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: The prognostic value of MRD detection in AML has been shown for PML-RARA, AML1-ETO, and CBFB-MYH11positive AML, a group which accounts for only 20–25% of AML. The largest group of AML is represented by cases with normal karyotype or unbalanced intermediate risk group karyotypes. In this group different molecular mutations occur, which are associated with heterogeneous clinical outcome. Thus, different MRD patterns can be anticipated. To expand the spectrum of molecular targets in intermediate risk AML, we used MLL-PTD, FLT3-LM, and NPM1 which are detectable in 45–50% of all intermediate risk group pts for MRD detection. In addition, overexpression of WT1 for MRD detection was used to even include pts. without detectable mutations. In total 996 samples (spl) of 234 patients (pts) were analysed by quantitative real time PCR. For MLL-PTD (321 spl of 78 pt), and WT1 (336 spl of 66 pts) universal assays were used. For NPM1 mutation specific assays (182 spl of 54 pts (42 x type A, 2 x type B, 7 x type D, 3 x rare not yet defined types)) and for FLT3-LM (161 spl of 18 selected pts) patient specific assays were used. All assays were RNA based. Sensitivity of the assays were between 1:100.000 to 1:1.000.000 for the FLT3-LM and NPM1, depending on pts specific assay. Sensitivity was 1:10.000 to 1:100.000 for MLL-PTD due to low background levels detectable in healthy controls. The sensitivity of WT1 was relatively low with 1:100 at most, as there was no high WT1 expressor at diagnosis in this cohort. With all markers the clinical course of the disease was clearly be reflected and all 84 relapses were detectable due to recurring high expression rates. In 17 cases, were samples 2–4 months before clinical relapse were available relapses were predictable based on increasing transcript levels. Five different follow up intervals (int) were defined: up to day 21; days 22–60; days 61–120; day 121–365, later. The log change from diagnosis to defined follow up intervals was analyzed. A rapid decline of median transcript ratios in the NPM1 group (int 1: 2 log; int 2 to 4: 4 log) was observed. Relapses in the NPM1 group occurred earliest after one year. AML with FLT3-LM similarly showed good responses with 1–2 log decreases in int 1 and 2 and 3 log in int 3 and 4. Also this group revealed the first relapses in int 5. The MLL-PTD group was characterized by slow response rates with only 0.2 log reduction in int 1 and 2 and hardly 3 log in int 3. In this group many relapses occurred in int 4 and 5. These data reflect the biological differences of these molecular subgroups: NPM1 as a favourable group, FLT3-LM as a slightly unfavourable group with good response rates but high relapse rates, and MLL-PTD as an entity with bad prognosis due to poor response rates and high relapse rates. Due to low sensitivity WT1 reflected only 0.2 log in interval 1 up to 2 log in interval 3. Seven cases were analyzed in parallel for WT1 and MLL-PTD, 6 for WT1 and FLT3-LM, and one for FLT3-LM and MLL-PTD. Although the correlation of parallel assessment was high (R=0.993) the median differences of log changes of FLT3-LM and NPM1 was one log larger than for MLL-PTD and three log larger than for WT1, depending on the initial sensitivity of the assays. In conclusion: MRD detection is feasible in the karyotypically intermediate risk group. it nicely reflects biological difference in this group NPM1, MLL-PTD and FLT3-LM are better MRD-markers than WT1, which may only be investigated in cases without any other available marker.

Description: We characterized the mutational status of the FLT3 tyrosine kinase domain (FLT3-TLD) in 3082 patients with newly diagnosed AML. FLT3-TKD mutations were detected in 147 of 3082 (4.8%) patients. Similar to the FLT3 juxtamembrane domain mutations (FLT3-LM), there was a high correlation of FLT3-TKD mutations with normal karyotype (88 of 1472; 6.0%). FLT3-TKD mutations were most frequent in the AML FAB subtypes M5b (15 of 114; 13.2%), M3v (6 of 51; 11.8%), and M4 (39 of 484; 8.1%). Similar to FLT3-LM, the FLT3-TKD mutations show elevated peripheral leukocytes compared with FLT3wt AML. FLT3-TKD had a high incidence in cases with NPM1 mutations (23 of 262; 8.8%), CEBPA mutations (6 of 76; 7.9%), and NRAS mutations (6 of 78; 7.7%). FLT3-TKD in combination with FLT3-LM (17 of 594 patients; 2.9%) and KITD816 (1 of 44; 2.3%) was rare. Unlike the FLT3-LM, which are associated with inferior survival, prognosis was not influenced by FLT3-TKD in the total cohort of 1720 cases, where follow-up data were available (97 FLT3-TKD; 1623 FLT3-WT). In t(15;17)/PML-RARA with FLT3-TKD mutations, in FLT3-LM/TKD double-mutated, and in MLL-PTD/TKD double-mutated cases prognosis was unfavorably influenced by FLT3-TKD mutations. In contrast, we found an additional favorable impact of FLT3-TKD on EFS in prognostically favorable AML with NPM1- or CEBPA mutations.

