- Email: [email protected]
601 Common mechanisms and signaling pathways in fibroblasts of cancer-prone genodermatoses
Melanoma and Other Skin Cancers | ABSTRACTS 600
601
Trends in incidence and tumour thickness of invasive cutaneous melanoma from 2000 to 2014 in an Eastern Hungarian melanoma centre EA Janka, K Ke´kedi, E Gelle´n, E´ Remenyik and G Emri Department of Dermatology, Faculty of Medicine University of Debrecen, Debrecen, Hungary In welfare societies the importance of malignant melanoma is increasing, since the incidence of melanoma is increasing worldwide especially among fair-skinned people. Assessing the characteristics of patients and their tumours may help to develop a more effective prevention strategy. The objective of this study was to assess the incidence, type and localization of melanomas diagnosed at the Department of Dermatology University of Debrecen in the period 2000-2014, to analyse the patient’s gender and age composition and the trend in tumour thickness over the examined time period. In the study we processed the data of 1567 tumours. For the purpose of determining the trend, the data were analysed for 3-year periods. The tumours were categorized according to the American Joint Committee on Cancer 7th edition (2009) TNM staging classification at the time of diagnosis. Our results showed a favourable tendency for tumour thickness in the second half of the 2000s, but this trend reversed during the 2012-2014 period, and the rate of tumours diagnosed at stage I began to decline, while the rate of those diagnosed at stage III began to increase. In addition, the incidence of melanoma was higher among females than among males but the difference was not significant. Nevertheless, the rate of new melanoma cases was significantly higher among older people. All this confirms the need to strengthen educational and screening programs in Hungary. Since cancer screening of a high-risk group is more cost-effective, based on our results it would be desirable to screen melanomas among those over 65 years of age. Most likely, this can only be achieved with the help of general practitioners.
Common mechanisms and signaling pathways in fibroblasts of cancer-prone genodermatoses F Dı´az1,2, C Leo´n1,2, F Garcı´a-Garcı´a3, E Chaco´n-Solano1,2, F Larcher1,2,4, J Carbonell3, JA Puig5, S Puig5, J Dopazo3 and M del Rı´o1,2,4 1 Bioengineering, Universidad Carlos III (UC3M), Madrid, Spain, 2 Instituto de Investigacio´n Sanitaria de la Fundacio´n Jime´nez Dı´az (IISFJD), Madrid, Spain, 3 Computational Genomics, CIBERER (U715), CIPF, Valencia, Spain, 4 CIEMAT-Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER U714), Madrid, Spain and 5 Melanoma Unit, Hospital Clinic & IDIBAPS, CIBERER U726, Barcelona, Spain Despite the genetic bases of many rare skin diseases having been elucidated in the past years, the mutations in the causal genes often fail to explain on their own the vast array of phenotypic manifestations in these pathologies. Recessive Dystrophic Epidermolysis Bullosa (RDEB), Kindler syndrome (KS) and Xeroderma Pigmentosum C (XPC) are three genodermatoses that share a number of features, including a predisposition to cancer, whose mechanisms are not yet fully understood. In this study we have investigated the transcriptional signature across these conditions to address the role of dermal environment in the development of the pathology. Fibroblasts isolated from several RDEB, XPC and KS patients, as well as healthy donors, have been studied using RNA-Seq technology. The analysis included a thorough examination of the differentially expressed genes, a functional enrichment analysis and the determination of the affected signaling circuits using computational models of signaling pathway activity. The results revealed a set of 227 genes and 42 signaling circuits commonly altered in all three conditions, along with unique mechanisms acting in each of them. The common signaling circuits and biomarkers uncovered by the analysis point towards a phenotype in which the fibroblasts of these conditions show similarities to cancerassociated fibroblasts, enabling a favorable environment for tumor development and progression. We anticipate this approach, focused on the mechanisms that govern cell behavior, to be useful in explaining obscure phenotypic traits of these genodermatoses and to increase the number of targets for effective therapeutic intervention.
