Dissertations / Theses on the topic 'Actinic keratosis'

The pyrrolobenzodiazepines (PBDs) are members of a family of DNA minor-groove binding anticancer agents. Some PBD compounds have very potent in vitro cytotoxicity and in vivo antitumour activities. Topical dosage forms in some cases offer more advantages than oral or parenteral dosage forms as more localised delivery of drug is possible with comparatively lower side effects. However, there are only a few anticancer topical formulations currently available for the treatment of skin cancer. Therefore, the main objective of this project was to develop topical formulations using PBD compounds for the palliative treatment of late stage melanoma patients, and for the treatment of non-melanoma skin cancers and potentially actinic keratosis. In order to develop suitable topical formulations for PBD compounds, a number of formulations were initially evaluated using anthramycin, a model PBD compound. In addition, the permeation and retention of the penetration enhancers (solvents) in human skin were also evaluated. Single solvent analysis results showed that Transcutol® and propylene glycol (PG) were the most highly permeable solvents. For all formulations, anthramycin absorption was comparatively higher in PG:PGML (propylene glycol monolaurate) (90:10). A focussed library of PBD C8-conjugates and a PBD-dimer (SJG-136) were also evaluated using dimethyl sulphoxide as a solvent. Potential topical efficacy was confirmed for a number of the compounds based on skin flux values and in vitro potency data. KMR-04-175 and KMR-28-24 were ranked as the most topically effective PBD conjugates. SJG-136 which has reached Phase II clinical trials, also showed higher skin absorption values compared with the other PBD compounds (except for KMR-28-24). This is the first report describing the preparation and evaluation of topical formulations of PBD compounds. The results are encouraging and suggest that topical formulations of PBD compounds could be efficacious for the topical treatment of skin cancers and potentially actinic keratosis.

Cutaneous squamous cell carcinoma (cSCC), premalignant actinic keratosis (AK) and Bowen’s disease (BD) are highly prevalent, heterogeneous keratinocytic skin lesions (KSLs). Discrepancies between clinical presentations and histologic analyses of KSLs frequently lead to misdiagnoses or delayed diagnoses. Biomarkers that can accurately stratify KSLs by their malignant potential are urgently needed to support a paradigm shift in skin cancer care to personalised, precision medicine. In this thesis, a liquid chromatography tandem mass spectrometry (MS) platform was employed to conduct comprehensive proteomic profiling of formalin-fixed and paraffin embedded samples of normal skin and KSLs. Using complementary MS approaches, namely information dependent acquisition (IDA) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS), 3574 proteins were quantified overall allowing the identification of novel protein signatures for KSL subtypes. Proteomic findings were further investigated in silico using transcriptome databases, and several interesting targets were confirmed by immunohistochemistry. Distinct proteome profiles corresponding to subcategories of cSCC and precursor lesions were found, demonstrating the potential of MS-based approaches to deliver reliable diagnostics and disease staging. The bioinformatic analysis provided new insights into molecular pathways disrupted in different KSLs. The successful application of a non-invasive tape-stripping method for proteome sampling of KSLs was also demonstrated. This work represents the most comprehensive proteome study of KSLs to date. The identification of deferentially altered proteins and molecular pathways between subtypes of KSLs will inform the development of diagnostic, therapeutic and disease staging strategies. Further exploration and implementation of the approaches described herein could have a major impact on patient outcomes and reduce the cost burden of KSLs.

Se pensaba que la progresión de una queratosis actínica (AK) a carcinoma escamoso infiltrante (SCC) de piel ocurría siempre y cuando la neoplasia intra-epidérmica ocupara todo el espesor de la epidermis como ocurre en la vía clásica (CP) descrita en el cáncer de cérvix. Sin embargo, el carcinoma escamoso infiltrante cutáneo puede aparecer directamente desde una displasia que sólo ocupe el tercio inferior de la epidermis (AK I, vía diferenciada o DP). Esta segunda vía de progresión se ha descrito en el SCC de vulva y de cavidad oral , cuyo comportamiento biológico es además más agresivo. Esta tesis empezó revisando todos los casos correspondientes a biopsias quirúrgicas, obtenidas mediante el BioBanco del Hospital Germans Trias y Pujol, de tres años consecutivos correspondientes al período 2004-2007. Seleccionamos 503 casos de SCC cutáneo, de los que finalmente estudiamos 196. La mayor parte mostraron AK I en superficie (63.8%) o en los bordes (77.9%), concluyendo así que la invasión directa desde AK I (DP) es la forma más frecuente de progresión a SCC cutáneo. Este estudio fue el primero que se propuso investigar la prevalencia de la CP y DP en la transformación de la AK en SCC infiltrante aportando evidencias de su existencia. Estos hallazgos se publicaron en J Eur Acad Dermatol Venereol. 2014 Oct; 29(5):991-7.). La siguiente etapa del estudio consistió en la realización de una matriz de tejidos (TMA) de las biopsias ya estudiadas y se segregaron los casos con SCC entre los originados por DP y los originados por CP. En total se realizaron ocho TMA que supusieron 756 cores a evaluar. Mediante el uso de técnicas de inmunohistoquímica se evidenció que la transición epitelio-mesénquima participa en la transformación de AK I en SCC (DP) mientras que una capacidad proliferativa mayor facilita la extensión intra-epidérmica en la vía clásica y se hallaron diferencias significativas en cuanto a la expresión de CD31 (angiogénesis) y MMP (metaloproteinasas) hallándose estos marcadores elevados en los tumores que progresan por DP, lo que junto con la transición epitelio-mesenquima podría facilitar la progresión local. Una parte de estos resultados han sido publicados en la revista J Eur Acad Dermatol Venereol. 2018 Apr; 32(4):581-586). Tambien se procedio al uso de técnicas de CISH para el estudio de miRNA hallándose los tumores surgidos por DP expresan mayores niveles de miRNA31 tanto en su intensidad como en su extensión. En la siguiente etapa se estudió la extensión de la atipia queratinocitaria por el epitelio de los anejos estando esta presente en el 25.9% de los casos y, de ellos, la infiltración del carcinoma escamoso directamente adyacente a la basal folicular estaba presente en el 58% de los casos. En consecuencia, seria altamente recomendable indicar la profundidad de la extensión folicular en el diagnóstico histológico de biopsias incisionales, dado el riesgo de recurrencia e infiltración que ello implica, así como las derivadas terapéuticas que conlleva. Los hallazgos han sido publicados en la revista J Eur Acad Dermatol Venereol. 2018 Oct; 32(10):1657-1661). Consideramos que la serie de estudios que conforman esta tesis proporcionan conocimiento nuevo sobre las vías de progresión del SCC cutáneo y de sus lesiones precursoras. Se ha establecido que existen al menos dos vías de progresión de SCC a AK, se ha introducido el concepto de DP en la carcinogénesis cutánea, se han hallado bases moleculares que explican la progresión a través de ambas vías y se ha constatado el riesgo de la extensión folicular en la AK. Todos estos estudios han supuesto en algunos ámbitos un cambio de paradigma y tienen relevancia tanto en el diagnóstico como en el tratamiento de las AK.
It was thought that the progression of an actinic keratosis (AK) to invasive squamous cell carcinoma (SCC) occurred as long as the intra-epidermal neoplasm occupied the entire thickness of the epidermis as in the classical pathway (PC) described in cervix cancer. However, cutaneous infiltrative squamous carcinoma can appear directly from a dysplasia that only occupies the lower third of the epidermis (AK I, differentiated pathweay or DP). This second pathway of progression has been described in SCC of the vulva and oral cavity, whose biological behavior is more aggressive. This thesis began by reviewing all cases corresponding to surgical biopsies, obtained through the BioBanco of the Germans Trias and Pujol Hospital, for three consecutive years corresponding to the period 2004-2007. We selected 503 cases of cutaneous SCC, of ​​which we finally studied 196. Most showed AK I on the surface (63.8%) or on the edges (77.9%), thus concluding that the direct invasion from AK I (DP) is the most frequent form of progression to cutaneous SCC. This study was the first one that was proposed to investigate the prevalence of CP and DP in the transformation of AK into infiltrating SCC, providing evidence of its existence. These findings were published in J Eur Acad Dermatol Venereol. 2014 Oct; 29 (5): 991-7.). The next stage of the study consisted in the realization of a tissue microarrays (TMA) of the biopsies already studied and the cases with SCC were segregated between those originated by DP and those originated by CP. In total, eight TMAs were performed, which involved 756 cores to be evaluated. Through the use of immunohistochemical techniques it was demonstrated that the epithelial-mesenchymal transition participates in the transformation of AK I into SCC (DP) while a greater proliferative capacity facilitates the intra-epidermal extension in the classical pathway and significant differences were found in to the expression of CD31 (angiogenesis) and MMP (metalloproteinases), these markers being elevated in tumors that progress through DP, which together with the epithelium-mesenchymal transition could facilitate local progression. A part of these results have been published in the journal J Eur Acad Dermatol Venereol. 2018 Apr; 32 (4): 581-586). We also proceeded to the use of CISH techniques for the study of miRNA, finding that tumors arising from DP express higher levels of miRNA31 both in their intensity and in their extension. In the next stage the extension of the keratinocyte atypia among adnexal structures was studied, being present in 25.9% of the cases and, of them, the infiltration of the squamous carcinoma directly adjacent to the follicular basal was present in 58% of the cases. Consequently, it would be highly advisable to indicate the depth of follicular extension in the histological diagnosis of incisional biopsies, given the risk of recurrence and infiltration that this implies, as well as the therapeutic derivatives that it entails. The findings have been published in the journal J Eur Acad Dermatol Venereol. 2018 Oct; 32 (10): 1657-1661). We consider that the series of studies that make up this thesis provide new knowledge about the pathways of cutaneous SCC progression and its precursor lesions. It has been established that there are at least two pathways of progression from SCC to AK, the concept of PD has been introduced in cutaneous carcinogenesis, molecular bases have been found that explain the progression through both pathways and the risk of follicular extension in the AK. All these studies have led to a paradigm shift in some areas and are relevant both in the diagnosis and treatment of AKs.

