ALBERT

Description: DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) mutations is one of the most frequent somatic mutations in acute myeloid leukemia (AML). Although DNMT3A mutations have been studied extensively, influence of mutations on AML prognosis and their role on the pathogenesis of AML remains not completely clear. Little is known about the significance of mutations burden in the time of diagnosis on the clinical and prognostic features of AML. Recently discovered persistence of DNMT3A mutations in complete remission after standard therapy indicates the presence of mutation in early pre-leukemic stem cells. In this aspect, it would be essential to analyze the existence of DNMT3A-positive cells after allogeneic stem cell transplantation (alloSCT). Using quantitative PCR, we analyzed mutations burden in diagnostic samples obtained from 65 AML patients with DNMT3A mutation. Then follow up samples were analyzed repeatedly, after induction, consolidation therapy, and after alloSCT with a mean follow-up of 33 months. Using well-established markers of minimal residual disease and donor engraftment, we monitored DNMT3A stability during complete molecular remission (CMR). In addition, we analyzed the combination of DNMT3A mutation with others mutations, such as FLT3, NPM1, IDH1, IDH,and others, at the time of diagnosis, as well as in relapse of AML. Finally, we analyzed the prognostic impact of DNMT3A mutations depending on the distinct combination of mutations with other genetic markers. For this, 150 matched-pairs AML patients without DNMT3A mutation were analyzed. We have found considerable variation of mutations burden (from 1% till 90%) in diagnostic samples. Although, the prognostic impact of mutations burden in diagnostic samples have not been verify, we have found a significant associations of higher mutations burden with M4/M5 FAB variant of AML (p=0,04) and presence of NPM1 mutation (p=0,023). Persistence of DNMT3A mutations in CMR after standard chemotherapy was found in all patients. The loss of correlation between DNMT3A and others could be explained by sub-clone architecture and clonal evolution of AML. After alloSCT, DNMT3A mutations were not discovered in patients with CMR and complete donor chimerism. This data suggests the removal of leukemic stem cells after alloSCT and indicate the importance of alloSCT for high risk AML patients. In relapse of leukemia, all samples showed an increasing of DNMT3A mutated alleles. In addition, matched-pairs analysis suggests that DNMT3A, particularly when accompanied by FLT3-ITD, is a significant prognostic factor for inferior outcome, even after alloSCT. We conclude that long-term quantitative monitoring of DNMT3A mutations could provide additional prognostic information and could explain the role of mutations in development and progression of AML. Disclosures Pezzutto: Cellgene, Novartis, Roche, Gilead: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: Over the last years, genome and exome sequencing approaches have increased our knowledge of molecular alterations in acute myeloid leukemia (AML). However, some important limitations still need to be addressed. First, insights into the spectrum of molecular alterations of patients with refractory AML are rare, partly due to the lack of remission samples as germline control. As these patients have a dismal prognosis, there remains an unmet need to improve therapeutic options and to identify druggable molecular lesions. Secondly, in AML patients achieving a complete remission (CR), preleukemic alterations may persist in CR and are underestimated in frequency and relevance. In this work, we investigated mesenchymal stromal cells (MSC) as germline control to decipher the spectrum of molecular alterations in refractory patients with induction failure and to disclose preleukemic hits in patients achieving CR. Patients and methods : Bone marrow (BM) aspirates at initial diagnosis (ID) were obtained from 18 AML patients (9 pts with subsequent induction failure and 9 pts that achieved CR after first induction). MSC were expanded to passage 4 and defined as CD73+/CD105+/CD271+/low/CD45-/CD33- plastic-adherent cells. For all patients, BM hematopoietic cells (BM-HC; n=18) as well as MSC (n=18) were analysed at the time of first diagnosis. All samples (n=45) were analysed by exome sequencing on a HiSeq2500 (100 bp paired end) with four samples per lane. For variant calling, MSC were used as germline control. We demanded a variant allele frequency (VAF) of 〉20%, coverage of 〉30 reads and translational consequences. In germline samples, the VAF had to be 〈 5%. For patients achieving CR, BM-HC at CR were also studied (n=9). We repeated the analysis with CR BM-HC as germline control and compared the two results. For all patients, clinical as well as molecular characteristics were available. Results: We obtained an average coverage of 96 reads per base for the protein coding regions. 