The comparative pathology of human and mouse mammary glands.

The mouse has emerged as a primary animal model for human breast cancer because the mammary glands of the two species are very similar in structure and function. In this regard the TDLU and LA have similar morphology. The mouse, infected by MMTV, develops "spontaneous" tumors with specific but limited tumor phenotypes. The advent of genetic manipulation has created transgenic mice that develop hyperplasias and tumors morphologically and cytochemically comparable to lesions in humans. Even experienced pathologists have difficulty distinguishing between lesions from the two species, and the morphological similarities support the utility of the mouse model in understanding human breast cancer. In this essay we review our experience with the histopathology of human and mouse mammary disease by comparing the normal gland with hyperplastic, dysplastic and neoplastic lesions of traditional and transgenic origin.

An internal X-cell pseudotumor in a black croaker (Cheilotrema saturnum).

Cells closely resembling X-cells were the primary cellular component of a large pseudotumor in the viscera of a black croaker (Cheilotrema saturnum). The fish was captured in coastal waters off San Diego, California and was maintained at the Scripps Aquarium. After 2 years in captivity the fish exhibited extreme abdominal swelling due to a visceral mass, which weighed approximately one-fifth the total body weight. The cells associated with the pseudotumor were identified as X-cells due to their great variability in size, vesicular nuclei which stained negative for DNA and their formation of a pseudotumor. This is the first report of an X-cell pseudotumor in the visceral cavity. We agree with the hypothesis that the X-cell is a protozoan, and the description here of X-cells in a visceral pseudotumor in a fish of the family Sciaenidae associates an additional type of lesion with this enigmatic parasite.

Human breast epithelial cells in serum-free collagen gel primary culture: growth, morphological, and immunocytochemical analysis.

Human breast epithelial cells derived from various sources (fibroadenoma, reduction mammoplasty, and mastectomy tissues from premenopausal patients) have been cultured in collagen gel matrix using serum-free medium. Response to various additives has been analyzed for growth-promoting effect when added to a basal medium containing insulin, cholera toxin, and BSA. A consistent observation has been the effect of EGF and cortisol in growth stimulation of human breast epithelial cells, while separately, each additive elicited only a small response. Under this condition, employing EGF and cortisol combinations, these cells gave rise to organized colonies consisting of clusters of cells, usually spherical, without any duct-like extensions. Ultrastructural and immunocytochemical studies, using a panel of monoclonal and polyclonal antibodies, have shown that cell types and features that can be identified in the original breast tissue can also be delineated in the progeny populations. The topographical feature, consisting of lumina surrounded by a single inner layer of epithelial cells and an outer layer of basal/myoepithelial cells, can be re-created in the collagen gel system starting from small clumps of cells.

Apocrine cystic metaplasia: subgross pathology and prevalence in cancer-associated versus random autopsy breasts.

Focal apocrine metaplasia was studied in 293 whole human breasts by a subgross sampling technique with histologic confirmation. There were 186 breasts obtained from random autopsies. Another 107 breasts were obtained that were cancer-associated; these breasts either contained an invasive cancer or were situated contralateral to a cancer-containing breast. The observations support a lobular origin for most if not all apocrine metaplasia and demonstrate some correlation between the presence of apocrine metaplasia and coincident invasive breast cancer on either the contralateral or ipsilateral side. When extensive, apocrine metaplasia appears to be a useful phenotypic marker for tissue at a modestly increased risk for breast cancer. There were no breast carcinomas of the apocrine type in this series, supporting the belief that apocrine metaplastic epithelium has little intrinsic malignant potential. Finally, the data support the hypothesis that most breast cysts arise from apocrine metaplastic lobules.

Different mitogenic and phenotypic responses of human breast epithelial cells grown in two versus three dimensions.

Human breast epithelial cells, derived from fibroadenomas, were cultured under conditions promoting growth in two-dimensions (2D) as monolayers using the collagen-coated dishes and in three-dimensions (3D) inside the collagen gel matrix. Both epidermal growth factor (EGF) and cortisol (F) were required for maximal stimulation in 3D growth, but only cortisol was required for 2D growth. The growth stimulation of exogenously added type IV collagen was no greater than that of type I as a substrate in both the 2D and 3D growth. Immunocytochemical staining, using a polyclonal actin antibody, showed homogeneous staining in all cells in 2D monolayers, whereas more restricted distribution was observed in 3D outgrowths in the collagen gel matrix. The same cells, when cultured in 2D vs 3D, elicit different responses and the original phenotypes may be better maintained in 3D.

The prevalence of carcinoma in situ in normal and cancer-associated breasts.

