Detection and Characterization of Circulating Tumor Cells in Patients with Merkel Cell Carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with increasing incidence and high mortality rates. MCC has recently become the subject of immune checkpoint therapy, but reliable biomarkers for estimating prognosis, risk stratification, and prediction of response are missing. METHODS: Circulating tumor cells (CTCs) were detected in peripheral blood from patients with MCC by use of the CellSearch® system. Moreover, CTCs of selected cases were characterized for Merkel cell polyomavirus (MCPyV), chromosomal aberrations, and programed death ligand 1 (PD-L1) production. RESULTS: Fifty-one patients were tested at first blood draw (baseline), and 16 patients had 2 or 3 consecutive measurements to detect CTCs. At baseline, ≥1 CTC (range, 1-790), >1, or ≥5 CTCs/7.5 mL were detected in 21 (41%), 17 (33%), and 6 (12%) patients, respectively. After a median follow-up of 21.1 months for 50 patients, detection of CTCs correlated with overall survival (≥1, P = 0.030; >1, P < 0.020; and ≥5 CTCs/7.5 mL, P < 0.0001). In multivariate Cox regression analysis, the detection of ≥5 CTCs/7.5 mL adjusted to age and sex compared to that of <5 was associated with a reduced overall survival (P = 0.001, hazard ratio = 17.8; 95% CI, 4.0-93.0). MCPyV DNA and genomic aberrations frequently found in MCC tissues could also be detected in single CTCs. Analyzed CTCs were PD-L1 negative or only weakly positive. CONCLUSIONS: The presence of CTCs is a prognostic factor of impaired clinical outcome, with the potential to monitor the progression of the disease in real time. Molecular characterization of CTCs might provide new insights into the biology of MCC.

Tight Junction Proteins Claudin-1 and Occludin Are Important for Cutaneous Wound Healing.

Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance.

Spontaneous lung metastasis formation of human Merkel cell carcinoma cell lines transplanted into scid mice.

Merkel cell carcinoma (MCC) is an aggressive skin cancer entity that frequently leads to rapid death due to its high propensity to metastasize. The etiology of most MCC cases is linked to Merkel cell polyomavirus (MCPyV), a virus which is monoclonally integrated in up to 95% of tumors. While there are presently no animal models to study the role of authentic MCPyV infection on transformation, tumorigenesis or metastasis formation, xenograft mouse models employing engrafted MCC-derived cell lines (MCCL) represent a promising approach to study certain aspects of MCC pathogenesis. Here, the two MCPyV-positive MCC cell lines WaGa and MKL-1 were subcutaneously engrafted in scid mice. Engraftment of both MCC cell lines resulted in the appearance of circulating tumor cells and metastasis formation, with WaGa-engrafted mice showing a significantly shorter survival time as well as increased numbers of spontaneous lung metastases compared to MKL-1 mice. Interestingly, explanted tumors compared to parental cell lines exhibit an upregulation of MCPyV sT-Antigen expression in all tumors, with WaGa tumors showing significantly higher sT-Antigen expression than MKL-1 tumors. RNA-Seq analysis of explanted tumors and parental cell lines furthermore revealed that in the more aggressive WaGa tumors, genes involved in inflammatory response, growth factor activity and Wnt signalling pathway are significantly upregulated, suggesting that sT-Antigen is the driver of the observed differences in metastasis formation.

Diverse regulation of claudin-1 and claudin-4 in atopic dermatitis.

