Journal articles on the topic 'NS1 truncation variants'

Abstract Somatic mutations are frequently found in patients with MDS. Germline (GL) alterations are less common, in part due to sequencing panels limited to mutations seen commonly in myeloid malignancies, with further exploration only pursued in patients with a clear indication of a familiar disease. It is common to have GL cases where family history is not as informative as we expect, given unreliable recollection by patients and premature mortality of family members. The recognition of pathogenic GL variants is difficult due to the tremendous numbers of inconsequential SNPs, unclear pathologic significance of most new/rare variants and the lack of paired tumor/GL DNA. GL variants may be affecting risk as i) predisposing factors to seemingly sporadic adult MDS or ii) known "leukemia genes" with variable penetrance or longer latency due to lesser functional impact than canonical defects seen in childhood. In addition, there is an ongoing debate as to the role of heterozygous variants of recessive traits serving as predisposition factors. Either way, GL variants may constitute a non-clonal first hit with a long latency until clinical manifestation. To determine the percentage of MDS cases whose etiology may be related to GL mutations, we screened for GL alterations in two sets of selected genes, i) genes often affected by somatic mutations in MDS (n=65), ii) genes known to predispose to bone marrow failure, leukemia or other cancers (n=105). We focused on Tier 1 variants only. We analyzed 766 patients diagnosed with MDS for the presence of GL variants in the previously described set of genes; a rational pipeline was developed. In brief, to determine which alterations were pathogenic, all variants with a VAF <30% were deemed somatic. From there, all variants with a CADD score less than 15 or with a benign annotation in ClinVar were removed. We then removed any remaining variants confirmed to be somatic in COSMIC or those tested as somatic by study of GL DNA. Questionable variants were validated by Sanger and targeted deep sequencing to exclude somatic lesions and technical artifacts. This strategy left a total of 829 alterations. We furthermore classified the variants as Tier 1 and Tier 2 variants. All frameshift/nonsense variants were classified as Tier 1, along with missense variants that were previously described as pathogenic or disease causative. A total of 283 variants were classified as Tier 1, and about half were frameshift/nonsense mutations. Focusing on genes that are frequently somatically mutated in MDS, GL variants were most commonly in TP53 (n=18), NF1 (n=17), RUNX1 (n=12), CEBPA (n=12), and ETV6 (n=6). GL mutations in these genes have been previously described as predisposition factors to MDS evolution. No Tier 1 variants were found in spliceosomal, cohesion complex genes or TET2 or ASXL1. DNA repair genes were frequently affected by Tier 1 variants; we found 24 FA, 16 ATR/ATM, and 25 BRCA1/2 variants. Genes associated with mismatch repair were also found to be recurrently mutated with 7 Tier 1 variants in MSH2/6. Most GL alterations in genes of the telomerase complex were classified as Tier 2 alterations, with the exception of a few frameshift alterations in POT1, DKC1, and TERT. We found 18 previously described DDX41 GL alterations, a number of already described CSF3R variants (N=20), and a reoccurring BARD1 nonsense mutation in 7 patients. Focusing on biallelic lesions of the same genes, GL variants can serve as bona fide ancestral non clonal hits with a less then random clonal succession. For example, multiple mutations, one somatic and one GL, of the same gene can be seen in patients with DDX41, RUNX1, and CSF3R. When assessing the frequency of GL truncating variants in the general population, we found DNA repair genes to be significantly more frequent in otherwise spontaneous adult MDS patients (p<.001). Despite the challenge in identifying germline-initatied MDS because of competing mortality risks, genetic factors likely play a role in adult patients with otherwise typical seemingly spontaneous MDS/AML. Some alterations include less penetrant alterations of oncogenic genes while others imply a generalized predisposition consistent with a mutator phenotype, HRD or genomic instability. Given our ability to obtain NGS information about myeloid neoplasms, it is essential to delineate not only somatic but also GL predisposition mutations that contribute to an individual's risk and their impact on genealogy. Disclosures Nazha: MEI: Consultancy. Carraway:FibroGen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Agios: