ANATOMIC PATHOLOGY Original Article
Potential Use of Monoclonal Antibodies in the Diagnostic Distinction of Gynecomastia from Breast Carcinoma in Men MARCELLA MOTTOLESE, PH.D., 1 GIULIO BIGOTTI, M.D.,2 ANTONELLA COLI, M.D.,2 CARLO VITUCCI, M.D.,3 AND PIER GIORGIO NATALI, M.D.4
demonstrated that MoAbs Bl.l, HMFG2, and MBrl displayed a strong reactivity with gynecomastia and carcinoma, but MoAbs B72.3 and B6.2 separated benign and malignant lesions in a high percentage of cases. When used in combination, the latter two reagents reacted with 96% of the carcinomas that were analyzed but labeled only 67% of gynecomastia cases. Thus, the conjoint use of these two reagents may enhance the use of FNA biopsy as a valuable tool in the presurgical diagnosis of breast nodules in men. (Key words: Breast carcinoma; Gynecomastia; Monoclonal antibodies; Fine-needle aspiration) Am J Clin Pathol 1991; 96:233-237
Carcinoma occurs infrequently in the male breast, and our knowledge of its biology and clinical behavior largely has been developed through a compilation of pooled experiences.1,2 Altered hormonal metabolism and antecedent gynecomastia appear to play a role in the development of the disease.3'4 Therefore, breast masses in men that are suspected to be malignant necessitate the differential diagnosis between gynecomastia and carcinoma.2 Fine-needle aspiration (FNA) cytology, which is applied widely in evaluating the nature of breast nodules in women, also could be a valuable tool in the clinical diagnosis of breast lesions in men. In most cases, conventional cytologic examination affords a definitive diagnosis, but the presence of dyscohesive, hyperchromatic cells in FNA from gynecomastia can mimic carcinoma and cause diagnostic confusion.5,6
We have demonstrated recently that the use of selected panels of monoclonal antibodies (MoAbs) to tumor-associated antigens (TAAs) can be a useful complement to conventional cytopathology7,8 and significantly improve the diagnostic accuracy of FNA of breast masses in women.9,10 To evaluate whether the same method could be helpful diagnostically in distinguishing gynecomastia from breast carcinoma in men, we analyzed the antigenic phenotype of 50 cases of gynecomastia and 30 cases of breast carcinoma in men, using a panel of five MoAbs that recognize distinct breast TAA (BTAA). The results of this retrospective study demonstrated that MoAbs B72.3 and B6.2 separated benign from malignant breast lesions in men in a high percentage of cases. Thus, these monoclonal antibodies may be useful tools in the presurgical cytodiagnosis of these rare carcinomas in men.
MATERIALS AND METHODS From the Departments of 'Pathology, ^Surgery, and 'Immunology, Regina Elena Cancer Institute, and the ^Department ofPathology, CathTissue Samples olic University of Sacred Heart, Rome, Italy. Received August 2, 1990; received revised manuscript and accepted for publication December 3, 1990. Supported by Tecnobiomedica and by Associazione per la Ricerca sul Cancro. Address reprint requests to Dr. Natali: Immunology Laboratory, Institute Regina Elena, Viale Regina Elena 291, Roma 00161, Italy. 233
Formalin-fixed, paraffin-embedded tissues from 50 cases of gynecomastia (mean patient age, 48.5 years; range, 16-74 years) and 30 breast carcinomas from men (mean patient age, 59.1 years; range, 23-77 years) were obtained from the E)epartments of Pathology at the Regina Elena
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Immunohistochemical (IHC) assays using the monoclonal antibodies (MoAbs) B72.3 and B6.2, recognizing two distinct and independently expressed breast tumor-associated antigens (BTAAs), recently have been shown to significantly improve the accuracy of cytodiagnosis of breast nodules by fine-needle aspiration (FNA). To evaluate whether the same method may be useful diagnostically in distinguishing gynecomastia from breast cancer in men, a retrospective avidin-biotin immunoperoxidase assay study was performed on 50 cases of gynecomastia and 30 cases of breast carcinoma in men, using a panel of five MoAbs known to recognize different BTAAs. The results of this study
234
ANATOMIC PATHOLOGY Article
Cancer Institute and the Catholic University of Rome. The breast tumors from men represented surgical biopsy specimens collected from 1978 to 1989. Five-micron sections from each block were mounted on gelatin-coated slides. Hematoxylin and eosin-stained sections were reviewed to confirm previous histologic diagnosis. Fine-needle aspiration biopsies from six breast nodules were performed with the use of a 21-gauge needle and a 20-mL disposable syringe mounted on a special holder (Cameco® 20 mL; Precision Dynamics, Burbank, CA). Cell smears werefixedin cold acetone for 10 minutes and stored at —20 °C. Our prior experience showed that this procedure caused no loss of reactivity for at least four months.
geneous" when the reactivity involved more than 50% of the tumor cell population. RESULTS Monoclonal Antibodies The MoAbs used in this study, with their relevant references, are listed in Table 1. They included five reagents that are known to recognize distinct BTAAs in women and were chosen on the basis of their ability to react with formalin-fixed, paraffin-embedded tissues. This characteristic permitted a retrospective analysis of gynecomastia and breast cancers in men.
