American Journal of Pathology, Vol. 13 1, No. 2, May 1 988

The Morphologic Effects ofSynthetic Reproductive on the Mammary Gland ofRhesus Monkeys , , Mestranol-Ethynerone, Chloroethynyl -Mestranol, and Acetate-Mestranol Combinations

F. A. TAVASSOLI, MD, From the Department of Gynecologic and Breast Pathology and the H. W. CASEY, COL, USAF, VC, Department of Veterinary Pathology, Armed Forces Institute of and H.J. NORRIS, MD Pathology, Washington, DC

A total of213 treated and 16 control monkeys compris- 22 of 52 animals (42%), including five identical to in- ing 12 experimental groups was evaluated for determi- traductal carcinoma in the human and one identical nation of the long-term (10 years) effects of various to lobular neoplasia. Of the 40 monkeys administered dosages of a variety ofsynthetic oral contraceptive ste- anagestone acetate and mestranol, 20 (50%) developed roids on the mammary glands ofrhesus monkeys. The proliferative atypias; the atypias ranged from mild to hormones included mestranol, ethynerone, a severe and included five resembling intraductal carci- combination ofmestranol and ethynerone, chlorethy- noma in human breast. The chloroethynyl norgestrel nyl norgestrel plus mestranol, and anagestone acetate and mestranol combination induced proliferative plus mestranol. Various degrees ofphysiologic lobular atypia in 25 of 52 monkeys (49%); six of these atypias hyperplasia and lactational changes were observed in were severe and indistinguishable from intraductal association with all of these steroid hormones; these carcinoma of the human breast; and one, if in the hu- changes appeared dose-dependent. Mestranol caused a man breast, would reflect a solid variant of an invasive proliferative atypia ranging from a minimal to a mod- carcinoma. Only 2 of the 16 control monkeys (12%) erate degree in 8 of 34 (23%) animals, but it was not developed proliferative atypias, and these were ofmin- dose-related. Eleven of 15 monkeys (73%) adminis- imal to mild degree. The occurrence of severe degrees tered ethynerone developed proliferative atypia, rang- ofatypia identical to intraductal carcinoma in the hu- ing in degree from minimal to severe, including one man breast and invasive carcinoma associated with invasive carcinoma and 2 lesions resembling intraduc- hormone administration suggests a carcinogenic tal carcinoma in the human. The mestranol and ethyn- effect. (AmJ Pathol 1988, 131:213-234) erone combination produced a proliferative atypia in

THE LONG-TERM effect of estrogen and estrogen- Supported by National Institute ofChild Health and Hu- progestin combinations on the human and monkey man Development Contract NO l-HD-2-2729. breast have not been studied systematically. A num- The investigators adhered to the principles described in ber of divergent opinions have appeared in the litera- "Guide for the Care and Use of Laboratory Animals" pre- ture regarding the association of oral contraceptive pared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, use with fibrocystic changes and benign and malig- National Research Council. Accepted for publication December 16, 1987. The opinions and assertions contained herein are the pri- Address reprint requests to F. A. Tavassoli, MD, Vice vate views of the authors and are not to be construed as Chairman, Gynecologic and Breast Pathology Department, official or as reflecting the views of the Department of the Armed Forces Institute of Pathology, Washington, DC Army or the Department ofDefense. 20306.

