Association of PD-L1, PD-L2, and Immune Response Markers in Matched Renal Clear Cell Carcinoma Primary and Metastatic Tissue Specimens

Arnab Basu, MD, MPH; Jennifer Holmes Yearley, DVM, PhD, DAVCP; Lakshmanan Annamalai, DVM, PhD; Christopher Pryzbycin, MD; Brian Rini, MD


Am J Clin Pathol. 2019;151(2):217-225. 

In This Article

Abstract and Introduction


Objectives: Immune checkpoint therapy has been promising in renal cell carcinoma, but no validated clinically relevant biomarkers exist. Metastatic deposits may have discordant biomarker expression.

Methods: Fifty matched pairs of primary and metastatic kidney tumors were evaluated via immunohistochemistry for immune checkpoint proteins PD-1, PD-L1, and PD-L2 and the T-cell and macrophage surface markers CD3, FOXP3, and CD163. Semiquantitative scores incorporating prevalence of both tumor and nontumor labeling were compared between metastatic and primary kidney tumor specimens.

Results: A large minority of patients had discordant expression of PD-1 (31.2%), PD-L1 (22.5%), or PD-L2 (21.5%) between primary and metastatic sites. The expression of the novel marker PD-L2 correlated with both PD-1 (r = 0.47, P = .02) and PD-L1 (r = 0.67, P < .001) in metastatic deposits.

Conclusions: This study demonstrates that renal clear cell carcinoma primary tumors and metastatic deposits have some discordance in the expression of PD-L1, PD-1, and PD-L2.


Metastatic renal cell carcinoma (RCC) has been a difficult disease to treat historically with minimal response to cytotoxic chemotherapy and high mortality rates. Immunotherapy had been used in RCC with only limited success such as with high-dose interleukin 2 (IL-2), which induces approximately 5% to 10% of patients into a durable remission.[1] Treatment outcomes have improved with the introduction of vascular endothelial growth factor (VEGF)–targeted therapies in the past decade, although durable responses are still uncommon. The understanding of the interplay between the tumor and the immune system has led to the development of immune checkpoint inhibitors that are active in many solid tumors, including RCC. Nivolumab, an anti–PD-1 antibody, was recently approved as a treatment for VEGF-refractory metastatic RCC. A phase III trial compared nivolumab with everolimus and showed a significant overall survival benefit.[2] Similarly, pembrolizumab, another PD-1 inhibitor, and atezolizumab, a PD-L1 antibody, are being investigated in combination with VEGF inhibitors with encouraging initial activity and ongoing phase 3 trials.[3] As immune checkpoint inhibitors and other targeted therapies continue development in RCC, there will be an increasing need to identify patients who are more likely to benefit to inform clinical decisions.

The expression of the PD-L1 ligand on the tumor cell surface is a logical choice as a marker of response to PD-1 inhibitors. In the abovementioned phase III trial of nivolumab, the hazard ratio for overall survival benefit of nivolumab over everolimus was nearly identical regardless of PD-L1 expression.[2] The anti–PD-L1 antibody atezolizumab also failed to show an association between response and PD-L1 expression in early phase studies.[4] Several hypotheses have been forwarded to explain this lack of association. One hypothesis is that there are considerable differences in expression of PD-L1 in metastatic lesions compared with primary tumors since metastatic deposits adapt under immune selection pressures. Some studies have demonstrated this to be the case in RCC. Discordant tumor cell PD-L1 staining between primary tumors and matched metastases was observed in 20% of clear cell kidney tumors in one analysis.[5] Similarly, PD-L1 expression has been found to be weakly correlated between metastasis and primary kidney tumors in another series.[6] Another hypothesis involves the presence of other clinically significant ligands apart from PD-L1. An example of such an alternative ligand is PD-L2.[7] The role of PD-L2 and its differential expression in metastatic sites and kidney cancer primaries has not been examined previously.

Macrophage and T-cell infiltration of tumors may also be important determinants of treatment response. Macrophage polarization into tolerance-promoting (M2) and antitumor phenotypes (M1) has been described.[8] M1 and M2 macrophages are identifiable by HLA-DR and CD163 staining, respectively.[9,10] T-cell subtypes involved in the tumor–immune system interaction include the regulatory and cytotoxic T cells. Regulatory T cells express FOXP3 and are active in increasing immune tolerance, resulting in muting of the immune-mediated anticancer response.[11] Regulatory T cells have been shown to influence outcome to IL-2 and sunitinib therapy in metastatic RCC.[12,13]

To gain further insight into expression of markers potentially relevant to checkpoint inhibition in RCC, matched primary and metastatic RCC tumors were characterized. The availability of matched samples could provide insight into intrapatient heterogeneity regarding biomarker expression. Such heterogeneity may have several implications, including which tissue is characterized prior to treatment selection and organ-specific clinical responses.