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PURPOSE The aim of the randomized trial, UKALL2003, was to adjust treatment intensity on the basis of minimal residual disease (MRD) stratification for children and young adults with acute lymphoblastic leukemia. We analyzed the 10-year randomized outcomes and the time for patients to be considered cured (ClinicalTrials.gov identifier: NCT00222612 ). METHODS A total of 3,113 patients were analyzed including 1,054 patients who underwent random assignment (521 MRD low-risk and 533 MRD high-risk patients). Time to cure was defined as the point at which the chance of relapse was < 1%. The median follow-up time was 10.98 (interquartile range, 9.19-13.02) years, and survival rates are quoted at 10 years. RESULTS In the low-risk group, the event-free survival was 91.7% (95% CI, 87.4 to 94.6) with one course of delayed intensification versus 93.7% (95% CI, 89.9 to 96.1) with two delayed intensifications (adjusted hazard ratio, 0.73; 95% CI, 0.38 to 1.40; P = .3). In the high-risk group, the event-free survival was 82.1% (95% CI, 76.9 to 86.2) with standard therapy versus 87.1% (95% CI, 82.4 to 90.6) with augmented therapy (adjusted hazard ratio, 0.68; 95% CI, 0.44 to 1.06; P = .09). Cytogenetic high-risk patients treated on augmented therapy had a lower relapse risk (22.1%; 95% CI, 15.1 to 31.6) versus standard therapy (52.4%; 95% CI, 28.9 to 80.1; P = .016). The initial risk of relapse differed significantly by sex, age, MRD, and genetics, but the risk of relapse for all subgroups quickly coalesced at around 6 years after diagnosis. CONCLUSION Long-term outcomes of the UKALL2003 trial confirm that low-risk patients can safely de-escalate therapy, while intensified therapy benefits patients with high-risk cytogenetics. Regardless of prognosis, the time to cure is similar across risk groups. This will facilitate communication to patients and families who pose the question “When am I/is my child cured?”

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in relapsed/refractory acute lymphoblastic leukemia (ALL)1–5, but toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, limits broader application. Moreover, 40–60% of patients relapse owing to poor CAR T cell persistence or emergence of CD19− clones. Some factors, including the choice of single-chain spacer6 and extracellular7 and costimulatory domains8, have a profound effect on CAR T cell function and persistence. However, little is known about the impact of CAR binding affinity. There is evidence of a ceiling above which increased immunoreceptor affinity may adversely affect T cell responses9–11. We generated a novel CD19 CAR (CAT) with a lower affinity than FMC63, the high-affinity binder used in many clinical studies1–4. CAT CAR T cells showed increased proliferation and cytotoxicity in vitro and had enhanced proliferative and in vivo antitumor activity compared with FMC63 CAR T cells. In a clinical study (CARPALL, NCT02443831 ), 12/14 patients with relapsed/refractory pediatric B cell acute lymphoblastic leukemia treated with CAT CAR T cells achieved molecular remission. Persistence was demonstrated in 11 of 14 patients at last follow-up, with enhanced CAR T cell expansion compared with published data. Toxicity was low, with no severe CRS. One-year overall and event-free survival were 63% and 46%, respectively. New low-affinity anti-CD19 CAR T cells exhibit peripheral expansion and persistence without inducing severe cytokine-response syndrome in pediatric patients with relapsed or refractory B cell acute lymphoblastic leukemia.

After how many years from diagnosis/completing treatment should children and young adults with acute lymphoblastic leukaemia be considered "cured"? Clinical trials generally report 5 year event free survival because the risk of relapse after that time-point is very low and this is assumed to mean cure. Indeed, the risk of relapse after four years from completing therapy was There were a total of 3113 patients eligible for analysis with a median follow up of 9.4 years (only 20 patients received cranial irradiation). Relative survival rates were estimated using nationwide population mortality rates as the baseline accounting for age, sex and calendar year (Office of National Statistics 2018 release). The relative survival of UKALL2003 patients compared to equivalent children in the UK population was 96% (95% CI 95.5-96.7). Relative survival did not differ by sex but was significantly worse for patients >10 years compared with younger patients (p To estimate the time to a cure, we calculated the conditional probability of relapse at 12 years for patients who had survived event to the start of each follow-up year. Although the initial risk of relapse differ significantly by sex, age, MRD and genetics the risk of relapse for all subgroups quickly coalesced at around 5-6 years (Figure 1). Furthermore, the time to cure, defined as a relapse risk In conclusion, the relative survival of young patients with good risk cytogenetics and EOI MRD negative status approaches that of their normal peers. However, regardless of prognosis, the time to cure is similar across risk groups. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The UKALL 2003 trial aimed to safely reduce treatment intensity in low-risk patients and intensify therapy in high-risk patients based on minimal residual disease (MRD) stratification. The MRD risk patients were randomly assigned to standard (Regimen A/B) or augmented (Regimen C) post remission therapy, whilst the MRD low risk patients were randomly allocated to receive one or a standard two delayed intensifications (DI). In the original analysis, the five-year event free survival in the MRD risk patients was superior in the augmented group whilst in the MRD low risk group there was no significant difference between one or two delayed intensifications. As late relapses may influence these results, particularly in the low-risk patients, we analysed ten-year outcomes for patients in the trial overall and by the randomisations. There were a total of a total of 3113 eligible patients for analysis. The median follow up time was 9.4 years. In the overall trial population, 10-year relapse risk was 10.7 % (95%CI 9.6-11.92 %), with a 10-year event free survival (EFS) of 84.8 % (95 % CI 83.5-86.1 %) and overall survival (OS) of 89.6% (95%CI 88.4-90.6%). There was a higher risk of relapse on univariate and multivariate cox analysis with male gender, increasing age, increasing white cell count, MRD (high vs low), NCI Risk Group (High vs standard) and immunophenotype (T vs B cell). All except gender were also significant on univariate and multivariate analysis for event free and overall survival. Cytogenetic high risk patients treated on regimen C had a lower 10 year relapse risk (22.1 % (95% CI 15.1-31.6) compared to those who remained on regimen A/B (52.4 % (95% CI 28.9-80.1, p=0.016), although the OS rates were not significantly different (75.3 % (95% CI 65.8-82.5) vs 66.7 % (95%CI 37.5-84.6), p=0.3). The ten year cumulative incidence of second tumours was 1.16 %( 95 % CI 0.74-1.82). 521 MRD low risk patients were randomised (260 assigned to one delayed intensification and 261 to two delayed intensifications). The 10-year EFS was 91.7 % (95% CI 85.7-94.0) with one course of delayed intensification vs 93.7 % (95% CI 90.0-96.1) with two delayed intensifications (adjusted hazard ratio 0.73, (95% CI 0.38-1.40) p=0.3). The 10-year overall survival was 97.1 % (95 % CI 94.0-98.6) with one delayed intensification and 97.6 % (95 % CI 94.7-98.9) with two delayed intensifications (adjusted hazard ratio 0.69 % (95 % CI 0.24-1.99) P=0.5. 533 MRD high risk patients were randomised (266 assigned standard therapy and 267 assigned to augmented therapy). The 10-year EFS (was 82.1% (95 % CI 76.9-86.2) with standard therapy vs 87.1 % (95 % CI 82.4-90.6) with augmented therapy (adjusted hazard ratio 0.68 (95 % CI 0.44-1.06) p=0.09). The 10-year OS was 87.9 % (95% CI 83.2-91.4) with standard therapy vs 90.7 % (95 % CI 86.4-93.7) (adjusted hazard ratio 0.74(95%CI 0.44-1.27) p=0.3. The loss of significance in EFS between 5 and 10 years was due to additional relapses since the original publication, in the augmented arm. Nevertheless, there remained a benefit for augmented therapy in reducing marrow relapses: cumulative incidence of marrow relapse was 10.4% (95% CI 7.2-14.9) in standard arm vs 5.9% (95% CI 3.6-9.6) (adjusted hazard ratio 0.55 (0.28-1.03) p=0.06. Long term outcome of UKALL 2003 confirms that low risk patients can safely de-escalate therapy and intensified therapy benefits high risk patients, especially those with high-risk cytogenetics. Disclosures Samarasinghe: AMGEN,JAZZ: Honoraria.

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in relapsed/refractory acute lymphoblastic leukemia (ALL)

The EBF1-PDGFRB gene fusion accounts for 0.5%) at week 14. Two UKALL 2011 patients, positive for EBF1-PDGFRB, received imatinib; 1 died 6 months after a matched unrelated bone marrow transplant as a result of undefined encephalopathy, and the other remained in remission 10 months after diagnosis.

Outcomes for teenage and young adult (TYA) patients with acute lymphoblastic leukaemia (ALL) who relapse on contemporary risk-adapted paediatric protocols are largely unknown and there is no consensus on optimal salvage strategies. We assessed the treatment and outcome of TYA patients (aged 16-24 years) recruited to the UKALL2003 trial, who relapsed following attainment of complete morphological remission. Forty-two of 223 patients (18·8%) relapsed, the majority (n = 26, 62%) on treatment. Thirty-eight (90%) patients received salvage treatment, with 22 (58%) achieving second remission (CR2) and 21 patients receiving an allogeneic haematopoietic cell transplant (alloHSCT). Post-relapse outcomes were poor with a 5-year overall survival (OS) of 23% (95% confidence interval; 11-37%). Outcomes for patients relapsing on active treatment were inferior to those relapsing after completing treatment (5-year OS 9% vs. 52%, log-rank P = 0·001). No patient with B cell ALL relapsing on treatment was alive at the end of the study period. TYA patients with ALL who relapse on the UK paediatric protocol, UKALL2003, are largely unsalvageable with conventional approaches aimed at achieving CR2 followed by alloHSCT. Future efforts should be aimed at identifying those patients who are destined to relapse and exploring novel treatment approaches for this high-risk group and for those who do relapse.

