Sylke Gellrich

Novel and highly recurrent chromosomal alterations in Sezary syndrome

This study was designed to identify highly recurrent genetic alterations typical of Sézary syndrome (Sz), an aggressive cutaneous T-cell lymphoma/leukemia, possibly revealing pathogenetic mechanisms and novel therapeutic targets. High-resolution array-based comparative genomic hybridization was done on malignant T cells from 20 patients. Expression levels of selected biologically relevant genes residing within loci with frequent copy number alteration were measured using quantitative PCR. Combined binary ratio labeling-fluorescence in situ hybridization karyotyping was done on malignant cells from five patients. Minimal common regions with copy number alteration occurring in at least 35% of patients harbored 15 bona fide oncogenes and 3 tumor suppressor genes. Based on the function of the identified oncogenes and tumor suppressor genes, at least three molecular mechanisms are relevant in the pathogenesis of Sz. First, gain of cMYC and loss of cMYC antagonists (MXI1 and MNT) were observed in 75% and 40% to 55% of patients, respectively, which were frequently associated with deregulated gene expression. The presence of cMYC/MAX protein heterodimers in Sézary cells was confirmed using a proximity ligation assay. Second, a region containing TP53 and genome maintenance genes (RPA1/HIC1) was lost in the majority of patients. Third, the interleukin 2 (IL-2) pathway was affected by gain of STAT3/STAT5 and IL-2 (receptor) genes in 75% and 30%, respectively, and loss of TCF8 and DUSP5 in at least 45% of patients. In sum, the Sz genome is characterized by gross chromosomal instability with highly recurrent gains and losses. Prominent among deregulated genes are those encoding cMYC, cMYC-regulating proteins, mediators of MYC-induced apoptosis, and IL-2 signaling pathway components.

CD56-positive haematological neoplasms of the skin: a multicentre study of the Cutaneous Lymphoma Project Group of the European Organisation for Research and Treatment of Cancer

Background: Cutaneous lymphomas expressing CD56, a neural cell adhesion molecule, are characterised in most cases by a highly aggressive clinical course and a poor prognosis. However, prognostic subsets within the CD56+ group have been difficult to identify due to the lack of uniform clinicopathological and immunophenotypical criteria. Methods: A multicentre study was conducted by the Cutaneous Lymphoma Task Force of the European Organisation for Research and Treatment of Cancer to define prognostic parameters and establish diagnostic and therapeutic guidelines for CD56+ haematological neoplasms presenting primarily in the skin. Results: Four different subtypes of lymphoproliferations with CD56 expression were identified: (1) haematodermic neoplasm; (2) skin infiltration as the first manifestation of CD56+ acute myeloid leukaemia; (3) nasal-type extranodal natural killer/T-cell lymphoma; and (4) “classical” cases of cutaneous T-cell lymphoma (CTCL) with co-expression of the CD56 molecule. Patients in the first three groups had a poor outcome (93% died) with a median survival rate of 11 months (95% CI 2–72 months), whereas all patients with CD56+ CTCL were alive at the last follow-up. Conclusion: Results show that CD56+ cutaneous lymphoproliferative disorders, with the exception of CD56+ CTCL have a very poor prognosis. It is therefore clinically important to separate CD56+ CTCL from the remaining CD56+ haematological disorders.

Analysis of VH-D-JH gene transcripts in B cells infiltrating the salivary glands and lymph node tissues of patients with Sjögren's syndrome

