NPM1-mutated acute myeloid leukemia (AML) is a provisional entity in the 2008 World Health Organization (WHO) classification of myeloid neoplasms. The significance of multilineage dysplasia (MLD) in NPM1-mutated AML is unclear. Thus, in the 2008 WHO classification, NPM1-mutated AML with MLD is classified as AML with myelodysplasia (MD)-related changes (MRCs). We evaluated morphologically 318 NPM1-mutated AML patients and found MLD in 23.3%. Except for a male predominance and a lower fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) incidence in the MLD(+) group, no differences were observed in age, sex, cytogenetics, and FLT3--tyrosine kinase domain between NPM1-mutated AML with and without MLD. NPM1-mutated AML with and without MLD showed overlapping immunophenotype (CD34 negativity) and gene expression profile (CD34 down-regulation, HOX genes up-regulation). Moreover, overall and event-free survival did not differ among NPM1-mutated AML patients independently of whether they were MLD(+) or MLD(-), the NPM1-mutated/FLT3-ITD negative genotype showing the better prognosis. Lack of MLD impact on survival was confirmed by multivariate analysis that highlighted FLT3-ITD as the only significant prognostic parameter in NPM1-mutated AML. Our findings indicate that NPM1 mutations rather than MLD dictate the distinctive features of NPM1-mutated AML. Thus, irrespective of MLD, NPM1-mutated AML represents one disease entity clearly distinct from AML with MRCs.

The World Health Organization classification of acute myeloid leukemia (AML) is hierarchically structured and integrates genetics, data on patients' history, and multilineage dysplasia (MLD). The category "AML with myelodysplastic syndrome (MDS)related changes" (AML-MRC) is separated from "AML not otherwise specified" (AML-NOS) by presence of MLD, MDS-related cytogenetics, or history of MDS or MDS/myeloproliferative neoplasm (MPN). We analyzed 408 adult patients categorized as AML-MRC or AML-NOS. Three-year event-free survival (EFS; median, 13.8 vs 16.0 months) and 3-year overall survival (OS; 45.8% vs 53.9%) did not differ significantly between patients with MLD versus without. However, MLD correlated with preexisting MDS (P < .001) and MDS-related cytogenetics (P = .035). Patients with MLD as sole AML-MRC criterion (AML-MLD-sole; n = 90) had less frequently FLT3 internal tandem duplication (P = .032) and lower median age than AML-NOS (n = 232). Contrarily, patients with AML-NOS combined with AML-MLD-sole (n = 323) had better 3-year EFS (16.9 vs 10.7 months; P = .005) and 3-year OS (55.8% vs 32.5%; P = .001) than patients with history of MDS or MDS/MPN or MDS-related cytogenetics (n = 85). Gene expression analysis showed distinct clusters for AML-MLD-sole combined with AML-NOS versus AML with MDS-related cytogenetics or MDS history. Thus, MLD alone showed no independent clinical effect, whereas cytogenetics and MDS history were prognostically relevant. (Blood. 2010;116(15):2742-2751)

BACKGROUND:Differences in survival have been reported between pediatric and adult acute lymphoblastic leukemia. The inferior prognosis in adult acute lymphoblastic leukemia is not fully understood but could be attributed, in part, to differences in genomic alterations found in adult as compared to in pediatric acute lymphoblastic leukemia. DESIGN AND METHODS:We compared two different sets of high-density single nucleotide polymorphism array genotyping data from 75 new diagnostic adult and 399 previously published diagnostic pediatric acute lymphoblastic leukemia samples. The patients' samples were randomly acquired from among Caucasian and Asian populations and hybridized to either Affymetrix 50K or 250K single nucleotide polymorphism arrays. The array data were investigated with Copy Number Analysis for GeneChips (CNAG) software for allele-specific copy number analysis. RESULTS:The high density single nucleotide polymorphism array analysis of 75 samples of adult acute lymphoblastic leukemia led to the identification of numerous cryptic and submicroscopic genomic lesions with a mean of 7.6 genomic alterations per sample. The patterns and frequencies of lesions detected in the adult samples largely reproduced known genomic hallmarks detected in previous single nucleotide polymorphism-array studies of pediatric acute lymphoblastic leukemia, such as common deletions of 3p14.2 (FHIT), 5q33.3 (EBF), 6q, 9p21.3 (CDKN2A/B), 9p13.2 (PAX5), 13q14.2 (RB1) and 17q11.2 (NF1). Some differences between adult and pediatric acute lymphoblastic leukemia were identified when the pediatric data set was partitioned into hyperdiploid and non-hyperdiploid cases and then compared to the nearly exclusively non-hyperdiploid adult samples. In this analysis, adult samples had a higher rate of deletions of chromosome 17p (TP53) and duplication of 17q. CONCLUSIONS:Our analysis of adult acute lymphoblastic leukemia cases led to the identification of new potential target lesions relevant for the pathogenesis of acute lymphoblastic leukemia. However, no unequivocal pattern of submicroscopic genomic alterations was found to separate adult acute lymphoblastic leukemia from pediatric acute lymphoblastic leukemia. Therefore, apart from different therapy regimen, differences of prognosis between adult and pediatric acute lymphoblastic leukemia are probably based on genetic subgroups according to cytogenetically detectable lesions but not focal genomic copy number microlesions.

