Material and Method: The breast carcinoma samples (n = 26) with medullary pattern (defined according to established criteria) were subjected to immunohistochemical assays of the following receptors: ER, PR, HER2/neu, Ki-67, p53, Bcl-2, VEGF, MMP1, E-cadherin, EGFR, Hsp70, Hsp90, CD20, CD3, CD4, CD8, CD68, CD163, CD56, CD138, MPO, S100, IgG, IgM, and PD-L1.

Results: IBC-NST with medullary pattern was found to have negative expression of ER, PR, and HER2/neu; strong positive expression of Kі-67, mutant р53, Bcl-2, E-cadherin, EGFR, and PD-L1; moderate positive expression of Hsp70 and Hsp90; and low or negative expression of VEGF and MMP1. Furthermore, there was pronounced variability in the qualitative composition of tumor immune infiltrates with regards to T-lymphocytes, B-lymphocytes, macrophages, plasmocytes, and granulocytes.

Conclusion: IBC-NST with medullary pattern has many unfavourable morphological and immunohistochemical prognostic characteristics, which are balanced against the pronounced protective properties of the tumor cells and the qualitative characteristics of the tumor microenvironment. These can lead to a favourable disease course despite the relatively adverse features of the carcinoma cells.

IBC-NST is well-defined and has a syncytial growth pattern with no glandular structures of high histological grade as well as prominent TILs ^2,5. Cases are mainly triplenegative without expression of the еstrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu). Pronounced proliferative activity of tumor cells and expression of tumor SUPPRESOR protein р53 are commonly observed ⁵. However, despite the potentially unfavourable histological features, IBCNST with medullary pattern has a relatively good prognosis ^2,6,7; notably, this has been associated with the qualitative features of the tumor microenvironment (TILs) and pronounced tumor cell adhesion (involving E-cadherin expression) and absence of matrix metalloproteinase-1 (MMP1) ^2,5,6.

The favourable course of this high-grade carcinoma has not been explained by the presence or absence of the abovementioned proteins. Further studies are required to explain the inconsistencies between the morphological and clinical features of IBC-NST with medullary pattern.

Immunohistochemical Assay
Paraffin-block samples stored for histological diagnosis were used in the immunohistochemical assay following the method detailed in our previous publication ⁸. The antibody panels (purchased from Thermo Scientific, Master Diagnóstica, and Abcam) shown in Table I were employed to establish the parenchymal and stromal components of the immunophenotype of IBC-NST with medullary pattern.

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Table I: Antibody panel used for immunohistochemical study.

Evaluation of Staining
Two pathologists independently analysed the immunohistochemical staining evaluations and reached a consensus decision when necessary. The reaction was considered positive if the cells had cytoplasmic and/or nuclear expression of heat-shock protein 70 (Hsp70) and 90 (Hsp90); cytoplasmic and/or membranous expression of CD20, CD3, CD4, CD8, CD163, CD56, and CD138; exclusively nuclear expression of ER, PR, Ki-67, and tumor SUPPRESOR protein p53; exclusively cytoplasmic expression of Bcl-2, VEGF, MMP1, CD68, MPO, S100, IgG, and IgM; and exclusively membranous expression of HER2/neu, EGFR, and E-cadherin. To determine the pattern of receptor expression, we used a simplified 3-tiered scoring system: “-” – negative expression (0% immunoreactive cells); “+” – low expression (5–25% immunoreactive cells); “++” – moderate expression (26–50% immunoreactive cells); “+++” – high expression (>50% immunoreactive cells). Only the p53 nuclear expression pattern was considered as positive or negative staining. Ki-67, Hsp70 and Hsp90 expression were scored in percentage. The percentage of receptor-positive cells among the total number of tumor cells was scored. The intensity of the stained cells was considered separately. PD-L1 expression was considered positive in the presence of more than 1% of carcinoma cells showing membranous staining of any intensity. This scoring sheet was discussed with and approved by two pathologists prior to the start of the study. No morphologic features of the cancer were taken into account in this scoring. Data processing was carried out using the GraphPad Prism 9 statistics software. Detection and evaluation of the links among indicators were carried out using the nonparametric Spearman’s rank correlation coefficient (r). A p-value of 0.05 (95% level of confidence) was considered statistically significant.

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Figure 1: IBC-NST with medullary pattern: a pronounced expression of Ki-67, p53, and bcl-2 in tumoral cells (A); immune microenvironment cells (Ki-67- and bcl-2-positive expression) (B). Immunohistochemical study of Ki-67, p53, and bcl-2; x200.

All cases of IBC-NST with medullary pattern had strong (immunoexpression in more than 50% of tumor cells) membranous expression of E-cadherin, EGFR, and PD-L1 in the cancer cells (Figure 2). Moderate positive nuclear and cytoplasmic expression of Hsp70 and Hsp90 was observed in 74±6.3% and 68±5.7% of the tumor cells, respectively. Simultaneously, the majority of the tumor cells were either negative or had low (weak expression in less than 25% of tumor cells) VEGF and MMP1 cytoplasmic expression (Table II).

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Figure 2: IBC-NST with medullary pattern: a pronounced expression of E-cadherin, EGFR, and PD-L1; moderate expression of hsp70 and hsp90; week expression of MMP1 and VEGF; negative expression of MPO in tumoral cells, positive in scattered cells of microenvironment. Immunohistochemical study of E-cadherin, EGFR, hsp70, hsp90, VEGF, MMP1, PD-L1, and MPO ; x400.

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Table II: Protein expressions in IBC-NST with medullary pattern.

