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 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: Key Points Anaplastic large-cell lymphoma has a unique miRNA signature. The miR-17∼92 is an important downstream effector of ALK oncogenic pathway.

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: Peripheral T-cell lymphoma (PTCL) is a group of clinically and pathologically heterogeneous non-Hodgkin lymphomas (NHL). Using gene expression profiling (GEP), we have defined molecular classifiers for PTCL subtypes reflecting their pathobiology and oncogenic pathways (Iqbal et al. 2014). We have also shown associations of specific mutations with the molecular subgroups (Wang et al. 2015). Although genomic information is increasing, the pathogenetic mechanisms of PTCLs remain largely unknown. Therefore, we analyzed copy number variation (CNV) and GEP to identify unique genetic abnormalities in the defined PTCL molecular subgroups. CNV data were generated on fresh frozen or formalin-fixed paraffin-embedded genomic DNA (n=114) on 3 Affymetrix platforms (SNP 6.0, 250K SNP, and OncoScan). Two published cohorts (PTCL-NOS, Hartmann et al. 2010; ALCL, Boi et al. 2013) were included for validation. The gene expression analysis, morphological review and clinical characteristics of these cases have been included in previous studies (Iqbal et al. 2010, 2014). Angioimmunoblastic T-cell lymphoma (AITL) represents 20% of all PTCL cases. The most recurrent CNV in AITL was chromosome (chr) 5 gain (39%), followed by chr 21 gain (21%). Interestingly, chr 21 gain co-occurred with chr 5 gain (p=0.003). No recurrent losses (≥20%) were identified among these cases. Molecularly re-classified AITL cases from morphologically classified PTCL-NOS cases showed concordant results with bonafide AITL cases. Of the commonly mutated genes, DNMT3A, IDH2, RHOA and TET2, only IDH2R172Kshowed a significant association (p=0.012) with chr 5 gain. GEP showed enrichment of gene signatures associated with oxidative phosphorylation (PGC-1α target genes) in cases with chr 5 gain. PTCL, not otherwise specified (PTCL-NOS) is the most common PTCL subtype and cannot be further sub-classified using conventional approaches; however, we have identified 2 molecular subgroups within PTCL-NOS, the GATA3 and TBX21 subgroups which are related to 2 distinct T-helper subsets (Iqbal et al. 2014), by employing GEP. Consistent with earlier observations (Hartmann et al. 2010), PTCL-NOS showed remarkably varied CNVs with nearly 50% of cases showing high CNV frequencies. When correlated with molecular subgroups, distinctive CNVs were observed in the molecular GATA3 and TBX21 subgroups. The GATA3 subgroup displayed a large assortment of CNVs. Complete or partial gain of chr 7 (57%) was the most recurrent gain in these cases. Losses affecting 17p, 10q and 9p21, encompassing tumor suppressors such as TP53 (57%), PTEN (43%) and CDKN2A (43%), were frequent in the GATA3 subgroup. The TBX21 subgroup had significantly fewer CNVs, as none were recurring (≥20%); but gains of 5p or 11p were observed in 14%. Additionally, PTCL-NOS cases with ≥10% abnormal genome had significantly poorer overall survival (p=0.012) compared to those with fewer abnormalities. This finding validates the GEP molecularly defined subgroups, as the GATA3 subgroup displayed more CNVs and has been associated with a worse prognosis compared to the TBX21 subgroup (Iqbal et al. 2014). We were able to distinguish CNVs characteristic of the different entities, including the co-occurrence of chr 5 and 21 gains specific in AITL. Gain of 1q (complete or partial) was identified in the GATA3 subgroup of PTCL-NOS and anaplastic lymphoma kinase (ALK) (-) ALCL with equal frequencies (~ 36%), but only 16% in ALK(+) ALCL. Complete or partial gain of chr 7 was also observed in ALCL, but at a considerably lower frequency than in the GATA3 subgroup. Additionally, gain of chr 18 or regions of 17q, and loss of 5q or regions on both arms of chr 9, were more frequent in the GATA3 subgroup compared to other entities. The TBX21 subgroup was primarily differentiated from the GATA3 subgroup by presence of fewer CNVs. Our analysis provides a framework for future investigations into the molecular pathogenesis of PTCL, and highlights potential candidate oncogenes and tumor suppressors deregulated by copy number aberrations. Comparative analysis revealed that certain chromosomal abnormalities are entity-specific. AITL cases with IDH2R172K also had trisomy 5 suggesting that these oncogenic events cooperate in malignant transformation. Thus, the complexity of PTCL is finally becoming clearer with the integration of high resolution molecular techniques for global genomic analysis. Disclosures No relevant conflicts of interest to declare.

