High expression of several tyrosine kinases and activation of the PI3K/AKT pathway in mediastinal large B cell lymphoma reveals further similarities to Hodgkin lymphoma.

Mediastinal large B-cell (MBL) and classical Hodgkin lymphoma (HL) have several pathogenic mechanisms in common. As we recently observed aberrant tyrosine kinase (TK) activities in HL, we now analysed also MBL for such activities. Indeed, MBL and HL were the only B-cell lymphomas where elevated cellular phospho-tyrosine contents were typical features. Three TKs, JAK2, RON and TIE1, not expressed in normal B cells, were each expressed in about 30% of MBL cases, and 75% of cases expressed at least one of the TKs. Among the intracellular pathways frequently triggered by TKs, the PI3K/AKT pathway was activated in about 40% of MBLs and essential for survival of MBL cell lines, whereas the RAF/mitogen-activated protein kinase pathway seemed to be inhibited. No activating mutations were detected in the three TKs in MBL cell lines and primary cases. RON and TIE1 were each also expressed in about 35% and JAK2 in about 53% of HL cases. JAK2 genomic gains are frequent in MBL and HL but we observed no strict correlation of JAK2 genomic status with JAK2 protein expression. In conclusion, aberrant TK activities are a further shared pathogenic mechanism of MBL and HL and may be interesting targets for therapeutic intervention.

Mismatch negativity: Alterations in adults from the general population who report subclinical psychotic symptoms.

BACKGROUND: Deficits of mismatch negativity (MMN) in schizophrenia and individuals at risk for psychosis have been replicated many times. Several studies have also demonstrated the occurrence of subclinical psychotic symptoms within the general population. However, none has yet investigated MMN in individuals from the general population who report subclinical psychotic symptoms. METHODS: The MMN to duration-, frequency-, and intensity deviants was recorded in 217 nonclinical individuals classified into a control group (n=72) and three subclinical groups: paranoid (n=44), psychotic (n=51), and mixed paranoid-psychotic (n=50). Amplitudes of MMN at frontocentral electrodes were referenced to average. Based on a three-source model of MMN generation, we conducted an MMN source analysis and compared the amplitudes of surface electrodes and sources among groups. RESULTS: We found no significant differences in MMN amplitudes of surface electrodes. However, significant differences in MMN generation among the four groups were revealed at the frontal source for duration-deviant stimuli (P=0.01). We also detected a trend-level difference (P=0.05) in MMN activity among those groups for frequency deviants at the frontal source. CONCLUSIONS: Individuals from the general population who report psychotic symptoms are a heterogeneous group. However, alterations exist in their frontal MMN activity. This increased activity might be an indicator of more sensitive perception regarding changes in the environment for individuals with subclinical psychotic symptoms.

Highly recurrent mutations of SGK1, DUSP2 and JUNB in nodular lymphocyte predominant Hodgkin lymphoma.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL)-a subtype of Hodgkin lymphoma (HL)-is characterized by a low content of tumor cells, the lymphocyte predominant (LP) cells. Transformation into diffuse large B-cell lymphoma (DLBCL) occurs in about 10% of patients. We performed whole-genome mutation analysis of the DLBCL components from two composite lymphomas consisting of clonally related NLPHL and DLBCL as a means to identify candidate tumor suppressor genes and oncogenes in NLPHL. The analysis of LP cells for selected mutations of the DLBCL revealed that most mutations are also present in the LP cells, indicating a close relationship between the two components. The analysis of 62 selected genes in NLPHL by targeted ultra-deep sequencing revealed three novel highly recurrently mutated genes (each mutated in ~50% of cases), that is, DUSP2, SGK1 and JUNB. SGK1 was expressed in the LP cells of primary NLPHL cases and in the NLPHL cell line DEV. Administration of an SGK1 inhibitor induced apoptosis in the NLPHL cell line DEV and the DLBCL cell line Farage, suggesting a pathogenetic role of SGK1 in the LP and DLBCL cells. In summary, the present study identifies SGK1, DUSP2 and JUNB as novel key players in the pathogenesis of NLPHL.

Complete dynamic repopulation of decellularized heart valves by application of defined physical signals-an in vitro study.

