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Identifying potential predictive and prognostic immune biomarkers in cutaneous melanoma
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RESUMO: O melanoma cutâneo (MC) é a forma mais agressiva de cancro da pele e constitui um problema crescente de saúde pública a nível mundial. Apesar de representar menos de 5% dos casos de cancro da pele, o MC é responsável por mais de 60% das mortes associadas a este tipo de cancro devido ao seu elevado potencial metastático. Os avanços nas terapias alvo e nos inibidores de checkpoints imunológicos (ICIs) transformaram o seguimento clínico do doente com MC, proporcionando responstas duradouras em alguns doentes. No entanto, cerca de metade dos doentes com MC continua sem responder a estas terapias, e continuam a faltar biomarcadores fiáveis para prever a resposta à terapêutica. Paralelamente, as atuais estratégias de estratificação de risco são insuficientes, uma vez que um número significativo de doentes inicialmente classificados como de baixo risco acaba por apresentar recorrência e progressão da doença. Assim, existe uma necessidade urgente de biomarcadores de prognóstico e preditivos, bem como de uma melhor compreensão dos mecanismos relacionados com a agressividade do MC e com a resistência à terapêutica, de modo a possibilitar o desenvolvimento de novas terapias.
Para abordar o primeiro desafio e identificar biomarcadores em circulação, baseados em características imunológicas, que permitam melhor estratificar os doentes com MC e a escolha da terapia mais adequada, analisámos por citometria de fluxo as células mononucleares do sangue periférico (PBMCs) de 54 doentes com MC, não tratados previamente (maioritariamente em estádio III/IV). A comparação das frequências das diferentes populações de células imunológicas e da expressão de marcadores funcionais com dadores saudáveis revelou perfis globalmente comparáveis, mas destacou elevada variabilidade entre os doentes e alterações funcionais envolvendo marcadores de ativação das células T no MC avançado. A análise de sobrevivência identificou características imunológicas com forte potencial de prognóstico. Uma maior expressão de HLA-DR e CD69 em células T CD4+ em circulação, juntamente com uma menor frequência de células T CD8+ circulantes, esteve associada a um aumento da sobrevivência global (SG), sendo que o marcador HLA-DR também se correlacionou com uma sobrevivência livre de eventos (SLE) mais prolongada. Modelos de prognóstico, obtidos através da regressão de Cox, que combinaram estas variáveis imunológicas com uma pontuação de risco clínico composta, estratificaram com precisão os doentes em função da SG (89,5% de sensibilidade; 72,7% de especificidade; AUC = 0.872, p < 0.0001) e do risco de progressão ou recidiva (75% de sensibilidade; 71,4% de especificidade; AUC = 0.763, p = 0.001), superando os modelos baseados apenas em características clinicopatológicas. Estes resultados identificaram a expressão de HLA-DR e CD69 em células T CD4+ em circulação e a frequência de células T CD8+ circulantes como potenciais biomarcadores prognósticos em MC.
Nos 43 doentes que posteriormente receberam tratamento com ICIs, a expressão basal de HLA-DR e CD69 em células T CD4+ circulantes também demostrou valor preditivo para o benefício clínico desta terapêutica. Um modelo de regressão logística preditivo que incorporou estes marcadores juntamente com variáveis clínicas demonstrou uma capacidade discriminatória moderada (AUC = 0.775, p = 0.0027), prevendo benefício clínico com 65,2% de sensibilidade e 88,9% de especificidade, e superando o modelo baseado apenas em variáveis clínicas. No entanto, a análise longitudinal mostrou utilidade limitada para monitorização em tempo real durante o tratamento. Estes resultados sugerem que a ativação sistémica de células T CD4+ pode constituir uma ferramenta para prever quais os doentes que beneficiarão dos ICIs antes de iniciarem o tratamento.
O perfil de expressão génica tumoral de 26 doentes com MC identificou ainda uma assinatura distinta relacionada com o interferão-gama (IFN-γ), associada a uma SG e SLE mais longas. Embora a sinalização do IFN-γ já tenha sido amplamente associada à resposta a ICIs, o seu papel como fator de prognóstico independente do tratamento não está tão bem estabelecido. Assim, estes resultados consolidaram os perfis de expressão génica relacionados com o IFN-γ como uma ferramente de prognóstico no MC. Além disso, destacam a importância da ativação imunológica tanto a nível sistémico como a nível tumoral na determinação dos desfechos clínicos, reforçando o valor de integrar biomarcadores provenientes do sangue e do tecido para captar de forma mais completa as interações entre o tumor e o sistema imunológico.
