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Background:Cutaneous squamous cell carcinoma (cSCC) is one of the most common malignancies in fair-skinned populations worldwide and its incidence is increasing. Despite previous observations of multiple genetic abnormalities in cSCC, the oncogenic process remains elusive. The purpose of this study was to elucidate key molecular events associated with progression from premalignant actinic keratoses (AKs) to invasive cSCC by transcriptome profiling.Methods:We combined laser capture microdissection with the Affymetrix HGU133 Plus 2.0 microarrays to profile 30 cSCC and 10 AKs.Results:We identified a core set of 196 genes that are differentially expressed between AK and cSCC, and are enriched for processes including epidermal differentiation, cell migration, cell-cycle regulation and metabolism. Gene set enrichment analysis highlighted a key role for the mitogen activated protein kinase (MAPK) pathway in cSCC compared with AK. Furthermore, the histological subtype of the tumour was shown to influence the expression profile.Conclusion:These data indicate that the MAPK pathway may be pivotal to the transition from AK to cSCC, thus representing a potential target for cSCC prevention. In addition, transcriptome differences identified between cSCC subtypes have important implications for future development of targeted therapies for this malignancy. [ABSTRACT FROM AUTHOR]

Background:Novel prognostic biomarkers and therapeutic strategies are urgently required for malignant melanoma. Ecto-5-prime-nucleotidase (NT5E; CD73) overexpression has been reported in several human cancers. The mechanism(s) underlying deregulated expression and the clinical consequences of changes in expression are not known.Methods:We used RT-PCR, qPCR, methylation-specific PCR and pyrosequencing to analyse expression and regulation of NT5E in malignant melanoma cell lines and primary and metastatic melanomas.Results:NT5E is subject to epigenetic regulation in melanoma. NT5E mRNA is downregulated by methylation-dependent transcriptional silencing in the melanoma cell lines SKMel2, SKMel23, WM35, Mel501, Mel505 and C81-61 and expression is reactivated by azacytidine. In contrast, the CpG island is unmethylated and the gene expressed in cultured normal melanocytes. In clinical cases of melanoma, methylation in the NT5E CpG island occurs in both primary and metastatic melanomas and correlates with transcriptional downregulation of NT5E mRNA. Relapse with metastatic disease, particularly to the visceral sites and brain, is more common in primary melanomas lacking NT5E methylation. Primary melanomas with methylation in NT5E show limited metastatic potential or more commonly metastasise predominantly to nodal sites rather than viscera and brain (P=0.01).Conclusion:Deregulation of NT5E expression in melanoma occurs via epigenetic changes in the NT5E CpG island. Confirmation of our results in larger clinical series would support the candidacy of NT5E as a clinical biomarker in melanoma, which could be applied in both primary and relapsed disease. Inhibition of NT5E may have therapeutic potential in melanoma, particularly in patients with more aggressive disease metastatic to viscera or the brain. [ABSTRACT FROM AUTHOR]

Identifying therapeutic targets for cancer treatment relies on consistent changes within particular types or sub-types of malignancy. The ability to define either consistent changes or sub-types of malignancy is often masked by tumor heterogeneity. To elucidate therapeutic targets in cutaneous squamous cell carcinoma (cSCC), the most frequent skin neoplasm with malignant potential, we have developed an integrated approach to gene expression profiling beginning with primary keratinocytes in culture. Candidate drivers of cSCC development were derived by first defining a set of in vitro cancer genes and then comparing their expression in a range of clinical data sets containing normal skin, cSCC and the benign hyper-proliferative condition psoriasis. A small interfering RNA (siRNA) screen of the resulting 21 upregulated genes has yielded targets capable of reducing xenograft tumor volume in vivo. Small-molecule inhibitors for one target, Polo-like kinase-1 (PLK1), are already in clinical trials for other malignancies, and our data show efficacy in cSCC. Another target, C20orf20, is identified as being overexpressed in cSCC, and siRNA-mediated knockdown induces apoptosis in vitro and reduces tumor growth in vivo. Thus, our approach has shown established and uncharacterized drivers of tumorigenesis with potent efficacy as therapeutic targets for the treatment of cSCC. [ABSTRACT FROM AUTHOR]