96% of the target region was covered at least 10-fold. The use of MSC as germline control allowed us to detect somatic mutations at initial diagnosis of refractory AML. In 9 refractory AML samples, we found 90 single nucleotide variants (SNV) and indels, which resulted in a median of 11 alterations per sample (range: 3-17). The spectrum of mutations showed an unexpectedly high rate of mutations in the spliceosome gene SRSF2 (3/9). Other recurrent mutations affected TET2 (2/9) and WT1 (2/9). Genes frequently mutated in non-selected AML were only present in one refractory patient (DNMT3A, RUNX1, IDH2, ASXL1, TP53, NRAS) or not found mutated (IDH1, KRAS). To uncover preleukemic alterations in AML patients achieving CR (n=9), we compared MSC and BM-HC at CR as germline controls. Using MSC as germline, we called 97 SNVs and indels (median: 11 per sample; range: 4-18) in the leukemic cells at ID. Thirty-three additional SNVs were called in the leukemic BM by using MSC as germline, whereas these would have been missed using BM-HC at CR as germline (median: 3 SNVs per sample, range: 0-7). These represent preleukemic hits persistent in CR with a VAF between 5% (lower bound) and 75%. Recurrently mutated genes included genes recently associated with clonal haematopoiesis in the elderly population: DNMT3A (3/9; VAF: 18%, 24%, 75%) and TET2 (2/9; VAF: 13%, 23%). In addition, mutations in ASXL1 (VAF: 14%), SRSF2 (VAF: 15%), and RUNX1 (VAF: 5%) persisted in at least one patient in CR. This unbiased approach also allowed us to identify lesions, which have not yet been associated with AML, but account for clonal events in remission. Candidates included genes linked to cancer like PROX1 (VAF: 5%), or ERBB2 (VAF: 35%), but also genes involved in NF-kB activation such as CARD8 (VAF: 30%), or NLRC3 (VAF: 10%). Conclusion: The use of MSC allows to unravel molecular lesions in refractory AML by exome sequencing. Refractory AML patients showed a high rate of mutations in the spliceosome gene SRSF2 that needs further investigations as potential therapeutic target for patients with treatment failure. Moreover, the comparison of two different germline controls (MSC and BM-HC in CR) allowed detecting persistent preleukemic alterations. In addition to known hits like in DNMT3A, TET2, or ASXL1, we systematically identified a broader spectrum of premalignant events that indicate clonal hematopoietic expansion and thereby may provide insights into leukemic transformation. Disclosures No relevant conflicts of interest to declare.

Description: Background: Approximately 5% of adult acute myeloid leukemia (AML) cases are associated with balanced translocations of chromosome 11q23, and AML with t(9;11)(p22;q23) is recognized as a distinct entity by the WHO Classification. Similarly, the presence of t(4;11)(q21;q23), which accounts for 8-10% of B-cell precursor acute lymphoblastic leukemia (ALL) in patients over the age of 20 years, defines a distinct entity termed "B-lymphoblastic leukemia with t(v;11q23)" according to the WHO Classification. On the molecular level, t(11q23) result in fusion of the KMT2A (also called MLL) gene, which encodes a histone 3 lysine 4 methyltransferase, to a broad spectrum of more than 70 partner genes. The prognosis of patients with relapsed/refractory KMT2A-rearranged leukemia is very poor, and new treatment approaches are needed. Using in vitro and in vivo experimental models, we previously identified cyclin dependent kinase 6 (CDK6) as a potential therapeutic target in KMT2A-rearranged leukemias (Placke et al. Blood. 2014;124:13-23). Aims: To evaluate the tolerability and efficacy of the small-molecule CDK4/6 inhibitor palbociclib in KMT2A-rearranged AML and ALL within a genotype-guided clinical trial (AMLSG 23-14; ClinicalTrials gov. Identifier NCT02310243). Methods: Patients with KMT2A-rearranged leukemia, either relapsed/refractory or newly diagnosed but ineligible for intensive chemotherapy, are enrolled. The study is a phase Ib/IIa trial with a safety/tolerability part in the phase Ib using the standard palbociclib dose of 125 mg once daily for 21 days in a 28-day cycle. Based on a 3+3 modified Fibonacci design, a dose deescalation to 100 mg and 75 mg in case of toxicity is possible in sequential cohorts. If no or only one limiting toxicity is observed among 6 patients at one dose level, this dose level will be taken forward to the phase IIa expansion part of the study. Limiting toxicities are defined as toxicities attributable to palbociclib, expected or unexpected. The expansion part of the study is based on Simon's optimal 2-stage design with 18 patients and 43 patients in the 2 stages. Results: The phase Ib of the study has been completed with recruitment of 6 patients with relapsed/refractory leukemia (AML, n=3; treatment-related AML, n=2; ALL, n=1; refractory to intensive chemotherapy, n=2; relapse, n=4 [following allogeneic stem cell transplantation, n=3; following chemotherapy, n=1]). Cytogenetic results were as follows: t(9;11), n=3; t(6;11), n=1; t(11;19), n=1; t(4;11), n=1. The median white blood cell count (WBC) at study inclusion was 7.05 G/l (range, 0.9-61.0). To control hyperleukocytosis, 3 patients