Relationship between the expressions of PD-L1 and tumor-infiltrating lymphocytes in oral squamous cell carcinoma

Tumor-infiltrating lymphocytes (TILs) are considered to represent immune reactions of the host to a malignant tumor. Programmed death receptor ligand-1 (PD-L1) is a surface protein that blocks the function of T lymphocytes and is expressed on cancer cells. Tumor-associated fibroblasts (TAFs), which influence tumor growth have also been reported to express PD-L1 and thus inhibit TILs. In the present study, we investigated the densities of CD4⁺/CD8⁺ TILs, PD-L1 expression of tumor cells and TAFs in oral squamous cell carcinoma (OSCC). Forty-five cases of OSCC were selected. We evaluated PD-L1 expression and the infiltration degree of each lymphocyte by immunohistochemical examination. These data were analyzed in connection with clinicopathological factors. Peritumoral CD8⁺ TILs were observed in every patient with OSCC, and their densities were correlated with lymph node metastasis (P < 0.001), tumor size (P = 0.003), and clinical stage (P < 0.001). PD-L1 expression on OSCC cells was observed in 39 cases and was associated with the lower density of intratumoral CD8⁺ TILs (P = 0.047). PD-L1 expression of tumors <4 cm in size was correlated with the histological grade of the tumor (P = 0.022). TAFs were positive for PD-L1 in 18 cases. Peritumoral TILs were significantly associated with tumor size, lymph node metastasis and clinical stage. Though PD-L1 expressed by OSCC cells did not affect patients’ survival, its correlation with decreased number of intratumoral TILs suggests that the development of a strategy to block the interactions of PD-L1 with TIL would be a useful tool for inhibiting tumor growth.     

Clinical Significance of PD-L1 Expression and CD8-Positive Tumor-Infiltrating Lymphocytes in Patients with Cavitary Lung Adenocarcinoma

Cavitary lung cancer is a rare type of lung cancer. Generally, the relationship between cavitary lung adenocarcinoma (LUAD) and specific immune checkpoints remains unknown. In this study, we aimed to detect the expression of programmed cell death ligand-1(PD-L1) and the density of CD8-positive (CD8⁺) tumor-infiltrating lymphocytes (TILs) to evaluate their clinicopathological significance in the case of patients with cavitary LUAD. This study included 65 patients with cavitary LUAD. Patient specimens were obtained from surgery. The expression of PD-L1 protein and CD8⁺ TIL status was detected by traditional immunohistochemistry and multiplex quantitative immunofluorescence technology. The correlation of PD-L1 expression and CD8⁺ TIL status was assessed along with clinicopathological parameters. This included evaluation of overall survival in cavitary LUAD patients based on the follow-up data. High expression of PD-L1 protein was detected in the cancerous tissues of cavitary LUAD patients, whose positive rate was recorded at 44.6% (29/65). PD-L1 expression level was significantly related with the lymph node (P = 0.001), TNM stage (P = 0.024), and CD8⁺ TIL status (rs = −0.272, P = 0.025). High PD-L1 expression indicated high mortality rate (P < 0.001); however, the CD8⁺ TIL group showed better survival in cavitary LUAD patients (P = 0.011). This phenotype, with high PD-L1 expression and low CD8⁺ TILs, can predict poorer overall survival of patients with cavitary LUAD compared with the other phenotypes. Moreover, CD8⁺ TIL level was an independent good prognosis factor. Initially, we reported that PD-L1 is upregulated in cavitary LUAD patients and that high expression of PD-L1 negatively correlates with CD8⁺ TIL status. High PD-L1 expression and low CD8⁺ TILs can predict decreased overall survival of patients with cavitary LUAD.     

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.     

