Mills Ch3 Breast

II

SECTION

Breast

3

Breast Laura C. Collins  ■  Stuart J. Schnitt

EMBRYOLOGY  69

THE ADULT MALE BREAST  80

ADOLESCENCE  70

BIOLOGIC MARKERS, IMMUNOPHENOTYPE, AND MOLECULAR BIOLOGY  80

THE ADULT FEMALE BREAST  71

Estrogen Receptor and Progesterone Receptor  80 Other Biomarkers and Immunophenotypic Features  81 Molecular Markers  81 CONCLUSION  81

PREGNANCY AND LACTATION  78

MENOPAUSE  79

BLOOD SUPPLY  79

REFERENCES  81

LYMPHATIC DRAINAGE  80

Advances in breast imaging have provided a variety of noninvasive means to assist in the evaluation of patients with breast disorders (1–4). Nevertheless, at the present time, histologic examination of tissue specimens remains the cornerstone for the diagnosis of breast diseases, and an understanding of normal breast histology is essential for accurate evaluation of such specimens. It should be noted, however, that what constitutes “normal” histology in the breast varies according to gender, age, menopausal status, phase of the menstrual cycle, pregnancy, and lac- tation, among other factors. Therefore, determination of whether a given breast specimen is normal or shows pathologic alterations must take these variables into con- sideration. EMBRYOLOGY Development of the human mammary gland begins during the 5th week of gestation, at which time thickenings of the ectoderm appear on the ventral surface of the fetus. These

mammary ridges, also known as milk lines, extend from the axilla to the groin. Except for a small area in the pectoral region, the bulk of these ridges normally regress as the fetus continues to develop. Failure of regression of other portions of the milk lines can result in the appearance in postnatal life of ectopic mammary tissue or accessory nipples anywhere along the milk lines; this phenomenon is most commonly encoun- tered in the axilla, inframammary fold, and vulva (5–7). The earliest stages of breast development are largely inde- pendent of sex steroid hormones (8). After the 15th week of gestation, the developing breast exhibits transient sensitivity to testosterone, which acts primarily on the mesenchyme. Under the influence of testosterone, the mesenchyme con- denses around an epithelial stalk on the chest wall to form the breast bud, the site of mammary gland development. Solid epithelial columns then develop within the mesen- chyme, and these ultimately give rise to the lobes or seg- ments of the mammary gland. Portions of the fetal papillary dermis encase the developing epithelial cords and eventu- ally give rise to the vascularized fibrous connective tissue that surrounds and invests the mammary ducts and lobules. The more collagen-rich reticular dermis extends into the breast to form the suspensory ligaments of Cooper, which attach the breast parenchyma to the skin. Portions of the mesenchyme differentiate into fat within the collagenous stroma between the 20th and 32nd weeks of gestation.

This chapter is an update of a previous version authored by Laura C. Collins and Stuart J. Schnitt.

69

70

SECTION II : Breast

TABLE 3.1 Major Steroid and Peptide Hormonal Influences on the Breast Hormone Effects Estrogen

Required for ductal growth and branching during adolescence Required for lobuloalveolar growth during pregnancy Required for induction of progesterone receptor Not necessary for maintenance of secretion or lactation Required for lobuloalveolar differentiation and growth Probable mitogen in normal estrogen-primed breast Not necessary for ductal growth and branching Stimulates breast mesenchyme during fetal development Causes mesenchymal destruction of mammary epithelium during critical period of testosterone sensitivity Enhanced ductal-alveolar growth Enhances protein synthesis in mammary epithelium Required for secretory activity (with glucocorticoids and prolactin) Stimulates epithelial growth after parturition Required for initiation and maintenance of lactation Enhances lobuloalveolar growth during pregnancy

Progesterone

During the last 8 weeks of gestation, the epithelial cords canalize and branch, forming lobuloalveolar structures as a result of mesenchymal paracrine effects. A depression in the epidermis, the mammary pit, forms at the convergence of the lactiferous ducts. The nipple forms by evagination of the mammary pit near the time of birth. During the last few weeks of gestation the fetal mam- mary gland is responsive to maternal and placental steroid hormones, and, as a result, the epithelial cells in the acinar units exhibit secretory activity. At the time of birth, with- drawal of the maternal and placental sex steroids stimulates prolactin secretion, which in turn stimulates colostrum secretion. Both male and female neonates exhibit palpa- ble enlargement of the breast bud. As the serum levels of maternal and placental sex steroid hormones and prolac- tin decline during the first month of life, secretory activ- ity ends, and the gland regresses and becomes inactive. At this stage, and until puberty, the breast consists primarily of lactiferous ducts that exhibit some branching without evi- dence of progressive alveolar differentiation, although some rudimentary lobular structures may persist. Another feature that may be seen in the fetal breast is extramedullary hematopoiesis, and this may persist in the periductal stroma until 4 months of age (Fig. 3.1) (9). ADOLESCENCE Adolescent breast development in the female begins with the onset of puberty and the cyclic secretion of estrogen and progesterone. However, a variety of other steroid and peptide hormones are also required for proper mammary gland development (Table 3.1) (8). The ducts elongate, branch, and develop a thickened epithelium primarily due to the influence of estrogen (Fig. 3.2) (10). The process FIGURE 3.1  Breast tissue from an infant showing ducts embedded in a loose connective tissue stroma. The stromal mononuclear cells are hematopoietic elements, indicative of persistent extramedullary hematopoiesis. (Courtesy of Theonia Boyd, MD, Children’s Hospital, Boston, MA)