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: Dose density during early induction has been demonstrated to be one of the prime determinants for antileukemic efficacy. The German AML-CG therefore pilots a dose dense induction regimen S-HAM (sequential HD-AraC [3g/m2/12h d1,2,8,9] and Mitoxantrone 10mg/m2 [d3,4,10,11] followed by pegfilgrastim) in which two induction cycles are applied over 11 – 12 days as compared to conventional double induction, in which two cycles are applied over 25 – 29 days - thereby increasing dose density ca. two-fold in the critical first weeks of treatment. In the past 2,5 years 168 patients with de-novo AML (excluding APL) have been recruited into the trial with a median age of 53 years (range 18 – 78). Of 136 patients evaluable for response the following results were achieved: CR 62%, CRi 22%, PL 7%, ED 9% - resulting in an overall response rate (ORR) of 84%. The early death rate (ED) of 9% and the toxicity profile compared favourably with a historical control group of the AML-CG 1999 study (de-novo AML, 〈 60 years, HAM-HAM double induction) which demonstrated an ED rate of 14% (ORR 68%, persistent leukemia (PL) 18%). The high antileukemic efficacy of S-HAM was also demonstrated by the fact that 89% of patients had a blast count of 〈 10% one week after therapy as compared to less than 48% of patients of the HAM-HAM double induction group. Whereas even for patients with unfavourable cytogenetics (including complex aberrations) a median overall survival of 13,5 months was reached (23% at 2 years), for patients with favourable karyotypes overall survival at 2 years was 81%and for patients with intermediate karyotypes 74% after S-HAM treatment. Importantly the compression of the two induction cycles into the first 11 – 12 days of treatment seems actually beneficial for normal hematopoesis as demonstrated by a significantly shortened duration of critical neutropenia of 30 days as compared to 45 days after conventionally timed double induction. This shortening of critical neutropenia by more than 2 weeks was highly relevant for the duration of hospital stay and hospital costs. In conclusion S-HAM with pegfilgrastim support is a highly effective regimen in primary de-novo AML with a very favourable safety profile and significantly shortened duration of neutropenia. This regimen will therefore constitute the (dose-dense) experimental arm for a randomized comparison with standard double induction in the next generation of the German AML-CG studies.

Description: Recent data indicate that mutations in exon 12 of the nucleophosmin (NPM1) gene characterize a distinct subgroup of adult and pediatric acute myeloid leukemia (AML). AML carrying NPM1 mutations account for about one-third of all adult AML, exhibit distinctive biological and clinical features and show a strong association to AML with normal karyotype (55% mutated). However, the role of NPM1 in leukemogenesis still remains elusive. Here we present data on a cohort of n=66 AML cases with normal karyotype analyzed by high-density whole genome expression microarrays (Affymetrix HG-U133 Plus 2.0). In parallel melting curve analysis was used to assess NPM1 mutational status: 41 cases were characterized as mutated (NPM1+) and 25 cases were unmutated (NPM1−). We first investigated the gene signature that discriminated NPM1+ from NPM1− cases. Genes that were significantly overexpressed comparing NPM1+ against NPM1– cases included a strong homeobox genes signature (HOXA1, HOXA5, HOXA7, HOXA9, HOXA10, HOXA11, HOXB2, HOXB4, HOXB5, HOXB6, HOXB7, MEIS1, and PBX3). A functional analysis (Gene Ontology) revealed a clear association of the group of overexpressed genes with the cell components nucleosome, chromatin, and the nuclear envelope-endoplasmatic reticulum network as well as involvement in the biological processes of nucleosome and chromatin assembly, establishment of protein transport and localization, and Notch signaling pathway. In contrast, the cellular processes completely differed when genes were investigated that were significantly underexpressed in NPM1+ cases compared to NPM1− cases. This group of genes encoded membrane-related proteins (gap junction, intercellular junction, signalosome complex) and proteins involved in cellular morphogenesis and cell communication. The differences in gene expression signatures between NPM1+ and NPM1− cases permit a robust classification approach by gene expression profiling. Support Vector Machine analysis resulted in 〉92% prediction accuracy of NPM1 mutation status (10-fold cross-validation). The sensitivity was very high for the positive detection of NPM1+ cases (〉97%). Using a 100-fold re-sampling approach and splitting the complete data set into a training set (n=44) and testing set (n=22) the following genes were most frequently selected as top discriminatory genes: HOXA5, HOXB4, HOXB5, HOXB6, MEIS1, PBX3, FGFR1, ADAM17, PRICKLE1, and TMPO. Interestingly, the classification was less accurate when also FLT3 internal tandem duplication mutation status was taken into account. The study cohort (n=66) then was distributed as follows: 19 NPM1+/FLT3+, 22 NPM1+/FLT3−, 4 NPM1−/FLT3+, and 21 NPM1−/FLT3− negative cases. Only 14 of 22 (64%) NPM1+/FLT3– cases were correctly predicted, with miscalls falling both into the group of NPM1+/FLT3+ and NPM1−/FLT3− cases. In conclusion, NPM1 mutations are the most frequent mutations in adult AML to date and their central prognostic role is increasingly recognized. Given the fact that they are nearly mutually exclusive with major recurrent genetic abnormalities and that they can be characterized by a distinctive gene expression program these data especially for of NPM1+/FLT3− AML with better outcome may support to classify this as a separate biological subgroup of AML with normal karyotype.

