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BACKGROUND: Children with high risk acute myeloid leukemia (AML) still experience consistently high rates of relapse. Survival after first relapse increased from 21% between 1987 and 1997 up to 39% in recent studies. However, since 2009, there have been no publications on subsequent large pediatric AML relapse trials. As the indications for HSCT during first-line treatment have been extended since then, the current survival of these patients at relapse remains unclear. Herein, we report outcome results from the BFM and COG study group, which represents the largest available dataset analyzed for post-relapse survival. PATIENTS AND METHODS: Pediatric patients with first relapse of AML (no Down syndrome, secondary leukemia or FAB M3) have been analyzed from two large study groups with patients from the United States, Canada, Australia, New Zealand, Germany, Austria, Czech Republic and Switzerland. Out of 1222 patients in the BFM cohort (AML-BFM study 2004, registry 2012 and study 2012), 350 experienced at least one relapse and 197 of those had a first relapse after closure of the last I-BFM relapse trial (04/2009 through 2017). Within the Children's Oncology Group (COG) Phase 3 trials (AAML0531 and AAML1031, n=2119) 852 pediatric patients suffered a relapse. Five-year probability of overall survival (pOS) and event-free survival (pEFS) were calculated according to Kaplan-Meier. EFS was calculated for the BFM cohort as time from relapse to the next event (second relapse, death, failure to achieve a second remission or secondary malignancy) or until last follow-up, while OS reflects the time from relapse until death or last follow-up. The Cox proportional hazards model was used for multivariate analysis of outcomes. Living patients were censored at last follow-up with a median follow-up after relapse of 4·2 years (BFM) and 4·8 years (COG). Data have been frozen at 03/27/2020 (BFM) and 03/31/2020 (COG). RESULTS: In the 197 patients with relapse after closure of the last BFM relapse trial (04/2009 through 2017) the pOS at 5 years was 42±4% (BFM). The 5-year pOS in patients relapsing after COG trials 2006-2018 was 35±2% (n=852). Patients experiencing a relapse between 2014 to 2017 had a pOS of 49±6% (BFM, n=78) and 40±3% (COG, n=333). Risk classification at initial diagnosis and a short time from diagnosis to relapse predicted a diminished survival probability in both cohorts (see Table 1). However, the absence of full hematopoietic regeneration of the bone marrow after re-induction did not predict survival: Within the BFM dataset, a subgroup analysis in all patients receiving DNX-FLA (n=156) have been performed. Initial characteristics are comparable to the total cohort. Among these patients 147 were evaluable for response (7 excluded due to early death before evaluation, 2 for insufficient data). Eighty-nine (57%) achieved a CR (n=69) or CRp (n=20) and 52 (33%) no response. Overall survival was superior for patients with a CR/CRp (54±6% (CR/CRp) vs. 32±7% (No CR/CRp); p=0·0064), but long-term survival was still possible even with a poor re-induction response. Patients with a CRp had a comparable survival to those with a CR after a second re-induction (pOS 60±11% (CRp) vs. pOS 52±7% (CR); p=0·57). Patients with >5% leukemic blasts (n=32) had the lowest survival (pOS 27±9%). The 5-year pEFS for this cohort was 29±4% (pEFS 50±6% (CR) vs. pEFS 50±11% (CRp)). The analysis of post-relapse treatment showed that the vast majority of patients who survive had a HSCT following relapse. By landmark analysis, survival was significantly higher in patients with subsequent HSCT compared to that of non-transplanted patients (BFM: pOS 53±4%, n=154 vs. pOS 5±5%, n=21; p(Mantel-Byar)=0·0002). CONCLUSION: This is the largest report to date on post relapse survival in children with AML. Our analysis confirmed previously described risk factors for poor survival while also highlighting new findings contrary to established standards. Strikingly, the absence of full hematopoietic regeneration of the bone marrow after re-induction did not predict survival at first relapse, thereby questioning the current value of the established International Working Group Criteria published by Cheson et al for response-evaluation in pediatric AML. As the international pediatric AML community embarks on collaborative efforts to evaluate new therapies in children with relapsed AML, a comprehensive review of post relapse survival is critical. Disclosures Bourquin: Servier: Other: Travel Support. Reinhardt:Novartis: Membership on an entity's Board of Directors or advisory committees; CLS Behring: Research Funding; bluebird