Targeting of B-cell receptor signalling in B-cell malignancies.

Pharmacological agents that inhibit enzymes of the B-cell receptor (BCR) pathway are of increasing importance in the treatment of B-cell malignancies. These include inhibitors of Bruton tyrosine kinase (BTK), phosphatidylinositol 3-kinase (PI3K), splenic tyrosine kinase and protein kinase Cß. Two agents are already approved in the USA and Europe: ibrutinib, a BTK inhibitor, for the treatment of chronic lymphatic leukaemia (CLL), mantle cell lymphoma (MCL) and Waldenström's macroglobulinemia; and idelalisib, a PI3Kδ inhibitor, for the treatment of CLL and follicular lymphoma. In addition, the role of these drugs in diffuse large B-cell lymphoma and marginal zone lymphoma is under investigation, as single agents and in combination with chemotherapy. In CLL, both ibrutinib and idelalisib have an established role as first-line therapy in patients with del(17p), and in MCL, ibrutinib is a standard option for patients relapsing after chemoimmunotherapy. Unexpected toxicities have been encountered when combining these potent new agents with other drugs, including chemotherapy and lenalidomide, and based on this experience the risks and benefits of novel combinations must be evaluated carefully. In this review, we summarize the efficacy and safety results with these inhibitors and discuss novel combinations that are under study and the future role of BCR inhibitors in these disorders.

Improved Myelination following Camp Leg Power, a Selective Motor Control Intervention for Children with Spastic Bilateral Cerebral Palsy: A Diffusion Tensor MRI Study.

BACKGROUND AND PURPOSE: Children with spastic cerebral palsy have motor deficits associated with periventricular leukomalacia indicating WM damage to the corticospinal tracts. We investigated whether practice of skilled lower extremity selective motor control movements would elicit neuroplasticity. MATERIALS AND METHODS: Twelve children with spastic bilateral cerebral palsy and periventricular leukomalacia born preterm (mean age, 11.5 years; age range, 7.3-16.6 years) participated in a lower extremity selective motor control intervention, Camp Leg Power. Activities promoted isolated joint movement including isokinetic knee exercises, ankle-controlled gaming, gait training, and sensorimotor activities (3 hours/day, 15 sessions, 1 month). DWI scans were collected pre- and postintervention. Tract-Based Spatial Statistics was used to analyze changes in fractional anisotropy, radial diffusivity, axial diffusivity, and mean diffusivity. RESULTS: Significantly reduced radial diffusivity (P < . 05) was found within corticospinal tract ROIs, including 28.4% of the left and 3.6% of the right posterior limb of the internal capsule and 14.1% of the left superior corona radiata. Reduced mean diffusivity was found within the same ROIs (13.3%, 11.6%, and 6.6%, respectively). Additionally, decreased radial diffusivity was observed in the left primary motor cortex. Additional WM tracts had decreased radial diffusivity and mean diffusivity, including the anterior limb of the internal capsule, external capsule, anterior corona radiata, and corpus callosum body and genu. CONCLUSIONS: Myelination of the corticospinal tracts improved following Camp Leg Power. Neighboring WM changes suggest recruitment of additional tracts involved in regulating neuroplasticity of the motor regions. Intensive practice of skilled lower extremity selective motor control movements promotes neuroplasticity in children with spastic bilateral cerebral palsy.

Stromal gene signatures in large-B-cell lymphomas.

BACKGROUND: The addition of rituximab to combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), or R-CHOP, has significantly improved the survival of patients with diffuse large-B-cell lymphoma. Whether gene-expression signatures correlate with survival after treatment of diffuse large-B-cell lymphoma is unclear. METHODS: We profiled gene expression in pretreatment biopsy specimens from 181 patients with diffuse large-B-cell lymphoma who received CHOP and 233 patients with this disease who received R-CHOP. A multivariate gene-expression-based survival-predictor model derived from a training group was tested in a validation group. RESULTS: A multivariate model created from three gene-expression signatures--termed "germinal-center B-cell," "stromal-1," and "stromal-2"--predicted survival both in patients who received CHOP and patients who received R-CHOP. The prognostically favorable stromal-1 signature reflected extracellular-matrix deposition and histiocytic infiltration. By contrast, the prognostically unfavorable stromal-2 signature reflected tumor blood-vessel density. CONCLUSIONS: Survival after treatment of diffuse large-B-cell lymphoma is influenced by differences in immune cells, fibrosis, and angiogenesis in the tumor microenvironment.

