Estudo retrospectivo sobre a análise das mutações por extração de DNA de melanoma e nevo melanocítico associado
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2015-04-30
Autores
Nascimento, Danielle Ioshimoto Shitara do [UNIFESP]
Orientadores
Bagatin, Edileia [UNIFESP]
Tipo
Tese de doutorado
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Fundamentos: Estudos clínicos e epidemiológicos sugerem que o melanoma pode surgir de novo ou estar associado a uma lesão prévia. Melanomas associados a nevos parecem ter prognóstico diferente dos melanomas de novo, quando considerada a espessura de Breslow, um dos fatores prognósticos. O papel das lesões melanocíticas benignas na gênese das células de melanoma permanece motivo de controvérsia: haveria colisão fortuita das duas entidades ou as células de melanoma e nevos poderiam estar clonalmente relacionadas, já que na maioria dos casos de melanomas associado a nevos as duas entidades compartilham o mesmo perfil genético em estudos moleculares. Objetivo: Avaliar se as mutações presentes no nevo melanocítico se mantêm nos melanomas associados, ou se o estado mutacional é distinto. Avaliar o perfil clinico-epidemiológico dos melanomas associados a nevo na cidade de São Paulo-Brasil. Materiais e métodos: Estudo retrospectivo, realizado em cooperação internacional Brasil-Espanha. Casos de melanomas associados a nevos de um laboratório especializado em Dermatopatologia, em São Paulo-SP foram selecionados, submetidos à coloração hematoxilina-eosina e à imunoistoquímica com anticorpos monoclonais MELAN_A, HMB45, Ki-67. Assim, o risco de viés de seleção das populações celulares foi minimizado, pois as células névicas e as de melanoma foram selecionadas combinando-se critérios morfológicos e imunoistoquímicos.Após seleção daqueles com distinção indiscutível entre populações celulares de melanoma e nevo, os casos brasileiros e os casos de origem na Unidade de Melanoma do Hospital Clínico e Barcelona, o material foi submetido à microdissecção à laser, amplificação e sequenciamento dos genes BRAF éxons 11 e 15, NRAS éxons 2 e 3, cKIT éxon 11, PPP6C éxon 7, RAC1 éxon 2 e STK19 éxon 2. Foram feitas duas avaliações mutacionais de cada gene para todos os melanomas e nevos, seja duas vezes por sequenciamento de Sanger ou por métodos moleculares distintos (i.e. Sanger e SNaPshot, ou Sanger e PCR alelo-específica nos casos de BRAF), de maneira que apenas as mutações confirmadas foram consideradas como positivas. Resultados: Do total de 135653 laudos histopatológicos (1995-2004), 1.190 melanomas foram selecionados. Melanomas associados a nevo corresponderam a 32,7% dos casos (390/1190), sendo a associação com nevo 1,52 vezes mais frequente em melanomas finos que em espessos (IC 95% [1,16;1,99], p<0,001). Melanoma disseminativo superficial foi o subtipo histológico mais frequente, enquanto nenhum caso de Lentigo Maligno foi associado a nevo. O Breslow mediano dos melanomas associados a nevos foi menor que o dos melanomas de novo. Dos 390 melanomas associados a nevos de origem na amostra brasileira, 34 casos foram selecionados para análise molecular. Da Unidade de melanoma do Hospital Clnico de Barcelona, 27 casos foram incluídos. Um caso foi excluído por ausência de amplificação do material do nevo associado. Em 39 casos, pelo menos um gene encontrava-se mutado. BRAF 15 V600E foi a mutação mais frequentemente encontrada em melanomas (28/60; 46,6%) e nevos 51.7% (31/60). Mutação NRAS éxon 3 (Q61K) foi observada em 21,7% (13/60) dos casos. Nenhum caso de mutação em BRAF éxon 11, NRAS éxon 2, RAC1, PPP6C ou STK19 ou cKIT foi observado. Sessenta e oito por cento (41/60) dos melanomas e nevos associados apresentaram perfil genômico semelhante para os 6 genes avaliados. Conclusão: Melanoma associado a nevo representa 1/3 dos melanomas em centro privado de Dermatopatologia, situado na região Sudeste do Brasil, cidade de São Paulo, sendo associado à exposição solar intermitente, melanomas do tipo disseminativo superficial e menor espessura de Breslow. A alta concordância de status mutacional entre melanoma e nevo corroboram a hipótese de que melanoma e nevo adjacente podem ser lesões clonalmente relacionadas. Este é, na literatura consultada, o primeiro estudo da América Latina, a maior série de casos de melanomas associados a nevos avaliada molecularmente, utilizando extenso painel de genes.
