Acute Myeloid Leukemia M2 Research Articles

Clinical Benefit of Maintenance Chemotherapy in Patients with Acute Myeloid Leukemia

Background: There is no standard treatment strategy for maintaining first complete remission (CR1) status after several cycles of consolidation chemotherapy in acute myeloid leukemia (AML) patients who are not suitable for transplantation. Maintenance chemotherapy has failed to document the cure rate or prolongation of survivals in patients with acute myeloid leukemia (AML) except for acute promyelocytic leukemia (APL). In this study, we retrospectively compared leukemia free (LFS) and overall survival (OS) of patients with or without maintenance therapy.Methods: Maintenance chemotherapy consisted of daily 6-mercaptopurine and weekly methotrexate per os and lasted for 2 years. Patients who maintained CR1 status after completion of consolidation therapy started maintenance chemotherapy.Results: Fifty two patients (not suitable for transplantation) who completed at least 1 cycle of consolidation therapy were eligible for analysis. Twenty seven patients agreed to administrate oral maintenance chemotherapy whereas 25 patients refused. Median age was 52 years and 24 patients were male. According to the FAB classification, 7.7, 9.6, 40.4, 38.5, 1.9 and 1.9% of patients are AML M0, M1, M2, M4, M6 and M7, respectively. Myelodysplastic syndrome (MDS) related and chemotherapy-related secondary AML patients were 5.6 and 9.6%, respectively. Favorable, intermediate and unfavorable cytogenetic risk groups were 32.7, 63.5 and 2%, respectively. Of 33 patients with intermediate risk, 84.8, 12.1 and 3% were normal karyotype, other not-defined and +8 alone, respectively. Two patients with unfavorable risk were complex karyotype and inv(3). There was no significant difference in the patients’ characteristics between non-maintenance and maintenance group. Almost of all patients (96.4%) received remission induction therapy with a same protocol (7-3 regimen). Relapse was observed in 27 patients (51.9%) after achieving CR1. Median LFS and OS was 28 (95% CI, 2–54) and 29 months (95% CI, 6–52), respectively. The OS was 19 (95% CI, 8–30) and 43 months (95% CI, 19–67) in non-maintenance and maintenance group, respectively (p = 0.044, Fig 1A), whereas LFS was not significantly different. In multivariate analysis, the presence of maintenance therapy was an independent prognostic factor for better LFS (p = 0.044) and OS (p = 0.042, Table 1.). In subgroup analysis (Table 2.)., statistically significant clinical benefit from maintenance chemotherapy was observed in patients with older age (>= 60 years) (p = 0.024), intermediate or unfavorable cytogenetic results (p = 0.004, Fig 1B.), initial higher WBC count (>= 50,000/mm3) (p = 0.005), secondary AML (p = 0.009), and receiving less than 2 cycles of consolidation therapy (p = 0.006).Conclusions: Despite limitation as retrospective analysis with small sample size, our data indicate that maintenance chemotherapy with oral 6-MP and MTX can prolong survivals of patients with AML (except APL) who are not suitable for transplantation as a post-remission therapy particularly with older age, intermediate or unfavorable cytogenetics, initial higher WBC count, secondary AML or receiving less than 2 cycles of consolidation therapy. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High Expression of Mir-15a Predicts Shorter Survival and Worse Response to Chemotherapy in Patients with Acute Myeloid Leukemia (AML)

Background: MicroRNAs (miRNAs) are small non-coding RNA molecules, that control gene expression by targeting messenger RNA (mRNA), via degradation or suppression of translation. Aberrant expression of microRNAs (miRs) has been proved to have a role in acute myeloid leukemias (AML)The aim of the study was to determine expression of miR-15a in acute myeloid leukemia patients before and after chemotherapy and its influence on patient clinical outcome.Methods: miRNAs from isolated leukemic cells were extracted using mirVanaTM miRNA Isolation kit (Ambion Inc., Carlsbad, CA, USA) following the manufacturer’s protocol. Reverse transcriptase (RT) reactions were performed for mature miRNA cDNA synthesis in separate tubes using specific stem-loop RT primers and TaqMan® MicroRNATM Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). After microRNA isolation, reverse transcriptase reactions were performed, followed by cDNA amplification. The relative amount of microRNA-15a was normalized according to the reference RNU48 level. Results were considered statistically significant with p-value < 0.05.Results: 95 patients (aged 60.2 ± 15.0, 22–90, Male = 61%) with newly diagnosed AML were included into the study. Samples of the bone marrow for miR-15a expression analysis were collected before start of chemotherapy and repeated after completed induction chemotherapy (40 patients). A control group of 20 matched patients was also taken into account. The analyzed group consisted of: 7 patients with AML M0, 34 had M1, 29 had M2, 14 had M4 and 11 had M5.AML patients has higher miR-15a expression than control group (p=0.005633). The risk of death in AML patients was higher in patients with higher miR-15a expression on diagnosis (p=0.0430), Fig.1. Patients with lower miR-15a expression were more likely to achieve complete remission after induction chemotherapy (p=0.0130). After successful chemotherapy we observed significant drop in miR-15a expression.Conclusions: We proved that miR-15a was upregulated in AML patients and its expression influenced remission achieving and death risk. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gemtuzumab Ozogamicin (GO) in Infants with Acute Myeloid Leukemia (AML) – Combined Results from the Children’s Oncology Group (COG) Trials, AAML03P1 and AAML0531

