Tumors of the Mammary Gland

17
Tumors of the Mammary Gland


Michael H. Goldschmidt,1 Laura Peña,2 and Valentina Zappulli3


1University of Pennsylvania, USA


2Complutense University of Madrid, Spain


3University of Padua, Italy


Epidemiology


Mammary neoplasms are very common in dogs, cats, and humans but are rare in most other species. Tumors are much more common in intact females and occur rarely in males. In the dog and cat, the incidence rate varies with geographic location and is directly related to ovariectomy and the age at which this is undertaken. In those countries where ovariectomy is infrequently performed, mammary neoplasms are exceedingly common and may represent 50–70% of all neoplasms in intact bitches.1 In the United States, the incidence of mammary neoplasia was originally reported to be 198 tumors per 100,000 dogs (males and females) per year and these accounted for 41.7% of all neoplasms in intact bitches. The same study also identified 257.7 malignant mammary tumors per 100,000 intact female dogs per year.2 More recent data from Sweden report an incidence of 111 tumors per 10,000 dog‐years at risk, mostly intact females, while in the United Kingdom, the reported incidence was 205 tumors per 100,000 dogs per year.3,4 In a lifetime study of Beagles in the United States, 71% of the females developed one or more mammary neoplasms.5 However, there are many other factors that influence the incidence, including breed popularity, husbandry, and socioeconomic factors.


The ratio of benign to malignant neoplasms in dogs is difficult to establish. Factors that influence the ratio are often directly linked to the experience of the pathologist; less‐experienced individuals tend to overdiagnose malignant neoplasms. In those countries where ovariectomy is commonly performed on dogs there is a lower overall incidence of mammary neoplasms but a higher incidence of malignant mammary neoplasms. In most studies 20–80% of canine mammary neoplasms submitted for surgical pathology evaluation are diagnosed as malignant.


Mammary neoplasms are less common in cats and the overall incidence of mammary tumors is low when compared to dogs. Mammary tumors are the third most common tumor, and make up 12% of all tumors irrespective of sex with an annual incidence of 25.4/100,000 cats per year.2 A study undertaken in northern Italy found that mammary tumors account for 16% of all tumors and 25% of tumors in female cats.6


The incidence of mammary tumors in other domestic animal species is very low as most food‐ and fiber‐producing species do not reach parity or an age at which mammary tumors are likely to develop. In humans, breast cancer is the most common cancer in women, with a very low incidence in men. There are numerous publications devoted to this most important topic.


Risk factors


Many factors contribute to the risk of developing a mammary neoplasm. We will only consider those factors that are directly associated with the development of mammary neoplasms in the dog and cat.


Age


Most mammary neoplasms occur in middle age to older dogs with a very low incidence in young dogs. The peak incidence is between 7 and 13 years old.7,8 In a recent study, benign neoplasms occured in younger dogs (mean 7–9 years) whereas malignant neoplasms occured in older dogs (mean 9–11 years).9 However, the age at which tumors develop will depend on the breed and size of the dog, as large breeds develop mammary neoplasms at a younger age than small breeds. The use of exogenous hormone, both progestins and estrogens, will also decrease the age at which mammary neoplasms develop.


Most cats with mammary neoplasms are diagnosed between 10 and 12 years old with an increased incidence after 9 years of age.2,10–13 “Tumors” in young cats are more commonly dysplastic lesions, specifically, mammary fibroadenomatous change (fibroepithelial hyperplasia/hypertrophy, mammary hypertrophy).


Ovariectomy and exogenous hormone exposure


Numerous studies have shown that ovariectomy undertaken at an early age has a profound influence on the subsequent development of mammary neoplasia. In dogs ovariectomized before their first heat cycle, the risk of developing a mammary neoplasm decreased by 99.5% and the risk decreased by 92% and 74% if ovariectomy was performed prior to the second and third heat cycles, with no statistically significant benefits if ovariectomy occured after the third cycle. Ovariectomy after the fourth heat cycle does not decrease the risk of developing a mammary neoplasm but does provide some protection. Other factors, such as pseudo‐pregnancy, pregnancy, or parity, do not significantly influence the risk of developing mammary neoplasms.14–16 Exogenous hormones, both progestins and estrogens increase the risk of mammary tumor development in dogs. Low doses of progestins promote the development of benign neoplasms, whereas a combination of progestins and estrogens appears to induce the development of malignant neoplasms.17


Several studies have shown that ovariectomy at a very young age significantly reduces the incidence of mammary neoplasm in older cats. The incidence is approximately 7‐fold higher in intact cats when compared to ovariectomized cats, and the risk of developing a mammary neoplasm increases with each heat cycle. Cats spayed prior to 6 months of age have a 91% reduction in the risk of mammary carcinoma development compared with intact cats. In those spayed prior to 1 year there is an 86% reduction in risk. There is no significant benefit after 2 years of age. Parity does not affect feline mammary carcinoma development.18


Exogenous progestins significantly increase the risk (3.4 times) of female and male cats developing mammary tumors. Most cases are dysplastic lesions (mammary fibroadenomatous change) that may show malignant progression with continued use of the progestins, and these cats may also develop neoplasms in more than one gland.19–22


Breed susceptibility


Mammary neoplasms tend to occur more commonly in small pure breeds although some larger breeds of dogs are at increased risk. It is unclear whether this represents an increased risk for small dogs or whether small dogs receive better veterinary care than large working dogs. However, different lines in the same breed were found to have a different risk of developing mammary neoplasms. Two maternal families in a beagle lifespan study had markedly different phenotypes, one susceptible and one resistant to mammary neoplasia. Neither p53 nor p185erB2 was the basis for this familial predisposition.23 A recent study (N = 292) found a predominance in small breed dogs, especially the Maltese, Yorkshire terrier, shih tzu and poodle, but no significant increase in the proportion of benign to malignant neoplasms in each breed.24


Mammary neoplasms are reported to occur more commonly in Siamese cats.25


Diet and obesity


Several observational studies have found a correlation between diet and the risk of developing mammary neoplasia. Dogs fed a diet high in red meat and that were obese at 1 year of age had an increased risk of developing mammary neoplasms.26 Another study reported that dogs that were thin at 9–12 months of age had a decreased risk of developing mammary neoplasms.15 There was a suggested association of longer survival times following removal of malignant mammary tumors in dogs fed a low‐fat, high‐protein diet.15 Whether these interesting observational studies can be validated or reproduced awaits additional investigations. However, in women there is thought to be a strong relationship between diet and the risk of developing breast cancer. Obesity, age at menarch, ingestion of animal fat and animal proteins have been implicated as nutritionally mediated risk factors.


Clinical presentation


Many dogs have more than one mammary neoplasm at the time of clinical presentation. The caudal mammary glands (MG5 and MG4) are most frequently affected, with a deceasing incidence in the more cranial glands (MG2 and MG1). Small neoplasms (<1 cm in diameter) are more likely to be benign whereas larger neoplasms (>3–5 cm in diameter) are more frequently malignant.8 However, all mammary nodules, irrespective of size, should be evaluated histologically to distinguish between benign and malignant tumors.


Most dogs with mammary neoplasms do not present with any signs of systemic illness. If the tumors metastasize the dog will show signs of cachexia and difficult respirations terminally. Apart from one or more palpable masses within the mammary glands, most dogs are healthy and may even be obese. Ulceration of the overlying epidermis may be found particularly over large masses that are traumatized. An exception to this is the “inflammatory mammary carcinoma” (Figure 17.1). In some instances these dogs are misdiagnosed as having a severe, acute, erosive, or ulcerative dermatitis. The affected areas of skin in the mammary region are erythematous, edematous, warm to the touch, and painful. Dogs with inflammatory mammary carcinomas may exhibit signs of systemic illness.27,28

Photo of inflammatory mammary carcinoma in a dog, with severe erosive and ulcerative dermatitis involving the inguinal and perivulvar skin.

Figure 17.1 Inflammatory mammary carcinoma, dog. Note the severe erosive and ulcerative dermatitis involving the inguinal and perivulvar skin. (See Figure 17.32A,B for the histopathology.)


