ALBERT

Description: Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children. Although it accounts for only 1-5% of NHL in adults, approximately 60% of the BL cases diagnosed each year in western countries occur in patients 〉40 years of age. Although adult and pediatric BL cases are indistinguishable by molecular classification, pediatric patients have a significantly better outcome than adults. While translocation of MYC to the immunoglobulin heavy or light chain genes is characteristic of pediatric and adult BL, genetic differences may contribute to the superior clinical outcome of childhood cases. Therefore, we aimed to identify the spectrum of additional genetic abnormalities that occur in adult and pediatric BL. Copy number analysis, gene expression profiling (GEP), and targeted sequencing of ~400 genes known to be mutated in NHLs were performed on a cohort of molecularly defined BL samples. Copy number abnormalities (CNAs) were identified by the Affymetrix 250k NspI SNP array in 73 BL tumors (28 adult, 45 pediatric), and sequencing was performed on 52 BLs (21 adult, 31 pediatric). Pediatric cases had fewer CNAs than adults. The most common focal abnormality identified was a gain on 13q31.3 encompassing MIR17HG. It was more frequent in adult compared to pediatric cases (35% vs 16%, p=0.085) and was associated with increased expression of miR-17~92 cluster members; and among adults, patients with this gain trended towards worse overall survival, though the number of cases with available information was small. Gain of 8q was found in ~20% of adult cases, but in no pediatric cases. Surprisingly, cases with 8q gain had significantly lower MYC mRNA expression (p〈 0.001) and lower protein expression. In cases with MYC gain 0/4 cases were positive for MYC protein expression by immunohistochemistry; in contrast,6/10 cases with no MYC gain were positive for MYC expression. This suggests that gain of 8q is driven by another gene in the region. Additional genetic alterations included gains of genomic loci encompassing MCL1 and MDM4 (1q21-24) and losses encompassing RB1, p53 and CDKN2A/CDKN2B. Pathway analysis of genes differentially expressed by CN status showed an enrichment of genes involved in cell cycle regulation, the p53 signaling pathway, and the ubiquitin proteasome pathway. The frequencies of mutations in commonly mutated genes including MYC, ID3, TP53, CCND3, DDX3X, ARID1A, and TCF3 were not significantly different in adult and pediatric BL. However, BCL2, (43%, p

Description: Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of diseases with poor prognosis and no standard therapy due to widely varying responses to treatments, thus new therapeutic targets need to be identified. Gene expression profiling (GEP) has helped to separate classes of these diseases to predict survival. Recent genetic studies on the two most common subtypes of PTCL in the Western world, angioimmunoblastic T-cell lymphoma (AITL) and PTCL, not otherwise specified (PTCL-NOS), have revealed recurrent mutations in the epigenetic modifiers TET2, IDH2, and DNMT3A; in the small GTPase RHOA; and rarely in the T-cell receptor (TCR) adaptor protein FYN. Because TCR stimulation is necessary for normal T-cell expansion, activating mutations in the stimulation (CD3/TCR) and costimulation (CD28) pathways could be involved in malignant lymphopoiesis. We performed whole-transcriptome sequencing on a small set of primary PTCL cases and found mutations in CD28, including an aspartate 124 mutation within a fusion transcript between CD28 and family member ICOS as well as a threonine 195 mutation. On targeted re-sequencing of 92 PTCL cases, we found two AITL cases and one ALK- anaplastic large cell lymphoma case with an aspartate 124 mutation (variant frequencies [VF]: 1.03% to 5.90%). We also found mutations at threonine 195 (two AITL, one PTCL-NOS, Tbx21 subtype; VF: 2.90% to 12.30%). Additionally, we found two recurrent mutations with low variant frequencies (

