Tesi sul tema "Skin carcinoma"

Exposure to ultraviolet radiation and immunosuppression are the principal causes of non-melanoma skin cancer (NMSC). One of the skin’s main functions is to act as a barrier against environmental insults and transepidermal water loss (TEWL) is a marker of skin barrier function. Previous phase 2 studies have shown nicotinamide (NAM) to reduce premalignant actinic keratoses. Topical NAM has been shown to reduce TEWL. The effects of oral NAM were evaluated in two clinical trials. The Oral Nicotinamide To Reduce Actinic Cancer (ONTRAC) study was a multicentre phase 3, double-blind, randomised controlled trial in 386 immune-competent participants with at least 2 NMSCs in the past 5 years. Participants were randomised to receive 500mg of NAM or placebo twice daily for 12 months, with assessment at 3-monthly intervals for 18 months. TEWL measurements were taken 3-monthly for 12 months in 292 participants at a single study site. The primary end point was the number of new histologically-confirmed NMSCs during the 12-month intervention period. A second, phase 2 pilot study was undertaken in 22 immunosuppressed renal transplant recipients who were randomised to receive 500mg of NAM or placebo twice daily for 6 months, with assessments at 2-monthly intervals. The ONTRAC study found a 23% relative rate reduction in new NMSCs in the NAM group compared to the placebo group (p=0.02). The estimated relative reduction in TEWL with NAM at 12 months was 6% on the forehead (p=0.04) and 8% on the limbs (p=0.04). The nicotinamide renal transplant pilot study found new NMSCs to be 35% lower in the NAM group than the placebo group (p=0.4). Oral NAM was well-tolerated and safe. The work described in this thesis provides evidence that NAM is a new chemopreventive agent for NMSC in high-risk immune-competent patients. Phase 3 studies are now warranted in immunosuppressed organ transplant recipients. Nicotinamide is also a potential new systemic agent for improving skin barrier function.

Non-melanoma skin cancer (NMSC) is the most common cancer in the world with a lifetime risk for development as high as 2 in 3 in Queensland, Australia. Mortality is quite low, representing an approximate 360 deaths in Australia annually but cost of treatment is extremely high, estimated at $232 million each year. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the two most common forms of NMSC. Although BCC generally do not have the propensity to metastasise, they are highly invasive and can be locally destructive. SCC on the other hand is invasive and has metastatic potential. SCC is generally derived from a precursor lesion, solar keratosis (SK), which is also considered to be a biomarker of BCC, SCC and malignant melanoma. According to one theory, SKs actually represent the first recognisable stage of SCC development and therefore may be indicative of the earliest stage of NMSC development. In addition to these common forms of NMSC, rarer forms such as keratoacanthoma (KA), which spontaneously regress, and SCC in situ, which rarely become invasive, may provide clues into protective mechanisms associated with prevention of development. Like all other cancers, NMSC arises from an accumulation of genetic abnormalities that result in severe cellular dysfunction. A number of genes have been proposed in the development of NMSC, including p53, CDKN2a, Bcl-2 and the Ras family of genes, which are typically associated with proliferative and differentiation processes. Also, a number of genetic disorders that predispose individuals to NMSC have also been identified. Genetic abnormalities in these genes may be a result of somatic mutations that may be promoted by environmental carcinogens. For NMSC, ultraviolet (UV) radiation is the primary environmental stimulus that acts upon skin to generate mutations. UV effects are 2-fold; the first being direct damage produced by UVB radiation and the second being indirect damage as a result of UVA-induced oxidative stress. In addition to mutations of genes that directly result in carcinogenesis, polymorphic variants of genes may also play a role in susceptibility to NMSC. These susceptibility genes may have immunogenic, detoxifying or transcriptional roles that could be involved in increased mutagenesis or activation of cancer causing genes. The purpose of this study was ultimately to identify further molecular based mechanisms associated with the development of non-melanoma skin cancer. Initially, this study aimed to examine the effects of aberrant chromosomal regions on NMSC development and also to identify candidate genes within these regions that may be implicated in the development and progression of NMSC. Also, based on chromosomal and functional implications, a number of candidate genes were assessed using association analysis to determine their involvement in susceptibility to the earliest stages of NMSC development. Implicated susceptibility genes were then further investigated to determine their response to UV radiation. Therefore the methodological approach of these studies was based on three broad technical applications of cytogenetic, association and expression analyses. Previous comparative genomic hybridisation (CGH) studies implicated the 18q chromosomal region in progression of SK to SCC and this region was therefore suspected of harbouring one or more tumour suppressor genes that were associated with a more malignant phenotype. Following on from this analysis, loss of heterozygosity (LOH) analysis was used for further delineation of this region and possibly to implicate candidate genes involved in progression. Additionally, CGH was used to investigate keratoacanthoma to determine aberrant regions that might be involved in progression and also regression of this NMSC. Genes that had potential functional roles in NMSC development and that were located in or near regions implicated by these cytogenetic analyses were further investigated using association analysis. Association analysis was performed using polymerase chain reaction and subsequent restriction enzyme digestion or GeneScan analysis to determine genotype and allele frequencies in an SK affected versus control population for polymorphisms within a number of candidate genes. This population was carefully phenotyped so that not only genotypic factors could be analysed but also their interaction with a number of phenotypic and environmental risk factors. Genes with polymorphisms that did show association with solar keratosis development were then examined functionally. Specifically, gene expression analysis was undertaken to investigate their response to UV radiation. Both UVA only and combined UVA/UVB treatments were used for short term irradiation and also for long term irradiation with recovery to determine differential effects of UV range and dose in human skin. Relative mRNA expression analysis of these genes was performed using quantitative real time reverse transcription polymerase chain reaction to determine if UV radiation imposed gene expression changes in the skin. A combination of these methodologies provided a wide basis for investigation of NMSC. Cytogenetic, association and expression analyses all allow for the identification of molecular risk factors that cause or are associated with NMSC development and progression. These analyses provided diverse results that implicated various molecular mechanisms in the development of NMSC. Cytogenetic analysis is a powerful technique, especially for the identification of a broad range of aberrations throughout the genome. This study employed LOH analysis to investigate an implicated region involved in progression to SCC and to attempt identification of candidate genes that may be involved in this process. LOH analysis was successfully performed on 9 SCCs, 5 SCCs in situ and 2 SKs using 8 microsatellite markers within the 18q region. Polymerase chain reaction (PCR) was used to amplify polymorphic regions of these markers and genotypic composition was determined for normal and cancerous tissue within the specimen. In heterozygote individuals, determined by analysis of normal tissue, the cancerous tissue was examined to determine if alleles within the implicated region had been lost. However, after analysis of multiple different samples, there was no LOH detected in any of the samples examined for this analysis. This does not necessarily reject a role for 18q, or genes within this region, as the localisation of candidate tumour suppressor genes within a small region may indicate a tighter region of involvement than was expected. As such, a more targeted study may further delineate this region and implicate candidate genes in progression of SK to the more malignant phenotype of SCC. Further CGH analysis of keratoacanthoma was also undertaken to identify aberrations associated with development and also regression of this skin cancer. CGH was performed using universal amplification and nick translation to incorporate a fluorescent dye. Differentially labelled normal and tumour DNA were then competitively hybridised to a normal metaphase spread and fluorescence emission indicated either amplification or deletion of specific chromosomal regions. In total, 6 KA samples were analysed, with 2 samples each from evolving, matured and regressing stages of KA development. In general, regressing KAs appeared to be more highly associated with deleted regions than evolving and matured KAs. Specifically, the 15q chromosomal region that was deleted in regressing KAs but amplified in evolving or matured KAs, may be significantly involved in the process of KA regression. Also various candidate genes that were being considered for analysis were located in or near some of these implicated regions, including GSTM1, GSTP1 and SSTR2. As such, these candidate genes were targeted for further investigation. A number of susceptibility genes that were located in or near aberrant regions implicated in NMSC development were investigated using association analysis. These genes included members of the somatostatin receptor family (SSTR1 and SSTR2), members of the glutathione-S-transferase (GST) family (GSTM1, GSTT1, GSTP1 and GSTZ1) and the vitamin D receptor (VDR). Studies detected a number of interesting interactions between genetic, environmental and phenotypic factors in the development of the early stages of non-melanoma skin cancer. Additionally, genes implicated in NMSC development were further investigated using expression analysis to determine response to UV radiation. Association analysis was initially performed on members of the somatostatin receptor family. Somatostatin is a growth inhibiting factor, amongst other things, that mediates its actions through the somatostatin receptors (SSTRs). The presence of these receptors (SSTR1-5) in tumour cells indicates a potential for somatostatin to bind and suppress growth, as well as allowing for therapeutic treatment with somatostatin analogues. Additionally, expression of these receptors in normal tissue, including skin, should allow for potential protection against tumour growth. The genes for SSTR1 and SSTR2 have been shown to contain dinucleotide repeat polymorphisms, and although these polymorphisms may not directly result in altered expression or binding potential, they may be linked to another functional polymorphism that does. Using association analysis the SSTR1 and SSTR2 genes were investigated to determine whether they play a role in the development of solar keratosis. Results showed that there were no significant differences between SSTR1 and SSTR2 polymorphism frequencies in the tested solar keratosis population (P = 0.10 and P = 0.883, respectively) as compared to an unaffected population. Hence, these studies do not support a role for the SSTR1 or SSTR2 genes in solar keratosis development. Further association analysis and subsequent expression analysis was also performed on members of the glutathione-S-transferase family. The GST enzymes play a role in the detoxification of a number of carcinogens and mutagens, including those produced by UV-induced oxidative stress. This study examined the role of GSTM1, GSTT1, GSTP1 and GSTZ1 gene polymorphisms in susceptibility to SK development. Association analysis was performed to detect allele and genotype frequency differences in SK affected and control populations using PCR and restriction enzyme digestion. No significant differences were detected in GSTP1 and GSTZ1 allele or genotype frequencies, however polymorphisms within both genes were found to be in linkage disequilibrium, as previously reported, and a new allelic variant of the GSTZ1 gene was identified. Significant associations between GSTM1 (P = 0.003) and GSTT1 (P = 0.039) genotypes and SK development were detected, with the null variants of both genes conferring an approximate 2-fold increase in risk for solar keratosis development (OR: 2.1; CI: 1.3-3.5 and OR: 2.3; CI: 1.0-5.0 for GSTM1 and GSTT1, respectively). For the GSTM1 gene, this risk was significantly higher in conjunction with high outdoor exposure (OR: 3.4; CI: 1.9-6.3) and although the GSTT1 gene showed a similar trend (OR: 2.9; CI: 1.1-7.7), this did not reach significance. The increased risk of SK development associated with these genes is likely due to a decreased ability of the skin to detoxify mutagenic compounds produced by UV-induced oxidative stress, and hence a decreased ability to protect against carcinogenesis. Implication of the GSTM1 and GSTT1 null variants in solar keratosis development prompted interest in analysis of gene expression changes in response to UV radiation. Due to the high homology of the GSTM1 gene with other GSTM genes, and therefore potential issues with primer specificity, the GSTT1 gene was focussed on for the expression studies. Real time reverse transcription PCR, incorporating SYBR green fluorescence and 18S as a comparative gene, was used to study GSTT1 gene expression changes in response to both UVA and combined UVA/UVB radiation. It was found that only short term UV radiation had an effect on GSTT1 expression changes, whereas no alteration of gene expression was seen after 4 and 12 hours of recovery from long term irradiation between irradiated and matched non-irradiated skin samples. This indicated that changes in gene expression for the GSTT1 gene apparently occur relatively quickly after exposure to UV radiation. Analysis of both UVA only and combined UVA/UVB short term irradiation indicated that an initial decrease in expression, followed by an increase was likely to represent translation into protein and subsequent transcription of mRNA, and in some cases a second decrease indicated further translation. Hence, it appears as though UV radiation does have a significant effect on the expression of at least one GST gene, and that UV radiation in combination with genetic variation of these genes may play a role in the development of NMSC. Finally, association and subsequent expression analysis was also performed on the vitamin D receptor. The hormonal form of vitamin D, 1a25 dihydroxyvitamin D3, has been shown to have numerous cancer-related effects, including antiproliferative, differentiation, proapoptotic and antiangiogenic effects. These effects are mediated through the binding of 1a25 dihydroxyvitamin D3 to the vitamin D receptor and subsequent transcriptional pathways. Polymorphisms within the VDR are known to regulate its transcription and therefore expression, which is linked to the ability of 1a25 dihydroxyvitamin D3 to bind. Association analysis of a 5’ initiation codon variant (Fok I) and two 3’ variants (Apa I and Taq I) was performed in SK affected and control populations. Although the Fok I variant showed no association with SK development, both the Apa I and Taq I variants were found to be associated with SK development (P = 0.043 and P = 0.012, respectively). In particular, the Aa and Tt genotypes were associated with increased risk of SK. These results were however more complicated, as shown by further analysis. This showed that genotypes containing at least one allele that conferred decreased VDR transcription (ie. AA/Aa and Tt/tt) increased risk of SK development by 2-fold in fair skinned individuals (OR: 2.1; CI: 1.2-3.7 and OR: 1.7; CI: 1.1-2.7 for Apa I and Taq I variants, respectively) but also found to decrease the risk of SK development by 2-fold in medium skinned individuals (OR: 0.5; CI: 0.3-1.0 for Apa I variants). Additionally, genotypes containing 2 alleles conferring decreased transcription of the VDR gene were found to further increase the risk for SK development in fair skinned individuals (OR: 2.5; CI: 1.4-4.5 and OR: 2.4; CI: 1.2-5.0 for Apa I and Taq I variants, respectively), indicating a possible additive effect for the alleles. The highly differential association of the VDR gene polymorphisms amongst phenotypes may reflect a combination between the ability of an individual to synthesise 1a25 dihydroxyvitamin D3 with the binding availability of the VDR. To further investigate the role of VDR in NMSC, expression analysis of the VDR gene was undertaken using real time reverse transcription PCR, with SYBR green fluorescence and 18S as a comparative gene, to examine expression pattern changes associated with UV radiation. It was found that short term irradiation, as well as long term irradiation and recovery were associated with gene expression changes. Short term irradiation resulted in patterns indicative of translation and subsequent transcription, whereas long term irradiated samples resulted in reduction of VDR expression that was recovered after an extended period of time. Thus, VDR expression is clearly influenced by UV exposure. It would be very interesting to see more specifically if particular VDR genotypes, which appear to play a role in NMSC risk, also are affected differentially by UV exposure. It is possible that VDR expression is reduced to limit excessive binding of 1a25 dihydroxyvitamin D3, although since both UVA and UVB radiation affect VDR expression, this may not be mediated the effect of 1a25 dihydroxyvitamin D3 but rather a different pathway resulting from a general UV response. In summary, the detection of a number of susceptibility genes involved in SK development and their subsequent expression analysis in response to UV radiation has given further insight into the molecular changes associated with NMSC. In fact, both detoxification genes (GSTM1 and GSTT1) and a transcription related gene (VDR), were found to confer susceptibility to solar keratosis, an early stage skin lesion with tumourigenic potential. This suggests that even the earliest stages of skin cancer are mediated through a wide range of effects. Additionally, expression changes related to these genes indicate that they are associated with the well known environmental carcinogen of UV radiation and that their effects may be mediated through a wide range of pathways. Although implication of the 18q region in SCC progression was not confirmed in this study, it is still likely to play a role in malignant transformation. The implication of this region, as well as the implication of susceptibility genes has vastly increased knowledge into processes associated with NMSC. Although additional analysis can confirm and further implicate these molecular alterations, this study has resulted in a more comprehensive understanding of NMSC that may ultimately be of benefit in terms of prognosis and treatment.

