Analysis of promoter hypermethylation of death-associated protein kinase and p16 tumor suppressor genes in actinic keratoses and squamous cell carcinomas of the skin.

Death-associated protein kinase is a serine/threonine protein kinase implicated in promoting apoptosis and tumor suppression, whereas p16 is a tumor suppressor gene that inhibits cyclin-dependent kinase 4 and 6 activity and arrests the cell cycle in the G1 phase. Hypermethylation of death-associated protein kinase or p16 gene with resultant gene inactivation has been described in a wide variety of human cancers. Promoter methylation of the death-associated protein kinase and p16 gene has been found in about 55% and 30% cases of head and neck squamous cell carcinoma respectively but has not yet been analyzed in cutaneous premalignant and malignant lesions. A total of 33 cases were examined for evidence of death-associated protein kinase and p16 hypermethylation and these consist of 9 cases of spongiotic dermatitis as nonneoplastic skin control, 9 cases of actinic keratosis, 8 cases of squamous cell carcinoma in situ, and 7 cases of invasive squamous cell carcinoma. Death-associated protein kinase promoter methylation was detected in 1 case of squamous cell carcinoma in situ and 1 case of nonneoplastic skin control but none of the cases of invasive squamous cell carcinoma or actinic keratosis. P16 promoter methylation was detected in 1 case of invasive squamous cell carcinoma and 1 case of nonneoplastic skin control but none of the cases of squamous cell carcinoma in situ or actinic keratosis. Promoter hypermethylation of the death-associated protein kinase and p16 genes does not appear to play an important role in the development of cutaneous squamous cell carcinoma. The data thus suggest that the mechanisms of ultraviolet-induced cutaneous carcinomas differ from those involved in the development of head and neck squamous cell carcinoma, a malignant disease induced by tobacco and alcohol exposure.     

Immunohistochemical comparison of p16 expression in actinic keratoses and squamous cell carcinomas of the skin.

There are approximately 200,000 new cases of cutaneous squamous cell carcinoma diagnosed each year in the United States, with between 1300 and 2300 deaths per year from metastatic disease. The tumor suppressor p16, encoded by the CDKN2/INK4a locus, has been reported mutated in >or=24% of squamous cell carcinomas. Mutations of the p16 gene have also been found in actinic keratoses, the first identifiable lesion in the continuum from normal skin to squamous cell carcinoma. We hypothesized that there may be an appreciable difference in expression of p16 between normal skin, actinic keratoses, squamous cell carcinoma in situ, and invasive squamous cell carcinoma. Ten actinic keratoses, 10 in situ squamous cell carcinomas, and 10 invasive squamous cell carcinomas were examined using the immunoperoxidase method with antigen retrieval for anti-p16(INK4a) antibody. All 10 actinic keratoses were positive for weak to moderate p16 staining in the lower third to lower half of the epidermis (especially the basal keratinocytes). This staining was significant when compared with the lack of staining seen in normal skin controls. Twenty percent of in situ squamous cell carcinomas had moderate to strong staining in only the lower half to lower two thirds of the epidermis, whereas 70% of the in situ squamous cell carcinomas exhibited full-thickness p16 staining, with no staining in the dermis. Thirty percent of invasive squamous cell carcinomas had full-thickness staining of the in situ component of the lesion, and 100% of invasive squamous cell carcinomas exhibited moderate to strong staining of the invasive component of the lesion. These findings indicate correlation between the increased expression of p16 during the progression of skin from actinic keratosis to in situ squamous cell carcinoma to invasive squamous cell carcinoma. These data may lend further support to the view of the actinic keratosis as a precursor lesion to squamous cell carcinoma.     

Quantitative assessment of Langerhans cells in actinic keratosis, Bowen's disease, keratoacanthoma, squamous cell carcinoma and basal cell carcinoma

The quantitative distribution of Langerhans cells (LC) was studied in a range of pre-neoplastic, in-situ and invasive neoplastic skin lesions using an antibody to S100 protein and the indirect immunoperoxidase technique. LC numbers were increased within the lesions of actinic keratosis, Bowen's disease, keratoacanthoma, squamous cell carcinoma and basal cell carcinoma. In all lesions except actinic keratosis the LC density was also significantly increased in the adjacent non-neoplastic epithelium. The increased LC density in neoplastic epithelium suggests either that LC are being retained within the abnormal epithelium for longer periods of time than normal or that increased numbers of LC are being actively attracted by factors produced by the neoplastic epithelium. While reduction of intraepithelial LC density may allow the initiation of neoplasia the increased density observed in this study suggests that at later stages of tumour growth LC may have a functional role in the host response to cutaneous neoplasia.     