Background: Infants <1 year of age with AML are at high risk of early death during induction, pulmonary and infectious toxicities, and treatment related mortality (TRM). Compared with older children, infants are more likely to have higher risk clinical and cytogenetic features. Targeted therapy is needed to improve the outcomes for infants with AML while minimizing toxicities.Objective: To determine if the addition of gemtuzumab ozogamicin (GO) to standard chemotherapy is safe, tolerable, and improves event free survival (EFS) in infants <1 year of age with AML.Methods: Infants >/= 1 month to <1 year of age with de novo AML or <1 month of age with progressive AML were eligible for enrollment on the COG trials AAML03P1 and AAML0531. Infants with acute promyelocytic leukemia, juvenile myelomonocytic leukemia, bone marrow failure syndromes, or Down syndrome were not eligible. The 5-course chemotherapy backbone was identical in both trials. GO 3 mg/m2/dose (0.1 mg/kg for patients with a body surface area <0.6 m2) was given on day 6 of Induction (Ind) I and day 7 of Intensification (Int) II to all patients on AAML03P1 and to randomized patients on the experimental arm of AAML0531. Patients on the control arm of AAML0531 were treated with standard therapy without GO (noGO). Stem cell transplant (SCT) was given following IntI to patients on AAML03P1 with a matched family donor (MFD) and to patients on AAML0531 with high risk (HR) disease or with intermediate risk (IR) disease and a MFD. SCT recipients only received one dose of GO. Patients were removed from protocol if the bone marrow had >/= 20% blasts after IndI on AAML03P1 or >/= 5% blasts after IndII on both studies. Early death (ED) was defined as death during IndI.Results: AAML03P1 enrolled 39 infants from 2003-2005 and AAML0531 enrolled 103 infants from 2006-2010 (Table 1). Median follow up was 5.02 years. The demographics and disease characteristics of infants enrolled on both studies were similar. Compared with enrolled children >/= 1 year of age, infants had higher frequencies of hepatomegaly (p<0.001), splenomegaly (p<0.001), hyperleukocytosis (WBC>100x103/µL, p=0.003) and French-American-British (FAB) M5 (p<0.001), and M7 (p<0.001) AML. Infants had less FAB M1 (p<0.001), M2 (p<0.001), and M4 (p=0.029) AML. There was no difference in CNS disease by age. The frequency of 11q23/MLL rearrangement was highest in infants 0-179 days of age (44%) and significantly decreased with increasing age (p<0.001). Favorable cytogenetics were rare in infants: t(8;21) was not found and inv(16)/t(16;16) was only found in 4 patients. FLT3-ITD HAR was absent in infants. The majority of infants (89%) fell into the IR group and infants were less likely to have had high (HR) or low risk (LR) disease when compared with children >/= 1 year. [Display omitted] The ED rate was higher in younger infants (7 ED, infants 0-179d) when compared with older infants (1 ED, infants 180-364d) (p=0.013). EDs were not increased in infants who received GO vs. noGO (5 vs. 3 ED, p=0.730). The complete remission (CR) rate for infants was 68% at the end of IndII. The 5 year EFS was lower in infants than children >/= 1 year (42 vs. 50%, p=0.001). The 5 year OS for infants was 62%, relapse risk (RR) was 44%, and TRM was 10%. Infants who received GO had improved OS, EFS, and RR, though the differences were not statistically significant (Table 2). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CBFA2T3-GLIS2 Fusion Is Prevalent in Younger Patients with Acute Myeloid Leukemia and Associated with High-Risk of Relapse and Poor Outcome: A Children’s Oncology Group Report

The cryptic CBA2T3-GLIS2 fusion generated by the inv(16)(p13.3q24.3) was initially identified in megakaryocytic leukemia and later implicated in other acute myeloid leukemia (AML) subtypes. Presence of this fusion may lead to altered expression of the potentially targetable sonic hedgehog and bone morphogenic protein pathways. We determined the prevalence of CBA2T3-GLIS2 in children treated on COG AAML03P1 and AAML0531 protocols, which collectively enrolled 1361 eligible children, adolescents, and young adults with de novo AML, and correlated the presence of this fusion with patient demographics, laboratory features, and clinical outcomes. We also determined the prevalence and clinical implications of CBA2T3-GLIS2 in 71 children with FAB M7 AML treated on 4 consecutive COG AML trials.Of the 1042 diagnostic samples available and tested for CBFA2T3-GLIS2, 45 (4.3%) were positive for the fusion. Fusion-positive patients were significantly younger than fusion-negative patients (2.1 vs. 10.3 years; P<0.001). CBFA2T3-GLIS2 was most prevalent in the youngest patients (10.6% for 0 to <2 year olds and 8.6% for 2 to <5 year olds) [Figure A]. In contrast, no fusion transcripts were identified in 299 unselected adult patients. All FAB subtypes were represented in fusion-positive patients. Overall, FAB M5 and M7 were equally prevalent in fusion-positive patients, and each subtype accounted for 20% of cases [Figure B]. There was a preponderance of MLL rearrangements (N=7) in fusion-positive patients, and 3 more patients had either t(8;21) or inv(16), and 25.6% (N=11) without karyotypic alterations (CN-AML). None of the fusion-positive patients had the t(7;12) or 12p abnormality. There were few common AML-associated mutations: 1 patient had FLT3ITD and 1 had the WT1 mutation (no NPM1 or biallelic CEBPA mutations were identified). [Display omitted] Rates of morphologic complete remission (CR) at the end of induction course 1 were similar for fusion-positive and -negative patients (68.9% vs. 77.7%; P=0.17). However, fusion-positive patients were more likely to have minimal residual disease (MRD) by flow cytometry at this time point (50% vs. 28.9%; P=0.006) with a correspondingly higher relapse rate (RR) from remission of 58% vs. 35% (p=0.005). Disease-free survival for those with and without fusion was 42% vs. 58% (p=0.060)In a subset analysis of 193 patients with CN-AML, the prevalence of CBFA2T3-GLIS2 was 4.7%. Fusion-positive patients were younger than fusion-negative patients (1.6 vs. 13.1 years; P<0.001) and more likely to have MRD at the end of induction (85.7% vs. 40.8%; P=0.043). CN-AML fusion-positive patients had significantly worse 5-year OS (36% vs. 67% P=0.025) and EFS (18% vs. 51%, P=0.017) than fusion-negative patients. All fusion-positive patients in CR had a higher 5-year RR than fusion-negative patients (88% vs. 33%; P<0.001), with a corresponding disease-free survival (DFS) of 13% vs. 59% (P<0.001).Implications of CBFA2T3-GLIS2 were evaluated in 71 cases of children with FAB M7 AML where the fusion was identified in 12 patients (17%). Fusion-positive FAB M7 patients had significantly lower CR rate than fusion-negative patients (33.3% vs. 77.6%, P=0.005) and all were MRD positive at the end of induction (100% vs. 30%, P=0.001). All FAB M7 fusion-positive patients relapsed (100% vs. 36%, P=0.007), with a DFS of 0% vs. 60% (p=0.013).As CBFA2T3-GLIS2 was most common in younger patients, we compared clinical implications in children <2 years of age. Although CR rates in fusion-positive and -negative patients were similar (68.2% vs. 74.9%; P=0.50), fusion-positive patients were more likely than fusion-negative patients to have MRD at the end of course 1 (63.2% vs. 24.8%, P=0.006). Five-year RR rates in fusion-positive and -negative patients were 71% vs. 39% (P=0.012), with corresponding DFS of 29% vs. 56% (P=0.030).This study provides a comprehensive evaluation of incidence and prognostic implications of the CBFA2T3-GLIS2 fusion in pediatric de novo AML. Along with MLL rearrangements, 12p abnormalities, and the recently described NUP98-JARID1A fusion, this cryptic inversion 16, as defined by presence of the CBFA2T3-GLIS2 fusion, represents a distinct, recurrent chromosomal abnormality associated with poor prognosis in infant AML and is a potential therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Clinical features and genetic analysis of 17 cases of glucose-6-phosphate dehydrogenase deficiency in Guizhou province

