JOURNAL
OF SURGICAL
RESEARCH
52,205208
(19%‘)
Ductal Carcinoma in Situ in Rat Mammary Gland KEITH A. CRIST, PH.D.,**’
BINA CHAUDHURI, M.D.,? SUNIL SHIVARAM, B.S.,* AND PRABIR K. CHAIJDHURI, M.D.*
Departments of *Surgery and TPathology, Medical College of Ohio, 3000 Arlington Avenue, P.O. Box 10008, Toledo, Ohio 43699-0008 Submitted
for publication
suggests the need for an animal model which mimics the human pathology. While tumor development induced by the direct-acting carcinogen N-nitroso-N-methylurea (NMU) has been well characterized and closely fits developmental patterns, hormone dependence, and metastatic involvement associated with human breast cancer [5,6], a model system for the study of intraductal carcinoma prior to the development of invasive disease has not previously been described. Histologic characterization of DCIS development is presented here along with selected observations on estrogen receptor status.
Antiestrogen therapy has been proposed as a treatment option for ductal carcinoma in situ (DCIS). However, its effectiveness has not been evaluated in the laboratory due to lack of an animal model. The present study was undertaken to evaluate the incidence, time span, and number of mammary glands involved with DCIS in a rat model treated with N-nitroso-N-methylurea (NMU). Sprague Dawley female rats 44 to 49 days old were treated with two iv doses of 5 mg NMU/ 100 g body wt given 7 days apart, initiated at diestrus. Animals were killed at intervals beginning 2 1 days following first injection. Breast tissues were evaluated following routine H&E stain. Standard histologic criteria were followed to establish the diagnosis of DCIS. The number of glands involved with DCIS increased from one to seven with time from first injection. This model demonstrates an incidence of 87% for DCIS and 0% for invasive Ca between 22 and 45 days following NMU injection. Nine rats were sacrificed between 50 to 60 days and five showed invasive carcinoma. This model appears suitable for studying the efficacy of hormone or chemoprevention in the progression of DCIS to invasive Ca. 0 1992 Academic Press, Inc.
MATERIALS
INTRODUCTION
While the mechanism governing progression through the histologic abnormalities of breast ductal epithelium to the stage of invasive carcinoma is unknown, the occurrence of ductal carcinoma in situ (DCIS) appears to serve as a marker for increased risk of ipsilateral invasive disease [ 11. Degree of associated risk for concurrent occult and future disease may depend on both size and subtype of the intraductal lesion [2]. At present, the optimal conservative therapy directed toward DCIS following detection of the initial tumor mass is controversial [3, 41. Availability of breast biopsy tissue for study of neoplastic development and treatment of DCIS is severely limited by the necessity of processing the entire specimen to rule out microscopic invasion. This clearly 1 To whom reprint
May 20, 1991
requests should be addressed.
AND
METHODS
Tumor induction. Sixty-five female 40-day-old Sprague-Dawley rats were singly housed in polycarbonate cages, maintained in a 12-hr light/dark environment, and fed a commercial pelleted diet ad libitum. Vaginal smears (0.9% NaCl lavage) were recorded beginning at 43 days of age as diestrus (leukocytic smear), proestrus (nucleated smear), or estrus (cornified smear). Between 44-49 days of age on a diestrus morning, rats were injected with 5 mg NMU/lOO g body wt (15 mg/ml, Sigma, St. Louis, MO; dissolved in 3% acetic aciddistilled water) by jugular vein. A second similar injection was given 7 days later on the opposite side. Rats were terminated at intervals beginning 22 days after the first NMU injection. Biopsies of each mammary gland, extending from the skin surface to either the abdominal or thoracic muscular wall, were placed in cryotubes and frozen immediately in liquid nitrogen. Frozen tissue from each of the 12 glands per rat was sectioned and stained with hematoxylin and eosin. One slide from the distal and one from the proximal aspect of each biopsy was submitted for histologic evaluation by a pathologist who was blinded to treatment group distribution of the samples. Ductal hyperplasia was distinguished from atypical ductal hyperplasia and carcinoma in situ. In no instance did jugular cut down preclude sampling of the first thoracic pair of glands. Histologic evaluation. We used the following criteria in defining ductal carcinoma in situ: (1) neoplastic cells must be confined within the ducts; (2) tumor cell nuclei show variation in size and shape, with some mitosis; (3)
205 All
Copyright 0 1992 rights of reproduction
0022404/92 $1.50 by Academic Press, Inc. in any form reserved.
