Annals of Diagnostic Pathology 18 (2014) 58–62
Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Immunohistochemical expression and prognostic relevance of Bmi-1, a stem cell factor, in epithelial ovarian cancer☆ Amal Abd El hafez, PhD a,⁎, Hend Ahmed EL-Hadaad, MD b a b
Pathology Department, Faculty of Medicine, Mansoura University, Egypt Clinical Oncology and Nuclear Medicine Department, Faculty of Medicine, Mansoura University, Egypt
a r t i c l e
i n f o
Keywords: Bmi-1 Epithelial ovarian cancer Immunohistochemistry Prognosis Survival
a b s t r a c t Ovarian cancer is the fourth most common cause of cancer-related death in women. Bmi-1 is a stem cell factor implicated in many human malignancies with poor outcome. Few published reports on the expression of Bmi1 in epithelial ovarian cancer were either experimental or performed on cell lines. This study evaluates the immunohistochemical expression of Bmi-1 protein in epithelial ovarian cancer tissue specimens and its relevance to the clinicopathologic prognostic variables and patient survival. Forty cases of epithelial ovarian cancer were selected according to the availability of paraffin-embedded tissue and the clinicopathologic and survival data. Immunohistochemistry was performed for anti–Bmi-1 antibody. Low and high Bmi-1 expression groups were compared with age, tumor stage, laterality, grade, histology, and patient survival. Bmi-1 expression was detected in 72.5% of cases, of which 42.5% had high expression. High Bmi-1 expression strongly associated with advanced International Federation of Gynecology and Obstetrics stages (P = .007), bilaterality (P = .01), and higher Gynecologic Oncology Group grades (P = .031) and carcinomas of serous histology (P = .027). It had no association with patient age. Bmi-1 expression displayed a significant inverse association with patient overall and mean survival (P = .006, P b .001). These observations suggested correlation between increased Bmi-1 expression and clinical progression in ovarian epithelial cancer. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Ovarian cancer is the fourth most common cause of cancer-related death in women and is the most lethal gynecological malignancy with 30% to 40% overall survival (OS) at 5 years [1]. Epithelial ovarian cancer comprises most malignant ovarian neoplasms in adult women, and different molecular genetic pathways participate in its development [2]. Because of its insidious onset, most patients are diagnosed with advanced-stage disease [3]. Clinically, ovarian cancer is characterized by an initial response to cytotoxic chemotherapy, followed frequently by recurrence and disease progression, which represents a major scientific and clinical barrier to the control of cancer. Thus, the development of new therapeutic strategies to combat ovarian cancer is needed [4]. Traditional cancer therapies typically target the rapidly dividing tumor cells; however, some cells of the tumor are spared. These spared cells, which are reported to be present within many tumor types, exhibit the potential to regenerate and are called cancer stem cells (CSCs) [5-9].
☆ Disclosure: No relevant financial affiliations or conflicts of interest to disclose. ⁎ Corresponding author: Pathology Department, Faculty of Medicine, Mansoura University, El-Gomhouria St, Mansoura-Dakahlia, Egypt. E-mail addresses: [email protected], [email protected] (A. Abd El hafez), [email protected] (A. Abd El hafez). 1092-9134/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anndiagpath.2013.11.004
There is increasing evidence that polycomb group (PcG) proteins (discovered in Drosophila as epigenetic gene silencers) play a crucial role in cancer development and recurrence [10]. Human B-cell specific moloney leukemia virus insertion-site 1 (Bmi-1), a member of the PcG family of transcription repressors, has emerged as a Myc-cooperating oncogene in murine lymphomas. In human, Bmi-1 gene localizes on short arm of chromosome 10 (10p11.23) and encodes a protein of 36.8 kDa [7,9]. Normally, Bmi-1 has an ubiquitous pattern of expression in almost all tissues with high expression levels in the brain, esophagus, salivary gland, thymus, kidney, lungs, gonads, placenta, blood, and bone marrow [9]. Bmi-1 controls the cell cycle by regulating the tumor suppressor proteins p16INK4a and p14ARF [11,12]. The p16INK4a protein inhibits binding of cyclin D to CDK4/6, resulting in the suppression of retinoblastoma activity and induction of cell cycle arrest [12,13], whereas p14ARF induces p53 and causes cell cycle arrest [12-14]. Thus, Bmi-1 promotes cell proliferation by suppressing p16INK4a/retinoblastoma and/or p14ARF/MDM2/p53 tumor suppressor pathways [13]. In addition, Bmi-1 is reported to play a critical role in tissue homeostasis by maintaining self-renewal and differentiation capacities and prevention of senescence through the activation of telomerase in adult stem cells [11,12,15]. The ability of CSCs to induce cancer recurrence after therapy has been attributed to the activation of different molecules including Bmi1 [16,17]. In addition, recent studies suggest that Bmi-1 acts as a stem cell factor that is involved in cancer initiation, progression, and