were treated with hydroxyurea during the first week of palbociclib and one patient with corticosteroids. No limiting toxicity occurred during the first 28-day cycle, the limiting-toxicity assessment period. White blood cell counts rapidly decreased after one week of palbociclib at a dose of 125 mg/day and remained low until week 3 (median, 1.6 G/l; range, 0.6-1.9). The median WBC after one week of drug holiday was 1.9 G/l (range, 1.3-7.3). Response assessment revealed one partial remission, 3 disease stabilizations, and 2 cases of progressive disease. Four patients completed further treatment cycles (median, 2; range 2-6), with one patient achieving a complete remission with incomplete hematologic recovery after cycle 2. This patient, a 76-year-old man with t(11;19)-positive de novo AML refractory to chemotherapy with daunorubicin and cytarabine, relapsed after cycle 6, and correlative laboratory studies are underway to determine potential resistance mechanisms. Conclusions: Palbociclib is well tolerated in patients with refractory/relapsed KMT2A-rearranged leukemia with no occurrence of limiting toxicities and has clinical activity in this prognostically unfavorable subset of AML/ALL. Therefore, the study will be taken forward to the efficacy part with accrual of further patients. In addition, the protocol is currently amended as a basket trial with inclusion of patients with locally advanced/metastatic chordoma based on preclinical evidence that CDK4/6 dependence represents a specific liability of chordoma cells that could be exploited for therapeutic benefit. Disclosures Lübbert: Celgene: Other: Travel Funding; Ratiopharm: Other: Study drug valproic acid; Janssen-Cilag: Other: Travel Funding, Research Funding. Schlenk:Amgen: Research Funding; Pfizer: Honoraria, Research Funding.

Description: Background: Measurable residual disease (MRD) can identify patients (pts) with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) in complete hematological remission (CR) at high risk of relapse even after allogeneic hematopoietic stem cell transplantation (HSCT). We have recently shown in 53 pts treated within the first cohort of the RELAZA2 trial that pre-emptive therapy with azacitidine (AZA) at the time of MRD-positivity (MRDpos) can successfully prevent imminent hematological relapse (Platzbecker et al. Lancet Oncol. 2018). We now report on the results of the second cohort of 41 pts undergoing MRD-guided treatment in the RELAZA2 trial (ClinicalTrials.gov NCT01462578) by the Study Alliance Leukemia (SAL). Methods: Between 2015 and 2018, 166 MDS/AML pts were screened and centrally monitored for MRD in bone marrow or peripheral blood at monthly intervals for a period of 2 years prospectively in 9 centers in Germany. Of these 166, 41 pts with either advanced MDS (n=6) or AML (n=35) in CR after either conventional chemotherapy only (n=13) or consecutive allogeneic HSCT (n=28) developed MRD above a threshold defining imminent hematological relapse. Still being in morphological CR, these pts pre-emptively received 6 cycles of AZA (75mg/m2, s.c. days 1-7), which was followed by a risk-adapted AZA-maintenance therapy based on MRD-response for up to 18 additional months. Pts developing a hematological relapse went off study. MRD was detected by either the quantification of NPM1 mutation level (n=19), leukemia-specific fusion genes DEK-NUP214 (n=1) or RUNX1/RUNX1T1 (n=2) or a sensitive donor chimerism analysis of sorted CD34(+)/CD117(+) peripheral blood cells (n=28) in pts undergoing allogeneic HSCT. Here, we report the analysis of the primary endpoint of the 41 pts in the second cohort as well as the data for the entire 94 pts who entered the treatment phase of the RELAZA-2 study. Results: At a median of 110 days (range 28-476) after start of screening, 41 (25%) out of 166 prospectively screened pts became MRDpos as defined by either a decrease of CD34(+)/CD117(+) donor chimerism to 1% (NPM1 n=18) while being still in hematological CR. All of these MRDpos pts started AZA-based pre-emptive treatment to prevent imminent hematological relapse. Six months after start of MRD-guided therapy, 25 out of 41 pts were still in CR (61%, 95%-CI 45-76%, p

Description: AML in the elderly has a very poor outcome. Despite advances in the characterization of molecular alterations in younger AML patients, comprehensive studies in elderly AML are still lacking to uncover their distinct underlying molecular alterations. In this project, we investigated genetic and epigenetic modifications to unravel the molecular background of this unfavorable disease. To capture a broad spectrum of relevant alterations in elderly AML, we performed genomic profiling by target enrichment of 555 candidate genes (mutated in cancer), followed by next generation sequencing on an Illumina HiSeq 1500 platform. Reads were mapped to NCBI hg19 RefSeq and SNV or INDELs were called if they were non-silent coding variations with a coverage of at least 〉 30x and a variant allele frequency higher than 20%. The mean coverage of the target sequence was 382±160x