Journal articles on the topic 'VPF B-Frames'

ABSTRACT By different approaches, we characterized the birnavirus blotched snakehead virus (BSNV). The sequence of genomic segment A revealed the presence of two open reading frames (ORFs): a large ORF with a 3,207-bp-long nucleotide sequence and a 417-nucleotide-long small ORF located within the N-terminal half of the large ORF, but in a different reading frame. The large ORF was found to encode a polyprotein cotranslationally processed by the viral protease VP4 to generate pVP2 (the VP2 precursor), a 71-amino-acid-long peptide ([X]), VP4, and VP3. The two cleavage sites at the [X]-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing. We showed that the processing of pVP2 generated VP2 and several small peptides (amino acids [aa] 418 to 460, 461 to 467, 468 to 474, and 475 to 486). Two of these peptides (aa 418 to 460 and 475 to 486) were positively identified in the viral particles with 10 additional peptides derived from further processing of the peptide aa 418 to 460. The results suggest that VP4 cleaves multiple Pro-X-Ala↓Ala motifs, with the notable exception of the VP4-VP3 junction. Replacement of the members of the predicted VP4 catalytic dyad (Ser-692 and Lys-729) confirmed their indispensability in the polyprotein processing. The genomic segment B sequence revealed a single large ORF encoding a putative polymerase, VP1. Our results demonstrate that BSNV should be considered a new aquatic birnavirus species, slightly more related to IBDV than to IPNV.

ABSTRACT Rotaviruses are a leading cause of viral acute gastroenteritis in humans and animals. They are grouped according to gene composition and antigenicity of VP6. Whereas group A, B, and C rotaviruses are found in humans and animals, group D rotaviruses have been exclusively detected in birds. Despite their broad distribution among chickens, no nucleotide sequence data exist so far. Here, the first complete genome sequence of a group D rotavirus (strain 05V0049) is presented, which was amplified using sequence-independent amplification strategies and degenerate primers. Open reading frames encoding homologues of rotavirus proteins VP1 to VP4, VP6, VP7, and NSP1 to NSP5 were identified. Amino acid sequence identities between the group D rotavirus and the group A, B, and C rotaviruses varied between 12.3% and 51.7%, 11.0% and 23.1%, and 9.5% and 46.9%, respectively. Segment 10 of the group D rotavirus has an additional open reading frame. Generally, phylogenetic analysis indicated a common evolution of group A, C, and D rotaviruses, separate from that of group B. However, the NSP4 sequence of group C has only very low identities in comparison with cogent sequences of all other groups. The avian group A NSP1 sequences are more closely related to those of group D than those of mammalian group A rotaviruses. Most interestingly, the nucleotide sequences at the termini of the 11 genome segments are identical between group D and group A rotaviruses. Further investigations should clarify whether these conserved structures allow an exchange of genome segments between group A and group D rotaviruses.

Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5′ untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency.

ABSTRACTHuman noroviruses are a leading cause of gastroenteritis across the globe, but the pathogenic mechanisms responsible for disease are not well established. The availability of a murine norovirus model system provides the opportunity to elucidate viral and host determinants of virulence in a natural host. For example, previous studies have revealed that the protruding domain of the murine norovirus capsid protein VP1, specifically residue 296 of VP1, regulates virulent infection. We identified a panel of nonsynonymous mutations in the open reading frame 2 (ORF2) gene encoding VP1 that arose in persistently infected mice and tested whether these mutations conferred phenotypic changes to viral replication and virulence. Consistent with previous studies, we demonstrate that a glutamic acid at position 296 results in attenuation. For the first time, we also demonstrate that a lysine at this position is sufficient to confer virulence on an otherwise attenuated murine norovirus strain. Moreover, our studies reveal a direct correlation between the efficiency of viral replication in B cells and virulence. These data are especially striking because mutations causing reduced B cell replication and attenuation had minimal effects on the ability of the virus to replicate in macrophages. Thus, norovirus infection of B cells may directly contribute to disease outcome.IMPORTANCEHuman noroviruses are a major global cause of disease, yet we know very little about their pathogenic mechanisms. The availability of a murine norovirus model system facilitates investigation of noroviruses in a natural host organism and the identification of viral and host determinants of pathogenesis. We have identified a panel of mutations arising in the viral capsid protein VP1 during persistent infection of mice. Our data reveal that the protruding domain of VP1 regulates the ability of the virus to replicate in B cells, and this directly correlates with virulence. Importantly, mutations impairing B cell infection had minimal effects on macrophage infection, revealing a potentially critical role for B cell infection in norovirus pathogenesis.

The major inner capsid protein (VP6) gene of the bovine group B rotavirus (GBR) Nemuro strain is 1269 nt in length and contains one open reading frame encoding 391 aa. Nucleotide and amino acid sequence identities of the Nemuro VP6 gene compared with the published corresponding human and rodent GBR genes were respectively 66–67 and 70–72 %, which are notably lower than those between human and rodent viruses (72–73 and 83–84 %, respectively). Overall identities of VP6 genes among GBRs were substantially lower than those among both group A rotaviruses (GARs) and group C rotaviruses (GCRs) derived from different species of mammals. These results demonstrate that bovine GBR is remarkably distinct from other GBRs and that GBRs from different species may have had a longer period of divergence than GARs and GCRs. Recombinant VP6 was generated with a baculovirus expression system and used for an ELISA to detect GBR antibodies. All 13 paired sera from adult cows with GBR-induced diarrhoea in the field showed antibody responses in the ELISA. In serological surveys of GBR infection using the ELISA, 47 % of cattle sera were positive for GBR antibodies, with a higher antibody prevalence in adults than in young cattle. In pigs, a high prevalence of GBR antibodies (97 %) was detected in sera from sows. These results suggest that GBR infection is common in cattle and pigs, notwithstanding the scarcity of reports of GBR detection in these species to date.

Phylogenetic analysis within the VP1 region now enables molecular typing of enteroviruses consistent with neutralization results. Three untypable isolates, 2776/82, 57/99 and 22/00, from Korea, North India and Bangladesh, respectively, showed within this region 98·0–99·0% amino acid identities. These were less than 77% to the previous enterovirus prototypes, but 91·5–92·5% to CA55-1988, the recently identified enterovirus 73 (EV73) prototype from California. All three strains were, however, most similar to CA64-4454, an EV73 prime strain, to which they shared 96·5–98·5% identity. Seven compared EV73 strains formed two clusters in the VP1 dendrogram, one cluster with strains from South and East Asia and CA64-4454, and the other with strains from Oman and California including the prototype. When sequencing the complete open reading frame of 2776/82, its non-structural region was found to be divergent from all human enterovirus B (HEV-B) strains, including CA55-1988, indicating that one or other strain was recombinant. Boot scanning of the genomes showed a recombination point within the P2 region. Therefore, part of this was sequenced for 57/99 and 22/00 and was found similar to 2776/82, while CA55-1988 was similar to coxsackievirus B3, demonstrating that CA55-1988 was the recombinant. Since all strains of EV73 isolated so far outside California originate from Asia, where it has a broad geographical distribution, it seems that EV73 may have been introduced to California from Asia. Further analysis of EV73 strains will reveal if the recombination occurred in the USA or in Asia and will help to elucidate the origin of this virus.