Many cats have one or more discrete masses within the glands. There is no predisposition for neoplasms to arise in any specific gland. The size of the tumor at the time of diagnosis is quite variable. As a rule larger tumors (>3 cm) are malignant, however, size of mammary tumors in cats does not correlate with biological behavior, even small tumors may be malignant. Many cases are detected at a later stage when the area becomes inflammed secondary to ulceration and infection, especially in long‐haired cats. Although a solitary neoplastic mass may be the only tumor found, there is often severe dysplasia of the uninvolved mammary glands.10,25


References



  1. 1. Moe, L. (2001) Population‐based incidence of mammary tumours in some dogs breeds. J Reprod Fertil Suppl 57:439–443.
  2. 2. Dorn, C.R., Taylor, D.O., Schneider, R., et al. (1968) Survey of animal neoplasms in Alameda and Contra Costa Counties, California. II. Cancer morbidity in dogs and cats from Alameda County. J Natl Cancer Inst 40:307–318.
  3. 3. Egenvall, A., Bonnett, B.N., Ohagen, P., et al. (2005) Incidence of and survival after mammary tumors in a population of over 80,000 insured female dogs in Sweden from 1995 to 2002. Prevent Vet Med 69:109–127.
  4. 4. Dobson, J.M., Samuel, S., Milstein, H., et al. (2002) Canine neoplasia in the UK: estimates of incidence rates from a population of insured dogs. J Small Anim Pract 43:240–246.
  5. 5. Benjamin, S.A., Lee, A.C., and Saunders, W.J. (1999) Classification and behavior of canine mammary epithelial neoplasms based on life‐span observations in beagles. Vet Pathol 36:423–436.
  6. 6. Vascellari, M., Baioni, E., Ru, G., et al. (2009) Animal tumour registry of two provinces in northern Italy: incidence of spontaneous tumours in dogs and cats. BMC Vet Res 5:39.
  7. 7. Schneider, R. (1970) Comparison of age, sex, and incidence rates in human and canine breast cancer. Cancer 26:419–426.
  8. 8. Goldschmidt, M., Shofer, F.S., and Smelstoys, J.A. (2001) Neoplastic lesions of the mammary gland In Pathobiology of the Aging Dog (eds. U. Mohr, W.W. Carlton, D.L. Dungworth, et al.). Iowa State University Press, Ames, IA, pp. 168–178.
  9. 9. Sorenmo, K.U., Kristiansen, V.M., Cofone, M.A., et al. (2009) Canine mammary gland tumours; a histological continuum from benign to malignant; clinical and histopathological evidence. Vet Comp Oncol 7:162–172.
  10. 10. Hayden, D.W. and Nielsen, S.W. (1971) Feline mammary tumours. J Small Anim Pract 12:687–698.
  11. 11. Hayes, A.A. and Mooney, S. (1985) Feline mammary tumors. Vet Clin North Am Small Anim Pract 15:513–520.
  12. 12. Misdorp, W., Romijn, A., and Hart, A.A. (1991) Feline mammary tumors: a case‐control study of hormonal factors. Anticancer Res 11:1793–1797.
  13. 13. Weyer, K., Head, K.W., Misdorp, W., and Hampe, J.F. (1972) Feline malignant mammary tumors. I. Morphology and biology: some comparisons with human and canine mammary carcinomas. J Natl Cancer Inst 49:1697–1704.
  14. 14. Schneider, R., Dorn, C.R., and Taylor, D.O. (1969) Factors influencing canine mammary cancer development and postsurgical survival. J Natl Cancer Inst 43:1249–1261.
  15. 15. Sonnenschein, E.G., Glickman, L.T., Goldschmidt, M.H., and McKee, L.J. (1991) Body conformation, diet, and risk of breast cancer in pet dogs: a case‐control study. Am J Epidemiol 133:694–703.
  16. 16. Brodey, R.S., Fidler, I.J., and Howson, A.E. (1966) The relationship of estrous irregularity, pseudopregnancy, and pregnancy to the development of canine mammary neoplasms. J Am Vet Med Assoc 149:1047–1049.
  17. 17. Misdorp, W. (1991) Progestagens and mammary tumours in dogs and cats. Acta Endocrinol (Copenhagen) 125:27–31.
  18. 18. Overley, B., Shofer, F.S., Goldschmidt, M.H., et al. (2005) Association between ovarihysterectomy and feline mammary carcinoma. J Vet Intern Med 19:560–563.
  19. 19. Skorupski, K.A., Overley, B., Shofer, F.S., et al. (2005) Clinical characteristics of mammary carcinoma in male cats. J Vet Intern Med 19:52–55.
  20. 20. Jacobs, T.M., Hoppe, B.R., Poehlmann, C.E., et al. (2010) Mammary adenocarcinomas in three male cats exposed to medroxyprogesterone acetate (1990–2006). J Feline Med Surg 12:169–174.
  21. 21. Loretti, A.P., Ilha, M.R., Ordas, J., and Martin de las Mulas, J. (2005) Clinical, pathological and immunohistochemical study of feline mammary fibroepithelial hyperplasia following a single injection of depot medroxyprogesterone acetate. J Feline Med Surg 7:43–52.
  22. 22. Mol, J.A., van Garderen, E., Rutteman, G.R., and Rijnberk. A. (1996) New insights in the molecular mechanism of progestin‐induced proliferation of mammary epithelium: induction of the local biosynthesis of growth hormone (GH) in the mammary glands of dogs, cats and humans. J Steroid Biochem Mol Biol 57:67–71.
  23. 23. Schafer, K.A., Kelly, G., Schrader, R., et al. (1998) A canine model of familial mammary gland neoplasia. Vet Pathol 35:168–177.
  24. 24. Im, K.S., Kim, I.H., Kim, N.H., et al. (2013) Breed‐related differences in altered BRCA1 expression, phenotype and subtype in malignant canine mammary tumors. Vet J 195:366–372.
  25. 25. Weijer, K., and Hart, A.A. (1983) Prognostic factors in feline mammary carcinoma. J Natl Cancer Inst 70:709–716.
  26. 26. Perez Alenza, D., Rutteman, G.R., Peña, L, et al. (1998) Relation between habitual diet and canine mammary tumors in a case‐control study. J Vet Intern Med 12:132–139.
  27. 27. Perez Alenza, M.D., Tabanera, E, and Peña, L. (2001) Inflammatory mammary carcinoma in dogs: 33 cases (1995–1999). J Am Vet Med Assoc 219:1110–1114.
  28. 28. Marconato, L., Romanelli, G., Stefanello, D., et al. (2009) Prognostic factors for dogs with mammary inflammatory carcinoma: 43 cases (2003–2008). J Am Vet Med Assoc 235:967–972.

Normal anatomy, histology, and immunohistochemistry


A review of these topics in the canine and the appropriate references was published in 2011.1,2


The mammary glands are modified subcutaneous apocrine sweat glands, found only in mammals, and have the important role of providing nourishment and passive immunity to the newborn. The mammary gland is a branching, ductal structure that is embedded in abundant fibrovascular and adipose tissue. The ductal system begins with the collecting (papillary) ducts of the nipple and end with secretory alveoli when the gland is fully differentiated. At birth, the glands are incompletely developed; their development starts at puberty and follows morphological changes with each estrus cycle. The last stages of development occur only during pregnancy.


In the canine and feline embryo the mammary glands appear as two linear thickenings or ridges (also referred as milk or mammary lines), detectable on the ventrolateral ectoderm and supported by specialized mesoderm. The ectodermal cells migrate along the mammary lines that extend from the axillary to the inguinal region, and coalesce into placodes. Each placode will give rise to a single gland. The ectodermal cells of the placode proliferate to form solid cords (mammary buds) that sprout into the underlying mesoderm as a branching structure. There are complex signaling pathways between the cells of the ectoderm and the mesoderm. The number of sprouts determines the number of openings (papillary duct orifices) that will develop on each nipple sheath by a process of cavitation. The nipple sheath is an area of specialized epithelium that gives rise to the raised teat in adults. Each teat usually has 4–7 papillary duct orifices in the cat and 7–16 (up to 22) in the dog. Each of these ducts will branch, forming a lobe of the adult gland that will act as an independent functional unit.