Purpose To determine the prevalence and prognostic association of immunoglobulin heavy chain (IGH@) translocations in acute lymphoblastic leukemia (ALL). Patients and Methods The cohort comprised 3,269 patients treated on either the UKALL2003 trial for children and adolescents (1 to 24 years old) or the UKALLXII trial for adolescents and adults (15 to 59 years old). High-throughput fluorescent in situ hybridization was used to detect IGH@ translocations. Results We identified IGH@ translocations in 5% of patients with ALL (159 of 3,269 patients), in patients with both B-cell (148 of 2,863 patients) and T-cell (11 of 408 patients) disease. Multiple partner genes were identified including CRLF2 (n = 35), five members of the CEPB gene family (n = 17), and ID4 (n = 11). The level of the IGH@-positive clone varied and indicated that some IGH@ translocations were primary events, whereas others were secondary aberrations often associated with other established aberrations. The age profile of patients with IGH@ translocations was distinctive, with a median age of 16 years and peak incidence of 11% among 20- to 24-year-old patients. Among patients with B-cell precursor ALL who were Philadelphia chromosome negative, those with an IGH@ translocation had an inferior overall survival compared with other patients (UKALL2003: hazard ratio, 2.37; 95% CI, 1.34 to 4.18; P = .003; UKALLXII: hazard ratio, 1.73; 95% CI, 1.22 to 2.47; P = .002). However, this adverse effect was not independent of age or minimal residual disease status and did not seem to be driven by an increased risk of relapse. Conclusion IGH@ translocations define a genetic feature that is frequent among adolescents and young adults with ALL. Although associated with an adverse outcome in adults, it is not an independent prognostic factor in children and adolescents.

Introduction As survival rates for acute lymphoblastic leukaemia (ALL) in children and young people improve, the need to understand and mitigate toxicity becomes increasingly important. The contribution of toxicity to mortality or significant morbidity is well described. However, the impact of dose reductions, drug omissions or delays in treatment which result from toxicity is poorly understood, other than the established association between effective count suppression during maintenance chemotherapy and a reduction in relapse rate. We report the significance of treatment delays and dose reductions in induction chemotherapy in the UKALL2003 trial. Methods UKALL2003 was a prospective multicenter phase 3 study recruiting patients aged 1-24 years with newly diagnosed Philadelphia chromosome negative ALL in the UK. Data regarding 4 different protocol deviations were prospectively collected at the end of each block of therapy; a) not given allocated regimen, b) 1 week in commencing the next treatment block. Univariable (UVA) and Multivariable (MVA) Cox regression was used to assess the association of each deviation with risk of relapse. To control for immortality bias, this was assessed at the end of each therapy block with only patients alive and relapse free at the end of the block included in the analysis. Patients with Down Syndrome were excluded from all analyses. Results Of 2991 patients without Down Syndrome completing induction chemotherapy, 9 patients did not receive the allocated regimen, 1558 patients received 90% of one or more drugs and delay of greater than 1 week was significantly associated with an increased risk of relapse with hazard ratios of 1.70 (95% CI 1.11 - 2.60, p=0.014) and 1.99 (95% CI 1.14 - 3.48, p=0.013) respectively. On MVA (complete cases, n=2464), including other factors of established prognostic significance, both deviations remained significantly associated with an increased risk of relapse (Table 1). The impact of a major deviation (defined here as dose reduction of >90% of one or more drugs or treatment delay of >1week) was seen in both the paediatric (90% of one or more drugs in induction were also more likely to have major deviations in subsequent blocks of therapy (p Conclusion Major deviations in the delivery of induction chemotherapy on UKALL2003 were significantly associated with a greater risk of further deviations in subsequent blocks of therapy and a higher risk of relapse. Further improvement in efficacy of ALL therapy in children and young people will require a greater understanding of which toxicities lead to major deviations in therapy and strategies developed to mitigate these risks. Disclosures Rowntree: Novartis: Consultancy.

We report the outcome for children and young people with Down syndrome-associated acute lymphoblastic leukaemia (DS-ALL) treated on a contemporary protocol. Compared with non-DS ALL, patients with DS-ALL had an inferior event-free survival (65·6% vs. 87·7% at 5 years; P < 0·00005) and overall survival (70·0% vs. 92·2%; P < 0·00005). Excess treatment-related mortality - was primarily responsible for the worse outcomes for DS-ALL (21·6% at 5 years, vs. 3·3%, P < 0·00005). Minimal residual disease (MRD) risk status was highly discriminant for relapse in DS patients with 0/28 relapses in the MRD low risk group. © 2014 John Wiley and Sons Ltd.

Purpose To evaluate the effect on outcome of intensifying therapy for patients with acute lymphoblastic leukemia (ALL) and an intrachromosomal amplification of chromosome 21 (iAMP21). Patients and Methods We report two cohorts of patients treated on Medical Research Council ALL97 or United Kingdom (UK) ALL2003. iAMP21 was identified retrospectively in ALL97 and was not used to guide therapy. However, in UKALL2003, iAMP21 was determined prospectively, and patients were allocated to the most intensive treatment arm (regimen C), which included augmented Berlin-Frankfurt-Munster consolidation, escalating Capizzi maintenance, double delayed intensification, and an option for first remission transplantation. The presence of iAMP21 was determined by fluorescence in situ hybridization using probes specific for the RUNX1 gene. Results iAMP21 was identified in 2% of patients with B-cell precursor ALL treated on UKALL2003 and ALL97. The event-free survival, relapse, and overall survival rates at 5 years for iAMP21 patients treated on ALL97 and UKALL2003 were 29% and 78%, 70% and 16%, and 67% and 89%, respectively (all P < .01). Patients treated on ALL97 had an increased risk of relapse compared with patients treated on UKALL2003 (hazard ratio, 7.2; 95% CI, 2.91 to 17.87; P < .001). Conclusion iAMP21 patients with ALL benefitted from receiving more intensive therapy in UKALL2003. In UKALL2011, they will continue to be treated as cytogenetic high risk, receive intensive chemotherapy (regimen C), and will only be recommended for transplantation if they do not achieve a complete remission by the end of induction therapy. This study illustrates how the discovery and characterization of disease-specific genetic aberrations can be used to tailor therapy more precisely.

Abstract 290 Multiplex Ligation-dependent Probe Amplification (MLPA) provides an accurate and reliable high throughput method to screen for copy number abnormalities (CNA) of the significant genes in BCP-ALL. We have screened a consecutive cohort of 1427 childhood BCP-ALL patients treated on UKALL97 and UKALL2003 using the P335 SALSA MLPA kit IKZF1 (MRC Holland, The Netherlands). We here report the association of CNA involving these genes with clinical features. The overall breakdown of CNA was CDKN2A/B 28% (395), ETV6 22% (312), PAX5 19% (272), IKZF1 14% (196), RB1 6% (92), BTG1 6% (89) and EBF1 2% (30), as well as the rearrangement, P2RY8-CRLF2 4% (63). The cohort comprised 762 (54%) males and 665 (46%) females. There was no shift in the gender balance according to CNA. The median age of the entire cohort was 5 years (range 1–23) with 24% of patients being ≥10 years. There was some correlation between the distribution of CNA and patient age. Patients with IKZF1 and CDKN2A/B deletions were significantly older at 7 years (p50×109/L (each p There is particular interest in IKZF1 and CRLF2 in relation to outcome in BCP-ALL. We have previously shown an intermediate outcome for patients with CRLF2 rearrangements. We here examined the prognostic effect of IKZF1 deletions in this patient cohort; but excluded Down syndrome and BCR-ABL1 positive patients from survival analyses. Among 469 ALL97 patients, 13% (63) had IKZF1 deletions and after a median follow-up time of 9.5 years, 49% (31) had relapsed and 40% (25) had died. In the NCI standard risk (SR) group, patients with IKZF1 deletions had a significantly inferior event free survival (EFS) at 5 years (61% v 85%, p=0.0006). However, the effect appeared to be largely confined to patients with intermediate risk cytogenetics (63% v 85%, p=0.025) rather than those with good risk cytogenetics (75% v 87%, p=0.2915). As expected the NCI high risk (HR) patients with IKZF1 deletions had an inferior EFS: 50% v 68%, p=0.0112. In ALL2003, 776 patients with a median follow-up time of 3.9 years were available for analysis. Among 11% (87) patients with IKZF1 deletions, 13% (11) had relapsed and 5% (4) died in remission, resulting in a significantly inferior 5 year EFS: 78% v 90% p=0.0025. Of note, 8/11 IKZF1 deleted patients who relapsed were MRD positive (>0.01%) at day 28 and only 1/22 patients with good risk cytogenetics and IKZF1 deletion relapsed. There was no evidence that the type of deletion (deletion of exons 4–7 vs others) was linked to outcome in either the ALL97 or ALL2003 cohort. In conclusion, we have shown that IKZF1, PAX5 and/or CDKN2A/B aberrations are significantly associated with NCI HR, which may point to an associated poor outcome. Indeed, we showed that patients with IKZF1 deletions had an inferior outcome overall. However, there was evidence to suggest that the effect was pleiotropic and, importantly, IKZF1 deleted patients treated on ALL2003 in general achieved a respectable EFS rate of 75% at 5 years. Although in most studies IKZF1 deletions have been associated with a poor prognosis in BCP-ALL, while the risk relating to CRLF2 has been variable, thus far these findings, including those from this study, do not support changes to treatment for patients with these abnormalities. Disclosures: No relevant conflicts of interest to declare.

Recent genomic studies have provided a refined genetic map of acute lymphoblastic leukemia (ALL) and increased the number of potential prognostic markers. Therefore, we integrated copy-number alteration data from the 8 most commonly deleted genes, subordinately, with established chromosomal abnormalities to derive a 2-tier genetic classification. The classification was developed using 809 ALL97/99 patients and validated using 742 United Kingdom (UK)ALL2003 patients. Good-risk (GR) genetic features included ETV6-RUNX1, high hyperdiploidy, normal copy-number status for all 8 genes, isolated deletions affecting ETV6/PAX5/BTG1, and ETV6 deletions with a single additional deletion of BTG1/PAX5/CDKN2A/B. All other genetic features were classified as poor risk (PR). Three-quarters of UKALL2003 patients had a GR genetic profile and a significantly improved event-free survival (EFS) (94%) compared with patients with a PR genetic profile (79%). This difference was driven by a lower relapse rate (4% vs 17%), was seen across all patient subgroups, and was independent of other risk factors. Even genetic GR patients with minimal residual disease (>0.01%) at day 29 had an EFS in excess of 90%. In conclusion, the integration of genomic and cytogenetic data defines 2 subgroups with distinct responses to treatment and identifies a large subset of children suitable for treatment deintensification.

