Abstract: DNA hypermethylation has long been implicated in the pathogenesis of myelodysplastic syndromes (MDS) and also highlighted by the frequent efficacy of demethylating agents to this disease. Meanwhile, recent genetic studies in MDS have revealed high frequency of somatic mutations involving epigenetic regulators, suggesting a causative link between gene mutations and epigenetic alterations in MDS. The accumulation of genetic and epigenetic alterations promotes tumorigenesis, hypomethylating agents such as Azacitidine exert their therapeutic effect through inhibition of DNA methylation. However, the relationship between patterns of epigenetic phenotypes and mutations, as well as their impact on therapy, has not been clarified. To address this issue, we performed genome-wide DNA methylation profiling (Infinium 450K) in combination with targeted-deep sequencing of 104 genes for somatic mutations in 291 patients with MDS. Beta-mixture quantile normalization was performed for correcting probe design bias in Illumina Infinium 450k DNA methylation data. Of the 〉 480,000 probes on the methylation chip, we selected probes using the following steps: (i) probes annotated with "Promotor_Associated" or "Promoter_Associated_Cell_type_specific; (ii) probes designed in "Island", "N_Shore" or "S_Shore"; (iii) removing probes designed on the X and Y chormosomes; (iv) removing probes with 〉 10% of missing value. Consensus clustering was performed utilizing the hierarchical clustering based on Ward and Pearson correlation algorithms with 1000 iterations on the top 0.5% (2,000) of probes showing high variation by median absolute deviation across the dataset using Bioconductor package Consensus cluster plus. The number of cluster was determined by relative change in area under cumulative distribution function curve by consensus clustering. Unsupervised clustering analysis of DNA methylation revealed 3 subtypes of MDS, M1-M3, showing discrete methylation profiles with characteristic gene mutations and cytogenetics. The M1 subtype (n=121) showed a high frequency of SF3B1 mutations, exhibiting the best clinical outcome, whereas the M2 subtype (n=106), characterized by frequent ASXL1, TP53 mutations and high-risk cytogenetics, showed the shortest overall survival with the hazard ratios of 3.4 (95% CI:1.9-6.0) and 2.2 (95% CI:1.2-4.0) compared to M1 and M3, respectively. Finally, the M3 subtype (n=64) was highly enriched (70% of cases) for biallelic alterations of TET2 and showed the highest level of CpG island methylation and showed an intermediate survival. In the current cohort, we had 47 patients who were treated with demethylating agents, including 11 responders and 36 non-responders. When DNA methylation status at diagnosis was evaluated in terms of response to demethylating agents, we identified 54 differentiated methylated genes showing 〉 20% difference in mean methylation levels between responders and non-responders (q 〈 0.1). Twenty-five genes more methylated in responders were enriched in functional pathways such as chemokine receptor and genes with EGF-like domain, whereas 29 less methylated gene in responders were in the gene set related to regulation of cell proliferation. Genetic alterations were also assessed how they affected treatment responses. In responders, TET2 mutated patients tended to more frequently respond (45% vs 34%), whereas patients with IDH1/2 and DNMT3A mutations were less frequently altered (0% vs 14%, 9% vs 14%) in responders, compared in non-responders. In conclusion, our combined genetic and methylation analysis unmasked previously unrecognized associations between gene mutations and DNA methylation, suggesting a causative link in between. We identified correlations between genetic/epigenetic profiles and the response to demethylating agents, which however, needs further investigation to clarify the mechanism of and predict response to demethylation agents in MDS. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kiyoi:Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.k.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Teijin Ltd.: Research Funding; Zenyaku Kogyo Company,Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Eisai Co.,Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Consultancy, Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Research Funding; Mochida Pharmaceutical Co.,Ltd.: Research Funding. Kobayashi:Gilead Sciences: Research Funding. Naoe:Toyama Chemical CO., LTD.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Miyazaki:Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding.