Consultancy, Speakers Bureau. Sekeres:Opsona: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Abstract TET2 mutations are the most common somatic genetic lesions in myeloid neoplasms. TET2 mutant clones have been found also in healthy individuals, increase with age, and convey an increased risk for myeloid clonal diseases. The TET2 gene is very polymorphic, with hundreds of single nucleotide polymorphisms (SNPs) of unknown clinical impact, but with some variants that may be pathogenically important. Similarly, somatic mutations affect all portions of the gene, and can be missense or truncating, homo-, hemi- and heterozygous. While the majority of TET2 mutations are ancestral, they can also be subclonal, implicating the clonal architecture in the consequences of TET2 lesions. This diversity may be hampering establishment of the clear prognostic impact of TET2 mutations. Taking advantage of a large cohort of patients (pts, N=4985 including 1616 MDS, 871 MDS/MPN, 1782 pAML, 304 sAML, 333 MPN, and 79 therapy-related MDS/AML/MDS-MPN) analyzed by targeted deep sequencing for TET2 and other common myeloid lesions, we examined the distribution and impact of TET2 mutations. DNA sequencing of all coding exons of TET2 and 61 other genes representing the most common somatic mutations in myeloid neoplasms. Nonsynonymous alterations not present in SNP database (dbSNP) were annotated as somatic mutations or SNPs if present in myeloid and T cells whenever available. Nonsynonymous alterations not in dbSNP or ExAC databases and not confirmed to be somatic were excluded. Overall, TET2 somatic mutations (TET2mut) were present in 920 pts (18%); 38% of MDS/MPN, 19% pAML, 16% MPN, 16% sAML, 12% MDS, and 13% of therapy related MDS/AML/MDS-MPN. Mutations included 16% missense, 33% frameshift deletions, 18% frameshift insertions, and 33% nonsense. TET2mut pts were older than those with TET2 wild type (TET2wt, 72 vs. 67 yrs, p<.001), had a higher presenting WBC (6 vs. 4 x103 /uL, p <.001), and lower blast % (3 vs. 7%, p =.03). Similar findings were observed in each myeloid subtype. Overall, median OS for TET2mut pts was similar to TET2wt (12 vs. 17 mo, p =.20). Median OS was similar in TET2mut pts compared to TET2wt in pts with MDS (23 vs. 23 mo, p =.77), MDS/MPN (15 vs. 21 mo, p=.1), pAML (9 vs. 14 mo, p =.77), sAML (6 vs. 9 mo, p =.07), and MPN (30 vs. 35 mo, p =.66). Neither the type of mutation (mis-, nonsense vs. truncating) nor location (catalytic domain vs. other) impacted the OS. Using variant allelic frequencies (VAF), we established a clonal hierarchy in individual cases; 24% of TET2 mutations were ancestral, 17% subclonal, and 59% codominant. TET2 mutations were ancestral in 23% of MDS samples, 29% of MDS/MPN, 25% of pAML, and 19% of sAML. Whether the mutation was ancestral or subclonal did not impact OS. The presence of TET2mut was associated with different mutations in each myeloid subtype. In MDS, TET2mut were associated with APC (p<.001), ASXL1 (p<.001), BCOR (p<.001), BCORL1 (p<.001), ETV6 (p<.04), SUZ12 (p<.001), RAD21 (p<.02), NF1 (p<.001), KDM6A (p<.001), ZRSR2 (p<.001), and U2AF1 (p=.02), in MDS/MPD correlated with ASXL1 (p<.04), NRAS (p<.02), and SRSF2 (p<.05), in pAML with JAK2 (p<.001), RUNX1 (p =.05), and CBL (p=.05), and in sAML with RUNX1 (p<.001), ASXL1 (p<.001), BCORL1 (p=.01), SUZ12 (p=.02), STAG2 (p=.05), and JAK2 (p<.001). When we next focused on germ line variants, we identified 2518 SNPs of TET2. All recurring SNPs were ranked according to the difference in their frequencies between pts and healthy controls. A large number of these SNPs were more common in our pts compared to controls, among them we identified 2 SNPs (both located in the dioxygenase domain) with a significantly higher frequency: SNP1 (OR 10.6, p<.0001), and SNP2 (OR 6.7, p=.02). We further investigated whether these SNPs were mutually exclusive or increased the risk for acquisition of somatic TET2 mutations; 91% of cases with SNP1 and 67% with SNP2 also acquired somatic mutations in TET2. In silico and crystallographic analyses showed that SNP1 is adjacent to the iron binding site (7th beta stand in the jelly roll motif) and is predicted to change the orientation of a-KG binding and thereby to be hypomorphic. SNP2 is located in a hot spot area known to be targeted by 3 recurrent somatic mutations. In conclusion, both somatic mutations as well as germ line variants affect TET2 in myeloid neoplasia. The interaction between clonal mutations and germ line lesions may lead to gain of function and thus a growth advantage. These mechanisms are currently being explored. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

