| Abstract|| |
Context: The c-erbB-2 proto-oncogene is a member of the epidermal growth factor receptor family and has been associated with a more aggressive breast tumor biology and resistance to some types of treatments. Aims: The aim is to investigate the correlation among bcl-2 and c-erbB-2 and the micronucleus (MN) formation in patients with early breast cancer (BC). Materials and Methods: This study was conducted between May 2010 and December 2011. We analyzed the MN frequencies in 15 patients with invasive breast carcinoma (IBC), 13 patients with intraductal proliferative lesion (IDPL) and 12 benign breast lesion (BBL). The sample consisted of 40 formalin-fixed, paraffin-embedded blocks of benign and malignant breast tissue. The specimens were evaluated for bcl-2 or c-erbB-2 immunoreactivity was semi-quantitatively evaluated in at least 1000 cells examined under the microscope at 40Χ magnification and recorded as the percentage of c-erbB-2 and bcl-2 positive tumor cells over the total number of cells examined in the same area. The percentage scores were subsequently categorized using the 5% cut-off point for positive staining. Results: The MN was significantly increased in IBC and in IDPL patients compared to BBL patients (3.82 ± 0.17 and 2.37 ± 0.52, respectively, vs. 1.61 ± 0.40, P < 0.001). On other hand, the MN frequencies in IBC patients were higher than those in IDPL patients (3.82 ± 0.17 vs. 2.37 ± 0.52, P < 0.01). c-erbB-2, had the highest record in IBC (60%), and the score was not observed in both IDPL and BBL: bcl-2 immunostaining was also assessed, the lowest recorded score was in IBC (46.66%) and the highest in both BBL and IDPL (100%). Furthermore, there was a significantly difference in the mean MN frequency between c-erbB-2 positive IBC patients (4.06 ± 0.48) and c-erbB-2 negative IBC patients (3.44 ± 0.39) (P < 0.05). Conclusions: Our results suggest that increased chromosome / DNA instabilities may be associated with the pathogenesis of early BC.
Keywords: Breast cancer, breast precancerous lesions, micronucleus test, c-erb2 gene, bcl-2 gene
|How to cite this article:|
Kabalar ME, Karaman A, Aylu B, Özmen SA, Erdem I. Genetic alterations in benign, preneoplastic and malignant breast lesions. Indian J Pathol Microbiol 2012;55:319-25
|How to cite this URL:|
Kabalar ME, Karaman A, Aylu B, Özmen SA, Erdem I. Genetic alterations in benign, preneoplastic and malignant breast lesions. Indian J Pathol Microbiol [serial online] 2012 [cited 2019 Jun 19];55:319-25. Available from: http://www.ijpmonline.org/text.asp?2012/55/3/319/101737
| Introduction|| |
Breast cancer (BC), like most other forms of malignancy, is mainly a multifactorial disease, occurring as a result of the combined effects of environmental and heritable factors. In only 5 -10% of the patients is the disease transmitted in a Mendelian fashion.  Family history is considered to be an important risk factor for the development of carcinoma of the breast.  It has been suggested that much of the increase in familial recurrence risk may be attributable to heritable factors. ,
c-erbB-2 overexpression or amplification is frequently, although not invariably, associated with a poor clinical outcome. ,, In recent times, c-erbB-2 gene amplification has been reported to correlate with the expression of the apoptosis-suppressing genes bcl-2 and bcl-xL in breast cancer.  Downregulation of bcl-2 and bcl-xL with antisense oligonucleotides increased the proapoptotic activity of trastuzumab.  This led to the hypothesis that combined targeting of c-erbB-2 and bcl-2 or bcl-xL mightrepresent a more effective breast cancer therapy compared to targeting only the individual factors. Only little is known about the relationship between c-erbB-2 and bcl-2 family members. Members of the bcl-2 family act as master regulators of mitochondrial homeostasis and apoptosis.  Transfection of c-erbB-2 into MCF-7 breast cancer cells has been shown to upregulate the expression of the bcl-2 family members. , At present, the prognostic value of overexpression or amplification of c-erbB-2 in breast cancer is controversial. , The expression of the antiapoptotic protein bcl-2 varies in breast cancer and seems to be associated with a favorable phenotype. 
