PD-1/PD-L1通路在乳腺癌治疗中的作用及意义

程序性死亡受体1（programmed death-1,PD-1,CD279）为共刺激受体CD28超家族成员,主要表达在活化T细胞、B细胞、单核细胞以及自然杀伤细胞表面。程序性死亡配体1（programmed death-ligand 1,PD-L1,CD274）为PD-1的一个重要配体,广泛表达于肿瘤细胞以及抗原呈递细胞（APC）表面。以PD-1/PD-L1为靶点的肿瘤免疫治疗为肿瘤治疗开辟了新的道路。PD-1通过与PD-L1和(或)PD-L2结合,抑制T细胞活化,诱导T细胞凋亡,在肿瘤免疫逃逸中起着重要的作用。目前PD-1/PD-L1信号通路成为免疫靶向治疗的新靶点,相关研究在非小细胞肺癌、晚期黑色素瘤等多种恶性肿瘤领域有重大进展,研究发现PD-L1 在多种肿瘤细胞包括乳腺癌中表达上调,提示 PD-1/PD-L1通路可能参与肿瘤的免疫逃逸。本文将针对PD-1/PD-L1通路在乳腺癌治疗中的作用及意义进行综述。     

Combination Treatment of the Oral CHK1 Inhibitor,SRA737, and Low-Dose Gemcitabine Enhances the Effect of Programmed Death Ligand 1 Blockade by Modulating the Immune Microenvironment in SCLC

IntroductionDespite the enthusiasm surrounding cancer immunotherapy, most SCLC patients show very modest response to immune checkpoint inhibitor monotherapy treatment. Therefore, there is growing interest in combining immune checkpoint blockade with chemotherapy and other treatments to enhance immune checkpoint blockade efficacy. Based on favorable clinical trial results, chemotherapy and immunotherapy combinations have been recently approved by the U.S. Food and Drug Administration for frontline treatment for SCLC.Methods and ResultsHere, we show that combined treatment of SRA737, an oral CHK1 inhibitor, and anti–programmed death ligand 1 (PD-L1) leads to an antitumor response in multiple cancer models, including SCLC. We further show that combining low, non-cytotoxic doses of gemcitabine with SRA737 + anti–PD-L1/anti–PD-1 significantly increased antitumorigenic CD8+ cytotoxic T cells, dendritic cells, and M1 macrophage populations in an SCLC model. This regimen also led to a significant decrease in immunosuppressive M2 macrophage and myeloid-derived suppressor cell populations, as well as an increase in the expression of the type I interferon beta 1 gene, IFNβ, and chemokines, CCL5 and CXCL10.ConclusionsGiven that anti–PD-L1/anti–PD-1 drugs have recently been approved as monotherapy and in combination with chemotherapy for the treatment of SCLC, and that the SRA737 + low dose gemcitabine regimen is currently in clinical trials for SCLC and other malignancies, our preclinical data provide a strong rational for combining this regimen with inhibitors of the PD-L1/PD-1 pathway.     

PD-1+ CD8+ T cells are exhausted in tumours and functional in draining lymph nodes of colorectal cancer patients

Background:

The blockade of PD-1–PD-L1 pathway is emerging as an effective therapeutic strategy for several advanced cancers. But the immune regulatory role of PD-1–PD-L1 pathway is not clear in colorectal cancer (CRC) patients. This study aims to evaluate the role of PD-1–PD-L1 pathway in CD8⁺ T-cell functions in tumour-draining lymph nodes (TDLNs) and tumours of CRC patients.

Methods:

PD-1 expression on CD8⁺ T cells was examined by flow cytometry, and PD-L1 expression in TDLNs and tumour tissues were examined by immunohistochemistry. Production of IFN-γ, IL-2 and expression of granzyme B, perforin in CD8⁺ T cells were detected by intracellular staining.

Results:

PD-1 expression is markedly upregulated on CD8⁺ T cells in TDLNs and tumours compared with that in peripheral blood. PD-1-expressing CD8⁺ T cells are competent for production of cytokine (IL-2 and IFN-γ) and perforin in the tumour-free lymph nodes (TFLNs), but exhibit exhausted phenotypes in tumours. In addition, PD-L1 is highly expressed in tumours rather than TFLNs, which is closely correlated with the impairment of IFN-γ production of tumour-infiltrating PD-1⁺ CD8⁺ T cells.

