High-risk Minimal Residual Disease Research Articles

High Dose Methotrexate Does Not Reduce the Risk of CNS Relapse in Children and Young Adults with Acute Lymphoblastic Leukaemia and Lymphoblastic Lymphoma. Results of the Randomised Phase III Study UKALL 2011

Introduction UKALL 2011 randomised children and young adults (up to 25) with Acute Lymphoblastic Leukaemia (ALL) or Lymphoblastic Lymphoma (LBL). The aims were to reduce induction toxicity (R1: Short [14 days] vs Standard [28 days] dexamethasone), CNS relapse risk (R2IM: HD MTX (HDM) vs standard interim maintenance (SIM)) and maintenance morbidity (R2pulses: Vincristine (VCR)/dexamethasone pulses or not). R1 results were presented at ASH 2017 and did not show a reduction in toxicity with short dexamethasone. Results of R2IM and R2pulses are reported here. Patients and Methods Patients were stratified by NCI risk, cytogenetics and end of induction MRD to receive Regimens A (NCI standard risk, MRD low risk), B (NCI high risk, MRD low risk) or C (Cytogenetic poor risk or MRD intermediate risk regardless of NCI risk). MRD high risk (end of consolidation MRD >0.5%, n=14) were not eligible for randomisation and received off protocol therapy. R2 was a factorial randomisation stratified by factors including NCI and MRD risk groups and resulted in 4 arms: HDM with pulses, HDM without pulses, standard interim maintenance (SIM) with pulses (standard of care) and SIM without pulses. SIM was for 2 months with oral mercaptopurine/MTX, monthly pulses and single IT MTX in Regimens A and B, and 5 doses of escalating IV MTX (Capizzi) + VCR + 2 doses of Pegylated asparaginase in Regimen C. HDM was given at a dose of 5g/m2 x 4 doses 2 weeks apart, low dose 6-MP and 2 doses of Pegylated asparaginase (Regimen C only). The primary endpoint for R2IM was the CNS relapse rate (CNSR) and for R2pulses was the bone marrow relapse rate (BMR) in ALL only; non-inferiority, 5% margin at 5 years. Event Free Survival (EFS) was considered a primary endpoint for both. Results Of the 2750 patients registered on trial between April 2012 and December 2018, 1902 were randomised to R1 (closed April 2017) and 1570 to R2. Median age 5 years (IQR: 3-11), 83% B-ALL, 12% T-ALL, 1% B-LBL and 4% T-ABL, 43% NCI high risk. Median follow-up for R1 is 76 months and R1 or R2 is 72 months. Overall, we found no difference in CNSR (HR: 0.99 (0.65-1.51), p=0.97, 5-year rates: 5.6% and 5.6%), or any other endpoints for R2IM. However, there was an interaction between the R1 and R2IM randomisations (p=0.006 for EFS) with inferior outcomes for patients treated with short dexamethasone followed by HDM, particularly in those who did not receive pulses (Figure 1). Limiting analyses to patients treated with standard dexamethasone (N=995, including those that received it after closure of R1) there was a significant reduction in BMR rate with HDM (HR 0.62 (95% CI: 0.40 - 0.95) p = 0.029) and a trend for improvements in EFS and OS (HRs: 0.75 (95%CI: 0.53 - 1.04), p=0.087 and 0.61 (0.37 - 1.03), p=0.067) but no difference in CNSR. In subgroup analyses there were no significant interactions, but the effect appeared stronger for B-lineage, NCI high risk and MRD low risk patients (Figure 2). Overall, the BMR for R2pulses was non-inferior (+2.1% increase BMR at 5 years for ALL patients (95% CI: -1.4% to 4.7%), HR 1.22 (95%CI: 0.89 - 1.67). Considering all four R2 treatment arms in standard dexamethasone only, standard IM without pulses has a lower EFS, whilst there is no appreciable benefit of pulses in HDM treated patients; 5-year EFS difference: -2.8% (95% CI: -7.4% to 4.4%) and BMR (ALL): +0.4% (-6.7% to 4.3%). Conclusions HDM does not improve CNSR within a UKALL treatment backbone, but pre-specified analyses suggest it may improve BMR for some sub-groups of B-lineage patients when given following standard dexamethasone induction. Although the 'no pulses' arm was non-inferior overall for BMR, further sub-group and toxicity analyses are to be performed and will presented at the meeting. Acknowledgements Children with Cancer, Blood Cancer (grant ref 09042) and Cancer Research UK funded the trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ten Year Outcomes of UKALL 2003: A Randomised Clinical Trial of Adjusting Treatment Intensity Based on Minimal Residual Disease

▪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. DisclosuresSamarasinghe: AMGEN,JAZZ: Honoraria.

Targeting JAK2 Gene Rearrangements with a Novel Kinase Inhibitor in a Preclinical Model of Pediatric Acute Lymphoblastic Leukemia

Background. Although the Event Free Survival for Childhood Acute lymphoblastic leukemia (ALL) reaches 85%, the remaining 15% of patients relapse, and 25-40% of them die. Novel molecular targets may increase the efficacy of therapy and reduce treatment toxicity. Among B-other ALL patients, JAK/STAT pathway alterations represent about 7% of the ‘Philadelphia-like’ cases. JAK2 gene encodes for a non-receptor tyrosine kinase fundamental for hematopoiesis, cellular proliferation and survival. In last years, JAK2 mutations have been widely studied in leukemia and lymphoma, whereas JAK2 fusion genes are still poorly characterized.Aim. This study aims to identify JAK2 fusion genes among BCP-ALL pediatric patients and develop a target strategy in in vitro and in vivo preclinical models.Methods. A targeted capture RNA Next Generation Sequencing strategy was applied to discover JAK2 fusion genes in a large cohort of PCR-based MRD high risk (HR) BCP-ALL pediatric patients. Fusions were validated by RT-PCR and/or FISH. Primary patients' cells have been in vivo expanded in NSG mice. We performed ex vivo and in vivo drug treatments with JAK2 inhibitors; phosphoflow and apoptosis-viability assays were performed in patients' blasts in co-culture with human bone marrow stroma.Results. We identified 10 pediatric cases carrying a JAK2 fusion gene with different partners in single cases, such as ATF7IP, ZEB2, MPRIP, BCR, TLE4, GIT2 and RAB7, in addition to PAX5, which was the only recurrent in three cases. Cells were available from 3 cases, carrying PAX5-JAK2, ATF7IP-JAK2 and ZEB2-JAK2, respectively. After in vivo expansion, we demonstrated that the JAK2 signaling pathway was active at basal level, through phosphorylation on Y1007-1008 JAK2 residues inside the catalytic activation loop, compared to cases wild type for JAK2 and CRLF2 (+70%, two-tailed P value 0.0355); a positive trend was also shown compared to primary cells with P2RY8-CRLF2 rearrangements and JAK2 mutation, as positive controls (+40% two-tailed P value 0.158). The JAK2 downstream effectors pS727-STAT3 and pY694-STAT5 were also activated.We thus setup a JAK2 targeted drug treatment using CHZ868, a new class-II tyrosine kinase inhibitor (TKI) (Novartis, Basel, CH). After 30 minutes of treatment, we appreciated a mean inhibition of -62% of Y1007-1008 JAK2 residues in PAX5-JAK2, -22% in ATF7IP-JAK2 and -35% in ZEB2- JAK2. Contemporarily, we observed a decrease of pS727-STAT3 (-35-50%) and pY694-STAT5 (-15-50%).After 48h monotherapy treatment by CHZ868, we detected apoptosis induction and cell viability decrease between 20- 75% at IC50. In combination with dexamethasone, we assessed a further decrease of viability between 10 to 95%. A biological variability among the three different patients was appreciated, according to the different partner genes.Exclusively for the PAX5-JAK2 fusion, we also performed treatments with the kinase inhibitor BIBF1120/Nintedanib, targeting LCK, which is activated downstream PAX5 fusions and we observed a 20% reduction of cell viability. Importantly, combination of BIBF1120 and CHZ868 showed a synergistic effect (-45%, at IC50). Moreover, we found that ruxolitinib caused autophagy as observed by higher levels of LC3-II compared to untreated cells (+ 45%, p<0.01), with consequent reduction of apoptosis induction. Indeed, active caspase 3 increased when ruxolitinib was given in combination with chloroquine, an autophagy inhibitor (+20% vs ruxolitinib alone, p<0.01). CHZ868 alone or in combination with chloroquine instead does not induce autophagy as LC3-II and active caspase 3 levels are the same of untreated cells.Finally, we demonstrated the in vivo efficacy of CHZ868 in patient derived xenograft model in presence of PAX5-JAK2 fusion. After two weeks of 30mg/Kg daily treatment of CHZ868, we observed a significant reduction of leukemic CD10+/CD19+ cells both in bone marrow (p<0.01, -43%), spleen (p<0.001, -72%), central nervous system (-40%) and peripheral blood (p<0.05, -46%), compared to vehicle mice. Further in vivo experiments are ongoing in other JAK2 fusion settings.Conclusion. CHZ868 is a promising candidate for treatment of BCP-ALL carrying JAK2 fusions, showing high efficacy and specificity, both ex vivo and in vivo. Further studies will include combination with standard chemotherapy drugs with the aim to maintain its efficacy by reducing the intensity and toxicity of chemotherapy. DisclosuresBiondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Colmmune: Honoraria; Amgen: Honoraria.