Monoclonal antibodies B72.3" 12 and B6.213 were purchased from Sorin Biomedica Company (Saluggia, Italy), and MoAb HMFG21415 was obtained from Oxoid Company (Milano, Italy). MoAbs MBrl16 and Bl.l 17 were provided by Dr. M. I. Colnaghi (National Cancer Institute, Milano, Italy) and Dr. J. Schlom (Laboratory of Tumor Immunology and Biology, National Cancer Institute [National Institutes of Health], Bethesda, MD), respectively. The immunoreactivities of these reagents were assessed repeatedly during the study, with the use of negative and positive control specimens. All MoAbs were used as purified reagents (B72.3 and B6.2) or as appropriately diluted ascites fluid preparations. Immunoperoxidase Technique The avidin-biotin peroxidase complex assay was performed on dewaxed, rehydrated, 5-fim sections of formalin-fixed, paraffin-embedded tissues or on FNA preparations with the use of a commercially available kit (IMMUCOLOR®, Sorin Biomedica). Slides were incubated with MoAbs to BTAA at 4 °C for at least 16 hours in a moist chamber. The protein concentration of the primary antibodies ranged from 25 to 50 fig/mh in Hanks' balanced salt solution (HBSS) containing 1% bovine serum albumin (BSA) (Sigma Chemical Company, St. Louis, MO). Bound peroxidase was visualized with the use of 3amino-9-ethylcarbazole (AEC) as the chromogenic substrate, for 8 minutes at room temperature. Slides then were rinsed with phosphate-buffered saline (PBS) (0.15 mol/L) and counterstained with Mayer's hematoxylin (BDH Chemicals, Ltd., Pool, United Kingdom). Finally, the sections were mounted in buffered glycerol. Immunohistochemical reactivity was scored as "heterogeneous" when 25-50% of the specimen was stained and "homo-
A.J.C.P. •
Among the five MoAbs used in this study, Bl.l, HMFG2, and MBrl displayed intense reactivity with 86100% of all cases of gynecomastia. MoAb B72.3 did not show detectable reactivity with benign lesions, and MoAb B6.2 reacted heterogeneously in only 3 of 50 cases (6%) of gynecomastia. The predominant staining pattern of MoAbs Bl.l, HMFG2, and MBrl was homogeneous and was restricted, in most instances, to the plasma membrane (Fig 1A). Among 30 breast carcinomas from men that were tested with our panel of reagents, a homogeneous pattern of reactivity was observed in 80-90% of the malignant lesions with MoAbs B72.3 and B6.2 (Fig. IB). Monoclonal antibody Bl.l reacted with 86.6% of these cases, and all of the breast carcinomas from men showed intense and predominantly cytoplasmic reactivity with MoAbs HMFG2 and MBrl (Table 2). When immunocytochemical analysis was performed on six FNA specimens from two malignant and four benign mammary tumors from men, we observed the same staining pattern as that seen in paraffin sections (Table 3). DISCUSSION Cancer of the male breast is a rare disease, accounting for approximately 1% of all reported cases of mammary TABLE 1. MONOCLONAL ANTIBODIES USED TO ANALYZE THE ANTIGENIC PHENOTYPE OF BREAST CARCINOMAS IN MEN MoAbs
Isotype
Antigen
Molecular Weight
References
Bl.l HMFG2 MBrl B72.3 B6.2
IgGl IgGl IgM IgGl IgGl
Glycoprotein Glycoprotein Glycolipid Glycoprotein Glycoprotein
180 X 10 3 daltons 220 X 10 3 daltons Unknown >10 6 daltons 90 X 103 daltons
17 14, 15 16 11, 12 13
1991
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Analysis of the Antigenic Phenotype of Gynecomastia and Breast Carcinoma in Men
Monoclonal Antibodies
235
MOTTOLESE ET AL. lmmunocytodiagnosis of Breast Tumors in Men
carcinoma. In contrast, gynecomastia is a common condition, showing a variable age of presentation, with the greatest prevalence in men in the third decade of life.2,318 Although the histopathologic features of the two lesions usually cause no diagnostic challenges, the clinical distinction between gynecomastia and breast carcinoma in men may be difficult. Presenting symptoms and signs may be identical in these conditions.19'20 In the current study, a retrospective avidin-biotin immunoperoxidase assay analysis was performed on 50 cases of gynecomastia and 30 examples of breast carcinoma
from men, using a panel of five MoAbs (B72.3, B6.2, HMFG2, MBrl, Bl.l). These reagents previously have been shown to react with benign and malignant breast lesions in women. Murine MoAbs B72.3 and B6.2 were generated by immunizing mice with membrane-enriched fractions of breast carcinoma metastases from the liver. The corresponding antigens appear to be expressed selectively by transformed female mammary epithelium in 75% and 80% of cases, respectively.""14 MoAb MBrl recognizes a cell membranous neutral glycolipidic antigen that is ex-
TABLE 2. ANTIGENIC PHENOTYPE OF GYNECOMASTIA AND BREAST CARCINOMA IN MEN USING A PANEL OF MONOCLONAL ANTIBODIES TO DISTINCT FEMALE BREAST TUMOR-ASSOCIATED ANTIGENS Monoclonal Antibodies Histologic Diagnosis
Immunohistochemical Reactivity
Gynecomastia Heterogeneousf Homogeneousf Carcinoma Heterogeneous^; Homogeneous^
HMFG2
MBrl
B72.3
B6.2
50/50(100%) 12 38 30/30(100%) 6 24
46/50 (92%) 12 34 30/30(100%) 3 27
0/50 (0%) 0 0 24/30 (80%) 2 22
3/50 (6%) 3 0 27/30 (90%) 5 22
Bl.l •43/50 (86%) 10 33 *23/50 (85.5%) 7 19
* Fraction positive. Subjacent column entries show number of cases with each staining pattern.
