ALBERT

Description: Key Points EZH2 mutations occur in more than 25% of follicular lymphoma patients. Mutations predominantly represent an early/clonal event in the pathogenesis of the disease.

Description: Introduction In 2012, whole-genome, whole-exome and global transcriptome sequencing studies unraveled the molecular complexity of Burkitt lymphoma (BL). Amongst a set of up to 70 recurrently mutated genes (Love C. et al., Nat. Genet. 2012), essential affected oncogenic pathways were discovered including mutations in MYC and the transcription factor TCF3 (Schmitz R. et al., Nature 2012), as well as inactivating mutations of ID3 (Richter J. et al., Nat. Genet. 2012). Aims (1) To validate the mutation frequency and mutational landscape of selected candidate target genes (ID3, MYC, TCF3, and TP53) in an independent cohort of 60 adult patients with Burkitt Lymphoma/Leukemia. (2) To investigate the clonal composition and associations with BCL2 rearrangements using a quantitative next-generation sequencing assay. Methods The patient cohort included 33 cases with Burkitt Leukemia (33/60, 55.0%) and 27 cases with Burkitt Lymphoma (27/60, 45.0%). 36 (60.0%) of these were single-hit lymphomas only harboring MYC-rearrangements, whereas 24 (40.0%) cases were multiple-hit lymphomas harboring in addition at least one of the following gene rearrangements: BCL2, BCL6, and/or CCND1. The median age was 60.8 years (range 5.5 - 82.7). All cases were comprehensively characterized by cytomorphology, cytogenetics and FISH, including evaluation for MYC-, BCL2-, BCL6-, and CCND1-rearrangements. Four candidate genes were sequenced using a quantitative deep-sequencing NGS assay (median coverage of 633 reads per mutation). The lower limit of detection of mutations was set at 2%. Amplicon sequencing libraries were prepared using genomic DNA extracted from mononuclear cells. The following regions were investigated: ID3 (exons 1-2), MYC (exons 2-3), TCF3 (exons 18), and TP53 (exons 4-11), respectively. Results In total, 112 mutations were detected in 39/60 (56.5%) patients. 9/112 (8.0%) were detected with a clone size of

Description: Abstract 70 Intrachromosomal amplification of chromosome 21 (iAMP21) represents a distinct cytogenetic subgroup of BCP-ALL, in which patients experience a high-risk of relapse on standard treatment protocols. The abnormal chromosome 21 defining iAMP21 has a heterogeneous, complex profile at the genomic level. This complexity has made it difficult to elucidate target genes or the initiating mechanism giving rise to iAMP21 using standard genomic approaches. In this study, detailed genomic and mutational analysis has highlighted potential novel targets in the development of iAMP21 BCP-ALL. DNA was available from 45 iAMP21 patient samples. Patient 1 was a 10 year old female; her diagnostic karyotype was 47,XX,+10,der(21)dup(21)(q?)r(21)(q?). Fluorescence in situ hybridization detected multiple copies of RUNX1, thus defining iAMP21. SNP 6.0 arrays indicated the characteristic genomic profile of chromosome 21, comprising a ∼30Mb (from 17–47Mb) region of copy number gain/amplification. Many of the breakpoints occurred within the Down Syndrome Critical Region (DSCR), specifically within the gene, DSCAM at 41.4Mb, and a telomeric deletion was identified with a breakpoint within the gene, TSPEAR at 45.9Mb. Chromosome 7 abnormalities were frequent, with one deletion including IKZF1 at 50.3Mb. The IKZF1, ETV6 and RB1 deletions seen by SNP 6.0 arrays were confirmed by Multiplex Ligation Probe-dependent Amplification (MLPA) and quantitative PCR. Whole-exome sequencing of the diagnostic and remission DNA of patient 1 identified 44 somatic mutations; 21 were computationally predicted to be potentially damaging to the function of the protein. The variant frequency of the individual somatic mutations ranged from 1.1% − 65.2%, indicating heterogeneity within the iAMP21 genome. The majority of the variants were detected at a frequency of

