Hyperplasia to Mammary Carcinoma in 7,1 2-Dimethylbenz(A)Anthracene-Treated LEW/Mai Rats

The Relationship of Terminal Duct Hyperplasia to Mammary Carcinoma in 7,1 2-Dimethylbenz(a)anthracene-Treated LEW/Mai Rats

Dallas M. Purnell, PhD

The evolution of dysplasias and carcinomas in the inguinal mammary glands of LEW/ Mai rats given 7,12-dimethylbenz(a)anthracene (DMBA) by gastric gavage was studied with the use of stained whole mounts. Two major dysplasias, hyperplastic terminal end buds (HEBs) and hyperplastic alveolar nodules (HANs), developed prior to mammary carcinomas. HEBs were present in the mammary glands of -70% of the rats within 1 week following DMBA. The percentage of rats with these lesions and the incidence of HEBs in the mammary gland decreased prior to the appearance of palpable and microscopic tumors. During the time when tumors first became evident (40-80 days), the percentage of rats with HEBs (11%) paraDeled the percentage of rats with mammary tumors (12%). The initial percentage of rats with HEBs (-70%) paralleled the final tumor incidence (71%) observed in DMBA-treated rats that were allowed to live until they developed tumors. The histologic features of HEBs resembled those of the carcinomas, and HEBs were present in the immediate vicinity of some of the microscopic and palpable tumors. With only one exception, the location of microscopic tumors in the mammary gland was consistent with their derivation from small terminal ducts. These data are compatible with a developmental relationship between HEBs and mammary carcinoma. HANs, on the other hand, developed relatively late (ie, 30 days) following DMBA administration and became more numerous with the passage of time. Over the period of time when mammary carcinomas first became evident, the percentage of rats with HANs (73%) was inconsistent with a developmental relationship between HANs and mammary carcinoma. This conclusion was supported by the absence of HANs in the vicinity of microscopic tumors, by the dissimilarity between the histologic features of HANs and mammary carcinomas, and by their absence from the mammary gland during the time when at least some of the mammary tumors must have arisen. The results implicate terminal duct hyperplasia in the histopathogenesis of rat mammary carcinomas. (Am J Pathol 98:311-324, 1980)

CONSIDERABLE CONTROVERSY surrounds the histopathogenesis of human breast carcinoma. In the opinion of many investigators, human breast carcinoma has as its site(s) of origin the terminal ducts, ducto-aci- nar junctions, acini (ductules), or some combination of these sites.'-4 How- ever, other researchers have noted the frequent association of ductal epithelial hyperplasia with breast carcinoma and have suggested that hyperplastic ductal lesions may evolve into mammary carcinoma. With the exception of virus-induced murine mammary tumors, which From the Department of Pathology, School of Medicine, University of Maryland, Baltimore, Maryland. Supported by Research Grant CA-23659 from the National Cancer Institute. Accepted for publication August 21, 1979. Address reprint requests to Dallas M. Purnell, PhD, Department of Pathology, University of Maryland School of Medicine, 660 W. Redwood Street, Baltimore, MD 21201. 0002-9440/80/0211-0311$01.00 311 ©) American Association of Pathologists 312 PURNELL American Journal of Pathology

arise from hyperplastic alveolar nodules (HANs),'0 a similar controversy exists concerning the histopathogenesis of mammary carcinoma in animals. Beuving 11,12 and others 13,14 have provided evidence that HANs induced by 7,12-dimethylbenz(a)anthracene (DMBA) in the Lewis rat are preneoplastic lesions, analogous to those that develop in the mouse mammary gland. Other investigators 15-17 have also associated HANs with mammary tumorigenesis in rats. On the other hand, studies by Russo et al 18 and by Sinha and Dao 19 in DMBA-treated Sprague-Dawley rats and Haslam and Bern 20 in Lewis rats have implicated terminal end bud hyperplasia (IDP), "duct" and terminal ductule hyperplasia (TDH), respec- tively, rather than HANs as early lesions important in rat mammary car- cinogenesis. Similarly, Middleton,21 in a still earlier study, provided evidence that most rat mammary carcinomas arise directly in ducts, ductules, and end buds without any intervening preneoplastic lesion, such as the HAN. In this paper, we describe our studies that support the origin of most mammary carcinomas in LEW/Mai rats from terminal duct hyperplasias (HEBs) that develop soon after DMBA administration.

