ALBERT

Description: Background: Mutations in the genes encoding NPM1, FLT3 (FLT3 ITD, FLT3 TKD), CEBPA, MLL (PTD) and NRAS have been identified as molecular markers in acute myeloid leukemia (AML) exhibiting a normal karyotype. Most recent studies focused on the prognostic value of single markers not taking into account their potential interactions. In addition, little is known about the predictive value of these markers on postremission therapy. Aims: To evaluate the prognostic impact of NPM1, FLT3, CEBPA, MLL and NRAS gene mutations on response to induction therapy, relapse-free (RFS) and overall survival (OS) as well as the predictive impact of these mutations on RFS and OS following different postremission therapies. Methods: Patients [16 to 60 years of age] were entered on four AMLSG treatment trials [AML-2/95, AML-1/99, AML HD93, AML HD98A]. As a consistent feature, in all four trials a genetic randomization was performed assigning all patients with an HLA-matched family donor (MRD) to allogeneic stem cell transplantation (SCT) in first complete remission (CR). Leukemia cells were analyzed for mutations in the above genes as previously described. Results: Between 1993 and 2004, 872 patients exhibiting a normal karyotype were registered. Median age was 48 years; median follow-up time was 49 months. Mutations were identified as follows (total number of samples analyzed; incidence of mutations): NPM1 (n=526; 53%), FLT3-ITD (n=531; 31%), FLT3-TKD (n=617; 11%), CEBPA (n=509; 14%), MLL-PTD (640; 8%), and NRAS (641; 13%). CR rate was 76%. In a logistic regression model, the NPM1+/FLT3-ITD- (p

Description: Acute myeloid leukemia (AML) with normal karyotype comprises a large number of molecularly distinct variants. For example the presence of internal tandem duplications (ITDs) of the FLT3 (fms-related tyrosine kinase 3) gene is associated with poor outcome, whereas mutations of the NPM1 (nucleophosmin) gene are prognostically favorable. However, this effect is mainly attributed to the NPM1-mutated/FLT3 ITD-negative AML cases. While NPM1-mutated cases are characterized by a distinct gene expression pattern, it remains unclear whether NPM1-mutated/FLT3 ITD-negative cases also display a characteristic signature, which might provide additional insights into the molecular basis for the good clinical outcome. Thus, we sought to identify a molecular profile for AML cases with NPM1-mutated/FLT3 ITD-negative normal karyotype disease. Towards this goal, we profiled gene expression of 138 samples of adult AML patients with normal karyotype using DNA microarray technology. All samples analyzed were derived from AML patients entered within the randomized multicenter treatment trial HD-98A of the German-Austrian AML Study Group (AMLSG). Based on supervised data analyses we were able to identify a 116-genes comprising expression pattern correlated with NPM1-mutated and FLT3 ITD-negative AML cases. In accordance with previous findings in NPM1-mutated cases (Alcalay et al. 2005, Verhaak et al. 2005), the NPM1-mutated/FLT3 ITD-negative pattern was also in part characterized by a prominent HOX gene cluster, which clearly separated the NPM1-wildtype from the NPM1-mutated cases. Similarly, the expression levels of BAALC and MN1 were correlated with the NPM1 mutational status, with NPM1-unmutated cases displaying higher BAALC and MN1 expression in our data set. However, as expected the newly defined signature also defined a NPM1-mutated group that did not contain many FLT3 ITD-positive samples. This group was characterized by several interesting genes including for example TLE1, which encodes a Groucho/TLE family protein. Groucho/TLE family proteins are transcriptional co-repressors, which mediate repression essential in embryonic development and are involved in regulation of Wnt signaling in adult tissue. Moreover, we identified several other genes of potential pathogenic relevance which also have been previously shown to be predictive in normal karyotype AML. Our findings support a distinct molecular mechanism associated with the favorable outcome of NPM1-mutated/FLT3 ITD-negative AML cases. Furthermore, the reported signature might contribute to improved risk stratification and clinical management of AML patients with normal karyotype disease.

