Peter Paschka

IDH1 and IDH2 Mutations Are Frequent Genetic Alterations in Acute Myeloid Leukemia and Confer Adverse Prognosis in Cytogenetically Normal Acute Myeloid Leukemia With NPM1 Mutation Without FLT3 Internal Tandem Duplication

PURPOSE To analyze the frequency and prognostic impact of isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) mutations in acute myeloid leukemia (AML). PATIENTS AND METHODS We studied 805 adults (age range, 16 to 60 years) with AML enrolled on German-Austrian AML Study Group (AMLSG) treatment trials AML HD98A and APL HD95 for mutations in exon 4 of IDH1 and IDH2. Patients were also studied for NPM1, FLT3, MLL, and CEBPA mutations. The median follow-up for survival was 6.3 years. RESULTS IDH mutations were found in 129 patients (16.0%) -IDH1 in 61 patients (7.6%), and IDH2 in 70 patients (8.7%). Two patients had both IDH1 and IDH2 mutations. All but one IDH1 mutation caused substitutions of residue R132; IDH2 mutations caused changes of R140 (n = 48) or R172 (n = 22). IDH mutations were associated with older age (P < .001; effect conferred by IDH2 only); lower WBC (P = .04); higher platelets (P < .001); cytogenetically normal (CN) -AML (P< .001); and NPM1 mutations, in particular with the genotype of mutated NPM1 without FLT3 internal tandem duplication (ITD; P < .001). In patients with CN-AML with the latter genotype, IDH mutations adversely impacted relapse-free survival (RFS; P = .02) and overall survival (P = .03), whereas outcome was not affected in patients with CN-AML who lacked this genotype. In CN-AML, multivariable analyses revealed a significant interaction between IDH mutation and the genotype of mutated NPM1 without FLT3-ITD (ie, the adverse impact of IDH mutation [RFS]; P = .046 was restricted to this patient subset). CONCLUSION IDH1 and IDH2 mutations are recurring genetic changes in AML. They constitute a poor prognostic factor in CN-AML with mutated NPM1 without FLT3-ITD, which allows refined risk stratification of this AML subset.

Adverse Prognostic Significance of KIT Mutations in Adult Acute Myeloid Leukemia With inv(16) and t(8;21): A Cancer and Leukemia Group B Study

PURPOSE To analyze the prognostic impact of mutated KIT (mutKIT) in core-binding factor acute myeloid leukemia (AML) with inv(16)(p13q22) and t(8;21)(q22;q22). PATIENTS AND METHODS Sixty-one adults with inv(16) and 49 adults with t(8;21), assigned to postremission therapy with repetitive cycles of higher dose cytarabine were analyzed for mutKIT in exon 17 (mutKIT17) and 8 (mutKIT8) by denaturing high-performance liquid chromatography and direct sequencing at diagnosis. The median follow-up was 5.3 years. RESULTS Among patients with inv(16), 29.5% had mutKIT (16% with mutKIT17 and 13% with sole mutKIT8). Among patients with t(8;21), 22% had mutKIT (18% with mutKIT17 and 4% with sole mutKIT8). Complete remission rates of patients with mutKIT and wild-type KIT (wtKIT) were similar in both cytogenetic groups. In inv(16), the cumulative incidence of relapse (CIR) was higher for patients with mutKIT (P = .05; 5-year CIR, 56% v 29%) and those with mutKIT17 (P = .002; 5-year CIR, 80% v 29%) compared with wtKIT patients. Once data were adjusted for sex, mutKIT predicted worse overall survival (OS). In t(8;21), mutKIT predicted higher CIR (P = .017; 5-year CIR, 70% v 36%), but did not influence OS. CONCLUSION We report for the first time that mutKIT, and particularly mutKIT17, confer higher relapse risk, and both mutKIT17 and mutKIT8 appear to adversely affect OS in AML with inv(16). We also confirm the adverse impact of mutKIT on relapse risk in t(8;21) AML. We suggest that patients with core-binding factor AML should be screened for mutKIT at diagnosis for both prognostic and therapeutic purposes, given that activated KIT potentially can be targeted with novel tyrosine kinase inhibitors.

