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ORIGINAL ARTICLE Table of Contents   
Year : 2010  |  Volume : 53  |  Issue : 4  |  Page : 676-680
Cytokeratin 5/6 expression in benign and malignant breast lesions


1 Sri Guru Ram Das Institute of Medical Sciences and Research, Vallah, Sri Amritsar, India
2 Kahlon Diagnostics and Cancer Research Center, Amritsar, India
3 Department of Pathology, Shri Ram Murti Smarak Institute of Medical Sciences, Bareilly, India

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Date of Web Publication27-Oct-2010
 

   Abstract 

Background: Cytokeratin s (CK) are used for the fingerprinting of carcinomas in general. In breast tissue, the luminal epithelial cells express CK 8/18, CK 7 and CK 19, while basal/myoepithelial cells express CK 5/6, CK 14 and CK 17. Material and Methods: Immunohistochemical staining for cytokeratin 5/6 was applied on cell block sections of 23 cases of benign and 25 cases of malignant breast lesions using avidin biotin peroxidase technique. The distribution and intensity of staining was recorded and graded semiquantitatively. Result: All benign lesions showed positive immunoreaction, with the staining index varying from 6-9, except lactating adenoma. The malignant lesions comprised three cases of ductal carcinoma in situ (DCIS) and 22 cases of infiltrating ductal carcinoma, not otherwise specified, IDC (NOS). None of the DCIS cases showed a positive immunoreaction. Among the IDC (NOS) lesions, six cases of grade III breast carcinoma exhibited a positive immunohistochemical reaction, the staining index of which varied from 2-6. The staining reaction in the malignant lesions was only cytoplasmic and the intensity was significantly less than that of benign lesions. Conclusion: CK 5/6 expression breast carcinoma implies a 'basal like' molecular phenotype and is associated with poor prognosis. This antibody is also used as a component of panels to differentiate benign and malignant breast lesions.

Keywords: Benign and malignant breast lesions, cytokeratin 5/6, immunohistochemistry

How to cite this article:
Bhalla A, Manjari M, Kahlon S K, Kumar P, Kalra N. Cytokeratin 5/6 expression in benign and malignant breast lesions. Indian J Pathol Microbiol 2010;53:676-80

How to cite this URL:
Bhalla A, Manjari M, Kahlon S K, Kumar P, Kalra N. Cytokeratin 5/6 expression in benign and malignant breast lesions. Indian J Pathol Microbiol [serial online] 2010 [cited 2019 Dec 10];53:676-80. Available from: http://www.ijpmonline.org/text.asp?2010/53/4/676/72026



   Introduction Top


Cytokeratin (CK), an intermediate filament protein, reflects the epithelial cell type, state of tissue growth, differentiation, functional status and is used for the fingerprinting of various carcinomas. [1] The normal resting breast tissue is composed of luminal cells which express CK 8/18, CK 7, CK 19. The basal/ myoepithelial cells express CK 5/6, CK 14, CK 17 and smooth muscle actin. A small subset of cells, comprising less than 5% of entire cell population, express CK 5. These cells are dispersed in the inner layer of ductal system and differentiate into myoepithelial or glandular cells via intermediary cells. [2],[3]


   Materials and Methods Top


Twenty three cases of benign and 25 cases of malignant breast lesions examined at the Surgical Pathology Laboratory constituted the material for the present study. The lesions were classified into various histological types. Histological grading, mitotic index, and lymph node involvement were noted.

Primary antibody: Monoclonal mouse antihuman Cytokeratin 5/6 clone D5/16 B4.

Poly-L-lysine hydrobromide (0.1%) for coating the slides.

Positive control section (for CK 5/6): Basal cell carcinoma.

Pressure cooker for target antigen retrieval.

DAB (3,3'-Diamino benzidine).

CK 5/6 immunoreactivity was expressed in the form of staining index [Table 1]. Cytoplasmic staining alone or along with membrane staining was taken as positive. [4]
Table 1 :Staining index


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   Observations Top


The benign lesions consisted of 12 cases of fibrocystic disease, nine cases of fibroadenoma, one case each of duct ectasia and lactating adenoma. The staining index of 22 cases varied from 6-9 and the single case which showed a negative immunohistochemical reaction was that of lactating adenoma [Table 2]. The staining intensity and proportion of immunopositive cells was more in foci of hyperplasia [Figure 1], adenosis and cystic change. The densely cellular areas exhibited both cytoplasmic and membranous staining. Staining index of fibroadenomas varied from 6-9. The foci with less cellularity showed cytoplasmic staining only, with a low staining index.
Table 2 :Benign breast lesions


