Markus Möbs

Genomic loss of the putative tumor suppressor gene E2A in human lymphoma

Loss of E2A, observed in more than 70% of patients with Sézary syndrome, which is a subtype of T cell lymphoma, results in altered expression of genes potentially relevant to oncogenesis.

Apoptosis Induction by SAHA in Cutaneous T-Cell Lymphoma Cells Is Related to Downregulation of c-FLIP and Enhanced TRAIL Signaling

Suberoylanilide hydroxamic acid (SAHA) has been approved for the treatment of cutaneous T-cell lymphoma (CTCL), but its mode of action remained largely elusive. As shown here in four CTCL cell lines, loss of cell viability correlated with significant time- and dose-dependent induction of apoptosis, whereas cytotoxicity was less pronounced. Both extrinsic and intrinsic apoptosis pathways were activated, as seen by processing of initiator caspases 8 and 9, loss of mitochondrial membrane potential, and cytochrome c release. Characteristically, antiapoptotic mediators such as Mcl-1, XIAP, survivin, and c-FLIP were downregulated. Consistent with its critical function, c-FLIP overexpression resulted in a significant decrease of SAHA-mediated apoptosis. Enhanced sensitivity to TRAIL (TNF-related apoptosis-inducing ligand) and enhanced TRAIL signaling was seen in CTCL cell lines with high sensitivity, whereas cell lines with moderate response were characterized by downregulation of TRAIL-R2 and weaker TRAIL expression. Comparable proapoptotic responses to SAHA and to the combination with TRAIL were seen in ex vivo tumor T cells of CTCL patients. Thus, activation of extrinsic apoptosis pathways, related to c-FLIP downregulation and enhanced TRAIL signaling, appeared as characteristic for CTCL cell responsiveness to SAHA. An improved understanding of the pathways may facilitate its targeted use and the selection of suitable combinations.

Nonsteroidal Anti-Inflammatory Drugs Induce Apoptosis in Cutaneous T-Cell Lymphoma Cells and Enhance Their Sensitivity for TNF-Related Apoptosis-Inducing Ligand

Cutaneous T-cell lymphomas (CTCL) form a heterogeneous group of non-Hodgkins lymphomas of the skin. In previous studies, we had characterized CTCL cells as resistant to the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which correlated to pronounced expression of the caspase-8/-10 inhibitor c-FLIP. For identification of proapoptotic strategies in CTCL cells and for overcoming their death ligand resistance, we investigated the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid, sodium salicylate, and diclofenac (DF). These drugs strongly enhanced apoptosis, as well as decreased CTCL cell proliferation and vitality, and DF furthermore sensitized for TRAIL-induced apoptosis. Full activation of the caspase cascade (caspase-3, -8, -9) and decreased mitochondrial membrane potential were characteristic for NSAID treatment, whereas cytochrome c release was seen only for DF. Downregulation of Mcl-1 and enhanced surface expression of TRAIL were seen in response to NSAIDs. Most characteristic for apoptosis induction was the downregulation of c-FLIP. In agreement with the critical role of c-FLIP for apoptosis deficiency of CTCL cells, its overexpression decreased NSAID-mediated apoptosis and its downregulation by small hairpin RNA-enhanced apoptosis. The study provides a rationale for the use of NSAIDs as a new therapeutic option for CTCL patients. Supporting this concept, ex vivo lymphoma cells of CTCL patients also revealed significant sensitivity for NSAID treatment.

Evaluation of T-cell clonality in archival skin biopsy samples of cutaneous T-cell lymphomas using the biomed-2 PCR protocol.

