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Figure S1 Recurrently mutated genes and comparison to TCGA cohort. Figure S2 Stabililty of recurrently mutated genes over disease course. Figure S3 Variant allele frequency plots from diagnosis to relapse for each individual patient. Figure S4 Mutation patterns of individual genes. Lines represent mutations in individual patients. Figure S5 Detection of the pre-existence of gained variants at initial diagnosis. Figure S6 Mutations in AML-associated functional pathways. Figure S7 Deletion of exons 3-10 of the KDM6A gene in the MM-6 cell line. The sister cell line MM-1 is not affected. Deletions were detected by quantitative MLPA analysis and the peak ratio for each KDM6A exon specific probe is shown. Graph represents the results from two independent experiments. Figure S8 H3K27 methylation in the AML cell lines MM-1 and MM-6. The global mono-, di- and tri-methylation of H3K27 in MM-1 and MM-6 was analyzed by Western blot and normalized to H3. The median of three independent experiments is shown. P-Values were calculated using a two-tailed, unpaired Student's t-test. Figure S9 The MM-6 cell line is resistant to cytarabine (Ara-C) but not to Daunorubicin or AC220. Error bars indicate mean {plus minus} s.d. of at least three independent experiments. **P75th percentile) with clinical outcome according to gender in the AMLCG-99 trial (NCT00266136). Figure S11 Proportion of transversions from diagnosis to relapse. Transversion frequencies are shown for lost (blue), stable (orange) and gained (red) mutations. Dots represent individual patients. Figure S12 Median coverage in targeted sequencing of persistent and non-persistent DNMT3A variant positions at complete remission (CR). Figure S13 Clinical outcome according to mutation persistence at remission.

Purpose: To study mechanisms of therapy resistance and disease progression, we analyzed the evolution of cytogenetically normal acute myeloid leukemia (CN-AML) based on somatic alterations.Experimental Design: We performed exome sequencing of matched diagnosis, remission, and relapse samples from 50 CN-AML patients treated with intensive chemotherapy. Mutation patterns were correlated with clinical parameters.Results: Evolutionary patterns correlated with clinical outcome. Gain of mutations was associated with late relapse. Alterations of epigenetic regulators were frequently gained at relapse with recurring alterations of KDM6A constituting a mechanism of cytarabine resistance. Low KDM6A expression correlated with adverse clinical outcome, particularly in male patients. At complete remission, persistent mutations representing preleukemic lesions were observed in 48% of patients. The persistence of DNMT3A mutations correlated with shorter time to relapse.Conclusions: Chemotherapy resistance might be acquired through gain of mutations. Insights into the evolution during therapy and disease progression lay the foundation for tailored approaches to treat or prevent relapse of CN-AML. Clin Cancer Res; 24(7); 1716–26. ©2018 AACR.

Background & Aims Patient-derived tumor organoids recapitulate the characteristics of colorectal cancer (CRC) and provide an ideal platform for preclinical evaluation of personalized treatment options. We aimed to model the acquisition of chemotolerance during first-line combination chemotherapy in metastatic CRC organoids. Methods We performed next-generation sequencing to study the evolution of KRAS wild-type CRC organoids during adaptation to irinotecan-based chemotherapy combined with epidermal growth factor receptor (EGFR) inhibition. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 protein (Cas9)-editing showed the specific effect of KRASG12D acquisition in drug-tolerant organoids. Compound treatment strategies involving Aurora kinase A (AURKA) inhibition were assessed for their capability to induce apoptosis in a drug-persister background. Immunohistochemical detection of AURKA was performed on a patient-matched cohort of primary tumors and derived liver metastases. Results Adaptation to combination chemotherapy was accompanied by transcriptomic rather than gene mutational alterations in CRC organoids. Drug-tolerant cells evaded apoptosis and up-regulated MYC (c-myelocytomatosis oncogene product)/E2F1 (E2 family transcription factor 1) and/or interferon-α–related gene expression. Introduction of KRASG12D further increased the resilience of drug-persister CRC organoids against combination therapy. AURKA inhibition restored an apoptotic response in drug-tolerant KRAS–wild-type organoids. In dual epidermal growth factor receptor (EGFR)– pathway blockade-primed CRC organoids expressing KRASG12D, AURKA inhibition augmented apoptosis in cases that had acquired increased c-MYC protein levels during chemotolerance development. In patient-matched CRC cohorts, AURKA expression was increased in primary tumors and derived liver metastases. Conclusions Our study emphasizes the potential of patient-derived CRC organoids in modeling chemotherapy tolerance ex vivo. The applied therapeutic strategy of dual EGFR pathway blockade in combination with AURKA inhibition may prove effective for second-line treatment of chemotolerant CRC liver metastases with acquired KRAS mutation and increased AURKA/c-MYC expression., Graphical abstract

Key Points Prediction of induction failure in AML is possible using cytogenetic data and a gene expression–based classifier.Integration of PS29MRC in the clinical routine or trials may be facilitated by gene expression analysis with the NanoString platform., Visual Abstract, Prediction of resistant disease at initial diagnosis of acute myeloid leukemia (AML) can be achieved with high accuracy using cytogenetic data and 29 gene expression markers (Predictive Score 29 Medical Research Council; PS29MRC). Our aim was to establish PS29MRC as a clinically usable assay by using the widely implemented NanoString platform and further validate the classifier in a more recently treated patient cohort. Analyses were performed on 351 patients with newly diagnosed AML intensively treated within the German AML Cooperative Group registry. As a continuous variable, PS29MRC performed best in predicting induction failure in comparison with previously published risk models. The classifier was strongly associated with overall survival. We were able to establish a previously defined cutoff that allows classifier dichotomization (PS29MRCdic). PS29MRCdic significantly identified induction failure with 59% sensitivity, 77% specificity, and 72% overall accuracy (odds ratio, 4.81; P = 4.15 × 10−10). PS29MRCdic was able to improve the European Leukemia Network 2017 (ELN-2017) risk classification within every category. The median overall survival with high PS29MRCdic was 1.8 years compared with 4.3 years for low-risk patients. In multivariate analysis including ELN-2017 and clinical and genetic markers, only age and PS29MRCdic were independent predictors of refractory disease. In patients aged ≥60 years, only PS29MRCdic remained as a significant variable. In summary, we confirmed PS29MRC as a valuable classifier to identify high-risk patients with AML. Risk classification can still be refined beyond ELN-2017, and predictive classifiers might facilitate clinical trials focusing on these high-risk patients with AML.

Identification of fusion genes in clinical routine is mostly based on cytogenetics and targeted molecular genetics, such as metaphase karyotyping, fluorescence in situ hybridization and reverse-transcriptase polymerase chain reaction. However, sequencing technologies are becoming more important in clinical routine as processing time and costs per sample decrease. To evaluate the performance of fusion gene detection by RNAsequencing compared to standard diagnostic techniques, we analyzed 806 RNA-sequencing samples from patients with acute myeloid leukemia using two state-of-the-art software tools, namely Arriba and FusionCatcher. RNA-sequencing detected 90% of fusion events that were reported by routine with high evidence, while samples in which RNA-sequencing failed to detect fusion genes had overall lower and inhomogeneous sequence coverage. Based on properties of known and unknown fusion events, we developed a workflow with integrated filtering strategies for the identification of robust fusion gene candidates by RNA-sequencing. Thereby, we detected known recurrent fusion events in 26 cases that were not reported by routine and found discrepancies in evidence for known fusion events between routine and RNA-sequencing in three cases. Moreover, we identified 157 fusion genes as novel robust candidates and comparison to entries from ChimerDB or Mitelman Database showed novel recurrence of fusion genes in 14 cases. Finally, we detected the novel recurrent fusion gene NRIP1- MIR99AHG resulting from inv(21)(q11.2;q21.1) in nine patients (1.1%) and LTN1-MX1 resulting from inv(21)(q21.3;q22.3) in two patients (0.25%). We demonstrated that NRIP1-MIR99AHG results in overexpression of the 3' region of MIR99AHG and the disruption of the tricistronic miRNA cluster miR-99a/let-7c/miR-125b-2. Interestingly, upregulation of MIR99AHG and deregulation of the miRNA cluster, residing in the MIR99AHG locus, are known mechanisms of leukemogenesis in acute megakaryoblastic leukemia. Our findings demonstrate that RNA-sequencing has a strong potential to improve the systematic detection of fusion genes in clinical applications and provides a valuable tool for fusion discovery.

