Histopathology I 979, 3, 407-4 I 9
Tubular carcinoma: a variant of secretory breast carcinoma V . E U S E B I , C. M . B E T T S & G . B U S S O L A T I Departments of Histopathology and Experimental Pathology Universilies of Bologna and Torino, Italy Accepted for publication 9 March 1979 EUSEBI V., BETTSC.M. & BUSSOLATI G . (1979) Histopathology 3, 407-419 Tubular carcinoma : a variant of secretory breast carcinoma
Fifteen cases of tubular carcinoma of the breast have been studied using histochemical methods for mucosubstances, immunocytochemical methods for casein and actin and conventional electron microscopy. Mucosubstances and casein were demonstrated lying freely in the lurnina of the tubules. Occasionally, mucosubstances and casein assumed the form of target-like intracytoplasmic 'inclusions' like those characteristically seen in lobular carcinoma. The neoplastic cells did not react with antisera specific against actin. Even at ultrastructural level no myoepithelial cells were observed, whilst villi were revealed along the tubular luminal surface. It appears that, in addition to distinctive biological, histological and ultrastructural features, tubular carcinoma has an almost constant histochemical pattern. This suggests a differentiation towards epithelial secretory cells engaged in intensive milk protein production which has also been shown to be a feature of lobular carcinoma. It is concluded that though lobular carcinoma and tubular carcinoma of the breast have been traditionally regarded as two distinct entities, they have certain similar functional characteristics and it is postulated that these two tumours could represent the extreme variants of the same entity: the infiltrative lobular carcinoma being the most undifferentiated and tubular carcinoma the most highly differentiated. Keywords: breast, carcinoma, tubular carcinoma, secretory carcinoma, casein
Introduction Tubular carcinoma of the breast became a well recognized entity with the description and delineation of the entity by Taylor & Norris (1970). Several studies in the literature have described the clinicopathological correlations (McDivitt, Stewart & Berg 1968, Carstens et al. r y p ) , the differential diagnosis from sclerosing adenosis (Jao, Recant & Swerdlow 1976) and the ultrastructural Address for correspondence: Dr V. Eusebi, Istituto Anatomia e Istologica Patologia, Policlinico S . Orsola, Via Massarenti 9, Bologna, Italy. 0309-0167/79/09oo-o407 Soz.00
0 1979 Blackwell Scientific Publications
407
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features of this tumour (Erlandson & Carstens 1973, Tobon & Salazar 1977, Harris & Ahmed 1977). Recently, Fisher et al. (1977) reported 24 cases of breast carcinoma combining features of both tubular and infiltrative lobular carcinoma. This variant was named tubulolobular invasive breast carcinoma, but Fisher et al. (1977) were uncertain whether this tumour should be regarded as a variant of tubular or lobular invasive carcinoma and stated that the decision as to its precise status might be more philosophical than one that could be decided entirely objectively. Breast carcinomas especially of lobular type have been shown to synthesize milk proteins, especially casein (Franchimont & Hendrik 1974, Bussolati, Pich & Alfani 1975, Bussolati & Pich 1975). such synthetic activity reflecting evidence of functional differentiation. We have previously employed immunocytochemical and electroncytochemical techniques to localize milk protein production at cellular level in lobular carcinoma (Eusebi et al. 1977). We therefore applied the same immunohistochemical techniques to investigate the nature and the secretory activity of tubular carcinoma of the breast.
Materials and methods Fifteen cases of pure tubular carcinoma of breast (McDivitt et al. 1968) were selected for study. The cases were obtained from different institutions and had been collected over a number of years. Follow-up information on the patients was available in all cases. HISTOLOGICAL A N D HISTOCHEMICAL METHODS
The tissues were fixed in 10% neutral formol saline and routinely embedded in paraffin wax. I n addition to haematoxylin and eosin (H & E) and Weigert-Van Gieson for elastic tissue, the sections were stained with periodic acid Schiff with and without diastase pretreatment, and with the alcian blue-periodic acid Schiff (AB/PAS) sequence according to Gad & Azzopardi (1975). I M MU N 0 C Y T 0C H E MI C A L ST A I N I N G FOR CASE I N
Casein was demonstrated by the immunofluorescence and immunoperoxidase methods as previously reported in detail (Eusebi et al. 1977). In addition to rabbit anti-total human casein antisera we also employed anti-sera specific for the major casein fraction : goat anti-human b-casein antisera and guinea-pig anti-human /)-casein antisera. I M M U N O C Y T O C H E M I C A L S T A I N I N G FOR A C T l N
Five cases were studied with anti-actin sera. The immunocytochemical staining method for myoepithelial cells as applied to formalin-fixed and paraffin embedded tissues, was that described recently by Bussolati et a/. (1978).
