Targeted panel sequencing in the routine diagnosis of mature T- and NK-cell lymphomas: report of 128 cases from two German reference centers

Diagnosing any of the more than 30 types of T-cell lymphomas is considered a challenging task for many pathologists and currently requires morphological expertise as well as the integration of clinical data, immunophenotype, flow cytometry and clonality analyses. Even considering all available information, some margin of doubt might remain using the current diagnostic procedures. In recent times, the genetic landscape of most T-cell lymphomas has been elucidated, showing a number of diagnostically relevant mutations. In addition, recent data indicate that some of these genetic alterations might bear prognostic and predictive value. Extensive genetic analyses, such as whole exome or large panel sequencing are still expensive and time consuming, therefore limiting their application in routine diagnostic. We therefore devoted our effort to develop a lean approach for genetic analysis of T-cell lymphomas, focusing on maximum efficiency rather than exhaustively covering all possible targets. Here we report the results generated with our small amplicon-based panel that could be used routinely on paraffin-embedded and even decalcified samples, on a single sample basis in parallel with other NGS-panels used in our routine diagnostic lab, in a relatively short time and with limited costs. We tested 128 available samples from two German reference centers as part of our routine work up (among which 116 T-cell lymphomas), which is the largest routine diagnostic series reported to date. Our results showed that this assay had a very high rate of technical success (97%) and could detect mutations in the majority (79%) of tested T-cell lymphoma samples

Molecular Cytogenetic Profiling Reveals Similarities and Differences Between Localized Nodal and Systemic Follicular Lymphomas

Recently, we have developed novel highly promising gene expression (GE) classifiers discriminating localized nodal (LFL) from systemic follicular lymphoma (SFL) with prognostic impact. However, few data are available in LFL especially concerning hotspot genetic alterations that are associated with the pathogenesis and prognosis of SFL. A total of 144 LFL and 527 SFL, enrolled in prospective clinical trials of the German Low Grade Lymphoma Study Group, were analyzed by fluorescence in situ hybridization to detect deletions in chromosomes 1p, 6q, and 17p as well as BCL2 translocations to determine their impact on clinical outcome of LFL patients. The frequency of chromosomal deletions in 1p and 17p was comparable between LFL and SFL, while 6q deletions and BCL2 translocations more frequently occurred in SFL. A higher proportion of 1p deletions was seen in BCL2-translocation–positive LFL, compared with BCL2-translocation–negative LFL. Deletions in chromosomes 1p, 6q, and 17p predicted clinical outcome of patients with SFL in the entire cohort, while only deletions in chromosome 1p retained its negative prognostic impact in R-CHOP–treated SFL. In contrast, no deletions in one of the investigated genetic loci predicted clinical outcome in LFL. Likewise, the presence or absence of BCL2 translocations had no prognostic impact in LFL. Despite representing a genetic portfolio closely resembling SFL, LFL showed some differences in deletion frequencies. BCL2 translocation and 6q deletion frequency differs between LFL and SFL and might contribute to distinct genetic profiles in LFL and SFL

A53T-alpha-synuclein-overexpression in the mouse nigrostriatal pathway leads to early increase of 14-3-3 epsilon and late increase of GFAP

Parkinson’s disease (PD) is a neurodegenerative disorder frequent at old age characterized by atrophy of the nigrostriatal projection. Overexpression and A53T-mutation of the presynaptic, vesicle-associated chaperone alpha-synuclein are known to cause early-onset autosomal dominant PD. We previously generated mice with transgenic overexpression of human A53T-alpha-synuclein (A53T-SNCA) in dopaminergic substantia nigra neurons as a model of early PD. To elucidate the early and late effects of A53T-alpha-synuclein on the proteome of dopaminergic nerve terminals in the striatum, we now investigated expression profiles of young and old mice using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and mass spectrometry. In total, 15 proteins were upregulated and 2 downregulated. Mice before the onset of motor anomalies showed an upregulation of the spot containing 14-3-3 proteins, in particular the epsilon isoform, as well as altered levels of chaperones, vesicle trafficking and bioenergetics proteins. In old mice, the persistent upregulation of 14-3-3 proteins was aggravated by an increase of glial fibrillary acidic protein (GFAP) suggesting astrogliosis due to initial neurodegeneration. Independent immunoblots corroborated GFAP upregulation and 14-3-3 upregulation for the epsilon isoform, and also detected significant eta and gamma changes. Only for 14-3-3 epsilon a corresponding mRNA increase was observed in midbrain, suggesting it is transcribed in dopaminergic perikarya and accumulates as protein in presynapses, together with A53T-SNCA. 14-3-3 proteins associate with alpha-synuclein in vitro and in pathognomonic Lewy bodies of PD brains. They act as chaperones in signaling, dopamine synthesis and stress response. Thus, their early dysregulation probably reflects a response to alpha-synuclein toxicity

Activity of tafasitamab in combination with rituximab in subtypes of aggressive lymphoma