and 98±2% of the target sequence had a read depth of at least 30x. We applied this sequencing strategy on diagnostic bone marrow samples of 100 AML patients enrolled on the Studienallianz Leukämie (SAL) registry. This cohort was composed of patients 65 to 90 years old (median 72 years), 76% were classified as de novo AML, 19% as secondary AML, 5% as therapy-related AML. In addition, we investigated the DNA methylation profile for this cohort with an Illumina 450k methylation array to further characterize the heterogeneity of elderly AML. Overall, 817 mutations were detected in 292 of the 555 candidate genes, 80 of these genes were mutated in more than two patients. A median number of 7 genes were mutated per patient (range: 1 to 23). Several known mutations were identified with a particular high frequency: DNMT3A 33%, TET2 24%, SRSF2 23%, ASXL1 21%, RUNX1 18%, IDH1 17%, NPM1 15%, IDH2 and BCOR 10% each. We also identified novel aberrations (not previously reported in AML) that provide new insights into the specificity of this disease, including mutations in the PI3K/mTOR pathway (PIK3C2B, MTOR), DNA damage proteins (BRCA2, ERCC2, FANCC, PMS1) and histone modifiers (EP300, JARID2, NSD1, MYST3). When compared to younger AML (less than 65 years, TCGA cohort NEJM 2013), elderly AML showed significantly higher mutation rates in ASXL1 (21% vs 1%), TET2 (24% vs 7%), RUNX1 (18% vs 7%), BCOR (10% vs 1%) and BRCA2 (8% vs 0%). In addition, we found a high rate of mutations in splicing regulators affecting 38% of elderly AML patients (SRSF2 23%, U2AF1 6%, SF3B1 5%, DDX5 3%, ZRSR2 2%), similarly distributed between de novo and secondary AML. Notably, 15% of elderly AML patients had mutations in the DNA repair genes TP53, NBN, ATM, FANCA, FANCC, likely responsible for drug resistance and unfavorable outcome: patients with DNA repair mutations had a median survival of only 4 months, compared to 16 months for patients without these mutations (p=5.99e-5); variations in these DNA repair proteins predicted poor overall survival independently of the TP53 mutational status (p=0.004). To describe the molecular heterogeneity of the disease we included 152 genes (mutated in more than 1 patient) to build a reactome functional interaction network and identified 9 different network modules. The first most prominent module comprised 15 mutual exclusively mutated genes including DNMT3A and genes of the DNA repair pathway (p=0.013; purple network in figure). The second most distinct module included 12 genes also being altered in a mutually exclusive manner (p=4.9e-4): NPM1, RNA splicing and transport genes (green network in figure). Together the alterations from these two modules affected 88% of the patients and showed no significant overlap (mutual exclusivity test p=7.5e-4). These findings indicate that elderly AML is characterized by two distinct molecular patterns, with patients frequently having one of the two modules altered (mutual-exclusive mutation plot of the two modules in figure). In conclusion, elderly AML harbors a high frequency of molecular alterations in spliceosome components, epigenetic regulators and in DNA repair factors, the latter being associated with poor prognosis. The characterization of recurrent mutations may guide the development of new strategies to adapt treatment for older AML patients. In this regard, the molecular categorization of elderly AML into two groups (DNA repair or RNA processing deficient) underscores the distinct biology and the need for molecularly driven therapeutic approaches. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Background In newly diagnosed acute myeloid leukemia (AML), the general recommendation is to start treatment immediately after the diagnosis has been made. This paradigm is based both on the observation that untreated acute leukemia has a poor prognosis and on retrospective analyses demonstrating a shorter survival in younger AML patients (pts) in whom treatment was delayed by more than 5 days (Sekeres et al., 2009). A more recent single-center analysis came to a different conclusion, showing no prognostic effect for the time from diagnosis to treatment (TDT; Bertoli et al., 2013). We explored the relationship between TDT and prognosis on a large set of real-world data from the AML registry of the Study Alliance Leukemia (SAL) and compared it to the published cohorts. Methods The SAL runs a transregional AML registry in 46 treatment centers across Germany (NCT03188874). All registered patients with an intensive induction treatment, a minimum follow-up time of 12 months and no acute promyelocytic leukemia were selected (n=2,200). Treatment start was defined by the first day of cytarabine, whereas single agent hydroxyurea (HU) was labeled as pretreatment. We analyzed the influence of TDT on complete remission (CR), early death (ED) and overall survival (OS) in univariable analyses for each day of treatment delay, in groups of 0-5, 6-10, 11-15 and 〉15 days of TDT, and by using the restricted cubic spline (RCS) method for data modelling. In order to adjust for