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Introduction: In acute myeloid leukemia (AML), the karyotype and the molecular mutation profile are the strongest parameters for classification and prognostication. Yet, diagnostic analyses rely on chromosome analysis and sequencing of a constantly growing number of genes. Aim: To evaluate whether whole exome sequencing (WES) can reliably identify copy number states and molecular mutations in a single-step procedure. Patients and Methods: The cohort included 24 AML with an aberrant karyotype at initial diagnosis (ID) who achieved cytogenetic remission (CR) after chemotherapy. Patients showed complex karyotype (n=6), 11q23/MLL-rearrangement (n=4), t(15;17)(q24;q21) (n=4), inv(16)(p13q22) (n=4), t(8;21)(q22;q22) (n=3), and 3q26/EVI1-rearrangement (n=3). For WES DNA was extracted from bone marrow and treated with the TruSeq Exome enrichment kit targeting 201,071 exons. 2x100 bp paired-end sequencing was performed on an Illumina HiSeq machine (Illumina, San Diego, CA) at Fasteris (Geneva, Switzerland). After mapping the sequenced reads with Burrows-Wheeler Aligner [Li&Durbin, Bioinformatics, 2009], variants where called with GATK [McKenna et al., Genome Res., 2010] and copy number variations (CNV) were detected by Excavator [Magi et al., 2013, Genome Biol.]. For validation of the detected variants, 21 leukemia related genes were screened by amplicon sequencing (Illumina MiSeq, or Roche 454, Branford, CT). Array-based comparative genomic hybridization (aCGH) using 12x270K microarrays (Roche NimbleGen, Madison, WI) or 4 x 180K microarray slides (Agilent Technologies, Santa Clara, CA) was performed on all samples. We called CNV using default settings as well as fixed thresholds on the probe medians (0.3 for gains and -0.5 for losses on probe medians and at least 10 probes per segment). Results: The targeted regions were covered by 86 reads on average, while 90% of the bases were covered by at least 15 reads. By comparing ID and CR we detected an average of 15 somatic single nucleotide variants and short indels per patient (range 4-25), affecting 303 genes in total, including genes involved in leukemogenesis. After excluding polymorphisms we screened the mutated genes for recurrence among all cases. Four genes were mutated in at least 3 samples: WT1 (n=5), TP53 (n=4), NRAS (n=3) and TNS1 (n=3). Fourteen genes were mutated in 2 samples: ASXL2, DSCAM, GATA2, IDH2, KIT, OR4C5, POU4F1, LOC93432, RPTOR, SMC1A, SYNE2, TET2, TTN and USP9X. Mutations in OR4C5, LOC93432, SYNE2, TTN and USP9X have not been associated with AML yet. They were rated as damaging according to the SIFT algorithm [Ng and Henikoff, Genome Res., 2003]. In a prior diagnostic work-up 21 different genes had been screened and revealed 16 mutations affecting 7 genes. WES identified 14 mutations correctly (the 2 remaining mutations were covered by reads only insufficiently) and did not call any mutation in genes classified as negative in the routine diagnostic work-up. We further compared CNV derived from WES and aCGH in all 24 patients. Gains and losses detected by aCGH involved 2.65 and 1.40 billion bp, respectively. 96% of bp involved in these CNV were also detected by WES. Of the regions in which WES could not reproduce CNV calls, 15% did not contain exons. WES called gains and losses covering in total 2.56 and 1.47 billion bp, respectively. With aCGH we detected 98% of the gains and 86% of the losses. Regions missed by aCGH did show concordant signal that did not pass the fixed thresholds. However, while relaxing the thresholds to default settings, aCGH reproduces 99% of the WES results. Thus, an excellent concordance was observed (R = 0.99, p < 2.2e-16). We further analysed 19 cytogenetically balanced rearrangements that caused 42 breakpoints in affected chromosomes in 17 patients. As most breakpoints occur in non-coding regions, WES in general is limited in detecting these balanced rearrangements. However, short CNV were detected by WES in 10 cases and confirmed by aCGH. Conclusion: WES was capable of delineating molecular mutation profiles and of robustly detecting copy number states in AML at diagnosis. We suggest that WES in combination with multiplex RT-PCR-based techniques for the detection of recurrent fusion transcripts is a promising approach for a future diagnostic work-up for AML classification and prognostication. This project has been funded by the Seventh Framework Programme (FP7/2007-2013) under grant agreement n. 306242. Disclosures Althammer: MLL Munich Leukemia Laboratory: Employment; Seventh Framework Programme (FP7/2007-2013): Research Funding. de Albuquerque:MLL Munich Leukemia Laboratory: Employment; Seventh Framework Programme (FP7/2007-2013): Research Funding. Nadarajah:MLL Munich Leukemia Laboratory: Employment; Seventh Framework Programme (FP7/2007-2013): Research Funding. Meggendorfer:MLL Munich Leukemia Laboratory: Employment; Seventh Framework Programme (FP7/2007-2013): Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Seventh Framework Programme (FP7/2007-2013): Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Seventh Framework Programme (FP7/2007-2013): Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Seventh Framework Programme (FP7/2007-2013): Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Seventh Framework Programme (FP7/2007-2013): Research Funding.