A. Abd El hafez, H.A. EL-Hadaad / Annals of Diagnostic Pathology 18 (2014) 58–62
chemoresistance. Therefore, Bmi-1 can be developed as a target for therapeutic agents that efficiently abolish chemoresistance in tumor cells [7,8,18]. Bmi-1 has been associated with a number of human malignancies including B-cell non-Hodgkin lymphoma; myelodysplastic syndrome; breast, gastric, nasopharyngeal, esophageal, colorectal, bladder, prostatic, and pancreatic carcinomas; non–small cell lung cancer; and neuroblastoma and has been shown to be a useful prognostic marker in many of these cancers [8,9,11,16,19-33]. Nonetheless, there are few published reports on the expression of Bmi-1 in ovarian cancer, and most of these studies were either experimental or performed on ovarian cancer cell lines [4,34,35]. Therefore, this study aims to assess the immunohistochemical expression of Bmi-1 protein in human epithelial ovarian cancer tissue specimens and to explore its relevance to the established clinicopathologic and prognostic variables and the patient survival. 2. Materials and methods 2.1. Patients and clinicopathologic evaluation Forty female patients with histologically confirmed primary ovarian epithelial cancers encompassing 5 serous borderline tumors, 21 serous carcinomas, 3 mucinous borderline tumors, 6 mucinous carcinomas, and 5 endometrioid carcinomas were investigated for Bmi-1 immunohistochemical expression. Cases were obtained from the archives of the Pathology Department, Faculty of Medicine, Mansoura University, Egypt, from January 2006 to December 2007. Inclusion criteria were the availability of tumor tissue paraffin blocks and the clinicopathologic data obtained from the medical records. Follow-up and OS data starting from the time primary surgery until the patients died or were lost to follow-up were obtained from Clinical Oncology and Nuclear Medicine Department in the same university. None of the patients had received preoperative radiation or chemotherapy. Routinely processed hematoxylin and eosin slides were reviewed to revaluate histopathologic type of ovarian cancer according to the World Health Organization classification, and tumors were graded according to Gynecologic Oncology Group (GOG) system. Combination of clinical and histopathologic information was adopted for staging according to International Federation of Gynecology and Obstetrics (FIGO) staging criteria. 2.2. Immunohistochemistry Immunohistochemical (IHC) staining was performed on formalinfixed, paraffin-embedded tissues. After deparaffinization and rehydration, 4- to 5-μm-thick sections on coated slides were heat pretreated in citrate buffer (pH 6 at 92°C) and immunostained using specific antibody against Bmi-1 gene protein (Anti-Bmi-1; ab97729; rabbit polyclonal antibody; Abcam Corporation (Cambridge, UK) product at dilution 1/50). Standard avidin-biotin-peroxidase technique was applied using diaminobenzidin for visualization and hematoxylin for counterstaining. Appropriate negative controls, consisting of histologic sections of each case processed without the addition of primary antibody, were prepared, along with a positive control sections prepared from human neuroblastoma tissue with confirmed Bmi-1 immunopositivity. 2.3. Evaluation of Bmi-1 staining Expression of Bmi-1 protein was visualized by observing the stained tissues under a light microscope. Immunostaining for Bmi-1 was defined as the presence of brown or yellowish brown granules in the nuclei, although occasionally, yellowish brown granules could also be seen in the cytoplasm. For evaluation of the Bmi-1 IHC staining in
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ovarian cancer tissues, a previously described [17,36], semiquantitative scoring was used, in which both staining intensity and positive areas were recorded. The intensity of Bmi-1 staining (negative, 1; weak, 2; moderate, 3; or strong, 4 scores) and the proportion of immunopositive cells (≤10%, 1; N10% to ≤ 50%, 2; N50% to ≤75%, 3; N75%, 4) were calculated. The 2 different scores of corresponding sample were then multiplied, so a staining index ranging from 1 to 16 points was obtained. Points equal or less than 4 was marked as (−); 5 to 8 points, as (+); 9 to 12 points, as (++); and 13 to 16 points, as (+++). For statistical analysis, (−) and (+) were counted as low expression of Bmi-1, whereas (++) and (+++) were counted as high (intensive) expression of Bmi-1 (Table 1). 2.4. Statistical analysis Statistical analysis was carried out with the SPSS version 16.0 (Chicago, USA). The association of Bmi-1 protein expression with ovarian carcinoma patients' clinicopathologic variables was assessed by the Pearson χ 2 test. The Student t test was used to compare mean OS time in relation to Bmi-1 expression. Survival curve was plotted by Kaplan-Meier method and compared by the log-rank test. P ≤ .05 was considered as statistically significant. 