Description: Up to 95% of all patients (pts) with polycythemia vera (PV) carry JAK2V617F mutations (V617Fmut). The underlying pathophysiologic event in the remaining 5% remained unclear. Recently, novel mutations in exon12 (exon12mut) of the JAK2 gene have been described. Thus far, 16 pts with 4 different mutation types (F537-K39delinsL, H538QK539L, N542-E543del, and K539L) have been identidied by two groups. A specific phenotype of isolated erythrocytosis and young age has been suggested by one group while another group found that besides pts with isolated erythrocythosis around 50% may present with typical criteria for PV. Thus, exon12mut are scarce with still unclear frequency. Therefore, we have applied melting curve screening covering codons 535-555 of exon12 on 211 pts with PV or suspected PV. DNA of all pts with aberrant curve was sequenced. We detected exon12mut in 10/211 (4.7%) of all pts. Of these 99 pts fulfilled diagnostic criteria of PV whereas 112 were suspected PV due to unclear polyglobulia. The frequency of exon12mut in V617Funmut (V617wt) PV was 10/99 pts (10.1%). No exon12mut was detected in pts with polyglobulia. In addition, 10 V617wt ET and 50 CMPD with elevated red blood cell counts were analyzed but turned out to be exon12 unmutated (exon12wt). 3 of the exon12mut pts had the previously described F537-K39delinsL, 2 the N542-E543del, 1 H538QK539K, 1 K539L. In addition, 2 new mutations were detected: a E543-D544del (2 pts) and a K539S mutation (1 pt). Quantification showed exon12mut in 10–30% of all PBC (7 pts) or BM cells (3 pts). 5 pts were analyzed at diagnoses and 5 pts 2–8 years (y) (median 4y) after initial diagnosis of PV. Treatment was phlebotomy and ASS only in all pts. In 1 pt a homozygous K539L mutation was found. Thus, unlike previously reported, the exon12mut like the V617F mutations can occur in a homozygous state. This pt was at advanced stage near to transformation to AML. While in PV overall the female/male ratio was 225/231 in the exon12 mutated cohort the female/male ratio was higher with 7/3. Age of onset was 16–75 y (median: 57.5 y). This is the same range as the exon12wt/V617wt pts (57.3 y) but younger than the V617Fmut PV (66.5 y). Hematocrit of the exon12mut pts was elevated with a median of 61% in males and 55% in females. Erythropoietin levels were very low with a median of 3.8 U/L. Other parameters were less conspicuous in exon12mut than in V617Fmut (given in medians): WBC: 6,100 vs. 12,200/μl; Hb: 150 vs. 158 g/l; platelets 282,000 vs. 483,000/μl. In detail, four pts had elevated WBC and platelets in addition to erythrocytosis whereas 6 pts had isolated erythrocytosis. Thus, exon12mut pts frequently show isolated polyglobulia (60%) which is rare in V617Fmut PV. In addition, none of the exon12mut pts had hepatomegaly, three pts had borderline splenomegaly. In conclusion, analysis for JAK2 exon 12 mutations is a new tool for diagnostics of PV. Based on this still small cohort it can be speculated that exon12mut in comparison to V617Fmut pts tend to occur more frequently in women, at younger age, have lower WBC and platelet counts and frequently isolated erythrocytosis.