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Figure 1 :Immunohistochemical staining with cytokeratin 5/6 in a case of usual ductal hyperplasia (H and E, ×400)

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Twenty five cases of malignant breast lesions were analyzed, which included three cases of Ductal carcinoma in situ (DCIS) and 22 cases of infiltrating ductal carcinoma, not otherwise specified IDC (NOS). The age of the IDC cases varied from 23-75 years. The size of the lesions varied from 3-9 centimeters and lymph node metastases was present in 11 cases [Table 3]. No immunohistochemical reaction was observed in any of the DCIS lesions. Among the IDC (NOS) cases, 19 cases were negative and six of grade III carcinoma showed positive immunoreaction. The staining characteristics of malignant lesions were different from those of benign lesions [Graph 1][Additional file 1]. The proportion of cells stained and staining intensity was considerably less as compared to the benign lesions. The cytoplasm was stained in a fine granular manner and membrane staining was absent [Figure 2].{Table 2}
Figure 2 :Immunohistochemical staining with cytokeratin 5/6 in a case of infiltrating ductal carcinoma showing staining of light intensity (H and E, ×400)

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In one particular case of infiltrating ductal carcinoma with squamous metaplasia, the keratin pearls exhibited a positive immunoreaction [Figure 3].
Figure 3 :Immunohistochemical staining with cytokeratin 5/6 in infiltrating ductal carcinoma with squamous metaplasia (H and E, ×400)

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On applying Chi Square test, the value obtained (35.81) for 6 degrees of freedom was higher than the table value (22.46) at 0.001 level. It may be thus concluded that the staining characteristics of the benign and malignant groups differ significantly.


   Discussion Top


The normal breast tissue consists of the following five distinct cell populations: Committed stem (progenitor) cells (CK5+), glandular precursor cells (CK 5+/ CK 8/18/19+), glandular end cells (CK 8/18/19+), myoepithelial precursor cells (CK 5/6+ / SMA+) and myoepithelial end cells (SMA+). [5]

CK 5 positive cells represent progenitor cells for both glandular and myoepithelial lineages of mammary epithelium. There is a gradual decrease of CK 5 expression, associated with an increase in expression of CK 8/18/19 in the glandular cells, and smooth muscle actin (SMA) in the myoepithelial cells along the pathways of differentiation. The terminally differentiated cells do not express CK 5. In the lactating breast, there is segregation of epithelial structures into CK 8/ 18 expressing secretory zone and the proliferative zone which harbors cells of both glandular (CK 8/18 +) and basal /myoepithelial (CK 5/6 +) type. Lactating adenoma is a benign tumor comprised of terminally differentiated secretory cells and is thus CK 5/6 negative. [6]

The proliferated luminal cells in benign lesions show a large number of CK 5/6 positive cells because of proliferation of both glandular and basal cells. Most malignancies are derived from differentiated glandular cells and do not reveal immunohistochemical staining with CK 5/6 leading, thus expaining its negativity in most lesions of atypical hyperplasia and ductal carcinoma in situ. [5],[7]

In the present study, all benign breast lesions, except lactating adenoma, consistently showed positive immunoreactions with high staining index. The lesions of intraductal carcinoma consistently showed a negative immunoreaction. Similar results were observed by other authors who reported a staining index of zero in majority of the cases of DCIS and atypical hyperplasia. [8]

The treatment and prognosis of DCIS and UDH (usual ductal hyperplasia) differs significantly, but the morphology often has overlapping features, thus giving rise to interobserver variability. Immunostaining with cytokeratin 5/6 can aid in reaching a definitive diagnosis. [7] Few studies have noted that AE1/AE3 and myoepithelial markers: smooth muscle actin, smooth muscle myosin heavy chain and p63 have higher specificity and sensitivity to differentiate benign and malignant breast lesions. Therefore, CK 5/6 has not gained much popularity but may be used as a component of a panel. [9]

This antibody is applied very frequently to help differentiate invasive from non invasive lesions. The common examples include radial scar versus Grade 1 invasive cancer, intraductal papilloma versus papillary intraductal carcinoma and microglandular adenosis versus tubular carcinoma. In radial scar the myoepithelial layer is retained around glandular structures and therefore these can be expected to show positive staining with CK 5/6 and myoepithelial cell markers. In foci of sclerosing adenosis, staining is heterogeneous. In low grade invasive carcinomas the myoepithelial layer is absent and CK 5/6 staining is negative.[8]