Abstract Myeloproliferative neoplasms (MPNs) are clonal malignant disorders characterized by the increased production of mature myeloid cells in blood. The classical MPNs include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Those pathologies are due to the acquisition of gain-of-function mutations leading to the constitutive activation of the cytokine receptor / JAK2 signaling pathway: JAK2V617F in 70% of cases and mutations in the thrombopoietin receptor (MPL) gene in 5% of cases. More recently, around fifty different mutations in the calreticulin (CALR) gene have been described in 30% of ET and PMF with two more frequent mutations called del52 (type 1) and ins5 (type 2). All the CALR mutations induce a frameshiflt to an alternative reading frame in the exon 9 leading to a new C-term tail of the protein with hydrophobic features, and the loss of the KDEL sequence, which is involved in its endoplasmic reticulum retention. The goal of this work was to understand the role of CALR mutants (del52, del46, del34, ins5, del19, del13) in human hematopoiesis. By studying the variant allele frequency (VAF) in 20 patients, we have shown that the CALR mutations are present in all blood mature cells not only in granulocytes and monocytes (CD14+) with a VAF >30% but also in B cells (CD19+), NK cells (CD56+) and in some cases in T cells (CD3+). Moreover, we have observed that CALR mutations are present in all hematopoietic progenitors including CD34+CD38-CD90+ (HSC), CD34+CD38-CD90- (immature progenitors) and CD34+CD38+ (committed progenitors) cell fractions after investigating the clonal architecture of the progenitors. CALR mutation was detectable in more than 40% of progenitor cells except in 2 patients (15 patients studied) and with, in some cases, no detectable wild type CALR progenitors. Homozygous CALR mutations were rare except in one case associated with disease progression. Whatever the VAF, there was no significant differences among the different progenitor types and granulocytes. Finally, we observed that all the associated mutations studied (TET2, PHF6, SYNE1, SCARA5, PIK3CD, SETD1B) in 6 patients postdated CALR mutations. We could also show in 15 patients samples that CALR mutants give a specific megakaryocytic progenitor (CFU-MK) spontaneous growth mediated both by MPL and JAK2 activation using specific inhibitors and short hairpin RNAs. The CFU-MK spontaneous growth correlated with a constitutive activation of JAK2/STAT3/5 pathway in megakaryocytes derived from in vitro cultures of CD34+ progenitors. In aggregate, these results show that all CALR mutants studied are present in all human hematopoietic cells including myeloid and lymphoid cells, give an early clonal advantage at the level of the HSC compartment and a specific increased growth of the megakaryocytic lineage via MPL/JAK2 activation. Disclosures No relevant conflicts of interest to declare.

Abstract The persistence of leukemic mutation(s) in AML patients who have achieved a morphologic complete remission (CR) after intensive induction chemotherapy is a strong predictor of early relapse and reduced overall survival (OS) (Klco JAMA, 2015; Morita, J Clin Oncol 2018; Jongen-Lavrencic, NEJM, 2018). There is no clinical consensus as to the optimal consolidation therapy for the ~50% of patients with intermediate-risk AML. The median relapse-free survival (RFS) for patients ≤60 years with ELN intermediate-risk disease is 0.8 years to 1.2 years, with a median OS of 1.2-2.1 years (Mrozek, J Clin Oncol, 2012). We have shown that intermediate-risk patients who clear all leukemia-associated mutations (LAMs) to a variant allele fraction (VAF) of <2.5% in first morphologic CR have a median event-free survival of 25.6 months, vs 8.8 months if they do not (HR 3.32). Median overall survival is 46.8 months if all LAMs are cleared, vs 19.3 months if they are not (HR 2.88). We hypothesized that improved post-remission risk stratification using LAM clearance can further refine risk assessment and optimize alloHCT decisions by identifying patients at lower risk of relapse, who might be expected to do well with standard chemotherapy. Here, we report the development of a pipeline to prospectively determine the persistence of LAMs after remission-induction, and return results in a clinically actionable time-frame. We perform enhanced exome sequencing (EES) of paired skin or buccal swab (normal tissue) and bone marrow DNA to comprehensively identify all LAMs at diagnosis (Day 0) and to assess their clearance post-induction (~Day 30). EES data are generated using a CLIA-compliant assay in the CLIA-licensed environment (CLE) lab at the McDonnell Genome Institute, and results are returned to the treating physician. Intermediate risk patients ≤60 years with clearance of all LAMs (VAFs <2.5%) are assigned to receive consolidation with high-dose cytarabine (HiDAC) (Cohort A). Patients with persistence of any mutation at a VAF ≥ 2.5% are assigned to the investigator's choice arm, and are treated with HiDAC and/or alloHCT at the discretion of the treating physician (Cohort B). This stratification is part of an ongoing clinical protocol (NCT02756962) whose primary objective is to determine whether the RFS of patients who have cleared all LAM(s) post-induction (VAFs <2.5%) and are treated with HiDAC alone (Cohort A) is significantly higher than expected from a historical intermediate risk group. Measurable residual disease testing by "difference from normal" flow cytometry (lower level of detection of 0.02%, Hematologics, Seattle WA) post-induction will be correlated with clearance or persistence of mutations and clinical outcomes. For the 23 patients sequenced to date, the mean turnaround time to issue sequencing results to the treating physician was 24 days from the time of the remission biopsy. All 23 patients had detectable LAMs at presentation (mean 28 per patient, range, 6 to 43) that could be used to track persistent disease in the day 30 remission sample. Eleven patients (48%) cleared all LAMs and received HiDAC only (Cohort A). There was no flow cytometric evidence of residual AML in Cohort A. Twelve patients (52%) had persistent LAMs (Cohort B, investigator's choice). The number of persistent leukemia-associated variants present in Cohort B ranged between 1 and 14. Surprisingly, 9 of the 12 patients with persistent LAMs by sequencing had no flow cytometric evidence of residual leukemia. Seven of 12 patients on the investigator's choice arm have received an alloHCT, and none have relapsed to date. The median follow-up for all subjects is 378 days (range, 59-683). Neither the median RFS (Fig. 1A) nor the median OS (Fig. 1B) has been reached for either cohort. While preliminary, these results suggest that patients who clear all LAMs to a VAF of <2.5% may have durable responses with HiDAC alone. The encouraging RFS seen in the investigator's choice arm (Cohort B) may reflect the decision to recommend transplant "upfront" in CR1 for patients who have molecular persistent disease. In summary, identifying persistent LAMs after induction chemotherapy is feasible in an actionable time-frame. Early data suggest that using LAM clearance post-induction may improve current risk-stratification for intermediate-risk AML. Accrual of patients and continued follow-up are ongoing. Disclosures Jacoby: NovoNordisk: Consultancy; Celgene: Speakers Bureau. Loken:Hematologics, Inc: Employment, Equity Ownership. Schroeder:Amgen Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Uy:GlycoMimetics: Consultancy; Curis: Consultancy. Vij:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansson: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kahl:Gilead: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy; CTI: Consultancy; ADC Therapeutics: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Acerta: Consultancy; Juno: Consultancy; Celgene: Consultancy.

Abstract *Contributed equally as first authors. **Contributed equally as senior authors. Recurrent mutations within EGR2, a versatile transcription factor involved in differentiation of hematopoietic cells, were recently reported in 8% of advanced-stage chronic lymphocytic leukemia (CLL) patients, where they appear to be associated with a worse outcome. EGR2 is activated through ERK phosphorylation upon B-cell receptor (BcR) stimulation, and we have previously shown that EGR2 -mutated CLL patients display altered expression of EGR2 down-stream target genes compared to wildtype (wt) patients, thereby pointing to a pathogenic role for EGR2 mutations in dysregulating BcR signaling. To gain further insight into the incidence and prognostic impact of EGR2 mutations in CLL, we screened samples from a well-characterized series of 1430 patients, either by Sanger sequencing (n=1019) or targeted deep-sequencing (n=370), both covering the recently reported EGR2 hotspot in exon 2. In addition, whole-exome data was available for an additional 43 patients. Different cohorts were included in our analysis ranging from 'general practice' CLL (33% IGHV-unmutated (U-CLL), 6% TP53 -aberrant (TP53abn), n=693), to adverse-prognostic CLL (89% U-CLL, 26% TP53abn, n=325), patients belonging to clinically aggressive stereotyped subsets #1-3 & #5-8 (n=342), patients relapsing after FCR therapy (n=41) and Richter transformed cases (n=31), thus reflecting the heterogeneous nature of CLL. Nineteen EGR2 mutations were detected by Sanger sequencing, while 22 additional mutations were identified with deep-sequencing using a 5% variant allele frequency (VAF) cutoff (median 39%, range 5.6-63.9%, median coverage 43,000X). With the exception of one in-frame deletion, all mutations were missense alterations located within the three zinc-finger domains. Significant enrichment of EGR2 mutations was observed in adverse-prognostic (18/325, 5.5%) and FCR-relapsing (4/41, 9.8%) CLL compared to the 'general practice' cohort (18/693, 2.6%, Figure 1A). A surprisingly low frequency was observed among clinically aggressive stereotyped subsets (5/342, 1.5%), although the cause for this observation is currently unknown. Finally, 2/31 (6.5%) cases with Richter transformation carried an EGR2 mutation. Of the 4 FCR-relapsing, EGR2 -mutated cases with available overtime samples, all demonstrated a significant expansion of the EGR2 -mutated clone at relapse (VAF-increase between 15-41%). In addition, subclonal levels of EGR2 hotspot mutations (VAF 0.5-5%) were detected in an additional 13/370 (3.5%) cases by deep-sequencing. The majority of EGR2 -mutated CLL patients (32/39, 82%) concerned U-CLL and the following aberrations co-occurred: 11q-deletions (n=10), TP53abn (n=6), NOTCH1 (n=3)or SF3B1 (n=3) mutations. EGR2 -mutated patients displayed a significantly worse overall survival compared to wt patients (median survival 59 vs. 141 months, p=0.003, using a conservative 10% VAF cutoff), and a poor outcome similar to cases with TP53abn (Figure 1B). In multivariate analysis (n=583), EGR2 status remained an independent factor (p=0.038), along with stage (p=0.048) and IGHV status (p<0.0001), while TP53abn and del(11q) showed borderline significant values (p=0.069 and p=0.059, respectively). To investigate the impact of EGR2 mutations in a homogeneously treated patient cohort, EGR2 mutation analysis of the UK CLL4 trial is underway. To date, 8/247 patients have been identified as EGR2 -mutated by deep-sequencing and they show a decrease of their median overall survival (42 vs. 77 months) compared to wt patients; however, this did not reach statistical significance, probably due to the low number of EGR2 -mutated cases. Final results of the UK CLL4 trial will be presented at the ASH meeting. In summary, EGR2 -mutant cases appear to constitute a novel poor-prognostic subgroup of CLL, with mutations occurring either as disease-initiating aberrations, i.e. in cases where mutations were found in the entire clone, or as subclonal driver events linked to progressive disease. The latter is reflected by the enrichment of EGR2 mutations in aggressive CLL and the association of EGR2 mutations with an overall dismal prognosis. Considering the potential role of mutated EGR2 in altering BcR signaling, it will be particularly relevant to study the efficacy of BcR inhibitors in this patient group. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Langerak: Roche: Other: Lab services in the field of MRD diagnostics provided by Dept of Immunology, Erasmus MC (Rotterdam); InVivoScribe: Patents & Royalties: Licensing of IP and Patent on BIOMED-2-based methods for PCR-based Clonality Diagnostics.; DAKO: Patents & Royalties: Licensing of IP and Patent on Split-Signal FISH. Royalties for Dept. of Immunology, Erasmus MC, Rotterdam, NL. Schuh:Acerta Pharma BV: Research Funding. Strefford:Roche: Research Funding.