Para abordar o segundo desafio de clarificar os mecanismos de agressividade do MC e da resistência às terapêuticas, estabelecemos e caracterizámos uma nova linha celular de MC, MelT79, derivada de um doente inicialmente diagnosticado em estádio IB (baixo risco), mas que apresentou progressão da doença até ao estádio IV, apesar de múltiplas estratégias terapêuticas. A análise citogenética revelou um cariótipo complexo com um genoma altamente desequilibrado, incluindo alterações em genes-chave como CDKN2A/B, SPRED1, e B2M, relacionados com a melanomagénese e a resistência à terapêutica. Fenotipicamente, MelT79 demonstrou uma elevada heterogeneidade e plasticidade fenotípica, exibindo características tanto do fenótipo melanocítico/proliferativo, como do fenótipo indiferenciado/invasivo, com expressão intermédia de MITF. Além disso, as células MelT79 são completamente amelanóticas, sugerindo alterações na biogénese dos melanosomas ou no seu transporte, em concordância com a evidência de que as células invasivas de MC frequentemente aumentam a secreção de melanina para favorecer um fenótipo menos rígido, e mais elástico e invasivo. A nível proteico, a expressão de MITF mostrou-se altamente heterogénea, confirmando a coexistência de múltiplos estados fenotípicos nesta linha celular, promovendo a resistência às terapias e a progressão do MC. Desse modo, MelT79 apresentou sensibilidade moderada a inibidores de BRAF e MEK quando comparada com outras linhas celulares comerciais, e o perfil imunológico circulante do doente MelT79 esteve associado a mau prognóstico e a benefício limitado dos ICIs. Importa salientar que as células MelT79 apresentaram uma mutação rara, RET S649L, nunca previamente descrita em MC, mas que também foi identificada numa metástase anterior, possivelmente conferindo uma vantagem seletiva, e salientando esta mutação como potencial alvo terapêutico. Ao refletir tanto a trajetória clínica do doente como as principais características biológicas de progressão do MC, a linha MelT79 constitui uma valiosa adição ao repertório de modelos celulares de MC derivados de doentes.
Em suma, os nossos resultados demonstram que a ativação imunológica sistémica, captada pela técnica simples de citometria de fluxo, e as assinaturas de expressão génica tumoral fornecem ferramentas com valor prognóstico e preditivo relevantes em MC. A identificação da expressão de HLA-DR e CD69 em células T CD4+ como biomarcadores candidatos reforça o papel central das células T CD4+ na imunidade antitumoral e na resposta aos ICIs. Além disso, o estabelecimento da linha celuar MelT79 fornece um modelo in vitro versátil para estudar mais profundamente a heterogeneidade do MC, as interações imunológicas, e a resistência às terapias. Por fim, mostramos que a imunidade sistémica e as características do tumor atuam em conjunto para moldar a progressão do MC, sugerindo que a adição da competência imunológica ao estádio e ao status mutacional pode permitir uma gestão mais personalizada dos doentes e melhorar os desfechos clínicos.
ABSTRACT: Cutaneous melanoma (CM) is the most aggressive form of skin cancer and represents a growing global public health issue. Despite representing less than 5% of skin cancer cases, it is responsible for over 60% of skin cancer-related deaths due to its high metastatic potential. Advances in targeted therapies and immune-checkpoint inhibitors (ICIs) have transformed clinical management, providing durable responses for some patients. However, approximately half of CM patients still fail to respond, and reliable biomarkers to predict therapeutic response remain lacking. In parallel, current risk stratification strategies are insufficient, as a significant number of patients initially classified as low-risk experience disease recurrence and progression. Thus, there is an urgent need for reliable prognostic and predictive biomarkers, along with a deeper understanding of the mechanisms underlying CM aggressiveness and therapeutic resistance to enable the development of new treatment strategies. To address the first challenge and identify circulating immune-based biomarkers for improved patient stratification and therapeutic decision-making, we analysed peripheral blood mononuclear cells (PBMCs) from 54 treatment-naïve CM patients (mainly stages III/IV) using flow cytometry. Comparison of immune cell frequencies and functional marker expression with healthy donors revealed broadly comparable profiles, but highlighted substantial inter-patient variability and functional shifts involving T cell activation markers in advanced CM. Survival analysis uncovered immune features with strong prognostic potential. Higher HLA-DR and CD69 expression on circulating CD4+ T cells, together with lower circulating CD8+ T cell