Clinical efficacy and biomarker analysis of dual PD-1/CTLA-4 blockade in recurrent/metastatic EBV-associated nasopharyngeal carcinoma

Abstract

Single-agent checkpoint inhibitor (CPI) activity in Epstein-Barr Virus (EBV) related nasopharyngeal carcinoma (NPC) is limited. Dual CPI shows increased activity in solid cancers. In this single-arm phase II trial (NCT03097939), 40 patients with recurrent/metastatic EBV-positive NPC who failed prior chemotherapy receive nivolumab 3 mg/kg every 2 weeks and ipilimumab 1 mg/kg every 6 weeks. Primary outcome of best overall response rate (BOR) and secondary outcomes (progression-free survival [PFS], clinical benefit rate, adverse events, duration of response, time to progression, overall survival [OS]) are reported. The BOR is 38% with median PFS and OS of 5.3 and 19.5 months, respectively. This regimen is well-tolerated and treatment-related adverse events requiring discontinuation are low. Biomarker analysis shows no correlation of outcomes to PD-L1 expression or tumor mutation burden. While the BOR does not meet pre-planned estimates, patients with low plasma EBV-DNA titre (<7800 IU/ml) trend to better response and PFS. Deep immunophenotyping of pre- and on-treatment tumor biopsies demonstrate early activation of the adaptive immune response, with T-cell cytotoxicity seen in responders prior to any clinically evident response. Immune-subpopulation profiling also identifies specific PD-1 and CTLA-4 expressing CD8 subpopulations that predict for response to combined immune checkpoint blockade in NPC.

Fig. 1. Details of clinical trial and patient cohort.

a Overview of the trial schema. b Consort diagram of trial.

Fig. 2. Details on patient response and outcome data.

a Extent and depth of response in trial cohort showing best tumor scan (Top) and changes in tumor size over time (Bottom). Not evaluable (NE) in gray, progressive disease (PD) in blue, partial response (PR) in red and stable disease (SD) in yellow. n = 40. b Progression-free (Top) and overall (Bottom) survivals of trial cohort. n = 40. Source data are provided as a Source Data file.

Fig. 3. Pre-treatment plasma circulating EBV-DNA levels and mutational landscape of tumors by whole-exome sequencing.

a Predictive ability of plasma circulating EBV-DNA levels for partial response to dual therapy (n = 26). b Progression-free survival for EBV-high (yellow) vs EBV-low (green) patients with two-sided log-rank p-value shown. c Summary of molecular assays performed and biomarker discovery workflow. Icons made by Freepik and monkik on www.flaticon.com. d Tumor co-mutation plot showing top recurrently mutated cancer genes in cohort. Tumor mutation burden and clinical covariates are shown. e Mutational signatures based on COSMIC signatures. Size of dot correlates with the strength of signature activity in each sample. n = 20, biologically independent samples.

Fig. 4. Gene expression analysis of tumor biopsy samples pre- and on-treatment.

a–c Results from differential expression analysis of on-treatment Nanostring IO360 data for PR vs PD outcome with (a) volcano plot and (b) heatmap highlighting key genes, and (c) pathway enrichment analysis of significantly differentially expressed genes. n_PR = 8, n_PD = 5. d Time to progression is positively correlated with markers of adaptive immune response (top) and negatively correlated with markers of proliferation and DNA damage response (bottom; r and two-sided p values determined using Pearson correlation statistical analysis). Each dot represents an individual sample. n = 18 (n_PR=7, n_SD = 6, n_PD = 5). e–f Gene expression changes associated with the interaction between treatment status (pre- vs on-treatment) and outcome (PR vs PD) and (f) their enriched pathways. n_PR = 8, n_PD = 5, biologically independent samples. a, b, e Statistical analyses for differential expression were performed using Wald-test, not corrected for multiple testing.

Fig. 5. Cell type profiling by Nanostring and multiplex IHC/IF staining.

Cell type profiling score (as determined using Nanostring IO360) of (a) Treg vs TILS, (b) CD8 vs Exhausted CD8, (c) Total TILS, and (d) CD8 vs Treg by outcome compared between pre- (left) and on-treatment (right) samples as indicated at the bottom of the graph. Pre-treatment = 22 (n_PR = 8, n_SD = 6, n_PD = 7), On-treatment=19 (n_PR = 8, n_SD = 6, n_PD = 5), biologically independent; two-sided Mann–Whitney U-test, adjusted for multiple comparisons, boxplots are shown with the boxes indicating quartiles with median at middle and the whiskers drawn at the lowest and highest points within 1.5 interquartile range of the lower and upper quartiles, respectively. Each dot represents an individual sample. (e) Representative images of 17 patients stained for DAPI (blue), PD-1 (cyan), FOXP3 (orange), CD39 (green), TCF1 (red), CD8 (yellow), CTLA-4 (magenta; 200x magnification), performed in 2 independent experiments. f Quantification of PD-1⁺ CTLA-4⁻ CD8⁺ and PD-1⁻ CTLA-4⁺ CD8⁺ cells as proportions of total CD8 cells. Tukey’s test, adjusted for multiple comparison. Data are presented as mean with SD. n_PR = 7, n_SD = 6, n_PD = 4. Each dot represents an individual sample. g Representative image of combined CD8 subpopulations described in (f) expressing either PD-1 or CTLA-4. h Correlation between PD-1/CTLA-4 score with TTP. n = 17, Spearman correlation r = 0.609, p = 0.0094. Each dot represents an individual sample. Source data are provided as a Source Data file.