Programmed cell death ligand-1 (PD-L1) expression combined with CD8 tumor infiltrating lymphocytes density in non-small cell lung cancer patients

Cancer immunotherapy targeting programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) pathway has shown promising results in treatment of non-small cell lung cancer (NSCLC) patients. T cells play a major role in tumor-associated immune response. This study aimed to investigate PD-L1 expression alone and combined with CD8 tumor infiltrating lymphocytes (TILs) density in relation to clinicopathologic parameters and survival in NSCLC patients. Immunohistochemical analysis was used to evaluate PD-L1 expression and CD8 TILs density in 55 NSCLC patients. PD-L1 immunopositivity was detected in 36 (65.5%) of NSCLC cases. PD-L1 expression was significantly related to high tumor grade (p value?=?0.038) and low CD8 TILs density (p value?=?0.004), whereas no significant relations were detected between PD-L1 expression and tumor stage (p value?=?0.121), overall survival (OS) (p value?=?0.428) and progression-free survival (PFS) (p value?=?0.439). Among PD-L1/CD8 TILs density groups, PD-L1⁺/CD8^Low group was significantly associated with high tumor grade compared to PD-L1^?/CD8^high group (pairwise p?=?0.016). PD-L1⁺/CD8^Low group was significantly related to advanced tumor stage compared to PD-L1⁺/CD8^high and PD-L1^?/CD8^Low groups (pairwise p?=?0.001 and 0.013 respectively). PD-L1^?/CD8^high group exhibited the best OS and PFS whereas PD-L1⁺/CD8^low group had the poorest OS and PFS (p value?=?0.032 and 0.001 respectively). Assessment of PD-L1 combined with CD8 TILs density, instead of PD-L1 alone, suggested important prognostic relevance in NSCLC patients.     

Chronologically changing patterns in the survival of korean patients with breast cancer and related clinical factors: a nationwide registry-based study

Purpose

The purpose of the study was to evaluate protein expression of PD-L1 and CD20 as prognostic biomarkers of patient outcome in inflammatory breast cancer (IBC) samples.

Methods

PD-L1 and CD20 protein expression was measured by immunohistochemistry in 221 pretreatment IBC biopsies. PD-L1 was assessed in tumor cells (PD-L1⁺ tumor cells) and tumor stromal infiltrating lymphocytes (PD-L1⁺ TILs); CD20 was scored in tumor-infiltrating B cells. Kaplan–Meier curves and Cox proportional hazard models were used for survival analysis.

Results

PD-L1⁺ tumor cells, PD-L1⁺ TILs, and CD20⁺ TILs were found in 8%, 66%, and 62% of IBC, respectively. PD-L1⁺ tumor cells strongly correlated with high TILs, pathological complete response (pCR), CD20⁺ TILs, but marginally with breast cancer-specific survival (BCSS, P?=?0.057). PD-L1⁺ TILs strongly correlated with high TILs, CD20⁺ TILs, and longer disease-free survival (DFS) in all IBC and in triple-negative (TN) IBC (P?<?0.035). IBC and TN IBC patients with tumors containing both CD20⁺ TILs and PD-L1⁺ TILs (CD20⁺TILs/PD-L1⁺TILs) showed longer DFS and improved BCSS (P?<?0.002) than patients lacking both, or those with either CD20⁺ TILs or PD-L1⁺ TILs alone. In multivariate analyses, CD20⁺TILs/PD-L1⁺TILs status was an independent prognostic factor for DFS in IBC (hazard ratio (HR): 0.53, 95% CI 0.37–0.77) and TN IBC (HR: 0.39 95% CI 0.17–0.88), and for BCSS in IBC (HR: 0.60 95% CI 0.43–0.85) and TN IBC (HR: 0.38 95% CI 0.17–0.83).

Conclusion

CD20⁺TILs/PD-L1⁺TILs status represents an independent favorable prognostic factor in IBC and TN IBC, suggesting a critical role for B cells in antitumor immune responses. Anti-PD-1/PD-L1 and B cell-activating immunotherapies should be explored in these settings.