Testosterone

Glucocorticoids Required for maximal ductal growth

Insulin

Prolactin

Human placental lactogen Able to substitute for prolactin in epithelial growth and differentiation Stimulates alveolar growth and lactogenesis in second half of pregnancy Growth hormone Required for ductal growth and branching during adolescence May contribute to lobuloacinar growth during pregnancy Thyroid hormone Increases epithelial response to prolactin May enhance lobuloacinar growth of ductal growth and branching is largely independent of progesterone. There is an increase in the density of peri- ductal connective tissue, also as a result of relative estrogen dominance. Deposition of stromal adipose tissue occurs, and it is this adipose tissue that is largely responsible for the enlargement and protrusion of the breast disk at this time. Cyclical exposure to progesterone following exposure to estrogen during ovulatory cycles promotes lobuloacinar growth, as well as connective tissue growth. Although the majority of breast development occurs during puberty, this process continues into the third decade, and terminal dif- ferentiation of the breast is only induced by pregnancy. Adapted from McCarty KS, Nath M. Breast. In: Sternberg SS, ed. Histology for Pathologists . Philadelphia, PA: Lippincott-Raven; 1997: 71–82.

71

CHAPTER 3:  Breast

A

B

FIGURE 3.2  Adolescent breast tissue composed of branching ducts with rudimentary lobule development (type 1 lobules). The stroma consists of a mixture of fibrous connective tissue and adipose tissue. A: Scanning magnification. B: High power.

The adolescent male breast is composed of fibroadipose tissue and ducts lined by low cuboidal cells.

the nonlactating adult breast, and the relative proportions of fibrous tissue and adipose tissue vary with age and among individuals (Fig. 3.3). The ductal-lobular system of the breast is arranged in the form of segments, or lobes. While these segments can be readily appreciated by injecting the ductal system with dyes or radiologic contrast agents (Fig. 3.4), they are ana- tomically poorly defined, and no obvious boundaries can be appreciated between these segments during surgery, upon gross inspection of mastectomy specimens, or on histologic examination. In addition, these segments show consider- able individual variation with regard to their extent and dis- tribution (11), and the ramifications of individual segments may overlap. The segmental nature of some neoplastic pro- cesses in the breast, particularly ductal carcinoma in situ, is now widely appreciated. This recognition, in conjunction with observations in developmental anatomy and morphol- ogy, has led to the development of the “sick lobe” hypoth- esis of breast cancer (12,13). This theory postulates that early breast carcinoma (ductal carcinoma in situ) is a lobar disease, often isolated to a single ductal system (or lobe). Thus, surgical resection of the involved lobe or segment is an important therapeutic goal. Unfortunately, since it is not possible for the surgeon to define intraoperatively the boundaries of the involved segment, performing a “segmen- tectomy” to remove the entirety of a diseased segment is at this time more of a theoretical concept than a practically attainable goal. Each segment consists of a branching structure that has been likened to a flowering tree (Fig. 3.5) (14). The lob- ules represent the flowers, draining into ductules and ducts (twigs and branches), which, in turn, drain into the collect- ing ducts (trunk) that open onto the surface of the nipple. Just below the nipple, the ducts expand to form lactiferous sinuses. The sinuses terminate in cone-shaped ampullae just below the surface of the nipple.