The BFM group (Blood Nov 2007; 110a: 585) has reported that monitoring of postconsolidation MRD kinetics identifies a very high risk (MRD VHR) group of children with ALL, who might benefit from novel therapy and first remission allogeneic transplantation. We have compared the effect of consolidation regimen on post-remission MRD kinetics using a standardised quality controlled RQ-PCR assay of Ig and TCR patient specific rearrangements with a reproducible quantitative range of 10−4 in an attempt to identify a MRD VHR group, similar to that reported by the BFM. MRC UKALL 2003 is a randomised trial testing whether treatment can be stratified by MRD at weeks 4 and 11 in children and young adults (up to age 25 years) with ALL from the UK and Ireland. Initially, patients are stratified by NCI criteria, cytogenetics and early morphological marrow response to receive three (Regimen A) or four (Regimen B, additional daunorubicin) drug induction. Patients with MRD >10−4 at end of induction (EOI) (MRD High Risk) are randomised to continue previously assigned therapy (regimen A or B) or receive the more intensive regimen C. The regimens are of escalating intensity (A→C) and include either CCG modified BFM (A), standard BFM (B) or augmented BFM (C) consolidation, the latter containing pegylated-asparaginase (Peg-ASP; Oncospar, Medac UK, 1000 units/m2 i.m. days 16 and 44) and vincristine (1.5mg/m2 i.v. days 16, 23, 44, 51) in addition to standard BFM consolidation. Only patients with high risk cytogenetics or day 28 BM3 marrow are eligible for CR1 transplant. Of the134 patients with MRD > 10–4 at EOI, 101 were MRD negative, and 33 had detectable disease post-consolidation therapy (16 low MRD positive; 5×10−4). Patients who received augmented BFM consolidation had more rapid clearance of MRD (Neg = 85%, low pos =7%, high pos =8%) than those who received CCG modified or standard BFM consolidation (Neg 65%, low pos =17%, high pos =17%) at week 11 – 15 of treatment (P=0.03 independent of age and white cell count). With median follow-up of 27 months, ten patients have relapsed, 7 in the negative group (3 year relapse Free Survival [RFS] 91.2% se=3.5) 1 in the low positive group (RFS 92.9% se = 6.9) and 2 in the high positive group (RFS 85.7% se = 9.4). The latter two were recipients of the lower intensity Regimens A (isolated CNS relapse) and B (isolated marrow relapse). The small numbers of post-consolidation high-positive patients do not permit us to identify a VHR group with a worse outcome than those patients with lower MRD levels. However, the early relapse risk (50% at 3 years). Differences in treatment protocol before and after consolidation may account for this disparity. In patients treated on the current UK childhood ALL protocol, we are unable to identify a VHR group on the basis of post-consolidation MRD. Augmented BFM consolidation obtains more rapid clearance of MRD than other regimens which, with longer follow-up, might be expected to translate into a better EFS.

L-asparaginase is an integral component of therapy for acute lymphoblastic leukemia. However, asparaginase-related complications, including the development of hypersensitivity reactions, can limit its use in individual patients. Of considerable concern in the setting of clinical allergy is the development of neutralizing antibodies and associated asparaginase inactivity. Also problematic in the use of asparaginase is the potential for the development of silent inactivation, with the formation of neutralizing antibodies and reduced asparaginase activity in the absence of a clinically evident allergic reaction. Here we present guidelines for the identification and management of clinical hypersensitivity and silent inactivation with Escherichia coli- and Erwinia chrysanthemi- derived asparaginase preparations. These guidelines were developed by a consensus panel of experts following a review of the available published data. We provide a consensus of expert opinions on the role of serum asparaginase level assessment, indications for switching asparaginase preparation, and monitoring after change in asparaginase preparation.

Purpose We sought to determine whether cranial radiotherapy (CRT) is necessary to prevent relapse in any subgroup of children with acute lymphoblastic leukemia (ALL). Patients and Methods We obtained aggregate data on relapse and survival outcomes for 16,623 patients age 1 to 18 years old with newly diagnosed ALL treated between 1996 and 2007 by 10 cooperative study groups from around the world. The proportion of patients eligible for prophylactic CRT varied from 0% to 33% by trial and was not related to the proportion eligible for allogeneic stem-cell transplantation in first complete remission. Using a random effects model, with CRT as a dichotomous covariate, we performed a single-arm meta-analysis to compare event-free survival and cumulative incidence of isolated or any CNS relapse and isolated bone marrow relapse in high-risk subgroups of patients who either did or did not receive CRT. Results Although there was significant heterogeneity in all outcome end points according to trial, CRT was associated with a reduced risk of relapse only in the small subgroup of patients with overt CNS disease at diagnosis, who had a significantly lower risk of isolated CNS relapse (4% with CRT v 17% without CRT; P = .02) and a trend toward lower risk of any CNS relapse (7% with CRT v 17% without CRT; P = .09). However, this group had a relatively high rate of events regardless of whether or not they received CRT (32% [95% CI, 26% to 39%] v 34% [95% CI, 19% to 54%]; P = .8). Conclusion CRT does not have an impact on the risk of relapse in children with ALL treated on contemporary protocols.

This study investigated the efficacy of a pre-emptive strategy based on the combination of Epstein-Barr virus (EBV) viraemia and poor T cell reconstitution in preventing post-transplant lymphoproliferative disease (PTLD) following T cell depleted stem cell transplant (SCT). EBV viral load and immune reconstitution were prospectively monitored in 70 consecutive children undergoing SCT following reduced intensity conditioning with alemtuzumab. Patients who developed significant EBV viraemia (40 000 copies/ml blood) were treated pre-emptively with rituximab if they were within 3 months of SCT or their CD3 count was0·3 × 10⁹ /l. Of 20/70 patients who developed significant EBV viraemia, 13 received pre-emptive rituximab. The incidence of PTLD was significantly reduced in the pre-emptive cohort compared to historical controls (1·4% vs. 21·7%, P = 0·003). This difference was more marked among viraemic patients (2·7% vs. 62·5%P0·0001). Patients treated with rituximab demonstrated significantly delayed B cell reconstitution at 1 year post-SCT but this was not associated with an increase in infectious mortality. In 6/6 patients3 months post-SCT who had a CD3 count0·3 × 10⁹ /l, reduced immunosuppression only resulted in successful resolution of EBV viraemia without PTLD. This strategy is safe and highly effective in preventing PTLD following T cell depleted SCT, and directs rituximab therapy to patients at highest risk of this complication.

Introduction Reported outcomes for children with Downs syndrome (DS) and acute lymphoblastic leukaemia (ALL) treated on our previous national study, UKALL 2003, were inferior compared to non-DS children. 5 year event free survival (EFS) for DS children was 65.6% vs. 87.7% for non-DS children and 5 year overall survival (OS) was 70.0% vs. 92.2% (British Journal of Haematology 2014; 165: 552-555). Excess treatment related mortality (TRM, 21.6% vs. 3.3% at 5 years) in children with DS-ALL was primarily due to infection. Similar results have been reported by other study groups. In our successor study, UKALL 2011, we employed several steps of de-escalation for DS-ALL patients compared to standard therapy. Here, we report that de-escalated therapy has resulted in a dramatic reduction in TRM for children with DS-ALL treated in the UK. Methods DS-ALL patients treated on UKALL 2011 had their treatment modified in the following respects: 1)NCI high risk patients did not receive anthracycline in induction unless they had a slow early response at day 15; 2) two years maintenance was given for both boys and girls; 3) no pulses were given in maintenance for MRD low risk patients. In addition, we recommended use of prophylactic antibiotics during induction and intensive phases of treatment. Fisher's exact test was used to compare induction mortality in the DS and non-DS pts. OS was defined as time from registration to death. EFS was defined as time from registration to first event (induction failure, relapse, second malignancy or death from any cause). The Kaplan-Meier method was used for survival estimation. Results The UKALL 2011 trial opened to recruitment on 26th April 2012 and has recruited 2362 patients, of whom 50 have Down syndrome, in the period up to 22nd February 2018. The study is open in 41 centres in UK and Ireland and included patients with both ALL and lymphoblastic lymphoma (LBL). The baseline characteristics of the patients recruited up to this date are shown in the table below. For DS-ALL, Day 29 MRD was available for 48 patients and was low risk in 25 cases (50%), risk in 19 (38 %) and no result was obtained in 4 (8 %). The therapy was generally well-tolerated, there have been no cases of therapy curtailment due to toxicity and only one patient due to receive intensified therapy per MRD risk assessment was unable to escalate due to toxicity. At a median follow-up of 32.7 months, there have been 4 events. These include two relapses (relapse rate: 4.1% (1.1-15.5) at 3 years) and two TRMs (one TRM in induction and the other in consolidation; both due to infection). Three year EFS is: 92.9% (95% CI: 79.1 - 97.7) with a three year OS of 95.9% (84.5 - 99.0). The remarkably low induction TRM for DS children on UKALL 2011 (1 death out of 50 patients, 2%), is comparable to that of non-DS children treated on the same protocol with a 3 drug induction (12 out of 1174 children, 1% induction TRM; induction TRM in DS-ALL vs non-DS-ALL p=0.42). It is also comparable to the induction TRM in DS-ALL noted in the latter half of the UKALL 2003 study (1/40, 2.5%) when the aforementioned treatment modifications were first implemented in the UK. Importantly, this TRM is a quarter of that when an historic cohort of DS-ALL children were treated with a 4-drug induction on our previous study (UKALL 2003, DS-ALL TRM 4/46 patients i.e. 8 %, p=0.19). Conclusion These preliminary results suggest that de-escalation of treatment is successful in reducing treatment related mortality for children with DS-ALL without increasing the risk of early relapse, however longer term follow-up is needed. Disclosures Ghorashian: Celgene: Other: travel support; Novartis: Honoraria. Hough:University College London Hospital's NHS Foundation Trust: Employment. Kearns:Pfizer: Consultancy, Research Funding; Galen: Research Funding. Vora:Jazz: Other: Advisory board; Medac: Other: Advisory board; Pfizer: Other: Advisory board; Amgen: Other: Advisory board; Novartis: Other: Advisory board.