Non-melanoma skin cancer (NMSC) is the most common cancer in the world with a lifetime risk for development as high as 2 in 3 in Queensland, Australia. Mortality is quite low, representing an approximate 360 deaths in Australia annually but cost of treatment is extremely high, estimated at $232 million each year. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the two most common forms of NMSC. Although BCC generally do not have the propensity to metastasise, they are highly invasive and can be locally destructive. SCC on the other hand is invasive and has metastatic potential. SCC is generally derived from a precursor lesion, solar keratosis (SK), which is also considered to be a biomarker of BCC, SCC and malignant melanoma. According to one theory, SKs actually represent the first recognisable stage of SCC development and therefore may be indicative of the earliest stage of NMSC development. In addition to these common forms of NMSC, rarer forms such as keratoacanthoma (KA), which spontaneously regress, and SCC in situ, which rarely become invasive, may provide clues into protective mechanisms associated with prevention of development. Like all other cancers, NMSC arises from an accumulation of genetic abnormalities that result in severe cellular dysfunction. A number of genes have been proposed in the development of NMSC, including p53, CDKN2a, Bcl-2 and the Ras family of genes, which are typically associated with proliferative and differentiation processes. Also, a number of genetic disorders that predispose individuals to NMSC have also been identified. Genetic abnormalities in these genes may be a result of somatic mutations that may be promoted by environmental carcinogens. For NMSC, ultraviolet (UV) radiation is the primary environmental stimulus that acts upon skin to generate mutations. UV effects are 2-fold; the first being direct damage produced by UVB radiation and the second being indirect damage as a result of UVA-induced oxidative stress. In addition to mutations of genes that directly result in carcinogenesis, polymorphic variants of genes may also play a role in susceptibility to NMSC. These susceptibility genes may have immunogenic, detoxifying or transcriptional roles that could be involved in increased mutagenesis or activation of cancer causing genes. The purpose of this study was ultimately to identify further molecular based mechanisms associated with the development of non-melanoma skin cancer. Initially, this study aimed to examine the effects of aberrant chromosomal regions on NMSC development and also to identify candidate genes within these regions that may be implicated in the development and progression of NMSC. Also, based on chromosomal and functional implications, a number of candidate genes were assessed using association analysis to determine their involvement in susceptibility to the earliest stages of NMSC development. Implicated susceptibility genes were then further investigated to determine their response to UV radiation. Therefore the methodological approach of these studies was based on three broad technical applications of cytogenetic, association and expression analyses. Previous comparative genomic hybridisation (CGH) studies implicated the 18q chromosomal region in progression of SK to SCC and this region was therefore suspected of harbouring one or more tumour suppressor genes that were associated with a more malignant phenotype. Following on from this analysis, loss of heterozygosity (LOH) analysis was used for further delineation of this region and possibly to implicate candidate genes involved in progression. Additionally, CGH was used to investigate keratoacanthoma to determine aberrant regions that might be involved in progression and also regression of this NMSC. Genes that had potential functional roles in NMSC development and that were located in or near regions implicated by these cytogenetic analyses were further investigated using association analysis. Association analysis was performed using polymerase chain reaction and subsequent restriction enzyme digestion or GeneScan analysis to determine genotype and allele frequencies in an SK affected versus control population for polymorphisms within a number of candidate genes. This population was carefully phenotyped so that not only genotypic factors could be analysed but also their interaction with a number of phenotypic and environmental risk factors. Genes with polymorphisms that did show association with solar keratosis development were then examined functionally. Specifically, gene expression analysis was undertaken to investigate their response to UV radiation. Both UVA only and combined UVA/UVB treatments were used for short term irradiation and also for long term irradiation with recovery to determine differential effects of UV range and dose in human skin. Relative mRNA expression analysis of these genes was performed using quantitative real time reverse transcription polymerase chain reaction to determine if UV radiation imposed gene expression changes in the skin. A combination of these methodologies provided a wide basis for investigation of NMSC. Cytogenetic, association and expression analyses all allow for the identification of molecular risk factors that cause or are associated with NMSC development and progression. These analyses provided diverse results that implicated various molecular mechanisms in the development of NMSC. Cytogenetic analysis is a powerful technique, especially for the identification of a broad range of aberrations throughout the genome. This study employed LOH analysis to investigate an implicated region involved in progression to SCC and to attempt identification of candidate genes that may be involved in this process. LOH analysis was successfully performed on 9 SCCs, 5 SCCs in situ and 2 SKs using 8 microsatellite markers within the 18q region. Polymerase chain reaction (PCR) was used to amplify polymorphic regions of these markers and genotypic composition was determined for normal and cancerous tissue within the specimen. In heterozygote individuals, determined by analysis of normal tissue, the cancerous tissue was examined to determine if alleles within the implicated region had been lost. However, after analysis of multiple different samples, there was no LOH detected in any of the samples examined for this analysis. This does not necessarily reject a role for 18q, or genes within this region, as the localisation of candidate tumour suppressor genes within a small region may indicate a tighter region of involvement than was expected. As such, a more targeted study may further delineate this region and implicate candidate genes in progression of SK to the more malignant phenotype of SCC. Further CGH analysis of keratoacanthoma was also undertaken to identify aberrations associated with development and also regression of this skin cancer. CGH was performed using universal amplification and nick translation to incorporate a fluorescent dye. Differentially labelled normal and tumour DNA were then competitively hybridised to a normal metaphase spread and fluorescence emission indicated either amplification or deletion of specific chromosomal regions. In total, 6 KA samples were analysed, with 2 samples each from evolving, matured and regressing stages of KA development. In general, regressing KAs appeared to be more highly associated with deleted regions than evolving and matured KAs. Specifically, the 15q chromosomal region that was deleted in regressing KAs but amplified in evolving or matured KAs, may be significantly involved in the process of KA regression. Also various candidate genes that were being considered for analysis were located in or near some of these implicated regions, including GSTM1, GSTP1 and SSTR2. As such, these candidate genes were targeted for further investigation. A number of susceptibility genes that were located in or near aberrant regions implicated in NMSC development were investigated using association analysis. These genes included members of the somatostatin receptor family (SSTR1 and SSTR2), members of the glutathione-S-transferase (GST) family (GSTM1, GSTT1, GSTP1 and GSTZ1) and the vitamin D receptor (VDR). Studies detected a number of interesting interactions between genetic, environmental and phenotypic factors in the development of the early stages of non-melanoma skin cancer. Additionally, genes implicated in NMSC development were further investigated using expression analysis to determine response to UV radiation. Association analysis was initially performed on members of the somatostatin receptor family. Somatostatin is a growth inhibiting factor, amongst other things, that mediates its actions through the somatostatin receptors (SSTRs). The presence of these receptors (SSTR1-5) in tumour cells indicates a potential for somatostatin to bind and suppress growth, as well as allowing for therapeutic treatment with somatostatin analogues. Additionally, expression of these receptors in normal tissue, including skin, should allow for potential protection against tumour growth. The genes for SSTR1 and SSTR2 have been shown to contain dinucleotide repeat polymorphisms, and although these polymorphisms may not directly result in altered expression or binding potential, they may be linked to another functional polymorphism that does. Using association analysis the SSTR1 and SSTR2 genes were investigated to determine whether they play a role in the development of solar keratosis. Results showed that there were no significant differences between SSTR1 and SSTR2 polymorphism frequencies in the tested solar keratosis population (P = 0.10 and P = 0.883, respectively) as compared to an unaffected population. Hence, these studies do not support a role for the SSTR1 or SSTR2 genes in solar keratosis development. Further association analysis and subsequent expression analysis was also performed on members of the glutathione-S-transferase family. The GST enzymes play a role in the detoxification of a number of carcinogens and mutagens, including those produced by UV-induced oxidative stress. This study examined the role of GSTM1, GSTT1, GSTP1 and GSTZ1 gene polymorphisms in susceptibility to SK development. Association analysis was performed to detect allele and genotype frequency differences in SK affected and control populations using PCR and restriction enzyme digestion. No significant differences were detected in GSTP1 and GSTZ1 allele or genotype frequencies, however polymorphisms within both genes were found to be in linkage disequilibrium, as previously reported, and a new allelic variant of the GSTZ1 gene was identified. Significant associations between GSTM1 (P = 0.003) and GSTT1 (P = 0.039) genotypes and SK development were detected, with the null variants of both genes conferring an approximate 2-fold increase in risk for solar keratosis development (OR: 2.1; CI: 1.3-3.5 and OR: 2.3; CI: 1.0-5.0 for GSTM1 and GSTT1, respectively). For the GSTM1 gene, this risk was significantly higher in conjunction with high outdoor exposure (OR: 3.4; CI: 1.9-6.3) and although the GSTT1 gene showed a similar trend (OR: 2.9; CI: 1.1-7.7), this did not reach significance. The increased risk of SK development associated with these genes is likely due to a decreased ability of the skin to detoxify mutagenic compounds produced by UV-induced oxidative stress, and hence a decreased ability to protect against carcinogenesis. Implication of the GSTM1 and GSTT1 null variants in solar keratosis development prompted interest in analysis of gene expression changes in response to UV radiation. Due to the high homology of the GSTM1 gene with other GSTM genes, and therefore potential issues with primer specificity, the GSTT1 gene was focussed on for the expression studies. Real time reverse transcription PCR, incorporating SYBR green fluorescence and 18S as a comparative gene, was used to study GSTT1 gene expression changes in response to both UVA and combined UVA/UVB radiation. It was found that only short term UV radiation had an effect on GSTT1 expression changes, whereas no alteration of gene expression was seen after 4 and 12 hours of recovery from long term irradiation between irradiated and matched non-irradiated skin samples. This indicated that changes in gene expression for the GSTT1 gene apparently occur relatively quickly after exposure to UV radiation. Analysis of both UVA only and combined UVA/UVB short term irradiation indicated that an initial decrease in expression, followed by an increase was likely to represent translation into protein and subsequent transcription of mRNA, and in some cases a second decrease indicated further translation. Hence, it appears as though UV radiation does have a significant effect on the expression of at least one GST gene, and that UV radiation in combination with genetic variation of these genes may play a role in the development of NMSC. Finally, association and subsequent expression analysis was also performed on the vitamin D receptor. The hormonal form of vitamin D, 1a25 dihydroxyvitamin D3, has been shown to have numerous cancer-related effects, including antiproliferative, differentiation, proapoptotic and antiangiogenic effects. These effects are mediated through the binding of 1a25 dihydroxyvitamin D3 to the vitamin D receptor and subsequent transcriptional pathways. Polymorphisms within the VDR are known to regulate its transcription and therefore expression, which is linked to the ability of 1a25 dihydroxyvitamin D3 to bind. Association analysis of a 5Â’ initiation codon variant (Fok I) and two 3Â’ variants (Apa I and Taq I) was performed in SK affected and control populations. Although the Fok I variant showed no association with SK development, both the Apa I and Taq I variants were found to be associated with SK development (P = 0.043 and P = 0.012, respectively). In particular, the Aa and Tt genotypes were associated with increased risk of SK. These results were however more complicated, as shown by further analysis. This showed that genotypes containing at least one allele that conferred decreased VDR transcription (ie. AA/Aa and Tt/tt) increased risk of SK development by 2-fold in fair skinned individuals (OR: 2.1; CI: 1.2-3.7 and OR: 1.7; CI: 1.1-2.7 for Apa I and Taq I variants, respectively) but also found to decrease the risk of SK development by 2-fold in medium skinned individuals (OR: 0.5; CI: 0.3-1.0 for Apa I variants). Additionally, genotypes containing 2 alleles conferring decreased transcription of the VDR gene were found to further increase the risk for SK development in fair skinned individuals (OR: 2.5; CI: 1.4-4.5 and OR: 2.4; CI: 1.2-5.0 for Apa I and Taq I variants, respectively), indicating a possible additive effect for the alleles. The highly differential association of the VDR gene polymorphisms amongst phenotypes may reflect a combination between the ability of an individual to synthesise 1a25 dihydroxyvitamin D3 with the binding availability of the VDR. To further investigate the role of VDR in NMSC, expression analysis of the VDR gene was undertaken using real time reverse transcription PCR, with SYBR green fluorescence and 18S as a comparative gene, to examine expression pattern changes associated with UV radiation. It was found that short term irradiation, as well as long term irradiation and recovery were associated with gene expression changes. Short term irradiation resulted in patterns indicative of translation and subsequent transcription, whereas long term irradiated samples resulted in reduction of VDR expression that was recovered after an extended period of time. Thus, VDR expression is clearly influenced by UV exposure. It would be very interesting to see more specifically if particular VDR genotypes, which appear to play a role in NMSC risk, also are affected differentially by UV exposure. It is possible that VDR expression is reduced to limit excessive binding of 1a25 dihydroxyvitamin D3, although since both UVA and UVB radiation affect VDR expression, this may not be mediated the effect of 1a25 dihydroxyvitamin D3 but rather a different pathway resulting from a general UV response. In summary, the detection of a number of susceptibility genes involved in SK development and their subsequent expression analysis in response to UV radiation has given further insight into the molecular changes associated with NMSC. In fact, both detoxification genes (GSTM1 and GSTT1) and a transcription related gene (VDR), were found to confer susceptibility to solar keratosis, an early stage skin lesion with tumourigenic potential. This suggests that even the earliest stages of skin cancer are mediated through a wide range of effects. Additionally, expression changes related to these genes indicate that they are associated with the well known environmental carcinogen of UV radiation and that their effects may be mediated through a wide range of pathways. Although implication of the 18q region in SCC progression was not confirmed in this study, it is still likely to play a role in malignant transformation. The implication of this region, as well as the implication of susceptibility genes has vastly increased knowledge into processes associated with NMSC. Although additional analysis can confirm and further implicate these molecular alterations, this study has resulted in a more comprehensive understanding of NMSC that may ultimately be of benefit in terms of prognosis and treatment.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

Ultraviolet radiation (UVR) is a major risk factor for development of skin cancer. UVR-induced DNA damage and a dysfunctional p53 protein are important steps in the development of squamous cell carcinoman in human skin (SCC). The aim of the present investigation was to analyze incidence trends of SCC in Sweden, quantify the risk of second primary cancer after SCC and further analyze the effects of UVR and p53 protein in human skin in vivo and in vitro. The effect of photoprotection by sunscreens was also evaluated.

