 16), CNS only (1 vs 4), other isolated (2 vs 3), BM+CNS (2 vs 5), BM+other (4 vs 4), death in CR (0 vs 2). The 6-year DFS rate from randomization was 90.6% (SE 2.1%) in the Pulses group and 82.8% (SE 2.8%) in the No Pulses group (hazard ratio (HR) = 0.54, 95% CI 0.32–0.94, 2-sided logrank p=0.027). The impact of pulses on DFS was similar in the PRED group (HR=0.56) and the DEX group (HR=0.59). In girls the treatment difference seemed to be more pronounced (HR=0.25, 99% CI 0.04–1.25; p=0.015) than in boys (HR=0.71, 99% CI 0.30–1.66; p=0.30), and also in those having been randomized to receive conventional duration of ASPA courses (HR=0.46, 99% CI 0.18–1.19; p=0.03) than in prolonged ASPA arm (HR=0.87, 99% CI 0.23–3.29; p=0.78). Grade 3–4 hepatic toxicity was lower in the Pulses group (30% vs 40%); incidence of grade 2–3 osteonecrosis was 4.4% (Pulses) vs 2% (No Pulses) and pancreatitis rate was 4.9% (Pulses) vs 2.9% (No Pulses). Conclusion: VCR+corticosteroid pulses, at long follow-up (median=6.3 yrs), significantly improved the DFS, particularly in pts having received conventional duration of ASPA. Pulses did not increase toxic effects. In future, for AR pts treated according to the BFM protocol, pulses should become a standard component of therapy.

Background: there is some biologic and clinical evidence that DEX is more efficient than PRED at similarly anti-inflammatory doses in the treatment of childhood ALL. Previous trials have called for PRED doses (40 mg/sm/d) lower than those of PRED traditionally used in EORTC trials. In order to check whether the claimed superiority of DEX might be dose-dependent, we conducted a randomized phase III trial comparing DEX (6 mg/sm/d) to PRED (60 mg/sm/d) (conventional dose) in induction therapy. Methods: the trial was opened in 12/1998 to all newly diagnosed children with ALL up to 18 y. of age. After the opening of the Interfant trial in 1999, children Results: between 12/1998 and 2/2008, a total of 1853 ALL pts were randomized; as of 2/2008, 1703 pts were evaluable for EFS analysis. At a median follow up of 4.3 y., the 5 y. EFS rate was 82.1% (SE 1.5%) for the pts in the DEX arm and 82.4% (SE 1.5%) in the PRED arm (hazard ratio (HR) = 0.99, 98.8% CI 0.72–1.36, p=0.94); the futility boundary was crossed. In precursor-B ALL pts, the 5 y. EFS rate was 84.2% (DEX) vs 84.1% (PRED) (HR=0.95, p=0.75) and in precursor-T ALL the 5y. EFS rate was 70.7% vs 72.0% (HR=1.06, p=0.83). Isolated and combined CNS relapse crude rates were 1.1% each (DEX) vs 1.6% and 2.2% (PRED) respectively (resp.). The 5-y CNS incidence rate was 2.9% (DEX) vs 4.9% (PRED), the non-CNS incidence rate was 10.9% (DEX) vs 9.4% (PRED), and the 5-yr death in CR incidence rate was 2.7% (DEX) vs 2.1% (PRED). During induction the incidence of grade 3–4 infection was very similar in the two arms. During post-induction consolidation (“protocol IB”), it was higher in the DEX arm (23.5% vs 21.1%), in particular in pts randomized to prolonged L-asparaginase arm: 29.1% (DEX) vs 21.8% (PRED). Conclusion: DEX and PRED, used at the doses of resp. 6 and 60 mg/sm/d during induction, had equal efficacy. DEX decreased by 2.0% the 5-yr CNS incidence rate but increased by 1.5% the non-CNS incidence rate and by 0.6% the death in CR incidence rate. DEX in induction entails a higher infection rate during the consolidation, particularly when L-asparaginase is pursued throughout this phase. The optimal dosage of either glucocorticoid, compatible with acceptable toxicity, remains to be determined.