Description: The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is a very aggressive lymphoma characterized by constitutive NF-kB activation, but whether miRNAs dysfunction contributes to this event, and their exact function and mechanism remain unclear. Starting from an integrative screening strategy, we revealed that there were some interactions between the NF-kB signaling and miR-17~92 cluster, which was essential for B-cell development and commonly gained and/or overexpressed in ABC-DLBCL. Several important NF-kB negative regulators including TNFAIP3 (A20), CYLD and Rnf11 were predicted and validated to be the direct targets of miR-17~92. Conditional knock-down of miR-17~92 using sponge could suppress NF-kB activity and elevate the A20, CYLD and Rnf11 expression in 293T cells. Furthermore, we demonstrated that enforced overexpression of miR-17~92 could also decrease the A20, CYLD and Rnf11 expression in ABC-DLBCL cells. Conditional overexpression of miR-17~92 could promote ABC-DLBCL cells growth, accelerate the cells G1/G0 phase to S phase transition, and suppress NF-kB inhibitor-induced apoptosis. Conversely, conditional knock-down of miR-17~92 could inhibit ABC-DLBCL cells growth and sensitize the cells to NF-kB inhibitor-induced apoptosis. The miR-17~92 could induce the IkB-a and NF-kB p65 phosphorylation, leading to the NF-kB activation and aberrant expression of NF-kB transcriptional target genes. However, miR-17~92 did not regulate the NF-kB p52/p100 phosphorylation. Overexpression of miR-17~92 enhanced K63-linked ubiquitination and reduced K48-linked ubiquitination of the TNFa receptor 1 complex including RIP1. Importantly, we found that high expression level of miR-17~92 was associated with poorer survival in ABC-DLBCL patients. Our results uncovered a novel mechanism for the canonical but not the non-canonical transcription factor NF-kB pathway by modulation of miR-17~92 in ABC-DLBCL, and suggested that targeting the miR-17~92 might be novel bio-therapeutic strategies, which could be single-agent or combined with NF-kB inhibitor treatment, for ABC-DLBCL patients. Figure 1. Inhibition of miR-17~92 blocks the activity of NF-kB in HER293T cells: (A) The schematic representation of reporter constructs involved in assay. (B) HEK293T cells were co-transfected with Dul-Luciferase reporter constructs and the miR-17~92 sponge plasmid. TNF-a stimulation or without stimulation for 18 h. Figure 1. Inhibition of miR-17~92 blocks the activity of NF-kB in HER293T cells: (A) The schematic representation of reporter constructs involved in assay. (B) HEK293T cells were co-transfected with Dul-Luciferase reporter constructs and the miR-17~92 sponge plasmid. TNF-a stimulation or without stimulation for 18 h. Figure 2. miR-17~92 directly regulates A20, CYLD and Rnf11 in ABC-DLBCL cells. (A) Fluorescence images of tranduced ABC-DLBCL cells. (B) Expression of sponge or miR-17~92 in tranduced ABC-DLBCL cells. (C) Inhibition of miR-17~92 increase the expression of A20, CYLD and Rnf11. Overexpression of miR-17~92 reduce the expression of A20, CYLD and Rnf11 in ABC-DLBCL cells. Figure 2. miR-17~92 directly regulates A20, CYLD and Rnf11 in ABC-DLBCL cells. (A) Fluorescence images of tranduced ABC-DLBCL cells. (B) Expression of sponge or miR-17~92 in tranduced ABC-DLBCL cells. (C) Inhibition of miR-17~92 increase the expression of A20, CYLD and Rnf11. Overexpression of miR-17~92 reduce the expression of A20, CYLD and Rnf11 in ABC-DLBCL cells. Figure 3. miR-17~92 modulate mediate NF-kB activity in ABC-DLBCL. (A) Immunoblot analysis of IkB-a, NF-kB p65, NF-kB p100/p52 and their phosphorylation. (B) Heat-map display of quantitative real-time RT-PCR measurements of six independent NF-kB transcriptional targets show significantly lower expression in sponge expressing cells and higher expression in miR-17~92 expressing cells. Figure 3. miR-17~92 modulate mediate NF-kB activity in ABC-DLBCL. (A) Immunoblot analysis of IkB-a, NF-kB p65, NF-kB p100/p52 and their phosphorylation. (B) Heat-map display of quantitative real-time RT-PCR measurements of six independent NF-kB transcriptional targets show significantly lower expression in sponge expressing cells and higher expression in miR-17~92 expressing cells. Disclosures No relevant conflicts of interest to declare.