Summary Background Stem-cell transplantation can cure primary immunodeficiencies. However, in patients with pre-existing organ toxicity, patients younger than 1 year, and those with DNA or telomere repair disorders, chemotherapy-based conditioning is poorly tolerated and results in major morbidity and mortality. We tested a novel antibody-based minimal-intensity conditioning (MIC) regimen to assess whether this approach allowed curative donor stem-cell engraftment without non-haemopoietic toxicity. Methods 16 high-risk patients underwent stem-cell transplantation for primary immunodeficiencies with an MIC regimen consisting of two rat anti-CD45 monoclonal antibodies YTH24.5 and YTH 54.12 for myelosuppression, and alemtuzumab (anti-CD52) and fludarabine, and low dose cyclophosphamide for immunosuppression. Donors were matched siblings (n=5), and matched (9) and mismatched (2) unrelated donors. Findings Antibody-based conditioning was well tolerated, with only two cases of grade 3 and no grade 4 toxicity. Rates of clinically significant acute (n=6, 36%) and chronic graft-versus-host disease (GVHD) (n=5, 31%) were acceptable. 15 of 16 patients (94%) engrafted, of whom 11 (69%) achieved full or high-level mixed chimerism in both lymphoid and myeloid lineages, and three achieved engraftment in the T-lymphoid lineage only. One patient needed retransplantation. At a median of 40 months post-transplant, 13 of 16 patients (81%) in this high-risk cohort were alive and cured from their underlying disease. Interpretation Monoclonal antibody-based conditioning seems well tolerated and can achieve curative engraftment even in patients with severe organ toxicity or DNA repair defects, or both. This novel approach represents a shift from the paradigm that intensive chemotherapy or radiotherapy, or both, is needed for donor stem-cell engraftment. This antibody-based conditioning regimen may reduce toxicity and late effects and enable SCT in virtually any primary immunodeficiency patient with a matched donor. Funding None.

Summary Some children with relapsed or high-risk acute leukaemia have an improved outcome if they have an allogeneic stem cell transplant, preferably from a sibling or well-matched unrelated donor. However, some children do not have these options or there is an urgent need to proceed to transplant because of disease status. We have investigated the role of haploidentical family members as donors in 34 patients with acute leukaemia (median age 11 years, range 1–16 years). Patients were conditioned with cyclophosphamide and total body irradiation (14·4 Gy in eight fractions) and received T-cell depleted peripheral blood stem cell grafts with a median CD34 cell dose of 13·8 × 106/kg (range 4·2–35·1) and 0·7 × 104 CD3-positive cells/kg. The actuarial survival at 2 years was 26% (13–41%, 95% CI). Eight patients have survived disease-free with a median follow up of 62 months. They have good performance status and a median lymphocyte count of 1·8 × 109/l. Relapse (14 patients) and adenoviral (six patients) or fungal infections (four patients) were the major causes of death. Haploidentical stem cell transplantation can produce medium term disease-free survival in a proportion of children with high-risk or relapsed acute leukaemia. None of the nine patients with acute myeloid leukaemia not in remission have survived.

Acute lymphoblastic leukaemia (ALL) responds well to chemotherapy and the majority of children and a significant proportion of adults are cured of their disease after primary therapy. However, a number of patients relapse and allogeneic transplantation following conditioning with chemotherapy and radiotherapy offers the possibility of long-term survival in a proportion of these patients. A significant number of patients with ALL develop disease that is refractory to further therapy. The infusion of unmodified donor lymphocytes (DLI) following relapse after allogeneic transplantation has been shown to be curative in patients with chronic myeloid leukaemia (CML). However, in ALL the success rate is much lower. The results of in vitro and limited in vivo studies suggest that it may be possible to manipulate lymphocytes from the transplant donor to produce cytotoxic T-lymphocytes (CTL) with increased effectiveness in killing patients' ALL cells. This may be done in a number of ways. For example, some strategies utilise the patients dendritic cells (DC) to present tumour antigens to donor lymphocytes and convert them into CTL either by pulsing DC taken in remission with ALL cells or lysate, fusing such 'normal' DC with ALL cells or using DC cultured from the patient's ALL cells. Other approaches include exploiting the expression of leukaemia-specific antigens such as the proteinase PR-3 or the zinc finger transcription factor Wilms tumour-1 protein (WT-1) to stimulate CTL responses. Alternatively, immunotherapeutic strategies might exploit differences in minor histocompatibility antigens such as HA-1 and HA-2 between donor and recipient. These are expressed solely on haemopoietic cells making them suitable targets for donor derived anti-leukaemic cells. In vivo studies to date suggest that educated T-cells may have a role to play in the treatment of relapsed and refractory ALL in the future.

Contains fulltext : 138929.pdf (Publisher’s version ) (Open Access) The outcomes of Central Nervous System (CNS) relapses in children with acute lymphoblastic leukaemia (ALL) treated in the ALL R3 trial, between January 2003 and March 2011 were analysed. Patients were risk stratified, to receive a matched donor allogeneic transplant or fractionated cranial irradiation with continued treatment for two years. A randomisation of Idarubicin with Mitoxantrone closed in December 2007 in favour of Mitoxantrone. The estimated 3-year progression free survival for combined and isolated CNS disease were 40.6% (25.1, 55.6) and 38.0% (26.2, 49.7) respectively. Univariate analysis showed a significantly better survival for age

Paediatric ALL is heterogeneous in terms of clinical presentation, response to therapy and underlying genetic mechanisms. The application of treatment stratification algorithms has resulted in survival rates exceeding 90%. However, current algorithms apply risk factors as binary variables using arbitrary thresholds and in an independent manner. This approach potentially distorts biological heterogeneity resulting in loss of prediction accuracy. Therefore, we sought to determine whether treating clinical risk factors and MRD response as continuous variables and integrating genetic features can refine prognostication. A total of 2542 patients, aged 1-24 years old, recruited to UKALL2003 were available for analysis. NCI standard/high patients were initially assigned to regimen A/B. Sow early responders and those with HR genetics were transferred to the more intensive regimen C. Minimal residual disease (MRD) was evaluated at the end of induction (EOI) by real-time quantitative PCR analysis of Ig/TCR rearrangements. Patients with EOI MRD >0.01% were randomised to stay on regimen A/B or move to C whereas patients with EOI MRD In order to examine MRD as a continuous variable, we log transformed the raw EOI MRD value assuming a minimum detection level of 0.00001 and assigned cases with undetectable MRD a value of one log below this threshold. The transformed MRD variable [t(MRD)] followed a log normal distribution (Figure 1). Importantly, this log normal distribution was distinct across the genetic subtypes (Figure 1, p Next we combined multiple significant risk factors from univariate analysis using forward selection modelling to fit a multivariate prognostic model. The final model included log(WCC) and t(MRD) as continuous variables and two binary genetic variables: GRcyto and HRcyto. Using the coefficients from this model, we constructed a linear model which enabled the calculation of individual risk scores. Figure 2 shows the correlation between an individual's risk score and their RR. To investigate the clinical relevance of this risk score, we generated five risk groups based on ascending RR (Table 2) which have significant differential outcomes (all p25% and together with the standard risk (SR) group capture >70% relapses and, importantly, >80% of HR relapses (very early/early relapses). Among the 136 VLR/LR patients who had MRD >0.01%, there was no difference in EFS between patients randomised to regimen A/B v C (97% v 99%, p=0.5). Equally among 470 VLR/LR patients with MRD In conclusion, we have showed that EOI MRD is log normally distributed but that the shape of the distribution depends on genetic subtype; suggesting differential MRD kinetics. In addition, we have shown that using a single MRD threshold does not reflect the biological heterogeneity that is a fundamental feature of the disease. Finally, we demonstrate that integrating clinical risk factors as continuous variables with genetics can better define prognostic subgroups. Disclosures No relevant conflicts of interest to declare.

The efficacy of allografting in acute lymphoblastic leukemia (ALL) is heavily influenced by remission status at the time of transplant. Using polymerase chain reaction (PCR)-based minimal residual disease (MRD) analysis, we have investigated retrospectively the impact of submicroscopic leukemia on outcome in 64 patients receiving allogeneic bone marrow transplantation (BMT) for childhood ALL. Remission BM specimens were taken 6 to 81 days (median, 23) before transplant. All patients received similar conditioning therapy; 50 received grafts from unrelated donors and 14 from related donors. Nineteen patients were transplanted in first complete remission (CR1) and 45 in second or subsequent CR. MRD was analyzed by PCR of Ig or T-cell receptor δ or γ rearrangements, electrophoresis, and allele-specific oligoprobing. Samples were rated high-level positive (clonal band evident after electrophoresis; sensitivity 10−2 to 10−3), low-level positive (MRD detected only after oligoprobing; sensitivity 10−3 to 10−5), or negative. Excluding 8 patients transplanted in CR2 for isolated extramedullary relapse (all MRD−), MRD was detected at high level in 12 patients, low level in 11, and was undetectable in 33. Two-year event-free survival for these groups was 0%, 36%, and 73%, respectively (P < .001). Follow-up in patients remaining in continuing remission is 20 to 96 months (median, 35). These results suggest that MRD analysis could be used routinely in this setting. This would allow identification of patients with resistant leukemia (who may benefit from innovative BMT protocols) and of those with more responsive disease (who may be candidates for randomized trials of BMT versus modern intensive relapse chemotherapy).

Detection of residual disease after completion of therapy or following bone marrow transplantation (BMT) in patients with acute promyelocytic leukaemia (APL) predicts relapse and is associated with a poor prognosis. Here we describe the successful treatment of residual disease post-transplant in APL using prolonged all-trans retinoic acid (ATRA) therapy in two children in whom autologous BMT (ABMT) had been performed in second complete remission (CR). ATRA treatment was well tolerated and found to be beneficial despite its prior use as a component of the initial induction protocol. ATRA therapy post-transplant led to molecular remission as determined by fluorescence in situ hybridization (FISH) as well as reverse transcriptase-polymerase chain reaction (RT-PCR) analyses and remission now exceeds 3.5 years in both patients. Overall, this study not only demonstrates that ATRA may successfully salvage APL patients with residual disease post-transplant, but also suggests a potential role for retinoids post-consolidation as a means of eliminating residual disease which could be beneficial even in patients previously exposed to ATRA as a component of the induction protocol.

We report a largely retrospective analysis of minimal residual disease (MRD) in a cohort of 66 children suffering from acute lymphoblastic leukaemia (ALL). All patients lacked high-risk features at diagnosis, i.e. the presenting white cell count was50 x 10(9)/l, age 1-16 years and translocations t(9;22) and t(4;11) were not present. All were treated according to either the MRC protocols UKALL X or XI. PCR of IgH, TCRdelta and TCRgamma gene rearrangements and allele-specific oligoprobing were employed for the detection of MRD. Sensitivity was at least 10(-4) in 78/82 (93%) probes examined. A total of 33 patients relapsed (seven on therapy and 26 off) and 33 remain in continuing complete remission (CCR) (median follow-up 69 months from diagnosis). Of those who remain in CCR, MRD was present in the bone marrow in 32%, 10% and 0% at 1, 3 and 5 months into therapy respectively. This is in marked contrast to the presence of MRD at these times in 82%, 60% and 41% of patients who relapsed (P0.001, P0.005 and P0.005). These results provide further evidence of a strong correlation between clearance of MRD early in therapy and clinical outcome in childhood ALL.