Background: The incidence of human papillomavirus-associated vulval neoplasia is increasing worldwide; yet the associated genetic changes remain poorly understood.Methods: We have used single-nucleotide polymorphism microarray analysis to perform the first high-resolution investigation of genome-wide allelic imbalance in vulval neoplasia. Our sample series comprised 21 high-grade vulval intraepithelial neoplasia and 6 vulval squamous cell carcinomas, with paired non-lesional samples used to adjust for normal copy number variation.Results: Overall the most common recurrent aberrations were gains at 1p and 20, with the most frequent deletions observed at 2q, 3p and 10. Copy-neutral loss of heterozygosity at 6p was a recurrent event in vulval intraepithelial neoplasia. The pattern of genetic alterations differed from the characteristic changes we previously identified in cutaneous squamous cell carcinomas. Vulval neoplasia samples did not exhibit gain at 5p, a frequent recurrent aberration in a series of cervical tumours analysed elsewhere using an identical protocol.Conclusion: This series of 27 vulval samples comprises the largest systematic genome-wide analysis of vulval neoplasia performed to date. Despite shared papillomavirus status and regional proximity, our data suggest that the frequency of certain genetic alterations may differ in vulval and cervical tumours. [ABSTRACT FROM AUTHOR]

The molecular mechanisms that underlie the development of squamous cell skin cancers (SSC) are poorly understood. We have used oligonucleotide microarrays to compare the differences in cellular gene expression between a series of keratinocyte cell that mimic disease progression with the aim of identifying genes that may potentially contribute towards squamous cell carcinoma (SCC) progression in vivo, and in particular to identify markers that may serve as potential therapeutic targets for SCC treatment. Gene expression differences were corroborated by polymerase chain reaction and Western blotting. We identified Axl, a receptor tyrosine kinase with transforming potential that has also been shown to have a role in cell survival, adhesion and chemotaxis, was upregulated in vitro in SCC-derived cells compared to premalignant cells. Extending the investigation to tumour biopsies showed that the Axl protein was overexpressed in vivo in a series of SCCs. [ABSTRACT FROM AUTHOR]

The few studies published on angiogenesis in lymphoma have raised the question of whether or not microvessel density (MVD) is associated with more aggressive disease and have reported the observation that in follicular lymphomas, vessels are mature rather than immature. We investigated MVD and the vascular phenotype within follicular or diffuse large B-cell lymphomas, reactive nodes and tonsils. Vascular phenotype was defined by the expression or loss of reactivity to the antibody LH39 (detecting the LH39 laminin epitope of the basement membrane in mature vessels) and by detection of alpha V beta 3 (expressed on immature vessels). In reactive nodes and in follicular lymphomas, MVD was higher in the paracortex than in germinal centres or in neoplastic follicles. However, in neoplastic follicles an increase in alpha V beta 3-positive endothelium suggested the activation of an angiogenic pathway different from that present in the reactive follicles. In large B-cell lymphomas, MVD was higher than in reactive and neoplastic follicles but lower than in the reactive paracortex. The number of immature vessels (LH39 negative) and of alpha V beta 3-positive vessels was higher than in reactive lymph nodes and follicular lymphoma suggesting that a switch to a different angiogenic pathway has occurred. Finally, we have demonstrated that within reactive and neoplastic follicles vascular regression is occurring, perhaps constraining the growth of reactive follicles alongside other phenomena such as apoptosis. Vascular regression was previously believed to occur in adults only in ovarian and endometrial tissue. We conclude that different types of angiogenesis are present in follicular lymphomas and large B-cell lymphomas. This has implications for possible future therapies. [ABSTRACT FROM AUTHOR]

We have previously described a group of non-small cell lung carcinomas without morphological evidence of neo-angiogenesis. In these tumours neoplastic cells fill up the alveoli and the only vessels present appear to belong to the trapped alveolar septa. In the present study we have characterised the phenotype of the vessels present in these non-angiogenic tumours, in normal lung and in angiogenic non-small cell lung carcinomas. The vessels, identified by the expression of CD31, were scored as mature when expressing the epitope LH39 in the basal membrane and as newly formed when expressing alphaVbeta3 on the endothelial cells and/or lacking LH39 expression. In the nine putative non-angiogenic cases examined, the vascular phenotype of all the vessels was the same as that of alveolar vessels in normal lung: LH39 positive and alphaVbeta3 variable or negative. Instead in 104 angiogenic tumours examined, only a minority of vessels (mean 13.1%; range 0--60%) expressed LH39, while alphaVbeta3 (in 45 cases) was strongly expressed on many vessels (mean 55.5%; range 5--90%). We conclude that in putative non-angiogenic tumours the vascular phenotype is that of normal vessels and there is no neo-angiogenesis. This type of cancer may be resistant to some anti-angiogenic therapy and different strategies need to be developed. [ABSTRACT FROM AUTHOR]