bio: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Despite attempts to improve the definitions of ambiguous lineage leukemia (ALAL) during the last 2 decades, general therapy recommendations are missing. Herein, we report a large cohort of children with ALAL and propose a treatment strategy. A retrospective multinational study (International Berlin-Frankfurt-Munster Study of Leukemias of Ambiguous Lineage [iBFM-AMBI2012]) of 233 cases of pediatric ALAL patients is presented. Survival statistics were used to compare the prognosis of subsets and types of treatment. Five-year event-free survival (EFS) of patients with acute lymphoblastic leukemia (ALL)-type primary therapy (80% ± 4%) was superior to that of children who received acute myeloid leukemia (AML)-type or combined-type treatment (36% ± 7.2% and 50% ± 12%, respectively). When ALL- or AML-specific gene fusions were excluded, 5-year EFS of CD19+ leukemia was 83% ± 5.3% on ALL-type primary treatment compared with 0% ± 0% and 28% ± 14% on AML-type and combined-type primary treatment, respectively. Superiority of ALL-type treatment was documented in single-population mixed phenotype ALAL (using World Health Organization and/or European Group for Immunophenotyping of Leukemia definitions) and bilineal ALAL. Treatment with ALL-type protocols is recommended for the majority of pediatric patients with ALAL, including cases with CD19+ ALAL. AML-type treatment is preferred in a minority of ALAL cases with CD19- and no other lymphoid features. No overall benefit of transplantation was documented, and it could be introduced in some patients with a poor response to treatment. As no clear indicator was found for a change in treatment type, this is to be considered only in cases with ≥5% blasts after remission induction. The results provide a basis for a prospective trial.

Acute lymphoblastic leukemia treatment leads to elimination of blasts and stepwise regeneration of normal hematopoiesis. Several studies identified prognostic relevance of minimal residual disease (MRD) in bone marrow (BM) before achieving complete remission (Giuseppe Basso et al., J Clin Oncol, 2009). Crucial question is how to assess BM quality at day 15 (d15) of ALL BFM protocols. In ALL BFM 2009 protocol good quality sample is defined as containing more than 2% erythroid precursors (EP) of nucleated cells. EP were defined as CD19neg(orCD7neg)CD45neg. Two pt cohorts were included in the study. First cohort (Coh2000) consisted of pts treated by AIEOP BFM ALL 2000, n=196 (177 BCP ALL, 19 T ALL, median follow-up 5.4 yrs, range 0.025-10). AIEOP BFM ALL 2000 study was a PCR MRD based protocol (assessment at day d33 and d78) and flow cytometric MRD (FC MRD) was assessed only on research basis at d15. Second cohort (Coh2009) consisted of pts treated by AIEOP BFM ALL 2009, n=331 (292 BCP ALL, 39 T ALL, median follow up 4.8 yrs; range 0.0027-7.6). In Coh2009, both PCR MRD (d33, d78) and FC MRD d15 were used for risk stratification. We asked following questions:What is the specificity and viability of EP defined by CD45 negativity? Is a definition based on bright expression CD71 more specific?Is the amount of EP different between B and T ALLs and between risk groups defined by FC at d15? What is the overall frequency of low EP at d15?What is the relationship between amount of EP and FC MRD at d15?Is there any prognostic relevance of low EP? Results:Population of EP was selected based on negativity of CD45 and a lineage marker (CD19 or CD7) among nucleated cells, which were defined as positive by a SYTO nucleic fluorescent dye. We found a high amount of non-viable cells defined by 4′,6-diamidino-2-phenylindole (DAPI) positivity (6.5-96%, median 55%). When we added bright CD71 into the EP definition (EP CD71++), the percentage of DAPI positivity was significantly lower (0-66%, median 9%) (p Conclusion: Sample quality is essential question in the assessment of MRD in BM. Although low EP may indicate poor BM aspiration quality, it may also result from other biological factors. At d15 BCP ALL, these factors include the interaction of normal BM cells with leukemia, patient's risk group, and type of corticosteroid used. EPs should be detected using an erythroid marker, such as CD71. However, new markers of BM quality, less influenced by leukemia treatment, are needed. Supported by Ministry of Health of CR, grant nr. 15-28525A, NV18-07-00430 and NV18-03-00343; Czech Science Foundation nr. P302/12/G101. Disclosures Brüggemann: PRMA: Consultancy; Incyte: Consultancy; Pfizer: Speakers Bureau; Roche: Speakers Bureau; Affimed: Research Funding; Regeneron: Research Funding; Amgen: Consultancy, Research Funding, Speakers Bureau.

