Bernhard J. Woermann

GATA2 zinc finger 1 mutations associated with biallelic CEBPA mutations define a unique genetic entity of acute myeloid leukemia

Cytogenetically normal acute myeloid leukemia (CN-AML) with biallelic CEBPA gene mutations (biCEPBA) represents a distinct disease entity with a favorable clinical outcome. So far, it is not known whether other genetic alterations cooperate with biCEBPA mutations during leukemogenesis. To identify additional mutations, we performed whole exome sequencing of 5 biCEBPA patients and detected somatic GATA2 zinc finger 1 (ZF1) mutations in 2 of 5 cases. Both GATA2 and CEBPA are transcription factors crucial for hematopoietic development. Inherited or acquired mutations in both genes have been associated with leukemogenesis. Further mutational screening detected novel GATA2 ZF1 mutations in 13 of 33 biCEBPA-positive CN-AML patients (13/33, 39.4%). No GATA2 mutations were found in 38 CN-AML patients with a monoallelic CEBPA mutation and in 89 CN-AML patients with wild-type CEBPA status. The presence of additional GATA2 mutations (n=10) did not significantly influence the clinical outcome of 26 biCEBPA-positive patients. In reporter gene assays, all tested GATA2 ZF1 mutants showed reduced capacity to enhance CEBPA-mediated activation of transcription, suggesting that the GATA2 ZF1 mutations may collaborate with biCEPBA mutations to deregulate target genes during malignant transformation. We thus provide evidence for a genetically distinct subgroup of CN-AML. The German AML cooperative group trials 1999 and 2008 are registered with the identifiers NCT00266136 and NCT01382147 at www.clinicaltrials.gov.

Combined Molecular and Clinical Prognostic Index for Relapse and Survival in Cytogenetically Normal Acute Myeloid Leukemia

PURPOSE Cytogenetically normal (CN) acute myeloid leukemia (AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician, it is difficult to summarize the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pretreatment patient and disease characteristics. PATIENTS AND METHODS Two prognostic indices for CN-AML (PINA), one regarding overall survival (OS; PINAOS) and the other regarding relapse-free survival (RFS; PINARFS), were derived from data of 572 patients with CN-AML treated within the AML Cooperative Group 99 study (www.aml-score.org). RESULTS On the basis of age (median, 60 years; range, 17 to 85 years), performance status, WBC count, and mutation status of NPM1, CEBPA, and FLT3-internal tandem duplication, patients were classified into the following three risk groups according to PINAOS and PINARFS: 29% of all patients and 32% of 381 responding patients had low-risk disease (5-year OS, 74%; 5-year RFS, 55%); 56% of all patients and 39% of responding patients had intermediate-risk disease (5-year OS, 28%; 5-year RFS, 27%), and 15% of all patients and 29% of responding patients had high-risk disease (5-year OS, 3%; 5-year RFS, 5%), respectively. PINAOS and PINARFS stratified outcome within European LeukemiaNet genetic groups. Both indices were confirmed on independent data from Cancer and Leukemia Group B/Alliance trials. CONCLUSION We have developed and validated, to our knowledge, the first prognostic indices specifically designed for adult patients of all ages with CN-AML that combine well-established molecular and clinical variables and that are easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care through risk-adapted therapy of CN-AML.

Allogeneic Transplantation Versus Chemotherapy as Postremission Therapy for Acute Myeloid Leukemia: A Prospective Matched Pairs Analysis

PURPOSE The majority of patients with acute myeloid leukemia (AML) who achieve complete remission (CR) relapse with conventional postremission chemotherapy. Allogeneic stem-cell transplantation (alloSCT) might improve survival at the expense of increased toxicity. It remains unknown for which patients alloSCT is preferable. PATIENTS AND METHODS We compared the outcome of 185 matched pairs of a large multicenter clinical trial (AMLCG99). Patients younger than 60 years who underwent alloSCT in first remission (CR1) were matched to patients who received conventional postremission therapy. The main matching criteria were AML type, cytogenetic risk group, patient age, and time in first CR. RESULTS In the overall pairwise compared AML population, the projected 7-year overall survival (OS) rate was 58% for the alloSCT and 46% for the conventional postremission treatment group (P = .037; log-rank test). Relapse-free survival (RFS) was 52% in the alloSCT group compared with 33% in the control group (P < .001). OS was significantly better for alloSCT in patient subgroups with nonfavorable chromosomal aberrations, patients older than 45 years, and patients with secondary AML or high-risk myelodysplastic syndrome. For the entire patient cohort, postremission therapy was an independent factor for OS (hazard ratio, 0.66; 95% CI, 0.49 to 0.89 for alloSCT v conventional chemotherapy), among age, cytogenetics, and bone marrow blasts after the first induction cycle. CONCLUSION AlloSCT is the most potent postremission therapy for AML and is particularly active for patients 45 to 59 years of age and/or those with high-risk cytogenetics.