Loss of T-cadherin (CDH13, H-cadherin) expression in cutaneous squamous cell carcinoma

We previously reported that T-cadherin (CDH13, H-cadherin), a unique cadherin molecule, was expressed on the basal cell layer in normal murine and human epidermis. In the present study, T-cadherin expression in archival human skin specimens comprising a spectrum of human squamous cell neoplasia was investigated. T-cadherin expression was observed in both normal epidermal basal cells and adnexal epithelial cells of formalin-fixed and paraffin-embedded tissue sections. Western immunoblotting also revealed that mature T-cadherin protein was expressed in cultured human skin tissue equivalent. Atypical keratinocytes in 27 of 53 specimens of actinic keratosis and 23 of 30 specimens of Bowen's disease expressed T-cadherin. In contrast, T-cadherin was focally expressed in 6 of 56 invasive cutaneous squamous cell carcinomas. To explore the molecular mechanism of down-regulation of T-cadherin expression in invasive squamous cell carcinoma, loss of heterozygosity, genetic alternations, and methylation status in the 5' region of the T-cadherin gene were investigated. Loss of heterozygosity at intron 1 of the T-cadherin gene was observed in 8 of 28 informative cases of invasive squamous cell carcinoma. Although no structural genomic alternations were found by sequence analysis, aberrant promoter methylation of the T-cadherin gene was found in 12 of 28 invasive squamous cell carcinomas. T-cadherin expression was restored in cultured A431 cells, in which aberrant methylation was found by treatment with the demethylating agent 5'-aza-2-deoxycytidine. These findings suggest that a combination of deletion and aberrant methylation of the T-cadherin gene may play a role in loss of gene expression in a considerable number of invasive cutaneous squamous cell carcinomas.     

Pagetoid variant of actinic keratosis with or without squamous cell carcinoma of sun-exposed skin: a lesion simulating extramammary Paget's disease

BACKGROUND: Extramammary Paget's disease usually occurs in anogenital skin. We present five cases of squamous cell carcinoma in situ of sun-exposed skin and non-squamous cell carcinoma in situ actinic keratosis that displayed atypical keratinocytes disposed in intraepithelial cell nests and immunohistochemical staining simulating extramammary Paget's disease. METHODS AND RESULTS: Two pilot cases--one squamous cell carcinoma in situ and one non-squamous cell carcinoma in situ actinic keratosis with formation of intra-epidermal nests of atypical keratinocytes with a pagetoid spread pattern--were encountered at our institution. Fifty-four consecutive cases of squamous cell carcinoma in situ including bowenoid actinic keratosis and 34 cases of non-squamous cell carcinoma in situ actinic keratosis were reviewed to identify pagetoid spread of atypical cells. Representative sections of all cases with pagetoid spread of atypical keratinocytes were submitted for special stains for mucin, and immunostaining for cytokeratin 7 (CK7), cytokeratin 20 (CK20), cytokeratin CAM 5.2 (CAM 5.2), carcinoembryonic antigen (CEA), vimentin and S100 protein. In the group of squamous cell carcinoma in situ, 10 cases displayed pagetoid spread of atypical keratinocytes with cytoplasm ranging from clear to pale and atypical hyperchromatic nuclei. One review squamous cell carcinoma in situ was multicentric with three separate lesions. The atypical keratinocytes tended to form well to poorly defined cell groups extending from the basal cell layer to the corneal layer. No similar cases were identified in the group of non-squamous cell carcinoma in situ actinic keratosis. Two pilot cases and three of 10 review cases with a total of seven separate lesions displayed a moderate to marked immunohistochemical reactivity for CK7 similar to extramammary Paget's disease. CEA immunoreactivity was also detected in two of these cases. In addition, two of 44 squamous cell carcinomas in situ without pagetoid spread of atypical keratinocytes showed a moderate reactivity for CK7 in very occasional atypical keratinocytes. The remaining seven squamous cell carcinomas in situ with pagetoid spread of atypical keratinocytes were not immunoreactive for CEA and CK7. Immunostaining for CK20, vimentin, S100 protein was negative in all atypical cells in all study cases. CONCLUSIONS: Actinic keratosis, particularly squamous cell carcinoma in situ of sun-exposed skin, may have histopathological and immunohistochemical features similar to extramammary Paget's disease and probably represents a variant of actinic keratosis. Awareness of the pagetoid variant of actinic keratosis arising in sun-exposed skin is helpful to avoid the over-diagnosis of extramammary Paget's disease.     