We found that the age-standardized incidence rate of SCC in Sweden increased substantially in both men and women during the period 1961-1995, especially in men and at chronically sun-exposed skin sites. Patients with SCC are also at increased risk of developing new primary cancers, especially in the skin, squamous cell epithelium, hematopoietic tissues and respiratory organs. In experimental studies in vivo and in vitro in human skin we observed that repair of UV-induced DNA damage appears to be more efficient in chronically sun-exposed skin despite a less uniform p53 response. Non-sun- exposed skin is more homogeneous with respect to the epidermal p53 response. Keratinocytes in skin exposed frequently to the sun may be prone to react more easily to cytotoxic stress. Two different modalities of photoprotection significantly reduced the amount of DNA damage and the number of p53-positive cells. In addition, we demonstrated that a well-defined system for in vitro culture of explanted skin provides an excellent alternative to in vivo experiments.

In conclusion, this study has increased our knowledge of SCC epidemiology in Sweden and of the effects of artificial and solar UVR and sunscreens on chronically sun-exposed and non-sun-exposed sites, respectively, of human skin.

BCC is the commonest cancer in European-derived populations and Australia has the highest recorded incidence in the world, creating enormous individual and societal cost in management of this disease. The incidence of this cancer has been increasing internationally, with evidence of a 1 to 2% rise in incidence in Australia per year over the last two decades. The main four epidemiological risk factors for the development of BCC are ultraviolet radiation (UVR) exposure, increasing age, male sex, and inability to tan. The pattern and timing of UVR exposure is important to BCC risk, with childhood and intermittent UVR exposure both associated with an increased risk. The complex of inherited characteristics making up an individual’s ‘sun sensitivity’ is also important in determining BCC risk. Very little is known about population genetic susceptibility to BCC outside of the rare genodermatosis Gorlin syndrome. Mutations in the tumour suppressor gene patched (PTCH) are responsible for this BCC predisposition syndrome and the molecular pathway and target genes of this highly conserved pathway are well described. Derangments in this pathway occur in sporadic BCC development, and the PTCH gene is an obvious candidate to contribute to non-syndromic susceptibility to BCC. The melanocortin 1 receptor (MC1R) locus is known to be involved in pigmentary traits and the cutaneous response to UVR, and variants have been associated with skin cancer risk. Many other genes have been considered with respect to population BCC risk and include p53, HPV, GSTs, and HLAs. There is preliminary evidence for specific familial aggregation of BCC, but very little known about the causes. 56 individuals who developed BCC under the age of 40 in the year 2000 were recruited from the Skin and Cancer Foundation of Australia’s database. This represents the youngest 7 – 8% of Australians with BCC from a database that captures approximately 10% of Sydney’s BCCs. 212 of their first degree relatives were also recruited, including 89 parents and 123 siblings of these 56 probands. All subjects were interviewed with respect to their cancer history and all reports of cancer verified with histopathological reports where possible. The oldest unaffected sibling for each proband (where available) was designated as an intra-family control. All cases and control siblings filled out a questionnaire regarding their pigmentary and sun sensitivity factors and underwent a skin examination by a trained examiner. Peripheral blood was collected from these cases and controls for genotyping of PTCH. All the exons of PTCH for which mutations have been documented in Gorlin patients were amplified using PCR. PCR products were screened for mutations using dHPLC, and all detectable variants sequenced. Prevalence of BCC and SCC for the Australian population was estimated from incidence data using a novel statistical approach. Familial aggregation of BCC, SCC and MM occurred within the 56 families studied here. The majority of families with aggregation of skin cancer had a combination of SCC and BCC, however nearly one fifth of families in this study had aggregation of BCC to the exclusion of SCC or MM, suggesting that BCCspecific risk factors are also likely to be at work. Skin cancer risks for first-degree relatives of people with early onset BCC were calculated: sisters and mothers of people with early-onset BCC had a 2-fold increased risk of BCC; brothers had a 5-fold increased risk of BCC; and sisters and fathers of people with early-onset BCC had over four times the prevalence of SCC than that expected. For melanoma, the increased risk was significant for male relatives only, with a 10-fold increased risk for brothers of people with early-onset BCC and 3-fold for fathers. On skin examination of cases and controls, several phenotypic factors were significantly associated with BCC risk. These included increasing risk of BCC with having fair, easyburning skin (ie decreasing skin phototype), and with having signs of cumulative sun damage to the skin in the form of actinic keratoses. Signs reflecting the combination of pigmentary characteristics and sun exposure - in the form of arm freckling and solar lentigines - also gave subjects a significantly increased risk BCC. Constitutive red-green reflectance of the skin was associated with decreased risk of BCC, as measured by spectrophotometery. Other non-significant trends were seen that may become significant in larger studies including associations of BCC with propensity to burn, moderate tanning ability and an inability to tan. No convincing trend for risk of BCC was seen with the pigmentary variables of hair or eye colour, and a non-significant reduced risk of BCC was associated with increasing numbers of seborrhoeic keratoses. Twenty PTCH exons (exons 2, 3, 5 to 18, and 20 to 23) were screened, accounting for 97% of the coding regions with published mutations in PTCH. Nine of these 20 exons were found to harbour single nucleotide polymorphisms (SNPs), seen on dHPLC as variant melting curves and confirmed on direct sequencing. SNPs frequencies were not significantly different to published population frequencies, or to Australian general population frequencies where SNP database population data was unavailable. Assuming a Poisson distribution, and having observed no mutations in a sample of 56, we can be 97.5% confident that if there are any PTCH mutations contributing to early-onset BCC in the Australian population, then their prevalence is less than 5.1%. Overall, this study provides evidence that familial aggregation of BCC is occurring, that first-degree relatives are at increased risk of all three types of skin cancer, and that a combination of environmental and genetic risk factors are likely to be responsible. The PTCH gene is excluded as a major cause of this increased susceptibility to BCC in particular and skin cancer in general. The weaknesses of the study design are explored, the possible clinical relevance of the data is examined, and future directions for research into the genetics of basal cell carcinoma are discussed.

Doctor of Philosophy(PhD)
BCC is the commonest cancer in European-derived populations and Australia has the highest recorded incidence in the world, creating enormous individual and societal cost in management of this disease. The incidence of this cancer has been increasing internationally, with evidence of a 1 to 2% rise in incidence in Australia per year over the last two decades. The main four epidemiological risk factors for the development of BCC are ultraviolet radiation (UVR) exposure, increasing age, male sex, and inability to tan. The pattern and timing of UVR exposure is important to BCC risk, with childhood and intermittent UVR exposure both associated with an increased risk. The complex of inherited characteristics making up an individual’s ‘sun sensitivity’ is also important in determining BCC risk. Very little is known about population genetic susceptibility to BCC outside of the rare genodermatosis Gorlin syndrome. Mutations in the tumour suppressor gene patched (PTCH) are responsible for this BCC predisposition syndrome and the molecular pathway and target genes of this highly conserved pathway are well described. Derangments in this pathway occur in sporadic BCC development, and the PTCH gene is an obvious candidate to contribute to non-syndromic susceptibility to BCC. The melanocortin 1 receptor (MC1R) locus is known to be involved in pigmentary traits and the cutaneous response to UVR, and variants have been associated with skin cancer risk. Many other genes have been considered with respect to population BCC risk and include p53, HPV, GSTs, and HLAs. There is preliminary evidence for specific familial aggregation of BCC, but very little known about the causes. 56 individuals who developed BCC under the age of 40 in the year 2000 were recruited from the Skin and Cancer Foundation of Australia’s database. This represents the youngest 7 – 8% of Australians with BCC from a database that captures approximately 10% of Sydney’s BCCs. 212 of their first degree relatives were also recruited, including 89 parents and 123 siblings of these 56 probands. All subjects were interviewed with respect to their cancer history and all reports of cancer verified with histopathological reports where possible. The oldest unaffected sibling for each proband (where available) was designated as an intra-family control. All cases and control siblings filled out a questionnaire regarding their pigmentary and sun sensitivity factors and underwent a skin examination by a trained examiner. Peripheral blood was collected from these cases and controls for genotyping of PTCH. All the exons of PTCH for which mutations have been documented in Gorlin patients were amplified using PCR. PCR products were screened for mutations using dHPLC, and all detectable variants sequenced. Prevalence of BCC and SCC for the Australian population was estimated from incidence data using a novel statistical approach. Familial aggregation of BCC, SCC and MM occurred within the 56 families studied here. The majority of families with aggregation of skin cancer had a combination of SCC and BCC, however nearly one fifth of families in this study had aggregation of BCC to the exclusion of SCC or MM, suggesting that BCCspecific risk factors are also likely to be at work. Skin cancer risks for first-degree relatives of people with early onset BCC were calculated: sisters and mothers of people with early-onset BCC had a 2-fold increased risk of BCC; brothers had a 5-fold increased risk of BCC; and sisters and fathers of people with early-onset BCC had over four times the prevalence of SCC than that expected. For melanoma, the increased risk was significant for male relatives only, with a 10-fold increased risk for brothers of people with early-onset BCC and 3-fold for fathers. On skin examination of cases and controls, several phenotypic factors were significantly associated with BCC risk. These included increasing risk of BCC with having fair, easyburning skin (ie decreasing skin phototype), and with having signs of cumulative sun damage to the skin in the form of actinic keratoses. Signs reflecting the combination of pigmentary characteristics and sun exposure - in the form of arm freckling and solar lentigines - also gave subjects a significantly increased risk BCC. Constitutive red-green reflectance of the skin was associated with decreased risk of BCC, as measured by spectrophotometery. Other non-significant trends were seen that may become significant in larger studies including associations of BCC with propensity to burn, moderate tanning ability and an inability to tan. No convincing trend for risk of BCC was seen with the pigmentary variables of hair or eye colour, and a non-significant reduced risk of BCC was associated with increasing numbers of seborrhoeic keratoses. Twenty PTCH exons (exons 2, 3, 5 to 18, and 20 to 23) were screened, accounting for 97% of the coding regions with published mutations in PTCH. Nine of these 20 exons were found to harbour single nucleotide polymorphisms (SNPs), seen on dHPLC as variant melting curves and confirmed on direct sequencing. SNPs frequencies were not significantly different to published population frequencies, or to Australian general population frequencies where SNP database population data was unavailable. Assuming a Poisson distribution, and having observed no mutations in a sample of 56, we can be 97.5% confident that if there are any PTCH mutations contributing to early-onset BCC in the Australian population, then their prevalence is less than 5.1%. Overall, this study provides evidence that familial aggregation of BCC is occurring, that first-degree relatives are at increased risk of all three types of skin cancer, and that a combination of environmental and genetic risk factors are likely to be responsible. The PTCH gene is excluded as a major cause of this increased susceptibility to BCC in particular and skin cancer in general. The weaknesses of the study design are explored, the possible clinical relevance of the data is examined, and future directions for research into the genetics of basal cell carcinoma are discussed.

The Aim of the project was to investigate cell senescence in basal cell carcinoma (BCC). Although the concept of oncogene-induced senescence (OIS) was originally confined to cultured cells, it is now well established that this mechanism has an important role in tumour biology. OIS represents a physiological response that restricts the progression of benign tumours into their malignant counterparts e.g. nevi to melanoma or adenoma to adenocarcinoma. Full malignancy is associated with the loss of important tumour suppressor genes including RETINOBLASTOMA and/or TP53. BCC of the skin is the most common skin tumour and is associated with mutational inactivation of the PTCH1 tumour suppressor gene (and less frequently oncogenic activation of SMOOTHENED). Although BCC does not appear to stem from precursor lesions - though mouse models of BCC display areas of basaloid hyperproliferation - and it is relatively stable at the genomic level, we sought to determine if these unique tumours display any characteristics of OIS. Human BCCs were positive for Senescence-associated β-galactosidase (SA-β-gal) activity (pH 6.0) and expressed known markers of senescence including DCR2, DEC1 (SHARP2) as well as the cell cycle inhibitors p15INK4b and p16INK4a. Interestingly, SA-β-gal activity was observed in stromal cells surrounding the tumour islands and this may account for why BCCs are difficult to culture in vitro as senescent cells are known to express increased levels of growth factors, cytokines and ECM proteins. To determine if OIS is associated with Hedgehog signalling in BCC, I employed a novel in vitro model of BCC created through PTCH1 suppression in human immortalised NEB1 keratinocytes. NEB1-shPTCH1 cells are viable and proliferative (albeit more slowly than control NEB1-shCON cells) and do not display SA-β-gal activity but they express higher levels of several senescent markers including DCR2 and p21WAF1. I also investigated senescence in a Mouse model of BCC in which one allele of Ptch1 is mutated and which on x-ray irradiation results in BCC formation. The expression of known markers of senescence including DCR2, DEC1 (SHARP2) as well as the cell cycle inhibitors p15INK4b, p16INK4a and p53 were all with the exception of p21WAF1 detected in these tumours. Together these data suggest that senescence is a characteristic of BCC and may explain why these tumours rarely metastasise.