Description: Background: c-MYC (MYC) is commonly dysregulated in aggressive B cell lymphomas. MYC associated lymphoma, especially Double Hit lymphoma (DHL) and Double-Expression Lymphoma (DEL) which are characterized by MYC and BCL2 dual overexpression usually present with the inferior outcome as rapid disease progression and poor response to standard chemotherapy regimen. Nevertheless, MYC is considered as an "undruggable" target and targeting strategies such as suppressing MYC transcription by bromodomain (BRD)-4 inhibitors have been widely investigated in both preclinical models and clinical trials. However, increasing evidence has shown that lymphoma cells displayed a wide range of resistance to BRD-4 inhibition, due to transcription adaptation or kinome reprogramming. Hence, alternative approaches for suppressing MYC or its function are urgently needed. Strategies directed against oncoprotein translation may efficiently repress key oncoproteins regardless of the abundant MYC mRNA due to genetic aberrations and secondary transcription up-regulated by MYC. Rocaglate is a class of natural products derived from plants of the Aglaia genus that have been demonstrated to potently inhibit protein translation initiation via eIF4A. The use of rocaglates for anti-cancer treatment was limited due to the scarcity and instability of these natural products e.g. Silvestrol. Recent chemical modification and screening studies have unveiled a few synthetic rocaglates that are more potent than Silvestrol such as SDS-1-021-(−), which unlocked the potential use of rocaglates for clinical applications. Methods and Results: To probe effective reagents for MYC-driven lymphoma, a screening of 50 drugs targeting common oncogenic pathways and tumorigenic machinery was performed in two isogenic B-lymphoma lines. Several protein translation inhibitors, such as mTOR kinase inhibitors (TORKi), and eIF4A inhibitor Silvestrol were identified as the most potent drugs in all of the tested cells. Further, we found that Silvestrol but not TORKi efficiently repressed MYC protein translation in MYC-driven B lymphoma cells, whereas neither of them inhibited BCL2 expression. Moreover, we demonstrated that eIF4E knockdown or eIF4E/eIF4G disruptor Briciclib did not significantly affect MYC expression, whereas eIF4A inhibitor hippuristanol and rocaglates derivate SDS-1-021-(−) diminished MYC expression similar to that observed in Silvestrol treated cells. By using dual luciferase assay, we demonstrated that rocaglates had stronger cap-dependent and IRES translation inhibition than TORKi in lymphoma cells. Next, by native RNA immunoprecipitation and siRNA knocking down, we found that rocaglates repressed MYC translation not only via eIF4A1 but also via eIF4A2, however, with different underlying mechanisms. Furthermore, to explore the molecular targets of rocaglates treatment in B cell lymphoma, we performed TMT-Mass Spectrometry which identified multiple oncoproteins including NEK2, MYC, MCL1, TCF3, BCL6, PLK1, AURKA, and WEE1 were significantly down-regulated by SDS-1-021-(−) treatment. Finally, we demonstrated that SDS-1-021-(−) is highly potent as a single agent and synergized with ABT199 at a low dose (0.2mg/kg) in PDX models with DHL/DEL. Brief summary : Our pre-clinical study provided strong evidence that rocaglates but not TORKi efficiently suppress MYC protein translation because 1). Rocaglates exhibit strong inhibition on both Cap- and IRES-dependent translation, 2). Rocaglates decrease PLK1 and AURKA/B thus destabilizing MYC protein. The synthetic rocaglate SDS-1-021-(−) is a potent agent that exhibits significant synergistic killing effect with ABT199 on DHL/DEL cells in the pre-clinical animal study. Figure Figure. Disclosures Lunning: TG Therapeutics: Consultancy; AbbVie: Consultancy; Genentech: Consultancy; Astra-Zeneca: Consultancy; Genzyme: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Gilead: Consultancy; Spectrum: Consultancy; Genentech: Consultancy; Seattle Genetics: Consultancy; Portola: Consultancy; Kite: Consultancy; Juno: Consultancy; Janssen: Consultancy; Verastem: Consultancy. Vose:Novartis: Honoraria, Research Funding; Epizyme: Honoraria; Incyte Corp.: Research Funding; Bristol Myers Squibb: Research Funding; Kite Pharma: Research Funding; Merck Sharp & Dohme Corp.: Research Funding; Abbvie: Honoraria; Legend Pharmaceuticals: Honoraria; Seattle Genetics, Inc.: Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding; Roche: Honoraria.