Šio tyrimo tikslas buvo įvertinti aktininės keratozės klinikinius ypatumus ir fotodinaminio gydymo skirtingomis raudonosios šviesos dozėmis veiks-mingumą. Aktininė keratozė – tai ikinavikinė odos liga, pasireiškianti ilgalaikio saulės poveikio srityse, o jos diagnozė nustatoma remiantis klinikiniu vaizdu. Histologinis tyrimas atliekamas tik neaiškiais atvejais. Vis tik pasitaiko diagnozavimo klaidų ir nesuta¬pimo atvejų tarp tyrėjų dermatologų bei dermatopatologų. Šiame darbe įvertinome ir nustatėme AK klinikinės raiškos ypatumus bei jų sąsajas su histologiniais požymiais, o taip pat AK klinikinio tyrimo jautrumą ir specifiškumą, remiantis histologinio tyrimo duomenimis. Nuo 0,025 iki 20 proc. atvejų AK transfor¬muojasi į plokščiųjų ląstelių karcinomą. Aktininę keratozę rekomenduojama gydyti, nes prognozuoti, kuri AK plis iš epidermio į gilesnius odos sluoksnius ir taps plokščiųjų ląstelių karcinoma, neįmanoma. Veiksmingas bei saugus būdas gydyti AK yra vietinio poveikio foto¬dinaminis gydymas, tačiau jo trūkumas – skausmas procedūros metu. Atlikę perspektyvųjį tyrimą, įvertinome aktininės keratozės klinikinės raiškos ypatumus po fotodinaminio gydymo su 5-aminolevulino rūgštimi. Nustatėme skausmą procedūros metu įtakojančius veiksnius. Nustatėme šio gydymo dviejomis raudonosios šviesos dozėmis veiksmingumą po dvejų metų stebėjimo bei AK atkrytį prognozuojančius veiksnius. Pateikėme praktines šio AK gydymo rekomendacijas.
The aim of this study was to evaluate clinical characteristics of actinic keratosis and the efficacy of photodynamic treatment with different red light doses. The diagnosis of AK is based upon its typical clinical aspects. However, there are data stating that diagnosing single AKs clinically is not precise and that there are even interobserver disagreements between dermatologists and dermatopathologists. In this study we assessed the clinical peculiarities of AK and its correlation with histological characteristics. We assessed the sensitivity and specificity of AK clinical diagnosis based on histopathological analysis. In 0.025% to 20% cases AK transforms into squamous cell carcinoma. It is impossible to predict which AK will spread from the epithelium into the deeper layers of the skin. Therefore, treatment of actinic keratosis is recommended in all cases. Topical photodynamic treatment is safe and effective therapy for AK but pain is the limitation of the procedure. We performed a prospective within-patient study and evaluated the clinical pecularities of actinic keratosis after photodynamic treatment. We assessed the factors that influence pain during the treatment. We assessed the efficacy of photodynamic treatment with different red light doses for actinic keratosis after two-years follow-up. Factors that influence the recurrence of AK were evaluated. Follow-up data for two years was used to formulate the practical recommendations of the study.

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
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Este estudo apresenta uma análise comparativa da taxa de proliferação celular e da expressão da enzima cicloxigenase-2 na pele de cães saudáveis e acometidos por ceratose actínica e carcinoma de células escamosas cutâneo (CCE). O objetivo foi avaliar o prognóstico destes cães utilizando-se a imunorreatividade a COX-2 e o índice de proliferação celular. Foram analisados 20 amostras de pele de cães, sendo 10 casos de ceratose actínica (G1) e 10 de CCE cutâneo (G2). Os dados epidemiológicos e as características clínicas e histopatológicas foram estudados. A taxa de proliferação celular foi avaliada por meio de índice mitótico (IM) e imunomarcação para MIB-1. A expressão da enzima cicloxigenase foi pesquisada por exame imunoistoquímico. Os cães da raça Pit Bull foram os mais acometidos em G1 e G2 e a média de idade foi 4,3 (±0,8) anos e 5,6 (±1,7) anos, respectivamente. Os valores médios do índice mitótico foram 0,4; 4,6 (±3,6) e 4,9 (±2,6) em G0, G1 e G2, respectivamente. Não houve diferença estatística entre G1 e G2, mas sim em relação ao G0. Houve imunomarcação para COX-2 em G1 e G2, a média dos escores foi 8,16 (±3,51) e 8,56 (±1,03), respectivamente. Não houve imunomarcação nas amostras de G0 e não houve diferença estatística entre G1 e G2, mas sim em relação ao G0 (p>0,05). A média da porcentagem de células imunomarcadas para Ki-67 em G0, G1 e G2 foi 0,4; 15,77 (± 8,81) e 17,71 (± 12,21), respectivamente. Não houve diferença entre G1 e G2, mas sim destes em relação ao G0 (p>0,05). A ceratose actínica se comportou como o CCE cutâneo, revelando não ser uma lesão pré-neoplásica, mas sim o estágio inicial da neoplasia propriamente dita.
This research presents a comparative analysis of cell proliferation index and cyclooxigenase-2 expression in skin of healthy and with actinic keratosis and cutaneous squamous cell carcinoma (SCC) dogs. The goal was to evaluate the prognosis of these dogs by COX-2 immunorreactivity and cell proliferation index. Skin sections from 20 dogs were evaluated, which 10 were actinic keratosis (G1) and 10 cutaneous SCC (G2). Epidemiological information, clinical and histopathological aspects were evaluated. Cell proliferation index hás been stimated by mitotic index (MI) and MIB-1 staining. Cyclooxigenase-2 expression has been analyzed by immunohistochemistry study. Pit Bull dogs were the main affected in G1 and G2 and the mean age was 4,3 (±0,8) years old and 5,6 (±1,7) years old, relatively. MI mean values were 0,4; 4,6 (±3,6) and 4,9 (±2,6) in G0, G1 and G2, relatively. There was no statistical difference between G1 and G2, but it did with G0. There was positive staining for COX-2 in G1 and G2, the score mean value was 8,16 (±3,51) and 8,56 (±1,03), relatively. There was no positive staining in G0 samples and neither statistical difference between G1 and G2, but it did with G0 (p>0,05). The positive cells for Ki-67 mean percentage in G0, G1 and G2 was 0,4; 15,77 (± 8,81) and 17,71 (± 12,21), relatively. There was no difference between G1 and G2, but it did with G0 (p>0,05). Actinic keratosis behavior was similar to cutaneous SSC, demonstrating that it is not a pre-neoplastic disease, but, in fact, the initial of neoplasm.

Orientador: Mário Roberto Hatayde
Banca: Mirela Tinucci Costa
Banca: Andrigo Barboza De Nardi
Resumo: Este estudo apresenta uma análise comparativa da taxa de proliferação celular e da expressão da enzima cicloxigenase-2 na pele de cães saudáveis e acometidos por ceratose actínica e carcinoma de células escamosas cutâneo (CCE). O objetivo foi avaliar o prognóstico destes cães utilizando-se a imunorreatividade a COX-2 e o índice de proliferação celular. Foram analisados 20 amostras de pele de cães, sendo 10 casos de ceratose actínica (G1) e 10 de CCE cutâneo (G2). Os dados epidemiológicos e as características clínicas e histopatológicas foram estudados. A taxa de proliferação celular foi avaliada por meio de índice mitótico (IM) e imunomarcação para MIB-1. A expressão da enzima cicloxigenase foi pesquisada por exame imunoistoquímico. Os cães da raça Pit Bull foram os mais acometidos em G1 e G2 e a média de idade foi 4,3 (±0,8) anos e 5,6 (±1,7) anos, respectivamente. Os valores médios do índice mitótico foram 0,4; 4,6 (±3,6) e 4,9 (±2,6) em G0, G1 e G2, respectivamente. Não houve diferença estatística entre G1 e G2, mas sim em relação ao G0. Houve imunomarcação para COX-2 em G1 e G2, a média dos escores foi 8,16 (±3,51) e 8,56 (±1,03), respectivamente. Não houve imunomarcação nas amostras de G0 e não houve diferença estatística entre G1 e G2, mas sim em relação ao G0 (p>0,05). A média da porcentagem de células imunomarcadas para Ki-67 em G0, G1 e G2 foi 0,4; 15,77 (± 8,81) e 17,71 (± 12,21), respectivamente. Não houve diferença entre G1 e G2, mas sim destes em relação ao G0 (p>0,05). A ceratose actínica se comportou como o CCE cutâneo, revelando não ser uma lesão pré-neoplásica, mas sim o estágio inicial da neoplasia propriamente dita.
Abstract: This research presents a comparative analysis of cell proliferation index and cyclooxigenase-2 expression in skin of healthy and with actinic keratosis and cutaneous squamous cell carcinoma (SCC) dogs. The goal was to evaluate the prognosis of these dogs by COX-2 immunorreactivity and cell proliferation index. Skin sections from 20 dogs were evaluated, which 10 were actinic keratosis (G1) and 10 cutaneous SCC (G2). Epidemiological information, clinical and histopathological aspects were evaluated. Cell proliferation index hás been stimated by mitotic index (MI) and MIB-1 staining. Cyclooxigenase-2 expression has been analyzed by immunohistochemistry study. Pit Bull dogs were the main affected in G1 and G2 and the mean age was 4,3 (±0,8) years old and 5,6 (±1,7) years old, relatively. MI mean values were 0,4; 4,6 (±3,6) and 4,9 (±2,6) in G0, G1 and G2, relatively. There was no statistical difference between G1 and G2, but it did with G0. There was positive staining for COX-2 in G1 and G2, the score mean value was 8,16 (±3,51) and 8,56 (±1,03), relatively. There was no positive staining in G0 samples and neither statistical difference between G1 and G2, but it did with G0 (p>0,05). The positive cells for Ki-67 mean percentage in G0, G1 and G2 was 0,4; 15,77 (± 8,81) and 17,71 (± 12,21), relatively. There was no difference between G1 and G2, but it did with G0 (p>0,05). Actinic keratosis behavior was similar to cutaneous SSC, demonstrating that it is not a pre-neoplastic disease, but, in fact, the initial of neoplasm.
Mestre

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.

