Results: The mIF panel demonstrated the distribution of TILs and PD-L1 in the FFPE tissue. Compared with each conventional IHC, we could evaluate the distribution of PD-L1, CD4+, CD8+, CD68+ cells in a slide. KEY WORDS: PD-L1, tumor infiltrating lymphocyte, multiplex immunohistochemistry, hypopharyngeal squamous cell carcinoma, head and neck.
The PD-1/PD-L1 axis is thought to regulate a pathway that allows cancers to escape immune surveillance. L1 is frequently overexpressed in many human cancers. 1) Antibody blockade of PD-1 with its ligand has shown favorable results against malignancies such as melanoma and non-small cell lung cancer. 2-4) These recent results have led to the testing of PD-1/PD-L1 inhibitors in head and neck squamous cell cancers (HNSCC).5-7). Studies performed in other tumors also revealed that clinical responses to immune checkpoint inhibitors are associated with increased immune inhibitory signals and increased tumor infiltrating lymphocytes (TIL).1,8-10) The understanding of this tumor microenvironment is made an important step in identifying evidence for the presence of distinct immunological phenotypes based on the presence or absence of certain immune cells. 11-12) For example, high myeloid cell density correlates with poor prognosis. 13) However, the correlation between macrophage density and patient survival is less significant than that of T cells, especially CD8+ T cells. 14).
Patients with hypopharyngeal squamous cell carcinomas (HPSCC) present with an advanced stage of the disease and have the worst prognosis among HNSCC, despite the concomitant use of treatments consisting of surgery and chemotherapy with or without radiotherapy.15) Currently, there are limited data on the prognostic values of PD-L1 or TIL expression in HPSCC patients. Both the location and density of TIL and their interrelationship with the PD-L1-positive tumor microenvironment are important considerations.12) The quantification method should be concise because the level of PD-1 on T cells may be associated with the state of differentiation and the level of function of T cells.16). This study analyzed PD-L1 expression, along with CD4+ and CD8+ T cell density, CD68+ density in hypopharyngeal squamous cell carcinomas using quantitative multispectral imaging (MSI).
Multiplex immunofluorescence (mIF) staining allows the simultaneous detection of multiple markers in the same tissue section in formalin-fixed and paraffin-embedded (FFPE) tumor tissue for a deeper understanding of the tumor microenvironment.17 We aimed to validate the mIF panels for application to FFPE HNSCC tissues using an array of immune marker antibodies, including those against PD-L1 and TILs, multispectral microscopy and image analysis software.
Study patients and FFPE tissue specimens
Multiplexed immunofluoresecence (mIF) and Quantification
The slide was then incubated with primary antibody for CD4 (NCL-L-CD4-368, Leica, United Kingdom, dilution 1:100) for 1 hour in a humidified chamber at room temperature, followed by detection using the OpalTM Polymer HRP Ms+ Rb set followed by visualization with Opal 620 TSA Plus (1:150). The slide was then incubated with primary antibody for CD68 (, dilution 1:100) for 1 hour in a humidified chamber at room temperature, followed by detection using the OpalTM Polymer HRP Ms+Rb kit followed by visualization with Opal 570 TSA Plus (1:150). The slide was then incubated with the final antibody, CD8 (4B11, NB100-65729, Novusbio, USA, dilution 1:300), for 1 hour in a humidified chamber at room temperature, followed by detection using the OpalTM Polymer HRP Ms +Rb kit.
Nuclei were then visualized with DAPI and the section was coverslipped using HIGHDEF® IHC fluoromount (ADI Enzo, USA). CD68-Opal 570 and DAPI) was used to establish the spectral library of fluorophores required for multispectral analysis (Fig. 1A). This spectral library provides the reference for target quantification, as the intensity of each fluorescent target is extracted from the multispectral data using linear unmixing. Immunostained sections were scanned using the Vectra 3.0 Automated Quantitative Pathology Imaging System at 20nm wavelength intervals from 420n to 720nm and combined these recordings to create a single stack image that captures the unique spectral signature of all mIF markers. retained.
The total number of CD274 (PD-L1), CD4, CD8 and CD68 positive cells was considered as identified as the total immune cell infiltrations in the tissue. The percentage of each subset of immune cells was calculated by dividing the absolute number of each subset by the total number of all these cells.
Analysis with Spotfire™ software and compartmentalization of the samples
CD68-Opal 570 and DAPI) was used to establish the spectral library of fluorophores required for multispectral analysis (Fig. 1A).
Statistical analysis
Ethics and Disclosures
The section was dichotomized into tumor and stroma to evaluate each component (Fig1D).
Patient characteristics
Multiplex IF Validation
TIL quantity and infiltration pattern
PD-L1 expression and PD-L1+CD8+ T-cell density
Distribution of CD4, CD8, CD68, PD-L1 according to tumor centers and stromata of 10 specimens of hypopharyngeal squamous cell carcinoma. CD4+ T cell densities in whole A sections, B tumor centers, and C stroma of hypopharyngeal squamous cell carcinoma specimens. CD68+ T cell densities in whole G sections, H tumor centers, and I stroma of hypopharyngeal squamous cell carcinoma specimens.
L1 expression in hypopharyngeal squamous cell carcinoma determined by a quantitative multispectral imaging system (Opal™) in A whole sections, B tumor centers and C stroma. In this study, we were able to demonstrate the distribution of CD4+, CD8+, CD68+, PD-L1 markers according to the tumor center and stroma of 10 HPSCC cases using multiplex immunofluorescence staining. Other studies on advanced head and neck cancer patients undergoing definitive chemoradiotherapy21) and neoadjuvant chemotherapy have shown similar results that patients with a higher number of tumor-infiltrating CD8+ TILs tend to survive longer.
There are also suggestions that further subgroup analysis of PD-L1 expression could help predict the prognosis of patients with higher CD8+ TIL density and could be used as a predictive biomarker. 20, 22-23) However, the immunologically hot tumor and cold tumor showed a different absolute number of TILs, including CD4+, CD8+, CD68+, and NK cells, instead of the absolute number of TILs. Thus, the relative distribution of these immune cells would be greater. et al in patients with HNSCC found that HPV+ patients had a significantly lower proportion of cells with high PD-1 expression compared to HPV patients. The association between PD-L1 expression and the prognosis of HNSCC patients still seems unclear.
Some studies show that higher levels of PD-L1 expression are associated with a poor prognosis in various types of cancer. 26-30) However, other studies question these results. 31-33) This study also found no significant association between PD-L1 expression and recurrence of HPSCC patients. This study showed the distribution of CD4+, CD8+, CD68+, PD-L1 markers according to the tumor center and stroma of 10 HPSCC cases using multiplex immunofluorescence staining, which can simultaneously analyze cells positive for multiple markers. Evidence for a role of the PD-1:PD-L1 pathway in the immune resistance of HPV-related squamous cell carcinoma of the head and neck.
Trends in incidence and prognosis are for head and neck cancer in the United States: a site-specific analysis of the SEER database e. PD-L1 expression on immune cells, but not on tumor cells, is a favorable prognostic factor for head and neck cancer. PD-1 status in CD8+ T cells associates with survival and anti-PD-1 therapeutic outcomes in head and neck cancer.
High PD-L1 expression in lung cancer may contribute to poor prognosis and tumor cell immunity by suppressing the maturation of tumor-infiltrating dendritic cells. Clinicopathological analysis of PD-L1 and PD-L2 expression in renal cell carcinoma: association with oncogenic proteins. PD-L1 mRNA expression is associated with increased plaque and better outcome in breast cancer noma.
Clinical relevance of immune parameters in the tumor or microenvironment of head and neck cancer.