Objective To explore the clinical and genetic characteristics of 17 cases with glucose-6-phosphate dehydrogenase(G6PD) deficiency in Guizhou, China. Methods The clinical features of 17 patients with G6PD deficiency were analyzed, DNA samples were obtained from the patients and some mothers, and the exons and flanking intronic sequences of the G6PD gene were analyzed by the polymerase chain reaction and sequencing. Results The cases had diverse phenotypes, these patients had acute haemolytic anaemia triggered by eating broad beans, infections, inges-tion of specific drugs or the neonatal period and chronic nonspherocytic haemolytic anaemia.Three cases of the patients had concomitant diseases for α Mediterranean anemia, acute myeloid leukemia M2 type and neonatal anal membrane stenosis, respectively.G1376T, G1388A and A95G were the commonest G6PD variants in patients in Guizhou, China.G1376T, G1388A and A95G mutations were observed in 82.4% cases.Two patients had only compound variants(c.1311 C>T, IVS11 nt 93 T>C). One case in the Rongjiang County, Guizhou Province had novel compound variants(c.G1388A, IVS10-10 T>G) in the world.A patient's mother in the Guiyang City, Guizhou Province, China had compound variants(c.1376 G>T, 1311 C>T, IVS11 nt 93 T>C) as a carrier. Conclusions G6PD deficiency has a wide range of clinical heterogeneity.A novel G6PD compound variant haplotype c. G1388A, IVS10-10 T>G was first found in the world, and the SNP spectrum of G6PD was enlarged.There may be a G6PD compound variant haplotype c. 1376 G>T, 1311 C>T, IVS11 nt 93 T>C in Guizhou. Key words: Glucose-6-phosphate dehydrogenase deficiency; Glucose-6-phosphate dehydrogenase gene; Clinical characteristics; Compound variant

Frequent coexistence of RAS mutations in RUNX1-mutated acute myeloid leukemia in Arab Asian children.

RUNX1 mutation plays an important role in adult leukemic transformation. However, its contribution to the development of childhood leukemia remains unclear. In the present study, we analyzed point mutations of RUNX1 gene in children and adolescents with acute myeloid leukemia (AML) from Iraq and Jordan. Bone marrow and/or peripheral blood samples were collected from 178 patients of Arab Asian ethnicity (aged ≤17 years) newly diagnosed with AML: 145 samples from Iraq and 33 samples from Jordan. Direct DNA sequencing was performed on six genes including RUNX1 gene (exons 3-8). RUNX1 point mutations were identified in 10 (5.6%) of 178 patients. One patient possessed biallelic mutations of RUNX1 gene. C-terminal area was the predominant site of RUNX1 mutations (eight in C-terminal and two in N-terminal). Patients with RUNX1 mutations were significantly older than those with wild-type of the gene. Additionally, AML M0 subtype was more frequently found in patients with RUNX1 mutations. Both RUNX1 mutations and RAS mutations were identified in 4 of 10 children. Three patients with RUNX1 mutation had FLT3-ITD. On the other hand, 36 (21.4%) and 25 (14.9%) of 168 patients with wild-type of the gene had a RAS mutation and FLT3-ITD, respectively. Eight of 10 patients with RUNX1 mutations died of hematological relapse. The incidence of RUNX1 mutations in Arab Asian children and adolescents with AML was 5.6%. Further studies are required to clarify whether RAS mutations contribute to the development of pediatric AML associated with RUNX1 mutations.

Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML.

Leukemic stem cells (LSCs) isolated from acute myeloid leukemia (AML) patients are more sensitive to nuclear factor κB (NF-κB) inhibition-induced cell death when compared with hematopoietic stem and progenitor cells (HSPCs) in in vitro culture. However, inadequate anti-leukemic activity of NF-κB inhibition in vivo suggests the presence of additional survival/proliferative signals that can compensate for NF-κB inhibition. AML subtypes M3, M4, and M5 cells produce endogenous tumor necrosis factor α (TNF). Although stimulating HSPC with TNF promotes necroptosis and apoptosis, similar treatment with AML cells (leukemic cells, LCs) results in an increase in survival and proliferation. We determined that TNF stimulation drives the JNK-AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC. We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF-JNK-AP1 signaling pathway. Our data suggest that co-inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

Acut monocytic leukemia with pituitary involvement; a case report

Abs tract Central nervous system involvement is rare in acute myeloid leukemia (AML) cases. Pituitary involvement seems much more rarely with unknown frequency. A 29-year-old male patient was admitted to our clinic with the complaints of visual disturbance and poor performance status. His medical history revealed AMLM5 diagnosed five years ago and allogeneic hematopoietic stem cell transplantation performed in 2010. During follow-up in remission, his complaints had begun. In our clinic, the patient was diagnosed with AML M5 relapse associated with panhypopituitarism and central diabetes insipidus. Chemoradiotherapy was initiated immediately and hormone replacement therapy started due to hypopituitarism. His complaints partially resolved. He is still being followed up with intermittent intrathecal and systemic chemotherapy. Although rare, leukemic infiltration of the pituitary gland should be evaluated in leukemic patients with visual disturbance, hypopituitarism or central diabetes insipidus. Turk Jem 2015; 19: 69-71

Identification of novel genomic aberrations in AML-M5 in a level of array CGH.

To assess the possible existence of unbalanced chromosomal abnormalities and delineate the characterization of copy number alterations (CNAs) of acute myeloid leukemia-M5 (AML-M5), R-banding karyotype, oligonucelotide array CGH and FISH were performed in 24 patients with AML-M5. A total of 117 CNAs with size ranging from 0.004 to 146.263 Mb was recognized in 12 of 24 cases, involving all chromosomes other than chromosome 1, 4, X and Y. Cryptic CNAs with size less than 5 Mb accounted for 59.8% of all the CNAs. 12 recurrent chromosomal alterations were mapped. Seven out of them were described in the previous AML studies and five were new candidate AML-M5 associated CNAs, including gains of 3q26.2-qter and 13q31.3 as well as losses of 2q24.2, 8p12 and 14q32. Amplication of 3q26.2-qter was the sole large recurrent chromosomal anomaly and the pathogenic mechanism in AML-M5 was possibly different from the classical recurrent 3q21q26 abnormality in AML. As a tumor suppressor gene, FOXN3, was singled out from the small recurrent CNA of 14q32, however, it is proved that deletion of FOXN3 is a common marker of myeloid leukemia rather than a specific marker for AML-M5 subtype. Moreover, the concurrent amplication of MLL and deletion of CDKN2A were noted and it might be associated with AML-M5. The number of CNA did not show a significant association with clinico-biological parameters and CR number of the 22 patients received chemotherapy. This study provided the evidence that array CGH served as a complementary platform for routine cytogenetic analysis to identify those cryptic alterations in the patients with AML-M5. As a subtype of AML, AML-M5 carries both common recurrent CNAs and unique CNAs, which may harbor novel oncogenes or tumor suppressor genes. Clarifying the role of these genes will contribute to the understanding of leukemogenic network of AML-M5.