OBJECTIVE: Cardiovascular tissue engineering is a novel concept to develop ideal heart valve substitutes. The objective of this study was to use decellularized porcine pulmonary valves, ovine cells and dynamic tissue culture to obtain viable and biomechanically stable constructs, resembling native aortic heart valves. METHODS: Endothelial cells and myofibroblasts were obtained from ovine carotid arteries. Porcine pulmonary valves were decellularized enzymatically, reseeded and cultured using a hydrodynamic bioreactor system over a time period of 9 or 16 days. Controls were grown over an equivalent time period under static conditions. Specimens of each valve were examined biochemically (cell proliferation, DNA, collagen, 4-hydroxyproline, elastin and glycosaminoglycans), histologically (hematoxylin-eosin, Movat-pentachrome and immunostains) and mechanically (radial and circumferential strength). RESULTS: Histology and biochemical assays demonstrated the removal of almost all cells after decellularization with preservation of the extracellular matrix. Recellularization under pulsatile conditions was significantly improved after 9 and 16 days compared to static conditions. Biochemical and mechanical analysis revealed a continuous increase of cell mass, collagen and elastin contents and strength under pulsatile culture conditions compared to significantly lower values in the static controls. CONCLUSION: This study demonstrated the superiority of the hydrodynamic approach of cellular reseeding to replace decellularized porcine heart valves with ovine cells with almost complete preservation of extracellular matrix integrity.

Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors.

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

Role of early B-cell factor 1 (EBF1) in Hodgkin lymphoma.

A hallmark of classical Hodgkin lymphoma (cHL) is that the B-cell-derived Hodgkin and Reed-Sternberg (HRS) tumor cells have largely lost the B-cell-typical gene expression program. The factors causing this 'reprogramming' of HRS cells are only partly understood. As early B-cell factor 1 (EBF1), a major B-cell transcription factor, is downregulated in HRS cells, we analyzed whether this downregulation contributes to the lost B-cell phenotype and tested the consequences of EBF1 re-expression in cHL cell lines. EBF1 re-expression caused an upregulation of B-cell genes, such as CD19, CD79A and CD79B, although the B-cell genes FOXO1 and PAX5 remained lowly expressed. The re-expression of CD19, CD79A and CD79B occurred largely without demethylation of promoter CpG motifs of these genes. In the cHL cell line L-1236 fitness decreased after EBF1 re-expression. These data show that EBF1 has the ability to reintroduce part of the B-cell signature in cHL cell lines. Loss of EBF1 expression in HRS cells therefore contributes to their lost B-cell phenotype. Notably, in the cHL cell line KM-H2 destructive mutations were found in one allele of EBF1, indicating that genetic lesions may sometimes have a role in impairing EBF1 expression.

SU-E-T-639: Dosimetric Evaluation of VMAT for the Treatment of NSCLC with SBRT.

PURPOSE: To demonstrate the dosimetric potential of volumetric modulated arc therapy (VMAT) for the treatment of patients with medically inoperable stage I/II non-small cell lung cancer (NSCLC) with stereotactic body radiation therapy (SBRT). METHODS: Fourteen patients treated with 3D-CRT with varying tumor locations, tumor sizes and dose fractionation schemes were chosen for study. The target prescription doses were 48 Gy in 4 fractions, 52.5 Gy in 5 fractions, 57.5 Gy in 5 fractions and 60 Gy in 3 fractions for 2, 5, 1 and 6 patients, respectively. VMAT treatment plans with a mix of 2-3 full and/or partial non-coplanar arcs with 5°-25° separations were retrospectively generated using Eclipse version 10.0. The 3D-CRT and VMAT plans were then evaluated by comparing their target dose, critical structure dose, high dose spillage, and low dose spillage as defined according to RTOG 0813 and RTOG 0236 protocols. RESULTS: The VMAT treatment plans yielded an average 9.6-33.7% reduction in dose to critical structures and an average 12.0-12.5% increase in conformity compared with the treated 3D-CRT plans. The D2cm improved with VMAT in 11 of 14 cases. The 3 that worsened were still within the acceptance criteria. Of the 14 3D-CRT plans, 7 had a D2cm minor deviation, while only one of the 14 VMAT plans had a D2cm minor deviation. The R50% improved in 13 of the 14 VMAT cases. The 1 case that worsened was still within the acceptance criteria of the RTOG protocol. Of the 14 3D-CRT plans, 7 had an R50% deviation. Only 1 of the 14 VMAT plans had an R50% deviation, but it was still improved compared to the 3D-CRT plan. CONCLUSIONS: In this cohort of patients, no dosimetric compromises resulted from planning SBRT treatments with VMAT relative to the 3D-CRT treatment plans actually used in their treatment.

Dysregulation of global microRNA expression in splenic marginal zone lymphoma and influence of chronic hepatitis C virus infection.

