Expression of CD274 mRNA Measured by qRT-PCR Correlates With PD-L1 Immunohistochemistry in Gastric and Urothelial Carcinoma

Abstract

Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) is widely used to predict the clinical responses to immune checkpoint inhibitors (ICIs). However, PD-L1 IHC suffers from the complexity of multiple testing platforms and different cutoff values caused by the current one drug-one diagnostic test co-development approach for ICIs. We aimed to test whether PD-L1 (CD274) mRNA expression levels measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) can represent PD-L1 IHC and predict responses to ICI. The FDA-approved PD-L1 IHC results with 22C3 pharmDx (gastric cancer) and SP142 (urothelial carcinoma) were compared with CD274 mRNA expression levels via qRT-PCR using the same formalin-fixed, paraffin-embedded tissue blocks from 59 gastric cancer and 41 urothelial carcinoma samples. CD274 mRNA expression was identified using three independent sets of primers and TaqMan® probes targeting exon 1-2, exon 3-4, and exon 5-6. CD274 mRNA levels in spanning exon 1-2, exon 3-4, and exon 5-6 junctions of CD274 correlated well with PD-L1 expression (r²=0.81, 0.65, and 0.59, respectively). The area under the curve of exon 1-2 was the highest (0.783), followed by exon 3-4 (0.701), and exon 5-6 (0.671) of the CD274 gene against the PD-L1 combined positive score cutoff of 10. When CD274 mRNA expression was matched for response to immunotherapy, the overall response rate was higher in patients with high CD274 mRNA levels with a cutoff of 0.0722 (gastric cancer) and 0.0480 (urothelial carcinoma) than in those with low CD274 mRNA expression (P < 0.001 and P = 0.018, respectively). These results show that CD274 mRNA levels predicted ICI responses in patients with gastric or urothelial carcinomas and could be used as alternatives for PD-L1 IHC.

Keywords: CD274; carcinoma; gastric; immunotherapy; mRNA expression; urothelial.

Figure 1

Representative PD-L1 immunohistochemical staining in GC and SP142 in UC. Combined positive scores of 95 (A), 25 (B) and 0 (C) in GCs with 22C3 pharmDx. Immune cell scores of 40 (D), 20 (E) and 0 (F) in UCs with Ventana PD-L1 (SP142) assay. Magnification in all images, 20×. GC, gastric cancer; UC, urothelial carcinoma.

Figure 2

Correlations between PD-L1 scores and CD274 mRNA expression. (A) Correlations between (A) PD-L1 scores and CD274 exons 1–2, 3–4, and 5–6 in all GC and UC. (B) PD-L1 combined positive score and CD274 mRNA expression in GC. (C) PD-L1 immune scores and CD274 mRNA expression in UC. GC, gastric cancer; PD-L1, programmed death-ligand 1; UC, urothelial carcinoma.

Figure 3

Results of qRT-PCR predicted responses to anti- PD-1 checkpoint blockade in GC and UC. (A) PD-L1 and CD274 mRNA expression per iRECIST ORR categories of responders (CR/PR) and non-responders (PD/SD). (B) Predictive performance of PD-L1 and CD274 mRNA expression determined from ROC curves in terms of ORR categories. (C) PD-L1 and CD274 mRNA expression levels per iRECIST DCR category of responders (CR/PR/SD) and non-responders with SD. (D) Predictive performance of PD-L1 and CD274 mRNA expression determined from ROC curves in terms of DCR category. CR, complete response; GC, gastric cancer; iRECIST, immune Response Evaluation Criteria in Solid Tumors; ORR, objective response rate; PD, progressive disease; PD-L1, programmed cell death ligand 1; PR, partial response; ROC, receiver operating characteristics; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; SD, stable disease; UC, urothelial carcinoma.

Figure 4

Survival outcomes and qRT-PCR results of GC treated with anti-PD-1/PD-L1. Kaplan–Meier curves of (A) PFS and (B) DSS of patients with GC treated with anti-PD-1/PD-L1 according to PD-L1 CPS cut-off 10 and CD274 mRNA expression determined by qRT-PCR with cut-off 0.0722. PFS, progression-free survival; DSS, disease-specific survival.