ALBERT

Description: Next generation sequencing (NGS) has been extensively used to characterize the molecular background in patients with AML. Several novel recurrent alterations have been discovered, such as the common mutations in the IDH-genes and in DNMT3A. We aimed to use this technology to characterize patients failing induction chemotherapy. To better understand mechanisms of resistance, we applied whole exome based NGS screening in younger AML patients failing conventional induction chemotherapy. In a group of 29 patients, two patients showed mutations in cMYC exon 2. Similar mutations have been reported in diffuse large B-cell lymphoma (DLBCL), Burkitt 's lymphoma and aggressive HIV associated lymphomas. These alterations cluster in the aminoterminal part of the protein and lead to stabilization of the MYC protein, MYC activation and evasion of TP53 mediated tumor surveillance. MYC mutations have only occasionally been described in AML before, but amplification of cMYC or activation of this important oncoprotein via other cellular pathways has been linked to aggressive disease and resistance to treatment. So, in order to better understand the prevalence and the prognostic implications of cMYC mutations in adult AML, we screened a set of more than 1200 patients with AML and advanced MDS for the presence of mutations in the cMYC- gene. Patients and Methods: We retrospectively characterized genomic DNA samples taken at the time of first diagnosis from 1281 patients with AML treated in the AML96 protocol of the Study Alliance Leukemia (SAL). Since all reported mutations cluster in cMYC exon 2, this region was analyzed using conventional Sanger sequencing. In cMYC-mutated patients, a set of 54 genes (Trusight Myeloid Panel) covering commonly mutated genes in myeloid disease was analyzed for alterations using next generation sequencing (NGS) on a MiSEQ instrument. Results: An exon 2 mutation in cMYC was found in 14/1281 (1.1%) of the patients, all mutations clustered in the the MbI-domain between codons 57 and 62, with codon P59 being the most frequently mutated position. Analysis of the allelic burden indicated that the mutations occurred early in the disease. Correlation with clinical, cytogenetic and molecular data showed, that the mutations were predominantly found in patients with normal or intermediate risk karyotype, but they were also seen in good risk and high risk patients. Strikingly, 9 of 14 patients (77%) showed an NPM1-mutation (p=.004) and 7 of 14 (50%) were FLT3-ITD mutated (p=.02). The extended NGS-characterization did not reveal any additional specific secondary alterations associated with this mutation. MYC mutations were found in all FAB-subtypes excluding FAB M0, no significant differences were seen for other clinical variables, including age, sex, white blood cell and bone marrow blast counts. In univariate as well as multivariate analysis, patients with cMYC mutations did not show any significant difference in the rate of complete remission (CR), the event free and the overall survival, even when restricted to subgroups such as patients with normal karyotype or with NPM1 mutations. Conclusions: This study showed that mutations in cMYC exon 2 are a rare but recurrent abnormality in AML. The mutations are significantly enriched in patients with mutant NPM1 and FLT3-ITD, but based on our analysis, they seem not to have any prognostic implications. Disclosures Platzbecker: Boehringer: Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Thiede:AgenDix GmBH: Equity Ownership; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 886 The diagnosis acute myeloid leukemia (AML) describes a heterogeneous group of myeloid stem cell disorders. Based on current concepts of disease development, the combination of at least two mutations is necessary for transformation, typically affecting transcription factors (e.g. RUNX1) blocking normal differentiation and growth promoting genes, e.g. receptor tyrosine kinases like FLT3. This model has been challenged by more recent results of genome-wide mutational analysis, which revealed a typical load of 8–12 mutations affecting several additional pathways, e.g. epigenetic regulation (DNMT3A, TET2 or IDH1). However, the sequence of acquisition and the individual impact of these mutations are largely unknown because these aspects are difficult to study. Here we describe a unique case of a donor cell leukemia giving unexpected insights into the development of AML in man. Case report and methods: In May 2004, a 51-year old male (P1) with an 8-year history of B-CLL received G-CSF mobilized peripheral blood stem cells after dose reduced conditioning from his HLA-identical