The precise molecular pathogenesis of splenic marginal zone lymphoma (SMZL) is still unknown. Clinical and epidemiological data suggest that chronic hepatitis C virus (HCV) infection may have an etiological role in a subset of cases.We performed a large-scale microRNA (miRNA) expression profiling analysis of 381 miRNAs by quantitative reverse transcription PCR (Q-RT-PCR) of 26 microdissected splenic tissue samples (7 HCV(+) SMZL; 8 HCV(-) SMZL and 11 non-neoplastic splenic controls). Single assay Q-RT-PCR and miRNA in situ hybridization (miRNA-ISH) were used to confirm the results in an independent cohort. Unsupervised hierarchical clustering of miRNA expression profiles demonstrated a distinct signature of SMZL compared with the normal splenic marginal zone. Supervised analysis revealed differentially expressed miRNAs, including miRNAs with previously recognized tumor suppressive or oncogenic potential. Five miRNAs were found significantly overexpressed in SMZL, including miR-21, miR-155 and miR-146a, whereas seven miRNAs showed significantly reduced expression, including miR-139, miR-345, miR-125a and miR-126. Furthermore, we identified miR-26b, a miRNA known to have tumor suppressive properties, as significantly downregulated in SMZL arising in HCV-positive patients (P=0.0016). In conclusion, there is a characteristic dysregulation of miRNA expression in SMZL with a possible implication in its molecular tumorigenesis.

STAT6 and STAT1 are essential antagonistic regulators of cell survival in classical Hodgkin lymphoma cell line.

Classical Hodgkin lymphoma (cHL) is a malignant lymphoid disorder characterized by aberrant activation of signaling pathways. Constitutive activation of several components of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been observed in Hodgkin and Reed/Sternberg cells, the tumor cells of cHL. In this study, we investigate the function of STAT6 in cHL cell lines and show that STAT6 promotes survival of these cells. Microarray expression analysis of STAT6-shRNA (short hairpin RNA)-expressing cHL cell lines was carried out to analyze the STAT6-mediated survival mechanism. Some of the identified genes with potentially important regulatory functions were also interleukin (IL)-4 dependently regulated in Ramos B cells and binding of STAT6 to the regulatory regions of several genes could be confirmed, indicating that these are direct STAT6 target genes. Importantly, STAT6 knockdown increased the expression and activation of STAT1 as well as the expression of known STAT1 target genes, indicating a cross-regulation between these signaling molecules. Forced expression of STAT1 was able to induce apoptosis in cHL cell line L1236. These findings indicate that both STAT6 and STAT1 can act as important antagonistic regulators in the pathogenesis of cHL.

Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma.

Anaplastic large cell lymphoma (ALCL) is a main type of T-cell lymphomas and comprises three distinct entities: systemic anaplastic lymphoma kinase (ALK) positive, systemic ALK(-) and cutaneous ALK(-) ALCL (cALCL). Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK(-) ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of five ALK(+) and four ALK(-) systemic ALCL, seven cALCL and sixteen cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4(+), CD8(+) or CD30(+) T-cell origin. Indeed, ALCL display a down-modulation of many T-cell characteristic molecules. All ALCL types show significant expression of NFkappaB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly, few genes are differentially expressed between systemic and cALCL despite their different clinical behaviour, and between ALK(-) ALCL and cHL despite their different cellular origin. ALK(+) ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.

Voltage dependence and ion selectivity of the mitochondrial channel, VDAC, are modified by succinic anhydride.

The mitochondrial protein VDAC forms voltage-dependent anion-selective channels in planar phospholipid membranes. When succinic anhydride was added to these membranes, it virtually eliminated VDAC's voltage-dependence and changed its selectivity from anion to cation. These results were obtained without large changes in open-channel conductance or in energy difference between the open and closed states in the absence of a field. Since succinic anhydride converts amino groups into carboxyl groups, we propose that amino groups are responsible for VDAC's voltage-dependence and anion selectivity. Perhaps the same charges are responsible for both functions.

The mitochondrial voltage-dependent channel, VDAC, is modified asymmetrically by succinic anhydride.

In the accompanying paper, succinic anhydride was shown to react with the outer mitochondrial membrane channel-forming protein, VDAC, resulting in the loss of its voltage dependence. In this paper, the anhydride was added to VDAC held in a particular conformational state by means of an applied electric field. VDAC was inserted into the membranes from the cis side and the anhydride was added either to the cis or trans side. Channels modified in the open state behaved similarly whether anhydride was added to the cis or trans side. Modifications of VDAC in either of the two closed states did not. Modifications resulting in the loss of voltage-dependence occurred primarily when anhydride was added to the negative side of the membrane irrespective of which closed state the VDAC was in indicating that the accessibility of the gating charges alternated between the cis and trans sides as the channel's conformation was changed from one closed state to the other. Despite the pronounced asymmetry, in general the resulting channels behaved in the same way in response to either positive or negative fields. A model consistent with the results is presented which proposes that the same gating charges are responsible for channel closure at both positive and negative fields.