Roland Schroers

Trisomy 19 is associated with trisomy 12 and mutated IGHV genes in B‐chronic lymphocytic leukaemia

The occurrence of trisomy 19 was investigated in 705 cases of B‐chronic lymphocytic leukaemia (CLL) by metaphase cytogenetic and/or fluorescence in situ hybridisation analyses. Trisomy 19 was detected in 11 cases (1·6%), all of which also carried a trisomy 12; nine of 10 had mutated IGHV genes. In contrast, B‐CLL cases with trisomy 12 lacking trisomy 19 mostly had unmutated IGHV genes. Karyotypes of the present study and the literature identified a strong correlation to trisomy 18 in addition to trisomy 12. Trisomy 19 seems to be a secondary event in B‐CLL with trisomy 12, mostly originating from mutated B cells.

Lentiviral Transduction of Human Dendritic Cells

Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that play a pivotal role in stimulating antigen-specific T cells in vivo. The cardinal properties of DCs are: the ability to take up, process, and present antigens; (2) the ability to migrate through different tissues into lymphoid organs; and (3) the ability to interact with and stimulate T cells (3). Because of their unique capability of generating primary CD4+ and CD8+ T-cell responses, DCs are of particular interest for immunotherapeutic approaches to infectious disease and cancer. There are many ways to load DCs with antigen and to subsequently induce specific immune responses in vivo and in vitro. One strategy relies on genetically modified peripheral blood mononuclear cell (PBMC)-derived DCs to establish expression of a gene that encodes a specific antigen. In contrast to pulsing DCs with antigenic peptides or proteins, genetic engineering of DCs has the advantages of providing multiple epitopes for major histocompatibility class (MHC) class I-and MHC class II-restricted immune responses as well as a continuous supply of antigen for presentation alone or together with immunomodulatory proteins (cytokines, for example).

B-cell Chronic Lymphocytic Leukemia with Aberrant CD8 Expression: Genetic and Immunophenotypic Analysis of Prognostic Factors

B-cell chronic lymphocytic leukemia (B-CLL) is phenotypically characterized by cell surface co-expression of CD19, CD20, CD5, and CD23. However, the concomitant presence of other antigens distinctive of a particular leukocyte subset, e.g. T cells, is an unusual finding in B-CLL. In the present report, a case of B-CLL with aberrant expression of the T-cell-associated antigen CD8 is described. Flow cytometric analysis of the patients cells demonstrated lack of CD38 expression, and cytogenetic analysis by FISH did not show any abnormalities of chromosomes 17p, 11q, and 12. Furthermore, the B-CLL cells expressed only low levels of the tyrosine kinase ZAP-70, which was in agreement with the mutated status of the immunoglobulin heavy-chain variable-region gene (IgVH). In summary, the determined profile of prognostic markers together with the highly stable and indolent disease in the described patient suggest a good prognosis of B-CLL with aberrant CD8 expression.

Multikinase inhibitor sorafenib exerts cytocidal efficacy against Non-Hodgkin lymphomas associated with inhibition of MAPK14 and AKT phosphorylation

Intracellular signal transduction by kinase‐mediated phosphorylation is essential for the survival and growth of lymphoma cells. This study analysed the multikinase inhibitor sorafenib for its cytotoxic activity against lymphoma cells. We found that sorafenib reduced cell viability at low micromolar concentrations in a time‐dependent manner in cell lines and primary cell suspensions representing major types of aggressive B‐ and T‐cell lymphomas. In cells surviving short term exposure, proliferative arrest occurred leading to complete loss of in vitro clonogenicity. Previously described sorafenib targets within the RAF kinase family were found to be expressed and phosphorylated in all cell lines, and sorafenib perturbed the activation of classical RAF/MEK/ERK pathway targets. However, using a global phoshoprotein array, the most consistent downstream effect of sorafenib in NHL cells was the inhibition of mitogen‐activated protein kinase 14 (MAPK14) and panAKT phosphorylation. In conclusion, sorafenib has significant in vitro efficacy against aggressive B‐ and T‐cell lymphoma cells, associated with inhibition of MAPK14 and panAKT.

Immunophenotypic and genetic characterization of a CD8 positive Mantle Cell Lymphoma in a patient with concomitant Mycosis Fungoides

Abstract:  Mantle cell lymphoma (MCL) is immunophenotypically characterized by cell surface co‐expression of CD19, CD20, CD5, IgM and FMC7. However, the concomitant presence of other antigens distinctive of a particular leukocyte subset, e.g. T‐lymphocytes, is an exceptional finding in MCL. Here, the first case of a blastic MCL in leukaemic phase with aberrant expression of the T‐cell associated antigen CD8 occurring in a patient with concomitant Mycosis fungoides is described. Comprehensive immunophenotypic analysis showed that the MCL cells expressed the typical B‐lymphocytic markers, were CD5 and CD8 positive, but did not express other T‐cell proteins, such as CD2, CD3, CD4, CD7, TCRαβ and TCRγδ. The MCL cells expressed both CD8α and CD8β chains indicating cell surface presence of CD8αβ heterodimers. Intriguingly, expression of the cytotoxic enzymes perforin and granzyme A was detected by RT‐PCR. Cytogenetic and molecular genetic analysis of the lymphoma cells confirmed cyclin D1 overexpression secondary to the t(11;14)(q13;32) chromosomal translocation. Furthermore, trisomy 11, trisomy 14 and extra copies of t(11;14) translocated chromosomes were detected in sub clones of the analyzed MCL cells. Clinically, an aggressive course of disease including cerebral lymphoma involvement was noted in the reported patient. Hence, systematic studies addressing the incidence, biology and clinical behavior of this form of MCL seem to be justified in future.

Characterization of HLA-DR-restricted T-cell epitopes derived from human proteinase 3.

Human proteinase 3 (PRTN3) is a leukemia-associated antigen specifically recognized by CD8+ cytotoxic T-lymphocytes (CTL). PRTN3 also has been shown to elicit both antibody responses and T-cell proliferation in patients with Wegeners granulomatosis. In order to improve current vaccines that aim to stimulate CTL without inducing harmful autoimmune disease, it is necessary to study the role of PRTN3-specific CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Since both TH and Treg cells recognize antigens in the context of HLA-class-II-molecules, identification of HLA-class-II-associated peptide-epitopes from self-antigens such as PRTN3 is required. Here, we analyzed T-cell responses against proteinase 3 using synthetic peptides predicted to serve as HLA-DR-restricted epitopes. We first screened a panel of ten epitope peptide candidates selected with the TEPITOPE program and found that nine out of ten peptides induced PRTN3 peptide-specific proliferation of T-cells with precursor frequencies of 0-1.1 x 10(-6). For one peptide-epitope, PRTN3(235), T-cell-clones were demonstrated to be capable of recognizing naturally processed protein antigen in a HLA-DR-restricted fashion. PRTN3(235)-specific T-cells could be stimulated from the blood of healthy individuals with multiple HLA-DR-genotypes. In summary, the identified PRTN3(235)-epitope can be used to study the role of CD4+ TH- and Treg-cells in immune responses against PRTN3 in leukemia patients and patients with Wegeners disease.