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological disorders that can progress to secondary acute myeloid leukemia (s-AML). We performed a comprehensive approach based on cytomorphology, cytogenetics and amplicon deep sequencing using a newly developed pan-myeloid gene panel in order to better clarify this process. Methods: Paired samples from 38 patients at initial diagnosis of MDS and at progression to s-AML were analyzed for the following genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3TKD,FLT3-ITD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1,WT1 and ZRSR2. With exception of RUNX1 and ZRSR2 (sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT)) and MLL-PTD and FLT3-ITD (analysed by PCR), all remainder genes were sequenced using a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA). A median coverage of 7,626 reads (range 174-12,256) per gene was achieved. The lower limit of detection was set at 3%. Results: At MDS diagnosis, patients were classified according to WHO: RARS (n=2), RCMD (n=3), RCMD-RS (n=8), RAEB-1 (n=12), RAEB-2 (n=12), and MDS with isolated 5q deletion (n=1). Progression to s-AML was defined by blasts >20% in bone marrow or peripheral blood. Transformation to AML occurred at a median time of 18 months (range 2 – 73) after MDS diagnosis. According to chromosome banding analysis and FISH, 14 (36.8%) MDS and 22 (57.9%) s-AML patients had an aberrant karyotype. Thirteen patients (34.2%; 8 with normal and 5 with already aberrant karyotype) gained chromosome abnormalities during progression to s-AML. All samples presented at least one mutation. The most frequent mutated genes were identical for MDS and s-AML: ASXL1 (36.8% / 42.1%); RUNX1 (36.8% / 39.5%); SF3B1 (23.7% / 23.7%); SRSF2 (47.4% / 47.4%) and TET2 (34.2% / 34.2%). Mutations in GATA1 and MPL were not found. Only one KIT-mutation was detected in a patient at MDS stage, while FLT3-TKD mutations were identified in s-AML samples only (7.9%; 3/38 patients). Results for MLL-PTD and FLT3-ITD were available for 58 samples (76.3%). The incidence of MLL-PTD was low (4.5%, 1/22 for MDS and 8.3%; 3/36 in s-AML). FLT3-ITD mutations were exclusively detected in 5 patients (14.3%) at s-AML stage. A total of 21 (55.3%) patients won at least 1 mutation during progression to s-AML. Surprisingly none of the gene mutation frequencies differed significantly between the MDS and s-AML stages. Therefore, we further compared this data set to an independent cohort of 944 newly diagnosed MDS patients (Haferlach T et al, Leukemia 2014). WHO diagnosis categories were comparable between both cohorts. However, mutation frequencies differed: in the present cohort the following mutations were more frequent than in the published cohort: GATA2 (5.3% vs 0.7%; P=0.044), IDH2 (18.4% vs 3.9%; P=0.001), RUNX1 (36.8% vs 10.6%; P= MDS patients with DNMT3Amut transformed faster to AML than DNMT3A wild-type (wt) cases (median of 8 vs 19 months; P= 0.022). Intriguingly, also SF3B1mut were associated with a faster s-AML progression compared to SF3B1wt (median 8 vs 19 months; P= 0.008). All mutations that were lost at the time of s-AML transformation were small clones (mutation load ≤10%), which hypothetically could have succumbed to more aggressive clones leading to transformation to AML. In detail, in 4 patients with mutation loss there was an increase in the loads of the remaining mutations, whereas their karyotype remained stable during s-AML transformation. On the other hand, the remaining 3 patients with mutation loss at the time of s-AML diagnosis had stable mutation loads in the remaining mutated genes but had a cytogenetic progression. Conclusions: 1) The molecular mutation profile remains stable between MDS and s-AML state in most of the cases and mutations were gained or lost in only a few cases. 2) FLT3-ITD was only detected at AML stage. 3) Mutations in GATA2, IDH2, RUNX1 and SRSF2 seem to predispose to s-AML transformation and mutations in DNMT3A and SF3B1 lead to a significantly faster s-AML progression. 