Abstract Introduction: Relapse of AML after initial remission occurs in about 30-50% of patients (pts) and is associated with dismal outcome. The majority of pts relapse within the first 2 years after diagnosis and their clonal evolution has been extensively studied. Late relapses, however, are very rare and therefore poorly characterized. We set out to study pts who experience relapse >3 years after initial diagnosis to depict the genetic profile and gain knowledge about the pathogenesis in these pts. Methods: We selected AML cases sent to our laboratory for diagnostic work-up from 2005 - 2015 and who relapsed > 3 years after initial diagnosis (Dx). Based on sample availability, whole exome sequencing (WES) was carried out for 31 pts at the time points of Dx and relapse as well as on matched remission samples that were available for 19 pts. Enrichment based library preparation was performed using the xGen Exome Research Panel and sequenced (2x151bp) on a NovaSeq instrument. Data was processed with BaseSpace using the BWA Enrichment app with BWA for Alignment (against hg19) and GATK for variant calling with default parameters. Data was subsequently loaded into BaseSpace Variant Interpreter to filter and prioritize variants of interest. Only passed protein changing variants were considered with an ExAC population frequency of less than 1% for further analysis. As control cohort, we identified patients that relapsed within 1 year (n=371) after Dx for comparison of genetic risk factors. Results: The cohort included 15 females and 16 males, aged 21 -75 years (median: 60 years). Relapse occurred 3.0 -8.1 years after Dx (median: 4.0 yrs). According to MRC criteria, pts were assigned to the following cytogenetic risk groups (cytogenetic data was not available for 2 pts): good risk, n=4 (14%); intermediate risk, n=22 (76%) and adverse risk, n=3 (10%). FLT3 internal tandem duplication (ITD) was identified in 5 of 29 tested pts (17%). When comparing pts that relapse within the first year after Dx to our cohort of pts with relapse after >3 years, we observed a trend towards a lower frequency of high-risk genetic parameters in the latter group (adverse risk cytogenetics 18% vs 10%; FLT3-ITD, 32% vs 17%; TP53 mutation 8% vs 0%). However, this trend was not statistically significant. Cytogenetic changes between Dx and relapse were observed in 13/27 (48%) of pts analyzed at both time points. In 8/13 (61%) of these cases, a single cytogenetic aberration was gained at relapse, while a new complex karyotype was observed in 2/13 (15%) of relapsed pts and in 1 pt with complex karyotype at diagnosis, additional aberrations were detected at relapse. Furthermore, we identified cytogenetically independent clones between Dx and relapse in 2/13 (15%) cases. Next, we analyzed the molecular genetic evolution in our cohort. Overall, WES identified a total of 106 mutations in 30 genes associated with myeloid neoplasia (Figure 1). Following genes were mutated in >10% of pts: NPM1 (13/31 of pts), FLT3 (10/31), IDH2, DNMT3A and RUNX1 (8/31, each), SRSF2 (6/31), BCOR (5/31), NRAS and IDH1 (4/31, each). When comparing the matched diagnostic and relapse samples, a total of 61 mutations were detectable at both time points. A total of 24 mutations present at Dx were lost at relapse. Most frequently, mutations in NRAS (n=4) and FLT3 (n=4) were not detectable in the relapse samples. Overall, 12/24 (50%) of mutations lost at relapse affected signaling pathways, followed by transcription factors and epigenetic modifiers (n=5, each). In contrast, 21 mutations were gained during disease progression. Ten of these mutations (48%) are predicted to result in truncation of a transcription factor with known role in normal hematopoiesis (RUNX1, n=4; ETV6 and BCOR, n= 2; WT1 and BCORL1, n=1). In addition, 4 mutations gained during disease progression affected tumor suppressor genes (NF1, n=2; TP53, n=1, ATM, n=1). In 6/19 (31%) of pts with available data mutations in epigenetic modifiers persisted in remission (DNMT3A, n=3; IDH1, SRSF2, and TET2, n=1). Conclusion: In our cohort of AML pts with relapses >3 years after Dx, we observed a high frequency of cases that lost mutations affecting signaling pathways during disease progression and gained mutations in hematopoietic transcription factors, indicating that the relapse clone in these patients is promoted by aberrant differentiation of hematopoietic stem cells. Disclosures Hartmann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Stengel: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.

