ALBERT

Beschreibung: 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

Beschreibung: 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

Beschreibung: Background: In a previous randomized trial (AML HD98B) of the AMLSG in elderly (〉60 yrs) patients with AML (excluding APL), we could demonstrate that all-trans retinoic acid (ATRA) given as adjunct to intensive chemotherapy significantly improved complete remission (CR) rate and overall survival (OS). Our hypothesis for this study was that this beneficial effect of ATRA may be confined to a specific genotypic subset of AML. Aims: To evaluate the impact of ATRA on clinical outcome in cytogenetic and molecular genetic subsets of AML. Methods: Between 1998 and 2004, a total of 372 patients were enrolled. 242 patients were randomized for ATRA as adjunct to intensive induction (idarubicin, cytarabine, etoposide) and first consolidation therapy (intermediate-dose cytarabine) (Schlenk et al., Leukemia 2004), followed by a second randomization between further intensive consolidation versus a one-year oral maintenance therapy (Schlenk et al., Leukemia 2006). After an interim analysis in 2003, first randomization was stopped; the following 130 patients received chemotherapy without ATRA. Data from conventional cytogenetics and from fluorescence in-situ hybridization were previously reported (Fröhling et al., Blood 2006). All available leukemia specimens were analyzed for mutations in the genes encoding NPM1, CEBPA, and FLT3. Results: Median age of the 372 patients was 67 years (range: 61 to 83 yrs); median follow-up time was 68 months. 67% of the patients had de novo AML, 33% had secondary AML. Incidences of mutations were as follows (no. of specimens analyzed): NPM1 mutation, 25% (n=242); FLT3 internal tandem duplication (ITD), 18% (n=263); FLT3 tyrosine kinase domain mutation, 5% (n=244); CEBPA mutation, 8% (CEBPA analyzed only in normal karyotype samples; n=109). Logistic regression analysis performed on all patients identified three significant variables for achievement of CR: the genotype NPM1mut (OR 2.6; 95%-CI, 1.2–5.5), non-adverse karyotype subsuming core-binding factor and cytogenetically normal AML (OR 2.7; 95%-CI, 1.4–4.9); and age (diff. of 10 years, OR 0.58; 95%-CI, 0.3–1.0). Cox proportional hazard model for OS was performed on patients who were up-front randomized for ATRA. This model revealed a significant interaction between ATRA and the NPM1mut/FLT3-ITDneg genotype, confining the beneficial effect of ATRA to patients in that subgroup (HR, 0.29; 95%-CI, 0.10–0.87). Additional variables were log(LDH) (HR, 2.6; 95%-CI, 1.4 – 4.6), age (HR, 1.6; 95%-CI, 1.2– 2.2) and non-adverse karyotype (HR, 0.7; 95%-CI, 0.5–1.0). Univariable analysis revealed a survival after 5 years in patients with the genotype NPM1mut/FLT3-ITDneg of 57% (95%-CI, 28%–78%) in the ATRA-arm (n=16) compared to only 6% (95%-CI, 0%–25%) in the standard-arm (n=14) (p=0.002). In contrast, there was no difference in survival in all other patients with a survival of 2% (95%-CI, 0%–7%) in both arms (p=0.23). Conclusions: The genotype NPM1mut/FLT3-ITDneg emerges as highly significant predictive factor for response to ATRA in elderly patients with AML. This finding is validated prospectively in the ongoing AMLSG 07–04 study randomizing for ATRA in younger adults (ClinicalTrials.gov Identifier: NCT00151242).