Abstract Background: Follicular lymphoma (FL) is the most common indolent NHL (iNHL), exhibits a variable clinical course, and remains largely incurable. The pathogenesis of FL is complex and involves over expression of Bcl2 via t(14;18) translocation, as well as copy number alterations, recurrent somatic mutations, and changes in the tumor microenvironment. In line with recent publications, we hypothesized that recurrent somatic genomic mutations in FL will be present and may impact FL development, progression, transformation, and clinical outcomes. Methods: To address this, we performed exome sequencing (NimbleGen SeqCap EZ V2.0) of tumor and normal frozen tissue pairs from 24 patients in a discovery cohort with untreated FL (12), relapsed FL (6), or transformed FL/iNHL (6). We developed a custom capture assay (NimbleGen) that targets 7.05 MB corresponding to the coding, 5' and 3' UTR regions of 1717 genes. The custom capture genes included somatic mutations identified in our exome discovery cohort (898 genes) or somatic mutations previously published to be recurrently mutated in B cell NHL (819 genes). Instrument data from the discovery cohort exome and re-sequenced custom capture were combined and analyzed using the McDonnell Genome Institute (MGI) somatic caller pipeline (5 SNV callers, 3 indel callers), filtered (minimum 20X coverage, minimum 2.5% VAF, maximum 10% normal VAF) and manually reviewed. Additionally, the 1717 custom capture strategy was used to sequence an extension cohort consisting of FFPE tumor samples from 80 patients with FL, achieving >20x coverage for >75% of the targeted region. All discovery and extension samples have clinical annotations that include FLIPI prognostic score, treatment, and clinical outcomes. Results: Combined analysis of exome and custom capture data for the discovery cohort yielded a robust data set with good sequence coverage of >78% of the targeted regions with at least 20x depth in all samples and a mean depth of 89x. Based upon somatic mutations identified and manually reviewed using this approach, we conservatively estimate 0.98 mutations per MB in FL. 23 genes were recurrently mutated in 3 or more cases, and an additional 75 genes recurrently mutated in 2 cases in the discovery cohort. Consistent with recent publications (Li H et. al., Blood, 2014; Green MR, PNAS, 2015; Yildiz M et al, Blood, 2015) we confirmed a number of genes that were highly recurrently mutated in FL [TNFRSF14 (50%), Bcl2 (25%), IRF8 (13%), TP53 (13%)] including chromatin modifying genes consisting of histone methyl transferases [KMT2D/MLL2 (58%), EZH2 (13%)], histone acetyltransferases [CREBBP (42%), EP300 (17%)], histone linkers [HIST1H1C (13%), HIST1H1E (8%), HIST1H2BO (8%), HIST1H3G (8%), HIST2H2AC (8%); collectively 42%]. We also confirmed (ATP6V1B2, 13%) and found unreported (ATP6AP2, 8%; ATP6V0A1, 4%; ATP6V1F, 4%) mutations in vacuolar ATPase proton pump genes and P5 or Ca++ ATPase genes (ATP13A2, 4%; ATP13A4, 4%, ATP2B4, 4%;). We confirmed (CD79B, 13%; BCL10, 8%) and found unreported (CD22, 13%) mutations in components of the B cell receptor signaling pathway. The previously unreported recurrent mutations in CD22 were consistent with loss-of function (2 missense, 1 nonsense, 1 frame shift deletion). As a negative regulator of BCR signaling, mutation of CD22 may represent a strategy of to enhance BCR signals in malignant germinal center B cells. We also identified members of the SWI/SNF complex mutated in 33% of this FL cohort: ARID1B (8%), BCL11A (4%), SMARCB1 (4%) in addition to previously reported members BCL7A (12%), SMARCA4 (8%), ARID1A (4%). Somatic mutations were also identified in the Notch pathway: DTX1 (29%), Notch2 (4%), Notch3 (4%), Notch4 (4%). We identified several genes that have not been reported as highly recurrent in FL CXCR4 (42%, mutation calls primarily in RNA), DMD (13%), DNAH9 (13%), FLG (13%), GON4L (13%), PCDH7 (13%), RLTPR (13%), SCN7A (13%), ST6GAL1 (13%). Conclusions: FL genomes harbor a large number of recurrent mutations, consistent with a role in the development and progression of this malignancy. Analysis of the extension cohort and association of recurrently mutated genes and pathways with clinical outcomes is ongoing and will be presented. Disclosures Bartlett: Gilead: Consultancy, Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Millennium: Research Funding; Colgene: Research Funding; Medimmune: Research Funding; Kite: Research Funding; Insight: Research Funding; Seattle Genetics: Consultancy, Research Funding; MERC: Research Funding; Dynavax: Research Funding; Idera: Research Funding; Portola: Research Funding; Bristol Meyers Squibb: Research Funding; Infinity: Research Funding; LAM Theapeutics: Research Funding.

