Transcriptional analysis reveals distinct gene expression profiles of three bowenoid papulosis patients.

Bowenoid papulosis (BP) is a benign and possibly carcinogenic disease associated with human papillomavirus (HPV) infection, which has been increasingly recognised and paid attention to in recent years, but the potential mechanisms still remain unclear. In our study, three patients who were diagnosed with BP were enrolled into our research. Skin biopsies were taken and were separated into two parts, one part was for HE staining and the others were for RNA-sequencing (RNA-seq). All the three patents were human papillomavirus (HPV) positive and HE staining revealed typical skin histopathological changes in BP, including dyskeratosis, hyperplasia and hypertrophy of the granular and spinous layers, atypical keratinocytes. RNA-seq analysis demonstrated that a total of 486 differentially expressed genes (DEGs) were detected between the skin tissues from BP and the controls, among which, 320 genes were significantly upregulated and 166 genes were dramatically downregulated. GO enrichment revealed that antigen binding, cell cycle, immune response and keratinisation to be the most notably altered pathways, whereas KEGG analysis indicated that cell cycle cytokine-cytokine receptor interaction, ECM receptor interaction and p53 signalling pathway to be the most significantly changed signalling pathways in BP. Furthermore, metabolism-associated enrichment analysis showed that cholesterol metabolism, metabolism of xenobiotics by cytochrome p450 and pyrimidine metabolism to be the most dramatically dysregulated metabolic pathways in BP as compared to normal controls. Our study revealed that inflammation, metabolism and cell proliferation signalling pathways might be the most important pathways for BP disease, targeted inhibiting of these signals might be a potential method for BP treatment.

From Bowen disease to cutaneous squamous cell carcinoma: eight markers were verified from transcriptomic and proteomic analyses.

BACKGROUND: Bowen's disease is a cutaneous squamous cell carcinoma (CSCC) in situ. If left untreated, BD may progress to invasive CSCC. CSCC is one of the most common cutaneous carcinoma in the elderly and the advanced, metastasis CSCC usually have a poor outcomes. However, the mechanisms of invasion and metastasis from Bowen's disease to CSCC is complicated and still unclear. OBJECTIVES: The aim of this study was to explore the biomarkers and molecular alterations in Bowen's disease development process via analyzing the proteomics changes in tissues of CSCC, Bowen disease and healthy skin. METHODS: A total of 7 individuals with CSCC (5 for proteomics study and 2 for validation), 7 individuals with Bowen disease (5 for proteomics study and 2 for validation) and 7 healthy controls (5 for proteomics study and 2 for validation) presented to the Department of Dermatology, Yijishan Hospital, the First Affiliated Hospital of Wannan Medical College between January 2021 and December 2021 were enrolled. The proteomics analysis was performed to screen differentially expressed proteins/gens (DEPs/DEGs) in the lesions of CSCC, Bowen disease and healthy skin tissues. The transcriptomic data (GSE32628) of CSCC was selected and downloaded from the GEO database. The common DEGs in our proteomics results and GSE32628 between CSCC and healthy skin tissues were selected. And then, the common DEGs which significantly up or down-regulated between CSCC and Bowen disease in our proteomics results were further screened to identify using Western blot methods in the validation group. CSCC A431 cells were transfected with SERPINB1 small interfering RNA (si-SERPINB1) or small interfering RNA negative control (si-NC). To explore the effect of SERPINB1 silencing on migration and invasion ability of A431 cells. RESULTS: A total of 501 proteins were differentially expressed between the CSCC and healthy skin tissues, with 332 up-regulated and 169 down-regulated at least 1.5-fold with a P value < 0.05. These DEPs involved multiple biological functions such as protein binding process, immune, inflammation, ribosome, protein digestion and absorption, ECM-receptor interaction, focal adhesion, PI3K-Akt signaling pathway and others. A total of 20 common DEGs (COL3A1, LUM, TNC, COL1A1, ALDH3A2, FSCN1, SERPINB4, SERPINB1, CD36, COL4A1, CSTB, GPX3, S100A7, ACTN1, SERPINB3, S100A8, RAB31, STAT1, SPRR1B, S100A9) between CSCC and healthy skin tissues in GSE32628 and our proteomics results were found. Besides, the proteins of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in CSCC were significantly up-regulated, while COL3A1, COL1A1 and CD36 were significantly down-regulated relative to Bowen disease in proteomics results. These proteins were mainly involved in multiple pathways, including Focal adhesion, ECM-receptor interaction, Human papillomavirus infection, PI3K-Akt signaling pathway, PPAR signaling pathway, AMPK signaling pathway and others. These eight proteins were selected for further validation. According to the Western blotting analysis, when compared with the Bowen disease and healthy skin tissues, we found that the relative expression levels of TNC, FSCN1, SERPINB1, ACTN1 and RAB31 in the CSCC were significantly increased, while COL1A1 and CD36 were significantly decreased, and the differences were statistically significant (P < 0.05). Furthermore, the relative expression levels of TNC, FSCN1, SERPINB1 in the Bowen disease were also significantly increased, while the COL3A1 were also significantly decreased relative to the healthy control. SERPINB1 siRNA inhibited the expression of SERPINB1 at mRNA and protein levels in the A431 cells. After interfering with the expression of SERPINB1, the migration and invasion ability in the A431 cells were significantly decreased (P < 0.05). CONCLUSIONS: This study highlights that eight proteins, TNC, FSCN1, SERPINB1, ACTN1, RAB31, COL3A1, COL1A1, CD36, were significantly associated with the mechanisms of invasion and metastasis in Bowen's disease.