Staining was topical or ^cytoplasmic.
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FlG. 1. A. Gynecomastia, showing homogeneous membranous cytoplasmic staining pattern with MoAb Bl.l (X200). B. Breast cancer from a man, showing intense cytoplasmic staining with MoAb B72.3 (X200). Mayer's hematoxylin counterstain.
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Article TABLE 3. IMMUNOCYTOCHEMICAL ANALYSIS OF FINENEEDLE ASPIRATES OF BREAST NODULES IN MEN USING A PANEL OF MONOCLONAL ANTIBODIES RECOGNIZING FEMALE BREAST TUMOR-ASSOCIATED ANTIGENS Immunocytochemical Reactivity Condition Gynecomastia Carcinoma
No. of Cases
Bl.l
4 2
3/4* 1/2*
HMFG2 MBrl B72.3 B6.2 4/4 2/2
4/4 2/2
0/4 1/2
0/4 2/2
• Fraction positive.
A.J.C.P. •
REFERENCES 1. Haagensen CD. Disease of the breast. Philadelphia: WB Saunders, 1986:976-989. 2. Siddiqui T, Weiner R, Moreb J, Marsh RD. Cancer of the male breast with prolonged survival. Cancer 1988;62:1632-1636. 3. Meyskens FL Jr, Tormey DC, Neifeld JP. Male breast cancer: a review. Cancer Treat Rev 1976;3:83-93. 4. Yap HY, Tashima CK, Blumenschein GR, Eckles NE. Male breast cancer. A natural history study. Cancer 1979;44:748-754. 5. Rone R, Ranzy I, Northcutt A. Gynecomastia: cytologic features and diagnostic pitfalls in aspiration biopsies. Acta Cytol 1986;30: 589. 6. Russin VL, Lachowicz C, Kline TS. Male breast lesions: gynecomastia and its distinction from carcinoma by aspiration biopsy cytology. Diagnostic Cytopathology 1989;3:243-247. 7. Mottolese M, Venturo I, Perrone Donnorso R, Gallo Curcio C, Rinaldi M, Natali PG. Use of selected combinations of monoclonal antibodies to tumor associated antigens in the diagnosis of neoplastic effusion of unknown origin. Eur J Cancer Clin Oncol 1988;24:1277-1284. 8. Mottolese M, Venturo I, Rinaldi M, et al. Combinations of monoclonal antibodies can distinguish primary lung tumors from metastatic lung tumors sampled by fine needle aspiration. Cancer 1989;64:2493-2500. 9. Natali PG, Mottolese M, Perrone Donnorso R, et al. Use of monoclonal antibodies to human breast TAA in the diagnosis of FNA
1991
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pressed by normal epithelium and roughly 80% of carcinomas of the breast.16 The MoAb HMFG2, produced by immunizing mice with human milk fat globule membrane and cultured human milk-producing cells, labels a group of high molecular weight (HMW) mucin glycoproteins expressed by more than 80% of normal breasts and benign mammary lesions and 95% of breast carcinomas.1415 MoAb Bl.l precipitates iodinated carcinoembryonic antigen, resulting in a radiolabeled peak at approximately 180 kD. This reagent reacted with 66% of the female mammary carcinomas that were tested.17 This IHC evaluation was conducted so that those reagents that might be useful in the cytologic diagnosis of breast masses in men could be selected. Fine-needle aspiration biopsy has been used increasingly in the presurgical assessment of breast cancer in women, whereas gynecomastia and mammary carcinoma in men have undergone only limited analysis with this diagnostic procedure.6,21,22 This is unfortunate, because aspirates from lesions of gynecomastia sometimes may be misinterpreted as malignant because of their cellularity, dyscohesion, and anisonucleosis.5,6,23 In previous studies, we have demonstrated that MoAbs B72.3 and B6.2, when used in combination, can significantly improve the diagnostic accuracy of FNA of female breast nodules10 and diminish false-negative interpretations. These findings were confirmed recently in a multicenter study.9 In the current series, we demonstrated that MoAbs Bl.l, MBrl, and HMFG2 showed an intense pattern of reactivity in benign and malignant breast nodules in men, as described in breast lesions in women.""17 Eighty percent to 100% of gynecomastia cases expressed BTAAs that were recognized by these three reagents, with a staining distribution that was associated with plasma membranes. When tested with the same MoAbs, breast cancers from men displayed a comparable percentage of reactivity, but with a more homogeneous and cytoplasmic staining pattern. The combination of MoAbs B72.3 and B6.2, which recognize BTAAs that are expressed independently by transformed cells, distinguished between benign and malignant lesions in men in 96% of cases. The same pattern