Description: Abstract 2539 Introduction: T-cell prolymphocytic leukemia (T-PLL) is a rare mature post-thymic T-cell neoplasm with aggressive clinical course and a median overall survival of less than one year. Due to the rareness of this disease (∼2% of cases of mature lymphocytic leukemias in adults) only few cases with cytogenetic and molecular genetic aberrations have been reported so far. However, almost 75% of T-PLL cases are reported to harbor chromosome 14 abnormalities involving the TCRA/D locus resulting in the aberrant activation of the proto-oncogenes TCL1A or MTCP1. Aim: Perform a comprehensive cytogenetic and molecular characterization of T-PLL. Patient and Methods: The cohort comprised 36 T-PLL cases diagnosed between 10/2005 and 07/2012 (23 male, 13 female patients). Median age was 71.0 yrs (range: 26.8–82.8 yrs). According to the WHO classification all T-PLL cases were characterized using immunophenotyping and cytomorphology. Patients were further investigated using chromosome banding analysis (CBA) (n=30), FISH for deletions of ATM (n=30), TP53 (n=29), and 13q (n=26), and CGH arrays (n=3, Human CGH 6×630K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI). Further, mutation analyses for BCOR and TP53 were performed using amplicon sequencing (Roche 454, Branford, CT). Results: Aberrant karyotypes were observed by CBA in 25/30 cases (83.3%). However, the 5 remaining cases with normal karyotype were due to insufficient proliferation of the T-PLL clone as gains, losses and rearrangements were detected in these 5 cases using FISH analyses. In more detail, combined CBA and FISH data revealed in 20/30 (66.7%) cases an inv(14)(q11q32)/t(14;14)(q11;q32)/TCRA/D-TCL1A (n=18) or t(X;14)(q27;q11)/TCRA/D-MTCP1 (n=2). Further, a gain of chromosome 8q and concomitant loss of 8p was observed in 13/30 (43.3%) cases. In addition, in 10/25 (40.0%) cases a 6q deletion and in 7/25 (28.0%) an 12p deletion were observed. Based on FISH data, deletions were detected of ATM in 19/30 (63.3%) cases, TP53 in 7/29 (24.1%), and 13q in 9/26 (34.6%) cases. In addition, 3 cases were studied using array CGH. Hereby, an intragenic deletion in the BCOR gene was observed in one patient. BCOR is a BCL6 corepressor and located on chromosome Xp11.4. BCOR mutations were recently described in cases with AML. BCOR mutation frequency was determined at 3.8% in AML with normal karyotype and mutations were associated with shorter overall and event-free survival. The deletion in BCOR identified in one of our cases and the TP53 deletions in 7 T-PLL cases prompted us to screen 35 cases for molecular mutations in these two genes. Overall, BCOR mutations were detected in 5/35 (14.3%) patients and TP53 mutations in 4/35 (11.4%) cases. In total, 7 missense, one frame-shift and one nonsense mutations were found. Median mutation load was 90.0% for BCOR (range: 38–100%) and 80.0% (59–87%) for TP53. Next, we performed correlation analyses between mutations in BCOR and TP53, rearrangements involving chromosome 14, deletions of ATM, TP53, 6q, 12p, and 13q and gain of 8q. Here, BCOR was not associated with any of these parameters. In contrast, TP53 mutations were accompanied in all 4 cases by TP53 deletions, while only 3/24 TP53 wild-type cases harbored a TP53 deletion (P=0.002). In addition, only one of 4 TP53 mutated cases harbored a chromosome 14 rearrangement while 18/25 (72%) TP53 wild-type cases did (P=0.105). In line with this result, TP53 deletions were also negatively associated with chromosome 14 rearrangements (2/7 vs 17/22, P=0.030). Further, all cases with a gain of 8q harbored a 14q rearrangement (13/13 vs 8/18 without gain of 8q, P=0.001). Conclusions: 1. CBA, FISH and mutation analysis of TP53 and BCOR revealed genetic abnormalities in all 36 analyzed cases. 2. The most frequent abnormality involved the TCRA/D locus (14q11) (20/30; 66.7%) activating the proto-oncogenes TCL1A on chromosome 14q32 (90.0%) or MTCP1 on chromosome Xq28 (10.0%). 3. Deletions were detected for ATM (63.3%), TP53 (24.1%), 6q (40.0%), 13q (34.6%), 12p (28.0%), and a gain was detected for the long arm of chromosome 8 (43.3%). 3. In addition to the detection of TP53 mutations in 11.4%, BCOR mutations were observed for the first time in a lymphatic malignancy with a mutation frequency of 14.3%. 4. The prognostic relevance of such cytogenetic and molecular genetic aberrations has to be determined in T-PLL, given that in myeloid malignancies both BCOR and TP53 are associated with shorter OS. Disclosures: 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.

