Supplementary Data from CBL Exon 8/9 Mutants Activate the FLT3 Pathway and Cluster in Core Binding Factor/11q Deletion Acute Myeloid Leukemia/Myelodysplastic Syndrome Subtypes

NPM1 mutations represent frequent genetic alterations in patients with acute myeloid leukemia (AML) associated with a favorable prognosis. Different types of NPM1 mutations have been described. The purpose of our study was to evaluate the relevance of different NPM1 mutation types with regard to clinical outcome. Our analyses were based on 349 NPM1-mutated AML patients treated in the AMLCG99 trial. Complete remission rates, overall survival and relapse-free survival were not significantly different between patients with NPM1 type A or rare type mutations. The NPM1 mutation type does not seem to play a role in risk stratification of cytogenetically normal AML.

In chronic lymphocytic leukemia (CLL) patients, disruptions of the TP53 tumor suppressor pathway by 17p13 deletion (del17p), somatic TP53 mutations, or downregulation of microRNA-34a have been associated with a poor prognosis. So far, the impact of the various TP53 defects has not been evaluated in a large cohort of previously treated and relapsed CLL patients. Here, we present the results of TP53 gene sequencing and fluorescence in situ hybridization for del17p in a phase 3 clinical trial (REACH [Rituximab in the Study of Relapsed Chronic Lymphocytic Leukemia]). Of the 457 patients, 52 had TP53 mutations and 37 had del17p. In 24 (46%) of the TP53 mutated patients, no del17p was found and in 9 of the del17p patients, no TP53 mutation was identified. Based on a predicted proportion of TP53 disruption, a complete disruption of TP53 function, either by a combination of point mutations and/or del17p, was associated with a high risk for disease progression. Progression-free survival of patients with a heterozygous TP53 mutation was not significantly different from patients with a completely intact TP53 locus. In addition, only a complete loss of TP53 function correlated with low microRNA-34a expression levels. This trial was registered at www.clinicaltrials.gov as #NCT00090051.

PURPOSECytogenetically 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 METHODSTwo 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).RESULTSOn 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.CONCLUSIONWe 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.

The impact of a FLT3-internal tandem duplication (FLT3ITD) on prognosis of patients with acute myeloid leukemia (AML) is dependent on the ratio of mutated to wild-type allele. In 648 normal karyotype (NK) AML patients, we found a significant independent effect of the quantitative FLT3ITD mRNA level—measured as (FLT3ITD/wtFLT3)/(FLT3ITD/wtFLT3 + 1)—on outcome. Moreover, this effect was clearly seen in 329 patients with a mutated NPM1 gene (NPM1+), but not in 319 patients without a NPM1 mutation (wtNPM1). In a multivariate Cox regression model, the quantitative FLT3ITD mRNA level showed an independent prognostic impact on overall survival (OS) and relapse-free survival (RFS) only in the NPM1+ subgroup (OS: hazard ratio, 5.9; [95% confidence interval [CI]: 3.1-11.2]; RFS: hazard ratio, 7.5 [95% CI: 3.4-16.5]). The FLT3ITD mRNA level contributes to relapse risk stratification and might help to guide postremission therapy in NPM1-mutated AML.