of reactivity also was observed in a few FNA specimens from tumors of the male breast that were analyzed with avidin-biotin peroxidase complex assays. These findings show that breast cancers in men and women have significant clinical and morphologic similarities19 and also share a comparable antigenic phenotype, as previously described by other authors using different MoAbs.24 However, to date, no study has provided information on the patterns of antibody reactivity in gynecomastia that would link it with carcinoma. This would be of clinical interest, because gynecomastia has been suggested as a condition predisposing men to breast cancer.1,2 Although clinical gynecomastia rarely is associated with breast carcinoma in general terms, microscopic features of the former lesion have been described in as many as 40% of men with breast cancer.19,20 This apparent discrepancy can be explained simply, by focusing on the wide difference in the prevalence of these two diseases. Of additional interest is the shared expression of the antigens identified by MoAbs B72.3 and B6.2 in malignant mammary epithelium in both women and men. These data may indicate that common pathogenetic mechanisms are operative in men and women with breast cancers, despite the origins of these neoplasms in different metabolic (hormonal) milieus. From a practical point of view, the expression of similar macromolecules in such tumors offers an objective criterion for the separation of gynecomastia from cancer of the male breast. If this IHC observation is confirmed in a larger series of cases, the use of FNA biopsy in the presurgical diagnosis of breast cancer in men may be applied more effectively.
MOTTOLESE ET AL. Immunocytodiagnosis of Breast Tumors in Men
10.
11.
12.
13.
14.
15.
17. Colcher D, Horan Hand P, Nuti M, Schlom J. Differential binding to human mammary and non mammary tumors of MoAb reactive with CEA. Cancer Invest 1983;1:127-138. 18. Hultborn R, Friberg S, Hultborn KA, Peterson LE, Raguhult I. Male breast carcinoma. II. A study of the total material reported to the Swedish Cancer Registry 1958-1967 with respect to treatment, prognostic factors and survival. Acta Oncologica 1987;26:327341. 19. Heller KS, Rosen PP, Schottenfeld D, Ashikari R, Kinne DW. Male breast cancer. A clinicopathologic study of 97 cases. Ann Surg 1978;188:60-65. 20. McDivitt RW, Stewart FW, Berg JW. Breast carcinoma in the male. In: Havlan I, Firminger MD, eds. Tumors of the breast. Atlas of tumor pathology, second series, vol 2. Washington, D.C.: Armed Forces Institute of Pathology, 1968:105-111. 21. Bhagat P, Kline TS. The male breast and malignant neoplasms. Diagnosis by aspiration biopsy cytology. Cancer 1990;65:23382341. 22. Kline TS. Handbook of fine needle aspiration biopsy cytology. New York: Churchill-Livingstone, 1988:207-243. 23. Stafford JR, Betsill WL. Fine needle aspiration cytologic diagnosis of gynecomastias. The Medical University of South Carolina experience, 1980 to 1985 [Abstract]. Acta Cytol (1985;29:948. 24. Lundy J, Mishriki Y, Viola MV, et al. A comparison of tumorrelated antigens in male and female breast cancer. Breast Cancer Res Treat 1986;7:91-96.
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16.