Telomeres and Telomerase in Cutaneous Squamous Cell Carcinoma.

The role of telomere biology and telomerase activation in skin cancers has been investigated in melanoma and basal cell carcinoma but limited evidence is available for cutaneous squamous cell carcinoma (cSCC). We will review the current knowledge on the role of telomere and telomerase pathway in cSCC pathogenesis. At the somatic level, both long and short telomere lengths have been described in cSCC. This telomere dichotomy is probably related to two different mechanisms of tumour initiation which determines two tumour subtypes. Telomere shortening is observed during the invasive progression from in situ forms of cSCC, such as Bowen's disease (BD) and actinic keratosis (AK), to invasive cSCC. At the germline level, controversial results have been reported on the association between constitutive telomere length and risk of cSCC. Approximately 75⁻85% of cSCC tumours are characterized by a high level of telomerase activity. Telomerase activation has been also reported in AKs and BD and in sun-damaged skin, thus supporting the hypothesis that UV modulates telomerase activity in the skin. Activating TERT promoter mutations have been identified in 32⁻70% of cSCCs, with the majority showing the UV-signature. No significant correlation was observed between TERT promoter mutations and cSCC clinico-pathological features. However, TERT promoter mutations have been recently suggested to be independent predictors of an adverse outcome. The attention on telomere biology and telomerase activity in cSCC is increasing for the potential implications in the development of effective tools for prognostic assessment and of therapeutic strategies in patients with cutaneous cSCC.

Investigation of Werner protein as an early DNA damage response in actinic keratosis, Bowen disease and squamous cell carcinoma.

BACKGROUND: Werner protein (WRN) has DNA helicase activity and participates in recombination, replication and repair of DNA. Loss-of-function mutations in WRN gives rise to genetic instability and diseases such as premature ageing and cancer. Upregulation of WRN promotes proliferation and survival of cancer cells. AIM: To evaluate the expression pattern of WRN in closely related skin cancers and their correlation with age, sex and UV exposure. METHODS: Immunohistochemistry was used to investigate expression of WRN in formalin-fixed, paraffin wax-embedded tissue specimens of 9 squamous cell carcinoma (SCC), 15 actinic keratosis (AK), 11 Bowen disease (BD) and 11 normal-appearing peripheral tissue samples, obtained from patients during surgical resections. RESULTS: WRN expression was significantly increased in BD, AK and SCC compared with normal controls, with the mean WRN staining score being highest in BD, followed by AK and SCC. However, age, sex and sun exposure were not associated with WRN expression. CONCLUSIONS: To our knowledge, this is the first report to date investigating the expression of WRN in skin cancers. The overtly high expression of WRN in premalignant lesions and in in situ cancer, with relatively low WRN expression in SCC, may indicate that WRN contributes as a checkpoint for early DNA damage response in skin tumorigenesis.

Mutations of the TERT promoter are common in basal cell carcinoma and squamous cell carcinoma.