Genome-Wide Association Study (GWAS) of High-Risk Minimal Residual Disease and Relapse in B-Cell Acute Lymphoblastic Leukemia on Trial AIEOP-BFM ALL 2000

▪A significant proportion of patients with pediatric acute lymphoblastic leukemia (ALL) still suffers from relapse and therapy-related toxicities. Thus, the focus of further improvement of therapy for them is not only cure, but minimizing both short-term and long-term therapy-associated toxicity. This can be achieved by better personalized adjustment of therapy to the risk of relapse. Continuing research has identified numerous features with prognostic potential, including clinical and genetic variables as well as early response to chemotherapy being associated with treatment outcome. While most research activities on genetic prognostic factors have focused on somatic features, relatively little is known on hereditary contributions to treatment response and outcome in pediatric ALL. Therefore, we conducted a genome-wide association study (GWAS) of high-risk minimal residual disease (MRD) and relapse in B cell precursor (BCP) ALL patients treated on trial AIEOP-BFM ALL 2000.On this trial, patients were stratified into three risk groups, mainly by MRD analyses. MRD standard-risk (MRD-SR) patients were MRD-negative on treatment days 33 and 78, MRD high-risk (MRD-HR) patients had residual disease of ≥5x10-4 on treatment day 78. All the remaining MRD results were considered MRD intermediate-risk (MRD-IR). Besides MRD, established high-risk parameters were also retained for stratification: prednisone poor-response, induction failure, positivity for t(4;11)/ MLL-AF4 . Treatment used standard drugs and, in some of the patients, cranial irradiation and/or hematopoietic stem cell transplantation. DNA isolated from remission samples was genotyped using Illumina Human1M-Duo BeadChips containing 1.048.711 single SNV markers. For GWAS analyses gPLINK v2.050, PLINK v.1.09 and RStatistics v3.3.1 were used. The analysis on MRD was conducted in 69 MRD-HR patients (cases) and in 1034 patients of the MRD-SR and MRD-IR groups (controls). The analysis on relapse employed 145 relapsed (cases) and 1007 non-relapsed patients (controls). After quality control assessment, the final analyses included 749.262 SNPs among 62 MRD-HR patients compared to 927 controls and 129 relapsed patients compared to 925 controls.In the analysis on MRD-HR five SNVs reached genome-wide significance at P ≤5×10−8. An intronic SNV (rs1570765) in STARD13 demonstrated the strongest association (P =1.73x10-10; odds ratio, OR=5.06). Two additional SNVs with P ≤5×10−8 in STARD13 were in linkage disequilibrium with rs1570765. The two remaining SNVs with P ≤5×10−8 were located in FEZ2 and NRIP1. Five additional SNVs demonstrated significant levels of ≤1×10−6 and were associatedwith the following genes: STARD13 , VIT and FICD . STARD13 is located on chromosome 13, belongs to the family of RhoGap proteins and is frequently lost in hepatocellular and breast carcinomas. Its RhoGAP domain activates members of the Ras superfamily which leads to a suppression of cell growth and differentiation (Lin et al. Oncogene 2010). In the GWAS approach on relapse three SNVs reached significant levels of ≤1×10−6 and were associatedwith the genes ASB3 (rs1549749, P =3.51x10-7; rs12465316, P =4.26x10-7) on chromosome 2 and NDN (rs9972595, P =1.04x10-6) on chromosome 15. Twenty additional SNVs demonstrated significance levels of ≤1×10−5. ASB3 codes for a member of the ankyrin repeat and SOCS box-containing (ASB) family of proteins containing ankyrin repeat sequence and a SOCS box domain. The latter serves to couple suppressor of cytokine signalling (SOCS) proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. NDN , a p53 target gene, codes for Necdin which was previously shown to regulate the quiescence and response to genotoxic stress of hematopoietic stem/progenitor cells (Asai et al., Blood 2012). Further replication results, an analysis of additive effects and multivariate analyses will be presented.In conclusion, our results demonstrate that germline genetic variation is associated with high levels of MRD after induction/consolidation treatment for ALL and relapse on an AIEOP-BFM ALL protocol. In line with previous studies (Yang et al., JAMA 2009), our data suggest that genome-wide approaches may be useful to identify novel and clinically relevant SNVs for further refinement of risk-adapted treatment strategies in pediatric ALL. DisclosuresSchrappe:JAZZ Pharma: Consultancy, Research Funding; SigmaTau: Consultancy, Research Funding; Medac: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Consultancy, Research Funding.

UKALL 2011: Randomised Trial Investigating a Short Induction Dexamethasone Schedule for Children and Young Adults with Acute Lymphoblastic Leukaemia

▪Dexamethasone is associated with a higher risk of infectious and non-infectious morbidity and mortality than prednisolone in induction therapy of children and young adults with Acute Lymphoblastic Leukaemia (ALL). We tested whether a 2 week induction dexamethasone schedule is less toxic than the standard 4 week schedule. Patients aged 1 -24 years with a new diagnosis of ALL or lymphoblastic lymphoma (excluding mature B lymphoma and ALL) in the UK and Ireland were randomly allocated to receive dexamethasone 5 mg/m2twice daily for 14 days (continuous in age <10 years and discontinuous week on/week off in age > 10 years) (Short) or 3 mg/m2 twice daily for 29 days followed by a 7 day wean (Standard). The primary end-point was a reduction in steroid related toxicity defined as any induction treatment related death, all serious adverse events (SAEs) within 8 weeks of the start of induction, and adverse events (AEs) which were classified as steroid related or contributory.At diagnosis, patients were stratified by NCI (Standard Risk = age <10 and white cell count <50 x 109/l, High Risk = age > 10 years or white cell count >50x 109/l) and cytogenetic risk to receive one of 3 escalating intensity treatment regimens (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 poor risk cytogenetics were transferred to Regimen C. Minimal Residual Disease (MRD) was measured at the end of induction (day 29) and end of consolidation (week 14) using a Real Time Quantitative PCR technique with a sensitivity of 0.01%. Patients were classified as MRD low risk (day 29 <0.005%), intermediate risk (day 29 > 0.005% - 5%, week 14 <0.5%) or high risk (day 29 > 5% or week 14 >0.5%).Between April 2012 and April 2017, 1904 eligible patients were randomly allocated to short (951) or standard dexamethasone (953) arms which were balanced for NCI risk, sex, immunophenotype and disease type (ALL or LBL). Analysis was intention to treat. Recruitment was stopped early on the recommendation of the data monitoring committee (IDMC) due to concerns about a non-significant excess of induction treatment related deaths in NCI standard risk patients with the short schedule (Short =9/503 (1.8%), Standard =4/506 (0.8%) (p =16) which led to a futility analysis demonstrating that there was <10% power of showing a significant improvement with short dexamethasone. At a median follow-up of 22.8 months, there is no statistically significant difference in steroid related toxicity, MRD response or relapse free survival between arms (Table).A shorter induction schedule of dexamethasone 10 mg/m2/day x 2 weeks is no less toxic than 6 mg/m2/day x 4 weeks in children and young adults with ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Prognostic Significance of Minimal Residual Disease in Multiple Myeloma in the Molecular Genetic Groups Msmart 3.0