In breast cancer, the clinical outcome is affected by a number of established prognostic factors, including age, tumor grade, estrogen receptor (ER) and progesterone receptor (PR) status. , In breast cancer specimens, bcl-2 expression is associated with markers of better differentiation, like lower grade lesions, ER positivity, and a low proliferation status.  Previous studies have shown the prognostic and predictive value of c-erbB-2 over-expression in node-positive breast cancer , and in metastatic disease. ,
Some studies have revealed reduced DNA repair capacity in peripheral blood mononuclear cells from BC patients, as evaluated by the chromosome aberration assay, ,, as well as by the micronucleus (MN) test. ,, The MN test in peripheral blood lymphocytes has been widely used as a biomarker of chromosomal damage both in vivo and in vitro., The frequency of MN is increased in untreated cancer patients and in subjects affected by cancer-prone congenital diseases, for example, Bloom syndrome or ataxia telengiectasia. , Furthermore, an association between MN and cancer has been reported.  The cause of this association may be structural chromosomal aberrations and aneuploidy. ,
We investigated the correlation among bcl-2 and c-erbB-2 and MN formation in patients with early breast cancer (BC).
| Materials and Methods|| |
This study was conducted between May 2010 and December 2011. Forty patients with diagnosed breast lesions of diagnosed were studied. The specimens were separated for each level and placed in 10% formalin solution.
The pathological specimens were reviewed independently by two pathologists. The pathologists were blinded to the subject's clinical history, and the results of the immunohistochemistry staining assay. The pathological reading was determined for each biopsy slide with an overall pathological diagnosis determined for each subject.
Permission was obtained from the Local Ethical Committee to collect breast tissues and all the patients gave informed consent to the research. The specimens (n = 40) were washed in phosphate-buffered saline (PBS) within five minutes and examined under a binocular dissecting microscope. Breast tissues were selected and fixed in freshly prepared 4% paraformaldehyde (4 g in 100 ml of 100 mmol / l PBS, pH 7.2) at 4°C for 24 hours. The breast masses were then embedded in paraffin wax with the breast tissues as close to the surface of the block as possible. Sections (5 μm) were cut and mounted on poly-L-lysine coated slides. Every tenth slide was stained with haematoxylin and eosin and examined in order to identify the breast tissue.
The slides containing breast tissue, were air-dried and heated for 20 minutes at 60°C before dewaxing, with xylene and rehydration. Two different mouse monoclonal antibodies were used in this study. Two negative controls were used, the primary antibody was replaced either with Tris-buffered saline (TBS), containing 0.1% bovine serum albumin (BSA), or by mouse immunoglobulin G (IgG) at the same concentration as the primary antibody. The positive controls were tissues known to express the antigen being studied. In order to maximize visualization of the gene product, each antibody was tested in a series of dilutions, with and without antigen retrieval (unmasking of epitopes after paraformaldehyde fixation and paraffin embedding) and in the presence and absence of enhancement steps. Antigen retrieval conditions (pH of the buffer, heating intensity and time) were altered in order to ascertain optimal antigen detection with minimal background staining for each antibody used.  For bcl-2, and c-erbB-2, optimization of immunohistochemical staining was achieved by pressure cooking the sections in 10 mmol / l citrate buffer (pH 6.0), for four minutes, at low pressure, in an aluminium pressure cooker. After dilution of the antibody in TBS containing 0.1% BSA, sections were incubated at room temperature for 60 minutes. The second antibody, a rabbit anti-mouse Ig antiserum (Dako Ltd., Bucks, UK) was then applied at 1 : 50 dilution for 30 minutes, at room temperature. This was followed by treatment with alkaline phosphatase anti-alkaline phosphatase complex (APAAP) (1 : 50 dilution for 30 minutes at room temperature, Dako Ltd.). To enhance the intensity of the APAAP labelling reaction, the second and third incubation steps were repeated (each step for 30 minutes). For development of alkaline phosphatase, the new fuchsin method was employed, with a substrate solution containing Tris-HCl, pH 8.8 (20 minutes, room temperature); 0.2 mmol / l levamisole was used to inhibit endogenous alkaline phosphatase. Finally, the sections were counterstained with haematoxylin for 20 seconds and mounted on Aquamount (BDH Laboratories, Poole, UK).