We focused on studying the qualitative composition of the tumor microenvironment of IBC-NST with medullary pattern. Immunohistochemical analysis of the expression of CD20 (B-lymphocytes), CD3 (T-lymphocytes), CD4 (T-helper cells), CD8 (T-killer cells), CD56 (natural killer cells), CD138 (plasmocytes), CD68, S100, and CD163 (macrophages), MPO (granulocytes), IgG, and IgM revealed the specific features of the immune infiltrate (Figures 2–4).

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Figure 3: IBC-NST with medullary pattern: Immunohistochemical study of CD20, CD3, CD4, CD8, CD56, CD138, IgG, and IgM ; x400.

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Figure 4: IBC-NST with medullary pattern: Immunohistochemical study of CD68, S100, and CD163; x400.

The tumor immune-infiltrate microenvironment mainly comprised T-lymphocytes (44% of immune cells) with a relatively low number of B-lymphocytes, macrophages, plasmocytes, and granulocytes (13%, 22%, 9%, and 3% of immune cells, respectively). T-helper cells (CD4+ cells), with a relatively small number of T-killer cells (CD8+ cells) and NK cells (CD56+ cells), were observed among the T-lymphocytes (Figure 3). The tumor microenvironment contained single and grouped plasmocytes, which reached a prevalence of about 8% of the immune-cell infiltrate in some cases. Most B-lymphocytes and plasmocytes showed IgG expression, and some showed IgM expression.

Macrophages (CD68+ cells) were present in significant numbers both among tumor cells and in the immune infiltrate (Figure 4). It should be noted that their active forms (S100+ cells) were mainly localised among the cancer cells. A significant population of M2 macrophages (CD163+ cells) was identified, and they were diffused throughout all the components of the neoplastic tissue.

The scores for each cell type in the tumor microenvironment showed the following ratios: CD3+:CD20+:CD138+:CD56+: CD68+:MPO = 0.44: 0.13: 0.09: 0.09: 0.22: 0.03.

Our study investigated the immunohistochemical features of IBC-NST with medullary pattern to explain the inconsistency between the morphological and clinical characteristics of this tumor type. We found that the cancer cells had pronounced proliferative activity (Kі-67 expression), and blocked both natural and drug-induced apoptosis (p53 and Bcl-2 expression), as well as the expression of unfavourable proteins (EGFR, Hsp70, Hsp90, and PD-L1) that induce progression of the neoplastic process ^8,9,11-13. At the same time, the cells were ER and PR-negative. In contrast, most of the tumor cells had no HER2/neu expression, low VEGF and MMP1 expression (weak expression in less than 25% of tumor cells), and high E-cadherin expression (strong expression in more than 50% of tumor cells), which indicated a favourable course of the carcinoma ^14-17.

Our results showed pronounced variability in the qualitative composition of the tumor microenvironment of IBC-NST with medullary pattern. This was represented mainly by TILs, which were predominantly T-lymphocytes and, in particular, CD4+ cells. Simultaneously, significant numbers of CD8+ T-cells, NK cells, В-lymphocytes, and plasmocytes were found in the immune infiltrate. Furthermore, the neoplastic tissue was infiltrated with a significant quantity of macrophages. Notably, most of the activated macrophages (S100+ cells) were in close contact with cancer cells. At the same, a significant number of M2 macrophages (CD163+ cells) were found in the tumor microenvironment, which could have an unfavourable adverse effect on the course of the neoplastic process ^5,9,18,19.

From the abovementioned findings, we concluded that the unique course of IBC-NST with medullary pattern is determined by several factors (Figure 5). This tumor type was characterized by a combination of both aggressive and favourable immunophenotypic features of cancer cells and the immune microenvironment. On the one hand, ER and PR were absent in the tumor tissue; there was a high proliferative index; overexpression of p53, Bcl-2, EGFR, hsp70, hsp90, and PD-L1; and significant numbers of M2 macrophages. On the other hand, there were insufficient amounts of pro-metastatic proteins (VEGF and MMP1); an absence of HER2/neu expression; and the severe adhesive capacity of tumor cells prevented local and remote tumor spread. The activation of cellular (macrophages, CD4+ and CD8+ T-cells, and NK cells) and humoral (activated macrophages, B-lymphocytes, CD4+ T-cells, plasmocytes, and IgG synthesis) local immune responses limited the spread of the disease.

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Figure 5: Interaction of protective and aggressive factors in IBCNST with medullary pattern.

In IBC-NST with medullary pattern, the favourable factors (protection indicators) mostly surpassed the adverse influences. This led to a favourable prognosis for the course of the malignant process, despite the numerous negative morphological and immunohistochemical features of the parenchymal and stromal tumor components.

In summary, IBC-NST with medullary pattern has many prognostically unfavourable morphological and immunohistochemical characteristics, which are balanced by the pronounced protective properties of the tumor cells and the qualitative traits of the tumor microenvironment. This leads to a favourable course for this carcinoma despite the relatively adverse features of the cancer cells.

CONFLICT of INTEREST
The authors declare that they have no competing interests.

ACKNOWLEDGMENTS
This research was supported by the Ministry of Education and Science of Ukraine Grant № 0121U100472 and research theme № 0119U100887.

INFORMED CONSENT
Written informed consent was obtained from patients who participated in this study.

AUTHORSHIP CONTRIBUTIONS
Concept: ML, YL, YS, AR, Design: ML, NH, AR, Data collection or processing: ML, NH, VS, YL, YS, Analysis of Interpretation: ML, NH, VS, YL, YS, AR, Literature search: ML, NH, VS, YL, YS, Writing: ML, NH, VS, YL, AR, Approval: ML, VS, AR.

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