Claudins 1, 2, 3, 4, 5 and 7 in solar keratosis and squamocellular carcinoma of the skin

Claudins are tight junction proteins regulating the paracellular permeability of cell layers. We investigated the expression of claudins 1, 2, 3, 4, 5 and 7 in a sample set consisting of a total of 93 cases representing normal skin, actinic keratoses and squamous cell carcinomas of the skin. There were several changes found in claudin expression. Claudin 1 appeared to be progressively decreased in solar keratosis and skin squamous cell carcinomas compared to normal skin while expression of claudin 2 was increased. With claudins 3 and 5 occasional immunoreactivity was found in squamous cell carcinomas. Claudins 4 and 7 were variably expressed in skin neoplasia compared to normal skin. According to the results expression of claudins 1 and 2 change in parallel with the severity of the epidermal preneoplastic and neoplastic lesions thus probably influencing the disturbed epithelial polarity characteristic of these lesions. Claudin 1 under- and claudin 2 overexpression also lead to a leakier epithelial barrier function of the skin with a resulting damage to skin epithelial resistance. Other claudins investigated in this study did not show progressive changes even though occasional overexpression of them was found in skin squamous cell carcinoma.     

Metalloproteinase-2 expression correlates with aggressiveness of cutaneous squamous cell carcinomas.

Matrix metalloproteinases compose a family of enzymes involved in degradation of the extracellular matrix. Tumor cells must penetrate the basement membrane and traverse the extracellular matrix in order to invade surrounding structures and metastasize to distant sites. Gelatinases, particularly gelatinase A (matrix metalloproteinase-2), demonstrate degradative activity against components of the basement membrane and may be involved in the progression of in situ squamous cell carcinoma lesions. Matrix metalloproteinase-2 overexpression has been correlated with tumor invasiveness and metastasis in a wide variety of cancer types, including squamous cell carcinoma arising on mucous membranes. However, correlation between matrix metalloproteinase-2 overexpression and the spread and prognosis of cutaneous squamous cell carcinoma has not been characterized in the literature at present. In this study, we used immunohistochemical techniques to examine the expression of matrix metalloproteinase-2 in 10 actinic keratosis, 15 in situ, 13 invasive, 13 primary (with documented metastatic disease), 11 metastatic, and 8 recurrent squamous cell carcinoma cases. We found that while the average staining intensity (scale 0-3+, 3+ being strongest) of actinic keratosis and in situ lesions was not statistically significant (0.87-1.3 and 0.75-1.4, respectively), the average staining intensity of invasive squamous cell carcinomas (1.6-2.5) was significantly greater than that of actinic keratosis and in situ lesions. Likewise, while the average staining intensity of primary squamous cell carcinomas and metastatic squamous cell carcinomas was not found to be statistically significant (3.1-3.5 and 3.1-3.8, respectively), the average staining intensity of these lesions was significantly higher than that of invasive lesions. We also found that the intensity of matrix metalloproteinase-2 staining correlates with cellular atypia, inflammation, neovascularization, and the invasive tumor front, as well as tumor aggressiveness, and may play a role in the pathogenesis, invasion, and metastasis of cutaneous squamous cell carcinoma.     

Angiogenesis in the progression of cutaneous squamous cell carcinoma: an immunohistochemical study of endothelial markers

OBJECTIVE:

To demonstrate the role of angiogenesis in the progression of cutaneous squamous cell carcinoma.