Pulmonary Presentation of Relapsed Acute Myeloid Leukemia

Extramedullary manifestations of acute myeloid leukemia (AML), often referred to as myeloid sarcoma (MS), occur relatively commonly in children with newly diagnosed or relapsed AML and have been associated with certain French-American-British morphologies and gene/chromosomal rearrangements. The most common locations of MS include the skin, orbit, skeleton, central nervous system, skin, and gut. Pulmonary MS is uncommon in adults and is extremely rare in children. We report the case of a 19-year-old man with French-American-British M5 AML, who before bone marrow transplant, presented with fever, hypotension, and respiratory symptoms that were ultimately attributed to pulmonary MS.

Pleural effusion as an unusual initial presentation of acute myeloid leukemia.

Pleural effusions in acute myeloid leukemia (AML) can have wide differential diagnosis, however AML presenting as pleural effusion with leukemic infiltration is rarely documented. A 22year old male presented with pleural effusion for 3months and subsequently diagnosed as AML M2, which prompted us for this communication.

Targeting CD13 (aminopeptidase-N) in turn downregulates ADAM17 by internalization in acute myeloid leukaemia cells.

Secreted matrix metalloproteinases (MMP)-2 and MMP-9 and membrane-anchored aminopeptidase-N/CD13 are abnormally expressed in human acute myeloid leukaemia (AML). We previously showed that CD13 ligation by anti-CD13 monoclonal antibodies can induce apoptosis in AML cells. Here, we assessed ADAM17 expression in primary blood blasts CD13+CD33+ from patients with AML. Primary AML cells expressed ADAM17 transcript and its surface expression was higher in subtype M4 (myelomonocytic) and M5 (monocytic) AML specimens than in M0 and M1/M2 (early and granulocytic) specimens. In AML cell lines defining distinct AML subfamilies (HL-60/M2, NB4/M3, THP-1/M5, U937/M5) and primary AML cells cultured ex vivo, anti-CD13 antibodies downregulated surface CD13 and ADAM17 without affecting MMP-2/-9 release. Knockdown of CD13 by siRNA prevented anti-CD13-mediated ADAM17 downregulation, indicating that CD13 is required for ADAM17 downregulation. Soluble ADAM17 was not detected in the medium of anti-CD13 treated cells, suggesting that ADAM17 was not shed. After ligation by anti-CD13, CD13 and ADAM17 were internalized. Subsequently, we found that ADAM17 interacts with CD13. We postulate that the interaction of ADAM17 with CD13 and its downregulation following CD13 engagement has important implications in AML for the known roles of ADAM17 in tumour-associated cell growth, migration and invasion.

CBFβ/MYH11 Hypomethylation Signature In Patients With Acute Myeloid Leukaemia

It has been shown that changes in DNA methylation pattern in patients with acute myeloid leukaemia (AML) may both reflect specific molecular abnormalities or characterize a group of patients without a known molecular aberration. Studying genes that are epigenetically deregulated in different groups of patients may contribute to a more detailed understanding of pathways involved in leukemic transformation.For 15 samples (14 AMLs of diverse clinical and genetic background and pool of CD34+ control samples) SureSelectXT Human Methyl-Seq system (Agilent) was used to interrogate DNA methylation of selected regions (84 megabases in total) of their genomes. Targeted bisulfite libraries were run on a HiSeqTM2000 sequencer (Illumina) using 105 bp paired-end sequencing reads. Methylation status of candidate regions was verified using 454 bisulfite pyrosequencing (Roche). The expression levels of selected genes were assessed with TaqMan Gene Expression Assays (Applied Biosystems).Targeted methylation data was correlated with previously obtained whole-genome bisulfite (WGBS) data for the cross-experiment (WGBS/target enrichment) samples (n=3), taking into account positions with minimal coverage of 10. There was a strong positive correlation (R≥0.97) for all three samples. Pairwise Pearson correlations were computed for all (targeted and WGBS) samples and formed the basis of a hierarchical clustering analysis. The cross-experiment replicates clustered together. From the clinical and molecular characteristics, only CBFβ/MYH11+ patients (n=2) clustered together in all clustering analysis performed (using CpG in/out CGIs or all CpG regardless of location). None of the other molecular abnormalities (i.e. DNMT3A mutations, MLL translocations, NPM1 mutations, FTL3/ITDs and CEPBα mutations) formed clusters. We did not observe any effect of clinical status of AML (de novo, secondary AML with dysplastic changes or relapsed AML). Based on this observation, we extracted genomic regions being uniquely differentially methylated for CBFβ/MYH11+ patients. There was a clear tendency towards hypomethylation in 125 regions out of 182 (69%) displaying lower DNA methylation levels compared to CD34+ pool of controls. All CBFβ/MYH11 differentially methylated regions (DMRs) were uploaded to GREAT (http://bejerano.stanford.edu/great/public/html) and enrichment for genes described as upregulated in inv(16) AML (CBFβ/MYH11+) was seen (reported in Valk et al., NEJM 2004, ID: VALK_AML_CLUSTER_9). This enrichment set comprised 10 genomic regions assigned for 6 genes – MN1, SPARC, ST18, DHRS3, FAM171A and BAHCC1.MN1, SPARC, ST18 and DHRS3 – were chosen for hypomethylation verification by 454 bisulfite pyrosequencing. Altogether 11 CBFβ/MYH11+ (inv(16)), 11 non-inv(16) AML M4, 6 other AML subtypes and 5 healthy control samples were examined by 454 pyrosequencing. Average levels of DNA methylation for selected regions were significantly lower for inv(16) versus non-inv(16) AML M4, other AML subtypes and healthy controls (P<0.0001). The 454 results support our theory about site specific CBFβ/MYH11 hypomethylation signature. MN1 gene was overexpressed in 82% of inv(16) AML versus 42% of non-inv(16) patients (χ2 test, P=0.009). Interestingly, MN1 overexpression in non-inv(16) patients was not accompanied with MN1 hypomethylation suggesting different mechanisms of gene deregulation in various AML subtypes.We found a new hypomethylation signature in CBFβ/MYH11+ patients. The described hypomethylated regions were assigned to genes known as being overexpressed in inv(16) patients. As for MN1 gene, we can speculate that different mechanism responsible for its overexpression in inv(16) versus non-inv(16) patients may explain discrepancy between its negative prognostic value in cytogenetically normal AML patients and generally good outcome of inv(16) patients.This study was supported by the project (Ministry of Health, Czech Republic) for conceptual development of research organization (00023736, IHBT) and was a part of the COST Action BM0801 (EuGESMA). Disclosures:No relevant conflicts of interest to declare.