206
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
52, NO. 3, MARCH~
1992
LXIS’I
7
w
ADH
m
DCIS
E’l’
AL.:
IXA’I’
MAMMAflY
lJlJc;‘l’AL
C;AHLlNUMA
26-28
31-33
37-38
45
DAYSFOLLOWING FIRSTNMU INJECTION Incidence of ductal carcinoma in situ and atypical ductal hyperplasia. Female Sprague-Dawley rats received 5 mg/lOO g body wt by jugular vein injection on a diestrus morning at 44-49 days of age. A second injection was given 7 days later. Values are means k SEM. No incidence of invasive disease was seen within the 22-45 day period. FIG. 2.
very
scanty
1
Incidence of Carcinoma with Age following NMU Induction
22-23 24-25 26-26 31-33 37-38 45 50-60 75-106 24-25
207
31’l‘U
TABLE
Days postinduction
22-23
1N
or no supporting
connective
tissue
within
the duct; (4) individual cell necrosis with central ductal necrosis; and (5) complete absence of stromal reaction and lymphocytic infiltration. Estrogen receptor determination. H222 Estrogen receptor primary antibody (generously provided by Abbott Labs) was biotinylated using Biotin-XX-NHS (Calbiothem) as described by Tse and Goldfarb [7]. Receptorantibody complex was detected by Avidin Peroxidase immunohistochemical staining using diaminobenzidine tetrahydrochloride as substrate (Vector Labs). Sections were counterstained with hematoxylin. Statistics. Data were analyzed by analysis of variance or McNemar’s test for paired data.
n 7
10 13 8 5 3 9 10
Number of rats with DCIS
Number of rats with invasive carcinoma
7 10 9 6 4 3 nd nd
0 0 0 0 0 0 5 9
Note. nd, Incidence of DCIS was not determined in rats where invasive disease was present.
incidence of atypical ductal hyperplasia or DCIS between 22 and 45 days postinduction, whether considered in terms of affected glands per rat (Fig. 2) or lesioned animals per number at risk (Table 1). Incidence of invasive carcinoma was 56% at 50-60 days and increased to 90% at 75-106 days postinduction. In those rats where lesioned glands were either abdominal or thoracic, abdominal lesions were more prevalent (Table 2). Immunohistochemical staining for ER appears localized to the nucleus with nearly 100% incidence of positively stained cells (Fig. 3). DISCUSSION
Our results demonstrate that an 87% incidence rate of DCIS involving three glands/rat can be achieved between 22 and 45 days post-NMU induction. Susceptibility to NMU administration and biologic characteristics of the resulting tumors are dependent on a number of factors including age, hormonal environment, and number of injections given. Rats at 35-50 days of age when mitotic activity is high in mammary terminal end buds appear most sensitive to initiating events caused by RESULTS NMU exposure [8,9]. Peak estradiol release during late diestrus or proestrus [lo] and the resulting increase in Diestrus induction of mammary tumors with NMU DNA synthesis 24 hr later [ll] tends to favor peak tuproduced well-defined DCIS lesions initially with a crib- mor incidence following injection timed to the proesriform pattern but progressing to comedo character as trus-estrus stages [12,13]. Diestrus induction, at a time illustrated in Fig. 1. In these lesions a variable degree of when estradiol is low, is associated with fewer tumors bridging of the cells within the duct was noted. The sup- per rat but enhanced growth rate for invasive lesions porting connective tissue was sparse and central necro- [12]. The impact of hormonal environment at the time of sis was common. The papillary histologic subtype was induction on tumor cells expressing ER receptor is less seen in only four of 160 total noninvasive carcinomas clear. An effect of cycle stage on the incidence rate of ER examined. There was no significant age effect on the positive tumors reported for multiple-dose induction has
FIG. 1. Rat mammary comedo ductal carcinoma in situ (A 200X, B 400X). Note hyperchromatic and irregular nuclei, individual cellular and central ductal necrosis with no stromal invasion or reaction. FIG. 3. Ductal carcinoma in situ stained for presence of nuclear estrogen receptor at 350X. Note the presence of unstained intraductal cells and the prominence of nuclear staining.