Description: Detection of minimal residual disease (MRD) in acute myeloid leukemia (AML) with specific gene fusions is an important tool for the assessment of response to treatment and the individual risk of relapse. We evaluated the predictive value of MLL/AF9 fusion-transcript quantification using real-time RT-PCR for disease relapse and verified the results with nested RT-PCR. A t(9;11) was identified in 27 younger AML patients (16–60 years) entered into the multicenter trials AML HD93 and AML HD98-A of the AML Study Group Ulm. One hundred seventy-eight samples (bone marrow [BM], n=84; peripheral blood [PB], n=79; leukapheresis product [LP], n=15) from 22 patients were available for real-time RT-PCR analysis. In 6 of these 22 patients, only samples from diagnosis were obtained, the remaining 16 patients were analyzed during and after therapy. The sensitivity of the real-time RT-PCR was 10−3 and of the nested RT-PCR 10−4 to 10−5. The MLL/AF9 copy number at diagnosis was not predictive of subsequent clinical outcome. Eight of the 16 evaluable patients became real-time RT-PCR-negative after double induction therapy. Six of these 8 patients are in continuous complete remission (CR), 1 died in CR from graft-versus-host disease, and 1 relapsed 11 months after diagnosis. Eight of 16 patients had detectable fusion-transcript levels after achieving complete hematologic remission. Six of these 8 patients relapsed after a median of 8.6 months (range, 3.6 to 13.5) from diagnosis, one patient died in CR 3 weeks after autologous BM transplantation, and one patient underwent allogeneic BM transplantation and is in continuous CR 16 months after diagnosis. Of 3 patients, who relapsed, we were able to analyze bone marrow 1 months before hematologic relapse occurred. Two of these 3 patients were real-time RT-PCR-negative after therapy and became positive 1 month before relapse, 1 patient was still real-time RT-PCR-positive after therapy and had increased MLL/AF9 copy numbers 1 month before relapse occurred. Five Patients, who were in continuous CR, were real-time RT-PCR-negative in all analyzed samples during follow up (median, 56 months; range, 13.8 to 68.9 months). One hundred seventy-two patient samples were examined by nested RT-PCR. Real-time RT-PCR-negative samples were also negative with nested RT-PCR, therefore nested RT-PCR did not enhance sensitivity of real-time PCR. We conclude that real-time RT-PCR is convenient for the detection of MRD in t(9;11)-positive AML and real-time RT-PCR positivity in at least one sample during therapy correlates with a high risk of relapse.

Description: Background: Mutations in the myeloid transcription factor CEBPA (CCAAT enhancer binding protein-alpha) have been implicated in 10–15% of cytogenetically normal (CN) acute myeloid leukemia (AML) patients (pts). At the molecular level, two types of heterozygous CEPBA mutations have been identified. First, nonsense mutations affecting the N-terminal region, preventing the expression of the full-length protein, and second, in-frame mutations located in the basic-region-leucine zipper domain, resulting in a decreased CEBPA DNA-binding or dimerization activity. Clinically, CN-AML with mutant CEBPA is associated with a favorable prognosis. Recently, two independent families were reported in whom several family members affected by AML carried heterozygous germline CEBPA mutations. All germline mutations were located in the N-terminus. In addition, somatically acquired C-terminal mutations were detected in one out of three and two out of four family members, respectively. These findings led to the hypothesis that CEBPA germline mutations predispose to AML and that additional somatically acquired mutations may contribute to the development of the disease. Aim: To screen CN-AML pts with somatically acquired CEBPA mutations for germline CEBPA mutations as predisposing events for the development of AML. Methods: Pts were entered in the AML HD98-A or AMLSG 07-04 multicenter treatment trials of the German-Austrian AML Study Group. Buccal mucosa was obtained using commercial FTA filter cards after informed consent; CEBPA mutation screening was performed as recently described. Results: Buccal DNA from 18 pts exhibiting CEBPA mutations in their leukemic cells (biallelic N-terminal and C-terminal, n=12; heterozygous C-terminal, n=5; heterozygous N-terminal, n=1) was available for analysis. In 16 pts, no CEBPA germline mutations could be detected. In one pt, the heterozygous C-terminal in-frame mutation (1609G〉A) that was identified in the diagnostic sample was also present in the germline DNA. In a second pt with an N-terminal deletion and a C-terminal insertion mutation in the diagnostic sample, the N-terminal mutation was also identified in the germline material. Interestingly, the daughter of this pt also developed AML at the age of three years, exhibiting the identical N-terminal germline mutation, while the t C-terminal mutation was acquired and different to that of her mother. None of the remaining family members who could be analyzed had CEBPA germline mutations or a history of leukemia. Conclusion: The detection of CEBPA germline mutations in another pedigree with familial AML further sustains the hypothesis that CEBPA germline mutations might be the predisposing event in the development of AML in these families. Since other AML-associated gene mutations are rarely detected in CEBPA mutated cases, somatic mutations in the second allele represent a possible ‘second hit’ in this molecularly defined AML subtype.

