Abstract
Gastric cancer is one of the most common malignant tumors and the second leading cause of cancer-related deaths in China. Although there is some progress in diagnose and treatment, the incidence of gastric cancer still keeps up increasing. In this study 40 patients with gastric cancer who underwent surgical operation is detected by immunohistochemistry. The positive rates of Bcl-2 and Ki67 protein expression in gastric cancer tissues were significantly higher than normal gastric mucous tissues. Correlation analysis showed that the expression of Bcl-2 is not correlated with that of Ki67. Positive expression of Bcl-2 or Ki67 did not correlate with age, gender, differentiation, stage and lymph node metastasis. These suggested that combination of Bcl-2 and Ki67 to detect gastric cancer is more effective.
Keywords: Bcl-2, Ki67, gastric cancer, immunohistochemistry
Introduction
Gastric cancer is one of the most common malignant tumors and causes approximately 800,000 deaths worldwide per year [1]. In China, the morbidity of gastric cancer has the first place among malignant tumors. Gastric cancer is the second leading cause of cancer-related deaths in China [2]. In recent years the incidence of gastric cancer still keeps up increasing, although there is some progress in diagnose and treatment [3]. The identification of prognostic factors in gastric cancer was essential for diagnosing patients and determining optimal therapeutic strategies.
Materials and methods
Patients
40 patients with gastric cancer who underwent surgical operation in the First Affiliated Hospital, Xi’an Medical University, 28 males and 12 females, were enrolled in the study. The study protocol was approved by the institutional ethics committee, and written informed consent was obtained from all participants.
Immunohistochemistry
The tissue specimens were formalin (10%) fixed and paraffin-embedded using standard technique. Each paraffin embedded tissue specimen was microtomed into four serial sections, of 4 µm thickness each. Two sections were used for H&E staining, one for Bcl-2 staining, and the other for Ki67 staining. Anti- Bcl-2 and -Ki67 monoclonal antibodies, and DAB reagent were purchased from Beijing Zhongshan Inc. China. The sections were stained with hematoxylin and DAB according to the manufacturer’s instructions.
Statistical analysis
Quantitative variables were compared using the Pearson’s chi-square test or Pearson’s relation probability test by analysis of variance. The statistical significance was accepted for P<0.05. All analyses were performed using the SPSS version 16.0 (SPSS, Chicago, IL).
Results
The expressions of Bcl-2 and Ki67 in gastric cancer
The cancer and normal tissues of 40 patients with gastric cancer were detected by immunohistochemistry. Immunochemical staining tests showed that Bcl-2 was mainly localized in the cytoplasm and Ki67 in nuclear of gastric cancer cells (Figure 1). The positive rates of Bcl-2 and Ki67 protein expression in gastric cancer tissues were both 70%. However, nothing of normal gastric mucous tissues express the protein of Ki67 and only 20% of normal tissues expressed Bcl-2, which differed significantly (P<0.05) from the corresponding positive rates in the cancer tissues.
Figure 1.
The expressions of Bcl-2 and Ki67 in gastric cancer were detected by immunohistochemistry (200×).
Correlation analysis showed that the expression of Bcl-2 is not correlated with that of Ki67 (P=0.234, Table 1). The total negative rate of Bcl-2 and Ki67 is only 5% (2/40), which reduce efficiently the missing diagnose of gastric cancer.
Table 1.
The correlation analysis of expression of Bcl-2 and Ki67
| Bcl-2 | r | P | ||
|---|---|---|---|---|
|
|
||||
| Positive | Negative | |||
| Ki67 | ||||
| Positive | 18 | 10 | 1.415 | 0.234 |
| Negative | 10 | 2 | ||
Overexpression of f Bcl-2 or Ki67 and clinic pathological factors
The relationship between expression of Bcl-2 or Ki67 and clinic pathological factors was investigated in gastric cancer tissues. Positive expression of Bcl-2 did not correlate with age, gender, differentiation, stage and lymph node metastasis (P>0.05, Table 2). In addition, Ki67expression did not correlate with the clinic pathological factors either (P>0.05, Table 3).