Abstract 136 Treatment modification according to Minimal Residual Disease (MRD) status at the end of induction and week 11 was investigated in a randomised control trial, UKALL 2003. Consecutively diagnosed children and young adults (up to age 25 years) with ALL were recruited from the UK and Republic of Ireland between October 2003 and June 2011. In the trial overall, we have observed a significant improvement in event-free survival (EFS) compared with its predecessor, ALL 97/99 (5-year EFS 87 vs 80%, log rank p Patients in the trial were initially stratified by NCI risk, blast karyotype, and morphological marrow response at day 8/15 of induction for allocation to one of 3 treatment regimens, escalating in intensity (A, B, C). NCI standard risk (SR) patients received a 3 drug induction (Regimen A) whilst high risk (HR) patients received 4 drugs including daunorubicin (Regimen B). Patients with a slow early response at day 8 (NCI HR) or day 15 (NCI SR) of induction and patients with poor risk cytogenetics were transferred to Regimen C. Patients without poor risk cytogenetics and who had a rapid early response were eligible for the MRD randomisations. MRD was measured using Real Time Quantitative PCR with a sensitivity of 10−4. Five hundred and thirty three patients who had an MRD >/= 10−4at day 29 of induction were entered into a randomisation comparing standard treatment (Regimens A or B = standard arm, n = 266) with the more intensive Regimen C (intensification arm, n = 267). Of these, 332 (62%) were NCI SR and 201 (38%) NCI HR. With follow-up to October 2011 (median 3 years 11 months), the 5 year EFS is significantly better for patients in the intensification arm (91%) compared with the standard arms (82%) due to a halving of relapse risk (OR 0.57, 95% CI 0.34 – 0.95, 2p = 0.03). The improved EFS translates into a trend for better overall survival (OR 0.57, 95% CI 0.31 – 1.05, p= 0.07). There was no heterogeneity in benefit of Regimen C for sub-groups defined by NCI risk, immunophenotype or cytogenetics. The risk of death in remission was not significantly different in the two arms (Intensification = 7, Standard = 9, OR 0.76, 2p = 0.6) but there was an excess of SAEs and AEs related to asparaginase (hypersensitivity, p=0.0003 and pancreatitis, p = 0.01) and intravenous methotrexate (mucositis, p=0.03 and vomiting, p = 0.03) in the intensification arm. This randomised study provides evidence that treatment intensification is of benefit to patients defined as high risk by MRD measured at day 29 of induction. The excess toxicity related to asparaginase and intravenous methotrexate did not result in an increase in treatment related mortality. Taken together with the results of the low risk randomisation, reported at ASH in 2010, UKALL 2003 has demonstrated that within a NCI directed CCG backbone, MRD based stratification provides the optimal approach to risk directed therapy. Disclosures: No relevant conflicts of interest to declare.

High hyperdiploidy (HeH) in acute lymphoblastic leukaemia (ALL) is defined by the non-random acquisition of multiple chromosomes, resulting in a modal number of 51-65 chromosomes. HeH is the most common primary abnormality in paediatric ALL and is associated with an excellent outcome. However, relapses do occur and prevalence of the subgroup means that they account for a sizeable proportion of relapsed cases. Hence identifying risk factors within this subtype remains important. Numerous studies have identified specific trisomies, modal numbers and structural rearrangements as prognostic factors. Despite these efforts, a consensus of the key risk factors has failed to emerge. We present a comprehensive analysis of 1181 children with HeH ALL, comprising discovery and validation cohorts: ALL97/99 (n=456) and UKALL2003 (n=725). The event-free survival (EFS), relapse risk (RR) and overall survival (OS) of HeH patients were: 84%/13%/93% and 91%/6%/95%, respectively. Initially, we examined previously reported risk factors in both cohorts separately. We did not observe any impact on outcome when patients were sub-divided using our copy number alteration (CNA) risk profile, which is based on the deletion status of the 8 most often affected genes. Similarly, there was no relationship between modal number and outcome whether we examined modal number as a continuous variable or in categories (51-53 v 54-57 v 58-65). Although the EFS hazard ratios (HZR) for patients with a double (+4, +10) or triple trisomy (+4, +10, +17) were HeH karyotypes harbour 5-19 additional chromosomes and although some chromosomes are more likely to be gained than others, there are thousands of combinations. In order to assess the optimal number of chromosomes required for predicting outcome, we used the discovery cohort to calculate the area under the curve (AUC) associated with each individual trisomy as well as all possible combinations of 2-6 chromosomes. This analysis revealed that the average AUC (an estimate for predictive power) increased with the number of chromosomes, but only up to 4, suggesting that there was no advantage in considering 5 or more chromosomes. Next, we derived EFS HZR from univariate Cox regression models for each chromosome and ranked them in order of p value. The HZR for the top 4 chromosomes were +18 (0.43, p0.01%) (56% v 47%, p=0.04) and IKZF1 deletions (12% v 2%, p=0.002). Factoring in EOI MRD, revealed that all UKALL2003 HeH patients had an excellent outcome except those with EOI MRD plus a poor risk HeH profile (table). This subset represented 33% of HeH patients but captured 71% (15/21) of the relapses recorded among UKALL2003 patients. In conclusion, we demonstrate that with the exception of EOI MRD the most important risk factor in HeH is the pattern of chromosomal gain. This result is in keeping with recent genomic studies, which have failed to identify a common underlying mutation, suggesting that the key driving event is disordered gene expression caused by the pattern of chromosomal gain. It is reassuring that the trisomies identified in this study have all previously been proposed as risk factors, providing a framework for further investigations aimed at elucidating precisely which genes are determining treatment response in HeH. Table.GroupHeH good riskHeH poor riskALL97/99Number of patients188268EFS (95% CI)91% (86-95)79% (74-84%)Hazard ratio (95% CI), p value12.81 (1.64-4.80%), p0.01%) Number of patients*95162EFS (95% CI)95%(88-98)83%(75-88)Hazard ratio (95% CI), p value13.55 (1.23-10.29), p=0.02EOI MRD ( Disclosures No relevant conflicts of interest to declare.

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.

The survival rate for infants is less than 50%. The fast majority of infant acute leukemias are characterized cytogenetically by balanced chromosomal translocations involving the mixed lineage leukemia (MLL) gene. Leukemic therapies that degrade the driver oncogene are associated with loss of cancer cell self-renewal and excellent cure rates. Therefore, therapy that degrades the MLL fusion gene would offer new hope to these patients. Recently it was shown that the Drosophila Trithorax gene, an analogue of the human MLL, is degraded by Radicicol. Radicicol is a natural compound and a well-known Hsp90 inhibitor. Here, we show that Radicicol is able to induce a dose dependent degradation of the MLL-fusion protein in a panel of human MLL rearranged cell lines and in human cord blood-derived MLL-AF9 immortalised myeloid cells. This drug induced degradation of the MLL-fusion gene results in down-regulation of the expression of MLL target genes, including HOXA9, MEIS1 and c-MYB. Functionally, this results in a loss of self-renewal of the leukemic stem cells, as shown by methylcellulose colony forming assays. Radicicol proved ineffective and too toxic for in vivo use. One of the best tolerated Hsp90 inhibitors is Ganetespib. It is currently in phase II/III clinical trials. We extended our Radicicol data to Ganetespib. Like Radicicol, Ganetespib induces MLL-fusion protein degradation and downregulation of MLL target genes. Treatment of MLL rearranged leukemia with Ganetespib results in a loss of leukemic stem cell activity. In conclusion, we show how the inactivation of the MLL-fusion and down-regulation of MLL target genes results in a block of leukemic stem cell self- renewal. We will validate these findings in a pre-clinical in vivo model in the near future. Disclosures No relevant conflicts of interest to declare.