Cell membrane reactivity for the c-erbB-2 oncoprotein was evaluated following a similar approach and the mean value was used to score each case. Tumors expressing c-erbB-2 in > 30% of the cancer cells were considered as positive. bcl-2 immunoreactivity was categorized in to four groups: Score 0: negative (no staining). Score 1: + weak positive (staining in 5 - 20% of the neoplastic cells). Score 2: ++ moderate positive (staining in 20 - 50% of the neoplastic cells). Score 3: +++ strong positive (staining in more than 50% of the neoplastic cells). In addition, for estrogen receptor (ER) and progesterone receptor (PR) expression the percentage of cancer cells showing a nuclear reactivity was recorded after inspection of all optical fields at 200X and the mean value was used to score each case. Tumors with expression of > 20% of cancer cells were considered to be positive.
The tumor grade was determined according to the modified Bloom-Richardson score. The grade is obtained by adding up the scores for tubule formation. Final grading scores were as follows (sum of point / final grade: 3 - 5 / I, 6 - 7 / II, and 8 - 9 / III). In addition, endolymphatic emboli, and status of lymph node metastasis was shown in patients with IBC.
We performed MN analysis in 15 females (mean age: 55.47 ± 13.83 years; age range: 38 - 88) patients with IBC: in 13 females (mean age: 35.62 ± 10.03 years; age range: 20 - 54) patients with IDPL: and in 12 females (mean age: 27.58 ± 6.67 years; age range: 15 - 42) patients with BBL. We selected patients from non-smoking and nonalcoholic subjects. None of the subjects had a history of viral infection, bacterial infection or any metabolic diseases. The patients had not been treated with anti-neoplastic therapy (chemotherapy or radiotherapy) during the last four months. The patient and control groups were chosen for their similar habits. The hospital Ethical Committee approved the human study. We obtained written in formed consent from each participant. All the patients were analyzed prior to treatment.
For MN analysis, 3 ml of heparinized blood was drawn from each individual. Lymphocyte cultures were established by adding 0.5 ml of whole blood to 5 ml of karyotyping medium (Biological Industries, Beit Haemek, Israel) with 2% phytohaemagglutinin M (PHA; Biological Industries) according to the standard techniques.  The cultures were incubated at 37°C for 72 hours. All the slides were coded and read blind.
Cytochalasin B (6 μg / ml, Sigma, USA) was added, after 44 hours of culture, to the block cytokinesis, allowing for the identification of lymphocytes dividing in the culture. The culture was kept at 37°C for 72 hours. Cells that had undergone the first mitosis were thus recognized as binucleated cells and were selectively screened for the presence of MN. The cells were then treated hypotonically with 0.075 molar KCl for five minutes, at room temperature, and fixed in methanol / acetic acid (3 : 1). The cells were dropped onto slides and stained with 5% Giemsa in a phosphate buffer (pH 6.8), for five minutes. About 1,000 binucleated cells (mean ± SD = 1004.58 ± 5.36, range = 993 - 1027) from each case were examined for MN by an experienced observer. 
The MN rates were analyzed statistically by the Mann-Whitney U-test. To evaluate the correlations between the age, sex, MN rates, c-erbB-2, bcl-2, ER , PR, tumor grade and status of the lymph node the coefficients of Spearman ρ correlation were calculated. A P value less than 0.05 was considered to be significant.
