| Abstract|| |
Background: The most common second primary cancer in women who survive breast cancer is colorectal cancer. Population cohort studies suggest that the risk of colorectal cancer is also high in female relatives of women with breast cancer compared to the general population. The histological distinction between a large hyperplastic polyp and a serrated adenoma is not straightforward and there is probably no sharp division between hyperplastic polyposis and 'serrated adenomatous polyposis'. Aim: The aim of the study was to define the distribution of Ki-67 and Bcl-2 protein in hyperplastic colorectal polyps of women with breast cancer. Materials and Methods: A cross-sectional, controlled study was conducted in 40 women with and without breast cancer who had hyperplastic colorectal polyps. The patients were divided into two groups: Group A (a control group of women without breast cancer, n = 20) and Group B (a study group of women with breast cancer, n = 20). The expression of Ki-67 and Bcl-2 protein was assessed on the basis of quantity of immunostaining, by counting antigen positive cells, in comparison with normal mucosa. Student's t-test and the chi-square test were used to analyse Ki-67 and Bcl-2 expression, respectively. Results: Statistical significance was established at P < 0.05. The mean percentage of Ki-67 stained nuclei in Groups A and B was 25.11 ± 2.07 and 41.51 ± 1.86, respectively (P < 0.003), whereas the percentage of cases with cells expressing Bcl-2 in Groups A and B was 15% and 85%, respectively (P < 0.002). Conclusions: In the present study, greater proliferative activity and greater expression of the antiapoptotic protein Bcl-2 was found in the hyperplastic colorectal polyps of women with breast cancer.
Keywords: Bcl-2, breast cancer, hyperplastic colorectal polyps, Ki-67
|How to cite this article:|
Gelincik I. Ki-67 and Bcl-2 in colorectal polyps with breast cancer. Indian J Pathol Microbiol 2011;54:307-11
| Introduction|| |
Most hyperplastic polyps are indistinguishable from their common counterparts, apart from their large size. As in the sporadic hyperplastic polyp, the proliferative zone is increased but remains confined to the lower crypt. There is abnormal retention of cells in the upper maturation zone associated with the characteristic appearance of serration. A small proportion contains foci of intraepithelial neoplasia (dysplasia) that may either resemble a tubular, tubulovillous, or villous adenoma, or retain a serrated architecture supporting a diagnosis of serrated adenoma. ,, The histological distinction between a large hyperplastic polyp and a serrated adenoma is not straightforward and there is probably no sharp division between hyperplastic polyposis and 'serrated adenomatous polyposis'.
Breast cancer is the most common malignant disease in women in Western Europe and North America.  Over the past few decades, there has been a considerable increase in the survival of women with breast cancer, principally due to early diagnosis and the growing use of adjuvant therapies. , Nevertheless, studies have reported a potential increase in the risk of developing a second primary cancer in patients who survive breast cancer. , With the exception of a contralateral breast tumor, the most common second primary cancer in women who survive breast cancer is colorectal cancer.  Population cohort studies suggest that the risk of colorectal cancer is also high in female relatives of women with breast cancer compared to the general population.  Ochsenkühn et al.  have also reported that women aged >65 years with breast cancer have an increased risk of developing colorectal adenomas. Hyperplastic polyps are traditionally considered non-neoplastic, but ras mutation is common, clonality has been demonstrated, and biochemical abnormalities and epidemiological associations that occur in colorectal adenomas and carcinomas have been found. ,, These lines of evidence suggest that hyperplastic polyps may be neoplastic but have a molecular pathogenesis that differs from the adenoma-adenocarcinoma sequence due to absence of inactivation of the APC/betacatenin pathway. The imbalance between cell proliferation and programmed cell death at colonic crypt level is a key phenomenon in the development of colorectal cancer. ,,
Expression of the Ki-67 protein and the antiapoptotic protein Bcl-2 have been the most frequently used biomolecular markers for the evaluation of cell proliferation and apoptosis, respectively.  The present study was evaluated proliferative and apoptotic activity of Ki-67 and Bcl-2 protein in 40 women with and without breast cancer who had hyperplastic colorectal polyps.
| Materials and Methods|| |
The present study was conducted in 40 women with and without breast cancer who had hyperplastic colorectal polyps and was evaluated proliferative and apoptotic activity of Ki-67 and Bcl-2 protein. The hyperplastic polyps were picked up on screening colonoscopy and they were asymptomatic. Those breast cancer patients who had a history of treatment for the disease were excluded from the study, while patients without breast cancer who had a history of any previous breast surgery or who were considered to have a high risk of developing breast cancer were also excluded. All the patients without breast cancer had been submitted to imaging tests (mammography and ultrasonography) with negative results for malignancy. None of the patients in the study had any history of colonoscopy or gastrointestinal surgery.
