Caracterización molecular del melanoma maligno mediante una aproximación genética y epigenética

El melanoma es un tumor procedente de los melanocitos cuya incidencia ha ido en ascenso en las últimas décadas. La clasificación clásica del melanoma establece cuatro subtipos principales: melanoma de extensión superficial, melanoma nodular, léntigo maligno melanoma, y melanoma lentiginoso acral. Esta clasificación se basa principalmente en la presentación clínica e histopatológica, y el patrón de crecimiento del melanoma; sin embargo, su utilidad es limitada ya que carece de valor pronóstico. El reciente desarrollo de nuevas tecnologías de secuenciación como la secuenciación masiva o next-generation sequencing (NGS), ha permitido realizar un cribado exhaustivo de las alteraciones moleculares implicadas en la etiopatogenia del melanoma, confirmando la implicación de genes previamente descritos (BRAF, NRAS, KIT, GNAQ, GNA11), e identificando nuevas mutaciones en otros genes (TERT, RAC1, ADAMTS18, EPHA7, ERBB4, PPP6C, TAF1L, NF1 e IDH1) que podrían tener implicaciones en el diagnóstico, pronóstico y tratamiento de los pacientes. . La mayoría de los estudios de NGS se basan en aproximaciones de alto coste y elevada complejidad, como WGS (Whole Genome Sequencing) y WES (Whole Exome Sequencing). La secuenciación de regiones exónicas específicas mediante la aplicación de paneles de genes específicos ha demostrado mayor utilidad en la práctica clínica por su menor coste y una mayor facilidad para la interpretación de los resultados; sin embargo, son escasos los diseños de paneles centrados únicamente en el estudio de los genes implicados en melanoma. Asimismo, se ha propuesto la implicación de los factores epigenéticos en la etiopatogenia del melanoma. Recientemente se han descrito hipermetilaciones anómalas en la región promotora de más de 100 genes, algunos de los cuales podrían tener valor pronóstico y de monitorización de respuesta. Respecto a la regulación post-transcripcional, se han identificado múltiples microRNAs implicados en la melanogénesis a partir de estudios de expresión en tejido y funcionales en líneas celulares. El objetivo de la presente Tesis Doctoral es realizar una caracterización molecular de una serie de melanomas mediante una aproximación genética y epigenética, para tratar de establecer una clasificación molecular que tenga valor diagnóstico, pronóstico y terapéutico. Para ello, se ha realizado un estudio observacional retrospectivo en 210 pacientes diagnosticados de melanoma. Se ha abordado el estudio de las alteraciones genéticas mediante NGS gracias al diseño de un panel específico de melanoma, así como el estudio de las alteraciones epigenéticas mediante el estudio de metilación en el promotor de genes supresores tumorales por MS-MLPA (amplificación múltiple empleando sonda dependiente de ligación sensible a metilación) y mediante el estudio de microRNAs mediante arrays de expresión y PCR cuantitativa en tiempo real (RT-PCR)

Association of TYR SNP rs1042602 with Melanoma Risk and Prognosis

Cutaneous melanoma is the most aggressive of skin tumors. In order to discover new biomarkers that could help us improve prognostic prediction in melanoma patients, we have searched for germline DNA variants associated with melanoma progression. Thus, after exome sequencing of a set of melanoma patients and healthy control individuals, we identified rs1042602, an SNP within TYR, as a good candidate. After genotyping rs1042602 in 1025 patients and 773 healthy donors, we found that the rs1042602-A allele was significantly associated with susceptibility to melanoma (CATT test: p = 0.0035). Interestingly, we also observed significant differences between patients with good and bad prognosis (5 years of follow-up) (n = 664) (CATT test for all samples p = 0.0384 and for men alone p = 0.0054). Disease-free-survival (DFS) analyses also showed that patients with the A allele had shorter DFS periods. In men, the association remained significant even in a multivariate Cox Proportional-hazards model, which was adjusted for age at diagnosis, Breslow thickness, ulceration and melanoma subtype (HR 0.4; 95% confidence interval (CI) 0.20–0.83; p = 0.0139). Based on our results, we propose that rs1042602-A is a risk allele for melanoma, which also seems to be responsible for a poorer prognosis of the disease, particularly in men

