Quantitative analysis of lymphocyte exhaustion markers in patients with localized clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a neoplastic disease associated with poor prognosis. Localized disease is successfully treated with nephrectomy; however, advanced disease often requires the combined use of immunotherapy and targeted therapy. To the best of our knowledge, there is no validated method to predict immunotherapy response and there is a lack of knowledge regarding the expression kinetics of exhaustion receptors in the early stages of ccRCC. In the present study, immunohistochemistry and flow cytometry were performed to evaluate the expression levels of five biomarkers associated with immune dysfunction [programmed cell death protein-1 (PD-1), lymphocyte activation gene 3 (LAG-3), T-cell immunoglobulin and mucin domain containing-3 (TIM-3), cytotoxic T lymphocyte-associated protein-4 (CTLA-4) and programmed death ligand-1 (PD-L1)] in 20 patients diagnosed with localized ccRCC. Immunohistochemistry demonstrated that ccRCC tissue was highly positive for CD3, with low expression of PD-L1 and CTLA-4. Based on flow cytometry, the infiltrating leukocytes were mostly CD8⁺ lymphocytes and monocytes. In tumors, decreased expression levels of LAG-3, TIM-3, CTLA-4 and PD-L1 were reported; however, expression levels of PD-1 remained unchanged. Sample stratification according to the nuclear grade demonstrated no changes in the evaluated markers. Finally, the 12-month follow-up of blood biomarkers revealed no change in the receptors, except for CTLA-4. The results of the present study suggested that immune exhaustion, defined as increased expression of exhaustion receptors in lymphocytes, was not present in tissues from patients with ccRCC with localized disease.