Independent association of PD‐L1 expression with noninactivated VHL clear cell renal cell carcinoma—A finding with therapeutic potential

Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor that is characterized in most cases by inactivation of the tumor suppressor gene VHL. The VHL/HIF/VEGF pathway thus plays a major role in angiogenesis and is currently targeted by anti‐angiogenic therapy. The emergence of resistance is leading to the use of targeted immunotherapy against immune checkpoint PD1/PDL1 that restores antitumor immune response. The correlation between VHL status and PD‐L1 expression has been little investigated. In this study, we retrospectively reviewed 98 consecutive cases of ccRCC and correlated PD‐L1 expression by immunohistochemistry (IHC) with clinical data (up to 10‐year follow‐up), pathological criteria, VEGF, PAR‐3, CAIX and PD‐1 expressions by IHC and complete VHL status (deletion, mutation and promoter hypermethylation). PD‐L1 expression was observed in 69 ccRCC (70.4%) and the corresponding patients had a worse prognosis, with a median specific survival of 52 months (p = 0.03). PD‐L1 expression was significantly associated with poor prognostic factors such as a higher ISUP nucleolar grade (p = 0.01), metastases at diagnosis (p = 0.01), a sarcomatoid component (p = 0.04), overexpression of VEGF (p = 0.006), and cytoplasmic PAR‐3 expression (p = 0.01). PD‐L1 expression was also associated with dense PD‐1 expression (p = 0.007) and with ccRCC with 0 or 1 alteration(s) (non‐inactivated VHL tumors; p = 0.007) that remained significant after multivariate analysis (p = 0.004 and p = 0.024, respectively). Interestingly, all wild‐type VHL tumors (no VHL gene alteration, 11.2%) expressed PD‐L1. In this study, we found PD‐L1 expression to be associated with noninactivated VHL tumors and in particular wild‐type VHL ccRCC, which may benefit from therapies inhibiting PD‐L1/PD‐1.     

High‐affinity PD‐1 molecules deliver improved interaction with PD‐L1 and PD‐L2

The inhibitory checkpoint molecule programmed death (PD)‐1 plays a vital role in maintaining immune homeostasis upon binding to its ligands, PD‐L1 and PD‐L2. Several recent studies have demonstrated that soluble PD‐1 (sPD‐1) can block the interaction between membrane PD‐1 and PD‐L1 to enhance the antitumor capability of T cells. However, the affinity of natural sPD‐1 binding to PD‐L1 is too low to permit therapeutic applications. Here, a PD‐1 variant with approximately 3000‐fold and 70‐fold affinity increase to bind PD‐L1 and PD‐L2, respectively, was generated through directed molecular evolution and phage display technology. Structural analysis showed that mutations at amino acid positions 124 and 132 of PD‐1 played major roles in enhancing the affinity of PD‐1 binding to its ligands. The high‐affinity PD‐1 mutant could compete with the binding of antibodies specific to PD‐L1 or PD‐L2 on cancer cells or dendritic cells, and it could enhance the proliferation and IFN‐γ release of activated lymphocytes. These features potentially qualify the high‐affinity PD‐1 variant as a unique candidate for the development of a new class of PD‐1 immune‐checkpoint blockade therapeutics.     

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.     

Claspin overexpression is associated with high‐grade histology and poor prognosis in renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most common human cancers. We previously reported that claspin is a key regulator in the progression of gastric cancer, and it likely plays an important role in cancer stem cells of gastric cancer. However, the significance of claspin in RCC has not been examined. First, we analyzed the expression and distribution of claspin in 95 RCC cases by immunohistochemistry. In the nonneoplastic kidney, the staining of claspin was either weak or absent, whereas RCC tissue showed nuclear staining. In total, claspin expression was detected in 45 (47%) of 95 RCC cases. The claspin staining appeared relatively stronger in high nuclear grade RCC than in low nuclear grade RCC. Claspin‐positive RCC cases were associated with higher T grade, tumor stage, nuclear grade, vein invasion, and poorer prognosis. CLSPN siRNA treatment decreased RCC cell proliferation. The levels of phosphorylated Erk and Akt were lower in CLSPN siRNA‐transfected RCC cells than in control cells. In addition, claspin was coexpressed with CD44, epidermal growth factor receptor, p53, and programmed death ligand‐1. These results suggest that claspin plays an important role in tumor progression in RCC and might be a prognostic marker and novel therapeutic target molecule.     