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.

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Ceratoses Actínicas (CA) são lesões de pele frequentes, induzidas primariamente pela luz ultravioleta e que surgem preferencialmente na superfície da pele foto exposta. Representam a manifestação inicial da proliferação de ceratinócitos atípicos intra-epiteliais com um potencial de 0,25% e 20% ao ano de evoluir para o carcinoma espinocelular (CEC) que é metastatizante. São diagnosticadas clinicamente e tratadas ambulatorialmente. Prevalência mundial, com maior distribuição em indivíduos de pele clara fototipo I e II de Fitzpatrick. Existem vários tratamentos (tópicos, cirúrgicos e sistêmicos), sendo a prevenção a modalidade mais importante. O ácido glicólico, primeiro de uma série de alfa hidroxiácidos, quando aplicado topicamente, em baixas concentrações (2% a 12%) nos veículos, cremes, géis ou loções não iônicas, age como epidermolítico e ceratolítico. Isto o torna um excelente ativo no tratamento que propomos. Nosso objetivo foi pesquisar a efetividade ou não de uma nova conduta terapêutica para CA. Avaliar a adesão do paciente ao tratamento. Observar a apresentação de efeitos adversos indesejáveis. O Estudo foi Clínico, Prospectivo, Transversal em pacientes atendidos no Ambulatório de Dermatologia Geriátrica do HU/CAS da UFJF, total de 50 indivíduos. Todos portadores de ceratoses actínicas na face. O resultado mostra que a resolução total das CA não foi observada em todos os pacientes, mas houve adesão completa ao medicamento, não sendo observados efeitos adversos na população em estudo. Concluímos que mesmo não sendo completamente eficaz para o desaparecimento das lesões levou a uma diminuição acentuada do número de lesões e do índice de gravidade das mesmas.
Actinic keratoses (AK) are common skin lesions, primarily induced by ultraviolet light, occurring mainly on the photoexposed skin surface. They represent the initial manifestation of proliferation of atypical intraepithelial keratinocytes with a potential of 0.25% - 20% per year progress to squamous cell carcinoma (SCC), which is metastasizing. They are clinically diagnosed and treated on an outpatient basis. They are prevalent worldwide, with higher distribution in light-skinned individuals of Fitzpatrick phototype I and II. There are several treatments (topical, surgical and systemic), prevention being the most important modality. Glycolic acid, the first of a series of alpha hydroxyacids, when applied topically in low concentrations (2% to 12%) in cream, gels or nonionic lotions, acts as an epidermolytic and keratolytic. This makes it an excellent asset in the treatment that we propose. Our objective was to investigate the effectiveness or otherwise of a new therapeutic approach to AK, assess patient adherence to treatment and observe the appearance of adverse reactions. The study was clinical, prospective, transversal in patients seen at the Geriatric Dermatology Clinic of HU / CAS UFJF, 50 individuals altogether. All patients had actinic keratoses on their face. The result show that total resolution of AK was not observed in all patients, but there was complete adherence to medication, adverse effects not being observed in the study population. We conclude that, although not completely effective for the disappearance of the lesions, it led to a marked decrease in the number of lesions and the severity index of these.

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INTRODUÇÃO: Rejuvenescimento fotodinâmico refere-se ao tratamento do fotoenvelhecimento e das queratoses actínicas. OBJETIVO: Avaliar a eficácia da terapia fotodinâmica com ácido aminolevulínico a 20% (ALA) ¾ tópico e luz intensa pulsada no tratamento das queratoses actínicas e do fotoenvelhecimento facial. MÉTODOS: Quarenta e três pacientes com idade média 71,3 anos, fototipos de pele I e IV divididos em: Controle (aplicação LIP/18J); Tratamento-I (ALA incubado por duas horas; ativado por duas passagens de LIP/16J); Tratamento-II (ALA; duas passagens de LIP/18J), foram avaliados por dois observadores, clínica e fotograficamente, no Pré, 48 horas, 8 e 12 semanas após, pela escala de Griffiths. Cinco queratoses actínicas foram marcadas como alvo para contagem no Pós- Tratamento, sendo uma submetida à biopsia antes e depois do tratamento, para avaliação anatomopatológica de atipias e perda de polaridade. RESULTADOS: Testes não-paramétricos mostraram melhora significativa (rugas finas, alterações texturais, pigmentação irregular) nos Grupos-Tratamento-I/II. No Grupo Controle, significância apenas para alterações pigmentares. Queratoses actínicas tiveram redução maior nos Grupos II (68, 4%) e I (51,2%) em relação ao Controle (5%). Houve redução significativa da gravidade das atipias e perda de polaridade nos Grupos-TratamentoI/II em relação ao controle. CONCLUSÃO: A terapia fotodinâmica, com ácido 5aminolevulínico e luz intensa pulsada, mostrou-se efetiva no tratamento do fotoenvelhecimento e das queratoses actínicas faciais. O tratamento das queratoses actínicas foi mais efetivo com uso de maior energia.
BACKGROUND: Photodynamic rejuvenation is the term used to the treatment of actinic keratosis and photodamage with photodynamic therapy. OBJECTIVE: Evaluate the efficacy of photodynamic therapy with aminolevulinic acid and intense pulsed light in the treatment of actinic keratosis and photodamage. METHODS: Fourty three patients mean age 71,3 years, were randomized in three groups: Control (IPL 18J); Treatment I (ALA+ IPL 16J); and Treatment II ( ALA+ IPL 18J) and evaluated clinically an photographically by two observers in Pre, 48hs, 8 weeks and 12 weeks Post-Treatment using Griffth’s Photonumeric Scale. Five actinic keratosis were marked as target in Pre-Treatment and biopsies Pre and Post-Treatment were done in one lesion to evaluate atypias and loss of cellular polarity. RESULTS: Non parametric tests showed marked improvement in mottled pigmentation, texture fine wrinkles in Groups Treatment I and II. In Control Group improvement only in mottled pigmentation. Actinic Keratosis clearance in Groups Treatment I and II were 51,2% and 68,4% respectively, whereas in Group Control it was 5%. In relation to Control Group, Groups Treatment I and II showed significant reduction of atypias and improvement in loss of cellular polarity. CONCLUSIONS: Photodynamic therapy with aminolevulinic acid and intense pulsed light is effective in the treatment of photodamage and actinic keratosis. The resolution of actinic keratosis was better with higher levels of energy.
TEDE
BV UNIFESP: Teses e dissertações

Orientador: Hélio Amante Miot
Resumo: O tratamento do campo de cancerização tem o intuito de evitar a progressão das lesões pré-malignas ou desenvolvimento de lesões subclínicas, como forma de prevenção da carcinogênese. Existem diversas propostas de tratamento, porém, há poucos estudos com nicotinamida oral, nenhum comparando a eficácia entre 5-fluorouracil (5FU) tópico com fotoproteção, ou na sua associação com a nicotinamida oral. Objetivos: Avaliação da eficácia da nicotinamida oral, versus 5FU tópico intermitente, e fotoproteção, no tratamento do campo de cancerização cutâneo e queratoses actínicas (QA). Casuística e métodos: Desenho: ensaio clínico randomizado (em blocos), controlado (intrasujeito), duplo cego, fatorial. Participantes: pacientes imunocompetentes, que continham entre três e dez QA cada antebraço, selecionados durante os atendimentos do ambulatório oncológico do serviço de dermatologia da Faculdade de Medicina de Botucatu – UNESP no período de março a setembro de 2016. Houve randomização em blocos e alocação em grupos dos pacientes e antebraços. Intervenção: um grupo recebeu nicotinamida 500mg, via oral, cada 12h, e o outro, placebo. Um dos antebraços recebeu 5FU 5% creme, noturno, 3x por semana, e ambos, filtro solar (FPS 30) diurno. A duração dos tratamentos foi de 120 dias. Os pacientes foram avaliados nos momentos: T0 e T120 para avaliação clínica (contagem de QA, escore de gravidade de QA e escore de fotoenvelhecimento) e biópsia de pele para avaliação do grau de neoplasia intraepitelial... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Background: The treatment of the skin field cancerization is aimed at preventing the progression of premalignant lesions or the development of subclinical lesions as a way of preventing carcinogenesis. There are several treatment proposals, however, there are few studies with oral nicotinamide, none comparing the efficacy of topical 5-fluorouracil (5FU) with photoprotection, or its association with oral nicotinamide. Objectives: To evaluate the efficacy of oral nicotinamide versus intermittent topical 5FU and photoprotection in the treatment of cutaneous cancer and actinic keratoses (AK). Casuistry and methods: Design: randomized (blocks), controlled (intrasubject), double blind, factorial clinical trial. Participants: immunocompetent patients, who had between 3 and 10 AK each forearm, selected during the visits of the oncology outpatient clinic of the dermatology department of Botucatu Medical School - UNESP from March to September, 2016. There was randomization in blocks and allocation in Groups of patients and forearms. Intervention: one group received nicotinamide 500mg, orally every 12h, and the other, placebo. One of the forearms received 5FU 5% cream, overnight, 3x per week, and both, daytime (SPF 30) sunscreen. The duration of the treatments was 120 days. The patients were evaluated at the moments: T0 and T120 for clinical evaluation (AK score, AK severity score and photoaging score) and skin biopsy for evaluation of the levels of keratinocyte intraepithelial neoplasi... (Complete abstract click electronic access below)
Mestre