Conclusions:

Our findings suggest a suppressive effect of PD-1 on CD8⁺ T-cell function in tumours, but not in TFLNs.     

PD-1/PD-L1拮抗剂在小细胞肺癌治疗中的研究进展

小细胞肺癌是以广泛转移和预后不良为特征的一种恶性肿瘤。虽然34%~85%的小细胞肺癌对化疗有效,但疾病进展迅速,后续的二线治疗效果很不理想,并且近几十年的治疗方案没有突破性进展。近几年生物治疗迅速发展,免疫治疗成为继靶向治疗后的又一次飞跃。程序性死亡蛋白-1(PD-1)与程序性死亡受体-配体1(PD-L1)结合,抑制了CD4^+和CD8^+T细胞的增殖和活化,负性调控机体免疫机制应答过程,从而介导肿瘤细胞的免疫逃逸,促进肿瘤生长。PD-1/PD-L1拮抗剂在SCLC中的临床前及临床研究中均获得了良好效果,让更多的小细胞肺癌患者受益,尽可能的延长生存时间。本文就PD-1/PD-L1拮抗剂在小细胞肺癌中的研究进展作一综述。     

Blockade of PD-L1 (B7-H1) augments human tumor-specific T cell responses in vitro

Human tumors frequently escape immune destruction, despite the presence of cytotoxic T cells (CTL) recognizing tumor-associated antigens (TAA). We have previously shown that programmed death ligand-1 (PD-L1), a recently identified ligand of the B7 superfamily, is expressed on murine tumors and can inhibit antitumor immune responses. To evaluate the clinical relevance of our animal model findings, we examined human tumors and tumor-specific T cells. We found PD-L1 to be constitutively expressed on human renal cell carcinoma (RCC) cell lines and upregulated on human melanoma cell lines upon exposure to interferon-gamma. Similarly, we found binding of anti-PD-L1 monoclonal antibody (mAb) on frozen sections from RCC and melanomas, but not on normal tissues. The corresponding inhibitory receptor of PD-L1, PD-1, revealed a higher expression on tumor-infiltrating lymphocytes than on peripheral blood lymphocytes (PBL) from melanoma patients upon specific antigen stimulation. Stimulation of PBL from healthy donors with peptide-loaded dendritic cells in the presence of anti-PD-L1 mAb altered neither the total T cell numbers after expansion, nor the percentage of peptide-specific CTL, when providing a T cell help by addition of cytokines. However, when stimulating TAA-specific CTL and T helper cells with Ag-pulsed dendritic cells in the absence of exogenous cytokines, PD-L1 blockade increased the cytokine production. Similar to the data achieved in the murine system, the blockade of PD-L1 on human tumors resulted in enhanced cytolytic activity of TAA-specific CTLs and cytokine production of TAA-specific T helper cells when interacting directly with the tumor. In summary, our data suggest that PD-L1/PD-1 interactions negatively regulate T cell effector functions predominantly in the absence of exogenous cytokine support, indicating an important role for this pathway in tumor evasion.     

PD-L1 is highly expressed in Enzalutamide resistant prostate cancer

Efficacy of Enzalutamide (ENZ) in castration resistant prostate cancer (CRPC) patients is short-lived. Immunotherapy like T cell checkpoint blockade may improve patient survival. However, when and where checkpoint molecules are expressed in CRPC and whether immune evasion is a mechanism of ENZ resistance remains unclear. Thus, we investigated whether clinically relevant immunotherapy targets, specifically PD-L1/2, PD-1 and CTLA-4, are upregulated in ENZ resistant (ENZR) patients and in a pre-clinical model of ENZ resistance. We show for the first time that patients progressing on ENZ had significantly increased PD-L1/2⁺ dendritic cells (DC) in blood compared to those naïve or responding to treatment, and a high frequency of PD-1⁺T cells. These data supported our pre-clinical results, in which we found significantly increased circulating PD-L1/2⁺ DCs in mice bearing ENZR tumors compared to CRPC, and ENZR tumors expressed significantly increased levels of tumor-intrinsic PD-L1. Importantly, the expression of PD-L1 on ENZR cells, or the ability to modulate PD-L1/2⁺ DC frequency, was unique to ENZR cell lines and xenografts that did not show classical activation of the androgen receptor. Overall, our results suggest that ENZ resistance is associated with the strong expression of anti-PD-1 therapy targets in circulating immune cells both in patients and in a pre-clinical model that is non-AR driven. Further evaluation of the contribution of tumor vs. immune cell PD-L1 expression in progression of CRPC to anti-androgen resistance and the utility of monitoring circulating cell PD-L1 pathway activity in CRPC patients to predict responsiveness to checkpoint immunotherapy, is warranted.     