INTRODUCTION:

Angiogenesis is a pivotal phenomenon in carcinogenesis. Its time course in cutaneous squamous cell carcinoma has not yet been fully established.

METHODS:

We studied the vascular bed in 29 solar keratoses, 30 superficially invasive squamous cell carcinomas and 30 invasive squamous cell carcinomas. The Chalkley method was used to quantify the microvascular area by comparing panendothelial (CD34) with neoangiogenesis (CD105) immunohistochemical markers. The vascular bed from non-neoplastic adjacent skin was evaluated in 8 solar keratoses, 10 superficially invasive squamous cell carcinomas and 10 invasive squamous cell carcinomas.

RESULTS:

The microvascular area in CD105-stained specimens significantly increased in parallel with cutaneous squamous cell carcinoma progression. However, no differences between groups were found in CD34 sections. Solar keratosis, superficially invasive squamous cell carcinoma and invasive squamous cell carcinoma samples showed significant increases in microvascular area for both CD34- and CD105-stained specimens compared with the respective adjacent skin.

DISCUSSION:

The angiogenic switch occurs early in the development of cutaneous squamous cell carcinoma, and the rate of neovascularization is parallel to tumor progression. In contrast to panendothelial markers, CD105 use allows a dynamic evaluation of tumor angiogenesis.

CONCLUSION:

This study demonstrated the dependence of skin carcinogenesis on angiogenesis.     

Assessment of cell proliferation in benign, premalignant and malignant skin lesions.

A deeper understanding of the variance of epidermal cell proliferation may eventually increase the reproducibility of diagnostic classification. A prospective study of 46 consecutive, unselected biopsies from benign (keratoacanthoma n=14), premalignant (actinic keratosis n=15 and Bowen disease n=10) and malignant (squamous cell carcinoma n=7) skin lesions was studied to assess the presence and extent of differences in expression of the proliferation marker Ki-67 using a monoclonal antibody directed against a c-DNA defined subsegment (MIB-1) and a noncross-linking, proprietary fixative BoonFix. MIB-1 was expressed in the adjacent, non-affected skin in a scattered to confluent linear pattern in the basal/suprabasal cell layer. In actinic keratosis, MIB-1 expression, in addition to basal/suprabasal layers, extended to mid-zones of the epidermis. An interesting feature in actinic keratosis as well as in Bowen disease was the expression of MIB-1 in the epithelium lining the hair follicles. In Bowen disease, MIB-1 was observed throughout the full thickness of the epidermis, unequivocally separating this entity from others under study. In invasive squamous cell carcinoma, MIB-1 expression was not consistent between and within cases. MIB-1 positivity was variably found in all layers of the epidermis, but showed a chaotic and haphazard pattern with total loss of polarity. Keratoacanthoma cases showed highly variable MIB-1 expression, ranging from no expression to expression in both basal/suprabasal and mid-zone layers of the epidermis. These results warrant further study of modulation of cell proliferation in actinic keratosis.     

Epigenetic events underlie the pathogenesis of sinonasal papillomas.

Benign inverted papillomas have been reported as monoclonal but lacking common genetic alterations identified in squamous cell carcinoma of the head and neck. Epigenetic changes alter the heritable state of gene expression and chromatin organization without change in DNA sequence. We investigated whether epigenetic events of aberrant promoter hypermethylation in genes known to be involved in squamous head and neck cancer underlie the pathogenesis of sinonasal papillomas. Ten formalin-fixed paraffin DNA samples from three inverted papilloma cases, two exophytic (everted) papilloma cases, and two cases with inverted and exophytic components were studied. DNA was obtained from microdissected areas of normal and papilloma areas and examined using a panel of 41 gene probes, designed to interrogate 35 unique genes for aberrant methylation status (22 genes) using the methylation-specific multiplex-ligation-specific polymerase assay. Methylation-specific PCR was employed to confirm aberrant methylation detected by the methylation-specific multiplex-ligation-specific polymerase assay. All seven cases indicated at least one epigenetic event of aberrant promoter hypermethylation. The CDKN2B gene was a consistent target of aberrant methylation in six of seven cases. Methylation-specific PCR confirmed hypermethylation of CDKN2B. Recurrent biopsies from two inverted papilloma cases had common epigenetic events. Promoter hypermethylation of CDKN2B was a consistent epigenetic event. Common epigenetic alterations in recurrent biopsies underscore a monoclonal origin for these lesions. Epigenetic events contribute to the underlying pathogenesis of benign inverted and exophytic papillomas. As a consistent target of aberrant promoter hypermethylation, CDKN2B may serve as an important epigenetic biomarker for gene reactivation studies.     