Description: The mechanism that may cause progression of myelodysplastic syndrom (MDS) to acute myeloid leukemia (AML) is genetically poorly defined and several different scenarios might account for this phenomenon. Previously, RUNX1 mutations (RUNX1+) but also cytogenetic aberrations and mutations of the RAS signal transduction pathway have been implicated in this process. Therefore, we analyzed a cohort of MDS (n = 179) and s-AML following MDS (n = 93). The MDS cohort consisted of RARS (n=2), RCMD (n=15), 5q- syndrome (n=2), CMML-1/-2 (n=13), RAEB-1 (n=48), RAEB-2 (n=58) and MDS not further classified (n=41). The entire coding region was screened for RUNX1+ by denaturing high performance liquid chromatography (DHPLC) and mutations were called by direct DNA sequencing. Furthermore, FLT3 was screened for length mutations (FLT3- LM, FLT3-ITD), NRAS for mutations in codons 12/13 and 61, MLL for partial tandem duplications (MLL-PTD) and NPM1 for exon-12 gene mutations. Cytogenetic analysis was done by chromosome banding and if needed for further clarification with FISH analysis. RUNX1+ were detected in 26 (14.5%) of 179 MDS cases and in 32 (34.4%) of 93 sAML. Thus the incidence of RUNX1+ in the MDS cohort was significantly lower compared to the incidence of RUNX1+ in s-AML following MDS (p

Description: In chromic myeloproliferative diseases (CMPD) CML can be identified by the presence of a BCR-ABL fusion gene. The genetic spectrum of the BCR-ABL negative disorders is diverse. The most frequent aberrations especially in PV, ET, and CIMF is the JAK2V617F mutation. Initially these two aberrations were considered to occur mutually exclusive and define different diseases. However, recently 3 reports documented coincidences of the JAK2V617F and BCR-ABL in individual cases. For further clarification we analyzed a cohort of 2317 cases which were screened for BCR-ABL and JAK2 in parallel due to suspected CMPD. Within this cohort 1249 (53.9%) were BCR-ABL-/JAK2V617F-, 119 (5.1%) were BCR-ABL+/JAK2V617F-, and 945 were BCR-ABL-/JAK2V617F+ (40.8%). Double positivity for BCR-ABL and JAK2V617F was detected in 4 cases (0.17%). Real-time PCR for BCR-ABL expression and the V617F was performed for all available timepoints during follow-up. In 2 cases only one timepoint was available and 2 pts were followed for 1 year and 7 months, respectively. After retrospective analysis of the clinical data different patterns of the mutations were detected: 1 pt: A 56-year old male patient was diagnosed with CML in chronic phase in 9/2004. Imatinib treatment led to a major molecular response, but in 10/2006 a marked thrombocytosis led to the diagnosis of a JAK2V617F positive CMPD. Retrospective real-time quantification revealed that the JAK2V617F stayed at the same level as was detected at diagnosis of CML and during one year of follow up whereas the %BCR-ABL/ABL decreased during treatment with imatinib over three log ranges. This indicated that the two mutations occurred in separate clones. 2 pt: A 45 year old male was diagnosed with CML in chronic phase in 9/2006. After 7 months of imatinib treatment RQ-PCR showed decrease of %BCR-ABL/ABL to 0.225 but JAK2V617F remained at a 30% level. This case resembled the first case and suggests different clones - one with BCR-ABL fusiongene and one with JAK2V617F. 3 pt: A 75-year old female, was diagnosed with BCR-ABL negative CMPD in 11/2000. Cytoreduction by hydroxurea (HU) was successful from 2004–2005, but in May 2006 the disease showed signs of acceleration. Molecular diagnostics showed a JAK2V617F and for the first time a BCR-ABL fusion gene, both with expression levels typical for untreated cases. Imatinib was not tolerated, and HU was started again. Over 7 months %BCR-ABL/ABL and %JAK2V617F/ABL remained at the same high level suggesting that one clone harbored both mutations. 4 pt: A 53 years old female was diagnosed with CMPD. 21 years later in 6/2005 molecular diagnostics was performed upon increasing thrombocytosis and showed BCR-ABL positivity and a homozygous JAK2V617F. Both mutations had high expression levels which persisted after 12 months of imatinib treatment. As in the third case the homozygous JAK2 mutational status and the high BCR-ABL expression suggested coexistence of both mutations in the same clone and resistance to imatinib. In conclusion, these four new cases support the idea that BCR-ABL and JAK2 mutations can occur in same patients in the same clone or in different clones and at different time points. It may be speculated that in these cases there is an unknown common antecedent defect.

Description: Dysplastic features can be detected in different cell lineages in myelodysplastic syndromes (MDS) by multiparameter flow cytometry (MFC). The aim of the present study has been the assessment of the flow cytometric detection of dysplastic features previously published to occur in MDS in relation to findings in cytomorphology (CM) and cytogenetics (CG). We analyzed 307 bone marrow samples from patients with suspected (n=130) or proven (n=177) MDS by MFC, CM, and CG in parallel. Blast counts as determined by CM and MFC, respectively, ranged from 0% to 21% (median, 3.5%) and from 0% to 23% (median, 3%; r=0.271, p