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This technique involves the use of rabbit anti-actin antibodies on sections previously treated for I hour at 37°C with 0.I % trypsin. ELECTRON MICROSCOPY
Two cases of tubular carcinoma were studied ultrastructurally : the formalin-fixed tissue was post-fixed in I % osmium tetroxide for I hour, dehydrated and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Siemens 102 electron microscope.
Results CLINICAL FINDINGS
All 15 patients were white females ranging in age from 38 to 72 years (mean age 51 years). They presented with small unilateral breast nodules situated on the left side in nine cases and on the right side in six cases. The upper outer quadrant of the breast was involved in eight cases whilst a parareolar lump was present in five cases and in two the lower outer quadrant was involved. The duration of symptoms varied from 4 weeks to 3 years. None of the patients had enlarged axillary lymph nodes. Mammograms were available in three cases; one was reported as positive for carcinoma and two as suspicious of malignancy. All patients underwent radical mastectomy. They were all alive between 2 months and 16 years after treatment, with a mean follow-up period of 3.5 years. One patient had two recurrences, 8 months and I year respectively after initial surgery, before the lesion was diagnosed as malignant. This patient died recently of heart failure 16 years after the first surgical excision. At necropsy no residual tumour was found. One patient was diagnosed as having lobular carcinoma in situ (LCIS) in the contralateral breast 6 months after mastectomy for tubular carcinoma. This patient refused any further treatment and remains alive and well 3 years after the mastectomy. None of the other 13 patients showed any evidence of tumour recurrence at their most recent examination.
Pathological findings GROSS EXAMINATION
The dimensions of the tumours were known in 1 0 cases and varied from 0.6 to 4.5 cm maximum diameter (mean of 1.3 cm). They were reported as firm in consistency, stellate in shape, with grey-white cut surfaces flecked with frequent elastotic yellow streaks.
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L I G H T MICROSCOPY
All tumoiirs had the typical histological pattern of the well differentiated tubular carcinoma as defined by McDivitt et al. (1968). The margins were irregularly radiating, sometimes penetrating the adjacent adipose tissue. The neoplastic elements were uniformly arranged as glandular structures displaying lumina of varying dimensions (Figure I). They were mostly formed by a single layer of cells, mainly cuboidal with round to ovoid nuclei and small nucleoli. When ovoid, the long axis of the nucleus was orientated circumferentially. The cytoplasm was eosinophilic showing an irregular luminal surface in nine cases and better defined cytoplasmic apical ‘snouts’ in five (Figure 2): in one case both features were prominent. Mitoses were very rare. Depending on the plane of sectioning, the lumina of the tubules appear round to ovoid with their long axes at variable angles rather than being arranged parallel to one another. Frequently the lumina contained eosinophilic material. Thirteen cases showed alcian blue (AB) positive intraluminal mucosubstances (Figure 3). Two cases were devoid of mucin. One case showed intense PAS-positive material in the centre of the lumina surrounded by AB-positive mucin. The quantity of mucosubstances present inside the lumina varied from case to case, being very scanty at one extreme and very abundant at the other. By contrast, we did not notice field changes of mucin pattern in different areas of the same tumour, the amount of mucin being fairly uniform in all tubules. When the tubular lumen appeared devoid of mucin in the H & E preparations, a thin rim of AB-positive material was found coating the luminal epithelial surfaces. Two cases displayed numerous cells with intracytoplasmic lumina (ICL) containing AB- and PAS-positive mucosubstances. The mucosubstances had a target-like appearance identical to that described by Gad & Azzopardi (1975) in lobular carcinoma of the breast. Stroma with plump fibroblasts and AB-positive material at pH 0.5 was observed in I I cases. The remaining four cases had a fibro-elastotic stroma. In addition, vascular and periductal elastosis was present in nine cases. Basophilic calcifications were found in three cases, mainly as psammoma bodies located inside the lumina. Lobular carcinoma in situ was present either within the confines of the tumour or at its margins in no fewer than four cases. Five cases showed in situ carcinoma o f ductal type (DCIS) and in two other cases both LCIS and DCIS were present simultaneously giving a total of seven cases with DCIS and six cases with LCIS. Three cases had axillary lymph nodal metastases. Three lymph nodes were involved in one case, two lymph nodes in another and a solitary lymph node in the third patient. In this last patient there were only two small neoplastic tubules located within the thickened fibrous capsule of a lymph node. All the metastatic deposits showed the morphological features of tubular carcinoma and they involved the proximal group of axillary lymph nodes only. I. Glandular structures displaying lumina of varying dimensions, irregularly orientated. Prominent focal elastosis (top) is also a feature of this tumour. H & E. x 60.