213 214 TAVASSOLI ET AL AJP * May 1988 nant breast lesions in humans.1-6 Various results have The sources of chloroethynyl norgestrel and mestra- been reported in a small number ofanimal studies re- nol were the Department of Health, Education, and garding the effect ofdifferent reproductive steroid hor- Welfare and the FDA, Washington, DC; Merck and mones.71-3 This study describes the effects of mestra- Company, Inc., Rahway, New Jersey; and Merck, nol, ethynerone alone, and combinations of mestra- Sharpe and Dohme, West Point, Pennsylvania. The nol with ethynerone, chlorethynyl norgestrel, or anagestone acetate plus mestranol (10:1) was received anagestone acetate on the mammary glands ofrhesus from the Ortho Research Foundation, Raritan, New monkey over a 10-year period. With the exception of Jersey. those dying from various infections, all monkeys re- The steroids and steroid combination compounds ceived hormones for 10 years for comparison with were administered orally in 28-day cycles consisting control monkeys. Morphologic effects observed in- of 21 days of administration followed by 7 days with- cluded physiologic lobular hyperplasia, lactational out the drug. The steroids were mixed by means of a change, and an atypical proliferation that was graded mortar and pestle with an applesaucelike food vehicle from minimal to severe. The nature and significance at varying concentrations so that a constant volume ofthese findings are discussed. of 1.0 ml/kg could be administered at all dosage levels. The monkeys usually consumed the substances. Materials and Methods When the food vehicle was refused, the monkeys re- ceived the dosage by gastric intubation. Individual All specimens from the 229 rhesus monkeys and daily doses were calculated on the basis of body the clinical data for this study were provided by the weights obtained once every 4 weeks. Food and Drug Administration, which initiated a se- The first group consisted of the control monkeys. ries of long-term toxicity studies with oral contracep- The animals were 16 immature rhesus females that tive steroids in 1968. The experimental design and weighed 1.8-3.6 kg at the start of the 10-year study. methods used were according to a common protocol The animals underwent necropsy when they reached used in long-term studies ofthe toxicity ofsteroid con- 10 years on the study. Three monkeys died during this traceptives with guidelines established by the Food period, at 5.0, 5.4, and 5.5 years; and 2 animals were and Drug Administration. The purpose of the study sacrificed at 8 years for special eye studies. The mam- was to determine the effect, if any, of mestranol and mary glands from all 5 of these monkeys were sam- various other synthetic steroids on the monkey breast. pled, and the findings included in the final results. The The steroids were synthesized and tested because of next group consisted ofmonkeys receiving mestranol; intentions to utilize them in oral contraceptive com- these were divided into subgroups A and B. Subgroup pounds for human use. The protocol recommended A consisted of 15 monkeys receiving 0.02 mg/kg/day 12 monkeys per group for each dosage level of each of mestranol (10 times the proposed human dose); steroid. and Subgroup B, of 19 monkeys receiving 0.1 mg/kg/ The light- and electron-microscopic appearance of day of mestranol (50 times the human dose). Eight 213 female rhesus monkey (Macaca mulatta) mam- monkeys in Subgroup A died ofinfections, at 3.1, 3.4, mary glands comprising 11 experimental groups was 4.4, 4.8, 6.0, 7.0, 7.7, and 9.3 years after initiation of studied after 10 years ofexposure to various synthetic the study. Thirteen monkeys in Subgroup B died in a steroid hormones. The hormones used were mestra- similar manner, at 0.25, 0.25, 0.5, 0.9, 1.6, 2.3, 2.7, nol, ethynerone (MK-665), ethynerone plus mestra- 3.8, 5.3, 6.1, 7.9, 8.4, and 9.0 years. This left only 7 nol, chloroethynyl norgestrel (WY-4355) plus mes- and 6 monkeys, respectively, that completed 10 years tranol, and anagestone acetate (ORF- 1658) plus of the study in each subgroup. The third group con- mestranol. The progestational compounds included sisted of 15 monkeys on 2.0 mg/kg/day ofethynerone halogenated 19-nortestosterone derivatives (chloro- (50 times the proposed human dose). Five of these ethynyl norgestrel and ethynerone) and a 1 7-acetoxy monkeys died of infections, at 0.27, 4.0, 4.6, 4.8, and derivative of (anagestone acetate). Mes- 7.6 years. A fourth group received a combination of tranol, 3-methyl ester ofethynyl estradiol, is the estro- ethynerone and mestranol and was divided into three gen component of several marketed oral contracep- subgroups. Subgroup A consisted of 16 monkeys on tives, while the other compounds are investigational 0.084 mg/kg/day ofa combination ofethynerone and progestins that have never been marketed. The mestranol; B, of20 monkeys receiving the same com- sources of mestranol and ethynerone were the Food pound at a dose of0.42 mg/kg/day; and C, of 16 mon- and Drug Administration (FDA), Washington, DC; keys receiving 2.1 mg/kg/day of the same combina- Merck and Company, Inc., Rahway, New Jersey; and tion. The dosages represent 2, 10, and 50 times the Merck, Sharpe and Dome, West Point, Pennsylvania. proposed human dose, and the ratio of progestin to Vol. 1 31 * No. 2 STEROID-INDUCED MAMMARY LESIONS 215 mestranol was 20:1. Three of the monkeys in Sub- Table 1 -Number of Monkeys That Died in Each Group and Status of Mammary Glands at Autopsy group A died, at 4.2, 4.5, and 10 years. Six monkeys in Subgroup B died, at 0.2, 0.3, 0.4, 1.1, 6.5, and 7.7 Contraceptive and Number Autopsy findings in dosage (mg/kg/day) dead mammary tissue years. Four monkeys in Subgroup C died, at 2.6, 8.9, 9.0, and 9.6 years. The monkeys in the fifth group re- Control, no hormones 5 Normal ceiving chloroethynyl norgestrel plus mestranol (20: 1) Mestranol (0.02) 8 1, moderate PA; 7 normal Mestranol (0.1) 13 2, lactational change were divided into three subgroups, namely, Subgroup Ethynerone (2.0) 5 4, lactational change; A, consisting of 17 monkeys; B, of 17 monkeys; and 1, autolyzed C, of 18 monkeys. These subgroups received 0.084, Ethynerone + mestranol 3 Normal (0.084) 0.42, and 2.1 mg/kg/day, respectively, representing 2, Ethynerone + mestranol 6 Focal PLH 10, and 50 times the proposed human doses, respec- (0.42) tively. Four monkeys were sacrificed or died from Ethynerone + mestranol 4 Lactational change and PLH (2.1) Subgroup A, at 1, 1.6, 1.8, and 9 years. Two monkeys Chloroethynyl norgestrel 4 4, lactational change; 1, mild died from Subgroup B, at 6 and 7.6 years. Nine mon- + mestranol (0.084) PA and PLH keys died in Subgroup C, at 0.3, 0.7, 3.0, 3.6, 4.6, 5.5, Chloroethynyl norgestrel 2 PLH + mestranol (0.42) 5.1, 6.6, and 8.1 years. This study was initiated in De- Chloroethynyl norgestrel 9 PLH and lactational change cember 1968 and terminated in April 1979. The last + mestranol (2.1) group of monkeys receiving anagestone acetate and Anagestone acetate 10 3, moderate PA and PLH; + mestranol (0.44) 7, focal PLH mestranol (10:1) were divided into two subgroups, A Anagestone acetate 16 1, severe PA; 1, moderate PA; and B, receiving 0.44 and 2.20 mg/kg/day, 10 and 50 + mestranol (2.20) 1, mild PA, PLH; 13, normal times the proposed human doses. These two sub- PA, proliferative atypia; PLH, physiologic lobular hyperplasia. groups of 20 monkeys each were originally part of an- other study but were incorporated into this study after 95 weeks after changes in technical procedures to con- riod. During this period, diarrhea was controlled with form to the protocol for the main study. This part of a combination of broad-spectrum antibiotics, and all the study was initiated in May 1968 and was termi- monkeys received an application of Para Bomb M nated in November 1978 for the lower dose (0.44 mg/ aerosol (Haver-Lockhart) for the control ofbody lice. kg/day) group. Mortality was high in this group, with An intrapalpebral injection of USDA Mammalian 10 monkeys dying at 0.13, 0.44, 0.7, 0.9, 2.3, 3.1, 3.9, Tuberculin was administered to all monkeys for signs 4.1, 7.0, and 8.3 years. Starting with 20 monkeys, 2 ofreaction. The tuberculin test was repeated monthly replacement monkeys were used for animals that died during the 13-week period and bimonthly for the du- at the higher dose (2.20 mg/kg/day) level. Thus, a total ration ofthe study. Prior to the administration ofany of42 monkeys were used in these two groups. Survival compound, the monkeys were given a physical exami- was poor in the higher dose group, with 16 monkeys nation that included palpation of mammary glands dying, at 0.04, 0.19, 0.4, 0.6, 0.965 1.8, 2.8, 5.0, 5.4, for the presence of nodules. Examinations of the 5.8, 5.9, 6.0 (2 monkeys), 6.6, 6.7, and 8.0 years. In mammary and/or contiguous tissues for the presence addition, 3 monkeys were sacrificed at 7.5 and 8.2 of nodules was conducted approximately once every years for special eye studies. The remaining 4 mon- 4 weeks for the duration of the study. Normal men- keys at the 2.2 mg/kg/day dosage level were sacrificed strual cycles were observed for control monkeys. at 8.6 years per request of sponsors, terminating the Progress reports on the 10-year study and details on study on the high-dose portion of this compound on the materials and methods have been published. "' Tis- November 1 1, 1976. sue removed for light microscopy consisted ofthe nip- Regardless of the time or cause of death and sacri- ples and approximately 2-3 cm of underlying mam- fice, autopsies were performed on all monkeys, and mary tissue. The laterality ofthe breast was not speci- samples were removed from the mammary glands for fied, and in a majority of cases only a single breast evaluation. The number ofmonkeys that died in each section was evaluated. group and the status of their mammary glands at au- Mammary glands were sampled randomly for ul- topsy for the entire study are shown in Table 1. The trastructural evaluation at the time of necropsy when monkeys were obtained from Woodard Asiatic Cor- the monkeys reached 10 years in the study. Multiple poration, Hartelust Thorsen, or Primate Imports Cor- tissue samples measuring 1-2 cm in aggregate were poration. They had no history of pregnancy or known taken from one or both breasts. These were cut into 1- hormone dysfunction and had not received steroids mm cubic blocks and fixed directly in glutaraldehyde, prior to initiation ofthe study. From their receipt, the postfixed in 3% buffered osmium tetroxide, and em- animals were observed for a 13-week conditioning pe- bedded in Epon after dehydration in graded alcohols 216 TAVASSOLI ET AL AJP * May 1988

