**9. Results**

Histopathological examination was performed in tumors obtained from 162 patients suffering from breast cancer. Among 162 breast cancer patients we identified 111 (68.5%) subjects with triple-negative breast cancer (TNBC was identified as ER-negative, PR-negative, and HER2-negative) and 51 (31.5%) subjects with non-triple-negative breast cancer. Mean age of patients with TNBC was 47.8 and of patients with non-TNBC 60.4 years.

cases—grade 3 (G3). Given the histological grade of malignancy, G2 and G3 tumors com-

71 (64.0)

41 (80.4)

**Table 1.** Relationship between immunohistochemical profile (TNBC/non-TNBC) and prognostic parameters invasive

**Prognostic parameters**

Tumor necrosis Histological type of invasive

0.036\* 99

positive negative p-value IDC-NST other

Triple-Negative Breast Cancer: Expression of Hypoxia-Inducible Factor 1α in Triple-Negative…

breast cancer

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(89.1)

45 (88.2) types

6 (11.8)

12 (10.9) p-value

91

0.858

In our study TNBC were most commonly assessed as G2 and G3 (52.2%; 45.1%), pT1 and pT2 (34.2%; 62.1%), and pN1, pN2 (45%; 41.4%). Respectively non-TNBC were most commonly assessed as G2 and G3 (47%; 47%), pT1 and pT2 (39.2%; 47%) and pN1 (52.9%). In our study a statistically significant association was found only between TNBC and non-TNBC tumor size (pT) (*p* = 0.0011). Furthermore in TNBC more commonly than in non-TNBC the presence of necrosis in the tumor mass was observed (36%; 19.6%) and statistically significant correlation between

TNBC and non-TNBC in the presence of necrosis was demonstrated (*p* = 0.036) (**Table 1**).

In all examined breast cancers we also assessed the expression of HIF-1α but not statistically significant relationship between TNBC and non-TNBC was revealed. TNBC more often presented HIF-1α expression (43.2%) than non-TNBC (35.2%). In both groups we investigated correlation between the HIF-1α expression and features such as: tumor size (pT), histological grade (G1– G3) and the presence of lymph node metastasis (pN1–pN3). While TNBC subgroup demonstrated significant correlation between HIF-1α expression and tumor size (pT1–pT4) (*p* = 0.021). Detailed data and relationships between different parameters are presented in **Tables 2** and **3**.

> **Frequency n = 162**

(100%)

(100%)

**Table 2.** Relationship between basic immunohistochemical profile (ER, PR, HER2) and expression HIF-1α in invasive

**HIF-1α expression**

Positive (>10%)

48 (43.2)

18 (35.2) p-value

0.339

Negative (<10%)

63 (56.8)

33 (64.8)

prised the largest group of triple-negative breast cancers.

**Immunohistochemistry – basal panel for diagnosis of breast** 

TNBC (ER-/PR-/HER2-) 111

non-TNBC (ER+/PR+/HER2+) 51

breast cancer with metastasis to lymph nodes (\*statistically significant result *p* < 0.05).

**cancer**

**n = 162**

(100%)

(100%)

40 (36.0)

10 (19.6)

breast cancer with metastasis to lymph bodes (\*statistically significant results *p* < 0.05).

**Immunohistochemistry Frequency**

TNBC 111

non-TNBC 51

Histopathological subtyping of the 111 triple-negative breast cancers identified 89.1% invasive ductal carcinomas of no special type (IDC-NST) (**Figure 1**) and 10.9% other special types of cancers: invasive lobular carcinomas, mixed ductal and lobular types, metaplastic carcinomas (**Table 1**).

All cases of triple-negative breast cancer were grouped according to histological grading: 3 (2.7%) cases were grade 1 (G1), 58 (52.2%) cases were identified as grade 2 (G2) and 50 (45.1%)

**Figure 1.** Triple negative breast cancer (TNBC) H&E.


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.

patients with TNBC was 47.8 and of patients with non-TNBC 60.4 years.

All statistical analyses were performed with SPSS software v12.0. The Chi-square (χ2

to assess the relationship between HIF-1α and degree of histological malignancy and clinical staging. The Fisher exact test was used when the expected cell counts were less than 5. The results were considered as statistically significant if the *p* value was less than 0.05 (*p* < 0.05).

Histopathological examination was performed in tumors obtained from 162 patients suffering from breast cancer. Among 162 breast cancer patients we identified 111 (68.5%) subjects with triple-negative breast cancer (TNBC was identified as ER-negative, PR-negative, and HER2-negative) and 51 (31.5%) subjects with non-triple-negative breast cancer. Mean age of

Histopathological subtyping of the 111 triple-negative breast cancers identified 89.1% invasive ductal carcinomas of no special type (IDC-NST) (**Figure 1**) and 10.9% other special types of cancers: invasive lobular carcinomas, mixed ductal and lobular types, metaplastic carcino-

All cases of triple-negative breast cancer were grouped according to histological grading: 3 (2.7%) cases were grade 1 (G1), 58 (52.2%) cases were identified as grade 2 (G2) and 50 (45.1%)

) was used

**8. Statistical analysis**

90 Breast Cancer and Surgery

**9. Results**

mas (**Table 1**).