sister, because he had relapsed after several lines of conventional therapy. He rapidly engrafted and showed complete donor chimerism (DC). In February 2012, he was admitted to the hospital with elevated WBC counts and circulating blasts. Bone marrow (BM) aspiration and morphology revealed an infiltration of the BM with 94% myeloid blasts (FAB M1). Cytogenetic and standard molecular assessment showed a normal female karyotype and NPM1 and FLT3-ITD mutations. STR-based analysis also revealed a persistent, 100% DC, thus the diagnosis of a donor cell AML was made, which developed almost 8 years after SCT. Interestingly, his sister, the donor (P2) had been also diagnosed with AML (FAB M2, cytogenetics: 47, XX,+8; NPM1 and FLT3-ITD neg.) only 3 months before her brother in Nov. 2011. Currently, P1 is in CR after re-SCT from an unrelated donor, whereas P2 relapsed and is scheduled for SCT after reinduction. Since DNA material of both individuals was available and due to this unique constellation, we performed next generation sequencing of whole exome enriched material using an Illumina HiSEQ 2000 platform after obtaining informed consent to compare both AMLs. Identified mutations were then confirmed using conventional Sanger sequencing and traced back by 454-based amplicon deep sequencing in a pre-SCT sample of the donor/P2 as well as several post SCT samples collected from P1 for the documentation of chimerism. Results: Comparison of the two AML-samples with a pre-SCT donor sample and a sample taken after 1.SCT as well as the HG19 and dbSNP135 releases revealed more than 100 unknown SNPs. Confirmation focused on cancer related changes or genes in critical pathways. In P1, in addition to the known NPM1 and FLT3-ITD mutations, we found somatic changes in CLCA1, PKHD1 and TET2, whereas in P2, we identified and confirmed somatic mutations in CDCA2, CBL, IDH1, NEK9 and PHF6. In addition, a typical DNMT3A R882C mutation was found in both leukemias. Interestingly, this mutation was also detectable by conventional Sanger sequencing in the pre-SCT sample of P2, but not in P2-germline DNA derived from buccal swaps. As shown in the figure, the 454-amplicon sequencing revealed a gradual increase of the TET2 (R1167G) mutational load over time in P1, and showed also that this mutation was present at low levels (4%) already in the pre-SCT sample of P2, but not in her final AML. FLT3-ITD and NPM1 mutations were detectable only in the AML-sample of P1, but not at any prior time points or in P2. Conclusions: These data indicate that mutations like the DNMT3A R882C can be present in normal appearing hematopoiesis at high levels years before the development of AML. The presence of the mutation in the absence of overt leukemia or MDS indicates that these mutations might not have a direct effect on the development of the disease, but favor the development of aberrant clones which then acquire additional changes in a latency phase. Other mutations (e.g. TET2) might give a small clonal advantage, but only the final acquisition of abnormalities like NPM1 and FLT3-ITD might transform this latency phase into a rapidly proliferating status, consistent with the “driver” status of these aberrations. The divergent mutational pattern found in the two AMLs emerging from the same DNMT3A-starting clone points to the high clonal diversity which might develop even within a single individual. Disclosures: Thiede: AgenDix GmbH: Employment, Equity Ownership.

Description: Mutations of the key myeloid transcription factor CCAAT/enhancer binding protein alpha (C/EBPa) are found in 5-10% of patients with acute myeloid leukemia (AML). Two mutational clusters exist, in the aminoterminal transcription activation domains (TAD1 or 2) and in the basic leucine zipper domain (bZIP) located at the carboxyterminal-part of the protein. Biallelic mutations (biCEBPA) have been found to be associated with improved outcome and are now included as an independent entity in the WHO-classification. In contrast, monoallelic CEBPA-mutations (moCEBPA) do not appear to provide prognostic information. We characterized a large cohort of AML patients for CEBPA mutations and further analyzed the mutational spectrum of mono- and biallelic CEBPA-mutant AML patients to better understand potential differences in the biology of these groups. Patients and Methods: Patients (including all age groups) analyzed had a newly diagnosed AML and were registered in clinical protocols of the Study Alliance Leukemia (SAL)(AML96, AML2003 or AML60+, SORAML) or the SAL-register. Screening for CEBPA mutations was done using PCR and capillary electrophoresis. All identified CEBPA mutations were confirmed using conventional Sanger sequencing and the samples were further analyzed using next generation sequencing (Trusight Myeloid Panel, Illumina) for the presence of associated alterations. Results: In the 4578 patients analyzed, 228 (5%) with CEBPA-mutations were identified. An initial analysis revealed substantial clinical differences between the different mutation subtypes. Patients with biCEBPA (n=111) were significantly younger (median age 46 yrs) than wt-CEBPA patients (median 57 yrs; p