Reciprocal IGH/14q32 translocations are detectable in 55-70% of patients with plasma cell myeloma; e.g., the adverse t(4;14)(p16;q32) fusing the IGH and FGFR3 genes (immunoglobulin heavy chain/fibroblast growth factor receptor 3). In a minority of patients with B-lineage chronic lymphocytic leukemia (CLL), reciprocal IGH/14q32 translocations have been reported as well. We describe the occurrence of a t(4;14)(p16;q32) in two lymphoma patients showing the immunophenotype of B-CLL, which, to our knowledge, is the first report on such an association. The first patient, a 72-year-old female, showed mature lymphocyte infiltration of the bone marrow and marked splenomegaly. Immunophenotyping revealed aberrant CD5 expression and light-chain lambda restriction on mature B-lymphocytes corresponding to a B-CLL. Interphase fluorescence in situ hybridization (FISH) plus chromosome banding revealed a t(4;14)(p16;q32) in addition to an unbalanced der(16)t(8;16)(q23;p13) and a del(8)(p11). The second patient, a male of 65 years, showed marked peripheral leukocytosis. Immunophenotyping revealed the phenotype of CLL/PLL (chronic lymphocytic leukemia/prolymphocytic leukemia). Again, FISH together with karyotyping revealed a t(4;14)(p16;q32). These two cases with an t(4;14), but the immunophenotype of B-CLL, demonstrate the genetic variability of B-cell lymphomas and the potential of specific metaphase cultivation techniques using oligonucleotides to increase our insights in the genetic pathways of these heterogeneous disorders.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course. The aim of this study was to evaluate whether a combination of genetic parameters can improve prediction of outcome irrespective of clinical stage. The prognostic impact of chromosome banding analysis (CBA) in addition to FISH and IgVH mutation status was evaluated. In total, 482 patients were analyzed, but evaluation of prognostic factors was restricted to 399 untreated cases. The prognostic significance of age, white blood cell (WBC) count, IgVH status, and TP53 and ATM deletions was confirmed. In addition, a prognostic impact of translocations involving the IGH@ locus (t(IgH)) and of a complex aberrant karyotype was found. On the basis of these results, we propose a scoring system for overall survival (OS) based on: age >or=65 years, WBC >or=20 x 10(9)/l, unmutated IgVH status, TP53 deletion, t(IgH), and the number of chromosome aberrations observed with CBA. Three risk groups showed considerable differences in OS (94.5% vs. 64.3% vs. 41.1% surviving at 5 years, P < 0.0001). Time to treatment (TTT) can be predicted best by unmutated IgVH status, ATM deletion, t(IgH), and number of chromosome aberrations. Four distinct subgroups were separated with median TTT of 110.7 months, 39.8 months, 19.5 months, and 3.8 months, respectively (P < 0.0001). In conclusion, cytogenetic data from CBA add prognostic information. The proposed scoring systems for OS and TTT based on a combination of genetic markers improve the separation of prognostic subgroups in CLL already early in the course of the disease.

Abstract Abstract 275 Cytogenetic abnormalities – translocations as well as deletions – involving the short arm of chromosome 12 are common in a wide variety of hematologic malignancies. However, data on the minimal deleted region and on expression of candidate genes in this region are limited. Therefore, we analyzed 590 myeloid malignancies with FISH probes flanking the breakpoints within the ETV6 gene which is located on the short arm of chromosome 12. 114 cases showed a deletion of the telomeric and the centromeric probe while in 7 cases a deletion of the probe localized centromeric of ETV6 was observed while the telomeric probe was retained, suggesting a small interstitial 12p deletion. These cases were further analysed using SNP microarrays (Affymetrix Genome-Wide Human SNP Array 6.0). The median size of the deletion was 1.6 Mb (range: 1.2 – 10.6 Mb). The minimal deleted region was narrowed down to 815 kb (physical map position start: 12005749 bp from pter; end: 12820773 bp from pter) and encompasses the following genes: BCL2L14, LRP6, MANSC1, DUSP16, CREBL2, GPR19, CDKN1B, APOLD1 and hsa-mir-613. The expression of these genes was further evaluated by gene expression microarrays (Affymetrix HG-U133 Plus 2.0) in 781 patients with hematological malignancies. Only three genes, CREBL2, MANSC1 and CDKN1B were expressed in more than 15% of cases. As CDKN1B plays an important role in multiple fundamental cellular processes, including cell proliferation, cell differentiation, and apoptosis and moreover is a putative tumor suppressor. Thus, it is an interesting candidate gene for playing an important pathogenetic role in cases with 12p deletions. Therefore, we first analyzed CDKN1B expression in more detail. The median CDKN1B expression intensity was 1,582 (range 83 – 4498) in 399 myeloid malignancies (286 AML, 113 MDS). Karyotypes in AML were t(15;17)(q22;q12) (n=15), t(8;21)(q22;q22) (n=16), inv(16)(p13q22) (n=7), t(11q23)/MLL-rearrangement (n=10), complex aberrant karyotype (n=53), normal karyotype (n=99), and various other chromosome abnormalities (n=86) and in MDS del(5q) sole (n=20), −7 sole (n=5), normal karyotype (n=46) and various other chromosome abnormalities (n=42). 