**7. Materials and methods**

Studies were conducted in a group of 162 patients with breast carcinoma with lymph node metastasis (111 triple-negative breast cancer and 51 non-triple-negative breast cancer) in the Department of Pathology, Military Medical Institute in Warsaw. Material for the study came from biopsies, excisional biopsies and modified radical mastectomies. Tumor samples were fixed in 10% buffered formalin phosphate. After 24-h fixation, material was dehydrated using alcohol in gradually increasing concentrations and embedded in paraffin. Paraffin blocks were cut into serial sections 4 μm in thickness. They were then stained using standard methods. The tumors were classified and graded according to the WHO and the Nottingham modification of the Scarff-Bloom-Richardson systems. In the sections stained with routine H&E method, the following determinations were carried out: type of neoplasm (WHO classification), tumor grade including tubule formation, and intensity of division as well as the degree of neoplastic cell differentiation and mitotic index as a mean number of mitotic figures in neoplastic cells counted in 10 fields of vision at a 400× magnification (surface field 0.17 mm2 ).

Paraffin sections on the slides covered with 2% saline solution in acetone at temperature of 42°C were used for immunohistochemical examination.

Routine tests were performed in order to determine immunohistochemical expression of basic profile of diagnostic markers, such as estrogen receptor (ER), progesterone receptor (PR) and HER2. Monoclonal antibodies against receptors for estrogen (Monoclonal Mouse Anti-Human Estrogen Receptor alpha, 1:50 dilution, Clone: 1D5, Code: IR654, DAKO) and progesterone (Monoclonal Mouse Anti-Human Progesteron Receptor, 1:400 dilution, Clone: PR636, Code: IR068, DAKO) were used in order to determine the expression of steroid receptors. Evaluation of the immunohistochemical markers was performed by two pathologists as follows: ER and PR were categorized as negative—(0%), low positive—(1–10%); nuclear staining in >10% of tumor cells was considered positive for ER and PR.

Besides playing a central role in the induction of inflammatory processes, interleukin 1β (IL-1β) was also identified as a factor important for progression of the tumor and stimulation of angiogenesis as well as being responsible for the increase in the invasiveness of cancer lesions. Recently, there has been considerable interest in understanding the non-hypoxic upregulation of the hypoxia-inducible factor HIF-1α by IL-1 in neoplastic cells since aberrant expression of HIF-1α correlates with tumor progression. Naldini et al. [48] studied the effect of IL-1β on cell migration and HIF-1α accumulation in the human invasive breast cancer cell line MDA-MB-231. It was found that hypoxia-independent induction of HIF-1α by IL-1β was associated with an increase in cell migration and a simultaneous increase in the activity of phosphorylated p38 MAPK and CXCR1 expression. Inhibition of HIF-1α by siRNA led to a significant reduction in CXCR1 expression and cell migration, confirming the role of HIF-1α in hypoxia-independent, IL-1β-induced migration of the MDA-MB-231 line cells. The results of the studies present a new role of IL-1 in breast cancer. The therapeutic approach focused on inhibition of IL-1β activity appears to be a new target for the research aimed at the development of novel meth-

The first aim of our study was to evaluate the expression of ER, PR and HER2 in order to extract a group of TNBC and non-TNBC. The second aim of this study was to evaluate the relationship between immunohistochemical expression of novel prognostic marker—HIF-1α—and

Studies were conducted in a group of 162 patients with breast carcinoma with lymph node metastasis (111 triple-negative breast cancer and 51 non-triple-negative breast cancer) in the Department of Pathology, Military Medical Institute in Warsaw. Material for the study came from biopsies, excisional biopsies and modified radical mastectomies. Tumor samples were fixed in 10% buffered formalin phosphate. After 24-h fixation, material was dehydrated using alcohol in gradually increasing concentrations and embedded in paraffin. Paraffin blocks were cut into serial sections 4 μm in thickness. They were then stained using standard methods. The tumors were classified and graded according to the WHO and the Nottingham modification of the Scarff-Bloom-Richardson systems. In the sections stained with routine H&E method, the following determinations were carried out: type of neoplasm (WHO classification), tumor grade including tubule formation, and intensity of division as well as the degree of neoplastic cell differentiation and mitotic index as a mean number of mitotic figures in neoplastic cells