Abstract Abstract 2173 The adaptive arm of the immune system - the T-cell compartment – may become compromised by inherited or acquired defects resulting in cancer, autoimmunity, or increased susceptibility to microbial infectious agents. A normal polyclonal T cell compartment comprises an estimated number of 2.5 × 10E7 individual T cell clones each expressing a unique antigen recognizing T cell receptor (TCR). The functionality of the T cell compartment is thus – at least partly - reflected by TCR sequence diversity. There is a medical need for rapid and robust diagnostic approaches that accurately monitor TCR diversity in patient samples, e.g. after bone marrow transplantation (BMT). Previously, complementarity determining region 3 (CDR3) size spectratyping in TCR β-chain subfamilies (Vβ), an immunoscopic technique, was employed for the analysis of T cell diversity. However, spectratyping is limited to the analysis of CDR3 length polymorphisms only. Underlying diversity of TCR Vβ sequences of equal length remain undetected. Furthermore, spectratyping is time consuming and consequently data can only be interpreted with a delay of weeks. To determine TCR diversity fast and accurately we developed next-generation-sequencing spectratyping (NGS-S), which employs high coverage, massive parallel Roche/454-sequencing of TCR Vβ-chain amplicons. Three different sample groups were analyzed in parallel by spectratyping and NGS-S: T cells from (1) healthy children (n=6), (2) children at diagnosis of severe aplastic anemia (n=7), and (3) children who underwent haploidentical BMT (n=7). In brief, RNA was extracted from bone marrow derived CD8+ cells and transcribed to cDNA. Amplicon libraries were generated by PCR employing two degenerated wobble primers (VP1, VP2), designed to cover most of the known TCR Vβ gene segments and a universal reverse primer (CP1) located in the conserved TCR region (Figure 1). The mean overall coverage of the CDR3 region achieved was 23.133 per patient.Figure 1:Workflow of NGS-SFigure 1:. Workflow of NGS-S For simultaneous characterization of these individual amplicons we generated the TCR Profiler (Figure 2). This new software tool automatically preprocessed raw sequencing data using a threshold quality value (q=30) to trim the 3' end of the TCR β-chain sequences. Rearranged germline TCR Vβ and Jβ genes were identified by Smith-Waterman local alignment against each human TCR β-chain germline gene of the IMGT/GENE-DB reference directory. Base call reliability was assured by incorporating phred-like quality values provided by the sequencer into the Smith-Waterman local alignment routine. A quality value, q, was computed for each base as the log-transformation of the probability p of a base being incorrectly called, q = − 10 x log10(p). Transformed into a reliability measure, r = 1- (1/10^(q/10)), q-values indicated the probability for each base to be correctly called. CDR3 regions were delimited using specific flanking amino acid sequence motifs. The 5' end motif varies dependently on the rearranged TCR Vβ gene and was identified using the IMGT/GENE-DB reference directory sequence set, whereas the 3' end motif [W/F]GXG (IUPAC code) is conserved in all TCR β-chains. CDR3 length was calculated including all amino acids between the two motifs. Screening for in-frame stop codons was done to exclude non-functional TCR β-chain transcripts.Figure 2:TCR-ProfilerFigure 2:. TCR-Profiler The TCR profiler identified on average 16165 of the input sequences as unique CDR3 sequences. Of these a mean of 9840 were predicted to be functionally rearranged. Whereas spectratyping gave a rough estimate of CDR3 size and allowed T cell deficient samples to be identified, NGS-S determined the exact length and sequence composition of the CDR3, identified the rearranged TCR Vβ and Jβ genes and the specific recombination (Figure 3A).Figure 3:Comparison of diversity detection by spectratyping and NGS-S (A). NGS-S, but not spectratyping, allows differentiation between diverse T cell pathologies (B).Figure 3:. Comparison of diversity detection by spectratyping and NGS-S (A). NGS-S, but not spectratyping, allows differentiation between diverse T cell pathologies (B). Utilization of specific genes, the resulting amino acid composition of the CDR3 region as well as its length and overall diversity were integrated into a novel NGS-S score. This score reliably differentiated not only between normal and T cell deficient samples, but also between the different T cell deficient groups (SAA and BMT) (Figure 3B). We conclude that NGS-S allows rapid and precise determination of TCR diversity in clinical samples. Disclosures: No relevant conflicts of interest to declare.

Introduction B-ALL with TP53 mutations (TP53MT) at diagnosis is associated with older age and frequent low-hypodiploid karyotype. We have previously shown that hyper-CVAD-based regimens negate the poor prognostic impact of TP53MT. However, the prognostic effect of TP53MT in B-ALL treated with mini-HCVD-inotuzumab with or without blinatumomab in the frontline and R-R settings have not been explored. Methods Older (≥60 yrs) pts with newly-diagnosed ALL and adults (≥18 yrs) with R-R ALL treated with mini-HCVD-inotuzumab ozogamicin with/without blinatumomab underwent testing for TP53MT. We compared the clinico-pathologic & outcome differences between TP53MT and TP53wt cases in both settings. Germline origin was inferred based on persistence of TP53MT at high variant allele frequency (VAF) during remission. Multiallelic (&gt;1) TP53 alteration was defined as a concurrent second TP53MT or deletion (TP53del). Results Of the 48 pts with newly diagnosed B-ALL, 19 (40%) pts had TP53MT at diagnosis; in 2 of 14 (14%), TP53MT was of germline origin. All pts were Philadelphia chromosome (Ph) negative; 1 pt had CRLF2 rearrangement (Ph-like immunophenotype); none had KMT2A rearrangement (KMT2Ar). Fifteen (83%) pts had 1 TP53MT, 2 had 2 mutations, and one pt had 3 mutations. The majority were missense (15, 83%), the rest included non-sense, frame-shift, and splice site mutation in 1 patient, each. The median TP53MT VAF was 46% (range, 4-80). Nine (56%) had multiallelic TP53 alteration, with 6 (38%) showing TP53del. Compared with TP53 wild-type (TP53wt), TP53MT had a higher frequency of low-hypodiploidy (40% vs. 8%, p=0.04), lower median platelet counts (34 vs. 55, p=0.02), and a trend for older age (median 71 vs. 68, p=0.09). There was no difference in morphologic response rates (89% vs. 100%; p=0.11) and minimal residual disease negativity [MRD neg] rates (94% vs. 75%; p=0.13) between mutated and wild type groups. Over a median follow-up (f/u) of 46 months, 21 (44%) patients died [9 (50%) mutated vs. 12 (44%) wild type]. There was no significant difference in the overall survival (median OS, 33 vs. 38 months, p=0.28; Fig 1A); the 3-year survival rates were 45% and 63% in TP53MT and TP53wt, respectively. Of the 47 pts with R-R B-ALL, 16 (34%) had TP53MT. Three (3/12, 25%) had germline TP53MT. All were Ph-negative, and 1 patient was Ph-like ALL. All 16 patients had a single TP53MT; 9 (56%) had multiallelic TP53 alteration due to concurrent TP53del. Majority were missense (12, 75%), the rest included 2 non-sense, 1 frame-shift and 1 in-frame duplication. The median TP53MT VAF was 33% (range, 1-78). Except for a higher frequency of low-hypodiploidy (30% vs. 0%, p=0.01), no other significant clinico-pathologic differences were noted between the TP53MT and TP53wt groups. Pts with TP53MT had a higher morphologic response rate (100% vs. 67%, p=0.02) with no difference in MRD neg rates (78% vs. 85%, p=0.65). Over a median f/u of 33 months, 15 TP53MT pts (94%) died compared to 14 TP53wt pts (45%; p=0.001). Pts with TP53MT had significantly shorter median survival (5 months vs not reached, p=0.0003; Fig 1B); the 3-year survival rates were 0% and 52% in TP53MT and TP53wt, respectively. Despite significant differences in outcome of TP53MT at diagnosis vs. relapse, there were no notable differences in TP53MT characteristics, except for enrichment of KMT2Ar in R-R (0% vs. 31%; p=0.04). We retrospectively reviewed the cytogenomic features at the time of diagnosis of 7 R-R B-ALL with TP53MT (non-germline). None had TP53delat diagnosis. TP53 NGS profiling at diagnosis on one KMT2Arpt was negative for TP53MT (1% sensitivity) and TP53del (Fig 1C). These findings suggest that TP53MT/TP53delclones (undetectable at diagnosis) expanded following chemotherapy and relapsed with cytogenetic evolution. TP53MT clones that survived the chemotherapy insult were inherently more aggressive than treatment naïve clones at diagnosis, and may explain the observed differences in outcome. Conclusions TP53MT is seen in up to 40% of B-ALL at diagnosis and ~35% at relapse, and are associated with low-hypodiploid karyotype. B-ALL with TP53MT at diagnosis shows no significant difference in OS compared to TP53wt in patients treated with frontline mini-HCVD-inotuzumab with or without blinatumomab regimen. In contrast, TP53MT confers a poor prognosis in pts with R-R disease. Sequencing studies on additional R/R TP53MT patients at diagnosis are underway. Figure 1 Disclosures Kantarjian: Pfizer: Honoraria, Research Funding; Astex: Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria; AbbVie: Honoraria, Research Funding; Cyclacel: Research Funding; Immunogen: Research Funding; Novartis: Research Funding; Agios: Honoraria, Research Funding; Ariad: Research Funding; BMS: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Research Funding; Daiichi-Sankyo: Research Funding. Short:Astellas: Research Funding; AstraZeneca: Consultancy; Takeda Oncology: Consultancy, Honoraria, Research Funding; Amgen: Honoraria. Ravandi:Xencor: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Macrogenics: Research Funding; Orsenix: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria. Konopleva:Sanofi: Research Funding; Kisoji: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Cellectis: Research Funding; Calithera: Research Funding; Forty-Seven: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; F. Hoffmann La-Roche: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Amgen: Consultancy; AbbVie: Consultancy, Research Funding; Agios: Research Funding; Ablynx: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Eli Lilly: Research Funding. Jain:Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Aprea Therapeutics: Research Funding; Incyte: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; BeiGene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Precision Bioscienes: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Fate Therapeutics: Research Funding; Cellectis: Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Research Funding; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Garcia-Manero:Bristol-Myers Squibb: Consultancy, Research Funding; Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Amphivena Therapeutics: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy; Acceleron Pharmaceuticals: Consultancy, Honoraria; H3 Biomedicine: Research Funding; Merck: Research Funding; Novartis: Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Onconova: Research Funding; AbbVie: Honoraria, Research Funding. Daver:Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kadia:Genentech: Honoraria, Research Funding; Incyte: Research Funding; Novartis: Honoraria; Celgene: Research Funding; BMS: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; JAZZ: Honoraria, Research Funding; Ascentage: Research Funding; Cellenkos: Research Funding; Astra Zeneca: Research Funding; Astellas: Research Funding; Cyclacel: Research Funding; Pulmotec: Research Funding. DiNardo:Daiichi Sankyo: Consultancy, Honoraria, Research Funding; ImmuneOnc: Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; MedImmune: Honoraria; Calithera: Research Funding; Jazz: Honoraria; Novartis: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria, Research Funding; Syros: Honoraria; Notable Labs: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria. O'Brien:Kite, Regeneron, Acerta: Research Funding; Gilead, Pharmacyclics, TG Therapeutics, Pfizer, Sunesis: Consultancy, Research Funding; Amgen, Astellas, Celgene, GlaxoSmithKline, Janssen Oncology, Aptose Biosciences Inc. Vaniam Group, AbbVie, Alexion, Verastem, Eisai, Juno Therapeutics, Vida Ventures: Consultancy. Jabbour:Adaptive Biotechnologies: Other: Advisory role, Research Funding; Genentech: Other: Advisory role, Research Funding; BMS: Other: Advisory role, Research Funding; Amgen: Other: Advisory role, Research Funding; Pfizer: Other: Advisory role, Research Funding; Takeda: Other: Advisory role, Research Funding; AbbVie: Other: Advisory role, Research Funding.

