Brief Communication: Production of Casein and the Presence of Estrogen Receptors in Human Breast Cancer 1, 2 A. Pich, M.D.,3 G. Bussolati, M.D.,4,5 and F. Oi Carlo, M.D. ABSTRACT-The relationship between the presence of estrogen receptors and casein (evaluated on a semiquantitative basis with a specific immunofluorescence method) was statistically analyzed in 50 cases of human breast carcinomas. No significant correlation was found between these two parameters, whereas a relationship was established between the production of casein and the degree of histologic differentiation. The results of this study, like those of other studies, revealed a lack of correlation between the presence of estrogen receptors and the degree of histologic differentiation.---J Natl Cancer Inst 58: 1483-1484, 1977.
The determination of estrogen-binding activity in breast carcinomas can be helpful in the selection of the appropriate treatment for patients with breast carcinoma (1). The presence of such activity indicates the persistence of a biologic characteristic typical of the normal mammary gland and theoretically could correspond to a lesser degree of neoplastic dedifferentiation. No consistent relationship between the level of estrogen receptors and the histologic grading of breast carcinomas has been found (1,2); this finding might, however, be interpreted as evidence that our accepted morphologic criteria of differentiation (3) (i.e., tubule formation, regularity of size, shape, and staining of nuclei, and a small number of mitoses) are insufficient and that other functional properties should be taken into account. One such property might be the production of milk proteins by the neoplastic cells. Experimental evidence in the rat indicates that the synthesis of milk proteins and especially of casein by cells of the R 3230 AC mammary adenocarcinoma is specifically stimulated by estradiol treatment (4, 5). Since it has recently been observed that casein production is a feature of some human breast cancers (6, 7), we have investigated the possibility of a correlation between the presence of estrogen receptors and the production of casein in a series of 50 unselected cases of human mammary carcinomas. MATERIALS AND METHODS
The neoplastic tissue was collected from the operating room. One sample of at least 500 mg was used for the detection of estrogen receptors, and a contiguous sample was fixed in 10% formalin for the assessment of the presence of casein. High-affinity estrogen receptors were measured according to the method of McGuire (8).
To simplify the data, the estrogen receptors were categorized as positive (present in quantities >3 fmoles/ mg of cytosol protein) or negative «3 fmoles/mg of cytosol protein). The presence of casein was demonstrated on tissue sections with the use of the immunocytochemical methVOL. 58, NO.5, MAY 1977
6,7
ods already described by Bussolati et al. (7) and Bussolati and Pich (9) employing antihuman casein antibodies. Appropriate controls for the specificity of the reaction were made as reported elsewhere (10) and included the use of antipurified human casein antibodies. The protein was detected in only a part of the tumors and was mainly present in intercellular spaces and cytoplasmic vesicles of the neoplastic cells. Employing a semiquantitative evaluation of the positive areas, we graded the tumors according to the amount of casein into categories (-), (+ -), and (+); (-) indicates that casein was not demonstrable, (+ -) indicates that the amount of casein was scanty, and (+) indicates that casein was present in most neoplastic areas and cells. RESULTS
The results, summarized in table 1, showed that there was not a significant association between the presence of casein and the presence or absence of estrogen receptors in human breast carcinomas. The association between the presence of casein and estrogen receptors was tested by chi-square test [for the 2 x 3 contingency table (11)] (table 1) and was shown to be not significant (0.IO:s:P:s:0.20) . TABLE