Abstract Different CSF3R mutations (CSF3RMT) result in aberrant G-CSF signaling pathways and are linked to a wide range of myeloid disorders. Loss-of-function mutations in its extracellular domain cause severe congenital neutropenia (SCN). Activating mutations in the juxtamembrane region have been associated with a variety of myeloid malignancies. Truncating mutations in the cytoplasmic domain are associated with SCN cases that progress to MDS or AML. In this study, we evaluate the extent to which different CSF3RMT associate with disease onset, progression to leukemia and neutrophil counts in patients (pts) diagnosed with myeloid malignancies. We identified CSF3RMT cases in a cohort of 1400 pts [median age 71 years (yrs)]. We analyzed somatic and germline mutational patterns, and cross-sectional correlation with other gene mutations in CSF3RMT. A stringent algorithm based on conserved amino acid residues and alterations of protein features was used to predict the pathogenic significance of CSF3RMT. We identified 44 CSF3RMT: 33 germline (CSF3RGL) and 11 somatic (CSF3RS) variants. Most CSF3RGL were found in pts (median age 63 yrs) with MDS or related conditions (87% of all mutant cases), conversely these mutations were present in 5% (n= 22/424) of MDS, 3% (n= 7/244) MDS/MPN and <1% (n= 3/392) of AML and in 1 out of 3 pts with aCML tested. Mutations were mostly missense and located between the cytoplasmic (58%: M696T, R698C (isoform III), D732N, P733T, S744F, Y752*, E808K), and extracellular (42%: C131Y, E149Q, A208V, Q216H, D320N, E405K, S413L, Y562H) domains. No mutations were detected in the juxtamembrane domain. Variants were grouped in Tier-1 (61%: C131Y, E149Q, A208V, Q216H, D320N, E405K, S413L, Y562H Y752*, E808K) and Tier-2 (variants with uncertain significance, 39%: S413L, M696T, R689C, D732N, P733T, S744F). E808K and R698C were the most common amino acid changes in Tier-1 (53%) and Tier-2 (44%), respectively. A total of 4/7 pts with E808K progressed to AML (but none with R698C), supporting previous observations that E808K (or E785K) represents a pathogenic variant predisposing to leukemia. A total of 46% (n=14) of pts with CSF3RGL had neutropenia [median 0.9x109/L (0.02-1.22x109/L)] at the time of sampling. Two pts diagnosed with a prior cancer manifested sustained neutropenia before the diagnosis of MDS and MDS/MPN. G-CSF was administered in 21% of pts. Alterations in -7/7q- were common (21%). Some pts also harbored other somatic mutations in NF1 (15%), DNMT3A (12%), SETBP1 (12%), or U2AF1 (12%). Of note, 1 patient carried mutations in WAS and GATA2 and another carried a mutation in VPS45, which have been previously associated with SCN/MDS. The patient with aCML harbored also a CSF3RS (T615A). Overall combined allelic burden in pts cohort was 2% vs. 1.6% expected allelic burden in control populations for the same variants (P=.02). CSF3R S were found in 11 pts (median age 71 yrs) with AML or MDS related conditions (73% of all mutant cases), conversely these mutations were present in 1.4% (n= 6/424) of AML, <1% in MDS (n= 2/244) and MDS/MPN (n= 1/392) and in 2/3 pts with aCML tested. Mutations were missense in 63% of pts, T618I being most recurrent (n=5/11; 45%). Frameshifts accounted for 36% of the mutations and were localized in the cytoplasmic domain (Q741*, Q749*, Y752*, Q768*). All mutations were heterozygous. At the time of sampling 3/11 pts had leukocytosis and 3/11 had neutropenia. Mutations were distributed between the juxtamembrane domain (55%) and the cytoplasmic domain (45%). Mutations in the extracellular domain were not detected. Pts with sAML mostly carried mutations in the juxtamembrane domain (67%), those with MDS carried only in cytoplasmic domain, and those with MDS/MPN or aCML carried mutations in both the juxtamembrane and extracellular domains. There was one somatic and one RUNX1GL mutation. The cytogenetic abnormalities -7/7q- were detected in 18% (2/11) of cases. Interestingly, T618I was found solely in pts with sAML. Focusing