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In this issue of Blood, Li et al(1) present an extensive in-depth genetic characterization of diagnostic, relapse, and remission samples from a cohort of 103 pediatric patients with acute lymphoblastic leukemia (ALL) treated according to the Shanghai Children's Medical Center ALL-2005 frontline protocol. Together with data obtained from 208 serial bone marrow samples collected during ALL therapy, their work suggests that relapse in a fraction of childhood ALL patients is driven by chemotherapy-induced mutations, which impact therapy response.

Introduction In Asians, NUDT15 genetic variants have been reported to be associated with low tolerance to 6-mercaptopurine (6-MP). However, variation in 6-MP tolerance is still observed among patients without the NUDT15 variants. Some patients required high doses of 6-MP to obtain treatment effect. We performed a genome-wide association study (GWAS) in Japanese to confirm reported associations as well as to identify additional genetic loci associated with variation of 6-MP tolerance in childhood acute lymphoblastic leukemia (ALL) patients. Methods We included ALL patients enrolled into a Tokyo Children's Cancer Study Group (TCCSG) trial who started maintenance therapy by normal protocol dose (30-50 mg/m2/day). Whole genome single nucleotide polymorphism (SNP) microarray genotyping was performed followed by whole genome SNP imputation (Japanese reference genome). Data for over six million SNPs were available for analysis. Association analysis was performed adjusting for age at diagnosis and considering average 6-MP dose during 168 days from maintenance therapy initiation as the outcome. Results A total of 224 patients were included in the analysis. The median age at diagnosis was 4.7 (range = 0.9-15.7) years old and boys comprised 56.3%. The average 6-MP dose during the 168 days of maintenance therapy was 41.1 (range = 13.3-78.5) mg/m2/day. Variants representing 10 genomic regions showed potential association (P < 5 x 10-6) with average 6-MP dose, in which one locus was genome-wide significant (13q14.2, rs116855232). Variant rs116855232 is located in the previously identified NUDT15 gene and represents the strongest association with average 6-MP dose within our Japanese study (β = -11.45, P = 8.5 x 10-10). In contrast, the TPMT locus, which is predictive of 6-MP intolerance in populations of European ancestry, was not associated with 6-MP dose in Japanese. Among the suggestive loci requiring further follow-up is a locus residing in AFF3 (2q11.2, P = 2.1 x 10-6), a gene previously implicated in childhood leukemia. Conclusion We have validated that NUDT15 rs116855232 is a locus with strong association with 6-MP tolerable dose, and it currently represents the most meaningful clinically predictive marker in Japanese. These findings indicated that NUDT15 genotyping should be considered prior to initiation of 6-MP treatment. Prospects for the identification of additional loci of 6-MP tolerance are high after further validation of suggestive loci observed in this Japanese study. Figure Disclosures No relevant conflicts of interest to declare.

In this issue of Blood, Ness et al report that, despite the omission of prophylactic cranial radiotherapy (CRT) in the treatment of acute lymphoblastic leukemia (ALL), adult survivors of childhood ALL remain at risk for impaired fitness, body composition, and energy balance.

Background: The detailed definition of causative genomic alterations is not only an indispensable prerequisite for the predictive and prognostic subdivision of childhood acute lymphoblastic leukemia (ALL) but increasingly also one, on which individualized treatment approaches will be based on. Apart from the already well-established genetic categories, the recent identification of several new classes of potentially relevant alterations together with the increasing availability of novel therapeutic options therefore necessitates a diagnostic workflow that is able to satisfy the ensuing clinical needs in a comprehensive manner. The two most interesting changes in this context are the therapeutically targetable recurrent but rare and heterogeneous tyrosine kinase and JAK2 pathway-activating (TKA) gene fusions and the more elusive cohort of apparently relapse risk-prone cases with hitherto only vaguely defined combinations of gene region-specific copy number alterations (CNA). Despite the availability of a multitude of applicable techniques, the fast and cost-efficient identification of the entire expectable abnormality patterns still remains a challenge, especially if one needs to perform the diagnostic work-up on a single case basis. We previously proposed that these diagnostic requirements could be covered best with a systematic hierarchical FISH screening approach for the identification of gene fusions together with array (combined SNP and non-polymorphic probes) analyses of genome-wide quantitative and qualitative large- and small-scale copy number aberrations (CNAs). Material and Methods: Since June 2015 we have therefore evaluated the feasibility of such a workflow in a prospective manner and screened so far 205 patients (i.e. 184 with B- and 21 with T-ALL, including 18 relapses with 14 diagnostic/relapse pairs) that were consecutively enrolled in the Austrian AIEOP-BFM 2009 treatment study. Cytogenetic preparations served as backup, since metaphase spreads were used for further FISH clarification of otherwise unresolvable complex rearrangements or ploidy patterns if deemed necessary. All identified gene fusions were subsequently validated with single or multiplex RT-PCR analyses. Results: FISH screening was positive in 90% (184/205) of cases and provided already the most essential diagnostic clues. CNAs were present in all T-ALL and 97% (179/184) of B-ALL cases, including 13 with an IKZF1pluspattern and three with ERG deletions, which both will be used das stratifying markers in the upcoming treatment trial. Taken together, our screening strategy allowed the unambiguous classification of the vast majority of B-ALLs: 70 hyperdiploid, 3 hypodiploid, 35 ETV6-RUNX1, 6 KMT2A-rearranged, 8 TCF3-PBX1, 2 BCR-ABL1, 4 dic(9;20), 5 iAMP21, 8 IGH-rearranged, 6 P2RY8-CRLF2, 3 PAX5-rearranged, 3 ZNF384-rearranged, 2 ETV6-rearranged, 3 TKA fusion-positive and 10 so called "B-other" cases without any apparent specific abnormality. RNA-sequencing analyses of these ten cases revealed that seven of them harbored a DUX4 gene rearrangement. Conclusions: Apart from its proven practical diagnostic value, our combined FISH/array approach has also several additional advantages, especially if one considers the amount of achievable information. Both procedures require only little amount of material and are highly standardized, reproducible and robust technologies. Moreover, arrays deliver DNA-sequence-based data, the coordinates of which can be efficiently stored, processed and analyzed. As such, they not only serve as a pure diagnostic tool but also as a valuable discovery platform. Disclosures No relevant conflicts of interest to declare.

CD200 is an immunomodulatory molecule overexpressed in childhood B cell precursor (BCP) acute lymphoblastic leukaemia (ALL). In minimal residual disease (MRD) low risk cases it has been shown that only CD200+ cells could engraft and self-renew in NOD.Cg-PrkdcscidIl2rgtm1Wjl (NSG) mice, making CD200 a very attractive target for these cases. Parthenolide (PTL) has been shown to eradicate CD34+/CD19+, CD34+/CD19- and CD34- leukaemia initiating cell (LIC) populations in BCP ALL. However, the effects of PTL on CD200 subpopulations are not known. In vivo responses to therapy can be restricted by components of the bone marrow microenvironment. Mesenchymal stem cells (MSC) have been shown to protect T-ALL cells from PTL by modulating reactive oxygen species (ROS) activity. This protection could be reversed with the use of sulfasalazine (SSZ), an inhibitor of the cystine uptake transporter xc-. SSZ blocks cysteine generation and release in MSC resulting in elevated ROS levels in T-ALL and increased sensitivity to PTL. This study investigated the effects of PTL and SSZ on unsorted, CD200+ and CD200- BCP-ALL subpopulations in the presence of MSC. Six MRD low risk and 4 risk cases were co-cultured on MSC, at a 2:1 ratio, 1 hour prior to addition of PTL at various doses up to 10mM. After 24 hours, cells were stained with Annexin V and PI and viability was assessed by flow cytometry. In the absence of MSC, low risk cases were slightly less sensitive to PTL with an IC50 of 2.23mM compared to risk ones (IC50 1.54mM, P=0.61). Interestingly, 4 of 6 low risk cases, which were less sensitive to PTL, had normal karyotypes, while the 2 responsive cases had t(12;21). When unsorted ALL cells were co-cultured with MSC, cell survival significantly increased following PTL treatment at doses up to 7.5mM. In low risk cases viability increased from 8.8±19.4% to 30.0±20.4% at 7.5mM PTL (P Disclosures No relevant conflicts of interest to declare.

To the editor: Killer-cell immunoglobulin-like receptors (KIRs) play a pivotal role in immunosurveillance and reduction of relapse after hematopoietic stem cell transplantation in acute leukemia.[1][1],[2][2] Outside the setting of hematopoietic stem cell transplantation, it was recently reported

In this issue of Blood , Gadner et al provides data from the third Histiocyte Society trial (LCH III).[1][1] ![Figure][2] (A) Response rates of initial therapy of LCH compared with childhood ALL and AML. (B) Overall 5-year survival percentages. RO+ is defined as patients with LCH that

In this issue of Blood , Lausten-Thomsen et al challenge the notion that TEL-AML1 transcripts are prevalent in newborns, raising questions about our understanding of the natural history of childhood ALL and the potential utility of newborn screening.[1][1] The t(12;21) translocation results in the

Survivors of childhood acute lymphoblastic leukemia (ALL) treated with cranial radiation therapy (CRT) are at risk for cognitive impairment, although whether impairment progresses with age into adulthood is unknown. We report change in intelligence for 102 adult survivors of childhood ALL (age range, 26.6-54.7 years) during a median interval of 28.5 years. Survivors demonstrated lower Performance intelligence (mean, 95.3; standard deviation, 16.5; P = .005) but not Verbal IQ (mean, 97.4; standard deviation, 15.44; P = .09) at initial testing. Verbal intelligence declined an average of 10.3 points (P < .0001) during the follow-up interval with no decline in Performance intelligence. Decline was associated with current attention problems (P = .002) but not gender, CRT dose, age at CRT exposure, or years between testing. Results suggest long-term survivors of childhood ALL treated with CRT are at risk for progressive decline in verbal intellect, which may be driven by attention deficits. This trial was registered at clinicaltrials.gov as no. NCT00760656.