208
JOURNAL
TABLE Distribution 22-45
present absent
RESEARCH:
2
20 3
Thoracic absent
36 10
16’ 7
not been observed when a single NMU injection is used [14,13]. Incidence of receptor positive tumors by either of these methods appears lower than when multiple injection protocols which ignore estrus cycling are used [15]. In the only other reports on preinvasive lesions of which we are aware, hormone dependence and sensitivity to retinoid therapy has been demonstrated [16, 171. However, in both cases, appearance of palpable tumors was the observed endpoint. The absence of invasive disease in the current model affords us the opportunity to evaluate effect of therapy on preneoplastic lesions in progression to DCIS and also to the stage of invasive carcinoma. There are many evident similarities between the human disease and tumors produced by this method [6]. Hyperplasia without atypia is evident in terminal end buds for both human and the rat which disappears following first pregnancy and lactation [ 18,9]. Hyperplasia with atypia is thought to be the earliest stage of cancer development although the mechanism of progression is unknown. All carcinomas in rats and 70% of those in women appear to arise from terminal ductal epithelium [l, 191. Sixty to 80% are found to express estrogen receptors [9, 201 which compares favorably with our results showing nearly complete nuclear staining of intraductal cells for ER activity. The more prevalent occurrence of abdominal tumors noted here in rats where the distribution was either abdominal or thoracic was unexpected and is at odds with the distribution reported by Russo and Russo [19] for 7,12dimethylbenz(a)anthracene (DMBA)-induced tumors in the rat. In addition to the differing carcinogens used for tumor induction, the most readily apparent difference is the end point for observation, being invasive lesions with DMBA and CIS lesions here. The number and prominence of terminal end buds in the thoracic glands suggests a lower level of differentiation and argues for an increased carcinogen susceptibility [19]. Confirmation of our observation with preinvasive lesions may suggest that progression to the invasive stage rather than tumor induction is more prevalent in the thoracic region. REFERENCES Carlson, R. W., and Stockdale, F. E. The clinical breast cancer. Annu. Rev. Med. 39: 453, 1988.
biology
of
52, NO. 3, MARCH
1992
2.
Lagios, M. D., Margolin, F. R., Westdahl, P. R., and Rose, M. R. Mammographically detected duct carcinoma in situ. Frequency of local recurrence following tylectomy and prognostic effect of nuclear grade on local recurrence. Cancer 63: 618,1989.
3.
Fisher, E. R., Sass, R., Fisher, B., Wickerham, L., Paik, S. M., and collaborating NSABP investigators. Pathologic findings from the national surgical adjuvant breast project (protocol 6) I. Intraductal carcinoma (DCIS). Cancer 57: 197,1986.
4.
Tinnemans, J. G. M., Wobbes, T., Holland, R., Hendriks, J. H. C. L., van der Sluis, R. F., and De Boer, H. H. M. Treatment and survival of female patients with nonpalpable breast carcinoma. Ann. Surg. 209: 249,1989. Rose, D. P., and Noonan, J. J. Influence of prolactin and growth hormone on rat mammary tumors induced by N-nitrosomethylurea. Cancer Res. 42: 35, 1982.
Total
a Values indicate number of rats with a given distribution. * Abdominal location significantly greater than thoracic (P < 0.05).
1.
VOL.
of DCIS Lesions in Rats Days Postinjection” Thoracic present
Abdominal Abdominal
OF SURGICAL
5.
6.