the influence of established prognostic variables on the outcomes, we used multivariable regression models and propensity score weighting. The influence of HU pretreatment on outcomes was investigated by introducing an interaction term between TDT and the presence of HU pretreatment. Results The median age was 59 years (y) (IQR 50-68), the proportion of pts with favorable, intermediate and adverse genetic risk according to ELN was 27%, 53%, and 20%; 〉95% of pts received induction treatment with standard 7+3. HU pretreatment was administered in 4% of pts. The median TDT was 3 days (IQR 2-6). Descriptive statistics after grouping of pts showed the highest median age and the lowest proportion of NPM1 mutated and favorable risk in the TDT group 11-15. Of all pts, 79% achieved a CR/CRi; unadjusted CR rates for the patient groups with TDT of 0-5, 6-10, 11-15 and 〉15 days were 80%, 77%, 74% and 76%, respectively (p=0.317). In multivariable analysis accounting for the influence of ELN risk, age, WBC, LDH, de novo versus secondary AML and ECOG, the OR for each additional day of TDT was 0.99 (95%-CI, 0.97-1.00; p=0.124). Four percent of pts died within the first 30 days from treatment start. The respective rates in the four TDT categories were 4.0%, 3.8%, 5.1% and 4.1% (p=0.960). In multivariable analysis, the OR for TDT was 1.01 (95%-CI, 0.98-1.05; p=0.549). After a median follow-up of 40 months, the 2-y OS of all pts was 51%. The unadjusted 2-y OS rates stratified by TDT of 0-5, 6-10, 11-15, 〉15 days were 52, 49, 46, and 51% (see Table 1 and Figure 1). The hazard ratio (HR) for each day of treatment delay was 1.00 (95%-CI; 0.99-1.01; p=0.317). In multivariable Cox regression analysis, the HR for TDT as continuous variable was 1.00 (95%-CI, 0.99-1.01; p=0.689). When OS was analyzed separately stratified for age ≤60 and 〉60 ys and for high versus lower initial WBC defined by a threshold of 50 x 109/L, no significant differences between TDT groups were observed. Multivariable models using TDT as a grouped variable or with RCS did not provide evidence for a significant influence of TDT on outcomes. Propensity score matching of pts in the four TDT groups did not reveal an influence on outcomes. The use of HU was not associated with CR, ED nor OS. Conclusion Our study on 2,200 newly diagnosed registry pts receiving consistent intensive induction with standard-dose cytarabine plus daunorubicin (7+3) suggests that TDT is not related to response or survival, neither in younger nor in older pts. Despite multivariable analyses, a bias towards longer TDT intervals in pts judged to be clinically stable by the treating physician cannot be excluded entirely. As treatment stratification in intensive first-line treatment of AML evolves, the TDT data suggests that it may be a safe and reasonable approach to wait for genetic and other laboratory test results in order to assign clinically stable pts to the best available treatment option before the start of intensive treatment. Disclosures Krämer: Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; BMS: Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hänel:Roche: Honoraria; Amgen: Honoraria; Celgene: Other: advisory board; Novartis: Honoraria; Takeda: Other: advisory board. Jost:Daiichi: Honoraria; Sanofi: Honoraria; Gilead: Other: travel grants; Jazz Pharmaceuticals: Honoraria. Brümmendorf:Merck: Consultancy; Janssen: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; University Hospital of the RWTH Aachen: Employment; Ariad: Consultancy. Krause:Siemens: Research Funding; Takeda: Honoraria; MSD: Honoraria; Gilead: Other: travel; Celgene Corporation: Other: Travel. Scholl:Novartis: Other: Project funding; Pfizer: Other: Advisory boards; Gilead: Other: Project funding; Daiichi Sankyo: Other: Advisory boards; AbbVie: Other: Advisory boards. Hochhaus:Pfizer: Research Funding; Novartis: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding. Kiani:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Middeke:Sanofi: Research Funding, Speakers Bureau; Roche: Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Gilead: Consultancy; Janssen: Consultancy, Speakers Bureau; MSD: Consultancy. Thiede:AgenDix GmbH: Employment, Equity Ownership; Novartis: Research Funding, Speakers Bureau; Bayer: Research Funding; Daiichi-Sankyo: Speakers Bureau. Stoelzel:JAZZ Pharmaceuticals: Consultancy; Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding. Platzbecker:Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