Abstract Abstract 3521 MicroRNA-34a (miR-34a), a direct downstream target of the tumor suppressor TP53 is upregulated in chronic lymphocytic leukemia (CLL) cells and leads to apoptosis and cell cycle arrest. Previous studies found low miR-34a expression levels in CLL patients with TP53 gene disruptions by either 17p13 deletions or TP53 mutations and this has been linked to chemotherapy resistance and poor prognosis. Alternatively, miR-34a expression levels might be influenced by a single nucleotide polymorphism 309 (SNP 309) in the intronic MDM2 promoter. In this work we retrospectively determined miR-34a expression levels and the TP53 status in previously treated CLL samples enrolled in an international phase III clinical study comparing Fludarabine and Cyclophosphamide with or without Rituximab (FC versus R-FC: REACH trial). MicroRNA profiling data (Affymetrix GeneChip miRNA 1.0 array) and TP53 mutation data (AmpliChip p53 Test and Sanger sequencing) were available for 275 of 546 patients at treatment begin. In this subgroup of patients, genotype data (Illumina 550k HumanHap array) were available for 265 of 275 patients. A Mann-Whitney Wilcoxon test was used to compare distributions across two groups for a continuous variable. Association of the clinical data with progression-free survival (PFS) was assessed by Cox proportional hazard models. Patients were stratified into the following three groups: patients with both a 17p13 deletion and a TP53 mutation and patients with known dominant negative mutations of TP53 (n=21) (complete disruption of TP53), patients with either a 17p13 deletion (n=8) or a TP53 mutation (n=10) (partial disruption of TP53), and patients without TP53 aberrations (n=236) (wildtype TP53 and no 17p13 deletion). The distribution of miR-34a expression levels (log2 transformed) was compared across these groups. Patients with a complete disruption of TP53 function (mean=2.1, sd=2.1) had significantly lower miR-34a expression levels compared to patients without TP53 aberrations (mean=6.8, sd=2.3, p <0.001). Very interestingly, patients with a complete disruption of TP53 function also had lower miR-34a expression levels compared to patients with partial loss of TP53 function either by TP53 mutation (mean=7.1, sd=2.3, p <0.001) or deletion of the 17p13 locus (mean=7.2, sd=1.5, p <0.001). Patients with partial loss of TP53 function showed relatively high expression levels of miR-34a and no significant difference in miR-34a expression levels in patients without TP53 aberrations. There was a high variability of miR-34a expression levels in the group of patients without TP53 aberrations, with several patients having a low miR-34a. In order to find out whether this variability could be explained in part by SNP309 in the MDM2 promoter, we stratified patients without TP53 aberrations and with available genotype data (n=208) according to the genotype for SNP309. Because SNP309 was not on the Illumina chip, we imputed its genotype by using a perfect proxy (SNP rs2279744, r-square=1.0 with SNP309, according to public Caucasian Hapmap genotype data). We then compared the miR-34a expression levels between groups defined by the SNP309GG genotype (n=28, mean=7.5, sd=2.1), the heterozygous GT variant (n=101, mean=6.9, sd=2.2) and the wildtype TT-genotype (n=79, mean=6.6, sd=2.5). We could not observe any significant difference in miR-34a expression levels associated with the SNP309 genotype. A multivariate survival analysis was then used to further assess whether miR-34a expression levels in the group of patients without TP53 aberrations was of prognostic relevance with regards to PFS. MiR-34a expression did not predict PFS in patients without TP53 aberrations, neither as a continuous variable (HR: 1.03 (0.95–1.1), p =0.5) nor as a binary variable (dichotomized by its median value: HR: 0.92 (0.7–1.3), p =0.6) after adjustment for treatment, age, Binet stage and IGVH mutational status. In previously treated CLL patients, only a complete loss of TP53 function correlates with low miR-34a expression levels. MiR-34a expression levels did not demonstrate prognostic significance in CLL patients without TP53 mutations and did not correlate with the presence or absence of MDM2 SNP309. Further studies are warranted to assess the functional and clinical role of miR-34a expression as prognostic factor in patients with a disruption of TP53 function. Disclosures: Dufour: Roche: Research Funding. Bohlander:Roche: Research Funding. Spiekermann:Roche: Research Funding. Schneider:Roche: Research Funding. Hiddemann:Roche: Research Funding. Truong:Roche: Employment. Patten:Roche: Employment. Wu:Roche: Employment. Dmoszynska:Roche: Honoraria. Robak:Centocor Ortho Biotech Research & Development: Research Funding. Geisler:Roche: Speakers Bureau. Dornan:Genentech: Employment. Lin:Genentech: Employment. Yeh:Genentech: Employment. Weisser:Roche: Employment. Duchateau-Nguyen:Roche: Employment. Palermo:Roche: Employment.

Abstract 2445This icon denotes a clinically relevant abstract

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.