Objectives: A small number of previous studies have demonstrated prognostic significance of MRD detection before and after allogeneic transplantation in children ALL. A major challenge of studies has been low patient numbers that were insufficient for multivariate analysis and assessment of specific post-transplant time points. To address this, the Westhafen Intercontinental Group (WIG) performed a retrospective analysis to investigate the impact of pre- and post-transplant MRD assessment in children with ALL. Patients and Methods: Patients (pts) treated in Europe (France, Germany, Italy, and Netherlands), North America (COG/PBMTC, Seattle, Minnesota, and Johns Hopkins) and Australia were included. All pts were registered either in local or national trials. Pts were transplantations between 09/99 and 05/16; analysis was performed in 06/16. Altogether 747 pts were included on whom 2960 MRD measurements were performed in bone marrow. MRD prior to transplant was available in 650 pts. MRD was assessed in bone marrow prior to transplant and on days +30, +60, +100, +180, +365 and beyond day +365 post-transplant either by flow cytometry or by RQ-PCR of TCR and IG gene rearrangements. Of these 747 pts, 466 (62%) were male and 281 (38%) were female. Pts received their transplant in CR1 (n=275, 37% ), CR2 (n=410, 55%), >CR3 (n=55, 7%), or non-remission (NR) (n=7, 1%). 586 patients (78%) had pre-B ALL, 145 pts (20%) had T-cell phenotype and 16 pts (2%) had bi-lineage or bi-phenotype AL. Grafts were matched sibling donors (MSD; n=227, 30%), matched unrelated donors (MUD; n=314, 42%), from mismatched donors (MMD, n=75, 10%), cord blood (n=128, 17%) or no data (n=3, 1%). 31 patients (4%) were younger than 2, 365 pts (49%) were ≥2 and ≤ 10, and 351 pts (47%) were older than 10 years at the time of transplant. After MRD assessment, pts were grouped for analysis according to the level of MRD in three groups: pts in whom no MRD was detectable were categorized "MRD negative", pts with MRD Results: The total group of pts showed a 4y-pEFS and a 4y-pOS of 55.0% and 60%; cumulative incidence (CI) of relapse (CIR) and CI of treatment related mortality (CI TRM) were 31% and 13%. The following pre transplant factors did not influence outcome: age, gender, relapse site, cytogenetics, donor, and stem cell source. MRD prior to transplant was available in 648 pts. MRD high, low and negative, patients showed a 4y-pEFS of 37%, 67%, and 62% (p MRD values post-transplant were analysed as time-dependent covariates. 4y-pEFS at day +30 for MRD high, MRD low and MRD negative pts were: 43%, 65%, and 59%, respectively (p=0.002). At Day +60 for MRD high, low and MRD negative pts: 36%, 47%, and 64%, respectively (p There was an interaction between MRD and aGVHD at all time-points investigated after transplantation. At day +30, patients with high MRD had a 2.54 fold increased risk to develop an event which is defined as relapse or TRM, where the risk for high MRD pts with aGVHD was 0.64. This was due to a lower relapse risk compared to MRD positive patients without aGVHD. Summary and Conclusion: This world wide study confirmed in the largest patient population investigated so far, MRD prior to transplant is a powerful predictor for survival after transplant. When analysed either for the different time points or for the highest MRD value post- transplant, MRD results were always significantly associated with pEFS. All pts who showed an MRD load of >10E-4 (0.01% by flow) at any time after transplant had an increased risk. Occurrence of aGVHD in MRD positive pts reduced the risk of relapse. Serial MRD detection after transplantation allows for the prediction of relapse. Disclosures Bader: Medac: Consultancy, Research Funding; Servier: Consultancy, Honoraria; Neovii Biotech: Research Funding; Riemser: Research Funding; Novartis: Consultancy, Honoraria. Verneris:Bimogen: Other: Advisory Board. Borowitz:Bristol-Myers Squibb: Research Funding; MedImmune: Research Funding; BD Biosciences: Research Funding; HTG Molecular: Consultancy. Peters:Medac: Consultancy; Novartis: Consultancy; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy. Grupp:Novartis: Consultancy, Research Funding; Pfizer: Consultancy. Pulsipher:Novartis: Consultancy, Other: Study Steering Committee; Jazz Pharmaceutical: Consultancy; Chimerix: Consultancy; Medac: Other: Housing support for conference.

In this issue of Blood , Oudin and colleagues report an increased prevalence of the metabolic syndrome (MS) in a cohort of adult survivors of childhood leukemia.[1][1] The coupling of a growing population of maturing childhood leukemia survivors with a syndrome that predisposes to cardiovascular

To the editor: Acute lymphoblastic leukemia (ALL) accounts for one-fifth of leukemia in adults.[1][1] The rates of second primary malignancies (SPMs) in childhood ALL are significantly higher than in the general population.[2][2],[3][3] However, there is a lack of information on SPMs in adult

To the editor: In Inside Blood 's provocatively titled “ ‘Lesser’ ALL: treat less or study less?,”[1][1] Dr Feusner questions whether, “in the current age of declining funding for research, is it worth it to study a disease in which at least 95% of the patients would be expected to

Background: Overall survival (OS) for childhood ALL treated on contemporary protocols is now > 90%. For low risk patients the risk of treatment related mortality (TRM) is now similar to the risk of death due to disease relapse. The only way to improve TRM and morbidity in this group is to identify even lower risk patients and reduce therapy. We analysed outcomes for patients with low risk MRD at end of induction (EOI) therapy treated on UKALL 2003, to identify a very low risk group who could potentially benefit from protocols that further reduce TRM. Methods: UKALL 2003 trial recruited 3126 patients aged 1-25 years with Philadelphia-negative ALL between Oct 1, 2003, and June 30, 2011. Treatment was initially stratified based on NCI risk and cytogenetic results. Subsequent treatment was directed by MRD from bone marrow (BM) measured using EuroMRD approved real-time quantitative PCR method for immunoglobulin and T-cell receptor gene rearrangements at EOI therapy. Analysis was performed on the 2666 patients with available MRD results and focused on patients with low risk MRD combined with other patient characteristics - cytogenetic risk group, white cell count (WCC) and age to identify those subgroups with extremely good event free survival (EFS). Low risk MRD at EOI was defined a level Results: Overall 52% (n=1391) of patients had MRD < 0.005% with the remaining 48% (n=1275) of patients with MRD ≥ 0.005%. 5yr OS 97.6 (95% CI 96.6-98.3) vs. 89.1% (87.2-90.7, p50 / age>10yrs). Long term survival remained excellent with 10 year EFS 93.9% (OS 98.2%). There were 32 relapses in the low risk MRD/ good risk cytogenetics group (relapse sites: 11 BM, 7 combined BM and central nervous system (CNS), 3 BM + other site, 6 isolated CNS and 5 other site, non BM/CNS), giving an overall relapse rate of 3.8% with a salvage rate of 81%. The relapse death rate was 0.7% (n=6) with TRM of 0.8% (n=7). The low risk group can be further sub divided into a group of 442 patients (16.6% of whole trial) with undetectable MRD at EOI and good risk cytogenetics who have a 5yr EFS of 97.4% (5yr OS 99.5%). Conclusion: This excellent long term outcome for a third of all patients with childhood ALL, low risk MRD and good risk cytogenetics in a large multi-center randomized controlled trial supports exploration of further reduction of therapy in this group to reduce TRM. This strategy is further supported by the observation that the rate of TRM is now similar the relapse death rate in this group, coupled with the very high salvage rates for those patients who do relapse. The superior OS for patients with undetectable MRD and good risk cytogenetics further highlights the additive strength of MRD to accurately predict outcome in the low risk cytogenetic group. Disclosures No relevant conflicts of interest to declare.

B cell precursor acute lymphoblastic leukemia (BCP-ALL) is one of the most common malignancies in children. In the period 1991-2013, the Dutch Childhood Oncology Group (DCOG) has completed three treatment trials for childhood ALL: ALL8, 9 and 10, each protocol with stratifications into risk-groups (details: www.skion.nl). Although the cure rates increased in these subsequent trials, relapses still occurred in a significant number of children. Since consecutive upfront treatment protocols usually change at multiple levels, genomic alterations that are associated with relapse may also be variable, which could provide insight into the biology underlying therapy failure and relapse. In this study, we characterized the genetic architecture of relapsed BCP-ALL patients within the context of these three Dutch upfront protocols. We identified 3 patient groups based on upfront treatment as follows: Group-1: patients treated upfront with high-amounts of corticosteroids (CS) and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). The number of high-risk patients that relapsed after ALL8 HR and ALL10 HR chemotherapy courses was too low to be included for analysis. We determined, at relapse, the presence of copy number alterations and sequence mutations in 21 recurrently affected genes involved in B-cell development, cell cycle regulation and RAS signaling, in 123 patients that relapsed after treatment in group-1 (n=56), group-2 (n=20) and group-3 (n=47). The number of CREBBP mutations in patients that relapsed after treatment according to group-1 (ALL9) was significantly lower compared to the other two groups, whereas B-cell development alterations were most common in patients that relapsed after treatment according to group-1, mainly due to a higher number of IKZF1 alterations (Figure 1). The high number of relapsed patients with leukemic clones carrying IKZF1 alterations in patients treated with high-amounts of CS and relatively mild additional chemotherapy is in line with our recent finding that IKZF1 is a key determinant of GC-induced apoptosis in normal and leukemic B-cells, and that loss of IKZF1 function confers resistance to dexamethasone, the major treatment component in group-1 (Marke et al., submitted). Additionally, in the group-2 patients treated with high-amounts of CS and highly intensive additional chemotherapy, a lower percentage IKZF1-deleted clones was detected at relapse, indicating that more GC-resistant, IKZF1-deleted clones are killed by the intense chemotherapy given in addition to CS in group-2 patients. Similarly, in the group-3 patients relapsing after treatment with lower amounts of CS and moderately-intensive additional chemotherapy, the percentage of surviving IKZF1-deleted clones was lower than in patients treated with high-amounts of CS. Taken together, our data indicate that the genetic architecture of relapsed BCP-ALL patients depends on the upfront treatment and, in addition, that the poor-prognostic feature of IKZF1-deletions may be more prominent in upfront treatment with high-amounts of CS and relatively mild additional chemotherapy. Figure 1. The frequency of genetic alterations in studied genes in patients that relapsed after treatment according to group-1, 2 and 3. Genes were grouped by their corresponding pathways. Group-1: patients treated upfront with high-amounts of CS and relatively mild additional chemotherapy (ALL9 NHR/HR); Group-2: patients treated with high-amounts of CS and intensive additional chemotherapy (ALL10 MR); Group-3: patients treated with low-amounts of CS and moderately-intensive additional chemotherapy (ALL8 SR/MR, ALL10 SR). Asterisk showed significant difference between upfront treatment groups, **p Disclosures No relevant conflicts of interest to declare.