Description: Background De novo acute myeloid leukemia (AML) is a malignant disorder of hematopoietic stem and progenitor cells usually characterized by a rapid clinical onset. Despite this clinical manifestation, recent evidence suggests that premalignant stem cells might be present in patients with AML, which can persist after chemotherapy in some patients and induce clonal hematopoiesis. Little is known about the prevalence of clonal persistence and about the molecular basis. In order to study the prevalence of this phenomenon and to better understand the underlying molecular mechanisms, we investigated AML patients in remission for clonal persistence of cells after chemotherapy. Patients and methods: All patients included in this analysis were treated within a prospective treatment protocol of the Study Alliance Leukemia (SAL). The primary study cohort consisted of 61 female patients with intermediate risk cytogenetics achieving hematologic complete remission (CR), whose DNA material was available at CR. Clonality analysis was based on X-chromosome inactivation testing using the HUMARA assay. DNA from diagnostic samples of patients presenting with evidence for X-chromosome skewing in CR was analyzed using amplicon based resequencing on a MiSeq next generation sequencing (NGS)-system for DNMT3A, ASXL1, ASXL2, TET1, TET2, EZH1, EZH2, IDH1 and IDH2. Results: Of the 61 patients included, 52 were heterozygous for the STR in the human androgen receptor gene. In CR, 22 of these 52 patients (42%) showed evidence for a skewed X-chromosome representation, indicating persistence of clonal hematopoiesis in remission. The NGS-based analysis of genes involved in epigenetic regulation revealed mutations in 13/22 (59%) of the patients. DNMT3A was most frequently mutated (11/13 patients), either alone or in combination with other alterations (TET2, EZH2). Interestingly, two patients showed somatic alterations in the TET1 gene. In remission, clonal persistence of these alterations was detected in all 13 patients with mutations at diagnosis at levels between 0.8 and 50% as documented using ultradeep-NGS. To get an idea on the prevalence of clonal persistence in other cytogenetic groups, we analyzed 22 low risk (i.e. CBF-leukemias) as well as 18 poor risk (-7, complex karyotype) patients using the HUMARA assay. Here we observed similar results, with 13/19 informative patients showing clonal persistence in low-risk group (68%) compared to 7/14 patients (50%) in the poor risk population. Since all these analyses were confined to female patients and potentially limited by the sensitivity of the HUMARA method, we went on to look for persistence of clonal molecular markers using more sensitive ultra-deep NGS. Because DNMT3A exon 23 was the common alteration in this initial analysis, we screened a cohort of 48 patients with mutations in NPM1 and comutations in DNMT3A. In this separate cohort, persistence of the DNMT3A mutations at CR or during follow-up (FU) was detected in 42 patients (87.5%) at levels between 0.5 and 50% (median 11.1%). No difference was seen between male and female patients, the median age was 51 years, persistence was seen even in young patients at 26 years of age. During FU, the DNMT3A VAF level rose further in all patients analyzed, arguing for a clonal advantage of the mutant cells. All patients with relapse and available material showed high levels of DNMT3A at time of relapse. However, correlation of DNMT3A mutant allele levels at CR1 with the incidence of relapse showed no significant impact of the VAF for the development of relapse. Conclusions: Our data indicate that clonal persistence of premalignant cells carrying clonal alterations in epigenetic regulator genes is a common phenomenon in patients in continuous CR. DNMT3A is the most common lesion persisting, the majority of patients with this mutations retain it at CR and during FU. These data indicate that de novo AML develops from preleukemic stem and progenitor cells in many patients. Preliminary data indicate that this persistence per se is not associated with inferior outcome. Disclosures Schuster: AgenDix GmbH: Employment. Thiede:AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.

