ALBERT

Description: FLT3 (fms-like tyrosine kinase 3) is constitutively activated in about 30% of patients with acute myeloid leukemia (AML) and represents a disease-specific molecular marker. Although FLT3-LM (length mutation) and TKD (tyrosine kinase domain) mutations have been considered to be mutually exclusive, 1% to 2% of patients carry both mutations. However, the functional and clinical significance of this observation is unclear. We demonstrate that FLT3-ITD-TKD dual mutants induce drug resistance toward PTK inhibitors and cytotoxic agents in in vitro model systems. As molecular mechanisms of resistance, we found that FLT3-ITD-TKD mutants cause hyperactivation of STAT5 (signal transducer and activator of transcription-5), leading to upregulation of Bcl-x(L) and RAD51 and arrest in the G2M phase of the cell cycle. Overexpression of Bcl-x(L) was identified as the critical mediator of drug resistance and recapitulates the PTK inhibitor and daunorubicin-resistant phenotype in FLT3-ITD cells. The combination of rapamycin, a selective mTOR inhibitor, and FLT3 PTK inhibitors restored the drug sensitivity in FLT3 dual mutant–expressing cells. Our data provide the molecular basis for understanding clinical FLT3 PTK inhibitor resistance and point to therapeutical strategies to overcome drug resistance in patients with AML.

Description: Accurate diagnosis and classification of leukemias are the bases for the appropriate management of patients. The diagnostic accuracy and efficiency of present methods may be improved by the use of microarrays for gene expression profiling. We analyzed gene expression profiles in 937 bone marrow and peripheral blood samples from 892 patients with all clinically relevant leukemia subtypes and from 45 nonleukemic controls by U133A and U133B GeneChip arrays. For each subgroup, differentially expressed genes were calculated. Class prediction was performed using support vector machines. Prediction accuracy was estimated by 10-fold cross-validation and was assessed for robustness in a 100-fold resampling approach using randomly chosen test sets consisting of one third of the samples. Applying the top 100 genes of each subgroup, an overall prediction accuracy of 95.1% was achieved that was confirmed by resampling (median, 93.8%; 95% confidence interval, 91.4%-95.8%). In particular, acute myeloid leukemia (AML) with t(15;17), AML with t(8;21), AML with inv(16), chronic lymphatic leukemia (CLL), and pro–B-cell acute lymphoblastic leukemia (pro–B-ALL) with t(11q23) were classified with 100% sensitivity and 100% specificity. Accordingly, cluster analysis completely separated all 13 subgroups analyzed. Gene expression profiling can predict all clinically relevant subentities of leukemia with high accuracy.