Orientador: Juliano Vilaverde Schmitt
Resumo: FUNDAMENTOS: As queratoses actínicas são lesões pré-malignas com risco de transformação para carcinoma espinocelular invasivo. OBJETIVOS: Correlacionar as características clínicas das queratoses actínicas dos antebraços e dorso das mãos com o grau de atipia do exame histológico (Keratinocyte Intraepidermal Neoplasia) e a expressão lesional e perilesional de p53 e Ki-67; desenvolver e validar uma escala de gravidade clínica que possa inferir o grau histológico das queratoses actínicas. MÉTODOS: Estudo transversal em que foram avaliadas clinicamente 162 queratoses actínicas quanto ao diâmetro, eritema, infiltração, hiperqueratose e exulceração e biopsiadas 34 lesões com diferentes padrões. As características clínicas foram correlacionadas com o grau de atipia histológica e expressão de p53 e Ki-67. RESULTADOS: As características clínicas apresentaram baixa consistência entre si (Cronbach=0,24). Apenas o diâmetro das lesões se correlacionou significativamente com o grau de atipia (p=0,04), e apenas o eritema, a hiperqueratose e o diâmetro se correlacionaram com as marcações imuno-histoquímicas. Foi desenvolvido um escore clínico incluindo o diâmetro, a hiperqueratose e a exulceração, o qual se correlacionou significativamente com o grau de atipia (Rho de Spearman=0,43; p=0,01). LIMITAÇÕES DO ESTUDO: O escore obtido não pode ser extrapolado para áreas que não sejam os antebraços e dorso das mãos; não houve seguimento prospectivo dos pacientes; não incluímos pacientes com genoderm... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: BACKGROUND: Actinic keratosis are pre-malignant lesions with risk of transformation for invasive squamous cell carcinoma. OBJECTIVES: To correlate the clinical features of actinic keratosis of the forearms and dorsum of the hands with its histological features (Keratinocyte Intraepidermal Neoplasia) and p53 and Ki-67 staining in the lesion and around the lesion; develop and validate a clinical severity scale that can infer the histological grade of actinic keratosis. METHODS: A cross-sectional study in which 162 actinic keratosis were clinically evalueted for diameter, erythema, infiltration, hyperkeratosis and exulceration, and 34 lesions with different patterns were biopsied. Clinical features were associated with degrees of atypia and p53 and Ki-67 staining. RESULTS: Clinical features were low in consistency (Cronbach=0.24). Only the diameter of the lesions was significantly correlated with the degree of atypia (p=0.04), and only erythema, hyperkeratosis and diameter were correlated with immunohistochemical staining. A clinical score including the diameter, hyperkeratosis and exulceration was developed, with significant correlation with the degree of atypia (Spearman's Rho=0.43; p=0.01). LIMITATIONS: The developed score can not be extrapolated to areas other than the forearms and back of the hands; there was no prospective follow-up of the patients; we did not include patients with genodermatosis or immunosuppressed patients. CONCLUSION: Clinical features of actinic kerato... (Complete abstract click electronic access below)
Mestre

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Fundamentos: Campo de cancerização cutâneo representa uma área com alterações genômicas induzidas pela radiação ultravioleta, cujo sinal de atividade são as queratoses actínicas (QA). Tratamentos que visem sua estabilização podem reduzir a incidência de QA e de tumores cutâneos não melanoma. Estudos em terapia fotodinâmica com metil aminolevulinato (TFD-MAL) no tratamento do campo de cancerização cutâneo mostram redução de até 89% na contagem de QA, já com colchicina tópica, há redução de até 78%. Até o momento, não há estudos comparando colchicina com TFD-MAL. Esse trabalho objetiva avaliar eficácia e segurança de colchicina 0,5% creme versus TFD-MAL no tratamento do campo de cancerização dos antebraços. Casuística e métodos: Ensaio clínico aberto, controlado, randomizado, envolvendo 36 pacientes do ambulatório de Dermatologia da UNESP-Botucatu, com 3-10 QAs em cada antebraço, tratados (cada antebraço) com creme de colchicina 0,5% (2x/dia por 10 dias) ou uma sessão de TFD-MAL; reavaliados após 60 dias. A avaliação clínica foi realizada pela contagem de QAs, seus subtipos clínicos, sua escala de gravidade e escala de fotoenvelhecimento dos antebraços. Avaliação histopatológica foi realizada pelo escore KIN (Keratinocyte Intraepithelial Neoplasia), atrofia epitelial e imunohistoquímica de p53 e Ki67. Todos os pacientes incluídos no estudo e randomizados fizeram parte da população ITT (intention to treat). Os dados do único dropout foram imputados como LOCF (last observed carried forward). Resultados: Foram incluídos 36 participantes: 50% eram do sexo masculino, com idade média (dp) de 70,9 (8,6) anos e predominância de pacientes fototipos I e II (89%). O clearance total de QAs ocorreu em 6 (17%) de COL e 7 (19%) de MAL-PDT (p=0,76), e clearance parcial (> 50%) foi alcançado em 16 (44%) dos antebraços COL e 24 (67%) MAL-PDT (p=0,07). Houve redução significativa no escore da escala de fotoenvelhecimento para COL e TFD-MAL (-6% vs -6%), escore de gravidade de QA (-65% vs -73%), subtipos clínicos das QAs presentes e contagem de QA (-45% vs -40%), após as intervenções (p<0,01), sem diferenças significativas entre os grupos. Houve redução no escore KIN em ambos os grupos (p<0,01), normalizado em 28% dos pacientes MAL-PDT e 20% em COL. Houve redução da atrofia epitelial após 60 dias (p<0,01), sem diferenças entre os tratamentos (p=0,58). Houve redução significativa do escore Ki67 em ambos os tratamentos: 15% em COL e 9% em MAL-PDT, sem diferença entre os grupos (p=0,32). Houve redução do escore de p53 em 27% no grupo COL, porém, um aumento de 4% na MAL-PDT (p<0,05). Efeitos adversos foram relatados em ambos os grupos: 69% em COL e 67% em TFD-MAL. Não houve evento adverso grave. Conclusão: Colchicina 0,5% creme e TFD-MAL são eficazes e seguros no tratamento do campo de cancerização cutâneo.
Background: Skin field cancerization is an area with genomic alterations induced by ultraviolet radiation, and actinic keratosis (AK) is a sign of its activity. Treatments to stabilize it may reduce the incidence of non-melanoma skin cancers and AK. Photodynamic therapy with methyl aminolevulinate (TFD-MAL) in the treatment of skin field cancerization shows a reduction of up to 89% in the AK count, and topical colchicine, up to 78% of reduction. To date, there are no studies comparing colchicine with PDT-MAL. The aim of this study was to evaluate the efficacy and safety of colchicine 0.5% cream versus PDT-MAL in skin field cancerization treatment of the forearms. Patients and methods: A randomized, controlled clinical trial involving 36 patients from the UNESP-Botucatu Dermatology outpatient clinic, with three to 10 AKs in each forearm, treated (each forearm) with colchicine cream (2x / day for 10 days) and one session of PDT-MAL. Reassessed after 60 days. Clinical evaluation was performed by AK count, its clinical subtypes, severity scale and forearm photoaging scale. Histopathological evaluation performed by KIN (Keratinocyte Intraepithelial Neoplasia), epithelial atrophy and immunohistochemistry of p53 and Ki67. All patients included in the study and randomized were part of the ITT population (intention to treat). The only dropout was analyzed through LOCF (last observation carried forward). Results: Thirty-six participants were included: 50% were males, mean age (SD) of 70.9 (8.6) years and predominance of phototype I and II patients (89%). Total clearance of AK occurred in 6 (17%) of COL and 7 (19%) of MAL-PDT (p = 0.76), and partial clearance (> 50%) was achieved in 16 (44%) of COL and 24 (67%) of MAL-PDT (p = 0.07). There was a significant reduction in the photodamage scale for COL and PDT-MAL (-6% vs -6%), AK severity score (-65% vs -73%), clinical subtypes of the present AK and AK (-45% vs -40%) after interventions (p <0.01), with no significant differences between the groups (p> 0.24). There was a reduction in KIN score in both groups (p <0.01), normalized in 28% of MAL-PDT patients and 20% in COL. There was reduction of epithelial atrophy after 60 days (p <0.01), with no differences between treatments (p = 0.58). There was a significant reduction of the Ki67 score in both treatments: 15% in COL and 9% in MAL-PDT, with no difference between groups (p = 0.32). There was a reduction of the p53 score in 27% in the COL group, but a 4% increase in MAL-PDT (p <0.05). Adverse effects were reported in both groups: 69% in COL and 67% in PDT-MAL. There was no serious adverse event. Conclusion: Colchicine 0.5% cream and TFD-MAL are effective and safe in skin field cancerization treatment.

