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: Background: B-cell development is a complex biological process that initiates in bone marrow and matures in lymph nodes. These hierarchical stages are tightly regulated by transcription factors and microRNAs (miRNAs). B-cells are prone to genetic alteration during these maturation stages due to DNA breaks required for B-cell receptor re-arrangement and additional changes that occur during the germinal center (GC)-reaction for class switch recombination and somatic hypermutation. The unwarranted genomic changes resulting from these processes can lead to B-cell malignancies with varied clinical and pathological characteristics. Of the lymphoma subtypes derived from mature B-cells, a subset of diffuse large B-cell lymphoma (DLBCL), primary mediastinal large cell lymphoma (PMBL), follicular lymphoma (FL) and Burkitt lymphoma (BL) are putatively derived from GC B-cells, with varied morphological, pathological and clinical characteristics. These tumor are characterized by distinct genetic abnormalities,[ t(14;18) in GCB-DLCBL, and FL or t(8;14)] in BL, and mutational profile. We postulate miRNA expression signatures, may also explain the wide-ranging clinico-pathological characteristics of these GC B- cell derived tumors. Methods: We performed a meta-analysis of miRNA profiles of DLBCL, BL, PBML, FL and EBV+DLBCL performed by us or others. (Iqbal 2015; 2012; Leich 2011; Andrade 2014). We also included lymphomas derived from naïve B-cells (MCL, SLL) and post GC B-cells (activated B cells like ABC-DLBCL), EBV (+) DLBCL, normal B-cell subsets and cell lines of distinct B-cell lineages. The majority of the cases have been characterized by gene expression profiling (GEP) with corresponding pathological clinical characteristics in above studies. The data was obtained using Taqman® human microRNA array (Applied Biosystems, CA) containing 380 miRNAs and analyzed using BRB-array Tools. Results In our initial miRNA expression profile of 249 lymphomas including GCB-DLBCL (n=34), ABC-DLBCL(n= 29), PMBL(n= 9), BL(n= 33), FL (n= 32), EBV+DLBCL (n=8); MCL(n=30) and SLL(n=12)showed distinct hierarchical clusters enriched with GEP defined subgroup, with few interspersed cases from other subgroups suggesting that miRNAs can complement and delineate GEP defined molecular subgroups, an observation consistent with earlier findings (Iqbal 2015). When we examined miRNAs, that are specifically associated with GC B- derived lymphomas, we observed a subset of miRNA including miR-146a, miR-142-3p, miR-17, miR19b, and miR106a were present at higher levels (top 5% in abundance by CT values) in GC B lymphomas. Of these, somatic mutations in miR142 have been recently reported in GC derived lymphomas (Alyssa, Leukemia 2016). These miRNAs also represented the most abundant miRNAs in centroblast, suggesting lineage specificity of miRNA signature in these lymphomas. However, miR222 highly expressed in centroblast was not expressed at higher levels in GCB-DLBCL and BL. A number of miRNAs specifically abundant in distinct lymphomas included (BL: miR-20a, miR-16), (GCB-DLBCL: miR-223, miR-24) and (FL: miR222). MiR-150 was highly expressed in NHLs putatively derived from naive B-cells like MCL or SLL, but absent from GC-derived NHLs. Consistent with this, miR-150 was present in naive B-cells, but not in centroblast, suggesting that miR-150 may have discrete function during early B-cell differentiation and late maturation stages. Within GC derived lymphoma, we examined miRNA profiles in clinically aggressive (BL); indolent lymphomas (FL) and intermediate aggressive (GCB-DLBCL) and their association with normal B-cell subsets, and indeed a number of miRNAs (ex. miR-150) were associated with indolent lymphomas, whereas a subset of miRNAs was present in aggressive lymphomas (ex. miR-16), suggesting these miRNAs may contribute the distinct clinical characteristics. Though we have identified unique miRNA profiles in these GC derived NHLs, the target genes of these miRNAs remains to be identified. Conclusion: We identified microRNAs that were differentially expressed between GC-derived DLBCL and other lymphomas, which may help explain distinguishing features of these lymphomas. Disclosures No relevant conflicts of interest to declare.