L-Asparaginase Sensitivity and Asparagine Synthetase Expression In Primary Tumor Cells From AML Patients

Abstract Introduction The rational of use of L-asparaginase (L-Aspa) is based on asparagine synthetase (ASNS) deficiency in leukemic cells. Its efficacy is due to its capacity to deplete plasmatic asparagine, starving the leukemic cells and subsequently inhibiting protein synthesis. It is a mainstay in the treatment of acute lymphoid leukemia (ALL), where it is established that resistance to treatment is in part related to detectable expression of ASNS (Aslanian et al., 2001; Su et al., 2008). In acute myeloid leukemia (AML), promising results have been obtained in clinical trials (Capizzi et al., 1988), with an improvement of complete remission rates from 18% to 54% in refractory patients &lt;60 year old and from 0% to 31% in refractory patients &gt;60 year old. More recently, it has been reported that leukemic cells from AML patients with M1, M4 and M5 FAB subtypes were more sensitive to L-Aspa (Okada et al., 2003).The aim of this study was to investigate the sensitivity to L-Aspa and the ASNS expression in an AML cell line (HL-60) and in primary leukemic cells from newly diagnosed AML patients. Materials and methods AML (HL-60) and ALL (MOLT-4) cell lines were grown according to ATCC recommendations. Primary leukemic cells from the bone marrow of 24 AML patients (median age: 69 years; range: 2-83) were harvested on EDTA and isolated by Ficoll density gradient within 72h. ASNS expression was determined by western-blot on isolated leukemic cells and expressed as a ratio ASNS/cyclophilin A. When a sufficient amount of leukemic cells was available, sensitivity to L-Aspa (expressed as an IC50 - concentration inhibiting 50% of cell viability) was evaluated in vitro by incubating various concentrations of L-Aspa (0.001 to 10 IU/mL) with the cells and by measuring the cell viability with a counting kit (CCK-8 viability assay) at day 4. Results Determination of IC50 for the HL-60 cell line demonstrated that these cells were equally sensitive to L-Aspa than the ALL cell line MOLT-4 in vitro (0.23 IU/mL versus 0.19 IU/mL, respectively). The expression of ASNS in the HL-60 cell line was low but higher than in MOLT-4 which is a well known ASNS deficient cell line. IC50 determination was possible on 17/24 patients. Seven displayed a high sensitivity to L-Aspa (IC50 &lt; 0.01 IU/mL) whereas 5 displayed a moderate sensitivity (IC50 &lt; 0.5 IU/mL). Remaining 5 patients were resistant to L-Aspa. The cells from the healthy subject were resistant (IC50 &gt; 10 IU/mL). To date, ASNS expression has been evaluated on 16 patients with ratio ranges from 0 to 2.27: six were negative, 4 low positive (&lt; 0.2), and 6 positive (&gt; 0.5, amongst them 4 were &gt; 1). No correlation was observed between ASNS expression and FAB grade. However, patients with blasts sensitive to L-Aspa had mainly M1 or M5 AML. Conclusion AML cell line HL-60 and 71% of primary cells from AML patients were found sensitive to L-Aspa. The sensitivity of cells from M1 and M5 AML patients is consistent with the findings of Okada et al. (2003). Globally, these results suggest that L-Aspa is effective for killing AML cells. Based on the epidemiology of AML subtypes (Selter et al., 2011) and our results, L-Aspa therapy may be beneficial for 50-70% of patients with AML. However, L-Aspa has only been used scarcely in the treatment of AML, mainly because of the commonly observed adverse effects that impair its use. A new formulation of L-Aspa encapsulated in homologous red blood cells was reported with a better safety profile, allowing its use even in elderly patients (Hunault et al., ASH 2012). A clinical study is currently recruiting patients unfit for intensive chemotherapy in order to evaluate its efficacy in combination with low-dose cytarabine (NCT01810705) in AML. In this study, L-Aspa sensitivity and ASNS expression in primary tumor cells at diagnosis will be explored to investigate the relationship with clinical response. Disclosures: Berlier: ERYTECH Pharma: Employment. Aguera:ERYTECH Pharma: Employment. Gallix:ERYTECH Pharma: Employment. Bertrand:ERYTECH Pharma: Principal Investigator Other. Thomas:ERYTECH Pharma: Principal Investigator Other; Orphan Europe: Consultancy. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

The Targeted Histone Deacetylase Inhibitor Tefinostat (CHR-2845) Shows Selective In Vitro Efficacy In Monocytoid-Lineage Acute Myeloid Leukaemia (AML)