Orientador: Hélio Amante Miot
Resumo: Fundamentos: Campo de cancerização cutâneo representa uma área com alterações genômicas induzidas pela radiação ultravioleta, cujo sinal de atividade são as queratoses actínicas (QA). Tratamentos que visem sua estabilização podem reduzir a incidência de QA e de tumores cutâneos não melanoma. Estudos em terapia fotodinâmica com metil aminolevulinato (TFD-MAL) no tratamento do campo de cancerização cutâneo mostram redução de até 89% na contagem de QA, já com colchicina tópica, há redução de até 78%. Até o momento, não há estudos comparando colchicina com TFD-MAL. Esse trabalho objetiva avaliar eficácia e segurança de colchicina 0,5% creme versus TFD-MAL no tratamento do campo de cancerização dos antebraços.Casuística e métodos: Ensaio clínico aberto, controlado, randomizado, envolvendo 36 pacientes do ambulatório de Dermatologia da UNESP-Botucatu, com 3-10 QAs em cada antebraço, tratados (cada antebraço) com creme de colchicina 0,5% (2x/dia por 10 dias) ou uma sessão de TFD-MAL; reavaliados após 60 dias. A avaliação clínica foi realizada pela contagem de QAs, seus subtipos clínicos, sua escala de gravidade e escala de fotoenvelhecimento dos antebraços. Avaliação histopatológica foi realizada pelo escore KIN (Keratinocyte Intraepithelial Neoplasia), atrofia epitelial e imunohistoquímica de p53 e Ki67. Todos os pacientes incluídos no estudo e randomizados fizeram parte da população ITT (intention to treat). Os dados do único dropout foram imputados como LOCF (last observed car... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

O conceito de campo de cancerização, em dermatologia, sugere que a pele fotodanificada tem maior potencial para o desenvolvimento de neoplasias cutâneas. A terapia fotodinâmica (TFD) é um método não invasivo para o tratamento de queratoses actínicas (QA) múltiplas, possibilitando a abordagem de todo o campo. Vinte e seis pacientes com múltiplas QAs na face foram submetidos a três sessões de TFD com metilaminolevulinato 16% (MAL) e luz vermelha, com intervalo de um mês. Biópsias foram realizadas antes e após três meses da última sessão e o material corado para hematoxilina-eosina e Weigert. O estudo imunohistoquímico foi feito para os marcadores: TP-53, pró-colageno I, Metaloproteinase-1 e Tenascina-C. A avaliação do fotoenvelhecimento global melhorou consideravelmente (p < 0,001) e a cura clínica das QAs foi de 89,5% ao final do estudo. Duas sessões mostraram ser equivalentes a três sessões de TFD. Diminuição significante do grau e extensão da atipia celular (p < 0,001), aumento das fibras colágenas (p = 0,001) e melhora do grau de elastose (p = 0,002) foram observadas. O estudo imunohistoquímico mostrou diminuição da expressão da TP-53 (p = 0,580), aumento de pró-colágeno I (p = 0,477) e de MMP-1 (p = 0,08), embora não houvesse diferença estatisticamente significante. Aumento significativo foi observado para Tenascina-C (p = 0,024). Múltiplas sessões de TFD com MAL induziram melhora clínica e histológica do campo de cancerização. A diminuição da severidade e extensão da atipia celular associada à menor expressão de TP-53 sugerem redução do potencial carcinogênico do campo
The field cancerization concept suggests that photodamaged skin has an increased risk for the development of malignant lesions. Topical photodynamic therapy (PDT) is a non-invasive therapeutic method for multiple actinic keratosis (AK), allowing the possibility of treating the entire surface. Twenty-six patients with photodamaged skin and multiple AKs on the face were submitted to three consecutive sessions of PDT with mehtylaminolevulinate 16% (MAL) and red light, one month apart. Biopsies were performed before and three months after the last treatment session, and stained for hematoxilin-eosin and Weigert. Immunohistochemestry study was performed for TP-53, pro-collagen I, Metalloproteinase-1 and Tenascin-C. The global score for photodamage improved considerably in all patients (p < 0.001). The AK clearance rate was 89.5% at the end of the study. Two treatments were similar to three MAL-PDT sessions. A significant decrease in keratinocytes atypia grade and amount was observed (p < 0.001). A significant increase in collagen deposition (p = 0.001) and improvement of solar elastosis (p = 0.002) were noticed. Immunohistochemical study showed decreased TP-53 expression although not statistically significant (p = 0.580), increased pro-collagen I and MMP-1 expressions (p = 0.477 and p = 0.08) again not statistically significant and a increased expression of Tenascin-C (p = 0.024), which was statistically significant. In conclusion, multiple sessions of MAL-PDT induced clinical and histological improvement of field cancerization. The decrease in severity and extension of keratinocytes atypia associated with a decreased expression of TP-53 suggest a reduced carcinogenic potential of the altered field

Für die Entstehung nicht-melanozytärer Hauttumore sind mehrere Risikofaktoren verantwortlich: UV-Exposition, Pigmentierung, Alter, Immunsuppression und möglicherweise Humane Papillomviren (HPV). Die molekularen Mechanismen der Tumorgenese des kutanen Plattenepithelkarzinoms (SCC) sowie der Präkanzerose Aktinische Keratose (AK) sind nur lückenhaft bekannt. Fokus dieser Arbeit ist die Untersuchung von SCC-Genexpressionsprofilen sowie der Einfluss kutaner HPV-Typen während der Karzinogenese bei immunkompetenten und immunsupprimierten, organtransplantierten Patienten. Durch Genexpressionsanalyse kutaner SCC, AK und normaler Haut konnten 118 differenziell exprimierte Gene in SCC mittels cDNA-Microarrays identifiziert werden. Bestätigt wurde die Expression von 11 aus 13 ausgewählten Genen (85%) mittels quantitativer real-time RT-PCR (qPCR), dabei konnte eine Korrelation der Genexpression mit der Progression der AK zum SCC für 3 Gene nachgewiesen werden. Dazu zählen das Gen Metalloproteinase-1, kodierend für ein Enzym, das in den Umbau von extrazellulärer Matrix involviert ist, das Protoonkogen RAB31 und das Tenascin-C (Tn-C) kodierende Gen Tn-C. Tn-C war im SCC-Gewebe an der Invasionsfront in Basalzellen sowie Keratinozyten im Stratum papillare und retikulare als Protein nachweisbar, nicht aber in normaler Haut. Die im Rahmen dieser Arbeit erstmalig nachgewiesene 2243 bp-Spleißvariante von Tn-C könnte aufgrund der primären Expression in SCC–Gewebe als diagnostischer Marker für SCC dienen. Diese Daten zeigen, dass simultane, multifaktorielle Dysregulationen von Genexpression und DNA-Reparatur, Zellzyklus und Proliferation, proteolytischen Enzymen und Adhäsionsmolekülen in SCC vorliegen. Ferner wurde die Expression von HPV in SCC und damit der kausale Zusammenhang einer HPV-Infektion mit der Hauttumorgenese untersucht. Das Infektionsmuster von SCC-Gewebe und normaler Haut mit spezifischen HPV-Typen erfolgte durch den Nachweis typenspezifischer HPV-DNA. Virale E6/E7-mRNA-Transkripte der kutanen HPV-Typen 8, 9 und 15 wurden in AK und SCC nachgewiesen. Dagegen konnten in HPV-DNA positiver, gesunder Haut oder Warzen keine HPV-Transkripte gefunden werden. Die Variantenanalyse des offenen Leserahmens von E6 identifizierte eine einzelne, bislang nicht beschriebene Punktmutation mit nicht bekannter Veränderung der Proteinstruktur. Die virale Aktivität der Onkogene E6 und E7 einiger kutaner Typen in AK und SCC weisen auf eine mögliche Rolle von HPV bei der kutanen Hautkarzinogenese hin.
During development of non-melanoma skin cancer, several risk factors are involved: UV-exposition, pigmentation, age, and potentially human papilloma virus (HPV). The molecular mechanisms underlying tumourgenesis in squamous cell carcinoma (SCC) and its pre-cancerosis actinic keratosis (AK) are not fully understood. In this study, the gene expression profile and HPV-infection status were analysed in SCC from immunocompetent and organ transplanted, immunocompromised patients.By global transcriptome analysis from cutaneous SCC, AK and healthy skin, 118 genes were identified differentially expressed in a cDNA-microarray. The expression of 11 out of 13 selected genes (85%) was investigated by real-time RT-PCR (qPCR) and the expression of three genes remarkably induced in SCC correlated with the progression to AK until SCC. These genes encoded for Metalloproteinase-1, which is involved in the remodelling of extracellular matrix, and the protooncogene RAB31 and Tenascin-C (Tn-C). Tn-C protein is expressed in SCC-tissue at the invasion front in basal cells and in keratinocytes in the Stratum papillare and retikulare, but not in healthy skin. This study, the 2243 bp Tn-C-specific splice-variant has for the first time detected in SCC, but not in normal skin. Thus it might serve as diagnostic marker of SCC progression. The data of the transcriptome analysis indicates that a simultaneous dysregulation of oncogene expression and DNA-repair, cell-cycle and proliferation, proteolysis and adhesion molecules exists in SCC. Additionally, the expression of HPV in SCC and thus the causal relationship between HPV-infection and tumourgenesis of SCC in immunocompromised patients was investigated. The HPV-infection pattern in SCC-tissue and normal skin was assessed by detection of DNA from cutaneous HPV-types. Viral E6/E7-mRNA-transcripts of the cutaneous HPV-types 8, 9, 15 were expressed selectively in AK and SCC. In contrast, no HPV-specific mRNA was present in HPV-DNA positive normal skin. The analysis of the open reading frame from the respective E6-protein genes unravelled one single pointmutation, which is not been characterized so far in terms of e.g. its impact on protein structure. The viral activity of the oncogenes E6 and E7 of cutaneous HPV-types indicates a potential function of HPV in the tumourgenesis of SCC in immunocompromised individuals.