Background: Melanoma origin has always been a debated subject and the precursor role of pigmented lesions as well. Considering epidemiological and histological studies, melanoma seems to arise either in association with a pre-existing nevus or de novo, without any associated lesion. Nevus-associated melanoma and de novo melanomas seem to have different prognosis, when considering prognostic factors such as Breslow thickness. The real significance of benign melanocytic lesions remains controversial: it could be a fortuitous collision or melanoma and nevus cells could be clonally related, since most of melanomas and associated nevus share the same genetic profile in some studies, supporting a nevusto-melanoma progression model which predicts conservation of the driver mutation. Objective: To evaluate if melanoma and associated nevus share the same mutational profile or if it is distinct. To evaluate the clinico-epidemiological profile of nevus-associated melanomas in São Paulo-Brazil. Methods: Retrospective international (Brazil-Spain) cooperation study (1995-2004). Melanomas with associated nevi from a specialized Dermatopathology center, from Sao Paulo City, Sao Paulo State, Brazil, and from the Melanoma Unit of the Hospital Clinic of Barcelona, Spain were routinely processed and stained with hematoxilin-eosin. Brazilian cases and doubtful spanish cases were submitted to immunohistochemistry with antibodies MELAN_A, HMB45, Ki-67. Thus, cell selection bias was minimised as melanoma and nevus cells were selected by combining morphology and immunohistochemistry. The brazilian cases and the ones from the Melanoma Unit of the Hospital Clinic of Barcelona, in which the distinction between melanoma and nevus cell populations was unmistakable, were submitted to laser microdissection, amplification and sequencing of the following genes: BRAF exons 11 and 15, NRAS exons 2 and 3, cKIT exon 11, PPP6C exon 7, RAC1 exon 2 e STK19 exon 2. Each gene was evaluated twice in all samples by sequencing or by sequencing and another confirmation method, allelespecific fluorescent Polymerase Chain Reaction (PCR) and capillary electrophoresis detection, or by SNaPshot Analysis. Only mutations confirmed via two different molecular methods or twice by sequencing were considered positive. Results: Out of a total of 135,653 brazilian pathological records from skin biopsies of a 10-year period, 1190 melanoma records were selected. Nevus-associated melanomas corresponded to 390 melanomas (32.7%), being the association of thin melanomas with a nevus 1.52 times the association observed with thick melanomas (>1.01mm) (CI 95% [1.16;1.99], p<0.001). Superficial spreading melanoma was the most frequent, while no Lentigo Maligna melanoma was associated with nevi. The median Breslow thickness of nevus-associated melanomas was lower than that of de novo melanomas. Of 390 nevus-associated melanomas, 34 cases were selected for molecular analysis. Twenty-seven cases from the Melanoma Unit Of the Hospital Clinic of Barcelona were included. One case was excluded for absence of amplification products for the associated nevus. In 39 out of 60 cases (65%) at least one gene was mutated. BRAF 15 V600E mutation was the most frequent mutation found in melanomas (28/60; 46.6%) and nevus (31/60; 51.7%). Thirteen NRAS exon 3 (Q61K) were observed. No BRAF exon 11, NRAS exon 2, cKIT, PPP6C, STK19 or RAC1 mutations were observed. Sixty eight percent (41/60) of melanomas and associated nevi demonstrated similar genomic profile for all six genes evaluated. Conclusion: Nevusassociated melanomas represented one third of melanomas in a private dermatopathology center in São Paulo State, situated in Southeast Brazil, being associated with intermittent sun exposure, superficial spreading melanomas and lower Breslow thickness. The results from the present study corroborate the hypothesis that melanoma and associated nevus may be clonally related, given the high agreement of mutational status between the two lesions. This is, up to our knowledge, the first case series evaluating the molecular status of nevus-associated melanomas in Latin America, as well as the largest in the literature comprising an extense panel of genes.