3. Results 3.1. Clinicopathologic characteristics As seen in Table 2, the study included 24 female patients 50 years or younger and 16 patients older than 50 years with mean and median ages of 46.5 and 48 years, respectively. Most patients (45%) were presented at FIGO stage I, and high percentage of carcinomas were at GOG grade 2 (30%) or 3 (27.5%). Approximately 42% of tumors were bilateral. Carcinomas with serous histology were the most frequent (52.5%), whereas borderline mucinous tumors were the least common (7.5%). Patients' survival ranged from 4 to 63 months with a mean survival of 33.3 months. 3.2. Bmi-1 expression in epithelial ovarian cancer Bmi-1 protein expression was detected in 29 (72.5%) of 40 of ovarian epithelial cancers (Fig. 1). In this study, we defined that (−) and (+) were considered as low expression of Bmi-1, whereas (++) and (+++) were considered as high expression of Bmi-1. According to this definition, the high Bmi-1 expression was detected in 17 (42.5%) of 40 of ovarian cancers (Table 1). 3.3. Association of Bmi-1 expression with the clinicopathologic variables Associations between Bmi-1 expression profile and the patients' clinicopathologic variables are presented in Table 2. High Bmi-1 expression was strongly associated with advanced FIGO cancer stages (P = .007), bilaterality (P = .01), higher GOG grades (P = .031), and carcinomas of serous histology (P = .027). However, no significant association was observed between Bmi-1 expression profile and patients' age (P = .433). 3.4. Association between Bmi-1 expression and patients' survival Bmi-1 expression displayed a significant inverse association with patient OS (P = .006). The mean and median survival times for patients with tumors having high expression of Bmi-1 were shorter than patients with tumors having low expression of Bmi-1 (21.8 and 21 months compared with 41.8 and 41 months, respectively) with a statistically significant difference between both groups (P b .001) (Table 2). The survival rate, assessed by the Kaplan-Meier method,
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Fig. 1. High Bmi-1 nuclear expression in borderline papillary serous tumor (A) and grade 1 papillary serous carcinoma (B). (C) High Bmi-1 nuclear and cytoplasmic expression in grade 3 papillary serous carcinoma. (D) Low Bmi-1 nuclear expression in endometrioid carcinoma (immunohistochemistry, ×200).
was 60.9% in the low expression group, whereas it was only 17.6% in the high expression group (Fig. 2).
Recently published data testify that Bmi-1 knockdown resulted in apoptosis in ovarian cancer cells but not in normal cells [27,34]. Moreover, Bmi-1 plays an important role in sensitization of
4. Discussion The development and progression of ovarian cancer are presumed to be a multistep process involving multiple genetic changes of p53, BRCA1, BRCA2, K-RAS, β-catenin, and PTEN [2]. However, it is important to identify a biological genetic molecular marker that is associated with pathophysiologic processes of human ovarian cancer to explain the high rates of recurrence and the mechanisms of drug resistance and improve the survival of patients with this often lethal disease [1,37]. Bmi-1 has been recently implicated in several aspects of cancer biology. In addition to its significant role in chemoresistance and tumor recurrence [38], Glinsky et al [39] demonstrated that both epithelial and nonepithelial malignancies displaying Bmi-1 gene signature have a probability to develop distant metastasis. Overexpression of Bmi-1 immortalizes human mammary epithelial cells and increases its motility and invasive properties [11,30]. In ovarian cancer, it is speculated that Bmi-1 as an oncoprotein participates in cellular multiplicative division and causes the down-regulation of p16INK4a, which prolongs the life period, promotes the proliferation, and decreases apoptosis rate of ovarian epithelial cancer cells [17,40].
Table 1 Expression of Bmi-1 in epithelial ovarian cancer Negative Bmi-1 staining index
Comparison groups
− (0-4)
Positive + (5-8) 12 (30%)
11 (27.5%)
++ (9-12) 5 (12.5%) 29 (72.5%)
+++ (13-16) 12 (30%)
Low Bmi-1 expression 23 (57.5%)
High Bmi-1 expression 17 (42.5%)
Table 2 Association of Bmi-1 expression with the clinicopathologic variables and survival of ovarian cancer patients Variable
Age b50 y ≥50 y Age range Mean (median) age Laterality Unilateral Bilateral Stage I II III IV Grade Borderline 1 2 3 Histology Borderline serous tumors Borderline mucinous tumors Serous carcinomas Mucinous carcinomas Endometrioid carcinomas OS OS range Mean (median) survival Total a
Bmi-1 expression
P
Low
High
24 (60%) 16 (40%) 21-66 y 46.5 (48) y
17 (70.8%) 6 (37.5%) 24-64 y 45.9 (45) y
7 (29.2%) 10 (62.5%) 21-66 y 47.3 (50) y
.433
23 (57.5%) 17 (42.5%)
17 (73.9%) 6 (35.3%)
6 (26.1%) 11 (64.7%)
.01a
18 (45%) 6 (15%) 10 (25%) 6 (15%)
14 (77.8%) 4 (66.7%) 5 (50%) 2 (33.3%)
4 (22.2%) 2 (33.3%) 5 (50%) 4 (66.7%)
.007a
8 (20%) 9 (22.5%) 12 (30%) 11 (27.5%)
7 (87.5%) 7 (77.8%) 6 (50%) 3 (27.3%)
1 (12.5%) 2 (22.2%) 6 (50%) 8 (72.2%)
.031a
5 (12.5%) 3 (7.5%)
4 (80%) 3 (100%)
1 (20%) 0 (0%)
.027a
21 (52.5%) 6 (15%) 5 (12.5%)
8 (38.1%) 4 (66.7%) 4 (80%)
13 (61.9%) 2 (33.3%) 1 (20%)
4-63 mo 33.3 (33) mo 40
5-63 mo 41.8 (41) mo 23 (57.5%)
4-55 mo 21.8 (21) mo 17 (42.5%)
P value is significant if ≤.05.