Age-dependent frequencies of NPM1 mutations and FLT3-ITD in patients with normal karyotype AML (NK-AML)

Prognosis of AML in elderly patients is poor due to adverse patient characteristics and comorbidities. In addition, disease-associated parameters reveal differences between older and younger patients with AML. Survival in normal karyotype AML (NK-AML) is influenced by different clinical and molecular markers. The aim of this work was to investigate the frequencies of molecular markers in patients with NK-AML with a focus on NPM1 mutations and FLT3-ITD in different age groups. In the present study, we analyzed the frequencies of mutations of NPM1 and FLT3-ITD in a cohort of 1,321 adult patients and 148 children with AML treated within the AMLCG99, the AML98, and AML04 trials and their distribution in different age groups. Additionally, the frequencies of mutations in CEBPA genes, FLT3-TKD, and MLL-PTD were analyzed in the cohort with NK-AML (n = 729). Our data show that the presence of mutations of NPM1 (from 60% to 40%) and FLT3-ITD (from 50% to 20%) significantly decreased with age in adult AML. Consequently, the proportion of NPM1−/FLT3-ITD− patients increased with age. The decreasing frequency of NPM1 mutations in elderly patients was paralleled by a reduced complete remission (CR) rate in the elderly of 55% compared to 80% in the younger patients. By contrast, the frequencies of other gene mutations, like FLT3-TKD and MLL-PTD, and mutations in CEBPA were not age-dependent. The decreasing frequency of the favorable NPM1 mutations with increasing age may partially explain the worse outcome in the elderly patients. Furthermore, the increasing amount of elderly patients without NPM1 mutations or FLT3-ITD suggests that other molecular and clinical risk factors may influence prognosis in this age group.

NPM1 but not FLT3-ITD mutations predict early blast cell clearance and CR rate in patients with normal karyotype AML (NK-AML) or high-risk myelodysplastic syndrome (MDS)

Mutations in the NPM1 gene represent the most frequent genetic alterations in patients with acute myeloid leukemia (AML) and are associated with a favorable outcome. In 690 normal karyotype (NK) AML patients the complete remission rates (CRs) and the percentage of patients with adequate in vivo blast cell reduction 1 week after the end of the first induction cycle were significantly higher in NPM1(+) (75% and 80%, respectively) than in NPM1(-) (57% and 57%, respectively) patients, but were unaffected by the FLT3-ITD status. Multivariate analyses revealed the presence of a NPM1 mutation as an independent positive prognostic factor for the achievement of an adequate day-16 blast clearance and a CR. In conclusion, NPM1(+) blast cells show a high in vivo sensitivity toward induction chemotherapy irrespective of the FLT3-ITD mutation status. These findings provide insight into the pathophysiology and help to understand the favorable clinical outcome of patients with NPM1(+) AML.

Isolated trisomy 13 defines a homogeneous AML subgroup with high frequency of mutations in spliceosome genes and poor prognosis

In acute myeloid leukemia (AML), isolated trisomy 13 (AML+13) is a rare chromosomal abnormality whose 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 performed exome sequencing, targeted candidate gene sequencing 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 = .006; median OS 9.3 vs. 14.8 months, P = .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%. 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 homogeneous 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 homogeneous subgroup with alterations in a few critical cellular pathways. Clinicaltrials.gov identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML60+: NCT00893373.

RUNX1 mutations in cytogenetically normal acute myeloid leukemia are associated with a poor prognosis and up-regulation of lymphoid genes