Angiogenesis, the formation of new vessels, has been demonstrated to be an indicator of prognosis in breast cancer patients. The extent of differentiation of the tumour vessels may affect access of peripheral white cells and egress or invasion of tumour cells. This has not been assessed in relation to tumour microvessel density or other variables and may be a marker of vascular remodelling. LH39 is a monoclonal antibody recognizing an epitope located at the lamina lucida of mature small veins and capillaries but not in newly formed vessels. To study vascular differentiation in breast tumours, we examined the vascular maturation index (VMI) in 12 normal and 50 breast carcinomas and this was correlated with different clinicopathological variables including angiogenesis. Mature vessels were defined by staining with antibodies to both LH39 and to CD31, using double immunohistochemistry, whereas immature vessels stained only for CD31. VMI was defined as the % fraction of mature vessels (LH39-positive) / total number of vessels (CD31-positive). The VMI was significantly higher in normal (54-68.5%; median 66.5%) than in tumours (0-47%; median 8.8%) (P = 0.0005). There was a significant inverse correlation between the tumour VMI and nodal status (Fisher's exact test,P = 0.01) and between high VMI and low thymidine phosphorylase (TP) expression (Mann-WhitneyU-test,P = 0.01). No significant association between VMI and tumour size, oestrogen receptor, epidermal growth factor receptor, grade, angiogenesis, patient age, or E-selectin was seen. There was a significant reduction in relapse-free survival (P = 0.01) with high angiogenesis. These findings show that the VMI gives new information on the mechanism of tumour angiogenesis independently from microvessel quantitation, there is a wide variation in the differentiation of tumour vasculature but the degree of capillary differentiation is not associated with quantitative angiogenesis. The VMI identifies a subset of patients... [ABSTRACT FROM AUTHOR]

Cytokeratin expression was studied in human middle ear cholesteatoma lesions, using a variety of immunohistological techniques and a wide range of polyclonal antisera and monoclonal antibodies against cytokeratin (CK) subgroups or individual CK polypeptides. The expression of the other cytoskeletal proteins, vimentin and desmin, was also investigated. Middle ear mucosa and epidermal tissues were used as reference tissues. Our investigations also included epithelial structures present in the cholesteatoma perimatrix and in dermal tissues. The results indicate that, compared with epidermal tissues, the expression profile of CKs in cholesteatoma matrix is representative of a hyperproliferative disease. Evaluating the presence of a marker of terminal keratinization - the 56.5 kD acidic CK n 10 - we found supportive evidence of a pronounced retardation of its expression, which did not parallel histological differentiation. In epidermal tissues, the first prickle cell layers are CK10 positive whereas in many cholesteatomas this finding was observed near the stratum granulosum only. Probing the early stages of keratinization -the 58 kD basic CK n 5 and the 50 kD acidic CK n 14 - we regularly observed an extended staining area in the cholesteatoma matrix. In epidermal reference tissues, only the basal and nearest suprabasal layers were convincingly labeled. As a rule, non-epidermal CKs did not belong to the cholesteatoma CK set. However, exceptions to that rule were noticed as a focal or more extended expression of one or more non-epidermal CKs in about half of the cases. Together with the extended CK5 topography, this is further evidence that CK expression is seriously affected by the diseased state. CK expression in the perimatrix is limited to mucous glands, either normal, atrophic or hyperplastic. CKs n 4, 5, 7, 14, 18 and 19, also displayed by middle ear mucosa, were consistently observed. Where ductal arrangements were present, CK10 was also detected, in analogy with the CK10 registration in ductal portions of mucous glands in the external ear canal skin. The absence of CK8 in mucous glands of the perimatrix, however, strongly differentiates these structures from the mucous gland acini and ducti in the external ear canal, where CK8 is systematically expressed. Vimentin staining was restricted to dendritic cells of the matrix (Langerhans cells) and to perimatrix fibroblasts, blood cells and vascular endothelium. Coexpression of CK and vimentin was not observed. [ABSTRACT FROM AUTHOR]

A cell line derived from the urothelium lining the ureter of a 12-year-old girl was immortalised using a temperature-sensitive SV40 large T-antigen gene construct, and designated UROtsa. Following immortalisation, UROtsa cells expressed SV40 large T-antigen, but did not acquire characteristics of neoplastic transformation, including growth in soft agar or the development of tumours in nude mice. Metaphase spreads had a normal chromosomal appearance and number. UROtsa cells remained permissive for cell growth at 39°C, indicating that they did not retain temperature sensitivity. UROtsa provides an in vitro model of 'normal' urothelium. [ABSTRACT FROM AUTHOR]