.006a b.001a
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Fig. 2. Kaplan-Meier curves with univariate analyses (log-rank P b .001) of patients with epithelial ovarian cancer, subdivided according to Bmi-1 protein expression. The cumulative 3-year survival rate was 60.9% in the low Bmi-1 protein expression group (n = 23), but it was only 17.6% in the high expression group (n =17).
chemoresistant ovarian cancer cells to cisplatin by regulating reactive oxygen species and glutathione levels and, thus, protects the ovarian cancer cells from chemotherapeutic insults [34]. Overexpression of polycomb protein Bmi-1 has been linked to an increasing number of cancer types, and the specificity of immunostaining of the anti–Bmi-1 antibody was confirmed [27,41]. In line with these findings, the current study showed that Bmi-1 is expressed in a high percentage (72.5%) of epithelial ovarian cancers. It is noteworthy that Bmi-1 is highly enriched in CSCs; however, not all Bmi-1expressing cells are CSCs [18]. In 2 prior studies [17,36], immunohistochemical examination of ovarian epithelial cancer specimens revealed positive expression of Bmi-1 in most of cases with high expression in 37% to 46.81% of ovarian carcinomas. In addition, Western blot studies showed high expression levels in approximately 74% of the ovarian cancer patient samples, thus suggesting an important role for Bmi-1 in ovarian cancer [4]. In accordance with these data, high Bmi-1 expression was evident in 42.5% of the present cohort. It is possible that there may be “threshold” of Bmi-1 protein expression, and when this “threshold” is overridden, this protein may start to function as an oncogene [29]. The cellular localization of Bmi-1 protein is quite mysterious. Bmi-1 protein seems to be localized in the nucleus of most breast cancer cells and in the cytoplasm of most noncancer cells [30]; however, Bmi-1 was observed in the cytoplasm of gastric lesions and oropharyngeal squamous cell carcinomas [9,28], opposite to its nuclear expression pattern in endometrial, cervical [41], esophageal squamous [27], and bladder [26] cancers. Supported with previous reports [17,36,41], this study revealed that Bmi-1 protein was mainly confined to the nucleus of the ovarian cancer cells, although occasionally positive staining was seen in the cytoplasm. It has been postulated that phosphorylation can explain differential subcellular localization of some of the polycomb family genes, as there is a rich proline/serine region at the carboxyl terminus of the Bmi-1 protein, where phosphorylation often occurs resulting in the nuclear-cytoplasm shuttling [30]. Another explanation is tissue specificity of Bmi-1 expression because Bmi-1 is a transcriptional factor and can translocate from nucleus to cytosol under some physiologic or pathologic conditions [28]. Data from several clinical studies show that abnormal expression of Bmi-1, in protein level as well as in gene level, is associated with
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poor prognostic markers and worse clinical outcome in diverse human cancers [16,22,26-28,30]. However, there is little information about the prognostic status and clinical outcome of Bmi-1 expression in ovarian cancer. In this study, high Bmi-1 protein expression in human epithelial ovarian cancer was significantly associated with advanced FIGO stages, tumor bilaterality, high tumor grades, and carcinomas of serous histology suggesting a correlation between increased Bmi-1 expression and clinical progression in ovarian epithelial cancer. Nonetheless, no significant difference in Bmi-1 expression was observed in relation to patient age. Among our cohort, Bmi-1 expression displayed a significant inverse association with patient survival; thus, patients with higher Bmi-1 expression had shorter survival time, and patients with lower Bmi-1 expression had a longer survival time; thus, high expression of Bmi-1 protein indicated poor prognosis for patients with ovarian cancer. These results were similar to those of previous studies of other human cancers [19-22,26] as well as with the few studies [17,36] that evaluated the expression of Bmi-1 by IHC in association with clinicopathologic and prognostic variables of ovarian cancer. In accordance with published literatures [26,27,35,36,41], our findings demonstrated that Bmi-1 can be used as a marker to identify subsets of ovarian cancer patients with more aggressive features and poor outcome and that higher Bmi-1 expression might contribute to the carcinogenesis of ovarian cancer. Therefore, further investigations are needed to confirm the prognostic relevance of Bmi-1 in ovarian epithelial cancer and the applicability of therapeutic targeting of Bmi-1 for attacking the “more aggressive” cancer cells causing recurrence and treatment resistance.