To the editor: A recent genome-wide association study of childhood acute lymphoblastic leukemia (ALL) by Xu et al[1][1] has identified a novel susceptibility locus encompassing BMI1 - PIP4K2A at chr10p12.31-12.2, bringing the number of known ALL risk loci to 5 (including ARID5B , IKZF1 , CEBPE, and

Asparaginase (ASNase) has long been considered to be an important element in the management of childhood ALL. Its antileukemic effect is thought to be related to a metabolic deficiency reflected by the blasts' incapability to synthesize asparagine (ASN) from aspartic acid. Treatment with ASNase aims

This study aimed at determining the prevalence of INK4deletions and their impact on outcome in 125 children with acute lymphoblastic leukemia (ALL) at first relapse using real-time quantitative polymerase chain reaction. Patients were enrolled into relapse trials ALL-REZ BFM (ALL-Relapse Berlin–Frankfurt–Münster) 90 and 96. The prevalence of p16INK4a and p15INK4bhomozygous deletions was 35% (44 of 125) and 30% (38 of 125), respectively. A highly significant association of both gene deletions was found with the 2 major adverse prognostic factors known for relapsed childhood ALL: T-cell immunophenotype and first remission duration. There was no correlation between INK4 deletions and probability of event-free survival. These findings argue against an independent prognostic role of INK4 deletions in relapsed childhood ALL.

Detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) predicts outcome. Previous studies were invariably based on relative quantification and did not investigate sample-inherent parameters that influence test accuracy, which makes comparisons and clinical conclusions cumbersome. Hence, we conducted a prospective, population-based MRD study in 108 sequentially recruited children with ALL uniformly treated with the ALL-Berlin-Frankfurt-Münster (ALL-BFM) 95 protocol in Austria (median follow-up of 40 months). Using sensitive, limited antibody panel flow cytometry applicable to 97% of patients, we investigated 329 bone marrow samples from 4 treatment time points. MRD was quantified by blast percentages among nucleated cells (NCs) and by absolute counts (per microliter). Covariables such as NC count, normal B cells, and an estimate of the test sensitivity were also recorded. Presence and distinct levels of MRD correlated with a high probability of early relapse at each of the time points studied. Sequential monitoring at day 33 and week 12 was most useful for predicting outcome independently from clinical risk groups: patients with persistent disease (≥ 1 blast/μL) had a 100% probability of relapse, compared to 6% in all others. Absolute MRD quantification was more appropriate than relative, due to considerable variations in total NC counts between samples. Regeneration of normal immature B cells after periods of rest from treatment limited the test sensitivity. In conclusion, MRD detection by flow cytometry is a strong and independent outcome indicator in childhood ALL. Standardization regarding absolute quantification on the basis of NCs and assessment during periods of continuous treatment promise to increase the accuracy, simplicity, and cost efficiency of the approach.

Osteonecrosis is a severe glucocorticoid-induced complication of acute lymphoblastic leukemia treatment. We prospectively screened children (n = 364) with magnetic resonance imaging of hips and knees, regardless of symptoms; the cumulative incidence of any (grade 1-4) versus symptomatic (grade 2-4) osteonecrosis was 71.8% versus 17.6%, respectively. We investigated whether age, race, sex, acute lymphoblastic leukemia treatment arm, body mass, serum lipids, albumin and cortisol levels, dexamethasone pharmacokinetics, and genome-wide germline genetic polymorphisms were associated with symptomatic osteonecrosis. Age more than 10 years (odds ratio, = 4.85; 95% confidence interval, 2.5-9.2; P = .00001) and more intensive treatment (odds ratio = 2.5; 95% confidence interval, 1.2-4.9; P = .011) were risk factors and included as covariates in all analyses. Lower albumin (P = .05) and elevated cholesterol (P = .02) associated with symptomatic osteonecrosis, and severe (grade 3 or 4) osteonecrosis was linked to poor dexamethasone clearance (P = .0005). Adjusting for clinical features, polymorphisms of ACP1 (eg, rs12714403, P = 1.9 × 10−6, odds ratio = 5.6; 95% confidence interval, 2.7-11.3), which regulates lipid levels and osteoblast differentiation, were associated with risk of osteonecrosis as well as with lower albumin and higher cholesterol. Overall, older age, lower albumin, higher lipid levels, and dexamethasone exposure were associated with osteonecrosis and may be linked by inherited genomic variation.

In this issue of Blood , Ensor et al describe the features associated with the deregulation of CRLF2 signaling in childhood B-cell precursor acute lymphoblastic leukemia. They conclude that CRLF2 deregulation is not an independent predictor of event-free survival.[1][1] Cytokine receptor-like

We thank Zuna et al and Greaves et al for their interest in our recent report in Blood [1][1] and for addressing the conflicting observations regarding levels of ETV6/RUNX1 (previously TEL/AML1 )–positive cells in healthy neonates.[2][2] Clarification of this issue is highly important both for the

Introduction: Considering that cytokines play an important role in immune response and that many infectious, autoimmune and malignant diseases are influenced by cytokine production, we hypothesized that genetically determined cytokine gene polymorphism might have an important influence on prognosis in pediatrics acute lymphoblastic leukemia (ALL). Methods: In this study, 95 pediatric ALL-patients were examined with regard to cytokine gene polymorphisms (TNFα, TGFβ, IL10 and IFNγ) and their potential association with prognosis. Moreover we analyzed the intracellular production of theses cytokines in patient T-cells. Results: TGFβ high-producer-haplotypes were associated with high-risk ALL-patients (Codon 10: T/T) and with the tendency of a reduced overall survival, whereas IL10 high-producer-genotypes were associated with a reduced relapse rate and a superior overall survival compared to IL-10-low-producer patients. Gene-polymorphisms of the pro-inflammatory cytokines IFNγ and TNFα did not show an impact on prognosis and risk-group of ALL in our cohort. On a functional basis TNFα and IFNγ expression of T-cells at initial diagnosis was significantly reduced in high-risk- and T-ALL-patients in comparison to healthy controls. Summary: Cytokine gene-polymorphisms of the regulatory/anti-inflammatory cytokines TGFβ and IL10, but not of the pro-inflammatory cytokines IFNγ and TNFα seem to have an impact on prognosis of pediatric ALL patients. Reduced capacity to produce pro-inflammatory cytokines at diagnosis may serve as a functional risk factor. These data may help in further risk stratification and adaptation of therapy-intensity. Disclosures No relevant conflicts of interest to declare.

BACKGROUND ALL is the most common form of childhood cancer with >80% cured with contemporary treatment protocols. Accurate risk stratification in childhood ALL is essential to avoid under- and over-treatment. Currently, we use presenting clinical, biological features, and minimal residual disease (MRD) quantitation to risk stratify patients. Although whole genome gene expression profiling (GEP) can accurately classify patients with ALL into various WHO 2008 defined subgroups, its value in predicting relapse remained to be defined. We hypothesized that global time-series GEPs of bone marrow (BM) samples at diagnosis and specific points during initial remission-induction therapy can measure the success of cytoreduction and be used for relapse prediction. METHODS We generated time-series GEPs from 181 children with de novo ALL at diagnosis and Day 8 of remission-induction therapy. We computed the time-series changes from diagnosis to Day 8 of remission-induction, termed Effective Response Metric (ERM-D8; Fig 1), that measures both the magnitude and direction of time-series change in multi-dimensional gene space towards the normal centroid, and we compared its ability to predict relapse against contemporary risk assignment methods including NCI criteria, cytogenetics and MRD. RESULTS Unsupervised hierarchical clustering of the first 96 patients revealed that 30.3% (10 of 33) patients whose diagnosis and day 8 GEP pairs clustered together in the same branch suffered a relapse compared to 7.5% (4 of 53) who clustered to different branches. This suggests that patients with minimal changes in GEP after 8 days of remission-induction therapy have a 4-fold significantly higher risk of relapse (p=5.4x10-3). To test this observation, we extended the study to 181 ERM-D8 in 8 batches. Using leave-one-batch-out cross-validation, we found that ERM-D8 is predictive of relapse; patients with favorable ERM-D8 (n=115) had a significantly lower 14.5% 5-year cumulative risk of relapse (CIR) compared to 44.5% with unfavorable ERM-D8 (n=66; p= 5.1x10-6; Fig 2). In multivariate analysis, ERM-D8 remained significant (Table 1; p=7.9x10-4, HR 3.19 [1.62-6.29]) after controlling for early clinical features of age (p= 0.37), WBC (p=0.05), cytogenetics (p=0.021) and Day 8 peripheral blast response (p=0.96). Even after adjusting for Day 33 MRD (p=5.4x10-3), ERM-D8 remained independently predictive of relapse (Table 1; p=8.5x10-3, HR 2.63[1.28-5.42]). ERM-D8 helped further refine Day 33 MRD risk stratification. Day 33 MRD negative patients with unfavorable ERM-D8 (n=29) had a 5 fold higher risk of relapse compared to patients with favorable ERM-D8 (n=60; 5-year CIR 21.7% vs 4.4; p=0.021). Similarly Day 33 MRD positive patients with unfavorable ERM-D8 (n=33) have double the risk of relapse compared to those with favorable ERM-D8 (n=45; 5-year CIR 62.8% vs 29.2%; p=1.01x10-3). ERM-D8 also improved risk stratification in patients with favorable cytogenetics. Favorable cytogenetics patients with unfavorable ERM-D8 (n=31) do significantly poorer than those with favorable ERM-D8 (n=51; 5-year CIR 36.4% vs 2.4%; p=1.04x10-4). CONCLUSION We describe a novel metric based on time-series GEP during first 8 days of remission-induction therapy that is independently predictive of risk of relapse even after adjusting for age, WBC, NCI risk, cytogenetics and MRD. | | Without Day 33 MRD | With Day 33 MRD | | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | ------------------ | --------------- | --------- | | p-value | HR | 95% CI | p-value | HR | 95% CI | | ERM-D8 unfavorable vs favorable* | 7.9x10-4 | 3.19 | 1.62~6.29 | 8.5x10-3 | 2.63 | 1.28~5.42 | | Cytogenetics unfavorable vs favorable and others** | 0.021 | 2.45 | 1.14~5.26 | 0.23 | 1.69 | 0.72~3.98 | | Age, years 10 vs 1~10 | 0.37 | 1.39 | 0.67~2.89 | 0.43 | 1.35 | 0.64~2.84 | | WBC, x109/L over 50 vs below 50 | 0.05 | 1.99 | 1.00~2.96 | 0.25 | 1.69 | 0.69~4.13 | | Day 8 Response poor vs good | 0.96 | 1.02 | 0.40~2.59 | 0.65 | 0.81 | 0.32~2.06 | | Day 33 MRD >=1.0x10-2 vs