602
603
Oxidative stress status in squamous cell carcinoma A Yoshifuku, K Fujii and T Kanekura Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan The oxidative stress (OS) is supposed to be a major pathogenesis of skin cancer, while the details of how OS affects skin carcinogenesis remains to be explained. To reveal this mechanism, OS status of actinic keratosis (AK), Bowen’s disease (BD) and cutaneous squamous cell carcinoma (SCC) were analyzed by immunohistochemistry. Totally, 258 patients, who were treated in our hospital between January 2009 and December 2014, were enrolled in this study. Oxidative stress to DNA, lipid peroxidation and protein oxidation were assessed by 8-hydroxydeoxyguanosine (8-OHdG), malondialdehyde (MDA) and dityrosine (DT), respectively, by immunohistochemical techniques. We found a significant overexpression of the 8-OHdG in AK and BD lesions compared with surrounding non-tumor tissue, healthy controls and SCC. On the other hand, MDA expression was increased in AK, BD and SCC lesions compared with controls, and the expression level was stronger in SCC than AK and BD. The expression level of DT was higher in AK, BD and SCC tissues than control, while the significant difference was not observed among the three lesions. In summary, DNA oxidation was observed in early stage of skin carcinogenesis; lipid peroxidation was associated with cancer progression; protein oxidation was observed in all stages of cutaneous SCC.
Expression of factors regulating cytokinesis in Bowen’s disease H Okada1, M Komine1,2, H Tsuda1, K Kamiya1 and M Ohtsuki1 1 Dermatology, Jichi Medical University, Shimotsuke, Japan and 2 Biochemistry, Jichi Medical University, Shimotsuke, Japan Bowen’s disease is a form of squamous cell carcinoma in situ, with full-thickness epidermal involvement with occasional multinucleated epithelial cells. Multinucleated cells imply defect in cytoplasmic division; cytokinesis. We have previously shown that loss of IL-33 in the epidermal keratinocytes caused decreased expression of epithelial cell transforming gene 2 (ECT2), resulting in decreased activation of Ras homologue gene family A (RhoA), and the formation of binucleated cells in culture. Loss of expression of ECT2 and RhoA have been reported to cause multinucleated cells in epidermal keratinocytes. We hypothesized that loss of expression of these molecules was involved in the formation of multinucleated cells in Bowen’s disease. We retrieved formalin-fixed, paraffin-embedded blocks of Bowen’s disease from the archives of Pathology Department in Jichi Medical University, sliced, and immunostained with anti-IL-33, anti-ECT2, and anti-RhoA antibodies. Tumor cells in Bowen’s disease showed decreased staining with IL-33, in contrast to pronounced nuclear staining in the surrounding hyperplastic epidermal keratinocytes. Normal epidermis showed diffuse or basal cytoplasmic staining with RhoA, while tumor cells showed decreased staining with RhoA in the lower portion where atypia is prominent, and cytoplasmic staining in the upper portion where atypia is not apparent. ECT2 showed basal cytoplasmic staining in the normal epidermis, while tumor cells showed decreased cytoplasmic staining in the lower potion, and cytoplasmic staining with occasional nuclear staining in the upper portion. Decreased expression of these factors in proliferating tumor cells could be involved in the multinucleation of tumor cells in Bowen’s disease.
604
605
Increased risk for vulvar melanoma among patients with lichen sclerosus N Hieta1, S Kurki2, M Rintala3, J Ristima¨ki3, S Hietanen3 and K Kivinen4 1 Dermatology, Turku University Hospital, Turku, Finland, 2 Auria Biobank, Turku, Finland, 3 Gynaecology, Turku University Hospital, Turku, Finland and 4 Laboratory of Molecular Genetics, Turku University Hospital, Turku, Finland The purpose of this study was to investigate the possible connection between lichen sclerosus (LS) and vulvar melanoma. LS is a chronic inflammatory skin disease presenting mainly on the anogenital area. The prevalence of LS is estimated as 1:300 to 1:900. The life-time risk for the development of vulvar SCC among female patients is 4-5%. There have also been anecdotal reports on vulvar melanoma among LS patients, but the risk has not been shown to be increased. 249 female patients with lichen sclerosus confirmed by vulvar biopsy and 10 patients with vulvar melanoma were identified using SNOMED Clinical Terms from the archives of the Department of Pathology / Auria Biobank in Turku University Hospital. Three vulvar melanomas were found among LS patients, giving a relative risk (RR) of 290 (p <0.001). Vulvar melanoma patients with lichen sclerosus had an average Breslow measure of 12.8 mm (range 9 mm to 19 mm), and Clark stage median was IV. Those patients without lichen sclerosus had an average Breslow of 10.5 mm (range 1.2 mm to 40 mm), and Clark median was IV. There was no statistically significant difference between the two groups in Breslow (p¼0.793) or Clark classification (p¼0.351). All patients except for one in the non-LS group had ulceration in the tumor. All the three patients with LS died of melanoma metastases by 2 years after the diagnosis, whereas only two out seven patients without LS had died by the end of follow-up. The difference was statistically significant (p<0.043). There is an increased incidence and worse prognosis of vulvar melanoma among patients with LS with no traditional prognostic factors to explain the difference.