Beschreibung: Background: Patients with primary refractory acute myeloid leukemia (AML) have a dismal outcome. Only allogeneic stem cell transplantion (SCT) currently offers the chance of cure to these patients. In order to improve outcome after allogeneic SCT, one important prerequisite is to increase response rates prior to SCT. Aims: To evaluate the impact of all-trans retinoic acid (ATRA) and gemtuzumab ozogamicin (GO) given as adjunct to high-dose cytarabine-based salvage therapy in younger adult patients with primary refractory AML on achievement of response. Consecutive allogeneic SCT was intended in all patients. Methods: Main inclusion criteria of the AMLSG 05-04 trial (NCT00143975) were refractory AML following one cycle of ICE (idarubicin, cytarabine, etoposide); and age 18 to 60 years. Dose and schedule of the GO-A-HAM regimen were as follows: GO 3mg/m2, day 1; cytarabine 3g/m2 bid., days 1–3; mitoxantrone 12mg/m2, days 2,3; ATRA 45mg/m2, days 3–5, 15mg/m2 days 6–28. Primary endpoint of the study was CR rate. Safety endpoints comprised early / hypoplastic (ED/HD) death rate, liver toxicity CTC grade 3–5, and rate of veno occlusive disease (VOD) after allogeneic SCT. Results: Between September 2004 and June 2007, 94 patients (median age, 48 yrs; range, 22 to 62) were enrolled. Distribution of cytogenetics was as follows: adverse, n=29 [abn(3q), −5/5q-, −7/7q-, abn(12p), abn(17p), complex]; other n=57 [core binding factor (n=3), cytogenetically normal AML (n=37), various aberrations (n=18)]. FLT3-ITD was present in 18 (22%) of 82 analyzed patients. Response to GO-A-HAM was as follows: CR, n=28 (30%); CRi, n=19 (20%); PR, n=11 (12%); refractory disease (RD), n= 34 (36%); and ED/HD, n=2 (2%). In a logistic regression analysis for achievement of CR, the only significant variable was adverse cytogenetics (OR 0.34, p=0.02). The rate of severe liver toxicity was 0%, the incidence of neutropenic fever was 52%, platelet and neutrophil recovery times from start of treatment were 21 and 22 days, respectively. Following GO-A-HAM, allogeneic SCT was actually performed in 60 patients (64%): matched related (n=14) or unrelated donor (n=42); haploidentical related donor, n=4. All SCT were performed within 3 months after GO-A-HAM, intermediate/severe VOD developed in 5 patients after SCT (9%, 95%-confidence interval (CI) 4–19%), mild VOD in 3 patients. Survival analyses revealed that patients with adverse cytogenetics and/or FLT3-ITD (n=45) had a significantly (p=0.001) inferior overall survival after one year of 38% compared to all other patients (n=39) of 81%. The proportions of patients receiving an allogeneic SCT were similar in both groups (68% and 66%, respectively). Conclusions: The GO-A-HAM regimen is feasible and effective as salvage therapy. However, cytogenetics still remains the most significant variable for achievement of response. Allogeneic SCT after GO-A-HAM was not associated with an increased VOD-rate.

Beschreibung: Background: The Wilms’ tumor 1 gene (WT1) encodes a zinc finger protein that functions as a transcriptional regulator. Although its role in haematopoiesis is still not clarified yet, disruption of WT1 function is discussed to promote stem cell proliferation and to induce a block in differentiation, the hallmarks of the two complementation groups of gene mutations that have been postulated for the development of acute myeloid leukemia (AML). In two small studies WT1 mutations were detected in 11% of cytogenetically normal (CN) AML and were associated with a lower complete remission (CR) rate and a higher rate of resistant disease (RD). Aims: To evaluate the incidence and clinical impact of WT1 mutations in the context of NPM1, FLT3-ITD (internal tandem duplication)/TKD (tyrosine kinase domain mutations at codon 835), CEBPA, MLL-PTD (partial tandem duplication), and NRAS mutation status in CN-AML. Methods: Bone marrow or peripheral blood samples from 279 younger adult patients (pts) 16 to 60 years of age with CN-AML (n=242 de novo, n=32 secondary, n=5 therapy-related) were studied. Pts had been entered on three AMLSG treatment trials (AML HD93, AML HD98A, AMLSG 07–04). WT1 gene mutation screening was performed using standard PCR-based direct sequencing of exons 1 to 10; mutation analyses for the other genes were performed as previously described. Results: WT1 mutations were identified in 37/279 (13%) CN-AMLs, predominantly clustering in exon 7 (25/37) and exon 9 (6/37), but