Objective: MLL gene rearrangement in pediatric acute lymphoblastic leukemia (ALL) was regarded as high risk factor because of the poorer outcome of overall survival(OS). Some reports further proved that patients with MLL/AF4 had the worse outcome than MLL rearranged with other partners. CCLG-ALL2008 protocol has been carried out in China for more than 5 years. However, there was no reports to evaluate its efficiency on pediatric ALL patients with MLL rearrangement. In this study, we analyzed the data of ALL patients to compare the outcome of patients with MLL rearrangement positive and negative treated by CCLG-2008 protocol. Methods During the period from 2009 to 2013, 379 patients were enrolled in this protocol, of which 19 cases were MLL rearrangement positive and treated with CCLG-ALL2008 protocol for high-risk (HR) group. The treatment efficiency was evaluated on the time points of day 7, day15, day 33 and 12th week after treatment, respectively. OS and treatment-related mortality (TRD) was calculated within high risk groups of MLL positive and negative. Results Patients with MLL rearrangement positive accounted for 5.01% of all patients. The characteristics and response to the treatment were illustrated in Table 1. Cases younger than 2 years old, with initial white blood cell (WBC) 50*109/L , or MRD more than 10-2 on day 33 had a lower OS (P0.05). Compared with the male, female patients had a better 5 year EFS (100% versus 50%±17%, P Conclusion Factors including female, older than 2 years of age, sensitivity to prednisone, are good prognostic index in predicting the outcome of 5 years of OS and EFS in childhood ALL with MLL gene rearrangement. Table 1 Clinical and laboratory characteristics of ALL patients with MLL positive Table 1. Clinical and laboratory characteristics of ALL patients with MLL positive Figure 1 Gender exerts influence on 5 years EFS in MLL rearrangement group Figure 1. Gender exerts influence on 5 years EFS in MLL rearrangement group Figure 2 Figure 2. Age influenced 5 years EFS in MLL rearrangement group Disclosures No relevant conflicts of interest to declare.

To the editor: Recently, Cox et al[1][1] described that only acute lymphoblastic leukemia (ALL) blasts with a CD19−CD133+ immune-phenotype are able to propagate human leukemia in immune-deficient nonobese diabetic severe combined immunodeficiency (NOD/scid) mice. We welcome this contribution to

Purpose Monitoring of minimal residual disease (MRD) after allogeneic stem cell transplantation (allo-SCT) by quantitative real-time PCR (qRT-PCR) of rearranged Ig- and TCR-genes may highlight patients with highest risk for relapse to whom pre-emptive treatment may be offered. Patients and Methods In the prospective phase 3 trial ALL-SCT-BFM-2003 (recruitment period 09/2003 to 09/2011; time point of analysis May 2013), MRD was assessed in bone marrow immediately on days +30, +60, +100, +200 and +365 post transplantation in 115 patients. Of these, 48 were male and 67 were female patients. All received a myeloablative conditioning regimen with TBI and VP 16. Patients received their transplant in CR2 (n=94), CR3 (n=21), CR4 (n=1) or NR (n=2). The transplantations were performed with bone marrow from matched sibling donors (MSD, n=23), matched unrelated donors (MUD, n=71) or with T-cell depleted stem cells from mismatched donors (MMD, n=21). Fifty-four patients were younger than 10 and 61 patients were older than 10 years at the time of transplant. Standardized quantification of MRD was performed according to the guidelines of the Euro-MRD-Group and MRD results were not released to the clinicians. Results The total group of patients showed a pEFS of 0.52±0.10; cumulative incidence of relapse (CIR) and cumulative incidence of treatment related mortality (CI TRM) was 0.41±0.11 and 0.19±0.09, respectively. In 76 patients, MRD could also be assessed prior to transplant: patients who were MRD negative (n=41) had a three year pEFS of 0.62±0.04, patients with a MRD load of 1E-3 survived their disease. MRD values post transplant were analyzed as time-dependent covariates. When analyzed either for the different time points or for the highest MRD value post transplant, MRD results had always significant influence on survival. Taken the highest MRD value post transplant, probabilities of pEFS and CIR were 0.65±0.11 and 0.23±0.09 for MRD negative patients (n=72), 0.36±0.18 resp. 0.75 ±0.18 for patients with MRD 10E-3 at any time after transplant finally developed relapse and died. However, 49% of patients who became MRD positive Conclusion Assessment of MRD post transplant allows the identification of patients with impending relapse. All patients who developed MRD >10E-3 at any time post transplant finally relapsed. Therefore, MRD may serve as an endpoint for further pre-emptive therapies. Acknowledgment This trial was supported by the “Deutsche Knochenmarkspender Datei” (DKMS). Disclosures: No relevant conflicts of interest to declare.

Abstract 4227 Asparaginase is an expensive drug, but important in childhood acute lymphoblastic leukemia (ALL). Due to increasing costs of treatment of childhood ALL more insight in costs of asparaginase preparations is desired. In order to compare pharmacoeconomic aspects of PEGasparaginase, Erwinia asparaginase and native E.coli asparaginase, we performed a cost-effectiveness analysis in Dutch Childhood Oncology Group (DCOG) ALL-10 medium risk group (MRG) intensification protocol. Between April 2005 and October 2009, MRG patients were included in this multi-center study. Treatment costs were calculated based on patient level data of 84 subjects (33 female), and were related to allergy to asparaginase. We have used 3 treatment scenarios for asparaginase of which 2 scenarios were hypothetical: PEGasparaginase as first line preparation and Erwinia asparaginase as second line used as scenario 1 (similar to the actual ALL-10 treatment schedule); native E.coli asparaginase as first line preparation and Erwinia asparaginase as second line used as hypothetical scenario 2; or native E.coli asparaginase as first line preparation, PEGasparaginase as second line and Erwinia asparaginase as third line preparation used as hypothetical scenario 3. Medical technology assessments techniques were used for this cost-effectiveness analysis. Decision tree analysis was used to compare costs of PEGasparaginase or Erwinia asparaginase to native E.coli asparaginase, while taking into account the incidence of allergy to asparaginase and the different associated costs. Sensitivity analyses (one-way and two-way) were conducted to account for uncertainty in the used prices and calculated costs. The total costs of the ALL-10 MRG intensification course of 30 weeks were $71,147 per patient. Subgroup analysis revealed that the costs were $57,893 in patients without PEGasparaginase allergy (N=64). The costs were significantly higher ($113,558) in case of PEGasparaginase allergy (N=20) necessitating a switch to Erwinia asparaginase. The total costs were also calculated based on two weeks of asparaginase exposure; in case of PEGasparaginase the costs were $ 1,930 and for Erwinia asparaginase $ 3,785 per two weeks of treatment. Decision tree analysis showed that the treatment costs were $ 70,402 when using native E.coli asparaginase as first line preparation in intensification (scenario 3) and $ 71,809 when using PEGasparaginase as first line preparation (scenario 1). Treatment costs using native E.coli asparaginase as first line followed by Erwinia asparaginase as second line (scenario 2) the costs would be significantly higher ($ 103,474). One-way sensitivity analysis showed that the subgroup with allergy to native E.coli asparaginase (scenario 2) had the largest range in treatment costs. When treatment costs were calculated with the new European price of Erwinia asparaginase (the price has been doubled in March 2011), both treatments with PEGasparaginase as first line preparation (scenario 1) or native E.coli asparaginase as first line preparation (scenario 3) would be less expensive ($ 100,199 or $ 103,089, respectively) compared to the native E.coli asparaginase scenario 2 ($ 190,284). Two-way sensitivity analysis revealed that treatment with PEGasparaginase (scenario 1) is less expensive than treatment with native E.coli asparaginase (scenarios 2) for allergy probabilities of PEGasparaginase ranging from zero to 0.8 with a fixed allergy rate of 0.65 (Veerman et al. (Lancet Oncol. 2009)). This also holds true if a fixed allergy rate for native E.coli asparaginase of 0.4 is used, which is frequently found in studies using less native E.coli asparaginase in intensification after native E.coli asparaginase in induction (Nachman et al. (JCO 1997) and Willer et al. (Blood 2011)). In conclusion, treatment with native E.coli asparaginase, followed by a switch to PEGasparaginase, and subsequently to Erwinia asparaginase in case of allergy carried similar overall costs compared to the treatment with PEGasparaginase as first line drug (followed by Erwinia asparaginase in case of allergy). However, PEGasparaginase is preferred, because it is administered less frequently, with less outpatient care visits and is less immunogenic compared to native E.coli asparaginase. The costs with PEGasparaginase are much lower compared to Erwinia asparaginase. Disclosures: No relevant conflicts of interest to declare.

