Search

Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

Follicular lymphoma (FL) is typically an indolent disease, but 30–40% of FL cases transform into an aggressive lymphoma (tFL) with a poor prognosis. To identify the genetic changes that drive this transformation, we sequenced the exomes of 12 cases with paired FL and tFL biopsies and identified 45 recurrently mutated genes in the FL–tFL data set and 39 in the tFL cases. We selected 496 genes of potential importance in transformation and sequenced them in 23 additional tFL cases. Integration of the mutation data with copy-number abnormality (CNA) data provided complementary information. We found recurrent mutations of miR-142, which has not been previously been reported to be mutated in FL/tFL. The genes most frequently mutated in tFL included KMT2D(MLL2), CREBBP, EZH2, BCL2and MEF2B.Many recurrently mutated genes are involved in epigenetic regulation, the Janus-activated kinase–signal transducer and activator of transcription (STAT) or the nuclear factor-κB pathways, immune surveillance and cell cycle regulation or are TFs involved in B-cell development. Of particular interest are mutations and CNAs affecting S1P-activated pathways through S1PR1 or S1PR2, which likely regulate lymphoma cell migration and survival outside of follicles. Our custom gene enrichment panel provides high depth of coverage for the study of clonal evolution or divergence.

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of mature T-cell neoplasms with a poor prognosis. Recently, mutations in TET2 and other epigenetic modifiers as well as RHOA have been identified in these diseases, particularly in angioimmunoblastic T-cell lymphoma (AITL). CD28 is the major co-stimulatory receptor in T cells which, upon binding ligand, induces sustained T-cell proliferation and cytokine production when combined with T-cell receptor stimulation. We have identified recurrent mutations in CD28 in PTCLs. Two residues—D124 and T195—were recurrently mutated in 11.3% of cases of AITL and in one case of PTCL, not otherwise specified (PTCL-NOS). Surface plasmon resonance analysis of mutations at these residues with predicted differential partner interactions showed increased affinity for ligand CD86 (residue D124) and increased affinity for intracellular adaptor proteins GRB2 and GADS/GRAP2 (residue T195). Molecular modeling studies on each of these mutations suggested how these mutants result in increased affinities. We found increased transcription of the CD28-responsive genes CD226and TNFAin cells expressing the T195P mutant in response to CD3 and CD86 co-stimulation and increased downstream activation of NF-?B by both D124V and T195P mutants, suggesting a potential therapeutic target in CD28-mutated PTCLs.

Autoimmune diseases such as multiple sclerosis are characterized by complex genetic traits and pathomechanisms that translate into clinical heterogeneity. This wide heterogeneity of multiple sclerosis as well as different biological responses to immunomodulatory drugs can be expected to contribute to differential treatment responses. Strategies that dissect the relationship between the treatment response and the biological characteristics in individual patients are valuable not only as a clinical tool, but also in leading to a better understanding of the disease. Here we address the in vitro and ex vivo RNA expression profile under one approved therapy of multiple sclerosis, interferon-beta (IFN-beta, Betaseron), by cDNA microarrays and demonstrate that non-responder and responder phenotypes to IFN-beta as assessed by longitudinal gadolinium-enhanced MRI scans and clinical disease activity differ in their ex vivo gene expression profile. These findings will help to better elucidate the mechanism of action of IFN-beta in relation to different disease patterns and eventually lead to optimized therapy.

The B-cell-type specificity of the immunoglobulin (Ig) heavy-chain and light-chain promoters is mediated by an octanucleotide (OCTA) element, ATGCAAAT, that is also a functional component of other RNA polymerase II promoters, such as snRNA and histone H2B promoters. Two nuclear proteins that bind specifically and with high affinity to the OCTA element have been identified. NF-A1 is present in a variety of cell types, whereas the presence of NF-A2 is essentially confined to B cells, leading to the hypothesis that NF-A2 activates cell-type-specific transcription of the Ig promoter and NF-A1 mediates the other responses of the OCTA element. Extracts of the B-cell line, BJA-B, contain high levels of NF-A2 and specifically transcribe Ig promoters. In contrast, extracts from HeLa cells transcribed the Ig promoter poorly. Surprisingly, addition of either affinity-enriched NF-A2 or NF-A1 to either a HeLa extract or a partially purified reaction system specifically stimulates the Ig promoter. This suggests that the constitutive OCTA-binding factor NF-A1 can activate transcription of the Ig promoter and that B-cell-specific transcription of this promoter, at least in vitro, is partially due to a quantitative difference in the amount of OCTA-binding protein. Because NF-A1 can stimulate Ig transcription, the inability of this factor to activate in vivo the Ig promoter to the same degree as the snRNA promoters probably reflects a difference in the context of the OCTA element in these two types of promoters.

