ALBERT

Description: Immunoglobulin variable heavy chain gene (VH) mutation status and VDJ rearrangement structure were analyzed in 141 patients with mantle cell lymphoma (MCL) and correlated with biologic and clinical characteristics; 29% of the MCLs displayed mutated VH using a 98% germline homology cutoff. Striking differences occurred in the VH mutation subgroups with respect to the use of specific V genes. Rearrangements involving V4-34 and V3-21 were almost exclusively unmutated, whereas rearrangements using V4-59 and V3-23 were typically mutated. Significant association occurred between mutated VH with shorter CDR3 lengths and the use of JH4b. V3-21 and V4-59 were involved in highly characteristic rearrangements, implying that antigen specificity might have been involved in MCL development. There was no evidence for isotype switch recombination or Bcl-6 expression in any MCL. ZAP70 expression was not different in VH-mutated or -unmutated MCL. Although the deletions 11q– and 17p– showed a balanced distribution, an overrepresentation was observed for trisomies +3q, +8q, and tetraploidy in the VH-unmutated subgroup and +12q in the VH-mutated subgroup. Clinically, mutated VH was associated with a higher rate of complete remission, but there was no correlation between VH mutation status and other clinical characteristics or overall survival.

Description: Clonal evolution in response to therapy is a central feature of disease relapse. This raises a fundamental question in cancer biology: what enables the relapse clone to replace the pre-treatment clone? In other words, is the increased fitness of the relapse clone due to a lower death rate during therapy (less sensitivity to therapy) or a higher growth rate following therapy (superior ability to compete during repopulation)? We sought to address this question in chronic lymphocytic leukemia (CLL), as its relatively indolent disease kinetics enable the study of serially collected samples from the same patient over time. We recently reported the genetic characterization of 278 samples from patients enrolled in the German CLL Study Group CLL8 trial (Nature, in press). These samples were collected prior to first therapy with FC or FCR, and studied using whole-exome sequencing (WES). From this cohort, we further analyzed by WES 59 patients (FC [n = 28] or FCR [n = 31]) at time of relapse. We found that clonal evolution is the rule rather than the exception (57 / 59 CLLs), with TP53 alterations found in relapse in 15 cases. This series constitutes a unique opportunity to dissect the clonal dynamics of treated CLL. We therefore quantified clone-specific death and growth rates by targeted deep sequencing of serial peripheral blood samples, beginning at pre-treatment and ending at relapse. Given the expected minimal mutation detection sensitivity (0.1-1%) by targeted deep sequencing, we only selected samples with 〉1% CLL cells by flow cytometry. Such samples were available for 23 of 59 patients, with a median of 6 samples/patient (range 3-10). Based on the mutations identified by WES in the pre-treatment and relapse samples, we designed patient-specific multiplexed assays for targeted deep sequencing (median sequencing depth - 6561). A series of normal samples were sequenced together with patient samples to account for sequencing errors. The measurements of the CLL cell fraction in the sample, by sequencing and by flow cytometry, were highly correlated (r=0.89, p

Description: Abstract 1586 Poster Board I-612 Background Mutations in the nucleophosmin 1 (NPM1) gene represent the most frequent gene mutations in acute myeloid leukemia (AML), with highest frequency (50-60%) in cytogenetically normal (CN)-AML. Several studies have shown the applicability and prognostic value of an NPM1 mutation (NPM1mut)-based assay for detection of minimal residual disease (MRD). So far, there are no studies evaluating the prognostic value of NPM1mut MRD levels in a large controlled cohort of AML patients (pts) enrolled on prospective clinical trials. Aims To evaluate the prognostic value of NPM1mut MRD levels in younger (16 to 60 years) AML pts harbouring NPM1 mutations type A, B or D, and to assess the influence of concurrent FLT3 internal tandem duplications (ITD). Methods All pts were enrolled in the prospective AMLSG 07-04 and AML HD98A treatment trials. Treatment comprised double induction therapy with ICE (idarubicin, cytarabine, etoposide) followed by high-dose cytarabine-based consolidation, autologous or allogeneic stem cell transplantation. Levels of NPM1mut expression ratios, defined as NPM1mut copies per 104ABL copies, were determined by RQ-PCR using TaqMan technology. Dilution series showed a maximum sensitivity of 10-6 and high specificity as no wildtype NPM1 could be detected. Results A total of 1079 samples, [bone marrow (BM), n=1062; peripheral blood, n= 17) from 212 pts were analyzed at diagnosis, after each treatment cycle, during follow-up and at relapse (median number of samples per pt, n=4; range, 1-16). NPM1mut expression ratios at diagnosis varied between 1.1×104 and 10.4×106 (median, 6.9×105). Pretreatment transcript levels were not associated with clinical characteristics (e.g., age, white cell counts, BM blasts) and did not impact on relapse-free (RFS) and overall survival (OS). Following the first induction cycle, the median decrease of the MRD level ratio normalized to pretreatment levels was 4.21×10-3, independent of the presence of concurrent FLT3-ITD (p=0.39). After the 2nd induction cycle, the median reduction of MRD levels was significantly stronger in the FLT3-ITDneg group (6.75×10-5) compared with the FLT3-ITDpos group (4.19×10-4) (p=0.003) and this differential effect was observed throughout consolidation therapy. For evaluation of the prognostic impact of NPM1mut MRD levels, we compared patients achieving PCR-negativity with those with positive values at different checkpoints. The first reliable checkpoint was after double-induction therapy: the cumulative incidence of relapse (CIR) at 4 years of PCR-negative patients (n=27) was 0% compared with 48% (SE, 4.4%, p

