Histologic tumor types (Table )
Tumors were categorized histologically using the recommendations of the Mouse Models of Human Cancers Consortium categorization scheme [17
] and the specific nomenclature applying to certain models [22
] or tumor types [21
] when applicable.
Spontaneous tumors were papillary adenocarcinomas with papillae lined by a single layer of cells (n = 6; Fig. ), papillary adenocarcinomas with papillae lined by two layers of cells (n = 2; Fig. ), glandular adenocarcinomas with EMT (n = 4; Fig. ), microacinar adenocarcinomas in C3H/HeJ mice (n = 8; Fig. ), type P tumors in C3H/HeJ mice (n = 3), adenomyoepitheliomas in BALBc/J mice (n = 5; Fig. ), and adenosquamous carcinomas (n = 7; Fig. ).
Papillary adenocarcinoma lined by an epithelium one cell thick in a BALB/cJ mouse: neoplastic cells lack myoepithelial differentiation. Myofibroblasts are present in the stroma of the neoplasm. Scale bar, 80 μm.
Papillary adenocarcinoma lined by an epithelium two cells thick: there is myoepithelial differentiation of the basal layer (a), whereas suprabasal cells have a luminal phenotype (b). Scale bar, 80 μm.
Epithelial to mesenchymal transition (EMT) in a spontaneous neoplasm: neoplastic cells with a spindloid phenotype are labeled strongly for vimentin in a glandular carcinoma with EMT in an SJL/J mouse. Scale bar, 80 μm.
Microacinar carcinoma in a C3H/HeJ mouse infected by the mouse mammary tumor virus. Microacinar carcinomas are characterized by a microacinar pattern with prominent myoepithelial differentiation. Scale bar, 80 μm.
Figure 5 Adenomyoepithelioma in a BALB/cJ mouse: individual cells (a, arrowhead), clusters of cells (a, arrow), and occasionally large areas (b) are not labeled by antibodies against terminal differentiation markers of myoepithelial (a) and luminal cells (b) of (more ...)
Figure 6 Adenosquamous carcinoma in a BALB/cJ mouse: squamous differentiation is commonly associated with the presence of keratohyalin (a, arrowhead) or trichohyalin (b, arrowhead) granules. Squamous differentiation is 'complete', with expression of markers of (more ...)
All Hras-induced (n = 4; Fig. ) and Neu-induced (n = 10) tumors as well as some Myc-induced (n = 4; Fig. ) and SV40-TAg-induced tumors (data not shown) had a solid pattern. Some SV40-TAg-induced (n = 5; Fig. ) and Myc-induced tumors (n = 9; Fig. ) as well as all Notch4-induced tumors (n = 6) had a glandular pattern. Some Myc-induced (n = 13; Fig. ), SV40-TAg-induced (n = 3; Fig. ), and Hras-induced (n = 3) carcinomas displayed areas of EMT. Some SV40-TAg-induced tumors (n = 4) had a papillary pattern with papillae lined by epithelial cells that piled up in a disorderly fashion (data not shown). Preneoplastic lesions of Tgfa-transgenic mice consisted of macrocysts (n = 8; Fig. ) and lactation-responsive plaques (n = 3; Fig. ). All Wnt1-induced tumors (n = 13; Fig. ) exhibited a type P tumor pattern.
Figure 7 Solid carcinoma from a mouse transgenic for Hras: expression of α-smooth muscle actin in carcinomas is restricted to stromal myofibroblasts and vascular smooth muscle fibers. A similar observation was made for carcinomas arising in mice transgenic (more ...)
Figure 8 Epithelial to mesenchymal transition (EMT) in a mouse transgenic for Myc: EMT is associated with expression of terminal differentiation markers of mesenchymal (a,b), myoepithelial (c,e,f) and luminal (d) cells, with de novo acquisition of a myoepithelial (more ...)
Epithelial to mesenchymal transition (EMT) in a mouse transgenic for SV40-TAg: EMT is associated with increased expression of luminal (a) and myoepithelial (b) terminal differentiation markers. Scale bars, 80 μm (a) and 160 μm (b).
Neoplasms from Tgfa-transgenic mice: myoepithelial differentiation is present in macrocysts (a, arrows<) and in lactation-responsive plaques (b, arrows). Scale bars, 80 μm (a) and 160 μm (b).
Figure 11 Influence of the background strain on protein expression in type P tumors: the expression of K5 (a,b) and K8/18 (not shown) is higher, and squamous differentiation (not shown) is more prominent in B6SJL-Wnt1 than in FVB/NJ-Wnt1 mice. Scale bar, 80 μm. (more ...)
All spontaneous type P tumors (n = 9), all microacinar carcinomas in C3H/HeJ mice (n = 8), and one of two papillary carcinomas in one C3H/HeJ mouse showed immunolabeling for MMTV (data not shown). Proteinaceous and/or lipid secretion was identified in a small number of tumors in most transgenic models and spontaneous tumor types (Fig. ), although it was observed most consistently in macrocysts and in adenosquamous carcinomas (Fig. ).
