Anti-PD-1 and anti-PD-l1 antibodies as immunotherapy against cancer: a structural perspective

ABSTRACT Programmed cell death protein 1 (PD-1) and its ligand, programmed death-ligand-1 (PD-L1), play key roles in the suppression of the cytotoxic activity of T cells. PD-L1 is overexpressed on various types of cancer cells, leading to immune evasion. In the past decade, therapeutic antibodies that target the PD-1/PD-L1 axis have been developed to inhibit the immune suppression triggered by these two proteins. At present, five antibodies (two anti-PD-1 and three anti-PD-L1) have received approval by regulatory agencies in the US and Europe. In this work, we aimed to review their clinical applications and adverse effects. Furthermore, using their reported crystal structures, we discuss the similarities and differences between the PD-1/PD-L1 interface and the epitopes that are recognized by the antibodies. Detailed analyses of the contact residues involved in the ligand-receptor and target-antibody interactions have shown partial overlap. Altogether, the data presented here demonstrate that: (1) in contrast to other therapeutic antibodies, anti-PD-1/PD-L1 has a wide range of clinical applications; (2) these targeted therapies are not exempt from adverse effects; and (3) the characterization of the structural domains that are recognized by the antibodies can guide the development of new PD-1- and PD-L1-blocking agents. (REV INVEST CLIN. 2021;73(1):8-16)

Prospective evaluation of EBUS-TBNA specimens for programmed death-ligand 1 expression in non-small cell lung cancer patients: a pilot study / Avaliação prospectiva de espécimes obtidos por meio de EBUS-TBNA quanto à expressão de programmed death-ligand 1 em pacientes com câncer pulmonar de células não pequenas: um estudo piloto

ABSTRACT Objective: EBUS-TBNA cytological sampling is routinely performed for pathological diagnosis, mediastinal staging, and molecular testing in lung cancer patients. EBUS-TBNA samples are not formally accepted for testing programmed death-ligand 1 (PD-L1) expression. The objective of the study was to compare the feasibility, reproducibility, and accuracy of PD-L1 expression assessment in cytological specimens and histological samples. Methods: We prospectively collected histological (transbronchial forceps biopsy) and cytological (EBUS-TBNA) samples from peribronchial neoplastic lesions during an endoscopic procedure at the same target lesion for the pathological diagnosis and molecular assessment of stage IV non-small cell lung cancer (NSCLC). Results: Fifteen patients underwent the procedure. Adequate cytological samples (at least 100 neoplastic cells) were obtained in 12 cases (92.3%). Assessment of PD-L1 expression was similar between histological and cytological samples (agreement rate = 92%). Sensitivity and diagnostic accuracy of EBUS-TBNA cytological specimens were 88.9% and 100%, respectively. Conclusions: The evaluation of PD-L1 expression in EBUS-TBNA cytological specimens is feasible and presents good reproducibility when compared with routine histological samples. EBUS-TBNA cytological samples could be used for the assessment of PD-L1 expression in patients with NSCLC as a minimally invasive approach in stage IV NSCLC cancer patients.

RESUMO Objetivo: A amostragem citológica por meio de EBUS-TBNA é realizada rotineiramente para diagnóstico anatomopatológico, estadiamento mediastinal e teste molecular em pacientes com câncer de pulmão. As amostras obtidas por meio de EBUS-TBNA não são formalmente aceitas para testar a expressão da proteína programmed death-ligand 1 (PD-L1, ligante de morte celular programada 1). O objetivo do estudo foi comparar a viabilidade, reprodutibilidade e precisão da avaliação da expressão de PD-L1 em espécimes citológicos e amostras histológicas. Métodos: Foram coletadas prospectivamente amostras histológicas (obtidas por meio de biópsia transbrônquica com pinça) e citológicas (obtidas por meio de EBUS-TBNA) de lesões neoplásicas peribrônquicas durante um procedimento endoscópico na mesma lesão-alvo para o diagnóstico anatomopatológico e avaliação molecular de câncer pulmonar de células não pequenas (CPCNP) em estágio IV. Resultados: Quinze pacientes foram submetidos ao procedimento. Amostras citológicas adequadas (pelo menos 100 células neoplásicas) foram obtidas em 12 casos (92,3%). A expressão de PD-L1 nas amostras histológicas e citológicas foi semelhante (taxa de concordância = 92%). A sensibilidade e precisão diagnóstica das amostras citológicas obtidas por meio de EBUS-TBNA foram de 88,9% e 100%, respectivamente. Conclusões: A avaliação da expressão de PD-L1 em espécimes citológicos obtidos por meio de EBUS-TBNA é viável e apresenta boa reprodutibilidade quando comparada com amostras histológicas rotineiras. Amostras citológicas obtidas por meio de EBUS-TBNA podem ser usadas para avaliar a expressão de PD-L1 como uma abordagem minimamente invasiva em pacientes com CPCNP em estágio IV.