changes, because these sections may not be represen- tative. When a lesion is present in these samples, how- ever, the finding is significant, even if samples re- moved for light microscopy lack similar changes. The monkeys that died at various times during the course of the study (Table 1) clearly did not have an equal chance of developing lesions, but they were included in the final analysis even though the incidences ofpro- liferative atypias and the degree of physiologic lacta- tional-type hyperplasia might have been higher had these monkeys survived to the end ofthe study. Physiologic lobular hyperplasia, secretory change, lactational change, eosinophilic cytoplasmic granu- larity, and apocrine metaplasia were recorded as mild, moderate, or marked. An atypical epithelial prolifera- Figure 1-Control. Minimal proliferation characterized by focal stratification tion, characterized by a monomorphic population of of three to four cells (or occasional epithelial mounds). (Toluidine blue, x1 60) cytologically atypical cells with an increased nucleus- to-cytoplasm ratio and hyperchromasia, was desig- nated as proliferative atypia because of its unknown and propylene oxide. Thick sections of at least six significance in the monkey. The cells were identical blocks were examined by light microscopy from each to those found in atypical intraductal hyperplasia and breast; and, depending on the findings, at least two cribriform and micropapillary variants of intraductal blocks were selected for thin sections from each carcinoma of the human breast. Four subdivisions or breast. Thin sections were stained with uranyl acetate grades ofthe proliferative atypia were identified: mini- and lead citrate and examined in a Zeiss 9S-2 electron mal, mild, moderate, and severe, based on the quan- microscope. Ifthe sample contained only a few breast tity and pattern ofproliferation. The earliest indicator lobules, supplementary tissue samples were retrieved of epithelial proliferation was a qualitative alteration from formalin-fixed tissue samples originally pre- in the cytologic appearance of the cells with diminu- served for light microscopy. In some instances, alter- tion of secretory material and subtle increase in ations found in samples examined by light micros- nucleus-to-cytoplasm ratio and the presence ofhyper- copy were not present in the samples taken for elec- chromasia. A minimal proliferation consisted offocal tron-microscopic evaluation, and vice versa. Due to cell stratification to three to four layers ofaltered cells, the small size ofthe samples prepared for electron mi- or epithelial mounds, as well as occasional minor tuft- croscopy, their evaluation alone is not the best way ing, which rarely extended midway through the lu- to determine the incidence of hormone-induced men ofeven the smaller (less than 1 mm in diameter) ducts (Figure 1). In the mild proliferative lesions, epi- thelial tufts and micropapillae formed by atypical mo- nomorphic round cells were more frequent; rarely, a partial cartwheel pattern was seen at one side ofa duct (Figures 2 and 3). A moderate degree of proliferation was characterized by irregular epithelial bridges in some generally smaller ducts in addition to tufts and micropapillae (Figure 4). Proliferative atypias of a moderate degree are comparable to atypical hyperpla- sia in the human mammary gland.'5 The severe prolif- erative atypias were characterized by a micropapillary or cribriform growth pattern identical to variants of intraductal carcinoma'6 iffound in human mammary gland (Figures 5 and 6). Also included among severe proliferative atypias were lesions with a pattern oftyp- ical human mammary comedo-type intraductal carci- noma with central necrosis. A solid pattern ofepithe- Figure 2-Anagestone acetate plus mestranol (0.44 mg/kg/day). Mild epi- thelial proliferation characterized by formation of epithelial tufts and micro- lial proliferation filling the terminal ductules within papillae. (Toluidine blue, x160) the lobules was present in 2 cases; this pattern was Vol. 1 31 * No. 2 STEROID-INDUCED MAMMARY LESIONS 217

Figure 3-Anagestone acetate plus mestranol (0.44 mg/kg/day). Mild hy- -WI perplasia-epithelial stratification and tufting. The few epithelial tufts barely Figure 5-Anagestone acetate plus mestranol (0.44/mg/kg/day). Severe meet, giving the appearance of epithelial bridges across the lumen of this proliferative atypia with well-formed cribriform growth pattern involving an small duct. Note the secretory activity in the adjacent lobule. (Toluidine blue, entire duct. (H&E, x250) X100)

Results comparable to lobular neoplasia in the human mam- mary gland and was also included in the group of se- Normal Anatomy A vere proliferative atypias (Figure 7). coarsely granu- Rhesus monkeys have one pair of pectoral mam- in lar, eosinophilic cell population also participated mary glands. Unlike the human breast, the in mammar- the proliferative atypias some of the ducts, includ- tissue in the monkey is diffusely spread in a thin sub- ing some ofthose with central necrosis (Figures 8 and cutaneous layer throughout the anterior chest wall 9). Proliferation of metaplastic apocrine cells (Figure and upper abdomen.15-16"18-20 The mammary layer 10) and hyperplasia ofa bland cell population in some lies between the skin and underlying pectoralis and the ducts were not included among proliferative abdominal muscles overlain by a layer of adipose tis- atypias. sue on either side, except at the region of the nipple, The number of monkeys studied at the ultrastruc- where the subcutaneous adipose tissue layer is thin tural level and the number ofcases and degrees ofpro- and disappears almost entirely, and in rare areas liferative atypia in each group are illustrated in Tables where the tissue is in direct contact with the pectoralis 2 and 3.

Figure 6-Anagestone acetate and mestranol (0.44 mg/kg/day). Severe Figure 4-Ethynerone plus mestranol (2.1 mg/kg/day). Moderate prolifera- proliferative atypia identical to cribriform intraductal carcinoma in human tive atypia, with multiple irregular epithelial bridges crossing the lumen, in breast. Note uniformity of the proliferating cells and persistence of myoepi- addition to tufts and micropapillae. (H&E, x250) thelial cell layer. (Toluidine blue, X400) 218 TAVASSOLI ET AL AJP * May 1988

found to have a nodule during palpation ofmammary glands and contiguous structures about 2 years prior to sacrifice, but the nodule had disappeared by the time of final physical examination prior to necropsy. None ofthe other animals had any palpable nodules. Histology The parenchyma of the mammary gland consisted of numerous lobules connected to one another by a duct system running parallel to the skin surface. The major ducts from different regions converged beneath the nipple to form sinuses from which four to seven main lactiferous ducts emerged to curve at a right an- gle into the nipple stroma. A few minor blunt-ended side branches were present in the major ducts in the lower half of the nipple. From the dilated sinuses, the Figure 7-Ethynerone plus mestranol (0.084 mg/kg/day). Severe prolifera- tive atypia characterized by an occlusive proliferation of a uniform cell popu- major ducts led to numerous terminal ducts which lation distending acini identical to lobular neoplasia in the human mammary ended in a grapelike cluster ofductules (acini) (Figure gland. (H&E, x250) 1 1). The clusters of ductules, or acini, together with the adjoining small "intralobular" segment ofthe ter- fascia. Highly variable in size and development within minal duct, formed the lobule. the species, the mammary gland is barely apparent or The duct system, including the intralobular duct- palpable in the adult female except during late preg- ules, was lined by two distinct cell types, one epithelial nancy and lactation, when some do show enlarge- and one myoepithelial, except for a short terminus at ment. There is generally one pair of nipples, which the nipple orifice, where the glandular epithelium was rarely exceed 1 cm in length and 5-6 mm in width replaced by squamous epithelium. The epithelial cells in quiescent glands. These become slightly larger and formed the inner layer and surrounded the lumen. pink or reddish during lactation. The areola has a Each cell was cuboidal to columnar, with the long axis minimal amount ofhair and also becomes pink or red of its nucleus perpendicular to the basement mem- during lactation.'6'18-20 brane. Myoepithelial cells formed a discontinuous layer between the epithelial cells and the basement Control Monkeys membrane. The long axis ofthe myoepithelial cell nu- Gross Findings clei was parallel to the basement membrane. The cy- toplasm was often inconspicuous or appeared as a The nipples appeared slightly enlarged in one mon- clear space around the naked-appearing nucleus. key at the time of necropsy. This monkey had been In toluidine blue-stained plastic sections, the myo- epithelial cells had spiderlike cytoplasmic processes extending along the surface of the basement mem- brane and separating the epithelial cells from the base- ment membrane (Figure 12). The cytoplasmic pro- cesses created gaps where the epithelial cells came in direct contact with the basement membrane, which surrounded the outer (myoepithelial) cell layer. More peripherally, a narrow space containing collagen fi- bers separated the basement membrane from the fi- broblasts, which formed a discontinuous fibroblastic layer encircling the ductule. Some epithelial cells within the ductules had in- tensely eosinophilic, granular cytoplasm, as illus- trated in Figure 8, but they lacked the typical luminal cytoplasmic projections of apocrine cells. The gran- ules were positive with the periodic acid-Schiff reac-