Description: Diffuse large B-cell lymphoma (DLBCL) displays significant heterogeneity with regard to clinical, pathological, and genetic features. Using gene expression profiling, we have delineated 3 molecular subgroups within DLBCL: germinal center B cells (GCB-DLBCL), activated B cells (ABC-DLBCL), and primary mediastinal B-cell lymphoma (PMBL),while these defined subgroups show characteristic mutation profiles and oncogenic pathways, a small set of cases of DLBCL still remain "unclassifiable" (UC). Burkitt lymphoma (BL) is characterized by the t(8;14)(q24;q32) with distinct morphological and immunophenotypic features. The diagnostic distinction between BL and DLBCL is challenging in a subset of cases that have overlapping morphological, immunohistochemical, and even molecular features, but is crucial for effective therapy. The identification of long non-coding RNA (lncRNA) has added another critical component to cancer biology. LncRNAs are defined as a distinct set of non-protein coding transcripts longer than 200 nucleotides. The functions of a few lncRNAs have been recently elucidated of which some are thought to regulate gene-specific transcription. The goal of the current study was to identify reliable lncRNA signatures for the BL and DLBCL subgroups and evaluate their usefulness as prognostic biomarkers. We examined the expression of lncRNAs from our earlier studies using Affymetrix-HG-U133 plus 2 arrays to distinguish unique gene expression profiles between BL and DLBCL (PMID: 27292966). In the initial analysis, we compared BL (n=77) with DLBCL (n=474) and identified 275 differentially expressed lncRNAs (p=0.005, fold change〉1.5). There was high expression of TCL6 and DDX-AS11 in BL. We confirmed the microarray results for TCL6 and DDX-AS11 by qRT-PCR in a subset of cases. We further tested whether expression of TCL6 and DDX-AS11 was regulated by the MYC oncogene and observed significant downregulation of these transcripts upon CRISPR/cas9 mediated deletion of the MYC promoter in the Raji cell line. We also sought to identify GCB-DLBCL and ABC-DLBCL associated lncRNAs. We observed 36 lncRNAs highly expressed in ABC-DLBCL and 40 lncRNAs highly expressed in GCB-DLBCL (P=0.005, fold change〉1.5). Of the differentially expressed lncRNAs, lnc00487 and DUBR were upregulated in GCB-DLBCL, whereas lnc00944 and FUT8 were upregulated ABC-DLBCL. The observed expression differences were validated in ABC-DLBCL and GCB-DLBCL cell lines. The differentially expressed LncRNAs were also validated in other DLBCL cohorts. LncRNA00487 expression was associated with superior clinical outcome in DLBCL series treated with Rituximab (R)-CHOP (p=0.01), and gene expression and overall survival (OS) were validated in another DLBCL series treated with R-CHOP (PMID:22437443). In the present study, we found that lncRNAs are differentially expressed in aggressive B cell lymphoma and could be useful as diagnostic or prognostic markers. They may play an important role in lymphoma biology and further studies of their functions are warranted. Disclosures No relevant conflicts of interest to declare.