Introduction Treatment outcomes for teenage and young adult (TYA) patients with Acute Lymphoblastic Leukaemia (ALL) lag behind those of younger patients when treated on equivalent protocols, with higher rates of toxicity and relapse reported for those aged >10 years. Outcomes of relapsed ALL are poor so more accurate identification of those destined to relapse is required. Whilst minimal residual disease (MRD) is a powerful tool to predict those at low risk and in whom treatment can be de-escalated, improved strategies are required for early identification of high risk patients. This study aimed to determine if the morphological early response assessment could be used as a predictor of relapse in TYA patients with ALL. Methods All patients treated on the national prospective UKALL2003 trial aged 10 - 24 years who had not relapsed by day 35 (i.e. could potentially have treatment escalated) were included in the study. Early response in patients aged >10 years was defined as percentage of blasts by morphology at day 8: rapid early responders had ≤ 25% blasts and slow early responders >25% blasts. We analysed the following patient characteristics which were previously associated with differential outcomes: age at diagnosis, presenting white cell count, rapid or slow early response (RER or SER); MRD status at day 28; immunophenotype (B/T/other) and cytogenetic risk group (high risk, good risk, poor risk, T-ALL, other). Cox regression was used to assess the association of these factors with time to relapse and logistic regression (LR) was used to assess their association with relapse by the end of treatment (EOT: 2.5 years for girls and 3.5 years for boys). Using backwards selection (cut off for inclusion: p=0.1) a multivariable LR model was established and a receiver operating characteristic curve (ROC) was used to assess how well it could predict relapse before the EOT. Results 820 consecutive patients were included in the analysis of whom 597 were aged 10 - 15 years and 223 were aged 16 - 24 years. 332 patients were classified as MRD high risk and 184 as SER on the basis of morphology. RER was significantly associated with a reduced risk of relapse even after adjustment for other factors including MRD, age, WBC and risk status (Hazard Ratio (HR) 0.64 (95% CI 0.42 - 0.97, p 697 patients with an outcome available at the EOT could be included in the LR analysis. The final multivariable model included day 28 MRD, day 8 response, age, WBC and cytogenetic risk group. The predictor generated by this model gives a ROC area under the curve of 81.2% (95% CI: 76.8 - 85.6) but there were no cut points which resulted in acceptable detection (DR) and false positive rates (FPR). As the intervention for the poor prognosis group would potentially include an allogeneic transplant with associated high mortality and morbidity a low FPR would be key to clinical utility. In order to achieve a clinically useful DR of 79.5% we would have to accept a false positive rate of 30.5%. As only 11% pf patients relapsed by EOT but 36% of patients fell into our bad prognosis group this would mean that for every 100 patients, ~27 would be transplanted unnecessarily. In the subgroup analysis of those aged 16 - 24 early response was not significantly associated with either time to relapse (HR RER: 0.74 (0.36 - 1.51, p = 0.4) or relapse by the EOT (Odds ratio RER: 0.63 (0.26 - 1.49, p =0.3) Conclusions Slow early response is significantly associated with both time to relapse and relapse by EOT in this patient cohort aged 10 - 24 years and this effect is independent of other prognostic factors including MRD. However, even when over-fit the prognostic model generated (which included SER) resulted in FPRs which were too high to be clinically useful. Future work should focus on novel alternative methods of relapse risk prediction for the adolescent patient with ALL. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: As the majority of children and young people now survive acute lymphoblastic leukaemia (ALL), the challenge has shifted to minimising treatment related toxicity. Osteonecrosis is one of the most serious complications of treatment, and can result in significant long term morbidity. In UKALL2003, which recruited patients between Oct 1, 2003 and June 30, 2011, all patients received dexamethasone 6mg/m2 for 29 days during induction and again for 5 days each month during maintenance in combination with Vincristine. Standard and intermediate risk patients were randomised to 1 or 2 delayed intensifications, each of which contained dexamethasone 10mg/m2 for 14 days in an alternate week schedule. Aim: To report the prevalence, management and long term outcomes of patients who developed osteonecrosis among 3126 patients aged 1-24 years old entered into UKALL2003. Methods: Patients with reported bone toxicity were identified by the central trials unit, which also provided information regarding age, sex, ethnicity and treatment. A questionnaire for each patient was sent to the relevant primary treatment centres (n=40) requesting information on patient demographics, diagnosis, management and outcomes of patients with osteonecrosis. Each primary treatment centre was also asked for details of previously unreported patients known to have osteonecrosis. Statistical analysis was undertaken using Chi-squared tests to identify significant differences in the prevalence of osteonecrosis according to age, sex, ethnicity, and treatment. Percentages were calculated as simple percentages. Results: We received an 83% response rate from primary treatment centres. 153 patients (5%) were reported as having developed osteonecrosis at some point after the start of treatment for ALL (including relapse therapy or stem cell transplant if required (n=5)), of which 38 were previously unreported to the clinical trials unit. Age demonstrated the strongest association with risk of osteonecrosis (p=0.01). Of patients aged Median time between onset of symptoms and diagnosis of osteonecrosis was 1 month, with 83% of patients diagnosed with MRI. The median time between diagnosis of ALL and onset of osteonecrosis symptoms was 14 months (range: minus 3 to 73 months). There were 438 areas of reported osteonecrosis in the 153 patients, with hips (157), knees (131), shoulders (61) and ankles (42) most commonly reported. Fractures were reported in 34 (22%) of patients who developed osteonecrosis. In the 153 patients with osteonecrosis, steroids were reported to have been discontinued in 93 (61%), bisphosphonates were given to 35 (23%), 10 of whom had also had fractures, 52 were given vitamin D (34%) and 64 (42%) required surgery. Hip replacements were required in 30 patients and of these, 29 were age >10 years at diagnosis of ALL (with a total of 839 patients aged >10 years in UKALL2003). There were 130 patients aged>18 years at diagnosis in UKALL2003, and of these, 4 required a hip replacement. With a median follow up of 70 months (range 27-124 months), 57 patients were reported to have no long term effects, 59 patients had minimal disability (able to carry out activities of daily living), 16 had significant disability, and 5 patients required a wheelchair at the time of response. 9 patients died from disease relapse, progression of ALL, sepsis or acute graft versus host disease. Outcome information was not available for a further 9 patients. Conclusion: This work highlights the impact of osteonecrosis as a cause of considerable morbidity for patients treated for ALL, with important implications for quality of life. Further, this work confirms that the greatest impact is on those aged over 10 years at diagnosis, with 1 in 30 patients in this group requiring hip replacement. Better strategies to monitor for and manage this condition are required to reduce the incidence, whilst maintaining overall and event free survival. Disclosures No relevant conflicts of interest to declare.

Background: Overall survival (OS) for children with ALL treated on contemporary protocols is now > 90%. To further improve outcomes, stratification must direct intensive treatment to those patients with the highest risk of relapse. Whilst minimal residual disease (MRD) has emerged as the most powerful predictor of outcome, its use in isolation is insufficient to identify those patients with the greatest risk. To address this, we performed a detailed analysis of the entire patient cohort from the recently reported large multi-center randomized controlled trial, UKALL 2003. Integration of end of induction (EOI) MRD with other predictive factors - cytogenetics, age and white cell count (WCC) - allowed the identification of very high risk groups that are likely to benefit from early treatment intensification or novel therapies. Methods: The 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 EOI MRD measured using EuroMRD approved real-time quantitative PCR for immunoglobulin and T-cell receptor gene rearrangements. Analysis was performed on 2666 patients with available MRD results and focused on the systematic integration of MRD results with other patient characteristics to identify subgroups with a very poor OS ( Results: Both MRD and cytogenetic risk group were independently predictive of survival. In particular, patients with high risk (HR) genetic abnormalities - low hypodiploidy, near haploidy, KMT2A (MLL) rearranged, iAMP21 and t(17;19)(q23;p13) (n=100) had a poor outcome with 5 yr OS of 76.0%. Combining these factors showed that MRD could stratify the HR cytogenetic subgroup with a significant difference in survival between those with low risk MRD and positive MRD (5yr OS 93.0 vs. 63.1%, p=0.002, n=43 vs. 57). Crucially, this demonstrates that HR cytogenetics are only truly poor risk in the context of poor treatment response. Importantly, there were almost no survivors following relapse in the HR cytogenetic/MRD positive group, whilst 50% of those with low risk MRD could be salvaged after relapse. In addition, whilst t(1;19)(q23;p13)was not considered a HR cytogenetic factor in this trial, combination with positive EOI MRD identified an additional subgroup of 20 patients with a 5yr OS of only 74.7% (vs. 96.7% in MRD low risk patients, p=0.003). Whilst WCC at presentation was also predictive of outcome, this difference was significant only in those with B-ALL and not T-ALL. In B-ALL, WCC>100 x109/L was associated with significantly poorer survival (5yr OS 95.1 vs. 84.0%, p200 x109/L (5yr OS 90.2 vs. 86.7%, p=0.283). Integration of MRD results allowed the identification of a very high risk group with B-ALL, WCC>100 and positive EOI MRD (n=80) which had a 5yr OS of only 71.3%. Further breakdown by cytogenetic risk group showed those patients with good risk cytogenetics (ETV6-RUNX1, high hyperdiploidy) (n=19) had a remarkably good survival despite WCC>100 (5yr OS 94%). In contrast, those with intermediate (n=34) or HR cytogenetics (n=15) had a very poor 5yr OS (65.7% and 60.0% respectively). Older age (≥10yr) was also associated with poorer outcomes compared to those Conclusions: Through the integration of EOI MRD with other risk factors in a large pediatric ALL cohort we have identified a population of patients with a very poor OS. When combined, these criteria select only 6% of all patients, but capture 35% of all deaths suggesting current treatment is suboptimal in this group. This information is essential for the planning of future trials to ensure that treatment intensification and novel therapies are targeted at those patients at greatest risk, whilst avoiding unnecessary treatment in those patients that already experience good outcomes. Disclosures No relevant conflicts of interest to declare.

The superior outcomes of teenage and young adult (TYA) patients with acute lymphoblastic leukemia (ALL) treated on contemporary pediatric minimal residual disease (MRD) directed chemotherapy regimens compared to adult ALL protocols are well established. However, there are limited data on the outcome of relapse following such regimens and consequently no consensus on optimal salvage strategies. UKALL 2003 was a prospective, randomised controlled trial investigating treatment modification according to MRD status at the end of induction in consecutively diagnosed children and young people with ALL recruited from the UK and Republic of Ireland between October 2003 and June 2011. The trial was initially open to children up to the age of 18 years but the upper age limit was increased to 20th birthday in 2006 and 25th birthday in 2007. Here we report the relapse outcomes of 16 - 24 year old TYA patients recruited to the trial. All TYA patients recruited to UKALL 2003 who subsequently relapsed were identified using the trial database, which also provided data regarding prognostic factors at presentation, MRD risk group, treatment allocation, site of and time to relapse and overall outcome. Individual centers were then contacted for follow up information including salvage chemotherapy, disease response, use of transplantation, subsequent outcome and cause of death. Of a total of 229 TYA patients recruited, 42 (18.3%) relapsed. The immunophenotype in those who subsequently relapsed was precursor B cell (B-ALL) in 30 patients and T cell (T-ALL) in 12. The median white cell count at diagnosis was 22x109/l and 1 patient had central nervous system (CNS) involvement. In B-ALL, 4 had good risk cytogenetics (all high hyperdiploid), 13 had intermediate risk, 6 had high risk (3 with MLL rearrangements), and 7 had unknown cytogenetics. End of induction MRD risk group was low risk in 2, intermediate risk in 11 and high risk in 29. The median time from diagnosis to relapse was 17 months (17.5 months for B-ALL and 14 months for T-ALL, p=0.1). The site of relapse was isolated bone marrow (BM) in 30 patients, combined BM and CNS in 4, combined BM and testicular in 1, and isolated CNS in 6 (T-ALL; 8 BM, 3 BM+CNS, 1 isolated CNS) (B-ALL; 23 BM, 1 BM+CNS, 1 BM + testicular, 5 isolated CNS). 36 of the 42 relapsed patients were given salvage chemotherapy, using a variety of regimens including R3 (n=9), Fludarabine/Cytarabine/Idarubicin based (n=16), and Clofarabine based (n=4). One patient received Blinatumamab and one entered the MARALL study (a Phase I/II study of the combination of Veltuzumab (anti-CD20) and Epratuzumab (anti-CD22) with intensive chemotherapy in patients with relapsed B-cell ALL). A second complete remission rate was achieved in 22 patients (61%) and a further 2 had an empty marrow without count recovery. 23 patients proceeded to an allogeneic hemopoietic stem cell transplant. The median overall survival (OS) from relapse was 213 days (actuarial 5 year OS of 18%). 9 patients remain alive; 8 in remission (1 unconfirmed) and 1 undergoing active treatment. The cause of death in the 33 patients who died was relapsed/refractory disease in 20, complications of re-induction treatment in 4, transplant related complications in 6 and unknown in 2. 1 further patient died of sepsis post transplant in the context of pancytopenia and suspected (though unproven) relapse. Those relapsing earlier than the median of 17 months had a shorter median survival and 5 year OS from relapse compared to those relapsing at >17 months (median 113.5 v 400 days, and OS 10% v 37%, p=0.0075). When this analysis was restricted to only the B-ALL cohort the effect was even more marked (median 151 v 595 days, OS 0% v 45%). No patient with B-ALL relapsing ≤24 months was salvageable. There were no differences in salvage approach between the early and late relapsing cohorts, nor differences in MRD risk. The late relapsing cohort did have more patients with good risk cytogenetics, and fewer with high risk cytogenetics. TYA patients with ALL who relapse despite intensive, contemporary MRD directed chemotherapy have an extremely poor outcome. Patients with B-ALL relapsing at 24 months from diagnosis or earlier are unsalvageable. Improving the outcomes of TYA ALL patients will depend on a) more accurate early identification of those destined to relapse and b) use of innovative therapies in those at high risk of relapse or with overt relapse. Disclosures No relevant conflicts of interest to declare.

