Jürgen Krauter

Mutations and Treatment Outcome in Cytogenetically Normal Acute Myeloid Leukemia

BACKGROUND Mutations occur in several genes in cytogenetically normal acute myeloid leukemia (AML) cells: the nucleophosmin gene (NPM1), the fms-related tyrosine kinase 3 gene (FLT3), the CCAAT/enhancer binding protein alpha gene (CEPBA), the myeloid-lymphoid or mixed-lineage leukemia gene (MLL), and the neuroblastoma RAS viral oncogene homolog (NRAS). We evaluated the associations of these mutations with clinical outcomes in patients. METHODS We compared the mutational status of the NPM1, FLT3, CEBPA, MLL, and NRAS genes in leukemia cells with the clinical outcome in 872 adults younger than 60 years of age with cytogenetically normal AML. Patients had been entered into one of four trials of therapy for AML. In each study, patients with an HLA-matched related donor were assigned to undergo stem-cell transplantation. RESULTS A total of 53% of patients had NPM1 mutations, 31% had FLT3 internal tandem duplications (ITDs), 11% had FLT3 tyrosine kinase-domain mutations, 13% had CEBPA mutations, 7% had MLL partial tandem duplications (PTDs), and 13% had NRAS mutations. The overall complete-remission rate was 77%. The genotype of mutant NPM1 without FLT3-ITD, the mutant CEBPA genotype, and younger age were each significantly associated with complete remission. Of the 663 patients who received postremission therapy, 150 underwent hematopoietic stem-cell transplantation from an HLA-matched related donor. Significant associations were found between the risk of relapse or the risk of death during complete remission and the leukemia genotype of mutant NPM1 without FLT3-ITD (hazard ratio, 0.44; 95% confidence interval [CI], 0.32 to 0.61), the mutant CEBPA genotype (hazard ratio, 0.48; 95% CI, 0.30 to 0.75), and the MLL-PTD genotype (hazard ratio, 1.56; 95% CI, 1.00 to 2.43), as well as receipt of a transplant from an HLA-matched related donor (hazard ratio, 0.60; 95% CI, 0.44 to 0.82). The benefit of the transplant was limited to the subgroup of patients with the prognostically adverse genotype FLT3-ITD or the genotype consisting of wild-type NPM1 and CEBPA without FLT3-ITD. CONCLUSIONS Genotypes defined by the mutational status of NPM1, FLT3, CEBPA, and MLL are associated with the outcome of treatment for patients with cytogenetically normal AML.

New insights to the MLL recombinome of acute leukemias

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements.

Incidence and Prognostic Influence of DNMT3A Mutations in Acute Myeloid Leukemia

PURPOSE To study the incidence and prognostic impact of mutations in DNA methyltransferase 3A (DNMT3A) in patients with acute myeloid leukemia. PATIENTS AND METHODS A total of 489 patients with AML were examined for mutations in DNMT3A by direct sequencing. The prognostic impact of DNMT3A mutations was evaluated in the context of other clinical prognostic markers and genetic risk factors (cytogenetic risk group; mutations in NPM1, FLT3, CEBPA, IDH1, IDH2, MLL1, NRAS, WT1, and WT1 SNPrs16754; expression levels of BAALC, ERG, EVI1, MLL5, MN1, and WT1). RESULTS DNMT3A mutations were found in 87 (17.8%) of 489 patients with AML who were younger than 60 years of age. Patients with DNMT3A mutations were older, had higher WBC and platelet counts, more often had a normal karyotype and mutations in NPM1, FLT3, and IDH1 genes, and had higher MLL5 expression levels as compared with patients with wild-type DNMT3A. Mutations in DNMT3A independently predicted a shorter overall survival (OS; hazard ratio [HR], 1.59; 95% CI, 1.15 to 2.21; P = .005) by multivariate analysis, but were not associated with relapse-free survival (RFS) or complete remission (CR) rate when the entire patient cohort was considered. In cytogenetically normal (CN) AML, 27.2% harbored DNMT3A mutations that independently predicted shorter OS (HR = 2.46; 95% CI, 1.58 to 3.83; P < .001) and lower CR rate (OR, 0.42; 95% CI, 0.21 to 0.84; P = .015), but not RFS (P = .32). Within patients with CN-AML, DNMT3A mutations had an unfavorable effect on OS, RFS, and CR rate in NPM1/FLT3-ITD high-risk but not in low-risk patients. CONCLUSION DNMT3A mutations are frequent in younger patients with AML and are associated with an unfavorable prognosis.

