|MINI SYMPOSIUM: BREAST- ARTICLE
|Year : 2012 | Volume
| Issue : 1 | Page : 60-65
Myoepithelial expression of Fas and strong nuclear expression of FasL in epithelial cells: A marker for risk stratification of breast cancer
KJ Ranade1, AV Nerurkar1, MD Phulpagar2, NV Shirsat3
1 Department of Biochemistry, T.N. Medical College, B.Y.L. Nair Hospital, Mumbai, India
2 Department of Pathology, T.N. Medical College, B.Y.L. Nair Hospital, Mumbai, India
3 Department of Neuro Oncology, Advanced Centre for Treatment, Research and Education in Cancer (Tata Memorial Centre), Kharghar, Navi Mumbai, India
|Date of Web Publication||25-Jul-2012|
A V Nerurkar
Department of Biochemistry, T.N. Medical College, B.Y.L. Nair Hospital, Mumbai
Source of Support: None, Conflict of Interest: None
Aim: The clinical significance of Fas and FasL in hormone-sensitive carcinomas has been reported. Our objective was to evaluate the expression of apoptosis-regulating genes Fas and FasL in Indian breast cancer and fibroadenoma patients in relation to hormone receptor status. Study Design: Retrospective. Materials and Methods: Paraffin-embedded tissue samples from 63 untreated female patients with invasive ductal carcinoma (IDC) and 32 female patients with fibroadenoma were studied. Expression of Fas and FasL was evaluated using immunohistochemical staining method. Statistical Analysis: Fisher's exact test and nonparametric correlation test (Spearman rank correlation test) were performed. Result: Fas was detected in 97% of the fibroadenomas and there was a slight decrease in levels of expression with histological grades of IDC. The expression of FasL was detected in 75% fibroadenomas and its expression increased in IDC. There was no correlation between Fas, FasL expression and hormone receptor status. Strong expression of Fas in myoepithelial cells was noted in 12 out of 32 fibroadenoma cases. Conclusion: Expression of Fas and FasL alone is unlikely to be important as a predictive factor as they express in both normal and malignant breast epithelium. But strong expression of Fas in myoepithelial cells along with strong nuclear expression of FasL in epithelial cells of fibroadenoma could be useful as an early predictive factor for onset of malignancy.
Keywords: Apoptosis, fibroadenoma, Fas, FasL, invasive ductal carcinoma
|How to cite this article:|
Ranade K J, Nerurkar A V, Phulpagar M D, Shirsat N V. Myoepithelial expression of Fas and strong nuclear expression of FasL in epithelial cells: A marker for risk stratification of breast cancer. Indian J Cancer 2012;49:60-5
|How to cite this URL:|
Ranade K J, Nerurkar A V, Phulpagar M D, Shirsat N V. Myoepithelial expression of Fas and strong nuclear expression of FasL in epithelial cells: A marker for risk stratification of breast cancer. Indian J Cancer [serial online] 2012 [cited 2020 Jul 10];49:60-5. Available from: http://www.indianjcancer.com/text.asp?2012/49/1/60/98921
| » Introduction|| |
Breast cancer is one of the most frequent cancers among women and the fifth most common cause of cancer death worldwide.  Invasive ductal carcinoma (IDC) accounts for about 80% of all breast cancers. Cancer cells develop multiple mechanisms to evade apoptosis either by inactivating proapoptotic factors or by upregulating antiapoptotic factors. For many years estrogen has been shown to be a mitogen-stimulating cell proliferation factor. It has been shown to prevent apoptosis through estrogen receptor-mediated mechanism and to be associated with the risk of developing breast cancer.