Abstract: Abstract 406 Introduction: Mutations in the spliceosome gene SF3B1 (splicing factor 3b, subunit 1; SF3B1 mut) are frequent in patients with myelodysplastic syndromes (MDS) and ring sideroblasts (RS). In contrast, in AML occurrence of SF3B1mut has been published to be comparatively low (2–5%). However, analysis of SF3B1 mut in AML with RS is lacking. We aimed to determine the frequency of SF3B1 mut in AML patients according to the percentage of RS and the association of SF3B1 mut with other genetic markers. Patients and Methods: 275 AML patients (115 f/160 m; median age: 71.2 years, range: 20.8 – 89.9 years) were analyzed for SF3B1 mut by Sanger sequencing of the coding region (exon 11 to 16). RS were detectable in 176/275 cases (RS in ≥15% of erythroid precursors: n=106; RS in 1–14%: n=70), 99 cases showed no RS. The cohort comprised 202 de novo AML (FAB: M0 n=27, M1 n=36, M2 n=55, M4 n=32, M5 n=1, M6 n=51), 65 s-AML and 8 t-AML patients. Data on other mutations were available as follows: FLT3-ITD n=242, FLT3-TKD n=173, MLL-PTD n=235, NPM1 n=230, RUNX1 n=199, CEBPA n=152, NRAS n=141, KRAS n=56, ASXL1 n=90, IDH1 n=78, IDH2 n=58, TP53 n=74, and DNMT3A n=52. Chromosome banding analysis (combined with FISH if needed) was performed in 262 cases. According to MRC criteria, favorable karyotypes were found in 6, intermediate in 169, and adverse in 87 cases. 120/262 (45.8%) cases had a normal karyotype (NK-AML). Results: Overall, in 44/275 (16.0%) patients SF3B1 mut were detected with a median mutation/wildtype ratio of 45% (range: 10 – 50%). The most frequent mutation was Lys700Glu (19/44, 43.2%) followed by Lys666Asn/Arg/Thr (15/44, 34.1%) and Arg625Cys/Leu (3/44, 6.8%) and other mutations found in single cases only. Patients without detectable RS had almost no SF3B1 mut in contrast to cases with RS (3/99, 3.0% vs 41/176, 23.3%, p 〈 0.001). Of note, all three patients with SF3B1 mut without RS were s-AML. SF3B1 mut were significantly more frequent in AML with RS ≥15% as compared to RS 1–14% (33/106, 31.1% vs 8/70, 11.4% p=0.003). The frequency of SF3B1 mut was significantly increasing by higher RS categories: group 1, RS 0–14%: SF3B1 mut in 6.5%; group 2, RS 15–34%: 17.9%; group 3, RS 35–54%: 45.4%, group 4, RS 55–74%: 41.2%, group 5, RS 75–100%: 54.5% (group 1 vs 2: p=0.017; 2 vs 3: p=0.020; comparison of the frequency of SF3B1 mut for all groups: p 〈 0.001). In line, SF3B1 mut had higher percentages of RS vs SF3B1 wt (mean: 37% vs 13%, p 〈 0.001), higher age (mean: 72 vs 69 years, p=0.044) and higher platelet counts (mean: 108 vs 71 x109/L, p=0.014). SF3B1 mut was not detected in any of the analyzed t-AMLs. Within cases with RS ≥15% the frequency of SF3B1 mut was only slightly higher in s-AML vs de novo AML (12/34, 35.3% vs 21/69, 30.4%, p=0.657), and likewise was the percentage of RS (mean: 45% vs 37%, p=0.137). In de novo AML, SF3B1 mut occurred more often in FAB M2 and M4 (M2: 13/55, 23.6%; M4 10/32, 31.3%; M2 and M4 combined vs others 23/87, 26.4% vs 4/115, 3.5%, p 〈 0.001). Frequency was lower in FAB M6 vs others (3/51, 5.9% vs 24/151, 15.9%, p=0.094) and M0 (1/27, 3.7% vs 26/175, 14.9%, p=0.138), whereas SF3B1 mut were mutually exclusive of FAB M1 (0/36, 0% vs 27/166, 16.3%, p=0.005). In intermediate MRC karyotypes frequency of SF3B1 mut was much higher vs all others (38/169, 22.5% vs 4/93, 4.3%, p 〈 0.001). In detail, SF3B1 mut showed high occurrence in NK patients (NK vs aberrant: 28/120, 23.3% vs 14/142, 9.9%, p=0.004) and within this subgroup a very high frequency of 48.9% (23/47) in cases with RS ≥15%. In contrast, SF3B1 mut were nearly mutually exclusive of complex karyotype (complex vs all others: 1/57, 1.8% vs 41/205, 20.0%, p 〈 0.001). Furthermore, SF3B1 mut were associated with FLT3-ITD (7/21, 33.3% vs 30/221, 13.6%, p=0.025) and RUNX1 mutations (19/65, 29.2% vs 12/134, 9.0%, p=0.001). Conclusions: So far, SF3B1 mut were considered to be mainly relevant for MDS. In this study, SF3B1 mut were found in 31.1% of AML with RS ≥15% and even more striking in 48.9% of AML-NK with RS ≥15%. SF3B1 mut were associated with higher age, AML FAB M2 and M4 subtypes, normal karyotype, FLT3-ITD and RUNX1 mutations. Our study adds both de novo and s-AML with RS ≥15% to the myeloid entities with frequent occurrence of SF3B1 mut and suggests analysis of a possible prognostic impact of SF3B1 mut in AML with increased RS. Disclosures: Jeromin: MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Bayer:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract: Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by varying degrees of cytopenias and a predisposition to acute myeloid leukemia (AML). With conspicuous clinical and biological heterogeneity in MDS, an optimized choice of treatment based on accurate diagnosis and risk stratification in individual patients is central to the current therapeutic strategy. Diagnosis and prognostication in patients with myelodysplastic syndromes (MDS) may be improved