Description: Natural killer/T cell lymphoma (NKTCL) is an aggressive subtype of non-Hodgkin lymphoma that is rare overall but has a higher prevalence in Chinese and Hispanic populations. Recent sequencing efforts have improved the understanding of the disease and revealed a number of genes that may drive the pathogenesis of NKTCL. These efforts were largely limited by the number of cases studied. Based on previously reported whole exome sequencing data in NKTCL and other lymphoid malignancies and on the frequency and potential biological significance of the mutant, we designed a 334-gene panel and sequenced 105 NKTCLs. We characterized single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations (CNAs) that may have pathogenic roles. Tumor DNA was isolated from 74 Chinese and 31 Hispanic NKTCL patients. For 7 of these, matched normal DNA was extracted from peripheral blood. The 334 genes in the custom panel were captured using the Agilent SureSelect platform, and the exons of these genes were sequenced on an Illumina HiSeq 2500 instrument. For cases without matched normal samples, we discriminated between somatic and germline variants with an in-house machine learning algorithm which is based on information of public variant databases, variant annotations, and sequencing statistics, and followed by manual inspection. CNAs were determined based on the untargeted sequences using an R package, CopywriteR. We identified a total of 479 SNVs and 113 indels in 155 genes in 102 out of 105 patients. Mutations in STAT3 and JAK3 were identified in 30% and 7% of the cases, respectively. The hotspot mutations Y640F, S614R, and G618R in the SH2 domain of STAT3 and A573V of the JH2 domain of JAK3 were observed in most of these cases. 46% of the cases harbored one or more mutations in epigenetic regulators ARID1A, EP300, MLL2, MLL3, and TET2. Transcription co-repressors BCOR and NCOR2 were mutated in 25 of the cases. 13% of the cases contained mutations in HLA-A or CIITA, most of which were inactivating, suggesting possible defects in immune surveillance. DDX3X and TP53 mutations were detected in 19% and 10% of the cases, respectively. TP53 mutations were mutually exclusive with mutations in DDX3X and, except for one case, with STAT3 also. MGA mutations, which were present in 12% of the cases, were usually concurrent with STAT3 mutations. The mutational profiles of Chinese and Hispanic cases were similar, and only CD58 mutations were significantly more common in Hispanic patients (p=0.03). Comparison between NKTCL with localized and systemic presentation demonstrated that CCR7 was mutated more frequently in systemic cases (p=0.02). Prognostic analysis identified EP300 mutations as a potential marker for poorer survival (p=0.01). Frequent copy loss on chromosome 6q was observed and PRDM1, which is present on 6q, was mutated in 10% of the cases. In summary, we identified the profile of mutated genes in a large series of NKTCL. Most of the genes have been previously reported to be mutated in NKTCL, but the frequencies for some of the mutants were quite different. Interestingly, the mutational profiles of NKTCL in Chinese and Hispanic patients were very similar despite the geographic differences. Mutations leading to abnormal activation of the JAK-STAT3 pathway and abnormal chromatin modification were highly prevalent. BCOR, DDX3X and TP53 mutations were also frequent. Variants in CCR7 and EP300 were potential markers for systemic disease and poor prognosis, respectively. Figure. Mutation profiles of genes with mutation frequency larger than 5%. Genes that are not expressed in normal NK cells were excluded. Figure. Mutation profiles of genes with mutation frequency larger than 5%. Genes that are not expressed in normal NK cells were excluded. Figure. Overall survival curves of cases with and without mutations in EP300. Figure. Overall survival curves of cases with and without mutations in EP300. Disclosures No relevant conflicts of interest to declare.