Telomerase is frequently expressed in cancer and contributes to carcinogenesis. Two recent publications report the identification of a set of recurrent mutations in melanoma in the promoter of the telomerase reverse transcriptase gene (TERT) that appears to be the result of mutagenesis from ultraviolet (UV) radiation. Both groups reported that the mutations increase the transcription of TERT. This prompted our search for similar mutations in two other UV-related skin cancers, basal cell carcinoma, and squamous cell carcinoma. We found that the activating TERT promoter mutations reported in melanoma are also frequent in squamous cell carcinoma (50%) and basal cell carcinoma, the latter including both sporadic tumors (78%) and tumors from patients with nevoid basal cell carcinoma syndrome (68%). These mutations were found in only 1 of 11 Bowen's disease (squamous cell carcinoma in situ) specimens, and in none of 15 non-malignant skin specimens and 57 blood specimens. The mutations were frequently homozygous or hemizygous, with little or no normal signal at the mutated positions. These data suggest that TERT promoter mutations are the most frequent putative oncogenic mutations in cutaneous cancer.

Cytogenetics of melanoma and nonmelanoma skin cancer.

Cytogenetic analysis of melanoma and nonmelanoma skin cancers has revealed recurrent aberrations, the frequency of which is reflective of malignant potential. Highly aberrant karyotypes are seen in melanoma, squamous cell carcinoma, actinic keratosis, Merkel cell carcinoma and cutaneous lymphomas with more stable karyotypes seen in basal cell carcinoma, keratoacanthoma, Bowen's disease and dermatofibrosarcoma protuberans. Some aberrations are common among a number of skin cancer types including rearrangements and numerical abnormalities of chromosome 1, -3p, +3q, partial or entire trisomy 6, trisomy 7, +8q, -9p, +9q, partial or entire loss of chromosome 10, -17p, +17q and partial or entire gain of chromosome 20. Combination of cytogenetic analysis with other molecular genetic techniques has enabled the identification of not only aberrant chromosomal regions, but also the genes that contribute to a malignant phenotype. This review provides a comprehensive summary of the pertinent cytogenetic aberrations associated with a variety of melanoma and nonmelanoma skin cancers.

Genomic landscape of multiple Bowen's disease using whole-exome sequencing.

The genomic landscape of Bowen's disease (BD), with multiple manifestations, has not yet been determined. This study aimed to investigate the genomic alterations in multiple BD. We performed whole-exome sequencing of BD lesions (n = 9) and matched germlines collected from three patients with multiple (≥3) BD to detect somatic and germline mutations. We found a median of 64 somatic mutations in each sample (range 20-267). UV-signature mutations accounted for 64.9% (median, range 26.0%-82.1%) of point mutations. Putative driver mutations were found in five BDs (RB1 p.R445*, ARID2 p.R274*, TP53 p.Y163D/p.Y205D/p.R342*, KMT2C p.R4549C) but not in the other four lesions. Somatic mutations were not shared between multiple BD lesions collected from the same patient, indicating a different clonal origin. We also found no known pathogenic germline mutations in cancer-related genes. The mutational signature analysis revealed that UV signatures (SBS7a/7b) and age-related signatures (SBS1/5) were the main active signatures. Copy number alterations (CNAs) were found in two BDs: one with extensive CNA regions (21.7% of the genome), including driver genes (PIK3CA/SOX2/TP63 and MYC gain, and CDKN2A loss), and the other with 1q gain. Our study revealed that multiple BD lesions harbor distinct genomic landscapes, suggesting that they have different risks of malignant progression.

Analysis of the 3'UTR of the prostaglandin synthetase-2 (PTGS-2/COX-2) gene in non-melanoma skin cancer after organ transplantation.

To define the potential involvement of polymorphisms in the 3'untranslated region (3'UTR) of the prostaglandin synthetase-2 (PTGS-2) gene to non-melanoma skin cancer (NMSC) predisposition after transplantation, we screened for genetic variant, relevant parts of this region. It contains binding sites for trans-acting factors, an alternative polyadenylation site and putative target sequences for miRNAs. Variant +8473T>C did not appear to play a functional role in the regulation of gene expression in human keratinocyte-transfected cells. In addition to the well-known +8473T>C, we identified four polymorphisms: +8293G>C, +10259T>G, +10267G>A and +10335G>A. No allele frequency differences were observed between cases and controls neither for +8473T>C nor for any of the identified polymorphisms, suggesting that polymorphisms in the 3'UTR of the PTGS2 gene are rare and unlikely to represent risk factor for NMSC after transplantation.

Combined analysis of cell growth and apoptosis-regulating proteins in HPVs associated anogenital tumors.