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Contents lists available at ScienceDirect
Annals of Diagnostic Pathology
Immunohistochemical expression and prognostic relevance of Bmi-1, a stem cell factor, in epithelial ovarian cancer☆ Amal Abd El hafez, PhD a,⁎, Hend Ahmed EL-Hadaad, MD b a b
Pathology Department, Faculty of Medicine, Mansoura University, Egypt Clinical Oncology and Nuclear Medicine Department, Faculty of Medicine, Mansoura University, Egypt
a r t i c l e
i n f o
Keywords: Bmi-1 Epithelial ovarian cancer Immunohistochemistry Prognosis Survival
a b s t r a c t Ovarian cancer is the fourth most common cause of cancer-related death in women. Bmi-1 is a stem cell factor implicated in many human malignancies with poor outcome. Few published reports on the expression of Bmi1 in epithelial ovarian cancer were either experimental or performed on cell lines. This study evaluates the immunohistochemical expression of Bmi-1 protein in epithelial ovarian cancer tissue specimens and its relevance to the clinicopathologic prognostic variables and patient survival. Forty cases of epithelial ovarian cancer were selected according to the availability of paraffin-embedded tissue and the clinicopathologic and survival data. Immunohistochemistry was performed for anti–Bmi-1 antibody. Low and high Bmi-1 expression groups were compared with age, tumor stage, laterality, grade, histology, and patient survival. Bmi-1 expression was detected in 72.5% of cases, of which 42.5% had high expression. High Bmi-1 expression strongly associated with advanced International Federation of Gynecology and Obstetrics stages (P = .007), bilaterality (P = .01), and higher Gynecologic Oncology Group grades (P = .031) and carcinomas of serous histology (P = .027). It had no association with patient age. Bmi-1 expression displayed a significant inverse association with patient overall and mean survival (P = .006, P b .001). These observations suggested correlation between increased Bmi-1 expression and clinical progression in ovarian epithelial cancer. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Ovarian cancer is the fourth most common cause of cancer-related death in women and is the most lethal gynecological malignancy with 30% to 40% overall survival (OS) at 5 years [1]. Epithelial ovarian cancer comprises most malignant ovarian neoplasms in adult women, and different molecular genetic pathways participate in its development [2]. Because of its insidious onset, most patients are diagnosed with advanced-stage disease [3]. Clinically, ovarian cancer is characterized by an initial response to cytotoxic chemotherapy, followed frequently by recurrence and disease progression, which represents a major scientific and clinical barrier to the control of cancer. Thus, the development of new therapeutic strategies to combat ovarian cancer is needed [4]. Traditional cancer therapies typically target the rapidly dividing tumor cells; however, some cells of the tumor are spared. These spared cells, which are reported to be present within many tumor types, exhibit the potential to regenerate and are called cancer stem cells (CSCs) [5-9].
☆ Disclosure: No relevant financial affiliations or conflicts of interest to disclose. ⁎ Corresponding author: Pathology Department, Faculty of Medicine, Mansoura University, El-Gomhouria St, Mansoura-Dakahlia, Egypt. E-mail addresses: [email protected], [email protected] (A. Abd El hafez), [email protected] (A. Abd El hafez). 1092-9134/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.anndiagpath.2013.11.004
There is increasing evidence that polycomb group (PcG) proteins (discovered in Drosophila as epigenetic gene silencers) play a crucial role in cancer development and recurrence [10]. Human B-cell specific moloney leukemia virus insertion-site 1 (Bmi-1), a member of the PcG family of transcription repressors, has emerged as a Myc-cooperating oncogene in murine lymphomas. In human, Bmi-1 gene localizes on short arm of chromosome 10 (10p11.23) and encodes a protein of 36.8 kDa [7,9]. Normally, Bmi-1 has an ubiquitous pattern of expression in almost all tissues with high expression levels in the brain, esophagus, salivary gland, thymus, kidney, lungs, gonads, placenta, blood, and bone marrow [9]. Bmi-1 controls the cell cycle by regulating the tumor suppressor proteins p16INK4a and p14ARF [11,12]. The p16INK4a protein inhibits binding of cyclin D to CDK4/6, resulting in the suppression of retinoblastoma activity and induction of cell cycle arrest [12,13], whereas p14ARF induces p53 and causes cell cycle arrest [12-14]. Thus, Bmi-1 promotes cell proliferation by suppressing p16INK4a/retinoblastoma and/or p14ARF/MDM2/p53 tumor suppressor pathways [13]. In addition, Bmi-1 is reported to play a critical role in tissue homeostasis by maintaining self-renewal and differentiation capacities and prevention of senescence through the activation of telomerase in adult stem cells [11,12,15]. The ability of CSCs to induce cancer recurrence after therapy has been attributed to the activation of different molecules including Bmi1 [16,17]. In addition, recent studies suggest that Bmi-1 acts as a stem cell factor that is involved in cancer initiation, progression, and