     

PD-L1 expression and immune cells in anaplastic carcinoma and poorly differentiated carcinoma of the human thyroid gland: A retrospective study

Anaplastic thyroid carcinoma (ATC) and poorly differentiated thyroid carcinoma (PDTC) have limited treatment options, and immune profiling may help select patients for immunotherapy. The prevalence and relevance of programmed death-1 ligand (PD-L1) expression and the presence of immune cells in ATC and PDTC has not yet been well established. The present study investigated PD-L1 expression (clone 22C3) and cells in the tumor microenvironment (TME), including tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs) and dendritic cells, in whole tissue sections of 15 cases of ATC and 13 cases of PDTC. Immunohistochemical PD-L1 expression using a tumor proportion score (TPS) with a 1% cut-off was detected in 9/15 (60%) of ATC cases and 1/13 (7.7%) of PDTC cases (P=0.006). PD-L1 expression in TILs was limited to the ATC group (73.3 vs. 0% in ATC and PDTC, respectively). In the ATC group, the TPS for tumor positive PD-L1 expression revealed a non-significant trend towards worse survival, but no difference was observed when investigating PD-L1 expression in TILs and TAMs. In addition to increased PD-L1 expression, all ATC cases exhibited significantly increased CD3⁺ and CD8⁺ T cells, CD68⁺ and CD163⁺ macrophages, and S100⁺ dendritic cells compared with the PDTC cases. Loss of mutL homolog 1 and PMS1 homolog 2 expression was observed in one ATC case with the highest PD-L1 expression, as well as in the only PDTC case positive for PD-L1. Notably, the latter was the only PDTC case exhibiting positivity for p53 and a cellular microenvironment similar to ATC. The current results indicated that PD-L1 expression was frequent in ATC, but rare in PDTC. In addition to PD-L1, the present study suggested that microsatellite instability may serve a role in both the TME and the identification of immunotherapy candidates among patients with PDTC.     

Gamma knife radiosurgery for trigeminal schwannoma: a 20-year experience with long-term treatment outcome

Background

Pituitary adenomas (PAs) are the second most common brain tumors, and mostly are benign tumors. However, there exists subtypes of PAs refractory to common treatments, and need novel therapy. Programmed death 1 (PD-1) blockade has shown durable objective response in a variety of malignancies, and the key predictive markers for this immunotherapy were PD-L1 and CD8⁺ tumor-infiltrating lymphocyte (TILs) expression. To evaluate the potential immunotherapy for PAs, we investigated the expression of these two immune markers in PAs.

Methods

Immunohistochemistry (IHC) was performed to detect the expression of PD-L1 and CD8⁺ TILs in PAs. The ratio of positive expression of PD-L1 and CD8⁺ TILs was compared with chi-squared tests among different subtypes of PAs. The association between their expression profile and clinical parameters was analyzed using a chi-squared test, or Fisher’s exact probability test when appropriate.

Results

One hundred and ninety one patients with PAs were retrospectively involved in this study, consisting of 106 non-functioning PAs (NF-PAs, 55.5%), 40 PRL-secreting PAs (PRL-PAs, 20.9%), 31 GH-secreting PAs (GH-PAs, 16.2%), 9 ACTH-secreting PAs (ACTH-PAs, 4.7%) and 5 plurihormonal adenomas (2.6%) respectively. 36.6% of them were PD-L1 positive and 86.9% were CD8⁺ TILs positive. The positive PD-L1 immunostaining presented more frequently in functioning PAs (58.8%), compared with that (34.3%) in nonfunctioning group (p?=?0.000). Moreover, the rates of PD-L1 expression were more associated with increased blood levels of PRL, GH, ACTH and cortisol. Contrastly, positive CD8⁺ TILs immunostaining was only correlated with elevated blood level of GH. For the analysis of immune markers with pathological results, PD-L1 expression was associated with PRL and GH immunostaining and higher Ki-67 index. But CD8⁺ TILs was only correlated with PRL immunostaining.

Conclusion

Our results showed that PD-L1 was frequently expressed in functioning PAs with association of aggressive behaviors in PAs. The immunotherapy could be a promising treatment option of PAs.

     

Immune Marker Profiling and Programmed Death Ligand 1 Expression Across NSCLC Mutations

Introduction

Programmed death 1/programmed death ligand 1 (PD-L1) axis inhibitors have been proven effective, especially in patients with tumors expressing PD-L1. Their clinical efficacy in patients with EGFR-activating mutations is still unclear, whereas KRAS mutations seem to be associated with good response.