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: Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas and approximately 30% of PTCLs are designated as not-otherwise specified (PTCL-NOS). Gene expression profiling (GEP) identified molecular classifiers for PTCL entities and identified 2 novel biological subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21), associated with T-cell differentiation subsets. To further investigate molecular oncogenesis, we performed microRNA expression profiling (miR-EP) in several molecular subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), PTCL-GATA3 and PTCL-TBX21 using formalin fixed paraffin embedded tissues. We also performed miR-EP of normal T-cell subsets polarized to represent different differentiation stages (TFH, TH1 and TH2). We performed miR-EP on 102 PTCL cases using either quantitative real time PCR (ABI, Biosystem) or ultra-sensitive direct miRNA counting (nCounter, NanoString). Corresponding GEP (mRNA) were available for 67 PTCL cases. Normal T-cells were polarized in-vitro with different cytokine milieu and examined by flow cytometry. We observed distinct miRNA profiles, with miRNA being uniquely expressed in TFH polarized cells (miR-26a-5p, miR-17-5p, miR-30d-5p, miR-22-3p, miR-222-3p, miR-142-3p, let-7i-5p and miR-29b-3p). In contrast, the TH1 lineage was enriched for expression of miR-155-5p, miR-146a-5p, miR-1246, miR-93-5p, miR-16-5p, miR-21-5p, miR-363-3p, miR-1260a, miR-186-5p, miR-148a-3p and miR-579-3p, whereas TH2 polarized cells expressed miR-181a-5p, let-7a-5p, miR-191-5p, miR-15b-5p, let-7d-5p, let-7b-5p, miR-140-5p, miR-98-5p, miR-423-5p and miR-630. Several of these miRNA expressed in the T-cells subsets showed corresponding expression in their respective PTCL entity such as miR-142-3p, let7i-5p, miR-21-5p and miR-29b-3p with AITL, miR-146-5p, miR-155-5p and miR-16-5p in PTCL-TBX21 and miR-181a-5p, miR-630 and let7a-5p in PTCL-GATA3. We also performed the MiRNA Enrichment Analysis and Annotation (miEAA) for miRNA signatures and observed an enrichment of miRNA regulating epigenetic modifications in TFH cells (p=0.028), whereas TH1 showed an enrichment of miRNA regulating IFN-g signaling (p=0.0024), and miRNA signatures in TH2 showed negative regulation of TGF-b signaling (p=0.023). Supervised analysis (p=0.05) of the miRNA profiles identified significant association of miR-126, miR-145, and let-7c-5p with AITL, when compared to other PTCLs. Similarly, miR-92a, miR-25, miR-636, miR-210, miR-222 and miR-491-5p significantly associated with PTCL-GATA3 and miRNA 126-3p, 145-5p, miR-26a-5p and miR-34a-5p associated with PTCL-TBX21. The miEAA for tumor miRNA signatures revealed enrichment of miRNAs regulating histone methylation (h3 k4 methylation) and chemokine receptor signaling in AITL, whereas miRNA regulating T-cell receptor were enriched in PTCL-TBX21 and TP53 signaling pathway in PTCL-GATA3. We validated the expression of miR-126 in AITL by qRT-PCR and also observed its increased expression in IL21 stimulated CD4+ T-cells. Ectopic expression of miR-126 resulted in a ~3 fold increased expression in T-cell lines and led to reduced proliferation and increased apoptosis with expression of T-cell exhaustion makers PD1 and TIM3. Computational algorithmic programs identified relevant biological targets of miR-126, including p85/PIK3R2, S1PR2 and DNMT3A that were further validated in-vitro. We observed an inverse correlation of miR-126 expression with S1PR2 expression (r=-0.64). S1PR2 is a crucial G protein-coupled receptor regulating B and T-cell migration in the germinal center (GC) reaction. Migration assays demonstrated significant decreases in T to B-cell migration, when B-cells (Raji) were co-cultured with Jurkat cells with ectopic expression of miR-126. With the GC reaction holding an important role in AITL, we investigated the biological significance of miRNA-126 in the context of the AITL microenvironment. High expression of miRNA-126 significantly associated with inferior survival in AITL (p=0.008) and significant differences in tumor microenvironment signatures. We identified distinct miRNA signatures for AITL and molecular subgroups of PTCL-NOS. Furthermore, elevated expression of miR-126 may contribute to the dysregulation and the homing of TFH cells in GC reaction through S1PR2 and warrants further mechanistic investigation. Disclosures No relevant conflicts of interest to declare.

Description: One-third of peripheral T-cell lymphomas are “not otherwise specified” (PTCL-NOS), but they have been subdivided into 2 subgroups based on gene expression profiling. Amador and colleagues generated an immunohistochemical algorithm that parallels the molecular separation of PTCL-NOS and provides useful prognostic information.