Description: Introduction: In T-ALL, in contrast to BCP-ALL, molecular subgroups are less well defined. Molecular subgroups are based on aberrant expression of oncogenes often resting upon structural aberrations (chromosomal translocation, copy number variations, point mutations). So far, comprehensive studies on molecular subgroups have been predominantly performed in pediatric patients. The clinical relevance of molecular characteristics is not taken into account for risk stratification and targeted therapeutic options for T-ALL patients are limited. Risk stratification is mainly based on immunophenotype and MRD response. Thus, in the German Multicenter Study Group for Adult ALL (GMALL) protocols early and mature T-ALL and molecular failure after first consolidation are high risk features. Whole RNA transcriptome sequencing (RNAseq) enables molecular subgroup allocation profiles with potential prognostic relevance. Herein, we investigated a large cohort of 161 adult T-ALL patients with RNAseq for molecular subgroups and their clinical implication. Patients and methods : RNAseq data (Illumina HiSeq 4000, 100 or 125 bp paired-end, average read count ~30 million reads/sample) were generated from 161 adult T-ALL patients from diagnostic bone marrow samples. For 84 of these T-ALL patients, we additionally investigated DNA methylation using Infinium 450k methylation bead arrays and mutation status using deep targeted DNA sequencing with a gene panel consisting of 206 genes (HiSeq 1500, 100 bp paired-end, average ~800 reads/bp). All patients were treated likewise within pediatric inspired protocols of the GMALL. Clinical follow up and outcome data were available for 129 patients aged between 18 and 55 years. Results: Based on oncogene expression, we could assign molecular subgroups to 160 out of the 161 T-ALL patients: HOXA: n=37 (23%), TLX1: n=37 (23%), TAL/LMO: n=33 (20%), LYL1/LMO2: n=31 (19%), TLX3: n=17 (11%) , NKX2-1: n=4 (2%), TAL2: n=1 (1%). Among others, gene fusion events incorporating TAL1 (n=16), HOXA (n=6), TLX1 (n=14), TLX3 (n=2) and NKX2 (n=2) were detected as underlying mechanisms. Age distribution revealed more TAL/LMO in the younger population (16-25 years: 35% versus 〉35 years: 3%; p=0.001) and more LYL1/LMO2 and HOXA in the older patients (16-25 years: 23% vs. 〉35 years: 40%; n.s.). DNA methylation analyses for 84 of the 161 patients support the molecular classification with four distinct subgroups demonstrating a homogenous methylation profile for the TLX1 driven subgroup (methylation cluster M3, n=30). In contrast, methylation cluster M2 (n=21) showed a hypomethylation in CpG islands and comprises the majority of TAL/LMO positive cases (19/21) including all samples with STIL-TAL1 fusions. M1 (n=25) and M4 (n=7) clusters comprised TLX3 (mainly cluster M1, 8/10), HOXA and LYL1/LMO2 cases. Mutation analysis showed a high rate of NOTCH1 (n=71%) mutations in the TLX1 subgroup and an increased rate of mutations in the JAK/STAT pathway (n=29%) and epigenetic regulators (n=50%) in HOXA and LYL1/LMO2 subgroups. Regarding clinical outcome, 126 out of 129 (98%) patients achieved a morphologically complete remission (CR), two patients failed CR, one patient died during induction therapy. Regarding MRD response, 12% of the investigated T-ALL patients showed a molecular failure after consolidation. Noteworthy, we found a molecular CR for 28/30 (93%) patients in the TLX1 subgroup, but only 11/19 (58%) respectively 2/6 (33%) in the HOXA and the LYL1/LMO2 subgroup. This finding results in exceptional five year-overall survival (5y-OS) of 93% in TLX1 patients. Together with the NKX2-1 subgroup (n=4, with 100% 5y-OS), these patients build a prognostically favourable risk group in the overall cohort (94% 5y-OS versus 76% in TAL-LMO patients versus 62% in all other subgroups, p=0.007). This result could not only be found in the overall cohort, but also within the already good risk subgroup of thymic T-ALL patients (93% vs. 75%, p=0.02). Conclusion: This is to our knowledge the largest cohort of adult T-ALL patients characterized by RNAseq on molecular level. Our findings point towards an age dependent distribution of molecular subgroups contributing to age-dependent outcome differences. Patients with TLX1 reveal a molecular subgroup with extraordinary good prognosis. Within thymic T-ALL, the subgroup of patients without TLX1 (~53%) has an inferior but still good prognosis. Disclosures Fiedler: Gilead: Other: support for meeting attendance; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Abbvie: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria; ARIAD/Incyte: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding; Daiichi Sankyo: Other: support for meeting attendance. Alakel:Pfizer: Consultancy. Schwartz:AMGEN: Other: personal fees and non-financial support; Novartis: Other: personal fees and non-financial support; Jazz Pharmaceuticals: Other: personal fees and non-financial support; Pfizer: Other: personal fees ; BTG Intl Inc: Other: personal fees ; Gilead Sciences: Other: personal fees and non-financial support ; MSD Sharp & Dohme: Other: personal fees ; Basilea: Other: non-financial suppor. Müller-Tidow:Pfizer: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Janssen-Cilag GmbH: Speakers Bureau; BiolineRx: Research Funding. Brüggemann:Regeneron: Research Funding; Affimed: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Incyte: Consultancy; Roche: Consultancy; Celgene: Consultancy. Goekbuget:Servier: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Gilead: Consultancy; Erytech: Consultancy; Kite: Consultancy; Jazz: Consultancy, Research Funding.