Gullino, P. M., Pettigrew, H. M., and Grantham, F. H. N-nitrosomethylurea as mammary gland carcinogen in rats. J. Natl. Cancer Inst. 54: 401,1975. 7. Tse, J., and Goldfarb, S. Immunohistochemical demonstration of estrophilin in mouse tissues using a biotinylated monoclonal antibody. J. Histochem. Cytochem. 36: 1527,1988. 8. Anisimov, V. N. Effect of age on dose-response relationship in carcinogenesis induced by single administration of N-nitrosomethylurea in female rats. J. Cancer Res. Clin. Oncol. 114: 628, 1988. 9. Grubbs, C. J., Pecknam, J. C., and Cato, K. D. Mammary carcinogenesis in rats in relation to age at time of N-nitroso-n-methylurea administration. J. Natl. Cancer Inst. 70: 209, 1983. 10. Nequin, L. G., Alvarez, J., and Schwartz, N. B. Steroid control of gonadotropin release. J. Steroid B&hem. 6: 1007, 1975. 11. Lan, N C., and Kaczenellenbogen, B. S. Temporal relationships between hormone receptor binding and biological responses in the uterus: Studies with short- and long-acting derivatives of estriol. Endocrinology 98: 220, 1976. 12. Braun, R. J., Pezzuto, J. M., Anderson, C. H., and Beattie, C. W. Estrous cycle status alters N-methyl-N-nitrosourea (NMU)-induced rat mammary tumor growth and regression. Cancer L&t. 48: 2051989. 13. Ratko, T. A., and Beattie, C. W. Estrous cycle modification of rat mammary tumor induction by a single dose of N-methyl-N-nitrosourea. Cancer Res. 46: 3042,1985. 14. Lindsey, W. F., Gupta, T. K. D., and Beattie, C. W. Influence of the estrous cycle during carcinogen exposure on nitrosomethylurea-induced rat mammary carcinoma. Cancer Res. 41: 3857, 1981. 15. Rose, D. P., Pruitt, B., Stauber, P., Erturk, E., and Bryan, G. T. Influence of dosage schedule on the biological characteristics of N-nitrosomethylurea-induced rat mammary tumors. Cancer Res. 40: 235,198O. 16. McCormick, D. L., Sowell, Z. L., Thompson, C. A., and Moon, R. C. Inhibition by retinoid and ovariectomy of additional primary malignancies in rats following surgical removal of the first mammary cancer. Cancer 51: 594,1983. 17. Grubbs, C. J., Peckham, J. C., and McDonough, K. D. Effect of ovarian hormones on the induction of l-methyl-l-nitrosoureainduced mammary cancer. Carcinogen&s 4: 495,1983. 18. Dawson, E. K. A histological study of the normal mamma in relation to tumour growth. I. Early development to maturity. Edinburgh Med. J. 41: 653, 1985. 19. Russo, J., and Russo, I. H. Developmental stage of the rat mammary gland as determinant of its susceptibility to 7,12dimethylbenz[a]anthracene. J. N&l. Cancer Inst. 54: 401,1975. 20. Sondergaard, G., Pedersen, K. O., and Paulsen, S. M. Estrogen receptor analyses in breast cancer: Comparison of monoclonal immunohistochemical and biochemical methods. Eur. J. Cancer Clin. Oncol. 25: 1425, 1989.
OF SURGICAL
RESEARCH
52,205208
(19%‘)
Ductal Carcinoma in Situ in Rat Mammary Gland KEITH A. CRIST, PH.D.,**’
BINA CHAUDHURI, M.D.,? SUNIL SHIVARAM, B.S.,* AND PRABIR K. CHAIJDHURI, M.D.*
Departments of *Surgery and TPathology, Medical College of Ohio, 3000 Arlington Avenue, P.O. Box 10008, Toledo, Ohio 43699-0008 Submitted
for publication
suggests the need for an animal model which mimics the human pathology. While tumor development induced by the direct-acting carcinogen N-nitroso-N-methylurea (NMU) has been well characterized and closely fits developmental patterns, hormone dependence, and metastatic involvement associated with human breast cancer [5,6], a model system for the study of intraductal carcinoma prior to the development of invasive disease has not previously been described. Histologic characterization of DCIS development is presented here along with selected observations on estrogen receptor status.
Antiestrogen therapy has been proposed as a treatment option for ductal carcinoma in situ (DCIS). However, its effectiveness has not been evaluated in the laboratory due to lack of an animal model. The present study was undertaken to evaluate the incidence, time span, and number of mammary glands involved with DCIS in a rat model treated with N-nitroso-N-methylurea (NMU). Sprague Dawley female rats 44 to 49 days old were treated with two iv doses of 5 mg NMU/ 100 g body wt given 7 days apart, initiated at diestrus. Animals were killed at intervals beginning 2 1 days following first injection. Breast tissues were evaluated following routine H&E stain. Standard histologic criteria were followed to establish the diagnosis of DCIS. The number of glands involved with DCIS increased from one to seven with time from first injection. This model demonstrates an incidence of 87% for DCIS and 0% for invasive Ca between 22 and 45 days following NMU injection. Nine rats were sacrificed between 50 to 60 days and five showed invasive carcinoma. This model appears suitable for studying the efficacy of hormone or chemoprevention in the progression of DCIS to invasive Ca. 0 1992 Academic Press, Inc.