Contexte : La thérapie photodynamique est un traitement efficace des kératoses actiniques, réparties en champs de cancérisation. Parmi les protocoles approuvés en Europe, le plus largement utilisé nécessite une illumination par lampe à LED. Cependant, la douleur pendant l’illumination et la reproductibilité du traitement sont deux facteurs limitants. Pour améliorer ce traitement, un textile lumineux a été développé. Nous avons évalué ce dispositif avec deux schémas d’illumination : Flexitheraligth à une irradiance de 12,3 mW/cm2 pour obtenir une fluence de 37 J/cm2 et Pho-Istos à une irradiance de 1,3 mW/cm2 pour obtenir une fluence 12J/cm2.Objectifs : Évaluer la non-infériorité, en termes d'efficacité du traitement photodynamique des kératoses actiniques par les dispositifs Flexitheralight et Phos-Istos par rapport au protocole conventionnel utilisant une lampe à LED rouge.Patients, matériels et méthodes : 2 études cliniques de phase II randomisées et contrôlées ont été menées. Nous avons inclus des patients atteints de kératoses actiniques de grade I-II sur le front et le cuir chevelu, traités par thérapie photodynamique au méthylaminolévulinate dans deux zones symétriques. Une zone a été traitée avec le protocole conventionnel, tandis que l'autre zone a été traitée avec le protocole Flexitheralight ou Phos-Istos selon l’étude. Le critère d'évaluation principal était le taux de réponse complète des lésions à trois mois (borne de non-infériorité de -10%). Les critères d'évaluation secondaires comprenaient la douleur signalée par le patient à la fin de l'irradiation évaluée par une échelle visuelle analogique.Résultats : L'étude Flexitheralight a inclus 25 patients traités avec le dispositif Flexitheralight d’un côté (n = 154 kératoses actiniques) et avec la lampe LED sur l’autre côté (n = 156 kératoses actiniques). Le taux de réponse complète avec le protocole Flexitheralight n'était pas inférieur à celui obtenu avec la lampe LED (respectivement 66,0% contre 59,1% ; différence absolue de 6,9% ; intervalle de confiance de 95% de -0,6% à 14,5%). La douleur était significativement moins importante avec le protocole Flexitheralight qu'avec la lampe à LED (moyenne ± écart-type: 0,4 ± 0,6 vs 5,0 ± 2,6; p <0,0001).L'étude Phos-Istos a concerné quarante-six patients traités avec le dispositif Phos-Istos sur une zone (n = 280 lésions) et avec la lampe LED sur la zone controlatérale (n = 280 lésions). Le taux de réponse complète à 3 mois du dispositif Phos-Istos n'était pas inférieur à celui de la lampe LED (79,3% vs 80,7%, respectivement ; différence absolue, -1,6%; intervalle de confiance unilatéral à 95%, - 4,5% à l'infini). Le score de douleur à la fin de l'illumination était significativement plus bas pour le dispositif Phos-Istos que pour la lampe LED (moyenne ± écart type: 0,3 ± 0,6 vs 7,4 ± 2,3; p <0,0001).Conclusions : Les dispositifs d’illumination textiles et les protocoles à faible irradiance ne sont donc pas inférieurs en termes d'efficacité au protocole conventionnel de traitement des kératoses actiniques du front et du cuir chevelu. La tolérance est cependant significativement améliorée
Background: Photodynamic therapy is an effective treatment for actinic keratosis, for patients with large areas of field cancerization. Among the approved protocols in Europe, the most widely used requires illumination with a LED lamp. However, pain during irradiation and the reproductibility are two limiting factors of this protocol. To overcome these limits, a light-emitting fabric-based device was developed. We evaluated these devices and two illumination schemes: Flexitheraligth at a fluence rate of 12.3 mW/cm2 to get a fluence of 37J/cm2 and Pho-Istos at a fluence rate of 1.3 mW/cm2 to get a fluence of 12J/cm2.Objectives: We aim to assess the non-inferiority, in terms of photodynamic therapy efficacy to treat actinic keratosis, of the Flexitheralight and Phos-Istos devices compared with the conventional protocol using the red LED lamp.Methods: Randomized, controlled, phase II clinical studies were performed. We included patients with grade I-II actinic keratosis of the forehead and scalp, treated with methyl aminolevulinate photodynamic therapy in two symmetrical areas. One area was treated with the conventional protocol, whereas the other area was treated with the Flexitheralight or the Phos-Istos protocol, depending on the study. The primary endpoint was the lesion complete response rate at three months (an absolute non-inferiority margin of -10% was used). The secondary endpoints included patient-reported pain at the end of the irradiation.Results: The Flexitheralight study included twenty-five patients treated with the Flexitheralight device on one area (n=154 actinic keratosis) and with the LED lamp on the contralateral area (n=156 actinic keratosis). The lesion complete response rate with the Flexitheralight protocol was non-inferior to that obtained with the LED lamp (66.0% vs. 59.1%, respectively; absolute difference, 6.9%; 95% confidence interval, -0.6% to 14.5%). Patient-reported pain was significantly lower with the Flexitheralight protocol than with the LED lamp (mean ± standard deviation: 0.4 ± 0.6 vs. 5.0 ± 2.6; p<0.0001).The Phos-Istos study included forty-six patients treated with the Phos-Istos device on one area (n=280 lesions) and with the LED lamp on the contralateral area (n=280 lesions). Three months following treatment, the lesion complete response rate of the Phos-Istos device was non-inferior to the LED lamp (79.3% vs. 80.7%, respectively; absolute difference, -1.6%; one-sided 95% confidence interval, -4.5% to infinity). The pain score at the end of illumination was significantly lower for the Phos-Istos device than for the LED lamp (mean±standard deviation: 0.3±0.6 vs. 7.4±2.3; p<0.0001).Conclusions: The light emitting fabrics and the low-irradiance protocols are non-inferior in terms of efficacy and superior in terms of tolerability to the conventional protocol for treating actinic keratoses of the forehead and scalp

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Fundação Amaral Carvalho
O câncer de pele é o tipo mais freqüente na população mundial. A sua lesão precursora é a queratose actínica (QA). Um dos grandes problemas associados com a QA é o fato de que as áreas atingidas podem ser extensas, tornando o tratamento demorado, ou no caso de alguns procedimentos químicos, demasiadamente agressivos ao paciente. A terapia fotodinâmica (TFD) tópica surge como uma opção no tratamento das QAs disseminadas. O objetivo do presente estudo foi avaliar in vivo o tempo ideal de oclusão do agente fotossensibilizador ácido 5-aminolevulínico (ALA) a 15% para tratamento das QA disseminadas em membros superiores pelo protótipo Kerato PDT. Foram selecionados 40 pacientes que apresentassem QAs disseminadas nos membros superiores. Estes foram submetidos à aplicação do ALA a 15% nos antebraços e mãos e em seguida a oclusão dos membros foi realizada. A partir disso, os pacientes foram divididos em dois grupos de acordo com tempo de oclusão, no qual os pacientes do grupo 1 (G1) permaneceram por 2 horas em oclusão e no grupo 2 (G2), por 4 horas. Na sessão seguinte, foi retirada a cobertura e realizada a irradiação da área por 40 minutos (40 J/cm2) pelo protótipo Kerato PDT a base de emissores LED. Foi realizada análise visual médica nos períodos de 30 e 90 dias seguindo-se escores de 0 (ausência de melhora) a 10 (melhora total das lesões). Os valores obtidos foram submetidos à análise estatística pelo Teste de Mann-Whitney com p<0,10. Os resultados mostraram que não houve diferença estatisticamente significativa entre os grupos. Pode-se concluir que tanto o tempo de 2 ou 4 horas de oclusão são eficientes para o tratamento das QAs disseminadas; porém o menor tempo permitiu melhor conforto para os pacientes
Skin cancer is the most incident neoplasm in world population. Actinic keratoses (AK) is considered a premalignant lesion. The AKs are often multiple, what can difficult the treatment. Photodynamic therapy (PDT) is an option for the treatment of widespread AK of the limbs. The aim of the present in vivo study was to evaluate the perfect time of occlusion of 5-aminolevulinic acid (ALA) 15% photosensitizing agent to treatment of widespread AKs by Kerato PDT. 40 patients with multiple AK on the upper limbs were selected. They were subjected to the following treatment: the ALA 15% was applied over the entire area (forearm and hand) and then the occlusion of the limbs was performed. After that, patients were divided into two groups according to duration of occlusion, in which patients in group 1 (G1) remained for 2 hours under occlusion and in group 2 (G2), for 4 hours. The next session the patients had their lesions submitted for 40 minutes (40 J/cm2) by prototype-based LED Kerato PDT. Medical visual analysis were performed in periods of 30 and 90 days througth scores from 0 (no improvement) to 10 (total improvement of the lesions). The values were statistically analyzed by Mann-Whitney test with p <0.10. The results showed no statistically significant difference between groups. It can be concluded that both times of occlusion are effective in the treatment of AK disseminated, but the minimum time allowed better comfort for patients

