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.

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.

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.

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.

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.

Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth.

To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.

The miRNA-17∼92 cluster mediates chemoresistance and enhances tumor growth in mantle cell lymphoma via PI3K/AKT pathway activation.

The median survival of patients with mantle cell lymphoma (MCL) ranges from 3 to 5 years with current chemotherapeutic regimens. A common secondary genomic alteration detected in MCL is chromosome 13q31-q32 gain/amplification, which targets a microRNA (miRNA) cluster, miR-17∼92. On the basis of gene expression profiling, we found that high level expression of C13orf25, the primary transcript from which these miRNAs are processed, was associated with poorer survival in patients with MCL (P=0.021). We demonstrated that the protein phosphatase PHLPP2, an important negative regulator of the PI3K/AKT pathway, was a direct target of miR-17∼92 miRNAs, in addition to PTEN and BIM. These proteins were down-modulated in MCL cells with overexpression of the miR-17∼92 cluster. Overexpression of miR-17∼92 activated the PI3K/AKT pathway and inhibited chemotherapy-induced apoptosis in MCL cell lines. Conversely, inhibition of miR-17∼92 expression suppressed the PI3K/AKT pathway and inhibited tumor growth in a xenograft MCL mouse model. Targeting the miR-17∼92 cluster may therefore provide a novel therapeutic approach for patients with MCL.

Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells.

Activating mutations in the KRAS gene are among the most prevalent genetic changes in human cancers. To identify synthetic lethal interactions in cancer cells harbouring mutant KRAS, we performed a large-scale screen in isogenic paired colon cancer cell lines that differ by a single allele of mutant KRAS using an inducible short hairpin RNA interference library. Snail2, a zinc finger transcriptional repressor encoded by the SNAI2 gene, was found to be selectively required for the long-term survival of cancer cells with mutant KRAS that have undergone epithelial-mesenchymal transition (EMT), a transdifferentiation event that is frequently seen in advanced tumours and is promoted by RAS activation. Snail2 expression is regulated by the RAS pathway and is required for EMT. Our findings support Snail2 as a possible target for the treatment of the broad spectrum of human cancers of epithelial origin with mutant RAS that have undergone EMT and are characterized by a high degree of chemoresistance and radioresistance.

Array-based DNA methylation profiling in follicular lymphoma.

Quantitative methylation profiling was performed using the Illumina GoldenGate Assay in untreated follicular lymphoma (FL) (164), paired pre- and post-transformation FL (20), benign haematopoietic (24) samples and purified B and T cells from two FL cases. Methylation values allowed separation of untreated FL samples from controls with one exception, based primarily on tumour-specific gains of methylation typically occurring within CpG islands. Genes that are targets for epigenetic repression in stem cells by Polycomb Repressor Complex 2 were significantly over-represented among hypermethylated genes. Methylation profiles were conserved in sequential FL and t-FL biopsies, suggesting that widespread methylation represents an early event in lymphomagenesis and may not contribute substantially to transformation. A significant (P<0.05) correlation between FL methylation values and reduced gene expression was shown for up to 28% of loci. Methylation changes occurred predominantly in B cells with variability in the amount of non-malignant tissue between samples preventing conclusive correlation with survival. This represents an important caveat in attributing prognostic relevance to methylation and future studies in cancer will optimally require purified tumour populations to address the impact of methylation on clinical outcome.

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.

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.

Expression profiling identifies responder and non-responder phenotypes to interferon-beta in multiple sclerosis.

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.

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.

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.

Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.

BACKGROUND: Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated. RESULTS: Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover. CONCLUSIONS: The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

Molecular features of B-cell lymphoma.

Malignant transformation of B cells can occur at various steps of lymphocyte development, starting from early B-cell progenitors up to mature B cells, which reflects the heterogeneity of B-cell malignancies with regard to their biologic and clinical behavior. The genetic characterization of B-cell neoplasms during the past two decades has elucidated the mechanisms underlying B-cell lymphomagenesis and led to a more precise definition of lymphoma subgroups. This progress is reflected in the upcoming World Health Organization classification for hematologic neoplasms, which stresses the diagnostic importance of recurrent genetic alterations in leukemias and lymphomas. In the recent past, several genes deregulated by such recurrent chromosomal aberrations have been identified. In addition, the recent introduction of microarray technology has now allowed a more global assessment of gene dysregulation in B-cell oncogenesis and provided a new means for more exactly defining the molecular hallmarks of distinct lymphoma subtypes. This review will focus on recently described molecular features of B-cell lymphomas discovered by the application of new molecular cytogenetic techniques, advanced breakpoint cloning strategies, and microarray approaches.

Signatures of the immune response.

A compendium of global gene expression measurements from DNA microarray analysis of immune cells identifies gene expression signatures defining various lineages, differentiation stages, and signaling pathways. Germinal center (GC) B cells represent a discrete stage of differentiation with a unique gene expression signature. This includes genes involved in proliferation, as evidenced by high expression of G2/M phase regulators and low expression of ribosomal and metabolic genes that are transcriptional targets of c-myc. GC B cells also lack expression of the NF-kappaB signature genes, which may favor apoptosis. Finally, the transcriptional repression signature of BCL-6 reveals how this factor can prevent terminal differentiation of B cells and cause B cell lymphomas.