The Fas and FasL system plays a key role in regulating apoptotic cell death and corruption of this signaling pathway has been shown to participate in immune escape and tumorogenesis.  The breast cancer cells can induce apoptosis of tumor-infiltrating lymphocytes through the expression of FasL,  thus can counterattack the immune system. This may be a mechanism of immune evasion in breast cancer. Tumor cells and tumor-infiltrating lymphocytes may express Fas and FasL in various proportions and their interplay may affect tumor behavior. In this study, expression of Fas and FasL in fibroadenoma and breast cancer cases has been studied, and its correlation with the hormone receptor status of the patients has been done.
| » Materials and Methods|| |
The present study was carried out on retrospective samples. Paraffin-embedded tissue samples between the years 2000 and 2006 were randomly selected for this study. Histopathologically proved and graded invasive ductal carcinoma (IDC) and fibroadenoma tissues were obtained from the Pathology Department of our institution after being fixed in 10% buffered formalin and embedded in paraffin for immunohistochemical analysis. None of the patients had received chemotherapy, radiotherapy, or immunotherapy at the time of sampling. This work was approved by the Institutional Ethics Committee. The clinical data of each patient was maintained, inclusive of age, sex and hormone receptor status.
Patients with cancerous lesions: The present study included 63 untreated female patients with invasive ductal carcinoma, between the age 22 to 50 (median age, 36 years). Histopathologically, the tumors were categorized as IDC I, IDC II and IDC III (21 cases each) using Bloom Richardson's scoring and grading.
Patients with benign breast lesions: Benign breast lesions (fibroadenoma) of 32 patients, between the age 18 to 52 (median age, 35 years), were studied.
Inclusion/ Exclusion criteria
All biopsies were collected from patients in a specified age group who had not received any therapy before surgery. Cases other than IDC and fibroadenoma were not included.
Tissue sections were cut at 5 μm and mounted on poly-l-lysine-coated glass slides (Sigma, St. Louis, MO, USA). Antigen retrieval was then performed by heating the slides immersed in citrate buffer, pH 6, in microwave oven.  Primary rabbit polyclonal antibodies against Fas (Sc-715) and FasL (Sc-834) were obtained from SantaCruz Biotechnology (Santa Cruz, USA) and used at a dilution of 1:100 in 1% bovine serum albumin BSA. Horseradish peroxidase-linked antirabbit Immunoglobulin G (NA 934V) obtained from GE Healthcare UK Lt. (Buckinghamshire, UK) was used as a secondary antibody, and the bound antibody was detected using 3, 3' diaminobenzidine as a substrate and Harris' hematoxylin as a counterstain. Positive and negative controls were included in each batch of slides. The correlation between the level of expression and the histological grade was analyzed using the Fisher's exact test. Nonparametric correlation test (Spearman rank correlation test) using Spearman correlation coefficient (r) was done between expressions and hormone receptor status.
Interpretation of slides: The immunohistochemical analysis of Fas and FasL expression in benign breast lesions and IDC of breast was carried out on the basis of the percentage of cells showing staining.
The level of expression was scored as follows:
0 = negative, less than 5% of cells staining
1+ = weak staining, between 6% and 25% of cells staining
2+ = moderate staining, between 26% and 50% of cells staining
3+ = medium strong staining, between 51% and 75% of cells staining
4+ = strong staining, more than 75% of cells staining
The scores of Estrogen Receptor (ER)/Progesterone Receptor (PR) status obtained were then correlated with Fas and FasL expression.
Statistical analysis was performed using Graphpad Instat 3 software. Fisher's exact test was used to find out the significant difference in expression of Fas, FasL in benign and malignant tissues. Spearman's correlation coefficient (r) was used to find out correlation between hormone receptor status and genetic expressions.
| » Results|| |
In benign breast disease 31 out of 32 cases (97%) showed Fas expression. Mainly Fas expressed in cytoplasm of the cells, however, the presence of cytoplasmic along with nuclear staining was observed in some of the cases studied. Some of the benign cases positive for Fas, expressed strong myoepithelium positivity (12/32) [Figure 1]a. In IDC I, II and III, Fas expression was observed in 86%, 81% and 76% respectively [Figure 1]b-d. In some positive cases, Fas expression was also observed in stroma cells and lymphocytes .The percentages of different levels of expression of Fas in benign and IDC cases have been illustrated in Graph 1a.