Tight junctions are important for skin barrier function. The tight junction protein claudin 1 (Cldn-1) has been reported to be down-regulated in nonlesional skin of atopic dermatitis (AD) patients. In contrast, we did not observe a significant down-regulation of Cldn-1 in nonlesional skin of the AD cohort used in this study. However, for the first time, a significant down-regulation of Cldn-1 in the upper and lower epidermal layers of lesional skin was detected. In addition, there was a significant up-regulation of Cldn-4 in nonlesional, but not lesional, AD skin. For occludin, no significant alterations were observed. In an AD-like allergic dermatitis mouse model, Cldn-1 down-regulation in eczema was significantly influenced by dermal inflammation, and significantly correlated with hallmarks of eczema (ie, increased keratinocyte proliferation, altered keratinocyte differentiation, increased epidermal thickness, and impaired barrier function). In human epidermal equivalents, the addition of IL-4, IL-13, and IL-31 resulted in a down-regulation of Cldn-1, and Cldn1 knockdown in keratinocytes resulted in abnormal differentiation. In summary, we provide the first evidence that Cldn-1 and Cldn-4 are differentially involved in AD pathogenesis. Our data suggest a role of Cldn-1 in AD eczema formation triggered by inflammation.

Immunohistochemical analyses point to epidermal origin of human Merkel cells.

Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, ß1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. ß1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.

Merkel cell polyomavirus detection in Merkel cell cancer tumors in Northern Germany using PCR and protein expression.

Merkel cell carcinoma is a highly malignant skin cancer which predominantly occurs in elderly and immunocompromised persons. The identification of the Merkel cell polyomavirus (MCPyV) has inaugurated a new understanding of Merkel cell carcinoma pathogenesis. The frequent detection of the virus in Merkel cell carcinoma tissue (70-90%), its monoclonal integration in the tumor cells and the expression of viral oncogenes highly suggest that MCPyV is causally linked to the pathogenesis of the majority of Merkel cell cancer (MCC) cases. Using qualitative and quantitative PCR together with immunohistochemical staining this study aimed at characterizing the presence of MCPyV sequences and viral early gene expression in a cohort of MCC cases (n = 32) selected in Northern Germany. 40-57% of the cases were identified as MCPyV positive with 40.6% of the cases positive by immunohistochemical staining and 51.6-57.6% positive by PCR. Interestingly, in the majority (64%) of LT-Antigen positive tumors only 25-50% of tumor cells express LT-Antigen. These data are in accord with published studies describing heterogeneity in MCPyV viral loads and suggest that detection of MCPyV in Merkel cell carcinoma by PCR should be undertaken using multiple primer pairs.

Tight junctions and differentiation--a chicken or the egg question?

Skin barrier function is indispensable to prevent the uncontrolled loss of water and solutes and to protect the body from external assaults. To fulfil this function, keratinocytes undergo a complex pathway of differentiation that terminates in the formation of the stratum corneum. Additionally, tight junctions (TJs), which are cell-cell junctions localized in the stratum granulosum, are involved in the barrier function of the skin. Important biological and clinical roles of TJs are strongly suggested by altered TJ protein levels and distribution in skin diseases like psoriasis, ichthyosis and atopic dermatitis. Because these skin diseases show alterations in differentiation and TJs, it was suggested that changes in TJs might simply be a consequence of altered differentiation. However, in this viewpoint, we like to argue that the situation is not as simple and depends on the specific microenvironment. We discuss three hypotheses regarding the interplay between TJs/TJ proteins and differentiation: (1) TJs/TJ proteins are influenced by differentiation, (2) differentiation is influenced by TJs/TJ proteins, and (3) TJs/TJ proteins and differentiation are independent of each other. In addition, the concept is introduced that both processes are going on at the same time, which means that while one specific TJ protein/barrier component might be influenced by differentiation, the other may influence differentiation.

Diagnostics of autoimmune bullous diseases in German dermatology departments.