One of the few pitfalls of diagnosis, while using the antibody is DCIS with spindle cells, a rare entity that mimics benign epithelial proliferations but consistently shows no immunostaining with CK 5/6. It may easily be confused with benign spindle cell lesions. [10] On the other hand; myoepithelial carcinoma of the breast is a malignant spindle cell lesion that exhibits a positive immunostaining reaction with cytokeratin 5. [11]

The other diagnostic pitfalls include benign cells with apocrine change, columnar cells in papillary lesions and columnar epithelial cells lining the periphery of the spaces in UDH. The cells in these three conditions do not stain with cytokeratin 5/6. Therefore a careful evaluation of the hematoxylin and eosin stained histopathological sections along with immunohistochemical analysis is of utmost importance. [7]

Breast cancer has been classified into distinct molecular subtypes based on gene expression profiling studies. Each subtype is a distinct entity with clinical, biological and therapeutic implications. The basal like group of tumors possess an expression signature similar to basal/ myoepithelial cells of breast and is reported to have transcriptomic similarities with tumors arising in breast cancer 1 (BRCA1) germline mutation carriers.

Basal like breast cancers are poorly differentiated, high grade infiltrating ductal carcinomas with large central acellular zones. They are an indicator of aggressive behavior and poor prognosis, independent of nodal status and tumor size. They show a specific pattern of distant metastases with an increased propensity for visceral metastases to brain and lung. [12]

Immunohistochemically, basal cell carcinomas are defined as oestrogen receptor (ER) negative, Her 2 negative and cytokeratin 5/6 and /or HER 1 positive and claimed to correlate best with the basal-like breast cancer gene expression profile. CK 5/6+ tumors do not express p27kip1, cyclin D1, but do express p53. This gene expression profile is similar to tumors with BRCA1 mutation. Current routine diagnostic practice does not specifically recognize these tumors. Patients with basal like cancer do not benefit from currently available targeted systemic therapy. [12] Amongst the cases of carcinoma breast, the present study showed positive immunostaining in 24% the cases. In various other studies, the positive immunoreaction ranged from < 5%, [6] to 21.1% Takei et al.[13]

This slightly higher percentage positivity of CK 5/6 in the present study can be attributed to different patient cohorts or a smaller study sample. This observation may reflect a different distribution in Asia/ India compared to the west.

All malignant cases showing a positive immunoreaction for cytokeratin 5/6 were three centimeters or more in size, showed necrosis on gross examination and were grade III tumors with a high mitotic index (0.3-1%). Four lesions out of the six exhibited metastatic lymph node involvement and three cases were those of recurrent breast lesions. Dalia AE Rehim et al[5] reported a positive correlation of tumor size, local and regional recurrence, distant metastases, poor outcome with CK 5/6 expression and an inverse correlation with patient age. Although recurrence and metastasis was seen in the CK 5/6 positive tumors in the present study, but no consistent correlation with size and age was revealed. Necrosis was a common factor in all the CK 5/6 positive tumors and similar findings were reported by Lakhani SR. [14]

Matt van de Rijn [15] observed that expression of basal type cytokeratins in node negative breast carcinoma was a prognostic factor independent of tumor size and tumor grade. It was associated with significantly shorter survival, but held no predictive value in patients with known lymph node metastases.

On the contrary, Takei et al.[13] report that there was no significant difference in overall survival and relapse free survival in tumors expressing high molecular weight keratin as compared to tumors not expressing it . No case of medullary carcinoma breast was reported in our study. However, Tot T [16] reported CK 5/6 positivity in 25% of the typical, 43% of the atypical and 20% of the metastatic medullary carcinomas.

Few studies have reported cases of breast cancer which were negative for both the luminal and basal markers. It can be attributed to loss of reaction due to differences in tissue handling, or due to non epithelial derivation or de-differentiation of an epithelial derived tumor to a more primitive subclass. A subtype of breast cancer display glandular precursor phenotype and show positive immunoreactions for both CK 8/18 and CK 5/6. [5],[6]


   Conclusion Top


A few grade III breast carcinoma cases, if positive for CK 5/6, imply a 'basal like' molecular phenotype and signify poor prognosis. These tumors require an aggressive intervention. The patients with this subtype of breast cancer, along with their first degree relatives must be subjected for BRCA1 mutation testing. Also, immunohistochemical staining with cytokeratin 5/6, as a component of panels along with AE1/ AE3 and myoepithelial markers: smooth muscle actin, smooth muscle myosin heavy chain and p63 helps to differentiate benign and malignant breast lesions in cases of interobserver variability.