Editor's choice: Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma

Background

Immune checkpoint inhibitors targeting programmed cell death 1 (PD1) or its ligand (PD-L1) showed activity in several cancer types.

Methods

We performed immunohistochemistry for CD3, CD8, CD20, HLA-DR, phosphatase and tensin homolog (PTEN), PD-1, and PD-L1 and pyrosequencing for assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status in 135 glioblastoma specimens (117 initial resection, 18 first local recurrence). PD-L1 gene expression was analyzed in 446 cases from The Cancer Genome Atlas.

Results

Diffuse/fibrillary PD-L1 expression of variable extent, with or without interspersed epithelioid tumor cells with membranous PD-L1 expression, was observed in 103 of 117 (88.0%) newly diagnosed and 13 of 18 (72.2%) recurrent glioblastoma specimens. Sparse-to-moderate density of tumor-infiltrating lymphocytes (TILs) was found in 85 of 117 (72.6%) specimens (CD3+ 78/117, 66.7%; CD8+ 52/117, 44.4%; CD20+ 27/117, 23.1%; PD1+ 34/117, 29.1%). PD1+ TIL density correlated positively with CD3+ (P < .001), CD8+ (P < .001), CD20+ TIL density (P < .001), and PTEN expression (P = .035). Enrichment of specimens with low PD-L1 gene expression levels was observed in the proneural and G-CIMP glioblastoma subtypes and in specimens with high PD-L1 gene expression in the mesenchymal subtype (P = 5.966e-10). No significant differences in PD-L1 expression or TIL density between initial and recurrent glioblastoma specimens or correlation of PD-L1 expression or TIL density with patient age or outcome were evident.

Conclusion

TILs and PD-L1 expression are detectable in the majority of glioblastoma samples but are not related to outcome. Because the target is present, a clinical study with specific immune checkpoint inhibitors seems to be warranted in glioblastoma.     

Evaluation of Immune Reaction and PD-L1 Expression Using Multiplex Immunohistochemistry in HER2-Positive Breast Cancer: The Association With Response to Anti-HER2 Neoadjuvant Therapy

Background

Immune reaction with tumor-infiltrating lymphocytes (TILs) has been extensively investigated in breast cancer. Programmed cell death 1 and its ligand (PD-L1) are key physiologic suppressors of cytotoxic immune reaction. However, the combination of TILs with PD-L1 expression has not been well studied in breast cancer.

Predictive factors of the tumor immunological microenvironment for long‐term follow‐up in early stage breast cancer

The aim of this research was to investigate the correlation of immunologic factors in the tumor environment of breast cancer, using immunohistological staining to evaluate the expression of programmed death 1/programmed death ligand 1 (PD‐1/PD‐L1), phosphatase and tensin homolog (PTEN), tumor infiltrating lymphocytes (TILs), and macrophages, and to analyze the association between the immunologic factors and clinical outcome for patients with early stage breast cancer (EBC). A total of 97 EBC patients who underwent standard surgery were investigated. Expression of PD‐1/PD‐L1 and PTEN and the density of CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages were evaluated by immunohistochemical analysis. The association between the immunologic factors and clinical outcome was statistically analyzed. The density of CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages and non‐expression of PTEN was significantly higher in cases of triple negative breast cancer. CD8⁺ TIL density and CD8⁺/PD‐L1⁺ expression were predictive factors for disease‐free survival and overall survival (OS). Human epidermal growth factor 2 (HER2)‐positive patients with PTEN expression and luminal/HER2‐negative patients without PD‐L1 expression had significantly longer OS compared to patients without PTEN expression (P = 0.049) and with PD‐L1 expression (P = 0.036), respectively. Furthermore, patients with PD‐L1⁺/CD8⁺ expression had worse median progression‐free survival (P = 0.022) and median OS (P = 0.037) compared with patients without PD‐L1⁺/CD8⁺ expression. The CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages were shown to infiltrate the tumor area of EBC. In particular, triple negative breast cancer had a higher rate of TIL infiltration within the tumor environment. Expression of PTEN and lack of PD‐L1 expression were associated with favorable survival in HER2‐positive and luminal/HER2‐negative EBC patients, respectively. The PD‐L1 expression combined with CD8⁺ density was significantly associated with an aggressive clinical outcome.     

