Automated brightfield multiplex immunohistochemistry to quantify biomarkers related to immune senescence: Relationships with survival in non-small cell lung cancer patients.

Abstract

e20500 Background: Elderly patients have an eroded immune characterized by a progressive decline in immune surveillance that favors infection and cancer development. Tumor cells can escape immune surveillance by upregulating inhibitory immune checkpoint such as PD-L1. High expression of PD-L1 was reported in association with CD8+T-cell exhaustion and increased levels of CD33+ myeloid-derived suppressor cells. Although low CD4/CD8 ratio is associated with increased mortality, the status of the CD4+T-cells as a clinical marker of immunosenescence is less well characterized in the field of aging. The aim of this study was to determine the presence of immunosenescence biomarkers according to age in non-small cell lung cancer (NSCLC) patients and to evaluate them as predictive biomarkers of patients’ outcome. Methods: One hundred NSCLC patients, matched by age (50 patients < 70 years, 50 patients ≥70 years) were included. An automated 4-Plex optical IHC assay was developed on the Discovery ULTRA automated stainer using monoclonal antibodies PD-L1 (SP263), CD4, CD8, and CD33. The stained slides were scanned with Nanozoomer HT 2.0 Scanner, and analyzed with Calopix software. Results: The CD4/CD8 ratio and PD-L1 expression in tumor and immune cells were significantly lower in elderly NSCLC patients ≥70 years than in age-paired patients, while absolute count of CD33+ was increased. Patients with CD4/CD8 ratio higher than two, high PD-L1 density and low CD33+ frequency achieved increase in median disease-free survival. Conclusions: Distribution of PD-L1, CD4, CD8, and CD33 cells was influenced by age in NSCLC patients. The proportion of CD8 + CD28- T cells, CD4+ T cells and CD4/CD8 ratio may be used as predictive biomarkers of anti-PD-L1 therapy efficacy in NSCLC patients. The automated 4-Plex IHC assay together with its respective digital analysis could serve as a tool for further characterizing tumors and their microenvironment and provide a better understanding of which patients may benefit from immunotherapy.

Reproducibility of PD-L1 assessment in non-small cell lung cancer—know your limits but never stop trying to exceed them

“…It would be beneficial to evaluate PD-L1 expression combined with multicolor IHC assays to better characterize the immune tumor microenvironment by staining cells such as CD8 T cells, macrophages, myeloid-derived suppressor cells, natural-killers or regulatory T cells (19). These new approaches may contribute to optimized biomarker assessment of clinical samples, as well as improvement of the predictive value of PD-L1 expression on both tumor cells and immune cells for immunotherapy.…”