I.-Frequency of occurrence ofcasein and estrogen receptors"
Estrogen receptor category Negative mg of tein) Positive mg of tein) Total
Grades of casein presence" (-)
(+ -)
(+)
No. of cases
(0-3 fmoles/ cytosol pro-
6 [3.64]
5 [5.46]
2 [3.90]
13
(>3 fmoles/ cytosol pro-
8 [10.36]
16 [15.54]
13 [11.10]
37
14
21
15
50
Test for significance of the 2 x 3 contingency table above: chisquare test = 3.370950 (0.10:sP:s0.20). b Numbers in brackets = the expected No. if grades of casein presence and estrogen receptor categories are independent. a
Taking a different approach, we attempted to correlate the degree of histologic differentiation [evaluated according to the criteria outlined by Scarff and Torloni (3)] and that of casein production. The results are summarized in table 2, where only the cases grading (+) and (-) for casein presence were reported. Most cases negative for casein (-) were undifferentiated grade III carciReceived June 30, 1976; accepted November 6, 1976. Supported in part by the Italian National Research Council. 3Istituto di Anatomia e Istologia Patologica (III Chair), Universita degli Studi di Torino, Via Santena 7,10126 Turin, Italy. 4Istituto di Anatomia e Istologia Patologica (II Chair), Universita degli Studi di Torino. 5 Address reprint requests to Dr. Bussolati. 6Istituto de Farmacologia, Universita degli Studi di Torino. 7We thank Professor A. Piazza for statistical evaluation. 1
2
1483
J NATL CANCER INST
1484 TABLE
PICH, BUSSOLATI, AND Dl CARLO
2.-Degree of histologic differentiation and casein production
Positive staining with anticasein antiserum
Histologic type of carcinomas a Grade
Infiltrating Muciparous lobular carcarcinoma cinoma
I II III 3(1) 11 (7) (-) 0 0 0 2(1) 2 (2) 1 (1) 4 (2) 6 (6) (+) 2 (2) 4 (2) 1(1) 9 (7) 13 (8) Total a Numbers in parentheses = No. of carcinomas positive for estrogen receptors. The data were analyzed by the Fisher's exact test, cumulating the No. of differentiated carcinomas; the association between histologic differentiation and casein production was of limited statistical significance (0.05$]>$0.10).
nomas, whereas most cases positive for casein displayed evidence of glandular differentiation. A statistical evaluation of the data obtained in these selected cases was of limited significance, given the small number of cases; it appeared, however, that the histologic differentiation was associated with casein production, whereas the presence of estrogen receptors failed to correlate with any of these characteristics. A correlation was observed in the two cases of infiltrating lobular carcinomas: These tumors were positive (+) for casein, were characterized by an intracytoplasmic localization of the protein, and were also positive for estrogen receptors. DISCUSSION
Our studies indicate that in human breast carcinomas the production of casein, a milk protein that can be localized and roughly evaluated in a semiquantitative approach with the use of specific immunocytochemical methods (7, 9), and the level of estrogen-binding activity do not seem associated. Instead, confirming previous reports (7), casein production appears to correlate with the degree of histologic differentiation of infiltrating carcinomas of the breast. The lack of association observed in our cases between the presence of estrogen receptors and the histologic differentiation agrees with the results found in (1,2). The behavior of the infiltrating lobular carcinomas seems different from that of other carcinomas, since our cases were positive for both estrogen receptors [a result that confirms previous observations by others (2) on this type of tumor] and casein. In conclusion, our data seem to indicate that there is a correlation between morphologic and functional characteristics of breast carcinomas and that these characteristics are not associated (at least in tumors of ductal, as opposed to lobular, origin) with the level of estrogen receptors.