Materials and Methods Animals Female LEW/Mai rats were obtained from Microbiological Associates, Inc, Bethesda, Maryland, at an age of 45 days. Rats were housed 5 per cage in an air-conditioned animal facility with a light: dark cycle of 12 hours: 12 hours. When rats were 52 days old, they were anesthetized with ether and given a single dose of DMBA (20 mg DMBA) (Sigma Chemical Co., St. Louis, Mo) dissolved in 1 ml sesame oil by gastric gavage. Rats were maintained as virgins throughout the experiments. Tissue Collection Rats were killed at 7-day intervals over the first 21 days after DMBA and then at 10-day intervals beginning 30 days after DMBA and continuing up to 80 days after DMBA (9 groups in all). Fifteen to 31 rats were killed at each interval (mean number of rats per group, 24; total number, 221). Both inguinal mammary glands were removed from the rats and fixed overnight in Bouin's fixative. The left inguinal mammary glands (one from each rat killed, total 221) were prepared as hematoxylin-stained whole mounts 22 to evaluate the incidence of dysplasias in the mammary gland at different times after exposure to DMBA. Mammary glands contralateral to those prepared as whole mounts were processed to hematoxylin-stained microtome sections for another study. In order to determine a final total tumor incidence, 14 rats were given DMBA and allowed to live until they developed mammary tumors. These rats were killed as tumors appeared, and all tumors were removed for study. Mammary Dysplasias The incidence of dysplasias in the mammary glands of rats killed at different times after DMBA treatment was determined according to the total number of lesions revealed at low Vol. 98, No. 2 TERMINAL DUCT HYPERPLASIA 313 February 1980 magnification per inguinal mammary gland whole mount. Data obtained from individual rats killed at comparable times after DMBA were aggregated and expressed as the mean number of dysplasias per positive inguinal mammary gland (±SE). The percentage of rats with one or more dysplasias per inguinal mammary gland at each time period after DMBA was also determined. To relate the histologic appearance of mammary dysplasias to their subgross appearance, selected lesions were photographed, dissected from the whole mounts, and embedded in paraffin. Five-micron parasagittal sections were cut from the lesions, stained with hematoxylin-eosin (H & E), and examined microscopically. Spatial Distribution of Mammary Dysplasias The incidence of dysplasias in different regions of the inguinal mammary gland was determined by means of the following technique: A line was drawn on the cover glass of the whole-mount preparation through the nipple area, perpendicular to the major axis of the gland, and a second line was drawn parallel to the nipple area through the estimated mid- point of the mammary gland. This procedure divided the inguinal mammary gland into four regions (cephalad, distal; cephalad, proximal; caudal, distal; and caudal, proximal). Distal and proximal refer to position in the mammary gland relative to the nipple. The total area of mammary gland in the cephalad and caudal regions was approximately equal, as judged by the number. of superimposed micrometer fields required to cover them. The presence of a small tag of the caudal mammary gland, removed along with the inguinal mammary gland, allowed accurate orientation of the mammary gland whole mounts in the cephalad-caudal axis. Dysplasias were counted in each of the regions with the aid of a low-magnification stereoscopic microscope equipped with a mechanical stage and eye- piece grid-micrometer. Counts of dysplasias in corresponding regions of the mammary gland from different rats were aggregated, and a mean incidence of dysplasias for each region was calculated. A total of 40 whole mounts were subjected to this procedure. Mammary Tumors The temporal appearance of mammary tumors in rats after DMBA treatment was determined by palpation and by examination of the whole mount preparations. In most cases tumors were 20 sq mm at the time of their discovery. Tumors in the inguinal mammary gland and those that developed in other mammary glands were removed and portions fixed in Bouin's fixative and formaldehyde (4%) and glutaraldehyde (1%) fixative, dehy- drated, and embedded in paraffin, and 5-I, sections were cut and stained with hematoxylin-eosin for microscopic study. Statistical Analysis Differences in mean dysplasia incidence between different regions of the inguinal mammary gland were tested for significance by one-way analysis of variance (ANOVA). The Newman-Keuls (NK) test 23 was used to identify which subgroups differed significantly from each other. Results Subgross and Microscopic Appearance of Mammary Dysplasias Two major dysplastic lesions were observed in the mammary glands of DMBA-treated LEW/Mai rats. These were hyperplastic end buds (HEBs) and hyperplastic alveolar nodules (HANs). Alveolar cysts were an occa- sional finding. HEBs were globular, dark-staining structures situated at the ends of terminal ducts or at the ends of lateral branches of terminal ducts (Figure 1). 314 PURNELL American Journal of Pathology