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chemoresistance. Therefore, Bmi-1 can be developed as a target for therapeutic agents that efficiently abolish chemoresistance in tumor cells [7,8,18]. Bmi-1 has been associated with a number of human malignancies including B-cell non-Hodgkin lymphoma; myelodysplastic syndrome; breast, gastric, nasopharyngeal, esophageal, colorectal, bladder, prostatic, and pancreatic carcinomas; non–small cell lung cancer; and neuroblastoma and has been shown to be a useful prognostic marker in many of these cancers [8,9,11,16,19-33]. Nonetheless, there are few published reports on the expression of Bmi-1 in ovarian cancer, and most of these studies were either experimental or performed on ovarian cancer cell lines [4,34,35]. Therefore, this study aims to assess the immunohistochemical expression of Bmi-1 protein in human epithelial ovarian cancer tissue specimens and to explore its relevance to the established clinicopathologic and prognostic variables and the patient survival. 2. Materials and methods 2.1. Patients and clinicopathologic evaluation Forty female patients with histologically confirmed primary ovarian epithelial cancers encompassing 5 serous borderline tumors, 21 serous carcinomas, 3 mucinous borderline tumors, 6 mucinous carcinomas, and 5 endometrioid carcinomas were investigated for Bmi-1 immunohistochemical expression. Cases were obtained from the archives of the Pathology Department, Faculty of Medicine, Mansoura University, Egypt, from January 2006 to December 2007. Inclusion criteria were the availability of tumor tissue paraffin blocks and the clinicopathologic data obtained from the medical records. Follow-up and OS data starting from the time primary surgery until the patients died or were lost to follow-up were obtained from Clinical Oncology and Nuclear Medicine Department in the same university. None of the patients had received preoperative radiation or chemotherapy. Routinely processed hematoxylin and eosin slides were reviewed to revaluate histopathologic type of ovarian cancer according to the World Health Organization classification, and tumors were graded according to Gynecologic Oncology Group (GOG) system. Combination of clinical and histopathologic information was adopted for staging according to International Federation of Gynecology and Obstetrics (FIGO) staging criteria. 2.2. Immunohistochemistry Immunohistochemical (IHC) staining was performed on formalinfixed, paraffin-embedded tissues. After deparaffinization and rehydration, 4- to 5-μm-thick sections on coated slides were heat pretreated in citrate buffer (pH 6 at 92°C) and immunostained using specific antibody against Bmi-1 gene protein (Anti-Bmi-1; ab97729; rabbit polyclonal antibody; Abcam Corporation (Cambridge, UK) product at dilution 1/50). Standard avidin-biotin-peroxidase technique was applied using diaminobenzidin for visualization and hematoxylin for counterstaining. Appropriate negative controls, consisting of histologic sections of each case processed without the addition of primary antibody, were prepared, along with a positive control sections prepared from human neuroblastoma tissue with confirmed Bmi-1 immunopositivity. 2.3. Evaluation of Bmi-1 staining Expression of Bmi-1 protein was visualized by observing the stained tissues under a light microscope. Immunostaining for Bmi-1 was defined as the presence of brown or yellowish brown granules in the nuclei, although occasionally, yellowish brown granules could also be seen in the cytoplasm. For evaluation of the Bmi-1 IHC staining in
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ovarian cancer tissues, a previously described [17,36], semiquantitative scoring was used, in which both staining intensity and positive areas were recorded. The intensity of Bmi-1 staining (negative, 1; weak, 2; moderate, 3; or strong, 4 scores) and the proportion of immunopositive cells (≤10%, 1; N10% to ≤ 50%, 2; N50% to ≤75%, 3; N75%, 4) were calculated. The 2 different scores of corresponding sample were then multiplied, so a staining index ranging from 1 to 16 points was obtained. Points equal or less than 4 was marked as (−); 5 to 8 points, as (+); 9 to 12 points, as (++); and 13 to 16 points, as (+++). For statistical analysis, (−) and (+) were counted as low expression of Bmi-1, whereas (++) and (+++) were counted as high (intensive) expression of Bmi-1 (Table 1). 2.4. Statistical analysis Statistical analysis was carried out with the SPSS version 16.0 (Chicago, USA). The association of Bmi-1 protein expression with ovarian carcinoma patients' clinicopathologic variables was assessed by the Pearson χ 2 test. The Student t test was used to compare mean OS time in relation to Bmi-1 expression. Survival curve was plotted by Kaplan-Meier method and compared by the log-rank test. P ≤ .05 was considered as statistically significant. 