Description: Background: Monitoring of measurable residual disease (MRD) in patients (pts) with advanced myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) who achieve complete remission (CR) can predict hematological relapse. Recently published data from the first cohort of the RELAZA2-trial have shown that pre-emptive therapy with azacitidine (AZA) can prevent or substantially delay an overt relapse in MRD-positive pts with MDS or AML (Platzbecker et al. Lancet Oncol. 2018). Aims: To evaluate outcome of the entire patient cohort of the RELAZA2-trial and determine whether MRD-guided pre-emptive AZA treatment could prevent relapse in molecularly defined cohorts. Methods: Between 12/2011 and 07/2018 380 pts with advanced MDS or AML, who had achieved CR after conventional chemotherapy or allogeneic hematopoietic stem-cell transplantation (allo-HCT) were prospectively screened for MRD in monthly intervals either in bone marrow (BM) or peripheral blood (PB). A total of 94 pts (AML, n=83; MDS, n=11) became MRD positive during 24 months from baseline by either quantitative PCR (qPCR) or analysis of CD34+ donor-chimerism and entered the treatment phase. Preemptive MRD-triggered treatment consisted of AZA 75 mg/m2 per day subcutaneously on days 1-7 of a 29-day cycle for up to 24 cycles. After six cycles, MRD status was reassessed and pts with MRD negativity were eligible for treatment de-escalation. Primary endpoint was relapse-free survival (RFS) six months after start of pre-emptive treatment. For mutational analysis next generation sequencing (NGS) with a panel of 54 genes was performed (Illumina Trusight Myeloid). Results: Median age was 60 yrs (range: 22-80 yrs); 52 (55%) of the pts were female. Prior therapy consisted of chemotherapy in 42 (45%) and allo-HCT in 52 (55%) of the pts. Cytogenetics could be analyzed in 93 (99%) of the 94 pts. Risk categorization according to ELN 2017 was favorable in 30 (37%), intermediate in 31 (38%) and adverse in 21 (26%) of the AML pts, respectively. Type of MDS was advanced in all 11 pts and all were previously transplanted. Fifty-two (61%) of 85 pts with available NPM1 status were positive. NGS on 64 (68%) pts with available DNA at the time of first diagnosis revealed additional mutations in DNMT3A (n=25), TET2 (n=15), FLT3-ITD (n=12), IDH1 (n=9), FLT3-TKD (n=8), ASXL1, NRAS, TP53 (n=7, each), IDH2 (n=6), PTPN11, WT1 (n=5, each), GATA2, U2AF1 (n=4, each), CBL (n=3), CEBPA, CSFR3, CUX1, EZH2, KIT, RAD21, RUNX1, SF3B, STAG2, ZRSR2 (n=2, each), and KRAS (n=1). MRD data were correlated with outcome in 45 pts for NPM1, in 3 for RUNX1-RUNX1T1, whereas CD34-donor-chimerism was analyzed in 39 pts (missing, n=7). There was a significant faster and deeper decline of MRD in PB as compared to BM (P=0.03). The same held true with regard to the increase of donor-chimerism, which was achieved faster in PB as compared to BM (P=0.05). Secondary molecular abnormalities (MAs) had no impact on MRD response as measured by qPCR, which was also true if MAs were categorized functionally. Similarly, additional chromosomal abnormalities had no impact on MRD response in both MRD methods. However, in pts with measurement of donor-chimerism ASXL1 mutations were a negative factor for MRD response (P

Description: MRD in ALL is defined as the detection of leukemic cells in bone marrow below the microscopic threshold in complete remission (CR). Patients (pts) with molecular failure (MolFail) or molecular relapse (MolRel) after induction/consolidation therapy are at a high risk for hematologic relapse. Targeted therapies should prevent hematologic relapse, reduce MRD load and provide a bridging strategy to allogeneic stem cell transplantation (SCT) and thereby improve overall outcome of these pts. In pts without (wo) SCT option reduction of MRD load is an essential goal as well. Blinatumomab is an antibody construct that redirects CD3+ T cells to CD19+ target cells, resulting in a serial lysis of CD19+ B cells. In a study in pts with MRD ≥10-3, 78% achieved complete MRD response (Gökbuget N et al., Blood 2018). The MolAct1 trial was initiated by the GMALL study group to evaluate the efficacy and tolerability of Blinatumomab in MRD+ ALL including those with MRD below 10-3 and pts with MRD after SCT. Adults (≥18 yrs) with CD19+, Ph-negative BCP ALL in CR after ≥ 3 chemotherapies with MRD ≥10-4 were eligible (NCT03109093). Recruitment of pts with MRD ≥10-3 was stopped after marketing authorization for this entity. After an amendment, which became effective after 44 recruited pts, pts with MRD below 10-4 or non-quantifiable (nq) MRD were eligible. Blinatumomab 28 μg/day was given as 4-wk infusion, followed by a 2-wk break (1 cycle). Responders could receive up to 4 cycles or undergo HSCT after ≥ 1 cycle. MRD after 1 cycle was the primary endpoint. MRD was centrally assessed by allele-specific quantitative real-time PCR of clonal rearrangements of immunoglobulin or T-cell receptor genes. For definition of MRD at inclusion and at response assessment see table 1. 