Pretreatment Cytogenetic Abnormalities Are Predictive of Induction Success, Cumulative Incidence of Relapse and Overall Survival in Patients >60 Years of Age with Newly Diagnosed Acute Myeloid Leukemia.

Abstract Purpose: Karyotype at diagnosis provides the most important prognostic information in younger adults with acute myeloid leukemia (AML). However, there are few data available looking in particular at patients (pts.) above 60 years of age. We prospectively analyzed 361 elderly pts. with newly diagnosed AML. All pts. were treated within the AMLHD98B treatment trial and received intensive induction and consolidation therapy. Pts. exhibiting a t(15;17) received an age-adjusted AIDA-regimen. Median follow-up time was 48 months. The median age was 67 years (range 60–85 years). Results: 160 pts. had a normal karyotype (44%); 48 pts. (13%) exhibited the balanced translocations t(8;21) (n=12), inv(16) (n=14), t(15;17) (n=11), or t(11q23) (n=11); in the absence of these balanced translocations, 73 pts. exhibited a single aberration, 179 pts. two aberrations, and 61 pts. a complex karyotype (≥3 aberrations; including 44 pts. with 5 or more aberrations). Analyses were normalized to the complete remission (CR) rate (52%), cumulative incidence of relapse (CIR) (77%) and overall survival (OS) (13%) after 4 years of pts. with normal karyotype. Pts. exhibiting a t(15;17) showed a significantly better CIR (29%) and OS (55%), whereas pts. with the other balanced translocations [t(8;21), inv(16)/t(16;16) and t(11q23)] did not differ from pts. with normal karyotype. The limited backward selected Cox-model for OS [t(15;17) excluded] revealed two risk groups: standard-risk [normal karyotype, t(8;21), inv(16), t(11q23), +8 and +11 in absence of a complex karyoytpe] and high-risk [all other aberrations]. The CR rates were 56% and 18%, and the OS-rates after 4 years for the standard- (n=223) and the high-risk group (n=127) were 15% and 0%, respectively. The MRC risk classification system for patients &amp;gt;55 years applied to our patients revealed CR- and OS-rates after 4 years of 73% and 19%, 47% and 12%, as well as 7% and 0% for the low (n=26), intermediate (n=282) and high risk groups (n=44), respectively [t(15;17) excluded]. In conclusion, our risk classification system identified a large high-risk group (36%) of elderly patients with AML who did not benefit from intensive chemotherapy.</jats:p

Integrative Analysis of NPM1 Mutation-Associated MicroRNA and Gene Expression Signatures Identifies Potential Leukemia-Relevant Genes in Acute Myeloid Leukemia.

Abstract Abstract 363 MicroRNAs (miRs) have been shown to control a wide range of biological functions such as differentiation, proliferation and apoptosis, either by translational repression, mRNA cleavage or miR mediated decay of the respective target mRNA. Deregulated miR expression has been associated with various human cancers, including acute myeloid leukemia (AML), a disease characterized by the accumulation of acquired genetic alterations in hematopoietic progenitor cells that lead to altered self-renewal, proliferation and differentiation. Mutations of the nucleophosmin (NPM1) gene could be identified as the most common genetic alteration in AML, mainly occurring in cytogenetically normal karyotype (CN-AML) cases. Furthermore, while NPM1 mutated cases show a favorable prognosis (in the absence of FLT3-ITD) and have been shown to possess a distinct gene expression profile (GEP), so far the biology underlying this aberration has still not been fully understood. In previous work, we profiled the miR expression in a cohort of 91 AML cases comprising all major cytogenetic and molecular genetic subgroups. Significance Analysis of Microarrays (SAM) revealed a distinct miR-signature associated with NPM1 mutation (NPM1mut) in CN-AML as also shown by other groups: 66 miRs were differentially expressed in NPM1mut compared to NPM1 wild-type (NPM1wt) cases. The vast majority of these miRs was strongly upregulated in NPM1mut CN-AML, whereas only few miRs were downregulated compared to NPM1wt cases. Therefore, overexpression of a distinct set of miRs seems to be an important characteristic of NPM1mut CN-AML, and the resulting deregulated expression of target genes of these NPM1mut signature miRs might contribute to leukemogenesis. To identify putative target genes of NPM1mut-associated miRs, we performed an integrative analysis of miR-expression and NPM1mut-related gene expression data in our cohort. First, we generated target gene lists for the core 33 overexpressed miRs of the NPM1mut signature by using the miRGator database. This resulted in a theoretical NPM1mut associated GEP. Then, a comparison of the theoretical with the measured NPM1mut GEP was performed in order to find putative targets whose mRNA levels are directly affected by the respective miRs. This approach revealed several promising candidate genes with known implication in tumorigenesis and/or leukemogenesis like APP, CCND1, IRF2, BCL2L1, MLL and KIT. Interestingly, these genes are putative targets of not only one, but several miRs (4 to 15) of the NPM1mut signature, thereby pointing towards a synergistic effect of these miRs. Validation of individual miR-target gene relations was carried out by qRT-PCR in cell lines transfected with the respective miR mimics, supplemented by Western Blot and 3'UTR-luciferase-reporter assays. This validation was successful, not only for already known miR-target gene connections, but also for novel candidates including e.g. CCND1, a cell cycle regulator, and interferon regulatory factor-2 (IRF2). IRF2 is known to show dysregulated expression in the majority of AML cases and has recently been described to be essential for preserving the self-renewal and multilineage differentiation capacity of hematopoietic stem cells (Sato et al., Nat Med 2009). Thus, our approach of combining miR expression information and GEP in NPM1mut CN-AML led to the identification of promising target genes with potential implication in leukemogenesis. Additional functional analyses of relevant miRs and target genes are currently in progress to further illuminate the mechanism of NPM1mut AML pathogenesis. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Chronic Myeloid Leukemia (CML) Cells Express Tumor Associated Antigens Eliciting Specific CD8+ T Cell Responses Despite of Deficient Expression of Costimulatory Molecules.

