*2.1. BMI-1 Expression—Qualitative Assessment*

The qualitative evaluation showed, at a glance, a heterogeneous expression in both groups, without a specific pattern for each group.

A double BMI-1 staining was found: a nuclear and cytoplasmic/membrane immunoexpression in EOC group. Strong expression of epithelial cells was observed in cases

with poor prognosis, such as high-grade serous and endometrioid carcinomas (HGSCs and HGECs), as well in clear cell ovarian carcinomas (COCs). A negative BMI-1 stroma expression in the endometrioid phenotype of EOC group was found, while positive stroma was dominant in the serous phenotype, clear cell and mixed subtypes. Relevant aspects of BMI-1 expression in EOC are presented in Figure 1. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 3 of 15 The qualitative evaluation showed, at a glance, a heterogeneous expression in both

In the NEOC group, the intensity of BMI-1 was predominantly moderate or strong in epithelial (nuclear or cytoplasmic/membrane immunoexpression) and stromal cells. Moderate and strong nuclear expression and weaker cytoplasmic expression was observed in cases with a serous phenotype and a more aggressive course, such as HGSC, while the endometrioid phenotype preserved a strong, diffuse, membrane BMI-1 staining. In undifferentiated carcinomas, BMI-1 expression was heterogeneous, displaying a weak cytoplasmic staining. Differences between BMI-1 expression in variable types of NEOC are illustrated in Figure 2. groups, without a specific pattern for each group. A double BMI-1 staining was found: a nuclear and cytoplasmic/membrane immunoexpression in EOC group. Strong expression of epithelial cells was observed in cases with poor prognosis, such as high-grade serous and endometrioid carcinomas (HGSCs and HGECs), as well in clear cell ovarian carcinomas (COCs). A negative BMI-1 stroma expression in the endometrioid phenotype of EOC group was found, while positive stroma was dominant in the serous phenotype, clear cell and mixed subtypes. Relevant aspects of BMI-1 expression in EOC are presented in Figure 1.

We also noted the lack of BMI-1 expression in normal ovary or ovarian surface, and its positivity in the normal tubal surface epithelium. In the NEOC group, the intensity of BMI-1 was predominantly moderate or strong in epithelial (nuclear or cytoplasmic/membrane immunoexpression) and stromal cells.

#### *2.2. BMI Expression—Semi-Quantitative Assessment* Moderate and strong nuclear expression and weaker cytoplasmic expression was

In the whole group of study, without division into EOC and NEOC categories, the BMI-1 semi-quantitative assessment showed the following: a high expression in 31 cases (65.96%) and a low expression in 16 cases (34.04%), in tumor cells, along with immunopositivity in 34 cases (72.34%), and immunonegativity in 13 cases (27.65%) in tumor stroma. The statistical analysis revealed significant correlations between BMI-1 expression in epithelial tumor cells (low/high) versus tumor stroma (negative/positive) (*p* = 0.01). observed in cases with a serous phenotype and a more aggressive course, such as HGSC, while the endometrioid phenotype preserved a strong, diffuse, membrane BMI-1 staining. In undifferentiated carcinomas, BMI-1 expression was heterogeneous, displaying a weak cytoplasmic staining. Differences between BMI-1 expression in variable types of NEOC are illustrated in Figure 2. We also noted the lack of BMI-1 expression in normal ovary or ovarian surface, and its positivity in the normal tubal surface epithelium.

(**c**) (**d**)

**Figure 1.** *Cont.*

**Figure 1.** (**a**–**f**) Histologic features and BMI-1 expression in EOC group in different ovarian tumor subtypes: (**a**,**b**) HGSC: (**a**) papillary growth, enlarged and irregular nuclei, prominent nucleoli, high cellular size and shape (hematoxylin and eosin–H&E, magnification 10×), (**b**) strong BMI-1 nuclear staining in epithelial tumor cells of HGSC (magnification 10×); (**c**,**d**) HGEC: (**c**) crowded back-to-back glands, lined by atypical columnar epithelium, and smooth luminal borders (H&E, magnification 10×), (**d**) weak BMI-1 cytoplasmic staining in epithelial tumor cells of HGEC (magnification 10×); (**e**,**f**) COC: (**e**) papillary and tubulocystic pattern, combined with clear and eosinophilic cells and stromal hyalinization (H&E, magnification 10×), (**f**) strong BMI-1 cytoplasmic staining of tumor cells and stroma in COC (magnification 10×). (**e**) (**f**) **Figure 1.** (**a**–**f**) Histologic features and BMI-1 expression in EOC group in different ovarian tumor subtypes: (**a**,**b**) HGSC: (**a**) papillary growth, enlarged and irregular nuclei, prominent nucleoli, high cellular size and shape (hematoxylin and eosin–H&E, magnification 10×), (**b**) strong BMI-1 nuclear staining in epithelial tumor cells of HGSC (magnification 10×); (**c**,**d**) HGEC: (**c**) crowded back-to-back glands, lined by atypical columnar epithelium, and smooth luminal borders (H&E, magnification 10×), (**d**) weak BMI-1 cytoplasmic staining in epithelial tumor cells of HGEC (magnification 10×); (**e**,**f**) COC: (**e**) papillary and tubulocystic pattern, combined with clear and eosinophilic cells and stromal hyalinization (H&E, magnification 10×), (**f**) strong BMI-1 cytoplasmic staining of tumor cells and stroma in COC (magnification 10×).