Description: Abstract 2400 Background: CEBPA mutations occur in 5–14% of patients with acute myeloid leukemia (AML). A difference in clinical outcome between single- (sm) and double-mutated (dm) cases has been reported, whereupon dm cases were shown to be associated with longer overall (OS) and event-free survival (EFS). Aims: 1. Determine the frequency and clinical impact of CEBPA sm and dm in a large AML cohort. 2. Evaluate the spectrum of additional molecular mutations in CEBPA dm AML and their prognostic influence. Patients and Methods: First, we investigated CEBPA mutations in a cohort of 997 AML cases (462 female, 535 male; median age: 66.8 years) by 454 deep-sequencing (454 Life Sciences, Branford, CT). The cohort included: t(15;17)(q22;q12) n=33; t(8;21)(q22;q22) n=39; inv(16)(p13q22) n=31; normal karyotype (NK) n=447; complex karyotype (CK) (≥4 abnormalities) n=116; other abnormalities n=331. Second, we investigated an additional cohort of 111 AML dm CEBPA cases for mutations in ASXL1, DNMT3A, FLT3-ITD, FLT3-TKD, GATA2, IDH1/2, KRAS, MLL-PTD, NPM1, NRAS, RUNX1, TET2, TP53, and WT1 using 454 sequencing, Sanger sequencing, conventional PCR and melting curve analyses. This cohort was composed of 60 female and 51 male cases; median age: 62.3 years; 76 cases showed NK, 19 aberrant karyotype (n=4 n.a.). Survival data was available in 90/111 (81.1%) cases. Results: 1. In total, CEBPA mutations were detected in 75/997 (7.5%) of cases (t(15;17)(q22;q12) n=2/33; NK n=52/447; CK n=1/116; other abnormalities n=20/331). Of the 75 patients with CEPBA mutations 31 (41.3%) were sm, while 44 (58.7%) were dm. Patients with dm CEPBA showed better outcome compared to sm cases (OS at 3 yrs: 78.9% vs 38.5%, P=0.014; EFS after 3 yrs: 53.9% vs 36.6%, P=0.108). OS and EFS of CEBPA sm cases were comparable to CEPBA wt cases (OS at 3 yrs: 38.5% and 43.6%, P=0.689, EFS at 3 yrs: 36.6% and 29.4%, P=0.678). OS of CEBPA dm cases was comparable to patients with t(15;17)(q22;q12) (OS after 3 yrs: 78.9% vs 86.1%, P=0.597). 2. In the cohort of 111 patients we detected 227 CEBPA mutations. In 106 (95.5%) cases two mutations, and in 5 (4.5%) cases three mutations were detected. The median mutation load was 42% (range: 2–98%). The majority of mutations were frame-shift (n=135) and in-frame (n=66). Further, missense (n=19) and nonsense (n=7) mutations were observed. Most cases showed one N- and one C-terminal mutation (92/111, 82.8%), 10 (9.0%) cases harbored two N-terminal mutations, and 4 (3.6%) cases showed two C-terminal mutations. In addition, two cases showed one N- and two C-terminal mutations, two cases two N- and one C-terminal mutations, and one case harbored three N-terminal mutations. In 92/111 (82.9%) cases we observed at least one additional mutation (mean: 1.6 mutations; range: 1–4): TET2 39/109 (35.8%), ASXL1 20/111 (18.0%), GATA2 20/111 (18.0%), WT1 14/111 (12.6%), DNMT3A 11/109 (10.1%), IDH1/2 9/111 (8.1%) (IDH1 n=2, IDH2 n=7), NRAS 9/111 (8.1%), RUNX1 7/111 (6.3%), FLT3-ITD 7/111 (6.3%), KRAS 4/109 (3.7%), NPM1 3/111 (2.7%), FLT3-TKD 2/110 (1.8%), MLL-PTD 1/111 (1.0%), and TP53 1/110 (1.0%). With respect to clinical outcome we observed no differences in OS for concomitant mutations in DNMT3A, FLT3-ITD, IDH1/2, NRAS, TET2 and WT1. Cases with additional GATA2 mutations showed longer survival than wt cases (OS at 3 yrs: 100% versus 73.4%, P=0.026, EFS at 3 yrs: 67.5% versus 48.5%, P=0.137). In contrast, cases harboring additional ASXL1 or RUNX1 mutations were associated with worse outcome (ASXL1: OS at 3 yrs: 32.8% versus 85.7%, P

Description: Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia. A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia. In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets. SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2 -mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004). ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36. We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding.