Epithelial to mesenchymal transition in cutaneous squamous cell carcinoma is correlated with COX-2 expression but not with the presence of stromal macrophages or CD10-expressing cells

Epithelial to mesenchymal transition (EMT) is an intricate process by which epithelial cells loose epithelial characteristics and acquire a mesenchymal-like phenotype. EMT and cyclooxygenase 2 (COX-2) expression are related to tumor invasion and metastasis. The tumor microenvironment plays a major role in tumor progression and the induction of EMT. Here, we investigated the relationship between EMT and COX-2 expression as well as tumor-associated macrophages (TAM) and CD10-positive stromal cells during the development of cutaneous squamous neoplastic lesion. We performed immunohistochemical staining for vimentin, E-cadherin, β-catenin, COX-2, CD68, and CD10 in 41 cases of squamous cell cancers (SCC), 20 of Bowen's disease, 30 of actinic keratosis, and 30 samples of normal skin. SCC cells showed significantly increased vimentin expression and reduced expression of membranous E-cadherin and β-catenin compared with cells in precursor lesions and in normal skin. COX-2 expression was also markedly increased in SCC cells. E-cadherin expression was positively correlated with β-catenin expression and inversely correlated with COX-2 expression in SCC cells. The number of TAM and CD10-positive stromal cells increased from the normal skin to precursor lesions and SCC cells. The number of TAM and of CD10-positive stromal cells did not correlate with the expression of E-cadherin, β-catenin, COX-2, and vimentin in SCC cells. We suggest that cutaneous SCC cells show EMT, which appears to be correlated with COX-2 expression but not with stromal CD10 expression and TAM.     

Signet-ring squamous cell carcinoma

Among carcinomas, signet-ring morphologic characteristics have been regarded as pathognomonic of adenocarcinoma. This report presents the case of a poorly differentiated cutaneous squamous cell carcinoma with a monodispersed, invasive signet-ring component. Kreyberg stains had negative results for mucin. Immunohistochemical analysis demonstrated that concentric rings in the signet-ring cells were composed of keratin. To the best of the authors' knowledge, this is the first report of signet-ring squamous cell carcinoma. Another feature that bears emphasis is that this squamous cell carcinoma led to the death of the patient, even though it originated in a field of actinic keratosis.     

Epstein-Barr virus and p16INK4A methylation in squamous cell carcinoma and precancerous lesions of the cervix uteri

Methylation of p16 is an important mechanism in cervical carcinogenesis. However, the relationship between cervical squamous cell carcinoma (SCC) and Epstein-Barr virus (EBV) remains controversial. Here, we explored whether EBV infection and/or p16 gene inactivation would play any role in cervical carcinogenesis. Eighty-two specimens included 41 invasive SCCs, 30 cervical intraepithelial neoplasm (CIN; CIN 1, 11 cases, CIN II, 3 cases, CIN III 16 cases) and 11 nonneoplastic cervices. EBV was detected by polymerase chain reaction (PCR) for EBNA-1 and in situ hybridization for EBER-1. The p16 methylation-status and the expression of p16 protein were studied by methylation-specific PCR and immunohistochemistry, respectively. The materials were divided into four groups: 1) nonneoplastic cervices, 2) CIN I, 3) CIN II-III and 4) invasive SCCs. p16 methylation and p16 immunoexpressions increased in CIN and invasive SCCs than nonneoplastic tissue. p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group. EBV was detected more frequently in CIN and SCCs than nonneoplastic cervices. In conclusion, a correlation between p16 methylation, p16 immunoreactivity and the detection of EBV strongly suggested that the cooperation of EBV and p16 gene may play a synergic effect on cell cycle deregulation.     

