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

Abstract

Introduction: Our previous studies detected focal disruptions in myoepithelial cell layers of several ducts with carcinoma in situ. The cell cluster overlying each of the myoepithelial disruptions showed a marked reduction in or a total loss of immunoreactivity for the estrogen receptor (ER). This is in contrast to the adjacent cells within the same duct, which were strongly immunoreactive for the ER. The current study attempts to confirm and expand previous observations on a larger scale.

Methods: Paraffin sections from 220 patients with ER-positive intraductal breast tumors were double immunostained with the same protocol previously used. Cross-sections of ducts lined by > or = 40 epithelial cells were examined for myoepithelial cell layer disruptions and for ER expression. In five selected cases, ER-negative cells overlying the disrupted myoepithelial cell layer and adjacent ER-positive cells within the same duct were separately microdissected and assessed for loss of heterozygosity and microsatellite instability.

Results: Of the 220 cases with 5698 duct cross-sections examined, 94 showed disrupted myoepithelial cell layers with 405 focal disruptions. Of the 94 cases, 79 (84%) contained only ER-negative cell clusters, nine (9.6%) contained both ER-negative and ER-positive cell clusters, and six (6.4%) contained only ER-positive cell clusters overlying disrupted myoepithelial cell layers. Of the 405 disruptions, 350 (86.4%) were overlain by ER-negative cell clusters and 55 (13.6%) were overlain by ER-positive cell clusters (P < 0.01). Microdissected ER-negative and ER-positive cells within the same duct from all five selected cases displayed a different frequency or pattern of loss of heterozygosity and/or microsatellite instability at 10 of the 15 DNA markers.

Conclusions: Cells overlying focally disrupted myoepithelial layers and their adjacent counterparts within the same duct displayed different immunohistochemical and molecular features. These features potentially represent an early sign of the formation of a biologically more aggressive cell clone and the myoepithelial cell layer breakdown possibly associated with tumor progression or invasion.

Figure 1

Disruptions of myoepithelial cell layers are independent of the size, length, architecture, and overall estrogen receptor (ER) negativity of the ducts. Paraffin-embedded breast tissue sections were double immunostained with antibodies to ER and smooth muscle actin, and were developed with 3,3'-diaminobenzidine (DAB; black or brown) and 3-amino-9-ethylcarbazole (AEC; red) chromogens, respectively. Distinct myoepithelial cell layers are seen in the following structures: (a) a large duct with ductal carcinoma in situ (DCIS), 100×; (b) a long normal duct, 40×; (c) a duct with intraductal hyperplasia, 100×; (d) intermediate grade ER-negative DCIS, 100×.

Figure 2

Myoepithelial cell layer disruptions and overlying small estrogen receptor (ER)-negative cell clusters (< 15 cells) in ER-positive ductal carcinoma in situ (DCIS). (a) H & E staining of a large duct with DCIS, 100×; (b) the adjacent section of (a) immunostained, 100×; (c) a higher magnification of (a); (d) a higher magnification of (b). The myoepithelial cell layer disruption and the overlying ER-negative cells are identified with an arrow.

Figure 3

Myoepithelial cell layer disruptions and overlying larger estrogen receptor (ER)-negative cell clusters (> 15 cells) in ER-positive neoplastic and hyperplastic ducts. All sections were immunostained for ER (brown) and smooth muscle actin (red). (a) Ductal carcinoma in situ, 100×; (b) two small ducts with atypical intraductal hyperplasia, 100×; (c) a hyperplastic duct, 100×; (d) two hyperplastic ducts, 200×. Myoepithelial cell layer disruptions and ER-negative cell clusters are identified with arrows.

Figure 4

Multiple myoepithelial cell layer disruptions and overlying estrogen receptor (ER)-negative cell clusters in ductal carcinoma in situ. (a) A paraffin section immunostained for ER (brown) and smooth muscle actin (SMA) (red), 200×; (b) a higher magnification of (a), 400×; (c) a paraffin section immunostained for ER (brown) and SMA (red), 200×; (d) a higher magnification of (c), 400×. Myoepithelial cell layer disruptions and ER-negative cell clusters are identified with arrows.

Figure 5

Myoepithelial cell layer disruptions and associated estrogen receptor (ER)-positive cells in hyperplastic ducts. All sections were immunostained immunostained for ER (brown) and smooth muscle actin (red). (a), (b) Two ER-positive hyperplastic ducts showing attenuated and disrupted myoepithelial cell layers, 200×. The disruptions are identified with arrows.

Figure 6

Myoepithelial cell layer disruptions and overlying estrogen receptor (ER)-negative cell clusters in normal and hyperplastic appearing ducts. All sections were immunostained immunostained for ER (brown) and smooth muscle actin (red). (a)–(c) Normal appearing ducts, 400×; (d)–(f) hyperplastic ducts, 400×. Myoepithelial cell layer disruptions and ER-negative cell clusters are identified with arrows.

Figure 7

Comparison of the loss of heterozygosity (LOH) pattern in estrogen receptor (ER)-negative cells and adjacent ER-positive cells within the same duct. (a) H & E staining of a duct with atypical intraductal hyperplasia, 200×; (b) the adjacent section of (a) immunostainined (before microdissection), 200×; (c) microdissection of ER-positive cells; (d) microdissection of ER-negative cells; (e) LOH at four selected DNA markers. Asterisks indicate the ER-negative cells and the ER-positive cells removed; arrows identify LOH.