Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that

Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with single-cell-resolution protein abundance measurements. Here, we introduce Deep Visual Proteomics (DVP), which combines artificial-intelligence-driven image analysis of cellular phenotypes with automated single-cell or single-nucleus laser microdissection and ultra-high-sensitivity mass spectrometry. DVP links protein abundance to complex cellular or subcellular phenotypes while preserving spatial context. By individually excising nuclei from cell culture, we classified distinct cell states with proteomic profiles defined by known and uncharacterized proteins. In an archived primary melanoma tissue, DVP identified spatially resolved proteome changes as normal melanocytes transition to fully invasive melanoma, revealing pathways that change in a spatial manner as cancer progresses, such as mRNA splicing dysregulation in metastatic vertical growth that coincides with reduced interferon signaling and antigen presentation. The ability of DVP to retain precise spatial proteomic information in the tissue context has implications for the molecular profiling of clinical samples.

Testicular germ cell cancers in young adult men derive from a precursor lesion called carcinoma in situ (CIS) of the testis. CIS cells were suggested to arise from primordial germ cells or gonocytes. However, direct studies on purified samples of CIS cells are lacking. To overcome this problem, we performed laser microdissection of CIS cells. Highly enriched cell populations were obtained and subjected to gene expression analysis. The expression profile of CIS cells was compared with microdissected gonocytes, oogonia, and cultured embryonic stem cells with and without genomic aberrations. Three samples of each tissue type were used for the analyses. Unique expression patterns for these developmentally very related cell types revealed that CIS cells were very similar to gonocytes because only five genes distinguished these two cell types. We did not find indications that CIS was derived from a meiotic cell, and the similarity to embryonic stem cells was modest compared with gonocytes. Thus, we provide new evidence that the molecular phenotype of CIS cells is similar to that of gonocytes. Our data are in line with the idea that CIS cells may be gonocytes that survived in the postnatal testis. We speculate that disturbed development of somatic cells in the fetal testis may play a role in allowing undifferentiated cells to survive in the postnatal testes. The further development of CIS into invasive germ cell tumors may depend on signals from their postpubertal niche of somatic cells, including hormones and growth factors from Leydig and Sertoli cells. [Cancer Res 2009;69(12):5241-50]

FOXP3 is a unique marker for CD4 þ CD25 þ regulatory T cells (Tregs). In solid tumours, high numbers of Tregs are associated with a poor prognosis. Knowledge about the implications of Tregs for the behaviour of haematological malignancies is limited. In this study, skin biopsies from 86 patients with mycosis fungoides (MF) and cutaneous T-cell lymphoma (CTCL) unspecified were analysed for the expression of FOXP3 on tumour cells and tumour-infiltrating Tregs. Labelling of above 10% of the neoplastic cells was seen in one case classified as an aggressive epidermotropic CD8 þ cytotoxic CTCL. In the remaining 85 cases, the atypical neoplastic infiltrate was either FOXP3 negative (n ¼ 80) or contained only very occasional weakly positive cells (n ¼ 5). By contrast, all biopsies showed varying numbers of strongly FOXP3 þ tumour-infiltrating Tregs. MF with early or infiltrated plaques had significantly higher numbers of FOXP3 þ Tregs than CTCL unspecified or advanced MF with tumours or transformation to large cell lymphoma. An analysis of all patients demonstrated that increasing numbers of FOXP3 þ Tregs were associated with improved survival in both MF and CTCL unspecified. In conclusion, our data indicate that the presence of FOXP3 þ Tregs in CTCL is associated with disease stage and patient survival.

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This paper reports that aggressive antibiotic treatment inhibits disease activity and lymphocyte proliferation in cutaneous T-cell lymphoma (CTCL). The study offers important evidence for a link between bacterial infection, activation of the immune system, and CTCL progression.

Sé zary syndrome (SS) is an aggressive variant of cutaneous T-cell lymphoma. During disease progression, immunodeficiency develops; however, the underlying molecular and cellular mechanisms are not fully understood. Here, we study the regulatory T cell (Treg) function and the expression of FOXP3 in SS. We demonstrate that malignant T cells in 8 of 15 patients stain positive with an anti-FOXP3 antibody. Western blotting analysis shows expression of two low molecular splice forms of FOXP3, but not of wild-type (wt) FOXP3. The malignant T cells produce interleukin-10 and TGF-b and suppress the growth of non-malignant T cells. The Treg phenotype and the production of suppressive cytokines are driven by aberrant activation of Jak3 independent of the FOXP3 splice forms. In contrast to wt FOXP3, the low molecular splice forms of FOXP3 have no inhibitory effect on nuclear factor-jB (NF-jB) activity in reporter assays which is in keeping with a constitutive NF-jB activity in the malignant T cells. In conclusion, we show that the malignant T cells express low molecular splice forms of FOXP3 and function as Tregs. Furthermore, we provide evidence that FOXP3 splice forms are functionally different from wt FOXP3 and not involved in the execution of the suppressive function. Thus, this is the first description of FOXP3 splice forms in human disease.

Deregulation of Notch signaling has been linked to the development of T-cell leukemias and several solid malignancies. Yet, it is unknown whether Notch signaling is involved in the pathogenesis of mycosis fungoides and Sézary syndrome, the most common subtypes of cutaneous T-cell lymphoma. By immunohistochemistry of 40 biopsies taken from skin lesions of mycosis fungoides and Sézary syndrome, we demonstrated prominent expression of Notch1 on tumor cells, especially in the more advanced stages. The γ-secretase inhibitor I blocked Notch signaling and potently induced apoptosis in cell lines derived from mycosis fungoides (MyLa) and Sézary syndrome (SeAx, HuT-78) and in primary leukemic Sézary cells. Specific down-regulation of Notch1 (but not Notch2 and Notch3) by siRNA induced apoptosis in SeAx. The mechanism of apoptosis involved the inhibition of nuclear factor-κB, which is the most important prosurvival pathway in cutaneous T-cell lymphoma. Our data show that Notch is present in cutaneous T-cell lymphoma and that its inhibition may provide a new way to treat cutaneous T-cell lymphoma.