THE ADULT FEMALE BREAST The size of the breast is greatly influenced by the indi- vidual’s body habitus since the breast is a major reposi- tory for fat; it can range in size from 30 g to more than 1,000 g. The breast lies on the anterior chest wall over the pectoralis major muscle and typically extends from the 2nd to the 6th rib in the vertical axis and from the sternal edge to the mid-axillary line in the horizontal axis. Breast tissue also projects into the axilla as the tail of Spence. The breast extends laterally over the serratus anterior muscle and inferiorly over the external oblique muscle and the superior rectus sheath. The breast lies within a space in the superficial fascia, which is continu- ous with the cervical fascia superiorly and the superficial abdominal fascia of Cooper inferiorly. The only bound- ary of the breast that is anatomically well defined is the deep surface where it abuts the pectoralis fascia. How- ever, despite this macroscopic demarcation, microscopic foci of glandular tissue may extend into and even through the pectoral fascia and may traverse the other anatomic boundaries described above. The clinical significance of this observation is that even total mastectomy does not result in removal of all glandular breast tissue. Bundles of dense fibrous connective tissue, the suspensory liga- ments of Cooper, extend from the skin to the pectoral fascia and provide support to the breast. The adult female breast consists of a series of ducts, ductules, and lobular acinar units embedded within a stroma that is composed of varying amounts of fibrous and adipose tissue. The stroma comprises the major portion of

72

SECTION II : Breast

A

B

FIGURE 3.3  The stroma is the predominant component of the nonlactating breast and consists of varying amounts of collagen and adipose tissue. A: Low-power view of breast with dense, fibrotic stroma. B: Low- power view of breast with predominantly fatty stroma.

The actual number of segments in the breast and their relationship to each other has long been a matter of debate. Most textbooks indicate that there are 15 to 20 ductal ori- fices on the nipple surface and suggest that this corresponds to the number of ductal systems, segments, or lobes in the breast (5,6,15,16). In contrast, a number of mammary duct

injection studies have suggested that there are only between 5 and 15 discrete breast ductal systems or segments in each breast. The discrepancy between the number of duc- tal orifices on the nipple and the actual number of breast segments or ductal systems may be explained by the fact that some of the orifices on the nipple represent openings of sebaceous glands or other nonductal tubular structures that do not contribute to the ductal-lobular anatomy of the breast. Another possibility is that some lactiferous ducts bifurcate immediately prior to entering the nipple or end blindly (16,17). The issue of anastomoses between ductal systems is also unresolved. One study indicated that, while ductal systems may lie in close proximity to one another and even intertwine within a particular quadrant, they do not interconnect (16). However, anastomoses between ductal systems have been reported by others (18).

FIGURE 3.4  Ductogram (galactogram). Performed by injecting con- trast material into an orifice of a lactiferous duct at the nipple, a duc- togram demonstrates the complex ramifications of a single mammary ductal system (also known as a segment or lobe).

FIGURE 3.5  Microanatomy of normal adult female breast tissue show- ing extralobular ducts, terminal ducts, and lobules, the latter composed of groups of small glandular structures, the acini.

73

CHAPTER 3:  Breast

A

B

FIGURE 3.6  The mammary ductal-lobular system is lined by a dual cell population, an inner epithelial cell layer and an outer layer of myoepithelial cells. A: High-power view of a lobule. The myoepithelial cells sur- rounding the acinar epithelial cells are variably conspicuous. B: High-power view of an extralobular duct, showing distinct epithelial and myoepithelial cell layers.

The epithelium throughout the ductal-lobular system is bilayered, consisting of an inner (luminal) epithelial cell layer and an outer (basal) myoepithelial cell layer. The importance of this double cell layer cannot be overempha- sized because it is one of the main guides used to distinguish benign from malignant lesions (19). The luminal epithelial cells of the resting breast ducts and lobules are cuboidal to columnar in shape and typically have pale eosinophilic cyto- plasm and relatively uniform oval nuclei. These epithelial cells express a variety of low—molecular-weight cytokera- tins, including cytokeratins 7, 8, 18, and 19 (20–24). The outer (or myoepithelial) cell layer, although always present, is variably distinctive (Fig. 3.6). Myoepithelial cells range in appearance from barely discernible, flattened cells with compressed nuclei to prominent epithelioid cells with

abundant clear cytoplasm. In some cases, the myoepithelial cells have a myoid appearance featuring a spindle cell shape and dense, eosinophilic cytoplasm, reminiscent of smooth muscle cells (Fig. 3.7). Even when inconspicuous on hema- toxylin- and eosin-stained sections, myoepithelial cells can readily be demonstrated using immunohistochemical stains for a variety of markers, including actins, calponin, smooth muscle myosin heavy chain, p63, CD10, and p75 among others (Fig. 3.8) (25–30). However, these markers vary in both sensitivity and specificity for myoepithelium as well as in their expression by location within the terminal duct lobular unit (TDLU). Myoepithelial cells express high— molecular-weight cytokeratins 5/6, 14, and 17 (20–24,31), but the expression of cytokeratin 14 is restricted to the myoepithelial cells of the large ducts and terminal ducts;

A

B

FIGURE 3.7  Myoepithelial cells can vary in their histologic appearance. A: Myoepithelial cells in this lobule show prominent cytoplasmic clearing. B: In this lobule, the myoepithelial cells show myoid features.

