Expression of T-Cell Exhaustion Molecules and Human Endogenous Retroviruses as Predictive Biomarkers for Response to Nivolumab in Metastatic Clear Cell Renal Cell Carcinoma

Abstract

Purpose: We sought to validate levels of CD8⁺ tumor-infiltrating cells (TIC) expressing PD-1 but not TIM-3 and LAG-3 (IF biomarker; Pignon and colleagues, 2019) and to investigate human endogenous retroviruses (hERV) as predictors of response to anti-PD-1 in a randomized trial of nivolumab (nivo) versus everolimus (evero) in patients with metastatic clear cell renal cell carcinoma (mccRCC; CheckMate-025).

Experimental design: Tumor tissues (nivo: n = 116, evero: n = 107) were analyzed by multiparametric immunofluorescence (IF) and qRT-PCR. Genomic/transcriptomic analyses were performed in a subset of samples. Clinical endpoints included objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and durable response rate (DRR, defined as complete response or partial response with a PFS ≥ 12 months).

Results: In the nivo (but not evero) arm, patients with high-IF biomarker density (24/116, 20.7%) had higher ORR (45.8% vs. 19.6%, P = 0.01) and DRR (33.3% vs. 14.1%, P = 0.03) and longer median PFS (9.6 vs. 3.7 months, P = 0.03) than patients with low-IF biomarker. By RNA sequencing, several inflammatory pathways (q < 0.1) and immune-related gene signature scores (q < 0.05) were enriched in the high-IF biomarker group. When combined with the IF biomarker, tumor cell (TC) PD-L1 expression (≥1%) further separated clinical outcomes in the nivo arm. ERVE-4 expression was associated with increased DRR and longer PFS in nivo-treated patients.

Conclusions: High levels of CD8⁺ TIC expressing PD-1 but not TIM-3 and LAG-3 and ERVE-4 expression predicted response to nivo (but not to evero) in patients with mccRCC. Combination of the IF biomarker with TC PD-L1 improved its predictive value, confirming our previous findings.

Figure 1.

Association of CD8⁺PD-1⁺TIM-3⁻LAG-3⁻ TIC density with clinical outcomes (PFS, OS) and with RNA-seq data. a, b Kaplan-Meier curves for PFS and OS per CD8⁺ PD-1⁺TIM-3⁻LAG-3⁻ TIC density at the optimized cutoff (Youden’s Index approach) in the nivo and evero arm. c, ssGSEA Hallmark gene sets enriched in the CD8⁺ PD-1⁺TIM-3⁻LAG-3⁻ TIC-High (orange) and CD8⁺PD-1⁺TIM-3⁻LAG-3⁻ TIC-Low (purple) tumors. Dotted lines indicate FDR q-value of 0.25 (light) and 0.1 (dark, significance level). d, Immune-related gene signature scores upregulated in CD8⁺PD-1⁺TIM-3⁻LAG-3⁻ TIC-High (orange) and CD8⁺PD-1⁺TIM-3⁻LAG-3⁻ TIC-Low (purple) tumors. Dotted lines indicate FDR q-value of 0.25 (light) and 0.05 (dark, significance level). e, Immune cell populations enriched in the CD8⁺PD-1⁺TIM-3⁻LAG-3⁻ TIC-High (orange) and CD8⁺PD-1⁺TIM-3⁻LAG-3⁻ TIC-Low (purple) cases by CIBERSORTx immune deconvolution of RNA-seq data. Dotted lines indicate FDR q-value of 0.25 (light) and 0.05 (dark, significance level).

Figure 2.

Kaplan-Meier curves for PFS per Tumor Cell (TC) PD-L1 expression levels (≥1%) combined with levels of CD8⁺PD-1⁺TIM-3⁻LAG-3⁻ TIC density (High versus Low). ^a In the evero arm, one patient with PD-L1 High/CD8⁺ PD1⁺TIM3⁻LAG3⁻ TIC Low was combined with the PD-L1 Low/CD8⁺ PD1⁺TIM3⁻LAG3⁻ TIC High group.

Figure 3.

Association of ERVE4 status with clinical outcomes (PFS, OS) and integration with WES and RNA-seq data. a, b Kaplan-Meier curves for PFS and OS per ERVE4 status at the optimized cutoff (Youden’s Index approach) in the nivo and evero arm. c, Most differentially expressed hERVs between ERVE4-positive and ERVE4-negative tumors using a database of about 1700 hERV transcripts. Horizontal red dotted line indicates FDR q-value of 0.25 (significance level). d, Immune-related gene signature scores upregulated in ERVE4-positive (orange) and ERVE4-negative (purple) tumors. Dotted lines indicate FDR q-value of 0.25 (light) and 0.05 (dark, significance level).