Description: Nucleoplasmin (NPM) mutations have recently been described as a new kind of mutation in AML. In the presented work we have analyzed 977 AML cases from different prognostic subgroups. Fifteen different mutations (type A:n=245 (78.3%), B:n=21 (6.7%), D: n=28 (11.4%), I:n=3 (1%), J:n=4 (1.3%), K:n=2, G,H, L, M, N, O, P, Q, R: n=1, each (0.3%) were detected in 313 cases. No mutation was found in inv(16) (n=89), t(8;21) (n=98), t(15;17) (n=17), t(11q23)/MLL (n=8), inv(3) (n=8), other reciprocal translocations (n=3), and complex aberrant karyotypes (n=59). In the normal karyotype group the incidence was 299/603 (49.6%) and in all others 13/90 (14.4%). In the latter cohort cases with NPM+ were: − Y (1 case), +4 alone (1 case), +4 and +8 (2 cases), +4 with multiple trisomies (1 case),+8 (1 case), multiple other trisomies (1 case), del(5q) (1 case), − 7 alone (2 cases), del(9q) (2 cases), unbalanced translocation (1 case). Data for evaluation of prognosis were available in 401 AML patients with normal karyotype treated within the AMLCG99 study. Results were calculated in relation to MLL-PTD, FLT3-LM, FLT3-TKD, NRAS, KIT, and CEBPA mutations and correlated to further clinical characteristics. NPM-mutated cases were frequently associated with FLT3 mutations but rarely with other mutations. The NPM-mutated group had a higher CR rate (70.5% vs. 54.7%, p=0.003), a trend to longer OS (median 1012 vs. 549 days, p=0.0762), and significantly longer EFS (median 428 vs. 336 days; p=0.0121). There was a clear favourable impact of NPM+/FLT3-LM- on OS and EFS (264/395 cases; 66.8%). This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM+/FLT3-LM+ double positive pts. was similar to NPM-/FLT3-LM+ cases. We further analysed stability for the NPM mutation at diagnosis and relapse and found stability in 14/15 paired samples. One case with AML M5 lost the NPM mutation at relapse. This case had also a shift from normal karyotype to 46,XX,del(7)(q22),der(7)t(7;21)(p11;q?) and a complete shift of immunophenotype indicating a therapy-related AML. In addition, we have analyzed the applicability of NPM mutations as targets for minimal residual disease (MRD) detection. In total, 82 samples of 22 selected cases were analyzed by use of quantitative real time PCR for the three most common mutation types. Fifteen of these cases had a type A, 3 a type B, and 4 a type D mutation. Samples at diagnosis had an NPM/ABL ratio of 267.6 (range: 54.9 to 3174.7) while each 10 NPM negative cases had a median ratio of 0.03 (assay for A), 0.0001 (assay for B), and 0.002 (assay for D), showing that these assays were nearly specific for the respective mutation. Using limited dilution series of NPM+ in NPM- cDNA a median sensitivity of 1:100.000 could be shown. In both analyzed cases with relapse these were early detectable by increasing NPM levels. Thus, NPM mutations can be used for highly sensitive PCR-based MRD detection in AML, especially if other markers are lacking, i.e. in normal karyotype. In conclusion, our data showa nearly exclusive association of NPM+ to the normal karyotype,a good prognosis of NPM+/FLT3-LM- in normal karyotype.an intermediate prognosis of NPM+/FLT3-LM+ in normal karyotypeNPM as a new marker for PCR-based MRD-detection.

Description: Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3–length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1–/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM– mutations are an independent predictor for a favorable outcome in AML with normal karyotype.

Description: Experimental data have shown that two of the most frequent genetic alterations in AML, the AML1-ETO (A/E) fusion gene and the FLT3 length mutation (FLT3-LM) are both mostly insufficient on their own to induce leukemia. These findings support the model that collaboration of two classes of genetic alterations, altering proliferation or differentiation, is necessary for leukemogenesis. When we first analyzed 135 patients with A/E positive AML, additional mutations affecting signal transduction were found in 38 % of all cases (FLT3-LM 10.3 %, KIT 8.1 % and NRAS 9.6 %). In contrast, none of the patient with A/E positive leukemia had alterations associated with transcriptional regulation such as MLL-PTD. To test the hypothesis that A/E collaborates with FLT3-LM in inducing acute leukemia, we transplanted mice with bone marrow (BM) cells retrovirally expressing A/E, FLT3-LM or both alterations. Mice transplanted with BM cells expressing A/E or FLT3-LM alone did not develop any disease. In contrast, mice (n=11) transplanted with BM cells expressing both alterations succumbed to an aggressive acute leukemia. Intriguingly, developing leukemias differed with regard to their phenotype with 7 animals developing AML and 4 animals developing ALL. Furthermore, the majority of AML cases showed simultaneous expression of lymphoid antigens as described in patients with A/E positive AML. The collaboration of A/E with FLT3-LM was depending on DNA binding activity of the fusion gene as the L148D point mutation in the Runx1 domain of the construct abrogated collaboration of A/E with the FLT3-LM in the CFU-S assay. Furthermore, inactivation of the kinase activity of the FLT3-LM (FLT3-LM K672R mutant) resulted in the complete loss of collaboration with the A/E fusion. Treatment of cells co-infected with A/E and FLT3-LM with the kinase inhibitor PKC412 resulted in a 62 % reduction of the CFU-S frequency. To further explore a possible contribution of retroviral insertional mutagenesis to the transformation process in this model, 10 retroviral integration sites were subcloned and sequenced from 4 leukemic mice: all 10 sites were unique with no indication of a common integration site associated with the leukemic transformation. Moreover, 5 sites were intergenic or not linked to known genes. The remaining sites were in introns in a 5′ to 3′ orientation most likely to lead to gene knockdown rather than activation. These data provide direct functional evidence for the oncogenic collaboration between A/E with a class of activating mutations, recurrently found in patients with t(8;21), and add experimental data to the clinical observation which demonstrated a significant inferior treatment outcome in patients with AML1-ETO and additional mutations of receptor tyrosine kinases.

