Expression of CD28, FAS, PD1, and CTLA4 molecules in the blood of women with triple-negative breast cancer.

BACKGROUND: Locally advanced triple-negative breast cancer (TNBC) represents a public health problem in Brazil. Its standard treatment consists of neoadjuvant chemotherapy (NAC). METHODS: This was a longitudinal study with follow-up performed between the years 2015 and 2017. Thirty women with locally advanced TNBC submitted to NAC, and 30 healthy were included. Peripheral blood samples were collected before NAC (Pre-NAC) and after NAC (Post-NAC). RESULTS: Patients with TNBC had elevated levels of CD28+ T, FAS+ T, CTLA4+ T, PD1+ T, CD28+CD4+ T, PD1+CD4+ T and CD8+ T and PD1+ CD8+ T cells compared to controls (p < 0.05). Patients with pathological complete response (pCR) had low FAS+ T cells, FAS+CD4+ T cells, and PD1+CD8+ T cells compared to the non-pCR (p < 0.05). Significant differences were observed in the levels of CD28+ T cells, FAS+ T and PD1+ T, CD4+ T, CD28+CD4+ T, FAS+CD4+ T, PD1+CD4+ T, CD8+ T, and PD1+CD8+ T cells between Pre-NAC and Post-NAC groups (p < 0.05). CONCLUSION: Alterations in the circulating FAS+CD4+ T and PD1+CD8+ T cell levels Pre-NAC are associated with pCR, suggesting potential predictive biomarkers of NAC response in TNBC. The largest changes in the cellular immune response profile Post-NAC showed that chemotherapy treatment can modulate the immune response and that it is associated with prognosis in TNBC.

Avaliação dos efeitos da quimioterapia neoadjuvante na resposta imune celular nos tumores de mama triplo-negativos localmente avançados / Evaluation of the effects of neoadjuvant chemotherapy on the cellular immune response in locally advanced triple-negative breast tumors

Introdução: O câncer de mama triplo-negativo (CMTN) localmente avançado representa um problema de saúde pública no Brasil; e seu tratamento padrão consiste em quimioterapia neoadjuvante, com taxa de sucesso extremamente variável a depender do acesso ao tratamento oncológico adequado e do tipo de resposta patológica. Na última década, o papel do sistema imune no câncer de mama tem ganhado espaço com a demonstração do impacto favorável do infiltrado tumoral de linfócitos (TILs) e expressão gênica de resposta imune, principalmente em tumores de alta taxa de proliferação e receptor de estrógeno negativo. Objetivo: avaliar a resposta imune celular em mulheres com câncer de mama triplo-negativo localmente avançado, e submetidas a quimioterapia neoadjuvante. Métodos: estudo longitudinal com seguimento realizado entre os anos de 2015 e 2017. O estudo foi realizado no Hospital de Câncer de Pernambuco (HCP) e Laboratório de Pesquisa Translacional do Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Foram incluídas 30 mulheres, entre 18 e 60 anos de idade, com diagnóstico de CMTN localmente avançado e submetidas a quimioterapia neoadjuvante; e como grupo controle de comparação, 30 mulheres saudáveis com idade entre 18 a 60 anos sem diagnóstico prévio ou atual, e histórico familiar de CM. Coletas de sangue periférico foram realizadas antes e após a quimioterapia neoadjuvante (NAC). As análises dos níveis percentuais de TCD3+, TCD4+ e TCD8+ com expressão de CD28, FAS, PD1 e CTLA4 foram realizadas por citometria de fluxo. O teste de múltiplas comparações não paramétrico de Kruskall-wallis foi utilizado na análise de associação. Para análise pareada pré e pós tratamento, foi utilizado o teste não paramétrico de Wilcoxon. Para análise de sobrevida livre de doença (SLD), foi utilizado o teste de Log-Rank para comparação entre os grupos. Foi adotado o nível de significância estatística de p<0.05. A análise estatística foi realizada através do programa graphpad prism v6.0. Resultados: Verificou-se níveis elevados de CD3+ T CD28+, CD4+ T CD28+, CD3+ T PD1+ e CD3+ T CTLA4+ no sangue periférico dos grupos de mulheres que obteve resposta patológica completa (RC) e parcial (RP) ao tratamento neoadjuvante quando comparado ao grupo controle (p<0,05). Foram observados níveis elevados de TCD4+FAS+ no grupo de pacientes com RP ao tratamento neoadjuvante quando comparado aos grupos com RC e controle (p<0,05). Foram observados níveis mais elevados de TCD8+PD1+ no grupo de pacientes com RP comparado aos grupos com RC e controle (p<0,05). Foram encontrados níveis elevados nos índices obtidos da relação CD4+ TCD28+/CD8+ TCD28+ nos grupos de mulheres que obteve RP quando comparado aos grupos com RC e de controle. Níveis elevados da relação CD4+ TPD1+/CD8+ TPD1+ nos grupos CMTN que obteve RC e RP ao tratamento neoadjuvante quando comparado ao grupo controle (p<0,05). Verificou-se diferença significativa na análise pareada dos níveis percentuais de TCD4+ TCD8+ antes e após NAC. Também se verificou diferença significativa dos níveis de TCD3+ e TCD4+ com expressão de CD28, FAS e PD1+ e de TCD8+PD1+ antes e após no sangue periférico das mulheres com CMTN antes e após NAC. A sobrevida livre de doença em 24 meses foi de 52,1% no grupo de pacientes com níveis percentuais de TCD3 < 68,0 e de 100% no grupo com CD3+ T ≥ 68,8 (p=0,007), e de 54,1% no grupo de pacientes com níveis percentuais de TCD4+FAS+ ≥ 44,5 e de 100% no grupo com TCD4+FAS+ < 44,5 (p=0,02). Com relação TCD4+ PD1+, a sobrevida livre de doença foi 69,5% no grupo com níveis percentuais de TCD4+ PD1+ ≥ 4,5 e de 100% no grupo TCD4+PD1+ < 4,5 (p=0,01). Conclusão: Conclui-se que os níveis TCD4+/FAS+ e TCD8+/PD1+ circulantes antes do tratamento estão associadas a RC, o que sugere ser potenciais biomarcadores preditivos de resposta a NAC. As alterações nos níveis circulantes de TCD3+, TCD4+FAS+ e TCD4+PD1+ após NAC estão a associadas a sobrevida livre de doença, o que sugere que essas populações celulares e os receptores de inibição da resposta imune, FAS e PD1, estão envolvidos no prognóstico do CMTN

