| Abstract|| |
Background: P-cadherin is cell-cell adhesion glycoprotein which can be used as a myoepithelial cell (MEC) marker in the breast lesions. MEC layer is retained in most benign lesions and loss of this outer layer is hallmark of infiltrating carcinomas in the breast. Aim: To evaluate the expression of P-cadherin as MEC marker in the differential diagnosis of benign and malignant breast lesions. Materials and Methods: Immunohistochemical staining was done using P-cadherin-specific antibody on formalin fixed paraffin-embedded sections of 25 benign and 15 malignant breast lumps. Results: All 25 cases of benign breast lesions showed positive P-cadherin immunostaining, while only 4 out of 15 cases of infiltrating ductal carcinoma showed positive immunostaining for P-cadherin. In the case of benign lesions, staining index varied from 4 to 6 or 7 to 9, while in case of malignant lesions, 11 cases showed staining index from 1 to 3. Only 4 out of 15 malignant cases had staining index from 4 to 6. None of them showed index from 7 to 9. Conclusions: P-cadherin as a MEC marker can be used in differentiating benign and malignant breast lesions.
Keywords: Breast lesions, differential diagnosis, immunohistochemistry, P-cadherin
|How to cite this article:|
Bhatia Y. P-cadherin as myoepithelial cell marker for differential diagnosis of benign and malignant breast lesions. Indian J Pathol Microbiol 2013;56:6-9
|How to cite this URL:|
Bhatia Y. P-cadherin as myoepithelial cell marker for differential diagnosis of benign and malignant breast lesions. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Dec 2];56:6-9. Available from: https://www.ijpmonline.org/text.asp?2013/56/1/6/116140
| Introduction|| |
Cadherins are cell-cell adhesion glycoproteins that form calcium-dependent intercellular junctions and play an essential role in morphogenesis, development, and maintenance of adult tissues and organs.  The cadherin family is subdivided into various subfamilies including classical E-, P-, and N-cadherin. The expression of P-cadherin is only restricted to basal or lower layers of stratified epithelia, including prostate and skin and also to breast myoepithelial cells (MECs).  Normal breast ducts and lobules are comprised of two epithelial layers. Outer MECs are spindle-shaped contractile, smooth muscle like cells.  Loss of this layer is hallmark of infiltrating carcinomas in breast as it is retained in most benign as well as in ductal carcinoma in situ (DCIS).  This study was conducted to assess the ability of immunohistochemical (IHC) marker, P-cadherin, to distinguish between benign and malignant breast lesions, through evaluating the P-cadherin expression in different benign and malignant lesions of breast.
| Materials and Methods|| |
This is a retrospective study of P-cadherin expression on cases of benign and malignant lesions of breast using IHC method. The paraffin-embedded blocks were taken from the Department of Pathology, Government Medical College, Amritsar and collected over a period of 3 years, 2007-2009. The total number of cases studied was 40, comprising of 25 benign and 15 malignant breast lesions. Normal breast tissue adjacent to the pathological area was taken as control.
Paraffin blocks were sectioned at 4 μm in thickness, mounted on freshly prepared 0.01% poly-l-lysine-coated slides. Slides were dried overnight at 37°C, de-waxed in xylene, and hydrated. Endogenous peroxidase activity was blocked by adding freshly prepared 0.3% H 2 O 2 in methanol for 10 min followed by three washings in Tris-buffered saline (TBS). Heat-induced antigen retrieval was used with pressure cooking for 2 min in 0.1 M citrate buffer (pH 6.0). They were washed with running water and then rinsed with TBS. Sections were incubated for 30 min at room temperature with primary antibodies, monoclonal mouse anti-P-cadherin clone 56C1 procured from the Diagnostic Biosystems, New Delhi, India. After washing thoroughly with TBS, the sections were incubated with secondary antibody for 1 h at room temperature. Sections were again thoroughly washed with TBS and incubated with tertiary antibody for another 1 h at room temperature, followed by rinsing with TBS. A drop of diaminobenzidine was then spread over the sections for 7 min and then it was rinsed in water. The sections were counter-stained with hematoxylin for 30-45 s before rinsing with running water for 3 min and dehydrated in increasing alcohol concentration and mounted.
Positive control section was normal breast tissue adjacent to pathological area. Negative control section was processed similarly by omission of primary antibody.
Interpretation of P-Cadherin reactivity
The P-cadherin reactivity was graded by determining the percentage of P-cadherin immunoreactive cells, i.e., brown cytoplasmic reactivity and intensity of staining.  Intensity scores (IS) of IHC reaction, as viewed under light microscope, are as follows:
0 - Negative
1 - Weak
2 - Medium
3 - High
Proportion score (PS) cells showing staining:
0 - No staining
1 - 1-10%
2 - 11-50%
3 - >50%
Total score (TS) = IS × PS (0-9)
o Negative Positive
o TS≤3 TS>3
The cases of breast lesions with P-cadherin expression were evaluated using Chi-square test with Yates' correction. Any P value less than 0.05 was considered statistically significant.
| Results|| |
The study included 40 cases of breast lesions classified as 25 cases of benign breast lesions and 15 cases of malignant breast lesions.