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**Figure 2.** (**a**–**h**) Histologic features and BMI-1 expression in NEOC group in different ovarian tumor subtypes: (**a**,**b**) MOC: (**a**) atypical mucin-producing tumor cells with an infiltrative pattern of invasion (H&E, magnification 10×), (**b**) negative BMI-1 staining in tumor stroma of MOC (magnification 10x); (**c**,**d**) HGSC: (**c**) variation in cellular size and shape, marked nuclear atypia, dense fibrous stroma, and inflammation around the tumor nests (H&E, magnification 10×), (**d**) strong BMI-1 cytoplasmic staining of tumor stroma in HGSC (magnification 10×); (**e**,**f**) LGSC: (**e**) micropapillary growth with minimal nuclear atypia in LGSC (H&E, magnification 10×), (**f**) moderate BMI-1 cytoplasmic staining of tumor cells and stroma in LGSC (magnification 10×); (**g**,**h**) LGEC: (**g**) papillary and glandular differentiation in LGEC (H&E, magnification 10×), (**h**) strong BMI-1 cytoplasmic staining of tumor cells and stroma in LGEC (magnification 10×). **Figure 2.** (**a**–**h**) Histologic features and BMI-1 expression in NEOC group in different ovarian tumor subtypes: (**a**,**b**) MOC: (**a**) atypical mucin-producing tumor cells with an infiltrative pattern of invasion (H&E, magnification 10×), (**b**) negative BMI-1 staining in tumor stroma of MOC (magnification 10x); (**c**,**d**) HGSC: (**c**) variation in cellular size and shape, marked nuclear atypia, dense fibrous stroma, and inflammation around the tumor nests (H&E, magnification 10×), (**d**) strong BMI-1 cytoplasmic staining of tumor stroma in HGSC (magnification 10×); (**e**,**f**) LGSC: (**e**) micropapillary growth with minimal nuclear atypia in LGSC (H&E, magnification 10×), (**f**) moderate BMI-1 cytoplasmic staining of tumor cells and stroma in LGSC (magnification 10×); (**g**,**h**) LGEC: (**g**) papillary and glandular differentiation in LGEC (H&E, magnification 10×), (**h**) strong BMI-1 cytoplasmic staining of tumor cells and stroma in LGEC (magnification 10×).

> *2.2. BMI Expression—Semi-Quantitative Assessment*  In the whole group of study, without division into EOC and NEOC categories, the The semi-quantitative expression of BMI-1 showed a different profile in the two analyzed groups.

> BMI-1 semi-quantitative assessment showed the following: a high expression in 31 cases (65.96%) and a low expression in 16 cases (34.04%), in tumor cells, along with immunopositivity in 34 cases (72.34%), and immunonegativity in 13 cases (27.65%) in tumor stroma. The statistical analysis revealed significant correlations between BMI-1 expression in epithelial tumor cells (low/high) versus tumor stroma (negative/positive) (*p* = 0.01). BMI-1 expression in epithelial tumor cells was mostly low or negative in the EOC group and predominantly positive in NEOC group. On the other hand, the cases of the EOC group expressed positive and negative stromal BMI-1 immunoreactions approximately equally, whereas the stromal BMI-1 expression was mainly strong in the NEOC group (Table 1). We noted statistically significant differences between the BMI-1 epithelial and stromal profiles in each group (Table 1).

> The semi-quantitative expression of BMI-1 showed a different profile in the two analyzed groups. BMI-1 expression in epithelial tumor cells was mostly low or negative in the EOC group and predominantly positive in NEOC group. On the other hand, the cases of the Comparing the epithelial and stromal BMI-1 expressions between the EOC and NEOC groups, we obtained statistically significant differences only for the epithelial component (*p* = 0.0002), not for the stromal one (*p* = 0.06).

#### EOC group expressed positive and negative stromal BMI-1 immunoreactions approximately equally, whereas the stromal BMI-1 expression was mainly strong in the *2.3. Relationship between BMI-1 Epithelial and Stromal Expression, and Clinicopathological Parameters in EOC*

NEOC group (Table 1). We noted statistically significant differences between the BMI-1 epithelial and stromal profiles in each group (Table 1) Comparing the epithelial and stromal BMI-1 expressions between the EOC and NEOC groups, we obtained statistically significant differences only for the epithelial component (*p* = 0.0002), not for the stromal one (*p* = 0.06). The results of the statistical analysis revealed a significant relationship between BMI-1 expression in tumor cells (low/high) and tumor grade (well and moderately differentiated versus poorly differentiated) (*p* = 0.04). On the other hand, stromal BMI-1 expression was significantly correlated with the median value of cancer antigen 125 (CA 125) (*<sup>p</sup>* = 0.03).No other significant differences were registered (Table 2).


**Table 1.** Correlations between the epithelial and stromal BMI-1 expression in the EOC and the NEOC groups.

**Table 2.** Correlations between BMI-1 expression in tumoral cells and clinicopathological parameters—EOC group.


LGSC (low-grade serous carcinoma); LGEC (low-grade endometrioid carcinoma); COC (clear cell ovarian carcinoma); MOC (mucinous ovarian carcinoma); HGSC (high-grade serous carcinoma); HGEC (high-grade endometrioid carcinoma); NED (no evident data about residual tumor).