also occurring in exons 1, 2, 3, and 8. Most of them were frameshift mutations resulting from insertions or deletions; 33 pts had heterozygous and 4 pts had homozygous mutations. Correlation of WT1 to the FLT3-ITD/TKD, NPM1, CEBPA, MLL-PTD, and NRAS mutation status revealed mutant (mut) WT1 to be significantly associated with FLT3-ITDpos (p=0.003) and CEBPAmut (p=0.02). In a multivariable logistic regression model on induction success, the genotype WT1mut was not significant (p=0.89); the only significant variable was the NPM1mut/FLT3-ITDneg genotype (p=0.003). In univariable analyses for overall (OS), event-free (EFS) and relapse-free (RFS) survival, there was no difference between the WT1wt and the WT1mut group. Multivariable Cox proportional hazard models for OS, EFS and RFS also revealed no significant impact of the genotype WT1mut (p=0.4, p=0.71, and p=0.81, respectively); significant variables were the genotypes NPM1mut/FLT3-ITDneg (p=0.007, p=0.0001, and p=0.01) and CEPBAmut (p=0.01, p=0.0003, and p=0.03); and in addition logarithm of white blood cell count for OS (p=0.02). Conclusion: In our study on a large series of molecularly well characterized CN-AML, WT1 mutations occurred in 13% of the pts and were significantly associated with the genotypes FLT3-ITDpos and CEBPAmut. However, in both univariable and multivariable analyses WT1 mutations did not impact on the prognosis of pts with CN-AML. Additional pts have to be investigated to determine whether WT1 mutation status might have an impact within distinct genotypes.

Beschreibung: Abstract 2175 Eight years from the start of a cooperation among five multicenter study groups in Germany, and three years after entering the last patient we here present updated therapeutic results. A total of 3171 patients 16–60 years of age with AML including primary and secondary AML and high-risk MDS, and excluding APL with t(15;17), were treated in the five study groups cooperating by a general upfront randomization allocating 10% of their patients to a common standard treatment and 90% to the study group own regimens before any treatment had been started. The standard treatment consisted of remission induction by two courses of standard dose araC over seven days with daunorubicin 60mg/m2/d on three days. Patients in complete remission received three monthly courses of high-dose araC 3g/m2q 12 h on day 1,3 and 5. In the participating study groups a total of 2866 patients eligible by the inclusion criteria of the AML Intergroup study were treated according to the study group own regimens with 828 patients in study group A, 373 in B, 235 in C, 808 in D, 622 in E, and 305 patients were treated according to the common standard. In an attempt to conduct treatment optimization trials the study group own strategies relied on a risk-oriented stratification among treatment options (group A), a randomized stratification (group D), or combinations of randomization and risk-adaption (B, C, E). High-dose araC was used up to cumulative 54g/m2 in groups A, E, and in the standard arm, and up to 36 to 40g/m2 in groups B, C, D. Marked differences were also seen in the use of allogeneic stem cell transplantation with 27% allografts in 1st CR in study group A, 33% in B, 19% in C, 23% in D, 25% in group E, and 21% in the common standard arm. Unlike the differences in the study groups strategies in the amounts of high-dose araC, and in the numbers of allografts in 1st remission, the risk profiles regarding age, secondary AML, cytogenetic groups, mutations in NPM1/FLT3-ITD, levels of WBC and LDH were concordant between the common standard arm and the study group own populations, except for single differences in secondary leukemia (group B), cytogenetics (C), and WBC (D). The study group itself was not an independent prognostic factor for any outcome. Similar patient populations could thus be evaluated to compare the treatment strategy of each study group with that of the common standard arm. The outcome of the standard treatment was 66.7% (95% CI 61.0–72.0) complete remissions, an overall survival of 44.3% (95% CI 37.9–50.9), and a relapse free survival of 44.9% (95% CI 36.9–52.5) at 5 years. Adjusted for prognostic baseline variables no significant difference in the remission rate and overall survival between the standard arm and any of the study groups were observed. Comparable patterns were also found for relapse free survival where the long-term results of the standard