Adult T cell leukemia/lymphoma (ATLL) is a frequently incurable disease associated with the human lymphotropic virus type I (HTLV-I). RNAi screening of ATLL lines revealed that their proliferation depends on BATF3 and IRF4, which cooperatively drive ATLL-specific gene expression. HBZ, the only HTLV-I encoded transcription factor that is expressed in all ATLL cases, binds to an ATLL-specific BATF3 super-enhancer and thereby regulates the expression of BATF3 and its downstream targets, including MYC. Inhibitors of bromo-domain-and-extra-terminal-domain (BET) chromatin proteins collapsed the transcriptional network directed by HBZ and BATF3, and were consequently toxic for ATLL cell lines, patient samples, and xenografts. Our study demonstrates that the HTLV-I oncogenic retrovirus exploits a regulatory module that can be attacked therapeutically with BET inhibitors.

A link between B-cell-receptor (BCR) signalling and human B-cell lymphomas has been inferred from the study of immunoglobulin genes in human lymphomas and from work on mouse models, but more evidence is required to confirm an oncogenic role. New work from Davis et al. shows that chronic activation of BCR signalling is required for the survival of the ABC DLBCL subset of B-cell lymphomas. Somatic mutations in CD29A and CD79B found in these lymphomas appear to contribute to BCR activation. The role of B-cell-receptor (BCR) signalling in human B cell lymphomas has been a long-standing question, with genetic and functional evidence for its oncogenic role in human lymphomas lacking. Here, a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like subtype of diffuse large B-cell lymphoma is described and analysed, with potential implications for future therapeutic strategies. A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas1,2 and by engineering mouse models3, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of ‘chronic active’ BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-κB pathway activity and survival in ABC DLBCL4. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-κB5, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton’s tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-κ, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules6 of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt’s lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

Introduction and methods: Gene expression profiling has been used to define three molecular subtypes of diffuse large B cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL).To investigate whether these DLBCL subtypes arise by distinct pathogenetic mechanisms, we analyzed 203 DLBCL biopsy samples by high resolution, genome-wide copy number analysis (aCGH) coupled with gene expression profiling.We defined minimal common regions (MCRs) that were recurrently altered in copy number and identified those that dysregulated the expression of their constituent genes.Statistical differences in MCR frequency between DLBCL subtypes were corrected for multiple hypothesis testing using a false discovery rate (FDR) calculation.Results: Of 258 MCRs that occurred in more than 5% of any DLBCL subtype, 31 were utilized differentially by the DLBCL subtypes (p<0.005,false discovery rate <0.05).Amplification of the oncogenic mir-17-92 microRNA cluster and deletion of the tumor suppressor PTEN were recurrent in GCB DLBCL but did not occur in ABC DLBCL.Conversely, deletion of the INK4a/ARF tumor suppressor locus, gain of chromosome arm 18q, and trisomy 3 occurred almost exclusively in ABC DLBCLs and were associated with inferior outcome within this subtype.BCL2 and NFATC1 are candidate oncogenes affected by the chromosome 18 aberrations.An amplicon on chromosome 19 was detected in 20% of ABC DLBCLs but in only 3% of GCB DLBCLs and PMBLs.The gene most upregulated by this amplicon was SPIB, which encodes an ETS family transcription factor.Knockdown of SPIB by RNA interference was toxic to ABC DLBCL cell lines, but not to GCB DLBCL, PMBL or myeloma cell lines, strongly implicating SPIB as an oncogene involved in the pathogenesis of ABC DLBCL.Conclusions: In summary, aCGH revealed novel copy number abnormalities in DLBCL that had significant different frequencies in the three DLBCL subtypes.These data provide genetic evidence that the molecular DLBCL subtypes are distinct diseases that utilize different oncogenic pathways.