Description: Peripheral T-cell lymphoma (PTCL) is a group of complex clinicopathological entities, often associated with an aggressive clinical course. Angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specified (PTCL-NOS) are the 2 most frequent categories, accounting for 〉50% of PTCLs. Gene expression profiling (GEP) defined molecular signatures for AITL and delineated biological and prognostic subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21). Genomic copy number (CN) analysis and targeted sequencing of these molecular subgroups revealed unique CN abnormalities (CNAs) and oncogenic pathways, indicating distinct oncogenic evolution. PTCL-GATA3 exhibited greater genomic complexity that was characterized by frequent loss or mutation of tumor suppressor genes targeting the CDKN2A/B-TP53 axis and PTEN-PI3K pathways. Co-occurring gains/amplifications of STAT3 and MYC occurred in PTCL-GATA3. Several CNAs, in particular loss of CDKN2A, exhibited prognostic significance in PTCL-NOS as a single entity and in the PTCL-GATA3 subgroup. The PTCL-TBX21 subgroup had fewer CNAs, primarily targeting cytotoxic effector genes, and was enriched in mutations of genes regulating DNA methylation. CNAs affecting metabolic processes regulating RNA/protein degradation and T-cell receptor signaling were common in both subgroups. AITL showed lower genomic complexity compared with other PTCL entities, with frequent co-occurring gains of chromosome 5 (chr5) and chr21 that were significantly associated with IDH2R172 mutation. CN losses were enriched in genes regulating PI3K–AKT–mTOR signaling in cases without IDH2 mutation. Overall, we demonstrated that novel GEP-defined PTCL subgroups likely evolve by distinct genetic pathways and provided biological rationale for therapies that may be investigated in future clinical trials.

Description: Key Points Adult-mBLs have distinct and more frequent DNA copy number abnormalities compared with pediatric-mBL. Comprehensive genomic analysis revealed that the BCR signaling pathway is a potential therapeutic target in adult-mBL.

Description: Background: Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL) with distinct pathological features and poor prognosis. Currently used chemotherapy is mostly unsuccessful with a 3-year overall survival of less than 30%. We and others have identified frequent mutations affecting IDH2 at arginine-172 (R172),TET2, DNMT3A and RHOA in AITL. The biochemical and functional consequences of IDH2R172 mutations in T cells have not been demonstrated. In this study, we performed targeted re-sequencing of epigenetic regulators, including IDH2, TET2 and DNMT3A in molecularly defined PTCL cases and analyzed the biochemical changes associated with IDH2R172 mutations as well as alterations in gene expression profiling (GEP), DNA methylation and histone modification that may improve our understanding of the pathogenetic mechanisms in AITL. Methods: We performed targeted re-sequencing of epigenetic regulators IDH2, TET2 and DNMT3A in AITL (n = 39) and PTCL subtypes (n = 53) with corresponding GEP. Due to lack of appropriate cell lines derived from AITL, we chose to use Jurkat T cells, a T-ALL cell line frequently used for T-cell functional studies and normal CD4+T cell to study the biochemical consequences of IDH2R172 mutations in T-cells. Liquid chromatography- tandem mass spectrometry was utilized for the detection of intracellular level of the 2-hydroxyglutarate and levels of 5-methycytosine and 5-hydroxymethycytosine in genomic DNA. Alterations of histone lysine tri-methylation were assessed by immunohistochemistry in AITL specimens and western blotting in vitro. Results: TET2 mutations appear to be the founder mutations in AITL with 82.1% (32/39) mutated cases and present as the major clone in the majority of mutant cases (75%; 24/32). TET2 mutations were also observed at a lower frequency in PTCL-NOS molecular subgroups (TBX21 (46%; 10/18); GATA3 (41%; 5/12)) and ALK negative-ALCL (33.3%, 4/12). The mutations in DNMT3A were observed at similar frequency in AITL (38.5%, 15/39) and PTCL-NOS subgroups [TBX21 (33%; 6/18); GATA3 (25%; 3/12)). However, IDH2R172 mutations were found predominantly in AITL (33.3%, 13/39), but rarely in PTCL-NOS subgroups (6.7%, 1/15 in PTCL-NOS). Remarkably, IDH2R172 mutant cases formed a unique cluster in unsupervised hierarchical clustering, and IDH2R172mutation defined a unique subset within AITL with a distinct gene expression signature. We observed that ectopic expression of IDH2R172K in the Jurkat T cell line led to a markedly increased level of intracellular 2-HG and up-regulation of the repressive histone methylation mark H3K27me3. Furthermore, a significant increase in 5-methylcytosine and corresponding decrease in 5-hydroxymethycytosine in genomic DNA was also observed in IDH2R172K transduced Jurkat and primary CD4+ T cells. Consistent with these findings, significant increase in aglobal DNA hypermethylation in proximal promoter regions and a global increase of the repressive histone mark H3K27me3 was observed in AITL harboring IDH2R172 mutants. Integrative analysis of GEP and promoter methylation identified several recurrently hypermethylated genes including negative regulators of the NF-kB pathway. Conclusion: IDH2R172 mutations define a unique subgroup of patients in angioimmunoblastic T-cell lymphoma with a distinct gene expression profile. IDH2R172 mutations are associated with global promoter hypermethylation in genomic DNA and trimethylation of H3K27 in AITL specimens. The current findings suggest that abnormal methylation associated with IDH2R172 mutations contribute to lymphomagenesis in AITL. Disclosures Fu: Nanostring: The author is a potential inventor on a patent application using Nanostring technology for the Lymph2Cx assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Greiner:Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties.