The Ras-related small GTP-binding proteins are involved in diverse cellular events, including cell signaling, proliferation, cytoskeletal organization, and secretion. The interconversion of the active, GTP-bound form of the protein to the inactive, GDP-bound form is influenced by two types of regulatory proteins, those that alter the intrinsic GTPase activity of the GTP-binding protein and those that affect the rate of GDP/GTP exchange. By utilizing a subtractive hybridization approach, we have isolated a human gene encoding Ly-GDI, a protein that has striking homology to the product of a previously cloned gene, Rho-GDI, which inhibits GDP/GTP exchange on the Rho family of GTPases. In contrast to Rho-GDI, which is ubiquitously expressed, Ly-GDI is expressed only in hematopoietic tissues and predominantly in B- and T-lymphocyte cell lines. The full-length Ly-GDI cDNA encodes a 27-kDa protein which binds to RhoA and inhibits GDP dissociation from RhoA. Stimulation of T lymphocytes with phorbol ester leads to phosphorylation of Ly-GDI, suggesting an involvement of Ly-GDI in lymphocyte activation pathways. Cell type-specific regulators of the Ras-like GTP-binding proteins may provide one mechanism by which different cell types respond uniquely to signals transduced through the same cell surface receptor or may provide a way by which the GTP-binding proteins can be uniquely engaged by tissue-restricted receptors.

We have examined the amino-terminal sequence of the kappa light chains of a set of monoclonal antibodies specific for one of the major antigenic determinants (Sb) on the influenza virus PR8[A/PR/8/34(H1N1)] hemagglutinin molecule. This set was believed to be structurally related from earlier serological analysis that typed these kappa chains as members of the variable (V) region V kappa 21 group [ Staudt , L. M. & Gerhard , W. (1983) J. Exp. Med. 157, 678-704]. Our sequence analysis confirms and extends this conclusion; all examples of this set belong to a subgroup of the V kappa 21 group, V kappa 21C . A special feature of this set of kappa light chains is that all examples were derived from the same mouse (designated H36 ). This sequence analysis along with the characterization of gene rearrangements at the kappa light chain loci of these hybridomas is consistent with the idea that certain members of this set are the progeny of one or two lymphocytes. Because of this potential clonal relationship, we can reach several conclusions about the diversity observed among these kappa light chains: (i) the diversity is due to somatic mutation, (ii) somatic mutations occur sequentially and accumulate in the first complementarity-determining region, and (iii) the extent of somatic variation in this sample is high, suggesting a somatic mutation rate of about 10(-3) per base pair per generation.

The DNA sequence motif ATTTGCAT (octamer) or its inverse complement has been identified as an evolutionarily conserved element in the promoter region of immunoglobulin genes. Two major DNA-binding proteins that bind in a sequence-specific manner to the octamer DNA sequence have been identified in mammalian species--a ubiquitously expressed protein (Oct-1) and a lymphoid-specific protein (Oct-2). During characterization of the promoter region of the chicken immunoglobulin light chain gene, we identified two homologous octamer-binding proteins in chicken B cells. When the cloning of the human gene for Oct-2 revealed it to be a member of a distinct family of homeobox genes, we sought to determine if the human Oct-2 cDNA could be used to identify homologous chicken homeobox genes. Using a human Oct-2 homeobox-specific DNA probe, we were able to identify 6-10 homeobox-containing genes in the chicken genome, demonstrating that the Oct-2-related subfamily of homeobox genes exists in avian species. Low-stringency screening of a chicken embryonic cDNA library allowed us to clone one of these genes. DNA sequence analysis revealed it to be the chicken homologue of the human Oct-1 gene. The predicted protein sequence of the chicken Oct-1 gene demonstrated that the gene for Oct-1 has been highly conserved during vertebrate evolution with an overall 96% amino acid sequence identity between the chicken and human proteins. The previously described POU domain (termed POU for its presence in the Pit-1, Oct-1/Oct-2, and Unc-86 genes) and homeobox domain are 100% conserved between the two protein products. Together, our data show that the POU-containing subfamily of homeobox genes have been highly conserved during vertebrate evolution, apparently as a result of selection for their DNA-binding and transcriptional regulatory properties.