BACKGROUND: The clinical course of human papillomavirus (HPV) associated with Bowenoid papulosis and condyloma acuminatum of anogenital tumors are still unknown. Here we evaluated molecules that are relevant to cellular proliferation and regulation of apoptosis in HPV associated anogenital tumors. METHODS: We investigated the levels of telomerase activity, and inhibitor of apoptosis proteins (IAPs) family (c-IAP1, c-IAP2, XIAP) and c-Myc mRNA expression levels in 20 specimens of Bowenoid papulosis and 36 specimens of condyloma acuminatum in anogenital areas. Overall, phosphorylated (p-) AKT, p-ribosomal protein S6 (S6) and p-4E-binding protein 1 (4EBP1) expression levels were examined by immunohistochemistry in anogenital tumors both with and without positive telomerase activity. RESULTS: Positive telomerase activity was detected in 41.7% of Bowenoid papulosis and 27.3% of condyloma acuminatum compared to normal skin (p < 0.001). In contrast, the expression levels of Bowenoid papulosis indicated that c-IAP1, c-IAP2 and XIAP mRNA were significantly upregulated compared to those in both condyloma acuminatum samples (p < 0.001, p < 0.001, p = 0.022, respectively) and normal skin (p < 0.001, p = 0.002, p = 0.034, respectively). Overall, 30% of Bowenoid papulosis with high risk HPV strongly promoted IAPs family and c-Myc but condyloma acuminatum did not significantly activate those genes. Immunohistochemically, p-Akt and p-S6 expressions were associated with positive telomerase activity but not with p-4EBP1 expression. CONCLUSION: Combined analysis of the IAPs family, c-Myc mRNA expression, telomerase activity levels and p-Akt/p-S6 expressions may provide clinically relevant molecular markers in HPV associated anogenital tumors.

Activation of fibroblast growth factor receptor 3 and oncogene-induced senescence in skin tumours.

BACKGROUND: The activation of oncogenes is an important step in tumorigenesis, and recently, oncogene-induced senescence (OIS) was proposed as a critical barrier against malignant transformation in normal primary cells. OBJECTIVES: The aim of this study was to examine the activation of fibroblast growth factor receptor 3 (FGFR3) as an oncogene product and OIS in human skin tumours. METHODS: We investigated the activation of FGFR3 and OIS by mutation and immunohistochemical analysis in skin tumours, including seborrhoeic keratosis, actinic keratosis (AK), Bowen's disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). RESULTS: Activated point mutations of FGFR3 were identified in four of 22 cases (18%) of seborrhoeic keratosis, but no mutation was detected in the other skin tumours. Twenty-seven of 31 cases (87%) of seborrhoeic keratosis showed moderately to strongly positive expression of the FGFR3 protein, but almost all the other skin tumours were negative. On the other hand, almost all the seborrhoeic keratoses showed negative immunoreactivity for antiphoshohistone H2AX (gamma-H2AX) as a marker of OIS, but 17 of 22 cases (77%) of AK were moderately to strongly positive. Immunoreactivity for gamma-H2AX was significantly greater in AK than in seborrhoeic keratosis, BD, BCC and SCC. CONCLUSIONS: The activation of FGFR3 might be a common feature in the tumorigenesis in seborrhoeic keratosis, although the activation does not induce a typical oncogenic signal in keratinocytes. In addition, OIS due to some oncogenic signals rather than activation of FGFR3 might be involved in the early skin carcinogenesis related to chronic ultraviolet radiation exposure.

[Merkel cell polyomavirus in the pathogenesis of non-melanoma skin cancer]. / Das Merkel-Zell-Polyomavirus in der Pathogenese nichtmelanozytärer Hauttumoren.

Merkel cell carcinoma (MCC) is a very aggressive neuroendocrine carcinoma of the skin. The recently identified Merkel cell polyomavirus (MCPyV) is present in the majority of MCCs. MCPyV clonally integrates in the tumor DNA and tumor-specific viral mutations are detected within the large T-antigen. To elucidate a possible role of MCPyV in the pathogenesis of other non-melanoma skin cancers (NMSC), i.e. squamous cell carcinoma, Bowen's disease and basal cell carcinoma we tested a group of these tumors in immunosuppressed and immunocompetent patients. In addition we tested MCPyV-positive tumors for viral mutations within the large T-antigen. MCPyV DNA was significantly more frequently detected in the NMSC of the immunosuppressed patients (p<0.001). No tumor specific mutations were found within the large T-antigen. The presence of the virus in tumor cells was confirmed by FISH analysis. Although MCPyV is present in the tumor cells of squamous cell carcinoma, Bowen's disease and basal cell skin carcinoma, further investigations into the role of MCPyV in the pathogenesis of these tumors is needed.

