Heiko Becker

IDH1 and IDH2 Gene Mutations Identify Novel Molecular Subsets Within De Novo Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

PURPOSE To analyze the frequency and associations with prognostic markers and outcome of mutations in IDH genes encoding isocitrate dehydrogenases in adult de novo cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS Diagnostic bone marrow or blood samples from 358 patients were analyzed for IDH1 and IDH2 mutations by DNA polymerase chain reaction amplification/sequencing. FLT3, NPM1, CEBPA, WT1, and MLL mutational analyses and gene- and microRNA-expression profiling were performed centrally. Results IDH mutations were found in 33% of the patients. IDH1 mutations were detected in 49 patients (14%; 47 with R132). IDH2 mutations, previously unreported in AML, were detected in 69 patients (19%; 13 with R172 and 56 with R140). R172 IDH2 mutations were mutually exclusive with all other prognostic mutations analyzed. Younger age (< 60 years), molecular low-risk (NPM1-mutated/FLT3-internal tandem duplication-negative) IDH1-mutated patients had shorter disease-free survival than molecular low-risk IDH1/IDH2-wild-type (wt) patients (P = .046). R172 IDH2-mutated patients had lower complete remission rates than IDH1/IDH2wt patients (P = .007). Distinctive microarray gene- and microRNA-expression profiles accurately predicted R172 IDH2 mutations. The highest expressed gene and microRNAs in R172 IDH2-mutated patients compared with the IDH1/IDH2wt patients were APP (previously associated with complex karyotype AML) and miR-1 and miR-133 (involved in embryonal stem-cell differentiation), respectively. CONCLUSION IDH1 and IDH2 mutations are recurrent in CN-AML and have an unfavorable impact on outcome. The R172 IDH2 mutations, previously unreported in AML, characterize a novel subset of CN-AML patients lacking other prognostic mutations and associate with unique gene- and microRNA-expression profiles that may lead to the discovery of novel, therapeutically targetable leukemogenic mechanisms.

miR-328 Functions as an RNA Decoy to Modulate hnRNP E2 Regulation of mRNA Translation in Leukemic Blasts

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNAs seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins.

Clinical response and miR-29b predictive significance in older AML patients treated with a 10-day schedule of decitabine

A phase II clinical trial with single-agent decitabine was conducted in older patients (≥60 years) with previously untreated acute myeloid leukemia (AML) who were not candidates for or who refused intensive chemotherapy. Subjects received low-dose decitabine at 20 mg/m2 i.v. over 1 h on days 1 to 10. Fifty-three subjects enrolled with a median age of 74 years (range, 60–85). Nineteen (36%) had antecedent hematologic disorder or therapy-related AML; 16 had complex karyotypes (≥3 abnormalities). The complete remission rate was 47% (n = 25), achieved after a median of three cycles of therapy. Nine additional subjects had no morphologic evidence of disease with incomplete count recovery, for an overall response rate of 64% (n = 34). Complete remission was achieved in 52% of subjects presenting with normal karyotype and in 50% of those with complex karyotypes. Median overall and disease-free survival durations were 55 and 46 weeks, respectively. Death within 30 days of initiation of treatment occurred in one subject (2%), death within 8 weeks in 15% of subjects. Given the DNA hypomethylating effect of decitabine, we examined the relationship of clinical response and pretreatment level of miR-29b, previously shown to target DNA methyltransferases. Higher levels of miR-29b were associated with clinical response (P = 0.02). In conclusion, this schedule of decitabine was highly active and well tolerated in this poor-risk cohort of older AML patients. Levels of miR-29b should be validated as a predictive factor for stratification of older AML patients to decitabine treatment.

Favorable Prognostic Impact of NPM1 Mutations in Older Patients With Cytogenetically Normal De Novo Acute Myeloid Leukemia and Associated Gene- and MicroRNA-Expression Signatures: A Cancer and Leukemia Group B Study

PURPOSE To analyze the prognostic significance of NPM1 mutations, and the associated gene- and microRNA-expression signatures in older patients with de novo, cytogenetically normal acute myeloid leukemia (CN-AML) treated with intensive chemotherapy. PATIENTS AND METHODS One hundred forty-eight adults age >or= 60 years with de novo CN-AML, enrolled onto Cancer and Leukemia Group B protocols 9720 and 10201, were studied at diagnosis for NPM1, FLT3, CEBPA, and WT1 mutations, and gene- and microRNA-expression profiles. RESULTS Patients with NPM1 mutations (56%) had higher complete remission (CR) rates (84% v 48%; P < .001) and longer disease-free survival (DFS; P = .047; 3-year rates, 23% v 10%) and overall survival (OS; P < .001; 3-year rates, 35% v 8%) than NPM1 wild-type patients. In multivariable analyses, NPM1 mutations remained independent predictors for higher CR rates (P < .001) and longer DFS (P = .004) and OS (P < .001), after adjustment for other prognostic clinical and molecular variables. Unexpectedly, the prognostic impact of NPM1 mutations was mainly observed in patients >or= 70 years. Gene- and microRNA-expression profiles associated with NPM1 mutations were similar across older patient age groups and similar to those in younger (< 60 years) patients with CN-AML. These profiles were characterized by upregulation of HOX genes and their embedded microRNAs and downregulation of the prognostically adverse MN1, BAALC, and ERG genes. CONCLUSION NPM1 mutations have favorable prognostic impact in older patients with CN-AML, especially those age >or= 70 years. The gene- and microRNA-expression profiles suggest that NPM1 mutations constitute a marker defining a biologically homogeneous entity in CN-AML that might be treated with specific and/or targeted therapies across age groups.