Description: Abstract 2387 AML1-ETO (AE) is the most frequent fusion gene in human AML. Previously, we and others have demonstrated that the fusion is not able to cause leukemia on its own in experimental murine models, but that it needs collaborative partners. Following the ‘two-hit model’ hypothesis that class I and class II mutations have to collaborate to induce leukemia, we could previously demonstrate that AE (class II) collaborates with FLT3-length mutation (class I) to generate AML in the murine bone marrow transplantation model (BMT model). We now demonstrate that AE can collaborate with mutations of its own class in inducing AML. We focussed our analyses on the TALE homeobox gene family, namely Meis1 and Meis2, as already known in the case of Meis1 to be a potent co-factor for Hox gene associated leukemias and being classified as other homeobox genes as a class II mutation. First, real-time RT - PCR confirmed that MEIS1 is expressed at high levels in a subgroup of AE positive AML patients. Furthermore, MEIS2 was highly and aberrantly expressed virtually in all AE patients (n=70) compared to normal bone marrow. Expression patterns of both MEIS genes correlated with their promoter methylation in the t(8;21) patients and cell lines. To test the functional relevance of MEIS expression in human AE patients, we analysed collaboration of AE with Meis genes in the BMT model: Meis1 and Meis2 were retrovirally expressed alone or in combination with AE in 5-FU treated primary murine bone marrow and injected into lethally irradiated recipients (AML1-ETO alone, n=10; EGFP control, n=7; AE+Meis1, n=14; AE+Meis2, n=4). None of the mice in the AE as well as in the control group developed disease. In contrast, mice transplanted with BM co-expressing AE+Meis1 and AE+Meis2 developed lethal disease after a median latency of 102 and 255 days respectively. AE+Meis1 induced MPS in three, AML in seven and ALL in three cases, whereas AE+Meis2 induced AML in all cases. Functional relevance of MEIS expression was further confirmed in human AML cell lines as shRNA mediated depletion of MEIS1 as well as MEIS2 impaired cell growth in AE positive AML cell lines and so far one primary AE AML sample. To understand the mechanisms of AE/MEIS collaboration co-immunoprecipitation assays were performed documenting weak binding of Meis1, but strong interaction of Meis2 with AE. ChIP-Seq is currently being performed to test whether DNA binding of MEIS genes alter in collaboration with AE. Taken together, we could demonstrate that AE collaborates with Meis genes in AML. It furthermore shows that a class II mutation can be converted into an overt oncogene by a mutation of its own class. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1275 Poster Board I-297 Core binding factor (CBF) leukemias, characterized by translocations t(8;21) or inv(16)/t(16;16) targeting the core binding factor, constitute acute myeloid leukemia (AML) subgroups with favorable prognosis. However, 40-50% of patients relapse, and the current classification system does not fully reflect the heterogeneity existing within the cytogenetic subgroups. Therefore, illuminating the biological mechanisms underlying these differences is important for an optimization of therapy. Previously, gene expression profiling (GEP) revealed two distinct CBF leukemia subgroups displaying significant outcome differences (Bullinger et al., Blood 2007). In order to further characterize these GEP defined CBF subgroups, we again used gene expression profiles to identify cell line models similar to the respective CBF cohorts. Treatment of these cell lines with cytarabine (araC) revealed a differential response to the drug as expected based on the expression patterns reflecting the CBF subgroups. In accordance, the cell lines resembling the inferior outcome CBF cohort (ME-1, MONO-MAC-1, OCI-AML2) were less sensitive to araC than those modeling the good prognostic subgroup (Kasumi-1, HEL, MV4-11). A previous gene set enrichment analysis had identified the pathways Caspase cascade in apoptosis and Role of mitochondria in apoptotic signaling among the most significant differentially regulated BioCarta pathways distinguishing the two CBF leukemia subgroups. Thus, we concluded that those pathways might be interesting targets for specific intervention, as deregulated apoptosis underlying the distinct subgroups should also result in a subgroup specific sensitivity to apoptotic stimuli. Therefore, we treated our model cell lines with the Smac mimetic BV6, which antagonizes inhibitor of apoptosis (IAP) proteins that are differentially expressed among our CBF cohorts. In general, sensitivity to BV6 treatment was higher in the cell lines corresponding to the subgroup with good outcome. Time-course experiments with the CBF leukemia cell line Kasumi-1 suggested a role for caspases in this response. Interestingly, combination treatment of araC and BV6 in Kasumi-1 showed a synergistic effect of these drugs, with the underlying mechanisms being currently further investigated. Based on the promising sensitivity to BV6 treatment in some cell lines, we next treated mononuclear cells (mostly leukemic blasts) derived from newly diagnosed AML patients with BV6 in vitro to evaluate BV6 potency in primary leukemia samples. Interestingly, in vitro BV6 treatment also discriminated AML cases into two distinct populations. Most patient samples were sensitive to BV6 monotherapy, but about one-third of cases were resistant even at higher BV6 dosage. GEP of BV6 sensitive patients (at 24h following either BV6 or DMSO treatment) provided insights into BV6-induced pathway alterations in the primary AML patient samples, which included apoptosis-related pathways. In contrast to the BV6 sensitive patients, GEP analyses of BV6 resistant cases revealed no differential regulation of apoptosis-related pathways in this cohort. These results provide evidence that targeting deregulated apoptosis pathways by Smac mimetics might represent a promising new therapeutic approach in AML and that GEP might be used to predict response to therapy, thereby enabling novel individual risk-adapted therapeutic approaches. Disclosures Vucic: Genentech, Inc.: Employment. Deshayes:Genentech, Inc.: Employment.

