ETV6 Mutations Research Articles

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy.

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Different Genomic Patterns in Patients with Primary Acute Myeloid Leukemia (AML) Compared to Secondary AML in Patients with Normal Karyotype

BackgroundFor decades, cytogenetic analysis has played an essential role in AML risk stratification. Among the 50% of AML patients (pts) with normal karyotype (NK), outcome can vary widely. More recently, whole genome sequencing (WGS) and whole exome sequencing (WES) have identified several recurrent mutations that play an important role in AML pathogenesis and impact outcome. Pts with secondary AML (sAML) have a particularly poor prognosis, are not as responsive to standard induction chemotherapy, and often are referred in first complete remission to hematopoietic stem cell transplantation. We hypothesized that different genomic patterns exist between primary AML (pAML) and sAML that can distinguish the two, and can alter treatment recommendations. To negate the impact of chromosomal abnormalities, we focused our analyses on pts with NK.MethodsWe performed WES and multi-amplicon targeted deep sequencing on samples from bone marrow and peripheral blood of pts diagnosed with sAML at our institution between 1/2003- 1/2013 and who had NK cytogenetics. We compared them to pts with NK primary AML (pAML) whose data were extracted from The Cancer Genome Atlas (TCGA). A panel of 62 gene mutations that has been described as recurrent mutations in myeloid malignancies was included. Mutations were considered individually and grouped based on their functional pathways: RNA splicing (SF3B1, U2AF1/2, SRSF2, ZRSR2), DNA methylation (TET2, DNMT3A, IDH1/2), chromatin modification (ASXL1, EZH2, MLL, SUZ12, KDM6A), transcription (RUNX1, CEBPA, NPM1, BCOR/BCORL1, SETBP1, ETV6), activating signaling (FLT3, JAK2), cohesion (STAG2, SMC3, RAD21), RAS superfamily (K/NRAS, NF1, PTPN11, CBL) and tumor suppressor genes (TP53, APC, WT1, PHF6). Using deep sequencing methodology for resequencing or targeted sequencing, variant allelic frequency (VAF) was measured for each mutation detected. VAF was adjusted by zygosity evaluated by SNP-array karyotyping. For confirmation of clonal architecture, serial sample sequencing and single colony PCR were applied. Differences were compared using Fisher-exact test and Mann-Whitney U test for categorical and continues variables respectively.Results:Of 143 pts included, 101 (71%) had pAML and 42 (29%) had sAML. Compared to pAML, sAML pts were older (59 vs 69 years, p <.001), and had lower white blood cell count (28 vs 3.5 X 109/L, p <.001). Median hemoglobin (10 vs 10) g/dl and platelet counts (57 vs 60) k/uL were similar between the two groups. With a median follow up of 26.4 months (mo, range, .93-95.4), median OS was shorter for sAML than for pAML (12.9 vs 16.2 mo, p= .03). Overall, the most common mutations were: NPM1 (35%), DNMT3A (27%), FLT3 (25%), RUNX1 (14%), IDH1 (12%), IDH2 (12%), STAG2 (12%), TET2 (11%), NRAS (8%), ASXL1 (8%), U2AF1 (8%), PTPN11 (7%), WT1 (6%), BCOR (5%), and PHF6 (5%). Mutations in SF3B1, U2AF1/2, BCOR/BCORL1, ETV6, ASXL1, JAK2, STAG2, and APC were more common in sAML compared to pAML, whereas mutations in DNMT3A, NPM1, CEBPA, and FLT3 were more common in pAML. Mutations in activated pathways in splicing machinery, transcription, chromatin modification, cohesion and RAS pathway were more prominent in sAML, while mutations in DNA methylation and signaling pathways occurred more frequently in pAML. Serial sample analyses at multiple time points demonstrated intra-tumor heterogeneity in most cases of sAML, which was supported by additional cross sectional analyses of VAF in multiple gene mutations in each case. These findings prompted us to evaluate secondary events in the cohort of pts whose sAML originated from an initial MDS stage, defined by ancestral mutations. Among genes frequently affected by mutations, TET2 and ASXL1 were identified as founder events, whereas STAG2, NRAS and PTPN11 were observed in subclonal sAML derived from founder MDS clones. In pAML, however, TET2 and ASXL1 mutations were found to be secondary lesions, while IDH1 and DNMT3A were identified as ancestral events.ConclusionClear genomic variations exist between sAML and pAML that suggest differences in the pathophysiology of both diseases. Specific therapies should be directed to the activated pathways according to the unique clonal hierarchy in each AML subtype. DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance of Myelofibrotic (MF) Changes in Myelodysplastic Syndromes (MDS): A Prospective Evaluation Including Mutational Analysis By Next-Generation Sequencing (NGS)