99 cases showed an expression intensity of CDKN1B below 1160 (first quartile) (83 AML, 16 MDS). In this cohort cases with t(8;21) (n=11), t(15;17) (n=11) or t(11q23)/MLL-rearrangement (n=6) were over-represented (Chi-square: p<0.0001, p<0.0001, and p=0.009, respectively), while cases with normal karyotype (n=28) were under-represented (Chi-square: p=0.048). In addition, an association of FLT3-TKD with low CDKN1B expression was observed (7/13 FLT3-TKD+ cases showed a low CDKN1 expression compared to 54/213 FLT3-TKD- cases, p=0.025), while no correlation to other molecular mutations was found (NPM1, FLT3-ITD, CEPBA, MLL-PTD). With respect to clinical data in AML, median overall survival (OS) and event-free survival (EFS) was longer in CDKN1B low expressers (not reached vs. 14.8 months; p=0.005 and 31 months vs. 9.7 months; p=0.013). In MDS, a tendency towards a longer OS was also observed (not reached vs. 55.9 months; p=0.29). For CREBL2 and MANSC1 no association of expression and survival was observed. In conclusion, the minimal deleted region on 12p in myeloid malignancies encompasses only 3 genes which are expressed in myeloid malignancies: CDKN1B, CREBL2 and MANSC1. Low CDKN1B expression is more frequently found in AML with t(8;21), t(15;17) or t(11q23)/MLL-rearrangement and is associated with a favorable outcome which might be due to a higher susceptibility to cytotoxic agents as low CDKN1B expression enhances cell cycle progression. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia, and primary myelofibrosis show an inherent tendency for transformation into leukemia (MPN-blast phase), which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, examined chromosomal abnormalities by high-resolution single nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters. Frequent genomic alterations were found in MPN after leukemic transformation with up to 3-fold more genomic changes per sample compared with samples in chronic phase (P < .001). We identified commonly altered regions involved in disease progression including not only established targets (ETV6, TP53, and RUNX1) but also new candidate genes on 7q, 16q, 19p, and 21q. Moreover, trisomy 8 or amplification of 8q24 (MYC) was almost exclusively detected in JAK2V617F(-) cases with MPN-blast phase. Remarkably, copy number-neutral loss of heterozygosity (CNN-LOH) on either 7q or 9p including homozygous JAK2V617F was related to decreased survival after leukemic transformation (P = .01 and P = .016, respectively). Our high-density SNP-array analysis of MPN genomes in the chronic compared with leukemic stage identified novel target genes and provided prognostic insights associated with the evolution to leukemia.

Abstract Abstract 1262 Poster Board I-284 14q deletions are rare aberrations in malignant neoplasia but recurrent in hematologic malignancies, where they occur most frequently in CLL and other mature B-cell neoplasms. The breakpoints and the size of the 14q deletion have not been exactly characterized in a larger cohort yet. Based on chromosome banding analysis we identified 47 cases with 14q deletion out of 3054 cases (1.5%) with mature B-cell neoplasms (CLL: 1863, CLL/PL: 92, other: 1099). 32 cases with 14q deletion were classified as CLL, 5 as CLL/PL and 10 as other mature B-cell neoplasms based on immunophenotyping or cytomorphology. Therefore, the incidence of 14q deletions was 1.7% in CLL, 4.7% in CLL/PL and 0.9% in other mature B-cell neoplasms. These cases were further analyzed by a panel of FISH probes localized in 14q22.1 (BAC RP11-218E20), 14q24.1 (BACs PR11-35D12, RP11-226F19), 14q32.33 (BAC RP11-521B24) (BlueGnome, Oxford, UK) and the IgH-locus (14q32.3). The study revealed that 14q deletions are heterogeneous in size. A breakpoint cluster on the centromeric side was identified in 14q24.1 between BAC PR11-35D12 and BAC RP11-226F19 (62% of cases), whereas the most frequent breakpoint on the telomeric side was within the IgH-locus (14q32.3) between the IgH3'-flanking probe and IgHV probe (45% of cases). In 16 cases (34%) breakpoints occurred within 14q24.1 and within 14q32.3. In 19% of patients the 14q deletion was the sole abnormality whereas 30% showed one, 15% two and 36% three or more additional aberrations. Trisomy 12 (45%) was the most frequent aberration accompanying 14q