Abstract Introduction: Diversity of the T-cell receptor (TCR) repertoire reflects the initial V(D)J recombination events as shaped by selection by self and foreign antigens. Next generation sequencing is a powerful method for profiling the TCR repertoire, including sequences encoding complementarity-determining region 3 (CDR3). Peripheral T-cell lymphoma (PTCL) is a group of malignancies that originate from mature T-cells. T-cell clonality of PTCL is routinely evaluated with a PCR-based method to detect TCR gamma and less frequently beta chain rearrangements using genomic DNA. However, there are limitations with this approach, chief among which is the lack of sequence information. To date, the TCR repertoire of different subtypes of PTCL remains poorly defined. Objective: The purpose of this study was to determine the utility of RNA-seq for assessing T-cell clonality and analyzing the TCR usage in PTCL samples. Methods: We analyzed RNA-seq data from 30 angioimmunoblastic T-cell lymphoma (AITL), 23 Anaplastic large cell lymphoma (ALCL), 10 PTCL-NOS, and 17 NKCL. Data from naïve T cells, TFH cells, and T-effector cells (CD4+ CD45RA− TCRβ+ PD-1lo CXCR5lo PSGL-1hi) were obtained from publicly available resources. Referenced TCR and immunoglobulin transcripts according to the International ImMunoGeneTics Information System (IMGT) database were quantified by Kallisto software. We divided the pattern of Vβ (T-cell receptor beta variable region) into three categories: monoclonal (mono- or bi-allelic), oligoclonal (3-4 dominant clones), and polyclonal. CDR3 sequences were extracted by MiXCR program. PCR of the gamma chain using genomic DNA was utilized to validate the clonality of selected cases. Single nucleotide variants (SNVs) were called from aligned RNA-seq data using Samtools and VarScan 2 programs. Results: Analysis of RNA-seq data identified preferential usage of TCR-Vβ, Dβ (diversity region), and Jβ (joining region), length diversity of CDR3, and usage of nontemplated bases. Dominant clones could be identified by transcriptome sequencing in most cases of AITL (21/30), ALCL (14/23), and PTCL-NOS (7/10). Median CDR3 length is 42 nucleotides (nt) in normal T cells, 41 nt in ALCL, 48 nt in PTCL-NOS, and 44 nt in AITL. In 30 AITL samples, 20 showed monoclonal Vβ with a single peak, and 9 showed polyclonal Vβ. One case had two dominant clones with different CDR3, only one of which was in frame, implying biallelic rearrangements. As many as 3511 clones supported by at least four reads could be detected in polyclonal cases. In monoclonal cases, the dominant clone varied between 11.8% and 92.8% of TCR with Vβ rearrangements. TRBV 20-1, which is the most commonly used segment in normal T cells, is also frequently used in the dominant clones in AITL. The monoclonal AITL cases showed mutation of TET2, RHOA, DNMT3A or IDH2 whereas most of the polyclonal cases were negative or had low VAF mutation suggesting low or absent of tumor infiltrate in the specimen sequenced. There is no obvious correlation of any of the mutations with Vβ usage. Clonal B cell expansion was noted in some AITL samples. The occurrence of a preferential TRBV9 expansion in PTCL-NOS was striking. More than half of ALCL samples (14/23) showed expression of clonal Vβ, but 3/14 dominant clones were out-of-frame. γ chain expression was very low in cells expressing TCRαβ, but both expression levels and clonality were higher in TCRγδ expressing tumors. NKCL did not express significant levels of TCR Vβ or Vγ genes. Discussion/Interpretation: Transcriptome sequencing is a useful tool for understanding the TCR repertoire in T cell lymphoma and for detecting clonality for diagnosis. Clonal, often out-of-frame, Vβ transcripts are detectable in most ALCL cases and preferential TRBV9 usage is found in PTCL-NOS. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction TP53 mutations in relapsed cases with pediatric acute lymphoblastic leukemia have been implicated in poor clinical outcomes. However, the prevalence and clinical significance of TP53 mutations at diagnosis have not been fully investigated. Such knowledge is essential for the care of patients, because treatment intensity is tailored to predictive prognosis, where increased attention has been directed toward de-escalation of treatment for the problem of long term effects and second malignancies in childhood cancer survivors. Methods Mutation status of TP53 was detected by targeted-capture sequencing of TP53 coding regions in 1,003 children with B-precursor ALL who had been treated in either of the two prospective clinical trials, JACLS (Japan Association of Childhood Leukemia Study) ALL-02 and TCCSG (Tokyo Children's Cancer Study Group) L04-16. Detection of common fusion genes, including BCR-ABL, ETV6-RUNX1, MLL-AF4, MLL-ENL, MLL-AF9, and TCF3-PBX1, were performed using qPCR assays. We designed SNP baits to analyze copy number status of chromosome 17, and also captured 662 probes tiling the entire IgH enhancer locus to identify IGH-DUX4 rearrangement. Result In total, 36 different non-silent coding TP53 mutations were identified in 30 (3%) patients, including 22 missense, 7 frameshift indel, 5 in-frame indel, and 2 nonsense mutations. All missense mutations were found in the core DNA-binding domain (n=21), except for one mutation, which affected the tetramerization motif. Variant allele frequencies (VAF) of TP53 mutations varied from 3% to 97% with 14 mutations showing < 10% VAFs. Showing a significant correlation with mutated TP53 (Odds ratio 20: 95%CI 6.4-61, P<0.001), loss of heterozygosity affecting the TP53 locus was observed in 11 (37%) cases and caused by del(17p) in most cases (n=10). We next evaluated clinical features of TP53-mutated cases. TP53 was most frequently mutated in Hypodiploid ALL (33% n=3), followed by MLL rearrangement (12% n=4), IGH-DUX4 (9% n=5), Others (3% n=8), TCF3-PBX1 (2% n=2), Hyperdiploid (2% n=6), and ETV6-RUNX1 (n=2 0.9%). TP53 mutations were not associated with age or white blood cell count at diagnosis. However, significantly more patients were categorized into National Cancer Institute (NCI) high risk (HR) category (Odds ratio 2.4: 95%CI 1.1-5.3, P = 0.03) and TP53 mutation was associated with a significantly shorter overall survival (OS) among NCI-HR patients (n = 16; HR for death, 6.3; 95% CI, 3.1-13; P<0.001). Five-year OS of NCI-HR patients with TP53 mutations was 44%, suggesting that early treatment intensification or alternative treatment strategies are warranted for these patients. TP53 mutations were also associated with a shorter OS in MLL rearrangement and IGH-DUX4 ALL. Particularly, 67% (n=4/6) of cases with any cause of death harbored TP53 mutation in IGH-DUX4 ALL. In contrast, TP53 mutation was not associated with shorter overall survival in NCI-SR cases. In Hyperdiploid ALL, 5 out of 6 cases with TP53 mutations were categorized into the NCI-SR category and were all alive. Prognostic impact of TP53 mutation was also investigated using recursive partitioning to generate a hierarchical prognostic model for OS by incorporating genetic subgroups and the NCI risk criteria. This model also demonstrated that the NCI risk criteria was the most important prognostic variable and TP53 mutation was used for stratification of patients only in the NCI-HR category. Conclusion TP53 mutations at diagnosis are common in Hypodiploid ALL and also found in a substantial fraction of MLL rearrangement and IGH-DUX4 ALL, where the mutations predict a poor prognosis. TP53 mutation is also found in NCI-SR cases but may not be associated with poor prognosis. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Abstract Children with acute myeloid leukemia (AML) have a dismal prognosis due to a high relapse rate; however, the molecular basis leading to relapsed pediatric AML has not yet been fully characterized. To define the spectrum of alterations common at relapse, we performed integrated profiling of 136 relapsed pediatric AML cases with RNA sequencing (RNA-seq), whole-genome sequencing, and target-capture sequencing. In addition to well-characterized fusion oncoproteins, such as those involving KMT2A (n=36, 26.5%) or NUP98 (n=18, 13.2%), we also identified somatic mutations in UBTF (upstream binding transcription factor) in 12 of 136 cases (8.8%) of this relapsed cohort. Somatic alterations of the UBTF gene, which encodes a nucleolar protein that is a component of the RNA Pol I pre-initiation complex to ribosomal DNA promoters, have rarely been observed in AML. In our cohort, all alterations can be described as heterozygous in-frame exon 13 tandem duplications (UBTF-TD), either at the 3' end of exon 13 of UBTF or of the entire exon 13 (Fig. A). As we noticed limited detection in our pipeline as a result of complex secondary indels alongside the duplications, we established a soft-clipped read-based screening method to detect UBTF-TD more efficiently. Applying the screening to RNA-seq data of 417 additional pediatric AMLs from previous studies and our clinical service, we identified 15 additional UBTF-TDs, many of which have not been previously reported. At the amino acid level, UBTF-TDs caused amino acid insertions of variable sizes (15-181 amino acids), duplicating a portion of high mobility group domain 4 (HMG4), which includes short leucine-rich sequences. UBTF-TD AMLs commonly occurred in early adolescence (median age: 12.6, range: 2.4-19.6), and 19 of the total 27 cases had either normal karyotype (n=12) or trisomy 8 (n=7). UBTF-TD is mutually exclusive from other recurrent fusion oncoproteins, such as NUP98 and KMT2A rearrangements (Fig. B), but frequently occurred with FLT3-ITD (44.4%) or WT1 mutations (40.7%). The median variant allele fraction (VAF) of the UBTF-TD was 48.0% (range: 9.7-66.7%). In four cases with data at multiple disease time points, the identical UBTF-TDs were present at high allele fractions at all time points, suggesting that UBTF-TD is a clonal alteration. tSNE analysis of the transcriptome dataset showed that UBTF-TD AMLs share a similar expression pattern with NPM1 mutant and NUP98-NSD1 AML subtypes, including NKX2-3 and HOXB cluster genes (Fig. C) . Altogether, these findings suggest that UBTF-TD is a unique subtype of pediatric AML. To address the impact of UBTF-TD expression in primary hematopoietic cells, we introduced UBTF-TD and UBTF wildtype expression vectors into cord blood CD34+ cells via lentiviral transduction. UBTF-TD expression promotes colony-forming activity and cell growth, yielding cells with a persistent blast-like morphology (Fig. D). Further, transcriptional profiling of these cells demonstrated expression of HOXB genes and NKX2-3, similar to UBTF-TD AMLs in patients, indicating that UBTF-TD is sufficient to induce the leukemic phenotype. To investigate the prevalence of UBTF-TDs in larger de novo AML cohorts, we applied the above UBTF-TD screening method to the available de novo AML cohorts of TCGA (n=151, adult), BeatAML (n=220, pediatric and adult), and AAML1031 (n=1035, pediatric). We identified UBTF-TDs in 4.3% (45/1035) of the pediatric AAML1031 cohort, while the alteration is less common (0.9%: 3/329, p=0.002) in the adult AML cohorts (Fig. E). In the AAML1031 cohort, UBTF-TDs remain mutually exclusive with known molecular subtypes of AML and commonly occur with FLT3-ITD (66.7%) and WT1 (40.0%) mutations and either normal karyotype or trisomy 8. The presence of UBTF-TDs in the AAML1031 cohort is associated with a poor outcome (Fig. F, median overall survival, 2.3 years) and MRD positivity; multivariate analysis revealed that UBTF-TD and WT1 are independent risk factors for overall survival within FLT3-ITD+ AMLs. In conclusion, we demonstrate UBTF-TD defines a unique subtype of AMLs that previously lacked a clear oncogenic driver. While independent of subtype-defining oncogenic fusions, UBTF-TD AMLs are associated with FLT3-ITD and WT1 mutations, adolescent age, and poor outcomes. These alterations have been under-recognized by standard bioinformatic approaches yet will be critical for future risk-stratification of pediatric AML. Figure 1 Figure 1. Disclosures Iacobucci: Amgen: Honoraria; Mission Bio: Honoraria. Miller: Johnson & Johnson's Janssen: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

ABSTRACTInfectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus. Segment A contains two overlapping open reading frames (ORFs), which encode viral proteins VP2, VP3, VP4, and VP5. Segment B contains one ORF and encodes the viral RNA-dependent RNA polymerase, VP1. IBDV ribonucleoprotein complexes are composed of VP1, VP3, and dsRNA and play a critical role in mediating viral replication and transcription during the virus life cycle. In the present study, we identified a cellular factor, VDAC1, which was upregulated during IBDV infection and found to mediate IBDV polymerase activity. VDAC1 senses IBDV infection by interacting with viral proteins VP1 and VP3. This association is caused by RNA bridging, and all three proteins colocalize in the cytoplasm. Furthermore, small interfering RNA (siRNA)-mediated downregulation ofVDAC1resulted in a reduction in viral polymerase activity and a subsequent decrease in viral yield. Moreover, overexpression of VDAC1 enhanced IBDV polymerase activity. We also found that the viral protein VP3 can replace segment A to execute polymerase activity. A previous study showed that mutations in the C terminus of VP3 directly influence the formation of VP1-VP3 complexes. Our immunoprecipitation experiments demonstrated that protein-protein interactions between VDAC1 and VP3 and between VDAC1 and VP1 play a role in stabilizing the interaction between VP3 and VP1, further promoting IBDV polymerase activity.IMPORTANCEThe cellular factor VDAC1 controls the entry and exit of mitochondrial metabolites and plays a pivotal role during intrinsic apoptosis by mediating the release of many apoptogenic molecules. Here we identify a novel role of VDAC1, showing that VDAC1 interacts with IBDV ribonucleoproteins (RNPs) and facilitates IBDV replication by enhancing IBDV polymerase activity through its ability to stabilize interactions in RNP complexes. To our knowledge, this is the first report that VDAC1 is specifically involved in regulating IBDV RNA polymerase activity, providing novel insight into virus-host interactions.

The article is the result of an educational research project of the National Technological Institute of Mexico that involves Virtual Learning Environments (AVA). The objective is to determine the impact of using a VPA as a tool in the teaching-learning process of the face-to-face classes of the Ciudad Valles Technology. The methodology is mixed, qualitative and quantitative. The quantitative number determines the number of teachers used by AVA, the number of students in a Blending Learning (B-Learning) course, the number of courses designed in Moodle. In the qualitative, the impact of B-Learning on students is analyzed; determining the degree of motivation and performance that is generated when using AVA and the ability to use information technology to generate their learning strategies. A composite and structural analysis of the AVA used in the institute is presented, the research hypothesis is disclosed: the use of an AVA as a b-learning strategy, improves the level of performance of student competencies compared to students who follow classroom learning strategies. The manner in which the research is conducted, the frame of reference is described and relevant results and conclusions are shown.