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.

Frequency and prognostic impact of mutations in SRSF2 , U2AF1 , and ZRSR2 in patients with myelodysplastic syndromes

Mutations in genes of the splicing machinery have been described recently in myelodysplastic syndromes (MDS). In the present study, we examined a cohort of 193 MDS patients for mutations in SRSF2, U2AF1 (synonym U2AF35), ZRSR2, and, as described previously, SF3B1, in the context of other molecular markers, including mutations in ASXL1, RUNX1, NRAS, TP53, IDH1, IDH2, NPM1, and DNMT3A. Mutations in SRSF2, U2AF1, ZRSR2, and SF3B1 were found in 24 (12.4%), 14 (7.3%), 6 (3.1%), and 28 (14.5%) patients, respectively, corresponding to a total of 67 of 193 MDS patients (34.7%). SRSF2 mutations were associated with RUNX1 (P < .001) and IDH1 (P = .013) mutations, whereas U2AF1 mutations were associated with ASXL1 (P = .005) and DNMT3A (P = .004) mutations. In univariate analysis, mutated SRSF2 predicted shorter overall survival and more frequent acute myeloid leukemia progression compared with wild-type SRSF2, whereas mutated U2AF1, ZRSR2, and SF3B1 had no impact on patient outcome. In multivariate analysis, SRSF2 remained an independent poor risk marker for overall survival (hazard ratio = 2.3; 95% confidence interval, 1.28-4.13; P = .017) and acute myeloid leukemia progression (hazard ratio = 2.83; 95% confidence interval, 1.31-6.12; P = .008). These results show a negative prognostic impact of SRSF2 mutations in MDS. SRSF2 mutations may become useful for clinical risk stratification and treatment decisions in the future.

Individual Patient Data–Based Meta-Analysis of Patients Aged 16 to 60 Years With Core Binding Factor Acute Myeloid Leukemia: A Survey of the German Acute Myeloid Leukemia Intergroup

PURPOSE To evaluate prognostic factors for relapse-free survival (RFS) and overall survival (OS) and to assess the impact of different postremission therapies in adult patients with core binding factor (CBF) acute myeloid leukemias (AML). PATIENTS AND METHODS Individual patient data-based meta-analysis was performed on 392 adults (median age, 42 years; range, 16 to 60 years) with CBF AML (t(8;21), n = 191; inv(16), n = 201) treated between 1993 and 2002 in prospective German AML treatment trials. RESULTS RFS was 60% and 58% and OS was 65% and 74% in the t(8;21) and inv(16) groups after 3 years, respectively. For postremission therapy, intention-to-treat analysis revealed no difference between intensive chemotherapy and autologous transplantation in the t(8;21) group and between chemotherapy, autologous, and allogeneic transplantation in the inv(16) group. In the t(8;21) group, significant prognostic variables for longer RFS and OS were lower WBC and higher platelet counts; loss of the Y chromosome in male patients was prognostic for shorter OS. In the inv(16) group, trisomy 22 was a significant prognostic variable for longer RFS. For patients who experienced relapse, second complete remission rate was significantly lower in patients with t(8;21), resulting in a significantly inferior survival duration after relapse compared with patients with inv(16). CONCLUSION We provide novel prognostic factors for CBF AML and show that patients with t(8;21) who experience relapse have an inferior survival duration.

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.

Prognostic impact, concurrent genetic mutations, and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients: further evidence for CEBPA double mutant AML as a distinctive disease entity.