HEBs occurred either as isolated lesions or as clusters of several lesions. Microtome sections of HEBs showed them to be small ducts, lined by hyperplastic epithelium containing four to five layers of cells (Figure 2). In some cases the hyperplastic epithelium was infolded, giving rise to a papillary appearance. The epithelial cells contained pale-staining, round to oval nuclei with scant chromatin and one or two dark-staining nucleoli. Mitoses were numerous and could be found in all of the cell layers com- posing the hyperplastic epithelium. Secretory activity was absent, and no inflammatory infiltrate was evident around these lesions. HANs were more complex than HEBs and occurred in a variety of forms. The simplest of these had the form of grape-like clusters of expanded, dark-staining ductules located at the ends of terminal branches of ducts. Large cystic dilations of some of the ductules in these structures were seen occasionally. Such regions were more translucent than the remainder of the HANs and contained a pink-staining material. The most frequently encountered HANs were larger, irregular, dark-staining structures that appeared to be made up of clusters of tightly spaced, dilated ductules (Figure 3). In some of these, the ductules were greatly expanded, forming translucent globular or antler-like cystic spaces that contained a pink-staining material. Less commonly, hyperplastic change involving most of the lobulo-alveolar epithelium in a localized region of the mammary gland was observed. Sections of HANs revealed a tissue composed of clusters of ductules with or without associated cystic areas (Figure 4). In the noncystic regions, ductules were lined with 1 and occasionally 2 layers of epithelial cells that displayed a moderate variation in nuclear size, shape, and staining intensity. Most nuclei had 1-2 nucleoli. Many of the cells lining the ductules were vacuolated, with evidence of secretory activity. The lumens of some ductules contained a pink-staining material. Mitoses were infrequent in HANs. Cells with the appearance of myoepithelial cells were evident at the periphery of many of the ductules. HANs were surrounded by scant connective tissue, and no inflammatory cell infiltrate was evident. Cystic areas of HAN were characterized by the presence of greatly dilated ductules lined in most cases by a single layer of cells containing round to oval, dark or pale-staining nuclei. One or two nucleoli were present in some of the nuclei. Focal areas of cystic epithelium having 2-3 cell layers were occasionally seen. Evidence of secretory activity was present in many regions of the cyst epithelium, and many cysts contained an eosinophilic material. Mitoses were uncommon in the cyst epithelium. No inflammatory cell infiltrate was present in the vicinity of the cysts. In contrast to HEBs and HANs, sections of normal-appearing lobulo-al- Vol. 98, No. 2 TERMINAL DUCT HYPERPLASIA 315 February 1980 veoli from DMBA-treated rats revealed ductules lined with 1-2 layers of cells, containing round to oval nuclei with granular, moderately dark- staining chromatin. Most of these nuclei lacked nucleoli. No evidence of secretory activity was present. Smaller cells with dark-staining, round nuclei were also occasionally seen among the larger epithelial cells of the ductules. Myoepithelial cells were present at the periphery of ductules. Extralobular ducts were lined by a single layer of cells with round or oval, moderately dark-staining nuclei, most of which lacked nucleoli.

Temporal Appearance of Mammary Dysplasias Table 1 shows the incidence of dysplasias in the inguinal mammary gland at different times after DMBA. Only HEBs were present in rats killed during the first 3 weeks after carcinogen treatment, and these lesions were most numerous during this period of time. The incidence of HEBs in rats killed 30 or more days after DMBA was lower. HANs were first observed in the mammary glands of rats killed 30 days after DMBA exposure. The incidence of HANs was constant from 30 to 50 days after DMBA exposure and then increased by about twofold. Table 2 shows the percentage and number of rats with HEBs, HANs, and mammary tumors at different times after exposure to DMBA. The percentage of rats with HEBs decreased prior to the appearance of mammary tumors at 40 days. In contrast, the percentage of rats with HANs increased prior to and during the time when tumors appeared. Mammary tumors (carcinomas) were present in 12% (15/123) of the rats killed 40 or more days after DMBA. Two of these were palpable 50 days after DMBA, and the remainder became palpable 60 or more days after the carcinogen. Of the total number of tumors, six were microscopic tu-

