European Journal of Cardiovascular Medicine

A number of cyclical hormonal changes takes place in a woman’s breast throughout her reproductive life. Breast diseases are showing a rising trend worldwide. A wide spectrum of disorders ranging from the self-limiting inflammatory lesion, benign breast lesion to life-threatening invasive carcinoma arises from the breast.

Benign breast diseases (BBD) comprise wide range of disorders from developmental abnormality, inflammatory lesions, epithelial and stromal proliferation to the neoplasm. BBD is the most common cause of breast problem among women with a prevalence rate of 68% among all breast lesions in India. It starts to rise in the second decade of life, peaks in the third decade and decline thereafter whereas the incidence of breast carcinoma rises as age advances. Because of its high incidence and cancerous potential of certain histological types, BBD needs to be given utmost attention.1

A BBD is more frequent than malignant ones. Thirty percent of women suffering from benign breast diseases require treatment at some point in their lives.2In most urban populations of India, carcinoma breast has a higher incidence compared to carcinoma cervix.3Several etiological factors: age, family history, genetics, alcohol, diet, obesity, sedentary lifestyle, endocrine factors are implicated. Current management of carcinoma of the breast is multi- modality treatment which includes surgery, radiotherapy, and chemotherapy and hormone therapy.

A triple breast assessment is done by clinical examination, radiological imaging (USG or mammography) and image result for fine needle aspiration cytology (FNAC) or core needle biopsy, allows the majority of patients with discrete BBDs to arrive at a diagnosis. An early diagnosis and treatment plan helps in reducing unnecessary anxiety about breast carcinoma and those with an increased risk of malignancy (atypical hyperplasia) are given prompt treatment thus improving prognosis.4 Diagnosis of benign and malignant epithelial lesions is done using hematoxylin-eosin microscopic sections alone. However in grey zone cases such as distinguishing usual ductal hyperplasia from atypical ductal hyperplasia/ductal carcinoma in situ (DCIS), diagnosing lobular vs. ductal or basal versus luminal, to identify true micro invasion, to improve sentinel lymph node staging, to localize metastatic carcinoma of unknown primary site as of breast origin, use of immunohistochemical stains are of great help in these difficult are as particularly when diagnosis can significantly impact on management and prognosis.5,6

Based on antigen–antibody recognition, immunohistochemistry is used for localizing specific antigens in tissue/cells. Cytokeratin an intermediate filament protein indicate epithelial cell type, state of tissue growth, differentiation, functional status and used for fingerprinting of various carcinomas. The normal breast tissue is composed of luminal cells that express CK 8/18, CK 7, and CK 19. The basal/ myoepithelial cells express CK5/6, CK 14, CK 17 and SMA. All benign breast lesions except lactating adenoma show positivity with CK5/6.7 the treatment and prognosis of DCIS and UDH differ significantly, but the morphology has overlapping features giving rise to interobserver variability. Immunostaining with CK 5/6 aid to reach a definitive diagnosis.7This antibody is applied very frequently to help differentiate invasive from non-invasive lesions, e.g. radial scar from grade I invasive carcinoma, microglandular adenosis from tubular carcinoma, intraductal Papilloma vs. papillary intra ductal carcinoma. Grade III breast carcinomas positive for CK5/6 imply basal-like molecular phenotype and require aggressive intervention. Immunohistochemical staining withCK5/6, as a component of panels along with AE1/AE3 and myoepithelial markers: smooth muscle actin, smooth muscle myosin heavy chain, p63 help differentiate benign and malignant breast lesion in case of inter-observer variability. Histomorphological study of breast carcinomas with CK5/6 IHC along with H-E stained section will be helpful in differential diagnosis of pre-invasive breast diseases thus becoming useful in routine usage.7

This is a cross-sectional study done in a hospital in Tumkur, Karnataka for a period of 1.5years.Total of 41 benign and 41 malignant biopsies of breast neoplasm from female patients were studied. Non-neoplastic lesions were excluded from the study.

After taking consent, detailed history of the patient including past history and family history were taken. Thorough physical examination of the patient was done to find out regarding position, size, shape, mobility, consistency of palpable breast mass, to know about skin changes over breast if any puckering, dimpling, nipple retraction ,discharge, peau-d-orange appearance or any lymph node metastasis.

Biopsy is done to confirm the diagnosis. Biopsy specimens were received in 10% formalin. In cases where mastectomy was done whole breast mass was received and grossing done as per standard procedures.