on associations between CSF3RMT and mutations in the class III receptor tyrosine kinases CSF1R, FLT3, and KIT we identified only FLT3 to be co-mutated with CSF3RMT. All 3 pts (2 CSF3RGL and 1 CSF3RS) with such co-mutations evolved to AML. In sum, we found that CSF3RGL do not commonly co-occur with CSF3RS, suggesting that the neutropenia observed at the sampling time most likely is causative of undetected GL variants and/or is representative of a long unrecognized disease. Disclosures Nazha: MEI: Consultancy. Carraway:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Speakers Bureau; FibroGen: Consultancy. Santini:Otsuka: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Abstract Introduction: Acute myeloid leukemia (AML) with inv(3)(q21q26) or t(3;3)(q21;q26) [inv(3)/t(3;3)] is associated with aberrant expression of the stem cell regulator EVI1 and dismal prognosis. Recently, we and others (Gröschel et al, Cell, 2014; Yamazaki et al, Cancer Cell, 2014) have shown, as a consequence of the inv(3)/t(3;3) rearrangements, that the proto-oncogene EVI1 is activated upon the structural repositioning of a distal GATA2 enhancer from 3q21 to EVI1, coinciding with loss of GATA2 expression from the rearranged allele. Notably, GATA2 deficiency has been shown to impair hematopoietic stem cell frequency and function (Lim et al, J. Clin. Invest., 2012) and Evi1 activation in inv(3) murine models is followed by leukemia onset after a long latency of 6 months (Yamazaki et al). We therefore hypothesize that additional cooperating genetic lesions, other than EVI1 activation and GATA2 deregulation, are required for full leukemic transformation. We sought to extend the molecular characterization of inv(3)/t(3;3) myeloid malignancies through next-generation sequencing. Methods: We selected 32 AML (including cell lines MUTZ-3 and UCSD-AML1), 4 CML-BC (including cell lines HNT-34 and MOLM-1), and 5 myelodysplastic syndrome (MDS) cases with inv(3)/t(3;3) rearrangements and performed whole transcriptome sequencing (n=40) as well as whole exome sequencing (WES, n=10) on the Illumina HiSeq 2500. For WES germline control we used DNA from cultured CD3+ T-cells from diagnostic bone marrow specimens. Variants were examined when recurrently detected in more than two patients or previously associated with leukemogenesis or cancer pathogenesis. Gene expression profiles (GEP) were constructed for 24 cases for subsequent analyses. Results: All examined variants, validated by Sanger sequencing, were assigned to mutational categories to discern patterns of mutations (Figure 1A). We observed that all 41 cases acquired at least one mutation belonging to the mutational categories reported as relevant for leukemia pathogenesis. Importantly, all AML and CML-BC patients, as well as 4 out of 5 MDS patients contained mutations in genes activating RAS/RTK signaling, amounting to 98% of all inv(3)/t(3;3) myeloid malignancies. An aggregate of 74% of all patients harbor mutually exclusive mutations in RAS-pathway associated genes; NRAS(27%), PTPN11(20%), KRAS(11%), and NF1(9%). Additionally, we found mutations in genes affecting signaling pathways involving RAS; FLT3(13%), BCR-ABL(11%), and KIT(2%) (Figure 1B). NPM1, CEBPA, and IDH1/2 mutations were absent in all cases. Furthermore, frequent mutations were found in transcription factor (32%), splice factor (29%), epigenetic modifier (29%), and tumor suppressor (10%) encoding genes (Figure 1A). GATA2 is the most commonly mutated transcription factor in inv(3)/t(3;3) myeloid malignancies (15%). As these mutations were found on the non-rearranged GATA2 allele, only mutant GATA2 is expressed due to GATA2 silencing mediated by the 3q21q26 rearrangement. Novel somatic truncating mutations and copy number loss were detected in the transcription factor FOXP1(10%). Additionally, we observed frequent mutations in the splice factor gene SF3B1(27%) in AML and MDS cases. Finally, the predominant monosomal karyotype within inv(3)/t(3;3) myeloid malignancies