Aikian A. Z., Kaidashev I. P.

PATHOMORPHOLOGY

Scentific article

The triple-negative subgroup of breast cancer (TNBC) (estrogen, progesterone-, and HER2-receptor negatives) often affects young women and usually has a worse prognosis as compared to other types of BC. Recently, a complex role of the immune system in the growth, elimination and metastasis became an object of increased attention, promising to provide clinical prognostic markers of progression, as well as targets for the treatment of a wide range of tumors, including TNBC. In BC in general and in TNBC in particular, tumor-associated macrophages (TAM) are mainly associated with a worse prognosis, but their composition, apparently, varies between individual cases of TNBC. Thus, the research of TAM in TNBC is relevant, and therefore the aim of this study was to examine the quantitative parameters and features of localization of CD68+ and CD163+M2-like TAMs, which infiltrate TNBC in the primary focus without metastases and in paired samples with metastases in the lymph nodes, as well as the pathomorphological characteristics of this type of BC to determine their contribution to metastasis. Biopsy samples and clinical data were obtained from patients undergoing treatment at Poltava Regional Clinical Dispensary. The study was approved by the Ethics Commission of Ukrainian Medical Stomatological Academy. Materials of the study were intraoperative tumor tissues and ipsilateral lymph nodes in radically removed mammary glands without neoadjuvant treatment. Immunohistochemical (IHC) characteristics of the removed tumors (HER2, ER, PR, Ki67) were used to distinguish the TNBC group. According to the meta-analysis, the use of CD68 as a biomarker for TAM for IHC evaluation has its priorities, as compared with the individual definition of CD163 or CD206. We analyzed two subgroups of TNBC, balanced by the N0/1 status, 3 samples in each, and 3 samples of metastases in the lymph nodes, respectively. The average age of patients was 64 years, from 52 to 76. Primary-metastatic BC was observed in all patients in the 2nd, N1 group. Morphological and IHC findings were obtained according to the criteria for determining the molecular and biological subtype. IHC studies for determining TAM and M2-like macrophages were performed using the streptavidin-peroxidase method. The evaluation of immunohistochemical coloring was performed by counting CD68+TAM and CD163+M2-like TAMs under the light microscope (Biolam, LOMO, Russia: lens ×40, eyepiece ×7) in 7-10 fields of view of the intensive ICH-reaction of each section, calculating the arithmetic mean, within the tumor nests and tumor stroma. The count included immunopositive cells with macrophage morphology. Microphotographs were obtained using the Leica DM500 microscope, Leica, Germany (lens × 4, 40). The statistical analysis was carried out using the GraphPad Prism 5 software, using nonparametric, parametric and simulation methods. The study is organized as a descriptive pathomorphological comparison between the two groups: N0 and N1, triple-negative BC, comparison of quantitative characteristics of CD68+ and CD163+M2-like fraction of TAM, as well as for verification of possible correlation relations between the quantitative characteristics of macrophages in the primary tumor and in the corresponding metastasis in the ipsilateral axillary lymph nodes. Increased level of infiltration of the primary focus of TNBC with CD68+TAM and CD163+M2-like macrophages is reliably associated with metastasis. It is assumed that the M1-like subpopulation of TAM most closely contacts with tumor nests in TNBC, which may be a prerequisite for the spread of the tumor, or metastasis. Primary premetastatic effects of TNBC may be related to the increase of not only M2, but also M1 macrophages. In metastatic lymph nodes, the amount of CD68+TAM was significantly lower than that of primary TNBC, whereas the quantitative indices of CD163+M2 correlated. Along with the localization and co-localization of CD68+ and CD163+M2-like TAMs, the results conform with the generally accepted paradigm that M1 TAM has an anti-tumoral but lower potential and M2 – the protumoral one, but this is shown for metastases in the regional lymph nodes in primary-metastatic TNBC. Precise quantitative TAM indices for the use in clinical practice have not been developed, although their pathogenetic significance and quantitative variations are reliable according to metastasis and other clinical data of TNBC.

tumor-associated macrophages, immunohistochemical studies, CD68+ and CD163+, triple-negative subgroup, breast cancer