The efficacy and safety of anti‐PD‐1/PD‐L1 antibodies combined with chemotherapy or CTLA4 antibody as a first‐line treatment for advanced lung cancer

Checkpoint inhibitors show promising efficacy in advanced lung cancer, especially in non‐small‐cell lung cancer (NSCLC). This meta‐analysis was conducted to explore the therapeutic efficacy and safety of anti‐PD‐1/PD‐L1 antibodies combined with chemotherapy or CTLA4 antibody as first‐line treatments for patients with advanced lung cancer. A systematic search was performed in databases for this system review and quantitative meta‐analysis. Twelve trials were finally enrolled in the meta‐analysis. Our analyses revealed that the combined overall response rate (ORR) and disease control rate (DCR) for immune checkpoint inhibitors combined with chemotherapy for the treatment of NSCLC were 47.0% (95% CI: 34.2%‐60.2%) and 80.9% (95% CI: 69.4%‐88.7%), respectively. The combined ORR and DCR for CTLA4 antibody combined with chemotherapy for the treatment of small‐cell lung cancer (SCLC) were 65.4% (61.1%‐69.5%) and 87.6% (84.5%‐90.2%), respectively. The combined six‐month progression‐free survival rates (PFSRs_6m) for NSCLC and SCLC were 50.2% (95% CI: 21.9%‐78.4%) and 30.7% (21.2%‐40.3%), respectively, and the OSRs_1y were 56.4% (39.1%‐73.7%) and 36.9% (33.3%‐40.5%), respectively. In addition, the combined ORR and DCR for the checkpoint inhibitors plus CTLA4 antibody treatment group in NSCLC were 29.6% (95% CI: 11.4%‐57.8%) and 48.7% (16.8%‐81.7%), respectively. In subgroup analyses, a significant improvement in PFS was observed in NSCLC and SCLC, with a combined hazard ratio and 95% confidence interval of 0.841 (0.737‐0.961) and 0.856 (0.756‐0.968), respectively. In summary, synergistic activity and an acceptable safety profile were observed with checkpoint inhibitor plus chemotherapy combination treatment in lung cancer.     

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.     

Nutlin‐3 enhances sorafenib efficacy in renal cell carcinoma

The renal cell carcinoma (RCC) is one of the top 10 cancers in USA. The renal tumors are highly angiogenic and are resistant to conventional interventions, particularly radiotherapy. The advent of multi‐specific tyrosine kinase inhibitor sorafenib has improved the progression‐free survival in RCC, but overall survival in recurrent and metastatic RCC is still a concern that has lead to characterization of combinatorial regimens. Hence, we studied the effect of combination of nutlin‐3, an MDM2 inhibitor, which increases p53 levels, and sorafenib in RCC. Sorafenib along with nutlin‐3 synergistically inhibited the cell survival and enhanced caspase‐3 cleavage leading to apoptosis in RCC. Nutlin‐3 and sorafenib were more effective in reducing the migration of RCC, in combination than as single agents. Sorafenib and nutlin‐3 decreased the phosphorylation of vascular endothelial growth factor receptor‐2 (VEGFR‐2) and ERK along with inducing p53 activity. The sorafenib and nutlin‐3 co‐treatment lead to enhanced levels of p53, p‐p53, and increase in the levels of p53 pro‐apoptotic effector PUMA, Bax, and decrease in the anti‐apoptotic Bcl‐2 levels. Importantly, our studies revealed that sorafenib alone can activate p53 in a concentration dependent manner. Thus, co‐treatment of nutlin‐3 with sorafenib leads to increased half‐life of p53, which in turn can be activated by sorafenib, to induce downstream pro‐apoptotic and anti‐proliferative effects. This is the first report showing the synergistic effect of sorafenib and nutlin‐3 while providing a strong clinical‐translational rationale for further testing of sorafenib and nutlin‐3 combinatorial regimen in human RCC. © 2011 Wiley Periodicals, Inc.     