Background: Cranial irradiation (XRT) in combination with intrathecal chemotherapy has markedly reduced the incidence of meningeal relapse in childhood acute lymphoblastic leukemia (ALL), and is still recommended for patients (pts) at high risk of relapse within the CNS. Because of late adverse effects of XRT, the previous CLG trial (EORTC 58881, 1989–1996) deleted XRT from the treatment regimen of all ALL patients. Nevertheless, the results (5-year event-free survival (EFS) rates) of 58881 trial were good in CNS-1 (72.1%), dubious CNS-2 (62.2%), surreptitious CNS-2 (64.6%) and particularly good in pts with CNS leukemia involvement (70.3%, SE 6.2%), emphasizing the importance of systemic chemotherapy in such pts, of whom, 50% were treated according to a very high risk group protocol (ASH, 2006). In order to validate this therapeutic strategy, we evaluate the prognostic significance of CNS status at diagnosis in ALL children enrolled from 12/1998 to 2/2008 in the EORTC CLG 58951 randomized phase III trial. Methods: Treatment design was according to BFM. Three randomizations were programmed: R1) the value of Dexamethasone (DEX) vs Prednisolone (PRED) during induction (all pts); R2) the value of prolonged (24 injections) vs conventional (12 injections) duration of L-asparaginase courses during consolidation and late intensification (all except very high risk pts); R3) the value of 6 (DEX or PRED + vincristine) pulses every 10 weeks in maintenance therapy (average risk pts). CNS-directed therapy consisted in i.v. methotrexate (MTX) (5 g/sqm over 24 hours) in 4 to 10 courses, according to the degree of initial CNS involvement, and intrathecal MTX. No radiotherapy was used. According to CNS status, pts were classified in 4 groups: CNS-1: no blasts and WBC5/μl, RBC Only CNS-3 pts were to receive 10 courses of i.v. MTX, but some of dubious (n=21) and surreptitious CNS-2 pts (n=38) did eventually receive 10 courses as well. Results: Between 12/1998 and 2/2008, a total of 1853 ALL pts were registered in 58951 trial. On 2/2008 a total of 1686 were evaluable for EFS; the distribution according to initial CNS status was: CNS-1 (n=1494, 88.6%), dubious CNS- 2 (n=100, 5.9%), surreptitious CNS-2 (n=59, 3.5%) and CNS-3 (n=33, 2%). The higher the degree of CNS involvement, the higher the incidence of unfavourable features: initial WBC□100000/μl, T-lineage, NCI high risk; very high risk (VHR) initial features (peripheral blasts ≥1000/μl post prephase, high-risk cytogenetics). In CNS-3 group, 10/33 (30%) pts were treated according to VHR protocol. Median follow-up was 4 years. CNS-1 Dubious CNS-2 Surrept. CNS-2 CNS-3 No CR 17 (1.1%) 0 (0%) 0 (0%) 1 (3.0%) CCR 1286 (86.1%) 84 (84.0%) 50 (84.7%) 22 (66.7%) Isolated CNS rel. 17 (1.1%) 3 (3.0%) 1 (1.7%) 2 (6.1%) Combined CNS r. 23 (1.5%) 1 (1.0%) 2 (3.4%) 2 (6.1%) Non-CNS relapse 20 (13.4%) 2 (2.0%) 1 (1.7%) 5 (15.2%) Death in CR 31 (2.1%) 2 (2.0%) 1 (1.7%) 1 (3.0%) 4-yr EFS (%, SE) 84.0% (1.1%) 88.2% (3.5%) 82.4% (5.4%) 55.1% (10.8%) 4-yr OS (%, SE) 90.7% (0.9%) 92.0% (2.9%) 88.0% (4.6%) 63.4% (12.8%) Conclusion: In comparison with previous EORTC 58881 trial, and according to CNS status, EFS, OS, and isolated and combined CNS relapses rates improved in 58951 trial, except for CNS-3 pts. These “disappointing” results in CNS-3 pts (representing 2% of the entire population), less heavily treated in 58951 than in 58881, confirm the importance of systemic chemotherapy in such pts.

