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

Materials and Methods

A search was conducted of the pathology database at the Cleveland Clinic Foundation for patients with the diagnosis of clear cell carcinoma between 1995 and 2016 for which both a nephrectomy specimen and metastatic tissue (inclusive of distant lymph nodes) were available for analysis. A cohort of 50 patients was selected based on availability of an adequate quantity of tissue for analysis. Data on tumor characteristics were extracted from original pathology reports. Clinical information was available and extracted by a review of the electronic medical record (A.B.).

Tumor Characteristics and Staging

Tumor pathology from both nephrectomy and metastasectomy specimens was reexamined by an expert genitourinary pathologist (C.P.). Information was collected on nuclear grade, lymphovascular invasion, renal sinus invasion, perinephric fat invasion, and sarcomatoid and/or rhabdoid differentiation. Nuclear grade was assigned using Fuhrman criteria.[14] For samples with heterogeneity in nuclear grade, the highest nuclear grade was recorded. A TNM stage was assigned based on standard cutoffs and notations for tumor size, nodal involvement, and metastatic disease sites using information from pathology reports, as well as from a combination of imaging studies and clinical history available from patient charts after physician review (A.B.).[15]

Tissue Processing and Staining

Tumor specimens were cut from formalin-fixed, paraffin-embedded (FFPE) tissue blocks, and 4-μm-thick FFPE tissue sections were used for immunostaining. The tissue sections were deparaffinized in xylene and rehydrated through graded ethanol to distilled water. For FoxP3, CD3, and CD163 immunostaining, heat-induced epitope retrieval (HIER) was performed by immersing the slides in citrate buffer for 4 minutes at 120°C. Endogenous peroxidase in tissues was blocked by incubation of slides in 3% hydrogen peroxide solution prior to incubation with primary antibody. After 60 minutes of incubation with the respective primary antibody (FoxP3, clone 236A/E7, Abcam; CD3, clone CD3-12, AbD Serotec; CD163, clone 10D6, Thermo Scientific), the sections were treated with EnVision anti-mouse/rabbit horseradish peroxidase–conjugated polymer (DAKO). The chromogenic substrate 3,3′-diaminobenzidine (DAB; DAKO) was used to localize antigen-antibody complexes. Tissue sections were counterstained with Mayer's hematoxylin (Sigma-Aldrich), cleared, and coverslipped with permanent mounting medium. For PD-1, PD-L1, and PD-L2 immunostaining, HIER was performed by immersing the slides in FLEX High pH target retrieval solution for 4 minutes at 97°C (cat. K8012; DAKO). Endogenous peroxidase in tissues was blocked by incubation of slides in 3% hydrogen peroxide solution prior to incubation with primary antibody (anti–PD-L1, clone 22C3 and anti–PD-L2, clone 3G2, Merck Research Laboratories, or anti–PD-1 clone NAT105, Cell Marque) for 60 minutes. Antigen-antibody binding was visualized via application of the FLEX+ polymer system (cat. K8012; DAKO) and application of DAB chromogen (cat. K4368; DAKO). These stained slides were counterstained with hematoxylin and coverslipped for review.

Immunohistochemistry Scoring

Scoring was conducted by an experienced pathologist (L.A.), with scores incorporating prevalence of both tumor cell and nontumor cell labeling using techniques described by us previously.[16] A semiquantitative 0 to 5 scoring system was applied such that 0 = no staining; 1 = rare individuated positive cells or only very small focus within or directly adjacent to tumor tissue; 2 = infrequent small clusters of positive cells within or directly adjacent to tumor tissue; 3 = single large cluster, multiple smaller clusters, or moderately dense diffuse infiltration, within or directly adjacent to tumor tissue; 4 = single very large dense cluster, multiple large clusters, or dense diffuse infiltration; and 5 = coalescing clusters, dense infiltration throughout the tumor tissue. Presence or absence of endothelial cell expression was evaluated specifically as a separate value. Image 1 demonstrates these patterns of staining and associated scores.

Image 1.

Immunohistochemical staining profile: renal primary vs metastases. A, Specimen 33: PD-L1 immunohistochemistry (IHC) primary tumor demonstrates a prominent marginal pattern with predominantly nontumor staining. IHC score assigned to this specimen was 3.5 (moderate to high). B, Specimen 33: PD-L1 IHC metastasis (lymph node) demonstrates strong staining in both tumor as well as infiltrating immune cells. Staining score assigned to this specimen was 5 (very high). C, Specimen 33: PD-1 IHC primary tumor demonstrates a heterogeneous distribution with a predominantly marginal pattern. IHC score assigned to this specimen was 3.5 (moderate to high). D, Specimen 33: PD-1 IHC metastasis (lymph node) demonstrates positivity in predominantly tumor cells. IHC score assigned to this specimen was 4 (high). E, Specimen 33: PD-L2 IHC primary tumor demonstrates no faint to scattered staining across tumor and infiltrating immune cells. IHC score assigned to this specimen was 1 (rare to trace). F, Specimen 33: PD-L2 IHC metastasis (lymph node) demonstrates positivity across tumor and infiltrating cells showing a discordance between primary tumor and metastatic site. IHC score assigned to this specimen was 4 (high). (All images ×20.)

Statistical Methods

All data analysis was performed using STATA v.12 (StataCorp LP). Data extracted from charts and staining processes were compiled into a database. Exploratory data analysis was conducted to document frequencies and measures of central tendency for summary statistics. Spearman correlation coefficients were obtained between tumor pathologic characteristics and the expression of PD-1, PD-L1, and PD-L2, as well as CD163, CD3, and FOXP3. Bonferroni corrections were made for multiple pairwise comparisons. A P value (two-sided) less than .05 was considered statistically significant.