Acute myeloid leukemia (AML) is a molecularly complex disease characterized by heterogeneous tumor genetic profiles and involving numerous pathogenic mechanisms and pathways. Integration of molecular data types across multiple patient cohorts may advance current genetic approaches for improved subclassification and understanding of the biology of the disease. Here, we analyzed genome-wide DNA methylation in 649 AML patients using Illumina arrays and identified a configuration of 13 subtypes (termed “epitypes”) using unbiased clustering. Integration of genetic data revealed that most epitypes were associated with a certain recurrent mutation (or combination) in a majority of patients, yet other epitypes were largely independent. Epitypes showed developmental blockage at discrete stages of myeloid differentiation, revealing epitypes that retain arrested hematopoietic stem-cell-like phenotypes. Detailed analyses of DNA methylation patterns identified unique patterns of aberrant hyper- and hypomethylation among epitypes, with variable involvement of transcription factors influencing promoter, enhancer, and repressed regions. Patients in epitypes with stem-cell-like methylation features showed inferior overall survival along with up-regulated stem cell gene expression signatures. We further identified a DNA methylation signature involving STAT motifs associated with FLT3-ITD mutations. Finally, DNA methylation signatures were stable at relapse for the large majority of patients, and rare epitype switching accompanied loss of the dominant epitype mutations and reversion to stem-cell-like methylation patterns. These results show that DNA methylation-based classification integrates important molecular features of AML to reveal the diverse pathogenic and biological aspects of the disease.

Resistance towards cancer treatment represents a major clinical obstacle, preventing cure of cancer patients. To gain mechanistic insights, we developed a model for acquired resistance to chemotherapy by treating mice carrying patient derived xenografts (PDX) of acute lymphoblastic leukemia with widely-used cytotoxic drugs for 18 consecutive weeks. In two distinct PDX samples, tumors initially responded to treatment, until stable disease and eventually tumor re-growth evolved under therapy, at highly similar kinetics between replicate mice. Notably, replicate tumors developed different mutations in TP53 and individual sets of chromosomal alterations, suggesting independent parallel clonal evolution rather than selection, driven by a combination of stochastic and deterministic processes. Transcriptome and proteome showed shared dysregulations between replicate tumors providing putative targets to overcome resistance. In vivo CRISPR/Cas9 dropout screens in PDX revealed broad dependency on BCL2, BRIP1 and COPS2. Accordingly, venetoclax re-sensitized derivative tumors towards chemotherapy, despite genomic heterogeneity, demonstrating direct translatability of the approach. Hence, despite the presence of multiple resistance-associated genomic alterations, effective rescue treatment for polychemotherapy-resistant tumors can be identified using functional testing in preclinical models.

The fusion genes CBFB/MYH11 and RUNX1/RUNX1T1 block differentiation through disruption of the core binding factor (CBF) complex and are found in 10–15% of adult de novo acute myeloid leukemia (AML) cases. This AML subtype is associated with a favorable prognosis; however, nearly half of CBF-rearranged patients cannot be cured with chemotherapy. This divergent outcome might be due to additional mutations, whose spectrum and prognostic relevance remains hardly defined. Here, we identify nonsilent mutations, which may collaborate with CBF-rearrangements during leukemogenesis by targeted sequencing of 129 genes in 292 adult CBF leukemia patients, and thus provide a comprehensive overview of the mutational spectrum (‘mutatome’) in CBF leukemia. Thereby, we detected fundamental differences between CBFB/MYH11- and RUNX1/RUNX1T1-rearranged patients with ASXL2, JAK2, JAK3, RAD21, TET2, and ZBTB7A being strongly correlated with the latter subgroup. We found prognostic relevance of mutations in genes previously known to be AML-associated such as KIT, SMC1A, and DHX15 and identified novel, recurrent mutations in NFE2 (3%), MN1 (4%), HERC1 (3%), and ZFHX4 (5%). Furthermore, age >60 years, nonprimary AML and loss of the Y-chromosomes are important predictors of survival. These findings are important for refinement of treatment stratification and development of targeted therapy approaches in CBF leukemia.

Based on the individual genetic profile, acute myeloid leukemia (AML) patients are classified into clinically meaningful molecular subtypes. However, the mutational profile within these groups is highly heterogeneous and multiple AML subclones may exist in a single patient in parallel. Distinct alterations of single cells may be key factors in providing the fitness to survive in this highly competitive environment. Although the majority of AML patients initially respond to induction chemotherapy and achieve a complete remission, most patients will eventually relapse. These points toward an evolutionary process transforming treatment‐sensitive cells into treatment‐resistant cells. As described by Charles Darwin, evolution by natural selection is the selection of individuals that are optimally adapted to their environment, based on the random acquisition of heritable changes. By changing their mutational profile, AML cell populations are able to adapt to the new environment defined by chemotherapy treatment, ultimately leading to cell survival and regrowth. In this review, we will summarize the current knowledge about clonal evolution in AML, describe different models of clonal evolution, and provide the methodological background that allows the detection of clonal evolution in individual AML patients. During the last years, numerous studies have focused on delineating the molecular patterns that are associated with AML relapse, each focusing on a particular genetic subgroup of AML. Finally, we will review the results of these studies in the light of Darwinian evolution and discuss open questions regarding the molecular background of relapse development.

In acute myeloid leukemia (AML), internal tandem duplications (ITDs) of FLT3 are frequent mutations associated with unfavorable prognosis. At diagnosis, the FLT3-ITD status is routinely assessed by fragment analysis, providing information about the length but not the position and sequence of the ITD. To overcome this limitation, we performed cDNA-based high-throughput amplicon sequencing (HTAS) in 250 FLT3-ITD positive AML patients, treated on German AML Cooperative Group (AMLCG) trials. FLT3-ITD status determined by routine diagnostics was confirmed by HTAS in 242 out of 250 patients (97%). The total number of ITDs detected by HTAS was higher than in routine diagnostics (n = 312 vs. n = 274). In particular, HTAS detected a higher number of ITDs per patient compared to fragment analysis, indicating higher sensitivity for subclonal ITDs. Patients with more than one ITD according to HTAS had a significantly shorter overall and relapse free survival. There was a close correlation between FLT3-ITD mRNA levels in fragment analysis and variant allele frequency in HTAS. However, the abundance of long ITDs (≥75nt) was underestimated by HTAS, as the size of the ITD affected the mappability of the corresponding sequence reads. In summary, this study demonstrates that HTAS is a feasible approach for FLT3-ITD detection in AML patients, delivering length, position, sequence and mutational burden of this alteration in a single assay with high sensitivity. Our findings provide insights into the clonal architecture of FLT3-ITD positive AML and have clinical implications.

Purpose: To study mechanisms of therapy resistance and disease progression, we analyzed the evolution of cytogenetically normal acute myeloid leukemia (CN-AML) based on somatic alterations. Experimental Design: We performed exome sequencing of matched diagnosis, remission, and relapse samples from 50 CN-AML patients treated with intensive chemotherapy. Mutation patterns were correlated with clinical parameters. Results: Evolutionary patterns correlated with clinical outcome. Gain of mutations was associated with late relapse. Alterations of epigenetic regulators were frequently gained at relapse with recurring alterations of KDM6A constituting a mechanism of cytarabine resistance. Low KDM6A expression correlated with adverse clinical outcome, particularly in male patients. At complete remission, persistent mutations representing preleukemic lesions were observed in 48% of patients. The persistence of DNMT3A mutations correlated with shorter time to relapse. Conclusions: Chemotherapy resistance might be acquired through gain of mutations. Insights into the evolution during therapy and disease progression lay the foundation for tailored approaches to treat or prevent relapse of CN-AML. Clin Cancer Res; 24(7); 1716–26. ©2018 AACR.