Genomic Classification and Prognosis in Acute Myeloid Leukemia

BACKGROUND Recent studies have provided a detailed census of genes that are mutated in acute myeloid leukemia (AML). Our next challenge is to understand how this genetic diversity defines the pathophysiology of AML and informs clinical practice. METHODS We enrolled a total of 1540 patients in three prospective trials of intensive therapy. Combining driver mutations in 111 cancer genes with cytogenetic and clinical data, we defined AML genomic subgroups and their relevance to clinical outcomes. RESULTS We identified 5234 driver mutations across 76 genes or genomic regions, with 2 or more drivers identified in 86% of the patients. Patterns of co-mutation compartmentalized the cohort into 11 classes, each with distinct diagnostic features and clinical outcomes. In addition to currently defined AML subgroups, three heterogeneous genomic categories emerged: AML with mutations in genes encoding chromatin, RNA-splicing regulators, or both (in 18% of patients); AML with TP53 mutations, chromosomal aneuploidies, or both (in 13%); and, provisionally, AML with IDH2(R172) mutations (in 1%). Patients with chromatin-spliceosome and TP53-aneuploidy AML had poor outcomes, with the various class-defining mutations contributing independently and additively to the outcome. In addition to class-defining lesions, other co-occurring driver mutations also had a substantial effect on overall survival. The prognostic effects of individual mutations were often significantly altered by the presence or absence of other driver mutations. Such gene-gene interactions were especially pronounced for NPM1-mutated AML, in which patterns of co-mutation identified groups with a favorable or adverse prognosis. These predictions require validation in prospective clinical trials. CONCLUSIONS The driver landscape in AML reveals distinct molecular subgroups that reflect discrete paths in the evolution of AML, informing disease classification and prognostic stratification. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT00146120.).

MicroRNA Expression in Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUND A role of microRNAs in cancer has recently been recognized. However, little is known about the role of microRNAs in acute myeloid leukemia (AML). METHODS Using microRNA expression profiling, we studied samples of leukemia cells from adults under the age of 60 years who had cytogenetically normal AML and high-risk molecular features--that is, an internal tandem duplication in the fms-related tyrosine kinase 3 gene (FLT3-ITD), a wild-type nucleophosmin (NPM1), or both. A microRNA signature that was associated with event-free survival was derived from a training group of 64 patients and tested in a validation group of 55 patients. For the latter, a microRNA compound covariate predictor (called a microRNA summary value) was computed on the basis of weighted levels of the microRNAs forming the outcome signature. RESULTS Of 305 microRNA probes, 12 (including 5 representing microRNA-181 family members) were associated with event-free survival in the training group (P<0.005). In the validation group, the microRNA summary value was inversely associated with event-free survival (P=0.03). In multivariable analysis, the microRNA summary value remained associated with event-free survival (P=0.04) after adjustment for the allelic ratio of FLT3-ITD to wild-type FLT3 and for the white-cell count. Using results of gene-expression microarray analysis, we found that expression levels of the microRNA-181 family were inversely correlated with expression levels of predicted target genes encoding proteins involved in pathways of innate immunity mediated by toll-like receptors and interleukin-1beta. CONCLUSIONS A microRNA signature in molecularly defined, high-risk, cytogenetically normal AML is associated with the clinical outcome and with target genes encoding proteins involved in specific innate-immunity pathways.