Description: The fusion transcript CBFB-MYH11 is the molecular correlate of inv(16)/t(16;16) and strictly associated with FAB subtype M4eo. This subgroup is associated with a favorable prognosis in AML. However, approximately 30% of the patients relapse. Our intention was to examine prognostic factors for the outcome within this subgroup. Therefore 153 CBFB-MYH11 positive AML patients were analyzed. The median age was 52 years (range 18–83), 80 patients were female, 73 were male. In 22 cases AML was therapy-related, in 131 cases a de novo AML was diagnosed. Inv(16) was detected in 138 and t(16;16) in 12 cases. In 3 cases neither inv(16) nor t(16;16) were detectable despite PCR and FISH positivity for CBFB-MYH11 suggesting cryptic rearrangements. The most frequent additional cytogenetic abnormalities were +8 (n=19), +9 (n=3), +21 (n=7), +22 (n=23). Cox regression analysis revealed that advanced age (OS: p=0.026; EFS: p=0.029) and increased CBFB-MYH11/ABL ratio at diagnosis (OS: 0.016, EFS: p=0.064) were associated with a worse prognosis. Using log rank test additional factors influencing survival were detected. These included: t(16;16) vs inv(16) (OS: n=8, censored 4, median 362 days vs n=118, censored 92, median not reached, p=0.018; EFS: n=8, censored 4, median 232 days vs n=118, censored 70, median 918 days, p=0.048) and trisomy 21 vs no additional aberrations (OS: n=6, censored 3, median 435 days vs n=74, censored 59, median not reached, p=0.024; EFS: n=6, censored 2, median 293 d vs n=74, censored 44, median 764 days, p=0.0047). Therapy related AML was associated with worse EFS than de novo AML (n=16, censored 6, median 371 days vs n=112, censored 70, median 1179 days, p=0.0167) and there was a trend towards worse OS (p=0.157 n=16, censored 10, median 764 days vs n=112, censored 88, median not reached). A multivariate analysis including t(16;16), age, CBFB-MYH11/ABL ratio, therapy related AML and +21 as covariates revealed t(16;16) and age as independent factor for OS (p=0.014 and p=0.015, respectively) and age, t(16;16), and +21 as independent factors for EFS (p=0.047, p=0.013, and p=0.016, respectively). There was no evidence that the additional aberrations +22 or +8 had an influence on survival. Taken together our data suggest that t(16;16) as compared to inv(16), trisomy 21 and age are associated with worse prognosis in patients with CBFB-MYH11 positive AML.