We evaluated concurrent gene mutations, clinical outcome, and gene expression signatures of CCAAT/enhancer binding protein alpha (CEBPA) double mutations (CEBPA(dm)) versus single mutations (CEBPA(sm)) in 1182 cytogenetically normal acute myeloid leukemia (AML) patients (16-60 years of age). We identified 151 (12.8%) patients with CEBPA mutations (91 CEBPA(dm) and 60 CEBPA(sm)). The incidence of germline mutations was 7% (5 of 71), including 3 C-terminal mutations. CEBPA(dm) patients had a lower frequency of concurrent mutations than CEBPA(sm) patients (P < .0001). Both, groups were associated with a favorable outcome compared with CEBPA(wt) (5-year overall survival [OS] 63% and 56% vs 39%; P < .0001 and P = .05, respectively). However, in multivariable analysis only CEBPA(dm) was a prognostic factor for favorable OS outcome (hazard ratio [HR] 0.36, P < .0001; event-free survival, HR 0.41, P < .0001; relapse-free survival, HR 0.55, P = .001). Outcome in CEBPA(sm) is dominated by concurrent NPM1 and/or FLT3 internal tandem duplication mutations. Unsupervised and supervised GEP analyses showed that CEBPA(dm) AML (n = 42), but not CEBPA(sm) AML (n = 18), expressed a unique gene signature. A 25-probe set prediction signature for CEBPA(dm) AML showed 100% sensitivity and specificity. Based on these findings, we propose that CEBPA(dm) should be clearly defined from CEBPA(sm) AML and considered as a separate entity in the classification of AML.

Monitoring of Minimal Residual Disease in NPM1-Mutated Acute Myeloid Leukemia: A Study From the German-Austrian Acute Myeloid Leukemia Study Group

PURPOSE To evaluate the prognostic value of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) with NPM1 mutation (NPM1(mut)). PATIENTS AND METHOD RNA-based real-time quantitative polymerase chain reaction (RQ-PCR) specific for the detection of six different NPM1(mut) types was applied to 1,682 samples (bone marrow, n = 1,272; blood, n = 410) serially obtained from 245 intensively treated younger adult patients who were 16 to 60 years old. RESULTS NPM1(mut) transcript levels as a continuous variable were significantly associated with prognosis after each treatment cycle. Achievement of RQ-PCR negativity after double induction therapy identified patients with a low cumulative incidence of relapse (CIR; 6.5% after 4 years) compared with RQ-PCR-positive patients (53.0%; P < .001); this translated into significant differences in overall survival (90% v 51%, respectively; P = .001). After completion of therapy, CIR was 15.7% in RQ-PCR-negative patients compared with 66.5% in RQ-PCR-positive patients (P < .001). Multivariable analyses after double induction and after completion of consolidation therapy revealed higher NPM1(mut) transcript levels as a significant factor for a higher risk of relapse and death. Serial post-treatment assessment of MRD allowed early detection of relapse in patients exceeding more than 200 NPM1(mut)/10(4) ABL copies. CONCLUSION We defined clinically relevant time points for NPM1(mut) MRD assessment that allow for the identification of patients with AML who are at high risk of relapse. Monitoring of NPM1(mut) transcript levels should be incorporated in future clinical trials to guide therapeutic decisions.

The impact of therapy-related acute myeloid leukemia (AML) on outcome in 2853 adult patients with newly diagnosed AML

To study the characteristics and clinical impact of therapy-related acute myeloid leukemia (t-AML). 200 patients (7.0%) had t-AML and 2653 de novo AML (93%). Patients with t-AML were older (P < .0001) and they had lower white blood counts (P = .003) compared with de novo AML patients; t-AML patients had abnormal cytogenetics more frequently, with overrepresentation of 11q23 translocations as well as adverse cytogenetics, including complex and monosomal karyotypes, and with underrepresentation of intermediate-risk karyotypes (P < .0001); t-AML patients had NPM1 mutations (P < .0001) and FLT3 internal tandem duplications (P = .0005) less frequently. Younger age at diagnosis of primary malignancy and treatment with intercalating agents as well as topoisomerase II inhibitors were associated with shorter latency periods to the occurrence of t-AML. In multivariable analyses, t-AML was an adverse prognostic factor for death in complete remission but not relapse in younger intensively treated patients (P < .0001 and P = .39, respectively), relapse but not death in complete remission in older, less intensively treated patients (P = .02 and P = .22, respectively) and overall survival in younger intensively treated patients (P = .01). In more intensively treated younger adults, treatment-related toxicity had a major negative impact on outcome, possibly reflecting cumulative toxicity of cancer treatment.