Acute leukemia (AL) of ambiguous lineage (AMBI-L) comprises up to 5% of AL cases in both children and adults. Although several definitions exist, a general treatment guideline has been missing. Single country studies usually report fewer than 50 cases of children or adults. Accordingly, the international iBFM AMBI2012 Study/Registry collected 275 AMBI-L cases in patients In total, 275 patients were included in the study. Of these, 240 fulfilled the definitions of biphenotypic/mixed phenotype AL, partially overlapping with cases in whom two clones had been identified (n=68) and 15 cases presented with undifferentiated AL. Most patients started their treatment with an ALL-type protocol (n=161), 79 with AML therapy, 27 with a combined regimen, including the Interfant protocols, 2 patients were not treated, 2 received other treatment, and in 4 patients such information was missing. The 5yEFS of the entire cohort was 56±3.7% and 5y overall survival was 67±3.3%. Patients treated by ALL-type protocols had superior 5 year event free survival (5yEFS) (70±4.6%, n=158) compared to those who started AML-type treatment (5yEFS: 40±6.4%, n=78) or hybrid ALL/AML treatment (5yEFS: 50±11%, n=27). Although protocol selection was likely biased, we recommend ALL treatment, when diagnostic findings, including molecular genetics, fail to indicate AML therapy. Although myeloperoxidase (MPO) has been used as the ultimate marker of myeloid lineage, patients who started with ALL-type treatment demonstrated a better prognosis even among cases classified as MPOpos/part pos (Fig. 1). These differences by initial choice of treatment are most prominent when CD19pos/part pos cases are analyzed regardless of the overall lineage (Fig. 2). This shows that at least for CD19pos/part pos cases in the absence of RUNX1/RUNX1T1 fusion, treatment should not start with current AML-type protocols. Until week 12, patients with higher leukemia burden were slightly overrepresented compared to non-AMBI ALL patients (data not shown). In addition, patients with higher residual disease had a much poorer prognosis. Thus, Prednisone poor and good responders (based on day 8 blood blast counts) had a 5yEFS of 50±9.7%, n=38 and 81±5.8%, n=82, respectively (p=0.005). By day 15 bone marrow (BM), only cutoffs of 10-4 and 10-3 were analyzed and neither showed significant associations with EFS. At the end of induction, patients with BM residual disease ≥10-3 had a 5yEFS of 51±10%, n=49 compared to 90±4.3% for those with lower levels, n=75 (p=0.0002). Especially higher residual disease at week 12 was associated with an extremely poor EFS (Fig. 3). Early identification of patients with inadequate response and designing alternative treatment for them is our important challenge. No overall benefit of transplantation was seen in patients who started on ALL treatment or hybrid ALL/AML treatment. Again, this may be caused by a biased selection of more severe cases for transplant. In patients who started with AML treatment, transplant appeared to improve prognosis (Fig. 4). This study provides the basis for improved treatment of future patients with AMBI-L, with more accurate diagnostics. OH, AL, IJ, EM and JS were supported by Czech Health Research Council 15-28525A. Disclosures Bleckmann: JazzPharma: Other: financial support of travel costs. Moricke:JazzPharma: Honoraria, Other: financial support of travel costs. Inaba:Arog: Research Funding. Kattamis:Novartis: Honoraria, Research Funding; ApoPharma: Honoraria. Reinhardt:Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Jazz Pharma: Other: Travel Accomodation; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Up to 5% of patients with acute leukemia (AL) are diagnosed as AL of ambiguous lineage. The ambiguous lineage ALs consist of mixed phenotype AL (MPAL, or biphenotypic AL, BAL), bilineal AL, switching AL and rare, undifferentiated ALs. From a molecular genetic point of view, they overlap with several molecular genetic subsets such as AL with MLL rearrangements or early T precursor