Molecular-biologic and immunohistochemical features of undifferentiated pleomorphic sarcomas

Abstract

Relevance. Undifferentiated pleomorphic sarcoma (UPS) is one of the most common subtypes of soft tissue sarcomas. The polymorphism of tumor cells and high degree of malignancy account for the aggressive potential of UPS. Due to the rarity of occurrence and high heterogeneity of UPS, the number of studies describing the cellular composition and molecular-­biological characteristics is very limited. Objective is to assess the cellular composition and gene expression of UPS. Materials and Methods. Biomaterial from 10 patients with UPS was analyzed in the study. In this study we used primary antibodies to CD163 (marker of M2 macrophages) and Fibroblast activation protein (FAP — marker of fibroblasts) and secondary Caprine-­Anti-­Rabbit IgG HRP were used. HRP-tagged secondary antibodies were manifested using DAB. Antibodies for automated BOND-III IHC stainer were used to evaluate the microenvironment: CD68‑marker of macrophages, CD19‑marker of B-lymphocytes, CD56‑marker of neuroendocrine tumors, metastasis protein, Ki67 antigen-­proliferation marker, Bcl‑2‑oncoprotein. Staining on an automated BOND-III IHC stainer was performed according to standard protocols. In homogenized samples of tumor tissue and peritumoral area with the number of cells 106/ml in order to assess the microenvironment of the tumor and surrounding tissue, cytofluorimetric study of the relative number of CD14+ and CD16+ monocytes, CD68+ macrophages, CD86+ M1 macrophages, CD163+ and CD206+ M2 macrophages, CD4+ helper T-lymphocytes and CD45+ leukocytes was performed on the MACS Quant Analyzer device. The mRNA expression levels of HIF1A, VEGF, MMP2, ARG1, NOS2, and EGFR were determined in tumor tissue and peritumoral samples by PCR. The RNA Solo RNA kit was used for RNA isolation, and the MMLV RT Kit was used for reverse transcription. The amplification reaction with real-time detection was performed on aDTprime Real-­Time Amplifier. Results and Discussion. The expression of CD56, FAP, CD68 is characteristic for UPS. Among the cells of the microenvironment, macrophages and CD16‑monocytes predominate in UPS. EGFR expression level is increased in tumor cells of the UPS compared to the peritumoral region. The expression levels of ARG1, NOS2, HIF1A, VEGF, and MMP2 in tumors have individual differences and are not specific to the UPS. Conclusion. In our study, we analyzed the cellular composition and gene expression in UPS samples. Further follow-up of patients is necessary to evaluate the clinical significance of each marker.

Keywords

• soft tissue sarcomas
• undifferentiated pleomorphic sarcomas
• tumor microenvironment
• cellular composition
• gene expression