The unpredictable oncogenic effects of contemporary treatment for childhood acute lymphoblastic leukemia (ALL) warrant continuous long-term monitoring of patients for second neoplasms. Bhatia and colleagues (page [4257][1]) in the Children's Cancer Group (CCG) reported cumulative rates of second

Abstract 4502 Introduction: As the prognosis of childhood acute lymphoblastic leukemia (ALL) with t(4;11) is generally poor, hematopoietic stem cell transplantation(HSCT) at 1st complete remission is recommended in most of Japanese ALL protocol. We analyzed outcomes of HSCT for childhood ALL with t(4;11) at our department, and assessed the outcomes with age and preconditioning at HSCT. Subjects: We retrospectively analyzed 15 patients who underwent HSCT between 1991 and 2011. The onset of ALL was less than one year old (infant ALL) in 9 patients and over one year old in 6 patients. The disease status at HSCT was 1st complete remission (CR1) in 10 patients, 2nd complete remission (CR2) in 2 patients, and more advanced stages in 3 patients. 13 patients underwent allogeneic HSCT and 2 patients underwent autologous HSCT at median age of 19 months. In allogeneic HSCT, the number of bone marrow transplantation from HLA matched sibling was 3 and 4 from unrelated donors, and that of cord blood transplantation was 6. Nine patients including 7 infant ALL underwent conventional stem cell transplantation (CST), 6 patients including one infant ALL underwent reduced intensity stem cell transplantation (RIST). Preconditioning of RIST was mostly consisted of fludarabine and melphalan with 3Gy of total body irradiation (TBI). Results: Of all 15 patients, 8 patients are alive in CR. Three out of 9 infant ALL and 5 out of 6 ALL patients, whose onset was above 1 year old, are alive in CR. Seven of 10 ALL patients who underwent HSCT in 1st CR are alive, on the other hand, only one of 5 patients in 2nd CR or non CR is alive. Five patients who underwent RIST over 1 year old are alive in CR. Discussion: HSCT for childhood ALL with t(4;11) in 1st CR could achieve better outcome than those in 2nd CR or non CR. The transplant outcomes with RIST was better than CST and it could be applied for ALL patients with t(4;11) whose onset is over one year old. Disclosures: No relevant conflicts of interest to declare.

Abstract 874 E. coli L-asparaginase (E. coli ASP) is an important component of treatment for childhood ALL, but is associated with multiple toxicities, including allergy, pancreatitis, and thrombosis. It is typically given intramuscularly (IM). Because most pediatric ALL patients have indwelling venous catheters, intravenous (IV) administration of asparaginase would be a more convenient and less painful option than IM injection. PEG-asparaginase (PEG), the polyethylene glycol conjugate of E. coli ASP, has a longer circulating half-life and so may be given less frequently. We have previously demonstrated that a single dose of PEG 2500 IU/m2 given IV is tolerable in children with ALL, with potentially therapeutic serum enzyme activity (≥ 0.1 IU/mL) maintained for at least 18 days in most patients.[Blood 2010;115:1351-3] On DFCI ALL Consortium Protocol 05-01, all patients (pts) with newly diagnosed ALL aged 1–18 years (yrs) who achieved complete remission were eligible to participate in a randomized comparison of IM E. coli ASP and IV PEG during the 30-week (wk) multi-agent post-induction Consolidation phase. Beginning at week 7 of therapy, pts received either IM E. Coli ASP 25000 IU/m2 weekly × 30 wks or IV PEG 2500 IU/m2 every 2 wks × 30 wks. Serum samples were obtained every 6 wks just prior to an ASP dose and were assayed for ASP enzyme activity by a validated biochemical assay. Between 2005–2010, 463 pts were enrolled in the randomized comparison. Median age was 5 yrs (range 1.2–17.9 yrs). There was no significant difference in presenting characteristics between the two arms, except that more pts on the E. coli ASP arm presented with a mediastinal mass (9% vs 3%, p=0.04). Median follow-up was 2.8 years. Median nadir serum ASP activity (NSAA) at each assayed timepoint during the Consolidation phase was significantly higher with IV PEG than with IM E. coli ASP (Table 1). An NSAA of ≥ 0.1 IU/mL was achieved in ≥ 95% of IV PEG pts compared with < 50% of IM E. coli ASP pts (p0.99 Allergy26 (11)20 (9)0.44 Pancreatitis25 (11)21 (9)0.64 Mild/Moderate13 (6)13 (6) Severe12 (5)8 (3) Thrombosis14 (6)21 (9)0.22Infection (bacteremia, invasive fungal disease)35 (15)46 (20)0.18 Disclosures: Silverman: Enzon Pharmaceuticals: Honoraria. Supko:Enzon Pharmaceuticals: Research Funding. Sallan:Enzon Pharmaceuticals: Honoraria.

Abstract 3064 Poster Board III-1 Both ETV6-RUNX1 (TEL-AML1)fusion and hyperdiploidy (>50 chromosomes) of lymphoblasts are favorable outcome predictors in childhood acute lymphoblastic leukemia (ALL). In 433 children with ALL diagnosed at our hospitals between 1997 and 2007 in Taiwan, the frequency of ETV6-RUNX1 fusion was 15.8%, and the frequency of hyperdiploidy (>50 chromosomes) was 14.1%, both were lower than those of the West. While ETV6-RUNX1 fusion had borderline favorable impact on outcome (p=0.053-0.061), hyperdiploidy showed significant favorable impact on event-free survival (91.1% vs 76.6 %, p= 0.016) in our patients. A meta-analysis from literature enrolled reports in which the case numbers and frequency of ETV6-RUNX1 fusion or hyperdiploidy in childhood ALL were described. It revealed that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East (Japan, Korea, Hong Kong, Chinese in Singapore, and Taiwan) was 14.2% (127/893, range 10-17%), significantly lower than 21.8% (152/697, range 19-27%) in the West (USA, Germany, Italy, France and Chile) (p < 0.0001). The frequency of hyperdiploidy in Japan and Taiwan was 15.2% (140/921, range 13-20%), significantly lower than 31.6% in the West (977/3,158, range 19-34%) (USA, UK and Germany) (p < 0.0001). So far as we know, there were several articles, including ours, addressing that the frequency of ETV6-RUNX1 fusion in childhood ALL was lower in a Far East country. This is the first meta-analysis to demonstrate that the frequency of ETV6-RUNX1 fusion in childhood ALL in Far East was lower than that in the West. There was no report on that the frequency of hyperdiploidy in Far East was lower than that in the West. This is also the first meta-analysis to demonstrate that the frequency of hyperdiploidy in childhood ALL in Far East is significantly lower than that in the West. The nature of these differences, probably due to racial, needs further study. In Far East, with both a lower frequency of ETV6-RUNX1 fusion, and a lower frequency of hyperdiploidy, it warrants renewed effort to cure a higher proportion of children with ALL. Disclosures No relevant conflicts of interest to declare.

To the Editor: We have recently reported a strong HLA class II association in childhood acute lymphoblastic leukemia (ALL) that is restricted to male patients.[1][1] The reason for this gender effect is currently unknown. Here we report another genetic association in childhood ALL with an HLA

Background: The beginnings of pediatric oncology in Bydgoszcz, Poland dates back to the year 1958. The first patient was treated with prednisolone only. Objective: Analysis of results of childhood acute lymphoblastic leukemia therapy in Bydgoszcz region in 1958–2008. Patients and methods: A total number of 570 children were diagnosed and treated over the period of 50 years, including: 215 children treated with different drugs and combinations in 1958–1972, 49 children treated with Memphis V–VII protocols in 1976–1983, 114 children treated with ALL-BFM83 or ALL-BFM86 protocols in 1983– 1995 and 192 children treated with ALL-BFM90, New York I–II or ALL-IC-2002 between 1995–2008. Results: A number of patients with „events” in respective time periods was: 205/215 (95.3%, in 1958–1972); 44/49 (89.8%, in 1976–1983), 54/114 (47.4%, in 1983–1995), 56/192 (29.2%, in 1995–2008). The values of 5-year pEFS in consecutive therapeutic periods were: p=0.012±0.008 (1958–1972), p=0.102±0.043 (in 1976–1983), p=0.526±0.047 (in 1983–1995), p=0.700±0.037 (in 1995–2008) (p Conclusions: Complex therapy based on international strategy of multiagent total therapy and supportive care, has led to an improved long-term survival in childhood ALL in Bydgoszcz region, Poland, from 1% in sixties up to 70% nowadays Figure 1. pEFS in ALL with respect to time period Figure 1. pEFS in ALL with respect to time period