Introduction Negative stereotyping of alternative diets such as veganism and other plant-based diets has been common in Australia, conventionally a meat-eating culture (OECD qtd. in Ting). Indeed, meat consumption in Australia is sanctioned by the ubiquity of advertising linking meat-eating to health, vitality and nation-building, and public challenges to such plant-based diets as veganism. In addition, state, commercial enterprises, and various community groups overtly resist challenges to Australian meat-eating norms and to the intensive animal husbandry practices that underpin it. Hence activists, who may contest not simply this norm but many of the customary industry practices that comprise Australia’s meat production, have been accused of promoting a vegan agenda and even of undermining the “Australian way of life”.If veganism meansa philosophy and way of living which seeks to exclude—as far as is possible and practicable—all forms of exploitation of, and cruelty to, animals for food, clothing or any other purpose; and by extension, promotes the development and use of animal-free alternatives for the benefit of humans, animals and the environment. In dietary terms it denotes the practice of dispensing with all products derived wholly or partly from animals. (Vegan Society)then our interest in this article lies in how a stereotyped label of veganism (and other associated attributes) is being used across Australian public spheres to challenge the work of animal activists as they call out factory farming for entrenched animal cruelty. This is carried out in three main parts. First, following an outline of our research approach, we examine the processes of stereotyping and the key dimensions of vegan stereotyping. Second, in the main part of the article, we reveal how opponents to such animal activist organisations as Animals Australia attempt to undermine activist calls for change by framing them as promoting an un-Australian vegan agenda. Finally, we consider how, despite such framing, that organisation is generating productive public debate around animal welfare, and, further, facilitating the creation of new activist identifications and identities.Research ApproachData collection involved searching for articles where Animals Australia and animal activism were yoked with veg*n (vegan and vegetarian), across the period May 2011 to 2016 (discussion peaked between May and June 2013). This period was of interest because it exposed a flare point with public discord being expressed between communities—namely between rural and urban consumers, farmers and animal activists, Coles Supermarkets (identified by The Australian Government the Treasury as one of two major supermarkets holding over 65% share of Australian food retail market) and their producers—and a consequent voicing of disquiet around Australian identity. We used purposive sampling (Waller, Farquharson, and Dempsey 67) to identify relevant materials as we knew in advance the case was “information-rich” (Patton 181) and would provide insightful information about a “troublesome” phenomenon (Emmel 6). Materials were collected from online news articles (30) and readers’ comments (167), online magazines (2) and websites (2) and readers’ comments (3), news items (Factiva 13), Australian Broadcasting Commission television (1) and radio (1), public blogs (2), and Facebook pages from involved organisations, specifically Australia’s National Farmers’ Federation (NFF, 155 posts) and Coles Supermarkets (29 posts). Many of these materials were explicitly responsive to a) Animals Australia’s Make It Possible campaign against Australian factory farming (launched and highly debated during this period), and b) Coles Supermarket’s short-lived partnership with Animals Australia in 2013. We utilised content analysis so as to make visible the most prominent and consistent stereotypes utilised in these various materials during the identified period. The approach allowed us to code and categorise materials so as to determine trends and patterns of words used, their relationships, and key structures and ways of speaking (Weerakkody). In addition, discourse analysis (Gee) was used in order to identify and track “language-in-use” so as to make visible the stereotyping deployed during the public reception of both the campaign and Animals Australia’s associated partnership with Coles. These methods enabled a “nuanced approach” (Coleman and Moss 12) with which to spot putdowns, innuendos, and stereotypical attitudes.Vegan StereotypingStereotypes creep into everyday language and are circulated and amplified through mainstream media, speeches by public figures, and social media. Stereotypes maintain their force through being reused and repurposed, making them difficult to eradicate due to their “cumulative effects” and influence (Harris and Sanborn 38; Inzlicht, Tullett, Legault, and Kang; Pickering). Over time stereotypes can become the lens through which we view “the world and social reality” (Harris and Sanborn 38; Inzlicht et al.). In summation, stereotyping:reduces identity categories to particular sets of deeds, attributes and attitudes (Whitley and Kite);informs individuals’ “cognitive investments” (Blum 267) by associating certain characteristics with particular groups;comprises symbolic and connotative codes that carry sets of traits, deeds, or beliefs (Cover; Rosello), and;becomes increasingly persuasive through regulating language and image use as well as identity categories (Cover; Pickering; Rosello).Not only is the “iterative force” (Rosello 35) of such associative stereotyping compounded due to its dissemination across digital media sites such as Facebook, YouTube, websites, and online news, but attempts to denounce it tend to increase its “persuasive power” (29). Indeed, stereotypes seem to refuse “to die” (23), remaining rooted in social and cultural memory (Whitley and Kite 10).As such, despite the fact that there is increasing interest in Australia and elsewhere in new food norms and plant-based diets (see, e.g., KPMG), as well as in vegan lifestyle options (Wright), studies still show that vegans remain a negatively stereotyped group. Previous studies have suggested that vegans mark a “symbolic threat” to Western, conventionally meat-eating cultures (MacInnis and Hodson 722; Stephens Griffin; Cole and Morgan). One key UK study of national newspapers, for instance, showed vegans continuing to be discredited in multiple ways as: 1) “self-evidently ridiculous”; 2) “ascetics”; 3) having a lifestyle difficult and impossible to maintain; 4) “faddist”; 5) “oversensitive”; and 6) “hostile extremists” (Cole and Morgan 140–47).For many Australians, veganism also appears anathema to their preferred culture and lifestyle of meat-eating. For instance, the NFF, Meat & Livestock Australia (MLA), and other farming bodies continue to frame veganism as marking an extreme form of lifestyle, as anti-farming and un-Australian. Such perspectives are also circulated through online rural news and readers’ comments, as will be discussed later in the article. Such representations are further exemplified by the MLA’s (Lamb, Australia Day, Celebrate Australia) Australia Day lamb advertising campaigns (Bembridge; Canning). For multiple consecutive years, the campaign presented vegans (and vegetarians) as being self-evidently ridiculous and faddish, representing them as mentally unhinged and fringe dwellers. Such stereotyping not only invokes “affective reactions” (Whitley and Kite 8)—including feelings of disgust towards individuals living such lifestyles or holding such values—but operates as “political baits” (Rosello 18) to shore-up or challenge certain social or political positions.Although such advertisements are arguably satirical, their repeated screening towards and on Australia Day highlights deeply held views about the normalcy of animal agriculture and meat-eating, “homogenizing” (Blum 276; Pickering) both meat-eaters and non-meat-eaters alike. Cultural stereotyping of this kind amplifies “social” as well as political schisms (Blum 276), and arguably discourages consumers—whether meat-eaters or non-meat-eaters—from advocating together around shared goals such as animal welfare and food safety. Additionally, given the rise of new food practices in Australia—including flexitarian, reducetarian, pescatarian, kangatarian (a niche form of ethical eating), vegivores, semi-vegetarian, vegetarian, veganism—alongside broader commitments to ethical consumption, such stereotyping suggests that consumers’ actual values and preferences are being disregarded in order to shore-up the normalcy of meat-eating.Animals Australia and the (So-Called) Vegan Agenda of Animal ActivismGiven these points, it is no surprise that there is a tacit belief in Australia that anyone labelled an animal activist must also be vegan. Within this context, we have chosen to primarily focus on the attitudes towards the campaigning work of Animals Australia—a not-for-profit organisation representing some 30 member groups and over 2 million individual supporters (Animals Australia, “Who Is”)—as this organisation has been charged as promoting a vegan agenda. Along with the RSPCA and Voiceless, Animals Australia represents one of the largest animal protection organisations within Australia (Chen). Its mission is to:Investigate, expose and raise community awareness of animal cruelty;Provide animals with the strongest representation possible to Government and other decision-makers;Educate, inspire, empower and enlist the support of the community to prevent and prohibit animal cruelty;Strengthen the animal protection movement. (Animals Australia, “Who Is”)In delivery of this mission, the organisation curates public rallies and protests, makes government and industry submissions, and utilises corporate outreach. Campaigning engages the Web, multiple forms of print and broadcast media, and social media.With regards to Animals Australia’s campaigns regarding factory farming—including the Make It Possible campaign (see fig. 1), launched in 2013 and key to the period we are investigating—the main message is that: the animals kept in these barren and constrictive conditions are “no different to our pets at home”; they are “highly intelligent creatures who feel pain, and who will respond to kindness and affection – if given the chance”; they are “someone, not something” (see the Make It Possible transcript). Campaigns deliberately strive to engender feelings of empathy and produce affect in viewers (see, e.g., van Gurp). Specifically they strive to produce mainstream recognition of the cruelties entrenched in factory farming practices and build community outrage against these practices so as to initiate