MATERIALS
INTRODUCTION
While the mechanism governing progression through the histologic abnormalities of breast ductal epithelium to the stage of invasive carcinoma is unknown, the occurrence of ductal carcinoma in situ (DCIS) appears to serve as a marker for increased risk of ipsilateral invasive disease [ 11. Degree of associated risk for concurrent occult and future disease may depend on both size and subtype of the intraductal lesion [2]. At present, the optimal conservative therapy directed toward DCIS following detection of the initial tumor mass is controversial [3, 41. Availability of breast biopsy tissue for study of neoplastic development and treatment of DCIS is severely limited by the necessity of processing the entire specimen to rule out microscopic invasion. This clearly 1 To whom reprint
May 20, 1991
requests should be addressed.
AND
METHODS
Tumor induction. Sixty-five female 40-day-old Sprague-Dawley rats were singly housed in polycarbonate cages, maintained in a 12-hr light/dark environment, and fed a commercial pelleted diet ad libitum. Vaginal smears (0.9% NaCl lavage) were recorded beginning at 43 days of age as diestrus (leukocytic smear), proestrus (nucleated smear), or estrus (cornified smear). Between 44-49 days of age on a diestrus morning, rats were injected with 5 mg NMU/lOO g body wt (15 mg/ml, Sigma, St. Louis, MO; dissolved in 3% acetic aciddistilled water) by jugular vein. A second similar injection was given 7 days later on the opposite side. Rats were terminated at intervals beginning 22 days after the first NMU injection. Biopsies of each mammary gland, extending from the skin surface to either the abdominal or thoracic muscular wall, were placed in cryotubes and frozen immediately in liquid nitrogen. Frozen tissue from each of the 12 glands per rat was sectioned and stained with hematoxylin and eosin. One slide from the distal and one from the proximal aspect of each biopsy was submitted for histologic evaluation by a pathologist who was blinded to treatment group distribution of the samples. Ductal hyperplasia was distinguished from atypical ductal hyperplasia and carcinoma in situ. In no instance did jugular cut down preclude sampling of the first thoracic pair of glands. Histologic evaluation. We used the following criteria in defining ductal carcinoma in situ: (1) neoplastic cells must be confined within the ducts; (2) tumor cell nuclei show variation in size and shape, with some mitosis; (3)
205 All
Copyright 0 1992 rights of reproduction
0022404/92 $1.50 by Academic Press, Inc. in any form reserved.
206
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
52, NO. 3, MARCH~
1992
LXIS’I
7
w
ADH
m
DCIS
E’l’
AL.:
IXA’I’
MAMMAflY
lJlJc;‘l’AL
C;AHLlNUMA
26-28
31-33
37-38
45
DAYSFOLLOWING FIRSTNMU INJECTION Incidence of ductal carcinoma in situ and atypical ductal hyperplasia. Female Sprague-Dawley rats received 5 mg/lOO g body wt by jugular vein injection on a diestrus morning at 44-49 days of age. A second injection was given 7 days later. Values are means k SEM. No incidence of invasive disease was seen within the 22-45 day period. FIG. 2.
very
scanty
1
Incidence of Carcinoma with Age following NMU Induction
22-23 24-25 26-26 31-33 37-38 45 50-60 75-106 24-25
207
31’l‘U
TABLE
Days postinduction
22-23
1N
or no supporting
connective
tissue
within
the duct; (4) individual cell necrosis with central ductal necrosis; and (5) complete absence of stromal reaction and lymphocytic infiltration. Estrogen receptor determination. H222 Estrogen receptor primary antibody (generously provided by Abbott Labs) was biotinylated using Biotin-XX-NHS (Calbiothem) as described by Tse and Goldfarb [7]. Receptorantibody complex was detected by Avidin Peroxidase immunohistochemical staining using diaminobenzidine tetrahydrochloride as substrate (Vector Labs). Sections were counterstained with hematoxylin. Statistics. Data were analyzed by analysis of variance or McNemar’s test for paired data.
n 7
10 13 8 5 3 9 10
Number of rats with DCIS
Number of rats with invasive carcinoma
7 10 9 6 4 3 nd nd
0 0 0 0 0 0 5 9
Note. nd, Incidence of DCIS was not determined in rats where invasive disease was present.