treatment were not further improved in any of the five study groups. We conclude that in unselected patients with AML a prospective comparison shows very similar outcomes of any current treatment strategy when compared with that of the common standard treatment. Present reliable and unbiased analyses became possible by a strictly prospective approach and intent-to-treat evaluation. These representative results can form a solid basis for novel therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 824 Background: Although acute myeloid leukemia (AML) with inv(16)(p13.1q22) or t(16;16)(p13.1;q22) [hereafter referred to as inv(16)] is considered as a favorable AML subset, 30-40% of the patients (pts) are not cured by the current treatment strategies. Various genetic markers have been assessed to identify pts who are at high risk to fail therapy. However, the results among the different studies were not fully consistent. Methods: We studied diagnostic bone marrow and/or blood specimens from 179 adult AML pts with inv(16) for secondary chromosome abnormalities and gene mutations in FLT3 [internal tandem duplications (ITD) and tyrosine kinase domain mutations (TKD)], KIT, JAK2 (V617F) and in N-and K-RAS. All pts were treated on one of 7 prospective protocols of the German-Austrian AML Study Group (AMLSG) including anthracycline- and cytarabine-based induction therapy, and consolidation therapy incorporating higher doses of cytarabine in various settings, but also autologous and allogeneic stem cell transplantation. Multivariable analyses were performed to assess the prognostic value of gene mutations on relapse-free (RFS) and overall survival (OS) and were stratified for treatment protocols. Results: At least one gene mutation was found in 84% of the pts with 21% of the pts having multiple mutations. Pts with gene mutations ≥2 showed no significant differences in WBC, incidence of trisomy 22 and age. Mutations were most frequent in RAS (53%), followed by mutations in KIT (37%) and FLT3 (17%) [FLT3-TKD (15%) and FLT3-ITD (4%)] genes. No mutations were detected in JAK2. Concurrent mutations of KIT and RAS in the same leukemia sample were less likely to occur (P=0.003) than expected based on their frequencies as single markers; no other significant interactions between the gene mutations were observed. Median age of the cohort was 41 years (yrs; range, 18-74 yrs), and median white blood count (WBC) was 38.3 × 109/l (range, 1.1 to 294.9 × 109/l). Median follow-up for survival according to Korn was 4.9 yrs [95%-confidence interval (CI), 4.3-6.0 yrs]. While one multivariable model was constructed using the mutations in KIT, FLT3 and RAS as covariates (model I), the other included the number of mutated genes (≥2 vs

Beschreibung: Abstract 237 Mutations in the nucleophosmin 1 (NPM1) gene represent one of the most frequent gene mutations in acute myeloid leukemia (AML), in particular in cytogenetically normal (CN)-AML. NPM1 mutations (NPM1mut) are considered as an early genetic event in the pathogenesis of AML. To address the role of clonal evolution from diagnosis to relapse in NPM1mut AML, we applied high-resolution genome-wide single nucleotide polymorphism (SNP) array analysis using the Affymetrix 6.0 platform to detect copy number alterations (CNAs) and uniparental disomies (UPDs) in paired samples from 42 patients. In addition, we determined NPM1 and FLT3 [internal tandem duplication (ITD) and tyrosine kinase domain (TKD)] mutation status in all samples. Blood or bone marrow samples obtained at complete morphologic remission were available for all patients to exclude germline copy number variations. At diagnosis, 29 cases (69%) had a normal karyotype by cytogenetics and no CNAs and UPDs by SNP analysis. In the 13 remaining cases, we found a total of 10 CNAs in 7 cases (19%), and 6 UPDs in 6 cases (14%): deletions of 9q21 (size range 0.9 to 17 Mb) were detected in 5 cases and were the only recurrent CNA; the only recurrent UPD affected the long arm of chromosome 13 in 4 cases, all resulting in homozygous FLT3-ITD mutations with FLT3-ITD/wildtype ratios 〉1; heterozygous FLT3-ITD and –TKD mutations were detected in 9 and 7 patients, respectively. At the time of relapse, the number of CNAs increased (34 CNAs in 16 cases, 38%) while the frequency of UPDs remained unchanged (6 UPDs in 6 cases, 14%). Of note, in 6 patients (14%) the NPM1 mutation was no longer detectable at the time of relapse; SNP analysis showed