The aim of this study was to culture human urothelium and generate enough cells for subsequent reconstructive surgery. Using a modification of the Rheinwald-Green method for the routine culture of keratinocytes from patients with burns, we successfully cultured 91% of 57 biopsies from the renal pelvis, ureter, bladder and urethra of paediatric patients. The cells could be split one to three up to 9 times at 7-10 day intervals, giving a surface area of 1000 cm after a 2 month culture period. Primary cultures could not be initiated in defined medium MCDB153, although cells initiated using the Rheinwald-Green method could subsequently be propagated in this medium. Cytokeratin patterns in vitro were similar to those in vivo in the expression of keratins 7, 18 and 19 (characteristic of simple epithelia) and keratin 13 (characteristic of non-cornified stratified epithelia). Cultured urothelium also expressed keratin 14 (characteristic of cornified stratified epithelium) in about 25% of cells and keratin 16 (characteristic of fast-growing cells). These findings indicate that urothelial cells can be propagated in vitro for autologous grafting, and the next step is to identify substrates suitable for urothelial cell growth and differentiation and surgical manipulation. [ABSTRACT FROM AUTHOR]

Keratinocytes were cultured on fibroblast-free dermal substitutes made of type I collagen film (collagen dermal substitute) and an extracellular matrix gel film (matrix dermal substitute), each of which was laid on a lyophilized type I collagen sponge. The morphology of the basal keratinocytes in these three-dimensional culture models of the skin was studied ultrastructurally and immunohistochemically to assess their differentiation to basal cells. The basal keratinocytes in the artificial epidermis cultured on the collagen dermal substitute showed poorly organized tonofibril networks and desmosomes. Neither the tonofibril-hemidesmosome complex nor the lamina densa were detected along the interface, where many cytoplasmic projections of basal keratinocytes were noted. There were no detectable antigens of type IV or VII collagen, LDA-1, or laminin in the interface. Bullous pemphigoid (BP) and 1-2B7B antigens and integrins were expressed along the cytoplasmic membrane and the projections of the basal keratinocytes. A high molecular weight keratin (keratin 1, 68 kDa, 34ΒB4) was detected only in part of the uppermost layers of this artificial epidermis. In contrast, basal keratinocytes in the artificial epidermis on the matrix dermal substitute developed tonofibril networks radiating to desmosomes and hemidesmosomes, under which a primitive lamina densa was present. Basement membrane zone antigens, such as type IV and VII collagens, LDA-1 and laminin were noted along the interface as were 1-2B7B and BP antigens and integrins. Laminin and type VII collagen were also detected along or in the membrane of the endoplasmic reticulum of basal keratinocytes. The high molecular weight keratin was detected in the suprabasal layers of this artificial epidermis, but the basal keratinocytes lacked expression of this epitope as does epidermis in vivo. The artificial epidermis cultured on the matrix dermal substitute had a basal layer of keratinocytes that could be characterized as basal cells, forming junctional structures resembling those of epidermis in vivo. [ABSTRACT FROM AUTHOR]

The mode of differentiation of seborrhoeic keratoses was investigated by immunohistochemical staining using cytokeratin (CK) polypeptide-specific monoclonal antibodies and an antibody specific for the particulate form of epidermal transglutaminase (ETgase), and by applying an anti-human involucrin serum. The role played by (E)Tgase was further evaluated using an activity assay based on the covalent attachment of monodansylcadaverine. Samples of uninvolved epidermis served as reference tissue. CK reactivities suggested that seborrhoeic keratoses is a hyperproliferative disease with an epidermal CK composition. CK5 and CK14 were prominent markers of basal and basaloid keratinocytes, whereas a decrease in staining occurred in advanced maturation stages and areas of terminal keratinization. In contrast, CK1 and CK10 were prominent markers of suprabasaloid differentiation stages and produced complementary stainings to those of CK5 and 14. Generally, CK10 staining was more impressive than CK1 staining and seemed to start before CK1 staining. In contrast to CK10 staining, cornified areas lost CK1 reactivity. These staining patterns were similar to those observed in uninvolved reference tissues. The epidermal CK subset was further supplemented with the 'hyperproliferative' CK6 and 16 which occur sequentially. Positive staining for CK6 was noted from basal and proximal basaloid cells onwards, whereas distal basaloid cells additionally showed CK16 staining. The presence of other non-epidermal CK polypeptides could not be shown. The competence for other differentiation markers belonging to the group of (E)Tgase and cornifying cell membranes also evolved with a typical epidermal pattern. (E)Tgase activity was restricted to advanced and terminal stages of keratinization and was dual in nature, i.e. a diffuse cytoplasmic staining occurred together with a prominent staining of cornifying cell membranes. Similarly, involucrin first detected in the cytosol of distal basaloid cells, was soon translocated to the cornifying cell membrane, reflecting its function as an ETgase substrate and precursor of the marginal band. Finally, the immunolocalization of the particulate form of ETgase was strikingly similar to the location of the first two markers. Taken together, the results allow us to conclude that seborrhoeic keratoses exhibits a hyperproliferative variant of the epidermal keratinization process. Maturation of basal keratinocytes is greatly retarded leading to an accumulation of basaloid cells which retain the molecular markers of basal cells in proximal areas, but progressively gain the molecular markers of advancing maturation in distal areas. [ABSTRACT FROM AUTHOR]