MDS with MF is not recognized as a distinct entity in 2008 WHO classification, and the prognostic role of MF remains controversial after several retrospective evaluations (Della Porta, 2009; Greenberg, 2012). In order to minimize inter-observer variability, a European Consensus (EC) has been reached for MF grade assignation (Thiele, 2005). IHC analysis of p53 protein BM accumulation has been proposed as prognosticator in MDS (Ramos, 2002), and more specifically in MDS with isolated 5q- (Jädersten, 2011; Saft, 2014) where it correlates with TP53 mutations. Several gene mutations have a prognostic role in MDS (Bejar,2011; Papaemmanuil, 2013; Haferlach, 2014), mutTP53 ranking as the most potent molecular prognosticator. By contrast, CALRmut, common in MPN, seems rare in MDS (Klampfl, 2013; Nangalia, 2013) albeit a focused evaluation taking into consideration EC grading has not been performed so far.The aim of this study was to evaluate the events that underlie MF changes in MDS by studying 77 MDS patients (52M/ 25F; median age 76, range 31-88; WHO-2008: RCUD 2, RARS 4, RCMD 40, RAEB-1 15, RAEB-2 7, 5q- 7, MDS-U 2), that were recruited prospectively since June 2006 and followed up to June 2104 in 7 GESMD centers. Diagnosis and cytogenetic evaluation followed GESMD SOP’s (Schanz’s cytogenetic score: Very good 6, Good 51, Intermediate 12, Poor 5, Very Poor 5). The study was approved by the IRB at each study site and all patients gave written informed consent. Median follow-up was 3.0 years (0.1-7.7). During this time, 49 patients (63.6%) died and 2 (2.6%) were lost to follow-up, while 20 (21.6%) progressed into acute leukemia.MF was evaluated independently by 2 pathologists following EC guidelines (MF-0 29, MF-1 31, MF-2 15, MF-3 2). IHC accumulation of p53 protein was evaluated as described previously and soluble p53 protein was analyzed in plasma by ELISA. Serum levels of 9 cytokines and chemokines were analyzed using cytometric bead arrays and a FACScanto flow cytometer (Becton-Dickinson). WT1 gene expression was evaluated by RT-PCR in PB and calculated as the WT1/GUSratio. The presence of mutations was evaluated in 67 patients. Sequencing was performed on a MISEQ NGS (Illumina) system using a panel of 111 genes previously related to MDS or MF. Sequencing results were analyzed by using the VariantStudio software package (Illumina), the threshold for mutation calling was set to 5%. Descriptive statistics and nonparametric statistical procedures were performed as appropriate, and univariate and multivariate analysis were performed for OS prediction.MF grade showed a statistically significant nonparametric correlation with the proportion of bone marrow erythroid precursors in BMA (-0.408), Hb levels (-0.271), number of PRBC transfused in the first 16 wks (0.302), ferritin levels (0.294), EPO levels (0.331) and p53 IHC score (0.231), as well as the presence of ALIPs (0.358), WT1/GUSratio (0.264) and serum levels of MIG (-0.251), but not with other covariates.Mutation incidence was highest for genes involved in splicing machinery (38.8%) as well as ASXL1 (18%). Mutated SF3B1 was associated with the absence of fibrosis (MF-0 23.1%, MF-1 7.4%, MF-2/3 0%; p=0.024) while the opposite was true for mutated ETV6 (MF-0 and MF-1 no mutations, MF-2/3 14.3%; p=0.041). Only 1 patient harbored a mutation in CALR. In our series, p53 protein accumulation and mutTP53 were not statistically linked. Mutations in GATA1 and SMC3 were more frequent in patients with IPSS-R High/Very High (25.0% vs. 3.6%, p=0.037 and 25.0% vs. 0%, p=0.005, respectively), but no single mutation was linked to Schanz’s cytogenetic score (Very Good/Good vs. Intermediate vs. Poor/Very Poor) with this sample size.Survival analysis showed MF-2 or higher as a relevant predictor for OS (median OS for MF-0/1 4.0 years [CI95% 1.4-6.6] vs. 1.2 [0-3.3] for MF-2/3; HR 2.3, CI95% 1.2-4.3, p=0.008), that was independent of IPSS-R in multivariate analysis (IPSS-R HR 2.47 [1.3-4.7], p=0.006; MF HR 2.18 [1.2-4.1], p=0.013).In conclusion, MF changes observed in MDS patients are not linked to mutations in CALR, cytogenetic score or IPSS-R, but to erythroid failure. Mutations in SF3B1 and ETV6 seem to diverge on their influence over MF. MF grade 2 or higher seems relevant for OS and independent of IPSS-R. DisclosuresNo relevant conflicts of interest to declare.

Acquisition of Signaling and ETV6 Mutations is Associated With Transformation to CMML

Acquisition of Signaling and ETV6 Mutations is Associated With Transformation to CMML

A targeted mutational landscape of angioimmunoblastic T-cell lymphoma

AbstractThe genetics of angioimmunoblastic T-cell lymphoma (AITL) are very poorly understood. We defined the mutational landscape of AITL across 219 genes in 85 cases from the United States and Europe. We identified ≥2 mutations in 34 genes, nearly all of which were not previously implicated in AITL. These included loss-of-function mutations in TP53 (n = 4), ETV6 (n = 3), CCND3 (n = 2), and EP300 (n = 5), as well as gain-of-function mutations in JAK2 (n = 2) and STAT3 (n = 4). TET2 was mutated in 65 (76%) AITLs, including 43 that harbored 2 or 3 TET2 mutations. DNMT3A mutations occurred in 28 (33%) AITLs; 100% of these also harbored TET2 mutations (P < .0001). Seventeen AITLs harbored IDH2 R172 substitutions, including 15 with TET2 mutations. In summary, AITL is characterized by high frequencies of overlapping mutations in epigenetic modifiers and targetable mutations in a subset of cases.