Description: Key Points IDH2 R172 mutations define a unique subgroup with distinct TFH-like gene expression signatures in AITL. IDH2 R172 mutations can induce DNA and repressive histone hypermethylation in AITL.

Description: 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.

Description: The Th1 and Th2 lineages of CD4+ T helper cells are essential for control of host infection. Both lineages respond to antigenic stimulation with distinct effector functions and cytokine profiles. Differential homing patterns permit localization within specific tissue sites where these cells interact with other immune cells to promote the immune response. Variability in T helper lineage homing is due, in part, to differing chemokine receptor expression patterns. This laboratory and others recently described another CD4+ T helper lineage, Th17. Following stimulation, Th17 cells also produce a unique cytokine profile, including interleukin (IL)-17, IL-21, and IL-22. The Th17 lineage has now been implicated in the pathogenesis of several human autoimmune diseases, including psoriasis and inflammatory bowel disease, and appears to be critical for the inflammation of both the skin and gastrointestinal tract, respectively, seen in these diseases. It is not well understood whether Th17 cells arise within the inflammatory milieu in these tissues, or whether these cells possess a distinct homing pattern. We have performed studies using in vitro polarized Th17 cells for the study of tissue homing patterns in vivo. Experiments were performed using the well-described HLA Class II-disparate C57BL/6 (B6) to B6.C-H-2bm12 (bm12) model. Previous studies have established CD4+ T cell-dependent inflammation in this model. Naïve CD4+ T cells from B6 mice were polarized to the Th17 lineage in vitro using standard techniques, including IL-6 and TGF-β. FACS analysis of the Th17 cells prior to adoptive transfer revealed IL-17-positive staining in 〉60% cells and IFN-γ-positivity in

Description: Abstract 1343 Poster Board I-365 Acute graft-versus-host disease (aGVHD) remains a major obstacle for successful allogeneic bone marrow transplantation (BMT). Pancytopenia is frequently observed in the patients with aGVHD and contributes to poor prognosis. However, the mechanisms for pancytopenia are not well understood. To study how hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) are affected by aGVHD, we have examined the kinetics and functional alterations of HSC and HPC in a haplo-identical BMT model, in which parent lymphocytes were transferred into F1 offerspings (P1→F1) for induction of aGVHD. 6×107 splenocytes together with 5×106 bone marrow nucleated cells (BMNCs) of B6.SJL (B6, CD45.1+) were transplanted into lethally irradiated B6 x Balb/c F1 mice (CB6F1, CD45.1/.2). Same numbers of BMNCs without splenocytes were transplanted into the irradiated recipients as controls. In this aGVHD model, the average pathological score for aGVHD was 3.2±0.3 at day 14 after transplantation. The donor-derived hematopoietic cells (CD45.1+) were isolated for phenotypic analyses and functional assays at different time points. As expected, reduced peripheral blood cell counts and bone marrow (BM) cellularity were observed in the aGVHD hosts. aGVHD hosts showed more severe deficiencies [(0.95±0.2) colony forming unit (CFU) /105 BMNCs and 1 cobblestone area forming cell (CAFC) /106 BMNCs] than the controls without aGVHD [(1.75±0.3) CFU /105 BMNCs and 1 CAFC/3×105 BMNCs]. The frequency of HSC-enriched population (CD150+CD48−Lin−) in the aGVHD hosts [(1.05±0.38)x10−5] was significantly lower than that in the controls [(1.97±0.21)x10−5] at day 14 (P=0.03). When normalized to the bone marrow cellularity, the absolute yield of this population was decreased 2.89-fold in the aGVHD hosts than that in the control group. To measure the long-term engraftment of HSCs isolated from aGVHD hosts, we performed the secondary BMT, in which 2 × 106 of donor-derived BMNCs (CD45.1+) isolated from the aGVHD hosts or control animals at day 14 after the induction of aGVHD were transplanted into lethally irradiated CB6F1 recipients. Unexpectedly, the multilineage engraftment of the hematopoietic cells from the aGVHD hosts was 2 times more than that of the control group [(21.24±4.21)% vs (9.14±2.54)%,P