deletion. In comparison to a cohort of 1579 cases with mature B-cell neoplasms without 14q deletion which were analyzed for IgVH mutation status in the same time period, patients with 14q deletions showed more often an unmutated IgVH status (39% vs. 61%, p=0.001). In the subgroup with breakpoint in 14q24.1 and 14q32.3 even 82% showed an unmutated IgVH-status. This subgroup was also associated with a CLL immunophenotype (81%). Clinical follow up data was available in 28 patients. CLL and CLL/PL with 14q deletion were compared to 390 CLL and CLL/PL cases without 14q deletion. While overall survival (OS) did not differ between both groups (OS at 3 yrs 86.4% vs. 88.7%, p=0.195), time to treatment (TTT) was significantly shorter in cases with 14q deletion (21.0 months vs. 80.1 months, p=0.015). Next, all 418 cases were assigned into 6 subgroups with known prognostic relevance: 1) 14q deletion, 2) 17p deletion, 3) 11q deletion, 4) trisomy 12, 5) 13q deletion as the sole abnormality, and 6) none of the mentioned abnormalities. OS at 3 yrs was 86.4%, 31.4%, 77.5%, 96.9%, 93.4% and 93.9%, respectively. For TTT the respective data were 21.0 months, 21.6 months, 20.9 months, not reached, 79.6 months and 147.0 months. In conclusion, 14q deletions are recurrent abnormalities in CLL and other mature B-cell neoplasms. Comparable to 13q deletions the size of 14q deletions varies. However, recurrent breakpoints in 14q24.1 and 14q32.3 were identified. Our data suggest to add FISH probes for 14q24.1 and for the IGH locus to the standard FISH panel in CLL and other mature B-cell neoplasms to pick up cases with 14q deletion, who would otherwise be assigned to “normal” FISH subgroup. In conclusion, 14q deletions are frequently accompanied by trisomy 12, an unmutated IgVH status and are associated with an intermediate overall survival and a shorter time to treatment comparable to other high risk cytogenetic aberrations (17p deletion, 11q deletion). Disclosures Reindl: MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract Abstract 1234 Poster Board I-256 CLL is a heterogeneous disease with a variable clinical course. In this study the prognostic power of chromosome banding analysis (CBA), interphase FISH and IgVH status was evaluated. In total 399 untreated cases were analyzed. First, we could confirm the prognostic significance of established parameters such as age (≥65 yrs), white blood cell count (≥20.000/μl), IgVH status, TP53 deletion and 11q deletion in our cohort. In addition, a negative prognostic impact of translocations involving the IgH locus, especially t(14;18)(q32;q21) and of the complexity of the karyotype measured by the number of clonal chromosome aberrations in CBA was observed. Furthermore it became obvious that some parameters discriminated better for overall survival and other for time to treatment. While the impact of the IgVH status on overall survival was low within the first 5 years after diagnosis (mutated 88.5% surviving vs unmutated 82.0% surviving, log rank test p=0.022), an unmutated IgVH status was strongly correlated with a shorter median time to treatment (18.3 months unmutated vs 110.7 months mutated, log rank test p<0.0001). On the other hand the impact of TP53 deletion was more pronounced on overall survival as compared to time to treatment. Age was associated with a shorter overall survival but was not significantly associated with time to treatment. Based on these results we propose a score for overall survival (OS) based on: age ≥65 yrs, WBC ≥20.000/μl, unmutated IgVH status, TP53 deletion, t(IgH), and the number of chromosome aberrations observed in CBA. Three respective risk groups showed considerable differences in OS (94.5% vs 64.3% vs 41.1% surviving at 5 yrs, p<0.0001). In contrast, time to treatment (TTT) was predicted best by unmutated IgVH status, ATM deletion, t(IgH) and number of chromosome aberrations. Four subgroups could be separated with median TTT of 110.7 months, 39.8 months, 19.5 months, and 3.8 months, respectively (p<0.0001). In conclusion, our data show that in combination with established prognostic markers such as an unmutated IgVH status, TP53/17p deletions or 11q deletions also the newly defined complexity of the karyotype measured by the number of chromosome aberrations has an important impact both on overall survival and also on time to treatment in CLL. These newly combined parameters translate into a more distinct separation of prognostic subgroups within the first years after diagnosis as compared to other prognostic systems using FISH data only or based on FISH data in combination with IgVH status. Prospective studies should evaluate the power for early stage CLL patients. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract 2618 This icon denotes an abstract that is clinically relevant.

