Results showed that all three cases spanned a morphologic spectrum ranging from columnar cell hyperplasia to ductal carcinoma in situ. Case 1 (columnar cell hyperplasia) contained two ducts of interest lined by about 500 epithelial cells surrounded by morphologically distinct ME cell layers on the H&E-stained section (Fig. ). The ME cells in about two-thirds of the ME layer showed distinct and strong SMA immunostaining, whereas the cells in about one-third of the layer were devoid of SMA immunostaining (Fig. ). The SMA-positive and SMA-negative cells were morphologically indistinguishable on H&E-stained sections. In double immunostained sections, these SMA-negative cells were also negative for SM-MHC, WT-1, CD10, and calponin (Fig. ). However, these SMA-negative cells were positive for maspin, CK5, CK14, and CK17 (Fig. ).
Figure 1 Immunostaining pattern of ME cells in columnar hyperplasia (case 1). (a) H&E staining; (b) immunostaining for SMA; (c-f) double immunostaining of SMA with SM-MHC, WT-1, CD10, and calponin, respectively, and the segment of the ME layer is negative (more ...)
Cases 2 and 3 contained ductal carcinoma in situ, of intermediate grade and low grade, respectively. The ducts were surrounded by morphologically distinct ME cell layers in H&E-stained sections (Fig. ). These ME cell layers were attenuated, consisting of elongated spindle cells with dark and compressed nuclei. The entire ME cell layer was devoid of SMA immunostaining (Fig. ). In double immunostained sections, these SMA-negative cells were also devoid of distinct immunostaining for any of the additional eight markers (Fig. ).
Figure 2 Immunostaining pattern of ME cells in ductal carcinoma in situ (case 2). (a) H&E staining; (b) immunostaining for SMA; (c-f) double immunostaining of SMA with maspin, CK5, CK14, and CK17, respectively, and the entire ME cell layer is negative (more ...)
The SMA-negative cells in all three cases showed distinct negativity to ER and CK8, in sharp contrast with the overlying epithelial cells that showed strong ER and CK8 positivities (data not shown). The distribution of these SMA-negative ME cells seemed to be independent of the ductal size, length, and architecture.
Our findings are consistent with those of a recent study showing that a vast majority of the ME cells in both normal and ductal carcinoma in situ
displayed distinct immunostaining to p63, SM-MHC, and calponin, whereas a single or a cluster of a few ME cells in some ducts failed to show immunoreactivity to all these three markers [28
]. However, our study differs from this study [28
] and previous studies [3
] in four aspects: first, the SMA-negative cells were segmented, accounting for at least one-third or all of the ME cells in involved ducts; second, we tested for more ME cell markers; third, the SMA-negative cells assessed were morphologically similar to their adjacent SMA-positive neighbors on H&E-stained sections; fourth, our focus is directed toward the elucidation of the detailed immunohistochemical profile of these SMA-negative cells identified in 3 of 175 examined cases. The distribution of different ME markers among normal, benign, and malignant breast lesions in the remaining cases will be presented separately. In our study, p63 was replaced with WT-1 for three main reasons: first, p63 is a nuclear protein, which is not easily identifiable in attenuated or compressed ME cells; second, previous studies have shown that this protein is also expressed in ME-cell-derived neoplasms and tumors with squamous cell differentiation [29
]; third, our preliminary study had showed that WT-1 had the same subcellular localization but seemed to be more specific for ME cells, compared with p63 [12
The mechanism of the loss of myoepithelial markers in some of the ME cells is unknown, but could result from the dynamic and reciprocal interactions between epithelial and ME cells. It has been documented that a variety of proteolytic enzymes produced by malignant epithelial cells could have substantial impacts on the physical integrity or functions of the subjacent ME cells and the basement membrane [30
]. In contrast, ME cells could influence the biological behavior of subjacent epithelial cells. Our recent studies have revealed the co-localization of maspin and WT-1 exclusively in mammary ME cells [12
]. In a vast majority of cases, the expression of these two proteins decreases linearly with tumor progression, and the loss of these proteins or focal disruptions in the ME cell layers leads to a significantly higher cell proliferation in the subjacent epithelial cells [12
]. In addition, these changes in ME cells could result from effects of certain chemical compounds.
It has been reported that the exposure to lambdacarrageenan could specifically result in filament disassembly and loss of ME cells [34
]. In contrast, exposure to oxytocin could substantially enhance ME cell differentiation and proliferation in mouse breasts [35
]. In addition, these SMA-negative ME cells might be newly formed through stem-cell-mediated proliferation and are in the transition to a terminally differentiated status. Our previous studies on animal models have shown that intercalated duct cells in adult rat submandibular glands are the common progenitor for both the acinar and granular duct cells [27
]. A recent study in human breast has revealed that CK5-positive cells are capable of giving rise to both glandular and ME cell lineages [21
]. In both cases the intermediate cells showed an unusual immunostaining pattern for several proteins [21
The total loss of all nine ME cell immunophenotypic markers suggests that these ME cells have genetic and biochemical properties differing from their SMA-positive counterparts. The significance and consequence of these changes in ME cells are unknown. However, given that the disruption of the ME cell layer and basement membrane is an absolute prerequisite for tumor invasion and metastasis, these alterations in ME cells might affect the biological behavior of the epithelial cells, making them prone to progression. Our assumption is in agreement with our previous findings, which have revealed that focal disruptions of ME cell layers could lead to a significantly higher proliferation rate in subjacent epithelial cells [12
]. Our assumption is also supported by a recent study showing that normal and tumor-derived ME cells differ substantially in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition [37
]. Further studies are currently in progress to elucidate the biological behavior and genetic profile of epithelial cells immediately subjacent to these SMA-negative ME cells.