4) Shifts in the karyotype were detected in more than one third of patients underlining its impact on s-AML transformation. Disclosures De Albuquerque: MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Introduction Azacitidine (aza) was the first drug to demonstrate a survival benefit for MDS patients (pts) in a randomized trial [Fenaux, Lancet Oncol 2009]. However, only about half of the pts respond and even after achieving complete remission (CR) and with sustained treatment, pts usually relapse. To date, limited data is available on prognostic factors influencing response. Therefore, until now we have analyzed 71 pts with MDS or AML, treated with at least 3 cycles of aza, to identify prognostic indicators that might help to improve treatment decisions. Patients and Methods 71 cases were included and characterized by cytogenetics and various molecular assays including sensitive next-generation sequencing for mutations in ASXL1, DNMT3A, EZH2, FLT3-LM, IDH1, IDH2, KRAS, MLL-PTD, NRAS, RUNX1, SF3B1, SRSF2, TET2, and TP53. Median age was 71 (50-84) years. 38 pts had MDS (8 RAEB I, 26 RAEB II, 4 RCMD), 28 had AML (6 dysplastic, 16 sAML/MDS, and 6 tAML/MDS), and 5 pts had MDS/MPD. The IPSS [Greenberg, Blood 1997] was low in one, intermediate (int) 1 in 4, int 2 in 26, and high in 14 pts. Regarding cytogenetic risk groups we found 39 low, 11 int and 20 high risk pts according to IPSS and 49 int vs 21 high risk pts according to AML criteria [Grimwade, Blood 2010]. Median time from diagnosis to treatment was 122 days (0-2348). With a median follow up of 1091 days from start of treatment median survival was 530 days (95% CI 412-874). Pts received a median number of 6 cycles (3-43). 12 pts have received allogeneic transplantation after aza treatment and were censored at that date. To assess the impact of biomarkers, Kaplan-Meier curves and Cox models were estimated from the first day of treatment. Variables were considered for multivariate analysis if the univariate p value was Results Regarding cytogenetics, 47% of pts exhibited a normal karyotype, 17% had chromosome 7 abnormalities, 13% of karyotypes included del5q, 13% +8, 7% del20q, and 13% were complex. All except one case showed molecular aberrations. The most frequently mutated gene was ASXL1 (42%), followed by RUNX1 (38%), SRSF2 (38%), TET2 (30%), TP53 (18%), DNMT3A (16%), IDH2 (13%), NRAS (13%), EZH2 (12%), SF3B1 (11%), IDH1 (9%), FLT3-ITD (8%), MLL-PTD (7%), and KRAS (6%). Responses were observed in 33 pts (46%; 4 CR, 3 marrow CR with hematological improvement, 8 partial remission, and 18 HI). When cytogenetic abnormalities were examined, responses were seen in 45% of pts with normal karyotype, in 50% of pts with chromosome 7 aberrations, in 33% with del5q, in 67% with +8, in 56% with complex karyotype, and in all 5 cases with del20q (p=0.02). Regarding molecular abnormalities response rate was highest in pts with mutations in NRAS (67%) and IDH2 (63%), followed by RUNX1 (56%), ASXL1 (54%), SF3B1 (43%), TP53 (42%), SRSF2 (36%), TET2 (30%), DNMT3A (30%), MLL-PTD (25%), KRAS (25%), FLT3-ITD (20%), IDH1 (17%), and EZH2 (13%). However none of these mutations had a significant impact on response rate, possibly due to limited statistical power. The only p-value Finally we analyzed the influence of prognostic markers on survival in aza treated pts. In univariate analysis potential candidate variables were: TP53 (p= In multivariate analysis TP53 retained a significant influence on survival (HR: 4.99 [CI: 2.20-11.31], p TP53 mutations negatively influenced survival, although response rates were comparable to other pts. Response duration was often short. However, pts might still benefit from aza treatment, if combination therapy (e.g. lenalidomide in del5q and TP53 positive pts) or allogeneic transplantation consolidate and prolong treatment results Conclusion After the analysis of 71 pts, TP53 mutations represent the only somatic mutations with a significant negative impact on survival in aza treated pts. The small group of pts with del20q might have higher response rates. The analysis of further pts is ongoing. Disclosures: Kuendgen: Celgene: Honoraria, Research Funding. Off Label Use: Azacitidine in AML >30% marrow blasts. Lauseker:Celgene: Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Alpermann:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Gattermann:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Germing:Celgene: Honoraria, Research Funding. Götze:Celgene Corp: Honoraria.