Abstract Abstract 2635 Poster Board II-611 Background: Cytogenetically normal acute myeloid leukemia (CN-AML) is associated with an intermediate outcome. A number of clinical and molecular risk factors have been characterized pointing to the heterogeneity of this group. The purpose of the study was to define a prognostic model based on pre-treatment patient characteristics to facilitate choice of therapy by definition of patient groups with different prognoses. Patients and methods: We evaluated four molecular markers (mutations of NPM1, CEBPA, MLL-PTD; FLT3-ITD mutant level; interaction term NPM1 and FLT3-ITD mutant level) and nine clinical parameters (white blood count (WBC), platelet count, hemoglobin level, lactase dehydrogenase (LDH) level, bone marrow blasts, de novo AML vs. non de novo AML, performance status, sex and age) at initial diagnosis in 648 patients with CN-AML treated in the AMLCG (German AML Cooperative Group) 1999 trial. The outcome parameter overall survival (OS) was calculated from randomization to death from any cause or to the latest follow-up date. Event-free survival (EFS) was defined as the period from the start of therapy until lack of a complete remission (CR), relapse of AML after CR or death without relapse. Relapse-free survival (RFS) was determined for responders from the first day of a CR until relapse or death without relapse. Univariate and multivariate Cox regression analyses for OS were performed. All parameters with p'0.05 in multivariate analyses after backward elimination and their regression coefficients were applied in the prognostic score. The minimal p-value approach was used to identify the risk groups with the greatest differences in OS. Results: In our patient cohort 84% had de novo AML. Median age was 60 years (17–85 years) and 70% had an ECOG score ≤1. Median platelet count was 57 G/l (5–643 G/l), median WBC was 18 G/l (0.1–798 G/l) and median hemoglobin level was 9.2 g/dl (4.2–16.4 g/dl). Mutations of NPM1, FLT3-ITD, MLL-PTD and CEBPA were present in 51%, 27%, 8% and 10% of patients, respectively. Median FLT3-ITD mutant level in FLT3-ITD mutated patients was 0.42 (0.02–1.00). Of 648 patients 377 had died. Median OS was 20 months with a median follow-up of 45 months. In the multivariate analyses for OS, the following parameters were significant: age (+10, years, HR: 1.3, p<0.001), WBC (10 fold, ×109/l, HR: 1.4, p<0.001), NPM1 (mutation vs. wild-type, HR: 0.35, p<0.001), CEBPA (mutation vs. wild-type, HR: 0.47, p=0.001), interaction term NPM1/FLT3-ITD mutant level (+1, HR: 4.5, p=0.006), performance status (ECOG 0,1 vs. ECOG 2-4, HR: 1.4, p=0.006) and platelet count (10 fold, ×109/l, HR: 0.70, p=0.016). After calculation of the prognostic score for each patient and definition of two cutpoints, we could identify three risk groups (median OS (N=590): not reached (n=169) vs. 22.7 months (n=220) vs. 8.4 months (n=201), p<0.001; median EFS (N=583): 42.3 months (n=168) vs. 7.6 months (n=216) vs. 3.2 months (n=199), p<0.001; median RFS (N=383): not reached (n=136) vs. 15.3 months (n=143) vs. 7.6 months (n=104), p<0.001). Furthermore this model was valid in both age subgroups (<60 years / ≥60 years). Interestingly, a subset of 31% of patients within the molecular favorable NPM1+/FLT3-ITD- risk group were assigned to the intermediate group according to our prognostic score and 31% of the low risk group were not NPM1+/FLT3-ITD-. Conclusions: We propose a new prognostic score based on pre-therapeutic clinical and well-established molecular markers that could be easily applied in the routine patient care setting for risk stratification and risk-adapted therapy. Further prospective validation is required to confirm the clinical relevance of this score. Disclosures: Unterhalt: Roche: travel support. Hoster:Roche: travel support.