of breast nodules: results of a multicenter study. Int J Cancer 1990;45:12-15. Nuti M, Mottolese M, Viora M, Perrone Donnorso R, Schlom J, Natali PG. Use of MoAbs to human breast TAA in FNA cytology. Int J Cancer 1986;37:493-498. Nuti M, Castagna M, Squartini F. Morphologic assessment of reactivity to MoAbs generated against breast cancer cells in mammary tissue removed for clinical dysplasia or cancer. Appl Pathol 1984;2: 117-127. Thor A, Ohuchi N, Szpak CA, Johnston WW, Schlom J. Distribution of oncofetal antigen tumor associated glycoprotein-72 defined by MoAb B72.3. Cancer Res 1986,46:3118-3124. Colcher D, Horan Hand P, Nuti M, Schlom J. A spectrum of MoAbs reactive with human mammary tumors. Proc Natl Acad Sci 1981;78:3199-3203. Burchell J, Durbin H, Taylor-Papadimitriou J. Complexity of expression of antigenic determinants recognized by MoAbs HMFG1 and HMFG2 in normal and malignant human mammary epithelial cells. J Immunol 1983;131:508-513. Griffiths AB, Burchell J, Gendler S, et al. Immunological analysis of mucin molecules expressed by normal and malignant mammary epithelial cells. Int J Cancer 1987;40:319-327. Canevari S, Fossati G, Balsari A, Sonnino S, Colnaghi MI. Immunochemical analysis of the determinant recognized by an MoAb (MBrl) which specifically binds to human mammary epithelial cells. Cancer Res 1983;43:1301-1305.
237
Potential Use of Monoclonal Antibodies in the Diagnostic Distinction of Gynecomastia from Breast Carcinoma in Men MARCELLA MOTTOLESE, PH.D., 1 GIULIO BIGOTTI, M.D.,2 ANTONELLA COLI, M.D.,2 CARLO VITUCCI, M.D.,3 AND PIER GIORGIO NATALI, M.D.4
demonstrated that MoAbs Bl.l, HMFG2, and MBrl displayed a strong reactivity with gynecomastia and carcinoma, but MoAbs B72.3 and B6.2 separated benign and malignant lesions in a high percentage of cases. When used in combination, the latter two reagents reacted with 96% of the carcinomas that were analyzed but labeled only 67% of gynecomastia cases. Thus, the conjoint use of these two reagents may enhance the use of FNA biopsy as a valuable tool in the presurgical diagnosis of breast nodules in men. (Key words: Breast carcinoma; Gynecomastia; Monoclonal antibodies; Fine-needle aspiration) Am J Clin Pathol 1991; 96:233-237
Carcinoma occurs infrequently in the male breast, and our knowledge of its biology and clinical behavior largely has been developed through a compilation of pooled experiences.1,2 Altered hormonal metabolism and antecedent gynecomastia appear to play a role in the development of the disease.3'4 Therefore, breast masses in men that are suspected to be malignant necessitate the differential diagnosis between gynecomastia and carcinoma.2 Fine-needle aspiration (FNA) cytology, which is applied widely in evaluating the nature of breast nodules in women, also could be a valuable tool in the clinical diagnosis of breast lesions in men. In most cases, conventional cytologic examination affords a definitive diagnosis, but the presence of dyscohesive, hyperchromatic cells in FNA from gynecomastia can mimic carcinoma and cause diagnostic confusion.5,6
We have demonstrated recently that the use of selected panels of monoclonal antibodies (MoAbs) to tumor-associated antigens (TAAs) can be a useful complement to conventional cytopathology7,8 and significantly improve the diagnostic accuracy of FNA of breast masses in women.9,10 To evaluate whether the same method could be helpful diagnostically in distinguishing gynecomastia from breast carcinoma in men, we analyzed the antigenic phenotype of 50 cases of gynecomastia and 30 cases of breast carcinoma in men, using a panel of five MoAbs that recognize distinct breast TAA (BTAA). The results of this retrospective study demonstrated that MoAbs B72.3 and B6.2 separated benign from malignant breast lesions in men in a high percentage of cases. Thus, these monoclonal antibodies may be useful tools in the presurgical cytodiagnosis of these rare carcinomas in men.
MATERIALS AND METHODS From the Departments of 'Pathology, ^Surgery, and 'Immunology, Regina Elena Cancer Institute, and the ^Department ofPathology, CathTissue Samples olic University of Sacred Heart, Rome, Italy. Received August 2, 1990; received revised manuscript and accepted for publication December 3, 1990. Supported by Tecnobiomedica and by Associazione per la Ricerca sul Cancro. Address reprint requests to Dr. Natali: Immunology Laboratory, Institute Regina Elena, Viale Regina Elena 291, Roma 00161, Italy. 233
Formalin-fixed, paraffin-embedded tissues from 50 cases of gynecomastia (mean patient age, 48.5 years; range, 16-74 years) and 30 breast carcinomas from men (mean patient age, 59.1 years; range, 23-77 years) were obtained from the E)epartments of Pathology at the Regina Elena
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Immunohistochemical (IHC) assays using the monoclonal antibodies (MoAbs) B72.3 and B6.2, recognizing two distinct and independently expressed breast tumor-associated antigens (BTAAs), recently have been shown to significantly improve the accuracy of cytodiagnosis of breast nodules by fine-needle aspiration (FNA). To evaluate whether the same method may be useful diagnostically in distinguishing gynecomastia from breast cancer in men, a retrospective avidin-biotin immunoperoxidase assay study was performed on 50 cases of gynecomastia and 30 cases of breast carcinoma in men, using a panel of five MoAbs known to recognize different BTAAs. The results of this study
234
ANATOMIC PATHOLOGY Article
Cancer Institute and the Catholic University of Rome. The breast tumors from men represented surgical biopsy specimens collected from 1978 to 1989. Five-micron sections from each block were mounted on gelatin-coated slides. Hematoxylin and eosin-stained sections were reviewed to confirm previous histologic diagnosis. Fine-needle aspiration biopsies from six breast nodules were performed with the use of a 21-gauge needle and a 20-mL disposable syringe mounted on a special holder (Cameco® 20 mL; Precision Dynamics, Burbank, CA). Cell smears werefixedin cold acetone for 10 minutes and stored at —20 °C. Our prior experience showed that this procedure caused no loss of reactivity for at least four months.
