Exploring predictive biomarkers of efficacy and survival with nivolumab treatment for unresectable/recurrent esophageal squamous cell carcinoma

Abstract

Background: Programmed cell death protein-1 (PD-1) blockade has improved survival for patients with esophageal squamous cell carcinoma (ESCC), but response rates are low. Biomarkers to predict who will benefit from PD-1 blockade are urgently needed.

Methods: This multicenter study involved 250 patients with recurrent/unresectable advanced ESCC receiving nivolumab as second- or later-line therapy. We assessed tumor-infiltrating T lymphocytes (TILs) and tertiary lymphoid structure (TLS) density using immunohistochemistry and hematoxylin/eosin staining in surgical specimens and pre-nivolumab endoscopic biopsies.

Results: In surgical specimens, clinical response (vs. non-response) to nivolumab correlated significantly with CD8⁺ lymphocyte count (160 vs. 95.2 cells/field, P = 0.0494), CD8/Foxp3 ratio (6.52 vs. 2.72, P = 0.0053), and TLS density (0.21/mm² vs. 0.10/mm², P = 0.0005). In terms of overall survival, multivariate analysis identified CD8/Foxp3 ratio (hazard ratio [HR] = 1.83, P = 0.0050) and TLS density (HR = 1.67, P = 0.0171 as independent prognostic parameters in surgical specimens. Similarly, in endoscopic biopsies, clinical response (vs. non-response) to nivolumab correlated significantly with CD8⁺ counts (254 cells/mm² vs. 124 cells/mm², P = 0.0344), CCR8⁺ lymphocyte count (62.6 cells/mm² vs. 140 cells/mm², P = 0.0355), CD8/Foxp3 ratio (2.09 vs. 0.89, P = 0.040), and CD8/CCR8 ratio (2.34 vs. 0.89, P = 0.0020). Multivariate analysis also identified CD8/CCR8 ratio in endoscopic biopsies (HR = 1.66, P = 0.0313) as an independent prognostic parameter.

Conclusions: CD8⁺ and CCR8⁺ cell counts, CD8/Foxp3 and CD8/CCR8 ratios, and TLS density may be predictive biomarkers of therapeutic efficacy and survival with PD-1 blockade for ESCC.

Keywords: Anti-PD-1 antibody; Biomarker; Esophageal squamous cell carcinoma; Tertiary lymphoid structure; Tumor-infiltrating T lymphocytes.

Fig. 1

Evaluations of CD3⁺, CD8⁺, Foxp3⁺, and CCR8⁺ lymphocyte counts and TLS density in endoscopic biopsy and surgical specimens. A Auto count of CD3⁺, CD8⁺, Foxp3⁺, and CCR8⁺ lymphocytes using HALO software (Indica Labs, Corrales, NM, USA). B Representative slide of CD8 immunostaining of an endoscopic biopsy and auto count of CD8⁺ lymphocytes. The total number of TILs, including CD3⁺, CD8⁺, Foxp3⁺, and CCR8⁺ lymphocytes, was automatically counted using HALO software and divided by the sum of all biopsy tumor areas, defined as the total number of positive cells counts per tumor area in the endoscopic biopsy (the area inside the green line) (/mm²). C The tumor area margin of 1000 μm in a surgical specimen (the area inside the yellow line), defined as the area 1000 µm outward from the boundary between the tumor tissue (the area inside the red line). D A heatmap created using HALO software; the top five TIL “hotspots” (5 tiles with the most TILs) were selected based on a heatmap of the tumor area margin of 1000 μm in a surgical specimen. E The tumor area margin of 1000 μm in a surgical specimen (the area inside the yellow line), defined as the area 1000 µm outward from the boundary between normal tissue and tumor tissue (the area inside the red line). We defined TLS density by calculating the number of TLSs per millimeter in H&E-stained sections. Scale bars: A = 100 µm, B = 500 µm, C = 2 mm, D = 1 mm, E = 2 mm (top), 1 mm (bottom)

Fig. 2

Representative slides of low and high staining densities for CD3⁺, CD8⁺, and Foxp3⁺ lymphocytes in a surgical specimen. A CD3; B CD8; C Foxp3. Scale bars: 100 µm

Fig. 3

Relationship between counts of CD3⁺, CD8 ⁺, and Foxp3⁺ lymphocytes and treatment response and survival with nivolumab in surgical specimens. A top: CD3⁺, CD8⁺, and Foxp3⁺ lymphocyte counts. A Middle: lymphocyte counts according to treatment response to nivolumab. A Bottom: CD8/Foxp3 ratio according to treatment response to nivolumab. Values in boxplots indicate medians. B Kaplan–Meier survival curves for Overall survival according to CD3⁺, CD8⁺, and Foxp3⁺ lymphocyte counts and CD8/Foxp3 ratio

Fig. 4

Relation between TLS expression and treatment response and prognosis with nivolumab. A TLS density (left) and TLS density according to treatment response to nivolumab (right); values in boxplots are medians. B Kaplan–Meier survival curves for progression-free and overall survival according to TLS density. C The response rates with nivolumab in three groups representing various combinations of high versus low TLS density and CD8/Foxp3 ratio

Fig. 5

Relation between CD3⁺, CD8⁺, Foxp3⁺, and CCR8⁺ lymphocyte counts in endoscopic biopsies and treatment response and survival with nivolumab. A Top: CD3⁺, CD8⁺, Foxp3⁺, and CCR8⁺ lymphocyte counts. A Middle: lymphocyte counts according to treatment response to nivolumab. A Bottom: CD8/Foxp3 and CD8/CCR8 ratios according to treatment response to nivolumab. Values in boxplots are medians. B Kaplan–Meier survival curves for Overall survival according to the lymphocyte counts and CD8/Foxp3 and CD8/CCR8 ratios