Figure
Figure 2. The tubules are formed by a single layer of cuboidal cells showing ‘apical snouts’. H & E. x 400.
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V .Eusebi, C.M . Belts and G.Bussolati
Figure 3. The small duct in the centre is formed by a two-layer epithelium which rests on a basement membrane. It is surrounded by neoplastic tubules formed by one layer of cells. The lumina are filled with AB-positive material and no basement membrane is evident. AB!PAS. x 2 5 0 .
Casein
All cases were positive for total casein (Figure 4) as well as for the p-casein fraction (Figure 5) using the immunofluorescence and immunoperoxidase staining methods. Although the positive staining for casein was also present freely dispersed within the luminal content, it was mostly present condensed along the luminal surface of the
Figure 4. Casein is observed free in the lumina and condensed along the luminal surface of the cells. Rabbit anti-total casein immunoperoxidase method. x 2 5 0 .
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Figure 5. All tubules contain casein deposited inside the lumina or coating the luminal cytoplasm. Goat anti-human p-casein immunofluorescence. x 100.
Figure 6. Casein coats the free cytoplasmic border of neoplastic cells and it is observed free in the lumen. Same method as Figure 5. x 400. E
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V.Eusebi, C.M .Betts and G.Bussolati
Figure 7. A normal lobule stained for actin with the irnmunofluorescence method: the external cells of the lobular ductules are heavily stained. x 100.
Figure 8. Normal preexisting ductules are surrounded by myoepithelial cells, whilst the neoplastic cells appear unstained. Anti-actin immunoperoxidase method. x 400.
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cells lining the tubules (Figure 6). The only two cases which were found to be negative for mucin, were positive for casein. Some ICL were seen inside the cytoplasm of neoplastic cells in three of the tumours but target-like ‘inclusions’ were most numerous in the two cases which also showed AB/PAS positive intracytoplasmic haloes and dots. Actin
Anti-actin sera stained only the myoepithelial cells of ducts and lobular terminal ductules (Figure 7) as well as the muscular cells in the vascular walls of normal residual breast tissue. All cells of neoplastic tubules appeared unstained and this negative reaction was even more striking when comparison was made with the normal structures present in the same sections (Figure 8).
Figure 9. Tubule formed by one layer of cells. The luminal surface shows a villous differentiation. No myoepithelial cells are present. E.M. x 3000.
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V.Eusebi, C.M.Betts and G.Bussolati
U L T R A S T R U C T U R A L FINDINGS
The neoplastic tubules were formed by a single layer of cuboidal cells with round nuclei showing a prominent nucleolus. The cytoplasm was rich in endoplasmic reticulum and the luminal surface showed numerous villi projecting towards the lumen. Desmosomes were only occasionally found. The neoplastic cells did not show any cytoplasmic filaments and there was no evidence of any myoepithelial differentiation (Figure 9). Discussion Tubular carcinoma of the breast is also referred to as orderly carcinoma (Carstens et al. 1972) or well differentiated carcinoma (Taylor & Norris 1970) and is reported to have a very good prognosis (Carstens el al. 1972). This behaviour is confirmed in the present series. It has characteristic histological and ultrastructural features which have led numerous investigators to regard it as a distinctive type of ductal breast carcinoma (Taylor & Norris 1970, Carstens el al. 1972, Tobon & Salazar 1977). The differentiation of neoplastic tubules with lumina which are sometimes elongated and orientated a t varying angles to one another; the cuboidal cells with or without cytoplasmic snouts; the round to ovoid nuclei with small nucleoli; the virtual lack of atypia and mitoses; the absence of myoepithelial cells both at immunohistochemical and ultrastructural level ; the AB-positive stroma associated with a frequent elastotic reaction (Tremblay 1974); and a high incidence of an associated in situ malignancy are all features of this entity. On histological grounds tubular carcinoma must be differentiated from ‘sclerosing adenosis with pseudoinfiltration’ as emphasized by McDivitt et al. (1968) and Carstens et al. (1972) and from the scleroelastotic lesions described by Hamper1 (1975) and stressed more recently by one of us (Eusebi, Grassigli & Grosso 1976). Tobon & Salazar (1977) reported that it was not uncommon to see myoepithelial cells either isolated or in continuous rows surrounding the neoplastic tubules. This observation was not supported by the ultrastructural studies of Erlandson & Carstens (1973) Jao el al. (1976) and Harris & Ahmed (1977). The present electronmicroscopic and immunohistochemical study also refutes the suggestion that myoepithelial cells are found in tubular carcinoma. Our evidence indicates that it is a purely epithelial tumour. All but two of our cases contained acid mucosubstances in the tubular lumina. When present, mucin tended to be observed uniformly in the majority of tubules of a given case, though the amount present varied widely between different cases. Two cases also showed target-like cytoplasmic inclusions with a PAS-positive core and AB-positive peripheral rim. All 15 cases showed the presence of intraluminal casein and in this respect casein synthesis appears to be a more constant finding than mucin production. Casein appoared free in the lumina and especially coating the cytoplasmic snouts which ultrastructurally showed a villous surface. These differences between mucin and casein synthesis are probably attributable not only to the highly sensitive method employed