Background: The addition of Rituximab to CHOP chemotherapy (R-CHOP) has significantly improved the overall survival of patients with diffuse large B cell lymphoma (DLBCL) by 10–15%. To evaluate the biological basis of survival for DLBCL, we profiled gene expression in biopsy samples from patients treated with R-CHOP. Methods: Whole genome Affymetrix U133 2.0 plus arrays were used to profile gene expression in pre-treatment biopsies from patients with de novo DLBCL who received CHOP (n=181) or R-CHOP (n=233). Samples were classified as germinal center B cell-like (GCB), activated B cell-like (ABC) or unclassified DLBCL. A multivariate gene expression-based survival predictor was created using CHOP cases as training and R-CHOP cases as validation set. Results: R-CHOP treated patients with GCB DLBCL had a more favorable survival than those with ABC DLBCL, with 3-year overall survival rates of 84% and 56%, respectively (p Conclusion: Survival in DLBCL is critically influenced by the composition of the tumor microenvironment, suggesting that therapeutic interference with microenvironmental interactions might further improve outcome in DLBCL. Specifically, inhibition of angiogenesis may be beneficial for a subset of patients with high relative expression of the stromal-2 signature and increased tumor blood vessel density.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous diagnostic category with at least three different molecular subtypes distinguishable by gene expression profiling, termed germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal B cell lymphoma (PMBL). We performed array comparative genomic hybridization (aCGH) in patient samples and cell lines representing different DLBCL subtypes to determine if they utilize distinct pathogenetic mechanisms. Using an array consisting of 386, 165 oligonucleotides (NimbleGen), we performed aCGH on 203 untreated de novo DLBCL samples and 30 DLBCL cell lines, and the same samples were profiled for gene expression using Affymetrix U133 plus arrays. Patient samples included 72 GCB DLBCLs, 74 ABC DLBCLs, 31 PMBLs, and 26 unclassified DLBCLs. Following segmentation of the aCGH data into intervals with a uniform copy number, segments were combined into minimal common regions (MCRs) that were recurrently altered in more than one sample. Statistical differences in MCR frequency between DLBCL subtypes were corrected for multiple hypothesis testing using a false discovery rate (FDR) calculation. The DLBCL subtypes differed in the frequency of MCRs residing at many chromosomal loci, and we used gene expression data to define potential target genes in these MCRs. The INK4a/ARF tumor suppressor locus on 9p21 was selectively lost in ABC DLBCL: homozygous deletions of INK4a/ARF was observed in 20% of ABC DLBCLs but in only 3% of GCB DLBCLs and never in PMBLs (FDR=4.5 E-3). Among ABC DLBCLs, loss of INK4a/ARF was associated with increased proliferation rate, as measured by a proliferation gene expression signature, and adverse survival (p=0.007, log rank test). 16% of ABC DLBCL cases had gain/amplification and overexpression of SPIB, a gene on 19q13 encoding an ETS family transcription factor that is characteristically expressed in ABC DLBCL. This copy number alteration was observed much less frequently in GCB DLBCL (3%) and never in PMBL (FDR=2.6 E-2). GCB DLBCLs had recurrent amplification and overexpression of C13orf25, which encodes the mir-17-92 polycistronic cluster of microRNAs that is transcriptionally activated by c-myc and cooperates with c-myc to accelerate tumor development. C13orf25 amplification was detected in 16% of GCB DLBCLs but in only 3% of PMBLs and never in ABC DLBCL (FDR=3.8 E-3). Recurrent amplification and overexpression of JAK2 on 9p24 was observed in 35% of PMBL cases, but only in 5% of GCB DLBCLs and 4% of ABC DLBCLs respectively (FDR=6.2 E-4). In summary, aCGH revealed copy number abnormalities in DLBCL that had strikingly different frequencies in the three DLBCL subtypes, supporting the hypothesis that these subtypes represent distinct diseases that utilize different oncogenic mechanisms. Our analysis specifically implicated the INK4a/ARF locus as a tumor suppressor and SPIB as an oncogene in ABC DLBCL, the mir-17-92 microRNA cluster as an oncogene in GCB DLBCL, and JAK2 as an oncogene in PMBL.