Out of 25 cases of benign lesions, no case showed negative result. All cases were positive for immunostaining with P-cadherin. Out of these cases, staining index for majority (80%) was between 4 and 6. The subgroup with staining index between 7 and 9 comprised 20% of cases. Majority of the cases with fibroadenoma (12 in number) showed staining index of 4-6. Rest of cases of fibroadenoma (3 in number) showed staining index of 7-9 [Figure 1]. This variation is more so due to variation in intensity of staining. Four cases of sclerosing adenosis showed staining index of 4-6. Among the two cases of fibrocystic disease, one showed staining index of 4-6, while other had staining index of 7-9. The only case of giant fibroadenoma showed staining index of 7-9 [Table 1].
|Figure 1: Immunohistochemical staining with P-cadherin in case of fi broadenoma of the breast showing positi ve immunostaining|
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|Table 1: P-cadherin immunohistochemical staining in benign breast lesions|
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Out of 15 malignant cases, 8 cases (53%) showed no staining. The staining index was zero [Figure 2]. Three cases (20%) showed staining index between 1 and 3 and four cases (27%) showed staining index of 4-6. None of the case had a staining index in the range of 7-9 [Table 2].
|Figure 2: Immunohistochemical staining with P-cadherin in case of infiltrating carcinoma of the breast showing negati ve immunostaining|
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|Table 2: P-cadherin immunohistochemical staining in malignant breast lesions|
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Chi-square test with Yates' correction was applied to the results of P-cadherin expression in the 40 cases of breast lesions [Table 3]. The two-tailed P value is less than 0.0001. Thus, the association between the type of lesions of breast and result of IHC staining with P-cadherin is considered to be statistically significant.
|Table 3: Comparison between results of P-cadherin immunostaining in benign and malignant lesions|
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| Discussion|| |
The epithelium throughout breast duct system is bilayered, consisting of an inner epithelial layer and an outer myoepithelial layer. The importance of this double cell layer cannot be overemphasized because it is one of the main guides used to distinguish benign from malignant lesions.  Despite its well-circumscribed morphologic features, because of variability of histological patterns and irregularity of growth pattern, epitheliosis can be confused with DCIS. Similarly, some forms of DCIS can be underdiagnosed because of the unusual regularity of the malignant proliferation.  The use of IHC method for MEC layer greatly aids in diagnosis of many pathological conditions.
In this study, we have made an effort to study the significance of expression of P-cadherin in various benign and malignant lesions of breast. P-cadherin was identified in all MECs of all breast tissues studied, with no differences between ducts and lobules, with overall strong staining. This study shows that P-cadherin expression can help in the differentiation between benign and malignant lesions. This is evidenced by the fact that P-cadherin immunoreactivity was seen in 100% of the benign breast lesions and only 27% of the malignant lesions submitted in this study [Table 1] and [Table 2]. This may be attributed to the fact that P-cadherin is cell-cell adhesion protein showing expression in MEC layer, so it shows positive results in benign lesions. Similar findings were reported by Palacios et al. in 2002. They studied anomalous expression of P-cadherin in breast lesions, especially carcinomas. It showed that P-cadherin was detected in 95.35% cases of normal breast and benign lesions.  A study conducted in 2003 by Kovαcs and Walker  also assessed the value of P-cadherin as myoepithelial marker in differential diagnosis of benign and malignant lesions of breast. All MECs in normal breast ducts, ductules, and lobules and sclerotic lesions showed strong staining for P-cadherin. There was no staining of tubular carcinomas. P-cadherin was detected in the MECs of ducts and lobules of all 10 samples from reduction mammoplasties. There was no difference in reactivity between large and small ducts or lobules. There were similar findings in normal breast tissue associated with the lesions.  These findings were similar to findings associated with our study. So as this study shows P-cadherin as a good MEC marker, so whenever confusion arises and diagnosis is not clear with routine staining, this can be used as a good marker to differentiate various lesions of the breast.
In our study, 75% of cases of fibroadenoma have staining index of 4-6 and rest of cases (25%) has staining index of 7-9. Most of cases showed cytoplasmic as well as membranous staining in >50% of tissue section (3+). Gama et al. studied expression of P-cadherin in mammary tissue to analyze the possible role of P-cadherin in mammary tissue. Its expression was examined immunohistochemically in 13 samples of normal ( n = 2) and hyperplastic ( n = 11) mammary tissues. In normal and hyperplastic mammary glands, P-cadherin was restricted to MECs, usually at sites of cell-to-cell contact.