Description: Abstract 417 Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic malignancy that is characterized by features of both a myeloproliferative neoplasm and a myelodysplastic syndrome. Here, we analyzed 81 CMML cases (45 CMML-1, 36 CMML-2). In chromosome banding analysis 59/76 (77.6%) patients showed a normal karyotype (data not availabel in 5 cases). Recurrent chromosome aberrations were trisomy 8 (n=6; 7.9%), monosomy 7 (n=3; 3.9%), and loss of the Y-chromosome (n=5; 6.6%). Fluorescence in situ hybridization (FISH) detected the deletion of one allele of the TET2 gene in 4/71 cases (5.6%). Thus, the majority of cases can not be genetically characterized by these techniques. Therefore, we applied next-generation sequencing (NGS) technology to investigate 7 candidate genes, represented by 43 PCR-products, at known mutational hotspot regions, i.e. CBL (exons 8 and 9), JAK2 (exons 12 and 14), MPL (exon 10), NRAS (exons 2 and 3), and KRAS (exons 2 and 3). In addition, complete coding regions were analyzed for RUNX1 (beta isoform) and TET2. NGS was performed using 454 FLX amplicon chemistry (Roche Diagnostics Corporation, Branford, CT). The median number of base pairs sequenced per patient was 9.24 Mb. For each target gene a median of 911 reads was generated (coverage range: 736-fold to 1606-fold). This approach allowed a high-sensitive detection of molecular mutations, e.g. detecting the JAK2 V617F mutation down to 1.16% of reads. In total, 146 variances were detected by this comprehensive molecular mutation screening (GS Amplicon Variant Analyzer software version 2.0.01). In 80.4% of variances consistent results were obtained after confirming NGS mutations with melting curve analysis and conventional sequencing. In the remaining discrepant variances (19.6%) NGS deep-sequencing outperformed conventional methods due to the higher sensitivity of the platform. After excluding 19 polymorphisms or silent mutations 127 distinct mutations in 61/81 patients (75.3%) were detected: CBL: n=21 point mutations and one deletion (18 bp) found in 20 cases (24%); JAK2: n=8 mutations (V617F) found in 8 cases (9.8%); MPL: no mutations found; NRAS: n=23 mutations found in 18 cases (22.2%); KRAS: n=12 mutations found in 10 cases (12.3%); RUNX1: n=6 point mutations and one deletion (14 bp) found in 7 cases (8.6%); and TET2: n=49 point mutations and 6 deletions (2-19 bp; 5/6 out-of-frame) found in 41 cases (50.6%). Furthermore, in 21 TET2-mutated cases 11 mutations previously described in the literature were detectable, whereas 28 cases carried novel mutations (n=28). In the cohort of TET2-mutated cases 17/41 (41.3%) patients harbored TET2 abnormalities as sole aberration. Interestingly, CBL mutations were found to be significantly associated with TET2 mutations (Fisher's exact test, p=0.008). In 17 of 20 (85.0%) CBL-mutated cases TET2 abnormalities were concomitantly observed. In contrast, no significant associations were found between any of the point mutations or deletions and the karyotype. There were also no associations observed between molecular aberrations and the diagnostic categories CMML-1 and CMML-2. With respect to clinical data a trend for better outcome was seen for patients that carried either or both TET2 and CBL mutations (median OS 130.4 vs. 17.3 months, alive at 2 yrs: 72.0% vs. 43.9%; p=0.13). In conclusion, 75.3% of CMMLs harbored at least one molecular aberration. In median 2 mutations per case were observed. Compared to limited data from the literature we detected not only a higher frequency of CBL mutations, but also add data on novel TET2 mutations. In particular, comprehensive NGS screening here for the first time has demonstrated its strength to further genetically characterize and delineate prognostic groups within this type of hematological malignancy. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kazak:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Weiss:MLL Munich Leukemia Laboratory: Employment. Dicker: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.