Altered expression of adhesion molecules and epithelial-mesenchymal transition in silica-induced rat lung carcinogenesis

Loss of the epithelial phenotype and disruption of adhesion molecules is a hallmark in the epithelial-mesenchymal transition (EMT) reported in several types of cancer. Most of the studies about the relevance of adhesion and junction molecules in lung cancer have been performed using established tumors or in vitro models. The sequential molecular events leading to EMT during lung cancer progression are still not well understood. We have used a rat model for multistep lung carcinogenesis to study the status of adherens and tight junction proteins and mesenchymal markers during EMT. After silica-induced chronic inflammation, rats sequentially develop epithelial hyperplasia, preneoplastic lesions, and tumors such as adenocarcinomas and squamous cell carcinomas. In comparison with normal and hyperplastic bronchiolar epithelium and with hyperplastic alveolar type II cells, the expression levels of E-cadherin, alpha-catenin and beta-catenin were significantly reduced in adenomatoid preneoplastic lesions and in late tumors. The loss of E-cadherin in tumors was associated with its promoter hypermethylation. alpha- and beta-catenin dysregulation lead to cytoplasmic accumulation in some carcinomas. No nuclear beta-catenin localization was found at any stage of any preneoplastic or neoplastic lesion. Zonula occludens protein-1 was markedly decreased in 66% of adenocarcinomas and in 100% squamous cell carcinomas. The mesenchymal-associated proteins N-cadherin and vimentin were analyzed as markers for EMT. N-cadherin was de novo expressed in 32% of adenocarcinomas and 33% of squamous cell carcinomas. Vimentin-positive tumor cells were found in 35% of adenocarcinomas and 88% of squamous cell carcinomas. Mesenchymal markers were absent in precursor lesions, both hyperplastic and adenomatoid. The present results show that silica-induced rat lung carcinogenesis is a good model to study EMT in vivo, and also provide in vivo evidence suggesting that the changes in cell-cell adhesion molecules are an early event in lung carcinogenesis, while EMT occurs at a later stage.     

Enhanced expression of retinoic acid-metabolizing enzyme CYP26A1 in sunlight-damaged human skin

Vitamin A deficiency (VAD) is associated with increased susceptibility to carcinogenesis. CYP26A1, the gene encoding a cytochrome P450 enzyme specifically involved in metabolic inactivation of retinoic acid (RA), the most active vitamin A derivative, has been shown to result in a state of functional VAD of the cell. Recently, we demonstrated that CYP26A1 efficiently promotes cell survival properties and eventually contributes to the carcinogenic process, implying roles as an oncogene. To clarify the possible association between VAD caused by CYP26A1 expression and the development of human epithelial neoplasia, we examined whether enhanced expression of CYP26A1 might be observed in various lesions of human skin. We report here that basal keratinocytes showed only weak positivity of CYP26A1 in sunlight-nonexposed areas, whereas strong positive staining was observed in skin from chronically sunexposed body areas and in epidermis that had the dysplastic changes known as actinic keratosis. However, we found no expression of constitutive CYP26A1 in skin malignancies such as squamous cell carcinomas. Our observation suggests an involvement of enhanced CYP26A1 expression causing a functional VAD state in skin that can potentially lead to neoplastic transformation of keratinocytes in an early phase during skin carcinogenesis.     

The expression of p63 and p53 in keratoacanthoma and intraepidermal and invasive neoplasms of the skin

p53 is a well-known tumor suppressor gene, and its mutation is a common event in intraepidermal and invasive neoplasms of the skin. p63 is a homologue of the tumor suppressor gene p53, which is expressed in human basal squamous epithelium, and despite its homology to p53, it is considered to act as an oncogene. We evaluated p63 and p53 expression in usual skin cancers, including basal cell carcinoma (BCC), squamous cell carcinoma (SCC), keratoacanthoma (KA), and intraepidermal neoplasms, including Bowen's disease (BD), actinic keratosis (AK), malignant melanoma in situ (MM in situ), and Paget's disease (PD) to clarify the putative role of p63 and p53 in the development and differential diagnosis of these lesions.     