Next-Generation Sequencing Of ASXL1, TP53, RUNX1, EZH2, and ETV6 Identifies a Significant Proportion Of Lower-Risk Myelodysplastic Syndromes With Poor Prognostic Indicators

IntroductionManagement of patients with myelodysplastic syndrome (MDS) is dependent in part on risk stratification by the International Prognostic Scoring System (IPSS). Patients with lower-risk disease as defined by low and intermediate-1 IPSS are predicted to have a relatively indolent clinical course and are usually offered less aggressive therapies, including transfusions, growth factors, or observation without treatment. In contrast, patients with higher-risk disease as defined by intermediate-2 and high IPSS are predicted to have a relatively aggressive clinical course and are usually treated with disease-modifying hypomethylating agents or considered for stem cell transplantation. However, despite IPSS risk stratification a subset of patients with lower-risk MDS will have more aggressive disease with shorter overall survival and increased risk of transformation to acute myeloid leukemia. Somatic mutations are common in MDS, but are not currently included in either the IPSS or revised IPSS (IPSS-R). Recent literature (Bejar et al. N Engl J Med 2011;364:2496-2506) revealed that mutations in ASXL1, TP53, RUNX1, EZH2, and ETV6 are indicators of poor overall survival in patients with MDS independent of IPSS and other established risk factors, and that mutations in one or more of these 5 genes essentially up-scores the IPSS to the next highest risk group. The aim of this study is to evaluate the frequency of mutations in these 5 genes using a clinically validated and sensitive next-generation sequencing (NGS) assay, and to determine the percentage of lower-risk MDS patients with a worse than expected prognosis who may potentially benefit from earlier treatment with disease-modifying therapy. MethodsDNA sequences of key exons in ASXL1, TP53, RUNX1, EZH2, and ETV6 were determined from the bone marrow aspirates of 200 MDS patients (29% low, 52% intermediate-1, 18% intermediate-2, and 1% high IPSS) using a clinically validated and sensitive (5% limit of detection) NGS assay on the Illumina MiSeq platform. Mutations were compared between duplicate samples and annotated using software that queried databases containing known somatic mutations and germline variants. ResultsSomatic mutations in one or more of the 5 genes were detected in 50.5% of MDS patients in this study. Mutations in ASXL1 were the most frequent (29.5%), followed by mutations in TP53 (15.5%), RUNX1 (9.5%), EZH2 (7%), and ETV6 (7%). While mutant allele percentage varied among the genes, 64% of MDS patients with ETV6 mutations had <20% mutant allele, followed by 37% for ASXL1, 29% for TP53, 21% for RUNX1, and 7% for EZH2. Mutations were detected in 45% (46/102) of MDS patients with normal cytogenetics and 60% (37/92) with abnormal cytogenetics. When stratifying by IPSS, 46% of MDS patients with lower-risk disease had a mutation, compared with 72% with higher-risk disease. When stratifying by IPSS-R, 41% of MDS patients with lower-risk disease (very low and low) had a mutation, compared with 77.5% with higher-risk disease (high and very high). ConclusionsSomatic mutations in ASXL1, TP53, RUNX1, EZH2, and ETV6 have previously been shown to be indicators of poor overall survival in patients with MDS independent of IPSS and other established risk factors. Using a clinically validated and sensitive NGS assay, 50.5% of MDS patients in this study were shown to have a mutation in one or more of the 5 genes. This percentage is higher than previously reported by Bejar et al (31.2%), and is likely explained by the higher sensitivity of NGS (5% limit of detection) as compared to Sanger sequencing (typically 20% limit of detection), which was utilized in the aforementioned study for ASXL1, EZH2, and ETV6. Mutations in one or more of the 5 genes were detected in 46% of MDS patients with lower-risk disease defined by IPSS, and a similar 41% with lower-risk disease defined by IPSS-R. Mutational profiling using NGS can thus help identify a significant proportion of lower-risk MDS patients with a worse than expected prognosis who may potentially benefit from earlier treatment with disease-modifying therapy. Disclosures:No relevant conflicts of interest to declare.

Diagnostic and Prognostic Utility Of a 26-Gene Panel For Deep-Sequencing Mutation Analysis In Myeloid Malignancies

▪ IntroductionMolecular 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. AimsTo test the utility of a pan-myeloid panel in routine diagnostics. MethodsWe 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%. ResultsThus 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<0.001). ConclusionA 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.

Comprehensive Analysis Of U2AF1 Mutations In 843 Patients With Myeloid Neoplasms With Respect To Other Genetic Alterations