A clone obtained from a differential display screen for cellular genes with altered expression during human immunodeficiency virus (HIV) infection matched the sequence for the human GLUT3 facilitative glucose transporter, a high-velocity-high-affinity facilitative transporter commonly expressed in neurons of the central nervous system. Northern (RNA) analysis showed that GLUT3 expression increased during infection. Flow cytometry showed that GLUT3 protein expression increased specifically in the HIV-infected cells; this increase correlated with increased 2-deoxyglucose transport in the HIV-infected culture. HIV infection therefore leads to increased expression of a glucose transporter normally expressed at high levels in other cell types and a corresponding increase in glucose transport activity. If HIV infection places increased metabolic demands on the host cell, changes in the expression of a cellular gene that plays an important role in cellular metabolism might provide a more favorable environment for viral replication.

This study focuses on 10 BALB/c anti-influenza virus (A/PR/8/34) hemagglutinin antibodies that have light chains encoded by the same variable region kappa chain (V kappa) gene, V kappa 21C. A comparison of antibodies from lymphocytes of independent origin reveals the contribution of germline diversity (combinatorial joining and association) to this response. Although combinatorial joining and association contribute to sequence diversity, they appear to have little effect on the fine specificity of these antibodies. Somatic mutation, in addition to contributing to the sequence diversity of these antibodies, creates differences in their fine specificity. The extent of mutation and its effect on fine specificity can be seen by comparing antibodies of lymphocytes from the same clone. These intraclonal comparisons also indicate that somatic mutation is an ongoing process occurring at a high rate (estimated to be at least 10(-3) mutations per base pair per division) in the expressed V region heavy chain (VH) and V kappa genes. Furthermore, both the nature and distribution of these mutations suggest that amino acid replacement mutations in the light but not the heavy chain are selected for by antigen.

Oct-2 is a transcription factor that binds specifically to octamer DNA motifs in the promoters of immunoglobulin and interleukin-2 genes. All tumor cell lines from the B-cell lineage and a few from the T-cell lineage express Oct-2. To address the role of Oct-2 in the T-cell lineage, we studied the expression of Oct-2 mRNA and protein in nontransformed human and mouse T cells. Oct-2 was found in CD4+ and CD8+ T cells prepared from human peripheral blood and in mouse lymph node T cells. In a T-cell clone specific for pigeon cytochrome c in the context of I-Ek, Oct-2 was induced by antigen stimulation, with the increase in Oct-2 protein seen first at 3 h after activation and continuing for at least 24 h. Oct-2 mRNA induction during antigen-driven T-cell activation was blocked by cyclosporin A, as well as by protein synthesis inhibitors. These results suggest that Oct-2 participates in transcriptional regulation during T-cell activation. The relatively delayed kinetics of Oct-2 induction suggests that Oct-2 mediates the changes in gene expression which occur many hours or days following antigen stimulation of T lymphocytes.

A negative element has previously been localized to a 57-base pair segment approximately 300 base pairs upstream of the human c-mycpromoter P1. Within this element, a 26-base pair region was protected in vitrofrom DNase I digestion with a HeLa cell nuclear factor(s). Two specific DNA-protein complexes were identified in gel retardation assays using HeLa cell nuclear extracts and an oligonucleotide probe spanning the footprinted region. Exonuclease and chemical footprint analyses suggested that the binding sites for both complexes are almost entirely overlapping. One of the complexes was eliminated by oligonucleotide competitors possessing known AP-1 binding sites. This same complex reacted strongly with anti-fosimmunoglobulin suggesting a role for c-fosin governing c-mycexpression. Precipitation of fosprotein bound to c-mycDNA that was immobilized on beads confirmed the involvement of c-fosin a specific complex with the c-mycupstream sequence. In contrast, the other complex seen by the c-mycprobe could not be competitively inhibited by AP-1 binding sites and was not affected by anti-fosantibody. Instead, this complex was efficiently eliminated by unlabeled oligonucleotides containing the octamer DNA motif found in immunoglobulin gene promoters. Purified octamer-binding proteins formed stable complexes with the 26-base pair c-mycsequences. These results demonstrate that degeneracy in the consensus recognition sequences of these distinct factors allows each of them to bind the c-mycnegative element. The interaction of known transcriptional activators with a negative element suggests that the same factors can mediate both transcriptional activation and repression.