PD‐L1 expression associated with worse survival outcome in malignant pleural mesothelioma

1 Aim

There is currently a need to identify prognostic biomarkers to assist in a risk adopted approach in treatment of malignant pleural mesothelioma (MPM). Expression of programmed death ligand 1 (PD‐L1) has been studied as a prognostic biomarker in a number of tumors given its central role in antitumoral immune response evasion. Four previously published analyses found PD‐L1 positivity to be an adverse survival prognostic factor in MPM. This study aims to further investigate the relationship between PD‐L1 expression in mesothelioma tissues and survival outcome.

2 Methods

Clinical data of MPM patients from a single institution between 2006 and 2016 were reviewed. Patient's archived tissues were stained with PD‐L1 (Clone Ventana SP263). PD‐L1 positivity was defined as > 1% membranous staining regardless of intensity.

3 Results

Data from fifty eight patients were analyzed. Median age was 73, majority was male (49, 84%) and had ECOG between 0 and 2 (46, 79%). Most common histopathological subtype was epithelioid (42, 72%), 9 (16%) biphasic subtype and 7 (12%) sarcomatoid. Thirty one patients (53%) received best supportive care and twenty seven patients (47%) received chemotherapy or combination treatment. Forty‐two patients had positive PD‐L1 expression (72.4%). The median survival time for PD‐L1 negative group is 15.5 months and 6 months for the positive group. Positive PD‐L1 expression is independently correlated with worse prognosis (HR = 2.02; 95% CI, 1.005–4.057; P‐value = 0.0484).

4 Conclusions

Our analysis found a higher percentage of MPM patients with positive PD‐L1 (> 1%) compared to other studies. Highly positive PD‐L1 expression was associated with statistically significantly lower median survival time.     

Correlation between sex and efficacy of immune checkpoint inhibitors (PD‐1 and CTLA‐4 inhibitors)

Immune checkpoint inhibitors (ICIs) exert the antitumor efficacy depending on immune response, which is affected by sex difference, where both biological and sociological factors are involved. The role of sex in ICI trials has been overlooked. How sex correlates with ICI efficacy is incompletely understood. Clinical trials evaluating ICI versus other therapies in male and female patients were included. The hazard ratio (HR) and 95% confidence interval (CI) of overall survival (OS) and progression‐free survival (PFS) were used. Six thousand and ninety‐six patients from 11 trials were included. More improvement of OS was observed in males (HR, 0.62; 95% CI, 0.53–0.71; p < 0.001) treated with ICI versus controls than females (HR, 0.74; 95% CI, 0.65–0.84; p < 0.001). ICIs improved PFS more in males (HR, 0.57; 95% CI, 0.43–0.71; p < 0.001) than females (HR, 0.71; 95% CI, 0.52–0.91; p < 0.001). The sex difference had more effect on the overall survival in melanoma patients versus NSCLC patients. Overall survival of patients treated with CTLA‐4 inhibitor was more influenced by sex variable compared with PD‐1 inhibitors. A significant sex‐related efficacy difference was observed between female and male melanoma patients. Although male patients had longer OS and PFS than females when treated with ICIs versus controls, the difference was not significant. Sex difference should be more considered in future clinical trials, guidelines and clinical practice.     

Circulating exosomes from esophageal squamous cell carcinoma mediate the generation of B10 and PD‐1high Breg cells

As one of the most frequently diagnosed cancers, esophageal squamous cell carcinoma (ESCC) remains the leading cause of malignancy‐related death worldwide. Many studies have focused on the potential role of cancer cells in educating B cells during cancer progression. Here, we aim to explore the role of circulating exosomes from ESCC in the generation of two main regulatory B (Breg) subsets, including interleukin‐10⁺ Bregs (B10) and programmed cell death (PD)‐1^high Bregs. Firstly, we observed an elevated percentage of B10 cells in peripheral blood of ESCC patients compared with healthy controls. Then we isolated and characterized exosomes from the peripheral blood of ESCC patients and an ESCC cell line. Exosomes from ESCC patients and the ESCC cell line suppressed the proliferation of B cells and induced the augmentation of B10 and PD‐1^high Breg cells. By comparing the long non‐coding RNA and mRNA expression profiles in exosomes from ESCC patients or healthy controls, we identified a series of differentially expressed genes. Finally, we undertook gene annotation and pathway enrichment analyses on differentially expressed genes to explore the potential mechanism underlying the modulatory role of cancer exosomes in B cells. Our findings contribute to the study on B cell‐mediated ESCC immunosuppression and shed light on the possible application of exosomes in anticancer therapies.     