Figure 8-Mestranol (0.1 mg/kg/day). Cells with coarse, eosinophilic gran- tion, and much of the positivity persisted after predi- ules were prominent in many ductules in some cases. (H&E, x250) gestion with diastase. There was no reaction with Vol. 1 31 * No. 2 STEROID-INDUCED MAMMARY LESIONS 219

Figure 9-Ethynerone (2.0 mg/kg/day). These eosinophilic, coarsely granular cells also participated in the proliferative process in the case with comedocarcinoma (arrows). (H&E, x160)

mucicarmine stains. These cells are either a form of lar. The major ducts and their branches were sur- apocrine metaplasia or a form of secretory alteration rounded by two distinct layers ofconnective tissue: an of the mammary epithelium. Secretory type changes inner vascular longitudinal layer running parallel to were minimal, ifpresent at all. the ducts and an outer circumferential collagenous The epithelial elements of the mammary glands layer. Beyond these two connective tissue layers, the were supported by loose connective tissue. This stroma contained abundant fibroblasts, myofi- stroma varied in different regions of the gland, de- broblasts, collagen fibers, blood vessels of various pending on whether it was interlobular or intralobu- sizes, and nerve fibers. Vessels and nerve fibers were

_~~~~~'

Figure 10-Ethynerone (2.0 mg/kg/day). The metaplastic apocrine cells are charac- terized by abundant finely granular, pink cy- toplasm with apical cytoplasmic protru- sions. (H&E, x250) 220 TAVASSOLI ET AL AJP * May 1988

Table 2-Number of Animals With a Proliferative Atypia Among Proliferative atypia was negligible in the control Those Studied by Electron Microscopy in Each Group group monkeys. In one monkey, however, some ducts Number of Number of showed minimal proliferative atypia in the form of Contraceptive and monkeys evaluated monkeys with dosage (mg/kg/day) by EM proliferative lesions epithelial stratification and occasional epithelial mounds 4-5 cells thick, none of which extended into Control, no hormones 7 0 lobules (Figure 13). In another case, a single ductule Mestranol (0.02) 6 3 Mestranol (0.1) 6 4 showed mild proliferative atypia. Although minimal, Ethynerone (2.0) 6 5 this finding indicates that an early stage of prolifera- Ethynerone + mestranol tive atypia can occur spontaneously in the absence of (0.084) 6 3 Ethynerone + mestranol exogenous steroid hormone stimulation. The ovaries (0.42) 6 4 and the endometrium from these 2 affected monkeys Ethynerone + mestranol were morphologically normal. (2.1) 8 5 Chloroethynyl norgestrel The mammary glands from the 3 monkeys that + mestranol (0.084) 9 5 died at 5.0, 5.4, and 5.5 years and the 2 that were sacri- Chloroethynyl norgestrel ficed at 8 years were essentially normal. + mestranol (0.42) 8 7 Chloroethynyl norgestrel Ultrastructure + mestranol (2.1) 7 7 Anagestone acetate and The entire duct system, from the lactiferous duct to mestranol (0.44) 9 6 the intralobular ductule, had the same basic ultra- Anagestone acetate and mestranol (2.20) 1 1 structural arrangement as that in the human breast.6 From the lumen outward, the continuous layer ofepi- The ratio of progestin to mestranol was 20:1 for ethynerone plus mestra- nol and chlorethynyl norgestrel plus mestranol and 0.1 for anagestone ace- thelial cells, the interrupted layer of myoepithelial tate plus mestranol. Dosages correspond to 2, 10, and 25 times the pro- cells, the lamina lucida, the basal lamina, the loose posed human dosage. connective tissue layer, and the exterior fibroblastic EM, electron microscopy. layers were evident (Figure 14). There were some differences in the number of collagen fibers and fre- present along the distribution of the major ducts as quency offibroblasts in the connective and supportive well as in the interlobular connective tissue. The intra- tissue found within the lobule and in the interlobular lobular stroma was less collagenous and more cellular area; collagen was more abundant in the interlobular than the interlobular stroma. Capillary spaces were stroma. present within the lobules. Nerve fibers were found Whether cuboidal or columnar, the epithelial cells rarely within lobules, although they were found with forming the luminal layer were oriented with the long slightly higher frequency adjacent to and apposing the axis oftheir nuclei perpendicular to the lumen and the lobules. The stroma of the nipple was rich in smooth basement membrane. Partly separated from the basal muscle cells and nerve fibers and was quite dense be- lamina by myoepithelial cells, the epithelial cell pro- cause of their presence. Small aggregates of lympho- cesses occasionally extended to the basal lamina, and cytes were found occasionally in the stroma around some protruded toward the stroma through the gap the ducts or the lobules and sometimes extended into between myoepithelial cells (Figure 15). Secretory the lumens. material was sparse in the quiescent (nongravid, non-

Table 3-Number of Cases and Degrees of Proliferative Atypia in Each Group Contraceptive and dosage Total number of (mg/kg/day) Minimal Mild Moderate Severe Total cases evaluated Control, no hormones 1 1 0 0 2 16 Mestranol (0.02) 1 1 2 0 4 15 Mestranol(0.1) 2 1 1 0 4 19 Ethynerone (2.0) 2 3 3 3 11 15 Ethynerone + mestranol (0.084) 0 3 2 2 7 16 Ethynerone + mestranol (0.42) 1 2 5 2 10 20 Ethynerone + mestranol (2.10) 0 1 2 2 5 16 Chloroethynyl norgestrel + mestranol (.084) 4 0 2 1 7 17 Chloroethynyl norgestrel + mestranol (0.42) 1 4 2 4 11 17 Chloroethynyl norgestrel + mestranol (2.1) 2 2 2 1 7 18 Anagestone acetate + mestranol (0.44) 0 3 7 3 13 18 Anagestone acetate + mestranol (2.20) 0 4 1 2 7 22 For definitions, see Materials and Methods. Vol. 1 31 * No. 2 STEROID-INDUCED MAMMARY LESIONS 221

:;11 T-".

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Figure 11-Control. Terminal duct leading to a lobule with numerous acini (ductules). Note the intralobular segment of the termi- nal duct and little or no secretory activity. (H&E, x175)

lactating) mammary epithelium. When present in cells usually had the long axis oftheir nucleus perpen- minimal amounts, the secretory material was distrib- dicular to that ofthe epithelial cells, but in some areas uted throughout the cytoplasm but was concentrated the long axis was parallel to that ofthe epithelial cells. toward the lumen, a feature particularly prominent in The latter orientation may have resulted from con- the granular, eosinophilic cells. traction, because myoepithelial cells can readily alter The myoepithelial cells were located between the their configuration. The nuclei were elongated with epithelial cells and the basement membrane as a dis- multiple deep indentations in contrast to the rounded continuous or a fenestrated layer. These cells were nuclei and shallow indentations ofthe epithelial cells. stellate, with elongated spiderlike cytoplasmic pro- The most distinctive feature ofthe myoepithelial cells cesses extending in various directions. Myoepithelial was their abundant cytoplasmic myofibrils with dense bodies. Another important feature of myoepithelial cells was the presence ofpinocytotic vesicles along the