This is a cross-sectional, controlled study carried out in 40 women with and without breast cancer who had hyperplastic colorectal polyps. The patients were divided into two groups: Group A (no breast cancer, control group, n=20) and Group B (breast cancer, study group, n=20). These groups were the statistically significant difference between the two groups was the size of the colorectal polyps [Table 1].
Immunohistochemistry for Ki-67 and Bcl-2
For the immunohistochemical evaluation of Ki-67 and Bcl-2 protein expression, previously the samples of hyperplastic colorectal polyps fixed in buffered formalin were cut into 3-μm-thick sections. Next, the sections were deparaffinized in xylol for 5 min, dehydrated in absolute ethanol and washed in buffered saline solution at pH 7.4 for 5 min. Subsequently, the sections were treated for 5 min with 3% hydrogen peroxide (H 2 O 2 ) diluted in buffered solution to block endogenous peroxide. For antigen recovery, the slides were placed in racks containing 0.21% citric acid (pH 6.0) and heated in a microwave oven for 15 min at maximum power. Phosphate-buffered saline containing Tween (PBS-Tween) was added to the slides after they had been allowed to cool for 20 min. The tissue samples were incubated overnight at 4-8 C with primary mouse anti-Ki-67 monoclonal antibody (clone C5 Mause MAb IgG2b, UK) and with mouse anti-Bcl-2 monoclonal antibody (clone Bcl-2/100/D5 IgG1, UK). The slides were then washed with PBS-Tween and instilled with secondary reagent (anti-mouse BA 2000, Vector, Burlingame, USA), incubated for 60 min at room temperature, washed again in PBS-Tween, and instilled with the ABC Elite detection system (PK 6100, Vector, Burlingame, USA), incubated for 45 min at room temperature, washed once again with PBS-Tween, instilled with DAB (Diaminobenzidine tetrahydrochloride, Ref. D5637, Sigma, St. Louis, USA) and incubated for 5 min. Finally, the slides were washed with distilled water, counterstained with hematoxylin, stained with ammoniacal solution, dehydrated with absolute ethanol, passed through Coplin jars containing xylol and mounted in Permount resin. The cells that expressed the Ki-67 and Bcl-2 proteins were identified by the dark brown coloring of the nucleus and cytoplasm, respectively.
For Ki-67 expression, a minimum of 400 cells were counted on each slide at a magnification of ×400, irrespective of whether they had been stained by anti-Ki-67 or not. Only crypts that were visible along their entire length with the base of the crypt touching the muscularis mucosa were evaluated. In each case, 8 to 10 colon crypts (mean, 9) were evaluated. The percentage of stained cells for each case was obtained from the ratio between the number of cells with stained nuclei and unstained nuclei multiplied by 100 (label index).
Bcl-2 immunoreaction was evaluated semi-quantitatively according to the criteria established by van Slooten and colleagues  taking the following parameters into consideration: Intensity of cell staining (I) and the fraction of stained cells (F). The intensity of cell staining was classified as: 0 (negative), 1 (weakly stained), 2 (moderately stained) and 3 (strongly stained). The fraction of stained cells was classified as: I (0-25%), II (25-75%) or III (75-100%). The final score was the result of the combination of the two parameters (I and F) and ranged from 0 to 6. Cases with a final score ≥3 were classified as positive for Bcl-2. In all cases, brownish staining in the cytoplasm was adopted as the standard for positivity. ,
Mann-Whitney's nonparametric test was used to evaluate the size of the polyps between the two groups.  Student's t-test was used to compare the means of the percentages of Ki-67-stained nuclei between the two groups and comparison of the proportions of cases with cells expressing Bcl-2 in hyperplastic colorectal polyps were performed using the chi-square test.  Significance was established at P < 0.05 throughout the study.
| Results|| |
Under light microscopy, there was a greater concentration of Ki-67-stained nuclei in the crypts of the hyperplastic colorectal polyps of patients with breast cancer (Group B) compared to those of Group A (controls). There were also more cases with a positive expression for Bcl-2 protein in the hyperplastic colorectal polyps of women in the study group as shown by a greater fraction of cells with intensely stained cytoplasm compared to the control group [Figure 1]. The mean percentage of Ki-67-stained nuclei was 25.11 ± 2.07 and 41.51 ± 1.86 in the crypts of the hyperplastic colorectal polyps of patients in Groups A (control) and B (study), respectively (P < 0.003) [Table 2]. With respect to Bcl-2 antigen expression, the number of cases with positive expression for Bcl-2 in the crypts of the hyperplastic colorectal polyps of patients in Groups A and B was 6/20 (30%) and 17/20 (85%), respectively (P < 0.002) [Table 3].