incidence of atypical ductal hyperplasia or DCIS between 22 and 45 days postinduction, whether considered in terms of affected glands per rat (Fig. 2) or lesioned animals per number at risk (Table 1). Incidence of invasive carcinoma was 56% at 50-60 days and increased to 90% at 75-106 days postinduction. In those rats where lesioned glands were either abdominal or thoracic, abdominal lesions were more prevalent (Table 2). Immunohistochemical staining for ER appears localized to the nucleus with nearly 100% incidence of positively stained cells (Fig. 3). DISCUSSION
Our results demonstrate that an 87% incidence rate of DCIS involving three glands/rat can be achieved between 22 and 45 days post-NMU induction. Susceptibility to NMU administration and biologic characteristics of the resulting tumors are dependent on a number of factors including age, hormonal environment, and number of injections given. Rats at 35-50 days of age when mitotic activity is high in mammary terminal end buds appear most sensitive to initiating events caused by RESULTS NMU exposure [8,9]. Peak estradiol release during late diestrus or proestrus [lo] and the resulting increase in Diestrus induction of mammary tumors with NMU DNA synthesis 24 hr later [ll] tends to favor peak tuproduced well-defined DCIS lesions initially with a crib- mor incidence following injection timed to the proesriform pattern but progressing to comedo character as trus-estrus stages [12,13]. Diestrus induction, at a time illustrated in Fig. 1. In these lesions a variable degree of when estradiol is low, is associated with fewer tumors bridging of the cells within the duct was noted. The sup- per rat but enhanced growth rate for invasive lesions porting connective tissue was sparse and central necro- [12]. The impact of hormonal environment at the time of sis was common. The papillary histologic subtype was induction on tumor cells expressing ER receptor is less seen in only four of 160 total noninvasive carcinomas clear. An effect of cycle stage on the incidence rate of ER examined. There was no significant age effect on the positive tumors reported for multiple-dose induction has
FIG. 1. Rat mammary comedo ductal carcinoma in situ (A 200X, B 400X). Note hyperchromatic and irregular nuclei, individual cellular and central ductal necrosis with no stromal invasion or reaction. FIG. 3. Ductal carcinoma in situ stained for presence of nuclear estrogen receptor at 350X. Note the presence of unstained intraductal cells and the prominence of nuclear staining.
208
JOURNAL
TABLE Distribution 22-45
present absent
RESEARCH:
2
20 3
Thoracic absent
36 10
16’ 7
not been observed when a single NMU injection is used [14,13]. Incidence of receptor positive tumors by either of these methods appears lower than when multiple injection protocols which ignore estrus cycling are used [15]. In the only other reports on preinvasive lesions of which we are aware, hormone dependence and sensitivity to retinoid therapy has been demonstrated [16, 171. However, in both cases, appearance of palpable tumors was the observed endpoint. The absence of invasive disease in the current model affords us the opportunity to evaluate effect of therapy on preneoplastic lesions in progression to DCIS and also to the stage of invasive carcinoma. There are many evident similarities between the human disease and tumors produced by this method [6]. Hyperplasia without atypia is evident in terminal end buds for both human and the rat which disappears following first pregnancy and lactation [ 18,9]. Hyperplasia with atypia is thought to be the earliest stage of cancer development although the mechanism of progression is unknown. All carcinomas in rats and 70% of those in women appear to arise from terminal ductal epithelium [l, 191. Sixty to 80% are found to express estrogen receptors [9, 201 which compares favorably with our results showing nearly complete nuclear staining of intraductal cells for ER activity. The more prevalent occurrence of abdominal tumors noted here in rats where the distribution was either abdominal or thoracic was unexpected and is at odds with the distribution reported by Russo and Russo [19] for 7,12dimethylbenz(a)anthracene (DMBA)-induced tumors in the rat. In addition to the differing carcinogens used for tumor induction, the most readily apparent difference is the end point for observation, being invasive lesions with DMBA and CIS lesions here. The number and prominence of terminal end buds in the thoracic glands suggests a lower level of differentiation and argues for an increased carcinogen susceptibility [19]. Confirmation of our observation with preinvasive lesions may suggest that progression to the invasive stage rather than tumor induction is more prevalent in the thoracic region. REFERENCES Carlson, R. W., and Stockdale, F. E. The clinical breast cancer. Annu. Rev. Med. 39: 453, 1988.
biology
of
52, NO. 3, MARCH
1992
2.
Lagios, M. D., Margolin, F. R., Westdahl, P. R., and Rose, M. R. Mammographically detected duct carcinoma in situ. Frequency of local recurrence following tylectomy and prognostic effect of nuclear grade on local recurrence. Cancer 63: 618,1989.