Resumo: O câncer de pele é o tipo mais freqüente na população mundial. A sua lesão precursora é a queratose actínica (QA). Um dos grandes problemas associados com a QA é o fato de que as áreas atingidas podem ser extensas, tornando o tratamento demorado, ou no caso de alguns procedimentos químicos, demasiadamente agressivos ao paciente. A terapia fotodinâmica (TFD) tópica surge como uma opção no tratamento das QAs disseminadas. O objetivo do presente estudo foi avaliar in vivo o tempo ideal de oclusão do agente fotossensibilizador ácido 5-aminolevulínico (ALA) a 15% para tratamento das QA disseminadas em membros superiores pelo protótipo Kerato PDT. Foram selecionados 40 pacientes que apresentassem QAs disseminadas nos membros superiores. Estes foram submetidos à aplicação do ALA a 15% nos antebraços e mãos e em seguida a oclusão dos membros foi realizada. A partir disso, os pacientes foram divididos em dois grupos de acordo com tempo de oclusão, no qual os pacientes do grupo 1 (G1) permaneceram por 2 horas em oclusão e no grupo 2 (G2), por 4 horas. Na sessão seguinte, foi retirada a cobertura e realizada a irradiação da área por 40 minutos (40 J/cm2) pelo protótipo Kerato PDT a base de emissores LED. Foi realizada análise visual médica nos períodos de 30 e 90 dias seguindo-se escores de 0 (ausência de melhora) a 10 (melhora total das lesões). Os valores obtidos foram submetidos à análise estatística pelo Teste de Mann-Whitney com p<0,10. Os resultados mostraram que não houve diferença estatisticamente significativa entre os grupos. Pode-se concluir que tanto o tempo de 2 ou 4 horas de oclusão são eficientes para o tratamento das QAs disseminadas; porém o menor tempo permitiu melhor conforto para os pacientes
Abstract: Skin cancer is the most incident neoplasm in world population. Actinic keratoses (AK) is considered a premalignant lesion. The AKs are often multiple, what can difficult the treatment. Photodynamic therapy (PDT) is an option for the treatment of widespread AK of the limbs. The aim of the present in vivo study was to evaluate the perfect time of occlusion of 5-aminolevulinic acid (ALA) 15% photosensitizing agent to treatment of widespread AKs by Kerato PDT. 40 patients with multiple AK on the upper limbs were selected. They were subjected to the following treatment: the ALA 15% was applied over the entire area (forearm and hand) and then the occlusion of the limbs was performed. After that, patients were divided into two groups according to duration of occlusion, in which patients in group 1 (G1) remained for 2 hours under occlusion and in group 2 (G2), for 4 hours. The next session the patients had their lesions submitted for 40 minutes (40 J/cm2) by prototype-based LED Kerato PDT. Medical visual analysis were performed in periods of 30 and 90 days througth scores from 0 (no improvement) to 10 (total improvement of the lesions). The values were statistically analyzed by Mann-Whitney test with p <0.10. The results showed no statistically significant difference between groups. It can be concluded that both times of occlusion are effective in the treatment of AK disseminated, but the minimum time allowed better comfort for patients
Orientador: Ana Gabriela Sálvio
Coorientador: Vanderlei Salvador Bagnato
Banca: Cristina Kurachi
Banca: Natalia Mayumi Lnada
Mestre

As Ceratoses Actínicas (CA) são lesões hiperceratóticas, displásicas de pele. Estão comumente localizadas em áreas expostas ao sol como couro cabeludo, face e antebraços. Acredita-se que a radiação ultravioleta (RUV) cumulativa seja o maior fator etiológico, considerando-se, também, a imunossupressão e infecção pelo papilomavírus humano(HPV) fatores contribuintes importantes. Existe o potencial de uma CA se transformar em Carcinoma Espinocelular (CEC) e, dessa forma, indica-se tratamento para as referidas lesões. Terapias tradicionais como crioterapia, curetagem e eletrocoagulação, medicações tópicas como 5-fluorouracil (5-FU) são ainda habituais. Porém novas opções como terapia fotodinâmica, diclofenaco e imiquimod 5% creme apresentam boa eficácia e perfil menor de efeitos colaterais, embora possam representar custo maior. A Terapia Fotodinâmica (TFD) envolve o uso de um agente fotossensibilizante, oxigênio e luz de comprimento de onda específico para causar morte celular. O fotossensibilizante geralmente utilizado é o ALA (ácido aminodeltalevulínico) ou seu éster metilaminolevulinato (MAL). No tecido lesionado, esses são convertidos em porfirinas fotoativas (PFAs) por enzimas da via biossintética do heme. A ativação é realizada por meio de luzes de comprimentos de onda que variam de 405 nm a 635 nm. Células displásicas ou neoplásicas produzem maior quantidade de porfirinas que os queratinócitos normais, sendo destruídas durante a aplicação da luz. O imiquimod 5% creme é um imunomodulador que estimula a resposta imune inata através da indução, síntese e liberação de citocinas. Isso resulta em efeitos antitumorais e antivirais indiretos. Seu uso tópico é eficaz e liberado para o tratamento de CA, Carcinoma Basocelular (CBC) superficial, Doença de Bowen (DB) e verrugas vulgares. Este trabalho tem como objetivo comparar duas recentes opções de tratamento para CA, a Terapia Fotodinâmica com metilaminolevulinato e o imiquimod 5% creme, por não existirem, na literatura atual, estudos comparativos dessas duas opções de tratamento. Foram selecionadas 12 pacientes com CAs que foram submetidas primeiramente à 1 sessão de TFD com MAL num lado da face e, 1 mês após, iniciaram o tratamento com imiquimod 5% creme aplicado no lado contralateral, duas vezes na semana, durante 16 semanas. A randomização foi realizada para determinar a hemiface para cada tratamento. Na primeira semana após a realização da TFD e mensalmente durante o tratamento com imiquimod, as pacientes foram avaliadas em relação aos efeitos colaterais dos tratamentos. Seis meses após entrarem no estudo, ambos os tratamentos foram analisados por um investigador cego para sua eficácia, tolerabilidade e seu resultado cosmético. Previamente ao tratamento as pacientes apresentaram um total de 245 lesões de CAs, sendo 120 lesões no lado submetido à TFD e 125 no lado tratado com imiquimod. Após o tratamento o número total de lesões diminuiu para 34 no lado tratado com TFD e para 30 no lado tratado com imiquimod, respectivamente. Não foram observadas diferenças estatisticamente significativas na eficácia de ambos os tratamentos e na frequência de efeitos colaterais. Entretanto, os pacientes, significativamente preferiram o tratamento com a terapia fotodinâmica, talvez pela rapidez do método, comparado com a aplicação tópica do creme de imiquimod a 5%.
Background: Actinic keratosis (AK) represents an initial process that may lead to in situ or invasive squamous cell carcinoma. The importance of its early diagnosis and treatment is well-established. There are several effective options available for the treatment of actinic keratosis, including topical imiquimod 5% cream and photodynamic therapy (PDT). Objetive: To compare the efficacy and patient preference between topical MAL-PDT and imiquimod 5% cream for the treatment of AK. Methods: Twelve patients, with a total of 245 lesions, underwent treatment with MAL-PDT and imiquimod 5% cream. Randomization was performed to determine the hemiface (right or left) for each therapy. First, patients were submitted to MAL-PDT. After one month, they started to use imiquimod on the opposite side of the face, twice a week, for 16 weeks. Six months after entering the study, both treatments were analyzed by a blinded investigator for their effectiveness, tolerability and cosmetic result. Results: Both treatments showed a good therapeutic response. 72% of the lesions treated with MAL-PDT were completely cleared, and 76% of those treated with imiquimod. The mean size of the residual lesions after the treatments was similar. Ten patients (83%) preferred MAL-PDT rather than imiquimod. (p: 0.03). Conclusions: Both MAL-PDT and imiquimod are effective in clearing AKs. Our results showed similar efficacy and good cosmetic outcomes with both treatments. However, a significant percentage of the subjects preferred MAL – PDT.

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O câncer de pele é o mais frequente tipo de câncer humano e mostra aumento de sua incidência nos últimos anos, o que o torna um crescente problema para a saúde pública mundial. Em muitos casos, antes do surgimento do carcinoma, instala-se uma lesão precursora, - a Ceratose Actínica, que pode evoluir para Carcinoma Espinocelular. Considerando o aumento de novos casos e recidivas frequentes, estudos tem sido intensificados no sentido de determinar os parâmetros com significado prognóstico na predição daqueles tumores que terão comportamento mais agressivo, que poderão recorrer e evoluir com metástases. O objetivo deste estudo foi avaliar a expressão dos marcadores de proliferação celular (PCNA, Ki-67) e apoptose (p53, Bcl-2), em portadores de Carcinoma Espinocelular e Ceratose Actínica. Foram estudadas amostras de 30 pacientes, submetidos à cirurgia reparadora, sendo dez portadores do Carcinoma Espinocelular, dez de Ceratose Actínica e dez indivíduos livres de lesões submetidos à blefaroplastia. A proteína p53 foi expressa em todos os casos estudados, com padrões quantitativos diferentes. O Bcl-2 foi expresso em baixa intensidade em seis casos de Ceratose Actínica, também baixa nas peles de blefaroplastia e negativo nos casos de Carcinoma Espinocelular. O PCNA exibiu expressão intensa em todas as amostras estudadas. O Ki-67 apresentou expressão variável nos casos de carcinoma e de ceratose e negativo na pele de pálpebra. A intensidade da expressão de PCNA, Ki-67, p53 e Bcl-2, associada a outros indicadores, tais como a análise histopatológica e a apresentação clínica, fornece informações sobre a provável progressão, levando a um diagnóstico mais seguro da neoplasia.
The skin cancer is the most frequent type of human cancer and shows an increase in its incidence in recent years, making it an important public health problem worldwide. In many cases before the onset of carcinoma install a precursor lesion, actinic keratosis, which can develop into squamous cell carcinoma. Considering the increase of new cases and relapses frequent, studies have been intensified in order to determine the parameters with prognostic significance in predicting those tumors which have more aggressive behavior, and can develop metastases. The objective was to evaluate the expression of markers of cell proliferation (PCNA, Ki-67) and apoptosis (p53,Bc1-2) in patients with squamous cell carcinoma and actinic keratosis. We studied samples from 30 patients, ten patients of squamous cell carcinoma, ten with actinic keratosis and ten samples free of lesions underwent blepharoplasty. The p53 protein was expressed in all cases with different quantitative patterns. The Bcl-2 was expressed at low intensity in six cases of actinic keratosis in the skins of blepharoplasty and negative in cases of squamous cell carcinoma. The PCNA showed intense expression in all samples. The Ki-67 showed variable expression in cases of keratosis and carcinoma and negative in the skin of eyelid. In conclusion the intensity of expression of PCNA, Ki-67, P53 and Bcl-2, associated with other indicators such as histopathological analysis and clinical presentation, provides information on the likely progression, leading to a more secure diagnosis of malignancy.

