The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18861号 / 医博第3972号 / 新制||医||1008(附属図書館) / 31812 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 野田 亮, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Submitted by Nadir Basilio (nadirsb@uninove.br) on 2015-07-27T14:33:27Z No. of bitstreams: 1 Erika Aparecida Felix de Barros.pdf: 3994975 bytes, checksum: 34211c9fc059d61aa8302afe5a482223 (MD5) / Made available in DSpace on 2015-07-27T14:33:27Z (GMT). No. of bitstreams: 1 Erika Aparecida Felix de Barros.pdf: 3994975 bytes, checksum: 34211c9fc059d61aa8302afe5a482223 (MD5) Previous issue date: 2014-12-18 / Prostate cancer (PCa) is the most common non-skin solid tumor in man in western countries. The prognosis is defined by the measurement of PSA, stage and Gleason score, however, none of these factors classics, even when combined, show good performance in prognosis definition, therefore molecular markers are being studied. Cytogenetic abnormalities, represented by chromosomal deletions and gains, are characteristic of oncogenesis and potentially can be used as a prognostic marker. Objectives: The study aimed to identify the cytogenetic alterations of aneuploidy in chromosomes 3 , 7 , 17 and 9p21 locus by FISH technique located in PCa underwent radical prostatectomy. As a secondary objective correlate the changes found with biochemical recurrence after surgical treatment and the classical prognostic factors of prostate adenocarcinoma. Methodology: For this case-control study 112 patients with clinically localized PCa who underwent radical prostatectomy were followed up postoperatively than ten years were analyzed. Samples of the primary tumor of each patient were available for tissue microarray matrix and cytogenetic changes were assessed by FISH technique. Results: Among the patients for the 9p21 locus, 6.4 % had lost one of the two alleles. In relation to the results obtained for chromosomes 3, 7 and 17 find deletion of one allele of 2.3, 1.2 and 1.8% of the cases respectively. No association between aneuploidy of these chromosomes with the Gleason score, pathological stage, serum PSA level or risk group . We observed that the loss of the 9p21 locus was associated with shorter time to recurrence (p 0.038 ). Conclusions: We found a low occurrence of aneuploidy detected by probes CEP 3 , 7 and 17 and LSI 9p in our series of tumors. We observed that the loss of the 9p21 locus was associated with worse prognosis in CaP located treated with surgery. / Introdução: O câncer de próstata (CaP) é o tumor sólido não cutâneo mais comum do homem em países ocidentais. O prognóstico é feito pela dosagem do PSA, estádio e escore de Gleason, porém, nenhum destes fatores clássicos, mesmo quando avaliados em conjunto, apresentam bom desempenho na determinação prognóstica, por isso marcadores moleculares estão sendo estudados. Alterações citogenéticas, representadas por ganhos e deleções cromossômicas, são características da oncogênese e potencialmente podem ser empregadas como marcador de prognóstico. Objetivos: O estudo teve como objetivo principal identificar as alterações citogenéticas de aneuploidia nos cromossomos 3, 7, 17 e lócus 9p21 pela técnica de FISH no CaP localizado submetido à prostatectomia radical. Como objetivo secundário correlacionamos as alterações encontradas com a recidiva bioquímica após tratamento cirúrgico e com os fatores prognósticos clássicos do adenocarcinoma de próstata. Metodologia: Para este estudo caso controle foram analisados 112 pacientes com diagnóstico de CaP clinicamente localizado submetidos à prostatectomia radical com seguimento pós operatório superior a dez anos. As amostras do tumor primário de cada paciente foram disponibilizadas em matriz de microarranjo tecidual e as alterações citogenéticas foram avaliadas através da técnica de FISH. Resultados: Dos pacientes avaliados para o lócus 9p21, 6,4% apresentaram perdas de um dos dois alelos. Em relação aos resultados obtidos para os cromossomos 3, 7 e 17 encontramos deleção de um dos alelos em 2,3; 1,2 e 1,8% dos casos respectivamente. Não observamos associação entre aneuploidia desses cromossomos com o escore de Gleason, estádio patológico, nível sérico de PSA ou grupo de risco. Observamos que a perda do lócus 9p21 esteve associado a menor tempo para recidiva (p 0,038). Conclusões: Encontramos baixa ocorrência de aneuploidia detectadas pelas sondas CEP 3, 7 e 17 e LSI 9p em nossa série de tumor. Observamos que a perda do lócus 9p21 esteve associado a pior prognóstico no CaP localizado tratado com cirurgia.