TET2 mutations improve the new European LeukemiaNet risk classification of acute myeloid leukemia: a Cancer and Leukemia Group B study.

PURPOSE To determine the frequency of TET2 mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in patients with primary cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS Four-hundred twenty-seven patients with CN-AML were analyzed for TET2 mutations by polymerase chain reaction and direct sequencing and for established prognostic gene mutations. Gene- and microRNA-expression profiles were derived using microarrays. RESULTS TET2 mutations, found in 23% of patients, were associated with older age (P < .001) and higher pretreatment WBC (P = .04) compared with wild-type TET2 (TET2-wt). In the European LeukemiaNet (ELN) favorable-risk group (patients with CN-AML who have mutated CEBPA and/or mutated NPM1 without FLT3 internal tandem duplication [FLT3-ITD]), TET2-mutated patients had shorter event-free survival (EFS; P < .001) because of a lower complete remission (CR) rate (P = .007), and shorter disease-free survival (DFS; P = .003), and also had shorter overall survival (P = .001) compared with TET2-wt patients. TET2 mutations were not associated with outcomes in the ELN intermediate-I-risk group (CN-AML with wild-type CEBPA and wild-type NPM1 and/or FLT3-ITD). In multivariable models, TET2 mutations were associated with shorter EFS (P = .004), lower CR rate (P = .03), and shorter DFS (P = .05) only among favorable-risk CN-AML patients. We identified a TET2 mutation-associated gene-expression signature in favorable-risk but not in intermediate-I-risk patients and found distinct mutation-associated microRNA signatures in both ELN groups. CONCLUSION TET2 mutations improve the ELN molecular-risk classification in primary CN-AML because of their adverse prognostic impact in an otherwise favorable-risk patient subset. Our data suggest that these patients may be candidates for alternative therapies.

Prognostic Significance of the European LeukemiaNet Standardized System for Reporting Cytogenetic and Molecular Alterations in Adults With Acute Myeloid Leukemia

PURPOSE To evaluate the prognostic significance of the international European LeukemiaNet (ELN) guidelines for reporting genetic alterations in acute myeloid leukemia (AML). PATIENTS AND METHODS We analyzed 1,550 adults with primary AML, treated on Cancer and Leukemia Group B first-line trials, who had pretreatment cytogenetics and, for cytogenetically normal patients, mutational status of NPM1, CEBPA, and FLT3 available. We compared complete remission (CR) rates, disease-free survival (DFS), and overall survival (OS) among patients classified into the four ELN genetic groups (favorable, intermediate-I, intermediate-II, adverse) separately for 818 younger (age < 60 years) and 732 older (age ≥ 60 years) patients. RESULTS The percentages of younger versus older patients in the favorable (41% v 20%; P < .001), intermediate-II (19% v 30%; P < .001), and adverse (22% v 31%; P < .001) genetic groups differed. The favorable group had the best and the adverse group the worst CR rates, DFS, and OS in both age groups. Both intermediate groups had significantly worse outcomes than the favorable but better than the adverse group. Intermediate-I and intermediate-II groups in older patients had similar outcomes, whereas the intermediate-II group in younger patients had better OS but not better CR rates or DFS than the intermediate-I group. The prognostic significance of ELN classification was confirmed by multivariable analyses. For each ELN group, older patients had worse outcomes than younger patients. CONCLUSION The ELN classification clearly separates the genetic groups by outcome, supporting its use for risk stratification in clinical trials. Because they have different proportions of genetic alterations and outcomes, younger and older patients should be reported separately when using the ELN classification.

ASXL1 mutations identify a high-risk subgroup of older patients with primary cytogenetically normal AML within the ELN Favorable genetic category

The associations of mutations in the enhancer of trithorax and polycomb family gene ASXL1 with pretreatment patient characteristics, outcomes, and gene-/microRNA-expression profiles in primary cytogenetically normal acute myeloid leukemia (CN-AML) are unknown. We analyzed 423 adult patients for ASXL1 mutations, other prognostic gene mutations, and gene-/microRNA-expression profiles. ASXL1 mutations were 5 times more common in older (≥ 60 years) patients (16.2%) than those younger than 60 years (3.2%; P < .001). Among older patients, ASXL1 mutations associated with wild-type NPM1 (P < .001), absence of FLT3-internal tandem duplications (P = .002), mutated CEBPA (P = .01), and with inferior complete remission (CR) rate (P = .04), disease-free survival (DFS; P = .03), overall survival (OS; P = .006), and event-free survival (EFS; P = .002). Within the European LeukemiaNet (ELN) genetic categories of older CN-AML, ASXL1 mutations associated with inferior CR rate (P = .02), OS (P < .001), and EFS (P < .001) among ELN Favorable, but not among ELN Intermediate-I patients. Multivariable analyses confirmed associations of ASXL1 mutations with unfavorable CR rate (P = .03), DFS (P < .001), OS (P < .001), and EFS (P < .001) among ELN Favorable patients. We identified an ASXL1 mutation-associated gene-expression signature, but no microRNA-expression signature. This first study of ASXL1 mutations in primary CN-AML demonstrates that ASXL1-mutated older patients, particularly within the ELN Favorable group, have unfavorable outcomes and may be candidates for experimental treatment approaches.