by high-throughput mutation/copy number profiling. Methods A total of 944 patients with various MDS subtypes were screened for gene mutations and deletions in 104 known/putative genes relevant to MDS using targeted deep-sequencing and/or array-based genomic hybridization. Impact of genetic lesions on overall survival (OS) was investigated by univariate analysis and a conventional Cox regression, in which the Least Absolute Shrinkage and Selection Operator (lasso) was used for selecting variables. The linear predictor from the Cox regression was then used to assign the patients into discrete risk groups. Prognostic models were constructed in a training set (n=611) and confirmed using an independent validation cohort (n=175). Results After excluding sequencing/mapping errors and known or possible polymorphisms, a total of 2,764 single nucleotide variants (SNVs) and insertions/deletions (indels) were called in 96 genes as high-probability somatic changes. A total of 47 genes were considered as statistically significantly mutated (p 〈 0.01). Only 6 genes (TET2, SF3B1, ASXL1, SRSF2, DNMT3A, and RUNX1) were mutated in 〉 10% of the cases. Less common mutations (2−10%) involved U2AF1, ZRSR2, STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS, PHF6, BRCC3, ETV6, and LAMB4. Intratumoral heterogeneity was evident in as many as 456 cases (48.3%), even though the small number of gene mutations available for evaluation was thought substantially to underestimate the real frequency. The number of observed intratumoral subpopulations tended to correlate with the number of detected mutations and therefore, advanced WHO subtypes and risk groups with poorer prognosis. Mean variant allele frequencies (VAFs) showed significant variations among major gene targets, suggesting the presence of clonogenic hierarchy among these common mutations during clonal evolution in MDS. The impact of these genetic lesions on clinical outcomes was initially investigated in 875 patients. In univariate analysis, 25 out of 48 genes tested significantly affected overall survival negatively (P 〈 0.05), and only SF3B1mutations were associated with a significantly better clinical outcome. Next, to evaluate the combined effect of these multiple gene mutations/deletions, together with common clinical/cytogenetic variables used for IPSS-R, OS was modeled by a conventional Cox regression. A total of 14 genes, together with age, gender, white blood cell counts, hemoglobin, platelet counts, cytogenetic score in IPSS-R, were finally selected for the Cox regression in a proportional hazard model and based on the linear predictor of the regression model, we constructed a prognostic model (novel molecular model), in which patients were classified into 4 risk groups showing significantly different OS (“low”, “intermediate”, “high”, and “very high risk”) with 3-year survival of 95.2%, 69.3%, 32.8%, and 5.3%, respectively (P 〈 0.001). These results demonstrated that the mutation/deletion status of a set of genes could be used as variables independent of clinical parameters to build a clinically relevant prognostic score. When applied to the validation cohort, the novel molecular model was even shown to outperform the IPSS-R. Conclusions Large-scale genetic and molecular profiling by cytogenetics, NGS and array-CGH not only provided novel insights into the pathogenesis and clonal evolution of MDS, but also helped to develop a powerful prognostic model based on gene mutations and other clinical variables that could be used for risk prediction. Molecular profiling of multiple target genes in MDS is feasible and provides an invaluable tool for improved diagnosis, biologic subclassification and especially prognostication for patients with MDS. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Bacher:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract: The BRAFV600E mutation was recently detected in hairy cell leukemia (HCL) by whole exome sequencing. To make use of this new marker for diagnosis and follow-up of HCL, we developed a BRAFV600Emut-specific quantitative real-time PCR assay and validated it in 117 HCL patients and 102 non-HCL/BRAFwt patients. The cut-off level to discriminate BRAFV600E-positive/-negative cases was set at 0.023% BRAFV600E/BRAFwt. A total of 115 of 117 HCL (98.3%) demonstrated percentage BRAFV600E/BRAFwt above the cut-off (mean, 29.6 ± 41.1). The remaining 2 of 117 HCL with lower percentage BRAFV600E/BRAFwt ratios were also BRAFwt by deep-sequencing technology. Sixteen HCL-variant patients showed percentage BRAFV600E/BRAFwt values corresponding to “non-HCL.” Follow-up studies in 19 HCL cases demonstrated a decrease of percentage BRAFV600E/BRAFwt during therapy. The log-reductions as determined by RT-PCR and immunophenotyping correlated significantly (P 〈 .001). In conclusion, we confirmed BRAFmut as a useful marker in HCL, its absence in HCL variant, and developed an RT-PCR-based assay to monitor minimal residual disease in HCL.

