Tumor cell expression of programmed cell death‐1 ligand 1 is a prognostic factor for malignant melanoma

BACKGROUND:

Melanoma tends to be refractory to various immunotherapies because of tumor‐induced immunosuppression. To investigate the mechanism underlining the immunosuppression of melanoma patients, the authors focused on programmed cell death‐1 (PD‐1)/PD‐1 ligand 1 (PD‐L1) interaction between tumor cells and T cells.

METHODS:

Melanoma specimens were collected from 59 primary tumors, 16 lymph nodes, and 4 lesions of in‐transit metastasis. Specimens stained with anti‐PD‐L1 monoclonal antibodies were digitalized to jpg files. To evaluate the intensity of PD‐L1 expression, histograms were used, and the red density (RD) was measured. PD‐1 expression on T cells was analyzed in blood samples from 10 patients who had stage IV melanoma and in 4 samples of in‐transit metastases.

RESULTS:

Twenty‐five patients comprised the “low” PD‐L1 expression group (RD value, <90), and 34 patients comprised the “high” group (RD value, ≥90). Breslow tumor thickness in the high‐expression group was significantly higher than in the low‐expression group. Univariate and multivariate analyses revealed that the overall survival rate of the high‐expression group was significantly lower than that of the low‐expression group. In all patients with stage IV disease who were examined, both CD8‐positive and CD4‐positive T cells had significantly higher PD‐1 expression levels in the peripheral blood. Tumor‐infiltrating T cells expressed high levels of PD‐1, and its expression was elevated further during the clinical course.

CONCLUSIONS:

The current results indicated that there is a correlation between the degree of PD‐L1 expression and the vertical growth of primary tumors in melanoma. Multivariate analysis demonstrated that PD‐L1 expression is an independent prognostic factor for melanoma. Cancer 2010. © 2010 American Cancer Society.     

Clinical significance of programmed death‐1 and programmed death‐ligand 1 expression in the tumor microenvironment of clear cell renal cell carcinoma

Recently, immunotherapy based on blocking immune checkpoints with programmed death‐1 (PD‐1) or PD‐ligand 1 (PD‐L1) Abs has been introduced for the treatment of advanced clear cell renal cell carcinoma (ccRCC), especially tumors resistant to vascular endothelial growth factor‐tyrosine kinase inhibitors (VEGF‐TKIs), but the significance of their expression in the tumor microenvironment is unclear. We investigated these immune checkpoint markers in tumor cells and tumor‐infiltrating immune cells (TIIC) in the tumor microenvironment of 100 untreated and 25 VEGF‐TKI‐treated primary ccRCC tissues. Upregulated expression of PD‐1 and PD‐L1 by TIIC, and PD‐L1 by tumor cells was associated with the histological grade and unfavorable prognosis of RCC patients. High PD‐1 and PD‐L1 expression by TIIC was associated with a poorer response to VEGF‐TKI, whereas PD‐L1 expression by tumor cells did not affect the efficacy of the treatment. Furthermore, increased PD‐1‐positive TIIC and PD‐L1‐positive TIIC were observed in tumors treated with VEGF‐TKIs compared with those in untreated tumors. Our data suggest that PD‐1 and PD‐L1 expression by TIIC in the tumor microenvironment is involved in treatment resistance, and that sequential therapy with immune checkpoint inhibitors could be a promising therapeutic strategy for ccRCC resistant to VEGF‐TKI treatment.     

Comparison of tumor‐infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients

The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2⁺, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8⁺ and CD4⁺ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2⁺ and TN breast cancers have a lower percentage of TILs and CD8⁺ and CD4⁺ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.     

Tumor‐reactive CD4+CD8αβ+ CD103+ αβT cells: A prevalent tumor‐reactive T‐cell subset in metastatic colorectal cancers

High level of T‐cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of this infiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype and functional features of tumor‐reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated from CRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivity of CRC TIL and tumor‐reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions (up to 18%) of double positive CD4⁺CD8αβ⁺ (DP) αβ T cells. Interestingly, a high proportion (16–20%) of this TIL subset displayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in most CRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DP TIL were polyclonal, restricted by HLA class‐I, proliferated poorly and secreted higher amounts of IL‐4 and IL‐13 than single positive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increased frequencies of tumor‐reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of this cancer. Based on their high secretion of IL‐4 and IL‐13 and on previously described roles of these cytokines in cancers, we postulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.     