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for casein detection but also to the excellent fixation essential for the optimal demonstration of mucin (Gad & Azzopardi 1975). In addition to the recognized distinctive biological, histological and ultrastructural features, tubular carcinoma appears to have an almost constant histochemical pattern which suggests a differentiation towards epithelial secretory cells engaged in intensive milk protein production. It has already been shown that in lobular carcinoma of the breast a large number of neoplastic cells produce mucosubstances (Gad & Azzopardi 1975) and, to an even greater extent, casein (Eusebi et al. 1977). Tubular carcinoma and lobular carcinoma share certain functional parameters of differentiation whilst infiltrating duct carcinoma shows a much more modest production of mucosubstances and casein (Gad & Azzopardi 1975, Pich, Bussolati & DiCarlo 1977). Lobular carcinoma in situ was present in six cases. This association is by no means unique to the present series because two of the 33 cases reported by Taylor & Norris (1970) and no fewer than five of the 35 cases reported by Carstens et al. (1972) showed in situ or infiltrating lobular carcinoma in association with the tubular carcinoma. This association between tubular and lobular carcinoma appears therefore to be more than a fortuitous one. In addition, one of our patients developed contralateral LCIS 6 months after the mastectomy for tubular carcinoma. Six of the 33 patients reported on by Taylor & Norris (1970) developed contralateral carcinomas, two of which were also tubular carcinomas. It appears, therefore, that tubular carcinoma has a high incidence of bilateral involvement which is also a well known feature of lobular carcinoma (Hutter & Foote 1969, Andersen 1974). Recently Haagensen et al. (1978) studied 38 carcinomas which developed subsequently to LCIS. Three cases were pure tubular carcinomas and in another two cases tubular carcinoma was associated with infiltrative lobular carcinoma. Furthermore, Martinez & Azzopardi (I979), in a study of 30 infiltrative lobular carcinomas have found, in seven cases, a further and separate invasive carcinoma in the same or the other breast. Four of these were tubular, two infiltrative lobular and one of uncertain classification. Lobular and tubular carcinoma are frequently associated together in the same or opposite breast; have a similarly high incidence of bilateral involvement; and in addition they share a similar functional differentiation. Both usually produce mucosubstances and casein and, at ultrastructural level, their constituent cells show an almost constant villous absorptive type of surface. Fisher et al. (1977) described the tubulo-lobular carcinoma as having histological and histochemical features of both infiltrative lobular and tubular carcinoma and they suggested that this entity could be regarded as an intermediate form of differentiation between the two structurally different tumours. The evidence presented by us points in the same direction and the concept that tubular carcinoma is necessarily a form of ductal carcinoma as currently believed needs reappraisal. Our findings suggest that both infiltrative lobular carcinoma and tubular carcinoma can be regarded either as divergent types of a specialized form of secretory breast carcinoma or, possibly inore likely, as two distinct variants of the same entity: the classical infiltrating lobular carcinoma representing the least differentiated variant
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whilst tubular carcinoma achieves a high degree of morphological as well as functional differentiation.
Acknowledgements The authors wish to thank Professor J.G.Azzopardi for critically reviewing this manuscript and Professor A.M.Mancini for assistance in its preparation. The authors are also grateful to Dr M.Monaco at the NCI, Bethesda, USA, for supplying the goat anti-human B-casein antisera and Dr G.F.Voglino at S . Anna Hospital, Torino for giving the guinea-pig anti-human B-casein antisera. Acknowledgement is also due to Dr Mary Osborn, at Max Plank Institute, Gottingen, West Germany, for supplying rabbit anti-actin antibodies. Mr M.Mariani has to be thanked for photographic assistance and Ms D.M.James for secretarial help. The work was supported by a CNR grant no. 79.00624.96, Ronie.