Abstract Specific immunotherapies for CML patients targeting T cell antigens might eliminate residual CML cells after therapy with imatinib or chemotherapy and might enhance a specific graft versus leukemia effect after allogeneic stem cell transplantation without aggravating the graft versus host disease. However, for effective specific immunotherapies in CML, extended studies on the expression, the function and the immunology of leukemia-associated antigens (LAAs) and on the LAA presentation by leukemia cells are required. Here, we investigated on expression and functional aspects of tumor/leukemia-associated antigens (TAAs/LAAs) in CML. Several LAAs are expressed and are therefore candidate structures for specific immunotherapies: bcr-abl (100%), G250 (24%), hTERT (53%), MPP11 (91%), NEWREN60 (94%), PRAME (62%), Proteinase3 (71%), RHAMM/CD168 (83%) and WT1 (53%), but not BAGE, MAGE-A1, SSX2 or NY-ESO-1. The expression of LAAs varied according to leukocyte subsets and the expression of RHAMM/CD168, Proteinase3 and PRAME was upregulated in accelerated phase and blast crisis. Quantitative expression of several TAAs/LAAs correlated with the clinical course. In flow cytometry, CD34+ CML progenitor cells typed positive for HLA-molecules, but were deficient for CD40, CD80, CD83 and CD86. This lack of costimulatory molecules might constitute a tumor escape mechanism. However, RHAMM/CD168-R3-specific T cell responses were demonstrated by ELISPOT analysis and specific lysis of R3-peptide pulsed T2 cells in chromium-51 release assays. These CD8+ cells could be phenotyped as CCR7-CD27-CD45RA+ early effector T cells by tetramer staining. In conclusion, several LAAs are expressed in CML and are therefore candidate structures for specific immunotherapies. CML progenitor cells express HLA-molecules, but lack costimulatory molecules. However, as CML patients show RHAMM/CD168-R3-specific early effector T cells, peptide vaccination might be therefore a promising approach to enhance specific immune responses in CML.</jats:p

Survival Prediction Acute Myeloid Leukemia (AML) Using a Combination of DNA Methylation Analysis and Gene Expression Profiling.