Figure 12-Control. The basic unit structure of the duct system. An inner (luminal) epithelial cell layer is surrounded by a discontinuous layer of myoep- ithelial cells. The lamina lucida, basal lamina, and loose connective tissue layer are represented by an amorphous band (arrow). The entire complex is Figure 13-Control. A minimal epithelial atypia in the form of epithelial strati- bound by the limiting fibroblastic layer. Note the variation in the shape and fication and occasional epithelial mounds found in one control monkey. appearance of the myoepithelial cells. (H&E, X630) (H&E, x400) 222 TAVASSOLI ET AL AJP * May 1988

Figure 14-Control. Ultrastructure of a ductular (acinar) unit complex in cross-sec- tion. The epithelial cells surround the lumen, their microvilli projecting into it. The myoepi- thelial cells are distributed in a discontinu- ous layer between the epithelial cells and the basement membrane. The space be- tween the basement membrane and the de- limiting fibroblast is partially filled with colla- gen fibers. Elongated cytoplasmic pro- cesses of the delimiting fibroblasts form a focally fenestrated ring around the ductule. Part of a vessel is present in the left lower edge of the field. (Uranyl acetate and lead citrate, x1 760) ....A x-. ..:"sff

~~~~~~~~~~~~, rft .::..ujj# ../~~~~~~~~~. X plasma membrane. The intercellular junctions were ules. Rarely, small capillaries were found between the mainly well-formed desmosomes; the cells attached to delimiting fibroblastic layer and the basal lamina. the basal lamina by hemidesmosomes. Elsewhere, the larger vessels had their own binding Within the epithelial cell layer of the ducts and fibroblastic layer. Fibroblasts, random collagen and ductules, there was an occasional cell with nuclear elastic fibers, and occasional lymphocytes were pres- characteristics and cellular configuration ofan epithe- ent between the delimiting fibroblast ofadjacent duct- lial cell and some cytoplasmic features (myofibrils) of ules. a myoepithelial cell and vice versa (Figure 16). These intermediate cells may have the to differen- capacity Summary of the Effects of Mestranol tiate into either epithelial or myoepithelial cells and replace degenerated or desquamated cells. Both subgroups (15 monkeys on 0.02 mg/kg/day Between the myoepithelial cell and the basal lamina and 19 monkeys on 0.1 mg/kg/day) showed physio- was a clear space, the lamina lucida, which was focally logic lobular hyperplasia with a particularly promi- traversed by the filaments of hemidesmosomes at- nent lactation noted in 2 monkeys at the higher dos- taching myoepithelial cells to the basal lamina. The age. Cells with coarse, eosinophilic granules in the cy- occasional epithelial cell apposed to the basal lamina toplasm were prominent in both groups. Proliferative was also separated from it by the lamina lucida, but atypias ranged from minimal to moderate, being pres- there were no junctional complexes apparent between ent in 8 of the 34 (23.5%) monkeys in the two sub- the two. The basal lamina completely encircled the groups. The physiologic and lactational changes in- ducts. A layer of loose connective tissue consisting of creased slightly with increasing dosage, but the prolif- an admixture of collagen with interspersed elastic fi- erative changes were not affected by the increasing bers surrounded the basal lamina. One or more delim- dosage, being present in 26.5% ofmonkeys in the low- iting fibroblasts (depending on the size of the ducts) dose group and 21% of monkeys in the high-dose with extremely attenuated long cytoplasmic processes group. encircled the loose connective tissue layer. These were At the ultrastructural level, both the proliferating similar to the delimiting fibroblasts described in the and nonproliferating epithelial cells were character- mammary gland ofthe human.2' ized by the presence ofabundant profiles ofrough en- Within the lobule, small vessels were found mainly doplasmic reticulum (RER). Secretory material was between the delimiting fibroblasts of different duct- abundant in the nonproliferating cells, particularly Vol. 1 31 * No. 2 STEROID-INDUCED MAMMARY LESIONS 223

Figure 15-Control. In a terminal duct, an epithelial cell is in direct contact with the basement membrane through a gap be- tween myoepithelial cells. (Uranyl acetate and lead citrate, x2300)

the lactating cells. The atypical proliferating cells were toplasmic granules. Proliferative atypia ranging from predominantly devoid ofsecretory material but some- minimal to severe, including an invasive carcinoma times contained abundant secretory material (Fig- (Figure 18) and 2 intraductal carcinomas (Figure 19), ure 17). was evident in the mammary glands of 11 of the 15 Tight junctions and desmosomes connected the in- (73%) ofthe monkeys. terdigitating cytoplasmic processes of adjacent cells. Ultrastructurally, normal and lactational cells were Interestingly, however, many of the myoepithelial identified in the nonproliferative ductules (acini). The cells in both the ducts with proliferative atypia and lactating cells had two types ofsecretory material with normal ducts also contained electron-dense granules varied sizes and electron densities corresponding to and lysozomes similar to those found in the epithelial the lipoproteinaceous and mucosubstances of these cells, even when the proliferative atypia cells showed cells. The lactational-type secretory change was much a paucity of secretory material. The basement mem- less pronounced or was absent in the epithelial cells branes and the epithelial-stromal junctions (ESJ)2' lining the ducts. Thus, even though the epithelial cells were intact. appear identical in the ducts and ductules of the rest- ing gland, under hormonal stimulation, the cells lin- Summary of the Effects of Ethynerone ing the ductules within lobules are predominantly en- gaged in production ofsecretory material, whereas the Ethynerone (2.0 mg/kg/day) produced pronounced cells lining the extralobular segment of the duct sys- physiologic lobular hyperplasia, lactational changes, tem show little or no secretory activity. The cells with and abundant cells with coarse, eosinophilic intracy- coarse, eosinophilic granules contained abundant 224 TAVASSOLI ET AL AJP * May 1988

Figure 16-Control. An intermediate cell with relatively electron-lucent cytoplasm, paucity of cytoplasmic organelles, but prominent perinuclear cytoplasmic microfi- brils. The cytoplasmic microfibrils were gen- erally absent in epithelial cells and when present were interpreted as implying an al- teration from or to intermediate cells. Note the elongated cytoplasmic processes and the deeply indented nucleus of the myoepi- thelial cell in contrast to the ovoid nucleus and the rounded configuration of the inter- mediate cell. (Uranyl acetate and lead ci- trate, X4910)

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electron-dense secretory material of relatively small ual myoepithelial cells, compared with the lesser de- and uniform size (Figure 20) with rare coalescence of grees ofatypical proliferation. The increased gap size, the secretory droplets, unlike the larger electron-dense however, seemed to reflect displacement of the myo- secretory droplets or the lipid droplets, which were less epithelial cells to other parts of the duct where they electron-dense and frequently coalesced in the lactat- were crowded together. The ESJ did not differ signifi- ing cells. Neither the lactating cells nor the cells with cantly in the severe proliferative atypias, whether ne- coarse, eosinophilic granules had apical cytoplasmic crosis was present or absent. In the lesion with a small projections, but microvilli were present in the luminal area ofstromal invasion (Figure 18), the invasive area side ofthe cytoplasm ofboth ofthese cells. There was was not present in the sections for electron micros- variation in the appearance of the secretory droplets copy, but was seen in four of the six levels examined in all cell types, however, with the eosinophilic, from the block processed for light microscopy. coarsely granular cells having the most uniform secre- RER was in tory granules. Abundant present all epi- Summary of Effects of Ethynerone and thelial cell variants. Mestranol Combination Proliferating atypical cells had abundant RER, al- most to the exclusion ofother cytoplasmic organelles, The degree of physiologic lobular hyperplasia and and ajagged outline, with numerous cytoplasmic pro- lactational secretory changes increased with increas- trusions creating an appearance ofintercellular fenes- ing doses in the three subgroups receiving 0.084, 0.42, trations. The cells in the severest proliferative atypias and 2.10 mg/kg/day of the compound, respectively. were not different qualitatively from those of the less The proliferative atypias were prominent but not sig- severe atypical proliferations, whether in the same an- nificantly altered by increasing dosage. Proliferative imal or in other monkeys. atypia was present in 22 of the 52 (42%) monkeys in In the most marked proliferative atypia, the myoep- this group (7 of these were in the low-dose, 10 in the ithelial cell layer had larger gaps between the individ- mid-dose and 5 in the high-dose group), being ofmod- Vol. 131 * No. 2 STEROID-INDUCED MAMMARY LESIONS 225