References ANDERSEN J.A. (1974) Multicentric and bilateral appearance of lobular carcinoma in situ of the breast. Acta Pathologica and Microbiologica Scandinauica Sect. A, 87, 730-734 BUSSOLATI G. & PICHA. (1975) Mammary and extra-mammary Paget’s disease: An immunocytochemical study. American Journal o/ Pathology 80, I 17-128 B ~ ~ S S O L G., A T IPICH A. & ALFANIV. (1975) Imrnunofluorescence detection of casein in human mammary dysplastic and neoplastic tissues. Virchow’s Archiu. Ahr. A Pathologische AnafomiePathology 365, I 5-2 I
BUSSOLATI G., BONFANTI S., WEBERK. & OSBORN M. (1978) Staining of the rnyopeithelial cells in fixed and embedded tissues by immunocytochemical techniques using antibodies to actin. Ricista di Istochimica Normale e Patologica 22, 387-389. CARSTENS P.H.F., Huvos A.G., FOOTE F.W. & ASHIKARI R. (1972) Tubular carcinoma of breast: a clinicopathologic study of 35 cases. American Journal of Clinical Pathology 58,231-238 ERLANDSON R.A. & CARSTENS P.H.B. (1973) Ultrastructure of tubular carcinoma of the breast. Cancer 29, 987-995 EUSEBI V., GRASSIGLI A. & GROSSO F. (1976) Lesioni focali sclero-elastotiche mammarie simulanti il carcinoma infiltrante. Pathologica 68, 507-5 I 8 EUSEBI V., PICHA., MACCHIORLAITE. & BUSSOLATI G . (1977) Morpho-functional differentiation in lobular carcinoma of the breast. Histopathology I, 301-314 FISHER E.R., GREGORIO R.M.,REDMOND C. & FISHER B. (1977) Tubulolobular invasive breast cancer: a variant of lobular invasive cancer. Human Pathology 8, 679-683 FRANCHIMONT P. & HENDRICK J.C. (1974) Radio-irnrnunoassay of casein in the serum of normal subjects and of patients with various malignancies. European Journal of Cancer 10, 725-730 GADA. & AZZOPARDI J.G. (1975) Lobular carcinoma of the breast: a special variant of mucinsecreting carcinoma. Journal of Clinical Pathology 28, 7 I I -7 I 6 HAAGENSEN C.D., LANEN., LArrEs R. & BODIANC. (1978) Lobular neoplasia (so-called lobular carcinoma in siru) of the breast. Cancer 42, 737-769 HAMPERL H. (1975) Strahlige Narben und obliterierende Mastopathie. Virchows Archic. Ahr. A Pathologische Anatomie-Pathology 369, 55-68 HARRISM. & AHMEDA. (1977) The ultrastructure of tubular carcinoma of the breast. Journal of Pathology I 23, 79-83
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HUTTERR.V.P. & FOOTE F.W. (1969) Lobular carcinoma in situ. Cancer 24, 1081-1085 JAOW., RECANT W. & SWERDLOW M.A. (1976) Comparative ultrastructure of tubular carcinoma and sclerosing adenosis of the breast. Cancer 38, 180-186 MART~NEZ V. & AZZOPARDI J.G. (1979) Invasive lobular carcinoma of the breast. Histopathology 3*in press M c D l v l n R.W., STEWART F.W. & BERGJ.W. (1968) Tumors of the breast. Atlas of Tumor Pathology, 2nd Series, Fascicle 2. Armed Forces Institute of Pathology, Washington D C G. & DI CARLOF. (1977) Production of casein and presence of oestrogen PICHA., BUSSOLATI receptors in human breast cancers. National Cancer Institute Journal 58, 1483-1484 ROSENP.P., MENDEZ-BOTET C.J., NISSELBAGM J.S., URBAN J.A., M I KV., ~ FRACCHIA A. & SCHWARTZ M.K. (1975) Pathological review of breast lesions analysed for oestrogen receptor protein. Cancer Research 35, 3 I 87-3 I 94 TAYLOR H.B. & NORRIS H.J. (1970) Well-differentiated carcinoma of the breast. Cancer 25,687-692. TOBONH. & SALAZAR H. (1977) Tubular carcinoma of the breast. Archiues of Pathology and Laboratory Medicine 101, 3 10-316 TREMBLAY G. (1974) Elastosis in tubular carcinoma of the breast. Archices ofPathology 98,302-307
Tubular carcinoma: a variant of secretory breast carcinoma V . E U S E B I , C. M . B E T T S & G . B U S S O L A T I Departments of Histopathology and Experimental Pathology Universilies of Bologna and Torino, Italy Accepted for publication 9 March 1979 EUSEBI V., BETTSC.M. & BUSSOLATI G . (1979) Histopathology 3, 407-419 Tubular carcinoma : a variant of secretory breast carcinoma