Abstract: Abstract 1394 Background: Acute erythroid leukemia (AEL) is characterized by a predominant erythroid population and is comprising 〈 5% of adult AML cases. Because of the relative rarity of AEL, few large studies have examined underlying clinical and genetic features. Aims: Molecular and cytogenetic characterization of AEL and identification of genes with prognostic impact. Patients and Methods: We studied an unselected cohort of 94 AEL patients including 32 female and 62 male cases; median age was 69.0 yrs (range: 21.3–88.3 yrs). Survival data was available in 73 cases; median survival was 15.9 months. First, chromosome banding analysis (n=94) was performed. In addition, all cases with normal karyotype (NK) were investigated by CGH arrays (n=32) (Human CGH 12×270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation screening for ASXL1 (n=87), CEBPA (n=94), DNMT3A (n=94), FLT3 (both internal tandem duplication (ITD) (n=93), and tyrosine-kinase domain (TKD) mutations (n=85)), IDH1 (n=93), IDH2 (n=65), NRAS (n=91), KRAS (n=93), MLL-PTD (n=79), NPM1 (n=94), RUNX1 (n=94), TP53 (n=94), and WT1 (n=90) was performed by 454 amplicon deep-sequencing (Roche, Branford, CT), Sanger sequencing or melting curve analyses. CGH array data analysis was performed using Nexus Copy Number 6.0 (BioDiscovery Inc, El Segundo, CA). Results: Cytogenetic data was available for all cases: 48 cases (51.1%) presented an intermediate-risk and 46 (48.9%) cases an unfavorable cytogenetic category according to the MRC Classification. By CGH array analysis 30/32 cases retained a NK, whereas in two cases small aberrations were detected: case 1: deletion of the CEBPA gene, case 2: duplication 11q13.3 to 11q25 including the ATM and MLL gene. Molecular mutations were detected in 85/94 patients (90.4%). 63.5% (54/85) of mutated patients carried one, whereas 36.5% (31/85) of cases harbored two (n=22) or more (n=9) mutations. In detail, TP53 was the most frequently mutated gene (41 cases, 43.6%). Other alterations were detected in NPM1 (15/94; 16.0%); DNMT3A (12/94; 12.8%); ASXL1 (8/87; 9.2%); MLL-PTD (7/79; 8.9%); RUNX1 (8/94; 8.5%); IDH1 (6/93; 6.5%); WT1 (5/90; 5.6%); IDH2 (3/65; 4.6%); NRAS (3/91; 3.3%); KRAS (3/93; 3.2%); FLT3-ITD (3/93, 3.2%), FLT3-TKD (3/85, 3.5%), and CEBPA (1/94). First, we were interested in any correlation with the respective karyotype and observed that NPM1, RUNX1, and WT1 mutations correlated with an intermediate-risk karyotype (NPM1: 15/48 vs 0/46, P 〈 0.001; RUNX1: 8/48 vs 0/46, P=0.006; WT1: 5/46 vs 0/44, P=0.056), whereas TP53mut correlated with the unfavorable karyotype (38/46 vs 3/48, P 〈 0.001). Within the cytogenetically adverse subset TP53mut were associated with complex karyotype (36/38 vs 2/8, P 〈 0.001). In addition, NPM1mut correlated with lower age (56±15 vs 67±13 yrs, P=0.002), whereas mutations in ASXL1, DNMT3A, and TP53 correlated with higher age (73±4 vs 64±15, P=0.001; 71±6 vs 65±14, P=0.015; 71±8 vs 61±15, P 〈 0.001). NPM1mut were associated with longer, and RUNX1mut and TP53mut with shorter OS (OS after 2 yrs: NPM1mut vs wt: 85.1% vs 28.3%, P=0.001; RUNX1mut vs wt: 0% vs 45.2%, P=0.007; TP53mut vs wt: 9.4% vs 61.6%, P=0.001). In the univariable Cox regression analyses mutations in NPM1 (HR 0.12; P=0.004), RUNX1 (HR 3.99; P=0.013), TP53 (HR 3.19; P=0.001), age (HR 4.24, P=0.001) and adverse cytogenetics (HR 2.98, P=0.002) were significantly associated with OS. Independent prognostic factors in multivariable Cox regression analysis were: age (HR 2.6, P=0.047) and RUNX1mut (HR 6.3, P=0.006). Of note, when separating MRC intermediate from MRC adverse cases, we confirmed the longer OS of NPM1 and shorter OS of RUNX1 mutated cases in comparison to NPM1, RUNX1 wt cases (OS after 2 yrs: NPM1mut vs wt: 85.1% vs 