RUNX1 Mutations in Acute Myeloid Leukemia: Results From a Comprehensive Genetic and Clinical Analysis From the AML Study Group

PURPOSE To evaluate frequency, biologic features, and clinical relevance of RUNX1 mutations in acute myeloid leukemia (AML). PATIENTS AND METHODS Diagnostic samples from 945 patients (age 18 to 60 years) were analyzed for RUNX1 mutations. In a subset of cases (n = 269), microarray gene expression analysis was performed. RESULTS Fifty-nine RUNX1 mutations were identified in 53 (5.6%) of 945 cases, predominantly in exons 3 (n = 11), 4 (n = 10), and 8 (n = 23). RUNX1 mutations clustered in the intermediate-risk cytogenetic group (46 of 640, 7.2%; cytogenetically normal, 34 of 538, 6.3%), whereas they were less frequent in adverse-risk cytogenetics (five of 109, 4.6%) and absent in core-binding-factor AML (0 of 77) and acute promyelocytic leukemia (0 of 61). RUNX1 mutations were associated with MLL-partial tandem duplications (P = .0007) and IDH1/IDH2 mutations (P = .03), inversely correlated with NPM1 (P < .0001), and in trend with CEBPA (P = .10) mutations. RUNX1 mutations were characterized by a distinct gene expression pattern; this RUNX1 mutation-derived signature was not exclusive for the mutation, but also included mostly adverse-risk AML [eg, 7q-, -7, inv(3), or t(3;3)]. RUNX1 mutations predicted for resistance to chemotherapy (rates of refractory disease 30% and 19%, P = .047, for RUNX1-mutated and wild-type patients, respectively), as well as inferior event-free survival (EFS; P < .0001), relapse-free survival (RFS, P = .022), and overall survival (P = .051). In multivariable analysis, RUNX1 mutations were an independent prognostic marker for shorter EFS (P = .007). Explorative subgroup analysis revealed that allogeneic hematopoietic stem-cell transplantation had a favorable impact on RFS in RUNX1-mutated patients (P < .0001). CONCLUSION AML with RUNX1 mutations are characterized by distinct genetic properties and are associated with resistance to therapy and inferior outcome.

Prognostic Significance of, and Gene and MicroRNA Expression Signatures Associated With, CEBPA Mutations in Cytogenetically Normal Acute Myeloid Leukemia With High-Risk Molecular Features: A Cancer and Leukemia Group B Study

PURPOSE To evaluate the prognostic significance of CEBPA mutations in the context of established molecular markers in cytogenetically normal (CN) acute myeloid leukemia (AML) and gain biologic insights into leukemogenesis of the CN-AML molecular high-risk subset (FLT3 internal tandem duplication [ITD] positive and/or NPM1 wild type) that has a significantly higher incidence of CEBPA mutations than the molecular low-risk subset (FLT3-ITD negative and NPM1 mutated). PATIENTS AND METHODS One hundred seventy-five adults age less than 60 years with untreated primary CN-AML were screened before treatment for CEBPA, FLT3, MLL, WT1, and NPM1 mutations and BAALC and ERG expression levels. Gene and microRNA (miRNA) expression profiles were obtained for the CN-AML molecular high-risk patients. RESULTS CEBPA mutations predicted better event-free (P = .007), disease-free (P = .014), and overall survival (P < .001) independently of other molecular and clinical prognosticators. Among patients with CEBPA mutations, 91% were in the CN-AML molecular high-risk group. Within this group, CEBPA mutations predicted better event-free (P < .001), disease-free (P = .004), and overall survival (P = .009) independently of other molecular and clinical characteristics and were associated with unique gene and miRNA expression profiles. The major features of these profiles were upregulation of genes (eg, GATA1, ZFPM1, EPOR, and GFI1B) and miRNAs (ie, the miR-181 family) involved in erythroid differentiation and downregulation of homeobox genes. CONCLUSION Pretreatment testing for CEBPA mutations identifies CN-AML patients with different outcomes, particularly in the molecular high-risk group, thus improving molecular risk-based classification of this large cytogenetic subset of AML. The gene and miRNA expression profiling provided insights into leukemogenesis of the CN-AML molecular high-risk group, indicating that CEBPA mutations are associated with partial erythroid differentiation.

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.