Background: Several large cohort studies–like from BFM and St Jude groups-have shown that minimal residual disease (MRD) is the single most important predictor of treatment outcome in childhood acute lymphoblastic leukemia. However, lingering concerns about clonal evolution of IgH/TCR rearrangements may result in false-negative MRD. Hence, the AIEOP-BFM ALL 2000 study utilized at least 2 sensitive MRD markers with at least 10−4 sensitivity for risk-stratification. This stringent minimum of 2 sensitive MRD markers criteria limits the applicability of MRD stratification to Methods: A total of 362 patients (B-lineage=333, T-lineage=29) were enrolled in Ma-Spore ALL 2003 from July 2002 to August 2008, with a median age of 5.61 years (range 0.14 to 15.29). The treatment protocol is based on the modified BFM ALL 2000 backbone without randomization. Risk-stratification is based primarily on MRD levels at end of induction (Day-33) and week 12, Day-8 PR, and presence of BCR-ABL or MLL. In patients whom we are unable to quantify MRD, are assigned IR risk group. Screening for IgH/TCR rearrangements is carried out using multiplex BIOMED-2 primers (JJM van Dongen et al 2003) with standardization with the European MRD Study Group, which we are an active member. Selection of MRD markers is based on frequency and stability of IgH/TCR rearrangements. MRD quantifications were carried out using Real-time PCR (LightCycler 1.0, Roche Diagnostics). We define three distinct treatment subgroups: Standard-Risk (SR) who had MRD 1×10−3; remaining patients formed Intermediate-Risk (IR). The SR patients received decelerated therapy while HR group was treated under intensified therapy or bone marrow transplant. Results: Of 362 patients enrolled, Ma-Spore risk stratification is possible in 360 patients (n=2 delayed treatment), out of which 239 have completed 2 years therapy, 15 had induction failures, and 22 had relapses (isolated BM=17, isolated CNS=4, isolated testis=1.) Patients stratified into SR=29% (n=106/360); IR=50% (n=180/360), and HR=21% (n=74/360). At the end of the study, 4-years overall survival (0S) was SR=94.2±2.5%; IR=95.3±1.6%; HR=66.2±6.5%; event-free survival (EFS) was SR=91.8±3%; IR=86.2±3.4%; HR=52.9±6.4%, and leukemia-free survival (LFS) was SR=94.8±2.6%; IR=90.5±3.2%; HR=62.7±6.6%. Within the HR subgroup, using multivariate analysis, high MRD levels at week 12 (>10−3) is significantly associated with adverse treatment outcome (OS=27.5±15.8%, EFS=17.9±14.4%; LFS=23.1±17.8%), whilst poor prednisolone responders alone in the absence of high risk MRD or high risk BCR-AL or MLL-AF4 did well (OS=84.1±7.4%, EFS=84.1±7.4%, LFS= no event). Of the 362 patients, at least 91% (n=309/339) of patients had at least 1 sensitive MRD marker; the remaining patients have no diagnostic samples (n=9), died before time-point 1(n=10), and default before time-point 1(n=4). For risk-stratification using only MRD levels, 45.7% of the patients (n=128/280) were low-risk with MRD10−3 at week 12; and the remaining 51.1% (n=143) were stratified as intermediate-risk MRD. Overall survival in MRD low-risk group was 93.2±2.5%, MRD intermediate-risk was 93.6±2.4% and MRD high-risk MRD was 45±18.8%. Conclusion: We have demonstrated that a simplified MRD methodology using a single PCR-based marker (IgH/TCR) can be successfully implemented to tailor therapy for childhood ALL without adversely affecting outcome. It is cost-effective and can be applied to a larger group of patients (91%). This is particularly useful for countries with limited resources to pursue risk stratification incorporating MRD. Deceleration of therapy in the SR group would also help to reduce long-term side effects and better quality life in these children.

Osteonecroses (ON) are common complications associated with the treatment of acute lymphoblastic leukemia (ALL). For symptomatic ON, incidences of 1.6% to 9.3% have been published in the literature. The range of incidence rates may be related to treatment, data acquisition methods and patients’ age distribution. Between 09/99 and 06/06, 3,048 protocol patients with ALL (age 1–17 years) were enrolled into the multicenter trial ALL-BFM 2000 (Germany, Austria, Switzerland). Data on symptomatic ON were prospectively collected and were reported in 111 patients (3.6%, 95% confidence interval (CI) 3.0–4.4%). The median age of the patients with ON was 12.8 years (range 1.3–17.9 years), the median time from ALL diagnosis until diagnosis of ON was 15 months (range 2–73 months; data available from 92 patients). The incidence in adolescents, in particular adolescent girls, was significantly higher than in younger children (age

In the AIEOP-BFM ALL2000 trial, childhood acute lymphoblastic leukemia (ALL) patients were stratified mainly according to minimal residual disease (MRD) levels at day 33 (Time Point 1, TP1) and 78 (TP2) of treatment, as detected by PCR amplification of clonotypic immunoglobulin and T-cell receptor gene rearrangements. Overall, the results confirmed that PCR-based MRD detection is an independent prognostic indicator which overrides classical risk factors. In this context, MRD measurement by flow cytometry (FCM) on day 15 has been evaluated as an earlier predictor of relapse-free outcome in children with ALL. To understand how these two methods could be applied in a MRD-based clinical protocol, we analyzed the correlation of methodologies in data sets in which PCR and FCM were simultaneously applied. We studied 3,618 BM samples from 1,570 patients (32.5% of all patients on trial) derived from day15 (n=478, 30.4%), 33 (n=1,570) and 78 (n=1,570) of remission induction therapy of the AIEOP-BFM ALL 2000 protocol. Patients were enrolled from September 2000 to June 2006 in Italy, Austria and Germany, and selection was based only on available samples for both PCR and FCM. As in most MRD-based protocols, RQ-PCR was performed on BM mononuclear cells after Ficoll gradient separation, while 4-colors FCM was done on 300,000 nucleated cells (NC) from the whole BM sample after red blood cells lyse-wash procedure. MRD levels ≥10−4 were considered as ‘positive’, while levels below this threshold, not-quantifiable or undetectable were classified as ‘negative’. Overall, qualitative concordance was observed in 2,704/3,618 samples (74.7%) measured at d15, d33 and d78. Concordances at each TP are indicated in the Table. Figure Figure Around 70% of discordant samples in all TPs were cases with low-positivity by PCR (10−4 log range) and negative by FCM, or viceversa. Concordance was also evaluated according to PCR-based MRD risk subgroups. Of note, 519/571 (90.9%) of Standard Risk by PCR-MRD (negative at d33 by 2 markers with sensitivity ≥10−4) were also FCM negative at d33; whereas 59/121 (48.8%) of cases PCR ≥10−3 at d78 (High Risk by PCR-MRD) were FCM negative. In order to evaluate the correlation of methodologies more exactly, we investigated similar cell preparations (same MNC divided for PCR and FCM), and we enhanced the resolution of FCM by using 7-colors and analyzing 500,000 MNC. Samples were collected at days 15, 33, 52, and 78 within a single center. Among a total of 266 samples, the concordance increased up to 87%. More specifically, 100 samples (37.6%) resulted undetectable by both methods; 56 samples were FCM-undetectable and PCR-positive

Survival in childhood ALL, in resource challenged nations, has not kept pace with cure rates of over 80% in developed countries. This descriptive study was designed to assess survival data and identify risk factors for adverse outcome. Data obtained from case records of 762 patients with ALL was analyzed. Information regarding the clinical-demographic profile, therapy and course of illness were recorded. Status and duration at last follow-up were utilized to generate Kaplan-Meier survival curves. The mean age at presentation was 5.7±0.23 years (M:F::3.2:1). Caretakers of 230 (30.2%) patients opted for no therapy. There were 68 and 60 deaths in induction and remission phases respectively. Besides these, 111 children either defaulted therapy (23) or were lost to follow up (88). Relapsed disease was documented in 125 cases. The 5 year overall and event-free survival was 46 and 34% respectively. The p values for survival analysis using the Log-rank test for groups formed according to the gender, age, symptom-diagnosis interval, TLC and platelet count were 0.19, 0.02, 0.08, 50000/microliter and platelet count

Asparaginase (ASP) is an effective antileukemic agent. However, hypersensitivity and/or allergic reactions (HA), estimated to occur in 2.5%–45% of patients are major limitations for its clinical use. The risk of developing hypersensitivity and/or an allergic reaction to ASP increases with higher doses, IV (vs. IM), continuous infusions and repeat treatment after prolonged abstinence. We surveyed the rate of HA in pediatric ALL in Israel, and identified additional factors of clinical significance. Methods: Data was gathered from the medical records of 245 children treated for ALL during the years 2000–2006 from 4 Pediatric Hematology-Oncology wards. All children were enrolled on the Israel National Leukemia Study 1998 or 2003, modified from BFM protocols. In Brief, patient received 8 doses of ASP in the induction and 4 additional doses in each reinduction, starting after a two-month period free of ASP. The uniform treatment used an E coli L-ASP preparation and was replaced with a pegylatedE coli ASP preparation in case of HA. The definition and severity of the HA was defined by the professional opinion of the treating medical team. Results: Forty-Eight of the 245 patients (19.6%) developed an HA reaction. 25 of the 48 were defined as severe. A wide range of HA was noticed between the treating centers (6.4%–29.4%). 16/48 of the children with HA were treated on a high-risk protocol (33% vs. 14% of the entire group, p Conclusions: The overall HA rate (with the noticed wide variations) and the high incidence at the beginning of the reinduction protocol are within the described literature, We believe that the lack of steroid treatment that precedes ASP in the reinduction contributes to the high rate of HA at this point. The findings regarding the PEG were unexpected. We found a relationship between the higher risk group and the development of HA as well as the severity. We believe that the differences observed between treating wards are due to unclear definitions of reaction and of severity .We have noticed a wide variability in executing the supporting treatment. We recommend a revision of the reinduction protocol to initiate steroids at the beginning of all treatment cycles and to consider the use of a different than E. coli source after a hypersensitivity or allergic reaction.

BACKGROUND: Dexamethasone (dex) may be more efficacious but also more toxic than prednisone (pred) in the treatment of childhood ALL. In order to compare the relative toxicities of these 2 steroids, patients (pts) on DFCI ALL Consortium Protocol 00–01 were randomized to receive dex or pred during post-induction therapy. METHODS: Pts received 5-day steroid pulses every 3 weeks for 2 years (yrs) (pred 40mg/m2/day or dex 6 mg/m2/day) beginning at week 7 of therapy. High Risk (HR) pts received 3 times these doses during the Intensification Phase (approximately 6 months). RESULTS: Between 2000–2004, 425 pts (ages 1–18 yrs) were randomized; 423 had evaluable skeletal toxicity data, including 241 Standard Risk (SR) and 182 HR pts. 211 received dex and 212 received pred. Within 26 months of enrollment, 55 pts (13%) experienced at least 1 bony event: 45 pts (11%) with fracture and 17 (4%) with osteonecrosis (ON). Bony events were more common in children ≥10 yrs of age (11% of those Skeletal Toxicity and Infection: Dexamethasone vs. Prednisone* Dexamethasone Prednisone P value *Depicted are the numbers of pts with at least 1 toxicity event of all pts within particular subgroups. Skeletal Toxicity All 28/211 (13.3%) 27/212 (12.7%) 0.89 Age < 10 yrs 14/160 (9%) 22/166 (13%) 0.21 Age ≥ 10 yrs 13/51 (25%) 5/46 (11%) 0.07 Infection All 37/200 (18.5%) 24/204 (11.8%) 0.07 Age < 10 yrs 26/154 (17%) 22/160 (14%) 0.53 Age ≥ 10 yrs 11/46 (24%) 2/44 (5%) 0.01 CONCLUSION: Dexamethasone was associated with increased risk of skeletal toxicity and infection in pts ≥10 yrs of age. Longer follow-up is needed to fully assess EFS differences between the randomized treatment groups.