IntroductionGenes affecting the splicing machinery have been found to be frequently mutated in MDS patients. U2AF1 codes for one of these splicing components, showing two distinct mutational hot spots at amino acids Ser34 and Gln157. Mutations in U2AF1 induce global abnormalities in RNA splicing, producing intron containing unspliced RNAs. U2AF1 has been shown to be most frequently mutated in MDS cases (7-11%), but was so far investigated only in small subsets of AML and MPN and was found rarely mutated. AimTo determine the frequency of U2AF1 mutations (U2AF1mut) in different myeloid entities and to evaluate the correlation of U2AF1mut with other gene mutations, cytogenetics and clinical features. Patients and MethodsThe total cohort consisted of 843 patients, whereof 74 were diagnosed as AML, 201 as MDS, 243 as MPN, and 325 as MDS/MPN overlap. 331 patients were female, 512 male. Cytogenetics was available in 830 patients and these were grouped by the following karyotypes: normal karyotype (n=561), +8 (n=39), -7 (n=15), del(20q) (n=95), -Y (n=29), other aberrations (n=59), and complex karyotype (n=32). Based on the previously described association of U2AF1mut with del(20q) there was an intended selection bias to this abnormality. Mutational analyses for U2AF1 were performed by either melting curve analyses or next generation sequencing. In subcohorts we investigated mutations in ASXL1 (n=505), CBL (n=647), CEBPA (n=68), CSF3R (n=213), DNMT3A (n=260), ETV6 (n=129), EZH2 (n=355), FLT3-ITD (n=352), FLT3-TKD (n=239), IDH1/2 (n=367 and 286, respectively), JAK2 (n=681), KITD816 (n=244), KRAS (n=393), MLL-PTD (n=384), MPLW515 (n=612), NPM1 (n=477), NRAS (n=509), RUNX1 (n=516), SETBP1 (n=336), SF3B1 (n=839), SRSF2 (n=784), TET2 (n=428), and TP53 (n=239) by Sanger sequencing, next generation sequencing, gene scan, or melting curve analysis. ResultsIn the total cohort we detected U2AF1 mutations in 55/843 (6.5%) patients, the two mutational hot spots were equally affected with 29 p.Ser34 and 26 p.Gln157 mutations, respectively. Mutation frequencies were 10.9% in MDS, 9.5% in AML, 7.1% in MDS/MPN overlap and 1.2% in MPN. U2AF1mut patients were older (median: 72.6 vs. 71.8 years; p=0.012), the mutation was more frequent in males (42/512 (8.2%) vs. 13/331 (3.9%) in females; p=0.015) and associated with lower hemoglobin levels (median: 9.5 vs. 11.0g/dL; p<0.001), and platelet counts (median: 78x109/L vs. 179x109/L; p=0.002). Regarding cytogenetics we found a high association of U2AF1mut to del(20q): in 18 of 95 cases (18.9%) with del(20q) a U2AF1 mutation was detected compared to 37 U2AF1mut in 735 cases (5.0%) with any other karyotype (p<0.001). This was true for AML (5/16 vs. 2/56; p=0.005), MDS (11/49 vs. 11/150; p=0.007) and MDS/MPN overlap cases (1/8 vs. 21/309; p=0.441). In contrast in MPN none of the 21 del(20q) patients showed a U2AF1 mutation compared to 18/74 in all other entities (p=0.01). Mutations in the two other genes of the splicing machinery, SF3B1 and SRSF2, occurred in 122/839 (14.5%) and 198/784 (25.3%) cases and were mutually exclusive with U2AF1mut. Only one case each showed an U2AF1mut and a SF3B1 (p=0.002) or SRSF2 (p<0.001) mutation. We furthermore analyzed a number of other gene mutations frequently mutated in myeloid entities and their association to U2AF1mut. There was no correlation to mutations in NPM1, FLT3-ITD and FLT3-TKD, MLL-PTD, and CEBPA in AML patients. In MDS patients there was also no correlation to mutations in ASXL1,ETV6, EZH2, TP53, RUNX1, NRAS, and KRAS. This was also true for JAK2, MPL, CBL, and TET2 mutations in MPN. However in MDS/MPN overlap patients U2AF1mut were more frequently found in cases with ASXL1mut (14/115 (12.2%) in ASXL1mut vs. 7/179 (3.9%) in ASXL1wt; p=0.01) and together with KITD816mut (3/10 (30%) in KITD816mut vs. 15/212 (7%) in KITD816wt; p=0.038). Conclusion1) U2AF1 is most frequently mutated in MDS, followed by AML and MDS/MPN overlap and in contrast rarely mutated in MPN. 2) U2AF1mut highly correlates with del(20q) in MDS, AML and MDS/MPN overlap but not in MPN cases. 3) In MDS/MPN overlap U2AF1mut associates significantly with ASXL1mut and KITD816mut. Disclosures:Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Jeromin: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. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Unbalanced Translocation t(5;17) Resulting In a TP53 Loss As Recurrent Aberration In Myelodysplastic Syndrome With Complex Karyotype