PD-1 blockade enhances cytokine-induced killer cell-mediated cytotoxicity in B-cell non-Hodgkin lymphoma cell lines

The clinical utility of immune checkpoint inhibitors, such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors used alone or in combination with other therapies, is currently gaining attention. In this particular scenario, the inclusion of cytokine-induced killer (CIK) cells has proven to be a novel therapeutic approach. CIK cells have shown anticancer activity in various hematopoietic malignancies, but their defined cytotoxicity in B-cell non-Hodgkin lymphoma (B-NHL) remains to be fully elucidated. The present study investigated the role of PD-1/PD-L1 blockades on the cytotoxic efficacy of CIK cells primarily in B-NHL cell lines. The current analysis revealed that CIK cells prompted cytotoxicity against B-NHL cell lines (DAUDI and SU-DHL-4), and a significant increase in PD-L1 expression was observed when CIK cells were co-cultured with B-NHL cells. Additionally, a combination of PD-1 and PD-L1 antibodies with CIK cells significantly decreased cell viability only in DAUDI cells. Furthermore, IFN-γ elevation was observed in both cell lines treated with CIK alone or with PD-1 antibody, but this tendency was not observed for PD-L1. Since PD-1 can suppress immune inactivation, whereas CD40L can promote it, the effects of CD40L blockade were also examined; however, no significant changes in cell viability were observed. Overall, the present in vitro data suggested that CIK cells exerted a cytotoxic function in B-NHL cells, and a combination of PD-1 inhibitors with CIK cells may provide a potential therapeutic option for this type of lymphoma. Nevertheless, in vivo experiments are warranted to undermine the extent to which PD-1 inhibitors may be used to enhance the antitumor activity of CIK cells in B-NHL.     

PD-1/PD-L1 expressions in medullary thyroid carcinoma: Clinicopathologic and prognostic analysis of Chinese population

IntroductionFew studies have focused on PD-L1 expression in medullary thyroid carcinoma (MTC). Expressions of PD-1 and PD-L1 and their clinicopathologic and prognostic relevance were therefore further investigated on a relatively large population of MTC patients.Materials and methodsSurgical specimens were obtained from 87 MTC patients during a median follow-up of 37.7 months. PD-1 and PD-L1 expressions on tumor and associated immune cells were studied immunohistochemically using >1% positive cells as a threshold for positivity. Their correlations with clinicopathologic and prognostic feature were analyzed.ResultsPD-1 and PD-L1 were positively stained in 22 and 19 MTC patients. Most PD-L1-positive cases (18/19) showed weak to moderate staining intensity. PD-1 and PD-L1 were co-expressed in 11 patients. PD-L1 positivity was significantly correlated with distant metastases at surgery (21.1% vs 1.5%, P = 0.007). Coexpression of PD-1 and PD-L1 in MTC was correlated with advanced pathologic TNM stage III/IV (P = 0.040) and distant metastases at surgery (P = 0.013). However, there was no other clinicopathologic and prognostic relevance regarding to PD-1, PD-L1 or their coexpression in our MTC patients.ConclusionPD-1/PD-L1 pathway was expressed in MTC patients and was significantly correlated with the distant metastases at surgery, which may shed light on PD-1/PD-L1 as a promising therapeutic target in MTC. Future better understanding of PD-1/PD-L1 expression and their relationship with immunotherapy response may provide direct evidence for management of refractory MTC.     