Background: The optimal approach for transplanting patients with primary immunodeficiency from a mismatched unrelated donor (MMUD) remains unclear. With reduced intensity conditioning, when bone marrow (BM) was used as the stem cell source in such patients we have previously observed a high rate of very low level donor chimerism (4 of 12 patients) requiring a second transplant procedure. The use of peripheral blood stem cells (PBSC) can overcome this but results in a high incidence of acute (≥ Gde II 10/21, Gde III-IV 5/21) and chronic (10/21, extensive 7/21) graft-versus-host disease (GVHD) (Rao et al in press). We hypothesized that the use of megadose CD34 selected stem cells from PB with addback of a T-cell dose akin to BM might facilitate engraftment through improved competition for the stem cell niche without severe GVHD. Methods: We prospectively analysed outcomes on 20 consecutive patients with primary immunodeficiency (SCID n=5, HLH n=3, Other PID n=12) transplanted from 8/10 (n=4) or 9/10 (n=16) HLA MMUD at our institution between 2011-2015. The mean age at transplant was 5.1 years. All patients received reduced toxicity conditioning regimens (12/20 Fludarabine/Melphalan/Alemtuzumab; 8/20 Fludarabine/Treosulphan/Alemtuzumab) with Cyclosporin (CsA) and mycophenolate mofetil (MMF) GvHD prophylaxis. CD34 selection of donor PBSC was performed using CliniMACs and the mean CD34+ dose was 20.1x106/kg. At day 0 either 108 CD3/kg (cohort 1, n=6) or 3x108 CD3/kg (cohort 2, n=14) T-cells from the CD34- fraction were infused with the graft. Mean follow up was 31.9 months. Results: In cohort 1, all patients engrafted and 2 developed high level donor mixed chimerism (both curative) in the myeloid and lymphoid lineages at last follow up. Two patients had Grade II acute GvHD and 1 had moderate chronic GvHD. In view of the slow immune reconstitution in this cohort, the T-cell addback dose was increased to 3x108/Kg in subsequent patients. In cohort 2, all patients achieved full donor haemopoiesis initially, 6/14 developed high levels of donor chimerism in both myeloid and lymphoid lineages later and 1/14 progressed with 10% donor T-cell engraftment but remains disease-free. The incidence of significant aGVHD was low (grade II n=4, no grade III or IV) and no patient developed cGvHD. Overall across both cohorts, 10 patients had viral reactivations and there were 5 deaths (3 viral complications, 1 pulmonary vasculopathy, 1 cGVHD lungs). The disease free survival at 2 years 7 months follow up was 70% which compares favorably with a previous cohort transplanted with unmanipulated BM as the stem cell source (Fig 1). Immune reconstitution was delayed (mean CD3 and CD19 counts at day 100 post-transplant was 245 x 109/L and 315 x 109/L) with similar kinetics in both cohorts (Fig 2), comparable to RIC transplant using unmanipulated BM. By 1 year post transplant 11/14 evaluable patients achieved normal CD3+T-cell numbers and 8/14 normal CD19+ B-cell counts. Conclusion: The use of megadose peripheral blood stem cells with T-cell addback in the mismatched unrelated donor transplant setting results in high rates of curative engraftment and a low incidence of acute and chronic GvHD following reduced toxicity conditioning. However, T-cell reconstitution remains delayed (presumably reflecting the effect of Alemtuzumab on the infused T-cells) with a high incidence of viral complications. Disclosures No relevant conflicts of interest to declare.

Chimeric antigen receptor (CAR)19 T-cells exhibit powerful anti-leukemic effects in patients with B cell malignancies. However, the complexity of production of patient bespoke T cell products is a major barrier to the broader application of this approach. We are investigating a novel strategy to enable "off-the-shelf"' therapy with mismatched donor CAR19 T cells. Transcription activator-like effector nucleases (TALEN)s can be used to overcome HLA barriers by eliminating the risk of graft-versus-host disease (GvHD) through disruption of T cell receptor expression, and by simultaneously targeting CD52, cells can be rendered insensitive to the lymphodepleting agent Alemtuzumab. Administration of Alemtuzumab can then be exploited to prevent host-mediated rejection of HLA mismatched CAR19 T cells. We manufactured a bank of such cells from volunteer donor T cells under GMP conditions on behalf of Cellectis S.A for final stage validation studies using a third generation self inactivating lentiviral vector encoding a 4g7 CAR19 (CD19 scFv- 41BB- CD3ζ) linked to RQR8, an abbreviated sort/suicide gene encoding both CD34 and CD20 epitopes. Cells were then electroporated with two pairs of TALEN mRNA for multiplex targeting of both the T cell receptor alpha constant chain locus, and the CD52 gene locus. Following ex-vivo expansion, cells still expressing TCR were depleted using CliniMacs alpha/beta TCR depletion, yielding a T cell product with An 11 month girl with high risk CD19+infant ALL (t(11;19) rearrangement) relapsed in bone marrow 3 months after a myeloablative 8/10 mismatched unrelated donor transplant. Leukaemic blasts expressed CD19 but were CD52negative. Her disease progressed despite treatment with Blinatumomab (70% blasts in marrow) and we were unable to generate donor-derived CAR19 T cells on an existing study. Following institutional ethics review, detailed counseling, and parental consent, the patient received cytoreduction with Vincristine, Dexamethasone and Asparaginase followed by lymphodepleting conditioning with Fludarabine 90mg/m2, Cyclophosphamide 1.5g/m2 and Alemtuzumab 1mg/kg. Immediately prior to infusion of UCART19 cells, the bone marrow showed persisting disease (0.5% FISH positive). She received a single dose (4.5x106/kg) of UCART19 T cells without any significant toxicity. To date there has been no significant perturbation of cytokine levels in peripheral blood, and no indication of cytokine release syndrome. Although profoundly lymphopenic, UCART19 T cells were detectable by qPCR in the circulation by day 14 and at increased levels in both blood (VCN 0.35) and marrow (VCN 0.22) on day 28. The patient exhibited signs of count recovery and the bone marrow, while hypoplastic, was in cytogenetic and molecular remission. Chimerism was 90% donor, and a clearly demarcated population (7%) of third party cells indicated persistence of UCART19. A residual persistence of 3% recipient cells in the marrow suggests that leukemic clearance was not mediated by transplant mediated alloreactivity. Within the short period of follow up available, our intervention comprising lymphodepletion and infusion of UCART19 T cells has induced molecular remission where all other treatments had failed. This first-in-man application of TALEN engineered cells provides early proof of concept evidence for a ready-made T cell strategy that will now be tested in early phase clinical trials. Disclosures Qasim: CATAPULT: Research Funding; CELLMEDICA: Research Funding; CALIMMUNE: Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding; CELLECTIS: Research Funding. Off Label Use: UCART19 T Cells are an unlicensed investigational medicinal product and in this case were used under MHRA special licence arrangements. Stafford:CELLECTIS: Research Funding. Peggs:Cellectis: Research Funding; Autolus: Consultancy, Equity Ownership. Thrasher:CATAPULT: Patents & Royalties, Research Funding; MILTENYI: Research Funding; AUTOLUS: Consultancy, Equity Ownership, Research Funding. Pule:AUTOLUS: Employment, Equity Ownership, Research Funding; CELLECTIS: Research Funding; AMGEN: Honoraria; UCLB: Patents & Royalties.

The single most frequent chromosomal translocation associated with childhood ALL is the t(12;21) rearrangement that creates a fusion gene between TEL (ETV6) and AML1 (RUNX1). Although TEL-AML1+ patients have very good prognoses, relapses occur in up to 20% of patients and many patients face long-term side effects of chemotherapy. Recent data has shown that TEL-AML1 has a direct role in inducing signal transducer and activator of transcription 3 (STAT3) activation in human t(12;21) leukemia. This activation has been shown to transcriptionally induce MYC and is critical for survival of TEL-AML+ leukemia cells. Here, we demonstrate that STAT3 also regulates SMAD7 gene expression. SMAD7 is an antagonist of TGF-β signaling, functioning through a negative feedback mechanism, but is also known to function in other biological pathways. Interestingly, SMAD7 has also been shown to play a role in promoting self-renewal of hematopoietic stem cells. We show that both pharmacological and mechanistic inhibition of STAT3 results in down regulation of SMAD7 gene expression in TEL-AML1+ cell lines. This result was specific to TEL-AML1+ cells and not found in cells of other ALL subtypes. To understand the role played by SMAD7 in TEL-AML1+ cells, we used lentiviral vectors expressing shRNA targeting SMAD7. Interestingly, SMAD7 silencing was found to inhibit proliferation of TEL-AML1+ cell lines, eventually leading to growth arrest and apoptosis. Furthermore, we have established that this effect is not mediated through TGF-β signalling. This poster highlights the results of RNA-seq performed on TEL-AML1+ cells with SMAD7 knockdown and in vivo xenograft model of SMAD7 shRNA in TEL-AML+ ALL. Disclosures No relevant conflicts of interest to declare.

