QUANTITATIVE CHARACTERISTICS OF CD68+ AND CD163+ MACROPHAGES IN THE PRIMARY FOCUS AND IN METASTATIC LESIONS OF REGIONAL LYMPH NODES IN TRIPLE-NEGATIVE INVASIVE BREAST CARCINOMA

Abstract

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.

Keywords

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