Ultra structural changes occurring in duct ectasia and periductal mastitis and their significance in etiopathogenesis

Introduction Duct ectasia (DE) and periductal mastitis (PDM) are the most common benign breast conditions seen in women. The etiopathogenesis of these entities is still not clear and most of the theories regarding the causation are based on the histological features as seen on light microscopy. The ultramicroscopic features associated with these conditions that may give more insight to the etiopathogenesis are unknown. Aim To study the ultrastructural changes occurring in mammary duct cones in patients with DE and PDM using Transmission Electron Microscopic (TEM). Method Major ducts removed by radical duct excision from 21 patients with final histopathological diagnosis of DE and PDM were subjected to TEM study with 2 normal duct samples as controls. Results The TEM features of DE were denudation of the epithelial cells with focal loss of microvilli, widening of the inter-epithelial junctions with focal disruption of the T bars, periductal collagenisation without inflammation, and features suggestive of Epithelial Mesenchymal Transition (EMT). PDM features are intact epithelial lining with proliferative epithelium and periductal collagenisation with inflammation. Conclusions Based on the TEM findings, we suggest that DE and PDM are two different entities. EMT a novel finding observed in DE in this study.

Introduction Nipple discharge among women is a common clinical entity. Duct ectasia (DE) / periductal masititis (PDM) is an important cause of this clinical condition. In spite of the enormous research, the etiopathogenesis and the structural pathological changes that take place in this condition are largely unknown. Literature is not clear as to whether DE and PDM are two different entities or different manifestations of a common clinical condition. A study of the sub cellular changes of DE and PDM may throw more light on these issues and may also provide insight into a specific target for local therapy if any, which is lacking so far.

Objective
To evaluate the ultra structural changes associated with DE and PDM by Transmission electron microscopic (TEM) study of mammary duct cones of patients with DE/PDM.

Materials and methods
Forty one consecutive patients with non bloody nipple discharge with clinical and radiological features suggestive of DE/PDM presenting to the surgical outpatient department of All India Institute of Medical Sciences, New Delhi were included in the study after obtaining Institute ethics committee's approval and informed, written consent. Patients who had the following presentations were diagnosed to have duct ectasia-Presence of thick, colored, or creamy nipple discharge, thickened subareolar ducts, nipple retraction, and absence of signs of inflammation with sonographic features of dilated subareolar ducts and mammographic features of prominent ducts appearing as conical opacities with apex of the cone towards the nipple with or without characteristic coarse calcifications (ring like, lying on the duct wall or circular or needle shaped when the duct contents are calcified). Those who had history suggestive of previous episodes of subareolar sepsis, presence of pus/serous nipple discharge, and signs of periductal inflammation, tender palpable subareolar ducts, nipple retraction, and mammary fistula with radiological features of dilated ducts and subareolar abscess were considered to have periductal mastitis. Excluded from the study were those patients who were unwilling to give consent, women with bloody nipple discharge and cytopathology of nipple discharge smear demonstrating atypical or malignant cells. A detailed history and thorough physical examination were done for all patients along with ultrasound of both the breasts and axillae. For women who were above 35 years digital ammography was done. Cytopathological examination of the nipple discharge smear was carried out in patients with active nipple discharge. Patients who had active subareolar sepsis were treated with antibiotics prior to the surgery. Major mammary duct excision, the surgical procedure of choice was performed in all [S1 File]. The excised ductal tissues were processed for both light and electron microscopy. The histopathological features for making the diagnosis of duct ectasia were 3 or more dilated ducts with thickened or sclerosed duct walls, periductal sclerosis, hyperelastosis with or withoutepithelial denudationand no or minimal inflammation. The histopathological features for reaching the diagnosis of periductal mastitis were dense periductal inflammation and fibrosis with or without focal dilatation of ducts. Twenty patients who had pathological diagnoses of mixed duct ectasia/periductal mastitis with fibrocystic disease (n = 12), papilloma (n = 3), papillomatosis (n = 3) and tuberculous mastitis (n = 2) were excluded from the electron microscopic study. Based on the final histopathological diagnoses, 21 patients with definitive diagnoses of pure DE (n = 9), PDM (n = 7) or mixed features of DE and PDM i.e. duct ectasia with features of peridcutal inflammation (n = 5) were included in the TEM study [S2 File]. The tissues were fixed in 2.5% glutaraldehyde and processed by automatic tissue processor. Several resin sections were cut at approximately 1 micron using glass knives made by the knife maker and an ultra microtome. The ultrastructural examination was performed on Philips Morgagni 268 transmission electron microscope and Technai G20 transmission electron microscope. The normal mammary ducts sampled from 2 patients who underwent reduction mammoplasty were taken as control. These two ladies did not suffer from any nipple discharge and were premenopausal with regular menstrual cycles.

