Probes for INS

Therapy with anti-PD-L1 immune check-point inhibitors is approved for several cancers, including advanced urothelial carcinomas. PD-L1 prevalence estimates vary widely in bladder cancer, and lack of correlation between expression and clinical outcomes and immunotherapyresponse may be attributed to methodological differences of the immunohistochemical reagents and procedures. We characterized PD-L1 expression in 235 urothelial carcinomas including 79 matched pairs of primary and metastatic cancers using a panel of four PD-L1 immunoassays in comparison with RNAscope assay using PD-L1-specific probe (CD274). The antibody panel included three FDA-approved clones (22C3 for pembrolizumab, 28.8 for nivolumab, SP142 for atezolizumab), and a commonly used clone E1L3N. Manual scoring of tissue microarrays was performed in each of 235 tumors (624 tissue cores) and compared to an automated image analysis. Expression of PD-L1 in tumor cells by ≥1 marker was detected in 41/142 (28.9%) primary tumors, 13/77 (16.9%) lymph nodes, and 2/16 (12.5%) distant metastases. In positive cases, high PD-L1 expression (>50% cells) was detected in 34.1% primary and 46.7% metastases. Concordant PD-L1 expression status was present in 71/79 (89.9%) cases of matched primary and metastatic urothelial carcinomas. PD-L1 sensitivity ranked from highest to lowest as follows: RNAscope, clone 28.8, 22C3, E1L3N, and SP142. Pairwise concordance correlation coefficients between the four antibodies in 624 tissue cores ranged from 0.76 to 0.9 for tumor cells and from 0.30 to 0.85 for immune cells. RNA and protein expression levels showed moderate to high agreement (0.72-0.87). Intra-tumor expression heterogeneity was low for both protein and RNA assays (interclass correlation coefficients: 0.86-0.94). Manual scores were highly concordant with automated Aperio scores (0.94-0.97). A significant subset of 56/235 (23.8%) urothelial carcinomas stained positive for PD-L1 with high concordance between all four antibodies and RNA ISH assay. Despite some heterogeneity in staining, the overall results are highly concordant suggesting diagnostic equivalence of tested assays.

ABASTRACT To determine PD1/PDL1 expression status in triple-negative breast cancer (TNBC) at both protein and mRNA levels, and to analyze the relationship between their expression and clinical parameters of the TNBC patients. Immunohistochemistry and RNAscope were used to semi quantitively evaluate PD1/PDL1 protein and mRNA expression in 195 TNBC cases on tissue microarrays. Tumor infiltrating lymphocyte (TILs) abundance was assessed using hematoxylin-eosin staining.Both tumor cells and TILs expressed PDL1. PDL1 protein and mRNA positivity was 6.7% and 74.4% respectively in tumor cells, and 31.3% and 50.9% respectively in TILs. PDL1 protein and mRNA expressions had no significant association with patient prognosis. PD1 protein was only detected in TILs (70.3% positivity). PD1 protein expression was significantly related to PDL1 expression, higher TIL abundance, Ki-67 index, basal-like subtypes, and distant metastasis. Furthermore, it was significantly associated with longer disease free survival (P<0.001) and overall survival (P = 0.004). There was no significant association between PD1 mRNA expression and clinicopathological characteristics. PD1/ PDL1 protein and mRNA expressions were inconsistent (kappa = 0.705 and 0.061, respectively). PD1 protein expression in TILs, but not PDL1 in tumor cells, was a favorable prognostic factor in TNBC. PD1/ PDL1 mRNA and protein expressions were inconsistent.