BACKGROUND: No consistent data are available on the currently employed diagnostic tools for autoimmune bullous diseases in Germany. The aim of this survey was to describe currently performed diagnostic methods for bullous autoimmune diseases in German dermatology departments. METHODS: A standardized questionnaire evaluated the available diagnostic methods i. e. direct immunofluorescence microscopy (IFM), indirect IFM, commercial ELISA systems, and non-commercial serological tests as well as the number of samples per year in all 34 university and 39 non-university dermatology departments. RESULTS: The overall return rate was 89 %, 100 % and 79 % for the university and non-university departments, respectively. Direct IFM was the most frequently used method and was applied in 98 % of the responding departments. In 74 % of the responding departments, indirect IFM was used mainly on monkey esophagus and human salt-split skin. Commercial ELISA systems were employed in 58 % of the clinics; all of them used anti-desmoglein ELISA, while anti-BP180 and anti-BP230 ELISA were established in 49 % and 48 % of departments, respectively. Non-commercial analytic methods were only performed in 22 % of the departments. CONCLUSIONS: The high return rate of this survey allows a relatively precise description of the current diagnostic methods used in German dermatology departments. Standard diagnostic tests are available nationwide and in bullous pemphigoid and pemphigus, the antigen-specific detection of autoantibodies is routinely performed in half of the departments. Rare disorders may be diagnosed by cooperation with some specialized centers.

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non-diabetic cells.

During early wound healing (WH) events Connexin 43 (Cx43) is down-regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down-regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non-diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non-diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non-diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non-diabetic cells. The cause of the discrepancy between diabetic and non-diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368-phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.

Intercellular adhering junctions with an asymmetric molecular composition: desmosomes connecting Merkel cells and keratinocytes.

Merkel cells (MCs) are special neuroendocrine epithelial cells that occur as individual cells or as cell groups within the confinements of a major epithelium formed and dominated by other epithelial cells. In the epidermis and some of its appendages MCs are mostly located in the basal cell layer, occasionally also in suprabasal layers and generally occur in linear arrays in outer root sheath cell layers of hair follicles. As MCs are connected to the adjacent keratinocytes by a series of adhering junctions (AJs), of which the desmosomes are the most prominent, these junctions represent heterotypic cell-cell connections, i.e. a kind of structure not yet elucidated in molecular terms. Therefore, we have studied these AJs in order to examine the molecular composition of the desmosomal halves. Using light- and electron-microscopic immunolocalization and keratin 20 as the MC-specific cell type marker we show that the plaques of the MC half of the desmosomes specifically and constitutively contain plakophilin Pkp2. This protein, however, is absent in the keratinocyte half of such heterotypic desmosomes which instead contains Pkp1 and/or Pkp3. We discuss the developmental, tissue-architectonic and functional importance of such asymmetric junctions in normal physiology as well as in diseases, in particular in the formation of distant tumor cell metastasis.

Expression of keratins in cutaneous epithelial tumors and related disorders--distribution and clinical significance.

Keratins are a highly diverse family of cytoskeletal proteins and important markers of epithelial cell differentiation. In this review, applying the new keratin nomenclature recently introduced, we summarize and discuss the distribution and significance of keratin patterns in cutaneous epithelial tumors in relation to the epithelial structures of normal human skin. The available literature data show that the analysis of keratin profiles broadens our understanding of the differentiation, nature and histogenetic origin of the various, highly singular epithelial tumors arising in the skin. Moreover, keratins may aid in histological diagnosis and, in certain instances, may be helpful for the recognition of tumor malignancy and aggressiveness. Furthermore, we briefly address the topic of keratin-related skin disorders.

Detection of Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma cell lines: cell morphology and growth phenotype do not reflect presence of the virus.

The recently discovered human polyomavirus (MCPyV) is frequently found in Merkel cell carcinoma (MCC) tissue and is believed to be causally linked to MCC pathogenesis. While cell lines established from MCC represent a valuable tool to study the contribution of MCPyV to MCC pathogenesis, hitherto only 1 MCPyV-positive line has been described. We have analyzed 7 MCC cell lines for the presence, integration pattern and copy number of MCPyV. In 5 cell lines, MCPyV specific sequences were detected. In 3 of these lines, multiple copies of viral genomes per cell were detected, and sequencing of PCR amplificates identified distinct mutations predicted to lead to the expression of a truncated large T-Antigen (LT-Ag). In 1 cell line, clonal integration of concatamerized viral genomes was confirmed by Southern Blotting. MCC cell lines are conventionally categorized as "classic" or "variant" and further divided into 4 subtypes, based on expression of neuroendocrine markers and morphology. While it has been suggested that the presence of MCPyV might promote a classic phenotype, such a notion is not supported by our data. Instead, we find MCPyV-positive as well as -negative lines of the classic variety, indicating that the distinguishing features are either inherently independent of viral infection or have become so in the course of tumorigenesis and/or cell line establishment. We therefore suggest a novel classification scheme based on MCPyV presence, integration patterns and T-Ag mutations. The cell lines described here extend the repertoire of available MCPyV-positive MCC-lines and should aid in the elucidation of the role of MCPyV in the pathogenesis of MCC.