64 pts with a median age of 44 (18-83) yrs were included from 19 centers and 60 were evaluable. 63 pts were treated in first CR (5 after SCT). Overall, 67% achieved MolCR, 10% had MolFail, 23% MolNE. MolNE identifies an intermediate response with different options clarified table 1. 81% of the pts included with MRD ≥10-4 had a molecular response i.e. MolCR or MRD 〈 10-4. No significant differences in terms of MRD response were observed according to MRD level at inclusion or other patient characteristics (table 1). 60 pts have completed study treatment (40 HSCT, 8 relapses during treatment, 4 completed 4 cycles wo SCT, 2 stopped earlier due to toxicities - 1 with subsequent SCT, 1 due to GvHD, 1 due to physicians' decision and 4 pts returned to standard treatment after 2 cycles). SCT pts had a median age of 42 (18-66) yrs and follow-up is available in 37 / 41 pts (29 CCR, 3 relapse, 5 death in CR). 16 relapses occurred: 8 during treatment (1 after MolCR, 5 MolNE1-3 and 2 with MolFail resp); after SCT in 3/41 pts; 5/11 with CR at end of treatment wo subsequent SCT. The median observation time of surviving pts is 12 (1-38) mo and the median survival is not reached. At 2 yrs the survival probability (OS) was 64%. OS was 70%, 64% and 43% in pts with MRD between 10-4-10-3, 10-3-10-2 and 〉10-2 at inclusion, resp (p〉.05; Fig.1). OS was 71% vs 54% in pts MolFail vs MolRel at inclusion (p〉.05). OS was 72%, 40% and 56% in pts with MolCR, MolFail and MolNE after cycle 1 resp (P=0.02; Fig.2). Overall, the results from previous trials were confirmed. In addition, it was demonstrated that pts with MRD between 10-4 and 10-3 had a similar response and a trend towards better outcome compared to pts with higher MRD levels 〉10-2. So far only 7 pts with MRD below 10-4 were included; more data are needed to evaluate the impact of Blinatumomab in this population; GMALL does currently not recommend SCT for these pts unless there is an indication due to other risk factors. Interestingly, a significant proportion of pts (23%) had an incomplete MRD response and the outcome results indicate that these pts together with those with MolFail may have an inferior survival compared to those with MolCR. The results underline that the clear definition of MRD categories and consideration of level and sensitivity is essential for interpretation, which is possible due to well defined standards for the PCR method used here. 68% of the pts received SCT in CR after Blinatumomab with a so far limited mortality (13%). Further follow-up is certainly required. The GMALL will continue to recruit patients with MRD levels below 10-3 in order to improve their chances for long-term survival This study was supported by Amgen Inc. Disclosures Goekbuget: Servier: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Erytech: Consultancy; Kite: Consultancy; Gilead: Consultancy. Schwartz:Pfizer: Other: personal fees ; AMGEN: Other: personal fees and non-financial support; BTG Intl Inc: Other: personal fees ; Gilead Sciences: Other: personal fees and non-financial support ; MSD Sharp & Dohme: Other: personal fees ; Basilea: Other: non-financial suppor; Novartis: Other: personal fees and non-financial support; Jazz Pharmaceuticals: Other: personal fees and non-financial support. Hüttmann:Lead Discovery Center GmbH: Consultancy; University Hospital Essen, University of Duisburg-Essen, Essen, Germany: Current Employment; Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Seattle Genetics: Research Funding; Roche: Other: Travel expenses; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Gilead: Honoraria. Viardot:Roche: Honoraria, Other: advisory board; Kite/Gilead: Honoraria, Other: advisory board; Novartis: Honoraria, Other: advisory board; Amgen: Honoraria, Other: advisory board. Fiedler:Daiichi Sankyo: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Abbvie: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria; ARIAD/Incyte: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding. Alakel:Pfizer: Consultancy. Stelljes:Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Subklewe:Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Morphosys: Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy; Roche AG: Consultancy, Research Funding. Wäsch:Pfizer: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Vucinic:Celgene: Honoraria. Fransecky:Amgen: Consultancy. Bargou:Amgen: Consultancy, Honoraria, Patents & Royalties; Gemoab: Consultancy, Honoraria; Cellex: Consultancy, Honoraria; Catalym: Consultancy, Honoraria. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Brüggemann:Regeneron: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy; Incyte: Consultancy; Roche: Consultancy; Affimed: Research Funding; Janssen: Consultancy, Honoraria. OffLabel Disclosure: Blinatumomab in MRD positive disease below 10-3