Ingrid Moll

UV-B-type mutations and chromosomal imbalances indicate common pathways for the development of Merkel and skin squamous cell carcinomas

Two developmentally highly divergent nonmelanoma skin cancers, the epidermal squamous cell carcinomas (SCC) and the neuroendocrine Merkel cell carcinomas (MCC), occur late in life at sun‐exposed body sites. To determine whether these similarities may indicate common genetic alterations, we studied the genetic profile of 10 MCCs and analyzed 6 derived cell lines and 5 skin SCC lines by comparative genomic hybridization (CGH) and molecular genetic analyses. Although the MCCs were highly divergent—only 3 of the 10 tumors exhibited common gains and losses—they shared gain of 8q21‐q22 and loss of 4p15‐pter with the genetically much more homogeneous SCC lines. In addition, 2 of 5 SCC and 2 of 6 MCC lines exhibited UV‐B‐type‐specific mutations in the p53 tumor‐suppressor gene and a high frequency (9/11) of CC→TT double base changes in codon 27 of the Harvey (Ha)‐ras gene. Since 45% of the tumor lines were homozygous for this nucleotide substitution compared to 14% of the controls and in 1 MCC patient the wild‐type allele was lost in the tumor, this novel polymorphism may contribute to tumor development. On the other hand, loss of 3p, characteristic for SCCs, was rare in MCCs. Although in 2 of 3 SCC lines 3p loss was correlated with reduced expression of the FHIT (fragile histidine triad) gene, the potential tumor suppressor mapped to 3p14.2 and 2 MCC lines with normal 3p showed aberrant or no FHIT transcripts. Taken together, in addition to the common UV‐B‐specific mutations in the p53 and Ha‐ras gene, MCCs and SCCs also share chromosomal imbalances that may point to a common environmental‐derived (e.g., UV‐A) oxidative damage.

CEACAM1 Expression in Cutaneous Malignant Melanoma Predicts the Development of Metastatic Disease

PURPOSE The cell adhesion molecule CEACAM1 is involved in intercellular adhesion and subsequent signal transduction events in a number of epithelia. CEACAM1 downregulation has been demonstrated in colorectal and prostate carcinomas. This study sought to analyze whether its expression in malignant melanoma is associated with metastasis. PATIENTS AND METHODS CEACAM1 expression was immunohistochemically evaluated in 100 primary cutaneous malignant melanomas and correlated with metastasis in a 10-year follow-up. Furthermore, CEACAM1 expression was analyzed in metastatic lesions (11 distant metastases and six sentinel lymph node metastases). Univariate Kaplan-Meier analysis and multivariate Cox proportional hazard regression analysis adjusted for standard prognostic indicators were performed to assess the prognostic relevance of CEACAM1 expression. RESULTS A total of 28 of 40 patients with CEACAM1-positive primary melanomas developed metastatic disease, compared with only six of 60 patients with CEACAM1-negative melanomas. Often, the strongest CEACAM1 expression was observed at the invading front. In addition, CEACAM1 expression was preserved in the metastatic lesions. Kaplan-Meier analysis revealed a highly significant association between CEACAM1 expression and metastasis (P <.0001); multivariate Cox regression analysis, including CEACAM1 expression status adjusted for tumor thickness, presence of ulceration, and mitotic rate, confirmed that CEACAM1 is an independent factor for the risk of metastasis and demonstrated that the predictive value of CEACAM1 expression is superior to that of tumor thickness. CONCLUSION Expression of the cell adhesion molecule CEACAM1 in the primary tumors in melanoma patients is associated with the subsequent development of metastatic disease. This raises the possibility of a functional role for this cell adhesion molecule in the metastatic spread it indicates.

Overexpression of the cell adhesion molecule L1 is associated with metastasis in cutaneous malignant melanoma

Modulation of cell adhesion molecule expression plays a key role in melanoma metastasis. In particular, the expression of the cell adhesion molecule L1 has been associated with the metastatic phenotype in a murine model of malignant melanoma. However, no such association between L1 expression and metastasis has been investigated in a clinical study. Therefore, L1 expression was determined immunohistochemically in 100 cases of malignant melanoma and correlated with metastasis in a 10-year retrospective study. Furthermore, nine distant metastases and five sentinel lymph node metastases were analysed for their L1 expression. Additionally, the expression of alpha2,3 sialic acid residues, which are recognised by the siglec domain of L1, was determined by Maackia amurensis agglutinin (MAA) lectin histochemistry. The log-rank test between Kaplan-Meier curves revealed a positive association between L1 expression and metastasis (P<0.0001) and multivariate Cox regression analysis adjusted for tumour thickness, ulceration and mitotic rate confirmed the prognostic power of L1 in malignant melanoma. As alpha2,3 sialic acid residues were absent in melanoma cells, homotypic adhesion between melanoma cells via their siglec domain can be excluded, suggesting a different adhesive function of L1 during melanoma metastasis. The functional role of L1 was further stressed by the fact that its expression was preserved in metastatic lesions.