74

SECTION II : Breast

A

B

both luminal epithelial cells and myoepithelial cells (34). The relationship of these putative “progenitor” cells to mam- mary stem cells is an unresolved issue (34,35). However, it is clear that the cells with the characteristic features of stem cells (i.e., self-renewal and the ability to differentiate down different cell lineages to form all of the cell types found in the mature tissue) do exist in the breast. Mammary stem cells appear to be important in both breast development (36) and mammary carcinogenesis (35,37,38). Phenotypic fea- tures that have been associated with the mammary stem cell population include lack of expression of ER and progester- one receptor (PR), high expression of CD44, low or absent expression of CD24 and expression of aldehyde dehydroge- nase 1 (ALDH1) (38,39), although the most accurate com- bination of markers to reliably identify mammary stem cells is unresolved at this time. A basal lamina consisting of type IV collagen and laminin surrounds the mammary ducts, ductules, and acini (21,40). This basal lamina is present outside of the myoepithelial FIGURE 3.8  Extralobular duct ( A ) and lobule ( B ) immunostained for p63. The myoepithelial cells show strong nuclear reactivity, whereas the epi- thelial cell nuclei are negative. C: Double immunostain for smooth muscle actin (red cytoplasmic staining) and p63 (brown nuclear staining) high- light the myoepithelial cells around this mammary duct. Note the lack of staining of the epithelial cells for both p63 and smooth muscle actin.

C

expression is not seen in myoepithelial cells of the intra- lobular ductules and acini (32). Normal breast luminal epithelium is composed of cells in various states of differentiation as demonstrated by a panel of biomarkers that includes estrogen receptor (ER), androgen receptor, vitamin D receptor, low- and high- molecular–weight cytokeratins, and Ki67. Interestingly, each of these cell types appears to correspond to breast cancers with a similar immunophenotype, raising the possi- bility that breast tumors have phenotypes or differentiation states which correspond to those of normal cells, akin to that seen in hematologic malignancies (33). A third cell type dispersed irregularly throughout the ductal-lobular system expresses high—molecular-weight cytokeratins 5 and 14 in the absence of expression of mark- ers of differentiated luminal epithelial cells (such as low– molecular-weight cytokeratins) or myoepithelial cells (such as smooth muscle actin). These cells have been postulated to represent progenitor cells capable of differentiating into

75

CHAPTER 3:  Breast

TDLU (14,41). Indeed, the only common lesion thought to arise from large- or medium-sized ducts rather than from the TDLU is solitary intraductal papilloma (Fig. 3.10). The normal lobule consists of a variable number of blind-ending terminal ductules, also called acini, each with its typical double cell layer. The lobular acini are invested by a loose, fibrovascular intralobular stroma with varying numbers of lymphocytes, plasma cells, macrophages, and mast cells. This specialized intralobular stroma is sharply demarcated from the surrounding denser, more highly collagenized, paucicellular interlobular stroma, and stro- mal adipose tissue (Fig. 3.11). One feature of note that is sometimes encountered in the extralobular stroma is the presence of multinucleated giant cells (42). Their signifi- cance is unknown; and, while they may present a disturbing appearance, they should not be mistaken for the malignant cells of an invasive carcinoma (Fig. 3.12). The size of mammary lobules and number of acini per lobule are extremely variable. Russo et al. have described four lobule types (43–45). Type 1 lobules are the most rudi- mentary and are most prevalent in prepubertal and nullipa- rous women, comprising 65% to 80% of the lobules in this group (Fig. 3.2). These lobules are comprised primarily of ducts with sprouting alveolar buds. However in practice, it is not possible to reliably distinguish type 1 lobules (i.e., those that have not fully developed) from those in which the number of acini is reduced due to involution. Type 1 lobules gradually evolve to more mature structures (type 2 and type 3 lobules) through the development of additional alveolar buds. The number of alveolar buds per lobule increases from approximately 11 in type 1 lobules to 47 and 80 in type 2 and 3 lobules, respectively. Recent data suggest that the histologic appearance of normal lobules may influence the risk of subsequent breast cancer. In par- ticular, women whose breast tissue exhibits predominantly type 1 lobules or lobules that have undergone involution have a reduced risk of subsequent breast cancer compared to women with predominantly type 3 lobules or those that have not undergone involution (46–48). While there is

FIGURE 3.9  Immunostain for type IV collagen highlights the basal lamina around the acini of a lobule.