Alteration of tight junction proteins is an early event in psoriasis: putative involvement of proinflammatory cytokines.

Psoriasis is an inflammatory skin disease characterized by hyperproliferation of keratinocytes, impaired barrier function, and pronounced infiltration of inflammatory cells. Tight junctions (TJs) are cell-cell junctions that form paracellular barriers for solutes and inflammatory cells. Altered localization of TJ proteins in the epidermis was described in plaque-type psoriasis. Here we show that localization of TJ proteins is already altered in early-stage psoriasis. Occludin, ZO-1, and claudin-4 are found in more layers than in normal epidermis, and claudin-1 and -7 are down-regulated in the basal and in the uppermost layers. In plaque-type psoriasis, the staining patterns of occludin and ZO-1 do not change, whereas the claudins are further down-regulated. Near transmigrating granulocytes, all TJ proteins except for junctional adhesion molecule-A are down-regulated. Treatment of cultured keratinocytes with interleukin-1beta and tumor necrosis factor-alpha, which are present at elevated levels in psoriatic skin, results in an increase of transepithelial resistance at early time points and a decrease at later time points. Injection of interleukin-1beta into an ex vivo skin model leads to an up-regulation of occludin and ZO-1, resembling TJ protein alteration in early psoriasis. Our results show for the first time that alteration of TJ proteins is an early event in psoriasis and is not the consequence of the more profound changes found in plaque-type psoriasis. Our data indicate that cytokines are involved in alterations of TJ proteins observed in psoriasis.

Evidence for distinct populations of human Merkel cells.

Merkel cells (MCs) are neuroendocrine cells of unknown origin located in the skin. They are identified at electron microscopic level by electron dense granules, at light microscopic level by the presence of cytokeratins 8, 18, 19 and 20. Contradictory reports concerning the presence of other molecules of epithelial as well as neural origin prompted us to investigate whether there are distinct populations of human MCs. Here, we show the heterogeneous expression of villin, N-CAM, NGF-R, and neurofilaments in MCs. Synaptophysin is found in all MCs but with different intensity, nestin is absent. Expression patterns vary between interfollicular epidermis, hair follicles and glabrous epidermis. We conclude that there are distinct populations of MCs, but all populations contain markers for epithelial as well as neural cells. Putative functions of the distinct populations are discussed.

Adjuvant photodynamic therapy does not prevent recurrence of condylomata acuminata after carbon dioxide laser ablation-A phase III, prospective, randomized, bicentric, double-blind study.

BACKGROUND: Recurrence after therapy for anogenital warts, or condylomata acuminata (CA), is common. Topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is efficient in the treatment of CA, but one problem with PDT is the limited penetration depth of photosensitizer and light. Pre-PDT vaporization of CA using a carbon dioxide (CO(2)) laser may enhance efficacy. OBJECTIVES: CO(2) laser ablation was followed by ALA-PDT in a phase III prospective randomized bicenter double-blind study to prevent recurrence of CA. MATERIALS AND METHODS: One hundred seventy-five patients with CA received CO(2) laser vaporization plus adjuvant ALA-PDT (n=84) or adjuvant placebo-PDT (n=91). A 20% ALA or placebo ointment was applied to the CA area 4 to 6 hours before CO(2) laser vaporization, followed by illumination with red light (600-740 nm, 100 mW/cm(2), 100 J/cm(2)). RESULTS: Cumulative recurrence rate 12 weeks after treatment was 50.0% in the ALA-PDT group, versus 52.7% in the placebo-PDT group (p=.72). No statistically significant difference between groups was detected with regard to recurrence rates up to 12 months after treatment. No major complications were observed. CONCLUSION: Adjuvant ALA-PDT of CA after CO(2) laser ablation was well tolerated, but no significant difference with regard to recurrence rate was observed from CO(2) laser vaporization alone.