Bladder cancer is a potentially curable malignancy; however in regards to the state of current therapy regimens, a plateau has been reached in both the non-muscle and muscle invasive types. To obtain effective treatment, and consequently a decreased mortality, it has become imperative to test and understand aspects affecting therapy response. The aim of this thesis is to illustrate a better understanding of clinical factors affecting therapy response using new drug combinations and new tumor markers alongside established risk criteria. In Paper I we reported the 5 year follow up from a multicenter, prospectively randomized study and we evaluated the 5-year outcomes of BCG alone compared to a combination of epirubicin and interferon-a2b in the treatment of patients with T1 bladder cancer. Treatment, tumor size and tumor status at second resection were independent variables associated with recurrence. Concomitant Cis was not predictive of failure of BCG therapy. Independent factor for treatment failure was remaining T1 stage at second resection. In Paper II &III we investigated the validity of emmprin, survivin and CCTα proteins as biomarkers for response and survival before neoadjuvant cisplatin chemotherapy. Bladder tumor specimens were obtained before therapy from a total of 250 patients with T1-T4 bladder cancer enrolled in 2 randomized trials comparing neoadjuvant chemotherapy before cystectomy with a surgery only arm. Protein expression was determined by immunohistochemistry (IHC). Patients in the chemotherapy cohort with negative emmprin and CCTα expression had significantly better overall survival (OS) than those with positive expression. In Paper IV primary end point was examining STMN1 as prognostic factor in bladder cancer.  Analysis was performed on three bladder cancer patient cohorts using IHC, western blot and a bladder cancer cell line. High levels of STMN1, expression correlated to shorter disease-specific survival and the growth and migration of the cells were significantly reduced when transfecting the cells with STMN1 siRNA. Conclusion Risk assessment and predictors of outcomes could help in individualized treatment and follow up.  Biomarkers will become more important for treatment choices in bladder cancer management.

<p>In this thesis, methods to study gene and protein expression in cells and tissues were developed and utilized in combination with protein-specific antibodies, with the overall objective to attain greater understanding of protein function.</p><p>To analyze protein expression in <i>in vitro</i> cultured cell lines, a cell microarray (CMA) was developed, facilitating antibody-based protein profiling of cell lines using immunohistochemistry (IHC). Staining patterns in cell lines were analyzed using image analysis, developed to automatically identify cells and immunohistochemical staining, providing qualitative and quantitative measurements of protein expression. Quantitative IHC data from CMAs stained with nearly 3000 antibodies was used to evaluate the adequacy of using cell lines as models for cancer tissue. We found that cell lines are homogenous with respect to protein expression profiles, and generally more alike each other, than corresponding cancer cells <i>in vivo</i>. However, we found variability between cell lines in regards to the level of retained tumor phenotypic traits, and identified cell lines with a preserved link to corresponding cancer, suggesting that some cell lines are appropriate model systems for specific tumor types. </p><p>Specific gene expression patterns were analyzed in vitiligo vulgaris and malignant melanoma. Transcriptional profiling of vitiligo melanocytes revealed dysregulation of genes involved in melanin biosynthesis and melanosome function, thus highlighting some mechanisms possibly involved in the pathogenesis of vitiligo. Two new potential markers for infiltrating malignant melanoma, Syntaxin-7 and Discs large homolog 5, were identified using antibody-based protein profiling of melanoma in a tissue microarray format. Both proteins were expressed with high specificity in melanocytic lesions, and loss of Syntaxin-7 expression was associated with more high-grade malignant melanomas.</p><p>In conclusion, the combination of antibody-based proteomics and microarray technology provided valuable information of expression patterns in cells and tissues, which can be used to better understand associations between protein signatures and disease.</p>