Immunohistochemical staining of palisading basal cells in Bowen's disease and basal involvement in actinic keratosis: contrasting staining patterns suggest different cells of origin.

Actinic keratosis (AK) and Bowen's disease (BD) are common patterns of in situ squamous cell carcinoma of the epidermis. In AK, atypical keratinocytes proliferate in the lower portion of the epidermis including the basal layer. In contrast, BD features atypical squamous cells in all portions of the epidermis but initially leaves basal cells in palisades along the basement membrane. To characterize immunohistochemically keratocyte proliferation in AK and Palisading Basal Cells (PBC) in BD, we stained microarray samples of 45 AK and 25 BD with Molecular Immunology Borstel (MIB-1). Subsequent immunostaining of full mounted sections examined 11 BD, 7 AK, and 4 examples of psoriasis for MIB-1 (as a proliferative marker) and p53 (as a cell cycle regulatory marker). AK stained for MIB-1 and p53 antibodies only in lower portion of epidermis and included the basal layer. BD with typical PBCs stained positive for both markers throughout the epidermis, except for the basal layer. Psoriatic biopsies stained positively for the 2 markers only in the basal and parabasal layers. Normal epidermis adjacent to the lesions in AK and BD biopsies stained sparsely in the basal layers. The correlation of different histologic patterns of epidermal involvement with different immunohistochemical patterns of stains argues for different cells of origin for BD versus AK. Lack of expression of proliferative antigens in palisading basal cells in BD provides evidence that PBCs are not the cell of origin for BD. Conversely in AK, expression of MIB-1 and p53 in basal cells argues that these cells play a role in histogenesis of AK.

Cyclin D1 promoter -56 and -54bp CpG un-methylation predicts invasive progression in arsenic-induced Bowen's disease.

BACKGROUND: Patients with arsenic-induced Bowen's disease (As-BD) are at risk of developing invasive cancers in the skin, lung, and urinary bladder. However, a longitudinal follow-up study on the association between As-BD and invasive cancers is still lacking. OBJECTIVES: This study aims to investigate the underlying molecular mechanisms of this malignant progression in the skin and internal organs. METHODS: This is a biopsy-based follow-up study. We tested the DNA histograms, Cyclin D1 (CCND1) protein expression and CCND1 promoter DNA methylation in 40 pathologically confirmed specimens from As-BD patients to correlate with individual's invasive cancer occurrence in the 5-year follow-up. RESULTS: Flow cytometric DNA histogram analysis of skin specimens showed aneuploid (n=15), G2/M arrest (n=22), and normal (n=3) DNA histograms. No patients with normal DNA histograms developed invasive cancers, whereas 13 developed invasive cancers in the aneuploid group and 2 developed invasive cancers in the G2/M arrest group. The aneuploid group showed a high risk of invasive cancer development. In all assessed aneuploid specimens, the CCND1 promoter hypomethylation was observed. Statistically, percentage of un-methylation more than 55.85% among 17 detected CpG sites showed extremely high predictive power in the occurrence of invasive arsenical cancers. Furthermore, the un-methylation at -56 and -54bp CpG sites was statistically significantly associated with invasive arsenical cancer development (p=1.29×10-5). CONCLUSIONS: As-BD lesions showing an aneuploid DNA histogram had a high risk of invasive cancer development. Un-methyaltion at -56 and -54bp CpG in the CCND1 promoter serves as a predictor for invasive progression in As-BD patients.

Subclonal Evolution of Cancer-Related Gene Mutations in p53 Immunopositive Patches in Human Skin.

Normal sun-exposed skin contains numerous epidermal patches that stain positive for p53 protein (p53 immunopositive patches, PIPs), which are considered potential early precursors of skin cancer. Although the TP53 gene is mutated in many PIPs, it is unclear whether PIPs contain any other cancer-related mutations. Here we report that PIPs, predominantly <3,000 p53 immunopositive cells in size, within normal chronically exposed skin contain mutations in multiple genes that are mutated in cutaneous squamous cell cancers. These mutations in the PIPs were not detected within the non-PIP epidermis of corresponding normal chronically exposed skin. Although some of these genetic alterations are clonal in the PIPs, many of the mutations are subclonal within these lesions. Similar mutations are seen in later precancers (actinic keratoses and Bowen's disease). Our results demonstrate that PIPs in chronically exposed skin contain multiple mutations in cancer-related genes. In addition, the results indicate that the clonal evolution of mutations that are seen within later precancerous lesions and in established malignancy can also occur in PIPs within normal human skin.