Table 2.
Relationship between expression of Bcl-2 and clinicopathological parameters in gastric cancer
| Variable | Cases | Positive (%) | Negative (%) | χ2 | P value |
|---|---|---|---|---|---|
| Gender | |||||
| Male | 28 | 20 (71%) | 8 (29%) | 0.006 | 0.940 |
| Female | 12 | 8 (67%) | 4 (33%) | ||
| Age | |||||
| ≤60 | 18 | 12 (67%) | 6 (33%) | 0.173 | 0.677 |
| >60 | 22 | 16 (73%) | 6 (27%) | ||
| Differentiation | |||||
| High | 12 | 10 (83%) | 2 (17%) | ||
| Median | 14 | 10 (71%) | 4 (29%) | ||
| Low | 14 | 8 (57%) | 6 (43%) | 1.033 | 0.309 |
| Stage | |||||
| I/II | 12 | 8 (67%) | 4 (33%) | 0.006 | 0.940 |
| III/IV | 28 | 20 (71%) | 8 (29%) | ||
| LN metastasis | |||||
| Positive | 26 | 16 (62%) | 10 (62%) | 1.512 | 0.219 |
| Negative | 14 | 12 (86%) | 2 (14%) |
Table 3.
Relationship between expression of Ki67 and clinicopathological parameters in gastric cancer
| Variable | Cases | Positive (%) | Negative (%) | χ2 | P value |
|---|---|---|---|---|---|
| Gender | |||||
| Male | 28 | 18 (64%) | 10 (36%) | 0.686 | 0.408 |
| Female | 12 | 10 (83%) | 2 (17%) | ||
| Age | |||||
| ≤60 | 18 | 12 (95%) | 6 (5%) | 0.005 | 0.945 |
| >60 | 22 | 16 (95%) | 6 (5%) | ||
| Differentiation | |||||
| High | 12 | 8(67%) | 4 (33%) | ||
| Median | 14 | 10 (71%) | 4 (29%) | ||
| Low | 14 | 10 (71%) | 4 (29%) | 0.027 | 0.870 |
| Stage | |||||
| I/II | 12 | 6 | 6 | 1.695 | 0.193 |
| III/IV | 28 | 20 | 8 | ||
| LN metastasis | |||||
| Positive | 26 | 18 | 8 | 0.584 | 0.445 |
| Negative | 14 | 8 | 6 |
Discussion
Bcl-2 is an anti-apoptotic protein, which localized in intracellular membranes, mostly in the outer mitochondrial membrane, nuclear membrane, and the endoplasmic reticulum [4]. Bcl-2 regulates ion channels, caspase status, and cytochrome c localization, and has an anti-apoptotic function [5].
Ki-67 is an antigen associated with cell proliferation [6]. Ki-67 is expressed during the proliferation and synthesis phases of the cell cycle (G1, S, G2, and M), and however it is not expressed during G0 phase [7].
Bcl-2 has been reported in a variety of human epithelial malignant tumors including gastric carcinoma [8]. Bcl-2 protein was not detected in chronic gastritis, but aberrant expression was found in gastric epithelial intestinal metaplasia and dysplasia. The overexpression of Bcl-2 protein is an early event in gastric tumorigenesis, before gastric dysplastic changes occur [9]. The expression of bcl-2 was an independent prognostic factor for patients with gastric cancer; it might be a candidate for the gastric cancer staging system [10]. Bcl-2 expression has an additional contribution in predicting response to this chemotherapy combination [11,12]. However, Martin-Arruti et al reported that Bcl-2 expression does correlate with worse prognosis [13].
The detection of expression of Ki67 in gastric cancer may provide useful prognostic information for patients with the disease [14-16]. The overexpression of Ki67 was negatively correlated with carcinoma differentiation [17]. The routine evaluation of Ki67 levels could be a useful tool in identification of patient with more aggressive disease and contribute to a better therapeutic approach [18]. A study showed that Bcl-2 and Ki-67 expression and apoptosis were not different among patients with and without a history of gastric cancer in first degree relatives8. Tsamandas et al reported that Bcl-2 expression did not correlate with Ki-67 [19].