Introduction Massively parallel next-generation sequencing (NGS) data have changed the landscape of molecular mutations in chronic lymphocytic leukemia (CLL). The number of molecular markers continues to constantly increase. As such, physicians and laboratories face a great unmet yet challenging need to test panels of genes at a high level of sensitivity. Aim To develop an assay that is easily adoptable to adjust gene targets and amplicons according to current state-of-the-art evidence regarding the published landscape of mutations in CLL. Methods We developed a sensitive deep-sequencing assay for routine diagnostics. In total, 13 genes with relevance in CLL providing in part adverse prognostic information were chosen: ATM, BIRC3, BRAF (V600), FBXW7, KLHL6, KRAS, NOTCH1 (PEST domain), NRAS, MYD88, POT1, SF3B1 (HEAT repeats), TP53, and XPO1. Targets of interest comprised either complete coding gene regions or hotspots. In summary, 323 amplicons were designed with a median length of 204 bp (range 150-240 bp), representing a total target sequence of 39.36 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient using a single-plex microdroplet-based assay (RainDance, Lexington, MA). Sequencing data was generated using the MiSeq instrument (Illumina, San Diego, CA) loading up to 10 patients per run. The total turn-around time of the assay was less than 5 days. As a proof-of-principle cohort, 18 clinically well-annotated CLL patients were analyzed during the evaluation phase. The median age was 78 years (range: 52 – 87 years). Results Using the 500 cycles sequencing by-synthesis chemistry, in median 7,262 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 7,476 reads (range: 5,595 - 10,337). (1) In this cohort of 18 cases, a total of 71 mutation analyses had already been previously performed for eight of the 13 genes using either capillary Sanger sequencing or alternative amplicon deep-sequencing assays (454 LifeSciences or Illumina MiSeq). In detail, in these 8 genes these 71 assays detected 56 known polymorphisms or mutations in ATM (n=8), BIRC3 (n=6), FBXW7 (n=4), MYD88 (n=4), NOTCH1 (n=10), SF3B1 (n=5), TP53 (n=14), and XPO1 (n=4) and 28 analyses revealed a wild-type status. When comparing these results with data obtained using the 13-gene NGS panel, in all 84/84 (100%) parallel assessments concordant results were obtained underlining the robustness of this assay. (2) Overall and extending the previous results, the comprehensive 13-gene NGS panel then detected in 18/18 patients a total of 46 mutations in 10 of the 13 genes with a range of 1-5 mutations per case (median: 2). The mutation types comprised 22 missense, 4 nonsense, 16 frame-shifts, 3 insertions and 1 splice-site alterations. In median, the coverage per variant was 10,390-fold, thus enabling a sensitive detection of mutations at a lower limit of detection set at 3%. The mutation burden ranged from 3.0% to 62.0%. 18/46 (39.13%) mutations were detected with a clone size Conclusion We demonstrated that microdroplet-based sample preparation enabled to robustly target 13 genes for next-generation sequencing in a routine diagnostics environment. This included also larger gene targets such as ATM, being represented by 119 amplicons. Thus, this approach provides the potential to screen for prognostically relevant mutations in all CLL patients in a fast and comprehensive way providing actionable information suitable to guide therapy. Disclosures: Kohlmann MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Introduction Molecular mutation analyses are performed in myeloid malignancies either in a stepwise procedure, i.e. one target gene after each other or are not performed at