Long-term ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) proves to be unfeasible as cellular differentiation occurs when HSPCs are detached from their supporting bone marrow stem cell niche. This issue renders it difficult to make use of the proliferation capacity of HSPCs to subsequently produce functional blood cells in relevant numbers, e.g. for cell therapy approaches. To circumvent this challenge, leukemia-associated chimeric transcription factors, including MLL fusion proteins, can be exploited for their pronounced ability to propel cell proliferation while preserving cell immaturity. By designing the protein's activity controllable, the immature state can be abolished at an arbitrary point in time enabling terminal differentiation. In this study, we employed the fusion gene mixed lineage leukemia/eleven nineteen leukemia (MLL-ENL) for engineering an inducible protein switch. For this purpose, we fused the coding sequence of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the transcription factor MLL-ENL and subsequently expressed the protein switch (DD-MLL-ENL) in human CD34+ HSPCs derived from adult healthy donors. In the presence of the specific ligand Shield1, DD-mediated protein degradation is prevented leading to massive and long-term expansion of HSPC-derived late monocytic precursors in the presence of IL-3, IL-6, SCF, FLT3-L, TPO and GM-CSF. The cells do not exhibit additional driver mutations, feature a normal karyotype and telomere length, and sustain immaturity that is strictly dependent on Shield1 supplementation every other day even after two years of ex vivo culture. Upon Shield1 deprivation, the cells completely lost self-renewal and colony-forming properties and spontaneously differentiated. By changing the cytokines to GM-CSF in combination with IFN-γ and LPS we differentiated the progenitor cells into macrophages (MΦ) (Fig. 1 A, B). Immunophenotypic characterization revealed upregulation of the monocyte/macrophage-associated surface markers CD14, CD80, CD86, CD163 and MHC class I and II, concordant with monocytic morphology as judged by cytospin preparations. Analysis of the transcription of selected inflammatory genes, including IL-6 and IL-10, revealed overlapping M1 and M2 macrophage characteristics. Furthermore, mRNA expression profiles using nCounter Systems technology covering a total of 770 myeloid innate immunity-related genes proves the cells' identity as differentiated phagocytes shown by upregulation of gene clusters involved in Fc receptor signaling, TLR signaling, antigen presentation and T cell activation. In functional assays, we demonstrated the ability of the obtained cells to migrate towards the chemokine CCL2 in a 3D chemotaxis assay, attach to VCAM-1 under flow and shear stress and produce reactive oxygen species. Regarding the cells' phagocytic capability, we could verify the uptake of bacterial particles as well as apoptotic cells in efferocytosis assays. Finally, we demonstrated IgG Fc region recognition and binding by the expressed Fcγ receptors enabling phagocytosis of lymphoblastic tumor cells, including Daudi, Raji and patient-derived MCL cells in an antibody-dependent manner using rituximab (RTX), daratumumab (Dara) and trastuzumab (Trast) as a negative control (Fig. 1C). Overall, we could demonstrate the conversion of a harmful leukemic transcription factor into a useful molecular tool for large-scale ex vivo production of functional blood cells. Such engineered controllable protein switches might have the potential to be employed as molecular tools to produce functional immune cells for cell-based immunotherapeutic approaches. Figure 1 Figure 1. Disclosures Redondo Monte: Minaris Regenerative Medicine: Current Employment. Beier: Alexion: Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Other: Travel reembursement. Weigert: Janssen: Speakers Bureau; Epizyme: Membership on an entity's Board of Directors or advisory committees; Roche: Research Funding. Greif: AstraZeneca: Honoraria.

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.

Neuroendocrine carcinomas (NECs) of the colorectum are rare but highly aggressive neoplasms. These tumors show some shared genetic alterations with colorectal adenocarcinomas, and most of them have adjacent glandular adenoma or adenocarcinoma components. However, genetic data on colorectal NECs still are sparse and insufficient for definite conclusions regarding their molecular origin. Based on morphological characterization, panel and whole-exome sequencing, we here present results from an in-depth analysis of a collection of 15 colorectal NECs with glandular components, 10 of which by definition were mixed adenoneuroendocrine carcinomas (MANECs). Among shared genetic alterations of both tumor components, we most frequently found TP53, KRAS and APC mutations that also had highest allele frequencies. Mutations exclusive to glandular or neuroendocrine components outnumbered shared mutations but occurred at lower allele frequencies. Our findings not only provide additional evidence for a common clonal origin of colorectal NECs and adjacent glandular tumor components, but strongly suggest their development through the classical adenoma-carcinoma sequence. Moreover, our data imply early separation of glandular and neuroendocrine components during malignant transformation with subsequent independent mutational evolution.

Deletions of the long arm of chromosome 9 [del(9q)] are a rare but recurring aberration in acute myeloid leukemia (AML). Del(9q) can be found as the sole abnormality or in combination with other cytogenetic aberrations such as t(8;21) and t(15;17). TLE1 and TLE4 were identified to be critical genes contained in the 9q region. We performed whole exome sequencing of 5 patients with del(9q) as the sole abnormality followed by targeted amplicon sequencing of 137 genes of 26 patients with del(9q) as sole or combined with other aberrations. We detected frequent mutations in NPM1 (10/26; 38%), DNMT3A (8/26; 31%), and WT1 (8/26; 31%) but only few FLT3-ITDs (2/26; 8%). All mutations affecting NPM1 and DNMT3A were exclusively identified in patients with del(9q) as the sole abnormality and were significantly more frequent compared to 111 patients classified as intermediate-II according to the European LeukemiaNet (10/14, 71% vs. 22/111, 20%; P

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm resulting from the malignant transformation of myeloid progenitors. Despite intensive chemotherapy leading to initial treatment responses, relapse caused by intrinsic or acquired drug resistance represents a major challenge. Here, we report that histone 3 lysine 27 demethylase KDM6A (UTX) is targeted by inactivating mutations and mutation-independent regulation in relapsed AML. Analyses of matched diagnosis and relapse specimens from individuals with KDM6A mutations showed an outgrowth of the KDM6A mutated tumor population at relapse. KDM6A expression is heterogeneously regulated and relapse-specific loss of KDM6A was observed in 45.7% of CN-AML patients. KDM6A-null myeloid leukemia cells were more resistant to treatment with the chemotherapeutic agents cytarabine (AraC) and daunorubicin. Inducible re-expression of KDM6A in KDM6A-null cell lines suppressed proliferation and sensitized cells again to AraC treatment. RNA expression analysis and functional studies revealed that resistance to AraC was conferred by downregulation of the nucleoside membrane transporter ENT1 (SLC29A1) by reduced H3K27 acetylation at the ENT1 locus. Our results show that loss of KDM6A provides cells with a selective advantage during chemotherapy, which ultimately leads to the observed outgrowth of clones with KDM6A mutations or reduced KDM6A expression at relapse.

High throughput sequencing approaches, including the analysis of exomes or gene panels, are widely used and established to detect tumor-specific sequence variants such as point mutations or small insertions/deletions. Beyond single nucleotide resolution, sequencing data also contain information on changes in sequence coverage between samples and thus allow the detection of somatic copy number alterations (CNAs) representing gain or loss of genomic material in tumor cells arising from aneuploidy, amplifications, or deletions. To test the feasibility of CNA detection in sequencing data we analyzed the exomes of 25 paired leukemia/remission samples from acute myeloid leukemia (AML) patients with well-defined chromosomal aberrations, detected by conventional chromosomal analysis and/or molecular cytogenetics assays. Thereby, we were able to confirm chromosomal aberrations including trisomies, monosomies, and partial chromosomal deletions in 20 out of 25 samples. Comparison of CNA detection using exome, custom gene panel, and SNP array analysis showed equivalent results in five patients with variable clone size. Gene panel analysis of AML samples without matched germline control samples resulted in confirmation of cytogenetic findings in 18 out of 22 cases. In all cases with discordant findings, small clone size (

Chromosomal rearrangements and specific aneuploidy patterns are initiating events and define subgroups in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here we analyzed 250 BCP-ALL cases and identified a novel subgroup (‘PAX5-plus’, n = 19) by distinct DNA methylation and gene expression profiles. All patients in this subgroup harbored mutations in the B-lineage transcription factor PAX5, with p.P80R as hotspot. Mutations either affected two independent codons, consistent with compound heterozygosity, or suffered LOH predominantly through chromosome 9p aberrations. These biallelic events resulted in disruption of PAX5 transcriptional programs regulating B-cell differentiation and tumor suppressor functions. Homozygous CDKN2A/B deletions and RAS-activating hotspot mutations were highly enriched as cooperating events in the genomic profile of PAX5-plus ALL. Together, this defined a specific pattern of triple alterations, exclusive to the novel subgroup. PAX5-plus ALL was observed in pediatric and adult patients. Although restricted by the limited sample size, a tendency for more favorable clinical outcome was observed, with 10 of 12 adult PAX5-plus patients achieving long-term survival. PAX5-plus represents the first BCP-ALL subgroup defined by sequence alterations in contrast to gross chromosomal events and exemplifies how deregulated differentiation (PAX5), impaired cell cycle control (CDKN2A/B) and sustained proliferative signaling (RAS) cooperatively drive leukemogenesis.