Background The RUNX1 (AML1) gene is a frequent mutational target in myelodysplastic syndromes and acute myeloid leukemia. Previous studies suggested that RUNX1 mutations may have pathological and prognostic implications. Design and Methods We screened 93 patients with cytogenetically normal acute myeloid leukemia for RUNX1 mutations by capillary sequencing of genomic DNA. Mutation status was then correlated with clinical data and gene expression profiles. Results We found that 15 out of 93 (16.1%) patients with cytogenetically normal acute myeloid leukemia had RUNX1 mutations. Seventy-three patients were enrolled in the AMLCG-99 trial and carried ten RUNX1 mutations (13.7%). Among these 73 patients RUNX1 mutations were significantly associated with older age, male sex, absence of NPM1 mutations and presence of MLL-partial tandem duplications. Moreover, RUNX1-mutated patients had a lower complete remission rate (30% versus 73% P=0.01), lower relapse-free survival rate (3-year relapse-free survival 0% versus 30.4%; P=0.002) and lower overall survival rate (3-year overall survival 0% versus 34.4%; P<0.001) than patients with wild-type RUNX1. RUNX1 mutations remained associated with shorter overall survival in a multivariate model including age and the European LeukemiaNet acute myeloid leukemia genetic classification as covariates. Patients with RUNX1 mutations showed a unique gene expression pattern with differential expression of 85 genes. The most prominently up-regulated genes in patients with RUNX1-mutated cytogenetically normal acute myeloid leukemia include lymphoid regulators such as HOP homeobox (HOPX), deoxynucleotidyltransferase (DNTT, terminal) and B-cell linker (BLNK), indicating lineage infidelity. Conclusions Our findings firmly establish that RUNX1 mutations are a marker of poor prognosis and provide insights into the pathogenesis of RUNX1 mutation-positive acute myeloid leukemia.

Exome sequencing identifies recurring FLT3 N676K mutations in core-binding factor leukemia

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.

Activating FLT3 Mutants Show Distinct Gain-of-Function Phenotypes In Vitro and a Characteristic Signaling Pathway Profile Associated with Prognosis in Acute Myeloid Leukemia

About 30% of patients with acute myeloid leukemia (AML) harbour mutations of the receptor tyrosine kinase FLT3, mostly internal tandem duplications (ITD) and point mutations of the second tyrosine kinase domain (TKD). It was the aim of this study to comprehensively analyze clinical and functional properties of various FLT3 mutants. In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with a worse relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a strong activating phenotype in Ba/F3 cells. In contrast, FLT3-TKD mutations and other point mutations – including two novel mutations – showed a weaker but clear gain-of-function phenotype with gradual increase in proliferation and protection from apoptosis. The pro-proliferative capacity of the investigated FLT3 mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD positive cell lines, in contrast to FLT3-non-ITD mutants, which displayed an enhanced signal of AKT and MAPK activation. Gene expression analysis revealed distinct difference between FLT3-ITD and FLT3-TKD for STAT5 target gene expression as well as deregulation of SOCS2, ENPP2, PRUNE2 and ART3. FLT3-ITD and FLT3 point mutations show a gain-of-function phenotype with distinct signalling properties in vitro. Although poor prognosis in AML is only associated with FLT3-ITD, all activating FLT3 mutations can contribute to leukemogenesis and are thus potential targets for therapeutic interventions.

Long-term follow-up of cytogenetically normal CEBPA-mutated AML

BackgroundThe aim of this study was to analyze the long-term survival of AML patients with CEBPA mutations.Patients and methodsWe investigated 88 AML patients with a median age of 61 years and (1) cytogenetically normal AML (CN-AML), (2) monoallelic (moCEBPA) or biallelic (biCEBPA) CEBPA mutation, and (3) intensive induction treatment. 60/88 patients have been described previously with a shorter follow-up.ResultsMedian follow-up time was 9.8 years (95% CI: 9.4-10.1 years) compared to 3.2 and 5.2 years in our former analyses. Patients with biCEBPA mutations survived significantly longer compared to those with moCEBPA (median overall survival (OS) 9.6 years vs. 1.7 years, p = 0.008). Patients ≤60 years and biCEBPA mutations showed a favorable prognosis with a 10-year OS rate of 81%.Both, bi- and moCEBPA- mutated groups had a low early death (d60) rate of 7% and 9%, respectively. Complete remission (CR) rates for biCEBPA- and moCEBPA- mutated patients were 82% vs. 70% (p = 0.17). biCEBPA- mutated patients showed a longer relapse free survival (RFS) (median RFS 9.4 years vs. 1.5 years, p = 0.021) and a lower cumulative incidence of relapse (CIR) compared to moCEBPA- mutated patients. These differences in OS and RFS were confirmed after adjustment for known clinical and molecular prognostic factors.ConclusionsIn this long-term observation we confirmed the favorable prognostic outcome of patients with biCEBPA mutations compared to moCEBPA- mutated CN-AML. The high probability of OS (81%) in younger patients is helpful to guide intensity of postremission therapy.