BackgroundDespite recent advances in the treatment of aggressive lymphomas, a significant fraction of patients still succumbs to their disease. Thus, novel therapies are urgently needed. As the anti-CD20 antibody rituximab and the CD19-targeting antibody tafasitamab share distinct modes of actions, we investigated if dual-targeting of aggressive lymphoma B-cells by combining rituximab and tafasitamab might increase cytotoxic effects.MethodsAntibody single and combination efficacy was determined investigating different modes of action including direct cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in in vitro and in vivo models of aggressive B-cell lymphoma comprising diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL).ResultsThree different sensitivity profiles to antibody monotherapy or combination treatment were observed in in vitro models: while 1/11 cell lines was primarily sensitive to tafasitamab and 2/11 to rituximab, the combination resulted in enhanced cell death in 8/11 cell lines in at least one mode of action. Treatment with either antibody or the combination resulted in decreased expression of the oncogenic transcription factor MYC and inhibition of AKT signaling, which mirrored the cell line-specific sensitivities to direct cytotoxicity. At last, the combination resulted in a synergistic survival benefit in a PBMC-humanized Ramos NOD/SCID mouse model.ConclusionThis study demonstrates that the combination of tafasitamab and rituximab improves efficacy compared to single-agent treatments in models of aggressive B-cell lymphoma in vitro and in vivo

Youth Unemployment in Western Europe: The Effects of Individual, Market, and Institutional Factors

Golsch K. Youth Unemployment in Western Europe: The Effects of Individual, Market, and Institutional Factors. In: Blossfeld H-P, Buchholz S, Bukodi E, Kurz K, eds. Young workers, globalization and the labor market: Comparing early working life in eleven countries. Cheltenham: Elgar; 2008: 29-49

Follicular Lymphoma in the 5th Edition of the WHO-Classification of Haematolymphoid Neoplasms—Updated Classification and New Biological Data

The conceptual description of Follicular lymphoma (FL) in the 5th edition of the World Health Organization (WHO) classification of haematolymphoid tumors (WHO-HAEM5) has undergone significant revision. The vast majority of FL (85%) with a follicular growth pattern are composed of centrocytes and centroblasts, harbor the t(14;18)(q32;q21) translocation and are now termed classic FL (cFL). They are set apart from three related subtypes, FL with predominantly follicular growth pattern, FL with unusual cytological features (uFL) and follicular large B-cell lymphoma (FLBCL). In contrast to the revised 4th edition of the WHO classification of haematolymphoid tumors (WHO-HAEM4R), grading of cFL is no longer mandatory. FL with a predominantly diffuse growth pattern had been previously recognized in WHO-HAEM4R. It frequently occurs as a large tumor in the inguinal region and is associated with CD23 expression. An absence of the IGH::BCL2 fusion and frequent STAT6 mutations along with 1p36 deletion or TNFRSF14 mutation is typical. The newly introduced subtype of uFL includes two subsets that significantly diverge from cFL: one with “blastoid” and one with “large centrocyte” variant cytological features. uFL more frequently displays variant immunophenotypic and genotypic features. FLBCL is largely identical to WHO-HAEM4R FL grade 3B and renaming was done for reasons of consistency throughout the classification. In-situ follicular B-cell neoplasm, pediatric-type FL, duodenal-type FL and primary cutaneous follicle center lymphoma are categorized as discrete entities. In addition, novel findings concerning underlying biological mechanisms in the pathogenesis of early and systemic follicular lymphoma will be presented

Follicular Lymphoma in the 5th Edition of the WHO-Classification of Haematolymphoid Neoplasms—Updated Classification and New Biological Data

The conceptual description of Follicular lymphoma (FL) in the 5th edition of the World Health Organization (WHO) classification of haematolymphoid tumors (WHO-HAEM5) has undergone significant revision. The vast majority of FL (85%) with a follicular growth pattern are composed of centrocytes and centroblasts, harbor the t(14;18)(q32;q21) translocation and are now termed classic FL (cFL). They are set apart from three related subtypes, FL with predominantly follicular growth pattern, FL with unusual cytological features (uFL) and follicular large B-cell lymphoma (FLBCL). In contrast to the revised 4th edition of the WHO classification of haematolymphoid tumors (WHO-HAEM4R), grading of cFL is no longer mandatory. FL with a predominantly diffuse growth pattern had been previously recognized in WHO-HAEM4R. It frequently occurs as a large tumor in the inguinal region and is associated with CD23 expression. An absence of the IGH::BCL2 fusion and frequent STAT6 mutations along with 1p36 deletion or TNFRSF14 mutation is typical. The newly introduced subtype of uFL includes two subsets that significantly diverge from cFL: one with “blastoid” and one with “large centrocyte” variant cytological features. uFL more frequently displays variant immunophenotypic and genotypic features. FLBCL is largely identical to WHO-HAEM4R FL grade 3B and renaming was done for reasons of consistency throughout the classification. In-situ follicular B-cell neoplasm, pediatric-type FL, duodenal-type FL and primary cutaneous follicle center lymphoma are categorized as discrete entities. In addition, novel findings concerning underlying biological mechanisms in the pathogenesis of early and systemic follicular lymphoma will be presented