Description: Background: In a previous randomized trial (AML HD98B) of the AMLSG in elderly (〉60 yrs) patients with AML (excluding APL), we could demonstrate that all-trans retinoic acid (ATRA) given as adjunct to intensive chemotherapy significantly improved complete remission (CR) rate and overall survival (OS). Our hypothesis for this study was that this beneficial effect of ATRA may be confined to a specific genotypic subset of AML. Aims: To evaluate the impact of ATRA on clinical outcome in cytogenetic and molecular genetic subsets of AML. Methods: Between 1998 and 2004, a total of 372 patients were enrolled. 242 patients were randomized for ATRA as adjunct to intensive induction (idarubicin, cytarabine, etoposide) and first consolidation therapy (intermediate-dose cytarabine) (Schlenk et al., Leukemia 2004), followed by a second randomization between further intensive consolidation versus a one-year oral maintenance therapy (Schlenk et al., Leukemia 2006). After an interim analysis in 2003, first randomization was stopped; the following 130 patients received chemotherapy without ATRA. Data from conventional cytogenetics and from fluorescence in-situ hybridization were previously reported (Fröhling et al., Blood 2006). All available leukemia specimens were analyzed for mutations in the genes encoding NPM1, CEBPA, and FLT3. Results: Median age of the 372 patients was 67 years (range: 61 to 83 yrs); median follow-up time was 68 months. 67% of the patients had de novo AML, 33% had secondary AML. Incidences of mutations were as follows (no. of specimens analyzed): NPM1 mutation, 25% (n=242); FLT3 internal tandem duplication (ITD), 18% (n=263); FLT3 tyrosine kinase domain mutation, 5% (n=244); CEBPA mutation, 8% (CEBPA analyzed only in normal karyotype samples; n=109). Logistic regression analysis performed on all patients identified three significant variables for achievement of CR: the genotype NPM1mut (OR 2.6; 95%-CI, 1.2–5.5), non-adverse karyotype subsuming core-binding factor and cytogenetically normal AML (OR 2.7; 95%-CI, 1.4–4.9); and age (diff. of 10 years, OR 0.58; 95%-CI, 0.3–1.0). Cox proportional hazard model for OS was performed on patients who were up-front randomized for ATRA. This model revealed a significant interaction between ATRA and the NPM1mut/FLT3-ITDneg genotype, confining the beneficial effect of ATRA to patients in that subgroup (HR, 0.29; 95%-CI, 0.10–0.87). Additional variables were log(LDH) (HR, 2.6; 95%-CI, 1.4 – 4.6), age (HR, 1.6; 95%-CI, 1.2– 2.2) and non-adverse karyotype (HR, 0.7; 95%-CI, 0.5–1.0). Univariable analysis revealed a survival after 5 years in patients with the genotype NPM1mut/FLT3-ITDneg of 57% (95%-CI, 28%–78%) in the ATRA-arm (n=16) compared to only 6% (95%-CI, 0%–25%) in the standard-arm (n=14) (p=0.002). In contrast, there was no difference in survival in all other patients with a survival of 2% (95%-CI, 0%–7%) in both arms (p=0.23). Conclusions: The genotype NPM1mut/FLT3-ITDneg emerges as highly significant predictive factor for response to ATRA in elderly patients with AML. This finding is validated prospectively in the ongoing AMLSG 07–04 study randomizing for ATRA in younger adults (ClinicalTrials.gov Identifier: NCT00151242).

Description: Differentiation arrest in acute myeloid leukemia (AML) results in part from dysregulation of histone acetylation and deacetylation, leading to transcriptional repression of differentiation-inducing genes. Transcriptional repression can be returned by histon deacetylation inhibitors, such as valproic acid (VPA). In September 2004, we initiated two randomized trials [AMLSG 07-04 (age 18–60 yrs), AMLSG 06-04 (age 〉60yrs)] to evaluate the impact of VPA as adjunct to standard induction therapy (idarubicin and cytarabine; additional etoposide in pts.

Description: Activating mutations in the juxtamembrane domain or the activation loop of the receptor tyrosine kinase FLT3 occur in many cases of acute myeloid leukemia (AML), but it is not known whether genomic alterations outside these regions contribute to leukemogenesis. High-throughput DNA sequence analysis has provided insights into the mutational profiles of various cancers and represents a promising strategy for the identification of novel therapeutic targets. However, recognizing the subset of genomic alterations that are functionally relevant has proven difficult. We used a high-throughput platform to interrogate the entire FLT3 coding sequence in a cohort of 222 adult AML patients without known FLT3 mutations and experimentally tested the functional consequences of each candidate leukemogenic allele by exogenous expression in BaF3 cells. DNA sequencing detected nine non-synonymous sequence variants in six exons that were not known single-nucleotide polymorphisms. Functional assessment of these alleles identified four novel activating mutations in the extracellular domain, the juxtamembrane domain, and the activation loop that induced constitutive kinase activity, differentially activated downstream signaling pathways, and conferred varying sensitivity to pharmacologic FLT3 inhibition. In contrast, the remaining five alleles, including mutations in highly conserved, key functional domains of FLT3, were not associated with increased kinase activity and aberrant signal transduction. These findings support the concept that acquired mutations in cancer may not contribute to malignant transformation, and underscore the importance of functional validation of candidate alleles discovered using high-throughput genomic screens, to distinguish between ‘driver’ mutations underlying cancer development, and biologically neutral ‘passenger’ alterations.