Immunohistochemical visualization of the signature of activated Hedgehog signaling pathway in cutaneous epithelial tumors.

Activation of the Hedgehog (HH) signaling pathway plays a critical role in the development of basal cell carcinoma (BCC). HH signaling activity is produced by nuclear translocation of transcription factors, glioma-associated oncogene homolog (GLI). Among three GLI subfamilies, GLI1 is the only full-length transcriptional activator, and its nuclear localization is recognized as a signature event in HH signaling activation. However, limited published work has investigated the nuclear staining of GLI1 protein in human tumor tissue samples by immunohistochemical analysis. In this study, we performed immunohistochemical staining of GLI1 in 382 cases of cutaneous epithelial tumors, including 196 BCC cases, using rabbit monoclonal antihuman GLI1 antibody (C68H3). As a result, 98.2% cases of BCC showed a diffuse and strong nuclear staining pattern regardless of the histological subtype. Positive staining was mainly restricted to the tumor nests, while the overlying epidermis was negative suggesting specificity of the antibody. In further analysis of other cutaneous epithelial tumors, 100% (4/4) cases of trichoblastoma, 15.1% (5/33) Bowen's disease, 3.5% (1/28) actinic keratosis and 12.5% (4/32) squamous cell carcinoma showed the nuclear staining pattern of GLI1. This suggested that HH signaling is also dysregulated in some other cutaneous malignant tumors. In conclusion, the C68H3 antibody is a useful tool for revealing activation of HH signaling in immunohistochemical analysis.

The higher incidence of squamous cell carcinoma in renal transplant recipients is associated with increased telomere lengths.

The incidence and aggressiveness of nonmelanoma skin cancers, including basal cell carcinoma and squamous cell carcinoma (SCC), in immunocompromised renal transplant recipients (RTRs) is dramatically higher (up to 100-fold) compared with the normal population. SCC lesions are also predominant in RTRs, in contrast to the normal population where basal cell carcinoma is more common. The mechanisms underlying this phenomenon are unknown, but effective treatments for these skin tumors would have a significant impact upon morbidity in this group of patients. The fundamental role of telomeres and telomerase in the development of most human cancers, including melanoma, is well established, but very few reports have assessed their function during the onset of nonmelanoma skin cancer. To assess whether telomere maintenance plays any role in the increased incidence of SCC in renal transplant patients, we analyzed both the telomere lengths and telomerase expression levels in 44 SCCs and 22 Bowen's disease (BD) samples (carcinoma in situ) from RTRs and nontransplant patients. Our findings provide statistically significant evidence that the telomeres are consistently longer in both BD RTR and SCC RTR lesions compared with their nontransplant counterparts. We also show by immunohistochemistry that there is a trend toward higher telomerase levels in both the BD RTR and SCC RTR lesions, although this was not statistically significant. Our data thus suggest that telomere lengthening may possibly be an early event in the development of SCC in renal transplant patients and demonstrate that telomere maintenance mechanisms should be further evaluated with respect to developing a future therapeutic strategy for these cancers.

Increased chromosome aberration frequencies in the Bowen's patients compared to non-cancerous skin lesions individuals exposed to arsenic.

Cytogenetic biomarkers are essential for assessing environmental exposure that can predict adverse human health effects such as cellular damage. Chromosomal aberrations are the most important cytogenetic end-points successfully used for the cancer risk assessment of populations occupationally or environmentally exposed to different toxic chemicals. Previous reports suggest that, increased frequency of chromosomal aberration (CA), in peripheral blood lymphocytes, is a predictor of cancer. Arsenic is a paradoxical human carcinogen, clastogen and aneugen. Despite of exposure at similar extent, only 15-20% of individuals show arsenic induced skin lesions including Bowen's disease (BD). Previously we have reported the significant increase in CA in the individuals with arsenic induced skin lesions when compared to individuals without any skin lesions, drinking arsenic contaminated water at similar extent. Presently, a matched case-control study was performed to examine whether biomarkers such as chromosomal aberrations can predict the development of arsenic induced Bowen's (in situ carcinoma) diseases. Chromosomal aberrations (both chromosome and chromatid types) and mitotic index were analyzed from the lymphocytes of 25 cases of Bowen's patient which was compared to matched control from the individuals with arsenic induced non-cancerous skin lesions such as raindrop pigmentation, keratosis of palm and sole, hypo and hyper pigmentation. Chromosomal aberrations/cell, chromosome type aberrations and total percentage of aberrant cells were significantly higher in cases compared to control (p<0.01). These results suggest that chromosomal aberrations can be used for cancer risk assessment of the population exposed to arsenic through drinking water.