Assessment of the role of genetic abnormalities and minimal residual disease (MRD) is an active developing area in hematology. The use of genetic methods makes it possible to predict the course of the disease and apply an individualized approach to antimyeloma therapy. At the same time, the identification of MRD after therapy determines possibility of relapse. Aim. To identify the prognostic potential of MRD in patients in the standard and high molecular risk groups. Materials and methods. We analyzed 72 patients with MM (median age was 59 years (range 37-80), male/female - 1.3:1). All patients received initial therapy with proteasome inhibitors and / or immunomodulators. High dose therapy (MEL200) and autologous stem cell transplantation (ASCT) was carried out 50 (69%) patients. Standard cytogenetic and FISH methods were used to stratify patients in risk groups of mSMART 3.0. The standard risk (SR) was established in 52 (72%) patients, the high risk (HR) - in 20 (28%) patients. The MFC MRD status of bone marrow was evaluated after 4-6 cycles of induction therapy or after ASCT with use of 5-colors flow cytometry. MRD-negative status (MRD-) was based on level of clonal plasma cells &amp;lt;10-4 in bone marrow sample. Results. The MRD- was reached in 36% (26/72) patients. The median of OS in MRD+ group was 104 months, in MRD- was 146 months (p=.01). The median of PFS in MRD+ group was 26 months, in MRD- was 70 months (p=.00021). 2-years PFS in MRD+ group was 56%, in MRD- group was 100% (p=.00021). We divided patients into the following groups for evaluation the effect of MRD on survival in risk groups: SR МRD+ 34/72 (47%), SR МRD- 18/72 (25%), HR МRD+ 12/72 (17%) and HR MFC МRD- 8/72 (11%). The median of OS in HR MRD+ group was 72 months, in SR MRD+ - 104 months, in HR MRD- - 146 months, in SR MRD- was not achieved (p=.02). The median of PFS in HR MRD+ group was 24 months, in SR MRD+ - 26 months, in HR MRD- - 68 months, in SR MRD- - 70 months (p=.003). The 2-years PFS in HR MRD+ group was 44%, in SR MRD+ group was 50% and in SR MRD- and HR MRD- groups were 100% (p=.003). Conclusion. The absence of MRD is the most important prognostic factor. The leveling of negative effect of genetic abnormalities become possible when the MRD-negative response status is achieved. Presumably, this is due to the elimination of clonal plasma cells owing to the use of optimal antimyeloma therapy which is based on the risk stratification. Disclosures Martynkevich: Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Shuvaev:Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Detection of measurable residual disease may better predict outcomes than mutations based on next-generation sequencing in acute myeloid leukaemia with biallelic mutations of CEBPA.

Acute myeloid leukaemia (AML) patients with biallelic mutations of CEBPA (bi CEBPA) have a 30-50% relapse rate. This study established the value of mutations based on next-generation sequencing (NGS) and multiparameter flow cytometric measurable residual disease (MFC-MRD) detection and compared the outcomes. From 2014 to 2018, 124 newly diagnosed bi CEBPA AML patients were treated. The median age was 37·5 (16-69) years. The 3-year cumulative incidence of relapse (CIR), relapse-free survival (RFS) and overall survival (OS) were 33·0%, 64·7% and 84·3%, respectively. Patients without additional mutations and with GATA2 mutations were defined as 'NGS low risk', which was the only favourable independent factor for CIR and RFS of pretreatment parameters. Patients with sustained positive MRD after two consolidation cycles and MRD negative losses at any time were defined as 'MRD high risk', which was the only poor independent factor for CIR, RFS and OS, including pretreatment and post-treatment parameters. In CR2 and non-remission patients who underwent allo-HSCT, superior OS was achieved. We conclude that NGS low risk was a favourable factor in the analysis of pretreatment parameters. MRD risk stratification was an independent prognostic factor in pretreatment and post-treatment parameters. Relapsed patients still have a favourable outcome followed by allo-HSCT.

Detection of Measurable Residual Disease May Better Predict the Outcome Rather Than Mutations Based on Next-Generation Sequencing in Acute Myeloid Leukemia with Biallelic Mutations of CEBPA

Objectives: Acute myeloid leukemia (AML) patients with biallelic mutations of CEBPA (bi CEBPA) have a 30-50% relapse rate after standard chemotherapy. This study established the value of multiparameter flow cytometric measurable residual disease (MFC-MRD) detection, mutations based on next-generation sequencing (NGS), and compares the outcomes of risk stratification treatment in adult bi CEBPA AML patients. Methods: From March 2014 to December 2018, 124 patients with newly diagnosed acute myeloid leukemia with bi CEBPA were treated with chemotherapy. Out of these, 93 patients were analyzed further by a sensitive NGS assay for mutations in 87 candidate genes. MRD was detected in all patients by MFC after each cycle of induction and consolidation chemotherapy and every 3 months later. Results: Among these patients, 73 patients were male (58.9%), and median age was 37.5 (16-69) years. The expression of CD34, CD117, CD7, HLA-DR, CD15, CD33 and CD13 in 124 patients were 120 (96.8%), 123 (99.2%), 112 (90.3%), 107 (86.3), 93 (75.0%), 115 (92.7%) and 107 (86.3%) respectively. The most common co-occurring mutations were found in the GATA2 (n=29/93, 31%), WT1 (n=20/93, 21.7%), NRAS (n = 13/93, 14.0%), CSF3R (n = 12/93, 12.9%), and TET2 (n = 9/93, 9.6%) genes. All patients (100%) achieved complete remission (CR) including 114 patients (91.9%) who achieved it with only 1 induction course. The median follow-up was 33.5 (4-69) months. The 3-year Cumulative incidence of relapse (CIR), disease-free survival (DFS) and overall survival (OS) were 32.8%, 64.7%, and 84.3%, respectively. Univariate analysis results shown that WT1 Wild-type was favorable factor of 3-year CIR (23.3% vs. 47.0%, P=0.022) and 3-year DFS (75.5% vs 53.0%; P=0.031), GATA2 Wild-type had good trend for lower CIR and longer DFS and CSF3R Wild-type had inferior trend for higher CIR and shorter DFS. Patients with sustained positive MRD status after 2 consolidation cycles and MRD negative status loses in any time defined as "MRD high risk" had higher CIR (3-year CIR, 100% vs 25.5%; P&amp;lt;0.001; 81.5% vs 6.4%; P&amp;lt;0.001, respectively) and worse DFS and OS (3-year DFS, 0% vs 71.5%; P&amp;lt;0.001; 16.8% vs 90.2%; P&amp;lt;0.001, respectively; 3-year OS, 39.0% vs 89.8%; P&amp;lt;0.001; 72.2% vs 96.5%; P=0.001, respectively ) than those with persistent negative MRD status that defined as "MRD low risk" (Figure). Multivariate analysis showed that MRD low risk was the only favorable factor of CIR, DFS and OS (CIR: HR=37.3, 95%CI: 8.6-161.3, P&amp;lt;0.001;. DFS: HR=24.6, 95%CI: 7.2-84.0, P&amp;lt;0.001; HR=28.2, 95%CI: 2.1-374.1, P=0.011; respectively). Thirty-two (91.4%) MRD high risk patients (P&amp;lt;0.001) relapsed at a median time of 8 months (range: 3-32 months) include 21 patients of MRD negative status loses in any time and 11 patients with positive MRD status after 2 consolidation cycles. In totally 35 relapsed patients, 3 patients give up, 19 (61.3%) patients achieved CR2 after induction again. 15 CR2 and 4 non-remission patients underwent allo-HSCT achieved superior 3-year OS (83.0% vs 5.1%; p= 0.034; 75.0% vs 0%; p=0.006; respectively) than chemotherapy. To assess the role of risk stratification treatment by transplant or non-transplant, the124 patients were divided into 2 groups by the unique risk factor: MRD high risk group (n=43, 34.7%) and MRD low risk group (n=81, 65.3%). In the MRD low risk group (median follow-up: 35 months; range: 4-65 months), there was no significant difference in probabilities of 3-year OS (92.3% vs. 96.9%, P=0.428) between the transplant and non-transplant cohorts. In the MRD high risk group (median follow-up: 30 months; range: 7-69 months), 3-year OS was significantly better in the transplant cohort than in the non-transplant cohort (84.0% vs. 44.3%, p=0.007).Conclusions: MRD high risk may better predict the outcome rather than mutations based on NGS in AML with bi CEBPA, and allo-HSCT may achieve superior survival in MRD high risk patients. Key words: Acute myeloid leukemia with biallelic CEBPA mutations; next-generation sequencing; multiparameter flow cytometric measurable residual disease; allogeneic stem cell transplantation; Chemotherapy Disclosures No relevant conflicts of interest to declare.