Most dogs develop five pairs (left (L) and right (R)) of mammary glands, although both four and six pairs have also been found. These are named as two thoracic (M1 and M2), two abdominal (M3 and M4), and one inguinal (M5). In the cat there are generally four mammary glands per side, referred to as axillary (M1), thoracic (M2), abdominal (M3), and inguinal (M4). They are also referred to as the cranial (T1) and caudal (T2) thoracic and cranial (A1), and caudal (A2) abdominal. In some cats a fifth inguinal (M5) gland may be present.


At birth, only the large ducts have developed and they extend a short distance from the teat into the underlying mesenchymal tissue. With puberty, the release of estrogens from the ovary activates cell proliferation at the terminal ends of the ducts (terminal end buds). The increased levels of progesterone during diestrus and pregnancy induce further development of ducts and the formation of lobules and alveoli (lobuloalveolar unit). Under the influence of gestational prolactin the presecretory alveolar cell differentiate into secretory alveolar cells, so that at parturition the mammary gland is characterized by a secretory ductal–lobular–alveolar structure. Ten days post‐partum, in the bitch, there is onset of alveolar regression that is nearly complete in 40 days, at which time only the pre‐existing ducts will be found. Changes during pseudo‐pregnancy are nearly identical, except that secretory activity is less well developed.


Normal histology


The teat is the terminal portion of the secretory system of the mammary gland. It is covered by epidermis. In dogs this lacks most of the dermal adnexal structures, whereas in cats hair follicles and large sebaceous and apocrine glands are present. Melanocytes may be found between basal epidermal keratinocytes. Teats of nonsecretory glands are very small and often hidden by hair in cats. Neoplasms of the epidermis, adnexa, melanocytes, and soft tissues are uncommon and discussed under tumors of the skin and soft tissues (Chapter 5) and are not classified as mammary neoplasms.


Multiple papillary ducts (teat or collecting ducts) open onto the teat surface in dogs; in the cat 2–3 major papillary orifices open at the apex whereas the remainder are more lateral (Figure 17.2A). Within the dermis of the teat and along the larger ductal system are longitudinal and transverse smooth muscle fibers and elastin fibers. The teat ducts are lined by a stratified squamous epithelium and surrounded by a circular smooth muscle sphincter. Each teat duct opens into the teat sinus that is lined by a bilayered cuboidal to columnar epithelium with flattened myoepithelial cells on the outside of the duct (Figure 17.2B). Neoplasms arising from the ducts and teat sinus are uncommon but discussed as a separate entity in the classification system. The large interlobular ducts (lactiferous ducts) empty into the distal teat sinus: these interlobular ducts are bilayered, lined by a cuboidal epithelium with an outer layer of fusiform myoepithelial cells. The distal smaller interlobular ducts are lined by a monolayer of cuboidal epithelium with fewer myoepithelial cells. The terminal interlobular ducts continue into the intralobular ducts that are lined by a single layer of cuboidal epithelium surrounded by discontinuous spindle‐shaped myoepithelial cells. The alveoli are lined by a single layer of epithelial cells and externally by star‐shaped myoepithelial cells. The secretory alveolar epithelium is tall, cuboidal to columnar with intracytoplasmic lipid droplets that accumulate within the alveolar lumina. Elongation and branching during ductal growth are achieved by the proliferation of epithelial cells that penetrate through gaps between myoepithelial cells.

Micrograph of normal nipple in a dog, illustrating the normal epidermis and numerous teat ducts and teat sinuses being ectatic and containing eosinophilic secretion. Micrograph of junction between normal teat duct and teat sinus with intraluminal secretion, and the teat duct and sinus lined by a stratified squamous and a bilayered cuboidal epithelium, respectively.

Figure 17.2 (A) Normal nipple, dog. The epidermis is normal and no adnexa are present. Numerous teat ducts and teat sinuses are ectatic and contain eosinophilic secretion. The surrounding stroma consists of collagen and smooth muscle bundles. (B) Junction between normal teat duct and teat sinus with intraluminal secretion. The teat duct is lined by a stratified squamous epithelium and the sinus by a bilayered cuboidal epithelium with peripheral myoepithelium. The dense collagenous stroma contains several smooth muscle trabeculae.


Both epithelial and myoepithelial cells reside on and produce a basement membrane that is composed mainly of collagen type IV, laminins, and heparin sulfate proteoglycans. The stroma (connective and adipose tissue, blood vessels, lymphatics, and nerves) of the mammary gland that supports the epithelial structures is derived from specialized mesoderm. This interstitial tissue can occasionally contain histiocytes, plasma cells, and small lymphocytes. It is subdivided into two different parts: the intralobular stroma is composed of more loosely arranged and finer collagen bundles, whereas the interlobular connective tissue separates the lobules and is formed by thicker and more tightly organized collagen (Figure 17.3). The amount of adipose tissue is variable.

Micrograph of mammary gland in a dog, with larger, elongated terminal interlobular ducts and smaller intralobular ducts surrounded by a more cellular and less dense collagenous stroma.

Figure 17.3 Mammary gland, dog. Normal. The larger, elongated terminal interlobular ducts and smaller intralobular ducts; the latter surrounded by a more cellular and less dense collagenous stroma.


Several publications describe the changes in histology of the mammary gland of bitches during the estrus cycle and with lactation.3–5 In the bitch there are specific hormonal and reproductive features that can affect the mammary gland histology and these must be considered when evaluating pathological changes of the gland. Estrous cycle stages in bitches can be classified as: pro‐estrus, an ovarian follicular period of 1–2 weeks; estrus of 1–2 weeks including ovulation and early luteinization; diestrus with fully developed and functioning corpora lutea lasting 2–3 months; and anestrus, a period of 3–5 months of relative ovarian quiescence.


Major changes of the canine mammary histology are identified in the following phases: pre‐pubertal, adult pro‐estrus, estrus, early and late diestrus, early and late anestrus.1,3–5 Please refer to these publications for the histologic changes that are associated with the canine estrus cycle.


Queens are unusual in their reproductive physiology. They can enter puberty at 4–12 months of age, and as late as 20 months in certain breeds. They are seasonally polyestrus and repeated matings trigger ovulation, inducing luteinizing hormone (LH) production. Spontaneous ovulation may also occur. Exogenous stimuli (mating or others) that affect the hypothalamo‐pituitary‐gonadal axis are necessary for the switch from FSH (follicle‐stimulating hormone) to LH synthesis to occur in the pituitary gland. Under FSH stimulation, follicles formation in the ovary produces estrogen (estrus), whereas with ovulation and corpus luteum formation progesterone will be synthesized. Interestrus intervals last approximately 10–15 days in non‐mated queens and 35–75 days in mated but nonpregnant queens. It is likely that mammary gland histology can be affected by reproductive changes, however no detailed studied have been conducted in female cats.


Neural innervation, vascularization, and lymph drainage


Branches of the genitofemoral and intercostal nerves innervate the mammary gland, are primarily associated with arteries and arterioles, and are predominantly peptidergic nerves, which may be involved in the regulation of local blood flow. In the nipple, noradrenergic and peptidergic fibers are found in the dermis in close proximity to the smooth muscle bundles, and may play a role in the afferent pathway of the milk ejection reflex. It is noteworthy, however, that large portions of the secretory parenchyma have no innervation; specifically, no peptidergic nerve fibers are found around alveoli or ducts.6


Hematic and lymphatic communications of the mammary glands are fundamental to understand the development of tumor metastases. In general, mammary carcinomas, the most common malignant mammary neoplasms in cats and dogs, metastasize via lymphatics whereas sarcomas, mostly canine mammary osteosarcomas, and fibrosarcoma, metastasize via blood vessels.