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Figure 17-Mestranol (0.002 mg/kg/day). The atypical proliferating cells appear relatively monotonous, with paucity of intracytoplasmic organelles and prominent interdigitation of the cytoplasmic processes. A few electron-dense secretory droplets are present in an occasional epithelial cell. Curiously, most myoepithelial cells contained multiple electron-dense bodies (arrows). (Uranyl acetate and lead citrate, x491 0)

Figure 18-Ethynerone (2.0 mg/kg/day). Early stromal invasion was identified in the case with comedocarcinoma illustrated in Figure 13. (H&E, x400)

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Figure 19-Ethynerone (2.0 mg/kg/day). -a_- Classic intraductal carcinoma with central necrosis (comedocarcinoma). (H&E, X250) W:

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erate to severe degree in 15. Of the 6 severe atypical proliferations, 5 were identical to intraductal car- cinoma in the human, and 1 corresponded to lob-

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t 20 Figure 20-Ethynerone (2.0 mg/kg/ ay) Collections of small electron- Wef *F ;=-' Cladense granules correspond to the * | necrcoarse,n _l eosinophilic granules. These were not membrane-bound. The mito- chondria had a fingerprint type of ap- pearance. (Uranyl acetate and lead ci- trate, X4910) Vol. 1 31 * No. 2 STEROID-INDUCED MAMMARY LESIONS 227

Figure 21-Chloroethynyl norgestrel plus metranol (0.42 mg/kg/day). The physiologi- cally hyperplastic lobules had an increased number of acini that were dilated and filled with secretory material in the presence of lactational activity.

monkeys receiving either mestranol or ethynerone changes in this group were more like those observed alone. in the monkeys receiving pure ethynerone than those Ultrastructurally, both the proliferative and non- receiving pure mestranol. Mestranol clearly does not proliferative atypias had prominent profiles of RER neutralize the effects ofethynerone. and abundant free ribosomes. The myoepithelial cells and endothelial cells ofinteracinar capillaries had par- Summary of the Effects of Chloroethynyl Norgestrel ticularly prominent pinocytotic vesicles, probably re- and Mestranol Combinations lated to the pronounced secretory lactational changes The degree of lactational secretory change and of in the epithelial cells of this group. Overall, the physiologic lobular hyperplasia (Figure 21) increased

Figure 22-Chloroethynyl norgestrel plus E mestranol (0.42 mg/kg/day). A solid carci- noma characterized by proliferation of atypi- cal cells expanding and producing conflu- ence of several ductules. (H&E, x1 60) This case may reflect an invasive carcinoma. 228 TAVASSOLI ET AL AJP * May 1988

Figure 23-Chloroethynyl norgestrel plus mestranol (0.42 mg/kg/day). Higher magni- fication of Figure 3 illustrating the prominent - nucleoli and mitotic figures in this solid carci- noma. (H&E, x250)

with increasing doses in subgroups receiving 0.084, keys in the low-dose (0.084 mg/kg/day), 11 of 17 0.42, and 2.1 mg/kg/day of the compound, respec- monkeys in the mid-dose (0.42 mg/kg/day), and 7 of tively. Proliferative atypia was present in 7 of 17 mon- the 18 monkeys in the high-dose (2.1 mg/kg/day)

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Figure 25-Chloroethynyl norgestrel plus mestranol (2.1 mg/kg/day). Lactational secretory cells contained large spaces filled with homogeneous lipoid secretory material and granular material of variable electron density in addition to the smaller electron-dense secretory droplets. The lumens are also filled with secretory material. Note a myoepithelial cell (arrow) lacking secretory activity. (Uranyl acetate and lead citrate, x4990) group. Nine monkeys in the high-dose group had died branes of adjacent cells. In some of the larger ducts, prior to the termination of the experiment, however. layers ofdisorderly stratified atypical proliferative epi- At least 6 cases ofthe severe atypical proliferations in thelium were present. The atypical epitheliuin formed the different subgroups would qualify as intraductal micropapillae and tufts (Figure 26). In adjoining ducts carcinoma if found in human breast, and a seventh and ductules, a cribriform pattern was present, com- probably is invasive because of its expansile growth posed of a relatively uniform cell population. Secre- and replacement of stroma (Figures 22 and 23). A tory material was sparse, when present, in the hyper- perilobular stromal proliferation and edema was evi- plastic cells. As in other epithelial cells of this group, dent in one case, resembling early fibroadenomatous the proliferating cells also contained abundant intra- hyperplasia similar to the lesion previously described cytoplasmic fibrils, but no dense bodies or pinocytotic in a monkey on ethynerone and mestranol combina- vesicles. Myoepithelial cells were present even in tion (2.1 mg/kg/day). ducts with intraductal carcinoma but appeared to Ultrastructurally, the areas unaffected by lacta- have an increased gap size in some areas. The ESJ was tional and hyperplastic changes were similar to the not altered, and the delimiting fibroblastic layer was normal breast of control animals. In the lactating intact. The endothelial cells lining the capillaries and ductules, some cells contained a uniform electron- other vessels had abundant electron-lucent cytoplasm dense secretory material (Figure 24) while others con- and unusually prominent pinocytotic vesicles. tained lipoid material as well (Figure 25). Many ofthe cells contained fine fibrils epithelial intracytoplasmic of the Effects of often in a perinuclear position, a feature not observed Summary Anagestone Acetate and in cells of previous groups. Microvilli were present in Mestranol Combinations the luminal side of the cells but were often blunted Seven monkeys in each ofthe two subgroups (0.44 when intraluminal secretion was present. Numerous and 2.20 mg/kg/day) developed prominent physio- well-developed desmosomes connected the cell mem- logic lobular hyperplasia and lactational changes; 230 TAVASSOLI ET AL AJP * May 1988

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Figure 26-Chloroethynyl norgestrel plus mestranol (0.42 mg/kg/day). The atypical proliferating cells show uniform appearance with irregularly indented nuclei, paucity of cytoplasmic organelles, and cytoplasmic processes interdigitating with those of adja- cent cells. Note the crowding of the myoepi- thelial cells in this portion of the duct. (Uranyl acetate and lead citrate, x1930)