Abstract Acute myeloid leukemia is classified by the presence or absence of recurrent cytogenetic aberrations. In order to improve diagnosis and therapy, more recently new studies have been performed to supplement the current classification with refined molecular information based on gene expression profiling. However, it has been established that expression levels of genes are often largely controlled by the state of cytosine methylation in the adjacent promoter region. Thus we were interested to evaluate the quantitative methylation levels for a previously identified predictive set of genes (Bullinger et al. 2004) using a novel technology based on a unique combination of base specific cleavage of single stranded nucleic acids with MALDI TOF detection. We have employed this new quantitative high throughput DNA methylation analysis technology to analyze 147 promoter regions in a total of 192 individuals. The resulting quantitative methylation data was analyzed using a semi-supervised approach to evaluate the quantitative methylation data as a predictor for patient survival. We used a first set of 96 individuals to train a statistical learning algorithm and a second set of 96 samples to validate the trained algorithm. The analysis revealed quantitative methylation patterns as a reliable predictor for survival (p &amp;lt; 0.001). Subsequently, we combined the methylation based predictive model with the results from the expression based predictor. The combination of both models yielded a superior predictive model for patient survival, which outperformed all clinical and cytogenetic risk stratification in the given sample set. The results of this work revealed a potential significance of DNA methylation in the pathophysiology of AML and suggest that DNA-methylation patterns might be useful molecular markers for patient survival prediction based on the fact that large-scale DNA methylation studies can now be performed with reasonable efforts in a limited amount of time. Therefore, these results lay the groundwork for future research which might ultimately enable individualized therapy based on improved molecular characterization of AML.</jats:p

Gains on Chromosome Arm 9q Represent a Novel and Independent Marker of Adverse Prognosis in Multiple Myeloma Patients Receiving Upfront High-Dose Chemotherapy and Autologous Stem Cell Transplantation.

Abstract Introduction: Genomic aberrations represent important prognostic markers in many hematological cancers. In multiple myeloma (MM), chromosome 13q and 17p deletions (13q−, 17p−) have emerged as important outcome predictors that indicate a dismal prognosis. Other chromosomal abnormalities have been discussed as prognostic markers in this disease but came not out as independent variables when they were tested in a multivariable fashion. However, the complexity of genomic rearrangements and the clinical heterogeneity seen in malignant plasma cell disorders argue against 13q− and 17p− as the sole genomic change of prognostic relevance. The significance of chromosome arm 1q, 9q, and 11q extra copies - three frequent genomic imbalances in MM - is undetermined. Material and Methods: 90 patients (pts.) treated with one or two cycles of high-dose chemotherapy (HD-CTX) followed by autologous stem cell transplantation (ASCT) at a single center were analyzed by tri-color FISH and five DNA probes mapping to chromosome bands 1q21, 9q34, 11q25, 13q14, and 17p13. A multivariable analysis (Cox proportional hazards regression model) including genetic and clinical variables was performed. Results: The most frequent aberrations in the present series were (in order of decreasing prevalence): +1q (n=39/78, 50.0%), +9q (n=38/78, 48.7%), 13q− (n=42/90, 46.6%), and +11q (n=39/85, 45.9%). 17p− was identified in only 3 out of 88 patients (3.4%), while +17p were present in 13 out of 88 patients (14.8%). The median follow-up time was 37 months (m) and the median event free survival (EFS) and overall survival (OS) time from first ASCT of the entire cohort was 26 m and 71 m, respectively. Multivariable analysis including genetic aberrations, ß2-microblobulin, albumin, LDH, Salmon/Durie stage, and age at time of diagnosis revealed +9q and 13q− as the only independent predictors of EFS (p=0.003 and p=0.01, respectively). The mEFS in patients with 13q− was 19.0 m and 20.7 m in patients with +9q. In patients with concurrent +9q and 13q−, mEFS was only 12.2 m. In patients lacking these two abnormalities, mEFS was not reached. OS was not significantly influenced by any genetic or clinical variable in our series, most likely due to effective salvage treatment after relapse. Conclusions: +9q represents a novel and independent marker of adverse prognosis in MM. A single FISH experiment applying two DNA probes allows easy and rapid assessment of outcome in patients with malignant plasma cell disorders.</jats:p

Impact of Pegfilgrastim and Dosing Schedule of Cytarabine on Hematological Reconstitution Times, Incidences of Severe Infection and Duration of Hospitalization after Consolidation Therapy with High-Dose Cytarabine in Acute Myeloid Leukaemia - Interim Results from AMLSG 07-04 Trial.