Abstract Abstract 3067 Poster Board III-4 During the last years it has been shown that PCR based detection of minimal residual disease (MRD) has high relevance for early detection of relapse and overall prognostication. This has been proven for several fusion transcripts but also for NPM1 as a target in normal karyotype AML (NK-AML). Other mutations frequently found in NK-AML are RUNX1 mutations (8-10%) and CEBPA mutations (10-15%). However, these mutations are distributed throughout the entire coding reading frames of CEBPA and RUNX1 making mutation analyses more laborious compared to analysis of genes with mutational hotspots like NPM1. In addition, it is nearly impossible to establish high sensitive real time PCR assays for every patient specific mutation. In contrast, DHPLC (denaturing high performance liquid chromatography) is a method that effectively can detect unknown mutations and for known mutations has a sensitivity of up to 1%. Therefore we analyzed the impact of DHPLC analysis for the applicability and value as predictive MRD analysis. At diagnosis mutation screening by DHPLC was performed first. Both genes were amplified each by four different PCR reactions that were subsequently analysed on a WAVE system (Transgenomic, Inc., Omaha, USA). Positive reactions were further characterized by sequencing. The respective fragment or fragments containing the mutations was/were subsequently also analyzed in the follow up samples. The sensitivity was dependent on the kind of the mutation and its position within the PCR fragment and was between 1 and 10% as estimated by limited dilution experiments. Paired diagnostic and relapse samples were available in 15 cases (12 RUNX1 and 3 CEPBA). The respective mutations were retained at relapse in all cases indicating the stability of both markers, rendering them eligible for follow up evaluation. Next, we analysed 30 patients with CEBPA mutation detected at diagnosis and further investigated 91 samples during follow up. 12 of these cases had two different mutations that were localized on two different DHPLC fragments and thus could be analysed in parallel. For RUNX1 we analysed 144 follow up samples of 60 patients that revealed one (n=51) or two (n=9) RUNX1 mutations at diagnosis. Six of the CEBPA mutated and 13 of the RUNX1 mutated cases had an FLT3-ITD in addition. The median follow up sample number per patient was 3 (range 2-13) and the median follow up time was 339 days (range: 57-3001 days). In the subsequent analysis both cohorts were combined. The follow up samples were simply rated as negative or positive. According to previous studies in fusion gene positive patients and NPM1 mutated patients the impact of the DHPLC results on survival was analysed for defined time intervals after start of treatment: interval 1 (days 18-60), interval 2 (days 61-120), interval 3 (day 121-365), interval 4 (days >365). DHPLC results within these intervals were as follows: interval 1 (positive: n=16; negative n=17), interval 2 (positive: n=14; negative n=19) interval 3 (positive: n=38; negative n=65), interval 4 (positive: n=22; negative n=38). Whenever in the follow up samples two different mutations were available (n=99), the results were shown to be concordant. The impact of the results was analysed by Kaplan Meier analysis. For overall survival a trend for significance was found for interval 1 (medians not reached; p=0.157) and interval 2 (medians not reached; p=0.090) and a significant impact for interval 3 (median: not reached vs. 981 days; p=0.015) and interval 4 (median not reached: vs. 285 days; p=0.048), demonstrating that negative DHPLC results correlate with longer OS. This effect could even more clearly be shown for event free survival with respective results for interval 1 (median: 463 vs 731; p=0.048), interval 2 (median: 499 vs 731 days; p=0.109) and interval 3 (p<0.001) (too few samples for interval 4). As neither age, WBC or pretreatment FLT3 status were significantly associated with outcome in this cohort a multivariate analysis could not be performed. These data clearly show that in the absence of sensitive markers for RQ-PCR low sensitive PCR can be very useful for follow up controls at least in RUNX1 and CEBPA mutated AML. Disclosures Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Lab: Employment. Sundermann:MLL Munich Leukemia Lab: Employment. Spiel:MLL Munich Leukemia Lab: Employment. Wendland:MLL Munich Leukemia Lab: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Lab: Equity Ownership. Kern:MLL Munich Leukemia Lab: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Summary A retrospective comparison of WT1 and BCR‐ABL1 expression was performed in 40 imatinib‐treated chronic myeloid leukaemia patients. The overall correlation of WT1 and BCR‐ABL1 was low. In two patients WT1 expression was increasing despite very low BCR‐ABL1 levels. As both revealed Ph‐negative aberrant clones, a second independent cohort of 20 cases, all with Ph‐negative clonal evolution, was analysed. High WT1 expression (5·0–177·0%) was detected in a case with +11 and in four of eight cases with +8, but not in cases with del(20q) or −Y. Thus, increasing WT1 levels in molecular responders may indicate Ph‐negative clonal cytogenetic evolution during imatinib treatment.