The differentiation state of skin epithelial cysts and some uncommon types of epithelial skin tumours was investigated by immunohistochemical staining, mainly using cytokeratin (CK) polypeptide-specific monoclonal antibodies. Samples of interfollicular epidermis, hair follicles and eccrine sweat glands were included as reference tissues. The CK reactivity in epidermoid cysts and milia is not restricted to CKs involved in epidermal-type differentiation, i.e. CK1, 5, 10 and 14, but in addition CK16, a hyperproliferative keratinocyte marker is suprabasally expressed. CK1 and 10 are other prominent suprabasal markers, while CK14 seems to be preferentially expressed in the basal cell layer. Of the non-epidermal CKs, only CK4 was focally or more extensively detected in about 50% of the cases. In terms of CK reactivity, keratinization of trichilemmal cysts corresponds to the keratinization of the anagen-phase hair follicle in the isthmus. The CK reactivity is again restricted to CK1, 5, 10, 14 and 16. However, the CK1 as well as CK10 reactivity is subject to serious limitations, since both CKs were only convincingly observed in foci of terminal differentiation. Eccrine hydrocystoma obligatorily expresses a complex CK set, including CK7, 8, 14, 18 and 19. This CK set perfectly corresponds to the CK composition observed in acini of eccrine sweat glands. In addition, a discontinuous CK4 and 16 reactivity was seen in about 50% of the sites, while CK1 and 10 displayed a strictly focal appearance. On the other hand, syringoma produces in its distinct structures, a CK pattern reminiscent of the one observed in eccrine sweat gland ducts and includes CK1, 5, 10, 14, 16 and 19. Finally, the CK expression pattern of pilomatricoma includes CK1, 8, 10, 14 and 19, and is reminiscent of the CK staining of hair bulb matrix cells differentiating in the keratogenous zone in the direction of hair cortex. The reactivity of CK1 and 10 was mainly restricted to foci of squamoid differentiation and also to transitional cells bordering on shadow cells, as far as it concerns CK10. Occasionally, CK7 and 16 were observed in individual cells or small cell groups. In our view these CK reactivity patterns are useful to judge the differentiation state reached in pathological conditions, but so far do not allow us unequivocally to determine the site of origin of these lesions. [ABSTRACT FROM AUTHOR]

The distribution of type VII collagen was examined in the normal human nervous system, in brain tumour biopsies and in glioma cell lines by immunohistochemistry and western blotting. In normal tissue, positivity was observed beneath choroid plexus epithelial cells and around pineal gland and pituitary gland cell nests, while other brain regions and peripheral nerves were negative. Expression was preserved in most related tumours (choroid plexus papilloma, pineoblastoma, pituitary adenoma). Scattered abnormal vessels showed neo-expression of type VII collagen in about half of the astrocytic and ependymal tumours. Glioma cells in situ were consistently negative for type VII collagen, whereas the glioblastoma cell lines were positive. Our results suggest that anchoring fibrils or at least epitopes of their major structural component are present in normal and pathological cerebral structures, indicating a unique distribution of type VII collagen in the nervous system. [ABSTRACT FROM AUTHOR]

Infantile myofibromatosis (IM) is a distinct but rare clinicopathological entity occurring in neonates and infants. It probably represents a rare soft-tissue tumour made up of undifferentiated myofibroblasts. Its recognition is important since it can be mistaken for a malignancy when, in fact, it has a generally benign prognosis with spontaneous regression. We describe the first case of an infant with IM presenting with acute intussusception due to gastrointestinal as well as the typical skin involvement. [ABSTRACT FROM AUTHOR]