industry change. Campaigns thus expressly challenge Australians to no longer support factory farmed animal products, and to identify with what we have elsewhere called everyday activist positions (Rodan and Mummery, “Animal Welfare”; “Make It Possible”). They do not, however, explicitly endorse a vegan position. Figure 1: Make It Possible (Animals Australia, campaign poster)Nonetheless, as has been noted, a common counter-tactic used within Australia by the industries targeted by such campaigns, has been to use well-known negative stereotypes to discredit not only the charges of systemic animal cruelty but the associated organisations. In our analysis, we found four prominent interconnected stereotypes utilised in both digital and print media to discredit the animal welfare objectives of Animals Australia. Together these cast the organisation as: 1) anti-meat-eating; 2) anti-farming; 3) promoting a vegan agenda; and 4) hostile extremists. These stereotypes are examined below.Anti-Meat-EatingThe most common stereotype attributed to Animals Australia from its campaigning is of being anti-meat-eating. This charge, with its associations with veganism, is clearly problematic for industries that facilitate meat-eating and within a culture that normalises meat-eating, as the following example expresses:They’re [Animals Australia] all about stopping things. They want to stop factory farming – whatever factory farming is – or they want to stop live exports. And in fact they’re not necessarily about: how do I improve animal welfare in the pig industry? Or how do I improve animal welfare in the live export industry? Because ultimately they are about a meat-free future world and we’re about a meat producing industry, so there’s not a lot of overlap, really between what we’re doing. (Andrew Spencer, Australian Pork Ltd., qtd. in Clark)Respondents engaging this stereotype also express their “outrage at Coles” (McCarthy) and Animals Australia for “pedalling [sic]” a pro-vegan agenda (Nash), their sense that Animals Australia is operating with ulterior motives (Flint) and criminal intent (Brown). They see cultural refocus as unnecessary and “an exercise in futility” (Harris).Anti-FarmingTo be anti-farming in Australia is generally considered to be un-Australian, with Glasgow suggesting that any criticism of “farming practices” in Australian society can be “interpreted as an attack on the moral integrity of farmers, amounting to cultural blasphemy” (200). Given its objectives, it is unsurprising that Animals Australia has been stereotyped as being “anti-farming”, a phrase additionally often used in conjunction with the charge of veganism. Although this comprises a misreading of veganism—given its focus on challenging animal exploitation in farming rather than entailing opposition to all farming—the NFF accused Animals Australia of being “blatantly anti-farming and proveganism” (Linegar qtd. in Nason) and as wanting “to see animal agriculture phased out” (National Farmers’ Federation). As expressed in more detail:One of the main factors for VFF and other farmers being offended is because of AA’s opinion and stand on ALL farming. AA wants all farming banned and us all become vegans. Is it any wonder a lot of people were upset? Add to that the proceeds going to AA which may have been used for their next criminal activity washed against the grain. If people want to stand against factory farming they have the opportunity not to purchase them. Surely not buying a product will have a far greater impact on factory farmed produce. Maybe the money could have been given to farmers? (Hunter)Such stereotyping reveals how strongly normalised animal agriculture is in Australia, as well as a tendency on the part of respondents to reframe the challenge of animal cruelty in some farming practices into a position supposedly challenging all farming practices.Promoting a Vegan AgendaAs is already clear, Animals Australia is often reproached for promoting a vegan agenda, which, it is further suggested, it keeps hidden from the Australian public. This viewpoint was evident in two key examples: a) the Australian public and organisations such as the NFF are presented as being “defenceless” against the “myopic vitriol of the vegan abolitionists” (Jonas); and b) Animals Australia is accused of accepting “loans from liberation groups” and being “supported by an army of animal rights lawyers” to promote a “hard core” veganism message (Bourke).Nobody likes to see any animals hurt, but pushing a vegan agenda and pushing bad attitudes by group members is not helping any animals and just serves to slow any progress both sides are trying to resolve. (V.c. Deb Ford)Along with undermining farmers’ “legitimate business” (Jooste), veganism was also considered to undermine Australia’s rural communities (Park qtd. in Malone).Hostile ExtremistsThe final stereotype linking veganism with Animals Australia was of hostile extremism (cf. Cole and Morgan). This means, for users, being inimical to Australian national values but, also, being akin to terrorists who engage in criminal activities antagonistic to Australia’s democratic society and economic livelihood (see, e.g., Greer; ABC News). It is the broad symbolic threat that “extremism” invokes that makes this stereotype particularly “infectious” (Rosello 19).The latest tag team attacks on our pork industry saw AL giving crash courses in how to become a career criminal for the severely impressionable, after attacks on the RSPCA against the teachings of Peter Singer and trying to bully the RSPCA into vegan functions menu. (Cattle Advocate)The “extremists” want that extended to dairy products, as well. The fact that this will cause the total annihilation of practically all animals, wild and domestic, doesn’t bother them in the least. (Brown)What is interesting about these last two dimensions of stereotyping is their displacement of violence. That is, rather than responding to the charge of animal cruelty, violence and extremism is attributed to those making the charge.Stereotypes and Symbolic Boundary ShiftingWhat is evident throughout these instances is how stereotyping as a “cognitive mechanism” is being used to build boundaries (Cherry 460): in the first instance, between “us” (the meat-eating majority) and “them” (the vegan minority aka animal activists); and secondly between human interest and livestock. This point is that animals may hold instrumental value and receive some protection through such, but any more stringent arguments for their protection at the expense of perceived human interests tend to be seen as wrong-headed (Sorenson; Munro).These boundaries are deeply entrenched in Western culture (Wimmer). They are also deeply problematic in the context of animal activism because they fragment publics, promote restrictive identities, and close down public debate (Lamont and Molnár). Boundary entrenching is clearly evident in the stereotyping work carried out by industry stakeholders where meat-eating and practices of industrialised animal agriculture are valorised and normalised. Challenging Australia’s meat production practices—irrespective of the reason given—is framed and belittled as entailing a vegan agenda, and further as contributing to the demise of farming and rural communities in Australia.More broadly, industry stakeholders are explicitly targeting the activist work by such organisations as Animals Australia as undermining the ‘Australian way of life’. In their reading, there is an irreconcilable boundary between human and animal interests and between an activist minority which is vegan, unreasonable, extremist and hostile to farming and the meat-eating majority which is representative of the Australian community and sustains the Australian economy. As discussed so far, such stereotyping and boundary making—even in their inaccuracies—can be pernicious in the way they entrench identities and divisions, and close the possibility for public debate.Rather than directly contesting the presuppositions and inaccuracies of such stereotyping, however, Animals Australia can be read as cultivating a process of symbolic boundary shifting. That is, rather than responding by simply underlining its own moderate position of challenging only intensive animal agriculture for systemic animal cruelty, Animals Australia uses its campaigns to develop “boundary blurring and crossing” tactics (Cherry 451, 459), specifically to dismantle and shift the symbolic boundaries conventionally in place between humans and non-human animals in the first instance, and between those non-human animals used for companionship and those used for food in the second (see fig. 2). Figure 2: That Ain’t No Way to Treat a Lady (Animals Australia, campaign image on back of taxi)Indeed, the symbolic boundaries between humans and animals left unquestioned in the preceding stereotyping are being profoundly shaken by Animals Australia with campaigns such as Make It Possible making morally relevant likenesses between humans and animals highly visible to mainstream Australians. Namely, the organisation works to interpellate viewers to exercise their own capacities for emotional identification and moral imagination, to identify with animals’ experiences and lives, and to act upon that identification to demand change.So, rather than reactively striving to refute the aforementioned stereotypes, organisations such as Animals Australia are modelling and facilitating symbolic boundary shifting by building broad, emotionally motivated, pathways through which Australians are being encouraged to refocus their own assumptions, practices and identities regarding animal experience, welfare and animal-human relations. Indeed the organisation has explicitly framed itself as speaking on behalf of not only animals but all caring Australians, suggesting thereby the possibility of a reframing of Australian national identity. Although such a tactic does not directly contest this negative stereotyping—direct contestation being, as noted, ineffective given the perniciousness of stereotyping—such work nonetheless dismantles the oppositional charge of such stereotyping in calling for all Australians to proudly be a little bit anti-meat-eating (when that meat is from factory farmed animals), a little bit anti-factory farming, a little bit pro-veg*n, and a little bit proud to consider themselves as caring about animal welfare.For Animals Australia, in other words, appealing to Australians to care about animal welfare and to act in support of that care, not only defuses the stereotypes targeting them but encourages the work of symbolic boundary shifting that is really at the heart of this dispute. 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