Prognostic value of intra‐tumoral CD8+/FoxP3+ lymphocyte ratio in patients with resected colorectal cancer liver metastasis

Background and Objectives

Patients with isolated colorectal‐cancer‐liver‐metastases (CRCLM) frequently undergo metastatectomy. Tumor‐infiltrating‐lymphocytes (TILs) have prognostic potential in the setting of primary colorectal cancer, however, their role in CRCLM is less studied. We aimed to study the spatial distribution and prognostic role of tumor‐infiltrating CD8⁺ cytotoxic T‐cells and FoxP3⁺ regulatory T‐cells at the metastatic site of CRCLM patients.

Methods

TILs were isolated from fresh metastatic tissues of 47 patients with CRCLM. Archived paraffin‐embedded tissue, from the same patients, was retrieved. CD8⁺ and FoxP3⁺ cells, both in the intra‐tumoral and the peri‐tumoral compartments, were measured by immunohistochemistry on full tissue sections. Proportions of cytotoxic T‐cells (CD8⁺) and regulatory T‐cells (CD4⁺CD25⁺FoxP3⁺), within CD45⁺TILs, were measured by flow‐cytometry.

Results

By immunohistochemistry, individual densities of intra‐tumoral or peri‐tumoral CD8⁺ and FoxP3⁺ cells were not prognostic of survival. However, the intra‐tumoral, but not the peri‐tumoral, CD8⁺/FoxP3⁺ ratio was an independent predictor of survival (HR 0.43, 95%CI 0.19‐0.95, P = 0.032). By flow cytometry, the intra‐tumoral CD8⁺/regulatory T‐cell ratio was also an independent predictor of survival (HR 0.45, 95%CI 0.20‐0.99, P = 0.044).

Conclusions

The ratio of cytotoxic (CD8⁺) to regulatory (FoxP3⁺) T‐cells, in the intra‐tumoral compartment, but not in the peri‐tumoral compartment, can predict survival after resection of CRCLM.     

Infiltration of tumor‐associated macrophages is involved in tumor programmed death‐ligand 1 expression in early lung adenocarcinoma

Programmed death‐ligand 1 (PD‐L1) is an immune modulator that promotes immunosuppression by binding to programmed death‐1 of T‐lymphocytes. Although tumor cell PD‐L1 expression has been shown to be associated with the clinical response to anti–PD‐L1 antibodies, its concise regulatory mechanisms remain elusive. In this study, we evaluated the associations of tumor PD‐L1 expression and immune cell infiltrating patterns in 146 cases of early lung adenocarcinoma (AC) to investigate the possible extrinsic regulation of tumor PD‐L1 by immune cells. Using immunohistochemistry, cell surface PD‐L1 expression in tumor cells was observed in 18.5% of stage 0‐IA lung AC patients. Tumor PD‐L1 positivity was significantly associated with stromal invasion, which was accompanied by increased tumor‐associated macrophages (TAM), CD8⁺ cytotoxic T cells and FoxP3⁺ regulatory T cells. Among these immune cells, TAM and CD8⁺ T cells significantly accumulated in PD‐L1‐positive carcinoma cell areas, which showed a tumor cell nest‐infiltrating pattern. Although CD8⁺ T cells are known to induce tumor PD‐L1 expression via interferon‐? production, the increased TAM within tumors were also associated with tumor cell PD‐L1 positivity, independently of CD8⁺ T cell infiltration. Our in vitro experiments revealed that PD‐L1 expression in lung cancer cell lines was significantly upregulated by co–culture with M2‐differentiated macrophages; expression of PD‐L1 was reduced to baseline levels following treatment with a transforming growth factor‐β inhibitor. These results demonstrated that tumor‐infiltrating TAM are extrinsic regulators of tumor PD‐L1 expression, indicating that combination therapy targeting both tumor PD‐L1 and stromal TAM might be a possible strategy for effective treatment of lung cancer.     

Immunological features of a lung cancer patient achieving an objective response with anti‐programmed death‐1 blockade therapy

The role of immune checkpoint inhibitors in metastatic lung cancer has been established in recent years and the pretherapeutic profiles of the tumor microenvironment in responders have been increasingly reported. The role of salvage surgery and the immune profiles of the posttherapeutic specimens in patients achieving an objective response have rarely been studied. We report a case of metastatic lung cancer treated by anti‐programmed death‐1 Ab followed by surgical resection. The immune status of the tumor was assessed, showing germinal center formation, memory B cell infiltration, and a high frequency of interferon gamma ‐secreting T cells.     