Specimens were then systematically examined. Gross details regarding site, size, surface, capsule, margins, calcification, consistency, necrosis, tumor area, and lymph-node involvement examined. Representative tissue section processed routinely by paraffin section for light microscopy.

After properly classifying the tumor histopathologically, paraffin blocks of the representative section were processed for Immunohistochemistry of CK5/6. Controls were run simultaneously.CK5/6 expression was evaluated on basis of extent and intensity of immunohistochemical expression in cytoplasm alone or along with membrane stain by microscopy using a scale from 0 to 3.8

Intensity                Proportion of immunopositive cell

0   No staining

1+Weak staining, 1+<10%immunopositivecells

2+Moderatestaining,           2+10–50%immunopositivecells

3+Strongstaining, 3+>50%immunopositivecells

Statistical Analysis

All data collected were entered into a master sheet and then fed into computer software for statistical analysis using Pearson’s Chi-square test.

The present study was undertaken to study the immunohistochemistry of CK5/6 performed on 82 breast biopsy specimens. The sample size consisted of 41 benign and 41 malignant lesions. In our study, benign lesions were commonest in age group of 21–30 years, and malignant lesions were common in 51–60years.Past studies have shown that benign breast lesions begin to rise during the 2nd decade of life and peaks in the 4th decade. Throughout a woman’s reproductive life both epithelial and stromal elements of breast lobule are under hormonal control. Any interference with these close interactions results in conditions grouped under benign breast disease. Also repeated development and involutional changes of menstruation, pregnancies are responsible for minor aberrations.

The incidence of breast cancer increases as age advances peaking at 70–80 years. Late menopause results in prolonged exposure to estrogens that increase risk. With increasing age breast tissue gets replaced by adipose tissue, fibrous connective tissue increase in density. Women with very dense breasts have an increased risk of carcinoma. Delayed involution and mammographic density result in accumulation of growth factors and hormones that cause excess cell division and damage to cells resulting in high breast cancer incidence.11 The risk for breast cancer also rises with increasing severity of BBD including hyperplasia with moderate to marked atypia in premenopausal women.

The most common benign neoplasm in our study was fibroadenoma (74%) in agreement with other available literature. Since they arise from lobules, they are seen predominantly in 15–25 years age group. Fibroadenomas also represent “Aberrations of normal development and involution”. A direct association has been noted between oral contraceptive usage before 20years age and risk of fibroadenoma. Mudholkar reported the highest incidence of fibroadenoma 87% which could be attributed to his study of 252 cases of breast neoplasm done for a long period of 5 years. In malignancy, commonest neoplasm was infiltrating ductal carcinoma-NOS (85%). The findings in our study correlated with others like Mudholkaretal., Bhallaetal., Kapoor et al.

In the present study maximum number of cases showed tumor size of more than 2cm and less than 5cm(T2).A possible reason for larger size of tumor is that this study was conducted in our hospital which is situated in a rural part of Karnataka where patients come at a later stage of the disease. Gross size of the tumor is an important prognostic factor in breast carcinoma, and with increasing size of the tumor there is an increased incidence of axillary lymph node metastases and decreased survival.

In the present study majority were grade II tumors (66%). Only 3 cases were grade III (7%) which correlated with other studies. Our study showed higher percentage of grade II tumors. A possible reason for this is patients presenting at a later stage of disease thus underlying the importance of regular breast self-examination. High-grade tumors significantly increase the frequency of lymph node metastases, developing more systemic recurrences, and bad prognosis.12

In our study 83% cases were negative for lymph node metastatic deposits; it may be due to the gross size of tumor. Most of the cases in our study are of sizeT2. The incidence of axillary lymph node metastasis and decreased chances of survival is proportional with increasing tumor size. Tumor size and axillary lymph node status are highly correlated but are also independent measures of outcome. Survival declines with increasing tumor size when nodal status is heldconstant.13 A number of nodes involved, amount of carcinoma measured by the microscopic size of the largest nodal metastases and presence or absence of extranodal spread, are also prognostically important.14