Immunotherapy treatment against cervical cancer

ABSTRACT Cervical cancer (CC) is one of the most common gynecological tumors and an important health problem, especially in developing countries. The vast majority of patients in early stages are cured of the disease with surgical treatment and with concomitant chemoradiotherapy in locally advanced stages. However, in patients with recurrent, persistent, or metastatic cervical CC, the effectiveness of treatment is limited, except for the combination of chemotherapy based on platinum doublets plus bevacizumab, the treatment that has achieved the best results to date. Programmed cell death-1/PD ligand-1 (PD-1/PD-L1) inhibitors could be a novel and cutting-edge therapeutic option to improve clinical outcomes in this group of patients. Thus far, there are a few Phase I/II clinical trials that have assessed the usefulness of pembrolizumab and nivolumab in this group of patients; these include the KEYNOTE 028, KEYNOTE 158, and CHECKMATE 358 trials, in which clinical benefit has been proven with PD-1/PD-L1 inhibitors in recurrent, persistent, or metastatic CC, as second-line treatment. There are also some ongoing trials that could provide further evidence on the PD-1/PD-L1 pathway as a therapeutic target in CC. In this review, we will focus on the usefulness of these PD-1/PDL1 inhibitors in CC, as well as on trials that are still in the recruitment phase, to confirm their effectiveness in this clinical setting.

Molecular profile of non-small cell lung cancer in northeastern Brazil / Perfil molecular do carcinoma pulmonar de células não pequenas no Nordeste brasileiro

ABSTRACT Objective: To investigate the histological subtypes and mutational profiles of non-small cell lung cancer in Brazil, looking for correlations among histological subtypes, expression of anaplastic lymphoma kinase (ALK), EGFR mutation status, and programmed death-ligand 1 (PD-L1) expression. Methods: We evaluated 173 specimens obtained from patients with lung adenocarcinoma in northeastern Brazil. Expression of PD-L1 and ALK was evaluated by immunohistochemistry; EGFR mutation status was evaluated by sequencing. We categorized the histological subtypes in accordance with the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Results: The most common histological subtypes of lung adenocarcinoma were solid predominant (in 46.8%), acinar predominant (in 37.0%), and lepidic predominant (in 9.8%). ALK expression was detected in 10.4% of the samples, and 22.0% of the tumors harbored EGFR mutations. The most common EGFR mutation was an exon 21 L858R point mutation (in 45.5%), followed by an exon 19 deletion (in 36.3%). The tumor proportion score for PD-L1 expression was ≥ 50% in 18.2% of the samples, 1-49% in 32.7%, and 0% in 49.5%. The solid predominant subtype was significantly associated with wild-type EGFR status (p = 0.047). Positivity for PD-L1 expression was not found to be significantly associated with ALK expression or EGFR mutation status. Conclusions: Our results suggest that the molecular profile of non-small cell lung cancer in northeastern Brazil differs from those of populations in other regions of the country, with ALK positivity being higher than the other biomarkers. Further studies including clinical and genetic information are required to confirm these differences, as well as studies focusing on populations living in different areas of the country.