The mammary glands are highly vascular, and veins are more extensive than arteries. In the dog mammary gland, perforating cranial branches of the internal thoracic artery and intercostal arteries supply the thoracic mammary gland (M1). M2 and M3 receive blood from the mammary branches of the cranial superficial epigastric artery, whereas M4 and M5 are supplied by the mammary branches of the caudal superficial epigastric artery and the cranial abdominal artery, and by branches of the external pudendal artery. Veins parallel the course of the arteries to a large extent in the dog. They drain blood from the caudal portion of M3 and from M4 and M5 into the external pudendal vein, while the cranial superficial epigastric vein receives blood from the cranial part of M3 and from M2 and M1.7–9


In the cat, the axillary (M1) and thoracic (M2) mammary glands are supplied by the perforating branches of the internal thoracic, the intercostal, and the lateral thoracic arteries. Abdominal (M3) glands receive blood from the cranial superficial epigastric artery whereas branches of the external pudendal artery supply the inguinal glands (M4). The veins of the feline mammary glands closely follow the arteries, except for some, which cross midline, allowing metastatic dissemination between paired glands. This is a unique feature of the cat mammary gland but is extremely uncommon.7–9


In the normal mammary glands both in dogs and cats lymph drainage from each gland on the same side may interconnect, but lymphatics do not connect across midline. Usually 1–3 main lymphatics leave each gland to drain to the nearest superficial lymph node. However drainage can be altered when mammary neoplasms occur in the glands. The normal and altered drainage have been described by several authors and are summarized in Table 17.1.10–18


Table 17.1 Direct normal and altered lymph drainage in canine and feline mammary glands


















































Mammary gland Normal lymph drainage Neoplastic lymph drainage
Canine
M1 cranial thoracic Axillary ln Axillary ln (sternal ln)
M2 caudal thoracic Axillary ln Axillary ln (sternal ln)
M3 cranial abdominal Axillary ln
Superficial inguinal ln		 Axillary ln
Superficial inguinal ln
Medial iliac ln
M4 caudal abdominal Superficial inguinal ln (iliac ln) Superficial inguinal ln
Axillary ln
M5 inguinal Superficial inguinal ln Superficial inguinal ln
Popliteal ln
Lymphatic vessels – medial thigh
Feline
M1 axillary Axillary ln (sternal ln)
M2 thoracic Axillary ln
Superficial inguinal ln (sternal ln)
M3 abdominal Axillary ln
Superficial inguinal ln (sternal ln)
M4 inguinal Superficial inguinal ln

ln, lymph node.


Each mammary gland has its own lymphatic plexus that anastomoses and encircles the base of the teat, extending into the parenchyma with the other plexuses. Common ipsilateral interconnections in dogs affect the caudal abdominal gland draining into the lymphatic plexus of the inguinal gland. Dogs with mammary carcinomas have abnormal connections of the neoplastic caudal mammary glands with the axillary lymph node and there may also be metastatic spread from neoplasms of the caudal glands to the subcutis of the inner thigh and to the popliteal lymph node.


In queens two separate lymphatic networks have been described that connect the two anterior glands (axillary and thoracic) and the two posterior glands (abdominal and inguinal), respectively.


Cell differentiation markers


In the normal mammary gland two different cell populations line the ducts and the alveoli. Their specific morphology and function can vary slightly depending on location and hormonal status. Generally, there is an inner layer of luminal epithelial cells that have a polygonal shape and a protective/secretory function, and an outer layer, referred to as the basal/myoepithelial cells because they exhibit an immunophenotype of both epithelial cells and smooth muscle cells, juxtaposed to the basement membrane. These usually have a spindle to stellate morphology and a contractile function. Differentiation between luminal epithelial cells and basal/myoepithelial cells can be accomplished using cytokeratin (CK) antibodies. In canine, feline and humans the normal mammary gland luminal epithelial cells express low molecular weight cytokeratins (CK7, CK8, CK18, CK19) and basal/myoepithelial cells variably express high molecular weight cytokeratins (CK5, CK6, CK7, CK14).1 All of these cytokeratins work well and are consistent in normal mammary tissues in dogs and cats (Table 17.2).


Table 17.2 Immunohistochemistry of normal canine and feline mammary gland
















Cell type Immunohistochemical staining
Luminal epithelial cells CK7, CK8, CK18, CK19
Basal cells CK5, CK6, CK7, CK14
Myoepithelium α‐Smooth muscle actin, p63, calponin, vimentin

In dogs and cats, luminal epithelial cells specifically express markers related to their protective function or hormonally induced secretory activity, such as epithelial cell adhesion molecules, MUC1 alpha‐6 integrin, and hormone receptors.1 Basal/myoepithelial cells have a dual phenotype of both epithelial and smooth muscle cells, hence they have been named myoepithelium, and are positive for other markers, including α‐smooth muscle actin, calponin, vimentin, p63, class 2 β‐tubulin, maspin, neutral endopeptidase (CD10), P‐cadherin, caveolin 1, GFAP, S100, and 14‐3‐3σ protein.1


This differential expression pattern of cell markers is also maintained during the development of mammary neoplasms. Neoplastic mammary subpopulations have been identified along with their role in mammary tumor histogenesis, metaplastic changes, subtype classification and prognosis.


The precise origin of the two major mammary lineages (luminal epithelial cells and basal/myoepithelial cells) is still poorly understood. A stem/progenitor cell in the mammary gland was first suggested in 1959.18 These cells are rare, and their exact location and phenotype still need to be precisely elucidated, but they are present during all stages of development and throughout the entire mammary tree. They can only be identified with immunohistochemistry (IHC). The main surface marker phenotypes associated with stem cell characteristics include CD133 and CD44. Studies have also identified bipotent mammary precursors that can differentiate between luminal epithelial cells and myoepithelial cells.19–23


Progenitor cells with stem cells properties have been identified in the mammary gland of dogs and cats. Specifically, cells with stem cell–like features (CD44+/CD24− and aldehyde dehydrogenase activity) have been isolated from normal and neoplastic canine mammary tissue and CD44‐positive cells with tumorigenic potential have also been identified in spontaneous feline mammary carcinomas.24–27 In addition to their role in mammary gland development, the potential implication of mammary stem/precursors cells in carcinogenesis is now widely accepted. Identification of normal and malignant stem/progenitor cells by the same markers support the hypothesis that these cells are the primary targets of neoplastic transformation. Current carcinogenesis theories suggest that tumor‐initiating cells (also referred to as cancer stem cells) are responsible for tumor formation, progression, and treatment failure.


The investigation of cell differentiation markers has been used to improve our knowledge of the histogenesis of canine mammary tumors. There are several hypotheses on the role of the myoepithelium in the genesis of complex mammary tumors (those with epithelial and myoepithelial proliferation) and mixed tumors (those with epithelial and myoepithelial proliferation and areas of bone, cartilage, and/or other mesenchymal elements), the most common mammary neoplasm in dogs. Historically, there were three hypotheses on the origin of the mesenchymal elements: (i) metaplasia from epithelial cells, (ii) metaplasia from stromal connective tissue, and (iii) metaplasia from basal/myoepithelial cells.28 Even if the specific mechanism is still not completely understood, several studies performed using different panels of basal markers reinforced the putative role of the myoepithelium in the metaplastic change to mesenchymal tissue.


In both complex and mixed tumors, those myoepithelial cells that were still present within the basement membrane maintained their normal IHC characteristics, whereas in those portions of the tumor where the myoepithelial cells were proliferating in the interstitium (outside the basement membrane) they showed decreased expression of CK14, CK5, SMA, calponin, and p63 and enhanced expression of vimentin. Proliferating interstitial myoepithelial cells may eventually become fibroblast‐like cells, showing only vimentin immunoreactivity. However, few of these cells and rare chondrocytes in mixed tumors retained expression of basal CKs, SMA, calponin, and p63, supporting the hypothesis that there was a metaplastic mesenchymal change of myoepithelial cells.3,29–35 This shift in the myoepithelial immunoprofile was associated with an increased expression of bone morphogenetic protein‐6 and chondromodulin‐I, proteins that may be involved in ectopic cartilage and bone formation.35 However, we do not know the role of the stromal connective tissue, such as interstitial myofibroblasts or fibroblasts in mesenchymal tissue formation, and how these cells might interact with the myoepithelial cells to produce the connective tissue.35


In dogs, p63 is most specific for myoepithelial cells as calponin and SMA cross‐react with stromal myofibroblasts.29,31 Expression of SMA, calponin, p63, and vimentin occurs in some simple canine mammary tumors with immunoreactivity of the myoepithelial cells and neoplastic epithelial cell population.30,32,36 Identifying which mammary tumors have myoepithelial cell differentiation may be important because participation of myoepithelial cells in malignant tumors of the mammary gland is considered a favorable prognostic indicator in dogs and women,28,37 as differentiated myoepithelial cells may be natural tumor suppressors because of their inhibitory effect on neoplastic cells, including tumor cell growth, invasion, and angiogenesis.22,38 There is also vimentin labeling of the luminal epithelial cells in canine mammary carcinomas,39–42 which has been associated with a poor prognosis and chemoresistance.