Making the most of RNAi Two papers this week highlight the impact of RNAi (RNA interference) in clinical medicine. Ngo et al . have developed a novel ‘Achilles heel' screen to identify genes that, if silenced, cause cancer cells to stop dividing. The novelty lies in a successful ‘negative’ screen that can reveal potential therapeutic targets that do not necessarily contain mutations or other alterations. Use of the screen on 2,500 genes in B-cell lymphoma cells identified three genes that were essential for cancer cell survival and growth of one particular B-cell lymphoma subtype. In particular the protein CARD11 looks a prime target. Zimmermann et al . report a significant step towards harnessing RNAi as a new class of drug treatment. They used systemic administration of small interfering RNA (siRNA) to silence a disease-causing gene in a non-human primate: it had previously been demonstrated in mice. Specifically, siRNA targeted against the gene for apolipoprotein B (ApoB) in cynomolgus monkeys successfully reduced in ApoB protein, serum cholesterol and low-density lipoprotein levels. This has implications for diseases associated with high cholesterol levels, such as coronary heart disease, and more broadly demonstrates that potential therapies may be developed against historically ‘non-druggable’ targets.

Gene expression profiling has been used to distinguish two major subtypes of diffuse large B cell lymphoma (DLBCL), termed germinal center B cell-like (GCB) DLBCL and activated B cell-like (ABC) DLBCL. Following CHOP-like chemotherapy, GCB and ABC DLBCLs had distinct 5-year survival rates of ∼60% and ∼30%, respectively. Prognostic gene expression signatures in CHOP-treated DLBCL include the lymph node signature, which reflects a non-malignant host response, the MHC class II signature, both favorable when expressed and the proliferation signature which is adverse when expressed. The addition of rituximab to CHOP chemotherapy (R-CHOP) has significantly improved the outcome for DLBCL patients. We therefore investigated, if gene expression signatures that predicted survival among DLBCL patients treated with CHOP remained predictive for DLBCL patients treated with R-CHOP. Gene expression profiling was performed on 156 samples from previously untreated patients with DLBCL using Affymetrix U133 plus arrays. All patients received rituximab and CHOP-like chemotherapy. Samples were classified as GCB DLBCL, ABC DLBCL, or unclassified, and were assessed for expression of the lymph node and proliferation signatures. A Cox-proportional hazards model was used to determine the association of these gene expression features with overall survival (OS). 71 DLBCL samples were classified as GCB DLBCL, 63 as ABC DLBCL, and 22 were unclassified. The addition of rituximab improved OS for both GCB and ABC DLBCL compared to historical controls treated with CHOP-like chemotherapy alone. After a median follow-up of 2.3 years, GCB DLBCL had a more favorable OS than ABC DLBCL, with 3-year OS rates of 86% vs. 68% (p = 0.014). The 3-year OS rate of unclassified DLBCLs was 69%. The lymph node signature was associated with favorable OS (p = 0.023) and the proliferation signature with inferior OS (p = 0.009), whereas the MHC class II signature was not associated with OS (p = 0.44). In summary, addition of rituximab to CHOP-like chemotherapy improved OS for both GCB and ABC DLBCL but ABC DLBCL remained inferior to GCB DLBCL. The prognostic value of the lymph node and proliferation signatures were maintained in the context of R-CHOP therapy. An understanding of the biological attributes of DLBCL tumors that are reflected in these gene expression signatures remains critical to our ability to improve survival of these patients.

BACKGROUNDThe distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is crucial because these two types of lymphoma require different treatments. We examined whether gene-expression profiling could reliably distinguish Burkitt's lymphoma from diffuse large-B-cell lymphoma.METHODSTumor-biopsy specimens from 303 patients with aggressive lymphomas were profiled for gene expression and were also classified according to morphology, immunohistochemistry, and detection of the t(8;14) c-myc translocation.RESULTSA classifier based on gene expression correctly identified all 25 pathologically verified cases of classic Burkitt's lymphoma. Burkitt's lymphoma was readily distinguished from diffuse large-B-cell lymphoma by the high level of expression of c-myc target genes, the expression of a subgroup of germinal-center B-cell genes, and the low level of expression of major-histocompatibility-complex class I genes and nuclear factor-kappaB target genes. Eight specimens with a pathological diagnosis of diffuse large-B-cell lymphoma had the typical gene-expression profile of Burkitt's lymphoma, suggesting they represent cases of Burkitt's lymphoma that are difficult to diagnose by current methods. Among 28 of the patients with a molecular diagnosis of Burkitt's lymphoma, the overall survival was superior among those who had received intensive chemotherapy regimens instead of lower-dose regimens.CONCLUSIONSGene-expression profiling is an accurate, quantitative method for distinguishing Burkitt's lymphoma from diffuse large-B-cell lymphoma.