| Results|| |
The associations of IBC, IDPL, and BBL with MN frequencies in groups are shown in [Table 1],[Table 2],[Table 3]. According to these results, MN was significantly increased in IBC patients, and in IDPL patients compared to BBL patients (3.82 ± 0.17 and 2.37 ± 0.52, respectively, vs. 1.61 ± 0.40, P < 0.001). The mean MN frequency of IBC patients was significantly higher than that of IDPL patients (3.82 ± 0.17 vs 2.37 ± 0.52 per metaphase, respectively; P < 0.01). Furthermore, there was a significantly difference in the mean MN frequency between c-erbB-2 positive IBC patients (4.06 ± 0.48) and c-erbB-2 negative IBC patients (3.44 ± 0.39) (P < 0.05). However, there was not difference in the mean MN frequency between bcl-2 positive IBC patients and bcl-2 negative IBC patients (P > 0.05). On the other hand, the MN frequencies did not correlate with the patients' age in the IBC patients (for each, P > 0.05). c-erb2, was with the highest record in IBC (60%) [Figure 1] and the score was not observed in both IDPL and BBL. bcl-2 immunostaining was also assessed, the lowest recorded score was in IBC (46.66 %) [Figure 2], and the highest in both IDPL and BBL (100%). Groups 2 and 3 were not statistically different in bcl-2 and c-erbB-2 status (P > 0.05).
Bcl-2 expression was found to be correlated with ER and PR expression (P < 0.01). However, c-erbB2 over-expression was found to be inversely correlated with bcl-2, ER and PR expression (P < 0.01). In addition, bcl-2(-) tumors were significantly correlated with the histological grade III (P < 0.01). In contrast, c-erbB-2(+) tumors were significantly correlated with the tumor grade III (P < 0.01). Furthermore, In c-erbB-2(+) patients axillary lymph node involvement was higher, and was lesser in bcl-2(+) (P < 0.01). Details are shown in [Table 1].
The micronuclei in the peripheral blood lymphocytes of a patient with IBC are shown in [Figure 3].
|Figure 3: The peripheral blood lymphocytes stained by Giemsa staining of the nuclei. The arrow shows the micronucleus [Giemsa, ×1000].|
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|Table 1: Age, age at diagnosis, and immunostaining of bcl-2, c-erbB-2, estrogen receptor (ER), progesterone receptor (PR), modifi ed Bloom- Richardson score (MBRS), angiolymphatic emboli (ALPE), axillary lymph node metastasis (ALNM) rates, and micronucleus (MN) frequencies for the patients with invasive breast carcinoma (IBC)|
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|Table 2: Age, age at diagnosis, immunostaining of bcl-2 and c-erbB-2 rates and micronucleus (MN) frequencies for the patients with intraductal proliferative lesion (IDPL)|
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|Table 3: Age, age at diagnosis, immunostaining of bcl-2 and c-erbB-2 rates and micronucleus (MN) frequencies for the patients with benign breast lesion (BBL)|
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| Discussion|| |
Breast cancer (BC) is a common type of malignancy in females, accounting for approximately 21% of all cancer cases in women worldwide.  Several DNA damage processing and repair pathways constitute a guard system that protects cells against genetic instability and tumorigenesis. Both genetic instability and impaired DNA restitution have been pointed out as factors underlying increased susceptibility to malignancy. , Apart from these rare syndromes, the deficient DNA repair capacity has been proposed to be a predisposing factor in familial BC and in some sporadic BC cases.  Genomic instability has also been described for various hereditary cancers including hereditary BC. ,
To better understand the relationship between the c-erbB-2 and bcl-2 family members, a group studied the extent to which the expression of bcl-2 family members is influenced by conditional the c-erbB-2 expression in the human breast cancer cell line MCF-7, and subsequently, analyzed the prognostic relevance of the identified genes, in patients with breast cancer. Inducible expression of NeuT, an oncogenic version of c-erbB-2, was achieved by the Tet-on system, , and c-erbB-2 expression levels were comparable with the breast tumors scoring 2+ / 3+. Overexpression of cytosol c-erbB-2 was also associated with gene amplification and the cytosol c-erbB-2 levels were significantly different between early, advanced and metastatic breast cancer.  They had reported that overexpression of cytosol c-erbB-2 was observed in recurrent breast cancer more frequently than in primary breast cancer (54 vs. 19%).  Furthermore, in another study, expression of c-erbB-2 was verified in nine (19.1%) of the ductal carcinomas in situ (P = 0.0001). Immunoexpression was not related to the extension of the lesions. There was no expression in the hyperplasias without atypias and adjacent tissues. 