Strong FGF1 signaling inhibits the proliferation, invasion and migration of murine angiosarcoma cell line ISOS-1 F Nakayama1, S Umeda1, M Fujita1, K Imadome1, M Kawano1, S Koike1, T Miura1, T Yasuda1, M Masuzawa2 and T Imai1 1 National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan and 2 School of Allied Health Sciences, Kitasato University, Sagamihara, Japan Angiosarcoma is a poor prognostic tumor which is treated with radiotherapy. FGF1 is a potential radioprotector; however, it may exacerbate the malignancy of tumors. However, the influence of FGF1 on the malignancy of angiosarcoma remains unknown. This study aimed at clarifying the effect of FGF1 signaling on the malignancy of murine angiosarcoma cell line ISOS-1. Highly stable FGF1 mutants were developed previously to increase mitogenic activity more than wild-type FGF1, so ISOS-1 cells were cultured in complete DMEM medium with each FGF1 mutant to examine their effects in the proliferation, migration, and invasion of ISOS-1 cells. FGF1 mutants with strong mitogenic activity reduced the colony formation, invasion, and migration of ISOS-1 cells, whereas they increased the radiosensitivity of ISOS-1 cells to X-rays in a mitogenic activity-dependent manner. In contrast, FGFR inhibitor canceled the inhibitory effects of FGF1 mutants on colony formation, invasion, and migration. In addition, the repression of FGFR1 by siRNA showed that strong FGFR1 signaling reduced the colony formation of ISOS-1 cells. On the other hand, FGF1 mutant reduced the activation of VEGFRs and EGFRs in ISOS-1 cells more than wild-type FGF1. Moreover, the co-inhibition of VEGFRs and EGFRs reduced the colony formation of ISOS-1 cells, although single inhibition of VEGFRs or EGFRs did not reduce it. These findings suggest that strong FGF1 signaling can inhibit proliferative and metastatic capability of angiosarcoma through down-regulation of both EGFR and VEGFR pathways. We conclude that FGF1 mutants are candidate radioprotectors for angiosarcoma radiotherapy with potential inhibitory effects on the malignancy of angiosarcoma.
www.jidonline.org S295
601
Trends in incidence and tumour thickness of invasive cutaneous melanoma from 2000 to 2014 in an Eastern Hungarian melanoma centre EA Janka, K Ke´kedi, E Gelle´n, E´ Remenyik and G Emri Department of Dermatology, Faculty of Medicine University of Debrecen, Debrecen, Hungary In welfare societies the importance of malignant melanoma is increasing, since the incidence of melanoma is increasing worldwide especially among fair-skinned people. Assessing the characteristics of patients and their tumours may help to develop a more effective prevention strategy. The objective of this study was to assess the incidence, type and localization of melanomas diagnosed at the Department of Dermatology University of Debrecen in the period 2000-2014, to analyse the patient’s gender and age composition and the trend in tumour thickness over the examined time period. In the study we processed the data of 1567 tumours. For the purpose of determining the trend, the data were analysed for 3-year periods. The tumours were categorized according to the American Joint Committee on Cancer 7th edition (2009) TNM staging classification at the time of diagnosis. Our results showed a favourable tendency for tumour thickness in the second half of the 2000s, but this trend reversed during the 2012-2014 period, and the rate of tumours diagnosed at stage I began to decline, while the rate of those diagnosed at stage III began to increase. In addition, the incidence of melanoma was higher among females than among males but the difference was not significant. Nevertheless, the rate of new melanoma cases was significantly higher among older people. All this confirms the need to strengthen educational and screening programs in Hungary. Since cancer screening of a high-risk group is more cost-effective, based on our results it would be desirable to screen melanomas among those over 65 years of age. Most likely, this can only be achieved with the help of general practitioners.