In acute myeloid leukemia (AML), DNA methylation is frequently altered and epigenetic regulators are commonly mutated. Here, we describe the effects of mutations in commonly mutated genes (NPM1, FLT3-ITD, DNMT3A, IDH1, IDH2, TET2, and WT1), including epigenetic regulators, on DNA methylation profiles and differential gene expression. Moreover, we show that subgroups of epigenetically homogeneous AML patients differ significantly in clinical outcome. We have characterized 212 AML patients treated on consecutive trials of the AMLCG study group using Illumina Infinium MethylationEPIC BeadChips, gene panel sequencing, and transcriptome sequencing. In order to detect differentially methylated CpG sites (dmCpGs) according to a particular mutation, we have selected sets of mutated and control samples for each gene of interest individually. We excluded samples with subclonal variants only and selected control samples based on matching karyotype and matching pattern of co-mutations. Mutations at the R882 hotspot of DNMT3A result in global hypomethylation while alterations of IDH1, IDH2, TET2, or WT1 lead to global hypermethylation. Still, subsets of dmCpGs are hypermethylated in DNMT3A-R882+ AML as well as hypomethylated in IDH1+, IDH2+, TET2+, and WT1+ AML. NPM1 mutations result both in hypo- and hypermethylation, while based on FLT3-ITD status, we could not detect significant changes in DNA methylation. Of note, we observed wildtype samples with a methylation profile highly matching that of mutated samples for most comparisons, suggesting alternative mechanisms. Moreover, mutations in IDH1, IDH2, TET2, and WT1 show substantial overlaps in dmCpGs, which is in line with their reported function, while the overlap with DNMT3A-R882 is rather small. Of note, we also detected overlaps in gene expression profiles by comparing test and control samples, in particular between AML with IDH1, IDH2, or WT1 mutations. The proto-oncogenes FOSB, FOSL2, and JUN are differentially expressed in IDH1+ AML, while in IDH2+ and WT1+ AML, members of the RPL and RPS gene families of ribosomal proteins are deregulated, known to alter FOS and JUN function. Unsupervised hierarchical clustering of all samples in our cohort results in two highly distinct epigenetic subgroups, each with three subclusters (Figure 1A). Of note, clusters are associated with distinct mutations. Most AML samples in clusters 1, 2, or 3 are mutated in NPM1, while clusters 4, 5, and 6 are mostly NPM1 wildtype. Still, the genetic profiles of subclusters differ based on the presence of mutations in IDH1/IDH2/TET2 (clusters 1 and 4), DNMT3A (cluster 2), and DNMT3A-R882/WT1 (cluster 3). Clusters 5 and 6 show only few mutations in DNMT3A, IDH1, IDH2, TET2, or WT1. Mutations in FLT3 are not associated with any cluster. Of note, epigenetic subgroups are also associated with differences in overall survival (OS) and event free survival (EFS) (Figure 1B). Clusters 1, 3, and 5 show significantly better outcome (median OS: 1113, 1046, and 1054 days; median EFS 513, 374, and 305 days) as compared to clusters 4 and 6 (median OS: 378 and 296 days; median EFS 103 and 70 days; p Although the epigenetic landscape of AML is complex, we identified nearly homogeneous subgroups, which are associated with but not limited to mutations in epigenetic regulators. As epigenetic subgroups show differences in clinical outcome, DNA methylation profiling has the potential to refine AML risk stratification. Disclosures Metzeler: Otsuka: Honoraria; Celgene: Honoraria, Research Funding; Daiichi Sankyo: Honoraria. Hiddemann:Vector Therapeutics: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria.

Accurate and complete genetic classification of AML is crucial for the prediction of clinical outcome and treatment stratification. Deciphering the spectrum of genetic abnormalities by polymerase chain reaction (PCR), karyotyping and fluorescence in situ hybridization (FISH) in routine diagnostics is the current gold standard, however, fusion genes might potentially be missed by these assays. Recently, several methods have been developed to improve the detection of gene fusion transcripts based on RNA sequencing data, providing robust results. To test the detection power and assess the applicability of RNA-Seq based methods in clinical diagnostics we applied two different algorithms, namely FusionCatcher (Nicorici D et al., bioRxiv, 2014) and Arriba (Uhrig S et al., DKFZ, https://github.com/suhrig/arriba), to the transcriptomes of 895 well-characterized AML samples from three independently sequenced cohorts: AMLCG (Herold T et al., Haematologica, 2018, n=261), DKTK (Greif PA et al., Clin Cancer Res, 2018 and unpublished data, n=166), BeatAML (Tyner JW et al., Nature 2018, n=468) and publicly available healthy control samples (SRA studies: SRP018028, SRP047126, SRP050146, SRP105369, SRP115911, SRP133442, n=38). According to karyotyping, 31% (277/895) of samples harbored chromosomal aberrations putatively causing gene fusions (i.e. translocations, interstitial deletions, duplications, inversions, insertions). Analyses by FISH and/or PCR confirmed these rearrangements in 51.3% (142/277) of samples, whereas fusion detection by the means of RNA-Seq showed evidence for fusion genes corresponding to these rearrangements in 60.3% (167/277) of samples. Chromosomal aberrations, identified by karyotyping, which are known to result in clinically relevant fusions (e.g. RUNX1-RUNX1T1, KMT2A fusions) were confirmed by FISH/PCR (AMLCG: n=27/27, DKTK: n=21/21, BeatAML: n=54/57) and RNA-Seq based methods (AMLCG: n=17/27, DKTK: n=21/21, BeatAML: n=56/57) in most of the cases. Of note, the AMLCG cohort was sequenced using the SENSE mRNA Library Prep Kit from Lexogen which seems to be not optimal for fusion detection. Furthermore, 19 samples (AMLCG: n=12, DKTK: n=4, BeatAML: n=3) were found to harbor known pathogenic fusions, described in previous studies, which were not reported by routine diagnostics: NUP98-NSD1 (n=11); CBFB-MYH11, RUNX1-RUNX1T1 and DEK-NUP214 (n=2 each); RUNX1-CBFA2T2 and RUNX1-CBFA2T3 (n=1 each). Reanalysis of six of these samples by PCR confirmed three fusions which were initially missed by routine diagnostics. In general, the amount of reported fusion events by RNA-Seq is high (on average 69 and 39 per sample as detected by FusionCatcher and Arriba respectively), even after applying the built-in filters, indicating a high false positive rate. To robustly identify putative novel fusions, we developed a filtering pipeline and incorporated two new filtering steps. The promiscuity score (PS) of a fusion measures the amount of further distinct fusion partners which were detected in the respective cohort for the 5' and 3' gene. The fusion transcript score (FTS) measures the relative abundance of a fusion transcript to its 5' and 3' partner gene. PS and FTS of known, clinically relevant fusions confirmed by FISH/PCR were used to define cut-offs. To further maximize specificity while maintaining sensitivity, we excluded fusion events which we detected in publicly available healthy samples and subsequently filtered for overlapping calls from FusionCatcher and Arriba (Fig. 1A). Additionally, we obtained further evidence for a fusion event by an elevated transcription of the 3' fusion partner. In case of a fusion event, the transcription of the 3' partner gene likely gets under the control of the promoter of the 5' partner gene. This results in an elevated transcription of genes which are otherwise transcribed at low levels (Fig. 1B-C). Thus, we identified five putatively novel recurrent fusion genes which were detected in two cohorts independently: NRIP1-MIR99AHG, LATS2-ZMYM2, ATP11A-ING1, MBP-SLC66A2, PRDM16-SKI (Fig. 1D-F). Although these events were called with high evidence, we aim at independent validation by complementary methods. In our study, we have not only demonstrated that the application of RNA-Seq to the detection of fusion genes is a valuable complement to diagnostic routine but also has the potential to discover novel putatively pathogenic fusions. Disclosures No relevant conflicts of interest to declare.

Heterozygous mutations in GATA2 underlie different syndromes, previously described as monocytopenia and mycobacterial avium complex infection (MonoMAC); dendritic cell, monocytes, B- and NK lymphocytes deficiency (DCML); lymphedema, deafness and myelodysplasia (Emberger syndrome) and familiar myelodysplastic syndrome/acute myeloid leukemia (MDS / AML). Onset and severity of clinical symptoms vary and preceding cytopenias are not always present. We describe a case of symptomatic DCML deficiency and rather discrete bone marrow findings due to GATA2 mutation. Exome sequencing revealed a somatic ASXL1 mutation and the patient underwent allogeneic stem cell transplantation successfully., Highlights • Allogeneic stem cell transplantation was performed for DCML caused by GATA2 mutation. • Genetic diagnostics were done by Sanger sequencing and whole exome sequencing. • We identified an ASXL1 mutation associated with high risk for leukemic transformation.