The phenomenon of epithelial–mesenchymal transition (EMT) reflects the final step of tumor dedifferentiation, with loss of epithelial characteristics and polarity, and acquisition of a mesenchymal phenotype with increased migratory behavior and metastatic capability.43,44 This finding has recently been described in association with the formation of microvascular channels by malignant mammary tumor cells (vasculogenic mimicry), a feature of highly aggressive tumors that invade dermal lymphatic vessels and with distant metastases (canine inflammatory mammary cancers).39


In women with breast cancer, use of breast cell differentiation markers in conjunction with ER, PR, and HER2 allow breast cancer cases to be divided into several subtypes, The more common subtypes are: (1) luminal tumors, expressing ER and/or PR receptors as well as luminal cytokeratins (CK7, CK8, CK18, CK19), (2) basal‐like tumors, negative for hormone receptors (ER, PR, HER2) and expressing basal markers (CK5, CK6, CK14, CK17, SMA, calponin, vimentin, and p63), and (3) HER2‐positive tumors, overexpressing the HER2 receptor.45–47 These breast cancer subtypes are associated with markedly different clinical outcomes, ranging from the best prognosis for the luminal group with a well‐differentiated glandular immunoprofile to the worst prognosis for the basal‐like phenotype, possibly reflecting a stem/progenitor cell origin.44,48


Applying similar IHC panels to canine mammary carcinomas has not proven useful to date because the panel used in breast cancers does not distinguish between the expression of the epithelial and the myoepithelial components, which is so frequently part of the neoplastic process in dogs.40,49,50


One use for IHC is for the evaluation of regional lymph nodes from dogs and/or cats with malignant mammary tumors that may have occult micrometastatic lesions which can be identified with a pan‐cytokeratin marker (AE1/AE3).51


References



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  17. 17. Patsikas, M.N., Papadopoulou, P.L., Charitanti, A., et al. (2010) Computed tomography and radiographic indirect lymphography for visualization of mammary lymphatic vessels and the sentinel lymph node in normal cats. Vet Radiol Ultrasound 51:299–304.
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Hormones and growth factors


Ovarian and exogenous steroid hormones, often used as contraceptives, are risk factors for the development of mammary tumors. The importance of sex hormones in canine and feline mammary cancer is apparent from several epidemiologic studies. The risk of mammary cancer decreases dramatically when animals have limited exposure to sex hormones: in bitches and queens, early ovariectomy greatly reduces that risk (see previous sections). Ovariectomy later in life has a minimal protective effect, although the possible beneficial effects of ovariectomy of bitches at the time of the mammary cancer extirpation on prognosis are still under study. Once a mammary tumor is present, the role of hormones relative to growth, progression to malignancy, metastasis, and prognosis is less well understood. Steroid hormones, peptide hormones, and growth factors are all known to promote the growth and development of normal mammary tissues and mammary neoplasia.


In the dog and cat, many of the studies of hormones and mammary neoplasia are contradictory. An overview of hormone receptor assessment in canine mammary tumors has recently been published and the reader is referred to this for additional information.1


Normal and neoplastic canine mammary tissue can act as a local source of steroids hormones where they may play an important role in growth and malignancy, by acting through specific receptors in an autocrine/paracrine manner.2–4 Several steroid hormones (progesterone, 17β‐estradiol, estrone sulfate, androstenedione, and dehydroepiandrosterone) are increased in tissue homogenates in malignant canine mammary tumors, especially in inflammatory mammary cancer, when compared to benign tumors and normal and dysplastic mammary glands.5


The study of steroidogenic enzymes in mammary cancer is directed at the possible use of enzyme inhibitors that will block local and external production of these hormones and, consequently, inhibit tumor growth. These enzyme inhibitors are considered potential new therapeutic agents for the treatment of hormone‐dependent cancers.6


There are no similar studies on the hormonal status of female cats with mammary tumors. The influence of exogenous and/or endogenous progesterone and increased serum progesterone levels has been well documented in cats with mammary hypertrophy/fibroadenomatous change.7,8


Steroid hormone receptors in canine mammary tumors


Hormones and growth factors act as growth promoters in the normal and neoplastic mammary gland by linking and binding to their receptors on target cells including estrogen receptor (ER), progesterone receptor (PR), prolactin receptor (PRLR), epidermal growth factor receptor (EGFR), androgen receptor (AR), luteinizing hormone releasing hormone receptor (LHRHR).9–31 A loss of PR and ER in malignant tumors when compared to benign lesions and normal mammary gland was demonstrated in the initial studies on canine mammary tumors, although this has not been a consistent finding in all studies.


Several monoclonal antibodies against human ER and PR have been used for the IHC localization of these hormone receptors in routinely processed formalin‐fixed, paraffin‐embedded (FFPE) samples, following a heat protocol to unmask the antigen.


Estrogen receptors and progesterone receptors


ER immunostaining is localized to the nuclei of normal, benign, and malignant epithelial cells.26–31 Myoepithelial cells may be positive28,31 or negative.30 Normal stromal and neoplastic stromal cells, including chondrocytes and osteocytes in mixed tumors are negative.28–30 PR immunostaining is found in the nucleus of normal and neoplastic epithelial cells27,30 and myoepithelial cells.27,32


Normal mammary glands are ER positive while some types of hyperplasia have decreased ER and PR expression, and ER and PR are decreased in malignant tumors.27


In canine mammary tumors ER expression has been found, in some studies, to decrease with age.28,29 Whether ER expression may be related to spay status, regularity of estrus, and a previous clinical history of pseudo‐pregnancy remains controversial, but PR expression is not associated with these characteristics.28,29 Mammary tumors in male dogs are strongly positive for ER and show less expression of PR.33


In malignant tumors, ER and/or PR immunostaining is higher in complex and mixed histologic subtypes than in simple histologic subtypes and higher in papillary adenocarcinomas than in solid tumors.28,30 Most inflammatory mammary carcinomas are ER negative while PR expression remains contradictory.34


Recommended guidelines for the standardization of ER and PR detection techniques and postanalytic standardization and interpretation have been recently published.1 Please refer to these for additional information.


Proliferation and other tumor markers


Low ER and PR are associated with greater neoplastic growth: proliferation indexes measured by PCNA (proliferating cell nuclear antigen) and Ki67 immunostaining are inversely correlated with ER scores,27,35–37 and PR immunoexpression is associated with a low mitotic count.29 Decreased ER expression is associated with decreased expression of LTBP‐4 (latent TGFb‐binding protein 4), a protein that has growth‐inhibitory effects, and is lost in carcinomas and metastases.36 ER may also play a role in apoptosis as a regulator of Bcl‐2 protein.37 Positive staining for FOXP3 (transcription factor forkhead box P3), a specific marker of natural T regulatory cells, was associated with loss of ER and PR expression.38 Negative expression of ER39 and PR40 is associated with loss of nuclear expression of BCRA1 (breast cancer type 1, responsible for hereditary breast cancer) and positive cytoplasmic and membrane expression of BRCA1.


Despite these interesting and encouraging findings, the use of these markers to predict prognoses and/or guide treatment options for dogs with mammary tumors still needs correlation with long‐term clinical follow‐up.