Description: Abstract 205 Introduction: Bendamustine has shown considerable activity in monotherapy for lymphoid malignancies including chronic lymphocytic leukemia (CLL). In vitro studies have demonstrated synergistic pro-apoptotic effects of bendamustine and the CD20 antibody rituximab (BR) in primary CLL cells. Encouraging results have also been obtained using the BR combination treatment in previously treated CLL. This multicenter phase II trial (CLL2M) is the first study assessing the efficacy and toxicity of bendamustine in combination with rituximab in previously untreated CLL patients (pts). Patients and Methods: Between March 2007 and September 2008 117 pts with untreated CLL requiring therapy were enrolled into the protocol. Bendamustine was given at a dose of 90 mg/m2 on day 1 and 2, combined with 375 mg/m2 rituximab for the first cycle and 500 mg/m2 for subsequent cycles. BR treatment was administered every 28 days for up to 6 courses. Blood samples were taken for analysis by fluorescence in situ hybridization (FISH), the IgVH mutational status and expression of ZAP70/CD38. Minimal residual disease (MRD) was evaluated in peripheral blood and bone marrow by 4-color flow cytometry. Results: Data on the entire study population of 117 pts (median age 64 years) with a total of 583 treatment cycles are available. As of June 2009 the median observation time was 15.4 months (mo). 11.1% of the pts presented with stage Binet A, 41.0% with Binet B and 47.9% with Binet C disease. A mean number of 5.0 courses were delivered. 114 pts were evaluable for toxicity, 110 for response and 113 for progression free survival (PFS). The most frequent adverse events based on 583 treatment cycles were myelosuppression and infections: grade 3/4 anemia occurred in 4.9%, grade 3/4 leukopenia in 14.6%, grade 3/4 neutropenia and thrombocytopenia in 6.5% and 6.1% of all given courses, respectively. 29 episodes of CTC grade 〉3 infections were documented (5.1% of all courses). Treatment related mortality occurred in 2.6% of the pts: one liver failure after attempt of suicide with antihistamines, one fatal pneumonia and one sepsis in neutropenia. The overall response rate was 90.9% with 32.7% (36 pts) clinical complete remissions (CR). A nodular partial remission (nPR) was achieved in 2.7% (3 pts) and a partial response (PR) in 55.5% of the pts (61 pts), respectively. 9.1% of the pts (10 pts) had stable disease (SD) whereas none of the pts was progressive (PD). After 18 mo 75.8% of the pts were still in remission, median PFS has not been reached. An MRD level below 10E-4 was observed after completion of therapy in 29 of 50 evaluable pts in peripheral blood, while 7 of 25 pts achieved MRD negativity in bone marrow. Differences in response were observed among the genetic subgroups: 19 of 21 pts with 11q- achieved a remission with 10 PR and 9 CR (ORR: 90.5%). Accordingly, 17 of 19 patients with +12 responded (14 PR, 3 CR, ORR: 89.5%). In the high-risk group with 17p-, 3 of 7 pts showed a partial response (ORR: 42.9%). 56 of 63 pts (ORR: 88.9%) with unmutated IgVH status responded to BR. Conclusion: Bendamustine plus rituximab (BR) is effective and safe in the first-line treatment of CLL. Major side effects (myelosuppression and infections) were infrequent. Based on these encouraging phase II data the German CLL Study group is presently investigating the efficacy of BR in comparison to fludarabine-based immunochemotherapy (FCR) in the first-line treatment of CLL within a randomized phase III trial (CLL10 protocol). Disclosures: Fischer: Roche: travel expenses. Cramer:Roche: travel grants. Stilgenbauer:Bayer: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding. Fink:Roche: . Boettcher:Roche: Research Funding. Ritgen:Roche: Research Funding. Kneba:Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Döhner:Roche: Research Funding. Eichhorst:Roche: Honoraria, Research Funding; Mundipharma: Research Funding; Hospira: Honoraria. Hallek:Roche: Consultancy, Honoraria, Research Funding. Wendtner:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer Schering: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