Proteotypic Classification of Spontaneous and Transgenic Mammary Neoplasms Based on Terminal Differentiation Markersa.
Terminal differentiation proteins expression patterns
The neoplasms were categorized into three groups: those with a pure luminal phenotype (simple carcinomas), those that constitutively expressed myoepithelial and luminal markers, and those that were characterized by EMT. Hampe and Misdorp [30
] defined the term complex as 'any type of neoplasm or proliferation composed of cells resembling both secretory epithelial and myoepithelial cells'. We therefore used the term 'constitutively complex carcinomas' to indicate the tumors in which luminal and myoepithelial cells were arranged as they would be in a normal mammary gland, suggesting that these two types of cells originated from the same progenitor cell. The tumors that expressed myoepithelial markers in the areas of EMT only are designated 'acquired complex carcinomas' to indicate that myoepithelial differentiation was absent in the areas representing the original phenotype of the neoplasms, before the occurrence of EMT.
Tumor categorization was then redesigned to take into account not only the architecture of the neoplastic process [17
] but also myoepithelial differentiation and EMT, which were not always apparent on H&E-stained sections. Keratins 5, 14, and 17, which are generally considered to be markers of myoepithelial cells [4
], were occasionally identified in suprabasal cells in a variety of neoplasms (Figs ,,). α-Smooth muscle actin, another marker of myoepithelial cells, was expressed by suprabasal cells only in MMTV-associated and Wnt1
-induced type P tumors (Fig. ), a feature previously noted by Li and colleagues [13
Figure 12 Type P tumors arise in mice infected by the mouse mammary tumor virus. Myoepithelial differentiation is prominent in ductal areas (a, arrowheads), whereas suprabasal cells have a luminal phenotype and label for K8/18 (b). Clusters and individual suprabasal (more ...)
Neoplasms with a pure luminal phenotype (simple carcinomas) were identified in all transgenic models of mammary carcinogenesis (Fig. , Table ) with the notable exception of Wnt1-induced carcinomas that had a constitutively complex phenotype. Most (six of nine) glandular carcinomas arising in mice transgenic for Myc showed a few areas of myoepithelial differentiation that were interpreted as early EMT rather than the neoplasms arising from a progenitor cell common to the luminal and myoepithelial phenotypes; hence these neoplasms with minimal myoepithelial differentiation were categorized as 'simple' carcinomas. The only spontaneous neoplasms composed exclusively of cells with a luminal phenotype were four of six papillary carcinomas that, on H&E sections, were characterized by papillae lined by an epithelium one cell thick.
Constitutively complex carcinomas consisted of spontaneous papillary carcinomas lined by an epithelium two cells thick (Fig. ), microacinar adenocarcinomas arising in MMTV-infected C3H/HeJ mice (Fig. ), type P tumors of MMTV-infected C3H/HeJ mice (Fig. ) and Wnt1-transgenic mice (Fig. ), adenomyoepitheliomas (Fig. ), adenosquamous carcinomas (Fig. ), and lactation-responsive plaques and most (six of eight) macrocysts of Tgfa-transgenic mice (Fig. ). Neoplastic cells with a myoepithelial phenotype were abundant and formed an almost continuous layer surrounding cells with a luminal phenotype in microacinar carcinomas (Fig. ) and in spontaneous papillary carcinomas lined by an epithelium two cells thick (Fig. ). In type P tumors, myoepithelial differentiation was most prominent in the areas of ductal metaplasia and was minimal or absent in the frond-like areas of the neoplasms (Fig. ). In adenosquamous carcinomas, myoepithelial differentiation was most prominent in the areas of squamous differentiation (Fig. ), and was often absent in the glandular areas.
In adenomyoepitheliomas (n
= 5), large proportions of suprabasal cells were labeled for K5, K6, K8/18, K14, and K17, with K1 being expressed in only two tumors, and with duct formation and cornification being identified in four and three neoplasms, respectively. The morphology of neoplastic cells in adenomyoepitheliomas varied, ranging from fusiform to cuboidal. The proteotypic pattern of these cells is not characteristic of any specific cell type, and these cells were categorized as 'cells with myoepithelial differentiation' when in contact with a basement membrane and as 'luminal cells' when not in contact with a basement membrane. Neoplastic cells in adenomyoepitheliomas were difficult to differentiate from stromal myofibroblasts and, as reported earlier [31
], they were negative for SMA.
In lactation-responsive plaques of Tgfa-transgenic mice, myoepithelial differentiation was most prominent in the areas of ductal differentiation located at the center of these lesions (Fig. ). The peripheral portions of lactation-responsive plaques, which morphologically resemble mammary acini, generally lacked myoepithelial differentiation. Macrocysts were predominantly composed of cells with a luminal phenotype (Table ) that occasionally rested directly on the basement membrane. Segmental portions of six of eight macrocysts had myoepithelial cells (Fig. ).