The mutational load and a T-cell inflamed tumour phenotype identify ovarian cancer patients rendering tumour-reactive T cells from PD-1+ tumour-infiltrating lymphocytes

Background Adoptive immunotherapy with tumour-infiltrating lymphocytes (TIL) may benefit from the use of selective markers, such as PD-1, for tumour-specific T-cell enrichment, and the identification of predictive factors that help identify those patients capable of rendering tumour-reactive TILs. We have investigated this in ovarian cancer (OC) patients as candidates for TIL therapy implementation.Methods PD-1⁻ and PD-1⁺ CD8 TILs were isolated from ovarian tumours and expanded cells were tested against autologous tumour cells. Baseline tumour samples were examined using flow cytometry, multiplexed immunofluorescence and Nanostring technology, for gene expression analyses, as well as a next-generation sequencing gene panel, for tumour mutational burden (TMB) calculation.Results Tumour-reactive TILs were detected in half of patients and were exclusively present in cells derived from the PD-1⁺ fraction. Importantly, a high TIL density in the fresh tumour, the presence of CD137⁺ cells within the PD-1⁺CD8⁺ TIL subset and their location in the tumour epithelium, together with a baseline T-cell-inflamed genetic signature and/or a high TMB, are features that identify patients rendering tumour-reactive TIL products.Conclusion We have demonstrated that PD-1 identifies ovarian tumour-specific CD8 TILs and has uncovered predictive factors that identify OC patients who are likely to render tumour-specific cells from PD-1⁺ TILs.Subject terms: Tumour immunology, Immunotherapy     

三阴性乳腺癌组织中PD-L1和CD8+ TIL的水平及其临床意义

目的：探讨程序性死亡蛋白-配体 1（programmed death ligand-1,PD-L1）和肿瘤浸润淋巴细胞（tumor-infiltrating lymphocyte, TIL）在三阴性乳腺癌（triple-negative breast cancer,TNBC）组织中的水平及其临床意义。方法：收集2015年1月至 2019年1月福建医科大学附属第二医院手术切除的 61 例 TNBC 患者的癌及癌旁组织石蜡标本,用免疫组化法检测癌组织中 PD-L1表达和CD8+ TIL的水平,用卡方检测方法分析TNBC 组织中PD-L1 和 CD8+ TIL 水平与患者临床病理特征及预后的关 系。结果：PD-L1和CD8+TIL在TNBC组织中的阳性率分别为63.9%（39/61）和32.8%（20/61）。PD-L1表达与TNBC患者的肿瘤 大小、淋巴结转移、病理分期、复发与否有明显关联（均P<0.05）,与患者的年龄、肿瘤分化程度、脉管侵犯以及Ki67表达水平无明 显关联（均P>0.05）;CD8+ TIL水平与TNBC 患者的肿瘤大小、肿瘤分化程度、淋巴结转移、病理分期、复发与否有明显关联 （均P<0.05）,与患者的年龄、脉管侵犯以及Ki67表达水平无明显关联（均P>0.05）。PD-L1和CD8+TIL水平与患者的无进展生存 期（PFS）及总生存期（OS）具有显著相关性（均P<0.05）,PD-L1+ 或者缺乏CD8+ TIL与患者更差的PFS及OS相关（均P<0.05）。结 论：TNBC组织中存在较高水平的PD-L1和CD8+TIL,PD-L1阳性表达或缺乏CD8+TIL与肿瘤侵袭性增加相关,也与患者更差的 PFS及OS相关。     

Programmed cell death-ligand 1 expression is associated with a favourable immune microenvironment and better overall survival in stage I pulmonary squamous cell carcinoma