CTLA-4、PD-1和PD-L1在小细胞肺癌外周血中的分布及临床意义

背景与目的 本研究旨在探索细胞毒性T淋巴细胞相关抗原(cytotoxic T lymphocyte associated anti-gen-4,CTLA-4)、程序坏死因子(programmed death 1,PD-1)和程序坏死因子配体(programmed death ligand 1,PD-L1)在小细胞肺癌(small cell lung cancer,SCLC)患者外周血中的分布情况,探索其免疫作用机制并评估其作为生物标志物的临床价值.方法 招募290例SCLC患者及60例健康志愿者,收集治疗前和治疗2nd周期末SCLC患者EDTA抗凝血2 mL.应用流式细胞仪检测CTLA-4、PD-1和PD-L1在外周血CD3、CD4、CD8及CD25的分布,分析其与临床病理特征的相关性;采用细胞免疫化学法和流式细胞法检测PD-L1在SCLC细胞系H446中的表达.结果 SCLC患者外周血中CTLA-4+细胞和PD-1+细胞水平分别为(1.56±1.24)%和(8.07±3.97)%、CTLA-4在CD3细胞和CD4细胞中的表达水平无明显差异,分别为(4.87±5.18)%和(3.85±2.60)%,均低于PD-1在CD3+或CD4+细胞中的表达(26.63±9.04)%和(20.79±9.41)%,与健康对照组相比,SCLC中CD4+CD25+CTLA-4+细胞水平明显升高(1.91±1.27)%vs(7.09±5.09)%,P<0.001;PD-1+(CD8+)细胞表达水平明显降低,分别为(22.56±4.21)%vs(11.47±5.85)%,P<0.001.CD4+CD25+CTLA-4+细胞或CD8+PD-1+细胞水平与患者的年龄、性别、吸烟状况、临床分期以及是否转移等因素无关(P>0.05).化疗两周期末CD4+CD25+CTLA-4+和CD8+PD-1+细胞的水平对比化疗前明显下降,分别为(5.11±2.60)%vs(6.94±4.91)%;(8.74±3.39)%vs(11.48±5.91)%,P值均<0.000,1,但与无疾病进展生存和总生存无显著相关性.PD-L1高表达于SCLC细胞系H446中并定位在细胞膜和细胞浆,但在外周血中未见表达.结论 本研究首次证实SCLC外周血中CTLA4高表达于调节性T细胞中,而PD-1低表达于效应性T细胞,该结果为揭示SCLC免疫监测点免疫逃逸机制提供了理论依据,可能作为一种新的无创性且可实时监测的生物标志物.     

免疫检查点PD-1／PD-L1通路在小细胞肺癌中的作用

小细胞肺癌（SCLC）主要的特点为生长迅速且早期易发生广泛转移。尽管SCLC对于化疗和放疗敏感,但几乎所有的患者均在治疗后发生复发转移,预后差。免疫检测点抑制剂,尤其程序化细胞死亡受体-1（PD-1）／程序化细胞死亡配体-1（PD-L1）拮抗剂在SCLC的临床前和临床研究中均获得了良好效果,并且能够延长患者生存。免疫检测点疗法作为一种新兴的方法在未来可能会改变SCLC治疗模式。此外,有限的数据显示出PD-L1表达可能成为筛选获益人群一个有效的生物标记物。本文总结PD-L1作为标记物的发展历程,并同时阐述PD-1／PD-L1抑制剂在SCLC治疗中的进展。     

Presence of innate lymphoid cells in pleural effusions of primary and metastatic tumors: Functional analysis and expression of PD-1 receptor