Common mechanisms and signaling pathways in fibroblasts of cancer-prone genodermatoses F Dı´az1,2, C Leo´n1,2, F Garcı´a-Garcı´a3, E Chaco´n-Solano1,2, F Larcher1,2,4, J Carbonell3, JA Puig5, S Puig5, J Dopazo3 and M del Rı´o1,2,4 1 Bioengineering, Universidad Carlos III (UC3M), Madrid, Spain, 2 Instituto de Investigacio´n Sanitaria de la Fundacio´n Jime´nez Dı´az (IISFJD), Madrid, Spain, 3 Computational Genomics, CIBERER (U715), CIPF, Valencia, Spain, 4 CIEMAT-Centro de Investigacio´n Biome´dica en Red de Enfermedades Raras (CIBERER U714), Madrid, Spain and 5 Melanoma Unit, Hospital Clinic & IDIBAPS, CIBERER U726, Barcelona, Spain Despite the genetic bases of many rare skin diseases having been elucidated in the past years, the mutations in the causal genes often fail to explain on their own the vast array of phenotypic manifestations in these pathologies. Recessive Dystrophic Epidermolysis Bullosa (RDEB), Kindler syndrome (KS) and Xeroderma Pigmentosum C (XPC) are three genodermatoses that share a number of features, including a predisposition to cancer, whose mechanisms are not yet fully understood. In this study we have investigated the transcriptional signature across these conditions to address the role of dermal environment in the development of the pathology. Fibroblasts isolated from several RDEB, XPC and KS patients, as well as healthy donors, have been studied using RNA-Seq technology. The analysis included a thorough examination of the differentially expressed genes, a functional enrichment analysis and the determination of the affected signaling circuits using computational models of signaling pathway activity. The results revealed a set of 227 genes and 42 signaling circuits commonly altered in all three conditions, along with unique mechanisms acting in each of them. The common signaling circuits and biomarkers uncovered by the analysis point towards a phenotype in which the fibroblasts of these conditions show similarities to cancerassociated fibroblasts, enabling a favorable environment for tumor development and progression. We anticipate this approach, focused on the mechanisms that govern cell behavior, to be useful in explaining obscure phenotypic traits of these genodermatoses and to increase the number of targets for effective therapeutic intervention.
602
603
Oxidative stress status in squamous cell carcinoma A Yoshifuku, K Fujii and T Kanekura Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan The oxidative stress (OS) is supposed to be a major pathogenesis of skin cancer, while the details of how OS affects skin carcinogenesis remains to be explained. To reveal this mechanism, OS status of actinic keratosis (AK), Bowen’s disease (BD) and cutaneous squamous cell carcinoma (SCC) were analyzed by immunohistochemistry. Totally, 258 patients, who were treated in our hospital between January 2009 and December 2014, were enrolled in this study. Oxidative stress to DNA, lipid peroxidation and protein oxidation were assessed by 8-hydroxydeoxyguanosine (8-OHdG), malondialdehyde (MDA) and dityrosine (DT), respectively, by immunohistochemical techniques. We found a significant overexpression of the 8-OHdG in AK and BD lesions compared with surrounding non-tumor tissue, healthy controls and SCC. On the other hand, MDA expression was increased in AK, BD and SCC lesions compared with controls, and the expression level was stronger in SCC than AK and BD. The expression level of DT was higher in AK, BD and SCC tissues than control, while the significant difference was not observed among the three lesions. In summary, DNA oxidation was observed in early stage of skin carcinogenesis; lipid peroxidation was associated with cancer progression; protein oxidation was observed in all stages of cutaneous SCC.
Expression of factors regulating cytokinesis in Bowen’s disease H Okada1, M Komine1,2, H Tsuda1, K Kamiya1 and M Ohtsuki1 1 Dermatology, Jichi Medical University, Shimotsuke, Japan and 2 Biochemistry, Jichi Medical University, Shimotsuke, Japan Bowen’s disease is a form of squamous cell carcinoma in situ, with full-thickness epidermal involvement with occasional multinucleated epithelial cells. Multinucleated cells imply defect in cytoplasmic division; cytokinesis. We have previously shown that loss of IL-33 in the epidermal keratinocytes caused decreased expression of epithelial cell transforming gene 2 (ECT2), resulting in decreased activation of Ras homologue gene family A (RhoA), and the formation of binucleated cells in culture. Loss of expression of ECT2 and RhoA have been reported to cause multinucleated cells in epidermal keratinocytes. We hypothesized that loss of expression of these molecules was involved in the formation of multinucleated cells in Bowen’s disease. We retrieved formalin-fixed, paraffin-embedded blocks of Bowen’s disease from the archives of Pathology Department in Jichi Medical University, sliced, and immunostained with anti-IL-33, anti-ECT2, and anti-RhoA antibodies. Tumor cells in Bowen’s disease showed decreased staining with IL-33, in contrast to pronounced nuclear staining in the surrounding hyperplastic epidermal keratinocytes. Normal epidermis showed diffuse or basal cytoplasmic staining with RhoA, while tumor cells showed decreased staining with RhoA in the lower portion where atypia is prominent, and cytoplasmic staining in the upper portion where atypia is not apparent. ECT2 showed basal cytoplasmic staining in the normal epidermis, while tumor cells showed decreased cytoplasmic staining in the lower potion, and cytoplasmic staining with occasional nuclear staining in the upper portion. Decreased expression of these factors in proliferating tumor cells could be involved in the multinucleation of tumor cells in Bowen’s disease.