J
NATL CANCER INST
This conclusion might be interpreted as evidence that, in the human mammary carcinomas, casein production is not under the influence of estradiol stimulation, a behavior different from that observed in the rat mammary carcinoma (4, 5). It is, however, known that estrogen responsiveness in human breast carcinomas does not identify with estrogen receptor positivity, since only part of the cases positive with the latter test respond to hormonal therapy (1). However, no in vitro tests are presently available to assess the former parameter; therefore, only future prospective studies correlating casein presence with responsiveness to hormonal therapy might bring meaningful data. Several investigations in organ cultures indicate that prolactin enhances the proliferation of some human breast cancers (12, 13) and the rate of synthesis of casein by rat mammary carcinoma cells (14); it can, therefore, be hypothesized that, in human breast cancers, milk protein synthesis and perhaps also histologic differentiation might be an index of prolactin dependence. REFERENCES
(1) MCGUIRE WL, CARBONE PP, VOLLMER EP, eds.: Estrogen Receptors in Human Breast Cancer, New York, Raven Press, 1975 (2) ROSEN PP, MENENDEZ-BoTET C], NISSELBAUM ]S, et al: Pathological review of breast lesions analyzed for estrogen receptor protein. Cancer Res 35:3187-3194, 1975 (3) SCARFF RW, TORLONI H: Histological typing of breast tumours. Geneva, Switzerland, World Health Organization, 1968 (4) HILF R: Milk-like fluid in a mammary adenocarcinoma: Biochemical characterization. Science 155:826-827, 1967 (5) TURKINGTON RW, RIDDLE M: Aquired hormonal dependence of milk protein synthesis in mammary carcinoma cells. Endocrinology 84:1213-1217,1969 (6) FRANCHIMONT P, HENDRICK ]C: Radio-immunoassay of casein in the serum of normal subjects and of patients with various malignancies. Eur] Cancer 10:725-730, 1974 (7) BUSSOLATI G, PICH A, ALFANI V: Immunofluorescence detection of casein in human mammary dysplastic and neoplastic tissues. Virchows Arch (Pathol Anat) 365:15-21,1975 (8) MCGUIRE WL: Estrogen receptors in human breast cancer.] Clin Invest 52:73-77, 1973 (9) BUSSOLATI G, PICH A: Mammary and extra-mammary Paget's disease. An immuno-cytochemica1 study. Am] PathoI80:117128, 1975 (10) PICH A, BUSSOLATI G, CARBONARA A: Immunocytochemical detection of casein and casein-like proteins in human tissues. ] Histochem Cytochem 24:940-947, 1976 (11) FISHER RA, ed.: Statistical Methods for Research Workers, chapt 4. Edinburgh, Oliver & Boyd, 1970, pp 85-113 (12) SALIM H, FLAX H, BRANDER W, et al: Prolactin dependence in human breast cancers. Lancet 2:1103-1105, 1972 (13) MAGIOS MD: Hormonally enhanced proliferation of human breast cancer in organ culture. Oncology 29:22-33, 1974 (14) TURKINGTON RW, RIDDLE M: Expression of differentiated function by mammary carcinoma cells in vitro. Cancer Res 30:127132, 1970
VOL. 58, NO.5, MAY 1977
The determination of estrogen-binding activity in breast carcinomas can be helpful in the selection of the appropriate treatment for patients with breast carcinoma (1). The presence of such activity indicates the persistence of a biologic characteristic typical of the normal mammary gland and theoretically could correspond to a lesser degree of neoplastic dedifferentiation. No consistent relationship between the level of estrogen receptors and the histologic grading of breast carcinomas has been found (1,2); this finding might, however, be interpreted as evidence that our accepted morphologic criteria of differentiation (3) (i.e., tubule formation, regularity of size, shape, and staining of nuclei, and a small number of mitoses) are insufficient and that other functional properties should be taken into account. One such property might be the production of milk proteins by the neoplastic cells. Experimental evidence in the rat indicates that the synthesis of milk proteins and especially of casein by cells of the R 3230 AC mammary adenocarcinoma is specifically stimulated by estradiol treatment (4, 5). Since it has recently been observed that casein production is a feature of some human breast cancers (6, 7), we have investigated the possibility of a correlation between the presence of estrogen receptors and the production of casein in a series of 50 unselected cases of human mammary carcinomas. MATERIALS AND METHODS
The neoplastic tissue was collected from the operating room. One sample of at least 500 mg was used for the detection of estrogen receptors, and a contiguous sample was fixed in 10% formalin for the assessment of the presence of casein. High-affinity estrogen receptors were measured according to the method of McGuire (8).