46.3%, P=0.027; RUNX1mut vs wt: 0% vs 69.0%, P 〈 0.001). Conclusions: (1) The frequency of cases with complex or other adverse karyotypes within the AEL cohort is very high (48.9%), (2) 93.7% of cases with NK also showed a NK using high-resolution CGH arrays. (3) Overall, a remarkably high mutation frequency of 90.4% was found. (4) NPM1 and RUNX1mut were exclusively detected in the cytogenetically intermediate-risk MRC, TP53 mut predominantly in the MRC adverse group and mainly in cases with complex karyotype. (5) In addition to chromosome banding analysis mutation screening of RUNX1 and NPM1 in cases with intermediate-risk karyotype should be considered for better prognostication. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Bacher:MLL Munich Leukemia Laboratory: Employment. Poetzinger:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Fasan:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Staller:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract: In patients with myelodysplastic syndromes (MDS), sole 20q deletion [del(20q)] is a recurrent favourable abnormality. We studied additional molecular and cytogenetic lesions and their prognostic impact in 305 MDS patients with del(20q) (229 males/76 females; 29–90 years). All patients were investigated by cytomorphology and chromosome banding analysis (CBA), subsets by fluorescence in situ hybridization, molecular mutation screening, and array comparative genomic hybridization ( aCGH ). By aCGH ( n  = 30), the minimal common deleted region (CDR) was flanked by PTPRT (20q13·11) and EYA2 (20q13·12). 210 (68·9%) patients had ‘early MDS’ without blast increase, 95 (31·1%) ‘advanced’ MDS with blast increase (5–19%). Additional chromosomal abnormalities (ACAs) were detected in 88/305 (28·9%) patients. Patients with advanced MDS more frequently had ACAs ( P  = 0·003) and had a higher mean number of ACAs ( P  =   0·020) and of molecular mutations ( P  =   0·060). Spliceosome mutations were frequent ( U2AF1 : n  = 31/155; 20·0%; SRSF2 : n  = 31/159; 19·5%; SF3B1 mut: n  = 8/159; 5·0%). ASXL1 mut (25/153; 16·3%) were associated with advanced MDS ( P  =   0·001). Presence of ≥3 ACAs ( P  =   0·003) and ASXL1 mut ( P  =   0·002) were associated with worse 2‐year survival. In conclusion, the cytogenetic subgroup of MDS with del(20q) has a good prognosis but may be further subclassified by additional cytogenetic and molecular lesions. U2AF1 mut is overrepresented in MDS with del(20q), and ASXL1 mut is prognostically adverse.

Abstract: In 2008, the World Health Organization introduced CEBPA (encoding the CCAAT/enhancer binding protein)–mutated acute myeloid leukemia (AML) as a provisional entity. However, the classification of CEBPA-mutated AML with multilineage dysplasia (MLD; ≥ 50% dysplastic cells in 2-3 lineages) remains to be clarified. In the present study, we investigated 108 CEBPA-mutated AML patients for the impact of MLD, karyotype, and additional mutations. MLD+ patients differed from MLD− patients only by lower mean WBC counts, not by biologic characteristics, cytogenetic risk profiles, or additional mutations. Survival was better for female patients, patients 〈 60 years of age, for intermediate versus adverse karyotypes, and, in the case of FLT3-ITD negativity, biallelic versus monoallelic/homozygous CEBPA mutations. In contrast, 2-year overall survival and event-free survival did not differ significantly between MLD+ and MLD− patients. By univariable Cox regression analysis, sex, age, WBC count, and cytogenetic risk category were related to overall survival, but MLD was not. Therefore, because dysplasia is not relevant for this subtype, CEBPA-mutated AML patients should be characterized only according to mutation status, cytogenetic risk group, or additional mutations.