Acute myeloid leukemia in the elderly is characterized by a distinct genetic and epigenetic landscape

Deletions of the long arm of chromosome 9 [del(9q)] are a rare but recurring aberration in acute myeloid leukemia (AML). Del(9q) can be found as the sole abnormality or in combination with other cytogenetic aberrations such as t(8;21) and t(15;17). TLE1 and TLE4 were identified to be critical genes contained in the 9q region. We performed whole exome sequencing of 5 patients with del(9q) as the sole abnormality followed by targeted amplicon sequencing of 137 genes of 26 patients with del(9q) as sole or combined with other aberrations. We detected frequent mutations in NPM1 (10/26; 38%), DNMT3A (8/26; 31%), and WT1 (8/26; 31%) but only few FLT3-ITDs (2/26; 8%). All mutations affecting NPM1 and DNMT3A were exclusively identified in patients with del(9q) as the sole abnormality and were significantly more frequent compared to 111 patients classified as intermediate-II according to the European LeukemiaNet (10/14, 71% vs. 22/111, 20%; P 0.001, 8/14, 57% vs. 26/111, 23%; P = 0.02). Furthermore, we identified DNMT3B to be rarely but recurrently targeted by truncating mutations in AML. Gene expression analysis of 13 patients with del(9q) and 454 patients with normal karyotype or various cytogenetic aberrations showed significant down regulation of TLE4 in patients with del(9q) (P = 0.02). Interestingly, downregulation of TLE4 was not limited to AML with del(9q), potentially representing a common mechanism in AML pathogenesis. Our comprehensive genetic analysis of the del(9q) subgroup reveals a unique mutational profile with the frequency of DNMT3A mutations in the del(9q) only subset being the highest reported so far in AML, indicating oncogenic cooperativity. © 2016 Wiley Periodicals, Inc.

The leukemia-associated fusion gene CBFB/MYH11 results from a pericentric inversion of chromosome 16, inv(16)(p13.1q22), or less commonly from a t(16;16)(p13.1;q22). Although this cytogenetic aberration is associated with a rather favorable prognosis in acute myeloid leukemia (AML), nearly half of patients eventually relapse after standard chemotherapy. To systematically analyze the clonal evolution in this AML-subgroup, we performed whole exome sequencing (WES) of 13 adult CBFB/MYH11-rearranged AML patients using matched diagnostic, remission and relapse samples ('triplets'). Thereby, we found 2-12 (median: 8) somatic sequence variants per patient at diagnosis and 2-13 (median: 4) mutations at relapse. These included mutations in genes known to cooperate with CBFB/MYH11 (e.g. RAS, FLT3, KIT) as well as in genes, which had not been associated with AML previously (MYO15A, EVPL, ROS1, FTCD and ASL). Next, we designed a custom targeted sequencing assay (Haloplex, Agilent), including the candidate genes from exome sequencing, as well as genes known to be recurrently mutated in AML (455 genes, 1.86 Mbp total target sequence) and performed targeted sequencing of 32 CBFB/MYH11-rearranged AML triplet samples (including the 13 triplets initially analyzed by WES) with a median read depth of 500. The results are summarized in Figure 1. Fourteen genes were found mutated in at least two patients at diagnosis and 9 genes at the timepoint of relapse. In all CBFB/MYH11-rearranged patients, more than one additional mutation was identified, each of them at a distinct variant allele-frequency, indicating clonal heterogeneity. All but one FLT3 TKD (D835 or N676) mutation were lost at relapse, whereas FLT3 ITDs were stable in 3 out of 7 patients. One FLT3 ITD was gained at relapse. The majority of RAS,KIT and CBL mutations were lost and none was acquired at relapse. Particularly, the loss of 6 out of 7 KIT exon 8 frameshift mutations was surprising since KIT exon 8 frameshift mutations were negative prognostic markers in a cohort of 162 patients with CBFB/MYH11 rearranged leukemia (OS: HR= 3.12, p= 0.001; Opatz et al. submitted). In contrast, mutations in WT1 and DNMT3A were all stable during relapse evolution and four patients gained mutations in these two genes. Furthermore, aberrations in CSF3R, BCORL1 and ZBTB7A were acquired at relapse. Of note, WT1 mutations causing a frameshift in exon 6 were found in 9% of adult de novo AML with CBFB/MYH11-rearrangement and have recently been characterized by our research group as negative prognostic marker for overall survival (HR: 2.93, p= 0.011) (Opatz et al. submitted). These findings are in line with the observed gain of WT1 mutations in 10% of relapsed cytogenetically normal AML patients (Greif et al., 2018, Clin Cancer Res) suggesting a common mechanism of disease progress across cytogenetic subgroups. Surprisingly, a mutation in ZBTB7A, a gene frequently altered in RUNX1/RUNX1T1 positiv leukemia (23%) but rarely in CBFB/MYH11 positiv leukemia (2%), was gained at relapse in one patient. Mutations in epigenetic modifiers, cohesion complex components and janus kinases are known cooperating events in RUNX1/RUNX1T1 rearranged leukemia and were, except for DNMT3A, not found in our CBFB/MYH11-positive cohort. The new recurring mutations in MYO15A (n=3), ROS1 (n=2), FTCD (n=2) and ASL (n=2) were partially lost at relapse, whereas EVPL (n=2) mutations were stable during the course of disease. In addition, we identified mutations in APC2, TP53 and ZFHX4 (gained at relapse), PTPN11, MECOM, BCOR, NPM1 and IDH2 (stable) as well as in ABL1 (lost at relapse) in individual patients. Taken together, our findings suggest that mutations in signaling pathway genes seem to be unstable during disease progression and may thus not be required for the evolution of relapse. The frequent loss of signaling gene mutations indicates that relapse might evolve from an early ancestral clone carrying the CBFB/MYH11 rearrangement only. Figure 1: Mutation profile of 32 patients with CBFB/MYH11-rearranged AML. The stability of recurrently mutated genes during the evolution of relapse is shown. Diagonal lines indicate two mutations in the respective gene. Figure 1 Figure 1. Disclosures Thiede: AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding. Middeke:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Stoelzel:Neovii: Speakers Bureau. Metzeler:Novartis: Consultancy; Celgene: Consultancy, Research Funding.

Over the past 6 years, next-generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identification of rare and common genetic variations. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry, and data analysis need to be overcome. Each workflow and sequencing platform have their particular problems and caveats, which need to be addressed. Regarding NGS, there is a variety of different enrichment methods, sequencing devices, or technologies as well as a multitude of analyzing software products available. In this manuscript, the authors focus on challenges in data analysis when employing different target enrichment methods and the best applications for each of them.

The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion., The t(8;21) translocation is often found in acute myeloid leukaemia but is not sufficient for development of the disease. In this study, the authors identify frequent mutations in the transcriptional repressor, ZBTB7A, in these patients and show that the mutations reduce DNA binding activity.

The contribution of molecular alterations in bone marrow mesenchymal stromal cells (BM-MSC) to the pathogenesis of acute myeloid leukemia (AML) is poorly understood. Thus we assessed genome-wide genetic, transcriptional and epigenetic alterations in BM-MSC derived from AML patients (AML BM-MSC). Whole-exome sequencing (WES) of AML BM-MSC samples from 21 patients revealed a non-specific pattern of genetic alterations in the stromal compartment. The only mutation present in AML BM-MSC at serial time points of diagnosis, complete remission and relapse was a mutation in the PLEC gene encoding for cytoskeleton key player Plectin in one AML patient. Healthy donor controls did not carry genetic alterations as determined by WES. Transcriptional profiling using RNA sequencing revealed deregulation of proteoglycans and adhesion molecules as well as cytokines in AML BM-MSC. Moreover, KEGG pathway enrichment analysis unravelled deregulated metabolic pathways and endocytosis in both transcriptional and DNA methylation signatures in AML BM-MSC. Taken together, we report molecular alterations in AML BM-MSC suggesting global changes in the AML BM microenvironment. Extended investigations of these altered niche components may contribute to the design of niche-directed therapies in AML.

High throughput sequencing approaches, including the analysis of exomes or gene panels, are widely used and established to detect tumor-specific sequence variants such as point mutations or small insertions/deletions. Beyond single nucleotide resolution, sequencing data also contain information on changes in sequence coverage between samples and thus allow the detection of somatic copy number alterations (CNAs) representing gain or loss of genomic material in tumor cells arising from aneuploidy, amplifications, or deletions. To test the feasibility of CNA detection in sequencing data we analyzed the exomes of 25 paired leukemia/remission samples from acute myeloid leukemia (AML) patients with well-defined chromosomal aberrations, detected by conventional chromosomal analysis and/or molecular cytogenetics assays. Thereby, we were able to confirm chromosomal aberrations including trisomies, monosomies, and partial chromosomal deletions in 20 out of 25 samples. Comparison of CNA detection using exome, custom gene panel, and SNP array analysis showed equivalent results in five patients with variable clone size. Gene panel analysis of AML samples without matched germline control samples resulted in confirmation of cytogenetic findings in 18 out of 22 cases. In all cases with discordant findings, small clone size (33%) was limiting for CNA detection. We detected CNAs consistent with cytogenetics in 83% of AML samples including highly correlated clone size estimation (R = 0.85), while six out of 65 cytogenetically normal AML samples exhibited CNAs apparently missed by routine cytogenetics. Overall, our results show that high throughput targeted sequencing data can be reliably used to detect copy number changes in the dominant AML clone. © 2016 Wiley Periodicals, Inc.