Clinical study on deletion of IKZF1 gene in 93 children with B-cell acute lymphoblastic leukemia

Objective To study the effect of IKZF1 gene deletion on the relapse and prognosis of children with B-cell acute lymphoblastic leukemia (B-ALL) , and provide a theoretical basis for the selection and intensity adjustment of chemotherapy in B-ALL children with IKZF1 gene. Methods From June 2014 to November 2016, a total of 93 children with B-ALL were included in the study. They were newly diagnosed in the Department of Hematology Oncology, Children′s Hospital of Kunming Medical University, and able to provide sufficient bone marrow DNA samples.They were divided into IKZF1 gene deletion group(n=18) and IKZF1 gene non-deletion group (n=75). The incidence of IKZF1 gene deletion was calculated. The gender, age, number of white blood cells count, early treatment response, central nervous system (CNS) classification, minimal residual disease(MRD) grouping, risk grouping, recurrence and survival constituent ratioes were compared between two groups. The 2-year disease-free survival (DFS) and overall survival(OS) rates of the two groups were calculated by Kaplan-Meier method, and the survival curves were plotted. The samples collection of this study were agreed from the guardians, and they signed the informed consent forms. The study protocol was approved by the Ethical Review Board of Investigation in Human Being of Kunming Medical University (approval No. CCLG-ALL-2015). Results ① The incidence of IKZF1 gene deletion was 19.4% (18/93). ② There were no significant differences in gender, age, CNS classification, early treatment response and risk grouping between the two groups (P>0.05). The proportion of children with white blood cell count ≥50×109/L and high-risk MRD in the IKZF1 gene deletion group were 38.9% (7/18) and 27.8% (5/18), respectively, which were significantly higher than those of IKZF1 non-deletion group with 13.3% (10/75) and 8.0% (6/75), respectively, and the differences were statistically significant (χ2=4.75, P=0.019; χ2=4.72, P=0.009). The recurrence rate of the IKZF1 gene deletion group was 33.3% (6/18), which was significantly higher than that of the IKZF1 non-deletion group with 9.3%(7/75), and the difference was also statistically significant (χ2=6.95, P=0.008). ③ The 2-year DFS and OS rates in the IKZF1 gene deletion group were (72.7±13.4)% and (81.8±11.6)%, respectively, which were significantly lower than those of IKZF1 non-deletion group with (92.3±3.0)% and (94.4±2.8)%, and the differences were significant (P<0.05). Conclusions Deletion of the IKZF1 gene is one of the high-risk predictors of recurrence in children with B-ALL. It should be used as a risk factor to guide the risk stratification of B-ALL children in order to adjust the intensity of chemotherapy method and improve their prognosis. Key words: Leukemia, B-cell; Gene deletion; Prognosis; Risk factors; IKZF1 gene; Child

Use of Morphological Early Response Data to Predict Relapse in Teenage and Young Adult (TYA) Patients with Acute Lymphoblastic Leukaemia (ALL)

IntroductionTreatment 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.MethodsAll 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.Results820 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<0.001).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)ConclusionsSlow 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. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Increased Frequency of Cells with Activated Ribosomal Protein S6 at Diagnosis Associates with MRD Positivity and Relapse in Childhood BCP ALL

Numerous studies have demonstrated the association between minimal residual disease (MRD) positivity and risk of eventual relapse for children with B cell precursor acute lymphoblastic leukemia (BCP ALL). Yet despite varied methods to detect MRD (flow cytometry, PCR, sequencing), we still lack knowledge of the biology of drug resistant cells that mediate relapse.Performing single cell proteomic analysis using mass cytometry in a clinically annotated cohort of diagnostic samples, we have identified a phosphorylated ribosomal protein S6 (prpS6) signaling phenotype associated with MRD positivity and relapse. This phenotype has been observed in matched diagnostic and MRD samples and notably, in matched diagnosis/relapse pairs, we have observed an enrichment for this population at relapse.Methods Diagnostic BCP ALL samples from patients treated on the AIEOP protocols or healthy bone marrow were assayed either in basal condition or after ex vivo treatment with tyrosine kinase inhibitors (Dasatinib, BEZ-235). Samples were analyzed using mass cytometry for expression of 40 functional and/or phenotyping proteins. Mass cytometry data was processed and analyzed using Cytobank. Statistical analysis was performed using GraphPad Prism software. Significance was determined using student's t-test or one way ANOVA.Results Of the nine functional proteins measured in this study, rpS6 demonstrated, in resting cells from patients, the most variable levels of phosphorylation. Across the cohort the frequency of cells with activated rpS6 in the basal state varied between 4%-86% (median 24.7%), depending on the patient. This level was significantly higher than in healthy bone marrow controls (median 10.7%; p=0.0009). In BCP ALL samples the cells with activated rpS6 expressed higher levels of CD19 (p=0.0001), IgH (p=0.0059), and PAX5 (p=0.0021) when compared to cells without activated rpS6, suggesting a more mature phenotype for the prpS6+ blasts. These cells also had significant activation of pCREB (p<0.0001). Treatment with BEZ-235, a PI3K/TORk inhibitor, or Dasatinib, dual ABL/SRC kinase inhibitor, was able to significantly decrease the frequency of cells with activated prpS6 by inhibiting the phosphorylation of rpS6 after 30-minute ex vivo treatment.Patients with intermediate or high risk MRD status (per clinical protocol definitions) had an increased frequency of cells with activated rpS6 at diagnosis compared with patients with standard risk MRD (30% vs 13.9%; p=0.02). For patients for whom long term follow up data was available (n=31), patients that went on to relapse (n=9) compared to those in remission (n=22) had a statistically significant increased frequency of cells with activated prpS6 (p=0.0391).To determine whether activation of prpS6 was important for cell survival, we identified two cells lines amongst 7 BCP ALL cell lines that contained this high basal prpS6 subpopulation: NALM6 and SUP-B15. Treatment of these cells with Dasatinib in combination with the BEZ-235 or the MEKi PD0325901 resulted in decreased viability (6%-25% in TKI treated vs. 84% in untreated) and decreased activation of prpS6 and pCREB at 72 hours. These results support the hypothesis that the prpS6 high phenotype is important for cell survival.Given the association of activated rpS6 at diagnosis with detectable MRD and relapse, we examined an additional cohort of 9 matched samples from diagnosis Day 8 and Day 15 post induction therapy. In these samples, it was noted that by Day 8, there was a significant enrichment for cells with activated rpS6 (p=0.0009). In paired diagnosis-relapse samples, in 5 of 6 pairs, there was an enrichment for cells with activated rpS6atrelapse, all suggesting that rpS6 is associated with cells that are drug resistant. Mean prpS6 was significantly higher at relapse compared to diagnosis (p=0.035). Further studies will be critical to determine whether the rpS6 signature is proximal to causal events driving resistance.Significance We describe biochemical features of a cellular population observed at diagnosis across genetic subtypesassociated with MRD positivity and relapse that is enriched in early MRD and relapse samples in BCP ALL. Ribosomal protein S6 is a target of PI3K, MEK/ERK signaling and the B cell receptor pathway. Inhibition of this signaling results in cell death in model cell lines and suggests a role for therapy directed at this target in BCP ALL. DisclosuresDavis:Fluidigm, Inc: Honoraria. Nolan:Fluidigm, Inc: Equity Ownership.