A complex karyotype, detected in approximately 10%-15% of patients with myelodysplastic syndrome (MDS), is associated with a very short median survival and a high risk of transformation into AML. The most frequent chromosome aberrations in complex karyotypes are deletion of 5q (del(5q)) and deletion of 17p (del(17p)) harboring the tumor suppressor gene TP53. It is still unclear, how complex karyotypes develop.We have identified an unbalanced translocation der(5)t(5;17)(q11∼q13;q11∼q13) in 199 patients with MDS or secondary AML after MDS. The cohort consists of 111 male (56 %) and 88 (44 %) female patients between 4 and 91 years of age (median age 68 years).In order to better understand the underlying pathomechanism of this aberration, we have investigated these cases in greater detail. For all patients, we performed cytogenetic banding analysis, fluorescence in-situ hybridization (FISH) and, in about one third, multicolor-FISH.In all patients, a complex aberrant karyotype with a median of 7 aberrations was observed, indicating high chromosomal instability. In 30 patients, clonal evolution was identified. To identify the breakpoints in 5q and 17q more precisely, array-CGH was performed in 7 patients. The breakpoints on 5q and 17p were located between the centromere of chromosome 5 and 5p15 and between the centromere of chromosome 17 and 17q22, respectively. The breakpoints were in gene-poor regions, suggesting that no fusion genes would result from these rearrangements. Notably, the breakpoints were all very close to the centromeric region and heterochromatin. It is known that an altered methylation of heterochromatic regions plays an important role in tumor development. Therefore, alterations of DNA methylation or histone modifications may be involved in the generation of the unbalanced translocation t(5;17).Using whole exome sequencing, we sought to define the mutational spectrum of complex karyotypes with t(5;17). In one patient we were able to analyse bone marrow cells from different time points: complex karyotype at diagnosis, complete remission and relapse with complex karyotype again. As possible candidate genes for driver mutations we identified mutations in the genes NF1, ETV6 (TEL) and KMT2C (MLL3). Of note, in this patient the allele frequencies of mutations affecting NF1 and KMT2C (MLL3) increased during the course of the disease, whereas the ETV6 (TEL) mutation found at diagnosis was lost at relapse indicating clonal evolution. Especially the identification of a mutation in NF1, a negative regulator of the RAS pathway, is of great significance. NF1 is encoded on 17q11.2. A possible underlying mechanism could be a downregulation of NF1 by a mutation of one allele and by a deletion evolved from the unbalanced translocation t(5;17) of the second allele. These data provide further evidence that the inactivation of NF1 seems to play an important role in clonal evolution and leukemic progression. Disclosures:No relevant conflicts of interest to declare.

Identification of Novel Somatic Mutations, Regions of Recurrent Loss of Heterozygosity (LOH) and Significant Clonal Evolution From Diagnosis to Relapse in Childhood AML Determined by Exome Capture Sequencing – an NCI/COG Target AML Study

AbstractAbstract 123▪▪This icon denotes a clinically relevant abstractComprehensive cataloguing of somatic changes in acute myeloid leukemia (AML) has revealed novel molecular pathways that contribute to the development and progression of the disease, however many lack prognostic significance. The challenge that lies ahead is to understand how these molecular changes relate to transcription and protein deregulation to influence treatment responses and outcomes. Also, some mutations found in adult AML are detected at lower frequency or not at all in pediatric AML.To better define the genomic profile of somatic mutations in childhood AML and evaluate the extent of clonal evolution from diagnosis to relapse, we performed whole exome sequencing in matched trios of specimens (diagnostic, germline and relapse) from 20 children with AML who lacked known karyotypic high-risk features. Candidate variants were verified by 454 sequencing. In total, 364 somatic variants were successfully verified, of which 263 were non-silent mutations (127 at diagnosis and 136 at relapse). Of the 195 unique nonsynonymous mutations present in 180 genes, mutations in 6 genes were detected in more than one patient, including ETV6, KIT, KRAS, NRAS in 2 patients and TET2 and WT1 in 3 patients. A polyphen prediction estimated 72% of the missense and nonsense mutations as possibly and/or probably damaging. Mutations identified in single patients included known genes implicated in leukemias (e.g., GATA2, IKZF1), as well novel mutations in genes not previously implicated in AML.Prevalence and clinical significance of ETV6 mutations were assessed in a large cohort of pediatric AML. The entire coding sequence of ETV6 gene was sequenced in 180 cases of pediatric AML. ETV6 mutations, including missense (N=5), Indels (N=4) or splice site mutations (N=1) were detected in 6% of the patients without karyotypic or molecular risk factors. Those with and without ETV6 mutations had an overall survival of 33% and 71%, respectively (p=0.006) with a corresponding relapse rate of 80% and 29% (p=0.004).We examined the clonal evolution of mutations from diagnosis to relapse by comparing the mutation profile at diagnosis to that in relapse. In the discovery phase, at diagnosis, 234 somatic mutations in 202 genes were detected, of which 174 were non-silent events, including 36 (21%) indels, 126 (72%) missense, 11 (6%) nonsense mutations and 1 (1%) splice site mutation. At relapse, 350 mutations in 309 genes were detected, of which only 112 mutations were detectable at diagnosis (29%). In verification phase, 71 mutations in 70 genes were identified only in diagnostic specimens, and 105 mutations in 104 genes were detected in relapse only, with an overlap of 192 variants, suggesting significant molecular evolution and loss or gain of genomic events from diagnosis to relapse. With secondary targeted deep sequencing the overlap of mutations present at diagnosis and relapse increased to 52%. Comparison of allele fractions between diagnosis and relapse revealed a significant enrichment of the variant from diagnosis to relapse with evolution of a minor clone at diagnosis to a more dominant presence at relapse. It also highlights that in a substantial subset of patients, novel genomic events may emerge, leading to disease relapse.In addition, analysis of the allele fractions of SNPs in the normal and tumors of these patients allowed us to detect regions of recurrent loss of heterozygosity (LOH). Six of 20 patients exhibited LOH in one or more regions. The most common region of LOH, found in 4 patients (20%) was the q-arm of chromosome 13 (3 with FLT3/ITD), including 10 genes with germ line variation across 4 patients, and in two patients the q-arm of chromosome 16 was involved. Two of the 4 patients had LOH only in the relapse specimen.This study highlights the potential impact of applying novel sequencing approaches to pediatric AML and not only identifies clinically significant somatic mutations, but defines genomic complexity in childhood AML by demonstrating significant clonal evolution from diagnosis to relapse. Mutations emerging at relapse may represent initially rare, chemotherapy-resistant events that cooperate with other mutations and lead to adverse outcome. Identifying mutations that may be therapeutically targeted early in the course of therapy may provide improved outcomes for patients. Disclosures:No relevant conflicts of interest to declare.