Recently, several European centers of lymphoma diagnosis and research developed various polymerase chain reaction (PCR) methods for clonality analysis in suspect T-cell and B-cell proliferations (Biomed-2 Concerted Action). They have mainly been applied to frozen material of systemic B-cell and T-cell malignancies. Thus far, only limited data exist with regard to cutaneous T-cell lymphoma (CTCL) and paraffin-embedded material. Thus, we applied the Biomed-2 T-cell receptor (TCR) γ and TCRβ PCR as well as an in-house TCRγ PCR to a collection of 107 archival skin samples (84 CTCL, 3 systemic TCL and 20 controls). As a result, the Biomed-2 TCRγ PCR revealed 81% clonality, the in-house TCRγ method revealed 86% clonality, and the Biomed-2 TCRβ revealed 78% clonality in CTCL samples generating at least the 300 bp fragment in the Biomed-2 control PCR. We found clonal TCRβ rearrangements in 5 of 17 CTCL samples that were polyclonal in the Biomed-2 TCRγ PCR. By combining all Biomed-2 assays, one or more clonal rearrangements were detected in 87% of CTCL and in all 3 systemic TCLs. By combining all TCR PCR assays applied here, clonality was shown in 90% of the CTCL cases. In conclusion, we showed that the Biomed-2 TCR PCR worked well with DNA from paraffin-embedded tissue, revealing a high-clonality detection rate in CTCL, and thus should be highly recommended for routine molecular analysis. In addition, the performance of our in-house TCRγ assay verifies our previously published findings on clonally expanded T-cells in CTCL.

The tumour suppressor p53 is frequently nonfunctional in Sézary syndrome.

Summary Background  Primary cutaneous T‐cell lymphomas (CTCLs) are a heterogeneous group with Sézary syndrome (SS) as one of the most aggressive variants. Recently, we identified a loss of E2A as a recurrent event in SS, which enhanced proliferation via upregulation of the proto‐oncogene MYC. MYC‐induced transformation usually requires deleterious alterations of key apoptotic genes including p53; however, p53 functionality and mutation status in SS are unclear.

Dimethyl fumarate restores apoptosis sensitivity and inhibits tumor growth and metastasis in CTCL by targeting NF-κB

Despite intensive efforts in recent years, a curative therapy for cutaneous T-cell lymphoma (CTCL) has not yet been developed. Therefore, the establishment of new therapeutic approaches with higher efficacy rates and milder side effects is strongly desired. A characteristic feature of the malignant T-cell population in CTCL is resistance toward cell death resulting from constitutive NF-κB activation. Therefore, NF-κB-dependent cell death resistance represents an interesting therapeutic target in CTCL because an NF-κB-directed therapy would leave bystander T cells widely unaffected. We investigated the effects of dimethyl fumarate (DMF) on CTCL cells in vitro and in vivo. DMF induced cell death in primary patient-derived CD4(+) cells and CTCL cell lines, but hardly in T cells from healthy donors. DMF-induced cell death was linked specifically to NF-κB inhibition. To study the impact of DMF in vivo, we developed 2 CTCL xenograft mouse models with different cutaneous localizations of the T-cell infiltrate. DMF treatment delayed the growth of CTCL tumors and prevented formation of distant metastases. In addition, DMF induced increased cell death in primary CTCL tumors and in liver metastases. In summary, DMF treatment represents a remarkable therapeutic option in CTCL because it restores CTCL apoptosis in vitro and in preclinical models in vivo and prevents spreading of the disease to distant sites. DMF treatment is of particular promise in CTCL because DMF is already in successful clinical use in the treatment of psoriasis and multiple sclerosis allowing fast translation into clinical studies in CTCL.

Molecular diagnostics in cutaneous lymphomas

Routine diagnosis of cutaneous lymphomas is based on its characteristic clinical and histopathologic features. However, the broad clinical and histological spectrum of the disease, especially of its early stages and rare variants, often complicates the differentiation between malignant and benign lymphoproliferative disorders. In these cases demonstration of a clonal Tor B-cell population by detection of identically rearranged immune receptor genes provides an adjunct. Using this approach, in combination with standardized PCR techniques according to the BIOMED-2 protocol delivers in most reliable and reproducible results with a high sensitivity. Here, we summarize important preand post-analytical aspects of clonality testing, and provide guidelines for interpretation of clonality testing results as well for the primary diagnosis of a cutaneous lymphoma and for staging analysis, e. g. corresponding blood and lymph nodes. Markus Möbs 1 , Lorenzo Cerroni 2 , Michael J. Flaig 3 , Dido Lenze 4 , Michael Hummel 4 , Chalid Assaf 1,5