The CALM/AF10 fusion gene is found in various hematological malignancies including acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant AML model as B220+ lymphoid cells with B-cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis, we inserted the CALM/AF10 fusion gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre-induced panhematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B-lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed coexpression of the B-cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of two to three additional mutations per leukemia, including activating mutations in known oncogenes such as FLT3 and PTPN11. Our results show that the COL for CALM/AF10 leukemia is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10+ leukemia.

Isolated trisomy 13 (AML+13) is a rare chromosomal abnormality in acute myeloid leukemia (AML), and its prognostic relevance is poorly characterized. We analyzed the clinical course of 34 AML+13 patients enrolled in the German AMLCG-1999 and SAL trials and studied their biological characteristics by exome sequencing, targeted sequencing of candidate genes and gene expression profiling. Relapse-free (RFS) and overall survival (OS) of AML+13 patients were inferior compared to other ELN Intermediate-II patients (n=855) (median RFS, 7.8 vs 14.1 months, p=0.006; median OS 9.3 vs. 14.8 months, p=0.004). Besides the known high frequency of RUNX1 mutations (75%), we identified mutations in spliceosome components in 88%, including SRSF2 codon 95 mutations in 81%, of AML+13 patients. Moreover, recurring mutations were detected in ASXL1 (44%) and BCOR (25%). Two patients carried mutations in CEBPZ, suggesting that CEBPZ is a novel recurrently mutated gene in AML. Gene expression analysis revealed a homogenous expression profile including upregulation of FOXO1 and FLT3 and downregulation of SPRY2. This is the most comprehensive clinical and biological characterization of AML+13 to date, and reveals a striking clustering of lesions in a few genes, defining AML+13 as a genetically homogenous leukemia subgroup with alterations in a few critical cellular pathways. These studies were registered at clinicaltrials.gov, identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML60+: NCT00893373.

The cadherin gene FAT1, located on chromosome 4q34-35 (ref. 1) within a region frequently deleted in human cancers,2 encodes a large protein with 34 extracellular cadherin repeats.3 In solid tumors, aberrant expression of FAT1 was found to be associated with disease progression.4 Although the gene was originally cloned from a human T-cell acute lymphoblastic leukemia (T-ALL) cell line,4 FAT1 just recently gained interest owing to its altered gene expression levels and the detection of somatic mutations identified by next-generation sequencing (NGS) in acute leukemia.2, 5, 6, 7 FAT1 was shown to be aberrantly expressed in pediatric patients with acute leukemia, whereas hematopoietic progenitors from healthy donors lacked FAT1 expression.5, 8 In addition, a recent report correlated high FAT1 expression with a higher probability of relapse in a small cohort of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) based on an in silico analysis comprising two BCP-ALL data sets including 32 and 27 patients.5

Novel target discovery is warranted to improve treatment in adult T-cell acute lymphoblastic leukemia (T-ALL) patients. We provide a comprehensive study on mutations to enhance the understanding of therapeutic targets and studied 81 adult T-ALL patients. NOTCH1 exhibitedthe highest mutation rate (53%). Mutation frequencies of FBXW7 (10%), WT1 (10%), JAK3 (12%), PHF6 (11%), and BCL11B (10%) were in line with previous reports. We identified recurrent alterations in transcription factors DNM2, and RELN, the WNT pathway associated cadherin FAT1, and in epigenetic regulators (MLL2, EZH2). Interestingly, we discovered novel recurrent mutations in the DNA repair complex member HERC1, in NOTCH2, and in the splicing factor ZRSR2. A frequently affected pathway was the JAK/STAT pathway (18%) and a significant proportion of T-ALL patients harboured mutations in epigenetic regulators (33%), both predominantly found in the unfavourable subgroup of early T-ALL. Importantly, adult T-ALL patients not only showed a highly heterogeneous mutational spectrum, but also variable subclonal allele frequencies implicated in therapy resistance and evolution of relapse. In conclusion, we provide novel insights in genetic alterations of signalling pathways (e.g. druggable by γ-secretase inhibitors, JAK inhibitors or EZH2 inhibitors), present in over 80% of all adult T-ALL patients, that could guide novel therapeutic approaches.

Even though two-thirds of acute myeloid leukemia (AML) patients respond to induction chemotherapy and achieve complete remission (CR), the majority of these patients will eventually relapse. The time from CR to relapse is an important clinical indicator of disease aggressiveness, as patients relapsing within the first 6 months after initial diagnosis have a poorer prognosis in terms of response to salvage therapy and overall survival compared to patients with a later relapse. To learn about the evolution during the course of disease, we analyzed the somatic mutation patterns from initial diagnosis to relapse in 50 cytogenetically normal (CN) AML patients. Based on the ELN classification, 38% of the patients (n=19) were assigned as "favorable" at diagnosis, all other patients were classified as "intermediate-I". ELN classification was associated with time to relapse as "intermediate-I" patients relapsed earlier than "favorable" patients (median 9.3 months vs. 16.1 months, p=0.008, log-rank test). Somatic alterations were detected by exome sequencing and confirmed by targeted amplicon sequencing of matched diagnostic, remission and relapse samples. FLT3-ITD and NPM1 mutation status were obtained from routine diagnostic tests as the reliable detection of these markers by NGS remains challenging. The vast majority of somatic alterations were present both at diagnosis and at relapse, hereafter referred to as stable mutations (70%, Fig. 1A). All patients in our cohort had ≥1 stable mutation with DNMT3A being the most stably altered gene. In 47 out of 50 patients (94%), we observed mutations that were only found at diagnosis or only at relapse. Based on the mutation patterns, four distinct 'evolutionary' subgroups of patients were defined (Fig. 1B): (I) patients with an identical mutation profile at diagnosis and at relapse ("stable", n=3, 6%), (II) patients who gained mutations at relapse ("stable + gain", n=24, 48%), (III) patients that lost mutations at relapse ("stable + loss", n=8, 16%), and (IV) patients with both loss and gain of mutations at relapse ("mixed", n=15, 30%). Mutations that were lost during the course of the disease were detected in e.g. PTPN11 or NRAS. Relapse-specific mutations were identified in e.g. IDH1/2, WT1, KPNB1 or KDM6A. Evolutionary subgroups showed differences in time to relapse (Fig. 1C). Patients with "stable + loss" relapsed earlier (median 4.1 months) than patients with gain of mutation at relapse (groups "stable + gain" and "mixed", median 12.2 months). All patients in the category "stable + loss" developed relapse within the first year after complete remission. The "stable" group of 3 patients showed an intermediate time to relapse (median 9.6 months), but was too small for a statistically valid comparison. Ultimately, the genetic evolution of CN-AML patients without gain of new mutations at relapse (categories "stable" and "stable + loss") was associated with significantly earlier relapse compared to patients that gained mutations at relapse (categories "stable + gain" and "mixed", Fig. 1D, p=0.001, log-rank test). Distinct predominant patterns of clonal evolution were observed in the ELN genetic groups, as only one patient of the "stable + loss" group was initially classified as "favorable". Interestingly, applying the ELN classification on relapse samples revealed a switch from "favorable" to "intermediate-I" in six patients, all with gain of mutations at relapse. This points towards more aggressive genetic profiles at relapse in these patients. The acquisition of mutations and/or the outgrowth of a resistant clone during/after chemotherapy might require a longer time or is per se associated with a longer time to relapse and a more favorable prognosis. Loss of mutations at relapse suggest the presence of two clones at diagnosis, with a chemotherapy resistant clone expanding after the eradication of a chemotherapy sensitive clone. As both clones share mutations and only the sensitive clone contains specific alterations, the resistant clone might be an ancestor of the sensitive clone. Taken together, in some patients the AML cells may require additional genetic alterations to become chemotherapy resistant, whereas in other patients the selective eradication of a sensitive clone is a potential mechanism underlying disease progression. Understanding the evolution of AML under selective pressure of chemotherapy is essential to cure or prevent AML relapse. Disclosures Hiddemann: Roche: Other: Grants; Genentech: Other: Grants; Roche: Membership on an entity's Board of Directors or advisory committees.