In this PhD thesis, two projects both aimed at understanding the molecular mechanisms underlying virus-induced tumour formation and progression in the skin were included. Specifically, two models of skin cancer were used: i) beta Human Papillomavirus (B-HPV) infection and keratinocyte carcinoma (KC); and ii) Merkel Cell Polyomavirus infection and Merkel Cell Carcinoma (MCC). Many evidences suggest a carcinogenic role of B-HPV in KC, especially in the immunosuppressed setting. HPV-associated cutaneous KC occurs mainly in sun-exposed areas of the body, and therefore these viruses are thought to cooperate with UV rays to induce cancer. Here, we used a new mouse model of immunosuppression, obtained by crossing the B-HPV8 transgenic mice with Rag2 deficient mice, to show a functional link between cutaneous PV VIIEIISIUm rerse so 0066 2010 1000 infection, immunosuppression, UVB exposure, and KC development. Our findings clearly demonstrate the concerted contribution of these factors in the accelerated development of skin cancer in an immunosuppressed setting. Within Polyomaviruses, MCPUV is the only family member proven to induce cancer, as it has been associated with the development of MCC. Recently, many studies have highlighted the contribution of tumor microenvironment (TME) in cancer progression suggesting the tumor-promoting role of cancer associated fibroblasts (CAFs) in many cancer types, but data describing CAFs and their molecular function in MCC are still missing. To define the role of CAFs in MCC cancerization, we have isolated CAFs from MCC patients and performed a set of in vivo experiments by injecting patient-derived CAFs along with the MCPVV positive MCC cell line MKL-1 in SCID mice. The results obtained with xenografts indicate a pivotal role for the TME, particularly CAFs in tumour development and metastasis formation

Genetic changes are hallmarks of cancer development involving the activation and/or inactivation of oncogenes and tumour suppressor genes, respectively. In non-melanoma skin cancer (NMSC) development, the initiation of genetic mutations results from exposure to solar ultraviolet radiation. Non-melanoma skin cancers are comprised of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Several related cutaneous lesions also exist, of which solar keratoses (SK) are widely accepted as a precursor dysplasia to SCC development. The study of recurrent genetic changes present within NMSC and SK should help reveal causative mutations in skin cancer development. Such analysis could also elucidate links in the genetic similarity of these dysplasia. The rapid screening of numerical changes in DNA sequence copy number throughout the entire genome has been made possible by the advent of comparative genomic hybridisation (CGH). This technique enables the identification of net gains and loss of genetic material within a tumour DNA sample. Chromosomal regions of recurrent gain or loss identify loci containing putative oncogenes and tumour suppressor genes, respectively with potential roles in NMSC tumourigenesis. Used in conjunction with tissue microdissection and universal degenerate PCR techniques this can enable the elucidation of aberrations in small histologically distinct regions of tumour. Such a technique can utilize archival material such as paraffin embedded tissue, which is the major source of neoplastic material available for cancer research. This study used the CGH technique to investigate aberrations in BCC, SCC and SK samples. The screening of copy number abnormalities (CNAs) in BCC revealed that although these tumours were close to diploid and generally genetically stable, they did contain several recurrent aberrations. The loss of genetic material at 9q was identified in a third of BCC tumours studied. This is characteristic of inactivation of the PTCH tumour suppressor gene, a known attribute in some sporadic BCC development. Validation of this loss was performed via loss of heterozygosity, demonstrating good concordance with the CGH data. In addition the over-representation of the 6p chromosome arm was revealed in 47% of biopsies. This novel CNA is also commonly observed in other cutaneous neoplasias, including Merkel cell carcinoma and malignant melanoma. This suggests a possible common mechanism in development and or promotion in these cutaneous dysplasias, the mechanisms of which have yet to be clearly defined. In contrast to BCC, numerical genetic aberrations in SCC and SK were much more frequent. Several regions of recurrent gain were commonly shared between both dysplasias including gain of 3q, 4p, 5p, 8q, 9q, 14q, 17p, 17q and 20q. Common chromosomal regions of loss included 3p, 8p, 9p, 11p, 13q and 17p. In addition loss of chromosome 18 was significantly observed in SCC in comparison to SK, a possible defining event in SK progression to SCC. The identification of shared genetic aberrations suggests a clonal and genetic relationship between the two lesions. This information further supports the notion for re-classification of SK to an SCC in situ or superficial SCC. Finally, the CNAs detected have been similarly observed in other squamous cell-derived tumours, for example cervical and head and neck SCC. This provides further evidence to common mechanisms involved in the initiation, development and progression of SCC neoplasia. This study has identified a number of recurrent chromosomal regions, some of which are novel in NMSC development. The further delineation of these loci should provide additional evidence of their significance and degree of involvement in NMSC tumourigenesis. The identification of the cancer-causing genes mapped to these loci will further demarcate the genetic mechanisms of BCC and SCC progression. An understanding of the events involved in skin cancer formation and progression should shed additional light on molecular targets for diagnostics, management and therapeutic treatment.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

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Introdução: O câncer de pele do tipo não melanoma apresenta uma incidência crescente em todo o mundo, causando um importante problema para a saúde pública. Pelas repercussões psicológicas, físicas e sociais que afetam os pacientes com câncer de pele não melanoma e pela necessidade de se avaliar os benefícios das modalidades de tratamento, torna-se importante disponibilizar para o Brasil, um instrumento específico para avaliação da qualidade de vida destes pacientes. Objetivo: Traduzir para a língua portuguesa, adaptar para a cultura brasileira e realizar a validação do instrumento The Skin Cancer Index. Métodos: The Skin Cancer Index é um instrumento específico composto por 15 itens divididos em três subescalas: emocional, social e a aparência. Foram realizadas a tradução, a adaptação cultural e testadas a consistência interna do instrumento, a reprodutibilidade e a validade de construção. Resultados: Nas etapas de tradução e adaptação cultural, três questões foram modificadas. Na consistência interna para as subescalas emocional, social e a aparência foram respectivamente de 0,78, 0,82 e 0,74. Na fase reprodutibilidade, a correlação linear de Pearson e o coeficiente de correlação intraclasse no primeiro momento foi de 0,991 e 0,984 e no segundo momento foi de 0,96 e 0,94. Na fase de validade de construção houve correlação do The Skin Cancer Index com o Short Form -12. Conclusão: The Skin Cancer Index foi traduzido e adaptado para a cultura brasileira e validado para avaliação da qualidade de vida de pacientes com câncer de pele não melanoma.
BV UNIFESP: Teses e dissertações

Mutations in RHBDF2, the gene encoding inactive rhomboid protein iRHOM2, result in the dominantly inherited condition Tylosis with oesophageal cancer (TOC). TOC causes plamoplantar keratoderma, oral precursor lesions and up to a 95 % life-time risk of oesophageal squamous cell carcinoma (SCC). The role of iRHOM2 in the epidermis is not well characterised, although we previously showed dysregulated epidermal growth factor receptor (EGFR) signalling and accelerated migration in TOC keratinocytes, and a role for iRHOM2 was shown in trafficking the metalloproteinase ADAM17. Substrates of ADAM17 include EGFR ligands and adhesion molecules. iRHOM2 localisation and function were investigated in frozen sections and keratinocyte cell lines from control and TOC epidermis. Although iRHOM2 was predicted to be an ER-membrane protein, it showed cell-surface expression in control epidermis, with variable localisation in TOC. Increased processing and activation of ADAM17 was seen in TOC keratinocytes compared with control cells, suggesting that increased ADAM17-mediated processing of EGFR ligands may cause the changes in EGFR signalling. Downstream of iRHOM2-ADAM17, Eph/Ephrin and NOTCH signalling also appeared affected. Additionally, desmosomes in TOC epidermis lacked the electron-dense midline of the mature desmosomes seen in normal skin; this was accompanied by increased processing of desmoglein 2, a substrate of ADAM17. Expression and localisation of iRHOM2 was also investigated in TOC and sporadic SCC. iRHOM2 expression varied between SCC cell lines, and appeared to correlate with ADAM17 and NOTCH1 expression in oesophageal SCC and head and neck SCC cells. In summary, iRHOM2 mutations in TOC appear to be gain-of-function in nature, resulting in increased ADAM17 processing and enhanced EGFR signalling. Questions remaining include the reason why iRHOM2 is found at the plasma membrane. Future study of the iRHOM2-ADAM17 pathway may provide additional insight into the mechanism of epidermal wound healing and the pathogenesis of oesophageal SCC.

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Thesis: M. Eng., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2018
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (page 51).
In this thesis we explore different machine learning techniques that are common in image classification to detect the presence of Basal Cell Carcinoma (BCC) in digital skin histological images. Since digital histology images are extremely large, we first focused on determining the presence of BCC at the patch level, using pre-trained deep convolutional neural networks as feature extractors to compensate for the size of our datasets. The experimental results show that our patch level classifiers obtained an area under the receiver operating characteristic curve (AUC) of 0.981. Finally, we used our patch classifiers to generate a bag of scores for a given whole slide image (WSI), and attempted multiple ways of combining these scores to produce a single significant score to predict the presence of BCC in the given WSI. Our best performing model obtained an AUC of 0.991 in 86 samples of digital skin biopsies, 43 of which had BCC.
by Tomás Alberto Calderón Gómez.
M. Eng.
M.Eng. Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science

Genetic changes are hallmarks of cancer development involving the activation and/or inactivation of oncogenes and tumour suppressor genes, respectively. In non-melanoma skin cancer (NMSC) development, the initiation of genetic mutations results from exposure to solar ultraviolet radiation. Non-melanoma skin cancers are comprised of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Several related cutaneous lesions also exist, of which solar keratoses (SK) are widely accepted as a precursor dysplasia to SCC development. The study of recurrent genetic changes present within NMSC and SK should help reveal causative mutations in skin cancer development. Such analysis could also elucidate links in the genetic similarity of these dysplasia. The rapid screening of numerical changes in DNA sequence copy number throughout the entire genome has been made possible by the advent of comparative genomic hybridisation (CGH). This technique enables the identification of net gains and loss of genetic material within a tumour DNA sample. Chromosomal regions of recurrent gain or loss identify loci containing putative oncogenes and tumour suppressor genes, respectively with potential roles in NMSC tumourigenesis. Used in conjunction with tissue microdissection and universal degenerate PCR techniques this can enable the elucidation of aberrations in small histologically distinct regions of tumour. Such a technique can utilize archival material such as paraffin embedded tissue, which is the major source of neoplastic material available for cancer research. This study used the CGH technique to investigate aberrations in BCC, SCC and SK samples. The screening of copy number abnormalities (CNAs) in BCC revealed that although these tumours were close to diploid and generally genetically stable, they did contain several recurrent aberrations. The loss of genetic material at 9q was identified in a third of BCC tumours studied. This is characteristic of inactivation of the PTCH tumour suppressor gene, a known attribute in some sporadic BCC development. Validation of this loss was performed via loss of heterozygosity, demonstrating good concordance with the CGH data. In addition the over-representation of the 6p chromosome arm was revealed in 47% of biopsies. This novel CNA is also commonly observed in other cutaneous neoplasias, including Merkel cell carcinoma and malignant melanoma. This suggests a possible common mechanism in development and or promotion in these cutaneous dysplasias, the mechanisms of which have yet to be clearly defined. In contrast to BCC, numerical genetic aberrations in SCC and SK were much more frequent. Several regions of recurrent gain were commonly shared between both dysplasias including gain of 3q, 4p, 5p, 8q, 9q, 14q, 17p, 17q and 20q. Common chromosomal regions of loss included 3p, 8p, 9p, 11p, 13q and 17p. In addition loss of chromosome 18 was significantly observed in SCC in comparison to SK, a possible defining event in SK progression to SCC. The identification of shared genetic aberrations suggests a clonal and genetic relationship between the two lesions. This information further supports the notion for re-classification of SK to an SCC in situ or superficial SCC. Finally, the CNAs detected have been similarly observed in other squamous cell-derived tumours, for example cervical and head and neck SCC. This provides further evidence to common mechanisms involved in the initiation, development and progression of SCC neoplasia. This study has identified a number of recurrent chromosomal regions, some of which are novel in NMSC development. The further delineation of these loci should provide additional evidence of their significance and degree of involvement in NMSC tumourigenesis. The identification of the cancer-causing genes mapped to these loci will further demarcate the genetic mechanisms of BCC and SCC progression. An understanding of the events involved in skin cancer formation and progression should shed additional light on molecular targets for diagnostics, management and therapeutic treatment.

There is an urgent need to define the key pathological driving events in human cutaneous squamous cell carcinoma (cSCC) in order to identify novel therapeutic targets. Already the second most common form of human non-melanoma skin cancer, the incidence of cSCC has continued to rise at epidemic proportions over the past two decades. Rarely, cSCC can be highly aggressive, causing significant soft-tissue defects in sun-exposed areas of skin and progressing to metastatic disease, which is usually associated with poor survival. The transforming growth factor-β (TGF-β) signalling pathway is known to play key regulatory roles in skin homeostasis and wound repair. Murine studies indicate that loss of TGF-β signalling is sufficient to drive cSCC, but conclusive evidence for a similar role in human cSCC remains elusive. Combining immunohistochemical and genetic studies of the TGF-β signalling pathway on human cSCC tissue, with a thorough examination of TGF-β responses in human primary cSCC cell lines in-vitro, this thesis aims to investigate the complex role of TGF-β signalling in human cSCC. An extensive tissue micro-array analysis demonstrated the consistent reduction of endogenous TGF-β signalling activity in human primary cSCC. This intriguingly correlated with higher risk thick tumours pathologically, indicating that TGF-β is likely to act primarily as a tumour suppressor in human cSCC and its reduction or loss may impart a significant growth advantage for cSCC tumour cells. This tumour suppressor effect was reflected in-vitro, whereby the majority of primary cSCC cell lines remained sensitive to TGF-β mediated growth arrest. Resistance to TGF-β tumour suppression was also identified, and mechanistically its main protagonist in cSCC cell lines appeared to be mutational loss of TGF-β receptors. Consolidating in-vitro findings, both whole exome sequencing and 454 pyrosequencing of human cSCC tissue revealed frequent functionally damaging mutations of both TGF-β type 1 and type 2 receptors, indicating that mutational loss of the TGF-β pathway may be a key driving event in human cSCC tumourigenesis. Perhaps most interestingly, mutational loss of TGF-β type 2 receptors in cSCC cell lines appeared to result in a novel pro-oncogenic dependence on TGF-β type 1 receptor kinase activity, highlighting not only the important paradoxical role of TGF-β mediated tumour promotion in cSCC, but also the potential for signalling crosstalk between alternative TGF-β superfamily members, namely Activin signalling, to drive tumourigenesis in the absence of active TGF-β signalling. Although further mechanistic studies are required to support this hypothesis, the mutational status of TGF-β type 2 receptors may not only provide a powerful prognostic tool for patients with cSCC, but also represent an important biomarker for the targeted use of TGF-β inhibitors in potentially aggressive disease where pro-tumourigenic responses could be driving disease progression.

Thesis (M.S.)--Missouri University of Science and Technology, 2009.
Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 13, 2009) Includes bibliographical references (p. 46).