The tumor microenvironment (TM) contains a wide variety of cell types and soluble factors capable of suppressing immune responses. While the presence of NK cells in pleural effusions (PE) has been documented, no information exists on the presence of other innate lymphoid cell (ILC) subsets and on the expression of programmed cell death-1 (PD-1) in NK and ILC. The presence of ILC was assessed in PE of 54 patients (n = 33 with mesothelioma, n = 15 with adenocarcinoma and n = 6 with inflammatory pleural diseases) by cell staining with suitable antibody combinations and cytofluorimetric analysis. The cytokine production of ILC isolated from both PE and autologous peripheral blood was analyzed upon cell stimulation and intracytoplasmic staining. We show that, in addition to NK cells, also ILC1, ILC2 and ILC3 are present in malignant PE and that the prevalent subset is ILC3. PE-ILC subsets produced their typical sets of cytokines upon activation. In addition, we analyzed the PD-1 expression on NK/ILC by multiparametric flow-cytometric analysis, while the expression of PD-1 ligand (PD-L1) was evaluated by immunohistochemical analysis. Both NK cells and ILC3 expressed functional PD-1, moreover, both tumor samples and malignant PE-derived tumor cell lines were PD-L1⁺ suggesting that the interaction between PD-1⁺ILC and PD-L1⁺tumor cells may hamper antitumor immune responses mediated by NK and ILC.     

Editor's choice: PD-L1 expression and prognostic impact in glioblastoma

Background

Therapeutic targeting of the immune checkpoints cytotoxic T-lymphocyte-associated molecule-4 (CTLA-4) and PD-1/PD-L1 has demonstrated tumor regression in clinical trials, and phase 2 trials are ongoing in glioblastoma (GBM). Previous reports have suggested that responses are more frequent in patients with tumors that express PD-L1; however, this has been disputed. At issue is the validation of PD-L1 biomarker assays and prognostic impact.

Methods

Using immunohistochemical analysis, we measured the incidence of PD-L1 expression in 94 patients with GBM. We categorized our results according to the total number of PD-L1-expressing cells within the GBMs and then validated this finding in ex vivo GBM flow cytometry with further analysis of the T cell populations. We then evaluated the association between PD-L1 expression and median survival time using the protein expression datasets and mRNA from The Cancer Genome Atlas.

Results

The median percentage of PD-L1-expressing cells in GBM by cell surface staining is 2.77% (range: 0%–86.6%; n = 92), which is similar to the percentage found by ex vivo flow cytometry. The majority of GBM patients (61%) had tumors with at least 1% or more PD-L1-positive cells, and 38% had at least 5% or greater PD-L1 expression. PD-L1 is commonly expressed on the GBM-infiltrating T cells. Expression of both PD-L1 and PD-1 are negative prognosticators for GBM outcome.

Conclusions

The incidence of PD-L1 expression in GBM patients is frequent but is confined to a minority subpopulation, similar to other malignancies that have been profiled for PD-L1 expression. Higher expression of PD-L1 is correlated with worse outcome.     

Programmed death-1 ligand 1 and 2 are highly expressed in pleomorphic carcinomas of the lung: Comparison of sarcomatous and carcinomatous areas

Pleomorphic carcinoma (PC) of the lung is a rare type of poorly differentiated non-small cell lung carcinoma (NSCLC) that belongs to sarcomatoid carcinoma (SC). It exhibits aggressive behaviour and resistance to chemotherapy and radiotherapy. Recently, immunotherapy targeting the programmed death-1 (PD-1)/PD ligand 1 (PD-L1) pathway has demonstrated favourable clinical outcomes in NSCLC. However, the expression patterns of PD-1-related molecules in pulmonary PC remain elusive.PD-L1 and PD-L2 expression was estimated in 41 cases of PC using immunohistochemistry. CD8⁺ and PD-1⁺ tumour-infiltrating lymphocytes (TILs) were also evaluated.PD-L1 and PD-L2 were highly expressed in pulmonary PCs (90.2% [37/41)]; 87.8% [36/41]). The amount of CD8⁺ or PD-1⁺ TILs and the ratio of PD-1⁺/CD8⁺ TILs in PC were higher in males, smokers and older patients. PD-L1-positive PCs were infiltrated by higher numbers of CD8⁺ TILs compared to PD-L1-negative cases (P = 0.006). Of note, PD-L1 expression in pulmonary PCs was significantly higher in sarcomatous areas than in the carcinomatous portion (P = 0.006). PC patients with a high ratio of PD-1⁺/CD8⁺ TILs showed a shorter progression-free survival (P = 0.036), whereas PD-L1 and PD-L2 expression had no prognostic implications.Our study demonstrates that pulmonary PCs very frequently express PD-L1 and PD-L2. Moreover, their expression is higher in sarcomatous cells than in carcinomatous areas. Thus, targeting the PD-1/PD-L1 pathway may represent a potential therapeutic candidate for this aggressive tumour.     