Analysis of p16 gene mutation, deletion and methylation in patients with arseniasis produced by indoor unventilated-stove coal usage in Guizhou, China.

The aim of this study was to determine p16 gene mutation, deletion, and promoter 5' CpG island hypermethylation in peripheral blood mononuclear leukocyte of patients with arseniasis as attributed to exposure to indoor unventilated coal stove. The role of the aberrant change of p16 gene in the induction and development of carcinogenesis in endemic arsenisiasis region in China was also examined. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP), multiplex PCR (mPCR), methylation-specific PCR (MSP), and sequencing techniques were performed to detect (1) mutation of the p16 gene exon 2, (2) homozygous deletion of the p16 gene exon 1 and exon 2, and (3) hypermethylation of the promoter CpG island in peripheral blood mononuclear leukocyte of patients with arseniasis. Results showed no mutation was found in exon 2 of p16 gene. The homozygous deletion frequency of p16 gene was 5 and 15% in control and arseniasis patients, respectively. The homozygous deletion occurred mainly in exon 2, with significant deletion frequencies of 9, 13, and 20% in mild, intermediate, and severe arseniasis groups. The significant homozygous deletion frequency was 9 and 39% in noncarcinoma and carcinoma individuals. The positive rate of p16 gene promoter CpG island hyermethylation was 42 and 2% in the exposed group and the control group, respectively. The positive rate was 26, 42, and 50% in mild, intermediate, and severe arseniasis. The marked different positive rate was 22 and 56% in noncarcinoma and carcinoma individuals, respectively. In conclusion, homozygous deletion and hypermethylation of p16 gene may play an important role in the initiation and development of manifestations seen in endemic arseniasis including carcinogenesis.

Significant Role of Collagen XVII And Integrin ß4 in Migration and Invasion of The Less Aggressive Squamous Cell Carcinoma Cells.

Collagen XVII and integrin α6ß4 have well-established roles as epithelial adhesion molecules. Their binding partner laminin 332 as well as integrin α6ß4 are largely recognized to promote invasion and metastasis in various cancers, and collagen XVII is essential for the survival of colon and lung cancer stem cells. We have studied the expression of laminin γ2, collagen XVII and integrin ß4 in tissue microarray samples of squamous cell carcinoma (SCC) and its precursors, actinic keratosis and Bowen's disease. The expression of laminin γ2 was highest in SCC samples, whereas the expression of collagen XVII and integrin ß4 varied greatly in SCC and its precursors. Collagen XVII and integrin ß4 were also expressed in SCC cell lines. Virus-mediated RNAi knockdown of collagen XVII and integrin ß4 reduced the migration of less aggressive SCC-25 cells in horizontal scratch wound healing assay. Additionally, in a 3D organotypic myoma invasion assay the loss of collagen XVII or integrin ß4 suppressed equally the migration and invasion of SCC-25 cells whereas there was no effect on the most aggressive HSC-3 cells. Variable expression patterns and results in migration and invasion assays suggest that collagen XVII and integrin ß4 contribute to SCC tumorigenesis.

Human papilloma virus 5-DNA in a carcinoma of an epidermodysplasia verruciformis patient infected with various human papillomavirus types.

An epidermodysplasia verruciformis patient suffering from generalized warts and a carcinoma at the forehead was found to be infected by at least six types and subtypes of human papillomaviruses. The central part of the carcinoma, however, harbored only human papillomavirus 5 DNA. The DNA persisted extrachromosomally in high genome copy number. In contrast to wart DNA preparations, a significant part of the viral sequences in the carcinoma was present as oligomers, at least part of them being concatemers as shown by S1 digest. The human papillomavirus 5 subtype from this carcinoma was compared with the two other carcinoma isolates described so far and proved to be rather similar.