LLocally advanced triple-negative breast cancer (TNBC) represents a public health problem in Brazil. Its standard treatment consists of neoadjuvant chemotherapy, with an extremely variable success rate depending on access to adequate cancer treatment and the type of pathological response. In the last decade, the role of the immune system in breast cancer has gained space with the demonstration of the favorable impact of lymphocyte tumor infiltration (TILs) and genetic expression of immune response, especially in tumors with high proliferation rate and estrogen receptor negative. Objective: to evaluate the cellular immune response in women with locally advanced triple-negative breast cancer, and to undergo neoadjuvant chemotherapy. METHODS: This was a longitudinal study with follow-up performed between the years 2015 and 2017. The study was conducted at the Hospital de Cancer de Pernambuco and Translational Research Laboratory of the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP). Thirty women aged between 18 and 60 years with a diagnosis of locally advanced CMTN and submitted to neoadjuvant chemotherapy were included, and 30 healthy women aged 18 to 60 years without previous or current diagnosis and family history of TNBC were included as control group. Peripheral blood samples were collected before and after neoadjuvant chemotherapy (NAC). The analysis of the percentage levels of CD3+ T, TCD4 + and TCD8 + with expression of CD28, FAS, PD1 and CTLA4 were performed by flow cytometry. The non-parametric Kruskall-wallis multiple-comparison test was used in association analysis. For pre-and post-treatment paired analysis, the non-parametric Wilcoxon test was used. For disease-free survival analysis (SLD), the Log-Rank test was used to compare the groups. The level of statistical significance of p <0.05 was adopted. Statistical analysis was performed using the graphpad prism v6.0 program (Graphpad software, San Diego, CA). Results: High levels of CD28+ CD3+ T, CD28+CD4+ T, PD1 +CD3+T and CD3+ T CTLA4 + were observed in the peripheral blood of the groups of women with complete pathological response (CPR) and no CPR to the neoadjuvant treatment when compared to the control group (p < 0.05). High levels of FAS+CD4+ T were observed in the group of patients with RP to the neoadjuvant treatment when compared to the CR and control groups (p <0.05). Higher levels of PD1+CD8+ T were observed in the group of patients with no CRP compared to the control and CRP groups (p <0.05). High levels were found in the indices obtained from the CD28+ CD4+T / CD28+ CD8 +T ratio in the groups of women who had when compared to the RC and control groups. High levels of the PD1 + CD4 + T / PD1+ CD8+ T ratio in the CMTN groups, which obtained CPR and no CPR to the neoadjuvant treatment when compared to the control group (p <0.05). There was a significant difference in the paired analysis of the percent levels of CD4+ T and CD8+ T before and after NAC. There was also a significant difference in CD3+ T and CD4+ T levels with expression of CD28, FAS and PD1 and PD1+ CD8+ T before and after NAC in the peripheral blood of women with CMTN. The 24-month disease-free survival rate was 52.1% in the group of patients with CD3+T <68.0 and 100% in the CD3+ T ≥ 68.8 (p=0.007) and 54.1 % in the group of patients with FAS+ CD4+T ≥ 44.5 and 100% in the group with FAS+ CD4+T <44.5 (p = 0.02). With respect to PD1+ CD4+T disease-free survival was 69.5% in the group with PD1+ CD4+T ≥ 4.5 and 100% in the PD1+ CD4+T <4.5 (p = 0.01) groups. Conclusion: It is concluded that pre-treatment FAS+ CD4+ T and PD1+ CD8+ T levels are associated with CPR, suggesting potential biomarkers predictive of NAC response. Changes in the circulating levels of CD3+ T, FAS+ CD4+T and PD1+ CD4 +T after NAC are associated with disease free survival, suggesting that these cellular populations and immune response inhibitory receptors, FAS and PD1, are involved in the CMTN prognosis