 
   References Top

1.Cooper D, Schermer A, Sun TT. Classification of human epithelia and their neoplasms using monoclonal antibodies to keratins: Straegies, applications, and limitations. Lab Invest 1985;52:243-56.  Back to cited text no. 1
    
2.Chu PG, Weiss LM. Keratin expression in human tissues and neoplasms. Histopathology 2002;40:403-39.  Back to cited text no. 2
    
3.Boecker W, Buerger H, Schmitz K, Ellis IA, van Diest PJ, Sinn HP, et al. Ductal epithelial proliferations of the breast: A biological continuum? Comparative genomic hybridization and high-molecular-weight cytokeratin expression patterns. J Pathol 2001;195:415-21.  Back to cited text no. 3
    
4.Foulkes WD, Stefansson IM, Chappuis PO, Bιgin LR, Goffin JR, Wong N, et al. Germline BRCA 1 mutations and a basal epithelial phenotype in breast cancer. J Natl Cancer Inst (Bethesda) 2003;95:1482-5.  Back to cited text no. 4
    
5.Abd El Rehim DM, Pinder SE, Paish CE, Bell J. Expression of luminal and basal cytokeratins in human breast carcinoma. J Pathol 2004;203:661-71.  Back to cited text no. 5
    
6.Bφcker W, Moll R, Poremba C, Holland R, Van Diest PJ, Dervan P, et al. Common adult stem cells in the human breast give rise to glandular and myoepithelial cell lineages: A new cell biological concept. Lab Investig 2002;82:737-45.  Back to cited text no. 6
    
7.Raju U, Crissman JD, Zarbo RJ, Gottlieb C. Epitheliosis of the breast: An immunohistochemical characterisation and comparison to malignant intraductal proliferations of the breast. Am J Surg Pathol 1990;14:939-47.  Back to cited text no. 7
    
8.Otterbach F, Bΰnkfalvi A, Bergner S, Decker T, Krech R, Boecker W. Cytokeratin 5/6 immunohistochemistry assists the differential diagnosis of atypical proliferations of the breast. Histopathology 2000;37:232-40.  Back to cited text no. 8
    
9.Heatley M, Maxwell P, Whiteside C, Toner P. Cytokeratin intermediate filament expression in benign and malignant breast disease. J Clin Pathol 1995;48:26-32.  Back to cited text no. 9
    
10.Tan PH, Lui GG, Chiang G, Yap WM, Poh WT, Bay BH. Ductal carcinoma in situ with spindle cells: A potential diagnostic pitfall in the evaluation of breast lesions. Histopathology 2004;45:343-51.  Back to cited text no. 10
    
11.Lakhani SR, Reis-Filho JS, Fulford L, Penault-Llorca F, van der Vijver M, Parry S, et al. Breast cancer linkage consortium: Prediction of BRCA 1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clin Cancer Res 2005;11:5175-80.  Back to cited text no. 11
    
12.Rakha EA, Reis-Filho JS, Ellis IO. Basal-like breast cancer: A critical review. J Clin Oncol 2008;26:2568-81.   Back to cited text no. 12
    
13.Takei H, Iino Y, Horiguchi J, Maemura M, Oyama T, Yokoe, et al. Low and high molecular weight cytokeratins in invasive breast carcinoma. Oncol Rep 1997;4:33-8.  Back to cited text no. 13
    
14.Lakhani SR, Reis-Filho JS, Fulford L, Penault-Llorca F, van der Vijver M, Parry S, et al. Breast cancer linkage consortium: Prediction of BRCA 1 status in patients with breast cancer using estrogen receptor and basal phenotype. Clin Cancer Res 2005;11:5175-80.  Back to cited text no. 14
    
15.van de Rijn M, Perou CM, Tibshirani R, Haas P, Kallioniemi O, Kononen J, et al. Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am J Pathol 2002;161:1991-6.  Back to cited text no. 15
    
16.Tot T. The cytokeratin profile of medullary carcinoma of the breast. Histopathology 2000;37:175-81.  Back to cited text no. 16
    

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Correspondence Address:
Amarpreet Bhalla
Department of Pathology, Sri Guru Ram Das Institute of Medical Sciences and Research, Vallah, Sri Amritsar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.72026

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    Figures

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    Tables

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