**Figure 1.** Triple negative breast cancer (TNBC) H&E.

**Table 1.** Relationship between immunohistochemical profile (TNBC/non-TNBC) and prognostic parameters invasive breast cancer with metastasis to lymph bodes (\*statistically significant results *p* < 0.05).

cases—grade 3 (G3). Given the histological grade of malignancy, G2 and G3 tumors comprised the largest group of triple-negative breast cancers.

In our study TNBC were most commonly assessed as G2 and G3 (52.2%; 45.1%), pT1 and pT2 (34.2%; 62.1%), and pN1, pN2 (45%; 41.4%). Respectively non-TNBC were most commonly assessed as G2 and G3 (47%; 47%), pT1 and pT2 (39.2%; 47%) and pN1 (52.9%). In our study a statistically significant association was found only between TNBC and non-TNBC tumor size (pT) (*p* = 0.0011). Furthermore in TNBC more commonly than in non-TNBC the presence of necrosis in the tumor mass was observed (36%; 19.6%) and statistically significant correlation between TNBC and non-TNBC in the presence of necrosis was demonstrated (*p* = 0.036) (**Table 1**).

In all examined breast cancers we also assessed the expression of HIF-1α but not statistically significant relationship between TNBC and non-TNBC was revealed. TNBC more often presented HIF-1α expression (43.2%) than non-TNBC (35.2%). In both groups we investigated correlation between the HIF-1α expression and features such as: tumor size (pT), histological grade (G1– G3) and the presence of lymph node metastasis (pN1–pN3). While TNBC subgroup demonstrated significant correlation between HIF-1α expression and tumor size (pT1–pT4) (*p* = 0.021). Detailed data and relationships between different parameters are presented in **Tables 2** and **3**.


**Table 2.** Relationship between basic immunohistochemical profile (ER, PR, HER2) and expression HIF-1α in invasive breast cancer with metastasis to lymph nodes (\*statistically significant result *p* < 0.05).


of TNBC, Dabbs et al. [59] found that all tested tumors showed high degree of histological malignancy. Choi et al. [60] obtained similar results, stating that in a group of triple-negative cancers 63.1% were G3 tumors. Research by Zhou et al. [61] also showed that triple-negative G2 (51.6%) and G3 (45.2%) cancers were most numerous. Osman et al. [55] confirmed in their study that G3 carcinomas (61.9%) comprised the largest group of triple-negative tumors, while

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93

There are conflicting reports on the prevalence of lymph node metastases at the time of diagnosis among patients with TNBC. In our study we found that women without metastases to regional lymph nodes (pN0) comprised the largest group of all investigated patients with invasive triple-negative breast cancer (56.7%); no statistically significant relationship between lymph node status and histological type of TNBC-IC (*p* > 0.05) was noted. Lymph node status among patients with TNBC was reported as follows: 19.81%—N1, 19.81%— N2, 3.6%—N3. The study also showed no association between tumor size and presence of lymph node metastasis in patients with TNBC, which stood in contradiction to the findings of Thike et al. [62] who had demonstrated a relationship between tumor size and presence of nodal metastases. In studies by Rao et al. [54] lymph node metastases were found in 37 of 50 patients with TNBC (74% of cases), and TNBC was associated with higher rates of node-positive cases, which was in agreement with the findings of Carey Rakha et al. [58]

In our study 30.9% of all tumors showed central necrosis. In TNBC more commonly than in non-TNBC the presence of necrosis was observed (36%; 19.6%). Yehia et al. [64] in their study divided breast cancers into three subgroups (TNBC, HER2+ and ER+/PR+). 15.3% of all tumors showed central fibrosis and tumor necrosis, which differed significantly among the three groups (*p* = 0.019). TNBC had the highest values among all groups even after adjusting the results for age. Respectively necrosis was observed in 25.8% TNBC, 9.4% HER2+ and 10.9% ER+/PR+ of cancers [64]. 62 TNBC, 64 HER2+, and 64 hormone-receptors positive breast cancers were evaluated also for HIF-1α expression. HIF-1α was expressed in 35.5% TNBC, 45.3% HER2+ and 25.0% ER+/PR+ (*p* = 0.055). In our study HIF-1α expression was observed in

Due to the fact that TNBC subtype frequently show morphologic evidence of hypoxia (central fibrosis and necrosis) [40, 41] an augmented expression of HIF-1α in tumors with a triplenegative phenotype was anticipated. In fact, this had been elegantly demonstrated through the preferential expression of HIF-1α in peri-necrotic tumor cells in TNBC and BRCA1

HIF-1α overexpression is an indicator of poor prognosis and significant survival time reduction in patients suffering from breast cancer [45]. HIF-1 upregulates transcription of angiogenic genes like EPO and vascular endothelial growth factor (VEGF), which induce sprouting of new vessels and in result they increase the risk of metastasis because they boost surface of contact between tumor cells and vasculature. HIF-1 induces transcription of cytoprotective proteins in malignant cells in hypoxic conditions. HIF-1α predicts poor prognosis breast

Sood et al. [56] pointed to G2 (47.22%) and G3 (38.89%) as most common tumors.

and Rakha et al. [63].

43.2% TNBC and 35.3% non-TNBC.

mutated breast cancers [42].

cancer [46, 47].

**Table 3.** Clinicopathological features of TNBC and their relationship to expression of novel breast cancer marker - HIF-1α (\*statistically significant result *p* < 0.05).