Prognostic models have clinical appeal to aid therapeutic decision making. In the UK, the Nottingham Prognostic Index (NPI) has been used, for over two decades, to inform patient management. However, it has been commented that NPI is not capable of identifying a subgroup of patients with a prognosis so good that adjuvant therapy with potential harmful side effects can be withheld safely. Tissue Microarray Analysis (TMA) now makes possible measurement of biological tissue microarray features of frozen biopsies from breast cancer tumours. These give an insight to the biology of tumour and hence could have the potential to enhance prognostic modelling. I therefore wished to investigate whether biomarkers can add value to clinical predictors to provide improved prognostic stratification in terms of Recurrence Free Survival (RFS). However, there are very many biomarkers that could be measured, they usually exhibit skewed distribution and missing values are common. The statistical issues raised are thus number of variables being tested, form of the association, imputation of missing data, and assessment of the stability and internal validity of the model. Therefore the specific aim of this study was to develop and to demonstrate performance of statistical modelling techniques that will be useful in circumstances where there is a surfeit of explanatory variables and missing data; in particular to achieve useful and parsimonious models while guarding against instability and overfitting. I also sought to identify a subgroup of patients with a prognosis so good that a decision can be made to avoid adjuvant therapy. I aimed to provide statistically robust answers to a set of clinical question and develop strategies to be used in such data sets that would be useful and acceptable to clinicians. A unique data set of 401 Estrogen Receptor positive (ER+) tamoxifen treated breast cancer patients with measurement for a large panel of biomarkers (72 in total) was available. Taking a statistical approach, I applied a multi-faceted screening process to select a limited set of potentially informative variables and to detect the appropriate form of the association, followed by multiple imputations of missing data and bootstrapping. In comparison with the NPI, the final joint model derived assigned patients into more appropriate risk groups (14% of recurred and 4% of non-recurred cases). The actuarial 7-year RFS rate for patients in the lowest risk quartile was 95% (95% C.I.: 89%, 100%). To evaluate an alternative approach, biological knowledge was incorporated into the process of model development. Model building began with the use of biological expertise to divide the variables into substantive biomarker sets on the basis of presumed role in the pathway to cancer progression. For each biomarker family, an informative and parsimonious index was generated by combining family variables, to be offered to the final model as intermediate predictor. In comparison with NPI, patients into more appropriate risk groups (21% of recurred and 11% of non-recurred patients). This model identified a low-risk group with 7-year RFS rate at 98% (95% C.I.: 96%, 100%).

Breast cancer (BC) is one of the leading causes of cancer-related deaths in Canadian women. Aggressive BCs (e.g. triple-negative subtype; TN) present a clinical challenge as defined biomarkers, particularly those indicative of unique cancer-associated signaling pathways, are needed to improve prognostication and prediction of therapeutic response. Overexpression of Src and its substrates, Ezrin and Tks5, have been associated with poor prognosis in many cancers. We have previously shown that Ezrin regulates proteolytic-independent invasion, while others have shown that Tks5 is associated with proteolytic-dependent invasion. Thus, expression of Ezrin versus Tks5 in BC cases may represent different invasion modalities. Additionally, immunofluorescence (IF)-based technologies may provide a more quantitative and objective approach for analysis of biomarker expression and subcellular compartmentalization compared to immunohistochemistry (IHC). In this study, I hypothesize that expression and subcellular localization of Src, Ezrin and Tks5, have improved prognostic significance in BCs, compared to current clinico-pathological parameters. To assess this, I optimized an IF-based automated quantification analysis (AQUA) system to measure subcellular expression in a 63-patient BC cohort and tested associations with clinico-pathological data. This thesis presents that: 1) Expression of Src and Ezrin increased, but that of Tks5 decreased in breast tumours compared to normal breast. 