these changes were focal and limited in the remaining At the ultrastructural level, the nonproliferative, se- monkeys. Proliferative atypia was present in 13 ofthe cretory, atypical proliferative and myepithelial cells 18 monkeys in the low-dose (0.44 mg/kg/day) sub- were essentially similar to the corresponding cells in group and in 7 of the 22 monkeys in the high-dose previous groups receiving hormones. In 1 case, how- (2.20 mg/kg/day) subgroup. Ten monkeys in the low- ever, the atypical cells had intracytoplasmic fibrils and dose subgroup and 3 in the high-dose subgroup had resembled the intermediate cells previously described moderate to severe proliferative atypias; the five se- in the control group (Figure 27). Although the myoep- vere atypias were comparable to cribriform intraduc- ithelial cell layer showed a focal increase in intercellu- tal carcinoma in human breast. There was high mor- lar gap size (Figure 28), the basement membrane and tality in both of these subgroups, 10 monkeys from the epithelial stromal junctions were not altered in the low-dose subgroup and 16 from the high-dose sub- any ofthe hyperplastic ducts. group dying prior to termination ofthe experiment. The extent and the degree ofthe proliferative atypia in both subgroups reflect the strong stimulatory effect Statistical Analysis of anagestone acetate on the proliferative activity of The total number of atypical proliferations ob- mammary ductal epithelium. Because 3 of the mon- served in each of the five major groups receiving hor- keys in the low-dose and 2 in the high-dose group de- mones was compared with the number of atypical veloped markedly advanced atypical proliferations proliferations in the control group with a two-sample identical to forms of human mammary intraductal normal test of proportions. Four of the five experi- carcinoma, this compound may be carcinogenic to mental drug groups had significantly greater number primates. oflesions than the control group (P < 0.025); the most Vol. 131 * No. 2 STEROID-INDUCED MAMMARY LESIONS 231

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Figure 27-Anagestone acetate and mes- tranol (0.44 mg/kg/day). In 1 case, perinu- clear cytoplasmic fibrils were prominent in epithelial cells that lacked significant secre- tory changes. (Uranyl acetate and lead ci- trate, x9100)

significant changes were in the group on ethynerone pensity to develop a mutation leading to proliferative (P < 0.0012). The fifth group of monkeys receiving atypia and neoplasia. It is equally likely, however, that mestranol alone did not exhibit a significantly greater some cells respond differently to prolonged hormonal number oflesions than the control group (P > 0. 125). stimulation by proceeding along a pathway toward Furthermore, a higher level of response (moderate to proliferative activity and atypia, rather than develop- severe atypia) was exhibited by the experimental ing physiologic hyperplasia with secretory products. groups, compared with the minimal to mild hyperpla- In intraductal proliferative activity, cell growth is per- sia observed in the control group (Table 3). pendicular to the lumen, unlike the lateral growth ob- served in the physiologic expansion of lobules. The Discussion paucity of intracytoplasmic secretory material in many cells within the proliferative atypia, situated Considering all monkeys that received steroid hor- against a background oflobules showing a physiologic mones, proliferative atypia was evident in 45% ofthe hyperplasia with various and often pronounced de- treated group, in contrast to only 12% of control ani- grees of secretory change, supports the concept of a mals. Also, the atypia of control animals was more differential response of cells to hormonal stimuli. Al- limited in extent and of a much lower grade than that though the secretory changes and physiologic lobular of monkeys receiving hormones. Whether or not the hyperplasia progressed with increasing dosage of the secretory lobular hyperplasia and the proliferative hormone, the proliferative atypia was accentuated atypia are related is not clear. Cells stimulated to un- with increasing dosages only up to the mid-range dergo a functional alteration may have a greater pro- dose levels in some groups, but a further increase 232 TAVASSOLI ET AL AJP * May 1988

Figure 28-Anagestone acetate and mestranol (0.44 mg/kg/day). The hyperplastic atypias sometimes formed a relatively solid growth. Note the variation in the nuclear indentations and nucleolar prominence in some cells. Only a single myoepithelial cell is apparent (arrow) because of the increase in the size of the intermyoepithelial cell gap. Compare with the crowded myoepithelial cells in Figure 26. Although these two figures are from different cases, in general, increase in size of the intermyoepithelial cell gap in part of a duct was associated with crowding of these cells in an adjacent area of the duct. The basement membrane persists. (Uranyl acetate and lead citrate, x1930) in the dose was not matched by a further increase in At the ultrastructural level, minor variations ex- atypia. isted between the proliferating cells in different groups The proliferative atypia in different groups (Table and to an even lesser extent within groups. This varia- 3) was morphologically similar at the light-micro- tion was generally noted in the quantity and variety scopic level and with rare exception was characterized ofintracytoplasmic organelles and to a lesser degree in by a proliferation of monomorphic cells, rather than the nuclear shape and chromatin distribution. There a pleomorphic cell population. At its most severe was, however, a rather striking uniformity of the cells form, the proliferative atypia was qualitatively identi- in any given case and area of proliferation. Unifor- cal to the cribriform and micropapillary variants of mity, in the form of a population of monomorphic intraductal carcinoma of human mammary glands,22 cells, rather than pleomorphism or anaplasia, was the being present in a total of 18 monkeys in various hallmark of the proliferating cells. Only in the com- groups. A comedocarcinoma with microinvasion edocarcinoma with invasion was there an admixture (Figure 18) developed in one animal receiving ethyn- of nonsecretory cells and secretory cells with abun- erone (2.0 mg/kg/day). Another case of solid intra- dant eosinophilic cytoplasmic granules in the areas of ductal carcinoma in a monkey receiving chloroethy- proliferation. Viral particles, previously observed in nyl norgestrel plus mestranol may also reflect an inva- mammary carcinomas of rhesus monkeys,23 were not sive carcinoma based on the extent ofstromal replace- found in any of the cases studied ultrastructurally. ment. One example oflobular neoplasia was observed There was a subtle increase in the size ofthe intermyo- in the ethynerone plus mestranol group. epithelial cell gaps in the more advanced proliferative Vol. 131 * No.2 STEROID-INDUCED MAMMARY LESIONS 233 atypias, but the basement membrane was intact, as monkey may assume even greater significance as an was the ESJ. This was most noticeable in the one ex- experimental model. ample of comedocarcinoma in which there was also Future studies should rely on mature monkeys-a some irregularity in the basement membrane where it factor not uniformly observed in the present study- was thinned, as well as some ofthe more severe atypi- although by the end ofthe 10-year period ofthe study, cal proliferations. The ESJ was undoubtedly pene- all monkeys were in a menstrual status. Also, because trated in the area of invasion; but because of its small the major differences between the proliferative lesions size, this alteration was not identified in the sample were observed between the low-dose (2 times the hu- obtained for ultrastructural evaluation. man dose) and the mid-dose (10 times the human The basic process of inducing or promoting prolif- dose), it would be valuable to evaluate the effect of a erative atypia took place whether the compound was stepwise increase between these levels. More extensive purely estrogenic, progestational, or a combination; sampling of the mammary glands is also recom- but variation in the degree and extent ofproliferation mended for obtaining a representative sample. Fur- was evident. This finding is not without precedent, as thermore, a larger number of monkeys are needed in complete growth and development of the mammary