cell layer and serves to demarcate the breast ductal-lobular system from the surrounding stroma (Fig. 3.9). Beyond the basal lamina, the extralobular ducts exhibit a zone of fibro- blasts and capillaries. Elastic tissue is normally present in variable amounts around ducts and is generally more promi- nent in older than in younger women. Elastic fibers are not typically seen around the terminal ducts or lobular acini. The lobule, together with its terminal duct, has been called the TDLU. This represents the structural and functional unit of the breast. During lactation, epithelial cells in both the ter- minal duct and lobule undergo secretory changes. Thus, the terminal ducts are responsible for both secretion and trans- port of the secretions to the extra-lobular portion of the ductal system (15). Subgross anatomic studies have shown that most lesions originally termed “ductal” (e.g., cysts, ductal epithelial hyperplasia, and ductal carcinoma in situ) actually arise from the TDLU, which “unfolds” with coalescence of the acini to produce larger structures resembling ducts. The majority of pathologic changes in the breast, including in situ and inva- sive carcinomas, are generally considered to arise from the

FIGURE 3.10  A schematic representation of the breast, indicating the sites of origin of patho- logic lesions. (Reprinted from Schnitt SJ, Millis RR, Hanby AM, et al. The breast. In: Mills SE, Carter D, Greeson JK, Oberman HA, Reuter VE, Stoler MH, eds. Sternberg’s Diagnostic Surgical Pathology . 4th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2004:323–398.)

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SECTION II : Breast

A

B

FIGURE 3.11  Intralobular and extralobular stroma. A: Low-power view of several lobules that are invested by loose, intralobular stroma. The interlobular stroma is composed primarily of dense collagen with admixed adipose tissue. B: Higher-power view contrasts loose intralobular stroma with more collagenized interlobular stroma.

some evidence to suggest the stage of involution is consis- tent across the breast and may provide an additional assess- ment of risk, the value of reporting the predominant lobular type in clinical practice remains to be determined (49). The lobules exhibit morphologic changes during the menstrual cycle, and these are seen in both the epithelial and stromal components (50–53). These changes are sum- marized in Table 3.2. While the changes that occur during the menstrual cycle are variable among lobules in the same breast, even among immediately adjacent lobules, a domi- nant morphologic pattern is typically present in each phase. However, these menstrual cycle–related changes are subtle when compared with the dramatic alterations seen during pregnancy and lactation and when compared with the men- strual cycle–related changes seen in the endometrium.

Occasionally, the TDLU epithelial cells show promi- nent clear cell change in the cytoplasm. This may be seen in both premenopausal and postmenopausal women and appears to be unrelated to pregnancy or exogenous hormone use (54). The nipple–areola complex is a circular area of skin that exhibits increased pigmentation and contains numerous sensory nerve endings. The nipple is placed centrally and is elevated above the surrounding areola. The tip of the nipple contains 15 to 20 orifices. However, as discussed earlier, the number of such openings may not correlate directly with the number of breast segments. In the nonlactating breast, these duct openings typically possess keratin plugs. The areola surface exhibits numerous small, rounded eleva- tions, the tubercles of Montgomery.

A

B

FIGURE 3.12  Multinucleated stromal giant cells. A: Low-power view showing multinucleated giant cells scat- tered in the stroma. B: High-power view illustrates cytologic detail. These cells have a mesenchymal phenotype. Despite their worrisome histologic appearance, they have no known clinical significance.

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CHAPTER 3:  Breast

TABLE 3.2 Histologic Changes in Lobules During the Menstrual Cycle Menstrual Cycle Phase Epithelium

Acinar Lumina

Intralobular Stroma

Largely closed and inapparent

Dense, cellular, with plump fibroblasts

Early follicular

Cells: single cell type (small, polygonal cells with pale eosinophilic cells); myoepithelial cells inconspicuous

Orientation: poor Secretion: none Mitoses/apoptosis: rare

Well-defined

Less cellular and more collagenized than in early luteal phase

Late follicular

Cells: three cell types, including luminal basophilic cells, intermediate pale cells (as seen in early follicular phase), and myoepithelial cells with clear cytoplasm Orientation: radial around lumen Secretion: none Mitoses/apoptosis: rare Cells: three cell types, including luminal basophilic cells with minimal apical snouting, intermediate pale cells, and myoepithelial cells with prominent cytoplasmic vacuolization and ballooning Orientation: radial around lumen Secretion: slight Mitoses/apoptosis: rare Cells: three cell types, including luminal basophilic cells with prominent apical snouting, intermediate pale cells and myoepithelial cells with prominent cytoplasmic vacuolization Orientation: radial around lumen Secretion: active apocrine secretion from luminal cells Mitoses/apoptosis: frequent (peak of mitotic activity) Cells: two cell types, including luminal basophilic cells with scant cytoplasm and less apical snouting than in late luteal phase, and myoepithelial cells with extensive cytoplasmic vacuolization Orientation: radial around lumen Secretion: resorbing Mitoses/apoptosis: rare