Sp1/NFκB/HDAC/miR-29b Regulatory Network in KIT-Driven Myeloid Leukemia

The biologic and clinical significance of KIT overexpression that associates with KIT gain-of-function mutations occurring in subsets of acute myeloid leukemia (AML) (i.e., core binding factor AML) is unknown. Here, we show that KIT mutations lead to MYC-dependent miR-29b repression and increased levels of the miR-29b target Sp1 in KIT-driven leukemia. Sp1 enhances its own expression by participating in a NFkappaB/HDAC complex that further represses miR-29b transcription. Upregulated Sp1 then binds NFkappaB and transactivates KIT. Therefore, activated KIT ultimately induces its own transcription. Our results provide evidence that the mechanisms of Sp1/NFkappaB/HDAC/miR-29b-dependent KIT overexpression contribute to leukemia growth and can be successfully targeted by pharmacological disruption of the Sp1/NFkappaB/HDAC complex or synthetic miR-29b treatment in KIT-driven AML.

Age-Related Prognostic Impact of Different Types of DNMT3A Mutations in Adults With Primary Cytogenetically Normal Acute Myeloid Leukemia

PURPOSE To determine the frequency of DNMT3A mutations, their associations with clinical and molecular characteristics and outcome, and the associated gene- and microRNA-expression signatures in primary cytogenetically normal acute myeloid leukemia (CN-AML). PATIENTS AND METHODS Four hundred fifteen previously untreated adults were analyzed for DNMT3A mutations and established prognostic gene mutations and expression markers. Gene- and microRNA-expression profiles were derived using microarrays. RESULTS Younger (< 60 years; n = 181) and older (≥ 60 years; n = 234) patients had similar frequencies of DNMT3A mutations (35.3% v 33.3%). Missense mutations affecting arginine codon 882 (R882-DNMT3A) were more common (n = 92; 62%) than those affecting other codons (non-R882-DNMT3A). DNMT3A-mutated patients did not differ regarding complete remission rate, but had shorter disease-free survival (DFS; P = .03) and, by trend, overall survival (OS; P = .07) than DNMT3A-wild-type patients. In multivariable analyses, DNMT3A mutations remained associated with shorter DFS (P = .01), but not with shorter OS. When analyzed separately, the two DNMT3A mutation types had different significance by age group. Younger patients with non-R882-DNMT3A mutations had shorter DFS (P = .002) and OS (P = .02), whereas older patients with R882-DNMT3A mutations had shorter DFS (P = .005) and OS (P = .002) after adjustment for other clinical and molecular prognosticators. Gene- and microRNA-expression signatures did not accurately predict DNMT3A mutational status. CONCLUSION DNMT3A mutations are frequent in CN-AML, and their clinical significance seems to be age dependent. DNMT3A-R882 mutations are associated with adverse prognosis in older patients, and non-R882-DNMT3A mutations are associated with adverse prognosis in younger patients. Low accuracy of gene- and microRNA-expression signatures in predicting DNMT3A mutation status suggested that the role of these mutations in AML remains to be elucidated.

FLT3 internal tandem duplication associates with adverse outcome and gene- and microRNA-expression signatures in patients 60 years of age or older with primary cytogenetically normal acute myeloid leukemia: a Cancer and Leukemia Group B study.

The clinical impact of FLT3-internal tandem duplications (ITDs), an adverse prognostic marker in adults aged < 60 years with primary cytogenetically normal acute myeloid leukemia (CN-AML), requires further investigation in older patients. In CN-AML patients aged ≥ 60 years treated on Cancer and Leukemia Group B frontline trials, we found that FLT3-ITD remained associated with shorter disease-free survival (P < .001; hazard ratio = 2.10) and overall survival (P < .001; hazard ratio = 1.97) in multivariable analyses. This impact on shorter disease-free survival and overall survival was in patients aged 60-69 (P < .001, each) rather than in those aged ≥ 70 years. An FLT3-ITD-associated gene-expression signature revealed overexpression of FLT3, homeobox genes (MEIS1, PBX3, HOXB3), and immunotherapeutic tar-gets (WT1, CD33) and underexpression of leukemia-associated (MLLT3, TAL1) and erythropoiesis-associated (GATA3, EPOR, ANK1, HEMGN) genes. An FLT3-ITD-associated microRNA-expression signature included overexpressed miR-155 and underexpressed miR-144 and miR-451. FLT3-ITD identifies older CN-AML patients with molecular high risk and is associated with gene- and microRNA-expression signatures that provide biologic insights for novel therapeutic approaches.