Description: Background Partial tandem duplication mutations of the Mixed Lineage Leukemia gene (MLL-PTD) can be found in about 10% of patients with AML, especially in patients with normal karyotype AML. The mutation generates a self-fusion within the N-terminal part of MLL and has been shown to be leukemogenic in mouse models. In patients, the presence of the mutation is associated with poor prognosis. Little is known on the molecular profile of patients with MLL-PTD and on the cooperating mutations. In order to identify accompanying molecular alterations, we performed whole exome sequencing (WES) of eight AML patients harbouring MLL-PTD mutations. Based on the observed alterations we then designed a custom amplicon panel and performed targeted resequencing in a cohort of 90 MLL-PTD mutated AML patients. Materials and Methods All patients included in this analysis were treated in prospective treatment protocols of the Study Alliance Leukemia (SAL). To enrich for malignant cells and to obtain germline reference material (T-cells), FACS sorting was performed on viable cells banked at diagnosis. After whole genome amplification of the primary DNA, whole exomes were enriched (TruSeq chemistry; Illumina), and paired-end sequenced using Illumina HiSeq2000 2x100 bp runs. Resulting data were mapped against human genome (Hg19). Only somatic single nucleotide variants (SNVs) were included in the final analysis. Based on the SNVs identified by whole exome sequencing (WES), a custom amplicon panel (TruSeq Custom Amplicon, TSCA, Illumina) for targeted resequencing was designed. The assay included either the entire coding region or mutational hot spots of 56 genes (Fig.1). In total, 700 targets were amplified in a single reaction for each patient and paired end sequenced on a MiSeq NGS system (Illumina). Paired end reads were BWA mapped against targeted regions and data analysis was done using the Sequence Pilot software package (JSI Medical Systems) with a 20% variant allele frequency (VAF) mutation calling cutoff. Only non-synonymous variants not specified as SNP in the db137 database and predicted as deleterious (Provean) were included in the final analysis. All variations were confirmed by Sanger sequencing. Results WES of eight MLL-PTD (7/8 FLT3-ITD negativ) patients revealed a total 490 SNVs (range 13-254 per patient). Most frequently mutated genes were DNMT3A, IDH1/2 and TET2. Somatic mutations were also found in genes rarely mutated in AML, such as ATM, GNAS, TET1 and EP300. Based on the WES-data, 90 MLL-PTD patients were screend for a panel of 56 genes using the TSCA assay, which revealed in total of 169 mutations. 18 genes were not found to be mutated and in 8 patients, no co-occurring mutations were identified. Due bad assay performance EP300, EZH1, JAK3, MLL2, MLL3 and NOTCH1 were excluded from the data analysis. Here again, the most frequently mutated genes were DNMT3A (34.4%), IDH1 (20.0%), IDH2R140 (18.9%), IDH2R172 (7.9%), TET2 (16.7%) and FLT3 (11.3%). Mutations were less frequently found in RUNX1 (8.9%) and ASXL1, SMC1A, U2AF1 (5.6% each) (Fig. 1). In addition to these known genes, most prevalent mutations were found in ATM (8.9%) as well as DNMT3B and TET1 (4.4% each). Overall, we oberserved a low frequency of mutations in typical class 1 genes such as NRAS, KRAS and FLT3, which was lower than reported in the TCGA data set. Conclusions This analysis in a large set of patients with MLL-PTD mutations did not reveal any new and specific individual mutation present in patients with this alteration. Instead, our finding of a very high prevalence of alterations in epigenetic regulator genes, found in more than 85% of patients with MLL-PTD, strongly argues for a particular disease biology in these patients. These findings might also implicate that treatment based on demethylating agents or histone-deacetylase inhibitors might be especially attractive in patients with MLL-PTD. Figure 1: Figure 1:. Distribution of mutations in MLL-PTD patients The assay included either the entire coding region or mutational hot spots of the following 56 genes; ASXL1, ATM, BCOR, BRAF, CBL, DDR1, DNMT1, DNMT3A, DNMT3B, EIF4A2, EP300, ETV6, EZH1, EZH2, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, JAK1, JAK2, JAK3, KDM4A, KDM5A, KDM5C, KDM6A, KIT, KRAS, MET, MLL, MLL2, MLL3, NOTCH1, NOTCH4, NPM1, NRAS, PDGFRA, PDGFRB, PHF6, PTEN, PTPN11, RAD21, RUNX1, SF3A1, SF3B4, SMC1A, SMC3, SMC4, TET1, TET2, TP53, U2AF1 and WT1. Disclosures Thiede: AgenDix GmbH: Equity Ownership, Research Funding; Illumina: Research Support, Research Support Other.