Abstract LBA-3 This icon denotes an abstract that is clinically relevant. Introduction: IDH1 is the gene coding for the soluble isocitrate dehydrogenease 1 (NADP+), which catalyzes the oxidative decaroxylation of isocitrate to 2-oxoglutarate. The gene has been shown to be frequently mutated in high-grade gliomas at residue p.R132, which is located in the substrate binding site of IDH1. So far, several other tumors have been analyzed without detection of the respective mutation (Bleeker et al., Human Mutation 2009). However, recently a next generation sequencing project found IDH1 mutated in 8.5% of AML with normal karyotype (Mardis et al., NEJM, 2009). Aim: To further evaluate the importance of IDH1R132 (IDH1mut) in AML we have analyzed a cohort of 999 comprehensively characterized AML cases. Methods: The respective base exchange was analysed by a LightCycler based melting curve assay with subsequent sequencing of the positive samples. Results: The cohort was comprised of 536 male and 463 female patients (median age: 65.9 years; range: 17.1- 93.3 years). 833 had de novo AML (83.4%), 122 AML following MDS (s-AML,12.1%) and 44 AML after previous treatment of different malignancies (t-AML, 4.4%). Karyotype was available in all cases: 681 had a normal karyotype (NK) AML, and 319 had chromosomal aberrations (t(15;17): n=29; inv(16): n=12, t(8;21): n=23, t(11q23): n=10, t(6;9): n=4, inv(3): n=3; -7: n=27, +8: n=29, +13: n=11, -Y: n=4; complex aberrant: n=60, others: n=106). Overall, in 93 pts IDH1 p.R132 mutations were detected (9.3%). Five different amino acid exchanges were observed: R132C (n=49), R132L (n=22), R132 H and G (n=7, each), and R132S (n=5). With respect to history of the patient IDH1mut were found in 80/833 of de novo AML (9.6%), 11/122 (9.0%) of s-AML, and 2/44 (4.5%) of t-AML, respectively (n.s.). More females (57/463, 12.3%) than males (36/536; 6.7%) had IDH1mut (p=0.003). Age was slightly higher in the mutated cases (63.9 vs. 61.9 years, n.s.). No differences were found for WBC count. IDH1mut were distributed differently between karyotypes: in NK 69/681 (10.1%) and in aberrant karyotypes 24/318 (7.5%). However, IDH1 was never mutated in inv(16), t(8;21), t(6;9), t(11q23), inv(3), or in complex aberrant karyotypes (n=112). In 2 of 27 cases (7.4%) with t(15;17) an IDH1 mutation was detected. Thus, the IDH1 mututations clustered in the intermediate risk karyotype group in comparison to the good or poor risk groups (91/771; 11.8% vs 2/134 (1.5%), p<0.001). The cohort was also characterized for several other molecular mutations. FLT3-ITD was present in 22% (212/954), FLT3-TKD in 6.7% (33/496), NPM1 in 35.4% (329/929), NRAS in 14.6% (48/328), MLL-PTD in 6.9% (64/932), CEBPA mutations in 7.4% (48/645) and RUNX1 mutations in 33.0% (99/299) of analysed cases, respectively. IHD1 mutations were found to be more frequent in NPM1 mutated than in NPM1wt cases (41/329; 12.4% vs 48/598; 8.0%, p= 0.019) and in those with MLL-PTD (11/64; 17.2% vs 77/867; 8.9%, p= 0.031). With lower frequencies IDH1mut were also detected together with RUNX1 mutations (n=8/99), CEBPA mutations (n=2/48), NRAS mutations (n=7/48), and FLT3-TKD (n=1/33). IDH1 was similarly distributed between FLT3-ITD mutated and unmutated cases (18/212; 8.5% vs. 72/744; 9.7%). In 22 (23.7%) of all IDH1mut AML no additional mutation was detected, whereas in 48 (51.6%) one additional, in 22 (23.7%) two additional and in one case three additional mutations were found. An unfavourable effect of IDH1mut on event free survival (EFS) was observed in the total group (median: 272 vs. 456 days; p=0.007) as well as in those with intermediate risk karyotype (median: 272 vs. 449 days; p=0.008). A shorter EFS of the IDH1mut was particularly seen in the NPM1wt cohort (median: 244 vs. 375 days; p=0.038) with a strong trend for an independent effect in a multivariate analysis (p=0.089). Conclusions: IDH1 mutations are frequent in AML and are prognostically unfavourable especially in the NPM1wt cohort. IDH1 mutations seem to be a new class of mutation probably complementing with the classical type 1 and type 2 mutations. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Ulke: MLL Munich Leukemia Lab: Employment. Kaya: MLL Munich Leukemia Lab: Employment. Weiss: MLL Munich Leukemia Laboratory: Employment. Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership.