Pathogenesis of Mycosis fungoides

Mycosis fungoides is the most common type of primary cutaneous lymphomas. The phenotype of the tumor cell corresponds to an effector/memory‐type of helper T cell which, given its repertoire of homing receptors, is specialized for recirculation through the skin. In recent years genetic analyses have uncovered various chromosomal aberrations in the tumour cells of mycosis fungoides. Their relevance to the pathogenesis and clinical appearance are discussed in the following.

IL32 Is Progressively Expressed in Mycosis Fungoides Independent of Helper T-cell 2 and Helper T-cell 9 Polarization

Ohmatsu and colleagues report the consistently high and increasing expression of IL32 in cutaneous T-cell lymphoma mycosis fungoides (MF) compared with benign inflammatory skin diseases, and these findings correlate with increases in IFNγ mRNA, suggesting that IL32 may be an autocrine cytokine in MF progression. Mycosis fungoides, the most common type of cutaneous T-cell lymphoma (CTCL), is characterized by a helper T-cell 2 (Th2) skewing with a mature CD4+ memory T-cell phenotype. Using skin samples from patients with mycosis fungoides (n = 21), healthy volunteers (n = 17), and individuals with atopic dermatitis (n = 17) and psoriasis (n = 9), we found IL32 mRNA expression significantly higher in mycosis fungoides samples than in samples from benign inflammatory skin diseases, and its expression increases with disease progression. By IHC and immunofluorescence, we confirmed IL32 protein expression in many CD3+CD4+ T cells and some epidermotropic T cells in mycosis fungoides lesions. MyLa cells (a mycosis fungoides cell line) express IL32, which, in turn, could promote cellular proliferation and viability in a dose-dependent fashion. IL32-treated MyLa and CTCL HH cells upregulated cell proliferation and survival genes. Of the major “polarizing” T-cell cytokines, only IFNγ mRNA increases with mycosis fungoides progression and positively correlates with IL32 mRNA expression. Th2 cytokines do not positively correlate with IL32 mRNA expression or mycosis fungoides progression. Furthermore, by flow cytometry, IL32 production by circulating activated T cells in healthy individuals was found in both IFNγ+ and IFNγ− cells but not in IL4+ or IL13+ cells. In conclusion, we have identified IL32+ cells as the likely tumor cells in mycosis fungoides, and demonstrated that IL32 mRNA expression increases with mycosis fungoides progression and is significantly higher than mRNA expression in other skin diseases, and that some IL32+ T cells are independent from the defined Th subsets. Thus, IL32 may play a unique role in mycosis fungoides progression as an autocrine cytokine. Cancer Immunol Res; 2(9); 890–900. ©2014 AACR.

Zanolimumab, a human monoclonal antibody targeting CD4 in the treatment of mycosis fungoides and Sézary syndrome

Background: The most common type of primary cutaneous T cell-lymphomas (CTCLs), which are characterised by a clonal proliferation of malignant skin-homing CD4+ lymphocytes, is mycosis fungoides (MF) and its rare leukaemic variant Sézary syndrome (SS). Objective: Zanolimumab is a high affinity human monoclonal IgG1k antibody, targeting the CD4-molecule. It exhibits cytotoxic and antiproliferative effects and has previously shown efficacy in CTCLs. Methods: Literature and reference research was done by using Pubmed and updates of ongoing studies were taken from American Society of Clinical Oncology (ASCO) and American Society of Hematology (ASH )annual meeting abstracts. Results: This article gives an overview about efficacy, tolerability and safety as well as chemistry, pharmacodynamics and pharmacokinetics of zanolimumab in the treatment of CTCLs.