Selective Motor Control is a Clinical Correlate of Brain Motor Tract Impairment in Children with Spastic Bilateral Cerebral Palsy.

BACKGROUND AND PURPOSE: Selective voluntary motor control is an important factor influencing gross motor function, interjoint coordination, and the outcome of hamstring-lengthening surgery in spastic cerebral palsy. Using DTI, we investigated whether selective voluntary motor control would show strong correlations with WM motor tract microstructure and whether selective voluntary motor control is more sensitive to global WM impairment than gross motor function. MATERIALS AND METHODS: Children with spastic bilateral cerebral palsy born preterm and typically developing children were recruited. The Selective Control Assessment of the Lower Extremity (SCALE) and Gross Motor Function Measure (GMFM) were assessed in participants with cerebral palsy. Participants underwent brain MR imaging to collect DWI data. Tract-Based Spatial Statistics was used to analyze the WM for between-group differences and correlations with SCALE and GMFM. ROI analyses compared motor regions. RESULTS: Twelve children with cerebral palsy (mean age, 11.5 years) and 12 typically developing children (mean age, 10.3 years) participated. Altered DTI outcomes were found throughout the whole brain for the cerebral palsy group. SCALE, developed to evaluate selective voluntary motor control in cerebral palsy, showed significant positive correlations with fractional anisotropy in more WM voxels throughout the whole brain and for motor regions, including the corticospinal tract and corpus callosum, compared with GMFM. A significant negative correlation between radial diffusivity and SCALE, but not GMFM, was found within the corpus callosum. CONCLUSIONS: SCALE was a more sensitive clinical correlate of motor and whole-brain WM tract impairment in children with spastic bilateral cerebral palsy, suggesting greater anisotropy and myelination in these regions for those with higher selective voluntary motor control.

Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. I. Significant variation in repertoire expression between individual mice.

The paratypic and idiotypic diversity of the BALB/c antibody response to the hemagglutinin (HA) of the influenza A/PR/8/34 virus (PR8) was investigated using a panel of 125 anti-HA hybridoma antibodies derived from 14 BALB/c mice. The paratypic diversity, as assessed by a fine specificity analysis using 51 related influenza viruses, was extensive: 104 distinct paratopes were observed. In three instances, antibodies with indistinguishable paratopes were isolated from two individual mice. A minimum estimate of the size of the adult BALB/c anti-HA paratypic repertoire, calculated from these data, is 1,500. The generation of this diverse repertoire was studied by screening the anti-HA hybridoma panel for the presence of idiotypes (Id) that are markers for variable (V) region sequences derived from related germ line V genes. Three cross-reactive Id (IdX) that are markers for the V(k)21C, V(k)21B, and V(k)21A, D, E, or F L chain subgroups were found, respectively on 16, 1, and 10 anti-HA hybridoma antibodies derived from seven individual BALB/c mice. Thus, the V(k)21 IdX(+) hybridomas constitute 22 percent of the anti-HA hybridoma panel. The V(k)21 IdX are also present on 8.6 percent of K-bearing immunoglobulin in normal BALB/c serum. This suggests that the V(k)21 group is used preferentially in the BALB/c anti-HA immune response. The generation of the anti-HA repertoire was further studied using large panels of anti-HA hybridomas derived from two individual adult BALB/c mice. Anti-idiotypic antisera were raised in rabbits against individual hybridomas from each mouse. One anti-Id serum defined a family of four idiotypically and paratypically related, but not identical, antibodies from mouse 36, which represented 31 percent of the hybridoma antibodies isolated from this mouse. None of the 112 anti-HA hybridoma antibodies derived from 13 other individual mice showed idiotypic cross-reactivity. Furthermore, this Id could not be detected in anti-PR8 antisera from 75 individual BALB/c mice. Another anti-Id serum defined a family of 27 idiotypically related antibodies from mouse 37, which represented 50 percent of the hybridoma antibodies isolated from this mouse. Only 1 of the 71 hybridoma antibodies isolated from 13 other individuals was idiotypically cross-reactive. These results demonstrate that individual adult BALB/c mice express paratypically and idiotypically distinct antibody repertoires to the HA of influenza virus PR8. Based on these observations, we suggest that somatic mutation plays an important role in the generation of the adult anti-HA repertoire. Mechanisms that could account for differences in repertoire expression among individual mice are discussed.