Two hundred ninety-two human breasts were examined in toto by a subgross sampling technique with histologic confirmation. The samples consisted of 185 breasts from random autopsies, 63 cancer-containing breasts, and 44 breasts contralateral to cancer-containing breasts. The method permits the identification and enumeration of essentially all of the dysplastic, hyperplastic, and neoplastic lesions present in each breast. Emphasis was on the prevalence within each sample category of ductal carcinoma in situ (DCIS), lobular carcinoma in situ (LCIS), and epithelial proliferative lesions with severe atypia, previously termed ALA 4 and ALB 4, which correspond to the clinicopathologic entities atypical ductal hyperplasia and atypical lobular hyperplasia, respectively. Additional primary foci of DCIS (unrelated to invasive breast carcinoma, if present) were found in 52.5 per cent of cancer-containing breasts, and were seen in 47.7 per cent of contralateral and 5.9 per cent of the breasts from random autopsies. Lobular carcinoma in situ was generally seen only in association with infiltrating carcinoma, usually of the ductal type. No LCIS was seen in the breasts from random autopsies. These trends are the same if the proliferative lesions with severe atypia are included with carcinoma in situ. The numbers of lesions were also markedly greater in affected cancer-associated breasts than in affected breasts obtained from autopsies. These findings suggest that LCIS, although a rare lesion in the general population, may be a significant marker for clinical carcinoma. They support previous studies showing a small percentage of women with undetected DCIS of uncertain clinical and biological potential. The multicentric nature of preinvasive breast carcinoma is further substantiated. Finally, when the prevalence and number of lesions are considered in association with the ages of the patients, the lower prevalence of such lesions in the older patients in each sample suggests that at least some DCIS and LCIS may be dependent on a premenopausal hormonal milieu for their continuing existence.

Subgross pathologic features and incidence of radial scars in the breast.

Eighty-three breasts obtained at random autopsy, which were presumed to be at low risk for development of cancer, and 107 cancer-associated breasts (containing cancer or contralateral to cancer-containing breasts), which were presumed to be at high risk, were studied in their entirety using a subgross slicer method with histologic confirmation. Twelve breasts (14 per cent) from the random autopsy series had from one to 13 radial scars each, with an average of 7.7 radial scars per involved breast; the average number of radial scars per breast for the entire series of 83 breasts from the autopsy series was 1.1. Twenty-eight breasts (26 per cent) of those from cancer-associated series had from one to 31 radial scars each, with an average of 15.5 per involved breast; the average number of radial scars per breast for the entire series of 107 cancer-associated breasts was 4.4. Radial scars are observed in breasts from patients in the same age--37 to about 85 years--in both series. Atypical epithelial hyperplasia ( epitheliosis ) was less frequent and less severe in breasts from the random autopsy series than in the cancer-associated breasts. Invasive carcinoma was not found in step sections of any of the radial scars; however, carcinoma in situ of the ductal type was found in the radiating arms of one. It is concluded that the presence of radial scars is at least a "marker" for enhanced tissue risk for cancer development, in much the same manner that some other mammary dysplastic features are "markers." The data do not support or definitely negate the hypothesis that tubular carcinoma develops in radial scars and subsequently evolves into the more common types of cancer, as proposed by some authors.

Primary culture of human mammary epithelial cells embedded in collagen gels.

Human mammary epithelial cells were dissociatd from mastectomy tissues. The contaminating fibroblasts were removed by the use of Percoll density-gradient centrifugation, which utilizes the difference in buoyant densities between epithelial cells and fibroblasts. A preparation highly enriched for mammary epithelial cells ws then embedded in collagen gel and cultured in Ham's F12 medium containing 12.5% horse serum, 2.5% fetal calf serum, 0.1 microgram cholera toxin/ml, an extract prepared from human male urine (L microgram protein/ml), and a hormone combination of 10 microgram insulin/ml, 10 microgram human placental lactogen/ml, 1 microgram aldosterone/ml, and 0.5 microgram hydrocortisone/ml. Sustained growth leading to an increase of tenfold to thirtyfold in cell number over the initial value was accomplished in primary culture, and this growth was maintained even after passage to secondary culture. Deletion of either the urine extract or the hormone combination resulted in less than optimal growth. Subsequent studies showed that hydrocortisone alone could replace the hormone combination. In addition, urine extract could be replaced by extracts prepared from human kidneys or brains. The collagen gel system provies a reproducible and consistent method for sustained three-dimensional growth of mammary epithelial cells from human breast tissue in primary as well as passaged cultures.

Correlative studies of the histological and radiographic appearance of the breast parenchyma.