Estrogen receptor β and androgen receptors


One‐third of canine mammary tumors expressed ERβ, which is higher in benign than in malignant tumors, and in malignant complex and mixed histologic subtypes than in simple subtypes.41 Higher expression of ERβ has been found in inflammatory mammary carcinomas than in noninflammatory mammary carcinomas.5 Little is known about androgen receptors (AR) in canine mammary tumors, although they have been detected by IHC in malignant mammary tumors.5


Steroid hormone receptors in feline mammary tumors


The literature regarding steroid receptors and epidemiological, clinical, and pathological characteristics of feline mammary tumors is still scant and controversial. ER and PR are present in the nuclei of normal, dysplastic, and neoplastic luminal epithelial cells, suprabasal or basal epithelial cells and stromal cells, while staining of myoepithelial cells is equivocal or negative.31,42,43


Dysplastic and benign lesions express both ER (76.82%) and PR (66.7%),42 although no ER or PR immunoreactivity has been detected in some preinvasive intraepithelial lesions.44 In feline fibroadenomatous change, all cases express PR and but only 50% of cases express ER.45


Although most carcinomas are ER negative, when ER is expressed it is more often in low‐grade carcinomas than intermediate and high‐grade carcinomas;31 more carcinomas are PR positive, but PR expression has not been related to lymphatic invasion.31


Insulin and growth hormone


The insulin/IGF/relaxin family, or insulin superfamily, are a group of related proteins that possess a variety of hormonal activities. Family members include insulin, insulin‐like growth factors (IGFs), and relaxin.


In dogs and cats, progestins stimulate the local production of growth hormone (GH) in the mammary gland. Progestin‐induced GH excess in the dog originates in the mammary gland, which is the major site of extrapituitary GH production.46,47 Mammary‐produced GH/IGF have a role in tumorigenesis and malignancy of the mammary gland and, together with other hormones and factors, may act in situ, stimulating the development and/or maintenance of canine mammary tumors in an endocrine, autocrine/paracrine manner.4,48–50 Locally produced GH acts through its receptor (GHR), which is found by IHC in normal and neoplastic canine mammary epithelial and myoepithelial cells and in the activated fibroblasts of desmoplastic tumor stroma.51


GH also promotes growth by increasing cell survival: GHR reduction decreases p‐ERK1/2 expression and causes an increase in apoptosis and a decrease in the number of cells in the S and G2M phases.52


Insulin receptor (INSR) and insulin‐like growth factors are expressed in the normal canine mammary gland and in adenomas but are decreased in metastatic carcinomas. Insulin‐like growth factor type I receptor (IGF‐IR) may be be associated with tumor hormone dependence and apoptosis,52 with increased IHC expression in malignant mammary tumors compared to benign tumors and normal mammary tissue.53


In the cat, progestin‐induced local synthesis of GH and IGF‐I in mammary epithelial cells may be one of the pathogenic mechanisms involved in the development of feline mammary fibroepithelial hyperplasia/fibroadenomatous change in cats.8 The IHC expression of PR, GH, and IGF‐I in feline fibroadenomatous change indicates that ligand‐activated progesterone receptors may induce the local synthesis of GH/IGF‐I, which may act as a proliferation promotors in an autocrine/paracrine manner.54


Prolactin


In the normal and neoplastic canine mammary gland, there is loss of PRLR expression.55


Epidermal growth factor and receptors


The EGF family of proteins may influence normal and neoplastic proliferation either independently or by interacting with steroid and peptide hormones that regulate mammary growth. Progesterone enhances EGF‐induced growth,56 and there is a strong correlation between tumor levels of EGF and progesterone, estradiol, and androgens in dogs with mammary tumors.4 EGF plays a role in canine mammary tumorigenesis, possibly interacting with steroid hormones and other factors by increasing proliferation, chemotaxis, and vascular endothelial growth factor (VEGF), and attenuating apoptosis.4,57


The overexpression of EGF receptors in breast cancer in women has been associated with a poor prognosis and therefore the development of targeted therapies. This family of EGF receptors includes four closely related tyrosine kinase receptors (EGFR or HER1 or c‐erbB1, HER2 or HER2/neu or c‐erbB2, HER3 or c‐erbB3, and HER4 or c‐erbB4), with targeted therapies with anti‐EGFR (cetuximab) and anti‐HER2 antibodies (trastuzumab). Canine HER1 and HER2 homologs are susceptible to cetuximab and trastuzumab targeting, since there is great amino acid homology for both receptors between the human and canine molecules.58


Epidermal growth factor receptor


EGFR IHC expression increases from benign to malignant mammary tumors and with increasing tumor size.59


HER2


Approximately 25–30% of human breast cancers overexpress HER2, and overexpression has been associated with the development of metastases and a poor prognosis. HER2 expression is not well established in canine and feline mammary tumors, although it has been extensively studied in the last years, but there is great variability of results among the studies.


HER2 has been detected by IHC in canine mammary tumors and feline mammary tumors but the results are very variable.1 This great variability of positive results, attributed to technical reasons, do not permit any definitive conclusion to be made regarding the relevance of HER2 in the pathogenesis and prognosis of canine and feline mammary tumors. There are recommended guidelines for IHC protocols and evaluation systems.1


Vascular endothelial growth factors and cyclooxygenase 2


VEGFs are a family of homodimeric proteins that include VEGF‐A (also known as VEGF), VEGF‐B, VEGF‐C, VEGF‐D, and PIGF (placental growth factor). VEGFs and their three tyrosine kinase receptors (VEGFR‐1, VEGFR‐2, and VEGFR‐3) are poorly studied in canine and feline mammary tumors.


VEGF is expressed by endothelial and neoplastic cells in canine and feline mammary tumors. VEGF enhances angiogenesis and also stimulates the proliferation of neoplastic cells in an autocrine/paracrine loop manner. The overexpression of VEGF is especially marked in canine inflammatory mammary carcinoma,60 and may be increased in malignant tumors in cats and dogs when compared to benign tumors.61,62


Cyclooxygenase (COX) enzymes, COX‐1 and COX‐2, catalyze prostaglandin formation from arachidonic acid. COX‐1 is constitutively expressed in all tissues, whereas COX‐2 has been implicated in the genesis of numerous cancers. COX‐2 may participate in different steps of the tumorigenic process such as invasiveness and metastasis, tumor proliferation, apoptosis inhibition, and tumor angiogenesis.


In canine mammary tumors, high expression of COX‐2 is related to increased malignancy, HER2 expression, VEGF expression, and angiogenesis.63,64


The little information pertaining to COX‐2 in feline mammary tumors indicates that COX‐2 is expressed in the majority of feline mammary carcinomas and COX‐2 overexpression correlates with ER‐negative status, increased PR, and VEGF.65


COX‐2 expression is related to a shorter overall survival in both species.64,65


Other hormones and growth factors


The role of the transforming growth factor beta (TGFβ) family in the carcinogenesis of canine mammary tumors shows that loss of TGFβ‐3 and LTBP‐4 expression, together with reduced TGFβR‐3 expression, may be associated with increased proliferative activity, which is similar to findings reported in human breast cancer.36