Immunohistochemistry indicated that the descriptive term 'papillary carcinoma' encompasses a variety of neoplasms. First, most (four of six) spontaneous papillary carcinomas of BALB/cJ mice (Fig. ) and all (four of four) papillary carcinomas with a pseudostratified epithelium in SV40-TAg-transgenic mice were exclusively composed of cells with a luminal phenotype (simple carcinomas). Cells with myoepithelial differentiation, identified in two of six spontaneous papillary carcinomas of BALB/cJ mice, were scarce and might represent entrapped remnants of the normal mammary gland, because they were located at the periphery of the neoplasm. In spontaneous papillary adenocarcinomas characterized by two layers of cells lining neoplastic papillae, the basal layer was continuous and had a myoepithelial phenotype (Fig. ), whereas cells of the luminal layer expressed K8/18 (Fig. ).
Type P tumors, adenomyoepitheliomas, and adenosquamous carcinomas were characterized by the presence of subpopulations of cells with a high mitotic index that did not express terminal differentiation markers, or expressed them only weakly. In type P tumors, these cells were grouped at the ends of the fronds, the morphology and immunohistochemistry of which mimicked terminal end buds of the pubertal mammary gland [29
Adenomyoepitheliomas (Fig. ) and adenosquamous carcinomas (Fig. ) were populated by individual cells and clusters of cells with a large open-faced nucleus and a moderate amount of pale cytoplasm with the appearance of ground glass. These cells were haphazardly distributed in the solid areas of adenomyoepitheliomas and in the viable epithelium enclosing cornified debris in adenosquamous carcinomas. Similar populations of neoplastic cells with low expression of terminal differentiation markers were not detected in the other neoplasms.
Carcinomas in B6SJL-Wnt1-and FBV-Wnt1-transgenic mice were essentially similar and had the classical appearance of a type P tumor. However, they differed slightly with regard to their proteotypic patterns: squamous differentiation and expression of K5 and K8/18 was higher in B6SJL-Wnt1-than in FBV-Wnt1-transgenic mice (Table ; Fig. ).
EMT, an important morphologic marker of tumor progression [32
], was detected in 23 tumors. It was most commonly observed in Myc
-induced carcinomas although it was also identified in Hras
-and in SV40-TAg-induced carcinomas, and in carcinomas spontaneously arising in SJL/J and PL/J mice. Various proportions of neoplastic cells with a mesenchymal phenotype expressed vimentin (Figs , ), K5 (Fig. ), K14 (Fig. ), and K17 (Figs , ). These cells consistently expressed SMA but could not be differentiated from myofibroblasts on the basis of the expression of this antigen alone (Fig. ). EMT was also associated with increased expression of K8/18 (Figs , ). In most cases (20 of 23), a small number of cells with a myoepithelial phenotype were present in the glandular or solid areas in the immediate vicinity of the areas of EMT (Fig. ).
Morphologic progression was also observed in microacinar adenocarcinomas associated with MMTV infection in C3H/HeJ mice, where it was characterized by focal to multifocal acquisition of a solid pattern. Acquisition of the solid pattern was associated with a loss of immunolabeling for MMTV proteins (data not shown) by neoplastic cells, but the expression of terminal differentiation markers remained unaltered.
-induced carcinomas did not display a morphologic alteration suggestive of tumor progression, although the tumors evaluated included both metastatic and non-metastatic neoplasms (data not shown). The markers tested in this study did not segregate the three cell types classically described in Neu
-induced tumors [7
]. EMT was not observed in Notch4
-induced carcinomas, although the glandular pattern of these neoplasms is reminiscent of Myc
-and SV40-TAg-induced carcinomas.
Three histologic features of mammary tumors have recently been related to the activation of specific pathways: myoepithelial differentiation has been associated with activation of the Wnt pathway [7
], squamous metaplasia has been attributed to β-catenin stabilization [34
], and alveolar differentiation and milk secretion are dependent on signal transducer and activator of transcription 5a [38
]. As expected, all Wnt1
-induced carcinomas and spontaneous type P tumors displayed myoepithelial differentiation. In addition, myoepithelial differentiation, as assessed by routine H&E histology or immunohistochemistry, was identified in spontaneous papillary carcinomas with a bi-stratified epithelium, MMTV-associated microacinar carcinomas, adenomyoepitheliomas, and adenosquamous carcinomas.
Squamous metaplasia associated with expression of the full array of terminal differentiation markers of the suprabasal layers of the epidermis was found only in adenosquamous carcinomas (Fig. ). However, squamous metaplasia was also identified in some MMTV-associated (7 of 9) and Wnt1-induced (12 of 13) type P tumors, Myc-induced carcinomas (7 of 19), adenomyoepitheliomas (3 of 5), and spontaneous carcinomas exhibiting EMT (1 of 4). In spite of areas histologically consistent with squamous differentiation, MMTV-associated and transgene-induced type P tumors express only a limited set of suprabasal markers of the epidermis (Table ). For example, K1 and K10, two markers of the stratum spinosum, were seldom expressed (4 of 30 and 6 of 30 carcinomas, respectively) in neoplasms other than adenosquamous carcinomas that exhibited cornification, whereas involucrin, a marker of the stratum corneum, was expressed in 19 of 30 such neoplasms.