Warburg phenotype in renal cell carcinoma: High expression of glucose‐transporter 1 (GLUT‐1) correlates with low CD8+ T‐cell infiltration in the tumor

Many tumor cells are characterized by a dysregulated glucose metabolism associated with increased glycolysis in the presence of oxygen (“Warburg Effect”). Here, we analyzed for the first time a possible link between glucose metabolism and immune cell infiltration in renal cell carcinoma (RCC). RCC specimens revealed a highly significant increase in the expression of lactate dehydrogenase A (LDHA) and glucose‐transporter 1 (GLUT‐1) compared to the corresponding normal kidney tissue on mRNA level. Accordingly, tumor cell lines of different origin such as RCC, melanoma and hepatocellular carcinoma strongly expressed LDHA and GLUT‐1 compared to their nonmalignant counterparts. In line with this finding, tumor cells secreted high amounts of lactate. High expression of GLUT‐1 and LDH5, a tetramer of 4 LDHA subunits, was confirmed by tissue microarray analysis of 249 RCC specimens. Overall, 55/79 (69.6%) and 46/71 (64.7%) cases of clear cell carcinoma showed a constitutive, but heterogeneous expression of GLUT‐1 and LDH5, respectively. The number of CD3⁺, CD8⁺ and FOXP3⁺ T cells was significantly elevated in RCC lesions compared to normal kidney epithelium, but effector molecules such as granzyme B and perforin were decreased in tumor infiltrating T cells. Of interest, further analysis revealed an inverse correlation between GLUT‐1 expression and the number of CD8⁺ T cells in RCC lesions. Together, our data suggest that an accelerated glucose metabolism in RCC tissue is associated with a low infiltration of CD8⁺ effector T cells. Targeting the glucose metabolism may represent an interesting tool to improve the efficacy of specific immunotherapeutic approaches in RCC.     

Accurate expression of PD‐L1/L2 in lung adenocarcinoma cells: A retrospective study by double immunohistochemistry

The percentage of programmed death ligand 1 (PD‐L1) positivity in cancer cells, named as the tumor proportion score, is considered to be a predictive biomarker for anti‐PD‐1/PD‐L1 therapy in lung cancer. PD‐L1 is expressed on not only cancer cells but also on immune cells, including macrophages. Although previous studies related to PD‐L1/2 expression in cancer tissues have been generally based on single immunohistochemistry (IHC), in the present study, we attempted to evaluate accurate PD‐L1/2 expression in cancer cells in lung adenocarcinoma cells using double IHC to also evaluate macrophages. Of the 231 patients, PD‐L1 expression was negative in 169 patients (73.2%), 1%‐49% positive in 47 patients (20.3%), and ≥50% positive in 15 patients (6.5%). Interestingly, PD‐L1 positivity was decreased when using double IHC compared with the estimation by single IHC. High PD‐L1 expression was associated with high‐grade cancer cells and in higher stage cancer. PD‐L2 was negative in 109 patients (47.2%), 1%‐49% positive in 50 patients (21.6%), and ≥50% positive in 72 patients (31.2%). The number of PD‐L2‐positive patients was increased in cases that had an epidermal growth factor receptor (EGFR) mutation and in lower stage cancer. Thirty‐five patients (15.2%) were positive for both PD‐L1 and PD‐L2, whereas 81 patients (35.1%) were negative for both PD‐L1 and PD‐L2. Log‐rank analysis showed that progression‐free survival and overall survival were significantly the longest in the PD‐L1‐negative and PD‐L2‐positive groups (P < .0001 and P = .0120). We observed lower PD‐L1 or PD‐L2 expression in lung adenocarcinoma than previously reported. Double IHC for macrophages may help clinicians to evaluate PD‐L1 or PD‐L2 expression specifically in cancer cells.