is contrasted by the low incidence of TP53 mutations (5%). No mutational pattern alluded to the high coincidence of monosomy 7 (68%), but inv(3)/t(3;3)/-7 cases were characterized by a strong down regulation of the transcription factor CUX1. In an effort to discriminate MDS and AML with inv(3)/t(3;3) we performed cluster and principle component analyses, revealing no cluster formation. Additionally, differential expression analysis was unable to differentiate AML from MDS, indicating strong homogeneity based on GEPs. Conclusion: Myeloid malignancies with inv(3)/t(3;3) harbor a common set of molecular alterations, i.e. EVI1 activation and GATA2 deregulation, conjugated by mutations activating signaling pathways. Mutational patterns and GEPs precluded the discrimination of MDS and AML with inv(3)/t(3;3), supporting the notion that these myeloid disorders should be categorized as AML irrespective of blast count. Exploiting signaling pathways therapeutically may prove a valuable adjunct to the scarce armamentarium of drugs effective in this malignancy. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract Genetic studies in myeloid neoplasms have revealed that somatic mutations and deletions frequently affect epigenetic regulation via DNA methylation and histone modification. One frequently affected epigenetic complex is polycomb repressive complex 2 (PRC2), which trimethylate Histone3Lysine27 (H3K27) to create a repression mark for the expression of a variety of genes that includes essential homeobox genes. Members of this complex include EZH2, EED and SUZ12, which exert methyltransferase activity, and perhaps indirectly also DNTM3A and ASXL1. The histone demethylase ubiquitously transcribed X (UTX) counters the enzymatic activity of PRC2 by removing di- and trimethyl groups from H3K27. It thus removes repressive marks. We were the first to report UTX mutations in a small portion of CMML and MDS cases. In another cohort, frequent somatic loss-of-function mutations in UTX were found in ALL. The goal of the present study was to understand the functional role of UTX and the consequences of its mutations on H3K27 methylation status, specifically with respect to their relevance to myeloid neoplasia. A total of 1,979 patients with various myeloid neoplasms (n = 1,686) and other hematologic disorders (n = 293) were screened for gene mutations in UTXand other reported driver genes relevant to myeloid neoplasms using whole exome sequencing and/or targeted deep-sequencing. Expression array analyses were performed on 200 samples. In addition, we also used sequencing and expression data from the de novo AML TCGA repository. UTXwas mutated in 55 (2.8%) cases out of 1,979 cases. The mutations were found in 2.5% of MDS, 3.1% de novo AML (including 8.3% CBF AML), 4.8% MDS/MPN, and 1.4% secondary AML (sAML). Of those, 77% were missense mutations and 23% nonsense, splice site and frameshift mutations, without an evident hot spot. The male-to-female ratio was 1.2 (55% vs. 45%). UTX gene expression was significantly higher in females than in males (p<.001). After gender adjustment and dichotomized using a threshold of expression of 2 standard deviations from the mean, low UTX expression levels were identified in 13/183 (7%) individuals. When we focused on clonal burden using variant allele frequencies (VAFs) to investigate clonal architecture within the tumor population, in 37 cases UTX constituted subclonal events and in 18 they were dominant. We then examined the molecular context of UTX lesions. Average mutation burden in patients with UTX mutations was higher than in WT UTX carriers (4 vs. 1.5, p<.001). UTX mutations co-occurred with other driver genetic mutations such as ASXL1, ZRSR2, CUX1, NF1. Surprisingly, EZH2 mutations are also enriched in UTX mutated cases although they function in the opposite direction of H3K27 trimethylation. Focusing on dominant clone and subclonal events in cases with these two mutations, EZH2mutations were enriched in cases with dominant