TP53 alterations in acute myeloid leukemia with complex karyotype correlate with specific copy number alterations, monosomal karyotype, and dismal outcome

To assess the frequency of TP53 alterations and their correlation with other genetic changes and outcome in acute myeloid leukemia with complex karyotype (CK-AML), we performed integrative analysis using TP53 mutational screening and array-based genomic profiling in 234 CK-AMLs. TP53 mutations were found in 141 of 234 (60%) and TP53 losses were identified in 94 of 234 (40%) CK-AMLs; in total, 164 of 234 (70%) cases had TP53 alterations. TP53-altered CK-AML were characterized by a higher degree of genomic complexity (aberrations per case, 14.30 vs 6.16; P < .0001) and by a higher frequency of specific copy number alterations, such as -5/5q-, -7/7q-, -16/16q-, -18/18q-, +1/+1p, and +11/+11q/amp11q13∼25; among CK-AMLs, TP53-altered more frequently exhibited a monosomal karyotype (MK). Patients with TP53 alterations were older and had significantly lower complete remission rates, inferior event-free, relapse-free, and overall survival. In multivariable analysis for overall survival, TP53 alterations, white blood cell counts, and age were the only significant factors. In conclusion, TP53 is the most frequently known altered gene in CK-AML. TP53 alterations are associated with older age, genomic complexity, specific DNA copy number alterations, MK, and dismal outcome. In multivariable analysis, TP53 alteration is the most important prognostic factor in CK-AML, outweighing all other variables, including the MK category.

Lack of clinical efficacy of imatinib in metastatic melanoma

This two-centre phase-II trial aimed at investigating the efficacy of imatinib in metastasised melanoma patients in correlation to the tumour expression profile of the imatinib targets c-kit and platelet-derived growth factor receptor (PDGF-R). The primary study end point was objective response according to RECIST, secondary end points were safety, overall and progression-free survival. In all, 18 patients with treatment-refractory advanced melanoma received imatinib 800 mg day−1. In 16 evaluable patients no objective responses could be observed. The median overall survival was 3.9 months, the median time to progression was 1.9 months. Tumour biopsy specimens were obtained from 12 patients prior to imatinib therapy and analysed for c-kit, PDGF-Rα and -Rβ expression by immunohistochemistry. In four cases, cell lines established from these tumour specimens were tested for the antiproliferative effects of imatinib and for functional mutations of genes encoding the imatinib target molecules. The tumour specimens stained positive for CD117/c-kit in nine out of 12 cases (75%), for PDGF-Rα in seven out of 12 cases (58%) and for PDGF-Rβ in eight out of 12 cases (67%). The melanoma cell lines showed a heterogenous expression of the imatinib target molecules without functional mutations in the corresponding amino-acid sequences. In vitro imatinib treatment of the cell lines showed no antiproliferative effect. In conclusion, this study did not reveal an efficacy of imatinib in advanced metastatic melanoma, regardless of the expression pattern of the imatinib target molecules c-kit and PDGF-R.

An 86-probe-set gene-expression signature predicts survival in cytogenetically normal acute myeloid leukemia.

Patients with cytogenetically normal acute myeloid leukemia (CN-AML) show heterogeneous treatment outcomes. We used gene-expression profiling to develop a gene signature that predicts overall survival (OS) in CN-AML. Based on data from 163 patients treated in the German AMLCG 1999 trial and analyzed on oligonucleotide microarrays, we used supervised principal component analysis to identify 86 probe sets (representing 66 different genes), which correlated with OS, and defined a prognostic score based on this signature. When applied to an independent cohort of 79 CN-AML patients, this continuous score remained a significant predictor for OS (hazard ratio [HR], 1.85; P = .002), event-free survival (HR = 1.73; P = .001), and relapse-free survival (HR = 1.76; P = .025). It kept its prognostic value in multivariate analyses adjusting for age, FLT3 ITD, and NPM1 status. In a validation cohort of 64 CN-AML patients treated on CALGB study 9621, the score also predicted OS (HR = 4.11; P < .001), event-free survival (HR = 2.90; P < .001), and relapse-free survival (HR = 3.14, P < .001) and retained its significance in a multivariate model for OS. In summary, we present a novel gene-expression signature that offers additional prognostic information for patients with CN-AML.