geneous" when the reactivity involved more than 50% of the tumor cell population. RESULTS Monoclonal Antibodies The MoAbs used in this study, with their relevant references, are listed in Table 1. They included five reagents that are known to recognize distinct BTAAs in women and were chosen on the basis of their ability to react with formalin-fixed, paraffin-embedded tissues. This characteristic permitted a retrospective analysis of gynecomastia and breast cancers in men.
Monoclonal antibodies B72.3" 12 and B6.213 were purchased from Sorin Biomedica Company (Saluggia, Italy), and MoAb HMFG21415 was obtained from Oxoid Company (Milano, Italy). MoAbs MBrl16 and Bl.l 17 were provided by Dr. M. I. Colnaghi (National Cancer Institute, Milano, Italy) and Dr. J. Schlom (Laboratory of Tumor Immunology and Biology, National Cancer Institute [National Institutes of Health], Bethesda, MD), respectively. The immunoreactivities of these reagents were assessed repeatedly during the study, with the use of negative and positive control specimens. All MoAbs were used as purified reagents (B72.3 and B6.2) or as appropriately diluted ascites fluid preparations. Immunoperoxidase Technique The avidin-biotin peroxidase complex assay was performed on dewaxed, rehydrated, 5-fim sections of formalin-fixed, paraffin-embedded tissues or on FNA preparations with the use of a commercially available kit (IMMUCOLOR®, Sorin Biomedica). Slides were incubated with MoAbs to BTAA at 4 °C for at least 16 hours in a moist chamber. The protein concentration of the primary antibodies ranged from 25 to 50 fig/mh in Hanks' balanced salt solution (HBSS) containing 1% bovine serum albumin (BSA) (Sigma Chemical Company, St. Louis, MO). Bound peroxidase was visualized with the use of 3amino-9-ethylcarbazole (AEC) as the chromogenic substrate, for 8 minutes at room temperature. Slides then were rinsed with phosphate-buffered saline (PBS) (0.15 mol/L) and counterstained with Mayer's hematoxylin (BDH Chemicals, Ltd., Pool, United Kingdom). Finally, the sections were mounted in buffered glycerol. Immunohistochemical reactivity was scored as "heterogeneous" when 25-50% of the specimen was stained and "homo-
A.J.C.P. •
Among the five MoAbs used in this study, Bl.l, HMFG2, and MBrl displayed intense reactivity with 86100% of all cases of gynecomastia. MoAb B72.3 did not show detectable reactivity with benign lesions, and MoAb B6.2 reacted heterogeneously in only 3 of 50 cases (6%) of gynecomastia. The predominant staining pattern of MoAbs Bl.l, HMFG2, and MBrl was homogeneous and was restricted, in most instances, to the plasma membrane (Fig 1A). Among 30 breast carcinomas from men that were tested with our panel of reagents, a homogeneous pattern of reactivity was observed in 80-90% of the malignant lesions with MoAbs B72.3 and B6.2 (Fig. IB). Monoclonal antibody Bl.l reacted with 86.6% of these cases, and all of the breast carcinomas from men showed intense and predominantly cytoplasmic reactivity with MoAbs HMFG2 and MBrl (Table 2). When immunocytochemical analysis was performed on six FNA specimens from two malignant and four benign mammary tumors from men, we observed the same staining pattern as that seen in paraffin sections (Table 3). DISCUSSION Cancer of the male breast is a rare disease, accounting for approximately 1% of all reported cases of mammary TABLE 1. MONOCLONAL ANTIBODIES USED TO ANALYZE THE ANTIGENIC PHENOTYPE OF BREAST CARCINOMAS IN MEN MoAbs
Isotype
Antigen
Molecular Weight
References
Bl.l HMFG2 MBrl B72.3 B6.2
IgGl IgGl IgM IgGl IgGl
Glycoprotein Glycoprotein Glycolipid Glycoprotein Glycoprotein
180 X 10 3 daltons 220 X 10 3 daltons Unknown >10 6 daltons 90 X 103 daltons
17 14, 15 16 11, 12 13
1991
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Analysis of the Antigenic Phenotype of Gynecomastia and Breast Carcinoma in Men
Monoclonal Antibodies
235
MOTTOLESE ET AL. lmmunocytodiagnosis of Breast Tumors in Men
carcinoma. In contrast, gynecomastia is a common condition, showing a variable age of presentation, with the greatest prevalence in men in the third decade of life.2,318 Although the histopathologic features of the two lesions usually cause no diagnostic challenges, the clinical distinction between gynecomastia and breast carcinoma in men may be difficult. Presenting symptoms and signs may be identical in these conditions.19'20 In the current study, a retrospective avidin-biotin immunoperoxidase assay analysis was performed on 50 cases of gynecomastia and 30 examples of breast carcinoma