RESUMO Objetivo: Investigar os subtipos histológicos e perfis de mutação do carcinoma pulmonar de células não pequenas no Brasil, bem como as correlações entre os subtipos histológicos, a expressão do gene anaplastic lymphoma kinase (ALK, quinase do linfoma anaplásico), o estado de mutação do gene EGFR e a expressão de programmed death-ligand 1 (PD-L1, ligante de morte celular programada 1). Métodos: Avaliamos 173 espécimes provenientes de pacientes com adenocarcinoma pulmonar no Nordeste brasileiro. A expressão de PD-L1 e ALK foi avaliada por meio de imuno-histoquímica, ao passo que o estado de mutação do EGFR foi avaliado por meio de sequenciamento. Os subtipos histológicos foram classificados de acordo com a International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society. Resultados: Os subtipos histológicos mais comuns de adenocarcinoma pulmonar foram o predominantemente sólido (em 46,8%), o predominantemente acinar (em 37,0%) e o predominantemente lepídico (em 9,8%). A expressão de ALK foi detectada em 10,4% das amostras, e 22,0% dos tumores apresentavam mutações do gene EGFR. As mutações mais comuns do EGFR foram a mutação pontual L858R no éxon 21 (em 45,5%) e a deleção do éxon 19 (em 36,3%). O tumor proportion score relativo à expressão de PD-L1 foi ≥ 50% em 18,2% das amostras, = 1-49% em 32,7% e = 0% em 49,5%. O subtipo predominantemente sólido relacionou-se significativamente com EGFR selvagem (p = 0,047). A expressão positiva de PD-L1 não se relacionou significativamente com a expressão de ALK ou o estado de mutação do EGFR. Conclusões: Nossos resultados sugerem que o perfil molecular do carcinoma pulmonar de células não pequenas no Nordeste brasileiro difere do de populações em outras regiões do país: a expressão positiva de ALK é maior que os demais biomarcadores. Mais estudos com informações clínicas e genéticas são necessários para confirmar essas diferenças, além de estudos que se concentrem em populações em diferentes áreas do país.

Análise imunoistoquímica da expressão de PD-L1 em células tumorais e células T nos carcinomas de células escamosas do pênis / Immunohistochemical analysis of PD-L1 expression in tumor cells and T cells in penile squamous cell carcinomas

Introdução: A neoplasia do pênis é uma doença rara, sendo mais frequente nos países em desenvolvimento. Representa 0,4%dos tumores malignos dos homens nos EUA e 2,1% no Brasil, sendo mais prevalente nas regiões norte e nordeste. O carcinoma epidermóide representa aproximadamente 95% das neoplasias do pênis e sua ocorrência está ligada à presença de fimose, higiene inadequada e infecções virais. O PD-1 e seus ligante PD-L1 são proteínas de membrana com papel crucial na regulação da resposta imune. O aumento da expressão de PD-L1 parece ser um mecanismo de evasão do sistema imune do hospedeiro pelas células tumorais e tem sido alvo de diversos estudos. Objetivos: estabelecer a frequência da Expressão da proteína PD-L1 e das células T nos carcinomas de pênis, correlacionando os aspectos demográficos e clínico-patológicos com a expressão doe as tendências do câncer de pênis no Estado do Ceará. Métodos: Tratou-se de estudo retrospectivo, transversal, clínico-patológico e imunoistoquímico de câncer de pênis em pacientes tratados e em acompanhamento no Hospital do Câncer Haroldo Juaçaba do Instituto do Câncer do Ceará (HHJ/ICC). A amostra constituiu-se de 84 pacientes com diagnóstico de câncer de pênis acompanhados no HHJ/ICC, entre 2002 e 2014. Os dados foram obtidos através de revisão dos prontuários e do preenchimento de formulário elaborado pelos pesquisadores. Foi preparado TMA a partir de amostras dos blocos de parafina dos tumores. A imunoistoquímica foi realizada a partir das lâminas do TMA. Os dados foram tabulados, armazenados e processados através do programa estatístico Statistical Package for the Social Science (SPSS) Versão 22.0. Resultados: A média de idade foi de 67,6 anos com a maioria oriunda do interior do Estado (65,5%) e casados (68,7%). Amputação parcial foi a cirurgia mais realizada (82,14%) e a maioria dos tumores tinha até 5cm (77,4%). Tivemos cerca de 31% pT1 e 21,5% pN positivo. O subtipo usual foi o prevalente (67,9%) e os carcinomas moderadamente diferenciados (61,9%) tiveram maior representatividade. Houve recidiva em cerca de 21% dos casos. Quanto ao PD-L1 houve expressão em 22,6% dos casos. Houve significância estatística para a associação entre a expressão de PD-L1 com Células Inflamatórias (p=0,000), CD4 (p=0,001), CD8 (p=0,012) e estádio pT (p=0,02). Também houve para a associação entre a expressão de Células Inflamatórias com CD4 (p=0,004), CD8 (p=0,000), estádio pT (p=0,001), grau histológico (p=0,03) e diferenciação histológica (p=0,04). Quanto ao prognostico, a recidiva (p=0,017), o estádio pT (p=0,027) e o grau histológico (p=0,020) mostraram-se como fatores de impacto na sobrevida. Conclusão: Trata-se de uma neoplasia que acomete mais a sexta década da vida. A recidiva, o estadiamento patológico (pT) e o grau histológico ratificaram-se como fatores prognósticos relevantes na sobrevida. A expressão de PD-L1 em tumores avançados reforça a importância de continuar a pesquisa para investigação adicional de anti-PD-1 e imunoterapêuticos anti-PD-L1 em pacientes com neoplasia peniana avançada. Reforça-se ainda que por se tratar de um problema de saúde pública, campanhas de conscientização quanto aos sintomas devem ser incentivadas para que se possa fazer o diagnóstico mais precocemente, o que poderia diminuir as taxas de incidência de tumores em estágio avançado e propiciar cirurgias menos mutilantes

