Differential Expression of TRAF1 Aids in the Distinction of Cutaneous CD30-Positive Lymphoproliferations

Lymphomatoid papulosis (LyP), primary cutaneous anaplastic large T-cell lymphoma (cALCL), and cutaneous infiltrates of systemic anaplastic large cell lymphoma (sALCL) are CD30-positive lymphoproliferative disorders of the skin that overlap clinically, histopathologically, immunophenotypically, and genetically but differ considerably in their prognosis. In particular, lesions of LyP regress spontaneously, whereas those of cALCL and sALCL persist and may progress and spread to extracutaneous sites. In contrast to patients with cALCL, LyP patients do not benefit from an aggressive radio- and/or chemotherapeutic approach. We generated a novel tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) antibody that recognizes a formalin-resistant epitope (Ber-TRAF1A) and investigated the expression of TRAF1, an intracellular component of TNFR signaling, in LyP and ALCL. We could show a strong TRAF1 expression in the tumor cells of most LyP cases (42/49, 84%). In contrast, tumor cells of primary and secondary cALCL revealed TRAF1 expression in only a few cases (3/41, 7%) as shown for sALCL without skin manifestation. The data indicate that TRAF1 expression reliably distinguishes LyP from primary or secondary cALCL. This might be of crucial diagnostic importance and has a strong impact on the treatment decision for patients with cALCL and LyP

Erforschung der Ätiopathogenese primär kutaner Lymphome mit Hilfe der Mikromanipulation und Einzelzell-PCR

Primär kutane Lymphome sind typische Krankheitsbilder in der Dermatologie. Obwohl diese Erkrankungen zu den seltenen Krankheiten zählen, sind sie jedoch von therapeutischem und wissenschaftlichen Interesse. Der erste Teil der Arbeit beschäftigt sich mit der Klassifikation und Therapie primär kutaner Lymphome. Die 1997 veröffentlichte EORTC-Klassifikation wird mit ihren wichtigsten Entitäten erläutert. Die EORTC-Klassifikation geht auf spezifische Besonderheiten der primär kutanen Lymphome ein und orientiert sich an der guten Prognose dieser Erkrankungen. Therapeutische Strategien wie der Einsatz von für kutane Lymphome typische Behandlungsmethoden (PUVA, Exzision, Radiatio) als auch gentechnisch hergestellte Medikamente wie therapeutische Antikörper und Vakzinen werden erklärt. Der zweiten Teil der Habilitationsschrift konzentriert sich auf experimentelle Arbeiten zur molekularbiologischen Untersuchung von primär kutanen Lymphomen. Im Mittelpunkt steht die Methode der Mikromanipulation und Einzelzell-PCR. Für die Mykosis fungoides konnte gezeigt werden, daß im initialen Ekzemstadium nur wenige klonale maligne T-Zellen in der Probe nachzuweisen sind. Mit Zunahme des Infiltrates (Plaque) sind die malignen Zellen in der Epidermis oder gruppiert in der Dermis lokalisiert. Im Tumorstadium dominieren die malignen Zellen das dermale Infiltrat (Gellrich S, J Invest Dermatol, 2000). Die Tumorzellen primär kutaner B-Zell-Lymphome weisen einen dem Keimzentrum ähnlichen Mutationsstatus, nämlich somatische Mutationen und intraklonale Diversifikation, auf (Gellrich S, J Invest Dermatol, 1997; Gellrich S, J Invest Dermatol, 2001). Die Daten sprechen für einen noch aktiven Mutationsmechanismus, sogenannte ongoing mutations (Golembowski S, Immunobiology, 2000). Eine Unterform der kutanen Lymphome stellen die primär kutanen CD30+ T-Zell Lymphome dar. In Untersuchungen mittels Mikromanipulation und Einzelzell-PCR wurden CD30+ Zellen aus primär kutanen CD30+ großzelligen Lymphomen hinsichtlich ihrer T-Zell-Klonalität untersucht. Dabei stellte sich heraus, daß nicht alle atypischen Zellen zur Tumorpopulation gehören. Es wird vermutet, daß ein unbekannter Stimulus zur Ausprägung der Zellmorphe und zur Expression des CD30-Moleküls führt (Gellrich S, J Invest Dermatol, 2003). Eine weitere Entität, bei welcher CD30+ Zellen eine Rolle spielen, ist die lymphomatoide Papulose. In den hier dargestellten Untersuchungen wurden CD30+ große atypische Zellen einzeln isoliert und anschließend mittels PCR für die Gene des T-Zell-Rezeptor-Gamma und des Immunglobulinrezeptors amplifiziert bei einem Patienten mit lymphomatoider Papulose und assoziierter Morbus Hodgkin-Erkankung. In zwei von drei Fällen waren diese CD30+ Zellen polyklonal. Die aus der Fragmentanalyse bekannte klonale T-Zell-Population konnte dagegen in CD3+CD30- kleinen Zellen gefunden werden. In einem dritten Fall enthielten die CD30+ Zellen klonale B-Zellen, welche die gleichen Immunglobulingene rearrangiert hatten wie Zellen aus einem zuvor bestehenden Hodgkin-Lymphom desselben Patienten. Diese Ergebnisse lassen vermuten, dass es für das Aufschießen und die Regredienz der Läsionen der lymphomatoiden Papulose einen Stimulus gibt und die klonalen T- und B-Zellen als Begleitinfiltrat ohne pathologische Bedeutung anzusehen sind. Insgesamt bilden die Daten dieser Arbeit eine Grundlage für eine Fortsetzung der Untersuchung zur Ätiopathogenese von primär kutanen Lymphomen und deren Therapie und bieten die Möglichkeiten vielfältiger wissenschaftlicher Kooperationen.Primary cutaneous lymphomas present with typical clinical features in dermatology. Although these diseases are rare, they particularly are of scientific and therapeutic interest. The first part of this work deals with the classification and treatment of primary cutaneous lymphomas (PCBCL). The 1997 published EORTC classification will be explained according to the good prognosis of PCBCL. Therapeutic strategies as well as typical procedures (PUVA, excision, irradiation) and gene-technically produced drugs (therapeutic antibodies, vaccination) are illustrated in detail. The second part of this publication focuses on the experimental molecular biological work-out, done in primary cutaneous lymphomas by means of micromanipulation and single cell PCR. For the mycosis fungoides could be shown that in the patch stage only a few malignant T cells can be detected. Increasing infiltrates (plaque-stage) are characterized by epidermotrop or dermally grouped atypical cells. In tumor stage dermal atypical T cells are predominating (Gellrich S, J Invest Dermatol, 2003). The tumor cells of primary cutaneous B cell lymphomas are comparable with the stage of mutation of follicle centre cells: somatic mutations and intraclonal diversity (Gellrich S, J Invest Dermatol, 1997; Gellrich S, J Invest Dermatol, 2001). The data indicate, that there may be an active mutation mechanism, the so-called ongoing mutations (Golembowski S, Immunobiology, 2000). One subgroup of PCBCL, are presented by the CD30positve entities. By means of micromanipulation and PCR, single cells were investigated due to T cell clonality. Not all atypical cells belong to the malignant population. It is supposed that an unknown stimulus leads to morphological features and CD30 expression (Gellrich S, J Invest Dermatol, 2003). Another CD30positive entity is reflected by the lymphomatoid papulosis. In these experiments large atypical CD30positve cells were isolated and have been investigated via PCR for T cell receptor g or immunoglobuline heavy and light chain gene rearrangement. The majority of the large CD30postive cells (two cases) belong to a polyclonal T cell population. In the opposite, the small CD3positive cells are the cells persisting within the T cell clone. In another case B cells with the same immunoglobulin gene rearrangement like in a preceding Hodgkin disease of the same patient could be detected. The data seem to underline the fact that reactive polyclonal CD30positive cells are triggered by an unkown stimulus with clonal bystander cells without any pathological significance. In summary, this work could be the basis for further investigations about the etiopathogenesis of PCBCL