Incidence trends for invasive and non-invasive disease are explored. A nested case control study of those women who progress from non-invasive disease to carcinoma is performed. The incidence of vulval carcinoma in Grampian remains stable. Incidence rates of all non-invasive chronic vulval skin disorders, regardless of underlying aetiology are rising dramatically. Age specific incidence rates suggest that there may be significant ascertainment bias in rising incidence rates for each of these skin disorders. No distinction could be found between the relative risks for women with vulval litchen sclerosus or vulval intraepithelial neoplasia developing subsequent carcinoma. Vulval squamous hyperplasia appears to be of low malignant potential. The malignant risk of vulval lichen sclerosus or vulval intraepithelial neoplasia within Grampian is currently in the order of 9% over ten years of follow-up. The presence of HPV 16 DNA does not appear to confer an increased risk of subsequent malignant transformation in women with biopsy-proven chronic vulval skin disorders. Although p-53 overexpression is not associated with an increased risk of subsequent malignant transformation, there are patterns of altered expression that may identify a high risk group of women. Those women that have been identified as suffering from a chronic vulval skin disorder before they develop invasive disease appear to have more favourable prognostic factors at presentation. The primary role of viral carcinogenesis in vulval carcinoma has been overestimated in the current literature. p53 aberrations may be a unifying factors in the biology of vulval carcinoma, with HPV acting as a co-factor in some cases. Aggressive surgical management for vulval intraepithelial neoplasia solely for the purposes of anticipating a rise in invasive disease rates should be resisted. An increase in resource allocation for the purpose of close surveillance of women with vulval lichen sclerosis or vulval intraepithelial neoplasia now seems justified.

The cancers of the skin, melanoma and the keratinocyte cancers, basal cell andsquamous cell carcinomas (BCC and SCC), are among the most common cancersin white populations. While ultraviolet radiation (UVR) is their principal cause,links with non-UVR-related factors have also been noted. Ultimately, theinteraction of these elements results in malignancy however, understanding oftheir specific contributions remains incomplete. This thesis reports findings fromsix studies aiming to investigate gaps in current knowledge of the role of UVR andnon-UVR-related risk factors on skin cancer. The papers are groupedaccording to the aspects of skin cancer epidemiology and aetiology they address. The first two papers address the descriptive epidemiology of melanoma inEngland, a country with low ambient solar UVR. They arise from ecologicalstudies using national melanoma registration data and document rising trends inmelanoma incidence by anatomic site (Paper 1), and by region of residence andsocio-economic deprivation (Paper 2). Their findings were consistent with thesuggestion that increases in recreational UVR exposure are driving rises inmelanoma rates. These results emphasise both the need to closely monitor UVRexposure and melanoma trends and the importance of public health campaigns. The second group of three papers considers the assessment of associations ofnutritional factors with keratinocyte cancer. Two studies use data from aprospective cohort to evaluate the relationship between dietary intake (Paper 3)and blood concentrations (Paper 4) of omega-3 and omega-6 polyunsaturatedfatty acids (PUFA) in relation to BCC and SCC risk. Associations with both PUFAtypes were observed. In addition, Paper 5, a three-way correlational assessment,demonstrated that questionnaire and blood circulating levels of omega-3 PUFAwere highly correlated with measures of skin bioavailability. Collectively, thesestudies give evidence for associations of these nutrients with skin cancer and forthe utility of both intake and biomarker measures for assessing the relationships. The final paper explores the relationship between a widely cited non-UVR riskfactor, namely scars and cancers of the skin. It reports a systematic review of allpublished observational studies quantifying this association. While innumerablecase reports were found, quantitative analyses were rare. The review identifieda major gap in the literature where knowledge of scar malignancies is notevidence-based, but rather founded mainly on cumulative anecdotal reporting. Taken together, this body of published work highlights the largely unrecognisedcomplexity of the aetiology of cancers of the skin. Future research must bebroad in scope in order to advance understanding of the interaction betweenUVR and other risk factors and to provide a base for health messages aimed atreducing the burden of these malignancies.

Kindlin-1 (Kin1) is an epithelial focal adhesion protein that plays a key role in integrin-mediated anchorage of cells to the extracellular matrix. Congenital loss of Kin1 leads to Kindler Syndrome (KS), whose symptoms include progressive epidermal atrophy, reduced keratinocyte proliferation, skin blistering and increased incidence of aggressive Squamous Cell Carcinoma (SCC). Objectives of this study were to examine the role of Kin1 in skin homeostasis and in the development of aggressive SCC in KS, as the molecular aetiologies for these pathologies are yet to be clearly understood. We first examined whether the recently discovered role of Kin1 in mitosis contributes to reduced keratinocyte proliferation observed in KS epidermis. We discovered that short-term loss of Kin1 in adult mouse epidermis reduced keratinocyte proliferation. We also found that Kin1 loss increased mitotic spindle misorientation that, according to the model of cell division in skin homeostasis, decreases cell proliferative potential, and, thus, may account for the reduced proliferation in our model. As spindle misorientation can stem from microtubule instability, we believe that the reduction in acetylated α-tubulin (ac-tub), a known marker of stable microtubules, that we also observed in mouse epidermis following Kin1 loss could account for the defective spindle orientation phenotype. The role of Kin1 in spindle orientation was also evident in vitro. Moreover, data from our lab revealed showed reduction in spindle ac-tub following Kin1 depletion, mirroring our in vivo observation. Additionally to orientation defects, in vitro depletion of Kin1 led to enhanced chromosome missegregation, which is likely to result from reduced microtubule stability due to low levels of ac-tub. We showed that role of Kin1 in spindle orientation and chromosome segregation is dependent on HDAC6, a known inhibitor of ac-tub. Overall, our results uncover an in vitro and in vivo role of Kin1 in mitotic spindle fidelity that could be crucial to skin homeostasis, and, when disturbed, may lead to reduced keratinocyte proliferation. Interestingly, our in vitro studies also revealed that in mitosis Kin1 and Kindlin-2 (Kin2) had overlapping, but also distinct roles, which is in line with various reports that show different biological functions for the two protein isoforms. Our next and final aim was to explore the roles of Kin1 in the development and progression of SCC, which would help us comprehend the reason behind the cancer's aggressive nature in KS. By employing in vitro and in vivo SCC growth assays and tumour immunohistochemical staining we found that absence of Kin1 in SCC cells and tumours enhanced proliferation and growth, and enhanced tumour vascularisation. RNA sequencing of tumour material revealed that lack of Kin1 increased expression of matrix metalloproteinases and chemokines, which have been implicated in tumour progression via promotion of angiogenesis and invasion in a plethora of studies, and of various angiogenesis markers. Together this provides an insight into the mechanisms via which Kin1 controls tumour microenvironment and, ultimately, SCC tumour growth and development. Overall, we report an in vitro and in vivo role for Kin1 in mitotic spindle stability, which affects a variety of mitotic processes and may be linked to reduced keratinocyte proliferation observed in epidermis of KS patients, thus contributing to skin homeostasis. Moreover, we describe a role for Kin1 in regulation of SCC tumour growth and progression, which may ultimately offer an explanation for the aggressive and life-threatening nature of SCC developed in KS.

A population based case-control study was designed to test the hypothesis that cases of squamous cell carcinoma (SCC) had lower concentration of cis linoleic (LA) in their red blood cell membranes and it would remain a predictor of SCC after adjusting for other risk factors. The probability of LA as a potential biomarker for SCC was investigated because it is the precursor of arachidonic acid (AA), a secondary messenger for proteins involved in cell growth and proliferation. Ras oncogene perpetually activates the two phospholipases A-2 and C, causing release of AA from the membrane phospholipids. Red blood cell membranes were used as the medium for assessing the concentration of cis linoleic acid because donation of blood was well accepted by the participants. Tissue availability, and the low cost of operation were the other factors to utilize red blood cell membranes. Cases who had confirmed pathological diagnosis of SCC between December 1991-October 1994 were identified and consecutively sampled from the Arizona Skin Cancer Registry. Controls were recruited from the same geographic area using a random digit dialing technique. Subjects, limited to White-Anglo European descendent, were frequency matched by their age (± 5 years). Participants were screened for use of steroids, prescribed non-steroid anti-inflammatory drugs and history of chronic illness. Data on sun exposure habits, medication usage, lifestyle habits, and family history of SCC were collected during a personal interview. A 15 cc blood sample was collected. The composition of fatty acids in the red blood cell membranes was analyzed using gas-liquid chromatography techniques. Statistical analysis revealed that concentration of LA was significantly lower in the red blood cell membranes of the cases than the controls. However, cases had higher concentration of linolenic and trans linoleic acids. The concentration of 17 other fatty acids were equal, indicating that the general dietary pattern of cases and controls were similar. Use of logistic regression statistics showed that LA remained a significant independent risk factor for SCC (OR = 2.83, 95% Cl = 1.38-5.97) after adjusting for other risk factors. Based on this preliminary research, it is cautiously suggested that LA could be used as a potential biomarker of SCC.

Dissertação de Mestrado Integrado em Medicina Veterinária
O Carcinoma Espinocelular (CEC) é uma neoplasia com origem nos queratinócitos e representa aproximadamente 5% das neoplasias cutâneas em cães. A variante cutânea do CEC apresenta-se preferencialmente em áreas de pele despigmentada com pouco pelo e acomete animais de ambos os sexos, com idade compreendida entre os 6 e os 10 anos. É uma neoplasia localmente invasiva, mas a ocorrência de metástases é pouco frequente. Vários factores etiológicos parecem dar origem ao CEC cutâneo. No entanto, a radiação ultravioleta (UV) e o papilomavírus (PV) são os mais frequentes. O aumento da altitude, diminuição da latitude (países tropicais) e depleção da camada do ozono aumentam a quantidade de raios UV que atingem a superfície terrestre. As lesões cutâneas causadas pela radiação UV iniciam-se com dermatite, progridem para queratose actínica (lesão pré-neoplásica) e esta pode evoluir para CEC. A radiação UV exerce efeito no gene supressor de tumores p53, levando à sua mutação. A lesão mais frequentemente causada pela radiação solar nas células da epiderme é a formação de dímeros de timina entre as bases pirimidínicas do ADN. No que diz respeito ao PV, quatro dos sete tipos deste vírus descritos em cães foram associados à transformação maligna para CEC. São eles: o Papilomavírus oral canino (PVOC), Canis familiaris Papilomavírus tipo 2 (CfPV2), Papilomavírus canino tipo 3 (PVC3) e o Papilomavírus canino tipo 4 (PVC4). Pensa-se que o PV actua como cofator de lesões provocadas por radiação solar, bloqueando a apoptose de células lesadas por raios UV. O PV pode também causar mutação direta em p53 ao inativá-lo. As lesões de CEC cutâneo podem manifestar-se de forma proliferativa ou de forma erosiva. O diagnóstico presuntivo é feito com base na anamnese e exame físico, mas o exame histopatológico após biópsia é essencial para chegar a um diagnóstico definitivo. A excisão cirúrgica com amplas margens é a terapêutica que obtem melhores resultados a longo prazo. A cirurgia combinada com radioterapia, implantes intralesionais de 5- fluorouracilo ou cisplatina, terapia fotodinâmica, anti-inflamatórios como o firocoxib, imunomoduladores como o imiquimode, eletroquimioterapia e retinoides (estes últimos em lesões pré-neoplásicas) também demonstraram ter efeitos benéficos. O prognóstico é favorável caso o diagnóstico seja feito precocemente.
ABSTRACT - Cutaneous Squamous Cell Carcinoma in dogs - The Squamous Cell Carcinoma (SCC) is a neoplasia with origin in the keratinocytes and represents approximately 5% of skin tumors in dogs. The cutaneous form of SCC presents mainly in areas of depigmented skin with little hair and affects animals of both sexes aged between 6 and 10 years. It is a locally invasive tumor but the occurrence of metastases is uncommon. Several etiological factors seem to lead to cutaneous SCC. However, ultraviolet radiation (UV) and papillomavirus (PV) are the most frequent ones. High altitude, lower latitude (tropical countries) and the ozone layer depletion increase the amount of UV rays that reach the earth's surface. The cutaneous injuries caused by UV radiation begin with dermatitis, that can progress to actinic keratosis (a pre-neoplastic injury) and this may evolve into SCC. UV radiation affects the p53 tumor suppressor gene, leading to its mutation. The most frequent injury caused by solar radiation on the epidermal cells is the formation of thymine dimers between the DNA pyrimidine bases. In what regards PV, four of the seven types that have been described in dogs are associated with malignant transformation into SCC. They are: the canine oral Papillomavirus (COPV), Canis familiaris Papillomavirus type 2 (CfPV2), canine Papillomavirus type 3 (CPV3) and canine Papillomavirus type 4 (CPV4). It is believed that the PV acts as cofactors of the injuries caused by solar radiation, blocking the apoptosis of the cells damaged by the UV rays. PV can also cause direct mutations in the p53, inactivating it. The lesions of cutaneous SCC can manifest in two forms, the proliferative and the erosive one. The presumptive diagnosis is based on the medical history and physical examination, but the histopathological examination after biopsy is essential to reach a definitive diagnosis. Surgical excision with wide margins is the treatment that gets better long term results. Surgery combined with radiation therapy, 5 - fluorouracil and cisplatin intralesional implants, photodynamic therapy, anti-inflammatory drugs such as firocoxib, immunomodulators such as imiquimod, electrochemotherapy and retinoids (the latter in pre-neoplastic injuries) also demonstrated to have beneficial effects. The prognosis is favorable if the diagnosis is made in the early stages.