PD-1/PD-L1通路在髓系肿瘤中的研究进展

在免疫系统中,为防止免疫反应过度,细胞表面会有抑制免疫反应的检查点,其中程序性死亡受体-１(programmed death-1,PD-1)与其程序性死亡配体-1(programmed death-ligand 1,PD-L1）就是其中的一对免疫检查点,肿瘤细胞通过表达PD-L1与免疫细胞上的PD-1受体结合,使得肿瘤细胞可以逃避免疫细胞的攻击。研究表明,PD-1及PD-L1在多种实体肿瘤组织及免疫细胞中存在过表达现象,导致抗肿瘤细胞功能消失,并且与不良预后相关,但在髓系肿瘤中的研究较少。因此本文从PD-1/PD-L1的生物学特点,目前在髓系肿瘤中的表达情况,以及传统化疗药物对该通路的作用作一综述,旨在为髓系肿瘤的诊疗提供新的靶点。     

EGFR-ERK pathway regulates CSN6 to contribute to PD-L1 expression in glioblastoma

Glioblastoma (GBM) is the most common and malignant brain tumor in adults. Recently, programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) checkpoint blockades have been applied for GBM treatment. However, the mechanism of PD-L1 upregulation in GBM is still unclear. COP9 signalosome 6 (CSN6) is crucial for maintaining the protein stabilization in cancer cells. In this study, we applied human GBM specimens and cell lines to investigate whether the EGFR-ERK pathway regulates CSN6 for PD-L1 upregulation. Data from The Cancer Genome Atlas dataset showed that high expression of EGFR, CSN6, and PD-L1 in patients with glioma was associated with poor prognosis. In 47 human GBM specimens, high expression of PD-L1 was associated with low amount of CD8⁺ T cell infiltration as well as the poor prognosis of patients. CSN6 was positively correlated with EGFR and PD-L1 expression in human GBM specimens. We treated two GBM cell lines (U87 and U251) with epidermal growth factor (EGF) in vitro, and found EGF-upregulated p-EGFR, p-ERK, CSN6, and PD-L1 expression in GBM cells. PD98059, the ERK blocker, inhibited upregulations of CSN6 and PD-L1 in EGF-treated cells. Inhibition of CSN6 by small interfering RNA decreased PD-L1 expression but also increased CHIP expression in GBM cells. When the cells were treated with EGF and cycloheximide (CHX), a protein synthesis inhibitor, EGF-reduced CHX-induced CSN6 and PD-L1 turnover in GBM cells. Furthermore, CSN6-mediated downregulation of PD-L1 was inhibited by MG132, a proteasome inhibitor in U87 cells. Thus, these results suggest that the EGFR-ERK pathway may upregulate CSN6, which may inhibit PD-L1 degradation and subsequently maintain PD-L1 stability in GBM.     

PD-L1的表达与妇科恶性肿瘤预后关系的研究进展

程序性死亡蛋白1(Programmed death protein 1,PD-1)是T细胞上主要存在的一种抑制性受体。程序性死亡配体-1(Programmed death ligand-1,PD-L1,又称B7-H1)在心、肝、肾、肺等多个器官上皮细胞和内皮细胞中广泛表达。肿瘤发生时机体内的肿瘤微环境会使浸润性T细胞高表达PD-1,同时肿瘤细胞中PD-L1的表达上调,与PD-1受体相结合,导致肿瘤微环境中PD-1通路持续开放,进而导致T细胞凋亡,肿瘤细胞则逃脱机体的免疫监视,发生肿瘤免疫逃逸。本文就PD-L1表达与妇科恶性肿瘤预后关系的研究进展进行综述。     

Regulatory T cells and the PD-L1/PD-1 pathway mediate immune suppression in malignant human brain tumors