A total of 143 breast biopsies were compared with xeroradiographic images. N1 breasts showed normal stroma, ducts, and lobules; P1 breasts had mild to moderate perioductal and perilobular fibrosis with some atypical lobules and other low-grade lesions; P2 was similar to P1 but with more fibrosis and higher-grade atypical lobules; and DY was generally similar to P2 but with more high-grade atypical lobules and extensive, confluent fibrosis. The highest grades of precancerous epithelial abnormality appear to be found in P2 and DY, seldom in P1, and very rarely in N1. Xeroradiographic and histological risk grades show very close correlation.

Pathology of hepatomas and other liver abnormalities in English sole (Parophrys vetulus) from the Duwamish River estuary, Seattle, Washington.

Liver abnormalities were found, by gross and histopathologic examination, in 92% of the English sole (Parophrys vetulus) from the Duwamish River Estuary, Seattle, Washington. Hepatomas were found in 32% (20 of 62) of the English sole. Other observed liver aberrations included increased fatty vacuolation, congestion, structure disarray, increased size and number of melanin-macrophage centers, centrolobular fatty degeneration and necrosis, increased amounts of perivascular connective tissue, intercellular melanin deposits, and hepatocellular hypertrophy often associated with the presence of bizarre nuclei and/or multiple nucleoli. Livers evidencing microscopic lesions were usually discolored. Livers containing hepatomas were often mottled yellow or tan and brown; occasionally, hepatomas were visible as tan or white nodules. Although the cause(s) of the liver abnormalities has not been conclusively identified, chemical analyses of Duwamish River English sole have detected polychlorinated biphenyl (PCB) levels of about 1.5 ppm (dry wt) in total body tissue. Many of the above-mentioned abnormalities, with the exception of hepatomas, have been observed in fish exposed to PCB's.

Biology of breast preneoplasia.

The literature dealing with mammary cancer and the presence of the identifiable preneoplastic lesions in the mammary gland of several species has been reviewed. A discrete morphologically identifiable lesion with a high malignant potential is considered preneoplastic. Lobuloalveolar lesions have been identified in the human mammary gland which fit most of the criteria for preneoplasia. The lesions, hyperplastic atypical lobules, are multicentric, have a high statistical association with breast cancer and demonstrate a morphological progression through dysplasia to frank carcinoma. Other breast lesions have a high statistical association but no morphological progression can be seen and their neoplastic potential remains unknown. The significance of these correlations is enhanced by the occurrence of homologous breast lesions in outbred and inbred animal models. Statistical correlation with breast cancer and aberrant morphology has been accepted as presumptive evidence of preneoplasia in some animals. Definitive proof of the neoplastic potential has been obtained in rodent models by transplantation of the suspected lesions and their subsequent development into cancers. Although a strong morphological and statistical analogy exists between the human and animal models, the causes and biological potential of the human lesions await experimental proof.

Persistent and atypical lobules in the human breast may be precancerous.

Persistent human mammary lobules (PL) remaining after the menopause, and certain atypical lobules (ALA) are morphologically similar to the common preneoplastic hyperplastic alveolar nodule (HAN) of mice of strains having a high incidence of mammary carcinoma. This and other evidence suggest that like the HAN of mice human PL and ALA are precancerous.

Preneoplastic lesions of the human mammary gland transplanted into the nude athymic mouse.

Morphologically normal lobules and atypical lobules postulated precancerous to ductal carcinoma were transplanted to test their biological behavior. Supravital staining disclosed the 1 to 4-mm microorgans. "Cleared" mammary fat pads of nude mice were optimal transplantation sites. Of the total of 217 transplants from 19 cancer-associated and 13 non-cancer-associated breast, 151 survived after 2 to 27 weeks. Of 61 surviving normal-appearing lobules from cancer-associated breasts transplanted without prior in vitro maintenance, 20 (30%) dedifferentiated, and of 48 surviving lobules from noncancerous breasts, 11 (20%) dedifferentiated. Fifteen of 28 histologically normal-appearing lobules (60%) obtained from cancer-associated breasts after age 50 dedifferentiated. Thirty of 36 atypical lobules isolated from the breast tissue were obtained from cancer-associated breasts and the 5 of those that dedifferentiated came from cancer-associated breasts. Twenty of 22 (90%) dedifferentiating transplants from cancer-associated breasts showed a vascularization response, whereas 3 of 7 (43%) from noncancer-associated breasts did so. If dedifferentiation in this experimental setting is indicative of a precancerous potential, the data on normal-appearing lobules obtained from cancer-associated breasts from women over age 50 suggest that these lobules carry the greatest precancerous potential. Such lobules probably belong to a type persisting after menopause and they may be hormonally autonomous. Such lobules might undergo further atypia in vivo and, eventually, cancerous transformation.