Table 1 -Incidence of Dysplasias in Inguinal Mammary Gland at Different Times After DMBA Administration Incidencet (Range) per Total dysplasias counted/total # positive mammary gland of positive mammary glands Days after DMBA* HEBs HANs HEBs HANs 7 10±5(1-55) 0 102/10 0 14 8±2(1-25) 0 120/15 0 21 11 ±3(2-34) 0 181/16 0 30 7 ± 2 (1-14) 3 ± 1 (1-8) 63/9 30/9 40 5 ± 2 (2-10) 3 ± 0.5(1-7) 24/5 38/12 50 5 ± 2 (2-7) 3 ± 0.4 (1-6) 14/3 40/14 60 - 7 ± 1 (1-19) 2/1 153/24 70 5 ± 5 (1-9) 7 ± 1 (1-25) 10/2 156/23 80 3 ± 1 (1-5) 7 ± 1 (1-19) 9/3 129/19 * 20 mg i.g. at 52 days of age. t Mean # ± SE. 316 PURNELL American Journal of Pathology

Table 2-Percentage (Number) Rats With Hyperplastic Terminal End Buds (HEBs), Hyperplastic Alveolar Nodules (HANs), and Mammary Tumors (MTs), Different Times After Exposure to DMBA % (No. rats with Frequency of Days after Palpable Microscopic DMBA* HEBs HANs MTs MTst MTst 7 67 (10/15) 0 (0/15) 0(0/15) 14 63 (15/24) 0 (0/24) 0(0/24) 21 55 (16/29) 0 (0/29) 0(0/29) 30 33 (9/27) 33 (9/27) 0(0/27) 40 21 (5/24) 50 (12/24) 4 (1/24) 0 1 50 14 (3/22) 64 (14/22) 9(2/22) 1 1 60 4 (1/25) 88 (22/25) 12 (3/25) 3 0 70 6 (2/31) 74 (23/31) 13(4/31) 3 1 80 14 (3/21) 90(19/21) 24(5/21) 2 3 40-80t 11 (14/123) 73 (90/123) 12 (15/123) 9 6 * 20 mg i.g. at 52 days of age. t Aggregated % (No.) rats with respective lesions betwen 40 and 80 days after DMBA. f All tumors were in the inguinal mammary gland. mors discovered on examination of the whole-mount preparations. The earliest of these was found in a rat killed 40 days after DMBA. With the exception of one tumor, which was located within a major duct near the nipple (Figure 5), the location of the microscopic tumors was consistent with their development within small terminal ducts. The percentage of rats killed between 40 and 80 days after DMBA with HEBs (11%, 14/123) paralleled the percentage of rats with mammary tumors (12%, 15/123). In contrast, the percentage of rats with HANs (73%, 90/123) showed no relation to the percentage of rats with tumors. These data suggest a developmental relationship between HEBs and mammary tumors. This conclusion was strengthened by the observation of several HEBs in the immediate vicinity of some of the microscopic tumors (Fig- ures 6 and 7).

Mammary Tumors Rats that were killed 40 to 80 days after exposure to DMBA had a total tumor incidence of 12% (15/123) (Table 2). This percentage was similar to that of rats that had HEBs killed during this period of time. Rats that were given DMBA and allowed to live until they developed tumors had a final tumor incidence of 71% (10/14), which was roughly equivalent to the percentage of rats with HEBs at 7 days (67%, 10/15) (Table 2). Sixty-four percent (16/25) of the rats developed unilateral inguinal tumors, 16% (4/25) developed unilateral axillary tumors, 12% (3/25) developed bilateral inguinal or axillary tumors, and 8% (2/24) developed both inguinal and ax- Vol. 98, No. 2 TERMINAL DUCT HYPERPLASIA 317 February 1980 illary tumors. No rats were observed with more than one tumor per mammary gland. Most of the mammary tumors were papillary or cribriform adenocarcinomas (Figure 8). The cells of such tumors were relatively uni- form in size and shape and appeared well differentiated. Mitoses were frequent, and evidence of secretory activity was absent in most cases. Inflam- matory cell infiltrate varied from moderate to nil. HEBs were occasionally observed in the immediate vicinity of large mammary tumors. In such cases, the cells of the HEBs were seen to resemble those of the carcinoma.