Objective. In patients with Sjogrens syndrome (SS), B lymphocytes have been found to infiltrate salivary glands, resulting in sialadenitis and keratoconjunctivitis. The disease is frequently associated with benign and neoplastic lymphoproliferation. The present study was undertaken to investigate whether clonal B cell expansion takes place in lymphocytic infiltrations of salivary glands under (auto- [?]) antigen stimulation, by analyzing in more detail the variable part (V H -D-J H ) of the immunoglobulin heavy chain genes expressed in these B cells. Methods. Biopsies of the labial salivary glands and lymph nodes were performed on 2 female patients with SS. The Ig gene rearrangements in these tissues were amplified by reverse transcriptase-polymerase chain reaction using specific primers. Results. A total of 94 V H -D-J H transcripts were cloned and sequenced. Our data suggest a polyclonal origin of the B cell infiltrates. In 92 of the transcripts, V H genes were modified by somatic mutation. Further analysis showed counterselection for replacement mutations within the framework regions, suggesting that those B cells were stimulated and selected for functional expression of a surface Ig. In labial salivary glands from both patients, clonally related B cells became evident. Members of 1 particular clone were found in both the lip and lymph node material. Conclusion. These data provide evidence, on the nucleotide sequence level, that an antigen-triggered clonal B cell expansion takes place in the salivary glands of patients with SS who do not have histologic evidence of developing lymphoma. It may be speculated that those B cell clones expand during disease progression, resulting in lymphomagenesis.

Systemic eight‐cycle anti‐CD20 monoclonal antibody (rituximab) therapy in primary cutaneous B‐cell lymphomas – an applicational observation

Background  Primary cutaneous B‐cell lymphomas (PCBCLs) are characterized by restriction to the skin and a variable but mostly favourable prognosis. Since 1997 the recombinant, chimeric anti‐CD20 antibody rituximab has been used in patients suffering from non‐Hodgkins B‐cell lymphomas. Different studies have shown that the effectiveness and safety in the treatment of patients with low‐grade follicular lymphoma is comparable to or even higher than the standard CHOP chemotherapy. So far it has been unclear whether an extended duration of therapy leads to a benefit for the patients with PCBCL.

Mimotopes for tumor-specific T lymphocytes in human cancer determined with combinatorial peptide libraries

Mimotopes of a tumor‐associated T cell epitope were determined using randomized and combinatorial peptide libraries and a CD8+ T cell clone specific for the cutaneous T cell lymphoma cell line MyLa. Antigen recognition by this clone was found to be HLA‐B8 restricted. More than 80 % of HLA‐matched patients with cutaneous T cell lymphoma had mimotope‐specific CD8+ T cells in their peripheral blood. Mimotope‐specific T cells isolated and expanded from a patient lysed MyLa cells in in vitro assays thus demonstrating their cytolytic capacity and tumor specificity. Mimotope vaccination of a patient without detectable mimotope‐specific T cells induced frequencies of these cells of up to 1.82 % of the peripheral blood CD8+ T cells.

Anti‐tumor immune responses and tumor regression induced with mimotopes of a tumor‐associated T cell epitope

Mimotopes provide an alternative to natural T cell epitopes for cancer immune therapy, as they can recruit and stimulate T cell repertoires that deviate from the repertoires engaged with the tumor and exposed to disease‐related immune suppression. Here, mimotopes of a shared tumor‐associated T cell epitope in cutaneous lymphoma were tested for their capacities to induce clinical and immunological responses in cancer patients. The mimotope sequences had been determined by a combinatorial peptide library approach without knowledge of the corresponding natural tumor‐associated antigen. Vaccination with these mimotopes together with helper T cell‐inducing antigens led to complete tumor remission in the two patients tested. After each booster vaccination, enhanced frequencies of mimotope‐specific CD8+ T cells were detected in the peripheral blood of the patients, and the CTL proved to be cytotoxic and tumoricidal when tested in vitro. These data provide a first indication of clinical efficacy of mimotopes in cancer patients.

Immunofluorescent and FISH analysis of skin biopsies

Cutaneous biopsies are traditionally studied for the expression of cellular markers by immunoenzymatic techniques. However, immunofluorescent analysis is a valuable, and largely overlooked, ancillary technique that can resolve questions arising from conventional immunostaining, since it allows pairs of antigens to be simultaneously visualized. Furthermore, a novel technique, based on a combination of immunoperoxidase and immunofluorescent staining, allows three markers to be demonstrated together. Fluorescent microscopy also allows skin biopsies from lymphoma cases to be analyzed for chromosomal abnormalities by the fluorescent in situ hybridization (FISH) technique, which is now applicable to routine biopsy samples. In this review, we describe the technical aspects of immunofluorescent and FISH analysis of routine cutaneous biopsy samples.