Abstract Introduction: Therapy using high-dose cytarabine (HiDAC) according to the CALGB scheme (3g/m2 bid. days 1,3,5) is recognized as a standard consolidation treatment for younger adult patients (pts) with AML. Pegfilgrastim (PF) has been shown to be effective in reducing the duration of neutropenia in treatment of solid tumors and it seems to be even more effective than Filgrastim in reducing the incidence of infection. Methods: The AMLSG 07-04 trial (NCT00151242) was initiated in September 2004 (age 18–60 yrs). Consolidation therapy for cytogenetic favorable- and intermediate-risk groups consists of 3 cycles of HiDAC (3g/m2 bid. days 1,3,5) with PF 6mg given at day 10 (1-3-5 schedule) or after amendment no. 2 of HiDAC (3g/m2 bid. days 1,2,3) with PF 6mg given at day 8 (1-2-3 schedule). As a control group, pts randomized from AMLSG into the German AML Intergroup protocol using the standard 1-3-5 schedule for consolidation therapy with allowed interventional application of G-CSF were used. Results: Data from 251 pts and a total of 584 cycles are available (137 pts and 324 cycles, 1-3-5 schedule; 78 pts and 185 cycles, 1-2-3 schedule; 36 pts and 75 cycles, German AML Intergroup standard arm). Data from all three consolidation cycles were pooled for the comparison between the AMLSG 07-04 1-3-5 schedule and the German AML Intergroup standard arm. The duration of leukopenia (LP) and neutropenia (NP) were significantly shorter in pts receiving PF within the AMLSG 07-04 trial compared to pts within the German AML Intergroup standard arm (intention-to-treat, p=0.08 and p=0.03; as-treated, p=0.01 and 0.008, respectively). This beneficial effect of PF on LP and NP increased with the number of cycles. This was paralleled by a lower incidence of infection ≥CTC grade 3 with 40% in the 1-3-5 schedule and 67% in the German AML Intergroup standard arm (p&amp;lt;0.0001). The comparison of the 1-2-3 schedule with the 1-3-5 schedule within the AMLSG 07-04 protocol revealed significantly shorter LP and NP in favor for the 1-2-3 schedule (p=0.03 and p=0.004, respectively). In median, pts after the 1-3-5 and the 1-2-3 schedule achieved a leukocyte count above 1.0/μl and a neutrophil count above 0.5/μl at day 20 and day 22 as well as 16 and 17, respectively. In a single center experience using out patient platelet and red blood cell support, the median time of hospitalization for the 1-3-5 (n=32 cycles) and the 1-2-3 (n=25) schedule could be reduced to 7.5 and 5 days with incidences for readmission of 33% and 12.5%, respectively. Conclusion: The administration of PF after HiDAC-based consolidation therapy in AML significantly shortened the duration of leuko- and neutropenia, reduced the rate of severe infections, and reduced the period of hospitalization.</jats:p

CEBPA Germline Mutation Screening in Cytogenetically Normal Acute Myeloid Leukemia with Somatically Acquired CEBPA Mutations.

Abstract 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&amp;gt;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.</jats:p

IL4 and CD40L Prevent Apoptosis of Chronic Lymphocytic Leukemia Cells Via Intracellular pSTAT6 and NFkB Signaling and Not Via Receptor Kinetics

Abstract Abstract 3893 Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental input for their extended survival in vivo, but the underlying molecular mechanism is still unclear. Compared to non-malignant B-cells, CLL cells are more responsive to contact dependent complex stimuli like coculture on bone marrow derived stromal cell lines of both human (p&lt;0.0001) and murine origin (p&lt;0.01), but also to soluble factors (human conditioned medium p&lt;0.0001, murine conditioned medium p&lt;0.001, all student′s t-test). In order to understand the intrinsic difference of the anti-apoptotic phenotype of CLL cells, the signalling circuitry of the malignant cells was modelled. Compared to candidate ligands like SDF-1 (at concentrations between 10–1000ng/ml), BAFF (250–1000ng/ml), APRIL (250–1000ng/ml) and soluble anti-IgM (1–25μg/ml), the factors CD40L (10–2000ng/ml) and IL4 (0.1–10ng/ml) were the most efficient ligands in rescuing CLL cells from spontaneous death in vitro. The dose response of IL4 and CD40L displayed different saturation and cooperativity between CLL cells and non-malignant B-cells. Using IL4, saturation was reached both for CLL cells and B-cells at 0.2pM, but at 52% survival (+/− 8%) for CLL cells and 28% (+/−7%) for B-cells, and the estimated dissociation constant Kd was 0.01pM for both ligands. For CD40L, CLL cell survival reached saturation at 40nM, while no saturation was reached for B-cells. Intriguingly, B-cells showed cooperativity in their response to CD40L, with a cooperativity coefficient of 2.0 and a Kd of 70pM, while cooperativity for CD40L was lost in CLL cells (Kd of only 2.6pM). This pointed towards distinct differences in ligand-receptor interactions or in downstream signaling between CLL cells and non-malignant B-cells. However, high-throughput spatial analysis with a microscope-coupled cytometer did not show differences of receptor quantity or receptor distribution between malignant and non-malignant cells. In contrast, quantity and phosphorylation levels of downstream signalling nodes like STAT6 (measured by flow cytometry and validated by Western-blot) and the activity of NF-kB (p65 binding to DNA measured by oligonucleotide-coupled ELISA) were higher in CLL cells compared to B-cells from healthy donors. Therefore, the defect in IL4 and CD40L signalling that leads to an enhanced survival in CLL cells is likely caused by changes in the intracellular circuitry. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Induction Therapy with Idarubicin and Etoposide Combined with Sequential or Concurrent Azacitidine In Patients with High-Risk Acute Myeloid Leukemia: Pilot-Phase of the AMLSG 12-09 Study