Overexpression of the B7-H1 (PD-L1) molecule in the tumor microenvironment (TME) is a major immune evasion mechanism in some patients with cancer, and antibody blockade of the B7-H1/PD-1 interaction can normalize compromised immunity without excessive side-effects. Using a genome-scale T cell activity array, we identified Siglec-15 as a critical immune suppressor. While only expressed on some myeloid cells normally, Siglec-15 is broadly upregulated on human cancer cells and tumor-infiltrating myeloid cells, and its expression is mutually exclusive to B7-H1, partially due to its induction by macrophage colony-stimulating factor and downregulation by IFN-γ. We demonstrate that Siglec-15 suppresses antigen-specific T cell responses in vitro and in vivo. Genetic ablation or antibody blockade of Siglec-15 amplifies anti-tumor immunity in the TME and inhibits tumor growth in some mouse models. Taken together, our results support Siglec-15 as a potential target for normalization cancer immunotherapy.

Abstract

PURPOSE:

Subsets of pituitary tumors exhibit an aggressive clinical courses and recur despite surgery, radiation, and chemotherapy. Because modulation of the immune response through inhibition of T-cell checkpoints has led to durable clinical responses in multiple malignancies, we explored whether pituitary adenomas express immune-related biomarkers that could suggest suitability for immunotherapy. Specifically, programmed death ligand 1 (PD-L1) has emerged as a potential biomarker whose expression may portend more favorable responses to immune checkpoint blockade therapies. We thus investigated the expression of PD-L1 in pituitary adenomas.

METHODS:

PD-L1 RNA and protein expression were evaluated in 48 pituitary tumors, including functioning and non-functioning adenomas as well as atypical and recurrent tumors. Tumor infiltrating lymphocyte populations were also assessed by immunohistochemistry.

RESULTS:

Pituitary tumors express variable levels of PD-L1 transcript and protein. PD-L1 RNA and protein expression were significantly increased in functioning (growth hormone and prolactin-expressing) pituitary adenomas compared to non-functioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary adenomas harbored higher levels of PD-L1 mRNA compared to recurrent tumors. Tumor infiltrating lymphocytes were observed in all pituitary tumors and were positively correlated with increased PD-L1 expression, particularly in the functional subtypes.

CONCLUSIONS:

Human pituitary adenomas harbor PD-L1 across subtypes, with significantly higher expression in functioning adenomas compared to non-functioning adenomas. This expression is accompanied by the presence of tumor infiltrating lymphocytes. These findings suggest the existence of an immune response to pituitary tumors and raise the possibility of considering checkpoint blockade immunotherapy in cases refractory to conventional management.

We co-assessed PD-L1 expression and CD8⁺ tumor-infiltrating lymphocytes in gastric cancer (GC), and categorized into 4 microenvironment immune types. Immunohistochemistry (PD-L1, CD8, Foxp3, E-cadherin, and p53), PD-L1 mRNA in situ hybridization (ISH), microsatellite instability (MSI), and EBV ISH were performed in 392 stage II/III GCs treated with curative surgery and fluoropyrimidine-based adjuvant chemotherapy, and two public genome databases were analyzed for validation. PD-L1⁺ was found in 98/392 GCs (25.0%). The proportions of immune types are as follows: PD-L1⁺/CD8^High, 22.7%; PD-L1⁻/CD8^Low, 22.7%; PD-L1⁺/CD8^Low, 2.3%; PD-L1⁻/CD8^High, 52.3%. PD-L1⁺/CD8^High type accounted for majority of EBV⁺ and MSI-high (MSI-H) GCs (92.0% and 66.7%, respectively), and genome analysis from public datasets demonstrated similar pattern. PD-L1⁻/CD8^High showed the best overall survival (OS) and PD-L1⁻/CD8^Low the worst (P < 0.001). PD-L1 expression alone was not associated with OS, however, PD-L1⁻/CD8^High type compared to PD-L1⁺/CD8^High was independent favorable prognostic factor of OS by multivariate analysis (P = 0.042). Adaptation of recent molecular classification based on EBV, MSI, E-cadherin, and p53 showed no significant survival differences. These findings support the close relationship between PD-L1/CD8 status based immune types and EBV⁺, MSI-H GCs, and their prognostic significance in stage II/III GCs.