Abstract Purpose: CBL is a negative regulator of activated receptor tyrosine kinases (RTK). In this study, we determined the frequency of CBL mutations in acute leukemias and evaluated the oncogenic potential of mutant CBL. Experimental Design: The cDNA of 300 acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) and acute lymphoblastic leukemia (ALL) patients and 82 human leukemic cell lines was screened for aberrations in the linker and RING finger domain of CBL. The oncogenic potential of identified mutants was evaluated in hematopoietic cells. Results: We identified 3 of 279 AML/MDS patients expressing CBL exon 8/9 deletion mutants. Three of four cases at diagnosis expressed deleted transcripts missing exon 8 or exon 8/9. In remission samples a weak or no expression of mutant CBL was detected. No aberrations were found in normal hematopoietic tissues. One of 116 sequenced AML/MDS cases carried a R420G missense mutation. All AML/MDS patients with identified CBL mutants belonged to the core binding factor and 11q deletion AML subtypes. Functionally, CBL negatively regulated FMS-like tyrosine kinase 3 (FLT3) activity and interacted with human FLT3 via the autophosphorylation sites Y589 and Y599 and colocalized in vivo. Expression of CBLΔexon8 and CBLΔexon8+9 in FLT3-WT-Ba/F3 cells induced growth factor–independent proliferation associated with autophosphorylation of FLT3 and activated the downstream targets signal transducer and activator of transcription 5 (STAT5) and protein kinase B (AKT). FLT3 ligand–dependent hyperproliferation of CBL mutant cells could be abrogated by treatment with the FLT3 PTK inhibitor PKC412 (midostaurin). Conclusion: CBL exon8/9 mutants occur in genetically defined AML/MDS subtypes and transform hematopoietic cells by constitutively activating the FLT3 pathway. This phenotype resembles the one of mutated RTKs and suggests that CBL mutant AML patients might benefit from treatment with FLT3 PTK inhibitors.

Nucleophosmin (NPM1) mutations in exon 12 represent the most frequent molecular aberrations in adult patients with acute myeloid leukaemia (AML). Molecular detection of NPM1 mutation A could be a useful marker for routine monitoring of minimal residual disease (MRD). We established a calibrator-normalized relative quantification real-time polymerase chain reaction (PCR) assay for NPM1 mutation A. ABL1 was used as a reference housekeeping gene and the NPM1 mutation A-containing OCI/AML3 cell line as a calibrator. Relative quantification was performed by calculating the NPM1 mutation A/ABL1 ratio which was normalized to the NPM1 mutation A/ABL1 ratio of OCI/AML3 calibrator cDNA. The assay showed a sensitivity of 10(-5). The clinical usefulness was evaluated by monitoring MRD in 51 AML patients with NPM1 mutation A. In 27 patients analysed at diagnosis and after induction treatment, NPM1 mutation A ratios showed a median log(10) reduction of 2.48, which correlated with response to therapy. Among the 51 patients, 21 relapsed and two lost the mutation. We established a sensitive, specific and reproducible assay for routine quantification and monitoring of NPM1 mutation A levels. However, clonal evolution was observed in 9.5% limiting the usefulness of the NPM1 mutation A mutation as a molecular marker in these patients.

The presence of CCAAT/enhancer binding protein alpha (CEBPA) gene mutations in patients with cytogenetically normal acute myeloid leukaemia (CN-AML) confers a favourable prognosis. Routine screening of all CN-AML patients for CEBPA mutations is therefore important for individual risk-adapted post-remission therapy and requires a fast and easy screening method. CEBPA mutations are distributed over the entire CEBPA gene and the functional and clinical consequences of the different mutations are still largely unknown. Therefore, we developed a multiplex polymerase chain reaction-based fragment length analysis mutation screening method for the entire CEBPA coding region. We initially evaluated our method by analysing 120 CN-AML samples both by fragment analysis and nucleotide sequencing and reached a sensitivity of 100% and a specificity of 90%. 349 CN-AML samples were subsequently screened for CEBPA mutations by fragment length analysis. Among a total of 469 CN-AML patient samples, 58 CEBPA mutations were detected in 38 CN-AML patients (8.1%). In conclusion, we established a fast and sensitive CEBPA mutation screening method suitable for inclusion in routine AML diagnostics.