Description: Abstract 660 Background: TP53 is the most frequently mutated gene in cancer. An association of TP53 mutations and adverse prognosis was shown in multiple malignancies. In AML, a high frequency of TP53 alterations has been reported in cases with complex karyotype. However, thus far, a comprehensive study analyzing the impact of TP53 alterations has been lacking. Aims: 1. Determine the frequency of TP53 mutations (TP53mut) and TP53 deletions (TP53del) in a large cohort of AML. 2. Analyze the relation of TP53mut and TP53del with cytogenetics and other molecular mutations. 3. Evaluate the impact of TP53 alterations on outcome. Patients and methods: In 1,000 AML patients (median age 66.8 yrs) the TP53 gene (exons 4–11) was analyzed to detect mutations by either DHPLC with subsequent direct Sanger sequencing (n=190) or a next-generation amplicon deep-sequencing assay (n=810) (454 Life Sciences, Branford, CT). All cases with available material (n=858) were analyzed by interphase FISH for TP53del. In all cases the karyotype was available and categorized according to the refined MRC classification (Grimwade et al., Blood 2010). Cases were also screened for mutations in NPM1 (n=966), CEPBA (n=997), RUNX1 (n=907), ASXL1 (n=937) as well as for FLT3-ITD (n=999) and MLL-PTD (n=952). Clinical follow-up data was available in 841 patients. Results: Frequency of TP53 mutations and deletions: In 115 patients (11.5%) a total of 131 TP53mut were detected. 99 patients showed one and 16 cases two TP53 mutations. Heterozygous deletions of the TP53 gene were detected by FISH in 55/858 (6.4%) patients. In 97/115 cases with TP53mut also the TP53del status was available: 41/97 (42.3%) cases harbored both a TP53mut and a TP53del. 32 of the 56 (57.1%) TP53mut cases without TP53del showed heterozygous and 24 (42.8%) homozygous TP53mut. 13/32 (40.6%) cases with heterozygous mutations harbored two distinct TP53mut, whereas only 19/32 (59.3%) were affected by one mutation suggesting a dominant negative effect of these mutations. In patients with homozygous mutations and no TP53del a copy neutral loss of heterozygosity (CN-LOH) can be assumed. In 2 of these patients SNP microarray data was available revealing in both cases a CN-LOH spanning from 17p11.2 to 17p13.3. Association with cytogenetics and other molecular markers: TP53mut were observed in 1/106 cases with favorable, 12/688 with intermediate (1.7%), and 17/90 (18.9%) with adverse cytogenetics. In cases with complex karyotype TP53mut frequency was 73.3% (85/116). TP53mut were mutually exclusive of CEPBA and NPM1 mutations. In patients harboring TP53mut FLT3-ITD, MLL-PTD, RUNX1 and ASXL1 mutations were detected at low frequencies (2.6%, 4.3%, 7.8% and 4.3%, respectively). TP53del were observed in 2/88 (2.3%) patients with favorable, in 6/601 (1.0%) with intermediate, and in 47/169 (27.8%) cases with adverse cytogenetics. 39/47 (83.0%) patients with adverse cytogenetics and TP53del harbored a complex karyotype. 41/55 (74.5%) cases with TP53del also harbored a TP53mut. Clinical impact: Median OS in patients with TP53mut (n=80) vs TP53 wild-type (wt) cases (n=761) was 4.6 vs 35.6 months (mo) (P

Description: Abstract 71 Acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) represents about half of all adult AML. NPM1 and CEBPA mutations define WHO provisional entities accounting for ∼60% of CN-AML, but the remaining cases (∼40%) remain poorly characterized. To address this issue, we carried out whole-exome-sequencing (WES) of leukemic and normal cells from one patient with CN-AML that lacked mutations in NPM1, CEBPA, FLT3-ITD, and MLL-PTD. Using this approach, we identified a clonal somatic mutation of BCOR, a gene located on chromosome Xp11.4, that was present in the leukemic but not normal cells of the index AML case. The BCOR (BCL6 co-repressor) gene encodes for an ubiquitously expressed nuclear protein that is involved in repressing the