Purpose: To assess the impact of addition of vinblastine (VLB) to standard chemotherapy induction courses, based on the NHL-BFM90 protocol, and then weekly for a total duration of treatment of one year in children with high-risk anaplastic large cell lymphoma (ALCL). Design: The ALCL99-VLB trial is a randomized trial involving most European groups and the Japanese group. This ALCL99-VLB trial is part of the factorial design ALCL99 trial also aiming to assess efficacy and safety of two methotrexate doses and administration schedules (ALCL99-MTX trial already reported). Chemotherapy was based on the NHL-BFM90 protocol. All patients received a 5-day cytoreductive prephase followed by 6 induction courses given every 21 days. Children with high-risk ALCL, defined as disease with mediastinal, lung, liver or spleen involvement or biopsy proven skin disease, were randomized before the second induction course to receive either no vinblastine (No-VLB arm, reference arm) or one vinblastine injection (6 mg/m²) during the five latter induction courses and then weekly as maintenance treatment (VLB-arm). The duration of chemotherapy was 4 months in the No-VLB-arm and 1 year in the VLB-arm. We report here the results of the final analysis, performed after 3 interim analyses, with a 2-sided p-value equal to .0412 and 95.88% confidence intervals [95.88%CI]. The main end-point was the Event-Free survival (EFS). The main analysis was performed on the intent-to-treat population. Results: Overall 217 patients (93% ALK+) were recruited between 11/1999 and 05/2006 in the VLB-trial: 110 in the VLB-arm and 107 in the No-VLB-arm. A major violation of protocol treatment was observed in 4 VLB-patients and 1 No-VLB patient respectively. In the VLB-arm, 100 patients started VLB maintenance treatment; their median total treatment duration was 53 weeks (range, 21 to 87 weeks) and the median dose intensity during maintenance treatment was approximately 4.9 mg/m2/week. At the time of this final analysis, all patients had a least 2-year follow-up, with a median follow-up equal to 4.7 years. A total of 66 events were reported, including 65 relapses or progressions and 1 treatment-related death as first event. No second malignancy has been reported so far. Compared to No-VLB patients, VLB-patients had a significantly lower risk of event during the first year, Hazard Ratio of event, HR=0.31 [0.15 to 0.67], p=.002, whereas they had an increased risk of event after the first year, HR=5.0 [1.65 to 15.0], p=.003. Consequently, EFS rates differed greatly at 1 year (74% in the No-VLB-arm versus 91% in the VLB-arm) whereas they were very similar at 2 years (70% versus 74%). Over the whole follow-up period, the EFS curves did not differ significantly, HR=0.91 [0.55 to 1.51], p=.70. Results were very similar after exclusion of the 6 patients for whom the diagnosis of ALCL was rejected after review. There was no significant heterogeneity in the VLB-effect on the EFS between the two MTX groups (p=.85) or between the countries (p=.29). There was no significant impact of the addition of VLB on the complete remission rate (85% versus 86%, p=.82) or on the overall survival (p=.61, with 95% versus 92% at 2 years). Toxicity during induction courses was very similar in both arms. Only 3 patients stopped VLB maintenance treatment due to toxicity. Conclusion: The addition of vinblastine during induction and as maintenance treatment significantly delayed occurrence of relapses but, with this duration of treatment of 1 year, did not reduce the risk of failure resulting in the absence of benefit when considering the overall EFS curves or the 2-year EFS.

