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


Baseline Characteristics

Forty-eight primary and metastatic tumor pairs were suitable for analysis of baseline characteristics (two patients were excluded based on lack of complete information in the electronic medical record). Metastatic lesions examined were representative of the most common metastatic sites in RCC and included lung, lymph node, and adrenal glands Table 1 . Other sites included the gastrointestinal tract, skin, non–lymph node soft tissue, and the genitourinary tract. Eighty percent of patients had advanced disease defined by a TNM stage III and greater at the time of nephrectomy, and 88% had a Fuhrman nuclear grade of 3 or higher. Most (57%) of the metastatectomies were performed within 12 months of the original nephrectomy, and 93% were obtained within 5 years of nephrectomy.

Expression of PD-1/PD-L1 and Association With Tumor Characteristics

In primary tumors, Fuhrman nuclear grade positively correlated with both increased prevalence of PD-1 staining in lymphocytes (r = 0.37, P = .02) but not with PD-L1 expression on the tumor (r = 0.30, P = .07). Fuhrman nuclear grade positively correlated with CD163 expression (r = 0.46, P < .01), indicating increasing macrophage concentration in tumors with higher nuclear grades. TNM stage of the primary tumor also positively correlated with PD-L1 expression of the primary tumors (r = 0.38, P = .02) Table 2 .

In metastatic sites, Fuhrman nuclear grade positively correlated with both increased prevalence of PD-1 staining in lymphocytes (r = 0.45, P < .01) and with PD-L1 expression on the metastatic tumor (r = 0.38, P = .02), as well as with increasing CD163 expression (r = 0.37, P = .02). Time to metastasis, as examined in years from nephrectomy, was not associated with expression of PD-1 or PD-L1 (data not shown).

Distribution of PD-L1 and PD-L2 Staining in Primary/Metastatic Sites

Based on immunohistochemistry (IHC) scores for PD-L1 and PD-L2 in primary tumors, 32 (64%) of 50 RCC primary tumors had no significant staining for PD-L1 or PD-L2, as defined by a semiquantitative score of 3 or more, while 13 of 50 stained for either of these two markers. Five of 50 tumors had significant scoring for both Figure 1 . Twenty-seven (54%) of 50 metastatic deposits did not significantly stain for either PD-L1 or PD-L2, while 14 of 50 stained positively for either of these ligands. Nine of 50 stained positive for both PD-L1 and PD-L2 on IHC staining. There appeared to be relatively low expression for PD-L1 and PD-L2 in lymph nodes compared with kidney, lung, and adrenal metastasis. Figure 1 presents the distribution of IHC scores for primary and metastatic sites for these markers.

Figure 1.

Staining scores for primary (A [n = 49], C [n = 50], E [n = 50], G [n = 50], I [n = 46], K [n = 50]) and metastatic (B [n = 49], D [n = 49], F [n = 49], H [n = 48], J [n = 46], L [n = 50]) deposits.

Concordance in PD-1, PD-L1, and PD-L2 Staining in Primary/Metastatic Sites

Thirty-three (69%) of 48 evaluable matched pairs were concordant for expression of PD-1 (ie, both sites were "positive" for the expression of PD-1 as defined by a score of 3 or higher). However, 31% of tumor pairs were discordant in their expression of PD-1 between primary tumors vs metastatic deposits. Thirteen of 50 primary tumors and 18 of 50 metastatic tumors were positive for PD-L1 expression. Thirty-eight (78%) of 49 evaluable matched pairs were concordant for the expression of PD-L1 while 11 were discordant. Finally, 39 (80%) of 49 tumor pairs were concordant for expression of PD-L2, while 10 pairs were discordant Table 3 .

Association of Immune Checkpoint Marker Expression Within Primary and Metastatic Tumors Based on IHC

IHC scores for PD-1 and PD-L1 in primary tumors were strongly positively related (r= 0.73, P < .001), and a similar positive correlation was observed between PD-1 IHC scores and CD3 IHC scores (r = 0.72, P < .001), confirming a PD-1–expressing infiltrating T-cell response to PD-L1–expressing tumor. PD-L1 IHC scores in the primary tumors also correlated positively with CD163 (r = 0.55, P > .001) and FOXP3 scores (r = 0.653 P < .01), suggesting that increasing PD-L1 expression leads to recruitment of M2 macrophages but also regulatory T cells. PD-L1 scores did not correlate with PD-L2 IHC scores (r = 0.22, P = 1) in the primary tumors. In metastatic tumors, IHC scores for PD-1 and PD-L1 were strongly related (r = 0.67, P < .001) as well as CD3 (r = 0.72, P < .001), confirming a similar PD-1–expressing T-cell response to PD-L1–expressing tumors. In contrast to primary tumors, PD-L1 scores correlated with PD-L2 scores in metastatic tumors (r = 0.62, P < .01) Table 4 and Table 5 .