Description: Acute myeloid leukemia (AML) with complex aberrant karyotype is a distinct biological entity. It is characterized by: 1) a sharp increase of incidence above age 50, 2) a characteristic pattern of chromosomal gains and especially losses, i.e. losses of 5q14q33, 7q32q35, and 17p13 translating into a reduced expression of genes located in these regions, 3) a unique gene expression pattern including an upregulation of genes involved in DNA repair, 4) a high incidence of TP53 deletions and/or mutations and 5) an overall unfavorable prognosis. So far, the pathogenetic role of the lost and gained chromosomal regions in AML with complex aberrant karyotype is unclear. In a first step we tested whether a correlation between genomic imbalances and changes in gene expression exists. Therefore, gene expression analysis was performed in 24 cases of AML with complex aberrant karyotype and in 57 AML with normal karyotype for comparison. Overall, genes located on 5q, which is deleted in the majority of cases, showed a significantly lower expression in AML with complex aberrant karyotype as compared to AML with normal karyotype. Furthermore, for each chromosomal band on chromosome 5 ratios were calculated between AML with complex aberrant karyotype and AML with normal karyotype. Ratios lower than 0.90 were found for all chromosome bands between 5q14 and 5q34 (range 0.64–0.88) with lowest values for 5q15 and 5q22 (0.64 and 0.68). Between 61% and 90% of genes located in one of the chromosome bands 5q14 to 5q34 showed a lower expression in AML with complex aberrant karyotype compared with AML with normal karyotype. An overall reduced expression of genes was also observed in other frequently lost regions such as 7q and 17p, while an overall higher expression of genes located in gained regions such as 1p, 8q and 11q was detected. However, not all genes located within a deleted or gained region showed an altered expression. In order to perform more precise correlations between the copy number of individual genes and their expression 33 cases with AML and complex aberrant karyotype were analyzed in parallel with conventional comparative hybridization, genomic arrays (Affymetrix 10K arrays) and gene expression arrays (Affymetrix U133A+B or 2.0 plus). The lost and gained regions detected in conventional CGH were confirmed by data obtained from the genomic arrays. In addition, gained and lost regions could be mapped more precisely. Interestingly, the genomic arrays revealed that even large deletions are truly continuous, although not all genes from the respective regions showed a lower expression. In contrast at the borders of amplifications amplified genes alternated with non-amplified genes. In conclusion, a detailed analysis on the genomic as well as on the gene expression level might lead to further insights into the pathogenesis of AML with complex aberrant karyotype and may also serve as a new diagnostic tool in the near future.

Description: The lack of somatic mutations of the immunoglobulin variable heavy chain (IgVH) gene has been established as poor prognostic marker for chronic lymphocytic leukemia (CLL) patients at early stage disease. Expression of the non receptor tyrosine kinase zeta chain associated protein (ZAP-70) was proposed as a surrogate marker for an unmutated IgVH, however, up to 30% discordant samples have been reported depending on the respective study. B cell receptor (BCR) mediated signaling is enhanced by ZAP-70 expression in CLL cells in vitro and ZAP-70 expression also tends to decrease the time from diagnosis to treatment irrespective of the IgVH status. Therefore, we wanted to identify differentially expressed genes between the ZAP-70 positive and negative CLLs by gene expression profiling of peripheral blood mononuclear cells (PBMCs) using Affymetrix microarrays (HG-U133 Plus 2.0). ZAP-70 expression was analyzed by quantitative real time PCR of CD19 purified (purity 〉 99%) PBMCs (n=62) using a LightCycler instrument. Expression of ZAP-70 mRNA was normalized against the housekeeping gene ABL and a relative quantitation against Jurkat T cells as a calibrator was performed. Results are expressed as normalized ratio and a cut-off of 0.5 normalized ratio gave the best correlation to the IgVH status with 77% concordant samples between ZAP-70 expression and the IgVH status. The discordant samples consisted of 5 unmutated IgVHs in the ZAP-70 negative group and 9 mutated in the ZAP-70 positive group. In a second step PBMCs of the same samples were analyzed by gene expression profiling and differentially expressed genes were identified by t-test. Among the two best genes that could be used in a classification algorithm (SVM) to distinguish between the 2 subsets with 92% accuracy were ZAP-70 and B cell scaffold protein with ankyrin repeats (BANK1). The expression of BANK1 was increased 3–4-fold in the ZAP-70 negative compared to the ZAP-70 positive CLL subset (P = 0,001). In the literature, BANK1 has been identified in human BCR expressing B cells and seems to be B cell restricted. In B cells the scaffolding protein BANK1 enhances BCR-mediated Ca2+-signaling, a signaling pathway that is also enhanced by ZAP-70 expression in CLL B cells. Based on these data we show that increased BANK1 expression correlates with a ZAP-70 negative status in CLL B cells. The functional consequences of BANK1 expression in the ZAP-70 negative subset of CLL B cells, which are usually associated with a more favorable prognosis, still need to be established further.