MiR‐15a/16 deficiency enhances anti‐tumor immunity of glioma‐infiltrating CD8+ T cells through targeting mTOR

MiR‐15a/16, a miRNA cluster located at chromosome 13q14, has been reported to act as an immune regulator in inflammatory disorders besides its aberrant expression in cancers. However, little is known about its regulation in tumor‐infiltrating immune cells. In our study, using an orthotropic GL261 mouse glioma model, we found that miR‐15a/16 deficiency in host inhibited tumor growth and prolonged mice survival, which might be associated with the accumulation of tumor‐infiltrating CD8+ T cells. More importantly, tumor‐infiltrating CD8+ T cells without miR‐15a/16 showed lower expression of PD‐1, Tim‐3 and LAG‐3, and stronger secretion of IFN‐γ, IL‐2 and TNF‐α than WT tumor‐infiltrating CD8+ T cells. Also, our in vitro experiments further confirmed that miR‐15a/16^?/? CD8+ T displayed higher active phenotypes, more cytokines secretion and faster expansion, compared to WT CD8+ T cells. Mechanismly, mTOR was identified as a target gene of miR‐15a/16 to negatively regulate the activation of CD8+ T cells. Taken together, these data suggest that miR‐15a/16 deficiency resists the exhaustion and maintains the activation of glioma‐infiltrating CD8+ T cells to alleviate glioma progression via targeting mTOR. Our findings provide evidence for the potential immunotherapy through targeting miR‐15a/16 in tumor‐infiltrating immune cells.     

PD‐1+ TIM‐3+ T cells in malignant ascites predict prognosis of gastrointestinal cancer

The liquid biopsy of ascites fluid could be an excellent source of tumor and microenvironment for the study of prognostic biomarkers because of its accessibility. Tumor‐infiltrating lymphocytes (TILs) can predict prognosis in multiple malignancies, including the response to immune checkpoint inhibitors, a breakthrough cancer therapy. However, TILs’ profiles from malignant ascites have not been extensively studied. Using flow cytometric analysis, we quantified the proportion of exhausted T cells and memory/naive/effector T‐cell subsets, among the CD4+ and CD8+ T‐cell populations of paired TILs and peripheral blood T cell samples (n = 22). The correlation between CD4+ and CD8+ subset profiles suggested that the combined analysis of CD4+ and CD8+ cells in malignant ascites was clinically significant. We found that cells positive for the exhaustion markers programmed cell death‐1 (PD‐1), and T‐cell immunoglobulin and mucin domain 3 (TIM‐3), and cells coexpressing PD‐1 and TIM‐3 abundantly exist among malignant ascites TILs. Furthermore, patients with high frequency of PD‐1+ TIM‐3+ cells among the CD4+ and CD8+ T‐cell population showed worse clinical outcome in multivariate analysis (n = 27). We propose that exhausted ascites TILs represent a clinically significant prognostic biomarker in advanced gastrointestinal cancer and represent an important target for immune checkpoint inhibitors.     

Multimarker phenotype predicts adverse survival in patients with lymph node-negative colorectal cancer

BACKGROUND: The heterogeneity of stage II colon cancer underlines the need for identifying high-risk, lymph node-negative patients. The objective of this study was to define a multimarker prognostic model of 5-year survival in patients with lymph node-negative, mismatch repair (MMR)-proficient colorectal cancer (CRC). METHODS: Immunohistochemistry for 13 tumor markers was performed on 587 lymph node-negative, MMR-proficient CRC samples by using a tissue microarray. Immunoreactivity was evaluated semiquantitatively. A receiver-operating characteristic-based approach was used to detect clinically relevant tumor markers and to determine cutoff scores for tumor positivity. Univariate and multivariate analyses stratified by pathologic T3 (pT3) or pT4 tumor classification were performed. RESULTS: In univariate analysis, the absence of CD8+ tumor infiltrating lymphocytes (TILs) (P < .001), loss of p27 (P = .006), positive urokinase-type plasminogen activator (uPA) expression (P = .002), and positive uPA receptor (uPAR) expression (P = .037) were associated with an adverse prognosis. In multivariate analysis, CD8 (P = .001), p27 (P = .031), and uPA (P = .014) were independent prognostic factors. The multimarker phenotype of negative CD8, loss of p27, and positive uPA expression led to significantly worse survival compared with all other combinations of these features. Stratified by pT3 or pT4 stage, CD8 (P = .006) and uPA (P = .011) had independent prognostic value. Combined CD8 negativity and uPA positivity led to a more adverse prognosis in both patients with pT3 tumors and patients with pT4 tumors (P < .001). No difference was observed in the length of survival between patients with pT3 tumors who had CD8 negativity and uPA positivity and patients with pT4 tumors (P = .267). CONCLUSIONS: The multimarker phenotype of the absence of CD8+ TILs, loss of p27, and positive uPA expression was predictive of an adverse prognosis in patients with lymph node-negative, MMR-proficient CRC. The current findings suggested that a subgroup of patients with high-risk, lymph node-negative pT3 tumors should be considered for adjuvant therapy.     