frequency, were associated with improved overall survival (OS), and HLA-DR further correlated with longer event-free survival (EFS). Prognostic Cox regression models combining these immune variables with a composite clinical risk score accurately stratified patients by OS (89.5% sensitivity, 72.7% specificity; AUC = 0.872, p < 0.0001) and progression or recurrence risk (75% sensitivity and 71.4% specificity; AUC = 0.763, p = 0.001), outperforming models based solely on clinicopathological features. These findings identified HLA-DR and CD69 expression on circulating CD4+ T cells and the frequency of circulating CD8+ T cells as potential prognostic biomarkers in CM. In the 43 patients who subsequently received ICI treatment, baseline HLA-DR and CD69 expression on circulating CD4+ T cells also predicted clinical benefit from ICIs. A predictive logistic regression model incorporating these markers with clinical covariates achieved moderate discrimination (AUC = 0.775, p = 0.0027), predicting benefit with 65.2% sensitivity and 88.9% specificity, and outperforming a model based on clinical variables alone. Longitudinal analysis, however, showed limited utility for real-time monitoring during treatment. These findings suggest that systemic CD4+ T cell activation provides a tool that helps predict which patients will respond to ICIs before treatment initiation. Tumour gene expression profiling in 26 CM patients further identified a distinct interferon-gamma (IFN-γ)-related signature associated with longer OS and EFS. While IFN-γ signalling has been widely associated with ICI response, its role as a prognostic factor independent of treatment is less well established. Therefore, these findings consolidated IFN-γ-related gene expression profiles as a prognostic tool in CM. Moreover, they highlight the importance of both systemic and tumour-level immune activation in shaping patient outcomes, reinforcing the value of integrating blood- and tissue-based biomarkers to fully capture tumour-immune interactions. To address the second challenge of clarifying mechanisms of CM aggressiveness and therapeutic resistance, we established and characterized a novel patient-derived CM cell line, MelT79, derived from a patient initially diagnosed at stage IB (low-risk) who nonetheless experienced disease progression to stage IV, despite multiple treatment strategies. Cytogenetic analysis revealed a complex karyotype with a highly unbalanced genome, including alterations affecting key genes such as CDKN2A/B, SPRED1, and B2M, implicated in melanomagenesis and therapy resistance. Phenotypically, MelT79 demonstrated high heterogeneity and phenotypic plasticity, displaying traits of both melanocytic/proliferative and undifferentiated/invasive phenotypes, with intermediate MITF expression. Moreover, MelT79 cells are completely amelanotic, suggesting impaired melanosome biogenesis or alterations in its trafficking, in agreement with evidence that invasive CM cells often upregulate melanin secretion to favour a less stiff, more elastic invasive phenotype. At the protein level, MITF expression was highly heterogeneous, confirming the coexistence of multiple phenotypic states within the culture, promoting resistance to therapy and CM progression. Accordingly, MelT79 exhibited moderate sensitivity to BRAF and MEK inhibitors when compared to other commercially available cell lines, and the circulating immune profile from the MelT79 patient was linked to poor prognosis and limited benefit from ICIs. Importantly, MelT79 carried a rare RET S649L mutation, not previously reported in CM, but also identified in an earlier metastatic lesion, possibly conferring a selective advantage, and highlighting this mutation as a potential therapeutic target. By capturing both the clinical trajectory of the patient and the biological hallmarks of CM progression, MelT79 is a valuable addition to the repertoire of patient-derived CM cell models. Our findings collectively demonstrate that systemic immune activation, captured through simple flow cytometry, and tumour-derived gene expression signatures both provide meaningful prognostic and predictive tools in CM. The identification of HLA-DR and CD69 expression on CD4+ T cells as candidate biomarkers underscores the central role of CD4+ T cells in anti-tumour immunity and ICI response. Furthermore, the establishment of the MelT79 cell line provides a versatile in vitro model to further study CM heterogeneity, immune interactions, and therapeutic resistance. Ultimately, we show that systemic immunity and tumour features jointly shape CM progression, suggesting that incorporating immune competence alongside stage and mutational status could enable more personalized patient management and improved patient outcomes.