the experimental groups to allow in-depth statistical ducts and lobules in prepubertal and oophorecto- analysis ofthe biologic events. mized animals, as well as marked ductal hyperplasias, Emphasis should not be placed on the development carcinomas, metaplasias, physiologic lobular hyper- or lack of development of transient palpable nodules plasia, and cystic changes, have been found in the in the experimental monkeys. Many ofthese nodules mammary glands of rhesus monkeys on continuous reflect physiologic lobular hyperplasia with secretory treatment with estrogens.3'8"10' 115,17 Progesterone, in and lactational change. Nodules tended to regress as large doses alone, can also cause development of a result of hormone accommodation. Reflecting a mammary glands in rhesus monkeys.'6 In this study, transient phenomenon, the nodules do not generally the proliferative atypia was more extensive and of imply a proliferative atypia or early carcinoma, be- higher degree in groups receiving synthetic progestins cause the latter are usually too small to be palpable. alone or in combination with mestranol. Interest- Intraductal hyperplasias and intraductal carcinomas ingly, whether the combinations have synergistic in the human breast, like the proliferative atypia ob- effect is not apparent, but they do not seem to nullify served in the treated monkeys, are generally not pal- each other. Secretory changes were also present in all pable or nodular. Thus, the absence of palpable nod- treatment groups, although they were most pro- ules is not an indication of lack of disease, as implied nounced in groups receiving ethynerone alone or in in previous reports.5'0 combination with mestranol. Individual monkeys have different thresholds of re- Although spontaneous tumors, benign or malig- sponse. Only certain monkeys within each group de- nant, have been reported rarely in the captive rhesus veloped proliferative atypias. In the human, breast monkey,20,24-27 only a relatively small number ofnon- cancer is relatively common, but its ultimate manifes- human primates have been observed through the du- tation is thought to be under the influence of multiple ration of their normal life span.25 Therefore, the pau- factors. Determination of the significance of a single city ofreported mammary carcinomas may not be an factor in the induction and promotion ofbreast carci- accurate reflection ofits incidence among nonhuman noma in the human is, therefore, a most difficult task. primates. If spontaneous mammary carcinomas are The fact that certain synthetic steroids not available indeed rare in the rhesus monkey, then tumors that for use in the human population induce carcinomas develop in association with administered drugs proba- and proliferative atypias similar to intraductal carci- bly are caused by the drug under investigation. In this nomas of the human mammary gland is sufficiently study, the synthetic steroids induced or promoted pro- worrisome that a long-term study of commercially liferative atypia and even mammary carcinoma; and available steroids in rhesus monkeys is recom- because the proliferative atypia produced in the mon- mended. Mestranol, which did not induce any severe keys is identical to severe atypias and early carcino- proliferative atypia, is the only steroid among those mas found in the human breast, the rhesus monkey evaluated that is currently used in some oral contra- should prove an excellent model for use in studying ceptive hormones. the initiation of proliferative atypia and mammary carcinoma. The significance ofatypical proliferations in the human breast has been appreciated relatively References recently28; and as our understanding of these lesions 1. LiVolsi VA, Stadel BV, Kelsey JL, Holford TR, White in the human improves, the findings in the rhesus C: Fibrocystic breast disease in oral contraceptive users: 234 TAVASSOLI ET AL AJP * May 1988 A histologic evaluation of epithelial atypia. N Engl J sia in animals: Pathologic aspects and the effects ofcon- Med 1978, 299:381-385 traceptive steroids. Recent Results Cancer Res 1979, 2. Pastides H, Kelsey JL, LiVolsi VA, Holford TR, Fischer 66:129-160 DB, Goldenberg IS: Oral contraceptive use and fibro- 15. Valerio MG: Mammary gland, Tumors: Pathology of cystic disease with special reference to histopathology. Laboratory Animals. Vol 2. Edited by K Benirschke, JNCI 1983, 71:5-9 FM Garner, TC Jones. New York, Springer-Verlag, 3. Fechner RE: The surgical pathology ofthe reproductive 1978, pp 1194-1213 system and breast during oral contraceptive therapy. 16. Buss DH: Mammary glands and lactation, Compara- Pathol Annu 1971, 6:299-319 tive Reproduction of Nonhuman Primates. Edited by 4. Olsson H, Olsson ML, Moller TR, Ranstam J, Holm ESE Hafez. Springfield, Charles C Thomas, 1971, pp P: Oral contraceptive use and breast cancer in young 315-333 women in Sweden. Lancet 1985, 1:748-749 17. Drill V, Martin DP, Hart ER, McConnell RG: Effect of 5. Sattin RW, Rubin GL, Wingo PA, Webster LA, Ory oral contraceptives on the mammary glands of Rhesus HW: Oral contraceptive use and the risk of breast can- monkeys: A preliminary report. JNCI 1974, 52:1655- cer: The cancer and steroid hormone study of the Cen- 1657 ters for Disease Control and the National Institute of 18. Macpherson EE, Montagna W: The mammary glands Child Health and Human Development. N Engl J Med ofRhesus monkeys. J Invest Dermatol 1974, 73:17-18 1986, 315:405-411 19. Schultz AH: Fetal growth and development of the rhe- 6. Trapido EJ, Brinton LA, Schairer C, Hoover R: Estro- sus monkey. Contributions to Embryology. Carnegie gen replacement therapy and benign breast disease. Institute ofWashington, Baltimore, 1948, 155:73-97 JNCI 1984, 73:1101-1105 20. Speert H: The normal and experimental development 7. Casey HW, Giles RC, Kwapien RP: Mammary neopla- of mammary glands in the rhesus monkey. Contribu- sia in animals: Pathologic aspects and the effects ofcon- tions to Embryology. Carnegie Institute ofWashington, traceptive steroids. Recent Results Cancer Res 1979, Baltimore, 1948, 208:9-65 66: 129-160 21. Ozello L: Ultrastructure of the human mammary 8. Folley SJ, Guthkelch AN, Zuckerman S: The mam- gland. Pathol Annu 1971, 1-59 mary glands of the Rhesus monkey under normal and 22. McDivitt RW, Stewart FW, Berg JW: Tumors of the experimental conditions. Proc R Soc Lond [B] 1938, breast, Atlas ofTumor Pathology, Second Series, Fasci- 126:469-491 cle Armed Forces Institute of Pa- 9. Geil RG, Lamar JK: FDA studies ofestrogen, progesto- 2, Washington, DC, gens, and estrogen/ combinations in the thology, 1968 dog and monkey. J Toxicol Environ Health 1977, 3: 23. Chopra HC, Mason MM: A new virus in a spontaneous 179-193 mammary tumor of a rhesus monkey. Cancer Res 10. Geschickter CF, Hartman CG: Mammary response to 1970, 30:2081-2086 prolonged estrogenic stimulation in the monkey. Can- 24. Brack VM: Carcinoma solidum simplex marnmae bei cer 1959, 12:767-781 einem Orang-Utan (Dygmaeus). Zentralbl Allg Pathol 11. Kirschstein RL, Rabson AS, Rusten GW: Infiltrating 1966, 109:474-480 duct carcinoma of the mammary gland of a rhesus 25. O'Conor GT: Cancer-a general review, Primates in monkey after administration of an oral contraceptive: Medicine. Vol 3. Basel and New York, Karger, 1969, A preliminary report. JNCI 1972, 48:551-556 pp 9-22 12. Pfeiffer CA, Allen EA: Attempts to produce cancer in 26. Ruch TC: Diseases of Laboratory Primates. Philadel- rhesus monkeys with carcinogenic hydrocarbons and phia, W. B. Saunders, 1959 estrogens. Cancer Res 1948, 8:97-127 27. Mason MM, Baker JR, Illievski VR: Histopathology of 13. Smoilovskaia EIa, Vadova AV, Padvalnaia MIa, a spontaneous tumor in a macaca mulatta in which Chachibaia IA: Carcinoma of mammary glands arising RNA virus particles were found (Abstr). Proc Am Assn in a monkey after hyperoestrinization and administra- Cancer Res 1970, 11:53 tion ofradioactive silver (110 Ag). Prob Oncol 1960, 6: 28. Page DL, Dupont WD, Rogers LW, Radoz MS: Atypi- 35-42 cal hyperplastic lesions of the female breast. Cancer 14. Casey HW, Giles RC, Kwapien RP: Mammary neopla- 1985, 55:2698-2708