Clinically Significant Mutations, Deletions and Translocations Involving ETV6 Identified by Whole Genome and Whole Exome Sequencing; Report From NCI/COG Target AML Initiative

AbstractAbstract 125▪▪This icon denotes a clinically relevant abstractDespite identification of genomic alterations in AML, clinically actionable variants are limited. Further, mutations identified in adult AML are less frequent or not found in childhood AML. TARGET AML initiative, a collaboration between National Cancer Institute Office of Cancer Genomics (NCI/OCG) and Children's Oncology Group (COG) provided resources to perform multi-platform analysis of a large number of pediatric AML cases in order to identify therapeutic targets in childhood AML. As part of this effort, diagnostic, remission and relapse specimens from a large cohort of children with AML were subjected to whole genome sequencing (WGS). In addition, all specimens underwent characterization by expression profiling, SNP genotyping, methylation profiling and miRNA sequencing. Specimens from a subset of patients also underwent whole exome sequencing (WES) and whole transcript sequencing (RNA Seq). Here we present data from an interim analysis of the data that identified clinically significant, novel somatic mutations, deletions and translocations involving the ETV6 gene in pediatric patients with AML. Non-silent somatic mutations of ETV6, including missense, splice site mutations and indels were identified by WGS and WES. Large deletions involving ETV6 gene were identified by WGS and SNP/CGH arrays, and novel fusion transcripts (cryptic translocations) involving ETV6 were identified by WGS and RNA Seq. We present the prevalence and clinical significance of genomic alterations of ETV6 that were identified as part of TARGET AML study.WGS and WES data were available from 54 and 22 pediatric AML patients, respectively. In these 76 selected cases, somatic mutations in ETV6 were identified and verified in 3 patients. Frequency validation was performed by sequencing of the entire coding region of the ETV6 gene in diagnostic specimens from 180 children with AML. Somatic mutations of ETV6 gene were identified in 6% of patients, of which none had cytogenetic or molecular risk features. Those with somatic ETV6 mutations had an overall survival (OS) of 20% vs. 76% for those without mutations (p=0.002) with a corresponding relapse risk (RR) of 80% vs. 29% (p=0.004), demonstrating the potential impact of ETV6 mutations in pediatric AML.Deletions involving ETV6 gene (del ETV6) were detected in 3 patients by WGS and confirmed by SNP genotyping array and by fluorescent in situ hybridization (FISH). Subsequent interrogation of the SNP genotyping data identified deletions of 12p13 involving ETV6 in 19/242 patients (8%) ranging from 0.25 Mb to >20 Mb. Those with del ETV6 lacked cytogenetic (−7, or −5/del5q) or molecular (FLT3/ITD) high-risk features, but 50% of those with del ETV6 had core binding factor (CBF) AML. All patients with del ETV6 achieved a morphologic complete remission (CR) and had no evidence of minimal residual disease at the end of induction chemotherapy. Relapse risk for patients with and without del ETV6 was 63% vs. 45% (p=0.3) with a corresponding disease-free survival (DFS) of 32% vs. 53% (p=0.2). Due to the high prevalence of CBF AML in patients with del ETV6, we inquired whether this alteration might impact outcome in patients with CBF AML. In patients with CBF AML, del ETV6 was associated with adverse outcome, where CBF patients with and without del ETV6 had an event-free survival of 0% vs. 63%, respectively (p=0.002). Of the CBF AML patients who achieved an initial CR, those with compared to those without a del ETV6 had a RR of 88% (vs. 38%, p=0.08) with a DFS of 0% (vs. 61%, p=0.009) respectively.Cryptic ETV6 translocations involving at least two distinct translocation partners represented a third class of ETV6 alterations identified by WGS and RNA Seq. FISH evaluation of select patients was available and identified 16 patients with positive FISH for ETV6 variants. Those who had ETV6 alterations by FISH had a median age at diagnosis of 3.7 years. Evaluation of remission induction rate as well as post remission outcome demonstrated that all but 3 of these 16 patients either failed to achieve a CR (N=4) or relapsed after an initial remission (82% failure rate). These data identified varying genomic alterations of ETV6 by whole genome sequencing that identify a significant proportion of pediatric AML cases with adverse outcome who may be targeted for altered therapy. Disclosures:No relevant conflicts of interest to declare.