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  29. 29. Chang, C.C., Tsai, M.H., Liao, J.W., et al. (2009) Evaluation of hormone receptor expression for use in predicting survival of female dogs with malignant mammary gland tumors. J Am Vet Med Assoc 235:391–396.
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  33. 33. Saba, C.F., Rogers, K.S., Newman, S.J., et al. (2007) Mammary gland tumors in male dogs. J Vet Intern Med 21:1056–1059.
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  35. 35. Dolka, I., Motyl, T., Malicka, R., et al. (2011) Relationship between receptors for insulin‐like growth factor‐I, steroid hormones and proliferative activity in canine mammary tumours. Pol J Vet Sci 14:245–251.
  36. 36. Klopfleisch, R., Schutze, M., and Gruber, A.D. (2010) Downregulation of transforming growth factor beta (TGF beta) and latent TGF beta binding protein (LTBP)‐4 expression in late stage canine mammary tumours. Vet J 186:379–384.
  37. 37. Yang, W.Y., Liu, C.H., Chang, C.J., et al. (2006) Proliferative activity, apoptosis and expression of oestrogen receptor and Bcl‐2 oncoprotein in canine mammary gland tumours. J Comp Pathol 134:70–79.
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  39. 39. Nieto, A., Perez‐Alenza, M.D., Del Castillo, N., et al. (2003) BRCA1 expression in canine mammary dysplasias and tumours: Relationship with prognostic variables. J Comp Pathol 128:260–268.
  40. 40. Im, K.S., Kim, I.H., Kim, N.H., et al. (2013) Breed‐related differences in altered BRCA1 expression, phenotype and subtype in malignant canine mammary tumors. Vet J 195:366–372.
  41. 41. Martin de las Mulas, J., Ordas, J., Millán, M.Y., et al. (2004) Immunohistochemical expression of estrogen receptor β in normal and tumoral canine mammary glands. Vet Pathol 41:269–272.
  42. 42. de las Mulas, J.M., van Niel, M., M., Millán, Y., et al. (2000) Immunohistochemical analysis of estrogen receptors in feline mammary gland benign and malignant lesions: comparison with biochemical assay. Domest Anim Endocrinol 18:111–125.
  43. 43. Martin De Las Mulas, J., Van Niel, M., Millán, Y., Ordás, J., et al. (2002) Progesterone receptors in normal, dysplastic and tumourous feline mammary glands. Comparison with oestrogen receptors status. Res Vet Sci 72:153–161.
  44. 44. Burrai, G.P., Mohammed, S.I., Miller, M.A., et al. (2010) Spontaneous feline mammary intraepithelial lesions as a model for human estrogen receptor‐ and progesterone receptor‐negative breast lesions. BMC Cancer 10:156.
  45. 45. Martin de las Mulas, J., Millan, Y., Bautista, M.J., et al. (2000) Oestrogen and progesterone receptors in feline fibroadenomatous change: an immunohistochemical study. Res Vet Sci 68:15–21.
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  47. 47. Mol, J.A., Selman, P.J., Sprang, E.P., et al. (1997) The role of progestins, insulin‐like growth factor (IGF) and IGF‐binding proteins in the normal and neoplastic mammary gland of the bitch: a review. J Reprod Fertil Suppl 51:339–344.
  48. 48. van Garderen, E., deWit, M., Voorhout, W.F., et al. (1997) Expression of growth hormone in canine mammary tissue and mammary tumors – evidence for a potential autocrine/paracrine stimulatory loop. Am J Pathol 150:1037–1047.
  49. 49. Queiroga, F.L., Perez‐Alenza, D., Silvan, G., et al. (2010) Serum and intratumoural GH and IGF‐I concentrations: Prognostic factors in the outcome of canine mammary cancer. Res Vet Sci 89:396–403.
  50. 50. Mol, J.A., Lantinga‐van Leeuwen, I., van Garderen, E., and Rijnberk, A. (2000) Progestin‐induced mammary growth hormone (GH) production. Adv Exp Med Biol 480:71–76.
  51. 51. van Garderen, E., van der Poel, H.J.A., Swennenhuis, J.F., et al. (1999) Expression and molecular characterization of the growth hormone receptor in canine mammary tissue and mammary tumors. Endocrinology 140:5907–5914.
  52. 52. Pawlowski, K.M., Popielarz, D., Szyszko, K., et al. (2012) Growth hormone receptor (GHR) RNAi decreases proliferation and enhances apoptosis in CMT‐U27 canine mammary carcinoma cell line. Vet Comp Oncol 10:2–15.
  53. 53. Illera, J.C., Silvan, G., Perez‐Alenza, M.D., and Peña, L. (2005) The possible role of IGF‐I and androgens in the development of canine inflammatory mammary carcinoma. In Hormonal Carcinogenesis, Vol. IV (eds. J.J. Li, S.A. Li, and A. Llombart‐Bosch). Springer, New York, pp. 436–442.
  54. 54. Ordas, J., Millan, Y., de los Monteros, A.E., et al. (2004) Immunohistochemical expression of progesterone receptors, growth hormone and insulin growth factor‐I in feline fibroadenomatous change. Res Vet Sci 76:227–233.
  55. 55. Michel, E., Feldmann, S.K., Boos, A., et al. (2012) Expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas. BMC Vet Res 8:72.
  56. 56. Modiano, J.F., Kokai, Y., Weiner, D.B., et al. (1991) Progesterone augments proliferation induced by epidermal growth factor in a feline mammary adenocarcinoma cell line. J Cell Biochem 45:196–206.
  57. 57. Kennedy, K.C., Qurollo, B.A., Rose, B.J., and Thamm, D.H. (2011) Epidermal growth factor enhances the malignant phenotype in canine mammary carcinoma cell lines. Vet Comp Oncol 9:196–206.
  58. 58. Singer, J., Weichselbaumer, M., Stockner, T., et al. (2012) Comparative oncology: ErbB‐1 and ErbB‐2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting. Mol Immunol 50:200–209.
  59. 59. Gama, A., Gärtner, F., Alves, A,, and Schmitt, F. (2009) Immunohistochemical expression of epidermal growth factor receptor (EGFR) in canine mammary tissues. Res Vet Sci 87:432–437.
  60. 60. Millanta, F., Caneschim V., Ressel, L., et al. (2010) Expression of vascular endothelial growth factor in canine inflammatory and non‐inflammatory mammary carcinoma. J Comp Pathol 142:36–42.
  61. 61. Millanta, F., Lazzeri, G., Vannozzi, I., et al. (2002) Correlation of vascular endothelial growth factor expression to overall survival in feline invasive mammary carcinomas. Vet Pathol 39:690–696.
  62. 62. Queiroga, F.L., Pires, I., Parente, M., et al. (2011) COX‐2 over‐expression correlates with VEGF and tumour angiogenesis in canine mammary cancer. Vet J 189:77–82.
  63. 63. Doré, M., Lanthier, I., and Sirois J. (2003) Cyclooxygenase‐2 expression in canine mammary tumors. Vet Pathol 40:207–212.
  64. 64. Lavalle, G.E.,, Bertagnolli, A.C., Tavares, W.L., and Cassali G.D. (2009) Cox‐2 Expression in canine mammary carcinomas: correlation with angiogenesis and overall survival. Vet Pathol 46:1275–1280.
  65. 65. Sayasith, K., Sirois, J., and Doré M. (2009) Molecular characterization of feline COX‐2 and expression in feline mammary carcinomas. Vet Pathol 46:423–429.

CLASSIFICATION OF MAMMARY TUMORS


Although some clinical signs (rapid growth, tumor size, ulceration, and fixation to skin and underlying tissues) may be an indication of malignancy, it is often impossible to differentiate between benign and malignant mammary tumors clinically in the dog. Cytological differentiation between benign and malignant canine mammary tumors is difficult; the accuracy of cytological differentiation is approximately 20%. Thus histopathology is the best method to determine diagnosis, including tumor type, benign versus malignant, grade and prognosis. The most significant criteria for differentiating benign from malignant mammary neoplasms based on H&E‐stained sections are described below. However, because of interobserver variability, significant differences remain in the threshold for the diagnosis of mammary carcinoma (Table 17.3).


Table 17.3 Differentiating benign from malignant mammary tumors




























Benign Malignant
Well circumscribed Poorly circumscribed
Smooth margins at periphery Irregular margins at periphery
Compact fibrous connective tissue at periphery Immature fibrous connective tissue at periphery
Necrosis is central and associated with loss of vascular supply Necrosis is multifocal and associated with rapid neoplastic cell proliferation
Nuclei are monomorphic Nuclei are pleomorphic
No lymphatic or vascular invasion Possible lymphatic or vascular invasion
No lymph node metastasis Possible lymph node metastasis

Molecular classification of canine and feline mammary carcinomas


Molecular subtypes in breast cancer in women were first described by Perou et al.1 by mapping the phenotypic diversity seen in breast cancer to a specific gene expression pattern. Other studies reported differences in the prognosis and chemotherapy response for the different subtypes.2


Breast cancer cases are divided into the following major types: (1) luminal tumors, expressing ER and/or PR receptors as well as luminal cytokeratins (CK7, CK8, CK18, CK19), subdivided into two subtypes: luminal A, expressing the luminal epithelial markers and with high expression of ER markers and lower expression of proliferation makers and luminal B, expressing the luminal epithelial markers, with lower expression of ER markers and higher expression of proliferation makers; (2) basal‐like tumors, negative for hormone receptors (ER, PR, HER2) and expressing basal markers (CK5, CK6, CK14, CK17, SMA, calponin, vimentin, and p63); (3) HER2‐positive tumors overexpressing the HER2 receptor; and (4) “normal‐like,” negative to all markers (Table 17.4).