Fifteen cases of tubular carcinoma of the breast have been studied using histochemical methods for mucosubstances, immunocytochemical methods for casein and actin and conventional electron microscopy. Mucosubstances and casein were demonstrated lying freely in the lurnina of the tubules. Occasionally, mucosubstances and casein assumed the form of target-like intracytoplasmic 'inclusions' like those characteristically seen in lobular carcinoma. The neoplastic cells did not react with antisera specific against actin. Even at ultrastructural level no myoepithelial cells were observed, whilst villi were revealed along the tubular luminal surface. It appears that, in addition to distinctive biological, histological and ultrastructural features, tubular carcinoma has an almost constant histochemical pattern. This suggests a differentiation towards epithelial secretory cells engaged in intensive milk protein production which has also been shown to be a feature of lobular carcinoma. It is concluded that though lobular carcinoma and tubular carcinoma of the breast have been traditionally regarded as two distinct entities, they have certain similar functional characteristics and it is postulated that these two tumours could represent the extreme variants of the same entity: the infiltrative lobular carcinoma being the most undifferentiated and tubular carcinoma the most highly differentiated. Keywords: breast, carcinoma, tubular carcinoma, secretory carcinoma, casein
Introduction Tubular carcinoma of the breast became a well recognized entity with the description and delineation of the entity by Taylor & Norris (1970). Several studies in the literature have described the clinicopathological correlations (McDivitt, Stewart & Berg 1968, Carstens et al. r y p ) , the differential diagnosis from sclerosing adenosis (Jao, Recant & Swerdlow 1976) and the ultrastructural Address for correspondence: Dr V. Eusebi, Istituto Anatomia e Istologica Patologia, Policlinico S . Orsola, Via Massarenti 9, Bologna, Italy. 0309-0167/79/09oo-o407 Soz.00
0 1979 Blackwell Scientific Publications
407
408
V.Eusebi, C.M.Betts atid G.Bussolati
features of this tumour (Erlandson & Carstens 1973, Tobon & Salazar 1977, Harris & Ahmed 1977). Recently, Fisher et al. (1977) reported 24 cases of breast carcinoma combining features of both tubular and infiltrative lobular carcinoma. This variant was named tubulolobular invasive breast carcinoma, but Fisher et al. (1977) were uncertain whether this tumour should be regarded as a variant of tubular or lobular invasive carcinoma and stated that the decision as to its precise status might be more philosophical than one that could be decided entirely objectively. Breast carcinomas especially of lobular type have been shown to synthesize milk proteins, especially casein (Franchimont & Hendrik 1974, Bussolati, Pich & Alfani 1975, Bussolati & Pich 1975). such synthetic activity reflecting evidence of functional differentiation. We have previously employed immunocytochemical and electroncytochemical techniques to localize milk protein production at cellular level in lobular carcinoma (Eusebi et al. 1977). We therefore applied the same immunohistochemical techniques to investigate the nature and the secretory activity of tubular carcinoma of the breast.
Materials and methods Fifteen cases of pure tubular carcinoma of breast (McDivitt et al. 1968) were selected for study. The cases were obtained from different institutions and had been collected over a number of years. Follow-up information on the patients was available in all cases. HISTOLOGICAL A N D HISTOCHEMICAL METHODS
The tissues were fixed in 10% neutral formol saline and routinely embedded in paraffin wax. I n addition to haematoxylin and eosin (H & E) and Weigert-Van Gieson for elastic tissue, the sections were stained with periodic acid Schiff with and without diastase pretreatment, and with the alcian blue-periodic acid Schiff (AB/PAS) sequence according to Gad & Azzopardi (1975). I M MU N 0 C Y T 0C H E MI C A L ST A I N I N G FOR CASE I N
Casein was demonstrated by the immunofluorescence and immunoperoxidase methods as previously reported in detail (Eusebi et al. 1977). In addition to rabbit anti-total human casein antisera we also employed anti-sera specific for the major casein fraction : goat anti-human b-casein antisera and guinea-pig anti-human /)-casein antisera. I M M U N O C Y T O C H E M I C A L S T A I N I N G FOR A C T l N
Five cases were studied with anti-actin sera. The immunocytochemical staining method for myoepithelial cells as applied to formalin-fixed and paraffin embedded tissues, was that described recently by Bussolati et a/. (1978).
Tubular carcinoma
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This technique involves the use of rabbit anti-actin antibodies on sections previously treated for I hour at 37°C with 0.I % trypsin. ELECTRON MICROSCOPY
Two cases of tubular carcinoma were studied ultrastructurally : the formalin-fixed tissue was post-fixed in I % osmium tetroxide for I hour, dehydrated and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Siemens 102 electron microscope.