PURPOSE:Risk stratification in acute myeloid leukemia (AML) is currently based on pretreatment characteristics. It remains to be established whether relapse risk can be better predicted through assessment of minimal residual disease (MRD). One proposed marker is the Wilms tumor gene WT1, which is overexpressed in most patients with AML, thus providing a putative target for immunotherapy, although in the absence of a standardized assay, its utility for MRD monitoring remains controversial. PATIENTS AND METHODS:Nine published and in-house real-time quantitative polymerase chain reaction WT1 assays were systematically evaluated within the European LeukemiaNet; the best-performing assay was applied to diagnostic AML samples (n = 620), follow-up samples from 129 patients treated with intensive combination chemotherapy, and 204 normal peripheral blood (PB) and bone marrow (BM) controls. RESULTS:Considering relative levels of expression detected in normal PB and BM, WT1 was sufficiently overexpressed to discriminate > or = 2-log reduction in transcripts in 46% and 13% of AML patients, according to the respective follow-up sample source. In this informative group, greater WT1 transcript reduction after induction predicted reduced relapse risk (hazard ratio, 0.54 per log reduction; 95% CI, 0.36 to 0.83; P = .004) that remained significant when adjusted for age, WBC count, and cytogenetics. Failure to reduce WT1 transcripts below the threshold limits defined in normal controls by the end of consolidation also predicted increased relapse risk (P = .004). CONCLUSION:Application of a standardized WT1 assay provides independent prognostic information in AML, lending support to incorporation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification, and to identify patients who may benefit from allogeneic transplantation.

Abstract Abstract 3270 Poster Board III-1 In chronic myeloid leukemia (CML), the progress from chronic phase (CP) to accelerated (AP)/blast phase (BP) is frequently accompanied by cytogenetic evolution within the Philadelphia (Ph+) positive clone. It was shown that additional chromosomal alterations follow non-random patterns with frequent occurrence of “major route” (e.g. +Ph, i(17q), +8) and “minor route” changes (e.g. -Y, -7 or +21). Corresponding to the pre-imatinib era, Ph+ clonal cytogenetic evolution was still identified to worsen prognosis in patients receiving imatinib. However, whether the introduction of TKIs changed the pattern of additional chromosome aberrations, was not studied so far. We here compared two subgroups: 1.) 245 CML patients treated with tyrosine kinase inhibitors (TKIs) with clonal evolution in the Ph+ clone investigated in the Munich Leukemia Laboratory (MLL) between 2005–2009. 2.) 500 CML cases published in the Mitelman Database (http://cgap.nci.nih.gov/Chromosomes/Mitelman) before the year 2000 (i.e. before the introduction of imatinib into the treatment of CML). The 245 patients from our cohort were selected for this study based on the occurrence of Ph+ clonal evolution at diagnosis or during the course of CML. Patients received imatinib or 2nd generation TKIs. First, analysis was performed for the Mitelman cohort and for the MLL cohort. Then, analysis was separated for those patients from the MLL cohort who showed Ph+ clonal evolution already at diagnosis of CML before start of TKIs (n=91), and for those patients who developed Ph+ cytogenetic alterations during TKIs (n=154). The patterns of chromosomal gains and losses were analyzed with the support of the CyDAS cytogenetic data analysis system (http://www.cydas.org/OnlineAnalysis/). All 4 cohorts showed comparable patterns of chromosomal gains/losses in addition to the Philadelphia translocation: Most frequent were +8, +Ph chromosome, +19, and i(17)(q10), and -Y. Less frequent were -7 and +21. Therefore, no difference between the patterns of abnormalities dating from the pre-imatinib era (data from the Mitelman database) or after the introduction of TKI (MLL cohort) was obvious. Also, there was no difference in cytogenetic patterns between patients who showed Ph+ clonal evolution at diagnosis of CML already and those who acquired them during TKI treatment. In conclusion, the patterns of cytogenetic alterations in the Ph+ clones in CML are similar as investigated in the pre-TKI and the TKI eras and therefore are independent, i.e. of the time point of analysis at diagnosis or during follow-up and also treatment of CML. Disclosures: Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Weiss:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract 2812 Poster Board II-788 From cytomorphological aspects, monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) are overlapping disorders. According to the WHO classification, a threshold of 10% of plasma cells (PCs) in bone marrow (BM) aspirates separates both categories. To clarify whether this separation is justified from cytogenetic aspects, we performed comparison of interphase fluorescence in situ hybridization (FISH) patterns in 691 patients with MGUS (n=381) or MM (n=310). Also, the results of cytomorphology and immunophenotyping using multiparameter flow cytometry (MFC) were correlated. 