Upregulation of programmed cell death ligand 1 promotes resistance response in non‐small‐cell lung cancer patients treated with neo‐adjuvant chemotherapy

To assess the association of the programmed cell death ligand 1 (PD‐L1) with cisplatin‐based neo‐adjuvant chemotherapy (NAC) response, we investigated the level of PD‐L1 and found increased PD‐L1 expression in chemo‐resistant tumors compared with chemo‐sensitive tumors according to RNA‐Seq analysis. In a cohort of 92 patients with NAC, the positive staining of PD‐L1 was correlated with TNM stage, lower sensitive‐response rates and shorter overall survival rates. In another 30 paired tumor specimens pre‐ and post‐chemotherapy, the patients with high PD‐L1 expression post‐chemotherapy had a worse outcome and higher stable disease rate. CD8⁺ tumor‐infiltrating lymphocytes were found to be related to chemosensitive response and better prognosis and negative PD‐L1 expression. Furthermore, in two patient‐derived xenograft models and cell lines A549 and PC‐9, cisplatin upregulated PD‐L1 expression, and the enhancement of PD‐L1 in cancer cell lines was in a drug dose‐dependent manner. Moreover, the depletion of PD‐L1 significantly reduced cisplatin resistance. When phosphatidylinositol 3‐kinase/protein kinase B signaling was inhibited by corresponding inhibitors, PD‐L1 expression was downregulated and apoptosis was upregulated in the cisplatin‐treated cancer cells. These results suggest that the upregulation of PD‐L1 promotes a resistance response in lung cancer cells that might be through activation of the phosphatidylinositol 3‐kinase/protein kinase B pathway and suppression of tumor‐infiltrating lymphocytes. The high expression of PD‐L1 after NAC could be an indication of therapeutic resistance and poor prognosis in patients with non‐small‐cell lung cancer.     

Programmed death‐ligand 1 is prognostic factor in esophageal squamous cell carcinoma and is associated with epidermal growth factor receptor

Programmed death‐ligand 1 (PD‐L1) expression either indicates immune inhibitory status or concurrent immune response. Although the relationship between PD‐L1 and clinical outcomes has been studied widely in recent years, its role in prognosis of esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we assessed the significance of PD‐L1 in ESCC and its association with epidermal growth factor receptor (EGFR) and radiation response. We found that PD‐L1 was present both on the surface of tumor cells and tumor‐infiltrating immune cells. Patients with tumor‐infiltrating immune cell PD‐L1 expression had better survival. PD‐L1 expression on immune cells was an independent prognostic factor for patients with ESCC. PD‐L1 expression either on tumor‐infiltrating immune cells or tumor cells was negatively associated with EGFR expression. EGFR/PD‐L1 pairs could separate the survival between EGFR low/PD‐L1 positive and EGFR high/PD‐L1 negative groups. In ESCC cell lines with EGFR high expression, PD‐L1 expression was induced significantly when EGFR signaling was activated by radiation and was dramatically inhibited by an EGFR tyrosine kinase inhibitor. In conclusion, tumor‐infiltrating immune cell PD‐L1 expression is an independent prognostic factor for ESCC, and the association between EGFR and PD‐L1 is vital to determining survival. It is important to consider radiotherapy‐induced imbalance of pro‐tumor and anti‐tumor immune response. A combination of radiotherapy and PD‐L1‐targeted therapy could be a promising therapeutic strategy for ESCC patients.     