Time-dependent inhibition of G protein-coupled receptor signaling by local anesthetics

BACKGROUND: Several beneficial effects of local anesthetics (LAs) were shown to be due to inhibition of G protein-coupled receptor signaling. Differences in exposure time might explain discrepancies in concentrations of LAs required to achieve these protective effects in vivo and in vitro (approximately 100-fold higher). Using Xenopus oocytes and human neutrophils, the authors studied time-dependent effects of LAs on G protein-coupled receptor signaling and characterized possible mechanisms and sites of action. METHODS: Measurement of agonist-induced Ca2+-activated Cl currents, using a two-electrode voltage clamp technique, and determination of superoxide anion production by cytochrome c assay were used to assess the effects of LAs on G protein-coupled receptor signaling in oocytes and primed and activated human neutrophils, respectively. Antisense knockdown of G alpha q protein and inhibition of various proteins within the signaling pathway served for defining mechanisms and sites of action more specifically. RESULTS: LAs attenuated G protein-coupled receptor signaling in both models in a time-dependent and reversible manner (lidocaine reduced lysophosphatidic acid signaling to 19 +/- 3% after 48 h and 25 +/- 2% after 6 h of control response in oocytes and human neutrophils, respectively). Whereas no effect was observed after extracellularly applied or intracellularly injected QX314, a lidocaine analog, using G alpha q-depleted oocytes, time-dependent inhibition also occurred after intracellular injection of QX314 into undepleted oocytes. Inhibition of phosphatases or protein kinases and agonist-independent G-protein stimulation, using guanosine 5'-O-3-thiotriphosphate or aluminum fluoride, did not affect time-dependent inhibition by LAs. CONCLUSION: Inhibition of G protein-coupled receptor signaling by LAs was found to be time dependent and reversible. Critically requiring G alpha q-protein function, this effect is located downstream of guanosine diphosphate-guanosine triphosphate exchange and is not dependent on increased guanosine triphosphatase activity, phosphatases, or protein kinase

Large B-Cell Lymphomas in the 5th Edition of the WHO-Classification of Haematolymphoid Neoplasms—Updated Classification and New Concepts

The family/class of the large B-cell lymphomas (LBCL) in the 5th edition of the World Health Organization (WHO) classification of haematolymphoid tumors (WHO-HAEM5) features only a few major changes as compared to the 4th edition. In most entities, there are only subtle changes, many of them only representing some minor modifications in diagnostic terms. Major changes have been made in the diffuse large B-cell lymphomas (DLBCL)/high-grade B-cell lymphomas (HGBL) associated with MYC and BCL2 and/or BCL6 rearrangements. This category now consists of MYC and BCL2 rearranged cases exclusively, while the MYC/BCL6 double hit lymphomas now constitute genetic subtypes of DLBCL, not otherwise specified (NOS) or of HGBL, NOS. Other major changes are the conceptual merger of lymphomas arising in immune-privileged sites and the description of LBCL arising in the setting of immune dysregulation/deficiency. In addition, novel findings concerning underlying biological mechanisms in the pathogenesis of the different entities are provided

The effects of S(-)-, R(+)-, and racemic bupivacaine on lysophosphatidate-induced priming of human neutrophils

Local anesthetics modulate inflammatory responses and may therefore be potentially useful in mitigating perioperative inflammatory injury. The inflammatory modulating effects of S(-)-bupivacaine are not known. Therefore, we compared the effects of S(-)-bupivacaine, R(+)-bupivacaine, and racemic bupivacaine on neutrophil function and receptor signaling. Priming (by lysophosphatidic acid [LPA]) and activation (by N-formylmethionine-leucyl-phenylalanine) of superoxide release by isolated human neutrophils was studied by using a cytochrome c-reduction assay. LPA receptor signaling in Xenopus oocytes was studied by using voltage clamp. All three local anesthetics were without effect on activation. S(-)-Bupivacaine inhibited priming more than did racemic bupivacaine; R(+)-bupivacaine was without effect. At 10(-4) M, S(-)-bupivacaine inhibited approximately 50%. Comparable results were obtained in our recombinant model, where S(-)-bupivacaine most effectively inhibited LPA signaling. Compared with racemic bupivacaine and other anesthetics, S(-)-bupivacaine appears particularly effective in suppressing neutrophil priming, a process responsible in part for the overactive neutrophil response. IMPLICATIONS: Overactive inflammatory responses underlie several perioperative disorders. Compared with racemic bupivacaine and other anesthetics, S(-)-bupivacaine appears particularly effective in suppressing neutrophil priming, a process responsible in part for the overactive neutrophil respons