3.
Fisher, E. R., Sass, R., Fisher, B., Wickerham, L., Paik, S. M., and collaborating NSABP investigators. Pathologic findings from the national surgical adjuvant breast project (protocol 6) I. Intraductal carcinoma (DCIS). Cancer 57: 197,1986.
4.
Tinnemans, J. G. M., Wobbes, T., Holland, R., Hendriks, J. H. C. L., van der Sluis, R. F., and De Boer, H. H. M. Treatment and survival of female patients with nonpalpable breast carcinoma. Ann. Surg. 209: 249,1989. Rose, D. P., and Noonan, J. J. Influence of prolactin and growth hormone on rat mammary tumors induced by N-nitrosomethylurea. Cancer Res. 42: 35, 1982.
Total
a Values indicate number of rats with a given distribution. * Abdominal location significantly greater than thoracic (P < 0.05).
1.
VOL.
of DCIS Lesions in Rats Days Postinjection” Thoracic present
Abdominal Abdominal
OF SURGICAL
5.
6.
Gullino, P. M., Pettigrew, H. M., and Grantham, F. H. N-nitrosomethylurea as mammary gland carcinogen in rats. J. Natl. Cancer Inst. 54: 401,1975. 7. Tse, J., and Goldfarb, S. Immunohistochemical demonstration of estrophilin in mouse tissues using a biotinylated monoclonal antibody. J. Histochem. Cytochem. 36: 1527,1988. 8. Anisimov, V. N. Effect of age on dose-response relationship in carcinogenesis induced by single administration of N-nitrosomethylurea in female rats. J. Cancer Res. Clin. Oncol. 114: 628, 1988. 9. Grubbs, C. J., Pecknam, J. C., and Cato, K. D. Mammary carcinogenesis in rats in relation to age at time of N-nitroso-n-methylurea administration. J. Natl. Cancer Inst. 70: 209, 1983. 10. Nequin, L. G., Alvarez, J., and Schwartz, N. B. Steroid control of gonadotropin release. J. Steroid B&hem. 6: 1007, 1975. 11. Lan, N C., and Kaczenellenbogen, B. S. Temporal relationships between hormone receptor binding and biological responses in the uterus: Studies with short- and long-acting derivatives of estriol. Endocrinology 98: 220, 1976. 12. Braun, R. J., Pezzuto, J. M., Anderson, C. H., and Beattie, C. W. Estrous cycle status alters N-methyl-N-nitrosourea (NMU)-induced rat mammary tumor growth and regression. Cancer L&t. 48: 2051989. 13. Ratko, T. A., and Beattie, C. W. Estrous cycle modification of rat mammary tumor induction by a single dose of N-methyl-N-nitrosourea. Cancer Res. 46: 3042,1985. 14. Lindsey, W. F., Gupta, T. K. D., and Beattie, C. W. Influence of the estrous cycle during carcinogen exposure on nitrosomethylurea-induced rat mammary carcinoma. Cancer Res. 41: 3857, 1981. 15. Rose, D. P., Pruitt, B., Stauber, P., Erturk, E., and Bryan, G. T. Influence of dosage schedule on the biological characteristics of N-nitrosomethylurea-induced rat mammary tumors. Cancer Res. 40: 235,198O. 16. McCormick, D. L., Sowell, Z. L., Thompson, C. A., and Moon, R. C. Inhibition by retinoid and ovariectomy of additional primary malignancies in rats following surgical removal of the first mammary cancer. Cancer 51: 594,1983. 17. Grubbs, C. J., Peckham, J. C., and McDonough, K. D. Effect of ovarian hormones on the induction of l-methyl-l-nitrosoureainduced mammary cancer. Carcinogen&s 4: 495,1983. 18. Dawson, E. K. A histological study of the normal mamma in relation to tumour growth. I. Early development to maturity. Edinburgh Med. J. 41: 653, 1985. 19. Russo, J., and Russo, I. H. Developmental stage of the rat mammary gland as determinant of its susceptibility to 7,12dimethylbenz[a]anthracene. J. N&l. Cancer Inst. 54: 401,1975. 20. Sondergaard, G., Pedersen, K. O., and Paulsen, S. M. Estrogen receptor analyses in breast cancer: Comparison of monoclonal immunohistochemical and biochemical methods. Eur. J. Cancer Clin. Oncol. 25: 1425, 1989.