Proteome-based analysis of serologically defined tumor-associated antigens in cutaneous lymphoma.

Information on specificities of serological responses against tumor cells in cutaneous lymphoma patients is relatively restricted. To advance the knowledge of serological immune responses against and to assess the scope of tumor antigenicity of cutaneous lymphoma, 1- and 2-dimensional Western blot analyses with sera from patients were combined with proteomics-based protein identification. Testing sera from 87 cutaneous lymphoma patients by 1-dimensional Western blot analysis, 64 cases of seroreactivity against lymphoma cells were found. The positive responses were relatively weak, restricted to few antigens in each case, and heterogeneous. To identify the antigens, proteins of the mycosis fungoides cell line MyLa and primary tumor cells were separated by 2-dimensional gel electrophoresis, Western-blotted and probed with heterogeneous and autologous patient sera. The antigens were identified from silver-stained replica gels by MALDI-TOF mass spectrometry. 14 different antigens were assigned and identified with this proteome-serological approach. Only one, vimentin, had been reported before, the other 13 are new antigens for cutaneous lymphomas

Phase II Clinical Trial of Intratumoral Application of TG1042 (Adenovirus-interferon-γ) in Patients With Advanced Cutaneous T-cell Lymphomas and Multilesional Cutaneous B-cell Lymphomas

Cutaneous lymphomas (CLs) are a heterogeneous group of lymphoproliferative disorders that are manageable by immunotherapy. Twenty-one patients were enrolled in a prospective open-label, dose-escalation multicenter study evaluating the effects of repeated TG1042 [adenovirus-interferon (IFN)-γ] intralesional injections in patients with primary CLs, of which 18 were of T-cell and 3 of B-cell type. Repeated intralesional therapy using TG1042 consistently results in local tumor regressions in about half of treated patients and one-third of patients also in regressions in noninjected distant lesions, likely reflecting the systemic immune activation after intralesional therapy. Treatment was well tolerated with few adverse events including injection site reactions, chills, lymphopenia, and fever. Immune monitoring in the peripheral blood demonstrated systemic immune activation and the induction of antibodies against tumor antigens in some patients without clear association with clinical responses. CLs, in particular B-cell lymphomas with high objective response rates, seem to be excellent targets for this type of immunotherapy