Abstract Abstract 2184 Background: Treatment outcome in patients with cytogenetically and/or molecularly defined high-risk acute myeloid leukemia (AML) is dismal with low complete remission (CR) rates after intensive induction therapy and 5-year overall survival of about 25% in patients 60 years and younger and far below 5% in patients above the age of 60 years. In younger patients, allogeneic hematopoietic stem cell transplantation (allo-HSCT) from matched related or unrelated donors results in significantly better clinical outcome especially if patients are transplanted early in first CR (Schlenk et al., J. Clin. Oncol. 2010, in press). Azacitidine is a demethylating agent showing promising results as a single agent in AML patients with bone marrow blast counts between 20 and 30%. Therefore, the randomized AMLSG 12-09 trial will evaluate the combination of idarubicin/etoposide chemotherapy combined with azacitidine instead of cytarabine as compared to induction with idarubicin/etoposide/cytarabine (ICE) in an attempt to increase CR rates in these high-risk patients. Aim: To evaluate feasibility of the investigational induction therapy with idarubicin and etoposide in combination with sequentially or concurrently administered subcutaneous (sc) azacitidine. Methods: Patients were treated according to the investigational treatment schedules of the AMLSG 12-09 protocol. Patients received idarubicin 12 mg/sqm on days 1, 3 and 5 and etoposide 100 mg/sqm on days 1, 2 and 3 (patients above the age of 65 years received idarubicin 12 mg/sqm and etoposide 100 mg/sqm only on days 1 and 3, respectively). Azacitidine 100 mg/sqm sc was added on days -5 to -1 in 7 patients (schedule A), days 1 to 5 in 6 patients (schedule B), and days 4 to 8 in 5 patients (schedule C). Results: 18 patients have been treated (13 males and 5 females). Median age was 62.5 years (range, 28–76). The cytogenetic and molecular risk profile of the 18 AML was as follows: Eight AML had MDS-related cytogenetic changes (WHO 2008) including five exhibiting a complex karyotype and two had 3q abnormalities; three AML had balanced t(v;11q23), and six exhibited a normal karyotype together with triple negative genotype (NPM1-wt, FLT3-wt and CEBPA-wt). In one case, there were no metaphases available, however molecularly NPM1-wt, FLT3-wt, CEBPA-wt, no core binding factor AML, no t(15;17) and or t(9;11) were present. Median WBC was 4.6/nl (range, 0–6-75/nl). Overall response to induction therapy was CR n=7, partial remission (PR) n=3, refractory disease (RD) n=7 and one patient died during induction therapy (ED). Moreover, two patients with RD achieved CR after additional cycles of single agent azacitidine treatment. Overall response rates (CR and PR) according to treatment schedule were 43% (3/7), 67% (4/6) and 80% (4/5) for schedules A, B and C, respectively. Most common azacitidine-related toxicity was local reactions at injection site not exceeding CTC-grade 2. As expected, fever in neutropenia was the most common severe toxicity (83%). In addition, one patient with history of epilepsy had seizures during induction therapy and one patient with history of Crohn‘s disease had mucositis CTC-grade 3. Allo-HSCT has been performed in three patients and is planned in five. After a median time of 7.5 months, 16 of 18 patients are alive. Conclusion: Azacitidine administered sc can be given safely either sequentially or concurrently in combination with idarubicine/etoposide induction chemotherapy. Response rate of this high-risk population appears promising and the toxicity profile was favorable. The question which schedule is the most effective will be addressed in the randomized AMLSG trial (NCT01180322) Disclosures: Stilgenbauer: Amgen: Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Sanofi Aventis: Research Funding. Döhner: Pfizer: Research Funding. Schlenk: Celgene, Pfizer, Novartis, Cephalon, Amgen: Research Funding. </jats:sec