Results
TEM study revealed the following features. The normal major mammary ducts showed a continuous epithelial layer (single or double layered) and myoepithelial cells interspersed in a discontinuous manner [Fig 1-A1]. Two types of epithelial cells lining the major mammary duct could be appreciated namely, the dark epithelial cells, being rich in electron dense ribosomes and the light epithelial cells [Fig 1-A2]. In the interepithelial junctions there were terminal bars [T bars] which were electron dense [Fig 1-A3]. The above mentioned findings correlated with the observations and the images documented in the TEM study of normal mammary ducts done by the various authors in the literature [1][2][3][4]. In

Discussion
There is no data available on the electron microscopic study of DE/PDM in literature. Hughes et al [5] have commented that there is no loss of microvilli in scanning electron microscope in cases of DE. However, no further details have been mentioned. Scanning electron microscopy as used by Hughes et al [5]describes only the surface details while TEM describes in detail about the intracellular changes which helps us to understand the pathogenetic mechanisms better. In DE, the changes observed suggested EMT. EMT is an interesting biological phenomenon, by which epithelial cells get transformed into mesenchymal cells and acquire new characteristics such as motility and collagen synthesis. The EMT in duct ectasia has been substantiated by demonstration of vimentin positive cells among the normal epithelial cells in patients with DE and presence of epithelial cells with elongated nuclei and cytoplasmic processeses containing fibril like material. Thus EMT seems to may play a key role in the pathogenesis of DE. Though periductal collagenisation is seen both in DE and PDM, in PDM, the collagenisation is associated with inflammation unlike in DE. Thus, it is evident that DE and PDM may be considered as two different entities with two different pathogenetic mechanisms.
The etiopathogenesis of DE and PDM are poorly understood. The classical concept as enunciated by Haagensen and Ewing [6] considers duct dilatation under hormonal influence as a primary event. This leads to epithelium ulceration, followed by leakage of secretion into the periductal tissue leading to inflammation, secondary bacterial infection and periductal fibrosis. An alternative explanation by Dixon et al considers DE and PDM as two different entities & PDM does not precede DE.
These controversies can be addressed by the ultramicroscopic features of DE and PDM observed in the present study. Distinct changes in the epithelial lining and in periductal tissue in DE and PDM were observed on electron microscopy and immuno-histochemistry. In this study, pure DE was seen in nine patients, pure PDM in six patients and a combination of both DE and PDM i.e. DE with periductal inflammation in six patients. The combination of both being present together could be explained by the fact that there could be secondary duct dilatation following periductal fibrosis.
Identification of EMT changes in patients of DE in our study adds mammary ducts to the list of organs where in EMT seems to play a role in pathologically induced fibrosis [7][8][9][10][11]. However, the signaling pathway involved in EMT of DE is yet to be studied. Success of BMP-6 & 7 (inhibitors of EMT induced fibrotic process) in reversal of renal fibrosis in animal models [12,13] is encouraging to conduct further research to identify the common switch of EMT in DE. Once the switch if any is identified, it may be turned off by molecular targeting and thuscure the disease.
Limited number of controls due to ethical reasons is a limitation of this study. However, the normal human major mammary duct ultrastructure that has been documented in the literature along with the present study controls aids us to compare the changes that have been observed in DE and PDM. Another limitation of the study which is common in any of the TEM study is that the small tissue dimension used may not be representative of the entire disease process.

Conclusions
As the electron microscopic findings of DE and PDM are distinct from one another, these can be considered as two different entities. The association of EMT in DE is a novel finding of the present study.