BackgroundProgrammed cell death-ligand 1 (PD-L1) is expressed in a subgroup of lung cancer that may benefit from immunotherapy. The interaction between PD-L1 expression and tumour infiltrating lymphocytes (TIL) remains poorly understood. This study investigated the expression of PD-L1 in surgically resected stage I pulmonary squamous cell carcinoma (SqCC) and correlated it with TILs in tumour microenvironments, common driver mutations, and clinical outcomes.Materials and methodsOne hundred and five patients with surgically resected stage I squamous cell carcinoma were examined. Paraffin-embedded tumour sections were stained with PD-L1 antibody. Tumours with moderate-to-strong membrane staining in ≥5% of tumour cells were scored as positive for PD-L1 expression. The driver mutation epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), and v-raf murine sarcoma viral oncogene homolog B (BRAF) were examined by direct sequencing, while anaplastic lymphoma kinase (ALK), phosphoinositide 3-kinase catalytic alpha (PI3KCA), and fibroblast growth factor receptor 1 (FGFR1) were analysed by immunohistochemistry. The correlations of PD-L1 expression with each subtype of TIL, driver mutations, clinicopathologic parameters, and clinical outcomes were analysed.ResultsThere was positive PD-L1 expression in 56.2% (59/105) of patients. PD-L1 expression was not associated with the common clinicopathologic features and mutations of EGFR, KRAS, BRAF, ALK, PI3KCA, and FGFR1. As regards TILs composition, tumour PD-L1 expression was significantly associated with increased tumour epithelial CD8+ T cells and stromal CD4+ T cells. Otherwise, PD-L1 (+) tumour cells were negatively correlated with PD-L1 (+) immune cells within tumour stroma. By multivariate analysis, tumour PD-L1 expression and increased CD4+ T cell infiltrations in the tumour stroma were independent predictors of better overall survival and had a trend of better disease-free survival.ConclusionsPD-L1 expression is associated with a favourable immune microenvironment in stage I pulmonary SqCC and correlates with better clinical outcome.     

Tumor infiltrating lymphocytes and PD-L1 expression in brain metastases of small cell lung cancer (SCLC)

Brain metastases (BM) are frequent in small cell lung cancer (SCLC). Novel insights into their pathobiology are needed for development of better therapies. We investigated tumor-infiltrating lymphocyte (TIL) subsets (CD3+, CD8+, CD45RO+, FOXP3+ and PD-1+) and expression of PD-L1 in a series of 32 SCLC BM specimens and four matched primary tumor specimens using immunohistochemistry. 30/32 (93.8?%) BM specimens showed TIL infiltration. CD3+ TILs were observed in 30/32 (93.8?%) BM specimens, CD8+ TILs in 25/32 (78.1?%), CD45RO+ TILs in 15/32 (46.9?%), FOXP3+ TILs in 15/32 (46.9?%) and PD-1+ TILs in 1/32 (3.1?%) BM specimens. Patients with infiltration of CD45RO+ TILS had a significantly longer median survival time (11 months; 95?% CI 0.000–26.148) as compared to patients without the presence of CD45RO+ TILs (5 months; 95?% CI 0.966–9.034; p?=?0.007; log rank test). Membranous PD-L1 on tumor cells was observed in 24/32 (75.0?%) BM specimens, with 11/32 (34.4?%) cases showing PD-L1 expression in over 5?% of viable BM tumor cells. PD-L1 expression on TILs was seen in 8/32 (25.0?%) and on tumor infiltrating macrophages in 9/32 (28.1?%) cases. Patients with PD-L1 expression on TILs presented with improved survival prognosis (6 versus 29 months; p?=?0.002; log rank test). Among matched primary tumors, all (4/4; 100?%) specimens showed TIL infiltration, while PD-L1 expression found in only 1/4 (25.0?%) specimen. TIL infiltration and PD-L1 expression are commonly found in SCLC BM and presence of CD45RO+ memory T-cells and PD-L1+ TILs in SCLC BM seem to associate with favorable survival times. Our data suggest an active immune microenvironment in SCLC BM that may be targetable by immune-modulating drugs.     

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.     