The brain is a specialized immune site representing a unique tumor microenvironment. The availability of fresh brain tumor material for ex vivo analysis is often limited because large parts of many brain tumors are resected using ultrasonic aspiration. We analyzed ultrasonic tumor aspirates as a biosource to study immune suppressive mechanisms in 83 human brain tumors. Lymphocyte infiltrates in brain tumor tissues and ultrasonic aspirates were comparable with respect to lymphocyte content and viability. Applying ultrasonic aspirates, we detected massive infiltration of CD4⁺FoxP3⁺CD25^high CD127^low regulatory T cells (Tregs) in glioblastomas (n = 29) and metastatic brain tumors (n = 20). No Treg accumulation was observed in benign tumors such as meningiomas (n = 10) and pituitary adenomas (n = 5). A significant Treg increase in blood was seen only in patients with metastatic brain tumors. Tregs in high-grade tumors exhibited an activated phenotype as indicated by decreased proliferation and elevated CTLA-4 and FoxP3 expression relative to blood Tregs. Functional analysis showed that the tumor-derived Tregs efficiently suppressed cytokine secretion and proliferation of autologous intratumoral lymphocytes. Most tumor-infiltrating Tregs were localized in close proximity to effector T cells, as visualized by immunohistochemistry. Furthermore, 61% of the malignant brain tumors expressed programmed death ligand-1 (PD-L1), while the inhibitory PD-1 receptor was expressed on CD4⁺ effector cells present in 26% of tumors. In conclusion, using ultrasonic tumor aspirates as a biosource we identified Tregs and the PD-L1/PD-1 pathway as immune suppressive mechanisms in malignant but not benign human brain tumors.     

LAG-3 Protein Expression in Non–Small Cell Lung Cancer and Its Relationship with PD-1/PD-L1 and Tumor-Infiltrating Lymphocytes

IntroductionImmunotherapy targeting the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) checkpoint has shown promising efficacy in patients with NSCLC. Lymphocyte activating 3 gene (LAG-3) is another important checkpoint, and its role in NSCLC is still not clear. In this study we investigated lymphocyte activing 3 (LAG-3) protein expression; its correlation with PD-1, PD-L1, and tumor-infiltrating lymphocytes (TILs); and its association with survival in NSCLC.MethodsThe expression of LAG-3 (EPR4392 [Abcam, Cambridge, MA]) protein was assessed in 55 NSCLC cell lines by immunohistochemistry. LAG-3, PD-1 (NAT 105 [Cell Marque, Rocklin, CA]), and PD-L1 (22C3 [Dako, Carpenteria, CA]) protein expression was evaluated by immunohistochemistry, and TIL abundance was scored in 139 surgically resected specimens from patients with NSCLC. We also verified results in samples from 62 patients with untreated NSCLC and detected a correlation between LAG-3 expression and EGFR and KRAS mutation and echinoderm microtubule associated protein like 4 gene (EML4)–anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement.ResultsLAG-3 was not expressed on any of the 55 NSCLC cell lines. However, LAG-3 was expressed on the TILs in 36 patients with NSCLC (25.9%). Sixty patient samples (43.2%) were positive for PD-1 on the TILs, and 25 (18.0%) were positive for PD-L1 on tumor cells. Neither LAG-3 nor PD-1 was expressed on the tumor cells. LAG-3 was overexpressed on the TILs in nonadenocarcinoma compared with in adenocarcinoma (p = 0.031). LAG-3 expression on TILs was significantly correlated with that of PD-1 on TILs (p < 0.001) and PD-L1 on tumor cells (p = 0.041) but not with TIL percentage (p = 0.244). With the logistic regression model, the ORs for LAG-3 were 0.320 (95% confidence interval [CI]: 0.110–0.929) and 4.364 (95% CI: 1.898–10.031) when nonadenocarcinoma was compared with adenocarcinoma and TILs that were negative for PD-1 were compared with those positive for PD-1. Recurrence-free survival was significantly different in patients whose TILs were LAG-3–negative as opposed to LAG-3–positive (1.91 years [95% CI: 0.76–3.06] versus 0.87 years [95% CI: 0.27–1.47] [p = 0.025]). Likewise, LAG-3 status of TILs (negative versus positive) did significantly affect overall survival (OS) (3.04 years [95% CI: 2.76–3.32] versus 1.08 years [95% CI: 0.42–1.74] [p = 0.039]). Using Kaplan-Meier analysis, we found that patients with both PD-L1–negative tumor cells and LAG-3–negative TILs have longer recurrence-free survival than patients who are either PD-L1– or LAG-3–positive or both PD-L1– and LAG-3–positive (2.09 years [95% CI: 0.90–3.28] versus 1.42 years [95% CI: 0.46–2.34] versus 0.67 years [95% CI: 0.00–1.45] [p = 0.007]). In the verification stage, high expression of LAG-3 was also significantly correlated with higher expression of PD-1 on TILs (p = 0.016) and PD-L1 on tumor cells (p = 0.014). There was no correlation between LAG-3 expression and EGFR (p = 0.325) and KRAS mutation (p = 1.000) and ALK fusion (p = 0.562).ConclusionsLAG-3 is expressed on TILs in tumor tissues of some patients with NSCLC. Its expression was higher in nonadenocarcinoma and correlated with PD-1/PD-L1 expression. LAG-3 positivity or both LAG-3 and PD-L1 positivity was correlated with early postoperative recurrence. LAG-3 was related to poor prognosis.     