Introduction In Acute Myeloid Leukemia (AML) internal tandem duplications (ITD) in the fms-related tyrosine kinase 3 (FLT3) are a frequent event associated with an unfavorable prognosis. At diagnosis, the FLT3-ITD status is routinely assessed by fragment analysis of PCR-amplified cDNA. However, this assay only provides information on the length but not on the position and sequence of the insertion. Therefore, it is attractive to overcome this limitation by the use of high-throughput amplicon sequencing (HTAS) as an alternative strategy for FLT3-ITD detection. To proof the feasibility and accuracy of this approach we performed HTAS on 260 AML patients, of which 250 were FLT3-ITD positive according to routine diagnostics. Patients and Methods All samples were obtained from patients treated on the German Acute Myeloid Leukemia Cooperative Group (AMLCG) trials 1999, 2004 and 2008. All patients received intensive induction therapy with curative intent. At diagnosis, patients had a median age of 60 years (range, 18-80 years). Additional molecular marker were screened routinely: NPM1 mutation (62%), KMT2A-PTD (8%), CEBPA mutations (8%). According to the ELN-classification patients clustered into the following groups: ELN intermediate I (70%), intermediate II (21%) or adverse (9%). A normal karyotype was observed in 71%, while 29% had an aberrant karyotpye. All patient samples were analyzed routinely by FLT3 fragment analysis. For HTAS of FLT3 we used custom FLT3 cDNA primers including barcode and adapter-sequences enabling a one-step PCR-protocol. Sequencing was performed (2x250bp paired end) on a MiSeq (Illumina). Per run, up to 96 samples were sequenced, yielding a median of 79,110 reads per amplicon (range: 31,996 - 259,783). As controls, cDNA from FLT3-ITD positive (Molm-13) and negative (HL60) cell lines were used. Sequencing data were aligned to the FLT3 cDNA reference (NM_004119.2) and FLT3-ITDs were called using Pindel software (version 0.2.5a7). Results Based on the HTAS results obtained from the FLT3 wild-type control HL60, we set the cut-off for ITD detection at a variant allele frequency (VAF) of 0.5%. Using this threshold, all patients which were assessed to be FLT3-ITD negative in diagnostic routine, were also negative in HTAS. FLT3-ITDs were detected by HTAS in 242 out of 250 (97%) patients who were FLT3-ITD positive patients according to routine fragment analysis. For six out of the 8 remaining patients, no valid ITD was detected by HTAS possibly due to one of the following reasons: a low mutational burden resulting in a VAF below the cut-off level (n=4) or a deletion near the ITD (n=2) potentially interfering with ITD-detection. In total, 308 ITDs were detected in 242 patients by HTAS while in these patients 282 ITDs were detected by routine diagnostics. Of note, HTAS missed 13 subclonal ITDs reported in routine, while 39 additional subclonal ITDs were detected by HTAS only. Overall, HTAS detected a higher number of ITDs per patient (mean: 1.27; range: 1-4) compared to fragment analysis (mean: 1.17; range: 1-3). Patients with more than one ITD according to HTAS showed a trend towards shorter overall and relapse free survival (p=0.105, p=0.104 respectively; Figure 1A). The ITD position (i.e. affected FLT3 domain) based on HTAS did not impact on clinical outcome. There was a significant correlation between the FLT3-ITD levels detected by fragment analysis and HTAS (Pearson, R=0.801, p=0.01; Figure 1B). High FLT3-ITD levels measured by fragment analysis had a significant impact on RFS, whereas this effect was not observed for FLT3-ITD levels measured by HTAS. The quantification of FLT3-ITD by HTAS might be hampered by underestimation of the VAFs of long ITDs that are less likely to be mapped correctly compared to shorter ITDs. Conclusion In summary, our study demonstrates the feasibility of HTAS for FLT3-ITD detection in AML. In particular, the identification of subclonal ITDs with high sensitivity provides additional information with potential prognostic value. Thus, HTAS may serve as a robust tool that could be implemented in future diagnostic routines. However, bioinformatic algorithms for ITD detection may need further improvement, e.g. to optimize ITD quantification and to facilitate the detection of ITDs in combination with deletions. Disclosures Hiddemann: Roche: Other: Grants; Genentech: Other: Grants; Roche: Membership on an entity's Board of Directors or advisory committees.

The t(8;21) and inv(16)/t(16;16) rearrangements affecting the core-binding factors RUNX1 and CBFB, respectively, are found in 15% to 20% of adult de novo acute myeloid leukemia (AML) cases and are associated with a favorable prognosis. Since the expression of the fusion genes CBFB / MYH11 or RUNX1 / RUNX1T1 alone is not sufficient to cause leukemia, we performed exome sequencing of an AML sample with an inv(16) to identify mutations, which may collaborate with the CBFB/MYH11 fusion during leukemogenesis. We discovered an N676K mutation in the adenosine triphosphate (ATP)-binding domain (tyrosine kinase domain 1 [TKD1]) of the fms-related tyrosine kinase 3 ( FLT3 ) gene. In a cohort of 84 de novo AML patients with a CBFB/MYH11 rearrangement and in 36 patients with a RUNX1/RUNX1T1 rearrangement, the FLT3 N676K mutation was identified in 5 and 1 patients, respectively (5 [6%] of 84; 1 [3%] of 36). The FLT3-N676K mutant alone leads to factor-independent growth in Ba/F3 cells and, together with a concurrent FLT3-ITD (internal tandem duplication), confers resistance to the FLT3 protein tyrosine kinase inhibitors (PTKIs) PKC412 and AC220. Gene expression analysis of AML patients with CBFB / MYH11 rearrangement and FLT3 N676K mutation showed a trend toward a specific expression profile. Ours is the first report of recurring FLT3 N676 mutations in core-binding factor (CBF) leukemias and suggests a defined subgroup of CBF leukemias. This trial was registered at www.clinicaltrials.gov as #NCT00266136.

Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is a high-risk subgroup of T-lineage ALL characterized by specific stem cell and myeloid features. In adult ETP-ALL, no comprehensive studies on the genetic background have been performed to elucidate molecular lesions of this distinct subgroup. We performed whole-exome sequencing of 5 paired ETP-ALL samples. In addition to mutations in genes known to be involved in leukemogenesis (ETV6, NOTCH1, JAK1, and NF1), we identified novel recurrent mutations in FAT1 (25%), FAT3 (20%), DNM2 (35%), and genes associated with epigenetic regulation (MLL2, BMI1, and DNMT3A). Importantly, we verified the high rate of DNMT3A mutations (16%) in a larger cohort of adult patients with ETP-ALL (10/68). Mutations in epigenetic regulators support clinical trials, including epigenetic-orientated therapies, for this high-risk subgroup. Interestingly, more than 60% of adult patients with ETP-ALL harbor at least a single genetic lesion in DNMT3A, FLT3, or NOTCH1 that may allow use of targeted therapies.

Acute lymphoblastic leukemia (ALL) consists of genetically heterogeneous cell subpopulations, but little is known about how genetic differences lead to functional differences between the clones. Of major clinical importance, aggressive, treatment-resistant and putatively relapse-inducing subclones need to be identified and require effective eradication by treatment. The most aggressive subpopulation likely determines prognosis and outcome in each patient. We aimed at characterizing on a functional as well as on a genetic level single stem cell clones derived from patients' samples growing in mice and to combine the results of both levels in order to learn which genetic characteristics are associated with adverse functional behavior. We transplanted primary tumor cells from a 5-year old girl with hyperdiploid ALL involving a trisomic X chromosome at first relapse into severely immune-compromised mice and lentivirally modified them to express the fluorochromes red, blue and green at different amounts and combinations (RGB marking, Weber et al., 2012). Eight single stem cell clones were generated by limiting dilution transplantation and their uniqueness was verified by ligation-mediated (LM) PCR. We functionally compared the single stem cell clones between each other by re-mixing them in a single mouse for in vivo assays; analysis was performed one-by-one for each clone by flow cytometry where they could be distinguished from each other using their unique color codes. Clones showed clear differences in proliferation rate with faster and slower growing clones, independently whether 2 or 5 clones were mixed. When mice harboring clone mixtures were treated with conventional chemotherapy, clonal composition changed markedly and resistant clones overgrew sensitive clones indicating selective clonal responses and clonal advantage. A clone which showed especially slow growth in vivo was most resistant to in vivo treatment with Glucocorticoids. The slowly proliferating, Glucocorticoid-resistant clone had lost the additional X chromosome, which was present in all other clones and the bulk and showed a distinct DNA-methylation pattern analyzed by 450K arrays (illumina). In exome analysis, the clone showed 11 unique alterations including a single nucleotide variant in the oncogene USP6. We are currently performing RNA sequencing analysis to assess the differential gene expression in the clones. Taken together, genetic multicolor marking PDX ALL cells in the individualized xenograft mouse model allowed generating viable single cell clones for genetic functional characterization in vivo. Within the heterogeneous tumor bulk, an subclone existed which showed slow tumor growth and drug resistance which was associated with distinct genetic characteristics. Our studies allow the challenging functional characterization of subclones in vivo in order to develop efficient novel treatment approaches to eliminate aggressive stem cell clones in ALL. Weber K, Thomaschewski M, Benten D, Fehse B., RGB marking with lentiviral vectors for multicolor clonal cell tracking. Nat Protoc. 2012 Apr 5;7(5):839-49. doi: 10.1038/nprot.2012.026. Disclosures No relevant conflicts of interest to declare.

Cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous disease with regard to genetic alterations and clinical outcome. Recent sequencing studies categorized the growing number of recurrently mutated genes into different functional groups, e.g. myeloid transcription factors, tumor suppressors, signal transducers, chromatin modifiers, cohesin-complex and spliceosome-complex. We set out to characterize mutations in genes linked to epigenetic regulation during the progression of CN-AML. Besides genes directly involved in chromatin modification (i.e. DNMT3A, TET2, MLL, ASXL1, KDM6A, KDM2A, NSD1 and EZH2), we also studied mutations in WT1 and IDH1/2 since they are known to inhibit TET2 function. Targeted sequencing of 46 genes related to leukemia (mean coverage >500x) was performed on matched diagnostic, remission and relapse samples of 50 patients with CN-AML (median age: 66, range: 21-89). We called somatic variants at diagnosis or at relapse and filtered for mutations with translational consequences, excluding known error-prone genes and common germline polymorphisms (dbSNP 138; MAF>=1%). At diagnosis, 36/50 patients (72%) carried a total of 48 mutations in epigenetic regulators (Figure 1). The majority of patients harbored a single mutation affecting this functional group, while 2 or 3 mutations were observed in 9 and 1 patient(s), respectively. The median variant allele frequency (VAF) of the mutations was 42% (range: 22-98%), indicating that mutations in epigenetic regulators are early events and are present in the founding clone. Of the 48 mutations detected at diagnosis, only 2 were lost at relapse, highlighting the stability of these lesions during disease progression. Moreover, in 12/50 patients (24%), mutations in epigenetic regulators were acquired at relapse. All but one of these patients already had a mutation in another epigenetic regulator at diagnosis. We did not identify patients who acquired DNMT3A, TET2 or ASXL1 mutations during disease progression. However, mutations in WT1, IDH1, and KDM6A were gained in several patients at relapse. In 4/13 cases, the gained mutations were already detectable at low levels at diagnosis (median VAF: 2.9%, range: 0.3-6%, mean coverage at the investigated sites: 629x, range: 85-1625x). We also evaluated the presence of these mutations in remission: In 18 out of 36 (50%) patients, some of the mutations affecting DNMT3A (n=14), TET2 (n=3) or IDH2 (n=2) were present at a VAF >5% (median: 22%, range: 9-75%) in cytomorphologically defined complete remission, suggesting the persistence of pre-leukemic clones with limited response to chemotherapy. Longer relapse-free survival was observed in patients with DNMT3A mutations that did not persist at remission (np-DNMT3A) in comparison to patients with persisting DNMT3A mutations (p-DNMT3A). Remarkably, the latter group was enriched for patients that also harbored FLT3 internal tandem duplications (ITDs) (10/14 versus 1/8; Fisher's exact test, p=0.02). The vast majority of p-DNMT3A showed alterations of R882, whereas mutations at other positions of DNMT3A tended to be undetectable in remission. When including the NPM1 status, only 1/8 patient with np-DNMT3A was triple mutated, compared to 11/14 patients with p-DNMT3A, suggesting that co-occurrence of DNMT3A, FLT3- ITD and NPM1 c is associated with p-DNMT3A (p=0.006). In summary, we show that a high proportion of patients (72%) with relapsing CN-AML is affected by mutations in genes linked to epigenetic regulation. The stability of these mutations between diagnosis and relapse in combination with their acquisition during disease progression, as well as the frequent persistence of DNMT3A, TET2 and IDH2 mutations during remission underscore the necessity for new therapeutic approaches. The striking association of DNMT3A R882 mutations with NPM1 c and FLT3 -ITD suggest a unique mechanism of oncogenic collaboration. Persistence of DNMT3A R882 mutations may indicate a fertile ground for relapse. Further studies will be required to clarify whether the actual relapse arises from a preleukemic clone harboring only the founder mutation or from residual leukemia cells containing several genetic lesions. Disclosures No relevant conflicts of interest to declare.

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.

Introduction: B cell precursor acute lymphoblastic leukemia (BCP-ALL) represents the largest immunophenotypically defined subgroup of adult ALL. Yet, targeted therapies are assigned only to the minority of patients, whereas druggable lesions for the majority of adult BCP-ALL are still unknown. Thus, it remains necessary to explore their genomic landscape to unravel molecular alterations suitable for targeted therapies. We analyzed the mutational pattern of 206 candidate genes in adult BCP-ALL patients at diagnosis and matched relapse samples, focusing on therapeutically targetable alterations and their clonal evolution. Patients and methods : We performed targeted resequencing on diagnostic samples of patients with BCR-ABL negative BCP-ALL (n=89) as well as matched relapse samples (n=53). The mean age at first diagnosis was 44 years (range 17 - 79). Relapses were categorized as early (> 18 months from diagnosis, n=33) or late (< 18 months, n=20). All patients were enrolled on trials of the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL). The targeted region comprised 206 genes known to be frequently mutated in leukemia and relevant in normal hematopoiesis. Customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to select the targeted regions. We performed 100-bp paired-end sequencing on an Illumina Genome HiSeq1500 sequencing system. For a variant call, we required at least a read depth of 30 and a variant allele frequency (VAF) of 10%. We obtained an average coverage of 825 reads for the target region with over 98 percent of the targeted region been covered with a minimum of 30 reads. After exclusion of polymorphism annotated in dbSNP135, 625 protein changing single nucleotide variations and small indels were identified. 139 of the 206 target genes were mutated at least once. Results: On average three (median 3.0, range 1-12) genes were mutated in the 89 diagnostic BCP-ALL samples. The most frequent mutations included alterations in NRAS (18%), PAX5 (16%), TP53 (9%), JAK1/2 (8%) and IKZF1 (6%). Categorizing mutated genes according to their functional annotation revealed that 44 (49%) of patients harbored at least one mutation affecting epigenetic regulation, followed by adhesion/matrix proteins (48%), transcription factors (46%), RAS pathway genes (29%), kinase signaling (28%), and p53/cell cycle regulation (26%). Four (KMT2D, SETD2, KDM6A, KDM6B) of the twelve most frequently mutated genes, affecting 30 patients (34%), were epigenetic regulators specifically involved in histone methylation. Mutations in these genes were nearly mutually exclusive, suggesting their functional redundancy in this context. For 53 of the 89 patients matched relapse samples were available, showing a median frequency of 4.0 (range 1-36) mutated genes per sample. The frequency of mutations across gene families was similar compared to diagnostic samples, except for those involved in epigenetic regulation that showed a higher mutation rate at relapse (55%). In 68% of matched relapse samples, mutational gains and losses indicated mechanisms of clonal evolution. Among 28 patients (53%), which gained at least one mutation, the Lysine-Specific Methyltransferase 2D (KMT2D) was the most frequently affected gene with 7 patients acquiring KMT2D alterations at relapse. Other methyl-transferases (SETD2, KMT2C) as well as demethylases (KDM5A, KDM6A, KDM6B) acquired novel mutations at relapse, contributing to a total of 11 patients (21%) that gained alterations in histone methylation regulators. Notably, mutations in methylation regulators were only gained, but not lost at relapse. Nine of these 11 patients suffered an early relapse, implicating a selection advantage for clones harboring mutations in methylation regulating genes and their involvement in a more aggressive course of the disease. Conclusion: We describe for the first time a highly heterogeneous genomic mutational spectrum in adult BCP-ALL. A high mutation rate at diagnosis and further increase at relapse identified regulators of histone methylation as a most prominent target of recurrent alterations. Functional studies are needed to determine the direct biological consequence of these alterations, facilitating targeted therapeutic interventions with epigenetically active compounds. Disclosures Baldus: Novartis: Research Funding.