Introduction: Penile neoplasia is a rare disease, being more frequent in developing countries. It represents 0.4% of the malignant tumors of the men in the USA and 2.1% in Brazil, being more prevalent in the north and northeast regions. Epidermoid carcinoma accounts for approximately 95% of penile neoplasms, and its occurrence is linked to the presence of phimosis, inadequate hygiene and viral infections. PD-1 and its PD-L1 linkers are membrane proteins with crucial role in the regulation of the immune response. Increased PD-L1 expression appears to be a mechanism for evading the host immune system by tumor cells and has been the subject of several studies. Objectives: To establish the frequency of PD-L1 protein and T-cell expression in penile carcinomas, correlating demographic and clinical-pathological aspects with the expression and trends of penile cancer in the State of Ceara. Methods: Retrospective, cross-sectional, clinical-pathological and immunohistochemical study of penile cancer in patients treated and followed up at Haroldo Juaçaba Cancer Hospital of the Cancer Institute of Ceara (HHJ / ICC). The sample consisted of 84 patients with a diagnosis of penile cancer in the HHJ / ICC, between 2002 and 2014. The data were obtained by reviewing the medical records and filling out a form prepared by the researchers. TMA was prepared from samples of the paraffin blocks of the tumors. Immunohistochemistry was performed from the TMA slides. Data were tabulated, stored and processed using the statistical program Statistical Package for Social Science (SPSS) Version 22.0. Results: The mean age was 67.6 years, with the majority coming from the interior of the State (65.5%) and married (68.7%). Partial amputation was the most performed surgery (82.14%) and the majority of tumors had up to 5 cm (77.4%). We had about 31% pT1 and 21.5% pN positive. The usual subtype was the prevalent one (67.9%) and the moderately differentiated carcinomas (61.9%) were more representative. There was recurrence in about 21% of the cases. Regarding PD-L1, there was 22.6% of cases. There was statistical significance for the association between PD-L1 expression with Inflammatory Cells (p = 0.000), CD4 (p = 0.001), CD8 (p = 0.012) and pT stage (p = 0.02). There was also an association between the expression of CD4 (p = 0.004), CD8 (p = 0.000), pT (p = 0.001), histological grade (p = 0.03) and histological differentiation, 04). As for the prognosis, the relapse (p = 0.017), the pT stage (p = 0.027) and the histological grade (p = 0.020) were shown as survival impact factors. Conclusion: This is a neoplasm that affects the sixth decade of life. Recurrence, pathological staging (pT) and histological grade were ratified as relevant prognostic factors in survival. The expression of PD-L1 in advanced tumors reinforces the importance of continuing the research for further investigation of anti-PD-1 and anti-PD-L1 immunotherapeutics in patients with advanced penile neoplasia. It is also reinforced that because it is a public health problem, awareness campaigns about the symptoms should be encouraged so that the diagnosis can be made earlier, which could reduce the incidence rates of advanced tumors and provide surgeries less mutilating