Various Germline Congenital Disorder Genes Are Somatically Mutated in Myeloid Malignancies

AbstractAbstract 1405Genes involved in congenital genetic cancer susceptibility syndromes are also targets of somatic mutations in various tumors. Examples include WT1, NF1, CBL, TP53 and MLL2 affected both in germ line as well as somatic mutations present in malignant disorders. To apply this approach to investigation of pathogenic mutations in myeloid malignancies, we selected 183 congenital disorders in which germline mutations of disease specific genes are reported to be pathogenic. Their main clinical presentations are skeletal abnormalities (N=54 disorders), skin abnormality (N=24), mental retardation (N=17) and hematological disorders (N=12). In total, we searched for mutations in 204 genes associated with these congenital disorders.We analyzed whole exome of various myeloid malignancies, including 60 cases with myelodysplastic syndromes (MDS), 29 MDS/MPN, 5 with MPN and 122 with acute myeloid leukemia (AML) and found somatic mutations in 62 genes, which also mutated in germ line in various congenital syndromes. Of those, the most frequently mutated genes were TP53 (25 cases) and WT1 (16 cases), associated with germline mutation of Li-Fraumeni syndrome and Wilms tumor, respectively. Some somatic mutations, for example, NF1 (R1276Q) and PTPN11 (D61N), were exactly the same as observed in corresponding congenital disorders (Neurofibromatosis or Noonan syndrome). One of the novel findings is that somatic SET binding protein 1 (SETBP1) mutations (D868N, G870S and I871T) were commonly observed in sAML and CMML, and were identical to germline mutations in Schinzel-Giedion syndrome (see designated abstract). We found recurrent somatic SETBP1 mutations in 15% of each CMML and sAML. Moreover, multiple genes pathogenic in Usher syndrome (congenital hearing and vision loss, complicated by vasoproliferative retinal tumor), were somatically mutated in various myeloid neoplasms. Out of 9 genes which are causative for this syndrome, 15 mutations of 6 genes (MYO7A, USH1C, CDH23, PCDH15, USH2A, and GPR98) were observed in 13 cases, including 2 frameshift and 13 missense mutations. These genes coordinate with each other to form a functional network. CDH23 and PCDH15 are cadherins and act as cell adhesion molecules. MYO7A are actin-based motor molecules with a variety of functions. USH1C serves as an anchor and codes for a scaffolding protein to form a complex with all the other proteins. Through the PDZ binding site, USH1C forms a complex with CDH23, which was the most frequently mutated gene in this family (1 frame shift and 3 misssense mutations). CDH23 mutations were observed in 2 cases with primary AML, sAML and MDS. Specifically, a somatic missense mutation G2771S of CDH23 in a secondary AML case was identical to germline of Usher syndrome. The second most frequently mutated gene, GPR98, is located in 5q14.3 locus; a small hemizygous clone found in del5q of an MDS case. In a serial sample analysis, this mutation increased to become the larger main clone during AML evolution. Moreover, in this case, an additional CDH23 mutation was acquired in the course of leukemic expansion. In such cases with Usher syndrome gene mutations, U2AF1, ZRSR2, EZH2, IDH2 and ETV6 mutations were also observed, suggesting pathogenic cooperation with these well-known tumor suppressor genes and oncogenes. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Makishima:Scott Hamilton CARES Initiative: Research Funding.

Clinical, immunophenotypic, cytogenetic, and molecular genetic features in 117 adult patients with mixed-phenotype acute leukemia defined by WHO-2008 classification

Among 4,780 consecutive adult acute lymphoblastic/myeloblastic leukemia patients, we identified 117 (2.4%) patients with mixed-phenotype acute leukemia fulfilling WHO 2008 criteria; these were classified as: Blymphoid+ myeloid (n=64), T-lymphoid+myeloid (n=38), B+T-lymphoid (n=14) and trilineage (n=1). Of 92 patients karyotyped, 59 were abnormal and were classified as: complex (22 of 92), t(9;22)(q34;q11) (14 of 92), monosomy 7 (7 of 92), polysomy 21 (7 of 92), t(v;11q23) (4 of 92), t(10;11)(p15;q21) (3 of 92), while STIL-TAL1 fusion was detected in one (T+My) patient. After investigating common acute leukemia-related mutations in 17 genes, 12 of 31 (39%) patients were found to have at least one mutation, classified with: IKZF1 deletion (4 of 31), and EZH2 (3 of 31), ASXL1 (3 of 31), ETV6 (2 of 31), NOTCH1 (1 of 31), and TET2 (1 of 31) mutations. Array-CGH revealed genomic deletions of CDKN2A (4 of 12), IKZF1 (3 of 12), MEF2C (2 of 12), BTG1 (2 of 12), together with BCOR, EBF1, K-RAS, LEF1, MBNL1, PBX3, and RUNX1 (one of 12 each). Our results indicate that mixed-phenotype acute leukemia is a complex entity with heterogeneous clinical, immunophenotypic, cytogenetic, and molecular genetic features.

ETV6 mutations in early immature human T cell leukemias

Early immature T cell acute lymphoblastic leukemias (T-ALLs) account for ~5-10% of pediatric T-ALLs and are associated with poor prognosis. However, the genetic defects that drive the biology of these tumors remain largely unknown. In this study, analysis of microarray gene expression signatures in adult T-ALL demonstrated a high prevalence of early immature leukemias and revealed a close relationship between these tumors and myeloid leukemias. Many adult immature T-ALLs harbored mutations in myeloid-specific oncogenes and tumor suppressors including IDH1, IDH2, DNMT3A, FLT3, and NRAS. Moreover, we identified ETV6 mutations as a novel genetic lesion uniquely present in immature adult T-ALL. Our results demonstrate that early immature adult T-ALL represents a heterogeneous category of leukemias characterized by the presence of overlapping myeloid and T-ALL characteristics, and highlight the potential role of ETV6 mutations in these tumors.