|Figure 1: (a) Photomicrographs of histological sections of hyperplastic colorectal polyps. Note the lower concentration of Ki-67-stained nuclei in the polyp cells of a patient from the control group (b) compared to a patient with breast cancer (c), Observe a negative immunohistochemical reaction for the Bcl-2 protein in the polyp cells of a patient from the control group (d), and a positive immunohistochemical reaction, as expressed by numerous cells with cytoplasms intensely stained of a patient from the study group. (IHC, ×400).|
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Mean percentage of Ki-67-stained nuclei in the hyperplastic colorectal polyps of patients in the control (A) and study (B) groups [Table 2].
The difference between the two groups was statistically significant (P < 0.003).
Percentage of cases with cells expressing Bcl-2 in the hyperplastic colorectal polyps of patients in Groups A (control) and B (study) [Table 3].
The difference between the two groups was statistically significant (P < 0.002).
| Discussion|| |
The findings of various population-based cohort studies suggest an increased risk of colorectal cancer in women with breast cancer compared to women in the general population and this risk varies, according to some studies, from marginally increased to higher relative risks. ,, All these studies are epidemiological and the subsequent risk of colorectal cancer in these patients remains unclear.  On the other hand, some investigators have shown that colorectal epithelial cell kinetics change in patients at increased risk for colon cancer.  However, it is probable that women with breast cancer and hyperplastic colorectal polyps have an increased risk of developing malignancy in these lesions compared to controls, and this probably involves a change in the expression of proteins related to cell proliferation and apoptosis. In the present study, the hyperplastic colorectal polyps of patients with breast cancer were found to have greatly increased cell proliferative activity as evaluated by Ki-67 protein expression and a markedly increased expression of the antiapoptotic protein Bcl-2 compared to hyperplastic polyps from women in the control group. Patients with history of any treatment for the breast cancer were excluded from the study, therefore none of them was receiving tamoxifen, since it has been shown in animal models that selective estrogen receptor modulators may decrease colonic cell growth. 
Colorectal epithelial cell proliferation is greater in individuals with a higher risk of colorectal neoplasias and consequently has been proposed as a biomarker for the prevention of colorectal cancer.  However, the ability of this measurement of proliferative activity to predict future adenomas or colorectal cancer remains unclear and requires further studies. Ki-67 protein was used in the present study in view of its sensitivity as a marker of cell proliferation since this protein is expressed in all the phases of the cell cycle except in the resting phase (G0). ,,,, Moreover, an increase in its expression has been associated with greater aggressivity of neoplasias. ,,,
Some investigators have divided colonic crypts into two or more parts for the evaluation of immunoreactivity, taking into consideration that it is in the lower third of the crypt that the germinative cells are found and it is in these cells that proliferative activity is greatest.  Nevertheless, in the present study, in order to evaluate all proliferative activity, the entire extension of crypts in the hyperplastic colorectal polyps (a mean of 8-10 crypts for each case) was studied.
With respect to Bcl-2, this protooncogene encodes the protein that protects cells from programmed cell death; this protein being expressed in the proliferative compartment of several tissues, including crypts in the hyperplastic colorectal polyps.  Bosari and colleagues  showed that the normal colorectal mucosa of patients who have premalignant or malignant colonic lesions positively expresses Bcl-2; however, the majority of carcinomas do not. Likewise, Flohil and colleagues  also demonstrated an increase in Bcl-2 expression in adenomatous colorectal polyps but not in carcinomas, suggesting that the Bcl-2 oncoprotein may play a role in colorectal tumorigenesis, probably in the early phases of the adenoma-carcinoma sequence. , Wherein BCL2 expression was found more frequently in hyperplastic and low dysplastic polyps with moderate and strong positivity compared to moderate and severe dysplasia. Even though, some clarity in this article is lacking, hyperplastic polyps with Bcl-2 are studied. 
To our knowledge, this is the first study to evaluate Ki-67 and Bcl-2 expression in the hyperplastic colorectal polyps of women with breast cancer. Although further studies are required, the present findings show that in relation to the controls, women with breast cancer had greater proliferative activity and a greater expression of the antiapoptotic protein Bcl-2 in the hyperplastic colorectal polyps, thereby possibly suggesting a higher risk for the development of premalignant and malignant lesions the present findings show an increase in Ki-67 and Bcl-2 protein expression in the hyperplastic colorectal polyps of women with breast cancer.
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Bolge Egitim ve Arastirma Hastanesi, Patoloji Klinigi Cat Yolu cad. Yildizkent-Erzurum
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]