278 females and 413 males (22.8-92.8 yrs) at first presentation of MM/MGUS were analyzed between 2005-2009 in our laboratory with a combination of cytomorphology, MFC, and FISH following magnetic activated cell sorting (MACS) of CD138+ PCs (Robosep, STEMCELL Technologies, Vancouver). According to cytomorphological WHO criteria, 381 patients were categorized as MGUS (median BM PCs, 5%), 310 as MM (median PCs, 18.5%). The number of FISH probes being applicable to samples was depending on the amount of plasma cells yielded from MACS procedure. In the MM pts, a median of 12 probes was applied (range, 0-22) which was slightly higher than in the MGUS patients (median: 11; 0-18) (p=0.00002). FISH procedure was hampered by insufficient PC numbers more frequently in MGUS (79/381, 20.7%) than in MM pts (29/310; 9.3%) (p=0.0004). In MM pts, FISH revealed a median of 2 and a maximum of 7 cytogenetic alterations per patient in contrast to a median of 0 and a maximum of 5 in MGUS (p<0.0001). In more detail, in MM the maximum number of gains of genetic material per patient was 7 (MGUS: 5), of losses 7 (MGUS: 3), and of reciprocal rearrangements 2 (MGUS: 1). Subsequently, cytogenetic alterations were compared in those 527 pts (260 MM and 267 MGUS pts; 76.3% of all pts), in whom PC numbers allowed performance of at least 5 FISH probes (t(11;14), t(4;14), t(14;16), 13q14, TP53 ). Abnormal FISH results were detected in 145/260 MM (55.8%) and 106/267 MGUS pts (39.7%) (p=0.0002). In MM, 31/260 (11.8%) had a t(4;14)/ IGH-FGFR3 in contrast to 5/268 (1.9%) in MGUS (p<0.0001). The t(11;14)/ IGH-CCND1 (MM: n=41; 15.6%; MGUS: n=50; 18.7%; n.s.) and t(14;16)/ IGH-MAF were similarly frequent in both cohorts (MM: n=8; 3.1%; MGUS: n=3; 1.1%; n.s.). Monosomy13/del(13)(q14) was more frequent in MM (n=103; 39.3%) when compared to MGUS (n=59; 22.1%, p=0.0001). Deletions of TP53 /17p13 were seen in 16 MM (6.1%) and in 6 MGUS pts (2.2%) (p=0.029). Notably, in 7 MGUS cases with <1% PCs in cytomorphology, FISH revealed genetic alterations in 3 cases. PCs as quantified by cytomorphology vs. MFC ranged from 0-96% vs. 0-84% (median 8.5 vs. 2.0), respectively, with a highly significant correlation between both methods (Pearson, r=0.712, p<0.0001). However, as previously reported, MFC detected lower numbers in general: the median ratio of PCs by cytomorphology:MFC amounted to 4.25 (range 0.00-178.00). In 12 MGUS cases (1.7%) as defined with cytomorphology, MFC did not detect any PCs. Conversely, in 5 MGUS cases, MFC detected PCs while cytomorphology did not. In conclusion, cytogenetic patterns showed higher genetic complexity in MM cases when compared to MGUS, and both the t(4;14) as -13/del(13)(q14) were significantly more frequent in MM when compared to MGUS. However, the cytogenetic alterations showed no specific pattern for MM or MGUS categories. Therefore, the overlaps being seen from morphological aspects do also exist on the genetic level. This suggests that a cytogenetically based categorization of these cases might correlate better with the clinical profiles than the cytomorphological separation of MM/MGUS. Finally, this study supports the performance of FISH in MGUS cases. The demonstrated detection of malignant/monoclonal PCs by MFC also in cases in which cytomorphology fails to diagnose MM/MGUS emphasizes the inclusion of MFC in a standard diagnostic procedure. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Weiss: MLL Munich Leukemia Laboratory: Employment. Schnittger: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership.

BACKGROUND:Because of limited reproducibility of morphologic features, the morphological categorization of initial myelodysplastic syndromes (MDS) cases remains a major task in a diagnostic setting. METHODS:To further evaluate the role of additional diagnostic methods for suspected early MDS, the authors analyzed 1965 cases with unclear cytopenia where at least cytomorphology and immunophenotyping were performed in parallel, combined with cytogenetics and molecular genetics. RESULTS:In 353 patients, both methods diagnosed malignant/nonmalignant disease other than MDS, and 557 patients had MDS-refractory anemia with excess of blasts/chronic myelomonocytic leukemia. The remaining 1055 patients (53.7%), where early MDS/reactive cytopenia had to be assumed, were categorized into 6 groups depending on cytomorphology/immunophenotyping results for or against MDS. In 659 of 1055 cases (62.4%) with suspected initial MDS, cytomorphology and immunophenotyping were concordant in the categorization of MDS/non-MDS. Cytogenetics, available in 951 of 1055 patients, revealed the highest frequency of aberrant karyotypes when both cytomorphology and immunophenotyping proposed MDS (63 of 227; 27.8%). But also in the groups where either cytomorphology or immunophenotyping showed evidence of MDS, aberrant karyotypes were found in 6% to 14% of patients. Even when both morphology and immunophenotyping showed no MDS, 11 of 208 (5.3%) had cytogenetic aberrations. RUNX1/AML1 mutation screening was positive in 15% in the latter group. NRAS, MLL-PTD, NPM1, and JAK2V617F were detected in low frequencies, confirming MDS diagnosis in the respective cases. CONCLUSIONS:This report outlines the power of a combined diagnostic approach for suspected initial cases of MDS including immunophenotyping, cytogenetics, and molecular genetics with selected markers in addition to cytomorphology. Diagnostic algorithms should be developed, and immunophenotyping should be further validated for this specific indication.