completely distinct CNAs/UPDs in 4 of these patients; 3 of these 4 cases had a small gain at 11q23 corresponding to MLL partial tandem duplications as confirmed by PCR. These findings suggest that these 4 cases were therapy-related AMLs (t-AML) rather than relapsed AML. The median interval from diagnosis to relapse/tAML in these 4 cases was 65 months compared with 9 months for the relapsed cases still having the NPM1 mutation. In the two remaining cases, genetic alterations were neither present at diagnosis nor at relapse. Analysis of other gene mutations (eg, IDH1 and 2, DNMT3A, ASXL1, p53) is currently under way to further elucidate the clonal origin of these cases. Of the 36 NPM1mut positive relapse samples, 15 maintained a “normal karyotype”, and 2 showed the CNAs already present at diagnosis; 19 relapse samples (53%) displayed clonal evolution with acquiring new (n=15) and/or loosing single aberrations (n=4): Acquired recurrent alterations comprised deletions of tumor suppressor genes [ETV6 (n=2), TP53 (n=2), NF1 (n=2), WT1 (n=2)], most of which are uncommon in de novo NPM1mut AML. All 6 UPDs detected in relapse samples affected 13q, of which 3 were already present at diagnosis. One patient with initial heterozygous FLT3-ITD mutation developed a homozygous state by acquiring UPD13q at relapse. Two cases with wild-type FLT3 at diagnosis acquired UPD13q at relapse. Of note, one UPD13q was not present in the corresponding relapse sample anymore. In conclusion, almost half (45%) of NPM1mut AML showed evolution to a more aberrant karyotype at relapse, including acquisition of high-risk genetic changes that may account for the adverse prognosis of relapsed patients. Conversely, other alterations such as UPD13q or del(9q) detected at diagnosis were not always present in relapse samples, implying that relapse had evolved from a more ancestral clone. In addition, our data suggest that in a proportion of cases t-AML rather than relapse had developed. Further analysis, such as gene mutation studies of paired diagnosis/ relapse samples, will provide more detailed information on clonal evolution events in the pathogenesis of NPM1mut AML. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 412 Background: A large proportion of patients are currently not eligible for genotype-adapted strategies in acute myeloid leukemia (AML), in particular those lacking specific genetic aberrations such as PML-RARA, CBFB-MYH11, RUNX1-RUNX1T1, NPM1 or activating FLT3 mutations. This subgroup of patients accounts for about one-third of all AML patients and mainly includes the large group of AML with myelodysplasia-related changes, AML with recurrent cytogenetic abnormalities [inv(3) or t(3;3), t(9;11), t(v;11q23)] and cytogenetically normal AML (CN-AML) with wild-type NPM1 and FLT3. Prognosis in this subgroup of patients is generally poor. Azacitidine has been shown to be active in AML with low blast counts frequently observed in AML with myelodysplasia-related changes and in CN-AML in the absence of specific gene mutations. Aims: To evaluate clinical efficacy of azacitidine in combination with intensive induction chemotherapy and in maintenance for two years as single agent in patients with AML who are not candidates for genotype-adapted treatment approaches. Methods: Patients with AML in the absence of specific genetic aberrations (PML-RARA, CBFB-MYH11, RUNX1-RUNX1T1, NPM1 mutation, activating FLT3 mutations) who are fit for intensive chemotherapy were eligible. Patients were up-front randomized for induction therapy into one standard arm and three experimental arms; i) ICE (standard arm), idarubicin (12 mg/m2/day, iv, days 1,3,5), cytarabine (100 mg/m2/day, cont. infusion, days 1–7), etoposide (100 mg/m2/day, iv, days 1,2,3); ii) AZA-prior, azacitidine (100 mg/m2/day, sc, days 1–5), idarubicin (12 mg/m2/day, iv, days 6, 8, 10), etoposide 100 mg/m2/day, iv, days 6,7,8); iii) AZA-concurrent, azacitidine (100 mg/m2/day, sc, days 1–5), idarubicin (12 mg/m2/day, iv, days 1,3,5), etoposide 100 mg/m2/day, iv, days 1,2,3); iv) AZA-after, idarubicin (12 mg/m2/day, iv, days 1,3,5), etoposide 100 mg/m2/day, iv, days 1,2,3), azacitidine (100 mg/m2/day, sc, days 4–8). After two induction cycles for patients achieving complete remission (CR), consolidation therapy was prioritized; first priority) allogeneic hematopoietic blood stem cell transplantation (HSCT) from matched related as well as unrelated donors, second priority) 3 courses