Loose

Open, enlarged compared to follicular phase, with slight secretion

Early luteal

Open, with secretion

Loose, edematous, congested blood vessels

Late luteal

Distended with secretion Dense, cellular

Menstrual

Adapted from McCarty KS, Nath M. Breast. In: Sternberg SS, ed. Histology for Pathologists . Philadelphia, PA: Lippincott-Raven; 1997: 71–82.

Both the nipple and areola are covered by keratiniz- ing, stratified squamous epithelium, and this extends for a short distance into the terminal portions of the lactifer- ous ducts. The epidermis of the nipple–areola complex may contain occasional clear cells that are cytologically benign and that must not be confused with Paget cells (Fig. 3.13) (55,56). Some of these cells represent clear keratinocytes, whereas others are thought to be derived from epidermally located mammary ductal epithelium (Toker cells) (56). The proximal ramifications of the mammary ductal sys- tem that are present in the dermis of the nipple typically have a pleated or serrated contour (Fig. 3.14). These ducts are surrounded by a stroma rich in circular and longitu- dinal smooth muscle bundles, collagen, and elastic fibers (Fig. 3.15). Occasionally, lobules may be seen in the nipple (57). Simple mammary ducts are also present throughout the dermis of the areola, even at its periphery, and these

FIGURE 3.13  Clear cells in nipple epidermis. In some cells, the clear- ing is extreme, with formation of large intracytoplasmic vacuoles. These cells should not be mistaken for the cells of Paget disease.

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SECTION II : Breast

FIGURE 3.14  Cross section through the nipple. The irregular, pleated, or serrated contour of the nipple ducts is evident.

FIGURE 3.15  High-power view of nipple dermis/stroma, demonstrating prominent bundles of smooth muscle fibers.

may extend to within less than 1 mm of the basal layer of the epidermis (58). While the nipple–areola complex lacks pilosebaceous units and hairs except at the periphery of the areola, the dermis contains numerous sebaceous glands. Some of these glands open directly onto the surface of the nipple and areola, whereas others drain into a lactiferous duct or share a common ostium with a lactiferous duct. The tubercles of Montgomery represent a unit consisting of a sebaceous apparatus and an associated lactiferous duct (Fig. 3.16) (59). During pregnancy, these tubercles become increas- ingly prominent. Apocrine sweat glands may also be seen in the dermis of the nipple and areola. Another finding that may occasionally be encountered within the breast parenchyma is the presence of intrama- mmary lymph nodes (60,61). These lymph nodes may be identified as an incidental finding in breast tissue removed

because of another abnormality, or they may be seen as den- sities on mammograms (62).

PREGNANCY AND LACTATION It is not until pregnancy that full development of the breast occurs in humans. During pregnancy, epithelial cell prolif- eration resumes. There is a dramatic increase in the number of lobules, as well as in the number of acinar units within each lobule secondary to epithelial cell proliferation and lobuloalveolar differentiation under the influence of estro- gen, progesterone, prolactin, and growth hormone; growth is further enhanced by adrenal glucocorticoids and insu- lin. This lobular development and expansion occurs at the expense of both the intralobular and extralobular stroma. By

A

B

FIGURE 3.16  Montgomery areolar tubercle. A: Low-power view. B: Higher-power view. These tubercles are units composed of a lactiferous duct and associated sebaceous gland.

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CHAPTER 3:  Breast

A

B

FIGURE 3.17  Lactating breast tissue. A: There are numerous acini in this lobule, and these are enlarged and dilated. There is minimal intervening stroma. B: Higher-power view illustrates prominent epithelial cell enlargement, cytoplasmic vacuolization, and protrusion of cells into the acinar lumen. Some of the cells have a hobnail appearance. Myoepithelial cells are inconspicuous.