GSTP1 promoter hypermethylation is an early event in breast carcinogenesis

Promoter hypermethylation in precursor lesions of the breast cancer may be biomarkers of cancer risk and targets for cancer chemoprevention. Pi-class glutathione-S-transferases (GSTP1) is inactivated by promoter hypermethylation in invasive breast cancers. However, little is known about epigenetic silencing of GSTP1 gene by promoter hypermethylation in precursor lesions. To determine the significance of GSTP1 promoter hypermethylation in breast carcinogenesis, methylation status of GSTP1 gene was studied by nested methylation-specific polymerase chain reaction, and GSTP1 expression was studied by immunohistochemistry in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), usual ductal hyperplasia (UDH), and normal breast tissue. GSTP1 promoter hypermethylation was detected in 4/24 (16.7%) of UDH, 18/49 (36.7%) of DCIS, and 14/36 (38.9%) of IDC. No hypermethylation was detected in normal breast tissues. GSTP1 promoter hypermethylation was found to be progressively elevated during breast carcinogenesis (p < 0.01). GSTP1 promoter hypermethylation was associated with loss of GSTP1 expression (p < 0.01 for UDH, p < 0.001 for DCIS and IDC). Our results suggest that GSTP1 promoter hypermethylation is an early event in breast carcinogenesis and appears to functionally silence GSTP1 expression. GSTP1 promoter hypermethylation in the precursor lesions of breast cancer may be used as a target for cancer chemoprevention.     

Extension of the typing in a general-primer-PCR reverse-line-blotting system to detect all 25 cutaneous beta human papillomaviruses

beta-Papillomaviruses (PV) seem to be involved in the pathogenesis of cutaneous squamous cell carcinoma and its early stage actinic keratosis. In this study, typing was extended of a previously described consensus primer-mediated beta- and gamma-cutaneous HPV PCR method followed by reverse-line-blotting (BGC-PCR/RLB) to detect all 25 known beta-PV and to examine their prevalence in actinic keratosis. The typing format of the BGC-PCR assay was extended by adding hybridization probes of six beta-PV (HPV 75, 76, 80, 92, 93, and 96) to the RLB system. Subsequently, tumor and normal skin tissues were collected from 75 patients with actinic keratosis, allowing typing for a total of 25 beta- and 5 gamma-types. The analytical sensitivity was between 10 copies (HPV 75, 80, 92, 93, and 96) and 100 copies (HPV 76). Except for that of HPV 76, none of the added probes showed any cross-hybridization with other beta-HPV. HPV DNA was detected in 45% of actinic keratosis and in 33% of normal skin by BGC-PCR, and at least one of the six added beta-types was present in 19% of actinic keratoses and in 13% of normal skin. Six beta-HPV types were added successfully to the typing format of the BGC-PCR/RLB system. The potential role of these types in the development of non-melanoma skin cancer awaits further studies.     

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5γ2 chain as well as α3, β1 subunits of α3β1 heterodimer and β4 subunit of integrin α6β4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of β1, β4 and α3 integrins, and SISCCs lacked β1, β4 and α3 integrins in the invasive front. AC cases were negative for the Ln-5γ2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5γ2 chain expression in the invasive front of the tumor. Integrin β1, β4 and α3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5γ2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype.     

Pulmonary preneoplasia – sequential molecular carcinogenetic events

Bronchial and bronchioloalveolar carcinogenesis is a multicentric and multistep process, leading to a sequential accumulation of molecular and genetic abnormalities, mainly due to exposure to tobacco carcinogens. Concomitantly, a series of morphological alterations of normal bronchial or bronchioloalveolar epithelium occur, resulting in preneoplastic and then neoplastic lesions. The three pulmonary preneoplastic changes recognized to date in the lung include bronchial squamous dysplasia and in situ carcinoma, preceding invasive squamous cell carcinoma and basaloid carcinoma, atypical adenomatous hyperplasia, a preneoplastic condition of bronchioloalveolar carcinoma, and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia, a proposed precursor for carcinoid tumours. Although the gradual accumulation of molecular alterations has been widely investigated in bronchial carcinogenesis, with the aim of determining new biomarkers for early lung cancer detection in high-risk patients and targeted chemoprevention, lung adenocarcinoma pathogenesis has been only recently highlighted, with the recent discovery of epidermal growth factor receptor mutation pathway in non-smokers. This review focuses on the current status of molecular pathology in lung cancer and pulmonary preneoplastic conditions.