Major progress has been made in the treatment of Acute Myeloid Leukaemia (AML). However, drugs that have been developed to treat leukaemia still fail in about half the patients. New treatments are urgently needed. In the last couple of years we have been working to understand the exact molecular changes in the blood forming cells that cause leukaemia. We, and others, have identified key proteins that these cells need to survive. These are known as the driving oncogenes. Here we describe a pipeline, implemented by us, to inactivate key driving transcription factors through drug repositioning. In this study we focus on the oncogene c-MYB, a transcription factor that has a critical role in AML. To achieve the goal of discovering drugs that pharmacologically target the driving transcription factors for inactivation we set up a technique for integrated analysis of gene expression datasets and transcription factor binding to DNA and combined this with pattern-matching software to mine the connectivity map database. Briefly, inducible expression/knockdown systems and next generation profiling allowed us to integrate cellular output (RNA-Seq or Genechip) and transcription factor binding (ChIP-Seq or ChIPip-on-Chip) of the driving c-MYB oncoprotein in leukaemia. This profile was used to mine the connectivity database for perturbagens of this profile. The top hit, of this integrated network-based analysis of transcription factor behaviour and perturbing agents, was taken forward for analysis. It was found to cause an acute reduction in the protein levels of MYB, preceding changes in the level of its mRNA. This reduction in c-MYB proteins level could be blocked with proteasome inhibitor MG132. Meaning we induced the proteolysis of the c-MYB oncogene. The reduction in c-MYB levels was accompanied by a significant anti-AML activity in an in vitro colony forming assay of both AML cell lines and primary patient samples. Interestingly, the drug had no significant effect on colony formation of CD34 positive cord blood cells. To investigate the specificity we tried to rescue the function of MYB. Through overexpressing a "stabilized" MYB mutant, lacking the negative regulatory domain we could, in large part, rescue the block in leukemic self-renewal. Meaning, its main effect on the block in AML stem cells is through targeting c-MYB oncogene. It is vital to know if this drug would be active in patients and we therefore required a model that reflected, as closely as possible, the disease progression in vivo. In order to achieve this, we established a robust xenograft model with human AML cell lines. Next, we genetically modified these cells with a luciferase-expressing lentiviral vector, so that they could be detected within the xenograft host using the IVIS III pre-clinical imaging system. Using this approach, we have shown that oral administration, even when used alone as a monotherapy, causes a strong block in the leukaemia progression (over a 300 fold difference in leukemic burden at day 17 of treatment vs control), resulting in prolonged survival of xenograft hosts. Here we describe a pipeline to discover drugs that inactivate key transcription factors important in AML. By combining our knowledge of transcription factor behaviour and drug repositioning, we discovered candidate drugs that inactivate key oncogenes. It's important to note that repositioning candidates have been through several stages of clinical development and therefore have well-known safety and pharmacokinetic profiles. Therefore, we hope that these candidates can be taken forward rapidly into clinical trials to improve the patient outcome in AML. Disclosures No relevant conflicts of interest to declare.

At acute lymphoblastic leukemia (ALL) relapse, about 40% of children can be salvaged with intensified multi-agent, high dose chemotherapy and in very high risk patients, with additional stem cell transplantation (SCT). To improve on this, the International BFM Study Group has led a consortium of 19 countries to develop the world's largest trial for relapsed ALL (IntReALL). Standard risk patients will be randomized to receive the ALL-REZ BFM 2002 or UK ALL R3 therapy and post induction will be randomised to receive the additional targeted anti-CD22 drug, Epratuzumab, during consolidation, to clear residual disease. Children with end of re-induction MRD positive bone marrow will undergo SCT following consolidation. Both ALL-REZ BFM 2002 and UK ALL R3 used MRD PCR-based quantification of clonal Ig/TCR rearrangements, with different cut offs (10-3 for ALL-REZ BFM 2002 and 10-4 for UK ALL R3) and this is the reference assay for IntReALL. However, flow MRD may also play a role for patients without PCR targets. Flow MRD relies on the discrimination of leukaemic blasts from hematogones and this can be hampered depending on the degree of haematopoietic regeneration which varies depending on the treatment protocol and is especially important after intensive induction treatment in relapsed protocols. Thus, prospective MRD quantification of patients entered onto the UKALLR3 and ALL-REZ BFM 2002 clinical trials was performed by a standardised, quality assured, 4-8colour Flow MRD assay in end of re-induction bone marrow aspirates, by laboratories in the IBFM FLOW consortium (n=221). Flow MRD in both treatment protocols was classed as a prospective biological add on study and not used for clinical decision making. Median MRD levels were 0.026 +/-9.9% SD for BFM versus 0.027+/-18% SD for UK protocols, with comparative MRD positivity rates of 45% versus 54%, respectively. Comparison with MRD levels as assessed by molecular analysis of antigen receptor gene rearrangements was performed in 170 samples (BFM,128; UK R3, 42). The Spearman rank correlation of all samples was 0.90 (p Although sample processing and quantification of MRD differ between PCR and FC MRD, in both re-induction protocols, there was good correlation of MRD levels assessed by flow cytometry and PCR, validating the use of Flow MRD as a method of choice in patients without PCR targets in the IntReALL trial. Flow MRD also has the advantage of enabling levels of CD22 to be assayed on MRD cells, prior to treatment with Epratuzumab. This research has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 278514 - IntReALL", Deutsche Kinderkrebsstiftung for its funding support of the ALL-REZ BFM 2002 clinical trial and the minimal residual disease studies by PCR and the Deutsche Jose Carreras Leukämiestiftung for support of the international principal investigator, Leukaemia and Lymphoma Research and North East Children's Cancer Research Fund, NT 13462-4, NV15-28525A, NV15-26588A, UNCE 204012. Disclosures No relevant conflicts of interest to declare.

Abstract 2573 Introduction: Polyethylene glycol conjugated L-asparaginase (PEG-ASNase) is used in varying doses between 1000–2500 U/m2 in the therapy of ALL and associated with a wide variation in pharmacokinetics. UKALL 2003 used intramuscular PEG-ASNase at 1000 U/m2. Patients & Methods: Patients enrolled in the UKALL 2003 trial (ISRCTN: 07355119), were consented for trough asparaginase activity analysis during therapy. National Cancer Institute (NCI) standard risk (SR) patients with rapid early response and non-high risk (HR) cytogenetics (Lancet Oncol. 2010;11:429) received a 3 drug induction (Dexamethasone, Vincristine & PEG-ASNase). All other patients received additional Daunorubicin. PEG-ASNase was given on days 4 and 18 of induction, and at least once post induction. Trough plasma asparaginase activity was measured by the indooxine method (Anal. Biochem. 2002;309:117). The lower limit of assay detection was 34 U/L. Adequate asparaginase activity was defined as a trough level of > 100 U/L. IgG and IgM antibodies to PEG-ASNase and native asparaginase were measured by indirect ELISA. Asparaginase activity was correlated with defined risk factors, minimal residual disease (MRD) at day 28 of induction and development of anti-asparaginase antibodies using the chi-squared test. Results: Between July 2008 to July 2011, 482 patients aged 1–25 years from 27 centres were recruited. Numbers of samples assayed in induction were 335 & 371 after first and second doses respectively. Overall, 86% (n=606/706) of samples had adequate activity during induction time points. There was > 10 fold variation in activity levels (Figure 1). Three hundred and nine out of 706 samples had activity > 3 times the therapeutic threshold, while 51/100 samples with inadequate activity had no detectable drug levels (median: below detection limit, range: < 34–99 U/L). Thus, increasing the dose of PEG-ASNase in induction is unlikely to benefit patients with inadequate activity. Compared to SR patients, NCI HR patients had a higher incidence of inadequate asparaginase activity in induction (p=0.002). Inadequate asparaginase activity correlated with high MRD (≥ 10−4) in SR patients (p=0.045), especially those with good risk cytogenetics (p=0.012), and in particular the high hyperdiploid subgroup (p=0.03). Inadequate asparaginase activity during induction did not correlate with MRD in HR patients (p=0.699), possibly because these patients received in addition Daunorubicin (Table 1). Results of serial asparaginase activity (at least one time point each in induction and post induction), measured in 282 patients are summarised in Table 2. Antibodies were detected in 18 of 81 patients tested. All had anti-PEG and 7 in addition also had anti-asparaginase antibodies. While all antibody positive patients had inadequate asparaginase activity at one time point, 17 had adequate activity prior to antibody detection, suggesting immune-mediated drug inactivation at re-exposure. Antibodies were not detected in 14/15 patients who had inadequate activity at first exposure, so the mechanism here remains unclear. The reported incidence of asparaginase toxicity in this study was 6.6% (n=32/482). This included hypersensitivity (n=17/482) that was almost exclusively seen in HR patients (n=16/17), thrombosis (n=10/482) and pancreatitis (n=5/482). Conclusions: Intramuscular PEG-ASNase given fortnightly at 1000 U/m2 during induction provides adequate asparaginase activity in 86% of patients. Monitoring asparaginase activity may benefit patients who receive 3 drug induction and improve the resolution of the current prognostic classification. Disclosures: Off Label Use: PEG-asparaginase. Essink:medac: Employment. Kuehnel:medac: Employment.

Treosulfan is an immuno-suppressive and myeloablative alkylating agent that has been introduced as a conditioning agent in stem cell transplantation (SCT). Most studies have been performed in adult patients with malignancy where a low incidence of regimen-related toxicity has been reported. We report the use of treosulfan in 32 consecutive children undergoing SCT for non-malignant disease. Patients received a total treosulfan dose of 36 or 42 g/m(2)/patient given in three daily, divided doses. A range of other conditioning agents and serotherapy was administered to patients who underwent family donor SCT (n = 11), or unrelated donor SCT (n = 21). One patient (3%) died early. Transplant morbidity was limited and mucositis was only mild. Dermatological toxicity was frequent but mild. Twenty-eight patients (87.5%) established donor cell engraftment. In 25 patients (78%) there was adequate, stable donor engraftment. Four patients have required additional transplant procedures to maintain adequate donor-derived haemopoiesis. Twenty-seven patients (84%) survive with a median follow up of 417 d. There were four late deaths due to progression of the underlying disease, graft-versus-host disease or infection. Treosulfan-based conditioning regimens achieve excellent engraftment with reduced regimen-related toxicity in children with non-malignant disease at high risk for both regimen-related toxicity and graft failure.