In vivo response to initial therapy, as assessed by determination of minimal residual disease after five and 12 weeks of treatment, has evolved as one of strong prognostic factors in children with acute lymphoblastic leukemia (ALL) treated according to the BFM regime. It is currently not known if the individual treatment response might be influenced by copy number alterations (CNA) leading to altered gene expression. We compared leukemic genomic profiles of 25 treatment sensitive (MRD-SR) and 25 resistant (MRD-HR) childhood ALL patients by means of high-resolution array-CGH. CNA were found in 46 patients (92%) of both treatment response groups. Microscopic alterations affecting the whole or nearly whole chromosome arm were frequently found, e.g. gain of 21 in 11/50, loss of 9p in 5/50, loss of 8p in 3/50, loss of 20q in 3/50 and loss of 7p in 2/50 or gain of 1q in 2/50. The most significant difference was a gain of chromosome 1q23-qter due to an unbalanced t(1;19), found in 10/25 MRD-SR patients, but in none of the MRD-HR patients (p

BACKGROUND: E.coli L-asparaginase (ASP) is an important component of multiagent chemotherapy for childhood ALL; however, allergic symptoms develop in 25–30% of patients (pts). Erwinia ASP is an alternative preparation which has been used after E.coli ASP allergy; however, it has not been previously shown how well E.coli ASP-allergic pts tolerate Erwinia ASP or whether they achieve therapeutic ASP levels. METHODS: On DFCI ALL Consortium Protocol 00–01 (2000–2005), newly diagnosed children with ALL (aged 1–18 years) received 30 weeks (wks) of IM ASP during consolidation beginning 7 weeks after diagnosis. All pts initially received weekly E.coli ASP. Nadir serum ASP concentrations were measured every 3 wks by a validated biochemical assay and antibodies to E.coli and Erwinia ASP were measured by ELISA every 6 wks. If E.coli ASP allergy developed, children received Erwinia ASP (25000 IU/m2/dose twice weekly) until 2003, when Erwinia ASP became unavailable. RESULTS: We analyzed the data of all 44 patients treated between 2000–2002 who received Erwinia ASP after E.coli ASP-allergy. Median age at diagnosis was 5.5 years. Median duration of E.coli ASP prior to development of allergy was 5 wks (range 1–23). 26 (59%) pts were positive for E.coli ASP antibodies. Erwinia ASP toxicities included: allergy in 15 (34%) pts (which occurred a median 8 wks after starting Erwinia, range 2–15.5), pancreatitis in 1 (2%) and insulin-requiring hyperglycemia in 1 (2%) pt. 18 (41%) pts became positive for Erwinia ASP antibodies, 4 of whom developed Erwinia ASP allergy. Highest nadir ASP concentrations observed after Erwinia ASP in the 44 E.coli-allergic pts are displayed in the Table (below). Prior data has suggested that a nadir serum ASP level of >=0.1 IU/mL is associated with asparagine depletion (therapeutic level). Nadir Serum ASP Levels after Erwinia ASP in E.coli ASP-allergic pts N Median (range) ASP Level (IU/mL) % with ASP Level >= 0.1 IU/mL All E.coli ASP allergic pts 44 0.231(0.00–3.33) 84% Subsequent allergy to Erwinia Yes 15 0.418 (0.00–3.33) 80% No 29 0.215 (0.00–0.93) 86% E.coli ASP antibody positive 26 0.171 (0.00–3.33) 73% negative 18 0.318 (0.10–0.93) 100% Erwinia ASP antibody Positive 18 0.171 (0.00–3.33) 72% negative 26 0.322 (0.00–0.93) 92% Highest ASP concentration with E.coli ASP prior to allergy Subtherapeutic(=0.1 IU/mL) 24 0.323 (0.00–3.33) 83% Excluding pts who switched to PEG when Erwinia became unavailable in 2003 (N=11), pts remained on Erwinia ASP for a median of 16 wks (range 2–28). 31 pts (70%) ultimately completed all planned 30 wks of ASP consolidation. CONCLUSIONS: We conclude that twice-weekly Erwinia ASP is well-tolerated and achieves detectable and potentially therapeutic serum ASP levels in the majority of E.coli ASP-allergic pts, including those with ASP antibodies (E.coli and/or Erwinia), pts who developed subsequent allergy to Erwinia ASP and pts who never achieved therapeutic nadir levels with prior E.coli ASP. Erwinia ASP should be considered as alternative therapy for pts with ALL who develop E.coli ASP allergy.

Adolescence has been claimed since the seventies to be associated to a a bad prognosis in childhood ALL. Out of 4658 patients with ALL, 258 adolescents (15–20 year old)(5.5%) were treated in the successive FRALLE 83, FRALLE 87–89, FRALLE 92 (pilot phase), FRALLE 93 and FRALLE 2000 protocols. The main characteristics were: a sex ratio of 1.8 (M/F), a B-lineage in 71% of the cases vs T lineage in 29%, and a median WBC of 12 G/L (9–1000). Translocation and fusion transcripts were searched for in 120 evaluable BCP-ALL: t(9;22)/BCR-ABL, 8 pts (6%); t(1;19)/E2A-PBX1, 12 pts (10%); t(4;11)/MLL-AF4, 4 pts (3%). Out of 75 evaluable pts t(12;21)/TEL-AML1 was found in only 4 pts (3%). 242 out of 258 adolescents were in CR at the end of induction therapy(EOI)(94%) without any significant difference according to the era. Nevertheless a major difference in the 3y and 5y EFS was found: Number of pts CR at EOI (%) 3y EFS (%) 5y EFS (%) 10y EFS (%) *: p=.04; **: p=.04 Eighties (F83, F87–89) 100 93 42 +/− 5 35 +/− 5 35 +/− 5 Nineties (F92, F93) 84 93 71 +/−5* 67 +/− 5** 67 +/− 5 2000– (F2000) 74 96 86 +/−5* 86 +/− 10** NYA The main modification introduced in the nineties was the adoption of a double delayed intensification for the good early responders. Autologous BMT or allogenic BMT were indicated in bad early responders (D8 poor prednisone response, D21 marrow M3 response) and/or unfavourable cytogenetics. The better results of the 2000 protocol can mainly be explained by the further intensification of chemotherapy between induction and delayed Intensification 1 and before delayed Intensification 2. These better results were obtained despite decreasing the indications of BMT (6 performed vs 20 in the nineties) and of CNS irradiation (100% in the nineties vs 35% in the current era, including the TBI for BMTs). Conclusions:excellent results can now be achieved in adolescents with ALLthis study emphasizes again the need to treat adolescents with ALL according to pediatric intensive protocols and not “adult-type” protocols, as we recently suggested (Boissel et al, J Clin Oncol 2003).Whether this could also be applied to young adults remains to be demonstrated but seems appealing.

Early response to therapy, including assessment of MRD is a powerful prognostic factor in childhood ALL. However, there are many other known prognostic factors, and the precise relationship among these and MRD has been incompletely explored. In COG protocol P9900, patients were assigned to one of 3 risk-directed therapies based on NCI risk group, as well as TEL-AML1 translocations (TEL) and simultaneous trisomies of chromosomes 4 and 10 (DT). Infants and Ph+ patients were excluded. Good risk patients (9904) comprised NCI SR patients with either TEL or DT without CNS disease. Poor risk patients (9906) included a subset of NCI HR patients with particularly unfavorable risk factors based largely on age and white count, while 9905 had both NCI SR and HR patients not eligible for 9904 or 9906, including NCI HR patients with TEL or DT. MRD was satisfactorily measured with at least .01% sensitivity by 4-color flow cytometry in the marrow at end of induction (D29) or in blood at Day 8 (D8). D29 MRD was associated with poor outcome in all studies, and in both HR and SR patients on 9905 as shown: Study 4 y EFS ± SE (n) p value MRD .01% 9904 Overall 94±1%(668) 75±6%(105) The proportion of D29 MRD positive DT(17.9%) patients was higher than that of TEL patients (9.4%) but MRD was also prognostic when each group was considered separately; similar findings were seen in HR TEL and DT patients. TEL or DT was associated with a significantly better prognosis in both HR and SR patients who were MRD negative, but were not favorable factors in MRD positive patients. D8 blood MRD was also prognostic in all studies, with higher MRD levels generally associated with poorer outcome. Patients who were both D29 and D8 MRD negative had a good outcome overall (93% 4 y EFS); combining all favorable risk factors, including TEL, DT, NCI risk group, D8 and D29 MRD identified a group of patients (about 13% of B-precursor ALL patients) with 98± 1% 4 y EFS. In multivariate analysis, D29 MRD was the most important prognostic factor. NCI Risk group, DT and D8 blood were also significant, but TEL and day 8 morphologic assessment of marrow were not. We conclude that MRD is a major prognostic factor in childhood ALL even accounting for other risk factors. The prognostic significance of TEL and DT depends on MRD status. The combination of good risk clinical and genetic factors, and absence of MRD in both D8 blood and D29 marrow identifies a group of patients with outstanding outcome on limited therapy.