from men, using a panel of five MoAbs (B72.3, B6.2, HMFG2, MBrl, Bl.l). These reagents previously have been shown to react with benign and malignant breast lesions in women. Murine MoAbs B72.3 and B6.2 were generated by immunizing mice with membrane-enriched fractions of breast carcinoma metastases from the liver. The corresponding antigens appear to be expressed selectively by transformed female mammary epithelium in 75% and 80% of cases, respectively.""14 MoAb MBrl recognizes a cell membranous neutral glycolipidic antigen that is ex-
TABLE 2. ANTIGENIC PHENOTYPE OF GYNECOMASTIA AND BREAST CARCINOMA IN MEN USING A PANEL OF MONOCLONAL ANTIBODIES TO DISTINCT FEMALE BREAST TUMOR-ASSOCIATED ANTIGENS Monoclonal Antibodies Histologic Diagnosis
Immunohistochemical Reactivity
Gynecomastia Heterogeneousf Homogeneousf Carcinoma Heterogeneous^; Homogeneous^
HMFG2
MBrl
B72.3
B6.2
50/50(100%) 12 38 30/30(100%) 6 24
46/50 (92%) 12 34 30/30(100%) 3 27
0/50 (0%) 0 0 24/30 (80%) 2 22
3/50 (6%) 3 0 27/30 (90%) 5 22
Bl.l •43/50 (86%) 10 33 *23/50 (85.5%) 7 19
* Fraction positive. Subjacent column entries show number of cases with each staining pattern.
Staining was topical or ^cytoplasmic.
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FlG. 1. A. Gynecomastia, showing homogeneous membranous cytoplasmic staining pattern with MoAb Bl.l (X200). B. Breast cancer from a man, showing intense cytoplasmic staining with MoAb B72.3 (X200). Mayer's hematoxylin counterstain.
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ANATOMIC PATHOLOGY
Article TABLE 3. IMMUNOCYTOCHEMICAL ANALYSIS OF FINENEEDLE ASPIRATES OF BREAST NODULES IN MEN USING A PANEL OF MONOCLONAL ANTIBODIES RECOGNIZING FEMALE BREAST TUMOR-ASSOCIATED ANTIGENS Immunocytochemical Reactivity Condition Gynecomastia Carcinoma
No. of Cases
Bl.l
4 2
3/4* 1/2*
HMFG2 MBrl B72.3 B6.2 4/4 2/2
4/4 2/2
0/4 1/2
0/4 2/2
• Fraction positive.
A.J.C.P. •
REFERENCES 1. Haagensen CD. Disease of the breast. Philadelphia: WB Saunders, 1986:976-989. 2. Siddiqui T, Weiner R, Moreb J, Marsh RD. Cancer of the male breast with prolonged survival. Cancer 1988;62:1632-1636. 3. Meyskens FL Jr, Tormey DC, Neifeld JP. Male breast cancer: a review. Cancer Treat Rev 1976;3:83-93. 4. Yap HY, Tashima CK, Blumenschein GR, Eckles NE. Male breast cancer. A natural history study. Cancer 1979;44:748-754. 5. Rone R, Ranzy I, Northcutt A. Gynecomastia: cytologic features and diagnostic pitfalls in aspiration biopsies. Acta Cytol 1986;30: 589. 6. Russin VL, Lachowicz C, Kline TS. Male breast lesions: gynecomastia and its distinction from carcinoma by aspiration biopsy cytology. Diagnostic Cytopathology 1989;3:243-247. 7. Mottolese M, Venturo I, Perrone Donnorso R, Gallo Curcio C, Rinaldi M, Natali PG. Use of selected combinations of monoclonal antibodies to tumor associated antigens in the diagnosis of neoplastic effusion of unknown origin. Eur J Cancer Clin Oncol 1988;24:1277-1284. 8. Mottolese M, Venturo I, Rinaldi M, et al. Combinations of monoclonal antibodies can distinguish primary lung tumors from metastatic lung tumors sampled by fine needle aspiration. Cancer 1989;64:2493-2500. 9. Natali PG, Mottolese M, Perrone Donnorso R, et al. Use of monoclonal antibodies to human breast TAA in the diagnosis of FNA
1991
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pressed by normal epithelium and roughly 80% of carcinomas of the breast.16 The MoAb HMFG2, produced by immunizing mice with human milk fat globule membrane and cultured human milk-producing cells, labels a group of high molecular weight (HMW) mucin glycoproteins expressed by more than 80% of normal breasts and benign mammary lesions and 95% of breast carcinomas.1415 MoAb Bl.l precipitates iodinated carcinoembryonic antigen, resulting in a radiolabeled peak at approximately 180 kD. This reagent reacted with 66% of the female mammary carcinomas that were tested.17 This IHC evaluation was conducted so that those reagents that might be useful in the cytologic diagnosis of breast masses in men could be selected. Fine-needle aspiration biopsy has been used increasingly in the presurgical assessment of breast cancer in women, whereas gynecomastia and mammary carcinoma in men have undergone only limited analysis with this diagnostic procedure.6,21,22 This is