Intermittent intense ultraviolet (UV) exposure represents an important aetiological factor in the development of malignant melanoma. The ability of UV radiation to cause tumour-initiating DNA mutations in melanocytes is now firmly established, but how the microenvironmental effects of UV radiation influence melanoma pathogenesis is not fully understood. Here we report that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression, independent of its tumour-initiating effects. UV irradiation enhanced the expansion of tumour cells along abluminal blood vessel surfaces and increased the number of lung metastases. This effect depended on the recruitment and activation of neutrophils, initiated by the release of high mobility group box 1 (HMGB1) from UV-damaged epidermal keratinocytes and driven by Toll-like receptor 4 (TLR4). The UV-induced neutrophilic inflammatory response stimulated angiogenesis and promoted the ability of melanoma cells to migrate towards endothelial cells and use selective motility cues on their surfaces. Our results not only reveal how UV irradiation of epidermal keratinocytes is sensed by the innate immune system, but also show that the resulting inflammatory response catalyses reciprocal melanoma-endothelial cell interactions leading to perivascular invasion, a phenomenon originally described as angiotropism in human melanomas by histopathologists. Angiotropism represents a hitherto underappreciated mechanism of metastasis that also increases the likelihood of intravasation and haematogenous dissemination. Consistent with our findings, ulcerated primary human melanomas with abundant neutrophils and reactive angiogenesis frequently show angiotropism and a high risk for metastases. Our work indicates that targeting the inflammation-induced phenotypic plasticity of melanoma cells and their association with endothelial cells represent rational strategies to specifically interfere with metastatic progression.

SUMMARY B cells infected by Epstein-Barr-Virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression EBV can spread from these cells and cause life threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV infected B cells in vivo, establish a pre-clinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a novel therapeutic approach.

CD40, a member of the tumor necrosis factor (TNF) receptor family, plays an essential role in T cell–dependent immune responses. Because CD40 is widely expressed on the surface of tumor cells in various B cell malignancies, deregulated CD40 signaling has been suggested to contribute to lymphomagenesis. In this study, we show that B cell-specific expression of a constitutively active CD40 receptor, in the form of a latent membrane protein 1 (LMP1)/CD40 chimeric protein, promoted an increase in the number of follicular and marginal zone B cells in secondary lymphoid organs in transgenic mice. The B cells displayed an activated phenotype, prolonged survival and increased proliferation, but were significantly impaired in T cell-dependent immune responses. Constitutive CD40 signaling in B cells induced selective and constitutive activation of the noncanonical NF-κB pathway and the mitogen-activated protein kinases Jnk and extracellular signal–regulated kinase. LMP1/CD40-expressing mice older than 12 mo developed B cell lymphomas of mono- or oligoclonal origin at high incidence, thus showing that the interplay of the signaling pathways induced by constitutive CD40 signaling is sufficient to initiate a tumorigenic process, ultimately leading to the development of B cell lymphomas.

The Epstein-Barr virus (EBV) protein LMP1 is considered to be a functional homologue of the CD40 receptor. However, in contrast to the latter, LMP1 is a constitutively active signaling molecule. To compare B cell-specific LMP1 and CD40 signaling in an unambiguous manner, we generated transgenic mice conditionally expressing a CD40/LMP1 fusion protein, which retained the LMP1 cytoplasmic tail but has lost the constitutive activity of LMP1 and needs to be activated by the CD40 ligand. We show that LMP1 signaling can completely substitute CD40 signaling in B cells, leading to normal B-cell development, activation, and immune responses including classswitch recombination, germinal center formation, and somatic hypermutation. In addition, the LMP1-signaling domain has a unique property in that it can induce class-switch recombination to IgG1 independent of cytokines. Thus, our data indicate that LMP1 has evolved to imitate T-helper cell function allowing activation, proliferation, and differentiation of EBVinfected B cells independent of T cells. IntroductionEpstein-Barr virus (EBV) is a ␥-herpes virus that preferentially infects human B lymphocytes. It has adapted to persist in B cells by encoding proteins mimicking cellular proteins that play important roles in B-cell physiology. 1 An example of the latter is the viral latent membrane protein 1 (LMP1), which is considered to be a functional homologue of the CD40 receptor. 2 CD40 is expressed on antigen-presenting cells including B cells, whereas the CD40 ligand (CD40L, CD154) is expressed mainly on activated T cells. The CD40 receptor belongs to the tumor necrosis factor receptor family, containing an extracellular portion with 4 cysteine-rich domains that mediate ligand binding. CD40-CD40L interactions play a crucial role in the T cell-dependent (TD) immune response. CD40 and CD40L knockout mice show defects in the immunoglobulin (Ig) class-switch recombination (CSR), the formation of germinal centers (GCs), somatic hypermutation (SHM) of Ig genes, and the establishment of B-cell memory. 3,4 In vitro stimulation of the CD40 receptor induces proliferation and activation of B cells, and mediates cytokine-dependent CSR. 5 LMP1 shows several functional similarities with the CD40 receptor. Both triggering of the CD40 receptor and LMP1 expression lead to activation, proliferation, and survival of B cells. 6 The intracellular domains of LMP1 and CD40 both interact with tumor necrosis factor receptor-associated factors (TRAFs) and activate overlapping signaling pathways, including extracellular signalrelated kinase (ERK), c-Jun N-terminal kinase (JNK), p38/MAPK, and NFB. 2 However, LMP1 and CD40 do not interact with exactly the same sets of molecules, indicating also some differences in their signaling properties. 7-10 Thus, both LMP1 and CD40 interact directly with TRAFs 1, 2, 3, and 5, but only CD40, and not LMP1, binds directly to TRAF 6. 7,11 Conversely, LMP1, but not CD40, binds to the tumor necrosis factor receptor-associated death domain protein (TRADD) and recept...

The increasing knowledge about genetic alterations and molecular biomarkers in cancer initiation and progression opens new possibilities for the treatment of various types of cancer. This requires the inclusion of sensitive, and preferably multiplex, methods for the detection of molecular genetic alterations in the toolbox of classic pathology. Multiplex ligation-dependent probe amplification (MLPA) is a multiplex polymerase chain reaction-based method that can detect changes in the gene copy number status, DNA methylation, and point mutations simultaneously. MLPA probes recognize target sequences of only 50 to 100 nucleotides in length. This makes it possible to use MLPA even on highly fragmented DNA, and allows the detection of small deletions encompassing only a single exon. MLPA is a reliable, cost-effective, and robust method that can be performed using a standard thermocycler and capillary electrophoresis equipment, generating results within 24 hours with a short hands-on working time. Up to 50 different genomic locations can be tested in a single reaction, which can be sufficient to detect those genetic alterations that are of diagnostic and prognostic significance in a certain tumor entity. In the last years, MLPA has been used successfully in tumor diagnostics and in cancer research. This review gives an overview on the collected experience of MLPA applications on tumor DNA, about the advantages but also potential pitfalls and limitations of this technique.