Background: Melanoma origin has always been a debated subject and the precursor role of pigmented lesions as well. Considering epidemiological and histological studies, melanoma seems to arise either in association with a pre-existing nevus or de novo, without any associated lesion. Nevus-associated melanoma and de novo melanomas seem to have different prognosis, when considering prognostic factors such as Breslow thickness. The real significance of benign melanocytic lesions remains controversial: it could be a fortuitous collision or melanoma and nevus cells could be clonally related, since most of melanomas and associated nevus share the same genetic profile in some studies, supporting a nevusto-melanoma progression model which predicts conservation of the driver mutation. Objective: To evaluate if melanoma and associated nevus share the same mutational profile or if it is distinct. To evaluate the clinico-epidemiological profile of nevus-associated melanomas in São Paulo-Brazil. Methods: Retrospective international (Brazil-Spain) cooperation study (1995-2004). Melanomas with associated nevi from a specialized Dermatopathology center, from Sao Paulo City, Sao Paulo State, Brazil, and from the Melanoma Unit of the Hospital Clinic of Barcelona, Spain were routinely processed and stained with hematoxilin-eosin. Brazilian cases and doubtful spanish cases were submitted to immunohistochemistry with antibodies MELAN_A, HMB45, Ki-67. Thus, cell selection bias was minimised as melanoma and nevus cells were selected by combining morphology and immunohistochemistry. The brazilian cases and the ones from the Melanoma Unit of the Hospital Clinic of Barcelona, in which the distinction between melanoma and nevus cell populations was unmistakable, were submitted to laser microdissection, amplification and sequencing of the following genes: BRAF exons 11 and 15, NRAS exons 2 and 3, cKIT exon 11, PPP6C exon 7, RAC1 exon 2 e STK19 exon 2. Each gene was evaluated twice in all samples by sequencing or by sequencing and another confirmation method, allelespecific fluorescent Polymerase Chain Reaction (PCR) and capillary electrophoresis detection, or by SNaPshot Analysis. Only mutations confirmed via two different molecular methods or twice by sequencing were considered positive. Results: Out of a total of 135,653 brazilian pathological records from skin biopsies of a 10-year period, 1190 melanoma records were selected. Nevus-associated melanomas corresponded to 390 melanomas (32.7%), being the association of thin melanomas with a nevus 1.52 times the association observed with thick melanomas (>1.01mm) (CI 95% [1.16;1.99], p<0.001). Superficial spreading melanoma was the most frequent, while no Lentigo Maligna melanoma was associated with nevi. The median Breslow thickness of nevus-associated melanomas was lower than that of de novo melanomas. Of 390 nevus-associated melanomas, 34 cases were selected for molecular analysis. Twenty-seven cases from the Melanoma Unit Of the Hospital Clinic of Barcelona were included. One case was excluded for absence of amplification products for the associated nevus. In 39 out of 60 cases (65%) at least one gene was mutated. BRAF 15 V600E mutation was the most frequent mutation found in melanomas (28/60; 46.6%) and nevus (31/60; 51.7%). Thirteen NRAS exon 3 (Q61K) were observed. No BRAF exon 11, NRAS exon 2, cKIT, PPP6C, STK19 or RAC1 mutations were observed. Sixty eight percent (41/60) of melanomas and associated nevi demonstrated similar genomic profile for all six genes evaluated. Conclusion: Nevusassociated melanomas represented one third of melanomas in a private dermatopathology center in São Paulo State, situated in Southeast Brazil, being associated with intermittent sun exposure, superficial spreading melanomas and lower Breslow thickness. The results from the present study corroborate the hypothesis that melanoma and associated nevus may be clonally related, given the high agreement of mutational status between the two lesions. This is, up to our knowledge, the first case series evaluating the molecular status of nevus-associated melanomas in Latin America, as well as the largest in the literature comprising an extense panel of genes.
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NASCIMENTO, Danielle Ioshimoto Shitara do. Estudo retrospectivo sobre a análise das mutações por extração de DNA de melanoma e nevo melanocítico associado. 2015. 189 f. Tese (Doutorado) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP), São Paulo, 2015.