3. Results 3.1. Clinicopathologic characteristics As seen in Table 2, the study included 24 female patients 50 years or younger and 16 patients older than 50 years with mean and median ages of 46.5 and 48 years, respectively. Most patients (45%) were presented at FIGO stage I, and high percentage of carcinomas were at GOG grade 2 (30%) or 3 (27.5%). Approximately 42% of tumors were bilateral. Carcinomas with serous histology were the most frequent (52.5%), whereas borderline mucinous tumors were the least common (7.5%). Patients' survival ranged from 4 to 63 months with a mean survival of 33.3 months. 3.2. Bmi-1 expression in epithelial ovarian cancer Bmi-1 protein expression was detected in 29 (72.5%) of 40 of ovarian epithelial cancers (Fig. 1). In this study, we defined that (−) and (+) were considered as low expression of Bmi-1, whereas (++) and (+++) were considered as high expression of Bmi-1. According to this definition, the high Bmi-1 expression was detected in 17 (42.5%) of 40 of ovarian cancers (Table 1). 3.3. Association of Bmi-1 expression with the clinicopathologic variables Associations between Bmi-1 expression profile and the patients' clinicopathologic variables are presented in Table 2. High Bmi-1 expression was strongly associated with advanced FIGO cancer stages (P = .007), bilaterality (P = .01), higher GOG grades (P = .031), and carcinomas of serous histology (P = .027). However, no significant association was observed between Bmi-1 expression profile and patients' age (P = .433). 3.4. Association between Bmi-1 expression and patients' survival Bmi-1 expression displayed a significant inverse association with patient OS (P = .006). The mean and median survival times for patients with tumors having high expression of Bmi-1 were shorter than patients with tumors having low expression of Bmi-1 (21.8 and 21 months compared with 41.8 and 41 months, respectively) with a statistically significant difference between both groups (P b .001) (Table 2). The survival rate, assessed by the Kaplan-Meier method,
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Fig. 1. High Bmi-1 nuclear expression in borderline papillary serous tumor (A) and grade 1 papillary serous carcinoma (B). (C) High Bmi-1 nuclear and cytoplasmic expression in grade 3 papillary serous carcinoma. (D) Low Bmi-1 nuclear expression in endometrioid carcinoma (immunohistochemistry, ×200).
was 60.9% in the low expression group, whereas it was only 17.6% in the high expression group (Fig. 2).
Recently published data testify that Bmi-1 knockdown resulted in apoptosis in ovarian cancer cells but not in normal cells [27,34]. Moreover, Bmi-1 plays an important role in sensitization of
4. Discussion The development and progression of ovarian cancer are presumed to be a multistep process involving multiple genetic changes of p53, BRCA1, BRCA2, K-RAS, β-catenin, and PTEN [2]. However, it is important to identify a biological genetic molecular marker that is associated with pathophysiologic processes of human ovarian cancer to explain the high rates of recurrence and the mechanisms of drug resistance and improve the survival of patients with this often lethal disease [1,37]. Bmi-1 has been recently implicated in several aspects of cancer biology. In addition to its significant role in chemoresistance and tumor recurrence [38], Glinsky et al [39] demonstrated that both epithelial and nonepithelial malignancies displaying Bmi-1 gene signature have a probability to develop distant metastasis. Overexpression of Bmi-1 immortalizes human mammary epithelial cells and increases its motility and invasive properties [11,30]. In ovarian cancer, it is speculated that Bmi-1 as an oncoprotein participates in cellular multiplicative division and causes the down-regulation of p16INK4a, which prolongs the life period, promotes the proliferation, and decreases apoptosis rate of ovarian epithelial cancer cells [17,40].
Table 1 Expression of Bmi-1 in epithelial ovarian cancer Negative Bmi-1 staining index
Comparison groups
− (0-4)
Positive + (5-8) 12 (30%)
11 (27.5%)
++ (9-12) 5 (12.5%) 29 (72.5%)
+++ (13-16) 12 (30%)
Low Bmi-1 expression 23 (57.5%)
High Bmi-1 expression 17 (42.5%)
Table 2 Association of Bmi-1 expression with the clinicopathologic variables and survival of ovarian cancer patients Variable
Age b50 y ≥50 y Age range Mean (median) age Laterality Unilateral Bilateral Stage I II III IV Grade Borderline 1 2 3 Histology Borderline serous tumors Borderline mucinous tumors Serous carcinomas Mucinous carcinomas Endometrioid carcinomas OS OS range Mean (median) survival Total a
Bmi-1 expression
P
Low
High
24 (60%) 16 (40%) 21-66 y 46.5 (48) y
17 (70.8%) 6 (37.5%) 24-64 y 45.9 (45) y
7 (29.2%) 10 (62.5%) 21-66 y 47.3 (50) y
.433
23 (57.5%) 17 (42.5%)
17 (73.9%) 6 (35.3%)
6 (26.1%) 11 (64.7%)
.01a
18 (45%) 6 (15%) 10 (25%) 6 (15%)
14 (77.8%) 4 (66.7%) 5 (50%) 2 (33.3%)
4 (22.2%) 2 (33.3%) 5 (50%) 4 (66.7%)
.007a
8 (20%) 9 (22.5%) 12 (30%) 11 (27.5%)
7 (87.5%) 7 (77.8%) 6 (50%) 3 (27.3%)
1 (12.5%) 2 (22.2%) 6 (50%) 8 (72.2%)
.031a
5 (12.5%) 3 (7.5%)
4 (80%) 3 (100%)
1 (20%) 0 (0%)
.027a
21 (52.5%) 6 (15%) 5 (12.5%)
8 (38.1%) 4 (66.7%) 4 (80%)
13 (61.9%) 2 (33.3%) 1 (20%)
4-63 mo 33.3 (33) mo 40
5-63 mo 41.8 (41) mo 23 (57.5%)
4-55 mo 21.8 (21) mo 17 (42.5%)
P value is significant if ≤.05.