MiR-138-5p Suppresses Cell Growth and Migration in Melanoma by Targeting Telomerase Reverse Transcriptase

Recent evidence suggests the existence of a miRNA regulatory network involving human telomerase reverse transcriptase gene (hTERT), with miR-138-5p playing a central role in many types of cancers. However, little is known about the regulation of hTERT expression by microRNA (miRNAs) in melanocytic tumors. Here, we investigated the effects of miR-138-5p in hTERT regulation in melanoma cells lines. In vitro studies demonstrated higher miR-138-5p and lower hTERT messenger RNA (mRNA) expression in human epidermal melanocytes, compared with melanoma cell lines (A2058, A375, SK-MEL-28) by quantitative polymerase chain reaction (qPCR) observing a negative correlation between them. A2058 melanoma cells were selected to be transfected with miR-138-5p mimic or inhibitor. Using luciferase assay, hTERT was identified as a direct target of this miRNA. Overexpression of miR-138-5p detected by Western blot revealed a decrease in hTERT protein expression (p = 0.012), and qPCR showed a reduction in telomerase activity (p p = 0.035) and migration abilities (p = 0.015) were observed in A2058-transfected cells using thiazolyl blue tetrazolium bromide and flow cytometry, respectively. This study identifies miR-138-5p as a crucial tumor suppressor miRNA involved in telomerase regulation. Targeting it as a combination therapy with immunotherapy or targeted therapies could be used in advanced melanoma treatment; however, more preclinical studies are necessary

Multiple Primary Melanomas: Retrospective Review in a Tertiary Care Hospital

Multiple primary melanomas (MPM) refer to the occurrence of more than one synchronous or metachronous melanoma in the same individual. The aim of this study was to identify the frequency of MPM and describe the clinical and histopathologic characteristics of patients with MPM. An observational single-center retrospective study was designed based on a cohort of melanoma patients followed in a tertiary care hospital. Fifty-eight (8.9%) patients developed MPM. Most patients were men (65.5%) and the median age at the time of diagnosis of the first melanoma was 71 years old. The median time of diagnosis of the second melanoma from the first melanoma was 10.9 months, and 77.6% of second melanomas were diagnosed within the first 5 years. In total, 29 (50%) and 28 (48.3%) first and second melanomas were located in the trunk, respectively. Concordance of anatomic site between primary and subsequent melanoma was found in 46.6% of the patients. Proportion of in situ melanomas was increasingly higher in subsequent melanomas (from 36.21% of first melanomas to 100% of fifth melanomas). An increasing rate of melanomas with histological regression was observed within subsequent melanomas (from 60.3% of first melanomas to 80% of third melanomas). Our results support the importance of careful long-term follow-up with total body examination in melanoma patients

Association of TYR SNP rs1042602 with Melanoma Risk and Prognosis

Cutaneous melanoma is the most aggressive of skin tumors. In order to discover new biomarkers that could help us improve prognostic prediction in melanoma patients, we have searched for germline DNA variants associated with melanoma progression. Thus, after exome sequencing of a set of melanoma patients and healthy control individuals, we identified rs1042602, an SNP within TYR, as a good candidate. After genotyping rs1042602 in 1025 patients and 773 healthy donors, we found that the rs1042602-A allele was significantly associated with susceptibility to melanoma (CATT test: p = 0.0035). Interestingly, we also observed significant differences between patients with good and bad prognosis (5 years of follow-up) (n = 664) (CATT test for all samples p = 0.0384 and for men alone p = 0.0054). Disease-free-survival (DFS) analyses also showed that patients with the A allele had shorter DFS periods. In men, the association remained significant even in a multivariate Cox Proportional-hazards model, which was adjusted for age at diagnosis, Breslow thickness, ulceration and melanoma subtype (HR 0.4; 95% confidence interval (CI) 0.20–0.83; p = 0.0139). Based on our results, we propose that rs1042602-A is a risk allele for melanoma, which also seems to be responsible for a poorer prognosis of the disease, particularly in men