AL. As no general treatment strategy exists, these patients have been variably treated with lymphoblastic (ALL)- , myeloid (AML)- or combined (hybrid) therapy, with or without stem cell transplant. They are often unreported as they are excluded from standard protocols. So far, attempts to shed more light on these patients has largely focused on definitions of ambiguous lineage AL. Only limited therapeutic observations have been possible in studies on this AL subset, usually reporting 50 or fewer pediatric/adult patients. In order to facilitate more detailed analyses, we have created an international study "iBFM AMBI2012 Study/Registry". In this study, patients under 18 years at diagnosis are eligible. Each center/country was asked to report all consecutive patients with ambiguous lineage AL, from a 2- to 13-year period ending May 31, 2015. The definitions included those with WHO and EGIL criteria and remained unchanged throughout the study. Complete information on type of treatment, follow up and immunophenotype was requested. Where available (n=101 at the time of this abstract uploading), raw cytometric FCS data files were stored centrally for review. Apart from the study itself, the central database served also as a basis for consulting individual patients during the diagnostic workup. Furthermore, data on fusion genes, cytogenetics, treatment response and availability of specimens for collateral studies were also collected. In total, 247 patients from Australia, Austria, Brazil, Czechia, Germany, Greece, Israel, Netherlands, NOPHO (Denmark, Estonia, Finland, Norway, Sweden, Iceland and Lithuania), PINDA (Chile), Poland, SAHOP (Argentina), Slovakia, St. Jude Children's Research Hospital (USA), Ukraine and United Kingdom are reported. Among those, 222 fulfilled the definitions of MPAL/BAL, partially overlapping with cases in whom two clones had been identified (n=47) and 14 cases presented with undifferentiated AL. Most of them, consistent with our general treatment guideline (Figure 1), started their treatment with an ALL type of protocol (n=150), 60 patients started on AML therapy, 8 patients received a combined regimen including the Interfant protocols, 2 patients were not treated, 13 received other treatment, and this information is missing in 9 patients (additional 5 pts. started on ALL treatment but their follow up information is incomplete). The 5 year event free survival of the entire cohort was 55±4% and its separation by first type of treatment is shown in Figure 2. In a collateral study, we set up a qPCR array on 90 genes that are characteristic of the lymphoid or myeloid lineages and/or are thought to be involved in their regulation. Using this array, sorted cells of granulocytic, monocytic, T, and B lineages at various stages of development (17 stages total) were analyzed and compared to samples of AL including 6 samples of MPAL of precursor B/myeloid phenotype. Although this array did not show a general deregulation in the MPAL genome compared to that in AL or healthy cells, subtle changes were seen such as decrease of CEBPE and LILRA2 gene expression, in comparison to classical B precursor ALL. Overall, our data shows that the general treatment guideline (Figure 1), which favors ALL treatment is justified by the outcome. However, although this study is larger than those published, caution is needed during its interpretation due to variations in diagnostics and treatment among the participating countries. Therefore, our data should be viewed as a basis for non-ambiguous treatment guidelines that will direct each patient to either ALL or AML treatment. These guidelines will be tested prospectively. In addition, the framework of this study is being used as a basis of consulting new AL cases with diagnostic uncertainties. Furthermore, it serves as a data resource for biologic studies. Supported by AZV 15-28525A, UNCE 204012, NT/14534-3, NT/13462-4, P302/12/G101. Disclosures Kattamis: Novartis: Research Funding, Speakers Bureau; ApoPharma: Speakers Bureau.