Prediction of outcomes by early treatment responses in childhood T-cell acute lymphoblastic leukemia: a retrospective study in China.

BackgroundEarly treatment responses are important prognostic factors in childhood T-cell acute lymphoblastic leukemia (T-ALL) patients. The predictive values of early treatment responses in Chinese childhood T-ALL patients were still unknown.MethodsFrom January 2003 to December 2012, 74 consecutive patients aged ≤15 years with newly diagnosed T-ALL were treated with BCH-2003 protocol or CCLG-2008 protocol in the Department of Pediatric, Institute of Hematology and Blood Diseases Hospital in China. Predictive values of early treatment responses, including prednisone response, bone marrow morphology at day 15 and day 33 during induction chemotherapy, and minimal residual disease (MRD) monitored by flow cytometry after induction therapy (time point 1, TP1) and before consolidation therapy (time point 2, TP2), were analyzed.ResultsThe 5-year event free survival (EFS) and overall survival (OS) rates for these patients were 62.5 % (SE, 6.4) and 62.7 % (SE, 6.6), respectively. Prednisone poor responder was strongly associated with increased chance of induction failure (14.8 %) and decreased survival rate (5 year EFS rate, 51.1 % (SE, 10.5)). Patients with ≥25 % blast cells in bone marrow at day 15 were more likely to have an inferior outcome. 93.2 % of the T-ALL patients achieved complete remission at day 33 while patients with resistant disease all died of disease progression. MRD ≥10−2 at TP1 or MRD ≥10−3 at TP2 was significantly related to dismal prognosis. Risk groups classified by MRD at two time points could stratify patients into different groups: 29.0 % of the patients were MRD standard risk (MRD < 10−4 at both time points) with 3-year EFS rate of 100 %, 29.0 % were MRD high risk (MRD ≥10−2 at TP1 or MRD ≥10−2 at TP2) with 3-year EFS rate of 55.6 % (SE, 16.6) , and the rest of patients were defined as MRD intermediate risk with 3-year EFS rate of 85.7 % (SE, 13.2).ConclusionOur study demonstrated that MRD was the most powerful predictor of treatment outcome in childhood T-ALL patients and conventional morphological assessments of treatment response still played important roles in predicting treatment outcome and tailoring treatment intensity especially in countries with inadequate skills or financial resources for MRD monitoring.Electronic supplementary materialThe online version of this article (doi:10.1186/s12887-015-0390-z) contains supplementary material, which is available to authorized users.

Thiopurine methyltransferase and treatment outcome in the UK acute lymphoblastic leukaemia trial ALL2003.

The influence of thiopurine methyltransferase (TPMT) genotype on treatment outcome was investigated in the United Kingdom childhood acute lymphoblastic leukaemia trial ALL2003, a trial in which treatment intensity was adjusted based on minimal residual disease (MRD). TPMT genotype was measured in 2387 patients (76% of trial entrants): 2190 were homozygous wild-type, 189 were heterozygous for low activity TPMT alleles (166 TPMT*1/*3A, 19 TPMT*1/*3C, 3 TPMT*1/*2 and 1 TPMT*1/*9) and 8 were TPMT deficient. In contrast to the preceding trial ALL97, there was no difference in event-free survival (EFS) between the TPMT genotypes. The 5-year EFS for heterozygous TPMT*1/*3A patients was the same in both trials (88%), but for the homozygous wild-type TPMT*1/*1 patients, EFS improved from 80% in ALL97% to 88% in ALL2003. Importantly, the unexplained worse outcome for heterozygous TPMT*1/*3C patients observed in ALL97 (5-year EFS 53%) was not seen in ALL2003 (5-year EFS 94%). In a multivariate Cox regression analysis the only significant factor affecting EFS was MRD status (hazard ratio for high-risk MRD patients 4·22, 95% confidence interval 2·97–5·99, P < 0·0001). In conclusion, refinements in risk stratification and treatment have reduced the influence of TPMT genotype on treatment outcome in a contemporary protocol.

Improved Outcome for Children and Young People with T-Acute Lymphoblastic Leukaemia: Results of the UKALL 2003 Trial