To simplify the data, the estrogen receptors were categorized as positive (present in quantities >3 fmoles/ mg of cytosol protein) or negative «3 fmoles/mg of cytosol protein). The presence of casein was demonstrated on tissue sections with the use of the immunocytochemical methVOL. 58, NO.5, MAY 1977
6,7
ods already described by Bussolati et al. (7) and Bussolati and Pich (9) employing antihuman casein antibodies. Appropriate controls for the specificity of the reaction were made as reported elsewhere (10) and included the use of antipurified human casein antibodies. The protein was detected in only a part of the tumors and was mainly present in intercellular spaces and cytoplasmic vesicles of the neoplastic cells. Employing a semiquantitative evaluation of the positive areas, we graded the tumors according to the amount of casein into categories (-), (+ -), and (+); (-) indicates that casein was not demonstrable, (+ -) indicates that the amount of casein was scanty, and (+) indicates that casein was present in most neoplastic areas and cells. RESULTS
The results, summarized in table 1, showed that there was not a significant association between the presence of casein and the presence or absence of estrogen receptors in human breast carcinomas. The association between the presence of casein and estrogen receptors was tested by chi-square test [for the 2 x 3 contingency table (11)] (table 1) and was shown to be not significant (0.IO:s:P:s:0.20) . TABLE
I.-Frequency of occurrence ofcasein and estrogen receptors"
Estrogen receptor category Negative mg of tein) Positive mg of tein) Total
Grades of casein presence" (-)
(+ -)
(+)
No. of cases
(0-3 fmoles/ cytosol pro-
6 [3.64]
5 [5.46]
2 [3.90]
13
(>3 fmoles/ cytosol pro-
8 [10.36]
16 [15.54]
13 [11.10]
37
14
21
15
50
Test for significance of the 2 x 3 contingency table above: chisquare test = 3.370950 (0.10:sP:s0.20). b Numbers in brackets = the expected No. if grades of casein presence and estrogen receptor categories are independent. a
Taking a different approach, we attempted to correlate the degree of histologic differentiation [evaluated according to the criteria outlined by Scarff and Torloni (3)] and that of casein production. The results are summarized in table 2, where only the cases grading (+) and (-) for casein presence were reported. Most cases negative for casein (-) were undifferentiated grade III carciReceived June 30, 1976; accepted November 6, 1976. Supported in part by the Italian National Research Council. 3Istituto di Anatomia e Istologia Patologica (III Chair), Universita degli Studi di Torino, Via Santena 7,10126 Turin, Italy. 4Istituto di Anatomia e Istologia Patologica (II Chair), Universita degli Studi di Torino. 5 Address reprint requests to Dr. Bussolati. 6Istituto de Farmacologia, Universita degli Studi di Torino. 7We thank Professor A. Piazza for statistical evaluation. 1
2
1483
J NATL CANCER INST
1484 TABLE
PICH, BUSSOLATI, AND Dl CARLO
2.-Degree of histologic differentiation and casein production
Positive staining with anticasein antiserum
Histologic type of carcinomas a Grade
Infiltrating Muciparous lobular carcarcinoma cinoma
I II III 3(1) 11 (7) (-) 0 0 0 2(1) 2 (2) 1 (1) 4 (2) 6 (6) (+) 2 (2) 4 (2) 1(1) 9 (7) 13 (8) Total a Numbers in parentheses = No. of carcinomas positive for estrogen receptors. The data were analyzed by the Fisher's exact test, cumulating the No. of differentiated carcinomas; the association between histologic differentiation and casein production was of limited statistical significance (0.05$]>$0.10).