Orientador: Antonio Santos Martins
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-07T06:49:57Z (GMT). No. of bitstreams: 1 ScanaviniJunior_RuiCarlos_M.pdf: 989083 bytes, checksum: 9b013db47c9ffb39e508299508218360 (MD5) Previous issue date: 2005
Resumo: O carcinoma espinocelular ou de células escamosas constitui a segunda neoplasia de pele mais freqüente e apresenta índice de cura superior a 90%, quando tratado na fase mais inicial. Tumores maiores e uma pequena fração dos tumores iniciais costumam apresentar evolução desfavorável, representada pelas recidivas loco-regional e a distância, apesar do tratamento inicial. Objetivo: Identificar, por análise retrospectiva, os fatores histológicos e clínicos associados à evolução adversa, identificando os tumores de alto risco. Material e método: Foram analisados trinta e cinco prontuários de pacientes submetidos ao tratamento cirúrgico do carcinoma espinocelular (CEC) cutâneo de cabeça e pescoço, sendo coletadas informações sobre espessura (milímetros), invasão perineural, grau de diferenciação e invasão angiolinfática. Realizou-se a análise estatística da associação desses fatores com as variáveis da evolução do CEC (recidiva local, metástase linfática, metástase a distância e óbito). Procedeu-se de forma similar na comparação das variáveis da evolução do CEC entre si. Admitiu-se diferença estatística de 5%. Resultados: Ocorreu associação estatística entre a recidiva local e invasão perineural; metástase linfática e invasão angiolinfática, espessura e invasão perineural; metástase a distância e invasão angiolinfática; óbito e invasão perineural, invasão angiolinfática, espessura tumoral. Entre as variáveis do CEC, verificou-se que a metástase linfática e a metástase a distância estiveram associadas ao óbito. Conclusão: Os fatores histológicos e clínicos observados na evolução permitem definir pacientes de alto risco para os quais o seguimento e novos protocolos terapêuticos prospectivos devem ser melhor estabelecidos. Unitermos: Carcinoma de células escamosas, pele, prognóstico
Abstract: Squamous cell carcinoma represents the second most common cancer of the skin. Most of the patients, especially with the small tumors, can be cured at rates that exceed 90%. Larger tumors and a subset of the small ones will present substantial risk of recurrence. Objective: To identify, by retrospective analysis, histological and clinical features associated with recurrence and metastasis. Material and method: There were analyzed thirty five records of patients treated by surgery for squamous cell carcinoma of the skin of the head and neck. From them we collected information about: tumor depth (millimeters), perineural invasion, grade (Broders) and angiolymphatic invasion. After that we verified the association of these features with the variants of prognosis: local recurrence, lymph node metastasis, distant metastasis and death caused by the disease. In a similar fashion we compared these variants of prognosis between them. Statistical significance was admitted at the level of 5%. Results: We found statistical significance between: local recurrence and perineural invasion; lymph node metastasis and perineural invasion, angiolymphatic invasion and depth; distant metastasis and angiolymphatic invasion; death and perineural invasion, depth and angiolymphatic invasion. Between the variants of prognosis we found association of death with distant and lymph node metastasis. Conclusion: Histological and clinical features of squamous cell carcinoma of the skin of the head and neck may help to define high risk patients. Prospective protocols should be established for better treatment results in these patients
Mestrado
Cirurgia
Mestre em Cirurgia

Topical photodynamic therapy (PDT) elicits a therapeutic response in both non melanoma skin cancer and immune-mediated skin disorders. Production of reactive oxygen species may result in direct cell death; however PDT-induced inflammatory and immune responses may also contribute to the therapeutic effects. The aims of my thesis were to examine for evidence of topical PDT-induced apoptosis and leucocyte trafficking, and the mechanisms underlying their mediation, in human skin.

Basal cell carcinoma (BCC) is a skin cancer of particular importance to the Australian community. Its rate of occurrence is highest in Queensland, where 1% to 2% of people are newly affected annually. This is an order of magnitude higher than corresponding incidence estimates in European and North American populations. Individuals with a sun-sensitive complexion are particularly susceptible because sun exposure is the single most important causative agent, as shown by the anatomic distribution of BCC which is in general consistent with the levels of sun exposure across body sites. A distinguishing feature of BCC is the occurrence of multiple primary tumours within individuals, synchronously or over time, and their diagnosis and treatment costs contribute substantially to the major public health burden caused by BCC. A primary knowledge gap about BCC pathogenesis however was an understanding of the true frequency of multiple BCC occurrences and their body distribution, and why a proportion of people do develop more than one BCC in their life. This research project sought to address this gap under an overarching research aim to better understand the detailed epidemiology of BCC with the ultimate goal of reducing the burden of this skin cancer through prevention. The particular aim was to document prospectively the rate of BCC occurrence and its associations with constitutional and environmental (solar) factors, all the while paying special attention to persons affected by more than one BCC. The study built on previous findings and recent developments in the field but set out to confirm and extend these and propose more adequate theories about the complex epidemiology of this cancer. Addressing these goals required a new approach to researching basal cell carcinoma, due to the need to account for the phenomenon of multiple incident BCCs per person. This was enabled by a 20 year community-based study of skin cancer in Australians that provided the methodological foundation for this thesis. Study participants were originally randomly selected in 1986 from the electoral register of all adult residents of the subtropical township of Nambour in Queensland, Australia. On various occasions during the study, participants were fully examined by dermatologists who documented cumulative photodamage as well as skin cancers. Participants completed standard questionnaires about skin cancer-related factors, and consented to have any diagnosed skin cancers notified to the investigators by regional pathology laboratories in Queensland. These methods allowed 100% ascertainment of histologically confirmed BCCs in this study population. 1339 participants had complete follow-up to the end of 2007. Statistical analyses in this thesis were carried out using SAS and SUDAAN statistical software packages. Modelling methods, including multivariate logistic regressions, allowed for repeated measures in terms of multiple BCCs per person. This innovative approach gave new findings on two levels, presented in five chapters as scientific papers: 1. Incidence of basal cell carcinoma multiplicity and detailed anatomic distribution: longitudinal study of an Australian population The incidence of people affected multiple times by BCC was 705 per 100,000 person years compared to an incidence rate of people singly affected of 935 per 100,000 person years. Among multiply and singly affected persons alike, site-specific BCC incidence rates were far highest on facial subsites, followed by upper limbs, trunk, and then lower limbs 2. Melanocytic nevi and basal cell carcinoma: is there an association? BCC risk was significantly increased in those with forearm nevi (Odds Ratios (OR) 1.43, 95% Confidence Intervals (CI) 1.09-1.89) compared to people without forearm nevi, especially among those who spent their time mainly outdoors (OR 1.6, 95%CI 1.1-2.3) compared to those who spent their time mainly indoors. Nevi on the back were not associated with BCC. 3. Clinical signs of photodamage are associated with basal cell carcinoma multiplicity and site: a 16-year longitudinal study Over a 16-year follow-up period, 58% of people affected by BCC developed more than one BCC. Among these people 60% developed BCCs across different anatomic sites. Participants with high numbers of solar keratoses, compared to people without solar keratoses, were most likely to experience the highest BCC counts overall (OR 3.3, 95%CI 1.4-13.5). Occurrences of BCC on the trunk (OR 3.3, 95%CI 1.4-7.6) and on the limbs (OR 3.7, 95%CI 2.0-7.0) were strongly associated with high numbers of solar keratoses on these sites. 4. Occurrence and determinants of basal cell carcinoma by histological subtype in an Australian community Among 1202 BCCs, 77% had a single growth pattern and 23% were of mixed histological composition. Among all BCCs the nodular followed by the superficial growth patterns were commonest. Risk of nodular and superficial BCCs on the head was raised if 5 or more solar keratoses were present on the face (OR 1.8, 95%CI 1.2-2.7 and OR 4.5, 95%CI 2.1-9.7 respectively) and similarly on the trunk in the presence of multiple solar keratoses on the trunk (OR 4.2, 95%CI 1.5-11.9 and OR 2.2, 95%CI 1.1-4.4 respectively). 5. Basal cell carcinoma and measures of cumulative sun exposure: an Australian longitudinal community-based study Dermal elastosis was more likely to be seen adjacent to head and neck BCCs than trunk BCCs (p=0.01). Severity of dermal elastosis increased on each site with increasing clinical signs of cutaneous sun damage on that site. BCCs that occurred without perilesional elastosis per se, were always found in an anatomic region with signs of photodamage. This thesis thus has identified the magnitude of the burden of multiple BCCs. It does not support the view that people affected by more than one BCC represent a distinct group of people who are prone to BCCs on certain body sites. The results also demonstrate that BCCs regardless of site, histology or order of occurrence are strongly associated with cumulative sun exposure causing photodamage to the skin, and hence challenge the view that BCCs occurring on body sites with typically low opportunities for sun exposure or of the superficial growth pattern are different in their association with the sun from those on typically sun-exposed sites, or nodular BCCs, respectively. Through dissemination in the scientific and medical literature, and to the community at large, these findings can ultimately assist in the primary and secondary prevention of BCC, perhaps especially in high-risk populations.

Previous data have unveiled a novel autoregulatory feedback loop between iASPP and p63 in the stratified epithelia; this involves two microRNAs, miR-574-3p and miR-720, and is critical for epidermal homeostasis. The iASPP oncoprotein, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is a key inhibitor of p53 and NF-κB and is highly expressed in many cancers. Non-melanoma skin cancer, comprising of cutaneous squamous carcinoma (cSCC) and basal cell carcinoma, is currently the most common malignancy in the UK. In view of this newly-identified iASPP-p63 axis, I hypothesised a potential role for dysregulation of this feedback loop in the pathogenesis of cSCC and aimed to assess the role of iASPP in human cSCC. Protein and mRNA expression patterns were assessed in a panel of 10 cSCC cell lines generated by our group. In addition, immunostaining of iASPP and p63 was performed in 107 cSCC clinical samples of variable differentiation status. The data reveal an overall increase in expression of iASPP and ΔNp63 in cSCC but also suggest a significant alteration of the cellular localisation of iASPP dependent on the differentiation status of the tumour. To further assess the effects mediated by the iASPP/p63 axis, iASPP and p63 have been silenced by RNAi technology in a subset of cSCC cell lines. Whilst data shows the direct effects of iASPP and p63 upon each other's expression are maintained in cSCC, epigenetic dysregulation of the feedback loop at the microRNA level may be occurring via a novel p63 regulator, miR-211-5p. Functionality of iASPP in cSCC (proliferation, apoptosis, cell motility/migration and invasiveness) provides evidence for a role of iASPP in preventing epithelial-mesenchymal transition in cSCC via a p63/miR-205-5. These findings provide potential future directions for development of clinical biomarkers and novel therapeutic targets for cSCC and may ultimately provide the tools for tackling the increasing morbidity and mortality associated with this malignancy.

Le carcinome spinocellulaire (SCC) est le 2ème cancer de la peau le plus fréquent avec plus d’un million de nouveaux patients diagnostiqués dans le monde chaque année. On retrouve également des SCCs associés à un pronostic plus sombre au niveau de la tête, du cou, de la cavité orale et de l’œsophage. Des travaux récents ont démontré l’existence de cellules souches cancéreuses (CSCs) dans les SCCs cutanés mais les mécanismes moléculaires contrôlant leurs fonctions restent indéterminés. Dans une première étude, nous avons montré que Sox2, un facteur de transcription (TF) associé aux cellules souches, est détecté de manière hétérogène dans une grande majorité des papillomes et des SCCs chez la souris et chez l’humain. La délétion conditionnelle de Sox2 dans l’épiderme réduit drastiquement l’apparition de tumeurs démontrant le rôle clé de Sox2 dans l’initiation tumorale. En utilisant une souris génétiquement modifiée Sox2-GFP knock-in, nous avons démontré que les cellules tumorales Sox2+ sont enrichies en cellules propagatrices de tumeurs dont la proportion augmente au fur et à mesure des transplantations sériées. L’ablation des cellules Sox2+ dans les papillomes et les SCCs conduit à une importante régression des tumeurs, indiquant que ces cellules ont un rôle crucial dans le maintien des tumeurs. La délétion conditionnelle de Sox2 dans des papillomes et SCCs préexistants provoque également une régression majeure des tumeurs, soulignant le rôle essentiel de Sox2 dans la régulation des fonctions des cellules tumorales. Une analyse transcriptionnelle et des expériences d’immunoprécipitation de chromatine nous ont permis de mettre en évidence un réseau de gènes associés à des fonctions essentielles des cellules tumorales et régulés par Sox2 dans les tumeurs primaires in vivo. Dans une 2ème étude, nous avons montré que les SCCs issus de l’épiderme inter-folliculaire (IFE) présentent en général un caractère différencié alors que ceux issus du follicule pileux (HF) présentent fréquemment des caractéristiques de transition épithélio-mésenchymateuse (EMT). En réalisant une analyse transcriptionnelle et épigénétique, nous avons démontré que les différentes cellules à l’origine expriment un réseau de gènes spécifiques et présentent une accessibilité différentielle à des sites de liaison d’importants TFs associés soit à un phénotype épithélial soit à l’EMT. Ces résultats démontrent que l’état transcriptionnel et épigénétique de la cellule à l’origine amorce spécifiquement les tumeurs vers le processus d’EMT. L’ensemble de ces résultats souligne des mécanismes cruciaux à l’établissement de l’hétérogénéité tumorale et seront essentiels pour parvenir à des pronostics affinés et au développement de nouvelles thérapies ciblées dans le traitement du cancer.
Skin squamous cell carcinoma (SCC) is the second most frequent skin cancer with more than a million new patients affected every year throughout the world. It is also the predominant cancer of the head, neck, oral cavity and esophagus, associated with a poor prognosis. Recent studies have identified cancer stem cells (CSCs) in skin SCC but the molecular mechanisms controlling their functions remain unclear. In a first study, we show that Sox2, a transcription factor (TF) associated with stemness, is expressed in a heterogeneous manner in the vast majority of benign and malignant skin tumors in mouse and human. Sox2 conditional deletion in the epidermis impairs tumor development showing that Sox2 plays a crucial role in tumor initiation. Using a Sox2-GFP knock-in mouse model, we show that Sox2-expressing tumor cells are greatly enriched in tumor-propagating cells, which further increase upon serial transplantations. Lineage ablation of Sox2-expressing cells in primary benign and malignant SCCs leads to tumor regression, consistent with the critical role of Sox2-expressing cells in tumor maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumor regression, supporting the essential role of Sox2 in regulating cancer cells functions. Using transcriptional profiling and chromatin immunoprecipitation, we uncovered a gene network controlling many cancer hallmarks regulated by Sox2 in primary tumour cells in vivo.In a second study, by targeting the same oncogenic mutations to distinct skin compartments, we show that interfollicular epidermis (IFE)-derived SCCs are generally well-differentiated, while hair follicle stem cells (HFSCs)-derived SCCs frequently exhibit features of epithelial-mesenchymal transition (EMT). Using transcriptional and epigenetic profiling, we show that IFE and HF tumor-initiating cells harbor distinct chromatin landscapes and gene regulatory networks associated with tumorigenesis and EMT. These different chromatin landscapes correlate with the differential accessibility of key epithelial and EMT TFs binding sites in the cancer cell of origin. These findings demonstrate that cell type-specific chromatin and transcriptional states differentially prime tumours towards EMT.Altogether, these results highlight crucial mechanisms for the establishment of tumor heterogeneity which will be relevant for better prognostic assessment and the development of novel targeted therapies for cancer treatment.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished

Les carcinomes épidermoïdes cutanés (CEC) sont des cancers invasifs du kératinocyte qui se développent suite à la subversion du système immunitaire. Une reprogrammation de l’immunité antitumorale par stimulation locale des cellules dendritiques (DC) et des macrophages pourrait être bénéfique. Dans ce contexte, l’objectif de ma thèse a été de comprendre l’hétérogénéité ontogénique, phénotypique et fonctionnelle de ces cellules immunitaires au cours de la carcinogenèse cutanée afin de développer des approches thérapeutiques les stimulant. Nous avons, d’abord, caractérisé les macrophages de la peau de souris et infiltrant les CEC chez l’homme. Nous avons identifié par cytométrie en flux spectrale une population de macrophages matures, autofluorescents et résidents de la peau de souris. Ces macrophages résidents présentent une polarisation fonctionnelle d’homéostasie et de réparation tissulaire. Dans les CEC humains, les macrophages autofluorescents semblent avoir les mêmes caractéristiques que leurs équivalents dans la peau de souris. Dans une seconde étude, nous avons mis en évidence que les DC associées aux tumeurs sont dysfonctionnelles. Une immunothérapie locale composée d’un agoniste de TLR9 et d’un anticorps bloquant la signalisation sous le récepteur à l’IL-10 induit la régression de tumeurs cutanées. Cette approche permet la reprogrammation fonctionnelle des DC et la génération de lymphocytes T CD8+ producteurs d’IFNγ, de TNFα et d’IL-17. Ces résultats mettent en évidence l’hétérogénéité fonctionnelle des cellules myéloïdes dans la peau et les CEC. Leur reprogrammation fonctionnelle permettrait le développement de nouvelles thérapeutiques contre les CEC chez l’homme
Skin Squamous cell carcinoma (sSCC) are invasive keratinocyte tumor that develop after immune system subversion. The reprogrammation of anti-tumoral immunity using local stimulation of dendritic cells (DC) and macrophages could be useful. In this line, the aim of my thesis was to understand ontogenic, phenotypic and functional heterogeneity of these cell subsets along skin carcinogenesis to develop new immunotherapies. First, we characterized skin macrophage subsets in mouse and those infiltrating sSCC in human. Using spectral flow cytometry, we identified matured autofluorescent tissue-resident macrophages. These macrophages are anti-inflammatory polarized. In human sSCC, autofluorescent macrophages seem to have same properties that their mouse counterparts. In second study, we identified tumor-infiltrating DC with altered functions. We used a local immunotherapy composed by a TLR9 agonist and blocking antibody against α-chain of IL-10 receptor. This combination induced tumor regression through DC reprogrammation and IFNγ+, TNFα+ IL17A+ T CD8+ lymphocyte generation. These results highlight functional myeloid heterogeneity in skin and sSCC. Their reprogrammation could promote the development of immunotherapies against sSCC in human

Purpose: Automatic border detection is the first and most crucial step for lesion segmentation and can be very challenging, due to several lesion characteristics. There are many melanoma border-detecting algorithms that perform poorly on dermoscopy images of basal cell carcinoma (BCC), which is the most common skin cancer. One of the reasons for poor lesion detection performance is that there are very few algorithms that detect BCC borders, because they are difficult to segment, even for dermatologists. This difficulty is due to low contrast, variation in lesion color and artifacts inside/outside the lesion. Segmentation that has adequate lesion-feature capture, with acceptable tolerance, will facilitate accurate feature segmentation, thereby maximizing classification accuracy. Methods: The main objective of this research was to develop an effective BCC border detecting algorithm whose accuracy is better than the existing melanoma border detectors that have been applied to BCCs. Fifteen auto-thresholding techniques were implemented for BCC lesion segmentation; but, only five were selected for use in algorithm development. A novel technique was developed to automatically expand BCC lesion borders, to completely circumscribe the lesion. Two error metrics were used that better measure Type II (false-negative) errors: Relative XOR error and Lesion Capture Ratio (a novel error metric). Results: On training and test sets of 1023 and 119 images, respectively, based on two error metrics, five thresholding-based algorithms outperformed two state-of-the-art melanoma segmentation techniques, in segmenting BCCs. Five algorithms generated borders that appreciably better matched dermatologists’ hand-drawn borders which were used as the “gold standard.” Conclusion: The five developed algorithms, which included solutions for image-vignetting correction and border expansion, to achieve dermatologist-like borders, provided more inclusive and therefore, feature-preserving border detection, favoring better BCC classification accuracy, for future work.

Basal cell carcinoma (BCC) of the skin is predominantly associated with mutational inactivation of the PTCH1 tumour suppressor gene resulting in constitutive activation of the Hedgehog (HH) developmental pathway. Tumour formation is linked to induction of the GLI (GLI1 and GLI2) transcription factors via a pathway that is thought to require the transmembrane protein SMOOTHENED (SMO) and accordingly, SMO is attracting much interest as a drug target in cancer therapy. However, although there has been a high degree of success in treating some BCCs with anti-SMO compounds, many tumours are only partially or unresponsive which indicates that SMO-independent mechanisms may contribute to tumour formation and/or viability. To further understand how loss of (or reduced) PTCH1 function contributes to BCC, RNAi (retroviral shRNA) was employed to suppress PTCH1 in NEB1 and N/Tert immortalised human keratinocyte cells. Compared to control (shCON) cells, PTCH1 knockdown (shPTCH1) cells displayed more compact colony formation as well as increased GLI1 (but not GLI2) expression however, whereas the increase of GLI1 was suppressed upon transfection with SMO siRNA in shPTCH1 cells, it was insensitive to the presence of the SMO antagonists Cyclopamine-KAAD and SANT1 in shCON cells. The reason for this is unclear but SMO levels were increased and more nuclear in shPTCH1 cells indicating that SMO may have nuclear signalling capability that is unresponsive to certain SMO antagonists. Indeed, cDNA microarray profiling revealed that 80% of the genes that were differentially expressed in NEB1-shPTCH1 cells (>2-fold, p<0.01) remained differentially expressed in the presence of Cyclopamine-KAAD; this includes the chemokines CXCL10 and CXCL11 which were recently shown to be over-expressed in BCC thus helping validate the efficacy of NEB1-shPTCH1 cells as an in vitro tumour model. In addition, functional gene grouping has predicted novel biological processes downstream of PTCH1 that may be important in BCC biology including NF-κB signalling. In summary, the data presented in this thesis suggests that the mechanism(s) through which the loss of PTCH1 leads to BCC formation may be more complex than has been inferred from previous studies.

Introduction: Head and Neck cutaneous Squamous Cell Carcinoma (HNcSCC) is one of the most common cancers in the world and is particularly prevalent in Australia. Regional metastasis remains one of the most important determinants of survival but predicting which lesions metastasise remains a challenge. Current staging systems have been shown to poorly stratify patient survival. Methodology: Three studies were initiated: (1) A prospective trial of patients with high risk primary HNcSCC underwent sentinel node biopsy (SNB) to identify subclinical metastasis. Patients were followed to determine the accuracy of SNB, overall rate of metastasis and clinicopathological predictors of metastasis. Retrospective reviews of patients with metastatic HNcSCC treated at a quaternary referral head and neck oncology service was undertaken to characterise the prognostic impact of (2) Soft tissue metastases (STM) and (3) Location (parotid v neck) of regional metastasis. Results: (1)105 high risk lesions underwent SNB with a total subclinical nodal metastasis rate of 14.3%. SNB identified 67% of patients with regional metastasis. The risk of metastasis is increased in the presence of perineural invasion and increasing depth of invasion and number of high-risk features. (2) A review of 200 patients with STM identified a poor five-year overall survival (OS) of 36% and that increasing number of deposits was a significant predictor of reduced survival on multivariable analysis. (3) A review of 535 patients with regional metastasis determined that multiple regional metastases and metastases to neck nodes were associated with reduced survival. Conclusion: Regionally metastatic HNcSCC is associated with a significant mortality rate in Australia. Methods to identify regional metastases early, before the development of multiple nodal metastases or STM, may improve survival rates. SNB is one such potential method; however, work is needed to better identify predictors of nodal metastases.

Cutaneous squamous cell carcinoma (SCC) is a common cancer yet its treatment is under-researched. The objective of this thesis was to develop a proposal for a randomised controlled trial (RCT) to address uncertainties relating to the management of the condition, and to ultimately improve the management of affected patients. Two systematic reviews were initially conducted to appraise the current evidence base for SCC treatments. Only one RCT was eligible for inclusion in the Cochrane systematic review; a small study which found no significant difference in time to recurrence between patients treated with post-operative 13-cis retinoic acid and interferon, and those not receiving adjuvant treatment. Systematic review and meta-analysis of observational studies included 118 studies. Pooled estimates of recurrence were lowest after cryotherapy and curettage and electrodesiccation, although lesions treated by these modalities were mostly small and low-risk. Although pooled recurrence after Mohs surgery appeared lower than after conventional excision or radiotherapy, the differences were not significant with overlapping 95% confidence intervals. For photodynamic therapy, pooled recurrence after apparently successful initial treatment was particularly high (26%). Evidence relating to the effectiveness of topical and systemic treatments was very limited. Estimates of recurrence were used to inform the sample size calculation for the proposed RCT. A survey of healthcare professionals was conducted to establish research priorities and identify clinically important management uncertainties from which initial trial scenarios were formulated. High-risk SCCs were identified as a research priority, with optimal surgical management and the role of adjuvant radiotherapy being key areas of uncertainty. Through multi-disciplinary collaboration, a proposal for a two-stage RCT has been developed; in the first stage, locoregional recurrence after conventional surgery with a controlled excision margin will be compared with Mohs surgery, and in the second stage locoregional recurrence will be compared between patients treated with adjuvant radiotherapy versus those receiving no adjuvant treatment. Feasibility work conducted during the development of the trial has involved: a) A retrospective analysis of SCCs treated over twelve-months to determine the number of patients and types of SCCs potentially eligible for recruitment into the proposed trial and to further inform the sample size calculation. Within five years of treatment 6% of 357 patients experienced local recurrence, 3% had regional recurrence and 1.5% died of their SCC. Comparison of the most recent American Joint Cancer Council (AJCC7) and an alternative Brigham and Women’s Hospital (BWH) classification showed that approximately 50% of SCCs were T2 in both schemes and eligible for entry into the first stage of the proposed trial. However, an additional BHW T2b substage better stratified outcomes dependent on the number of risk features, and indicated that 19% of all SCCs would potentially be also eligible for the second stage of the trial. b) A questionnaire and focus group study to assess the acceptability of the RCT and to identify possible barriers to recruitment. Participants had a desire to be better informed about SCC, wanting information relating to the trial to be provided in a variety of formats. 71% of participants were hypothetically willing to be randomised into the surgical stage of the proposed trial but had more concerns about the second stage involving adjuvant radiotherapy. Lack of equipoise and confusion about the concept of randomisation will need to be carefully addressed when presenting the trial to participants. The proposed trial will be the first to directly compare treatments for the types of SCC seen commonly in clinical practice. For the trial to be adequately powered, an estimated 5400 participants will need to be recruited, so a multi-centre, multi-disciplinary approach will be necessary.

This thesis conducted in-depth exploration of the aetiology of basal cell carcinoma (BCC) according to the anatomic site on which the tumour arises. It compared phenotypic characteristics, environmental and genetic risk factors, between people who develop incident BCCs on habitually sun-protected body sites (trunk) and those who had only ever had BCCs on habitually sun-exposed sites (head). The findings suggested that the pathophysiology of BCC differs between lesions arising on different anatomic sites and subtypes. Truncal BCCs arise because of relatively low dose cumulative UVR exposure of the trunk in people who have had a greater opportunity for truncal exposure.

Depuis sa découverte, le gène TP63 a soulevé un intérêt considérable grâce à son rôle majeur dans la morphogenèse des épithélia. La quasi-totalité de nos connaissances dérive de l’observation des phénotypes des souris déficientes qui présentent une absence d’épithélia pluristratifiés, due à un défaut d'expression des complexes d'adhérence cellule-matrice extra cellulaire et cellules-cellules. De plus, TP63 est amplifié et surexprimé dans environ 25% des carcinomes épidermoïdes de l’œsophage et est quasi absent des adénocarcinomes du même organe. Dans ce travail nous avons étudié l’expression de p63 dans la muqueuse œsophagienne, et nous avons montré que p63 exerce un rôle de régulateur de l’expression des complexes d’adhérence intra-épithéliaux lors de la transition entre les cellules basales/suprabasales hautement prolifératives et les couches les plus différenciées incapables de proliférer. Puis, nous avons étudié le rôle possible de p63 dans la formation de la métaplasie intestinale, une lésion précurseur de l’adénocarcinome. Dans ce contexte, nous avons établi qu’un traitement reconstituant le stress acido-biliaire induit une perte d’expression de p63 secondaire à une dégradation par le protéasome dans des cellules primaires et des lignées dérivées de carcinomes œsophagiens. Enfin, à l’aide d’un modèle de peau reconstruite, nous avons montré l’implication de p63 dans la stratification épithéliale, dans la prolifération, la différenciation et les interactions épithélium-mésenchyme. Ces réstultats clarifient le rôle de TP63 comme un ongène potentiel dans le carcinome épidermoïde de l’œsophage et comme potentiel suppresseur dans l’adénocarcinome
Since its discovery in 1998, the TP63 gene has raised considerable interest due to its major role in epithelial morphogenesis. The vast majority of our current knowledge is based on the phenotypes of TP63 deficient mice, which show a lack of stratified epithelia associated with defects in the expression of cell-cell and cell-matrix adhesion complexes. In addition, TP63 systematically overexpressed in squamous cell carcinomas and amplified in about 25% of œsophageal squamous cell carcinomas, whereas it is barely detectable in adenocarcinomas that arise in the same organ. This work analyzes the expression of p63 in normal œsophageal mucosa and demonstrates its regulatory role in the expression of cell-cell adhesion complexes at the transition between highly proliferative basal/suprabasal layers and more differentiated, non-proliferative superficial layers. Next, the role of p63 in the formation of intestinal metaplasia, a precursor of adenocarcinoma, is addressed in experiments reconstituting in vtro the effects of acid-bile gastro-oesophageal reflux. We show that this form of stress induces a loss of p63 in cell lines derived from oesophageal cancers, due to its rapid proteasome-dependent degradation. Finally, we have used an in vitro skin reconstruction model to demonstrate the involvement of p63 in the process of epidermal stratification, proliferation, differentiation, and epithelium-mesenchyme interactions. These results clarify the role of TP63 as a potential oncogene in œsophageal squamous cell carcinoma, and as a potential suppressor in adenocarcinoma

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via regulation of HOXD10. This report was soon challenged by another group who reported that miR-10b expression in a large patient cohort correlated inversely and significantly with tumor size, grade, and vascular invasion, but did not correlate with development of distant metastases or survival. These latter data suggest that miR-10b may impede specific functions associated with breast cancer progression. In this thesis, I present my analysis of miR-10b function in breast carcinoma cells, which revealed that it suppresses their migration and invasion. To define a mechanism that accounts for this suppressive function, I identified T-lymphoma invasion and metastasis 1 (TIAM1), a guanine nucleotide exchange factor for Rac1, as a miR-10b target and demonstrated that miR-10b inhibits TIAM1-dependent Rac1 activation, migration, and invasion. In addition, I identified the VEGF receptor fms-related tyrosine kinase 1 (FLT-1) as a second target of miR-10b and discovered a novel function for FLT-1 in promoting breast carcinoma cell migration and invasion. My results show, for the first time, that Rac activation can be regulated by a specific miRNA and provide a novel mechanism for the regulation of TIAM1 and FLT-1 in breast cancer. These data support the conclusion from clinical data that miR-10b expression correlates inversely with breast cancer progression, and suggest that miR-10b functions to impede breast carcinoma progression by regulating key target genes involved in cell motility.