PD-1/PD-L1 regulates Treg differentiation in pregnancy-induced hypertension

Pregnancy-induced hypertension (PIH) causes significant maternal and fetal morbidity and mortality. A decreased number of regulatory T (Treg) cells is associated with the pathogenesis of PIH. The programmed cell death-1 (PD-1)/PD-ligand 1 (PD-L1) pathway is critical to normal pregnancy (NP) by promoting Treg cell development. However, the relationship between PD-1/PD-L1 and Treg differentiation in PIH has not been fully elucidated. In this study, venous blood was obtained from 20 NP and 58 PIH patients. Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood. The levels of Treg-related cytokines (TGF-β, IL-10, and IL-35) in serum and PBMCs were measured by ELISA. The percentage of Treg cells in PBMCs was assessed by flow cytometry. The mRNA levels of Treg-specific transcription factor Foxp3 in PBMCs, and PD-1 and PD-L1 in Treg cells were detected by qRT-PCR. The protein levels of PD-1 and PD-L1 in Treg cells were evaluated by western blot. The serum levels of TGF-β, IL-10, IL-35, and Foxp3 mRNA expression and CD4+CD25+ Treg cell percentage in PBMCs were decreased in PIH. Furthermore, a significant increase of PD-1 in Treg cells was found in PIH compared with NP. In addition, PD-L1 Fc, an activator of PD-1/PD-L1 pathway, increased Treg cell percentage, enhanced Foxp3 mRNA expression, and elevated levels of TGF-β, IL-10, and IL-35 in PBMCs. However, anti-PD-L1 mAb exerted a reverse effect. These findings revealed that PD-L1 Fc had a favorable effect on Treg cell differentiation, indicating a potential therapeutic value of PD-1/PD-L1 pathway for PIH treatment.

Metastatic Lymphoepithelioma-like Hepatocellular Carcinoma: a Potential Diagnostic Pitfall and Demonstration of Pd-l1 Expression

Abstract: Lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) is a rare primary hepatic neoplasm with female predominance and relatively good prognosis. We report a 73-year-old female with chronic hepatitis B who developed metastatic lesions 5 years after underwent resection for LEL-HCC. The metastatic lesions showed a spectrum of morphologic findings, which could be mistaken for other entities such as lymphoma, particularly in lesions with single-cell infiltrative pattern and abundant tumor-infiltrating lymphocytes. Immunohistochemical study to confirm the origin of the neoplastic cells is important to make the diagnosis. We also highlighted the clinicopathologic correlation and potential therapeutic implication of programmed death ligand-1 expression in LEL-HCC.

Effects of lung cancer cell-associated B7-H1 on T-cell proliferation in vitro and in vivo

B7 homolog 1 (B7-H1) is the most potent immunoinhibitory molecule in the B7 family. In this study, we examined the effects of tumor-associated B7-H1 on T-cell proliferation in lung cancer. The expression of B7-H1 in human adenocarcinoma A549 and mouse Lewis lung carcinoma (LLC) cells were examined by flow cytometry. To assess the in vitro effect of tumor-associated B7-H1 on T-cell proliferation, we isolated T cells from peripheral blood mononuclear cells (PBMCs) of healthy individuals, labeled them with carboxyfluorescein succinimidyl ester, and co-cultured them with A549 cells in the absence or presence of anti-B7-H1 antibody. For in vivo analysis, LLC cells were subcutaneously injected into mice treated or not with anti-B7-H1 antibody. T-cell proliferation in both in vitro and in vivo assays was analyzed by flow cytometry. In vitro, co-culturing T cells with A549 cells significantly inhibited the proliferation of the former compared with the proliferation of T cells alone (P<0.01), and the addition of B7-H1 blocking antibody dramatically reversed the inhibition of T-cell proliferation by A549 cells. Similarly, in mice bearing LLC-derived xenograft tumors, in vivo administration of anti-B7-H1 antibody significantly increased the total number of spleen and tumor T cells compared to levels in control mice that did not receive anti-B7-H1 antibody. Functionally, in vivo administration of anti-B7-H1 antibody markedly reduced tumor growth. Tumor-associated B7-H1 may facilitate immune evasion by inhibiting T-cell proliferation. Targeting of this mechanism offers a promising therapy for cancer immunotherapy.