Box 23-1. GENE EXPRESSION PORTRAITS OF BREAST CARCINOMAS
Until recently, changes occurring in cancer cells were studied one at a time or in small groups in small sets of tumors. New microarray technologies ("gene chips") have enabled
investigators to simultaneously detect and quantify the expression of large numbers of genes (potentially all genes) in different tumors (see Box 7-1 , Chapter 7).
A major advantage of gene arrays is the ability to analyze a multitude of changes in cancer cells (i.e., a "molecular portrait") to discern overall patterns that would not be possible to
detect by conventional techniques. An example of the type of data that may be generated from such assays is illustrated in a simplified form in the figure on the facing page. The results
for 26 breast carcinomas (each corresponding to one column) for 28 genes (represented by each row) are displayed. A relative increased quantity of mRNA (relative to a reference
standard) is shown by red, a relative decreased quantity by green, and an average amount by black. Also shown in the figure are the histology of the different tumor types and the
expression of selected proteins detected by immunohistochemistry (ER, HER2, e-cadherin, and basal keratin).
Microarray studies, such as this one and others, have identified breast cancer subtypes previously identified by morphology (e.g., lobular carcinomas), by protein expression (e.g., ERpositive
and HER2/neu-positive carcinomas), and by germ-line mutations (e.g., BRCA1 and BRCA2 carcinomas). In addition, new subtypes that were not previously well defined have
been identified (e.g., the basal-like carcinomas). In the figure, results are not shown for many other tumor subtypes, such as tubular, mucinous, and medullary carcinomas, because these
are relatively rare and too few cases have been examined to allow firm conclusions.
mRNA levels do not always correspond to changes in protein expression. The quantity of protein within a cell depends not only on the amount and rate of transcription and translation,
but also on protein degradation and the rate of transport out of the cell. Therefore, other assays are necessary to determine actual protein content. Immunohistochemistry (IHC) uses
antibodies to detect proteins on tissue sections. Whereas tissue used for mRNA profiling may include both tumor and stromal cells, IHC has the advantage of being able to identify the
cell type expressing the protein and the specific cellular location of the protein.
Estrogen Receptor-Positive Carcinomas.Seventy per cent to 80% of breast carcinomas express ER and are thought to arise from intrinsically ER-positive luminal cells. ER-positive
ductal carcinomas ("no special type") are usually well to moderately differentiated and often show tubule formation. Most special types of breast cancer (i.e. lobular, tubular, mucinous,
and papillary) are also ER-positive. In the microarray data illustrated, the group of ductal carcinomas, in general, show normal or overexpression of the ER-related gene cluster and
luminal keratins, and exhibit low levels of mRNAs from the groups of genes characteristic of the other tumor types. In the lower part of the figure, IHC on one representative ductal
carcinoma demonstrates that ER is present in the nucleus, e-cadherin on the cell membrane, and that HER2/neu and basal keratins are undetectable.
In contrast to traditional IHC assays that determine the expression of only ER and a single gene under its regulation, PR, mRNA profiling provides information about many other ERregulated
genes. Using this type of assay, it might be possible to identify the cancers that express ER but fail to respond to hormonal treatments due to disruption of the signaling
pathway, resulting in low expression of other ER-regulated genes.
Lobular carcinomas can be identified by the distinctive morphologic pattern of infiltration as single cells or loosely cohesive cell clusters. This appearance has been linked to the loss of
the normal cell adhesion molecule e-cadherin, which is retained in most other carcinomas within the ER-positive group. By expression profiling, the lobular carcinomas cluster together
and are most closely related to the other ER-positive carcinomas. The absence of e-cadherin can be seen by both diminished mRNA and the absence of the protein by IHC.
Estrogen Receptor-Negative Carcinomas.These carcinomas may arise owing to loss of ER expression or from normally ER-negative cells. Expression profiling identifies two major
types of ER-negative carcinomas.
HER2-Positive Carcinomas. This group of carcinomas was previously identified by overexpression of the HER2/neu protein. In the majority of carcinomas, the mechanism of overexpression
is amplification of the gene resulting in increased transcription into mRNA and protein translation. Breast cancers are routinely assayed for HER2/neu gene and protein using
FISH or IHC ( Figs. 23-27C and B , respectively) in order to predict clinical responses to antibodies targeted to the protein. These carcinomas tend to be poorly differentiated.
The expression profile reveals not only increased copies of HER2/neu mRNA, but also increased transcription of other adjacent genes that are amplified within this segment of DNA.
These carcinomas do not overexpress the genes that are characteristic of the other subtypes of cancers in this array (e.g. ER and basal keratins), but do express e-cadherin.
Basal-like Carcinomas. This group of carcinomas is distinguished by the expression of keratins that are more typical of myoepithelial cells or potential breast progenitor cells; it has not
been previously well characterized. Because the myoepithelial cell is located in the basal area of the lobules and ducts, in the absence of knowing the specific cell of origin, this group of
carcinomas was termed basal-like. In addition to the expression of specific keratins, they also show expression of other genes in common with myoepithelial cells (e.g., p-cadherin) as
well as numerous genes related to cell proliferation. This group of carcinomas does not express ER or ER-related genes or HER2/neu, as can be seen by the array data and by IHC.
Carcinomas arising in women with BRCA1 mutations also cluster with this group. BRCA1 carcinomas are similar to basal-like carcinomas in being poorly differentiated, lacking ER and
HER2/neu expression, and expressing basal-like keratins. However, most women with basal-like carcinomas do not have germ-line BRCA1 mutations.