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 Figure

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B cell-like (ABC), and primary mediastinal (PM) DLBCL. The BCL6 gene on chromosome 3q27 is a transcriptional repressor and is required for GC formation and function. Two molecular alterations involving the BCL6 gene are commonly observed in DLBCL: chromosomal translocations and mutations in the 5′ non-coding region. The functional consequences of BCL6 translocation and mutation have not been studied in the context of DLBCL subgroups. Therefore, we examined the frequency of translocations and the spectrum of BCL6 mutations in exon 1 and intron 1 in different DLBCL subgroups. We correlated these findings with BCL6 mRNA and protein expression, as well as the expression of BCL6 target genes. Fluorescence in situ hybridization (FISH) using a break-apart probe detected BCL6 translocations in 24 of 112 (21%) DLBCL cases. Surprisingly, the frequency of BCL6 translocation was higher in the ABC subgroup than the GCB subgroup (10 of 40; 25% vs 5 of 44; 11%, respectively) and the PM subgroup had the highest incidence (4 of 8; 50%). Expression of BCL6 protein was detected by immunohistochemistry in 75 of 138 cases (54%), including 15 of 44 (34%) in the ABC subgroup, 46 of 57 (80%) in the GCB subgroup and 4 of 10 (40%) in the PM subgroup. A good correlation of protein and mRNA expression, as assessed by cDNA microarrays (NEJM346:1937–47; 2002) was observed in the GCB subgroup but not in the other subgroups. BCL6 mutations were detected in 71 of 128 cases (55%) of DLBCL with a higher frequency in the GCB subgroup (34 of 50; 68%) and PM subgroup (10 of 12; 90%) than the ABC subgroup (14 of 43; 32%). Interestingly, there was a distinctive pattern of distribution of BCL6 mutations in the DLBCL subgroups. For example, 11 of 100 (11%) of the mutations targeted exon 1 and 8 of these 11 mutants were observed in the GCB subgroup. They preferentially affected the BCL6, STAT1 or predicted CEBP binding sites. High BCL6 mRNA and protein expression was generally associated with mutants affecting the 3′ BCL6 binding sites. We also examined the expression of 25 known BCL6 target genes in the different subgroups of DLBCL and observed the repression of this set of genes mainly in the GCB but not in the ABC subgroup, and the presence of a translocation did not correlate with target gene suppression. In conclusion, the frequency of BCL6 translocation and mutation is distinctly different in the DLBCL subgroups and BCL6 expression has different regulatory influences on target genes in different subgroups of DLBCL. Exon 1 mutations preferentially occur in the GCB subgroup and affect transcription factor binding sites. However, BCL6 translocations did not show good correlation with BCL6 expression or functional repression of target genes. The influence of the reciprocal partner genes in the translocations on the pathogenesis of DLBCL warrants further investigation.

Clinical management differs significantly for the various types of non-Hodgkin lymphoma (NHL), and the diagnosis of these lymphomas can be challenging in some cases. Further, existing NHL categories include subgroups that can differ substantially in gene expression, response to therapy and overall survival. We have created a custom oligonucleotide microarray, named LymphDx, which could prove clinically useful for molecular diagnosis and outcome prediction in NHL. Biopsy specimens were obtained from 559 patients with a variety of lymphomas and lymphoproliferative conditions. Gene expression profiles of these samples were obtained using Affymetrix U133 A and B microarrays. The 2653 genes on LymphDx were chosen to include:(1)Genes most differentially expressed among NHL types based on Affymetrix U133 or Lymphochip microarrays (2)Genes predicting length of survival in diffuse large B cell lymphoma(DLBCL), follicular lymphoma(FL) and mantle cell lymphoma(MCL) (3)Genes encoded in the EBV and HHV-8 viral genomes (4)Genes encoding all known surface markers, kinases, cytokines and their receptors, as well as oncogenes, tumor suppressors, and other genes relevant to lymphoma. The LymphDx microarray was used to profile gene expression in 434 biopsy samples. These data were used to create a diagnostic algorithm that can distinguish various NHL types and benign follicular hyperplasia(FH) based on gene expression. The algorithm classifies a sample into one of the following categories: Burkitt’s lymphoma(BL), DLBCL, FL, MCL, small lymphocytic lymphoma(SLL) or FH. The algorithm further distinguishes the 3 recognized DLBCL subgroups: germinal center B cell-like, activated B cell-like or primary mediastinal lymphoma. Using a leave one out, cross validation strategy, the algorithm was found to agree well with the pathology diagnosis (see Figure). Some samples were deemed unclassified when their gene expression did not adequately match with that of any of the NHL categories. For a few samples, the gene expression-based diagnosis and the pathology diagnosis were discordant. Pathology review showed that two NHL types coexisted (eg FL and DLBCL) in many of these cases, potentially explaining the results of the diagnostic algorithm. LymphDx could also reliably predict the overall survival of patients with DLBCL, FL and MCL. Prospective evaluation of the LymphDx microarray is warranted since it could be used to provide objective molecular diagnostic, and prognostic information for patients with NHL. Figure Figure