2) Src and Ezrin localized to the apical regions of normal breast epithelia but shifted to the cytoplasm in breast tumours. Tks5 exhibited a granular basal expression in normal breast epithelia, and is weakly expressed in tumour cellular compartments. 3) In our 63-patient cohort, Src and Ezrin had significant correlations with multiple clinico-pathological parameters, including TN status and lymphovascular invasion. 4) Clinico-pathological associations with IF-based AQUA scoring are directly comparable to conventional manual IHC scoring. Our study supports the role of Src and Ezrin as potential prognostic biomarkers for BC. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-07-09 12:51:09.527

O objetivo deste estudo foi verificar o comportamento morfológico e a expressão das proteínas S-100, Melan-A, HMB-45, Ki-67, PCNA e p53, em 25 neoplasias melanocíticas de equinos. Informações clínicas (gênero, raça, pelagem, idade e localização da lesão) e morfológicas (características celulares, pigmentares, nucleares, de nucléolo, de melanófagos, presença de invasão e necrose) dos animais foram coletadas. Para a expressão das proteínas por imunoistoquímica foi confeccionado um bloco de microarranjo de tecidos das amostras teciduais, juntamente com os controles positivos das reações. A avaliação da expressão das proteínas S100, HMB-45 e Melan-A foi baseada em um escore, e a das proteínas Ki-67, PCNA e p53 foi feita por contagem de células. Animais SRD (16/25, 64%), de raça Lusitana (6/25, 24%), Árabe (2/25, 8%) e Sueca (1/25, 4%) fizeram parte deste estudo, todos tordilhos e a maioria machos (18/25, 72%). A idade dos animais variou de 4 a 24 anos (média de 13 anos). A região perianal (13/25, 52%) foi a que mais apresentou neoplasias. Na análise morfológica houve predomínio de neoplasias com celularidade moderada (52%) e intensa (40%), distribuição difusa e em feixes (52%), ausência de figuras de mitose (96,0%) e predomínio de células epitelióides e fusiformes no mesmo tumor (80%). A atipia nuclear era discreta (48%) e moderada (44%), com núcleos de formato arredondado e alongado em um mesmo tumor (76%) e cromatina dispersa (60%). Os nucléolos eram múltiplos e, em sua maioria, proeminentes (88%). Observou-se predomínio de células tumorais de pigmentação intensa (68%), distribuição difusa e localização em derme (100%). A maioria dos casos apresentou alta celularidade de macrógafos (64%) e distribuição difusa (96%). Quanto à expressão de proteínas para melanócitos por imunoistoquímica, 44% dos casos apresentaram expressão moderada a forte de S100, 56% apresentaram expressão fraca de HMB-45 e 64% apresentaram expressão negativa de Melan-A. Houve positividade de 72% dos casos para pelo menos dois dos anticorpos citados acima. Os anticorpos de proliferação celular Ki-67 e PCNA tiveram média de acima. Os anticorpos de proliferação celular Ki-67 e PCNA tiveram média de positividade de 0,0005% e 15,7%, respectivamente. A análise da expressão de p53 teve média de 6,1% de positividade. Houve associação estatisticamente positiva entre a celularidade dos macrófagos com S100 e com p53. Em conclusão: 1. os dados clínicos obtidos reproduzem o comportamento biológico das neoplasias melanocíticas em equinos, exceto pela idade dos animais; 2. as neoplasias equinas se assemelham a nevos azuis celulares em humanos e melanocitomas em cães; 3. o microarranjo de tecidos mostrou-se uma maneira econômica, rápida e com menos variáveis técnicas; 4. a utilização de um painel de anticorpos de melanócitos é pertinente na diferenciação entre tumores melanocíticos e não melanocíticos, reproduzindo o painel diagnóstico utilizado em literatura humana e canina; 5. o índice de proliferação celular encontrado sugere que os dois anticorpos (Ki-67 e PCNA) podem ser usados na contagem de células em atividade mitótica e 6. a proteína p53 tem maior relação com a parada do ciclo celular que a observada em outros estudos em equinos, podendo indicar um comportamento biológico diferente do apresentado em cães e humanos. / The aim of this study was to evaluate the morphological behavior, and expression of the following proteins: S-100, Melan-A, HMB-45, Ki-67, PCNA and p53, in 25 equine melanocytic neoplasms. Clinical (gender, breed, coat color, age and lesions location) and morphological (cellular, pigment, nuclear, nucleoli, melanophages, invasion and necrosis) data were collected. A tissue microarray