Spatial Distribution of Dysplasias and Microscopic Tumors in the Mammary Gland Table 3 shows the incidence of dysplasias and their total number in different regions of the inguinal mammary gland. The incidence of HEBs in the cephalad, distal portion of the mammary gland was significantly higher than the incidence of HEBs in other regions of the gland. Of the total HEBs encountered, 94% (108/115) were found in the cephalad half of the gland, and most (84%) of these in the cephalad, distal portion. The incidence of HANs was significantly higher in the cephalad, distal, and proximal portions of the mammary gland than in other regions of the gland. Eighty-six percent (176/204) of the total HANs counted were located in the cephalad half of the mammary gland, with the majority (56%) in the distal cephalad region.

Table 3-Incidence of Dysplasias in Different Regions of the Inguinal Mammary Gland of DMBA* Treated Rats Mean No. ± SE (range) dysplasias in Total No. (%) dysplasias region indicated in region indicated Region HEBst HANs: HEBs HANs I Cephalad, distal 8 ± 1 4 ± 0.4 97(84) 114(56) (0-22) (0-18) 11 Cephalad, proximal 1 ± 0.3 2 ± 0.3 11 (10) 62(30) (0-3) (0-11) IlIl Caudal, proximal 0.5 ± 0.4 1 ± 0.2 6 (5) 18 (9) (0-6) (0-4) IV Caudal, distal 0.1 +0.1 0.4 ±0.2 1 (1) 10 (5) (0-1) (0-3) Total 115 (100.0) 204 (100.0) * 20 mg i.g. at 52 days of age. t F = 18.08, P < 0.01 (ANOVA). Incidence of HEBs in IV, III, and 11 significantly different from 1, P < 0.05 (NK). Differences between 11, Ill, and IV not significant (P > 0.05). Results based on data from 13 rats killed 7-21 days after exposure to DMBA. t F = 13.02, P < 0.01 (ANOVA). Incidence of HANs in IV significantly different from 11 and 1, P < 0.05 (NK); IlIl compared with 11, P 0.05, and P < 0.05 (NK) compared with I: Incidence in 11 significantly different compared with 1, P < 0.05 (NK). Results based on data from 27 rats killed 30-80 days after DMBA exposure. 318 PURNELL American Journal of Pathology