Tefinostat (CHR-2845) is a novel monocyte/macrophage-targeted histone deacetylase inhibitor (HDACi) that is cleaved to an active acid, CHR-2847, by an intracellular esterase (human carboxylesterase-1, hCE-1), found only in cells of monocytoid lineage and hepatocytes. The clinical uptake of HDAC inhibition to date has been restricted by systemic toxicities including gastrointestinal disturbance, thrombocytopenia and fatigue. Accumulation of CHR-2847 in hCE-1-expressing cells results in a 20-100-fold increase in targeted anti-proliferative potency, considerably widening the potential therapeutic window in malignancies involving cells of monocytoid lineage (AML-M4, AML-M5 and CMML) by sparing the systemic toxicological effects associated with non-selective HDAC inhibition.The in vitro efficacy of tefinostat was assessed in primary AMLs using stored mononuclear cells obtained at diagnosis from 70 AML patients. Dose-dependent induction of apoptosis and significant growth inhibitory effects were seen in M4 /M5 AMLs (median IC50; 1.1µM+/-1.8) compared to non-M4/M5 FAB types (median IC50 5.1µM +/-4.7) (p=0.007). This potency and monocytoid specificity was not reproduced when using an alternative HDACi, tefinostat analogue CHR-8185 which is not cleaved by hCE-1.hCE-1 protein expression in patient samples was measured by both intracellular flow cytometry and immunoblotting, with highest levels seen in M4/M5 patients. This observation was validated by microarray analysis of hCE-1 mRNA in a further 130 AML samples with M4/M5 AMLs showing significant overexpression compared to normal bone marrow CD34+ cells (p=0.009). High levels of hCE-1 expression were found to drive a significant increase in tefinostat efficacy as measured by growth inhibition assays (p=0.001), and also strongly correlated with expression of the mature monocytoid marker CD14+.Sub-population analysis by flow cytometry revealed variable sensitivity to tefinostat within AML blasts, with CD14+ expressing cells showing maximum growth inhibition. This CD14+ response was accompanied by an induction of intracellular protein acetylation at nanomolar concentrations in tefinostat-responsive samples. Tefinostat-sensitive samples also showed strong induction of the cell cycle arrest and DNA damage sensor protein pH2AX, which is a potential biomarker of patient responsiveness.Importantly, no growth inhibitory effects were seen in normal bone marrow cells (n=5) exposed to AML-toxic doses of tefinostat while, in comparison, equivalent concentrations of the non-hCE-1-dependent analogue CHR-8185 caused considerable cytotoxicity, again emphasising the potential for expansion of the clinical therapeutic window using an hCE-1-dependent agent.In vitro synergy was demonstrated in combination experiments with tefinostat and cytarabine (median Combination Index value=0.68) which is likely to be a logical combination for future clinical evaluation.In summary, monocytoid targeting of HDACi activity was achieved using tefinostat in primary AML samples of monocytoid lineage, with minimal toxicity to normal bone marrow cells at equimolar concentrations. Given the absence of significant toxicity seen in a recently-published phase 1 study of tefinostat in patients with advanced haematological malignancies, further larger scale clinical evaluation of this compound is warranted in haematological malignancies involving cells of monocytoid lineage. Disclosures:Zabkiewicz:Chroma Therapeutics: Research Funding. Gilmour:Chroma Therapeutics: Research Funding. Hills:Chroma Therapeutics: Research Funding. Bone:Chroma Therapeutics: Employment. Davidson:Chroma Therapeutics: Employment. Burnett:Chroma Therapeutics: Research Funding. Knapper:Chroma Therapeutics: Research Funding.

Tumor Suppressor RASSF2 Is Downregulated By The RUNX1-ETO Fusion Protein In t(8;21)+ Acute Myeloid Leukemia

IntroductionThe t(8;21) chromosomal translocation is one of the most common chromosomal translocations associated with acute myeloid leukemia (AML), present in greater than 10% of de novo AML cases. Most of these t(8;21) AML cases are classified as FAB subtype M2. This translocation results in the formation of a stable fusion protein made up of portions of the RUNX1 (aka AML1) and ETO (aka MTG8 and RUNX1T1) proteins. RUNX1 is a transcription factor that is essential for regulating the differentiation of hematopoietic cells, and the fusion protein retains its DNA-binding domain. Additionally, ETO contains four Nervy homology (NH) domains which facilitate a number of protein-protein interactions, notably with the NCOR2/SMRT co-repressor complex. The identification of individual genes or biological pathways which are specifically disrupted in the presence of RUNX1-ETO will provide further molecular insight into the pathogenesis of t(8;21)+ AML and lead to the possibility for improved treatment for these patients. Methods/ResultsWe analyzed publicly available gene expression microarray datasets (Oncomine, TCGA) to search for genes whose expression was significantly altered in the blood of t(8;21)+ AML patients as compared to non-t(8;21) FAB subtype M2 AML and to CD34+ cells in healthy controls. One such gene that was consistently significantly downregulated in t(8;21)+ patients was Ras-association domain family member 2 (RASSF2). RASSF2 is a putative tumor suppressor that is capable of mediating apoptosis (in a Ras dependent manner) through its interactions with the MST1/2 kinases and the cancer-specific apoptotic protein Par-4. RASSF2 has previously been shown to be frequently downregulated via hypermethylation in a wide variety of solid tumors, however little is known about its function in leukemia. Here we demonstrate that RASSF2 is a potentially interesting target for downregulation by the RUNX1-ETO fusion protein. Gene expression analysis by RT-qPCR in leukemia cell lines confirmed that RASSF2 is significantly downregulated in both Kasumi-1 and SKNO t(8;21)+ cell lines as compared to a similar non-t(8;21) HL-60 line. We found that exogenous expression of AML1-ETO in HL-60 leukemia cells induces a rapid downregulation of RASSF2, further supporting that it is a target of this leukemogenic fusion protein. Over-expression of RASSF2 in leukemia cells significantly inhibits their proliferative capability, indicating an important biological effect of RASSF2 in blood cells. Finally, over-expression of RASSF2 significantly inhibits the long-term self-renewal capability of RUNX1-ETO expressing hematopoietic cells as measured by their serial replating ability in a colony formation assay. DiscussionBased on the analysis of patient data and our own experiments it appears that RASSF2 is a direct target for downregulation by the AML1-ETO fusion protein. Due to its potential involvement as a mediator of apoptosis in important oncogenic signaling pathways RASSF2 is a strong candidate for further investigation in the context of t(8;21)+ AML pathogenesis. In particular, it will be interesting to continue to investigate the relationship between RASSF2 and apoptotic protein Par-4, as several lines of evidence suggest Par-4 to be therapeutically relevant due to its ability to selectively induce apoptosis in cancer cells. Disclosures:No relevant conflicts of interest to declare.

AML Cells Utilize TNF-Driven JNK Signaling As a Critical NF-κB-Independent Survival Signal