the end of the first trimester, there is grossly evident breast enlargement, superficial venous dilatation, and increased pigmentation of the areola. During the second and third trimesters, lobular growth continues, and the acinar units begin to appear monolayered. The myoepithelial cells in the acini are difficult to discern at this time due to the increase in size and volume of the epithe- lial cells, but they remain clearly evident in the extralobular ducts. The cytoplasm of the epithelial cells becomes vacu- olated, and secretion accumulates in the greatly expanded lobules. After parturition, the lactating breast is character- ized by distension of the lobular acini as a result of abundant accumulated secretory material and prominent epithelial cell cytoplasmic vacuolization. Many of the epithelial cells have a bulbous or hobnail appearance and protrude into the acinar lumina (Fig. 3.17). Myoepithelial cells remain attenuated and inconspicuous. The florid changes seen in pregnancy and lactation can be alarming to the inexperienced observer; areas of infarction, which occasionally occur in the pregnant breast, may compound the problem (63). When lactation ceases, the lobules involute and return to their normal resting appearance. Involution usually pro- ceeds unevenly and takes several months. Involuting lob- ules are irregular in contour and are frequently infiltrated by lymphocytes and plasma cells (64,65). Occasionally, an isolated lobule showing secretory changes may be seen in the breasts of women who are not pregnant; this phenom- enon may occur in the nulliparous woman as well.

and atrophy of the mammary TDLUs, with reduction in the size and complexity of the acini, and there is loss of the specialized intralobular stroma (66,67). Ducts may become variably ectatic. The postmenopausal breast is characterized by a marked reduction in glandular tissue and collagenous stroma, often with a concomi- tant increase in stromal adipose tissue. The end stage of menopausal involution is typified by remnants of the TDLUs, typically composed of ducts with atrophic acini, surrounded by hyalinized connective tissue or embedded within adipose tissue with little or no surrounding stroma (Fig. 3.18). BLOOD SUPPLY The principal arterial supply to the breast is provided by the internal mammary and lateral thoracic arteries. Perfo- rating branches of the internal mammary artery provide the blood supply to approximately 60% of the breast, mainly the medial and central portions. Approximately 30% of the breast, mainly the upper and outer portions, receives blood from the lateral thoracic artery. Branches of the thoracoacromial, intercostal, subscapular, and thoracodor- sal arteries make minor contributions to the mammary blood supply (7). Venous drainage of the breast, as in other locations, shows considerable individual variation but largely follows the arterial system. There is a superficial venous complex that runs transversely from lateral to medial in the subcuta- neous tissue. These vessels then drain into the internal tho- racic vein. Deep venous drainage of the breast is via three routes: the perforating branches of the internal thoracic vein, branches of the axillary vein, and tributaries of the

MENOPAUSE During the postmenopausal period, with the reduction of estrogen and progesterone levels, there is involution

80

SECTION II : Breast

A

B

FIGURE 3.18  Postmenopausal breast tissue. A: This sample consists primarily of fatty stroma with a few atro- phic ductules. B: In this specimen, a few residual, atrophic lobular acini are evident in a fibrotic stroma, which has replaced the normal, loose intralobular stroma.

intercostal veins, which drain posteriorly into the vertebral veins and the vertebral plexus (8,68).

THE ADULT MALE BREAST The adult male breast, like the female breast, is composed of glandular epithelial elements embedded in a stroma that is composed of varying amounts of collagen and adipose tis- sue. However, in contrast to the adult female breast, the epithelial elements of the male breast normally consist of branching ducts without lobule formation.

LYMPHATIC DRAINAGE Lymphatic drainage of the breast occurs through four routes: cutaneous, axillary, internal thoracic, and pos- terior intercostal lymphatics. The cutaneous lymphatic drainage system consists of both a superficial plexus of channels that lie within the dermis overlying the breast and a deeper network of lymphatic channels that runs with the mammary ducts in the subareolar area. Most of these cutaneous channels drain to the ipsilateral axilla. Cutaneous lymphatics from the inferior aspect of the breast may drain to the epigastric plexus and ultimately to the lymphatic channels of the liver and intra-abdominal lymph nodes. There are three lymphatic drainage pathways in the mammary parenchyma. The most important drainage basin for lymphatic flow from the breast is the axilla, and the axillary lymph nodes receive the vast majority of the lymph drained. The internal thoracic lymphatic route car- ries less than 10% of the lymphatic flow from the breast and ultimately terminates in the internal mammary lymph nodes (7). Drainage eventually empties into the great veins via the thoracic duct, the lower cervical nodes, or the jugular–subclavian confluence. The third and least impor- tant route of mammary lymphatic drainage is the poste- rior intercostal lymphatics, which drain into the posterior intercostal lymph nodes. An understanding of the lym- phatic drainage of the breast is of particular importance in the current era of sentinel lymph node biopsy since this explains the occasional finding of sentinel lymph nodes outside of the axilla (5,7,8).