Despite improvements in front-line therapy for acute lymphoblastic leukemia (ALL), treatment after relapse remains a critical challenge. Examination of outcome after relapse in a large, recently treated, and unselected population provides a useful benchmark for evaluating new approaches. Survival after relapse was tracked on Children’s Cancer Group (CCG) ALL trials. Relapses were categorized by site (isolated or combined bone marrow relapse (MR); isolated central nervous system relapse (CNSR); isolated testicular relapse (TR)), and time from diagnosis to relapse (early < 18 months; intermediate between 18 and 36 months; late > 36 months). We examined the impact of age, sex, ethnicity, initial risk assignment (standard risk (SR) or higher risk (HR)), immunophenotype, length of first remission (CR1), and site of relapse on outcome in multivariate analyses. A total of 9,585 infants, children, and adolescents with newly diagnosed acute lymphoblastic leukemia were enrolled on CCG trials conducted between 1988 and 2002. We examined survival after relapse for 1,874 children with MR, CNSR, or TR. Survival after relapse by site and time to relapse Site MR CNSR TR Time to Relapse n *survival(%) n *survival(%) n *survival(%) *3-year survival Early 498 10 175 49 15 50 Intermediate 378 23 179 73 35 57 Late 488 53 52 77 54 70 Overall survival for any bone marrow relapse was best for late MR and worst for early MR. Early MR was linked to HR presenting features and T-cell disease. Survival after intermediate or late MR was superior for SR patients (p Importantly, in the same multivariate analysis, patients treated on more contemporary trials (CCG1952CCG1953CCG1961, and 1962) had inferior survival rates after relapse when compared to patients treated on earlier trials (CCG 1881CCG 1882CCG 1883CCG 1891CCG 1901, and 1922) (p

Introduction: In a recent French FRALLE versus LALA comparison, we have demonstrated a large benefit in outcome when adolescents and young adults were treated in a pediatric rather than an adult ALL protocol (Boissel JCO 2003). Similar provocative results have been observed in three other pediatric versus adult ALL studies (Stock ASH 2000, de Bont Leukemia 2004, Testi ASH 2004). One explanation may be the larger amounts of steroids, vincristine (VCR), and L-asparaginase (L-aspa) administered in pediatric patients. The GRAALL-2003 study was thus designed to offer a pediatric approach in adults with Ph-negative ALL until 60 years of age. Methods: Treatment included a 5-drug induction, high dose-intensity consolidation blocks, delayed intensification, and 2-year maintenance. The comparison with the former LALA-94 protocol showed a 8.6-fold, 3.7-fold, and 16-fold increase in cumulative doses of prednisone, VCR, and L-aspa, respectively. One difference with childhood ALL therapy remained indication of allogeneic SCT in first CR which was offered to all patients with high-risk factor and a donor. In addition, induction was reinforced with a hyper-cyclophosphamide sequence (HyperC) in case of poor early response (cortico- and/or chemoresistant ALL). In the present report, 212 GRAALL-2003 patients with Philadelphia-negative ALL aged 15–55 years with a median follow-up of 18 months were compared to 712 patients previously treated in the LALA-94 trial. Results: Cohorts were comparable in terms of prognostic factors. CR rate was significantly higher in GRAALL patients (93 vs 88%, P=0.02) due to a reduction in resistant disease (0.5 vs 8%, P Conclusion: In conclusion, a pediatric approach benefits to younger adults with ALL.

Early response to therapy is the most important prognostic factor for childhood ALL. CCG investigators have shown that Day-7 and Day-14 BM blast counts were prognostically important although there is great inter-observer variability. BFM group have shown that day 8 prednisolone (PRED) response is highly predictive of the treatment outcome. While gene expression profiling (GEP) of diagnostic marrow can discern a pattern of PRED sensitivity as determined by in vitro MTT assay, the accuracy was low at only 70%. We hypothesized that changes in global GEP after therapy have a higher likelihood to predict response as the signatures of sensitivity and resistance may be unmasked during the therapy. We prospectively studied the changes in GEP using Affymetrix HG-U133A or Plus 2 chips on paired BM samples before and after 7-day course of PRED and one dose IT MTX in 58 patients with newly diagnosed or relapsed ALL. Unsupervised hierarchical clustering revealed that pre- and post- PRED samples in the patients still tended to cluster together, indicating that expression profiles of molecular subgroups were still most important. To remove intrinsic influence of molecular subtypes and identify potential signatures independent of genetic abnormalities, we subtracted Day-0 GEP from its paired Day-8 profile and retained probe sets with significant changes (≥ 10-fold). To avoid the ambiguity of variation in BM blast counting at Day-8, we divided the samples into a stringently reproducible group where “Good” PRED response was defined as that Day-8 blast count in PBL < 109/L and BM lymphoblasts ≤ 30% (n=16). “Poor” response was when Day 8 PBL ≥ 109/L (n=11). This stringently reproducible group (n=27) formed the training group to help define a distinct signature while the rest (n=31 pairs) were used as a blinded test set. 54 and 19 discriminating genes were identified by 2 independent statistical methods respectively, and an integrated predictor model was constructed based on shortlisted entries. This model predicted the PRED response with 100% accuracy for the training set using the leave-one-out cross validation but was less accurate in predicting the BM blast count in blinded test set. But intriguingly, in the blinded test set, this model predicted correctly 19 out of 21 reliable “Good” PRED responses are in CCR (91%), while among 8 predicted as “Poor” responses, only 2 are in CCR (25%). This suggests that as gene expression profiling as early as day 8 of PRED could discern the beginning of leukaemia cell death even before morphological changes are discernable and is highly correlated to eventual outcome. In conclusion, we have shown that analyses on the relative changes of gene expression profile can identify real genetic signatures indicating the sensitivity to PRED administration which is highly correlated with outcome.

More than 800 children with acute lymphoblastic leukaemia (ALL) are treated every year according to ALL IC-BFM 2002 protocol, which was designed by the International-BFM Group as a parallel to MRD-based ALL/AIEOP BFM 2000 study. The ALL-IC BFM 2002 risk group stratification comprises blast proportion in peripheral blood (PB) after 7 days of prednisone and one IT-MTX (prednisone response) and bone marrow (BM) morphology evaluation at days 15 and 33 of therapy together with age, initial WBC and presence of BCR/ABL and MLL/AF4 fusion. One of the aims of the ALL IC-BFM 2002 study is the comparison of this risk group assessment to the MRD-based criteria used in ALL-BFM 2000. We analyzed a total of 203 patients treated according to the ALL IC-BFM 2002 in the Czech Republic, Israel, Hong Kong and Uruguay for the presence of clonal antigen receptor rearrangements. MRD was evaluated in 175 patients at several time-points of therapy including mandatory points at week 5 and 12, which are used in the ALL/AIEOP BFM 2000 stratification. In total, 654 follow-up BM specimens and 80 PB samples were tested. In the univariate analysis, a good molecular response defined as MRD negativity at both week 5 and 12 was associated with the age of 1–6 years (p=0.0001), WBC

Objective: We conducted a randomized study to compare the effectiveness of two maintenance regimens, namely the 6-MP/MTX-based regimen (A-arm) and the LSA2L2 type regimen (B-arm). Patients and Methods: A total of 218 newly diagnosed patients between 1 and 15 years of age were enrolled from August 1996 to August 2002. The patients characteristics was as follows: median age, 5; T-cell/B-cell precursor phenotype, 22/196; median WBC, 8,100/μl (range, 600–809, 200). The high-risk group (HR) was defined as having a WBC of over 50,000/μl, a T-cell precursor phenotype, CNS disease, hepatosplenomegaly of more than 5 cm under the costal margin while the standard-risk group (SR) was defined as having no HR features. Any cases with t(9;22) and t(4;11) were excluded from the present protocol. After successful induction treatment, the patients received early intensification, consolidation and late intensification treatment. Before late intensification, the patients were randomly assigned to receive one of two maintenance regimens: the A-arm consisted of daily 6-MP with weekly MTX pulsed with DEX and VCR for 2 weeks irrespective of the risk group. The B-arm consisted of 4 blocks of 6-MP with CPM, 6-MP with CA, 6-MP with VCR and PSL, and intermediate-dose (ID)-MTX in the SR group, while in the HR group, high-dose CA was substituted for ID-MTX. The total treatment duration was 3 years. Eleven cases with t(9:22) and t(4;11), 5 with a disagreement to randomization, while the other 6 did not meet the eligibility criteria and thus were excluded from the protocol. As a result, 196 cases were evaluable (98 cases for each risk-group). Of these, 95 and 94 cases, excluding 7 who developed relapses before randomization, were assigned to the A and B-arms, respectively. Results: CR was successfully achieved in 195 of 196 patients (99%). Relapse occurred in 36 cases (isolated BM, 30; others, 6). No toxic death was observed during the entire treatment period. The event-free survival (EFS) rates in all 196 eligible cases, SR and HR cases were 75.5±5.1 (SE) %, 88.1±3.6% and 62.5±7.5% (p=.0004), respectively. The EFS rates for SR were 86.8±5.6% in the A-arm and 90.6±4.6% in the B-arm (p=ns). In the HR cases, the EFS rate in the A-arm was tended to be superior to that in the B-arm (81.2±5.6% and 52.3±13.1%, p=.13). Conclusion: No significant difference was observed in the EFS rates between the two maintenance regimens. However, the late adverse effects of each regimen should be carefully evaluated by a long-term follow-up of the patients.

Purpose To analyze the main reason of failure in children with ALL at a single institute which is at most developed city of developing country China. Method All the ALL patients who was diagnosed at our hospital from 1998.10 to 2003.12 were analyzed. The date was created from our department tumor registry database. Patient was divided into 3 groups, high, middle and low risk, depends on 1.Age >=10 year, 2. 50×109/L>WBC=100×109/L, 8.t(9;22), 9.12 year, 10. Early response (1) Pred.test day 8,pripherial blast >=1,000/μl, (2) Induction day19–21 or day 35 bone marrow blast >=5%. Anyone from item 1 to 5 was middle risk factor, item 6 to 9 was high risk factor. The patients not receiving any therapy after ALL diagnosis were accounted as early protocol compliance failure, receiving therapy less than 15 days were middle protocol compliance failure, giving up therapy or losing follow-up after 15 day with stable disease or CR were accounted as late compliance failure. Results Total 224 ALL were diagnosed, of them 38 patients went home with no any therapy. That means early protocol compliance failure was 17%. Of the remained 186 patients, 26 patients(12%) belonged to middle protocol compliance failure and 6 (3%) was late compliance failure. So total protocol compliance failure was 31%. The main reason of compliance failure was lacking financial support. Within the 160 patients who received more than 15 days therapy, 50(31%) was high risk, 51(32%) middle risk, and 52(33%) low risk, another 7 at un-know risk group. Of them, 48 patients relapsed(40) or not reached CR(8), 10 died of complication(mainly infection), total treatment failure was 48(30%). Conclusion Besides the treatment failure, protocol compliance failure is a important reason for childhood ALL survival failure in developing country.