Treatment of cutaneous T-cell lymphomas.

Primary cutaneous T-cell lymphomas (CTCL), representing a heterogeneous group of non-Hodgkins lymphomas (NHL), can be defined as clonal proliferation of skin-infiltrating T lymphocytes primarily presenting in the cutaneous compartment. They show a considerable variation in clinical presentation, histology, immunophenotype, and prognosis, which is best reflected by the proposal of the Cutaneous Lymphoma Study Group of the European Organization for Research and Treatment of Cancer (EORTC). Due to the heterogeneity of CTCL and the lack of curative therapy regimens, multiple strategies have been proposed for the management of the different CTCL entities. This includes topical application of corticosteroids, nitrogen mustard or carmustine (BCNU), radiotherapy, including total skin electron beam irradiation, photo(chemo)therapy, biological response modifiers, cytostatic chemotherapy, and combined regimens. More recently, fusion proteins and peptide vaccines have been introduced in the management of CTCL. Classification, staging, and treatment modalities are discussed in detail and summarized in a stage-adapted therapy regimen for CTCL.

Clonal evolution in a primary cutaneous follicle center B cell lymphoma revealed by single cell analysis in sequential biopsies.

B cell neoplasias descending from germinal center cells harbor the hallmark of intraclonal diversity resulting from ongoing mutation in the variable parts of their immunoglobulin-encoding genes. To characterize a primary cutaneous follicle center B cell lymphoma in more detail, we analyzed the respective VH and VL genes in single cells mobilized from four sequential biopsies, three taken from the skin and one obtained after internal dissemination from a retrobulbar infiltrate. The lymphoma cells were found to contain V5-51/D6-12/JH5b (heavy chain) and A27/Jkappa2 (light chain) gene rearrangements detected on both the genomic and the transcriptional level. To provide an accurate mutation analysis, the specific VH gene counterpart (V5-51UK) was cloned from the patients germline. Analyzing 226 single cells, we found: (i) complete nucleotide identity when VH and VL genes of lymphoma cells from one particular biopsy were compared among each other; (ii) intraclonal diversity due to ongoing mutation comparing the sequences obtained from sequential biopsies; (iii) both VH and VL genes to be highly mutated. Deducing from the sequence data, we propose a scenario of the clonal evolution of the B cell tumor in this patient. From the molecular-biological point of view, this primary cutaneous follicle center B cell lymphoma shows the features of a germinal center cell lymphoma. To draw this conclusion from single cell PCR data, however, a sample of sequential biopsies had to be analyzed.

Mainly unmutated VH genes rearranged in B cells forming germinal centers in a cutaneous pleomorphic T‐cell lymphoma

B Cells in skin lesions of a pleomorphic cutaneous T‐cell lymphoma with reactive germinal center hyperplasia were analyzed for their immunoglobulin VHDJH gene rearrangements by micromanipulation and single cell polymerase chain reaction (PCR) analysis. In B lymphocytes located in germinal center‐like structures, we found in 11/16 different VHDJH rearrangements completely Unmutaied VH genes, suggesting that those cells did not undergo antigen‐driven selection. Two VH genes showed more than 98% germ‐line identity. In only three cells VH segments were somatically mutated to a higher extent, but two of these rearrangements were non‐productive. These results diller markedly from what we have previously detected in B cells present in mycosis fungoides, another entity of cutaneous T‐cell lymphomas where the Ig gene repertoire resembles the situation in peripheral blood with a significantly higher proportion of mutated VH genes. When investigating the large atypical B cells strongly expressing CD30 which were detected within the T‐cell zone outside the germinal centers. we found again, in most cases, that the rearranged VH genes were completely unmutated. The B cells were of polyclonal origin. Due to this comparable Ig gene repertoire and mutational pattern, we suggest that these cells descend from the germinal center centroblasts, which migrated into the T‐cell zone and obviously became stimulated to express the CD30 marker. The mieromanipulation technique and molecular analysis on the single cell level may provide an important Input into our understanding of the mechanisms of immune regulation in cutaneous lymphomas.