碩士
國立臺灣大學
光電工程學研究所
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Actinic keratosis (AK) is a common cutaneous neoplasm consisting of proliferation of atypical epidermal keratinocytes resulting from severe solar damage. AKs are important because of their potential to develop into invasive cutaneous squamous cell carcinoma (SCC) with the highest incidence in the aged population. AK patients usually have multiple AKs. To study the diagnosis of AK, in vivo harmonic generation microscopy (HGM) was proposed as the imaging tool. HGM provides submicron resolution crucial for histopathological examination and causes little optical damage in bio-tissues. With HGM, this thesis is aimed at seeking out the consistent cell morphology in comparison with the H&E stained histopathological images. Five Asian volunteers (2 females and 3 males) aged from 70 to 93 years old with AK were enrolled in this study. It contained 1 ex vivo and 4 in vivo examinations. Due to the clinical demands, we also developed movable HGM system with light guide in this study. HGM and H&E images of AK both show parakeratosis and hyperkeratosis in the stratum corneum (SC), irregular acanthosis, abnormal architecture and pleomorphism of cells and nuclei in the stratum spinosum (SS), crowding of keratinocytes, abnormal architecture and pleomorphism of cells and nuclei in the stratum basale (SB), and solar elastosis in the dermis. Moreover, we also found dendritic-like cells in AK through HGM images. Our case study indicates that noninvasive HGM may become an alternative to invasive biopsy in the diagnosis of AKs and also helps physician to improve follow-up treatment assessment.

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introduction: Nowadays, medicinal plants are reaching a spotlight, with their biopharmacological properties taking a centre stage in human health. Birch is a tree from the Betulaceae family, which has been used empirically since ancestral times, due to its wide therapeutic actions. Through unceasing investigation, researchers have been able to gather the necessary scientific evidence to prove such therapeutic properties, sustaining current uses. Nevertheless, the latest advances also allowed to unveil new ones. Objective: To write a critical review, aiming to compile and describe the main scientifically sustained biopharmacological properties of birch tree and of its main phytoconstituents: betulin and betulinic acid. Methods: Scientific articles that fulfil the objectives were selected from Pubmed and Embase databases. They were reviewed and the major benefits of birch were organized according to therapeutic properties.Results: The main effects of birch were grouped into: anti-microbiological, nephro-urinary, dermatological, anti-inflammatory, endocrine-metabolic, neuroprotective and oncological. The collected information shows enough scientific evidence to support such properties, reflecting birch’s potential therapeutic application. Conclusion: The state-of-play concerning birch supports its empiric and ancestral use. Moreover, new applications have also been discovered, while the safety and efficacy of this medicinal plant were confirmed. Through biotechnological progress, researchers revealed the underlying mechanisms of action and, at the same time, improved some limitations of these bioactives, specially their decreased bioavailability. Although more studies, particularly in vivo, are still needed to corroborate these findings, today’s scientific evidence allows us to believe that, in the coming years, a clinical application of birch might actually be achieved.
Introdução: No mundo atual, as plantas medicinais têm ganho um crescente destaque, com as suas diversas propriedades biofarmacológicas a assumirem um papel cada vez mais importante. A Bétula é uma árvore pertencente à família Betulaceae, que, dadas as suas vastas ações terapêuticas, era utilizada empiricamente em tempos ancestrais. Com a extensa investigação desenvolvida nesta área e o atual avanço biotecnológico, tais ações vieram a ser sustentadas e outras tantas viriam a ser evidenciadas, motivo pelo qual é ainda hoje utilizada mundialmente. Objetivo: Redigir uma revisão crítica que tem como finalidade indagar e reunir as principais e cientificamente sustentadas atividades biofarmacológicas da Bétula e seus principais fitoconstituintes, nomeadamente a betulina e o ácido betulínico. Métodos: Através de uma pesquisa eletrónica nas bases de dados PubMed e Embase, foram recolhidos artigos que cumprissem os objetivos. Procedeu-se à sua revisão, e à organização dos principais benefícios associados a esta planta por temas. Resultados: Tendo em conta a informação disponível, foi possível agrupar os principais efeitos associados a esta planta em: antimicrobiológicos, nefrourinários, dermatológicos, anti-inflamatórios, endócrino-metabólicos, neuroprotetores e oncoprotetores. A evidência científica revela resultados promissores em todas estas áreas, fazendo desta planta medicinal, e respetivos bioativos, potenciais agentes terapêuticos. Conclusão: A informação científica atual, além de suportar o uso empírico e ancestral desta árvore, e de desvendar outras tantas aplicações, sustenta também o seu bom perfil de segurança e elevada eficácia. A evolução biotecnológica permitiu estudar os mecanismos subjacentes a estes efeitos e melhorar limitações relativas à biodisponibilidade destes bioativos, ambicionando, um dia, a sua aplicação clínica. Contudo, são necessários mais estudos, nomeadamente in vivo que comprovem tais achados.

Το καρκίνωμα εκ πλακώδους επιθηλίου ή πλακώδες καρκίνωμα (ΠΚ) του δέρματος είναι ο δεύτερος πιο συχνός καρκίνος του δέρματος και εμφανίζεται συνήθως σε έδαφος ακτινικής υπερκεράτωσης (ΑΚ). Τα νεοπλασματικά κύτταρα εκφράζουν μια ποικιλία αντιγόνων προσελκύοντας με αυτό τον τρόπο λεμφοκύτταρα, τα διηθούντα τον όγκο λεμφοκύτταρα (Tumor Ιnfiltrating Lymphocytes-TILs) στο μικροπεριβάλλον του όγκου. Με βάση το ανοσοφαινοτυπικό τους προφίλ η πλειοψηφία των TILs εκφράζει το μόριο CD3 και αφορά σε Τ-λεμφοκύτταρα. Αυτά με την σειρά τους διαχωρίζονται σε CD8+ Τ-κυτταροτοξικά λεμφοκύτταρα και CD4+ Τ-λεμφοκύτταρα. Η έκφραση του δείκτη CD25+ επιτρέπει τον διαχωρισμό των CD4+ λεμφοκυττάρων σε δύο επιπλέον υποομάδες, τα T-επικουρικά (CD4+/CD25-) και τα Τ-ρυθμιστικά (CD4+/CD25+) κύτταρα. Ο πιο αξιόπιστος δείκτης για την ανίχνευση των Τ-ρυθμιστικών κυττάρων (Tregs) θεωρείται ο Forkhead box P3 (Foxp3). Ο αυξημένος αριθμός των TILs έχει συσχετισθεί με την πρόγνωση και τη θεραπευτική αντιμετώπιση σε μια ποικιλία νεοπλασμάτων, περιλαμβανομένων και καρκίνων του δέρματος όπως το μελάνωμα. Η μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης, ωστόσο, δεν έχει ακόμα αποσαφηνισθεί και ποικίλλει στα διάφορα είδη νεοπλασμάτων. Στα πλαίσια της παρούσας διπλωματικής εργασίας η ανίχνευση των Τ-λεμφοκυττάρων έγινε με ανοσοϊστοχημική μέθοδο με τη χρήση των κατάλληλων αντισωμάτων (CD3, CD4, CD8, Foxp3). Η παρουσία των κυττάρων αξιολογήθηκε με τη χρήση φωτονικού μικροσκοπίου με δυο διαφορετικά ημιποσοτικά συστήματα βαθμολόγησης για όλους τους μελετηθέντες Τ-κυτταρικούς υποπληθυσμούς καθώς και με ένα τρίτο σύστημα ποσοτικής καταμέτρησης για τα Τregs. Σκοπός ήταν να γίνει σύγκριση των μεθόδων ώστε να βρεθεί η καταλληλότερη μεθοδολογία αξιολόγησης της λεμφοκυτταρικής διήθησης στο πλακώδες καρκίνωμα του δέρματος, να υπολογιστεί ο αριθμός των Tregs στις μελετούμενες οντότητες χρησιμοποιώντας το βέλτιστο σύστημα βαθμολόγησης και να αναζητηθούν διαφορές στην πυκνότητα των λεμφοκυτταρικών υποπληθυσμών μεταξύ των οντοτήτων, οι οποίες μπορεί να έχουν παθογενετική ή θεραπευτική κλινική σημασία.
Squamous cell carcinoma (SCC) of the skin is the second most common skin cancer. It usually develops in a background of actinic keratosis (AK). Neoplastic cells express a variety of antigens, attracting lymphocytes in the tumor microenvironment (Tumor Ιnfiltrating Lymphocytes-TILs). Based on the immunophenotypic profile, the majority of TILs expresses the CD3 molecule, so they are T-lymphocytes. These in turn are divided into CD8+ cytotoxic T-lymphocytes and CD4+ T-lymphocytes. The expression of CD25 enables the separation of CD4+ lymphocytes in two further subgroups, the T-helper (CD4+/CD25-) and T-regulatory (CD4+/CD25+) cells. The most reliable marker for the detection of T-regulatory cells (Tregs) is considered to be the Forkhead box P3 (Foxp3). The increased number of TILs is associated with prognosis and treatment in a variety of tumors, including skin cancers, such as melanoma. The evaluation methology of lymphocytic infiltration, however, is not yet clarified and varies in different types of tumors. In the context of this thesis, the detection of T-lymphocytes was performed by immunohistochemistry using the appropriate antibodies (CD3, CD4, CD8, Foxp3). The presence of the cells was evaluated using light microscopy with two different semiquantitative scoring systems, for all the T-lymphocyte subpopulations, as well as with a third system of quantification of Tregs. The purpose was to compare the methods in order to find the most suitable methodology for the evaluation of the lymphocytic infiltration in squamous cell carcinoma of the skin, to evaluate the number of Tregs in the studied entities using the best method and examine for differences in the density of lymphocyte subpopulations.