Molecular-biologic and immunohistochemical features of undifferentiated pleomorphic sarcomas

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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 a DTprime 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.

About the authors

Anna M. Kosyreva

RUDN University; Avtsyn Research Institute of Human Morphology of Petrovsky National Research Centre of Surgery

Email: enar2017@yandex.ru
ORCID iD: 0000-0002-6182-1799
SPIN-code: 5421-5520
Moscow, Russian Federation

Enar D. Jumaniyazova

RUDN University

Author for correspondence.
Email: enar2017@yandex.ru
ORCID iD: 0000-0002-8226-0433
SPIN-code: 1780-5326
Moscow, Russian Federation

Dzhuliia Sh. Dzhalilova

RUDN University; Avtsyn Research Institute of Human Morphology of Petrovsky National Research Centre of Surgery

Email: enar2017@yandex.ru
ORCID iD: 0000-0002-1337-7160
SPIN-code: 3660-5827
Moscow, Russian Federation

Alexandra V. Sentyabreva

RUDN University; Avtsyn Research Institute of Human Morphology of Petrovsky National Research Centre of Surgery

Email: enar2017@yandex.ru
ORCID iD: 0000-0001-5064-219X
SPIN-code: 6966-9959
Moscow, Russian Federation

Ekaterina A. Miroshnichenko

RUDN University; Avtsyn Research Institute of Human Morphology of Petrovsky National Research Centre of Surgery

Email: enar2017@yandex.ru
ORCID iD: 0000-0002-0020-958X
SPIN-code: 2436-4104
Moscow, Russian Federation

Timur I. Fetisov

N.N. Blokhin Russian Cancer Research Center

Email: enar2017@yandex.ru
ORCID iD: 0000-0002-5082-9883
SPIN-code: 6890-8393
Moscow, Russian Federation

Anastasia V. Lokhonina

RUDN University; National Medical Research Center of Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov

Email: enar2017@yandex.ru
ORCID iD: 0000-0001-8077-2307
SPIN-code: 4521-2250
Moscow, Russian Federation

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Supplementary files

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1. Figure 1. Heterogeneity of Ki‑67 (A, Б) and Bcl-2 (В—Д) expression on histologic sections of UPS. A — Ki‑67 expression more than 50 % of cells (x200, Ob.3); Б — Ki‑67 expression less than 30 % of cells (x200, Ob.1); В — marked expression of Bcl-2 (x200, Ob.5); Г — low expression of Bcl-2 (x200, Ob.7); Д — negative reaction with antibodies to Bcl-2 (x200, Ob.10)

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2. Figure 2. CD68+ macrophages (A, B) and CD19+ lymphocytes (В–Д) in UPS. A — high number of CD68+ macrophages (x200, Ob.5); Б — giant CD68+ cells (x400, Ob.10); В — CD163+ macrophages (x400, Ob.7); Г — negative reaction with antibodies to CD163+ (x200, Ob.10)

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3. Figure 3. CD56+ tumor cells (А, Б), CD163+ macrophages (В, Г) and FAP+ fibroblasts (Д, Е) in UPS. А — marked expression of CD56+ (x400, Ob.1); Б — moderate expression of CD56+ in tumor cells (x400, Ob.7); В — cluster of CD19+ lymphocytes (x200, Ob.9); Г — single CD19+ lymphocytes (x400, Ob.1); Д — high number of FAP+ fibroblasts (x400, Ob.4); Е — moderate number of FAP+ fibroblasts (x400; Ob.9)

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4. Figure 4. UPS microenvironment. A — relative number of CD4 — helper T-lymphocytes, CD45 — leukocytes, CD14 — monocytes, CD16 — monocytes, CD68 — macrophages, CD86 — M1 macrophages, CD163 — M2 macrophages, CD206 — M2 macrophages. Б — relative number of lymphocytes and monocytes in 8 patients

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5. Figure 5. Expression levels of EGFR, ARG1, NOS2, HIF1A, VEGF and MMP2 in the peritumoral region (ПО) and in tumor samples of UPS. Mann-Whitney criterion, *p < 0.05

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Copyright (c) 2024 Kosyreva A.M., Jumaniyazova E.D., Dzhalilova D.S., Sentyabreva A.V., Miroshnichenko E.A., Fetisov T.I., Lokhonina A.V.

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