Conclusions.mRNA expression profiling is a powerful tool for investigating breast carcinomas. Analogous arrays to analyze DNA and protein expression profiles are under
development. In addition to identifying tumor types, as in this example, mRNA arrays have been used for predicting prognosis and response to therapy, examining tumor changes after
therapy, and classifying hereditary carcinomas. Although it might not be feasible to perform transcriptional profiling on every clinical case of breast cancer, these studies will generate
information that will lead to better diagnostic, prognostic, and therapeutic tests that are applicable to all patients.
Figure 23-Selected data from mRNA expression profiling (26 carcinomas and 28 genes) are shown in the top half of the figure. Each vertical column represents one carcinoma (and
shows information acquired from one "gene chip") and each horizontal row represents the data for a gene (identified at the left). Red indicates an increase, green a decrease, and black no
change in mRNA relative to a standard. Cluster analysis was used to group carcinomas with similar expression patterns and the groups are identified as basal-like, HER2 positive, and
the ER-positive lobular and ductal carcinomas. The most important gene clusters are identified on the right. These carcinomas have typical morphologic appearances as shown in the
middle row of images (H&E). In the lower half of the figure, mRNA expression patterns are correlated with changes in protein expression by using antibodies to detect antigens within
tissues. The presence of a protein is indicated by a brown reaction product within the tumor cells and can be localized to a subcellular site (estrogen receptor-nuclear; HER2/neu and ecadherin-
membrane; basal keratin-cytoplasmic). The array data are courtesy of Dr. Andrea Richardson, Brigham and Women's Hospital, Boston, MA, as modified from Signoretti S, Di
Marcotullio L, Richardson A, et al.: Oncogenic role of the ubiquitin ligase subunit Skp2 in human breast cancer, J Clin Invest 110:633–641, 2002.
Figure 23-15The normal breast is maintained by a complex set of interactions among luminal cells, myoepithelial cells, the basement membrane, and stromal cells (illustrated to the left
of the figure). Morphologic changes are displayed according to the risk for subsequent invasive carcinoma (top row of pictures). The seven categories of changes in biologic functions
that must occur in successful malignant cells are shown in colored boxes. The changes need not occur in a specific order but accumulate until cells acquire malignant potential. The
association of these changes with premalignant breast lesions suggests that the earliest events are related to evasion of growth-inhibiting signals, evasion of apoptosis, and selfsufficiency
in growth signals. Hereditary carcinomas arise from cells that have germ line mutations that alter DNA repair and/or normal signals for apoptosis and therefore require fewer
acquired changes. Luminal cells likely give rise to the majority of cancers, but myoepithelial cells can also undergo malignant transformation. Changes in the malignant cells are
accompanied by alterations in the supporting myoepithelial and stromal cells due to a combination of genetic and epigenetic events and disruption of the normal intercellular signaling
pathways. The final alteration, invasion of stroma, is the least well understood. It has been difficult to identify biologic changes that are specific to invasive carcinomas. It is possible that
invasion is a result of the loss of the ability of myoepithelial and stromal cells to maintain the basement membrane rather than a gain of function by the malignant cells.
Figure 23-16 A, This mammogram reveals multiple clusters of small, irregular calcifications in a segmental distribution. Suspicious calcifications must be biopsied, as 20% to 30% will
prove to be due to DCIS. B, Comedo DCIS fills several adjacent ducts (or completely replaced lobules) and is characterized by large central zones of necrosis with calcified debris. This
type of DCIS is most frequently detected as radiologic calcifications. Less commonly, the surrounding desmoplastic response results in an ill-defined palpable mass or a mammographic
density.
Figure 23-17Noncomedo DCIS. A, Cribriform DCIS comprises cells forming round, regular ("cookie cutter") spaces. The lumens are often filled with calcifying secretory material. B,
This solid DCIS has almost completely filled and distorted this lobule with only a few remaining luminal cells visible. This type of DCIS is not usually associated with calcifications and
may be clinically occult.
Figure 23-18Noncomedo DCIS. A, Papillary DCIS. Delicate fibrovascular cores extend into a duct and are lined by a monomorphic population of tall columnar cells. Myoepithelial
cells are absent. B, Micropapillary DCIS. The papillae are connected to the duct wall by a narrow base and often have bulbous or complex outgrowths. The papillae are solid and do not
have fibrovascular cores.
Figure 23-19Paget disease of the nipple. DCIS arising within the ductal system of the breast can extend up the lactiferous ducts into nipple skin without crossing the basement
membrane. The malignant cells disrupt the normally tight squamous epithelial cell barrier, allowing extracellular fluid to seep out and form an oozing scaly crust over the nipple skin.
Figure 23-20Lobular carcinoma in situ. A monomorphic population of small, rounded, loosely cohesive cells fills and expands the acini of a lobule. The underlying lobular architecture
can still be recognized.
Figure 23-21Invasive ductal carcinoma. A, This mammogram shows a density with an irregular border. There is a small, superimposed, incidental calcification. (Courtesy of Dr. Jack
Meyer, Brigham and Women's Hospital, Boston, MA.) Over 90% of such masses will prove to be invasive carcinomas. Rarely, complex sclerosing lesions, prior surgical scars, and
fibromatosis may present in this fashion. B, An irregular dense white mass is present within yellow adipose tissue. The pathologic gross differential diagnosis is the same as the
radiologic differential diagnosis.
TABLE 23-4-- Distribution of Histologic Types of Breast Cancer