In 45% of patients with de novo acute myeloid leukemia (AML) no cytogenetic abnormalities can be detected (normal karyotype AML, NK-AML). Recently, several molecular markers with prognostic significance have been described which define distinct subgroups of NK-AML patients. Mutations in the CEBPA gene have shown to occur at about 8% of NK-AML in Western countries and confer a favorable prognosis. The C/EBPα protein, a member of the family of basic region leucine zipper (bZIP) transcriptional regulators, is important for normal granulocytic differentiation and is frequently disrupted in AML. We retrospectively analyzed bone-marrow samples of 442 patients with de novo NK-AML for the presence of CEBPA mutations and established a fast and sensitive screening method. A multiplex-PCR-gene scanning assay for combined detection of CEBPA and NPM mutation has recently been described in which, however, the primers did not cover the whole CEBPA gene. CEBPA mutations have been found to be distributed over the entire CEBPA gene and their functional and clinical consequences are not yet clear. Therefore, we designed a rapid CEBPA specific multiplex PCR-gene scanning assay covering the entire coding region of the CEBPA gene. Four primer pairs were designed, fluorescently labeled and included in 2 multiplex PCR reactions. The PCR products were electrophoresed on a genetic analyzer and the amplicon sizes were compared to wildtype CEBPA of U937 cell line by fragment length analysis. In order to evaluate our method, we analyzed 120 patient samples in parallel by both multiplex PCR and sequencing analysis. Using sequencing analysis as a gold standard, all of the CEBPA mutations could be detected by multiplex PCR and fragment length analysis. Thus, our multiplex PCR assay reached a sensitivity of 100%. The specificity was 89% due to the false positive detection of a 6 basepair duplication polymorphism in 2.5% of patients (Wouters BJ et al, Blood, 2007). 322 patient samples were subsequently screened for CEBPA mutations by the multiplex PCR assay. In case of alterations in the fragment length analysis, the relevant CEBPA region was sequenced to identify the exact type of mutation. Among 442 patients with NK-AML, 32 patients (7%) showed CEBPA mutations. Taken together, we identified 47 mutations in 32 patients, of which 17 patients had a single CEBPA mutation and 15 patients had more than one CEBPA mutation. We identified 30 out of frame insertion/deletion nonsense mutations resulting in an N-terminal stopcodon and 14 in frame insertion/deletion mutations as well as 3 out of frame insertion/deletion mutations in the C-terminal bZIP region. The only limitation of this method might be that single basepair substitutions, which do not affect the length of the amplicon, cannot be detected. Substitutions in the CEBPA gene are, however, rare events and often silent. In 120 sequenced AML patients we did not find any non-silent substitution. We established a fast and sensitive screening method suitable for large-scale detection of CEBPA mutation and applicable for inclusion in routine AML diagnostics. This is so far the largest reported analysis of CEBPA mutations in patients with NK-AML and might provide further insights into the functional and clinical relevance of the different types of CEBPA mutations and their correlation to other molecular markers in NK-AML.

Background: Prognosis of AML is influenced by different clinical and molecular alterations. We performed a multivariate analysis including five molecular markers NPM1, FLT-ITD, CEBPA, FLT-TKD and MLL-PTD combined with clinical parameters at initial diagnosis to refine risk stratification.

FLT3-internal tandem duplications (FLT3-ITDs) comprise a heterogeneous group of mutations in patients with acute leukemias that are prognostically important. To characterize the mechanism of transformation by FLT3-ITDs, we sequenced the juxtamembrane region (JM) of FLT3 from 284 patients with acute leukemias. The length of FLT3-ITDs varied from 2 to 42 amino acids (AAs) with a median of 17 AAs. The analysis of duplicated AAs showed that in the majority of patients, the duplications localize between AAs 591 to 599 (YVDFREYEY). Arginine 595 (R595) within this region is duplicated in 77% of patients. Single duplication of R595 in FLT3 conferred factor-independent growth to Ba/F3 cells and activated STAT5. Moreover, deletion or substitution of the duplicated R595 in 2 FLT3-ITD constructs as well as the deletion of wild-type R595 in FLT3-ITD substantially reduced the transforming potential and STAT5 activation, pointing to a critical role of the positive charge of R595 in stabilizing the active confirmation of FLT3-ITDs. Deletion of R595 in FLT3-WT nearly abrogated the ligand-dependent activation of FLT3-WT. Our data provide important insights into the molecular mechanism of transformation by FLT3-ITDs and show that duplication of R595 is important for the leukemic potential of FLT3-ITDs.

Background: Approximately 45% of AML patients have a normal karyptype (NK-AML) and an intermediate clinical prognosis. As only 20–42% of these patients show long-term survival, it is important to identify prognostic markers to distinguish patients' outcome more precisely. Mutations in the FLT3 gene such as internal tandem duplications (ITD) in the juxtamembrane domain and point mutations in the tyrosine kinase domain (TKD) are the second most common abnormalties in AML patients. For FLT3-ITD it is well known that patients have an unfavourable prognosis. Up to now there are not enough reliable data to determine the prognostic impact of FLT3-TKD mutations.