activity of BCL6 and other transcriptional factors. BCOR is a key transcriptional regulator of early embryonic development, mesenchymal stem cell function and hemopoiesis. Germline mutations of BCOR are responsible for the oculo-facio-cardio-dental (OFCD) genetic syndrome that is inherited in an X-linked pattern and comprises microphtalmia, dysmorphic appearance, dental abnormalities (radiculomegaly), hammer-toe deformity and cardiac defects. WES findings in the index case were subsequently validated and further studied in a total cohort of 514 AML patients. We first performed deep-sequencing analyses of all exons of the BCOR gene in an initial set of 82 AML cases that were selected because they showed the same genetic characteristics of our index patient (i.e. normal karyotype without NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Disruptive BCOR mutations (i.e., nonsense mutations, out-of-frame small indels, and consensus splice-site mutations) were detected in 14/82 (17.1%) of these cases. We next assessed the frequency of BCOR mutations in a series of unselected CN-AML patients (n=262) and found that they occurred in 4.2% of cases, mostly showing the typical features of BCOR-mutated cases (absence of NPM1, CEBPA, FLT3-ITD and MLL-PTD mutations). Almost mutual exclusion of BCOR and NPM1 mutations was further confirmed in a separate series of 71 NPM1-mutated only AML patients. No BCOR mutations were observed in the 89 AML cases with recurrent cytogenetic abnormalities investigated, including t(8;21)(q22;q22) (n= 29), inv(16)(p13q22) (n=40), t(15;17)(q22;q12) (n=10), and t(11q23)/MLL (n=10), and in the 10 patients with double CEBPA-mutated AML studied. BCOR mutations were: i) scattered across the whole length of the coding sequence with no hotspots identified; ii) somatic in origin and disruptive molecular events similar to germline BCOR mutations causing the OFCD genetic syndrome; iii) associated with markedly decreased BCOR mRNA levels, absence of full-length BCOR and absent or low expression of a truncated BCOR protein; iv) almost mutually exclusive with NPM1 (only 1.5% of the 197 NPM1-mutated AML investigated carried BCOR mutations); v) rarely associated with FLT3-ITD; and vi) frequently associated with DNMT3A and RUNX1 mutations, suggesting cooperation with the respective mutated pathways. Clinically, BCOR mutations correlated with poor outcome among the cohort of 160 CN-AML patients evaluated (28.0% versus 66.3% overall survival at 2 years, P=0.024). We also searched for BCOR mutations in the human AML cell lines OCI-AML2, OCI-AML3, KG1a, U937, HL-60, HL-60R, HB4, AML193, and MVP-11. Only HL-60 and HL-60R (a ATRA-resistant derivative of HL-60) carried a BCOR mutation that consisted of a hemizygous G to T transition at position 4616 in exon 10, leading to the Glu1442X nonsense mutation. Western blot analysis of HL-60 cells resulted in the absence of the full-length BCOR protein (predicted MW: 192 kDa) and presence of a low intensity 156 kDa band likely corresponding to a truncated BCOR protein. In conclusion, our results implicate for the first time BCOR in the pathogenesis of CN-AML and suggest it may act as tumor suppressor gene. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: Abstract 458 The first mutation detected in BCR-ABL1 negative myeloproliferative neoplasms (MPN) was JAK2V617F that revolutionized diagnostics of MPN during the last five years. However, although this genetic marker is useful to discriminate MPN from reactive disorders, it is not specific for one entity. In addition, approximately 5% of all polycythemia vera (PV) and 50% of essential thrombocytosis (ET) and primary myelofibrosis (PMF) are not JAK2V617F mutated. In these entities other activating mutations, e.g. MPLW515 mutations or JAK2exon12 mutations, cover additional small proportions of patients without JAK2V617F mutation. To further improve the molecular genetic characterization of