1. Stovgaard ES, Nielsen D, Hogdall E, Balslev E. Triple negative breast cancer – prognostic role of immune-related factors: a systematic review. Acta Oncol.2018 Jan;57(1):74-82. DOI: 10.1080/0284186X.2017.1400180. Epub 2017 Nov 23. 
2. Zhao X, Qu J, Sun Y, Wang J, Liu X, Wang F, et al. Prognostic significance of tumor-associated macrophages in breast cancer: a meta-analysis of the literature. Oncotarget.2017 May 2;8(18):30576-86. DOI: 10.18632/oncotarget.15736
3. Ruffell B, Au A, Rugo HS, Esserman LJ, Hwang ES, Coussens LM. Leukocyte composition of human breast cancer. Proc Natl Acad Sci USA. 2012;109:2796-801. [PMC free article] [PubMed].
4. Yoshihiro Komohara, Koji Ohnishi. Possible functions of CD169-positive sinus macrophages in lymph nodes in anti-tumor immune responses. Motohiro Takeya Cancer Sci. 2017 Mar;108(3):290-5. Published online 2017 Mar 7. DOI: 10.1111/cas.13137
5. Stewart DA, Yang Y, Makowski L. Basal-like breast cancer cells induce phenotypic and genomic changes in macrophages. Mol Cancer Res. 2012;10:727-38.
6. Yang M, Li Z, Ren M, Li S, Zhang L, Zhang X, et al. Stromal Infiltration of Tumor-Associated Macrophages Conferring Poor Prognosis of Patients with Basal-Like Breast Carcinoma. J Cancer.2018 Jun 6;9(13):2308-16. DOI: 10.7150/jca.25155. eCollection 2018. 
7. Yang M, Ma B, Shao H, Clark AM, Wells A. Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells. MC Cancer.2016 Jul 7;16:419. DOI: 10.1186/s12885-016-2411-1. 
8. Klingen TA, Chen Y, Aas H, Wik E, Akslen LA. Tumor-associated macrophages are strongly related to vascular invasion, non-luminal subtypes, and interval breast cancer. Hum Pathol. 2017 Nov;69:72-80. DOI: 10.1016/j.humpath.2017.09.001. Epub 2017 Sep 18
9. Chen H, Chen X, Lin H. Expression of Tumor-Related Macrophages and Cytokines After Surgery of Triple-Negative Breast Cancer Patients and its Implications. Wang J, Med Sci Monit.2016 Jan 11;22:115-20.
10. Yuan ZY, Luo RZ, Peng RJ, Wang SS, Xue C. High infiltration of tumor-associated macrophages in triple-negative breast cancer is associated with a higher risk of distant metastasis. Onco Targets Ther.2014 Aug 21;7:1475-80. DOI: 10.2147/OTT.S61838. e Collection 2014
11. Harney AS, Arwert EN, Entenberg D, Wang Y, Guo P, Qian BZ, et al. Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage-Derived VEGFA. Cancer Discov.2015 Sep;5(9):932-43. DOI: 10.1158/2159-8290.CD-15-0012. Epub 2015 Aug 12. 
12. Chitty JL, Filipe EC, Lucas MC, Herrmann D, Cox TR, Timpson P. Recent advances in understanding the complexities of metastasis. F1000Research. 2018;7:F1000 Faculty Rev-1169. DOI: 10.12688/f1000research.15064.1
13. Ju Yang, Xiaojing Li, Xiuping Liu, Yongjuan Liu. The role of tumor-associated macrophages in breast carcinoma invasion and metastasis. Int J Clin Exp Pathol. 2015;8(6):6656-64. 
14. Kim O-H, Kang G-H, Noh H. Proangiogenic TIE2+/CD31+ Macrophages Are the Predominant Population of Tumor-Associated Macrophages Infiltrating Metastatic Lymph Nodes. Molecules and Cells. 2013;36(5):432-8. DOI: 10.1007/s10059-013-0194-7
15. Heba Sailem, Faraz K. Mardakheh, Mar Arias Garcia, Christopher J. Tape, Mitch Dowsett. A Histology-Genomic Integration Analysis Rachael Natrajan, Microenvironmental Heterogeneity Parallels Breast Cancer Progression. PLoS Med. 2016 Feb;13(2):e1001961. Published online 2016 Feb 16. DOI: 10.1371/journal.pmed.1001961PMCID: PMC4755617 PMID: 26881778
16. Aras S, Zaidi MR. TAMeless traitors: macrophages in cancer progression and metastasis. British Journal of Cancer. 2017;117(11):1583-91. DOI: 10.1038/bjc.2017.356
17. Laoui D, Van Overmeire E, Di Conza G, Aldeni C, Keirsse J, Morias Y, et al. Tumor hypoxia does not drive differentiation of tumor-associated macrophages but rather fine-tunes the M2-like macrophage population. Cancer Res. 2014 Jan 1;74(1):24-30. DOI: 10.1158/0008-5472.CAN-13-1196. Epub 2013 Nov 12.
18. Norton KA, Jin K, Popel AS. Modeling trip, fibroblasts and tumor vasculature. J Theor Biol. 2018 Sep 7;452:56-68. DOI: 10.1016/j.jtbi.2018.05.003. Epub 2018 May 8.

«Bulletin of problems biology and medicine» Issue 4 Part 2 (147), 2018 year, 313-319 pages, index UDK 618.19-006.04/.44:611.018

10.29254/2077-4214-2018-4-2-147-313-319