604
605
Increased risk for vulvar melanoma among patients with lichen sclerosus N Hieta1, S Kurki2, M Rintala3, J Ristima¨ki3, S Hietanen3 and K Kivinen4 1 Dermatology, Turku University Hospital, Turku, Finland, 2 Auria Biobank, Turku, Finland, 3 Gynaecology, Turku University Hospital, Turku, Finland and 4 Laboratory of Molecular Genetics, Turku University Hospital, Turku, Finland The purpose of this study was to investigate the possible connection between lichen sclerosus (LS) and vulvar melanoma. LS is a chronic inflammatory skin disease presenting mainly on the anogenital area. The prevalence of LS is estimated as 1:300 to 1:900. The life-time risk for the development of vulvar SCC among female patients is 4-5%. There have also been anecdotal reports on vulvar melanoma among LS patients, but the risk has not been shown to be increased. 249 female patients with lichen sclerosus confirmed by vulvar biopsy and 10 patients with vulvar melanoma were identified using SNOMED Clinical Terms from the archives of the Department of Pathology / Auria Biobank in Turku University Hospital. Three vulvar melanomas were found among LS patients, giving a relative risk (RR) of 290 (p <0.001). Vulvar melanoma patients with lichen sclerosus had an average Breslow measure of 12.8 mm (range 9 mm to 19 mm), and Clark stage median was IV. Those patients without lichen sclerosus had an average Breslow of 10.5 mm (range 1.2 mm to 40 mm), and Clark median was IV. There was no statistically significant difference between the two groups in Breslow (p¼0.793) or Clark classification (p¼0.351). All patients except for one in the non-LS group had ulceration in the tumor. All the three patients with LS died of melanoma metastases by 2 years after the diagnosis, whereas only two out seven patients without LS had died by the end of follow-up. The difference was statistically significant (p<0.043). There is an increased incidence and worse prognosis of vulvar melanoma among patients with LS with no traditional prognostic factors to explain the difference.
Strong FGF1 signaling inhibits the proliferation, invasion and migration of murine angiosarcoma cell line ISOS-1 F Nakayama1, S Umeda1, M Fujita1, K Imadome1, M Kawano1, S Koike1, T Miura1, T Yasuda1, M Masuzawa2 and T Imai1 1 National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology, Chiba, Japan and 2 School of Allied Health Sciences, Kitasato University, Sagamihara, Japan Angiosarcoma is a poor prognostic tumor which is treated with radiotherapy. FGF1 is a potential radioprotector; however, it may exacerbate the malignancy of tumors. However, the influence of FGF1 on the malignancy of angiosarcoma remains unknown. This study aimed at clarifying the effect of FGF1 signaling on the malignancy of murine angiosarcoma cell line ISOS-1. Highly stable FGF1 mutants were developed previously to increase mitogenic activity more than wild-type FGF1, so ISOS-1 cells were cultured in complete DMEM medium with each FGF1 mutant to examine their effects in the proliferation, migration, and invasion of ISOS-1 cells. FGF1 mutants with strong mitogenic activity reduced the colony formation, invasion, and migration of ISOS-1 cells, whereas they increased the radiosensitivity of ISOS-1 cells to X-rays in a mitogenic activity-dependent manner. In contrast, FGFR inhibitor canceled the inhibitory effects of FGF1 mutants on colony formation, invasion, and migration. In addition, the repression of FGFR1 by siRNA showed that strong FGFR1 signaling reduced the colony formation of ISOS-1 cells. On the other hand, FGF1 mutant reduced the activation of VEGFRs and EGFRs in ISOS-1 cells more than wild-type FGF1. Moreover, the co-inhibition of VEGFRs and EGFRs reduced the colony formation of ISOS-1 cells, although single inhibition of VEGFRs or EGFRs did not reduce it. These findings suggest that strong FGF1 signaling can inhibit proliferative and metastatic capability of angiosarcoma through down-regulation of both EGFR and VEGFR pathways. We conclude that FGF1 mutants are candidate radioprotectors for angiosarcoma radiotherapy with potential inhibitory effects on the malignancy of angiosarcoma.
www.jidonline.org S295