The in situ local immune response,tumour senescence and proliferation in colorectal cancer

Background:

Immune cell infiltrates are important determinants of colorectal cancer (CRC) outcome. Their presence may be driven by tumour or host-specific factors. From previous studies in mice, senescence, a state of cell cycle arrest, may moderate tumour progression through upregulation of antitumour immune responses. The relationships between senescence and immune infiltrates have not previously been studied in humans. We explore whether a marker of senescence (p16^ink4a) in combination with low level expression of a proliferation marker (ki-67) relate to T cell infiltrates in CRC, and whether p16^ink4a, Ki-67 and immune infiltrates have similar prognostic value.

Methods:

Immunostaining of p16^inka and Ki-67 was performed within a CRC tissue microarray. Nuclear p16^inka and Ki-67 were categorised as high/low. T-cell markers, CD3, CD45RO, CD8 and FOXP3 were scored separately as high/low grade in three areas of the tumour: the invasive margin (IM), tumour stroma and cancer cell nests (CCNs).

Results:

Two hundred and thirty stage I–III cancers were studied. High nuclear p16^ink4a was expressed in 63% and high proliferation (Ki-67 >15%) in 61%. p16^ink4a expression was associated with reduced CD45RO+ cells at the IM (P<0.05) and within the stroma (P<0.05) and reduced CD8+ cells at the IM (P<0.01). A low Ki-67 proliferative index was associated with reduced density of CD3+ cells in CCNs (P<0.01), reduced CD45RO+ cells at the IM (P<0.05) and within the CCNs (P<0.001), reduced FOXP3+ cells at the IM (P<0.001), within the stroma (P=0.001) and within CCNs (P<0.001) and reduced CD8+ cells at the IM (P<0.05) and within the CCNs (P<0.05). Tumours with both a low proliferative index and expression of p16^ink4a demonstrated similar consistent relationships with reduced densities of T-cell infiltrates. On multivariate analysis, TNM stage (P<0.001), low CD3 cells at the IM (P=0.014), low CD8 cells at the IM (P=0.037), low proliferation (Ki-67; P=0.013) and low senescence (p16^ink4a; P=0.002) were independently associated with poorer cancer survival.

Conclusion:

Senescence, proliferation and immune cell infiltrates are independent prognostic factors in CRC. Although related to survival, p16^ink4a-associated senescence is not associated with an upregulation of antitumour T-cell responses.     

三阴性乳腺癌组织中LAG-3、PD-1、PD-L1的表达和TIL计数的临床意义

目的:探讨三阴性乳腺癌（triple-negative breast cancer,TNBC）组织中淋巴细胞激活基因-3(lymphocyte-activation gene-3,LAG-3)、程序性死亡受体-1(programmed death-1,PD-1)、程序性死亡受体-1配体(programmed death ligand-1,PD-L1)的表达和肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte,TIL)计数的临床意义。方法:免疫组织化学法检测62例原发性TNBC组织和30例癌旁组织中LAG-3、PD-1、PD-L1的表达,苏木素-伊红法染色并计数TIL,分析四者与TNBC临床病理特征及预后的关系。结果:TNBC组织中LAG-3、PD-1和PD-L1表达显著高于癌旁组织（P＜0.01）;LAG-3表达与TNBC患者的淋巴结转移、Ki-67、PD-1、PD-L1表达和TIL计数有关（P＜0.05）;单因素分析显示,TNBC患者DFS与淋巴结转移、TNM分期、Ki-67、TIL、LAG-3+PD-1+、LAG-3+PD-L1+、LAG-3+TILlow有关（P＜0.05）;多因素分析显示,TNM分期、LAG-3+PD-L1+和TIL是TNBC患者预后的独立危险因素（P＜0.05）。结论:TNM分期、LAG-3+PD-L1+和TIL与TNBC患者不良预后有关,特别是LAG-3和PD-L1结合共表达,可为免疫检查点双重阻滞在TNBC患者的临床应用中提供更多的理论依据。     

Predictive relevance of PD-L1 expression combined with CD8+ TIL density in stage III non-small cell lung cancer patients receiving concurrent chemoradiotherapy