Description: Loss of chromosome 7 (−7) or deletion of the long arm (7q−) are recurring chromosome abnormalities in myeloid leukemias. The association of −7/7q− with myeloid leukemia suggests that these regions contain novel tumor suppressor gene(s), whose loss of function contribute to leukemic transformation or tumor progression. Based on chromosome banding analysis, two critical regions have been identified, one in band q22 and another in bands q32-q35. Presently there are no data available on the molecular delineation of the distal critical region. In this study we analyzed bone marrow and blood samples from 13 patients with myeloid leukemia (de novo myelodysplastic syndrome [MDS] , n = 3; de novo acute myeloid leukemia [AML], n = 9; therapy-related (t-) AML, n = 1) which, on chromosome banding analysis, exhibited deletions (n = 12) or in one case a balanced translocation involving bands 7q31-qter using fluorescence in situ hybridization (FISH). As probes we used representative clones from a contig map of yeast artificial chromosome (YAC) clones that spans chromosome bands 7q31.1-qter. In the 12 cases with loss of 7q material, we identified a commonly deleted region of approximately 4 to 5 megabasepairs in size encompassing the distal part of 7q35 and the proximal part of 7q36. Furthermore, the breakpoint of the reciprocal translocation from the patient with t-AML was localized to a 1,300-kb sized YAC clone that maps to the proximal boundary of the commonly deleted region. Interestingly, in this case both homologs of chromosome 7 were affected: one was lost (−7) and the second exhibited the t(7q35). The identification and delineation of translocation and deletion breakpoints provides the first step toward the identification of the gene(s) involved in the pathogenesis of 7q35-q36 aberrations in myeloid disorders.

Description: In acute myeloid leukemia (AML), cyotgenetics is used to stratify cases for appropriate risk-adapted therapy. The largest subset of patients, those with normal karyotype AML (NK-AML), comprises a heterogeneous group with intermediate prognosis. To improve outcome prediction for this group, we carried out gene-expression profiling of a set of 65 clinically-annotated NK-AML cases. In exploratory studies, an outcome predictor derived from semi-supervised analysis comprised genes mainly correlated with the presence of FLT3 (fms-related tyrosine kinase 3) internal tandem duplication (ITD) activating mutation, itself a prognostic factor. We therefore sought to directly define and characterize a gene-expression predictor of FLT3-ITD mutation status. Using a supervised analysis (Prediction Analysis of Microarrays), we identified an optimal 25-gene FLT3 signature, which in an independent set of 73 NK-AML cases was a significant predictor of clinical outcome, notably outperforming FLT3-ITD mutation status itself. We speculate that the signature identifies cases with alternative genetic or epigenetic changes that phenocopy FLT3-ITD, and the signature genes provide a starting point to dissect these pathways. Our findings underscore the prognostic relevance of FLT3-ITD in NK-AML, and indicate the potential clinical utility of a gene-expression based measure of clinically-relevant FLT3 pathway activation.

Description: To assess the prognostic relevance of mutations in the NPM1 gene encoding a nucleocytoplasmic shuttle protein in younger adults with acute myeloid leukemia (AML) and normal cytogenetics, sequencing of NPM1 exon 12 was performed in diagnostic samples from 300 patients entered into 2 consecutive multicenter trials of the AML Study Group (AMLSG). Treatment included intensive double-induction therapy and consolidation therapy with high cumulative doses of high-dose cytarabine. NPM1 mutations were identified in 48% of the patients including 12 novel sequence variants, all leading to a frameshift in the C-terminus of the nucleophosmin 1 (NPM1) protein. Mutant NPM1 was associated with specific clinical, phenotypical, and genetic features. Statistical analysis revealed a significant interaction of NPM1 and FLT3 internal tandem duplications (ITDs). NPM1 mutations predicted for better response to induction therapy and for favorable overall survival (OS) only in the absence of FLT3 ITD. Multivariable analysis for OS revealed combined NPM1-mutated/FLT3 ITD–negative status, CEBPA mutation status, availability of a human leukocyte antigen (HLA)–compatible donor, secondary AML, and lactate dehydrogenase (LDH) as prognostic factors. In conclusion, NPM1 mutations in the absence of FLT3 ITD define a distinct molecular and prognostic subclass of young-adult AML patients with normal cytogenetics.