.006a b.001a
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Fig. 2. Kaplan-Meier curves with univariate analyses (log-rank P b .001) of patients with epithelial ovarian cancer, subdivided according to Bmi-1 protein expression. The cumulative 3-year survival rate was 60.9% in the low Bmi-1 protein expression group (n = 23), but it was only 17.6% in the high expression group (n =17).
chemoresistant ovarian cancer cells to cisplatin by regulating reactive oxygen species and glutathione levels and, thus, protects the ovarian cancer cells from chemotherapeutic insults [34]. Overexpression of polycomb protein Bmi-1 has been linked to an increasing number of cancer types, and the specificity of immunostaining of the anti–Bmi-1 antibody was confirmed [27,41]. In line with these findings, the current study showed that Bmi-1 is expressed in a high percentage (72.5%) of epithelial ovarian cancers. It is noteworthy that Bmi-1 is highly enriched in CSCs; however, not all Bmi-1expressing cells are CSCs [18]. In 2 prior studies [17,36], immunohistochemical examination of ovarian epithelial cancer specimens revealed positive expression of Bmi-1 in most of cases with high expression in 37% to 46.81% of ovarian carcinomas. In addition, Western blot studies showed high expression levels in approximately 74% of the ovarian cancer patient samples, thus suggesting an important role for Bmi-1 in ovarian cancer [4]. In accordance with these data, high Bmi-1 expression was evident in 42.5% of the present cohort. It is possible that there may be “threshold” of Bmi-1 protein expression, and when this “threshold” is overridden, this protein may start to function as an oncogene [29]. The cellular localization of Bmi-1 protein is quite mysterious. Bmi-1 protein seems to be localized in the nucleus of most breast cancer cells and in the cytoplasm of most noncancer cells [30]; however, Bmi-1 was observed in the cytoplasm of gastric lesions and oropharyngeal squamous cell carcinomas [9,28], opposite to its nuclear expression pattern in endometrial, cervical [41], esophageal squamous [27], and bladder [26] cancers. Supported with previous reports [17,36,41], this study revealed that Bmi-1 protein was mainly confined to the nucleus of the ovarian cancer cells, although occasionally positive staining was seen in the cytoplasm. It has been postulated that phosphorylation can explain differential subcellular localization of some of the polycomb family genes, as there is a rich proline/serine region at the carboxyl terminus of the Bmi-1 protein, where phosphorylation often occurs resulting in the nuclear-cytoplasm shuttling [30]. Another explanation is tissue specificity of Bmi-1 expression because Bmi-1 is a transcriptional factor and can translocate from nucleus to cytosol under some physiologic or pathologic conditions [28]. Data from several clinical studies show that abnormal expression of Bmi-1, in protein level as well as in gene level, is associated with
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poor prognostic markers and worse clinical outcome in diverse human cancers [16,22,26-28,30]. However, there is little information about the prognostic status and clinical outcome of Bmi-1 expression in ovarian cancer. In this study, high Bmi-1 protein expression in human epithelial ovarian cancer was significantly associated with advanced FIGO stages, tumor bilaterality, high tumor grades, and carcinomas of serous histology suggesting a correlation between increased Bmi-1 expression and clinical progression in ovarian epithelial cancer. Nonetheless, no significant difference in Bmi-1 expression was observed in relation to patient age. Among our cohort, Bmi-1 expression displayed a significant inverse association with patient survival; thus, patients with higher Bmi-1 expression had shorter survival time, and patients with lower Bmi-1 expression had a longer survival time; thus, high expression of Bmi-1 protein indicated poor prognosis for patients with ovarian cancer. These results were similar to those of previous studies of other human cancers [19-22,26] as well as with the few studies [17,36] that evaluated the expression of Bmi-1 by IHC in association with clinicopathologic and prognostic variables of ovarian cancer. In accordance with published literatures [26,27,35,36,41], our findings demonstrated that Bmi-1 can be used as a marker to identify subsets of ovarian cancer patients with more aggressive features and poor outcome and that higher Bmi-1 expression might contribute to the carcinogenesis of ovarian cancer. Therefore, further investigations are needed to confirm the prognostic relevance of Bmi-1 in ovarian epithelial cancer and the applicability of therapeutic targeting of Bmi-1 for attacking the “more aggressive” cancer cells causing recurrence and treatment resistance.
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