block, embedded in paraffin, with equine tissue samples and positive controls, was elaborated for protein expression through immunohistochemistry. The evaluation of S100, HMB-45 and Melan-A was based on a score, and for Ki-67, PCNA and p53 it was based on cellular count. Breeds were: Mixed breed (16/25, 64%), Lusitano (6/25, 24%), Arab (2/25, 8%) and Swedich (1/25, 4%). All animals were gray and the majority males (18/25, 72%). Age varied from 4 to 24 years old (mean=13 years). The perianal region (13/25, 52%) was the most common location. Morphological analysis have shown neoplasms with predominantly moderate (52%) and intense (40%) cellularity, diffuse and fascicles distribution (52%), no mitoses figures (96%) and predominance of epithelioid and spindle cells in the same tumor (80%). There was discrete (48%) and moderate (44%) nuclear atypia, round and elongated nucleus in the same tumor (76%), and disperse chromatin (60%). Nucleoli were multiple and prominent in the majority of cases (88%). Tumor cells with diffuse and intense pigmentation, with dermal location (100%) were predominant. High cellularity of macrophages (64%) with diffuse distribution (96%) was mostly seen. The protein expression for melanocytes have shown 44% of moderate to strong expression for S100 protein, 56% of weak expression for HMB-45 protein and 64% of negative expression for Melan-A protein. It was found positivity for more than two antibodies in 72% of equine melanocytic neoplasms. The proliferation antibodies Ki-67 and PCNA had mean positivity of 0,0005% and 15,7%, respectively. The p53 expression had mean positivity of 6,1%. Macrophages cellularity was statistically associated with S100 and p53. In conclusion: 1. clinical data obtain reproduce the biological behavior of equine Macrophages celllularity was statistically associated with S100 and p53. In conclusion: 1. clinical data obtain reproduce the biological behavior of equine melanocytic neoplasms, excepting the animals age; 2. equine melanocytic neoplasms assemble to human cellular blue nevi and dogs melanocytoma; 3. the tissue microarray was shown to be an economic, rapid and less variable technique; 4. using a panel for antibodies for melanocytes is relevant to differentiate melanocytic and not melanocytic tumors, reproducing the diagnosis panel used in human and canine literature; 5. the proliferation index found suggests that both antibodies (Ki-67 and PCNA) could be used in mitotic activity cell count; and 6. p53 protein has more relation with cellular cycle stop than in other equine studies, probably indicating a different biological behavior than the presented in humans and dogs.

O objetivo deste estudo foi verificar o comportamento morfológico e a expressão das proteínas S-100, Melan-A, HMB-45, Ki-67, PCNA e p53, em 25 neoplasias melanocíticas de equinos. Informações clínicas (gênero, raça, pelagem, idade e localização da lesão) e morfológicas (características celulares, pigmentares, nucleares, de nucléolo, de melanófagos, presença de invasão e necrose) dos animais foram coletadas. Para a expressão das proteínas por imunoistoquímica foi confeccionado um bloco de microarranjo de tecidos das amostras teciduais, juntamente com os controles positivos das reações. A avaliação da expressão das proteínas S100, HMB-45 e Melan-A foi baseada em um escore, e a das proteínas Ki-67, PCNA e p53 foi feita por contagem de células. Animais SRD (16/25, 64%), de raça Lusitana (6/25, 24%), Árabe (2/25, 8%) e Sueca (1/25, 4%) fizeram parte deste estudo, todos tordilhos e a maioria machos (18/25, 72%). A idade dos animais variou de 4 a 24 anos (média de 13 anos). A região perianal (13/25, 52%) foi a que mais apresentou neoplasias. Na análise morfológica houve predomínio de neoplasias com celularidade moderada (52%) e intensa (40%), distribuição difusa e em feixes (52%), ausência de figuras de mitose (96,0%) e predomínio de células epitelióides e fusiformes no mesmo tumor (80%). A atipia nuclear era discreta (48%) e moderada (44%), com núcleos de formato arredondado e alongado em um mesmo tumor (76%) e cromatina dispersa (60%). Os nucléolos eram múltiplos e, em sua maioria, proeminentes (88%). Observou-se predomínio de células tumorais de pigmentação intensa (68%), distribuição difusa e localização em derme (100%). A maioria dos casos apresentou alta celularidade de macrógafos (64%) e distribuição difusa (96%). Quanto à expressão de proteínas para melanócitos por imunoistoquímica, 44% dos casos apresentaram expressão moderada