Objectives: to compare the impact of a different dose and schedule of administration of MTX on the outcome of pediatric patients with ALCL Background: BFM-K2 protocol with 6 alternating courses A (MTX-ifosfamide-etoposide-cytarabine-dexamethasone) and B (MTX-cyclophosphamide-doxorubicin-dexamethasone) has been shown to be highly efficient in childhood ALCL. In this protocol MTX was given at a dose of 1g/m2 in 24 hour infusion with intra-thecal injection (IT). Methods: ALCL99 study is a multicentric international prospective study for childhood ALCL, involving most European groups and Japan, comparing BFM-K2 chemotherapy with MTX 1 g/m2 in 24 hour infusion with IT (M1-arm) and BFM-K2 with MTX 3g/m2 in 3 hours infusion without IT (M3-arm). Children classified as high risk patients (HR) due to mediastinum, lung, liver, spleen involvement or skin lesions were eligible for a second randomisation, testing the impact of adding Vinblastine during induction treatment and weekly as a maintenance treatment for a total of one year (factorial design). Endpoints: Event-free Survival (EFS), Overall survival (OS), CNS relapse, toxicity. Results: 375 patients recruited between November 99 and December 2005, from 175 centres, 12 countries: 187 in the M1-arm and 188 in the M3-arm. Median age: 11.3y (range 4 mo–20y). National or international pathology review already available for 86% patients, 91% Alk+. 232 (62%) classified as HR patients (114 M1, 118 M3), 266 (75%) as St Jude stage 3 or 4 (132 M1, 134 M3). Overall 92% of patients randomised in R1 received protocol treatment, in terms of number of induction courses, dose of MTX and IT injection. Median follow-up: 31 months. Outcome: EFS curves were superimposable with 2-y EFS 73% (95%CI, 66–80%) and 73% (65–79%) in M1 and M3 arms respectively, hazard ratio=1.0 (0.60 to 1.7), p=.98, and 2-y OS 88% (82–93%) and 94% (89–97%) respectively. CNS relapses: 2 versus 0 (p=.25). Toxicity assessed in 2313 induction courses: haematological toxicity grade 4 was observed after 82% M1 and 68% M3 courses (p Conclusion: Outcome appears very similar in both groups whereas severe toxicity was more frequent in the MTX 1g/m2 24h arm as compared to the MTX 3g/m2 3h arm. Thus in childhood ALCL, there is no benefit of using MTX 1 g/m2 in 24 hour infusion with IT as compared to MTX 3g/m2 in 3 hours infusion without IT.

The Philadelphia translocation, encoding the BCR-ABL1 (BCR-ABL) fusion gene, is typically found in chronic myeloid leukemia (CML) and precursor B-cell acute lymphoblastic leukemia (B-ALL), but is exceptionally rare in T-cell acute lymphoblastic leukemia (T-ALL). To study the potential involvement of ABL1 gene rearrangements in T-cell malignancies, we screened 90 T-ALL cases by fluorescence in situ hybridization (FISH), using BCR and ABL1 probes. No BCR-ABL1 fusion signals were observed, confirming the low frequency of this rearrangement in T-ALL, but we did observe marked amplification (> 10 signals per nucleus) ABL1 of in 5 of 90 (5.5 %) T-ALL patients. Amplification of ABL1 occurred on small extrachromosomal elements that were not detectable by conventional cytogenetics. and hence are referred to as episomes. FISH, and array-CGH analyses delineated the amplicon as a 500 kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (CAN). Molecular analysis led to the identification of a NUP214-ABL1 fusion gene, which is generated as result of the circularization of the genomic region between ABL1 and NUP214 to form the episomes. This is the first example of an oncogenic fusion gene generated by extrachromosomal amplification. The NUP214-ABL1 transcript was detected in 5 patients with ABL1 amplification, in 5 of 85 (5.8 %) additional T-ALL patients, and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib mesylate (STI-571). The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased expression TLX1 of (HOX11) or TLX3 (HOX11L2), and with deletion of CDKN2A (p16), consistent with a multi-step pathogenesis of T-ALL. Our results identify a novel mechanism for the generation of a fusion gene on extrachromosomal elements, and indicate the importance of activated tyrosine kinase signaling in the pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of T-ALL patients that could benefit from imatinib treatment.

We read with interest the paper of Dordelmann et al[1][1] on prednisone (PDN) response as the strongest predictor value of outcome in infant acute lymphoblastic leukemia (ALL). Between June 1989 and November 1998, 60 (3.1%) infants, among 1963 children less than 18 years of age, were registered