Soluble forms of programmed cell death receptor pd-1 and its ligand pd-l1 in breast cancer

Introduction. Immunotherapy for breast cancer is not a universal tool and requires careful selection of patients for its implementation. When using immune checkpoint inhibitors PD-1/PD-L1, determining the IHC status of the tumor is sometimes insufficient and the results of the expression of these proteins in tumor cells are contradictory. In the last decade, research interest has increased in soluble forms of PD-1/PD-L1, which can be identified in the serum or plasma of cancer patients, while the concentrations of these proteins differ in patients with various malignancies and change during tumor progression, which can be used to monitor the course of the disease and assess the prognosis of the disease.

The purpose of the work is to analyze the clinical and laboratory significance of soluble forms of the PD-1 receptor and its ligand in the blood serum of patients with breast cancer, taking into account the main clinical and morphological characteristics of the disease.

Material and methods. An analysis of the content of sPD-1 and sPD-L1 in the blood serum of 77 patients (average age 52.3 years) with newly diagnosed breast cancer at various stages and in 49 healthy women (average age 47.9 years) who made up the control group was carried out. The concentration of sPD-L1 and sPD-1 in blood serum obtained according to standard methods before the start of specific treatment was determined using reagent kits for direct enzyme-linked immunosorbent assay "Human PD-L1 Platinum ELISA" and "Human PD-1 ELISA kit" (Affymetrix, eBioscience, USA) in accordance with the manufacturer's instructions. Measurements were carried out on an automatic enzyme immunoassay analyzer BEP 2000 Advance (Siemens Healthcare Diagnostics, Germany). Statistical analysis of the obtained results was carried out using GraphPad Prizm v. 10. When comparing indicators and analyzing their relationships, nonparametric Mann–Whitney and Kruskal–Wallis tests and Spearman's rank correlation coefficient were used. Differences were considered statistically significant at p < 0.05.

Results. It was found that the median concentration of sPD-L1 in the blood serum of patients with breast cancer was statistically significantly higher (49.2 pg/ml) than the control (9.2 pg/ml) (p < 0.0001), while the level of sPD-1 on the contrary, it is statistically significantly lower in patients with breast cancer (7.5 pg/ml) compared to the control group (47.1 pg/ml; p < 0.0001). The levels of markers in the blood serum of patients with unilateral and primary multiple breast cancer were practically the same. Analysis of diagnostic significance showed that at an sPD-1 concentration of 18.1 pg/ml, the sensitivity and specificity of the method are 98% (AUC – area under the curve 0.998 with 95% CI 0.993–1.000; p < 0.0001), which indicates promise further study of this protein in breast cancer. For sPD-L1, it was demonstrated that the maximum sensitivity and specificity of the test (92% and 80%, respectively) was achieved at a cut-off value of 24 pg/ml (AUC – area under the curve 0.954 with 95% CI 0.923–0.985; p < 0.0001). It was shown that in the group of patients with the HER2+ subtype of breast cancer, the content of sPD-L1 is almost 2 times higher compared to other subtypes of this disease and 10 times higher compared to the control group. With a threshold value of sPD-L1 equal to the median content in this group of patients (96.2 pg/ml), the sensitivity and specificity of the method were (86% and 100%, respectively). Patients with this receptor type of tumor constitute the smallest group, and therefore the results obtained require validation on a larger sample. The analysis did not reveal correlations between the levels of the studied proteins and the clinical and morphological characteristics of breast cancer, with the exception of a direct association between the content of sPD-1 in the blood serum and the proliferative status of the tumor.

Conclusions. The detected changes in the production of soluble forms of the sPD-1 receptor and its ligand sPD-L1 in patients with breast cancer may indicate the immunogenicity of these tumors. At the same time, repeated analysis of sPD-1 and sPD-L1 in the blood serum of patients is possible, which can be used not only in the diagnosis of these neoplasms, but also in monitoring the disease with repeated studies of these biomarkers in the case of immunotherapy with immune checkpoint inhibitors PD-1/PD-L1. All this requires further research.