Results CLINICAL FINDINGS
All 15 patients were white females ranging in age from 38 to 72 years (mean age 51 years). They presented with small unilateral breast nodules situated on the left side in nine cases and on the right side in six cases. The upper outer quadrant of the breast was involved in eight cases whilst a parareolar lump was present in five cases and in two the lower outer quadrant was involved. The duration of symptoms varied from 4 weeks to 3 years. None of the patients had enlarged axillary lymph nodes. Mammograms were available in three cases; one was reported as positive for carcinoma and two as suspicious of malignancy. All patients underwent radical mastectomy. They were all alive between 2 months and 16 years after treatment, with a mean follow-up period of 3.5 years. One patient had two recurrences, 8 months and I year respectively after initial surgery, before the lesion was diagnosed as malignant. This patient died recently of heart failure 16 years after the first surgical excision. At necropsy no residual tumour was found. One patient was diagnosed as having lobular carcinoma in situ (LCIS) in the contralateral breast 6 months after mastectomy for tubular carcinoma. This patient refused any further treatment and remains alive and well 3 years after the mastectomy. None of the other 13 patients showed any evidence of tumour recurrence at their most recent examination.
Pathological findings GROSS EXAMINATION
The dimensions of the tumours were known in 1 0 cases and varied from 0.6 to 4.5 cm maximum diameter (mean of 1.3 cm). They were reported as firm in consistency, stellate in shape, with grey-white cut surfaces flecked with frequent elastotic yellow streaks.
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L I G H T MICROSCOPY
All tumoiirs had the typical histological pattern of the well differentiated tubular carcinoma as defined by McDivitt et al. (1968). The margins were irregularly radiating, sometimes penetrating the adjacent adipose tissue. The neoplastic elements were uniformly arranged as glandular structures displaying lumina of varying dimensions (Figure I). They were mostly formed by a single layer of cells, mainly cuboidal with round to ovoid nuclei and small nucleoli. When ovoid, the long axis of the nucleus was orientated circumferentially. The cytoplasm was eosinophilic showing an irregular luminal surface in nine cases and better defined cytoplasmic apical ‘snouts’ in five (Figure 2): in one case both features were prominent. Mitoses were very rare. Depending on the plane of sectioning, the lumina of the tubules appear round to ovoid with their long axes at variable angles rather than being arranged parallel to one another. Frequently the lumina contained eosinophilic material. Thirteen cases showed alcian blue (AB) positive intraluminal mucosubstances (Figure 3). Two cases were devoid of mucin. One case showed intense PAS-positive material in the centre of the lumina surrounded by AB-positive mucin. The quantity of mucosubstances present inside the lumina varied from case to case, being very scanty at one extreme and very abundant at the other. By contrast, we did not notice field changes of mucin pattern in different areas of the same tumour, the amount of mucin being fairly uniform in all tubules. When the tubular lumen appeared devoid of mucin in the H & E preparations, a thin rim of AB-positive material was found coating the luminal epithelial surfaces. Two cases displayed numerous cells with intracytoplasmic lumina (ICL) containing AB- and PAS-positive mucosubstances. The mucosubstances had a target-like appearance identical to that described by Gad & Azzopardi (1975) in lobular carcinoma of the breast. Stroma with plump fibroblasts and AB-positive material at pH 0.5 was observed in I I cases. The remaining four cases had a fibro-elastotic stroma. In addition, vascular and periductal elastosis was present in nine cases. Basophilic calcifications were found in three cases, mainly as psammoma bodies located inside the lumina. Lobular carcinoma in situ was present either within the confines of the tumour or at its margins in no fewer than four cases. Five cases showed in situ carcinoma o f ductal type (DCIS) and in two other cases both LCIS and DCIS were present simultaneously giving a total of seven cases with DCIS and six cases with LCIS. Three cases had axillary lymph nodal metastases. Three lymph nodes were involved in one case, two lymph nodes in another and a solitary lymph node in the third patient. In this last patient there were only two small neoplastic tubules located within the thickened fibrous capsule of a lymph node. All the metastatic deposits showed the morphological features of tubular carcinoma and they involved the proximal group of axillary lymph nodes only. I. Glandular structures displaying lumina of varying dimensions, irregularly orientated. Prominent focal elastosis (top) is also a feature of this tumour. H & E. x 60.
Figure
Figure 2. The tubules are formed by a single layer of cuboidal cells showing ‘apical snouts’. H & E. x 400.