▪T-ALL accounts for around 15% of paediatric ALL and carries a less favourable prognosis than B-ALL due to a higher relapse rate (RR) and poor response to salvage therapy. We sought to determine if the outcome for patients with T-ALL had improved on a contemporary, response based treatment protocol, UKALL 2003, which did not include pre-emptive cranial radiotherapy or high dose methotrexate. NCI standard risk patients received a 3 drug induction with dexamethasone, vincristine and pegylated asparaginase (1000 units/m2) (regimen A). NCI high risk (HR) patients also received daunorubicin (regimen B). Early morphological marrow response was measured on day 8 for NCI HR patients and day 15 for NCI standard risk patients except patients > 16 years. Rapid early responders were stratified by minimal residual disease (MRD) at day 29 which was measured by RQ-PCR. MRD negative patients were randomised to standard or reduced intensity therapy. MRD high risk (>0.01%) patients were randomised to standard therapy (regimen A/B) or an intensified regime consisting of augmented BFM consolidation, escalating Capizzi methotrexate and 2 delayed intensifications (regimen C). Patients with high risk cytogenetics or a slow early morphological response received regimen C, regardless of MRD. Regimen A and B patients received 4 doses of pegylated asparaginase (2 in induction and 1 in each DI) and Regimen C patients received an additional 8 doses.Of 3126 patients in the trial, 388 (12.4%) had T-ALL. Of those, 73% were male with a median age of 10 yrs (range 1-24). Median WCC was 94x109/L (range 1-881) and 85% were HR by NCI criteria. 76% had a rapid early response (RER), defined as <25% bone marrow blasts. MRD was evaluable for 72% of patients. MRD at day 29 was ≥0.1% in 37%, detectable at a level of <0.1% in 22% and undetectable in 41%. Compared to the trial population as a whole, patients with T-ALL were more often stratified to receive the higher intensity regimens. T-ALL 11%, 52% and 37% treated on Regimens A, B and C compared with 51%, 26% and 23% for trial overall.With a median follow up of 5 yrs 7 mths (range 1 yr 4 mths – 9 yrs 11 mths), patients with T-ALL had an inferior outcome compared to those with B-ALL due to a higher risk of relapse (p=0.0003) and death in remission (p=0.04); 5 year event free survival (EFS) 81.2% (95% CI 77.3-85.1) vs 88.1% (86.6-89.5), overall survival (OS) 86.4% (82.9-89.9) vs 92.4% (91.4-93.4), RR 13.5% (10.0-17.0) vs 8.2% (7.0-9.4) and death in remission 4.3% (2.3-6.3) vs 2.4% (1.8-3.0). The site of excess relapses in T-ALL was both bone marrow (10.1% (7.0-13.2) vs 5.9% (4.9-6.9)) and isolated CNS (3.5% (1.5-5.5) vs 1.9% (1.3-2.5)). However, the CNS relapse rate is comparable to other protocols containing cranial radiotherapy and high dose methotrexate. The excess deaths in remission are a reflection of the higher intensity regimens that a majority of T-ALL patients received. In contrast to B-ALL (data not shown), increasing WCC was not a risk factor for relapse in T-ALL. 5 year EFS for T-ALL patients with a WCC <100 was 85% vs 81% for those with a WCC ≥100. CNS relapse rate was not different between the two groups, 5% vs 8% (p=0.3). There was no difference in outcome between those with a WCC of 100-300 and ≥300. Response to induction was the strongest predictor of relapse. Patients with a slow early response had a high relapse risk (Hazard Ratio=2.31 (1.11-4.84), p = 0.03 in multivariate analysis) despite receiving Regimen C. Patients with MRD ≥0.1% had a 5 year EFS of 71.5% compared to 89.6% for the MRD <0.1% group and 90.3% for the MRD negative group (log rank p(trend)=0.001), RR 23.2% vs 7.1% vs 8% (p=0.003). There was a trend for a worse outcome in the patients aged ≥16 although this was not statistically significant, 5 year EFS 74.1% vs 82.8% (log rank p=0.7), RR 20.8 vs 11.9%, (p=0.09). Compared with our previous trial, UKALL97/99, outcome for T-ALL patients has improved with a higher EFS, 81.7% vs 72.8% at 5 years (log rank p=0.08), due mainly to a non-significantly lower relapse rate 13.5% vs 20.6% (p=0.09).T-ALL outcomes have improved with better risk stratification and the use of dexamethasone and pegylated asparaginase. Pre-emptive cranial radiotherapy or high dose methotrexate are not essential to preventing CNS relapse. The outcome for patients with T-ALL, however, remains inferior to those with B-ALL highlighting the need for novel therapies for patients with high risk T-lineage disease. DisclosuresNo relevant conflicts of interest to declare.

Augmented post-remission therapy for a minimal residual disease-defined high-risk subgroup of children and young people with clinical standard-risk and intermediate-risk acute lymphoblastic leukaemia (UKALL 2003): a randomised controlled trial

No randomised study has shown whether stratification of treatment by minimal residual disease (MRD) response improves outcome in children and young people with acute lymphoblastic leukaemia (ALL). We assessed whether children and young people with clinical standard and intermediate-risk ALL who have persistent MRD at the end of induction therapy benefit from augmented post-remission therapy. Between Oct 1, 2003, and June 30, 2011, we enrolled eligible patients aged 1-24 years and initially categorised them into clinical standard-risk, intermediate-risk, and high-risk groups on the basis of a combination of National Cancer Institute criteria, cytogenetics, and early morphological response to induction therapy. Clinical standard-risk and intermediate-risk patients with MRD of 0·01% or higher at day 29 of induction (MRD high risk) were randomly assigned (1:1) to standard therapy (treatment regimens A and B) or augmented post-remission therapy (regimen C). Compared with standard therapy, the augmented treatment regimen (regimen C) included an additional eight doses of pegylated asparaginase, 18 doses of vincristine, and escalated-dose intravenous methotrexate without folinic acid rescue during interim maintenance courses. Computer randomisation was used for treatment allocation and was balanced for sex, age (<10 years vs ≥10 years), and white blood cell count at diagnosis (<50 × 10(9)/L vs ≥50 × 10(9)/L) by minimisation. Patients, clinicians, and data analysts were not masked to treatment allocation. The primary outcomes were event-free survival and overall survival. Analyses were by intention to treat. This trial is registered with Current Controlled Trials, number ISRCTN07355119. 533 MRD high-risk patients were randomly assigned to receive standard (n=266) or augmented (n=267) post-remission therapy. After a median follow-up of 70 months (IQR 52-91), 5-year event-free survival was better in the augmented treatment group (89·6% [95% CI 85·9-93·3]) than in the standard group (82·8% [78·1-87·5]; odds ratio [OR] 0·61 [95% CI 0·39-0·98], p=0·04). Overall survival at 5 years was numerically, but not significantly, higher in the augmented treatment group (92·9% [95% CI 89·8-96·0]) than in the standard therapy group (88·9% [85·0-92·8]; OR 0·67 [95% CI 0·38-1·17], p=0·16). More adverse events occurred in the augmented treatment group than in the standard group (asparaginase-related hypersensitivity in 18 [6·7%] in the augmented group vs two [0·8%] in the standard group and asparaginase-related pancreatitis in eight [3·0%] vs one [0·4%]; intravenous methotrexate-related mucositis in 11 [4·1%] vs three [1·1%] and methotrexate-related stomatitis in 48 [18·0%] vs 12 [4·5%]). Our findings suggest that children and young people with acute lymphoblastic leukaemia and 0·01% or more MRD at the end of remission induction therapy could benefit from augmented post-remission therapy. However, the asparaginase and intravenous methotrexate used in the augmented treatment regimen is associated with more adverse events than is the standard post-remission treatment regimen. Medical Research Council and Leukaemia and Lymphoma Research.

Low PKCα expression within the MRD-HR stratum defines a new subgroup of childhood T-ALL with very poor outcome.

Pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) outcome has improved in the last decades, yet one patient in every four still relapses. Except treatment response and immunophenotype, few markers are reliably prognostic in pediatric T-ALL patients. Aiming to improve T-ALL risk stratification, we investigated a new candidate biomarker with potential prognostic relevance. A phosphoproteomic screening of 98 pediatric T-ALL samples at diagnosis had been performed using the high-throughput Reverse Phase Protein Arrays technique, which led to the identification of PKCαS657 as an activated protein with a broad variation among T-ALL samples. To evaluate PKCα potential as a prognostic biomarker, PKCα expression was analyzed using RQ-PCR in a cohort of 173 patients, representative of ALL2000-ALLR2006 AIEOP study. A threshold of PKCα expression with the highest discrimination for incidence of relapse was identified. Patients with PKCα down-regulation, compared to patients with PKCα levels above the threshold, presented a markedly increased cumulative incidence of relapse (43.8% vs. 10.9%, P<0.001), as well as a worse 4-year overall survival (66% vs. 87.9%, P=0.002) and event-free survival (53.1% vs. 85.2%, P=0.002). In particular, low PKCα expression identified cases with extremely poor outcome within the high-risk minimal residual disease (MRD) stratum, their incidence of relapse being of 69% vs. 15% in the high PKCα levels group. In a multivariate analysis adjusting for main prognostic features, PKCα proved to be an independent prognostic factor related to incidence of relapse. Very high risk patients within the high-risk MRD stratum, identified by PKCα expression, could be proposed for experimental therapeutic protocols.