Intra-Patient Heterogeneity of Circulating Tumor Cells and Circulating Tumor DNA in Blood of Melanoma Patients.

Despite remarkable progress in melanoma therapy, the exceptional heterogeneity of the disease has prevented the development of reliable companion biomarkers for the prediction or monitoring of therapy responses. Here, we show that difficulties in detecting blood-based markers, like circulating tumor cells (CTC), might arise from the translation of the mutational heterogeneity of melanoma cells towards their surface marker expression. We provide a unique method, which enables the molecular characterization of clinically relevant CTC subsets, as well as circulating tumor DNA (ctDNA), from a single blood sample. The study demonstrates the benefit of a combined analysis of ctDNA and CTC counts in melanoma patients, revealing that CTC subsets and ctDNA provide synergistic real-time information on the mutational status, RNA and protein expression of melanoma cells in individual patients, in relation to clinical outcome.

CD34(+) fibrocytes in melanocytic nevi and malignant melanomas of the skin.

CD34(+) fibrocytes are constitutive elements of the human connective tissue. The stroma associated with invasive carcinomas is characterized by a stereotypic loss of CD34(+) fibrocytes and a phenotype change towards CD34(-) alpha-Smooth muscle actin (SMA)(+) myofibroblasts. Secreted protein acidic and rich in cysteine (SPARC) is an important mediator of tumor-associated stromal remodeling. Melanocytic lesions of the skin have not been investigated as to this aspect up to now. Thus, we investigated a total of 20 malignant melanomas and 29 melanocytic nevi. The normal dermis and benign melanocytic nevi showed numerous CD34(+) fibrocytes, whereas malignant melanomas were devoid of this cell type. alpha-SMA-positive myofibroblasts were absent from the normal dermis, melanocytic nevi, and malignant melanomas. SPARC was positive in malignant melanoma cells and negative in their associated stroma, while all melanocytic nevi were completely negative. The stromal phenotype of malignant melanomas (CD34(-) alpha-SMA(-)) differs from that of invasive carcinomas (CD34(-) alpha-SMA(+)) suggesting different pathogenic mechanisms involved in tumor-associated stromal remodeling. SPARC expression appears to be closely related to malignancy in melanocytic lesions.

The different structures containing tight junction proteins in epidermal and other stratified epithelial cells, including squamous cell metaplasia.

In stratified squamous epithelia constituent proteins of tight junctions (TJs) are not restricted to the zonula occludens-related structures of the uppermost living cell layer such as the stratum granulosum of the epidermis but TJ membrane proteins such as occludin and certain members of the claudin family as well as TJ plaque proteins, notably cingulin and protein ZO-1, have also been identified by immunofluorescence and immunoelectron microscopy in more basal layers where they form special cell-cell-connecting structures such as the "lamellated" and the "sandwich" junctions. In the present study, we describe another TJ protein-containing structure, the very small puncta occludentia ("stud junctions"), as the smallest identifiable TJ-like unit that occurs in most, perhaps all strata. We have also determined the specific distributions of TJ proteins in the cell layers of squamous cell metaplasias of the human bronchial tract. Moreover, we show that the occludin-related tetraspanin protein tricellulin-alpha connects and seals the membranes of adjacent "three corner" cell structures of the uppermost layer in keratinocytes growing in culture. We hypothesize the possible occurrence of tricellulin-beta in more basal cell layers of keratinocyte cultures and the general occurrence of different tricellulin splice forms in stratified epithelia in situ, and discuss the possible functions of TJ proteins in stratified epithelia and tumors derived therefrom.