ETV6 Is An Early T-Cell Progenitor (ETP) Specific Tumor Suppressor Gene in Adult T-ALL

Abstract 406T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease in which genetic lesions coordinately affect cell proliferation, differentiation and survival of thymocytes. Although pediatric T-ALL has been extensively studied on the genomic level during the last decade, large scale genomic studies in adult T-ALL are sparse. Importantly, significant differences in treatment outcome are present between pediatric and adult T-ALL.Here we performed integrative analysis of gene expression, copy number alterations and mutation analysis on 58 adult T-ALL samples treated in the Eastern Cooperative Oncology Group (ECOG) E2993 protocol. Unsupervised and consensus clustering of microarray gene expression data in this series revealed the presence of 2 stable gene expression clusters resembling immature/ETP (n=30) and typical (n=28) T-ALL gene signatures. Indeed, gene set enrichment analysis (GSEA) confirmed that our ETP/immature adult T-ALL signature closely resembled the previously published ETP profiles. In this adult patient cohort, the immature/ETP patient samples were associated with poor outcome (5 year OS 36% for ETP and 58% for non ETP TALL, P=0.088). Additional supervised analyses revealed differential expression of ERAP2, a gene involved in the processing of MHC peptides, between early relapse and long time survivor adult T-ALL patients. Consistently low ERAP2 expression was associated poor prognosis in adult T-ALL (OS 26%, 36% and 78% for 1st 2nd and 3rd terciles respectively, P=0.016). In addition, absence of TCR gamma deletion (p=0.013) as well as the absence of biallelic CDKN2A/CDKN2B deletion (p=0.009) were shown to be poor prognostic markers in adult T-ALL.To gain additional insight in the biology of adult ETP T-ALL, we performed GSEA analysis using the immature/ETP gene expression profiles. This analysis revealed that the adult ETP T-ALL signature is more closely related to acute myeloid leukemia (AML) profiles as compared to ALL signatures. Given this, we hypothesized that myeloid specific genetic alterations might be uniquely present in ETP adult T-ALL. Mutation analysis of AML oncogenes and tumor suppressor genes revealed the presence of myeloid mutations targeting IDH1, IDH2, DNMT3A, FLT3 and N-RAS in 13/30 (43%) of adult ETP T-ALL cases, whereas only 1/28 (3.5%) typical adult T-ALL showed a mutation in N-RAS. Although these ETP adult T-ALL samples seem to have myeloid properties, they still retain T-lymphoid characteristics including activational NOTCH1 mutations in 12/30 (40%) of ETP adult T-ALLs. Of note, the myeloid specific genetic alterations were preferentially present in NOTCH1 wild type ETP adult T-ALL samples.Next, we used a systems biology approach to identify master regulators that might be able to drive the ETP signature identified in adult T-ALL. For this, we interrogated a T-ALL interactome that was constructed from gene expression profiles of 228 human T-ALL samples. This master regulator analysis identified MN1 as the key transcription factor driving the ETP signature. The MN1 gene was initially identified as fusion partner of TEL (ETV6) in patients with AML. Notably, MN1-TEL expression in multipotent progenitors induces both AML and T-ALL and mutation analysis of ETV6 in adult T-ALL revealed the presence of 10 ETV6 mutants (9 frameshift and 1 splice site mutation) in adult T-ALL. Analysis of bone marrow remission genomic DNA confirmed the somatic origin of ETV6 mutations (3 frameshift and 1 splice site) in each of the 4 patient samples with available material. Most notably, ETV6 mutations were exclusively present in the ETP subtype of adult T-ALL. In addition, 8/10 (80%) of ETV6 mutant ETP T-ALL cases were characterized mutations in NOTCH1, suggesting a specific interaction between the oncogenic NOTCH1 programs and the mutational loss of ETV6 in early T-cell transformation.In conclusion, our integrative genomic analyses in adult T-ALL unraveled a high prevalence of immature/early T-cell progenitor (ETP) T-ALLs, associates the pathogenesis of ETP-TALL with myeloid mutations and identifies ETV6 truncating alleles as the first ETP specific gene mutation that strongly interact with NOTCH1 in T-cell transformation. Disclosures:No relevant conflicts of interest to declare.

ETV6 mutations and loss in AML-M0

ETV6 (ETS translocation-variant gene 6, located on chromosome 12p), also known as TEL, encodes a transcription repressor belonging to the E26 transforming specific (ETS) family of DNA-binding proteins. ETV6 is known as a proto-oncogene involved in translocation with over 40 partners.1 In acute myeloid leukemia (AML) only a few rare translocations result in transforming fusion proteins,1 indicating that the oncogenic role of ETV6 does not play a major part in AML. However, abnormalities of the short arm of chromosome 12 (12p) are found in about 5% of AML and myelodysplastic syndromes. Most abnormalities consist of total or partial loss of 12p usually affecting ETV6 and CDKN1B, implicating these genes as tumor-suppressor genes.1, 2, 3, 4 Recently, heterozygous mutations of ETV6, resulting in loss of repressor activity, were found in AML, adding to the view that ETV6 might have tumor-suppressor characteristics.5