PD-1/PD-L1 抑制剂在肿瘤免疫治疗中的研究进展*

肿瘤免疫治疗是目前肿瘤领域的研究热点,是一种疗效显著的肿瘤治疗模式。程序性死亡受体PD- 1（programmed cell death-1）是一种重要的免疫抑制分子,主要在激活的T 细胞和B 细胞中表达。肿瘤细胞中高表达PD- 1 的配体PD-L1,导致肿瘤微环境中PD- 1 通路持续激活。PD- 1/PD-L 1 抑制剂可以阻断PD- 1 与PD-L1 的结合,阻断负向调控信号,使T 细胞恢复活性,从而增强免疫应答。近期研究发现PD- 1 和PD-L1 的抑制剂在多种肿瘤类型中疗效显著。本文对PD- 1/PD-L 1 抑制剂的现况及其对不同肿瘤类型临床疗效的研究进展进行综述。      

PD-L1 expression in small cell neuroendocrine carcinomas

Small cell lung cancer and extrapulmonary small cell carcinomas are the most aggressive type of neuroendocrine carcinomas. Clinical treatment relies on conventional chemotherapy and radiotherapy; relapses are frequent. The PD-1/PD-L1/PD-L2 pathway is a major target of anti-tumour immunotherapy. Aberrant PD-L1 or PD-L2 expression may cause local immune-suppression. Here we investigated expression of PD-1 and its ligands by immunohistochemistry and RNA-seq in small cell carcinomas.PD-L1 and PD-1 protein expression were analysed in 94 clinical cases of small cell carcinomas (61 pulmonary, 33 extrapulmonary) by immunohistochemistry using two different monoclonal antibodies (5H1, E1L3N). RNA expression was profiled by RNA-seq in 43 clinical cases.None of the small cell carcinomas showed PD-L1 protein expression in tumour cells. PD-L1 and PD-1 expression was noticed in the stroma: Using immunohistochemistry, 18.5% of cases (17/92) showed PD-L1 expression in tumour-infiltrating macrophages and 48% showed PD-1 positive lymphocytes (45/94). RNA-seq showed moderate PD-L1 gene expression in 37.2% (16/43). PD-L1 was correlated with macrophage and T-cell markers. The second PD-1 ligand PD-L2 was expressed in 27.9% (12/43) and showed similar correlations.Thus, the PD-1/PD-L1 pathway seems activated in a fraction of small cell carcinomas. The carcinoma cells were negative in all cases, PD-L1 was expressed in tumour-infiltrating macrophages and was correlated with tumour-infiltrating lymphocytes. Patients with stromal PD-L1/PD-L2 expression may respond to anti-PD-1 treatment. Thus, evaluation of the composition of the tumour microenvironment should be included in clinical trials. Besides conventional immunohistochemistry, RNA-seq seems suitable for detection of PD-L1/PD-L2 expression and might prove to be more sensitive.