of high-dose cytarabine followed by two-year maintenance therapy with azacitidine as single agent (50 mg/m2/day, sc, days 1–5, every 4 weeks) in patients initially randomized to experimental treatment. The primary endpoint was achievement of CR. The statistical design of the study was based on the Simon's optimal two-stage design applied for each arm separately. The null hypothesis was CR-rate equal or below 0.40 whereas the alternative hypothesis was a CR rate of at least 0.55 with a power of 80% and a level of significance of 5%. Thus, in each arm at least 12 of 26 patients with response to induction therapy were necessary after the first to proceed to the second stage. Results: During the first stage of the study 104 patients were randomized; median age was 62.5 years (range 18–82), 46% were female. Data on cytogenetics showed intermediate risk karyotype in 67% (n=50) including CN-AML (n=31) and high-risk karyotype in 33% (n=25). The most frequent serious adverse events were grade 3/4 infection with an overall incidence of 25% and ranging from 20 to 34% in the different treatment arms. The number of responding patients in the treatment arms AZA-prior and AZA-concurrent after the first stage of the study were 11 of 26 (42%) and 10 of 26 (38%)Both arms, AZA-prior and were terminated accordingly. In contrast, the treatment arms ICE and AZA-after were carried forward to the second stage of patient recruitment since responding patients at that time were 14 of 26 (54%) in both arms. In total, 100 patients each have been enrolled in both treatment arms, ICE and AZA-after, with CR-rates of 59% and 52%, respectively (p=0.39). To date, 60 patients received an allogeneic HSCT (n=36 matched unrelated donors, n=23 matched related donors, n=1 haploidentical family donor). Maintenance treatment was started in 12 patients. Conclusion: Induction therapy with ICE or idarubicin, etoposide followed by azacitidine (AZA-after) appears equally effective in producing CR in patients with AML who are not candidates for genotype-adapted treatment approaches. An amendment perpetuating the treatment arms ICE and AZA-after within a phase-III concept is planned. Disclosures: Schlenk: Celgene: Research Funding. Off Label Use: Azacitidine combined with intensive chemotherapy.

Beschreibung: Abstract 412 Background: Somatic mutations of the ASXL1 (Additional Sex Comb-Like 1) gene on chromosome 20q11.1 were identified in various myeloid malignancies with the highest incidence reported in CMML (∼40%) and lower frequencies in MDS, AML, CML, and myeloproliferative neoplasia. The ASXL1 protein has been suggested to act as chromatin modifier and the highly conserved C-terminal plant homeo-domain (PHD) finger is presumably critical for its function. ASXL1 mutations cluster in exon 12 and are mainly frameshift mutations predicted to remove the PHD domain. We and others (Paschka et al., Haematologica 2011;96(s2);425; Chou et al., Blood 2010;116:4086–94) have recently reported first results on the unfavorable prognostic impact of ASXL1 mutations in AML. However, the clinical relevance of these mutations still needs to be elucidated in larger AML cohorts. Methods: Mutational analyses of ASXL1 were performed on diagnostic samples from 1429 patients with AML aged 18 to 61 years. All patients were intensively treated on one of two AMLSG trials [AML HD98A (n=745), Schlenk et al., J Clin Oncol. 2010;28:4642–8; AMLSG 07–04(n=684), NCT00151242]. GeneScan-based fragment analysis of several amplicons spanning ASXL1 exon 12 was used to screen for ASXL1 mutations. Samples showing altered GeneScan profiles were amplified in a second PCR reaction, and the amplicons were sequenced to confirm the mutation and to determine the mutation type. Patients were also assessed for the presence of NPM1, FLT3 (ITD and TKD), CEBPA, IDH1/2, RUNX1, and DNMT3A mutations by standard PCR-based methods. Results: ASXL1 mutations were detected in 90 (5.9%) of 1429 patients. All mutations were heterozygous frameshifts predicted to cause loss of the PHD finger. The most common mutation was a duplication of guanine at position 1934 (c.1934dup) identified in 59% (53/90) of the mutated cases. Three other ASXL1 mutations were detected in more than one patient: c.1900_1922del (n=16), c.1934del (n=5), c.1960dup (n=3). The majority of mutations (91%) clustered within or around a glycine-rich protein domain spanning amino acids 642–685. Patients with ASXL1 mutations were older (P