Characterization of the immune cell profile in metastatic nasopharyngeal carcinoma treated with chemotherapy and immune checkpoint inhibitors

Abstract

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer, and immune checkpoint inhibitors (ICIs) have shown efficacy in its treatment. The combination of chemotherapy and ICIs represents a new trend in the standard care for metastatic NPC. In this study, we aim to clarify the immune cell profile and related prognostic factors in the ICI-based treatment of metastatic NPC. Programmed cell death ligand 1 (PD-L1) expression was measured in 81 metastatic tissue samples that had not received prior ICI treatment. The combined positive score (CPS) was positive in 58.0% of the samples, with a statistically significant correlation to median overall survival (OS) (CPS ≥ 1 vs. CPS < 1: 28 vs. 16 months, P = 0.004). For the combination treatment of metastatic NPC, 62 patients were enrolled in a retrospective analysis, yielding a median OS of 39.3 months. The objective response rate for this combination therapy was 71%, with a complete response rate of 45.2%. With a cutoff value of 4.8 for the pre-treated neutrophil-lymphocyte ratio (NLR) in peripheral blood (PB), the difference in median OS was statistically significant (P = 0.021). Thirty-seven patients received local treatment following the combination therapy of ICIs and chemotherapy, which provided additional survival benefits. Most hyper-responders exhibited a prolonged low NLR (< 3), a high total lymphocyte count, and an undetectable or stable EBV DNA load in PB during treatment. Peripheral blood mononuclear cells (PBMCs) from most patients receiving the combination treatment were rich in PD-1+CD8+ lymphocytes, which showed high expression of both IFN-γ and Granzyme B, demonstrating the ability to kill the EBV-positive NPC cell line and xenografts in vitro and in vivo. Responders also displayed increased levels of CD4+CD45RA-CCR7-CD28+CD57- cells (effector memory cell subset) in peripheral blood. These results indicate that in the context of combined chemotherapy and ICIs, high PD-L1 expression in pre-treated metastatic tumor tissue, a low NLR before treatment, a decrease in NLR after treatment, and local treatment can provide significant benefits for patients with metastatic NPC.

Keywords: Epstein-Barr virus; Nasopharyngeal carcinoma; flow cytometry; immune checkpoint inhibitor; neutrophil-lymphocyte ratio; programmed death ligand 1.

Figure 1

PD-L1 expression in metastatic NPC tissue. (A) PD-L1 expression in metastatic tissue identified with IHC staining. Kaplan-Meier curves of overall survival: (B) CPS ≥ 1 vs. CPS < 1, P = 0.004; (C) TPS ≥ 1% vs. TPS < 1%, P = 0.929.

Figure 2

The clinical response and parameters of combined chemotherapy, ICIs, and local treatment in metastatic NPC. (A) A 48 y/o female patient was diagnosed as having NPC with lung metastasis. She was treated with combined chemotherapy and ICIs and a good partial response was achieved. Kaplan-Meier curves of overall survival: (B) treatment response, P < 0.001; (C) re-sponder vs. non-responder, P = 0.002; (D) pre-treatment NLR of cutoff value: 4.8, P = 0.021; and (E) local treatment, P = 0.008. Abbreviations: CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease.

Figure 3

Changes in neutrophil-lymphocyte ratio (NLR): (A) among responders and non-responders; and (B) regarding response and local treatment status.

Figure 4

The peripheral blood NLR, total lymphocyte count, and plasma EBV DNA load in met-astatic NPC patients receiving ICI-based treatment. Patients A-G were hyper-responders (OS > 24 months, CR or PR, and ICI treatment > 6 cycles), patient H was a responder but with high NLR and low absolute lymphocyte count, and patient I was a non-responder.

Figure 5

Flow cytometry analysis of amplified PBMCs. A. Lymphocytes were dominant and rich in CD8+ T cells. B. CD4 and CD8 T cells expressed activation-induced markers such as ICOS and the inhibitory markers TIM3, PD-L1, and PD-1. MFI: mean fluorescence intensity. C. In CD8+ T cells, about 33.9% expressed both IFN-γ and Granzyme B.

Figure 6

Ex vivo immune cell functional test. (A-E) Patient A: (A) In vitro functional assay of amplified PBMCs from Patient A, determining the IC₅₀ of NPC-B13 cell line. In vivo functional test of amplified PBMCs from ICI-treated Patient A in NPC PDX-B13 model, showing (B) gross tumor, (C) tumor volume, (D) tumor weight, and (E) CD8+ lymphocyte infiltrated around the PDX tumor. (F-H) Patient B: (F) In vitro functional assay of amplified PBMCs from Patient B, determining the IC₅₀ of NPC-B13 cell line. In vivo functional test of amplified PBMCs from ICI-treated Patient B in NPC PDX-B13 model, showing (G) gross tumor, (H) tumor weight.

Figure 7

Increase in CD4+CD45RA-CCR7-CD28+CD57- cells in the responder group. A. t-SNE plots showing clustering of live T cells (CD3+) from non-responders and responders. B. The percentages of clusters 6 and 9 among T cells are displayed. C. Heatmap illustrating the expres-sion of selected markers within these clusters. D. Initial gating of T cells based on CD4+CD45RA-CCR7- expression; representative gates for CD57 and CD28 expression are shown in the left panel. The cumulative data representing the percentage of cells in the CD4+CD45RA-CCR7- effector memory (EM) cell subset is presented in the right panel.