nomas, whereas most cases positive for casein displayed evidence of glandular differentiation. A statistical evaluation of the data obtained in these selected cases was of limited significance, given the small number of cases; it appeared, however, that the histologic differentiation was associated with casein production, whereas the presence of estrogen receptors failed to correlate with any of these characteristics. A correlation was observed in the two cases of infiltrating lobular carcinomas: These tumors were positive (+) for casein, were characterized by an intracytoplasmic localization of the protein, and were also positive for estrogen receptors. DISCUSSION
Our studies indicate that in human breast carcinomas the production of casein, a milk protein that can be localized and roughly evaluated in a semiquantitative approach with the use of specific immunocytochemical methods (7, 9), and the level of estrogen-binding activity do not seem associated. Instead, confirming previous reports (7), casein production appears to correlate with the degree of histologic differentiation of infiltrating carcinomas of the breast. The lack of association observed in our cases between the presence of estrogen receptors and the histologic differentiation agrees with the results found in (1,2). The behavior of the infiltrating lobular carcinomas seems different from that of other carcinomas, since our cases were positive for both estrogen receptors [a result that confirms previous observations by others (2) on this type of tumor] and casein. In conclusion, our data seem to indicate that there is a correlation between morphologic and functional characteristics of breast carcinomas and that these characteristics are not associated (at least in tumors of ductal, as opposed to lobular, origin) with the level of estrogen receptors.
J
NATL CANCER INST
This conclusion might be interpreted as evidence that, in the human mammary carcinomas, casein production is not under the influence of estradiol stimulation, a behavior different from that observed in the rat mammary carcinoma (4, 5). It is, however, known that estrogen responsiveness in human breast carcinomas does not identify with estrogen receptor positivity, since only part of the cases positive with the latter test respond to hormonal therapy (1). However, no in vitro tests are presently available to assess the former parameter; therefore, only future prospective studies correlating casein presence with responsiveness to hormonal therapy might bring meaningful data. Several investigations in organ cultures indicate that prolactin enhances the proliferation of some human breast cancers (12, 13) and the rate of synthesis of casein by rat mammary carcinoma cells (14); it can, therefore, be hypothesized that, in human breast cancers, milk protein synthesis and perhaps also histologic differentiation might be an index of prolactin dependence. REFERENCES
(1) MCGUIRE WL, CARBONE PP, VOLLMER EP, eds.: Estrogen Receptors in Human Breast Cancer, New York, Raven Press, 1975 (2) ROSEN PP, MENENDEZ-BoTET C], NISSELBAUM ]S, et al: Pathological review of breast lesions analyzed for estrogen receptor protein. Cancer Res 35:3187-3194, 1975 (3) SCARFF RW, TORLONI H: Histological typing of breast tumours. Geneva, Switzerland, World Health Organization, 1968 (4) HILF R: Milk-like fluid in a mammary adenocarcinoma: Biochemical characterization. Science 155:826-827, 1967 (5) TURKINGTON RW, RIDDLE M: Aquired hormonal dependence of milk protein synthesis in mammary carcinoma cells. Endocrinology 84:1213-1217,1969 (6) FRANCHIMONT P, HENDRICK ]C: Radio-immunoassay of casein in the serum of normal subjects and of patients with various malignancies. Eur] Cancer 10:725-730, 1974 (7) BUSSOLATI G, PICH A, ALFANI V: Immunofluorescence detection of casein in human mammary dysplastic and neoplastic tissues. Virchows Arch (Pathol Anat) 365:15-21,1975 (8) MCGUIRE WL: Estrogen receptors in human breast cancer.] Clin Invest 52:73-77, 1973 (9) BUSSOLATI G, PICH A: Mammary and extra-mammary Paget's disease. An immuno-cytochemica1 study. Am] PathoI80:117128, 1975 (10) PICH A, BUSSOLATI G, CARBONARA A: Immunocytochemical detection of casein and casein-like proteins in human tissues. ] Histochem Cytochem 24:940-947, 1976 (11) FISHER RA, ed.: Statistical Methods for Research Workers, chapt 4. Edinburgh, Oliver & Boyd, 1970, pp 85-113 (12) SALIM H, FLAX H, BRANDER W, et al: Prolactin dependence in human breast cancers. Lancet 2:1103-1105, 1972 (13) MAGIOS MD: Hormonally enhanced proliferation of human breast cancer in organ culture. Oncology 29:22-33, 1974 (14) TURKINGTON RW, RIDDLE M: Expression of differentiated function by mammary carcinoma cells in vitro. Cancer Res 30:127132, 1970
VOL. 58, NO.5, MAY 1977