In this study, we assessed the expression of Bcl-2 or Ki67 in gastric cancer tissues. Our data showed that the positive rates of Bcl-2 and Ki67 protein expression in gastric cancer tissues were higher than normal gastric mucous tissues. Correlation analysis showed that the expression of Bcl-2 is not correlated with that of Ki67. Positive expression of Bcl-2 or Ki67 did not correlate with age, gender, differentiation, stage and lymph node metastasis. The total negative rate of Bcl-2 and Ki67 is only 5%, which reduce efficiently the missing diagnose of gastric cancer.
Acknowledgements
This study was supported by Scientific Research Program Funded by Shaanxi Provincial Education Department (Program No. 12JK0765), Scientific Research Program Funded by Xi’an Medical University (Program No. 11FZ07) and Scientific Research Program Funded by The First Affiliated Hospital of Xi’an Medical University (Program No. XYFY10-04).
Disclosure of conflict of interest
None.
References
- 1.Global Facts and Figures 2007. Available at: www. cancer.org. Accessed May 1, 2011.
- 2.Sun GR, Dong XY, He QS, Qu H, Wang YM. Expression and clinical significance of CK19 and CK20 expressions in transverse mesocolon biopsies from patients with gastric carcinoma. Cell Biochem Biophys. 2012;62:361–364. doi: 10.1007/s12013-011-9293-2. [DOI] [PubMed] [Google Scholar]
- 3.Shen X, Zheng JY, Shi H, Zhang Z, Wang WZ. Survivin knockdown enhances gastric cancer cell sensitivity to radiation and chemotherapy in vitro and in nude mice. Am J Med Sci. 2012;344:52–58. doi: 10.1097/MAJ.0b013e318239c4ee. [DOI] [PubMed] [Google Scholar]
- 4.Antonsson B. Bax and other pro-apoptotic Bcl-2 family killer-proteins and their victim, the mitochondrion. Cell Tissue Res. 2001;306:347–361. doi: 10.1007/s00441-001-0472-0. [DOI] [PubMed] [Google Scholar]
- 5.Karam JA, Lotan Y, Karakiewicz PI, Ashfaq R, Sagalowsky AI, Roehrborn CG, Shariat SF. Use of combined apoptosis biomarkers for prediction of bladder cancer recurrence and mortality after radical cystectomy. Lancet Oncol. 2007;8:128–136. doi: 10.1016/S1470-2045(07)70002-5. [DOI] [PubMed] [Google Scholar]
- 6.Morimoto K, Kim SJ, Tanei T, Shimazu K, Tanji Y, Taguchi T, Tamaki Y, Terada N, Noguchi S. Stem cell marker aldehyde dehydrogenase 1-positive breast cancers are characterized by negative estrogen receptor, positive human epidermal growth factor receptor type 2, and high Ki67 expression. Cancer Sci. 2009;100:1062–1068. doi: 10.1111/j.1349-7006.2009.01151.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Gerdes J, Li L, Schlueter C, Duchrow M, Wohlenberg C, Gerlach C, Stahmer I, Kloth S, Brandt E, Flad HD. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 1991;138:867–873. [PMC free article] [PubMed] [Google Scholar]
- 8.Erkan G, Gonul II, Kandilci U, Dursun A. Evaluation of apoptosis along with BCL-2 and Ki-67 expression in patients with intestinal metaplasia. Pathol Res Pract. 2012;208:89–93. doi: 10.1016/j.prp.2011.12.002. [DOI] [PubMed] [Google Scholar]
- 9.Anagnostopoulos GK, Stefanou D, Arkoumani E, Sakorafas G, Pavlakis G, Arvanitidis D, Tsianos E, Agnantis NJ. Bax and Bcl-2 protein expression in gastric precancerous lesions: immunohistochemical study. J Gastroenterol Hepatol. 2005;20:1674–1678. doi: 10.1111/j.1440-1746.2005.04057.x. [DOI] [PubMed] [Google Scholar]