Outcomes Of Teenagers and Young Adults On The UKALL 2003 Paediatric Trial For Children and Young People With Acute Lymphoblastic Leukaemia

▪ BackgroundOutcomes for adults with acute lymphoblastic leukaemia (ALL) have traditionally been inferior to outcomes for children, with differences in survival being noted from adolescence onwards. The reasons for the declining cure rates with increasing age are likely to be multi factorial but the choice of treatment protocol has been shown to play an important role in outcomes for teenagers and young adults (TYAs) with ALL. Over the past decade there has been increasing evidence of better outcomes for TYAs treated on paediatric regimens compared to adult regimens. Historically, 15-17 year olds treated on the adult UKALL XII trial had about 15% lower 5-year probability of both overall survival (OS) and event-free survival (EFS) compared to those treated on the corresponding paediatric ALL 97/99 trial during the same period, with the EFS difference not accounted for by age, gender, WBC or Ph status. (Ramanujachar et al. Pediatr Blood Cancer. 2007 Mar;48(3):254-61).UKALL 2003 was a randomised controlled trial investigating treatment modification according to Minimal Residual Disease (MRD) status at the end of induction and week 11, in consecutively diagnosed children and young people with Philadelphia negative 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 outcomes for the 16 – 24 year old teenage and young adult (TYA) patients recruited to the trial from 2003-2011. ResultsA total of 229 out of 3126 patients entered were aged 16-24 years (7.3%), although only 56 patients were over 20 years of age at diagnosis. With follow up to October 2012, the 5-year EFS for the 16-24 year old cohort was 72% (95% CI: 66-79%) compared to an EFS for 1-10 year olds and 10-15 year olds of 90% (88-91%) and 84% (80-87%) respectively, p(trend)<0.0001. At the last analysis, 37 TYA patients had relapsed with a 5-year cumulative risk of relapse of 21% (14-27%), compared to 11% (8-14%) for the 10-15 year olds and 7% (6-9%) for the 1-10 year olds, p(trend)<0.0001.Within UKALL 2003 there was no significant increase in reported toxicities in the 16-24 year age group compared to the 10-15 year age group. The induction death rate was 2.2% for 16-24 year olds, 2.1% for 10-15 year olds and 1.2% for under 10 year olds. 53% of TYA patients had one or more serious adverse event (SAE) reported compared to 56% for 10-15 year olds and 31% for under 10 years. The SAEs reported were primarily infections and expected toxicities related to steroids, pegylated asparaginase and methotrexate. There was a trend (p=0.05) towards an increase in deaths in remission in the TYA age group at 6.1% compared to 3.1% in the 10-15 year olds.Outcomes in TYAs were analysed by MRD status as previously described (Vora et al. Lancet Oncol. 2013 Mar;14(3):199-209). There was an increase in the proportion of TYA patients with persistent MRD positivity compared to the younger patient groups; 49% were MRD high risk, 24% were MRD low risk and 27% MRD indeterminate compared to 32%, 36% and 32% respectively for patients under 16 years, p<0.0001. MRD was highly predictive of outcome in the TYA age group, as in the trial overall. Five-year EFS for those aged 16-24 was 64% (95% CI: 54-74%) for MRD high risk patients, 71% (58-84%) for MRD indeterminate and 93% (86-100%) for MRD low risk; MRD high risk vs low risk, p=0.0003. The TYA EFS compares favourably with the 10-15 year old cohort with EFS of 74% (68-81%), 83% (77-88%) and 95% (91-99%) respectively. ConclusionThis large randomised study provides evidence that TYAs with ALL can be treated on a national paediatric trial with toxicities similar to those seen in younger teenagers. Outcomes are significantly improved using this approach in TYAs compared to the historical, transplant focused adult ALL regimens. MRD based risk stratification in this age group remains a valid approach with low risk patients having an unprecedented 93% EFS at 5 years. Further intensification of therapy for MRD high risk patients, possibly including transplantation may be required for the MRD high risk TYA patients and this question is being examined in the next national trial, UKALL 2011. Disclosures:No relevant conflicts of interest to declare.

Philadelphia-Like Signature In Childhood Acute Lymphoblastic Leukemia: The AIEOP Experience

▪ BackgroundDespite the current risk-based stratification and therapies, up to 20% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) experience relapse. A major research effort is dedicated to identifying poor prognostic subgroups in the larger subset of patients with intermediate-risk disease, where most relapses occur. Recently, two groups in the US and the Netherlands independently identified a novel BCP-ALL subtype negative for the BCR/ABL fusion gene but with a gene-expression profile similar to Philadelphia chromosome-positive (Ph+) ALL. This ‘Ph-like' (or ‘BCR/ABL-like') subtype encompasses 10-15% of BCP-ALL patients, predicts high incidence of relapses and defines a candidate subgroup for targeted treatment with tyrosine kinase inhibitors and alternative drugs. AimAim of this project was to identify BCP-ALL Ph-like cases and their prognosis in patients treated in Study Protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) MethodsIn the context of the Microarray Innovations in LEukemia (MILE) study, gene expression profiling was successfully performed on 400 Italian childhood BCP-ALL cases enrolled in AIEOP-BFM ALL2000/R2006 protocols, from whom material was available. The MILE classifier recognized the most common genetic subgroups, namely t(4;11), t(12;21), t(1;19) or t(9;22) positive, or high hyperdiploid (DNA index≥1.16). Out of 400 BCP-ALL cases, 143 negative for common fusion transcripts, non-high hyperdiploid (DNA index<1.16) and non-Down Syndrome, were defined as ‘B-others'. ResultsUsing signatures of known ALL subgroups, the likelihood to be close to one of the ‘known' classes was defined for each of the B-others samples. Specifically, 43 B-other cases (43/143=30.1%, about 10% of total BCP-ALL cohort) presented as a cluster with a gene expression signature close to the BCR/ABL signature, therefore referred to as ‘Ph-like'. Among B-others, Ph-like cases had a significantly increased proportion of males, age>10 years and WBC>20x109/L. The 5y event-free survival (EFS) of Ph-like patients was 54.8% (SE 8.2) vs 83.1% (3.9) in the remaining B-others patients (p<0.001), mostly due to an increased cumulative incidence of relapse (CIR: 33.9% (7.4) vs 14.9% (3.7); p=0.009). Notably, Ph-like patients did not differ for prednisone (PDN) and minimal residual disease (MRD) response or final risk stratification. Indeed, out of 36/43 stratified by MRD, only 7 patients had high-risk MRD, while 20 were MRD intermediate and 9 even MRD standard risk. Only 3 non-HR cases by MRD were then classified as HR based on PDN response. Of relevance, the significant difference in 5y EFS was confirmed in the 33 Ph-like patients with no HR features (59.8% (9.2) vs 88.5% (3.9) in the remaining B-others patients with similar features (p<0.001), with a significantly increased CIR (32.1% (8.4) vs 10.2% (3.7); p=0.005). Among 43 Ph-like cases, 1 was resistant and 1 died during the induction phase. Out of the 14 relapses, 10 were isolated medullary, and 4 combined with an extramedullary site (no CNS relapses). Two patients died in remission and no secondary malignancies occurred during the observation period. Overall, 25/43 (58.1%) Ph-like patients are alive in complete remission versus 84/100 (84.0%) in B-others, non Ph-like patients. Conclusions and perspectivesWe identified a ‘Ph-like' subgroup in the Italian cohort of children with BCP-ALL, associated to a poor outcome. Ph-like patients, independently of the other known risk features, could be considered eligible for alternative treatments. The genetic characterization of this subgroup is ongoing within the AIEOP-BFM research trial cooperative group, which is aimed at further defining the pathogenetic mechanisms and identifying the therapeutic translation. Disclosures:No relevant conflicts of interest to declare.