Abstract 872 Introduction: ALL IC-BFM 2002 is one of the most successful projects developed by the I-BFM-SG, which is one of the world largest societies involving national leukemia groups. The trial evaluated in a randomized manner the impact on outcome of intensified late reinduction in the context of a newly developed risk stratification. The main goal of the trial was improvement of outcome of children with ALL in each of the 3 risk groups (RG). Patients & methods: From Nov 2002-Nov 2007, 5060 eligible pts aged 0–18 yrs with newly diagnosed ALL from Argentina (1270), Chile (558), Croatia (122), Cuba (151), Czech Republic (291), Hong Kong (155), Hungary (259), Israel (292), Poland (908), Serbia (266), Slovakia (137), Slovenia (36), Ukraine (421), Uruguay (96), and Moscow (98) were enrolled in the trial (http:clinicaltrials.gov “NCT00764907”). Stratification into 3 RGs was based on early treatment response (evaluated in PB on day 8 and in BM on days 15 and 33), age, initial WBC, and presence/absence of BCR/ABL or MLL/AF4. Standard Risk (SR) criteria were: < 1,000 blasts/μL in PB day 8 after 7 days of oral prednisone with 1 intrathecal methotrexate (IT-MTX) and age ≥ 1 yr and < 6 yr and initial WBC < 20,000/μL and M1 or M2 marrow on day 15 and M1 marrow on day 33 (all criteria must be fulfilled). Intermediate Risk (IR) criteria were: < 1,000 blasts/μL in PB day 8 and age < 1 yr or ≥ 6 yr and/or WBC ≥ 20,000/μL and M1 or M2 marrow on day 15 and M1 marrow on day 33 (or SR criteria but M3 marrow on day 15 and M1 marrow on day 33). High Risk (HR) criteria were: IR and M3 marrow on day 15, PB on day 8 ≥ 1,000 blasts/μL, M2 or M3 marrow on day 33, translocation t(9;22) [BCR/ABL] or t(4;11) [MLL/AF4] (at least one criterion must be fulfilled). The majority of infants < 1 yr were treated in studies Interfant 99 and Interfant 06. Treatment consisted of protocol I‘/I, SR/IR consolidation with 6-MP and MD MTX 2g/m2 × 4 (with additional IT-MTX in maintenance) for BCP-ALL, 6-MP and HD MTX 5g/m2 × 4 for T-ALL, HR consolidation with 3 HR polychemotherapy blocks. A randomized question was asked in late intensification: SR: would 2 shorter elements (protocol III × 2) be more effective than 1 longer (protocol II × 1) even though the cumulative dose of most drugs is not increased? IR: could the risk of failure be reduced by a third reintensification element (protocol III × 3)? HR: could the use of 3 reintensification elements (protocol III × 3) achieve the same or better results than the HR approach applied in BFM (3 HR blocks + protocol II × 1) or AIEOP (protocol II × 2)? Chemotherapy was concluded by maintenance therapy (6-MP/MTX), making up a total of 2 yrs overall treatment. Prophylactic CNS radiotherapy 12 Gy was applied in T-ALL and HR pts. Results: With median follow-up 4.9 yr, the 5-yr EFS was 74 ± 1% and 5-yr OS 82 ± 1% for the whole group of 5060 pts. The CR rate was 97%, 255 (5%) children died in remission. The 5-yr EFS/OS was 81 ± 1%/90 ± 1% in 1564 SR pts (30.9% of all pts), 75 ± 1%/83 ± 1% in 2650 IR pts (52.4%) and 55 ± 2%/62 ± 2% in 846 HR pts (16.7%). Randomization rate was 79% of those patients who survived for at least 20 weeks (planned timepoint of randomization). None of the experimental arms achieved significantly better EFS compared to standard treatment. CI of relapses at 5 yr was 18 ± 1% overall, CI for isolated BM relapse was 12 ± 1%, isolated and combined CNS relapse 4 ± 0.3%, isolated and combined testicular relapse 2 ± 0.2%. Secondary malignancy was diagnosed so far in 26 patients (5-yr CI 0.6 ± 0.1%). Significantly better EFS was achieved in BCP-ALL(75 ± 1%) in comparison with T-ALL (69 ± 1%), girls vs. boys (76 ± 1% vs 72 ± 1%), children aged < 10 yr vs ≥ 10 yr (77 ± 1% vs 65 ± 1%), M1/M2 BM D15 vs. M3(76 ± 1% vs 50 ± 3%). 140 pts with Ph+ALL achieved a EFS of 47 ± 4% Allogeneic HSCT in CR1 was done in 139 pts with 5 -yr DFS of 64 ± 4%. Conclusions: Although the experimental arm was no better than the traditional one across individual RGs, the majority of participating countries, many of them were new-comers to this intensive therapy, improved their treatment results against previous national studies. The trial confirmed the validity and feasibility of a simple risk stratification of ALL applied in a complex and heterogeneous multinational environment. Despite the great differences between individual countries, the trial set a firm stage for willing national leukemia groups to run collaborative clinical trials in ALL under the umbrella of I-BFM-SG. Supported by MSM0021620813 and MZ0FNM2005. Disclosures: No relevant conflicts of interest to declare.