|Figure 1: Representative photomicrographs of the staining for Fas (a-d) and FasL (e-h) in case of fibrobroadenoma (a and e), IDC I (b and f), IDC II (c and g) and IDC III (d and h). Immunochemical staining shows positive staining in brown (DAB as substrate), counterstained with hematoxylin. All photographs were taken with digital camera at ×40 magnification under identical conditions. (a) showing strong myoepithelial positivity|
Click here to view
FasL expression was observed in 75% (24/32) [Figure 1]e of the benign cases. FasL staining was localized to cytoplasm in some cases, and in some cases FasL was seen in both the cytoplasm and nucleus of the cells. In case of immunohistochemical analysis of FasL in carcinoma cases, overall 90% (57/63) cases expressed FasL. IDC I, IDC II and IDC III showed 86%, 90% and 95% positivity respectively [Figure 1]f-h. The percentages of different levels of expression of FasL in benign and IDC cases are illustrated in Graph 1b. FasL expression was not observed in myoepithelial cells of any of the benign cases. When Fas and FasL were correlated, we observed statistical positive correlation between Fas and FasL in malignant lesions (spearman r = 0.3856 and P =0.0018, 95% confidence interval: 0.1452 to 0.5833).
Out of 63 IDC cases, ER [Table 1] and PR [Table 2] status was obtained from 47 cases, and correlative studies between hormone receptor status and immunohistochemical staining of Fas and FasL were done in these 47 cases only.
When statistical correlation between hormone receptor status and expression of Fas, FasL was performed it was found that Fas and FasL were independent of hormone receptor status.
| » Discussion|| |
Fas and its natural ligand FasL are members of the tumor necrosis family and expressed on cellular membranes, being involved in the induction of programmed cell death or apoptosis. , Reduced expression of Fas and/or increased expression of FasL is known to exist in many cancers including breast cancer, ,,, so the Fas/FasL system may play a role in the course of the disease. Lack of cell surface Fas expression is one of the main routes of apoptotic resistance in the tumor formation and progression.  Functional mutations and polymorphisms in the Fas gene that impair apoptotic signal transduction are associated with susceptibility to various types of cancer. , It has been shown that breast cancer cells can induce the apoptosis of tumor-infiltrating lymphocytes(TILs) through the expression of FasL, which can counterattack the immune system and may be the mechanism of immune evasion in breast cancer.  This might be suggestive of a relation between FasL expression by cancer and systemic immunosuppression. ,
In the present study, the expression of Fas and FasL in fibroadenoma, a benign breast disease and in different grades of IDC was observed. It was seen that expression of Fas was maximum in benign cases and its expression decreased in malignant breast cases. Ninety-seven percent of benign cases showed Fas expression whereas in IDC I, II and III, Fas expression was observed in 86%, 81% and 76% (average 75%) respectively. Thus we observed slightly decreased Fas expression with increased grade of IDC. Our results are consistent with those obtained by Mottolese et al., who also observed decreased Fas expression in malignant cases compared to benign cases. Bebenek et al., observed decreased Fas expression in breast cancer patients with perilymphatic infiltration of fat. Analysis revealed that perilymphatic fat infiltration shortened overall survival rate in breast cancer. But Bebenek et al., further stated that decreased expression of Fas in breast cancer patients with perilymphatic infiltration cannot be an independent predictor of survival. In our study, we observed both strong and mild expression of Fas in benign as well as malignant tissues. Jaberipour et al.,  compared the Fas expression in low-grade, high-grade and healthy population. They observed 3-4fold increased Fas expression in the peripheral blood cells of lower grade IDC patients in comparison with healthy individuals. They also concluded that Fas gene expression decreased in high-grade tumors.
In case of immunohistochemical analysis of FasL, 75% of benign cases expressed FasL. Whereas in IDC I, II and III, 86%, 90% and 95% positivity was observed respectively. Thus we observed an upregulated expression of FasL in breast carcinoma compared to benign cases and its expression was found to increase slightly with the histological grade. Our results are consistent with the results observed by Mallauer et al., who have shown that FasL expression is frequently upregulated in breast cancer.  Further El-Sarha AI et al., observed that FasL expression directly correlated with distant metastasis and poor overall survival.
In the present study 12 out of 32 benign cases expressed strong Fas in myoepithelial cells. Out of these 12 cases, 10 cases showed uniform Fas expression in epithelial cells also. Strong cytoplasmic and nuclear expression of FasL was observed in the epithelial cells of these cases. Follow-up study for these cases showed that 3 out of 12 fibroadenoma turned into breast cancer (25%). The above mentioned pattern was also observed in benign tissue adjacent to malignant tissue in two cases.