MPN research focuses on the identification of novel mutations and, recently, CBL, TET2, and EZH2 genes were identified to be mutated in MPN. We here report on our single centre experience in applying these markers in a daily diagnostic work flow comprizing a total cohort of 18,547 cases with suspected MPN that were investigated between 8/2005 und 8/2010 with individual patient specific combinations of these markers as soon as published. Thus, the most frequently tested marker was JAK2V617F that was applied in 17,027 pts. In 6,622/17,027 (38.9%) a definite diagnosis of MPN could be made or confirmed on the basis of the detection of JAK2V617F mutation. More detailed, the percentage of JAK2V617F positive cases varied depending on the suspected diagnoses: In patients with cytomorphologically confirmed or suspected ET 581/891 (65.2%) were JAK2V617F positive, in PMF: 168/290 (57.9%), in PV: 800/942 (84.9%), in MPN-U: 51/212 (24.0%), in CMML: 38/383 (9.9%), in “MPN” not further specified by the referring physician: 4741/11249 (42.1%), and in those with unexplained leukocytosis/thrombocytosis/splenomegaly or suspected hematologic malignancy: 139/2492 (5.6%). Many of the before mentioned cases were suspected MPN and therefore analyzed for both JAK2V617F and BCR-ABL1. Thus, in 9,924 pts BCR-ABL1 and JAK2V617F testing were performed in parallel. As such, in 541/9,924 (5.5%) analyses BCR-ABL1 positive CML was identified and 3,558 cases were JAK2V617F mutated (35.9%). Only 8 pts were BCR-ABL1/JAK2V617F double positive (0.08%), thus this is a very rare event. In cases with JAK2V617F negative PV in a second step JAK2exon12 mutation was analyzed and 27/147 (18.3%) were tested positive. JAK2V617F negative ET or PMF were analyzed in a second step for MPLW515 mutations. In ET 24/258 (9.3%) and in PMF 14/164 (8.5%) cases were tested positive. JAK2exon12 or MPLW515 were never concomitantly detected with JAK2V617 in our cohort (parallel assessments: n=3,769). PCR for detection of FIP1L1-PDGFRA was performed in 1,086 cases with suspected HES/CEL or unclear eosinophilia but only 26 (2.4%) were tested positive and a CEL could be diagnosed. However, in 36/130 (27.7%) FIP1L1-PDGFRA negative cases a KITD816V mutation was detected and thus a diagnosis of mastocytosis could be established. In addition, confirmation of mastocytosis was achieved in further 326/731 (44.6%) pts with suspected mastocytosis, three of these pts had a JAK2V617F mutation in addition. Further analyses were recently done on selected well characterized cohorts of MPN: CBL mutations were analyzed in 623 cases and tested positive in 54 (8.7%): 26/199 CMML (13.0%), 1/25 PMF, 27/293 MPN-U (9.2%), but never were detected in ET (n=61) or PV (n=45). TET2 sequencing detected mutations in 56/191 (29.3%) of pts analyzed: ET: 6/28 (21.4%), PMF: 4/12 (33.3%), PV: 10/31 (32.3%), CMML: 17/22 (77.3%) cases, MPN-U: 17/86: (19.8%), HES: 1/9 cases, Mastocytosis: 1/3 cases. Thus, TET2 mutations are widely spread in different entities and were frequently associated with other mutations: JAK2V617F: n=16, JAK2exon12: n=1, MPLW515: n=2, CBL: n=5, FIP1L1-PDGFRA: n=1, KITD816V: n=1, and EZH2: n=2. Finally, EZH2 sequence analysis detected mutations in 4/68 (5.9%) cases (1/16 PV, 2/11 PMF, 1/17 MPN-U, 0/20 ET, 0/4 CEL). In conclusion, these data show that the analysis of molecular mutations greatly improved the diagnostic work up of MPN in the last 5 years. The detection of some mutations (JAK2exon12, MPLW515, CBL) are useful to further subclassify MPNs. Others (JAK2V617F, TET2, EZH2) are widely distributed and are helpful for classification and also to discriminate MPN from reactive disorders. The individual power of each marker for prognostication in MPN remains to be defined in future studies. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Grossmann: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. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.