Inter-tumor heterogeneity of PD-L1 expression in non-small cell lung cancer

Abstract

Background: The Dako PD-L1 immunohistochemistry (IHC) 22C3 pharmDx and the Dako 28-8 IHC pharmDx assays were approved by the US Food and Drug Administration, as a companion diagnostic test for pembrolizumab (Keytruda, Merk, Kenilworth, NJ, USA) and a complementary diagnostic test for nivolumab (Opdivo, Bristol Meyer Squibb, New York, NY, USA) in non-small cell lung cancer (NSCLC), respectively. Increased PD-L1 expression levels can be associated with greater therapeutic efficacy of pembrolizumab relative to other anti-PD-1 agents. However, in treatment decision making, little is known about which tissue (primary or metastatic lesion) should be stained by 22C3 antibody. We investigated the relationship between PD-L1 expression in primary tumors and paired metastatic lymph nodes using the 22C3 assay, and evaluated the concordance between the 22C3 and 28-8 assays.

Methods: PD-L1 expression was evaluated in cells from primary tumors and paired metastatic lymph nodes using the 22C3 and 28-8 IHC assays. Total 35 patients with primary tumor and paired metastatic lymph node were enrolled into this study, and all samples were surgically resected, formalin-fixed, and paraffin-embedded NSCLC tissues. Tumor cells exhibiting complete or partial membrane staining, were considered as PD-L1 positive. On the basis of tumor proportion score (TPS), all samples were classified as no expression (TPS: <1%), low expression (TPS: 1-49%), or high expression (TPS: ≥50%).

Results: TPS distribution was markedly different between primary tumors and paired metastatic lymph nodes. In 22C3 IHC assay, TPS similar to that of metastatic lymph nodes was demonstrated in 10 primary tumors, and concordance rate between them was 28.6%. Concurrently, in 28-8 IHC assay, 11 primary tumors had TPS similar to that of metastatic lymph nodes, with a concordance rate of 31.4%.

Conclusions: TPS concordance rates (for both 22C3 and 28-8 antibodies) between primary tumors and paired lymph nodes were low. Inter-tumor heterogeneity of PD-L1 expression is an important issue for clinical oncologists during treatment planning.

Keywords: Lung cancer; heterogeneity; immunotherapy; programmed death-ligand 1 (PD-L1); tumor proportion score (TPS).

Figure 1

Scoring system of PD-L1 22C3 immunohistochemistry assay. At least 100 viable tumor cells were evaluated to judge PD-L1 immunohistochemistry positivity. PD-L1 positivity was defined as staining of the tumor cell membrane excluding cytoplasmic staining. All slides were evaluated at magnifications of 10×, 20×, and 40×. Each representative image demonstrated <1% positivity (A), 1–49% positivity (B), and >50% positivity (C). TPS, tumor proportion score.

Figure 2

Tumor proportion score (TPS) of primary tumors and their paired metastatic lymph nodes. All cases were classified into 3 groups by PD-L1 expression using the Dako PD-L1 IHC 22C3 or 28-8 pharmDx assays; high expression (≥50%), low expression (1–49%), and no expression (<1%) are represented in red, green, and blue, respectively. (A) Pie chart representing TPS categories in primary tumors stained using the 22C3 antibody; (B) pie chart representing TPS categories in primary tumors stained with the 28-8 antibody. Primary tumors demonstrated similar TPS values in both assays; (C) pie chart representing TPS categories in metastatic lymph nodes using the 22C3 antibody; (D) last pie chart representing TPS categories in metastatic lymph nodes using the 28-8 IHC antibody. Both assays resulted in similar TPS percentages between primary tumors and lymph nodes. However, there was no similarity observed in TPS values between primary tumors and paired lymph nodes for both 22C3 and 28-8 IHC assays.

Figure 3

The five scatter plots illustrate the distribution of PD-L1 expression. (A) Each colored dot represents PD-L1 expression; blue (primary tumors stained by 22C3), green (lymph nodes stained by 22C3), red (primary tumors stained by 28-8), and yellow (lymph nodes stained by 28-8); (B) the scatter plot illustrates PD-L1 expression distribution in primary tumors stained by either 22C3 or 28-8 antibodies; (C) the scatter plot depicts a similar correlation of PD-L1 expression in lymph nodes between the 22C3 and 28-8 assays; (D) no correlation of PD-L1 expression was demonstrated between primary tumors and lymph nodes when performing the 22C3 assay; (E) similarly, in the 28-8 assay, there was no statistical association of PD-L1 expression between primary tumors and lymph nodes.

Figure 4

A representative example demonstrating strongly positive staining in primary tumors but no staining in paired metastatic lymph nodes, when using both the 22C3 and 28-8 assays.