With the exception of one tumor (Figure 5) that could not be classified, all of the microscopic tumors were situated in the cephalad half of the inguinal mammary gland. HEBs, but not HANs, were observed in the immediate vicinity of some of these tumors (Figures 6 and 7). Discussion The present study has examined the evolution of mammary dysplasias and carcinomas in virgin LEW/Mai rats exposed to DMBA. Our data show that two distinct types of dysplasias (HEBs and HANs) appear in the rat mammary gland prior to mammary carcinomas. HEBs appeared within 7 days following DMBA administration and were numerous over the first 3 weeks after the administration of carcinogen. However, the percentage of rats with HEBs and the incidence of HEBs in the inguinal mammary gland decreased prior to the emergence of palpable and microscopic tumors. This observation, together with the agreement between the percentage of rats with HEBs and those with mammary tumors during the time when mammary tumors were apparent (Table 2) and the agreement between the initial percentage of rats with HEBs and the final tumor incidence, was consistent with a developmental relationship between HEBs and mammary carcinoma. With one exception, the location of microscopic tumors in the mammary gland was also consistent with their development from small terminal ducts, and HEBs were present in the immediate vicinity of some of these tumors. HANs, on the other hand, appeared relatively late, ie, at 30 days, and then became numerous with increasing time after DMBA. Both the percentage of rats with HANs and the incidence of HANs in the inguinal mammary gland during the time when mammary tumors were present were inconsistent with a relationship between HANs and mammary carcinoma. This conclusion was strengthened by the absence of HANs from the mammary gland during the time when at least some of the mammary tumors in our study must have arisen and by the fact that most of the mammary tumors observed in this study were papillary or cribriform adenocarcinomas with high mitotic activity and little evidence of secretory activity, features that were common to HEBs but not to HANs. Finally, HANs were not seen in the immediate vicinity of microscopic tumors, an observation that also argued against a developmental association between HANs and mammary carcinoma. The role of dysplasia in mammary tumorigenesis in the rat is con- troversial. Beuving 11,12 presented evidence in Lewis rats that DMBA-induced HANs are premalignant lesions. His study showed that adenocarcinomas arose in -20% of HAN transplants studied, whereas none Vol. 98, No. 2 TERMINAL DUCT HYPERPLASIA 319 February 1980 developed in similarly transplanted normal mammary ducts obtained from carcinogen-treated rats. Rivera et al 13 reported a 10% incidence of carcinomas in transplanted Lewis HANs. They also showed that administration of DMBA to rats bearing transplanted HANs increased the incidence of carcinomas in these outgrowths from 10% to 50-100%, depend- ing on when the carcinogen was administered relative to establishment of the HAN outgrowth. In contrast, untreated outgrowths of normal mammary gland failed to develop tumors, and the incidence of tumors in DMBA-treated rats carrying transplants of normal mammary gland was very low, compared with those carrying HAN transplants. More recently, Maiorana and Gullino,14 using the rabbit iris assay, reported the angiogenic capacity of a serially transplanted Lewis rat HAN (line T18), which was described as having a high neoplastic potential. They obtained a positive angiogenic response in 13-21% of the T18 HAN transplants tested, which was significantly higher than the angiogenic response obtained with normal mammary epithelium but lower than the response obtained with tumor tissue. Since unquestionably preneoplastic mouse HANs also have an angiogenic capacity that is intermediate between normal and neoplastic mammary tissue,24 the above data could be taken to indicate that at least some of the HANs that develop in DMBA-treated Lewis rats are preneoplastic lesions analogous to those of mice. The preceding result should, however, be cautiously considered, because the same investigators failed to obtain a positive angiogenic response when primary rat HANs were studied in the same system. Haslam and Bern 26 have suggested that TDHs, which appear to be equivalent to what we have called HEBs and others 18 have called IDPs, are early DMBA-induced lesions that progress to mammary carcinoma in the Lewis rat. This conclusion was based on the time course in the incidence of TDH, the agreement between the number of TDHs and the number of mammary tumors induced by DMBA, and transplantation data that indicated that approximately 50% of TDHs gave rise to carcinomas. Transplanted HANs, on the other hand, gave rise to a low incidence (-5%) of mammary tumors, which was not significantly different from that obtained with randomly transplanted mammary duct tissue obtained from DMBA-treated rats. They concluded that DMBA-induced HANs in Lewis rats are not significant preneoplastic lesions. Similar conclusions concerning the relationship of ductule hyperplasia to the genesis of rat mammary carcinoma have been reached by other investigators, including Russo et al 18 and Sinha and Dao '9 in their studies with Sprague-Dawley rats and by Middleton 21 in a study that utilized several strains of rats. Although further definition of the relative neoplastic potential of differ- 320 PURNELL American Journal of Pathology ent carcinogen-induced mammary dysplasias in the LEW/Mai rat must await transplantation studies, the data reported here support the conclusions of other investigators 18-21 concerning the probable involvement of small duct hyperplasias in the histopathogenesis of DMBA-induced rat mammary carcinoma. References 1. Wellings SR, Jensen HM, Marcum RG: An atlas of subgross pathology of the human breast with special reference to possible precancerous lesions. J Natl Cancer Inst 55:231-273, 1975 2. Wellings SR, Jensen HM: On the origin and progression of ductal carcinoma in the human breast. J Natl Cancer Inst 50:1111-1118, 1973 3. Cheatle GL: Desquamative and dysgenetic epithelial hyperplasias in the breast: Their situation and characteristics: Their likeness to lesions induced by tar. Br J Surg 13:509-532, 1926 4. Muir R: The evolution of carcinoma of the mamma. J Pathol Bacteriol 52:155-172, 1941 5. Fisher ER: Ultrastructure of the human breast and its disorders. Am J Clin Pathol 66:291-375, 1976 6. Glaager HS, Martin JE: Early phases in the development of breast cancer. Cancer 24:1170-1178, 1969 7. Humphrey LJ, Swerdlow M: Relationship of benign breast disease to carcinoma of the breast. Surgery 52:841-846, 1962 8. Ryan JA, Coady CJ: Intraductal epithelial proliferation in the human breast: A comparative study. Can J Surg 5:12-19, 1962 9. Fisher ER, Shoemaker RH, Sabnis A: Relationship of hyperplasia to cancer in 3- methylcholanthrene-induced mammary tumorogenesis. Lab Invest 33:33-42, 1975 10. DeOme KB, Faulkin LJ Jr, Bern HA, Blair PB: Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 19:515-520, 1959 11. Beuving LJ: Mammary tumor formation within outgrowths of transplanted hyperplastic nodules from carcinogen-treated rats. J Natl Cancer Inst 40:1287-1291, 1968 12. Beuving LJ: Responsiveness of carcinogen-induced hyperplastic alveolar nodules in Lewis rats to mammary gland growth-regulatory mechanisms. J Natl Cancer Inst 43:1191-1196, 1969 13. Rivera EM, Walbridge M, Hill SD: Tumor development in transplants of rat mammary hyperplastic alveolar nodules. Proc Am Assoc Cancer Res 18:203, 1977 14. Maiorana A, Gullino PM: Acquisition of angiogenic capacity and neoplastic trans- formation in the rat mammary gland. Cancer Res 38:4409-4414, 1978 15. Tuba J, Rawlinson HE, Fraser MS, Jeske I: Hyperplastic nodules in rat mammary glands following feeding of 4-acetylaminobiphenyl. Can J Med Sci 31:95-98, 1953 16. Beuving LJ, Faulkin LJ Jr, DeOme KB, Bergs VV: Hyperplastic lesions in the mammary glands of Sprague-Dawley rats after 7,12-dimethylbenz(a)anthracene treatment. J Natl Cancer Inst 39:423-429, 1967 17. Beuving LJ, Bern HA, DeOme KG: Occurrence and transplantation of carcinogen- induced hyperplastic nodules in Fischer rats. J Natl Cancer Inst 39:431-447, 1967 18. Russo J, Saby J, Isenberg WM, Russo IH: Pathogenesis of mammary carcinomas induced in rats by 7,12-dimethylbenz(a)anthracene. J Natl Cancer Inst 59:435-445, 1977 19. Sinha D, Dao TL: Hyperplastic alveolar nodules of the rat mammary gland: Tu- mor-producing capability in vivo and in vitro: Cancer Lett 2:153-160, 1977 Vol. 98, No. 2 TERMINAL DUCT HYPERPLASIA 321 February 1980