Beside contractile functions, myoepithelial cells also play a key role in the organizational development of the mammary gland and that loss/or change of myoepithelial cell function is a key step in the development of breast cancer.  It was Barsky et al., who showed that myoepithelial cells exhibit many antitumerogeic properties such as the ability to inhibit tumor cell invasion, angiogenesis and cell cycle arrest.  Strong Fas expression in myoepithelial cells and simultaneous strong FasL expression in epithelial cells of fibroadenoma may indicate the early apoptosis of myoepithelial cells which have a function in tumor suppression.
Further, in our study all four phenotypes-Fas positive/FasL positive, Fas positive/FasL negative, Fas negative/FasL positive and Fas negative/FasL negative-were observed in benign and malignant cases. Similar phenotypes were also observed by Sjostrum et al., in primary tumor tissues and involved lymph nodes. They could not detect much difference in Fas expression between tumor tissues and involved lymph nodes, however, FasL expression was shown to be reduced in involved lymph nodes. Statistically, positive correlation between Fas and FasL was also observed in malignant cases (spearman r = 0.3858 and P =0.0018, 95% confidence interval: 0.1452 to 0.5833). However, no such correlation was observed in the benign cases studied, but inverse relation between Fas and FasL expression was observed in some of the benign cases studied. Our results are consistent with the findings of Sjostrum et al.,, which stated that Fas correlated positively with FasL in breast carcinoma. When these gene expressions were correlated with the ER/PR status of the patients, it was observed that Fas and FasL were independent of the ER/PR status.
It has been shown that fibroadenoma is a long-term risk factor for breast cancer, especially with family history. , In the present study the presence of strong myoepithelial expression of Fas and simultaneous cytoplasmic and nuclear expression of FasL in epithelial cells could be suggestive as an early predictive factor or signal for onset of malignancy. However, follow-up studies with a greater sample size are required to conclude significantly.
| » Acknowledgment|| |
We sincerely acknowledge Mr. Umesh Kadam, technician A.C.T.R.E.C. for the technical help provided by him.
| » References|| |
|1.||World Health Organization (February 2006). Fact sheet No. 297. Cancer Retrieved on 2007- 04-26. |
|2.||Zhang X, Miao X, Sun T, Tan W, Qu S, Xiong P, et al. Functional polymorphisms in cell death pathway genes FAS and FASL contribute to risk of lung cancer. Med Genet 2005;42:479-84. |
|3.||Cheng B. Association between up-regulation of Fas ligand expression and apoptosis of tumor-infiltrating lymphocytes in human breast cancer. J Huazhong Univ Sci Technolog Med Sci 2006;26:573-5. |
|4.||Holm R, Nesland JM. Microwave oven inimmunohistochemistry: An important tool for determination of antigens in formalin-fixed and paraffin-embedded tissue. Tidsskr Nor Laegeforen 1994;114:2392-4. |
|5.||Puiu L, Petrakou E, Apostolidou A, Athanassiadou A, Psiouri L, Papachatzopoulou A, et al. Lack of Fas (APO-1/CD95) gene structural alterations or transcript variant ratio changes in breast cancer. Cancer Lett 2003;194:91-7 |
|6.||Crew KD, Gammon MD, Terry MB, Zhang FF, Agrawal M, Eng SM, et al. Genetic polymorphisms in the apoptosis-associated genes FAS and FASL and breast cancer risk. Carcinogenesis 2007;28:2548-51. |
|7.||Gormus U, Ergen A, Yaylim-Eraltan I, Yilmaz H, Turna A, Bozkurt N, et al. Fas-1377 A/G polymorphism in lung cancer. In Vivo 2007;21:663-6. |