Characteristics and Outcomes Of Patients With Down Syndrome Associated Acute Lymphoblastic Leukaemia Treated On UKALL 2003

Children with Down Syndrome (DS) are at significantly increased risk of developing acute leukaemia, both AML and ALL. Whilst outcomes for AML are excellent, ALL associated with DS (DS-ALL) confers an inferior survival due to a combination of increased relapse risk and high treatment related mortality (TRM). We report the outcome for children and young adults with DS-ALL treated on a modern, intensive, minimal residual disease (MRD) directed protocol, UKALL2003.Treatment was not modified for DS-ALL patients at the start of the trial. NCI standard risk patients received a 3 drug induction with dexamethasone, vincristine and pegylated asparaginase. NCI high risk patients also received daunorubicin. MRD was measured at day 29 and MRD negative patients were randomised to standard or reduced intensity therapy. MRD high risk patients were randomised to standard therapy or an intensified regime consisting of augmented BFM consolidation, escalating Capizzi methotrexate and 2 delayed intensifications. Six years after the trial opened, an unacceptably high TRM in DS patients, (10 deaths out of 46 patients), prompted modifications to their treatment. DS patients were excluded from randomisations and, regardless of NCI risk, received a three drug induction with the addition of daunorubicin at day 15 for those with a slow morphological marrow response (>25% blasts). Patients with positive MRD at day 29 or those with a slow day 15 morphological response were treated with augmented therapy described above, in the absence of established significant comorbidity. All patients received only one delayed intensification and maintenance was shortened to 2 years for boys (standard duration 3 years).Strict supportive care measures during intensive phases of treatment were mandated.Of 3126 eligible patients in the trial, 85 (2.7%) had DS with median age 4 years (range 1-23 years). DS-ALL patients were more likely than non-DS-ALL patients to be male (68% vs 56%, p=0.03) and were almost exclusively B-cell precursor ALL (99 % vs 87%, p=0.001), but there were no significant differences by age, WBC or NCI risk. Nine of 80 DS-ALL patients from whom data on cytogenetic abnormalities was available had high hyperdiploidy (11% vs 30% in non-DS-ALL, p=0.0003), and 13 had an ETV6/RUNX1 fusion (16% vs 25%, p=0.06).With a median follow-up of 4 years 10 months, patients with DS-ALL had an inferior EFS compared to non-DS-ALL patients (65.3% (95% CI 54.5-76.1%) at 5 years vs. 87.8% (86.4-89.2%); OR=8.90 (95% CI: 4.53-17.48) p<0.00005). Of the 85 DS-ALL patients, 25 have died, 7 due to relapsed disease, 2 due to induction failure and death with active disease and 16 due to TRM during first line therapy. TRM deaths occurred throughout treatment (induction = 5, CR = 11) and were not confined to the most intensive phases. In all cases, deaths were due to infection, most commonly bacterial sepsis (75%) and viral pneumonitis (25%). The excess TRM was primarily responsible for the worse EFS for DS patients, as their risk of relapse (16.4% at 5 years ) was not significantly worse (p=0.1) than for non-DS patients (8.8%).In contrast to other recently reported studies, we report excess TRM as the main cause of treatment failure in DS-ALL, possibly due to the intensity of our treatment protocol which included dexamethasone and pegylated asparaginase for all patients. There have been fewer treatment related deaths in induction after introducing the treatment modifications in 2009 (4/46 vs 1/41) without an apparent increase in relapse risk. Improvements in outcome for this vulnerable group of patients will require such risk stratified treatment modifications, better supportive care and innovative agents that can replace the more intensive elements of current treatment schedules. Disclosures:No relevant conflicts of interest to declare.

Hematopoietic Cell Transplantation for Children with High Risk Acute Lymphoblastic Leukemia in First Complete Remission: A Report From the Italian Association of Pediatric Hematology and Oncology (AIEOP) Registry.

AbstractAbstract 3036Children with acute lymphoblastic leukemia (ALL) in first complete remission (CR1) and high risk (HR) characteristics can benefit from allogeneic hematopoietic stem cell transplantation (HSCT). We carried out a retrospective, multicenter study to analyze the outcome of 211 consecutive ALL pediatric patients who received either related or unrelated (UD) HSCT for ALL in CR1 and were reported to the Italian Association of Pediatric Hematology and Oncology (AIEOP)-HSCT Registry between 1990 and 2008. Sixty-nine patients (33%) were transplanted between 1990 and 1999, 58 (27%) between 2000 and 2005, and 84 (40%) between 2005 and 2008. A matched family donor (MFD) was employed in 138 patients (65%) and an UD in 73 (35%).The 10-year probability of overall survival and disease-free survival (DFS) was 63.4% (95% CI, 57–70) and 61% (95% CI, 54–68), respectively. In univariate analyses, the donor type had an impact on DFS only for patients transplanted between 1990 and 1999 (MFD: 65% [95% CI, 53–77], UD: 33% [95% CI, 3–64], p=0.06). There were no differences between MFD and UD for patients who underwent HSCT after 2000. DFS was better in patients with grade 0-II aGvHD than in those with grade III-IV aGvHD (65% [95% CI, 58–62] and 40% [95% CI, 22–58], p=0.002). In multivariate analyses, the occurrence of grade IV aGvHD (RR=3.8 [95% CI, 1.58–9.20], p=0.002) was an independent factor associated with worse DFS. On the contrary, the occurrence of grade I and II aGvHD (RR=0.54 [95% CI, 0.29–0.99], p=0.05; RR=0.56 [95% CI 0.30–0.98], p=0.07) were independent favorable prognostic variables for DFS.The 10-year cumulative incidence of relapse incidence (RI) was 24% (95% CI, 19–30). In univariate analyses patients who experienced grade 0-I aGvHD (30% [95% CI, 23–40]) had higher RI than patients with grade II-IV aGvHD (15% [95% CI, 9–25) (p=0.013). In multivariate analysis, the occurrence of aGvHD remained an independent prognostic variable for RI.The 10-year cumulative incidence of transplant-related mortality (TRM) was 15% (95% CI, 11–21). In univariate analyses, TRM was lower for children aged 1–9 years at diagnosis as compared with those aged 10–14 years or older than 15 years (8% [95% CI, 4–15], 18% [95% CI, 11–31], and 54% [95% IC, 34–84], respectively, p<0.00001). The impact of donor type on TRM was only observed for patients transplanted between 1990 and 1999 (UD: 44%, [95% IC, 21–92], MFD: 8% [95% IC, 4–19], p=0.0043). Patients with grade III and IV aGvHD had TRM of 32% (95% CI, 16–61) and 82% (95% CI, 62–100), respectively, versus 13% (95% CI, 7–25), 3% (95% CI, 1–14) and 12% (95% CI, 6–26) of patients with grade II, I and 0 aGvHD, respectively (p<0.00001). In multivariate analysis the strongest predictors of TRM were grade IV aGvHD (RR 18.1 [95% IC, 4.37–75.3], p<0.00001) and UD donor for HSCT performed between 1990 and 1999 (RR 4.83 [95% IC, 1.43–16.3], p=0.01).In this study, 27 and 73 out of the 100 patients investigated had minimal residual disease (MRD)-intermediate risk (IR) and MRD-high risk (HR) features, respectively. The 27 with MRD-IR were given the allograft for the presence of other characteristics rendering them classifiable as HR. The probability of DFS and RI of MRD-IR and MRD-HR was comparable. Thus, although the number of patients investigated was limited, our results seem to suggest that HSCT could reduce or, at best, abrogate the effects of MRD on patient outcome. Our results suggest that after 1999 transplant outcomes are remarkably similar in recipients of UD and MFD. No advantage of total body irradiation (TBI) over chemotherapy in the conditioning regimen in terms of DFS, RI and TRM was found. The benefit offered by the occurrence of GvHD in terms of reduction of disease recurrence was, however, offset by a higher incidence of TRM. Indeed, taking patients who did not have aGvHD as the reference group, a better probability of DFS was observed only in patients who developed grade I-II aGvHD, this suggesting that only GvHD of mild/moderate severity can favorably impact on disease outcome.In conclusion, our data support the choice of performing allogeneic HSCT in pediatric and adolescent patients with HR ALL in CR1 from either MFD or UD. Randomized prospective cooperative group studies are desirable to establish the role of TBI-based conditioning in these patients. Disclosures:No relevant conflicts of interest to declare.