Leukemic Stem Cells (LSCs) isolated from Acute Myeloid Leukemia (AML) patients are highly sensitive to NF-κB inhibition-induced cell death in in vitro culture when compared to normal hematopoietic stem/progenitor cells (HSPCs). This suggests blocking NF-κB could be an effective strategy for treating AML. However, NF-κB inhibitor treatment alone is unable to clear AML tumors in vivo. We propose this is most likely due to elements within the niche microenvironment stimulating compensatory survival/proliferation signals in AML cells that can overcome NF-κB inhibition.We utilize a multi-faceted AML model that includes a murine system created by MLL-AF9 transduced hematopoietic cells, several established human AML cell lines, and primary AML tumors isolated from patients. We observed elevated Tumor Necrosis Factor-α (TNF) levels in the peripheral blood of AML M3, 4, and 5 patients. Our studies suggest this TNF is produced directly by the AML cells, and that AML cells utilize TNF to stimulate JNK as a survival signal parallel to NF-κB in leukemic cells and a death signal in HSPCs.In healthy HSPCs, stimulation with exogenous TNF induces RIP1/3-mediated necroptosis and caspase-mediated apoptosis, resulting in cell death and loss of normal hematopoietic function. Inactivation of JNK signaling can partially rescue TNF-mediated loss of function in HSPCs. Additionally, blocking NF-κB in HSPCs sensitizes them to TNF-mediated cell death, which can also be partially prevented by inactivation of JNK signaling. These data suggest that JNK acts as a TNF-mediated death signal in HSPCs.In AML, we found TNF signaling promotes the in vitro growth of leukemic cells as well as enhancing the in vivo development of AML tumors by stimulation of both NF-κB and JNK signaling in parallel. We determined that TNF-mediated JNK signaling in leukemic cells results in the propagation of a critical survival signal. These cells convert TNF-JNK stimulation from a death to survival signal by limiting JNK activity duration as well as upregulating the expression of the AP1 family transcription factor c-Jun. Therefore, TNF-JNK signaling in leukemic cells does not drive apoptotic cell death, but instead drives c-Jun activity and the subsequent production of the anti-apoptotic genes c-Flip and Mcl-1.We found inactivation of any part of the TNF-JNK-AP1 signaling axis can repress the growth of leukemic cells in vitro and delay leukemogenesis in vivo. We also show that blocking any portion of the TNF-JNK-AP1 signaling axis sensitizes TNF-expressing leukemic cells, including LSCs, to NF-κB inhibitor treatment while at the same time protecting HSPCs from such treatment.In conclusion, our studies suggest that the inadequate ability of NF-κB inhibition to clear AML tumors in vivo is due to the TNF-mediated activation of the JNK-AP1 signaling axis that can compensate for NF-κB signal repression in LSCs. We found that many types of AMLs produce TNF, and this TNF acts both in an autocrine fashion to promote LSC survival and self-renewal through parallel activation of NF-κB and JNK-AP1, as well as in a paracrine fashion to repress normal hematopoiesis. Therefore, we propose that inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more thorough treatment for AML patients with elevated peripheral blood TNF. Disclosures:No relevant conflicts of interest to declare.

Prevalence Of FLT3 Mutations In Acute Myeloid Leukemia: A Multicenter Latin America Study

In acute myeloid leukemia (AML), FLT3 mutations are associated with a poor prognosis, particularly the internal tandem duplication (ITD/FLT3). In Latin America there are few epidemiological data about these mutations.This study assessed the prevalence of FLT3 mutations in patients with AML at four reference hematology centers from Latin America. We included 138 samples of patients attending the Hematology Service of three Mexican University Hospitals (Monterrey,México D.F. and Puebla) and one Colombian center (Medellín) with a diagnosis of AML from different morphologic subgroups according to the French-American-British classification from 2006 to 2011. AML was diagnosed by morphology according to the FAB classification, by immunohistochemical staining and/or by immunophenotype according to each particular case. For sample processing DNA was extracted from peripheral blood or bone marrow with the automatic Maxwell®16 System (Promega Corporation, Madison, WI) using the principle of cellular lyses and binding nucleic acids to magnetized silice particles or the QIAAmp DNA Blood Kit (QIAGEN, Mexico City). The quality and DNA concentration was evaluated with the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). Then internal tandem duplication and kinase domain mutations detection was performed with the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA), through amplification of exons 14,15 with specific primers of FLT3 gen region, using the Seeplex® FLT3 Genotyping kit (Seegene, Rockville, MD, USA) or according to Kottaridis et al (1). Later, an electrophoretic analysis of the amplified products was made in a 2% agarose gel stained with ethidium bromide or in polyacrylamide gel electrophoresis (PAGE) and were observed by transilumination. In the Puebla samples the products were analyzed by capillary electrophoresis (ABI3130, Applied Biosystems, Foster City, CA). For detection of D835X mutations the exon 20 amplicom was subjected to digestion with EcoRV and analyzed by 4.5% PAGE. The patients were cytogenetically classified into three risk groups: favorable, intermediate, and adverse. ResultsWe analyzed 138 samples of AML patients and found FLT3 mutations in 28 patients, for a prevalence of 20.3%. The median age was 47 years (5-96). Only four patients had the KD FLT3 mutation (3% of total population). The FLT3 mutation positive group was older than the negative (47 vs. 39 years), had higher WBC/mm3(66.0 vs. 56.4 x 109/dl), higher hemoglobin values (9.3 vs. 8.6 g/dl), and lower platelet counts (72.6 vs. 92.5 x109/dl), although there were not statistically significant differences. Thirteen patients had AML M2, nine the monocytic variety, four had M3 and two M1. On cytogenetics 25% , 62.5% and 12.5% had favorable, intermediate and unfavorable risk karyotypes respectively. The rate response to standard Induction Chemotherapy was 78.3 % for the FLT3 positive group vs. 74.1 % for the non-mutated group. Nineteen of 28 patients (67.8%) with FLT3 mutations died, compared to 47 of 100 (42.72%) in those without the mutation. The median survival was 4.9 months for the FLT3 mutated group vs 20.4 months for the FLT3 negative group, P= 0.009. The cytogenetic intermediate risk group (62.5%) was further analyzed and no statistically significant difference in overall survival between FLT3 non-mutated and FLT3 mutated patients was found (P= 0.22). Younger patients (<55 years) had a higher mortality in the FLT3 positive group (P = 0.023).The presence or absence of the FLT3 mutation in patients who had the morphologic subtype M3 did not impact mortality (P = 0.28), but in non M3 subtypes, it did (P= 0.017). As conclusion, in this Latin American population the FLT3 mutation conferred a bad prognosis.