|8.||Reesink-Peters N, Hougardy BM, van den Heuvel FA, Ten Hoor KA, Hollema H, Boezen HM, et al. Death receptors and ligands in cervical carcinogenesis: Immunohistochemal study. Gynecol Oncol 2005;96:705-13. |
|9.||Xia C, Zhao H, Tang X. The expressions of Fas and FasL in human invasive ductal carcinomas and their surrounding tissues. Zhonghua Wai Ke Za Zhi 1999;37:648-50. |
|10.||Kykalos S, Mathaiou S, Karayiannakis AJ, Patsouras D, Lambropoulou M, Simopoulos C. Tissue Expression of the Proteins Fas and Fas Ligand in Colorectal Cancer and Liver Metastases. J Gastrointest Cancer 2011 [Epub ahead of print] |
|11.||Shao P, Ding Q, Qin C, Wang M, Tang J, Zhu J. Functional polymorphisms in cell death pathway genes FAS andFAS ligand and risk of prostate cancer in a Chinese population. Prostate 2011;71:1122-30 . |
|12.||Cao Y, Miao XP, Huang MY, Deng L, Lin DX, Zeng YX, et al. Polymorphisms of death pathway genes FAS and FASL and risk of nasopharyngeal carcinoma. Mol Carcinog 2010;49:944-50. |
|13.||Müschen M, Moers C, Warskulat U, Niederacher D, Betz B, Even J, et al. CD95 ligand expression in dedifferentiated breast cancer. J Pathol 1999;189:378-86. |
|14.||Müschen M, Moers C, Warskulat U, Even J, Niederacher D, Beckmann MW. CD95 ligand expression as a mechanism of immune escape in breast cancer. Immunology 2000;99:69-77. |
|15.||Mottolese M, Buglioni S, Bracalenti C, Cardarelli MA, Ciabocco L, Giannarelli D, et al. Prognostic relevance of altered Fas(cd95) system in human breast cancer. Int J Cancer 2000;89:127-32. |
|16.||Bebenek M, Dus D, Kozlak J. Fas expression in primary breast cancer is related to neoplastic infiltration of perilymphatic fat. Adv Med Sci 2008;53:49-53. |
|17.||Jaberipour M, Habibagahi M, Hosseini A, Abbasi M, Sobhani-Lari A, Talei A, et al. Detection of B cell lymphoma 2, tumor protein 53, and FAS gene transcripts in blood cells of patients with breast cancer. Indian J Cancer 2010;47:412-7. |
|18.||Müllauer L, Mosberger I, Grusch M, Rudas M, Chott A. Fas ligand is expressed in normal breast epithelial cells and is frequently up-regulated in breast cancer. J Pathol 2000;190:20-30. |
|19.||El-Sarha AI, Magour GM, Zaki SM, El-Sammak MY. Serum sFas and tumor tissue FasL negatively correlated with survival in Egyptian patients suffering from breast ductal carcinoma. Pathol Oncol Res 2009;15:241-50. |
|20.||Adriance MC, Inman JL, Petersen OW, Bissell MJ. Myoepithelial cells: Good fences make good neighbors. Breast Cancer Res 2005;7:190-7. |
|21.||Barsky SH, Karlin NJ. Mechanisms of disease: Breast tumor pathogenesis and the role of the myoepithelial cell. Nat Clin Pract Oncol 2006;3:138-51. |
|22.||Sjostrom-Mattson J, Von Boguslawski K, Bengtsson NO, Mjaaland I, Salmenkivi K, Blomqvist C. The expression of p53, bcl-2, bax, fas and fasL in the primary tumour and lymph node metastases of breast cancer. Acta Oncol 2009;48:1137-43. |
|23.||Sjostrum J, Blomqvist C, von Boguslawski K, Bengtsson NO, Mjaaland I, Malmström P, et al. The predictive value of bcl-2, bax, bcl-xL, bag-1, fas, and fasL for chemotherapy response in advanced breast cancer. Clin Cancer Res 2002;8:811-6. |
|24.||Ragnarsson GB, Mikaelsdottir EK, Vidarsson H, Jónasson JG, Olafsdottir K, Kristjansdottir K, et al. Intracellular Fas ligand in normal and malignant breast epithelium does not induce apoptosis in Fas-sensitive cells. Br J Cancer 2000;83:1715-21. |
|25.||E-Wakeel H, Umpleby HC. Systematic review of fibroadenoma as a risk factor for breast cancer. Breast 2003;5:302-7. |
|26.||Dupont WD, Page DL, Parl FF, Vnencak-Jones CL, Plummer WD Jr, Rados MS, et al. Long-term risk of breast cancer in women with fibroadenoma. N Engl J Med 1994;331:10-5. |
[Table 1], [Table 2]