a forte de S100, 56% apresentaram expressão fraca de HMB-45 e 64% apresentaram expressão negativa de Melan-A. Houve positividade de 72% dos casos para pelo menos dois dos anticorpos citados acima. Os anticorpos de proliferação celular Ki-67 e PCNA tiveram média de acima. Os anticorpos de proliferação celular Ki-67 e PCNA tiveram média de positividade de 0,0005% e 15,7%, respectivamente. A análise da expressão de p53 teve média de 6,1% de positividade. Houve associação estatisticamente positiva entre a celularidade dos macrófagos com S100 e com p53. Em conclusão: 1. os dados clínicos obtidos reproduzem o comportamento biológico das neoplasias melanocíticas em equinos, exceto pela idade dos animais; 2. as neoplasias equinas se assemelham a nevos azuis celulares em humanos e melanocitomas em cães; 3. o microarranjo de tecidos mostrou-se uma maneira econômica, rápida e com menos variáveis técnicas; 4. a utilização de um painel de anticorpos de melanócitos é pertinente na diferenciação entre tumores melanocíticos e não melanocíticos, reproduzindo o painel diagnóstico utilizado em literatura humana e canina; 5. o índice de proliferação celular encontrado sugere que os dois anticorpos (Ki-67 e PCNA) podem ser usados na contagem de células em atividade mitótica e 6. a proteína p53 tem maior relação com a parada do ciclo celular que a observada em outros estudos em equinos, podendo indicar um comportamento biológico diferente do apresentado em cães e humanos. / The aim of this study was to evaluate the morphological behavior, and expression of the following proteins: S-100, Melan-A, HMB-45, Ki-67, PCNA and p53, in 25 equine melanocytic neoplasms. Clinical (gender, breed, coat color, age and lesions location) and morphological (cellular, pigment, nuclear, nucleoli, melanophages, invasion and necrosis) data were collected. A tissue microarray block, embedded in paraffin, with equine tissue samples and positive controls, was elaborated for protein expression through immunohistochemistry. The evaluation of S100, HMB-45 and Melan-A was based on a score, and for Ki-67, PCNA and p53 it was based on cellular count. Breeds were: Mixed breed (16/25, 64%), Lusitano (6/25, 24%), Arab (2/25, 8%) and Swedich (1/25, 4%). All animals were gray and the majority males (18/25, 72%). Age varied from 4 to 24 years old (mean=13 years). The perianal region (13/25, 52%) was the most common location. Morphological analysis have shown neoplasms with predominantly moderate (52%) and intense (40%) cellularity, diffuse and fascicles distribution (52%), no mitoses figures (96%) and predominance of epithelioid and spindle cells in the same tumor (80%). There was discrete (48%) and moderate (44%) nuclear atypia, round and elongated nucleus in the same tumor (76%), and disperse chromatin (60%). Nucleoli were multiple and prominent in the majority of cases (88%). Tumor cells with diffuse and intense pigmentation, with dermal location (100%) were predominant. High cellularity of macrophages (64%) with diffuse distribution (96%) was mostly seen. The protein expression for melanocytes have shown 44% of moderate to strong expression for S100 protein, 56% of weak expression for HMB-45 protein and 64% of negative expression for Melan-A protein. It was found positivity for more than two antibodies in 72% of equine melanocytic neoplasms. The proliferation antibodies Ki-67 and PCNA had mean positivity of 0,0005% and 15,7%, respectively. The p53 expression had mean positivity of 6,1%. Macrophages cellularity was statistically associated with S100 and p53. In conclusion: 1. clinical data obtain reproduce the biological behavior of equine Macrophages celllularity was statistically associated with S100 and p53. In conclusion: 1. clinical data obtain reproduce the biological behavior of equine melanocytic neoplasms, excepting the animals age; 2. equine melanocytic neoplasms assemble to human cellular blue nevi and dogs melanocytoma; 3. the tissue microarray was shown to be an economic, rapid and less variable technique; 4. using a panel for antibodies for melanocytes is relevant to differentiate melanocytic and not melanocytic tumors, reproducing the diagnosis panel used in human and canine literature; 5. the proliferation index found suggests that both antibodies (Ki-67 and PCNA) could be used in mitotic activity cell count; and 6. p53 protein has more relation with cellular cycle stop than in other equine studies, probably indicating a different biological behavior than the presented in humans and dogs.