Genomics has provided a lever to pry open lymphoid cells and examine their regulatory biology. The large body of available gene expression data has also allowed us to define the of coordinately expressed genes, termed gene expression signatures, which characterize the states of cellular physiology that reflect cellular differentiation, activation of signaling pathways, and the action of transcription factors. Gene expression signatures that reflect the action of individual transcription factors can be defined by perturbing transcription factor function using RNA interference (RNAi), small-molecule inhibition, and dominant-negative approaches. We have used this methodology to define gene expression signatures of various transcription factors controlling B-cell differentiation and activation, including BCL-6, B lymphocyte-induced maturation protein-1 (Blimp-1), X-box binding protein-1 (XBP1), nuclear factor-kappa B (NF-kappa B), and c-myc. We have also curated a wide variety of gene expression signatures from the literature and assembled these into a signature database. Statistical methods can define whether any signature in this database is differentially expressed in independent biological samples, an approach we have used to gain mechanistic insights into the origin and clinical behavior of B-cell lymphomas. We also discuss the use of genomic-scale RNAi libraries to identify genes and pathways that may serve as therapeutic targets in B-cell malignancies.

Background Burkitt lymphoma(BL) is a potentially curable, aggressive lymphoma. The distinction between BL and diffuse large B-cell lymphoma (DLBCL) is important because they differ significantly in clinical management. The distinction can be difficult because DLBCL can resemble BL in morphology, immunophenotype and cytogenetics. We investigated whether gene expression profiling (GEP) could create a molecular definition of BL that can reliably distinguish it from DLBCL. Methods Biopsy samples were collected from 312 patients with a diagnosis of sporadic BL or Burkitt-like lymphoma, or DLBCL. All cases were reviewed by a panel of expert hematopathologists. GEP of all the samples was carried out using a specialized oligonucleotide microarray. We constructed a predictor using 197 genes to distinguish BL from each molecular subtype of DLBCL. Leave-one-out cross-validation was used to evaluate the predictor’s performance. Chemotherapy treatments were grouped into either CHOP-like(CHOP, CNOP) or intensive(BFM, CODOX-M IVAC, regimens requiring stem cell rescue). Results After pathology review, the samples were reclassified as:classic BL(25 cases), atypical BL(19), DLBCL(261), and unclassifiable lymphoma(7). All classic BL and 18/19 cases of atypical BL shared a profile that was strikingly different from that of all the molecular subtypes of DLBCL, including those DLBCL cases that have a c-myc translocation. C-myc and its target genes, and genes related to germinal center differentiation were expressed at high levels in BL. NF-kB and its target genes and MHC class-I genes were expressed at very low levels in BL. Interestingly, 10 cases that were DLBCL by pathology were classified as BL by the predictor. The diagnosis of BL was supported by FISH analysis indicating a c-myc translocation. Among adults identified as having BL by the predictor (with full clinical data in N=15), overall survival was markedly superior for those receiving intensive regimens compared to CHOP-like regimens(Fig 1). The groups were similar with regard to age, stage, performance status and sites of involvement. Conclusion This study demonstrates that the molecular characteristics of BL can be used to accurately distinguish it from DLBCL. Importantly, a subgroup of BL was identified by the predictor that could not be diagnosed as BL by conventional criteria. The ability of the predictor to identify patients who benefit from aggressive therapies suggests that it will be useful in the diagnosis and management of patients with Burkitt lymphoma. ![Figure][1] Figure [1]: pending:yes