20. Haslam SZ, Bern HA: Histopathogenesis of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors. Proc Natl Acad Sci USA 74:4020-4024, 1977 21. Middleton PJ: The histogenesis of mammary tumours induced in the rat by chemical carcinogens. Br J Cancer 19:830-839, 1965 22. Purnell DM, Stowers DJ: Mammary gland hyperplastic alveolar noduligenesis in the LEW/Mai rat in relation to mitotic activity, estrous cycle status, and age. J Natl Cancer Inst 61:1339-1346, 1978 23. Winer BI: Statistical Principles in Experimental Design. Second edition. New York, McGraw-Hill, 1971, pp 216-218 24. Gimbrone MA Jr, Gullino PM: Neovascularization induced by intraocular xeno- grafts of normal, preneoplastic, and neoplastic mouse mammary tissues. J Natl Can- cer Inst 56:305-318, 1976 Acknowledgment The author is grateful to Ms. Deborah J. Stowers, BA, for her excellent technical assistance. 322 PURNELL American Journal of Pathology

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Figure I-Whole mount of an inguinal mammary gland from a rat killed 21 days after intragastric DMBA. Several HEBs are present at upper right. (Hematoxylin, xl1 5) Figure 2--Microtome section from the area illustrated in Figure 1. Shown is a cross-section of one HEB. Note hyperplastic epithelium, narrow lumen, and mitoses. Epithelium shows no evidence of secretory activity. (H&E, x500) Figure 3-Whole mount of an inguinal main mary gland from a rat killed 80 days after intragastric DMBA, showing HAN. (Hematoxylin, x31 2) Figure 4--Microtome section of the HAN shown in Figure 3. The structure is composed of groups of dilated ductules lined by epithelium showing secretory activity (H&E, x_500) 5 6

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Figure 5-Whole mount of an inguinal mammary gland from a rat killed 80 days after intragastric DMBA. Shown is a microscopic tumor located within a major duct near the nipple. The tumor is extending along the bifurcation of the duct. (Hematoxylin, x25) Figure 6-Whole mount of an inguinal mammary gland from a rat killed 40 days after intragastric DMBA. Shown is a microscopic tumor (upper right) that appears to arise from terminal duct and several HEBs (lower left) near the tumor. (Hematoxylin, x 15) Figure 7-Whole mount of an inguinal mammary gland from a rat killed 80 days after intragastric DMBA. Present is a microscopic tumor (upper right) and several associated HEBs (lower left). (Hematoxylin, x 15) Figure 8-Microtome section of a mammary tumor found in an inguinal mammary gland of a rat killed 70 days after intragastric DMBA. The histologic appearance is that of papillary adenocarcinoma. (H&E, x 125)