Clinical implications of immune checkpoint markers and immune infiltrates in patients with thymic neuroendocrine neoplasms

Abstract

The potential response of immune checkpoint blockade (ICB) in thymic neuroendocrine neoplasms (T-NEN) is largely unknown and full of great expectations. The expression of immune checkpoint molecules and immune infiltrates greatly determine the response to ICB. However, studies regarding the immune landscape in T-NEN are scarce. This work was aimed to characterize the immune landscape and its association with clinical characteristics in T-NEN. The expression of programmed cell death protein 1 (PD-1) and its ligand, programmed death ligand-1 (PD-L1), and the density of tumor-infiltrating lymphocytes (TILs), monocytes, and granulocytes were determined by immunohistochemical (IHC) staining on tumor tissues from T-NEN. Immune landscapes were delineated and correlated with clinicopathological factors. We found that T-NEN with increased immune cell infiltration and enhanced expression of PD-1/PD-L1 tended to have restricted tumor size and less metastases. A higher density of CD8⁺ TILs was associated with a significantly lower rate of bone metastasis. In addition, we presented three cases of T-NEN who progressed after multiple lines of therapies and received ICB for alternative treatment. ICB elicited durable partial responses with satisfactory safety in two patients with atypical carcinoid, but showed resistance in 1 patient with large cell neuroendocrine carcinoma. This innovative study delineated for the first time the heterogeneous immune landscape in T-NEN and identified CD8⁺ TILs as a potential marker to predict bone metastasis. An "immune-inflamed" landscape with the presence of TILs predominated in T-NEN, making T-NEN a potentially favorable target for ICB treatment. Further judicious designs of "tailor-made" clinical trials of ICB in T-NEN are urgently needed.

Keywords: immune checkpoint blockade; immune infiltrates; programmed death ligand-1; programmed death-1; thymic neuroendocrine neoplasms.

Figure 1

Expression pattern of immune checkpoint markers PD-L1 and PD-1 in T-NEN. Representative PD-L1 IHC staining images of T-NEN are showed. (A) Different levels of PD-L1 staining (brown) on tumor cells are showed. (B) Different levels of staining (brown) on immune cells are showed. (C) Proportion of stratified PD-L1 intensity according to TPS and IPS evaluation is displayed. (D) Different infiltration levels of PD-1-positive cells are shown with representative IHC staining images. (E) Proportion of stratified PD-1 intensity in T-NEN is displayed. Remark: Representative IHC staining images are shown in an area of 0.09 mm² (0.3 mm×0.3 mm) captured from each slide.

Figure 2

Patterns of different immune cell infiltrations in T-NEN. (A) Different infiltration levels of CD4⁺ TILs, CD8⁺ TILs, CD14⁺ monocytes, and CD15⁺ granulocytes in T-NEN are shown with representative IHC-stained images. (B) Proportion of stratified immune cell infiltration levels in T-NEN is displayed. (C) Classification of the immune landscape according to the PD-L1 overall/TILs pattern in T-NEN.

Figure 3

CD8⁺ TILs are a potential immunological marker for predicting bone metastasis in T-NEN. (A) Representative ¹⁸F-FDG PET/CT scan imaging of T-NEN patients with or without bone metastasis. (B) Representative IHC images showing CD8⁺ TILs deposition in T-NEN with and without bone metastasis. (C) Level of CD8⁺ TILs (hotspot of CD8⁺ cells/mm²) was quantified in each case. Scatter plots of CD8⁺ hotspot scores in patients with bone metastasis (n = 16) and without bone metastasis (n = 35) were displayed and comparison analysis was performed using the unpaired t-test. Data represent the mean ± SEM, ^** p < 0.01. (D) ROC was plotted to assess the predictive capacity of CD8⁺ TILs to predict bone metastasis.

Figure 4

Clinical practice of ICB with toripalimab in patients with metastatic T-NEN. (A) Representative IHC images showing CD8, PD-1, and PD-L1 expression patterns in case No.1, 2, and 3. (B) CT scans at baseline (pre-anti-PD-1 treatment) and re-examinations after anti-PD-1 treatment at indicated time points are shown. The arrow points to the lesions (with red marks) that changed significantly upon toripalimab treatment.

Figure 5

Graphical abstract of immunologically”hot”and”cold”tumors in T-NEN. Immunologically “hot” T-NEN with increased immune cell infiltration and enhanced expression of PD-1/PD-L1 tended to have restricted tumor size and less metastases, and would be more sensitive to immunotherapy. Immunologically “cold” T-NEN with limited immune cell infiltration and lacking expression of PD-1/PD-L1 tended to grow more aggressively and develop more metastases, especially to bones, and would be more resistant to immunotherapy.