ABSTRACT: Cutaneous melanoma (CM) is the most aggressive form of skin cancer and represents a growing global public health issue. Despite representing less than 5% of skin cancer cases, it is responsible for over 60% of skin cancer-related deaths due to its high metastatic potential. Advances in targeted therapies and immune-checkpoint inhibitors (ICIs) have transformed clinical management, providing durable responses for some patients. However, approximately half of CM patients still fail to respond, and reliable biomarkers to predict therapeutic response remain lacking. In parallel, current risk stratification strategies are insufficient, as a significant number of patients initially classified as low-risk experience disease recurrence and progression. Thus, there is an urgent need for reliable prognostic and predictive biomarkers, along with a deeper understanding of the mechanisms underlying CM aggressiveness and therapeutic resistance to enable the development of new treatment strategies. To address the first challenge and identify circulating immune-based biomarkers for improved patient stratification and therapeutic decision-making, we analysed peripheral blood mononuclear cells (PBMCs) from 54 treatment-naïve CM patients (mainly stages III/IV) using flow cytometry. Comparison of immune cell frequencies and functional marker expression with healthy donors revealed broadly comparable profiles, but highlighted substantial inter-patient variability and functional shifts involving T cell activation markers in advanced CM. Survival analysis uncovered immune features with strong prognostic potential. Higher HLA-DR and CD69 expression on circulating CD4+ T cells, together with lower circulating CD8+ T cell frequency, were associated with improved overall survival (OS), and HLA-DR further correlated with longer event-free survival (EFS). Prognostic Cox regression models combining these immune variables with a composite clinical risk score accurately stratified patients by OS (89.5% sensitivity, 72.7% specificity; AUC = 0.872, p < 0.0001) and progression or recurrence risk (75% sensitivity and 71.4% specificity; AUC = 0.763, p = 0.001), outperforming models based solely on clinicopathological features. These findings identified HLA-DR and CD69 expression on circulating CD4+ T cells and the frequency of circulating CD8+ T cells as potential prognostic biomarkers in CM. In the 43 patients who subsequently received ICI treatment, baseline HLA-DR and CD69 expression on circulating CD4+ T cells also predicted clinical benefit from ICIs. A predictive logistic regression model incorporating these markers with clinical covariates achieved moderate discrimination (AUC = 0.775, p = 0.0027), predicting benefit with 65.2% sensitivity and 88.9% specificity, and outperforming a model based on clinical variables alone. Longitudinal analysis, however, showed limited utility for real-time monitoring during treatment. These findings suggest that systemic CD4+ T cell activation provides a tool that helps predict which patients will respond to ICIs before treatment initiation. Tumour gene expression profiling in 26 CM patients further identified a distinct interferon-gamma (IFN-γ)-related signature associated with longer OS and EFS. While IFN-γ signalling has been widely associated with ICI response, its role as a prognostic factor independent of treatment is less well established. Therefore, these findings consolidated IFN-γ-related gene expression profiles as a prognostic tool in CM. Moreover, they highlight the importance of both systemic and tumour-level immune activation in shaping patient outcomes, reinforcing the value of integrating blood- and tissue-based biomarkers to fully capture tumour-immune interactions. To address the second challenge of clarifying mechanisms of CM aggressiveness and therapeutic resistance, we established and characterized a novel patient-derived CM cell line, MelT79, derived from a patient initially diagnosed at stage IB (low-risk) who nonetheless experienced disease progression to stage IV, despite multiple treatment strategies. Cytogenetic analysis revealed a complex karyotype with a highly unbalanced genome, including alterations affecting key genes such as CDKN2A/B, SPRED1, and B2M, implicated in melanomagenesis and therapy resistance. Phenotypically, MelT79 demonstrated high heterogeneity and phenotypic plasticity, displaying traits of both melanocytic/proliferative and undifferentiated/invasive phenotypes, with intermediate MITF expression. Moreover, MelT79 cells are completely amelanotic, suggesting impaired melanosome biogenesis or alterations in its trafficking, in agreement with evidence that invasive CM cells often upregulate melanin secretion to favour a less stiff, more elastic invasive phenotype. At the protein level, MITF expression was highly heterogeneous, confirming the coexistence of multiple phenotypic states within the culture, promoting resistance to therapy and CM progression. Accordingly, MelT79 exhibited moderate sensitivity to BRAF and MEK inhibitors when compared to other commercially available cell lines, and the circulating immune profile from the MelT79 patient was linked to poor prognosis and limited benefit from ICIs. Importantly, MelT79 carried a rare RET S649L mutation, not previously reported in CM, but also identified in an earlier metastatic lesion, possibly conferring a selective advantage, and highlighting this mutation as a potential therapeutic target. By capturing both the clinical trajectory of the patient and the biological hallmarks of CM progression, MelT79 is a valuable addition to the repertoire of patient-derived CM cell models. Our findings collectively demonstrate that systemic immune activation, captured through simple flow cytometry, and tumour-derived gene expression signatures both provide meaningful prognostic and predictive tools in CM. The identification of HLA-DR and CD69 expression on CD4+ T cells as candidate biomarkers underscores the central role of CD4+ T cells in anti-tumour immunity and ICI response. Furthermore, the establishment of the MelT79 cell line provides a versatile in vitro model to further study CM heterogeneity, immune interactions, and therapeutic resistance. Ultimately, we show that systemic immunity and tumour features jointly shape CM progression, suggesting that incorporating immune competence alongside stage and mutational status could enable more personalized patient management and improved patient outcomes.
Descrição
Palavras-chave
Cutaneous melanoma Immune-checkpoint inhibitors Immunotherapy Prognostic biomarkers Predictive biomarkers CD4+ T cell activation HLA-DR CD69 CD8+ T cells IFN-γ signalling Primary cell culture Targeted therapy Phenotypic plasticity Intra-tumour heterogeneity