Sealing the live part of the skin: The integrated meshwork of desmosomes, tight junctions and curvilinear ridge structures in the cells of the uppermost granular layer of the human epidermis

In the literature the question of whether a system structurally and functionally related to the barrier function of the tight junctions (TJs) of polarized epithelia exists in the epidermis has been and still is controversially discussed. We have systematically addressed this question in a study of the granular layer of fetal and adult human epidermis, combining different light and electron microscopic methods. We show that the lateral membranes of the cells of the stratum granulosum are connected by an extended subapical complex system integrating desmosomes and TJ structures identified as sites of close membrane-membrane contact and as regions of membrane-to-membrane apposition that in immunoelectron microscopy are positive for TJ marker proteins, notably occludin, indicative of an extended, probably continuous TJ barrier. In addition, we have noted in freeze-fractures of the apical membrane attaching this layer to the basalmost membrane of the stratum corneum an extended system integrating desmosomes with intramembraneous ridge configurations that appear as strands, circles, lariats or complex meshworks showing numerous continuities with the desmosomes. In some regions this system interconnecting desmosomes with curvilinear ridge structures occupies the major part of the plasma membrane. The molecular organizations and possible functional contributions of both structural systems positioned at the border between the living portion of the epidermis and the corneal layer are discussed, in particular in relation to the formation of a stable association between the two layers and of a barrier to the paracellular flow of molecules and particles. It is also discussed whether similar structures occur in other keratinizing stratified squamous epithelia, in squamous metaplasias and in tumors derived from such tissues.

Developmental timing of hair follicle and dorsal skin innervation in mice

The innervation of hair follicles offers an intriguing, yet hardly studied model for the dissection of the stepwise innervation during cutaneous morphogenesis. We have used immunofluorescence and a panel of neuronal markers to characterize the developmental choreography of C57BL/6 mouse backskin innervation. The development of murine skin innervation occurs in successive waves. The first cutaneous nerve fibers appeared before any morphological evidence of hair follicle development at embryonic day 15 (E15). Stage 1 and 2 developing hair follicles were already associated with nerve fibers at E16. These fibers approached a location where later in development the follicular (neural) network A (FNA) is located on fully developed pelage hair follicles. Prior to birth (E18), some nerve fibers had penetrated the epidermis, and an additional set of perifollicular nerve fibers arranged itself around the isthmus and bulge region of stage 5 hair follicles, to develop into the follicular (neural) network B (FNB). By the day of birth (P1), the neuropeptides substance P and calcitonin gene‐related peptide became detectable in subcutaneous and dermal nerve fibers first. Newly formed hair follicles on E18 and P1 displayed the same innervation pattern seen in the first wave of hair follicle development. Just prior to epidermal penetration of hair shafts (P5), peptide histidine methionine‐IR nerve fibers became detectable and epidermal innervation peaked; such innervation decreased after penetration (P7– P17). Last, tyrosine hydroxylase‐IR and neuropeptide Y‐IR became readily detectable. This sequence of developing innervation consistently correlates with hair follicle development, indicating a close interdependence of neuronal and epithelial morphogenesis. J. Comp. Neurol. 448:28–52, 2002.

Clusters of Perifollicular Macrophages in Normal Murine Skin: Physiological Degeneration of Selected Hair Follicles by Programmed Organ Deletion

In back skin sections from adolescent C57BL/6 mice, regularly distributed, perifollicular inflammatory cell clusters (PICC) were found located around the distal noncycling portion of about 2% of all hair follicles examined. The PICC and the affected hair follicles were characterized during spontaneously developed or induced hair cycle stages, using antibodies against MHC Class II,F4/80, ER-MP23, NLDC 145, CD4, CD8, γδTCR, IL-1 receptor, and ICAM-1. PICC consisted predominantly of macrophages (MAC), accompanied by a few CD4+ cells, whereas γδTCR+ and CD8+ cells were absent. During anagen and catagen, some of the PICC+ hair follicles showed variable degenerative phenomena reminiscent of scarring alopecia: thickened basement membrane, ectopic MHC II expression, MAC infiltration into the follicle epithelium, and signs of keratinocyte apoptosis. Loss of distal outer root sheath keratinocytes was detected in 10% of PICC+ hair follicles (0.2% of all hair follicles). Because PICC were located in the vicinity of the bulge region, MAC-dependent damage to follicle stem cells might eventually lead to follicle degeneration. These perifollicular MAC clusters around selected hair follicles may indicate the existence of a physiological program of MAC-dependent controlled follicle degeneration by which damaged or malfunctioning follicles are removed by programmed organ deletion (POD).