BIOLOGIC MARKERS, IMMUNOPHENOTYPE, AND MOLECULAR BIOLOGY

Estrogen Receptor and Progesterone Receptor It is now known that there are at least two different ERs, ER α and ER β ; ER α has been far more extensively studied. Using immunohistochemistry, ER α expression can be dem- onstrated in the nuclei of both ductal and lobular epithe- lial cells, with a higher proportion in lobules than in ducts. However, even in the lobules, only a small proportion of the cells show ER α immunoreactivity. Most often, ER α - positive cells in the lobules are distributed singly, admixed with and surrounded by ER α -negative cells (Fig. 3.19) (69). Furthermore, there is considerable heterogeneity in stain- ing for ER α among lobules in the same breast. Of interest, in breast tissue from premenopausal women, there is gen- erally an inverse relationship between expression of ER α and markers of cell proliferation. In particular, most ER α - positive cells do not show expression of the proliferation- related antigen Ki-67, and Ki-67–positive cells are typically ER α -negative. The proportion of ER α -positive cells gradu- ally increases with age but remains relatively stable after the menopause. The incidence of lobules showing contiguous

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breast epithelium may show HER2 protein overexpression, p53 protein accumulation, or p53 mutations. Of note p53 alterations have been identified more frequently in normal epithelium adjacent to breast carcinomas that are ER, PR and HER2 negative (“triple negative”), and/or in BRCA1 mutation carriers raising the possibility that this “p53 sig- nature” may confer a predisposition to p53-associated— high-grade carcinomas (76,77). The antiapoptotic protein bcl-2 is consistently expressed by normal breast epithelial cells (78). S-100 pro- tein is strongly expressed by normal myoepithelial cells and variably expressed by mammary epithelial cells (79). Epi- thelial cells also show variable expression for casein (80), α -lactalbumin (81), gross cystic disease fluid protein-15 (82), mammaglobin (83), GATA-3 and c-Kit (CD117) (84), among other proteins. As noted earlier, cytokeratins 7, 8, 18, and 19 (20–24) are typically expressed by epithelial cells, whereas myoepithelial cells express cytokeratins 5/6, 14, and 17 (20–24,31). Molecular Markers The ability to evaluate DNA, RNA, and protein using the modern tools of molecular biology, particularly when guided by such techniques as laser capture microdissection (85), are enhancing our understanding of breast tumorigenesis and may even serve to redefine what constitutes “normal.” For example, a number of studies have shown that histo- logically normal TDLUs can exhibit an abnormal geno- type, characterized by loss of heterozygosity (86,87), allelic imbalance (88,89) at various chromosomal loci, or altered gene expression profiles (90). At this time, however, the sig- nificance of these genetic and molecular alterations in his- tologically normal breast tissue remains to be determined. Studies of normal breast tissue using these techniques will also help to further define the presence and nature of pro- genitor cells or stem cells and their role in breast develop- ment and carcinogenesis (23,91–93), as well as patterns of gene and protein expressions that distinguish normal from abnormal breast tissue and cells (94–97). CONCLUSION The histologic features of the normal breast are dynamic and vary with age and hormonal milieu, among other fac- tors. An understanding of normal breast histology is essen- tial to permit the reliable distinction between physiologic changes and pathologic alterations.

FIGURE 3.19  Immunostain for estrogen receptor- α (ER α ) in a normal lobule. A minority of epithelial cells show nuclear staining.

patches of ER α -positive cells also increases with age and with involutional changes (69). In addition, the proportion of ER α -positive proliferating cells increases with age (69). In premenopausal women, ER α expression varies with the phase of the menstrual cycle, being higher in the follicular than in the luteal phase (70). Myoepithelial cells do not show ER α immunoreactivity (33). A second form of ER, ER β , is also expressed in nor- mal breast tissue. Expression of ER β has been observed not only in epithelial cells of ducts and lobules, but also in myoepithelial cells, endothelial cells, and stromal cells (70–73). The expression of this form of ER does not seem to vary with the phase of the menstrual cycle. It has been speculated that the relative levels of ER β and ER α may be important in determining the risk of breast cancer devel- opment, and that higher levels of ER β relative to ER α are protective against neoplastic progression in the breast (71). However, additional studies are needed to more clearly elucidate the role of ER β in normal breast physiol- ogy and in breast cancer pathogenesis and to determine which ER β isoform provides the greatest specificity in this regard (74). Expression of PR has not been as extensively studied in normal breast tissue as has ER. Like ER α , PR is expressed in the nuclei of ductal and lobular epithelium. However, in contrast to ER α expression, PR expression does not seem to vary with the menstrual cycle phase (70).

Other Biomarkers and Immunophenotypic Features

Expression of a wide variety of biomarkers has been studied in benign breast tissue (75) and a comprehensive review of these is beyond the scope of this chapter. However, a cou- ple of these merits brief mention, though the clinical sig- nificance of the findings is as yet uncertain. Rarely, normal

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