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

The human skin provides us with an excellent protective barrier and possesses a remarkable ability of constant renewal. Basal cell carcinoma is the most common type of skin cancer. The aim of this project was to verify results from an earlier study investigating the molecular differences between basal cell carcinoma (BCC) and basal cells of normal human epidermis. In that study microdissection of cell populations from BCC and basal cells of normal epidermis respectively was performed in five cases of confirmed BCC. Following RNA extraction and amplification, a gene expression analysis was performed using a 46 k human cDNA microarray. Comparison of expression profiles showed a differential expression of approximately 300 genes in BCC. An upregulation of signaling pathways previously known to be of importance in BCC development could be observed, as well as a downregulation of differentiation markers, MHC class II molecules, and proteins active in scavenging of oxygen radicals. We wanted to confirm these findings for a number of selected genes, using real time PCR. The focal point of this project was microdissection of cells from BCC and subsequent isolation of RNA. Microdissection based methods offer a possibility of selecting well defined cell populations for further analysis by using a focused laser beam. Initially tests in order to optimize the method were also performed, concerning the dehydration process and choice of slides used in microdissection. Isolation of RNA may, as we experienced, be associated with problems due to destruction of RNA by degrading enzymes.

Non-melanoma skin cancer (NMSC), comprised of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), is the most common cancer in Caucasians. Ultraviolet radiation (UVR) exposure is the most important environmental risk factor for both BCC and SCC development. However, the precise relationship between UVR and the risk of NMSC is complex, and the relationship may differ by skin cancer type. It has been hypothesized that intermittent patterns and childhood sunlight exposure are important for BCC while continuous (chronic) and lifelong (i.e. childhood and adulthood) sunlight exposure is important for SCC. Epidemiologic studies have demonstrated that cutaneous human papillomavirus (HPV) infection may also be a risk factor for developing NMSC. However, the pathway by which cutaneous HPV is associated with NMSC remains unclear. It is hypothesized that UVR exposure may interact synergistically with cutaneous HPV in NMSC development. The goal of the research study was to evaluate the relationship between levels of sunlight exposure and BCC and SCC and to investigate differences in sunlight-associated BCC and SCC risk by genus-specific cutaneous HPV serostatus. To address these goals, we conducted a clinic based case-control study of histologically confirmed BCC and SCC cases recruited from a university dermatology clinic and controls with no history of cancer and screened negative for current skin cancer. Logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI) for the associations between measures of sunlight exposure and BCC and SCC. Multiplicative interactions were tested by placing an interaction term for the product of genus-specific HPV seroreactivity and sunlight related factors in the logistic regression models. Measures of both intermittent and continuous patterns of sunlight exposure were associated with both types of skin cancer (i.e. BCC and SCC). Specifically, history of blistering sunburn (a marker of intermittent sunlight exposure) and occupational sunlight exposure (i.e. having a job in the sun for at least 3 months for >10 years) were both associated with BCC and SCC. The major differences in patterns of sunlight exposure between BCC and SCC were observed for sunlight exposure in one's thirties. Additionally, sunlight exposure in one's twenties was associated with SCC, regardless of pattern of exposure; similar associations were not observed for BCC. Measures of timing of sunlight exposure consistently demonstrated that childhood/adolescent sunlight exposure was more important for SCC than BCC. These included number of moles on the forearms and entire body (measure of increased childhood sunlight exposure), and younger age at first and tanning bed use. Younger age at first blistering sunburn was statistically significantly associated with both BCC and SCC. NMSC cases were more likely to be seropositive for cutaneous HPV antibodies compared to controls. Compared to tanning, having a propensity to sun burn (p=0.006), or poor tanning ability (p=0.003) were significantly associated with a higher seroprevalence to genus beta HPV types within SCC cases. Statistically significant interactions were observed between poor tanning ability and genus-specific seropositivity with NMSC. Specifically, the associations between poor tanning ability and BCC (p interaction=0.02) and SCC (p interaction=0.01) were significantly stronger among individuals that were seropositive for antibodies to genus alpha HPV types. Similarly, the association between poor tanning ability and SCC was stronger among those seropositive for genus beta HPV types (p interaction=0.001). No additional significant interactions were observed for BCC or SCC between cutaneous sensitivity, history of blistering sunburn, or cumulative sunlight exposure and genus-specific seroreactivity. In conclusion, associations with patterns of sunlight exposure appeared to be similar between BCC and SCC cases. With the exception of age at first blistering sunburn, factors measuring timing of sunlight exposure demonstrated stronger and statistically significant relationships with SCC. Additionally, of the sunlight related factors measured, only the associations between poor tanning ability and BCC and SCC were significantly modified by HPV seropositivity to types in genera alpha or beta.

Melanoma is one of the most aggressive types of cancer. It originates from the transformation of melanocytes present in the epidermal/dermal junction of the human skin. It is commonly accepted that melanomagenesis is influenced by the interaction of environmental factors, genetic factors, as well as tumor-host interactions. DNA photoproducts induced by UV radiation are, in normal cells, repaired by the nucleotide excision repair (NER) pathway. The prominent role of NER in cancer resistance is well exemplified by patients with Xeroderma Pigmentosum (XP). This disease results from mutations in the components of the NER pathway, such as XPA and XPC proteins. In humans, NER pathway disruption leads to the development of skin cancers, including melanoma. Similar to humans afflicted with XP, Xpa and Xpc deficient mice show high sensibility to UV light, leading to skin cancer development, except melanoma. The Endothelin 3 (Edn3) signaling pathway is essential for proliferation, survival and migration of melanocyte precursor cells. Excessive production of Edn3 leads to the accumulation of large numbers of melanocytes in the mouse skin, where they are not normally found. In humans, Edn3 signaling pathway has also been implicated in melanoma progression and its metastatic potential. The goal of this study was the development of the first UV-induced melanoma mouse model dependent on the over-expression of Edn3 in the skin. The UV-induced melanoma mouse model reported here is distinguishable from all previous published models by two features: melanocytes are not transformed a priori and melanomagenesis arises only upon neonatal UV exposure. In this model, melanomagenesis depends on the presence of Edn3 in the skin. Disruption of the NER pathway due to the lack of Xpa or Xpc proteins was not essential for melanomagenesis; however, it enhanced melanoma penetrance and decreased melanoma latency after one single neonatal erythemal UV dose. Exposure to a second dose of UV at six weeks of age did not change time of appearance or penetrance of melanomas in this mouse model. Thus, a combination of neonatal UV exposure with excessive Edn3 in the tumor microenvironment is sufficient for melanomagenesis in mice; furthermore, NER deficiency exacerbates this process.

L'autofluorescenza (AF) è definita come l'emissione di fluorescenza osservata quando determinate molecole sono eccitate da raggi UV o luce visibile di lunghezze d'onda adeguate. Quando una molecola viene illuminata ad una lunghezza d'onda di eccitazione, assorbirà questa energia e verrà attivata dal suo stato fondamentale a quello eccitato. La molecola (fluoroforo) può quindi rilassarsi dallo stato eccitato a fondamentale generando energia sotto forma di fluorescenza, a lunghezze d'onda di emissione più lunghe di quella di eccitazione. I fluorofori endogeni sono molecole ampiamente distribuite in cellule e tessuti, come proteine ​​contenenti aminoacidi aromatici, flavine e lipopigmenti. I principali fluorofori della cute sana si trovano nell'epitelio (ad es. cheratina, nicotinamide adenine dinucleotide o NADH e flavin adenine dinucleotide o FAD) e nella sottomucosa (ad es. collagene ed elastina). Queste molecole, quando irradiate tra le lunghezze d'onda da 375 a 440 nm, mostrano fluorescenza nell'intervallo spettrale del verde. Il non-melanoma skin cancer (NMSC) è il tumore maligno più comune al mondo. Lo sviluppo dei NMSC è accompagnato da cambiamenti istopatologici nell'epidermide come perdita di maturazione cellulare, alterazione della produzione di cheratina, ispessimento generale dello strato epiteliale e alterazioni biochimiche (riduzione del NADH). I NMSC sono anche accompagnati da cambiamenti istopatologici nello stroma e nella sottomucosa sottostanti, tra cui la neovascolarizzazione e la distruzione del legame crociato di collagene da parte delle proteasi. Queste alterazioni portano ad una generale riduzione dell’AF dovuta all'alterazione della distribuzione dei fluorocromi e in particolare al NADH e al collagene. Negli ultimi due decenni, gli studi riguardanti ll’AF cellulare e tissutale hanno avuto un notevole aumento. Sono stati condotti studi sull’AF sia in vitro che in vivo, per lo studio dei tessuti normali e per la discriminazione tra tessuti normali e lesioni neoplastiche di mucosa orale, cute, esofago, colon, polmone, bronchi, cervello e vescica. I metodi utilizzati sono sia il direct visual fluorescence examination (DVFE) sia lo spettrofotometria. In particolare, il DVFE è stato ampiamente utilizzato per studi clinici sulla mucosa orale. Per quanto riguarda l’AF della cute, questa è stata studiata più frequentemente usando lo spettrofotometria. Il principio è la scansione e l'analisi della luce emessa dalla cute dopo l'esposizione a una fonte di luce attivante. Tuttavia, ad oggi non sono emersi metodi che fossero traducibili nella pratica clinica. L'obiettivo principale di questo studio è quello di analizzare la correlazione tra la misurazione spettrale dell’AF cutanea e le caratteristiche istopatologiche della cute maligna e pre-maligna nel campo dei NMSC. Dopo la rimozione chirurgica, verrà eseguita una valutazione ex vivo dell’AF. Il campione verrà irradiato con una sonda che emette una luce nello spettro blu (lunghezza d'onda 400-440 nm) e la fluorescenza emessa dal tessuto verrà misurata mediante uno spettrofotometro in modalità spot standardizzata. Eventuali modifiche rilevate verranno riportate sul campione chirurgico con l'applicazione di un repere. Verrà eseguito un esame istopatologico della lesione e eventuali cambiamenti nel pattern di fluorescenza saranno correlati con possibili alterazioni del pattern istopatologico, facendo riferimento ai reperi chirurgici. Le alterazioni delle misure spettrali sono correlate alle alterazioni istopatologiche dei NMSC. La misurazione spettrale può essere un nuovo supporto per la diagnosi precoce dei NMSC, una guida per le biopsie incisionali mirate, uno strumento per la definizione dei margini chirurgici intraoperatori e per il follow-up dei pazienti trattati.
Autofluorescence (AF) is defined as the fluorescence emission observed when certain cell molecules are excited by UV or visible light of suitable wavelenghts. When a biologic molecule is illuminated at an excitation wavelength within the absorption spectrum of that molecule, it will absorb this energy and be activated from its ground state to an excited state. The molecule (fluorophore) can then relax back from the excited to the ground state by generating energy in the form of fluorescence, at emission wavelengths, which are longer than that of the excitation wavelength. The most important endogenous fluorophores are molecules widely distributed in cells and tissues, like proteins containing aromatic aminoacids, flavins and lipopigments. The main fluorophores of healthy skin are located in the epithelium (eg. keratin, nicotinamide adenine dinucleotide or NADH and flavin adenine dinucleotide or FAD) and the submucosa (e.g. collagen and elastin). These molecules when irradiated between the wavelengths from 375 and 440 nm, show fluorescence in the green spectral range. Nonmelanoma skin cancer (NMSC) is the most common malignancy worldwide. The developement of NMSC is accompanied by histopathological changes in epidermis such as loss of cellular maturation, alteration in keratin production, overall thickening of the epithelial layer and biochemical alterations (NADH decrease). NMSC is also accompanied by histopathological changes in the underlying stroma and submucosa, including neovascularization and destruction of the collagen cross-link by proteases. These alterations lead to a general decrease in AF due the alteration in distribution of the fluorochromes and in particular to NADH and collagen. In the last two decades, studies concerning cell and tissue AF has had a dramatic increase. AF studies have been performed both in vitro and in vivo, for the study of normal tissue and for the discrimination between normal tissues and neoplastic lesions of oral mucosa, skin, esophagus, colon, lung, bronchi, brain and bladder. The methods used are both direct visual fluorescence examination (DVFE) and spectrophotometry. In particular, DVFE has been widely used for clinical studies on oral mucosa. Regarding AF of the skin, this has been studied more frequently by using spectrophotometry. The principle is scanning and analyzing reflected light from the skin after exposure to an activating light source. AF spectroscopy is a very sensitive technique for quantitative measurements of tissue constituents. However, to date no methods have emerged that can be translated into clinical practice. The primary objective of this study is to investigate the correlation between spectral mesurement of cutaneous AF and the histopathological characteristics of malignant and pre-malignant skin in NMSC. Following surgical removal of the cancer, an ex vivo evaluation of the AF will be performed. The specimen will be irradiated with a probe that emits a light in the blue spectrum (wavelength 400-440 nm) and the fluorescence emitted by the tissue will be measured using a spectrophotometer in a standardized spot modality. Any changes detected will be reported on the surgical specimen with the application of a surgical mark. Histopathological examination of the lesion will be performed and any changes in the fluorescence pattern will be correlated with possible alterations in the histopathological pattern, referring to surgical marks. Alterations in AF spectral measurement correlate with histopathological alterations in NMSC. the spectral measurement can be a new support for the early diagnosis of NMSCs, a guide for the targeted incisional biopsies, a tool for the definition of the intraoperative surgical margins, and for the follow-up of treated patients.