In another study, there was not any advantage to determine c-erbB-2 as a prognostic factor on the overall survival or on relapse-free survival.  Similarly, no statistically significant differences were detected between the c-erbB-2 negative and positive groups, with regard to a five-year disease-free survival (41 and 27%, P = 0.11) and overall survival (60 and 45%, P = 0.33). They found no differences between c-erb2 negative and positive groups regarding disease-free and overall survival.  In this study, c-erb2 had the highest record in BC (63.64%) and the score was not observed in both premalignant lesions or benign lesions.
The bcl-2 is responsible for the inhibition of apoptosis, and is therefore, important for longer cell survival. According to Yanez et al.,  bcl-2 plays an important role in the development of pleomorphic adenoma (PA), as all cases examined were positive for this protein. Bcl-2 staining in this study was positive in about one of the three PA cases, which was in agrement with the other studies, indicating the possible role of bcl-2 as an anti-apoptotic agent. Bcl-2 immunoreactivity was found mainly in the cells surrounding the mesenchmal structures. In this study, bcl-2 immunostaining was also assessed, the lowest recorded score was in BC (45.45 %) and the highest in both premalignant lesion and benign lesion (100%). Groups 2 and 3 were not statistically different for bcl-2 and c-erbB-2 status (P > 0.05).
Previous studies have shown a strong correlation between c-erbB-2 protein over-expression and ER and PR negativity and high tumor grade. ,, In our study, c-erbB-2 positive cases usually had ER(-), PR(-), high tumor grade, and axillary lymph node (+) (P < 0.01). In the previous studies, the expression of bcl-2 in breast cancer was found to be associated with favorable prognostic factors, such as, smaller tumor size, ER positivity, and low nuclear grade. ,, In our study, bcl-2 positive cases usually had ER(+), PR(+), and low tumor grade, and axillary lymph node (-) (P < 0.01). As a result, the bcl-2 and c-erbB-2 status, gives important clues about the biological behavior of tumors. This will provide guidance for the prognosis of the tumor.
There were numerous case-control studies on breast cancer in which the micronucleus test was used to compare the repair capacity between BC cases and controls. , In almost all of them, a proportion of 20 - 50% of the patients could be identified, which showed an increased MN frequency exceeding the ninetieth percentile of the distribution in the controls. Complex inheritance was generally assumed for DNA repair capacity, as indicated by the numerous association studies carried out on cancer cases and the genes involved in DNA repair. The data available for MN frequencies appeared to be compatible with the assumption of continuous variation, when cases were interpreted as a separate group, with an overall slightly reduced repair capacity. In our study, MN was significantly increased in IBC patients, and in IDPL patients compared to BBL patients (3.82 ± 0.17 and 2.37 ± 0.52, respectively, vs. 1.61 ± 0.40, P < 0.001). The mean MN frequency of IBC patients was significantly higher than that of IDPL patients (3.82 ± 0.17 vs. 2.37 ± 0.52 per metaphase, respectively; P < 0.01).
A recently published cohort study linking the frequency of micronuclei in lymphocytes of healthy subjects to the risk of cancer, reported stomach cancer among the sites more specifically associated to micronuclei frequency.  Similar findings have also been reported for pre-neoplastic lesions of the colon  and cervix.  In particular, the higher risks for stomach and intestinal cancers, are in agreement with the literature, which emphasizes the role of chromosome rearrangements in the early stages of these tumors. ,
In our study, there was a difference in mean MN frequency between c-erbB-2 positive IBC patients and c-erbB-2 negative IBC patients (P < 0.05). However, there was no difference in the mean MN frequency between bcl-2 positive IBC patients and bcl-2 negative IBC patients. Our study, which showed increased MN frequencies in the lymphocytes of IBC patients, could support these observations, as the induction of changes in DNA that lead to mutations play a role in carcinogenicity.
These results suggests that genomic instability is present in the early stages of BC. Thus, MN is a promising biomarker for assessing the risk in the process of BC. Therefore, MN may be benefical in the early detection and prevention programs.
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Department of Medical Genetics, Erzurum Nenehatun Obstetrics and Gynecology Hospital, 25070 Erzurum
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]