BackgroundExpression of programmed cell death-ligand 1 (PD-L1) is known to be a mechanism whereby cancer can escape immune surveillance, but little is known about factors predictive of efficacy in patients with locally advanced non-small cell lung cancer (NSCLC). We investigated the predictive relevance of PD-L1 expression and CD8+ tumour-infiltrating lymphocytes (TILs) density in patients with locally advanced NSCLC receiving concurrent chemoradiotherapy (CCRT).MethodsWe retrospectively reviewed 74 consecutive patients with stage III NSCLC who had received CCRT. PD-L1 expression and CD8+ TIL density were evaluated by immunohistochemical analysis.ResultsUnivariate and multivariate analyses demonstrated that CD8+ TIL density was an independent and significant predictive factor for progression-free survival (PFS) and OS, whereas PD-L1 expression was not correlated with PFS and OS. Sub-analysis revealed that the PD-L1+/CD8 low group had the shortest PFS (8.6 months, p = 0.02) and OS (13.9 months, p = 0.11), and that the PD-L1-/CD8 high group had the longest prognosis (median PFS and OS were not reached) by Kaplan-Meier curves of the four sub-groups.ConclusionsAmong stage III NSCLC patients who received CCRT, there was a trend for poor survival in those who expressed PD-L1. Our analysis indicated that a combination of lack of PD-L1 expression and CD8+ TIL density was significantly associated with favourable survival in these patients. It is proposed that PD-L1 expression in combination with CD8+ TIL density could be a useful predictive biomarker in patients with stage III NSCLC.     

Prognostic value of the density of tumor-infiltrating lymphocytes in colorectal cancer liver metastases

Tumor-infiltrating lymphocytes (TILs) have been reported to reflect the anti-tumor immune status of patients and to be correlated with their prognosis and therapeutic outcomes. However, the characteristics of the local immune status in metastatic tumors is poorly understood, as primary tumors have been the focus in most previous studies. In addition, the local immune status may be influenced by preoperative chemotherapy. The present study aimed therefore to investigate the relationship between the degree of TIL infiltration and the prognosis in patients with curative resection of colorectal cancer liver metastases and to examine the effects of preoperative chemotherapy on the function of immune cells. A total of 108 patients who underwent curative resection of colorectal cancer liver metastases in our department between May 1996 and January 2017 were enrolled in the present study. Peripheral blood samples were obtained within two weeks before surgery. TIL infiltration was evaluated by immunohistochemical staining of surgically resected specimens of liver metastases using anti-CD8/CD3 antibodies. The mean number of TILs in five different fields was calculated, and patients were classified into a high-TIL group and a low-TIL group. Furthermore, patients were divided into three groups as follows: i) A group of patients who did not receive preoperative chemotherapy; ii) a group of patients who received short-term preoperative chemotherapy for <6 months; and iii) a group of patients who received long-term preoperative chemotherapy for ≥6 months. The results demonstrated that the density of TILs in colorectal liver metastases was not correlated with the absolute peripheral lymphocyte count in all patients. Furthermore, the degree of CD8⁺TIL infiltration in liver metastases was significantly lower in the recurrence group compared with the recurrence-free group following hepatectomy. In all patients with colorectal liver metastases, the degree of CD8⁺TIL infiltration was significantly associated with the relapse-free and overall survival. In patients without preoperative chemotherapy, the degree of CD8⁺TIL infiltration was significantly associated with the relapse-free survival, and a high CD8⁺TIL presence tended to have a better effect on the overall survival than a low CD8⁺TIL presence. In the short-term chemotherapy group, the degree of CD8⁺TIL infiltration was significantly associated with the relapse-free and overall survival. In the long-term chemotherapy group, there were no significant differences between the high- and low- CD8⁺TIL groups in the relapse-free and overall survival. In contrast to CD8⁺TILs, CD3⁺TILs showed a poor prognostic ability. In summary, the degree of CD8⁺TIL infiltration in colorectal cancer liver metastases may be correlated with patient prognosis. However, in patients who received long-term chemotherapy before surgery, the degree of TIL infiltration was not necessarily associated with prognosis as the anti-tumor effects of TILs may decrease. The degree of CD8⁺TIL infiltration may therefore be considered as a useful prognostic factor in patients with colorectal liver metastases, but the prognostic accuracy may decrease in patients who received long-term chemotherapy.