unfortunate, because aspirates from lesions of gynecomastia sometimes may be misinterpreted as malignant because of their cellularity, dyscohesion, and anisonucleosis.5,6,23 In previous studies, we have demonstrated that MoAbs B72.3 and B6.2, when used in combination, can significantly improve the diagnostic accuracy of FNA of female breast nodules10 and diminish false-negative interpretations. These findings were confirmed recently in a multicenter study.9 In the current series, we demonstrated that MoAbs Bl.l, MBrl, and HMFG2 showed an intense pattern of reactivity in benign and malignant breast nodules in men, as described in breast lesions in women.""17 Eighty percent to 100% of gynecomastia cases expressed BTAAs that were recognized by these three reagents, with a staining distribution that was associated with plasma membranes. When tested with the same MoAbs, breast cancers from men displayed a comparable percentage of reactivity, but with a more homogeneous and cytoplasmic staining pattern. The combination of MoAbs B72.3 and B6.2, which recognize BTAAs that are expressed independently by transformed cells, distinguished between benign and malignant lesions in men in 96% of cases. The same pattern
of reactivity also was observed in a few FNA specimens from tumors of the male breast that were analyzed with avidin-biotin peroxidase complex assays. These findings show that breast cancers in men and women have significant clinical and morphologic similarities19 and also share a comparable antigenic phenotype, as previously described by other authors using different MoAbs.24 However, to date, no study has provided information on the patterns of antibody reactivity in gynecomastia that would link it with carcinoma. This would be of clinical interest, because gynecomastia has been suggested as a condition predisposing men to breast cancer.1,2 Although clinical gynecomastia rarely is associated with breast carcinoma in general terms, microscopic features of the former lesion have been described in as many as 40% of men with breast cancer.19,20 This apparent discrepancy can be explained simply, by focusing on the wide difference in the prevalence of these two diseases. Of additional interest is the shared expression of the antigens identified by MoAbs B72.3 and B6.2 in malignant mammary epithelium in both women and men. These data may indicate that common pathogenetic mechanisms are operative in men and women with breast cancers, despite the origins of these neoplasms in different metabolic (hormonal) milieus. From a practical point of view, the expression of similar macromolecules in such tumors offers an objective criterion for the separation of gynecomastia from cancer of the male breast. If this IHC observation is confirmed in a larger series of cases, the use of FNA biopsy in the presurgical diagnosis of breast cancer in men may be applied more effectively.
MOTTOLESE ET AL. Immunocytodiagnosis of Breast Tumors in Men
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of breast nodules: results of a multicenter study. Int J Cancer 1990;45:12-15. Nuti M, Mottolese M, Viora M, Perrone Donnorso R, Schlom J, Natali PG. Use of MoAbs to human breast TAA in FNA cytology. Int J Cancer 1986;37:493-498. Nuti M, Castagna M, Squartini F. Morphologic assessment of reactivity to MoAbs generated against breast cancer cells in mammary tissue removed for clinical dysplasia or cancer. Appl Pathol 1984;2: 117-127. Thor A, Ohuchi N, Szpak CA, Johnston WW, Schlom J. Distribution of oncofetal antigen tumor associated glycoprotein-72 defined by MoAb B72.3. Cancer Res 1986,46:3118-3124. Colcher D, Horan Hand P, Nuti M, Schlom J. A spectrum of MoAbs reactive with human mammary tumors. Proc Natl Acad Sci 1981;78:3199-3203. Burchell J, Durbin H, Taylor-Papadimitriou J. Complexity of expression of antigenic determinants recognized by MoAbs HMFG1 and HMFG2 in normal and malignant human mammary epithelial cells. J Immunol 1983;131:508-513. Griffiths AB, Burchell J, Gendler S, et al. Immunological analysis of mucin molecules expressed by normal and malignant mammary epithelial cells. Int J Cancer 1987;40:319-327. Canevari S, Fossati G, Balsari A, Sonnino S, Colnaghi MI. Immunochemical analysis of the determinant recognized by an MoAb (MBrl) which specifically binds to human mammary epithelial cells. Cancer Res 1983;43:1301-1305.
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