Constitutive nuclear factor kappaB activity is required for survival of activated B cell-like diffuse large B cell lymphoma cells.

Gene expression profiling has revealed that diffuse large B cell lymphoma (DLBCL) consists of at least two distinct diseases. Patients with one DLBCL subtype, termed activated B cell-like (ABC) DLBCL, have a distinctly inferior prognosis. An untapped potential of gene expression profiling is its ability to identify pathogenic signaling pathways in cancer that are amenable to therapeutic attack. The gene expression profiles of ABC DLBCLs were notable for the high expression of target genes of the nuclear factor (NF)-kappaB transcription factors, raising the possibility that constitutive activity of the NF-kappaB pathway may contribute to the poor prognosis of these patients. Two cell line models of ABC DLBCL had high nuclear NF-kappaB DNA binding activity, constitutive IkappaB kinase (IKK) activity, and rapid IkappaB(alpha) degradation that was not seen in cell lines representing the other DLBCL subtype, germinal center B-like (GCB) DLBCL. Retroviral transduction of a super-repressor form of IkappaBalpha or dominant negative forms of IKKbeta was toxic to ABC DLBCL cells but not GCB DLBCL cells. DNA content analysis showed that NF-kappaB inhibition caused both cell death and G1-phase growth arrest. These findings establish the NF-kappaB pathway as a new molecular target for drug development in the most clinically intractable subtype of DLBCL and demonstrate that the two DLBCL subtypes defined by gene expression profiling utilize distinct pathogenetic mechanisms.

Inter- and intraclonal diversity in the antibody response to influenza hemagglutinin.

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.

Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.

The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression "signature," irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

Repression of the IgH enhancer in teratocarcinoma cells associated with a novel octamer factor.

Embryonal carcinoma (EC) cell lines are models for early cells in mouse embryogenesis. A 300-base pair fragment of the heavy chain enhancer was inactive in F9 EC cells, unlike in other nonlymphoid cells where it has significant activity. Alterations of the octamer motif increased enhancer activity. Nuclear extracts from F9 cells contained an octamer binding protein (NF-A3) that was unique to EC cells; the amount of NF-A3 decreased upon differentiation. It is proposed that NF-A3 represses specific regulatory sequences that contain the octamer motif. Thus, the same DNA sequence mediates either negative or positive transcriptional effects, depending on the cell type.

Oct-3 and mammalian development: correction of discussion.

The title of the 5 June report on page 1445 by R. C. deL. Milton et al. should have been "Total chemical synthesis of a D-enzyme: The enantiomers of HIV-1 protease show reciprocal chiral substrate specificity." Figure 3 in the same report (p. 1447) was inadvertently printed upside down. The labels "L-HIV protease" and "D-HIV protease" were therefore under the wrong illustrations. The correct figure is printed below. [See figure in the PDF file]

Control of inflammation, cytokine expression, and germinal center formation by BCL-6.

The gene encoding the BCL-6 transcriptional repressor is frequently translocated and mutated in diffuse large cell lymphoma. Mice with a disrupted BCL-6 gene developed myocarditis and pulmonary vasculitis, had no germinal centers, and had increased expression of T helper cell type 2 cytokines. The BCL-6 DNA recognition motif resembled sites bound by the STAT (signal transducers and activators of transcription) transcription factors, which mediate cytokine signaling. BCL-6 could repress interleukin-4 (IL-4)-induced transcription when bound to a site recognized by the IL-4-responsive transcription factor Stat6. Thus, dysregulation of STAT-responsive genes may underlie the inflammatory disease in BCL-6-deficient mice and participate in lymphoid malignancies.

Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif.

An octamer DNA sequence plays a critical role in directing transcription of immunoglobulin genes in B lymphocytes. A new technique of direct binding of radioactive DNA was used to screen a complementary DNA expression library from the BJAB cell line in lambda gt11 phage to derive molecular cDNA clones representing a putative B lymphocyte-specific octamer binding protein. The plaques were screened with DNA containing four copies of the octamer sequence and positive phage recombinants were identified. The fusion protein produced on inducing a lysogen of one phage bound to a monomeric octamer probe. The cDNA insert from this phage hybridized to messenger RNA found in B lymphocytes, but not in most other cells. Thus, this cDNA derives from a gene (oct-2) that specifies an octamer binding protein expressed preferentially in B lymphocytes, proving that, for at least one gene, a cell-specific transcription factor exists and its amount is controlled through messenger RNA availability.

The transcriptional program in the response of human fibroblasts to serum.

The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

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 is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

Integrating genomic alterations in diffuse large B-cell lymphoma identifies new relevant pathways and potential therapeutic targets.

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.

Genomic-scale gene expression profiling of normal and malignant immune cells.

Gene expression variation is critical for the normal development and physiology of immune cells. Using cDNA microarrays, a systematic, genomic-scale view of gene expression in immune cells at many stages of differentiation and activation can be obtained. From the high vantagepoint provided by this technology, the gene expression physiology of immune cells appears remarkably ordered and logical. Each stage of lymphocyte differentiation can be defined by a characteristic gene expression signature. Genes that are co-regulated over hundreds of experimental conditions often encode functionally related proteins. Gene expression profiles also provide unprecedented ability to define the molecular and functional relationships between normal and malignant lymphocyte cell populations.

Induction of the POU domain transcription factor Oct-2 during T-cell activation by cognate 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.

Combined copy number and mutation analysis identifies oncogenic pathways associated with transformation of follicular lymphoma.

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, BCL2 and 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.

Recurrent activating mutations of CD28 in peripheral T-cell lymphomas.

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 CD226 and TNFA in cells expressing the T195P mutant in response to CD3 and CD86 co-stimulation and increased downstream activation of NF-κB by both D124V and T195P mutants, suggesting a potential therapeutic target in CD28-mutated PTCLs.

Transcriptional repression by the proto-oncogene BCL-6.

In up to 45% of reported cases of the non-Hodgkin's lymphoma, diffuse large cell lymphoma, there are translocations of the BCL-6 gene, which are presumed to deregulate its expression. The BCL-6 protein, which is unmutated in these lymphomas, contains six Krüppel-like zinc fingers at its carboxy terminus and a 121 amino acid domain at its amino terminus, termed the POZ domain, which bears homology with amino terminal domains in a subset zinc finger transcription factors. In this study, we tested whether BCL-6 regulates transcription and if the POZ domain has a role in this function. The BCL-6 POZ domain, when fused to the GAL4 DNA binding domain, strongly repressed transcriptional activation initiated from several different promoters including the SV40 enhancer/promoter. Repression was also observed when the fusion protein was bound at a distance of 200 bp 5' of the promoter. When the GAL4/BCL6 POZ domain fusion protein was expressed in yeast, it was able to homodimerize in the nucleus. Nevertheless, in contrast with mammalian cells, the fusion protein did not repress transcription. To test the ability of the full length BC1-6 protein to repress transcription when bound to DNA through its zinc finger DNA binding domain, high affinity BCL-6 binding sites were selected from a pool of random oligonucleotides. Full length BCL-6 was able to strongly repress transcription when bound to its cognate site cloned upstream of the thymidine kinase promoter. This repression was mediated, in large measure, by the POZ domain, although a variant of BCL-6 lacking the POZ domain was able to repress transcription modestly. The ability of BCL-6 to function as a transcriptional repressor may contribute to its ability to transform B lymphocytes in diffuse large cell lymphoma.