Paraffin sections on the slides covered with 2% saline solution in acetone at temperature of

).

clinicopathological features for patients with triple-negative breast cancer.

counted in 10 fields of vision at a 400× magnification (surface field 0.17 mm2

42°C were used for immunohistochemical examination.

ods to treat invasive breast cancer [48].

**7. Materials and methods**

**6. Aim**

88 Breast Cancer and Surgery

The study was conducted as follows: sections were incubated at 60°C overnight and subsequently dewaxed. The next step involved revealing the epitope by heating the slides in a buffer for 40 min. Subsequently, preparations were left at room temperature for 20 min. Preparations were rinsed in buffer and endogenous peroxidase was blocked by washing in 3% H2 O2 for 10 min. In the next step, preparations were incubated with an appropriate antibody for 30 min. After incubation, preparations were rinsed in buffer for 10 min, and then incubated with the reagent (Visualization Reagent) for 30 min. After incubation with the reagent, preparations were washed in TBS (Tris-Buffered Saline, Code: S1968) with pH 7.6 for 10 min, and then incubated with 3,3′-diaminobenidine (DAB) (Substrate—Chromogen Solution) for 10 min to visualize the color of the reaction. At the end of the procedure, preparations were stained with hematoxylin.

HER2 expression was determined using HerceptTest™ DAKO test (Code: K5204). It enabled detection of HER2 expression using a polyclonal antibody against this protein (Rb A—Hu HER2—Rabbit Anti-Human HER2 Protein). Antigen retrieval for HER2 using HerceptTest was performed by immersing and incubating the slides in 10-mmol/L citrate buffer in a calibrated water bath (95–99°C) for 40 min (±1 min). After decanting the epitope-retrieving solution, sections were rinsed in the wash buffer and later, soaked in the buffer for 5–20 min before staining. The slides were loaded onto the autostainer using the HercepTest program, as described in the manufacturer's insert. In the autostainer, the slides were rinsed, placed in 200 μL of peroxidase-blocking reagent for 5 min, rinsed, placed in 200 μL of primary anti-HER2 protein (or negative control reagent) for 30 min, rinsed twice and immersed in 200 μL of substrate chromogen solution – DAB for 10 min. The slides were counterstained with hematoxylin and finally coverslipped. HER2 results were determined based on the maximum area of staining intensity according to the instruction in the package insert and the ASCO/CAP guidelines as follows: strong, circumferential membranous, staining in >30% of invasive carcinoma cell was scored 3+, moderate, circumferential, membranous staining in ≥10% of invasive tumor cells or 3+ staining in ≤30% of cells was designated as 2+ staining, weak and incomplete membranous staining in invasive tumor cells was scored 1+ and no staining was scored 0. Tumors with 0 and 1+ staining were considered negative.

A total of 162 cases of breast cancer with metastasis to lymph nodes were assessed for expression of HIF-1α (Monoclonal Mouse Anti-Human HIF-1α 1:50 dilution, Clone:28b, Santa Cruz Biotechnology®, Inc.). A visualization system ImmunoCruz™ Mouse ABC Staining System (Santa Cruz Biotechnology®, Inc.) was subsequently applied; tumor-cell immunoreactivity was scored according to both the extent of nuclear staining—relative number of HIF-1α positive cells, and the intensity of the reaction: [−] not detected; [+] <1% positive cells; [+] 1–10% weakly to moderately stained cells; [++] 1–10% intensively stained cells or 10–50% weakly stained cells; [+++] 10–50% positive cells with moderate to marked staining; [++++] >50% positive cells [49]. Positive controls were HIF-1α immunoreactive breast cancer tissues. Negative controls were prepared with omission of primary antibodies.