all, e.g. in low-risk MDS. A comprehensive pan-myeloid panel to simultaneously target mutations in 26 genes allows a comprehensive analysis with the perspective to detect disease defining mutations in the majority of patients. Aims To test the utility of a pan-myeloid panel in routine diagnostics. Methods We developed sensitive next-generation deep-sequencing (NGS) assays comprising in total 26 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. With the exception of RUNX1, which was sequenced on the 454 Life Sciences NGS platform (Branford, CT), all remainder genes were studied using a combination of a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA). The assay's turn-around time was less than 6 days, loading up to eight patients per sequencing run. In summary, 389 amplicons were designed with a median length of 206 bp (range 150-240 bp), representing a total target sequence of 78.15 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient, purified from isolated mononuclear cells. Using the 500 cycles sequencing-by-synthesis chemistry in median 7.644 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 7,626 reads (range 174-12,256). The lower limit of detection was set at a cut-off of 3%. Results Thus far, 191 prospectively collected cases have been analyzed during routine operations. In all cases the assay was successfully performed. Mutations (range 0-7) have been found in 119/191 (62.3%) cases. The major disease categories were as follows: MDS (n=76), suspected MDS (n=28), MDS/MPN (n=10), reactive bone marrow conditions (n=46), AML (n=8), CML (n=3), other conditions (n=20). We first were interested to address the utility of the panel in MDS when the analysis was restricted to the five prognostically relevant predictors of poor overall survival according to Bejar et al. (N Engl J Med. 2011;364:2496-506), i.e. ASXL1, ETV6, EZH2, RUNX1, and TP53. In detail, 69 cases with MDS were studied and in 42.0% (29/69) of cases mutations had been detected in these five genes while 40 patients showed no mutation. Interestingly, upon extending the analysis to the remainder 21 genes, at least one more mutation was discovered in 72.5% (29/40) of these cases, thereby extending the number of cases with at least one mutation to 84.1% (58/69) of patients. Of note, in 65.5% (19/29) of these latter cases, spliceosome mutations occurred in a mutually exclusive manner (SRSF2, SF3B1, U2AF1), thus also detecting mutations conferring a favorable clinical outcome, i.e. SF3B1 alterations. We next studied in more detail 28 cases with suspected MDS according to cytomorphology, i.e. cases with dysplastic features not sufficient to diagnose MDS. When again in a first step the five predictors of poor overall survival according to Bejar et al. were analyzed, mutations in ASXL1, ETV6, EZH2, RUNX1, and TP53 were observed in 25.0% of cases (7/28). In the group of 75.0% (21/28) of samples with no mutations according to Bejar et al., 28.6% (6/21) of cases harbored a mutation in the group of the 21 remainder genes analyzed simultaneously in the gene panel assay. Thus, in total the number of cases with at least one mutation increased to 46.4% (13/28) of patients. Of note, 6 of the 13 suspected MDS cases with mutations had a normal karyotype. In summary, with respect to correlations between these two cohorts, we observed that morphologically confirmed MDS cases (n=69) showed a higher number of mutated genes compared to “suspected MDS” cases (n=28) (1.88 vs 0.71; p Conclusion A pan-myeloid screening assay using NGS allows to address 26 relevant gene mutations in myeloid malignancies with diagnostic or prognostic impact. This approach is scalable and adoptable to accommodate the inclusion of novel gene targets according to the latest evidence from the literature. Importantly, given the broad spectrum of mutations in myeloid diseases covered by such a panel, mutations can be identified in the majority of patients and enable to support a more comprehensive classification in these complex diseases. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.