Which Are the Cells of Origin in Merkel Cell Carcinoma

Merkel cell carcinoma (MCC), a highly aggressive skin tumour with increasing incidence, is associated with the newly discovered Merkel cell polyomavirus (MCPyV). Studies on MCC and MCPyV as well as other risk factors have significantly increased our knowledge of MCC pathogenesis, but the cells of origin, which could be important targets in future therapies, are still unknown. Merkel cells (MCs), the neuroendocrine cells of the skin, were believed to be at the origin of MCC due to their phenotypic similarities. However, for several reasons, for example, heterogeneous differentiation of MCCs and postmitotic character of MCs, it is not very likely that MCC develops from differentiated MCs. Skin stem cells, probably from the epidermal lineage, are more likely to be cells of origin in MCC. Future studies will have to address these questions more directly in order to identify the physiological cells which are transformed to MCC cells.

PAS-positive loops and networks as a prognostic indicator in cutaneous malignant melanoma

Recently, microvascular channels, as detected by PAS histochemistry, were positively correlated with poor prognosis in uveal malignant melanoma. Since uveal melanomas are not penetrated by lymphatic vessels, while cutaneous melanomas are, the question arises as to whether these loops and networks are also of prognostic relevance in cutaneous melanoma. Histochemically and immunohistochemically detected loops and networks in 100 cases of cutaneous malignant melanoma were correlated with the occurrence of metastasis in a 10‐year follow‐up study. To detect these patterns, the significance of various methods (PAS reaction with/without nuclear counterstain, anti‐laminin immunohistochemistry) was investigated. The presence of loops and networks was a highly significant prognostic marker (p<0.0001) for metastasis in cutaneous malignant melanoma. The presence of these patterns proved to have higher prognostic relevance for metastasis than Breslows tumour thickness, especially for stage IB and stage IIA tumours (intermediate thickness/risk). PAS reaction without nuclear counterstain proved to be the best method to detect these patterns. Compared with the conventional staging of Breslows tumour thickness, and especially so for stage IB and IIA melanomas, the determination of PAS‐positive loops and networks in cutaneous malignant melanoma provides additional prognostic information. Copyright

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.

Epidermal adhesion molecules and basement membrane components as target structures of autoimmunity.

Abstract Intraepidermal and dermal-epidermal cohesion are of paramount importance for the integrity of the skin. Some constituent molecules of keratinocyte adhesion complexes and basement membrane-associated structures are the targets of antibody-mediated autoimmune reactions that give rise to various (muco-)cutaneous blistering diseases. The current state of our knowledge about these molecules – along with the main clinical, histological, and immunohistochemical features of the corresponding autoimmune diseases and their pathogenetic mechanisms – comprise the subjects surveyed in this review. Among the desmosomal cadherins (desmogleins and desmocollins) that mediate epidermal cell–cell adhesion, it has been demonstrated that desmoglein 1 and desmoglein 3 are the autoantigens of pemphigus foliaceus and pemphigus vulgaris, respectively, both diseases that result in intraepidermal blistering. Further, desmocollin autoantibodies may be involved in IgA pemphigus. Paraneoplastic pemphigus is associated with autoantibodies directed against the desmosomal plaque protein, desmoplakin. Of the constituents of hemidesmosomes, the plaque protein, BP230 (BPAG1), and the collagen-like transmembrane protein, BP180 (BPAG2), are the autoantigens of bullous pemphigoid and pemphigoid gestationis, the manifestations of both of which include subepidermal blistering. Several diseases arise from autoimmune reactions against certain proteins associated with the basement membrane located beneath hemidesmosomes, for example laminin 5 (cicatricial pemphigoid), ladinin (LAD-1; linear IgA disease), uncein, and collagen VII (epidermolysis bullosa acquisita), the last of which is the constituent protein of the anchoring fibrils. Such recent advances in the elucidation of the molecular nature of autoantigens may serve as the basis for the development of novel molecule-based therapeutic strategies.