In our study all benign breast lesions showed positive expression for CK 5/6; the staining index of benign lesions varied from 5to 9.It correlated with other studies. Bhalla etal. reported positive immunoexpression in all benign cases with a staining index of 6–9 except in Lactating Adenoma. Akhtaretal. reported stain index of5–8.15In the present study staining intensity and proportion of immunopositive cells was more in areas of hyperplasia, adenosis, and cystic change. Highest stain index was seen in fibrocystic with ductal epithelial hyperplasia. Most case showed cytoplasmic and membranous staining. Most fibroadenomas showed stain index of 6. In benign phyllodes, there were areas of low cellularity showing cytoplasmic staining only with a stain index 5. In the present study 38 cases of infiltrating carcinomas showed negative CK 5/6 expression. Only 3 cases showed weak positive expression. Findings correlated with other studies. Bhallaetal. reported in his study 6 out of 22 cases showed positive immunoreactions. Otterbach etal. also reported only 3 out of 39 infiltrating breast carcinomas showed CK 5/6 expression.16Most malignancies are derived from differentiated glandular cells and do not reveal immunohistochemical staining with CK 5/6. Lack of CK5/6 expression in neoplastic cells of atypical hyperplasia, in-situ carcinomas and infiltrating carcinomas could be attributed to its arising from CK 5/6-negative glandular precursor cells.16 Also, the myoepithelial cell is absent in most invasive cancers helping in differentiation of in-situ carcinomas from invasive cancers.17 Lacroix et al reported that the expression rate of CK5/6 in UDH, ADH, IDC was 62.9%,10.0% and 0 respectively.18CK5/6 was positive virtually in most benign ductal lesions and was expressed in glandular epithelium and myoepithelial cells. In DCIS, almost all the proliferating cells CK5/6 was negative. In atypical ductal hyperplasia, only partially proliferating cells were positive. IDC ,no CK 5/6 was detectable with complete lack of expression. Poorly differentiated carcinomas showed weak positive expression CK5/6 because these high-grade IDC that are basal-like breast cancers possess an expression signature similar to basal/myoepithelial cells of the breast.7

In our study,28 cases were of size T2(>2 cm and <5cm) and showed CK5/6 stain index 0.A p value was insignificant. No consistent relation could be obtained. Herranz M reported CK 5/6 positive IDCs had bigger tumor size (p= 0.005) and concluded that CK 5/6 positivity was associated with lower age, greater size, distant metastases, grade 3, Ki-67 positive and p53 positive.19All these support associations between CK5/6 positivity and basal-like tumors and it can define patient subgroups with different risks. Rehim et al. also reported a positive correlation between CK 5/6 positivity and greater tumor size(p<0.001).21Bhallaetal.(p>0.05),Akhtar etal. (p >0.05) found no consistent relation with tumor size and CK 5/6 positivity .

All grade III infiltrating carcinoma showed weak positive immunohistochemical expression which correlated with other studies. Bhalla et al. reported all grade III malignant cases showed weak positive immunoreactions (p<0.05). Akhtar et al. also reported all 6 cases of breast carcinoma positive for CK5/6 expression were grade III and triple negative (p<0.05). Rehim et al. reported a significant association between grade III carcinomas and CK5/6 positivity(p<0.001),he stated that invasive grade III carcinomas with extensive central necrosis had basal phenotype and aggressive behavior. But in low-grade invasive carcinoma myoepithelial layer is absent and so staining negative. Herranz reported CK 5/6 positive IDCs were more frequently grade III (p = 0: <0, 0000). CK 5/6 positivity in poorly differentiated carcinomas is because these high-grade IDC that are basal-like breast cancers possess an expression signature similar to basal/ myoepithelial cells of the breast7 .

In the present study out of 7,only 3cases with positive lymph node deposits showed stain index of 2. The findings were consistent with other studies. Rehim etal. reported the inverse correlation between CK 5/6 positivity and nodal metastasis (p = 0.127). Akhtar etal. reported a positive correlation between CK 5/6 positivity and lymph node metastasis (p <0.05). Dent et al. reported lymph node positivity in 55% cases of triple negative carcinomas while among other breast carcinomas 40%lymph node positivity.21The larger sample size is required to establish conclusive results.

With the current burden of breast neoplasm, the precision of diagnosis is of utmost importance to provide accurate treatment to patients. Immunohistochemistry (IHC) is an integral part of pathology. Although H & E stain remains gold standard method for diagnosis, IHC provide vital information in differentiation in grey-zone cases.

CK 5/6, as a component of panels along with AE1/ AE3 and myoepithelial markers: smooth muscle actin, smooth muscle myosin heavy chain and p63 help differentiate benign and malignant breast lesions in cases of interobserver variability. Grade III breast carcinoma cases, if positive for CK5/6, imply ‘basal-like ’molecular phenotype and signify a poor prognosis. These tumors require aggressive intervention. The patients with this subtype of breast cancer must be subjected to BRCA1 mutation testing. Thus with CK5/6, we can help to provide prognostic information and better treatment modalities.

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