Table 17.4 Molecular classification of canine and feline mammary tumors








































Molecular type Luminal cell marker ER+ and/or PR+ HER2+ Basal cell marker
Luminal A Yes Yes No Yes/no
Luminal B Yes Yes Yes Yes/no
Basal‐like No No No Yes
HER2 overexpressing Yes/no No Yes Yes/no
“Normal‐like” No No No No

Luminal markers: cytokeratins (CK) CK7, CK8, CK18, CK19.


Basal markers: CK5/6, CK14, p63, P‐cadherin.


Human epidermal growth factor receptor 2 (HER2).


Luminal A and B can be also differentiated using Ki67 instead of HER2: Luminal A = ER+ and/or PR+ and Ki67 <14%; Luminal B = ER+ and/or PR+ and Ki67 >14%.


Triple‐negative breast cancer (TNBC) = ER− and PR− and HER2−, regardless of basal marker.


These breast cancer subtypes are associated with markedly different clinical outcomes, ranging from the best prognosis for the luminal A phenotype to the worst prognosis for the basal‐like phenotype, the latter possibly reflecting a stem/progenitor cell origin. In women with breast cancer these markers are used along with the histopathologic subtype, tumor grade, and clinical stage of the tumor in determining treatments. Molecular subtypes can be identified by IHC or PCR, however, IHC is a more feasible diagnostic tool in routine diagnosis than PCR.


Recently there have been several attempts to classify canine3–5 and feline6 mammary carcinomas by their molecular subtypes using an IHC panel. This panel consisted of several molecular markers that identifird five phenotypes: luminal A, luminal B, basal‐like type (triple negative), HER2‐overexpressing, as well as the “normal” phenotype.


The proportions of the different molecular subtypes found in canine mammary carcinomas vary depending on the study, probably due to the panel of antibodies used. In the reported studies, the proportion of canine mammary carcinomas were: luminal A carcinomas 29.0%5 to 44.8%3; luminal B carcinomas 13.5%3 to 49.0%5; basal‐like carcinomas 24.5%4 to 29.2%3; HER2‐overexpressing carcinomas 0%5 to 24.46%4; and “normal‐like” 3.1%4 of cases.


Molecular subtypes are correlated with histological type, grade, and survival in dogs.3 The basal subtype is associated with simple carcinomas, frequently solid carcinomas, while complex carcinomas are mostly luminal A; grade I and II carcinomas are luminal A, while grade III carcinomas are mostly of the basal‐like phenotype. The basal‐like subtype is also significantly associated with shorter overall survival and disease‐free interval. However, the results are contradictory and differ between studies and additional investigations using standardized protocols for IHC and interpretation of the IHC results need to be undertaken.


In the cat, only three molecular subtypes of mammary carcinomas have been found using IHC on primary mammary carcinomas and their associated lymph node metastases: luminal B, HER2‐overexpressing, and basal‐like.6 Whether the absence of the luminal A subtype is due to the selection in this study of highly malignant metastatic cases or is a characteristic of all feline mammary carcinomas remains to be elucidated.


References



  1. 1. Perou, C.M., Sorlie, T., Eisen, M.B., et al. (2000) Molecular portraits of human breast tumours. Nature 406:747–752.
  2. 2. Sorlie, T., Perou, C.M., Tibshirani, R., et al. (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98:10869–10874.
  3. 3. Gama, A., Alves A., and Schmitt, F. (2008) Identification of molecular phenotypes in canine mammary carcinomas with clinical implications: application of the human classification. Virchows Arch 453:123–132.
  4. 4. Im, K.S., Kim, N.H., Lim, H.Y., et al. (2014) Analysis of a new histological and molecular‐based classification of canine mammary neoplasia. Vet Pathol 51:549–559.
  5. 5. Sassi, F., Benazzi, C., Castellani, G., and Sarli, G.(2010) Molecular‐based tumour subtypes of canine mammary carcinomas assessed by immunohistochemistry. BMC Vet Res 6:5.
  6. 6. Brunetti, B., Asproni, P., Beha, G., et al. (2013) Molecular phenotype in mammary tumours of queens: correlation between primary tumour and lymph node metastasis. J Comp Pathol 148:206–213.

Grading of canine mammary tumors


The histological grading system is a useful and important prognostic indicator of canine mammary neoplasms. In human breast cancer, the most prevalent system used worldwide is the Elston and Ellis numeric method,1 which has been adapted for use in veterinary medicine and assesses three morphological features: tubule formation, nuclear pleomorphism, and mitotic counts.


A canine‐adapted histological grading method (referred to as the Peña method) was recently published;2 this system considers the heterogenicity of canine mammary tumors, how to assess complex and mixed tumors and is adapted to assess the great variability of nuclei and nucleoli (Table 17.5).


Table 17.5 Histologic grading of canine mammary neoplasms


Source A: Peña, L., De Andres, P.J., et al. (2013) Prognostic value of histological grading in noninflammatory canine mammary carcinomas in a prospective study with two‐year follow‐up: Relationship with clinical and histological characteristics. Vet Pathol 50:94–105. Reproduced with permission of SAGE Publications.






















































A.

Points
A. Tubule formationa 1 Formation of tubules in >75%
2 Formation of tubules in 10–75% (moderate formation of tubular arrangements admixed with areas of solid growth)
3 Formation of tubules in (<10%) (minimal or no tubule formation)
B. Nuclear pleomorphismb 1 Uniform or regular small nucleus, and occasional nucleoli
2 Moderate degree of variation in nuclear size and shape, hyperchromatic nucleus, presence of nucleoli (some of which can be prominent)
3 Marked variation in nuclear size, hyperchromatic nucleus, often with one or more prominent nucleoli
C. Mitoses per 10 HPFc 1 0–9 mitoses/10 HPF
2 10–19 mitoses/10 HPF
3 images 20 mitoses/10 HPF
B. Histologic malignancy grade
Total score (A + B + C) Grade of malignancy
3–5 I (low)
6–7 II (intermediate)
8–9 III (high)

a In complex and mixed tumors, the percentage of tubular formation is scored considering only epithelial areas. In malignant myoepithelioma tubular formation is 2. In heterogeneous canine mammary carcinomas, tubular scoring should be assessed in the most representative malignant area.


b In complex and mixed tumors, nuclear pleomorphism is evaluated in all the malignant components.


c HPF, high power field. The fields are selected at the periphery or the most mitotically active parts of the sample (not only epithelial cells). Diameter of the field of view = 0.53 mm ± 0.02 mm.


This grading system is a useful prognostic tool, facilitates histological interpretation, and offers uniform criteria for veterinary pathologists to follow.2 Tumor grade in conjunction with histologic subtype is predictive of biological behavior of mammary carcinomas.3


References



  1. 1. Elston, C.W. and Ellis, I.O. (1991) Pathologic prognostic factors in breast cancer: I. The value of histological grade in breast cancer: experience from a large study with long‐term follow‐up. Histopathology 19:403–410.
  2. 2. Peña, L., De Andres, P.J., Clemente, M., et al. (2013) Prognostic value of histological grading in noninflammatory canine mammary carcinomas in a prospective study with two‐year follow‐up: Relationship with clinical and histological characteristics. Vet Pathol 50:94–105.
  3. 3. Rasotto, R., Zappulli, V., Castagnaro, M., and Goldschmidt, M.H. (2012) A retrospective study of those histopathologic parameters predictive of invasion of the lymphatic system by canine mammary carcinomas. Vet Pathol 49:330–340.

CLASSIFICATION OF CANINE MAMMARY TUMORS


The classification described in Box 17.1 is the same as that published in Veterinary Pathology in 2011.1 The reader is also referred to this publication for a review of previous classifications of canine mammary tumors and for significant references.1–3

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Mar 30, 2020 | Posted by in INTERNAL MEDICINE | Comments Off on Tumors of the Mammary Gland

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