卵巢癌患者外周血T淋巴细胞的表达分析

目的探讨卵巢癌患者外周血T淋巴细胞的表达变化情况,分析CD+4CD+25表达与预后的关系。方法选择2009年2月到2012年11月收治的35例卵巢癌患者作为试验组,另选择35例同期体检排除肿瘤及免疫相关性疾病的健康人作为对照组,检测两组患者外周血中T淋巴细胞的表达水平。试验组同期分离肿瘤淋巴细胞进行免疫染色分析。结果对照组和试验组外周血淋巴细胞中CD+4CD+25的表达量分别为(5.36±0.63)%和(44.12±6.23)%,差异有统计学意义(P<0.05)。外周血淋巴细胞CD+4CD+25比例与卵巢癌患者年龄、NCCN分期、组织学类型和淋巴结转移均无关(P>0.05)。CD+4CD+25高表达组卵巢癌患者总生存期低于CD+4CD+25低表达组(P<0.05)。结论卵巢癌患者外周血CD+4CD+25淋巴细胞多呈现高表达,与预后密切相关,可成为卵巢癌预后指标和免疫治疗的生物靶点。     

Myeloid-derived suppressor cell infiltration is associated with a poor prognosis in patients with hepatocellular carcinoma

The clinicopathological features of myeloid-derived suppressor cell (MDSC) and CD8⁺ T-cell infiltration in hepatocellular carcinoma (HCC) are poorly understood. The present study examined MDSC and CD8⁺ T-cell infiltration in surgically resected primary HCC specimens and investigated the association of MDSC and CD8⁺ T-cell infiltration with clinicopathological features and patient outcomes. Using a database of 466 patients who underwent hepatic resection for HCC, immunohistochemical staining of CD33 (an MDSC marker) and CD8 was performed. High infiltration of MDSCs within the tumor was observed in patients with a poorer Barcelona Clinic Liver Cancer stage, larger tumor size, more poorly differentiated HCC, and greater presence of portal venous thrombosis, microscopic vascular thrombosis and macroscopic intrahepatic metastasis. MDSC infiltration and CD8⁺ T-cell infiltration were independent predictors of recurrence-free survival and overall survival, respectively. Stratification based on the MDSC and CD8⁺ T-cell status of the tumors was also associated with recurrence-free survival (10 year-recurrence-free survival; MDSC^highCD8⁺ T-cell^Low, 3.68%; others, 25.7%) and overall survival (10 year-overall survival; MDSC^highCD8⁺ T-cell^Low, 12.0%; others, 56.7%). In conclusion, the present large cohort study revealed that high MDSC infiltration was associated with a poor clinical outcome in patients with HCC. Furthermore, the combination of the MDSC and tumor-infiltrating CD8⁺ T-cell status enabled further classification of patients based on their outcomes.     

Differences in the immunosurveillance pattern associated with DNA mismatch repair status between right‐sided and left‐sided colorectal cancer

Tumor location and immunity play important roles in the progression of colorectal cancer (CRC). This study aimed to investigate the differences in the immunosurveillance pattern between right‐ and left‐sided CRC and analyze their association with clinicopathologic features, including mismatch repair (MMR) status. We included surgically resected stage II/III CRC cases and evaluated the immunohistochemical findings of HLA class I, HLA class II, programmed cell death‐ligand 1 (PD‐L1), PD‐1, CTLA‐4, CD3, CD4, CD8, TIA‐1, T‐bet, GATA3, RORγT, Foxp3, and CD163. A total of 117 patients were included in the analyses; of these, 30 and 87 had right‐ and left‐sided cancer, respectively. Tumor immunity varied according to the tumor location in the overall cohort. Analysis of the tumors excluding those with DNA mismatch repair (MMR) deficiency also revealed that tumor immunity differed according to the tumor location. In right‐sided colon cancer (CC), high expression of Foxp3 (P = .0055) and TIA‐1 (P = .0396) were associated with significantly better disease‐free survival (DFS). High CD8 (P = .0808) and CD3 (P = .0863) expression tended to have better DFS. Furthermore, in left‐sided CRC, only high PD‐L1 expression in the stroma (P = .0426) was associated with better DFS. In multivariate analysis, high Foxp3 expression in right‐sided CC was an independent prognostic factor for DFS (hazard ratio, 7.6445; 95% confidence interval, 1.2091‐150.35; P = .0284). In conclusion, the immunosurveillance pattern differs between right‐ and left‐sided CRC, even after adjusting for MMR deficiency.