Analysis Of Micro-RNA-142 Mutations In a Cohort Of 944 Patients With MDS and AML

BackgroundMicroRNAs are short (20-40 nucleotides) non-coding RNA molecules that are responsible for the post-transcriptional regulation of gene expression. Aberrant expression of MicroRNAs has been associated with various malignancies. Specifically, downregulation of MicroRNA-142 (miR-142) has been shown to occur in acute myeloid leukemia (AML). Interestingly, also gene mutations in miR-142 have been recently described in de novo AML. So far, little is known about mutations in miR-142 in myeloid malignancies. The aim of this study was to analyze mutations in the miR-142 in a large cohort of 944 patients with AML and myelodysplastic syndrome (MDS). Patients and MethodsThe patient group consisted of 425 de novo AML patients (excluding AML M3) who entered the multicenter treatment trials AML SHG 0199 or AML SHG 0295, 326 patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for secondary acute myeloid leukemia after a prior diagnosis of MDS (sAML) (n=170) or primary MDS (n=156), and 193 primary MDS patients not undergoing intensive therapy or allogeneic HSCT. The genomic region of the miR-142 gene, containing miR-142-5p and miR-142-3p, was sequenced by Sanger sequencing. Patient samples were also assessed for other frequently mutated genes in AML and MDS. Results and DiscussionWe identified five patients with mutations in miR-142. All mutations were heterozygous point mutations affecting the seed region of miR-142-3p, thereby potentially changing the target specificity of miR-142. Mutations in miR-142 occurred in male and female patients. Of the five patients with mutations in miR-142, only one patient carried the diagnosis of de novo AML (0.2% in de novo AML), while two patients were diagnosed with sAML (1.2% in sAML) and two patients had MDS (0.56% in MDS, corresponding to 0.77% in MDS/AML from MDS). Apart from one patient who underwent allogeneic transplantation for sAML, all other patients with follow-up died of the disease in less than a year. 3 patients had normal cytogenetics, while one patient had a complex karyotype and one patient had a trisomy 8 with translocation t(1;4). No mutated patient showed aberrations typically associated with de novo AML (RUNX1/RUNX1T1, CBFB/MYH11, FLT3-ITD, NPM1 mutations or CEBPA mutations). However, myelodysplasia-related gene mutations such as mutations in the splicing genes or chromatin remodelling genes were found in two patients (one patient with mutated ASXL1 and SRSF2, one patient with mutated U2AF1). Furthermore, one patient had a concomitant mutation in NRAS and IDH1. Thus, the associated mutational profile suggests that miR-142 mutations play a role in the pathogenesis of MDS rather than de novo AML. ConclusionMicroRNA-142 is recurrently but infrequently mutated in MDS and secondary AML evolving from MDS, and some mutations co-occur with MDS-related gene aberrations. As miR-142 mutations affect the seed region of the miRNA the target specificity is likely changed, and the miRNA may lose its tumor suppressor function, which has been implicated from functional studies. Disclosures:Platzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Acute Myeloid Leukemia With t(8;21)/RUNX1-RUNX1T1 demonstrate a High Number Of Secondary Genetic Lesions: Frequency and Impact On Clinical Outcome

▪ BackgroundTranslocation t(8;21) with the resulting RUNX1-RUNX1T1 rearrangement is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML). Although it is generally associated with a favourable prognosis, many additional genetic lesions may impact on outcome. AimTo assess the frequency and clinical impact of additional mutations and chromosomal aberrations in AML with t(8;21)/RUNX1-RUNX1T1. MethodsWe analyzed 139 patients (pts) who were referred to our laboratory for diagnosis of AML between 2005 and 2012 (65 females, 74 males; median age 53.3 years, range 18.6 - 83.8 years). All pts were proven to have t(8;21)/RUNX1-RUNX1T1 by a combination of chromosome banding analysis, fluorescence in situ hybridisation and RT-PCR. Analysis of mutations in ASXL1, FLT3-TKD, KIT (D816, exon8-11), NPM1, IDH1 and IDH2, KRAS, NRAS, CBL, and JAK2 as well as of MLL-PTD and FLT3-ITD was performed in all pts. Results107/139 pts were classified according to FAB criteria (77.0%). 34/107 had AML M1 (31.8%) and 73/107 AML M2 (68.2%). 117/139 had de novo AML (84.2%), 22/139 had therapy-related AML (t-AML) (15.8%). 69/139 (49.6%) pts had at least one molecular alteration in addition to RUNX1-RUNX1T1, 23/69 (33.3%) had two or more additional mutations. Most common were mutations (mut) in KIT (23/139; 16.5%), followed by NRAS (18/139; 12.9%) and ASXL1 (16/139; 11.5%). FLT3-ITD and mutations in FLT3-TKD, CBL, and KRAS were found in 4.3% - 5.0% of all pts, whereas mutations in IDH2 and JAK2 were detectable in 3.6% and 2.9%, respectively. IDH1 mutations were found in only 0.7% (1/139). NPM1mut and MLL-PTD were mutually exclusive of RUNX1-RUNX1T1. FLT3-ITD as well as FLT3-TKD were exclusive of ASXL1 mutations. With exception of FLT3-ITD, which was only present in de novo AML, there was no difference in mutation frequencies between de novo AML and t-AML.69.8% (97/139) pts had at least one chromosomal aberration in addition to t(8;21)(q22;q22). Most frequent was the loss of either X- or Y-chromosome (together 46.8%), followed by del(9q) (15.1%), and trisomy 8 (5.8%). FLT3-ITD, FLT3-TKD and trisomy 8 were found to be mutually exclusive. The number of secondary chromosomal aberrations did not differ significantly between pts with de novo AML and t-AML, showing only a trend towards higher frequency of -Y, del(9q), and trisomy 8 in pts with t-AML.Survival was calculated in pts who received intensive treatment (n=111/139, 79.9%; median follow-up 26.9 months; 2-year survival rate 73.4%). With exception of KITD816 mutation, which had a negative impact on overall survival in pts with de novo AML (2-year survival rate 64.2% vs. 82.3%, p=0.03), none of the other 13 mutations significantly influenced outcome, not even in case of 2 or more coexistent mutations. Also, no influence of additional chromosomal aberrations on survival was found.In selected cases (n=21/139), we compared dynamic changes in the patterns of genetic lesions at diagnosis and at relapse. In 14/21 (66.7%) pts the initial molecular mutation pattern changed at relapse. Mutations commonly gained at relapse were KIT mutations (6/21, 28.6%), followed by ASXL1 and IDH1R132 (each 2/21, 9.5%). FLT3-ITD, CBL, NRAS and JAK2 mutations emerged in 1/21 patients (4.8%) each. Loss of a mutation at relapse has been observed in KIT, ASXL1, and NRAS (each 2/21, 9.5%), as well as in KRAS, FLT3-ITD and FLT3-TKD (each 1/21, 4.8%). Concerning chromosomal alterations at relapse, 7/21 pts (33.3%) showed a change of their initial cytogenetic pattern, mostly shifting to a more complex karyotype (gain of chromosomal aberrations: 5/21, 23.8%; loss of chromosomal aberrations: 2/21, 9.5%). In all cases, t(8;21)(q22;q22)/RUNX1-RUNX1T1 remained stable at time of relapse. Conclusions1) 50% of t(8;21)/RUNX1-RUNX1T1 positive pts had at least one additional molecular mutation and almost 70% showed additional chromosomal abnormalities. 2) KIT was the most frequent additional molecular mutation, followed by NRAS and ASXL1. 3) The only additional genetic marker with a significant adverse prognostic impact was KITD816 mutation. Disclosures:Krauth:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.