Abstract: In patients with myelodysplastic syndromes (MDS), a 20q deletion [del(20q)] is considered to define a cytogenetic subgroup and, if present as a sole cytogenetic aberration, is associated with a favorable prognosis according to the revised IPSS (IPSS-R). However, associated genetic lesions and their prognostic impact in MDS with del(20q) remain to be clarified. Study Design We studied 305 MDS patients with del(20q) for associated molecular and cytogenetic markers and clinical outcomes. All patients (229 males/76 females; median age, 74 yrs) were investigated by cytomorphology (CM) and chromosome banding analysis (CBA). Additionally, subsets were characterized by fluorescence in situ hybridization (FISH), molecular genetics including high-throughput sequencing (NGS), and array comparative genomic hybridization (aCGH). Results A total of 210 (68.9%) patients had “early” MDS with blasts below 5%, and 95 (31.1%) “advanced” MDS i.e. RAEB-1/-2. When only patients with a sole del(20q) (n=217) were considered, 161 (74.2%) had early MDS, 56 (25.8%) had advanced MDS. This confirmed the preponderance of good-risk MDS subtypes in patients with del(20q). Additional chromosomal aberrations (ACAs), e.g. -Y, del(5q), -7/del(7q), and +8, were detected in 88/305 (28.9%) patients. Cytogenetic complexity was higher in advanced MDS patients as they showed a higher frequency of ACAs than early MDS (39/95; 41.1% vs. 49/210; 23.3%; P=0.003) and a higher mean number of ACAs (mean±SD, 1.3±2.7 vs. 0.6±1.5; P=0.020). The minimal commonly deleted region (CDR) was determined using aCGH (n=30 investigated) and was located from position 41,067,253 to 45,700,000 on 20q13.11-q13.12 (4.6 Mb in size). The flanking genes of the minimal CDR were PTPRT (receptor-type tyrosine-protein phosphatase T) on 20q13.11 and EYA2 (Eyes Absent Homolog 2) on 20q13.12. A total of 159 patients (104 early MDS, 55 advanced MDS) were investigated for additional molecular mutations, 82 patients (51.6%) had at least one mutation. The mean number of molecular mutations per patient was higher in advanced MDS as compared to early MDS (0.9±0.9 vs. 0.6±0.7; P=0.060). In detail, spliceosome mutations were frequent (U2AF1: n=28/140 investigated; 20.0%; SRSF2: n=29/159; 18.2%; SF3B1: n=8/159; 5.0%) and were mutually exclusive and equally distributed in early und advanced MDS. ASXL1mut which were found in 25/153 cases (16.3%) investigated in the total cohort showed a significantly lower frequency in early MDS as compared to advanced MDS (9/100; 9.0%; vs. 16/53; 30.2%; P=0.001). Lower mutation frequencies we observed for RUNX1 (14/157; 8.9%) and MLL-PTD (2/134; 1.5%). The 2-year overall survival (2-yr OS) was better in patients with early MDS as compared to advanced MDS (92.3% vs. 73.7%; P=n.s.). Patients with a sole del(20q) showed similar 2-yr OS to patients with del(20q) and occurrence of ACAs (87.1% vs 81.1%, P=n.s.). Patients with ASXL1mut had significantly worse 2-yr OS as compared to wild-type ASXL1 (45.5% vs 87.9%; P=0.002). The SRSF2 and U2AF1 mutation status did not significantly impact survival. Conclusion The cytogenetic subgroup of MDS with del(20q) has a good prognosis but may be further subclassified by additional cytogenetic and molecular lesions. U2AF1 mutations were overrepresented in MDS with del(20q) as compared to published frequencies in overall MDS. ASXL1 mutations were relevant for disease progression. Therefore, analyses of molecular mutations may contribute to prognostication and to therapeutic decisions in patients with MDS and del(20q). Disclosures: Bacher: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Krauth:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.