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: The CLL4 trial evaluated 1st line treatment with F or FC among 375 CLL patients up to 65 years (Eichhorst et al., Blood, 2006). Updated follow-up (median 52.8 months (mo) for alive patients) revealed 50.4% events for progression-free survival (PFS) and 24.5% for overall survival (OS). Comparing the treatment arms, significant differences were observed in favor of FC for CR (19.9% vs 5.9%, p7% in 47.9%. β2-MG and TK (both n=261) were 〉5 mg/L in 13.8% and 〉 10U/L in 73.0%, respectively. The distributions of these biologic markers and the major clinical characteristics (age: median 59 (42–65) years, sex: 73.1% male, stage: Binet A 9.2%, B 55.4%, C 35.4%, LDH 〉 250U/L: 39.2%, lymphocytes 〉 50/nl: 57.1%, comorbidities 〉1: 21.4%, and creatinine clearance were not significantly different between treatment arms. Outcome was analyzed for subgroups defined by the clinical and biologic parameters in univariate analyses for the entire population (i.e. both treatment arms combined). PFS was significantly shorter for 11q-, 17p- (Fig.1), TP53 mutation, elevated TK and LDH. OS was significantly shorter for the same parameters and unmutated VH, elevated β2-MG, and 〉1 comorbidities. All except 2 patients with 17p- had TP53 mutations. There were 13 additional cases with TP53 mutation without 17p-. OS was similarly poor for TP53 mutation without 17p- as compared to 17p- and all other cases (OS at 48 mo: 25.9%, 21.4%, and 84.6%, respectively, p7% (p=.011), and β2MG

Description: Conventional chromosome analysis (CCA) and interphase fluorescence in situ hybridization (FISH) was performed in 42 patients with mantle-cell lymphoma (MCL), with BCL1 rearrangement. The t(11;14)(q13;q32) or 11q abnormalities were detected by CCA in 34 cases, 20 of which had additional aberrations. A normal karyotype was observed in 8 cases. Probes detecting the chromosome aberrations that were observed in at least 3 cases by CCA, ie, +12, 13q14 deletion, and 17p deletion, were used for interphase FISH analysis. FISH detected total or partial +12, 13q14 deletion and 17p- in 28.5%, 52.4%, and 26% of the cases, respectively. The presence of these anomalies was not a function of karyotype complexity. Based on the results of CCA/FISH, three groups of increasing karyotype complexity were recognized: group 1, including 11 patients without detectable aberrations in addition to BCL1 rearrangement; group 2, including 14 patients with 1 to 2 additional anomalies; and group 3, including 17 patients with three or more additional anomalies. Clinical parameters associated with shorter survival were male sex (P= .006) and primary lymph-node involvement compared with primary bone marrow involvement (P = .015). Trisomy 12 was the only single cytogenetic parameter predictive of a poor prognosis (P = .006) and the best prognostic indicator was the derived measure of karyotype complexity (P 〈 .0001), which maintained statistical significance in multivariate analysis (P〈 .0001). We arrived at the following conclusions: 13q14 deletion occurs at a high incidence in MCL; 17p deletion and total/partial +12 are relatively frequent events in MCL, the latter aberration being associated with a shorter survival; and the degree of karyotype complexity has a strong impact on prognosis in this neoplasia.