UTX clone. This suggests that they play important roles in the cancer's pathogenesis. To clarify the role of UTX in the maintenance of leukemia, genomic knockouts of UTX were developed in human leukemic cell lines using the CRISPR-Cas9 system. RNA sequencing revealed that knockout cell enrichment for developmental regulators such as Hox genes. In addition, we made knockdowns of human CD34+ cells using short hairpin RNAs against UTX. The cells showed enhanced colony formation and increased replating efficiency consistent with retained clonogenicity. The truncating pattern of UTX mutations in myeloid neoplasms suggests that they are loss-of-function hits. Missense mutations thus need to be confirmed. Functional analysis in vitro shows that low expression of UTX may have functionally equivalent consequences. If so, an additional 7% of patients may have low UTX expression, and may thus phenocopy patients with UTX mutations. Combined, a total of ~10% out of myeloid neoplasm patients may harbor UTX deficiency. Epigenetic modifying drugs related to H3K27 such as inhibitors of EZH2and histone deacetylases are in development. It is controversial to which patients they should be applied. Our findings could be key to a deeper understanding of epigenetic alterations, drug function, and response. Disclosures Makishima: The Yasuda Medical Foundation: Research Funding. Mukherjee:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

PurposeThe contribution of rare genetic variation in the development of soft-tissue sarcoma (STS) remains underexplored. To address this gap, we conducted a whole-exome case-control and somatic-germline interaction study to identify and characterise STS susceptible genes.MethodsThe study involved 219 STS cases from The Cancer Genome Atlas and 3507 controls. All cases and controls were matched genetically onEuropean ancestry based on the 1000 Genomes project. Cross-platform technological stratification was performed with XPAT and gene-based association tests with VAAST 2.ResultsNF1 exhibited the strongest genome-wide signal across the six subtypes, with p=1×10−5. We also observed nominally significant association signals for three additional genes of interest, TP53 (p=0.0025), RB1 (p=0.0281), and MSH2 (p=0.0085). BAG1, which has not previously been implicated in STS, exhibited the strongest genome-wide signal after NF1, with p=6×10−5. The association signals for NF1 and MSH2 were driven primarily by truncating variants, with ORs of 39 (95% CI: 7.1 to 220) for NF1 and 33 (95% CI: 2.4 to 460) for MSH2. In contrast, the association signals for RB1 and BAG1 were driven primarily by predicted damaging missense variants, with estimated ORs of 12 (95% CI: 2.4 to 59) for RB1 and 20 (95% CI: 1.4 to 300) for BAG1.ConclusionsOur results confirm that pathogenic variants in NF1, RB1 and TP53 confer large increases in the risk of developing multiple STS subtypes, provide support for the role of MSH2 in STS susceptibility and identify BAG1 as a novel candidate STS risk gene.

Abstract Background Dedifferentiated liposarcoma (DDLPS), which accounts for an estimated 15–20% of liposarcomas, is a high-grade and aggressive malignant neoplasm, exhibiting a poor response to available therapeutic agents. However, genetic alteration profiles of DDLPS as well as the role of NF1 mutations have not been studied extensively. Case presentation The current study reports a patient presenting with rapidly growing DDLPS accompanied by multiple lung and pleural metastases, in whom whole-exome sequencing revealed a NF1 truncating mutation of the known pathogenic variant, c.C7486T, p.R2496X, as well as multiple copy number alterations (CNAs), including the well-known 12q13–15 amplification, and multiple chromothripsis events encompassing potential cancer-related genes. Conclusions Our results suggest that, in addition to the 12q13–15 amplification, NF1 inactivation mutation and other CNAs may contribute to DDLPS tumorigenesis accompanied by aggressive clinical features.