In this study, the staining intensity was diffuse and medium, comparable to that of positive control (2+). In few cases, only membranous staining was seen. The areas showing hyperplasia, cystic change or adenosis showed maximum positivity. In a study done in 2005 on cadherins and mammary gland, P-cadherin was expressed both in epithelial and MECs of normal breast tissue as well as benign lesions. Its expression was lost in carcinomas. 
Out of 15 malignant cases, 8 cases (53%) showed no staining (staining index was zero). Four cases (27%) of them were immunopositive for P-cadherin with all cases having staining index between 4 and 6. Three cases (20%) had staining index in the range of 1-3. Similar findings have been reported by Palacios et al. in 2002. They studied anomalous expression of P-cadherin in breast carcinoma. P-cadherin expression was detected in 9 out of 45 cases (20%) of infiltrating ductal carcinomas not otherwise specified. 
The proportion of staining was 3+ in three of the positive case and 2+ in the other one. The staining characteristics of the malignant lesions were different from benign lesions.
There have been studies showing P-cadherin-positive staining in basal-like subtype of breast carcinomas. This can be source of confusion but it is necessary to evaluate these findings in combination with routine staining findings. 
It has been observed in a study conducted in 2003 that P-cadherin is a highly sensitive marker for MECs, with exceptions in few cases of benign and malignant proliferations which were also stained. The distinct staining of MECs for P-cadherin and the lack of staining for myofibroblasts, when differentiating between a radial scar and a tubular carcinoma, show its advantage over smooth muscle actin.  Thus, P-cadherin should be considered a helpful tool in the differential diagnosis of breast lesions.
| Conclusion|| |
This study shows that P-cadherin expression strongly correlates with the type of breast lesion, which can help in the differentiation between benign and malignant lesions whenever there is confusion on routine staining. This is evidenced by the fact that P-cadherin immunoreactivity was seen in 100% of benign cases, whereas only 27% of malignant cases were P-cadherin immunoreactive. Thus, P-cadherin is a highly sensitive marker for MECs and should be considered a useful marker in the differential diagnosis of breast lesions wherever there is confusion in diagnosis with routine methods.
| References|| |
|1.||Paredes J, Correia AL, Ribeiro AS, Albergaria A, Milanezi F, Schmitt FC. P-cadherin expression in breast cancer: A review. Breast Cancer Res 2007;9:214. |
|2.||Lo Muzio L, Campisi G, Farina A, Rubini C, Pannone G, Serpico R, et al. P-cadherin expression and survival rate in oral squamous cell carcinoma: An immunohistochemical study. BMC Cancer 2005;5:63. |
|3.||Adriance MC, Inman JL, Petersen OW, Bissell MJ. Myoepithelial cells: Good fences make good neighbors. Breast Cancer Res 2005;7:190-7. |
|4.||Man YG, Tai L, Barner R, Vang R, Saenger JS, Shekitka KM, et al. Cell clusters overlying focally disrupted mammary myoepithelial cell layers and adjacent cells within the same duct display different immunohistochemical and genetic features: Implications for tumor progression and invasion. Breast Cancer Res 2003;5:R231-41. |
|5.||Collett K, Stefansson IM, Eide J, Braaten A, Wang H, Eide GE, et al. A basal epithelial phenotype is more frequent in interval breast cancers compared with screen detected tumors. Cancer Epidemiol Biomarkers Prev 2005;14:1108-12. |
|6.||Mills SE, Sternberg S. Breast. In: Mills SC, editor. Histology for Pathologists. 3 rd ed. New York: Lippincott Williams and Wilkins; 2004. p. 10-53. |
|7.||Silverstein MJ, Lagios MD, Craig PH, Waisman JR, Lewinsky BS, Colburn WJ, et al. A prognostic index for ductal carcinoma in situ of the breast. Cancer 1996;77:2267-74. |
|8.||Palacios J, Benito N, Pizarro A, Suárez A, Espada J, Cano A, et al. Anomalous expression of P-cadherin in breast carcinoma. Correlation with E-cadherin expression and pathological features. Am J Pathol 1995;146:605-12. |
|9.||Kovács A, Walker RA. P-cadherin as a marker in the differential diagnosis of breast lesions. J Clin Pathol 2003;56:139-41. |
|10.||Gama A, Paredes J, Albergaria A, Gartner F, Schmitt F. P-cadherin expression in canine mammary tissues. J Comp Pathol 2004;130:13-20. |
|11.||Knudsen KA, Wheelock MJ. Cadherins and the mammary gland. J Cell Biochem 2005;95:488-96. |
|12.||Liu N, Yu Q, Liu TJ, Gebreamlak EP, Wang SL, Zhang RJ, et al. P-cadherin expression and basal-like subtype in breast cancers. Med Oncol 2012;29:2606-12. |
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Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]