- 10.Liu X, Cai H, Huang H, Long Z, Shi Y, Wang Y. The prognostic significance of apoptosis-related biological markers in Chinese gastric cancer patients. PLoS One. 2011;6:e29670. doi: 10.1371/journal.pone.0029670. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Yildirim M, Suren D, Goktas S, Dilli UD, Kaya C, Copuroglu R, Yildiz M, Sezer C. The predictive role of Bcl-2 expression in operable locally advanced or metastatic gastric carcinoma. J BUON. 2012;17:106–109. [PubMed] [Google Scholar]
- 12.Korbakis D, Scorilas A. Quantitative expression analysis of the apoptosis-related genes BCL2, BAX and BCL2L12 in gastricadenocarcinoma cells following treatment with the anticancer drugs cisplatin, etoposide and taxol. Tumour Biol. 2012;33:865–875. doi: 10.1007/s13277-011-0313-z. [DOI] [PubMed] [Google Scholar]
- 13.Martin-Arruti M, Vaquero M, Díaz de Otazu R, Zabalza I, Ballesteros J, Roncador G, García-Orad A. Bcl-2 and BLIMP-1 expression predict worse prognosis in gastric diffuse large B cell lymphoma (DLCBL) while other markers for nodal DLBCL are not useful. Histopathology. 2012;60:785–792. doi: 10.1111/j.1365-2559.2011.04160.x. [DOI] [PubMed] [Google Scholar]
- 14.Al-Moundhri MS, Nirmala V, Al-Hadabi I, Al-Mawaly K, Burney I, Al-Nabhani M, Thomas V, Ganguly SS, Grant C. The prognostic significance of p53, p27 kip1, p21 waf1, HER-2/neu, and Ki67 proteins expression in gastric cancer: a clinicopathological and immunohistochemical study of 121 Arab patients. J Surg Oncol. 2005;91:243–252. doi: 10.1002/jso.20324. [DOI] [PubMed] [Google Scholar]
- 15.Zheng Y, Wang L, Zhang JP, Yang JY, Zhao ZM, Zhang XY. Expression of p53, c-erbB-2 and Ki67 in intestinal metaplasia and gastric carcinoma. World J Gastroenterol. 2010;16:339–344. doi: 10.3748/wjg.v16.i3.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Docea AO, Mitruţ P, Grigore D, Pirici D, Călina DC, Gofiţă E. Immunohistochemical expression of TGF beta (TGF-β), TGF beta receptor 1 (TGFBR1), and Ki67 in intestinal variant of gastric adenocarcinomas. Rom J Morphol Embryol. 2012;53:683–692. [PubMed] [Google Scholar]
- 17.Chen L, Li X, Wang GL, Wang Y, Zhu YY, Zhu J. Clinicopathological significance of overexpression of TSPAN1, Ki67 and CD34 in gastric carcinoma. Tumori. 2008;94:531–538. doi: 10.1177/030089160809400415. [DOI] [PubMed] [Google Scholar]
- 18.Tzanakis NE, Peros G, Karakitsos P, Giannopoulos GA, Efstathiou SP, Rallis G, Tsigris C, Kostakis A, Nikiteas NI. Prognostic significance of p53 and Ki67 proteins expression in Greek gastric cancer patients. Acta Chir Belg. 2009;109:606–611. doi: 10.1080/00015458.2009.11680496. [DOI] [PubMed] [Google Scholar]
- 19.Tsamandas AC, Kardamakis D, Tsiamalos P, Liava A, Tzelepi V, Vassiliou V, Petsas T, Vagenas K, Zolota V, Scopa CD. The potential role of Bcl-2 expression, apoptosis and cell proliferation (Ki-67 expression) in cases of gastric carcinoma and correlation with classic prognostic factors and patient outcome. Anticancer Res. 2009;29:703–709. [PubMed] [Google Scholar]