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V .Eusebi, C.M . Belts and G.Bussolati
Figure 3. The small duct in the centre is formed by a two-layer epithelium which rests on a basement membrane. It is surrounded by neoplastic tubules formed by one layer of cells. The lumina are filled with AB-positive material and no basement membrane is evident. AB!PAS. x 2 5 0 .
Casein
All cases were positive for total casein (Figure 4) as well as for the p-casein fraction (Figure 5) using the immunofluorescence and immunoperoxidase staining methods. Although the positive staining for casein was also present freely dispersed within the luminal content, it was mostly present condensed along the luminal surface of the
Figure 4. Casein is observed free in the lumina and condensed along the luminal surface of the cells. Rabbit anti-total casein immunoperoxidase method. x 2 5 0 .
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Figure 5. All tubules contain casein deposited inside the lumina or coating the luminal cytoplasm. Goat anti-human p-casein immunofluorescence. x 100.
Figure 6. Casein coats the free cytoplasmic border of neoplastic cells and it is observed free in the lumen. Same method as Figure 5. x 400. E
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V.Eusebi, C.M .Betts and G.Bussolati
Figure 7. A normal lobule stained for actin with the irnmunofluorescence method: the external cells of the lobular ductules are heavily stained. x 100.
Figure 8. Normal preexisting ductules are surrounded by myoepithelial cells, whilst the neoplastic cells appear unstained. Anti-actin immunoperoxidase method. x 400.
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cells lining the tubules (Figure 6). The only two cases which were found to be negative for mucin, were positive for casein. Some ICL were seen inside the cytoplasm of neoplastic cells in three of the tumours but target-like ‘inclusions’ were most numerous in the two cases which also showed AB/PAS positive intracytoplasmic haloes and dots. Actin
Anti-actin sera stained only the myoepithelial cells of ducts and lobular terminal ductules (Figure 7) as well as the muscular cells in the vascular walls of normal residual breast tissue. All cells of neoplastic tubules appeared unstained and this negative reaction was even more striking when comparison was made with the normal structures present in the same sections (Figure 8).
Figure 9. Tubule formed by one layer of cells. The luminal surface shows a villous differentiation. No myoepithelial cells are present. E.M. x 3000.
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U L T R A S T R U C T U R A L FINDINGS
The neoplastic tubules were formed by a single layer of cuboidal cells with round nuclei showing a prominent nucleolus. The cytoplasm was rich in endoplasmic reticulum and the luminal surface showed numerous villi projecting towards the lumen. Desmosomes were only occasionally found. The neoplastic cells did not show any cytoplasmic filaments and there was no evidence of any myoepithelial differentiation (Figure 9). Discussion Tubular carcinoma of the breast is also referred to as orderly carcinoma (Carstens et al. 1972) or well differentiated carcinoma (Taylor & Norris 1970) and is reported to have a very good prognosis (Carstens el al. 1972). This behaviour is confirmed in the present series. It has characteristic histological and ultrastructural features which have led numerous investigators to regard it as a distinctive type of ductal breast carcinoma (Taylor & Norris 1970, Carstens el al. 1972, Tobon & Salazar 1977). The differentiation of neoplastic tubules with lumina which are sometimes elongated and orientated a t varying angles to one another; the cuboidal cells with or without cytoplasmic snouts; the round to ovoid nuclei with small nucleoli; the virtual lack of atypia and mitoses; the absence of myoepithelial cells both at immunohistochemical and ultrastructural level ; the AB-positive stroma associated with a frequent elastotic reaction (Tremblay 1974); and a high incidence of an associated in situ malignancy are all features of this entity. On histological grounds tubular carcinoma must be differentiated from ‘sclerosing adenosis with pseudoinfiltration’ as emphasized by McDivitt et al. (1968) and Carstens et al. (1972) and from the scleroelastotic lesions described by Hamper1 (1975) and stressed more recently by one of us (Eusebi, Grassigli & Grosso 1976). Tobon & Salazar (1977) reported that it was not uncommon to see myoepithelial cells either isolated or in continuous rows surrounding the neoplastic tubules. This observation was not supported by the ultrastructural studies of Erlandson & Carstens (1973) Jao el al. (1976) and Harris & Ahmed (1977). The present electronmicroscopic and immunohistochemical study also refutes the suggestion that myoepithelial cells are found in tubular carcinoma. Our evidence indicates that it is a purely epithelial tumour. All but two of our cases contained acid mucosubstances in the tubular lumina. When present, mucin tended to be observed uniformly in the majority of tubules of a given case, though the amount present varied widely between different cases. Two cases also showed target-like cytoplasmic inclusions with a PAS-positive core and AB-positive peripheral rim. All 15 cases showed the presence of intraluminal casein and in this respect casein synthesis appears to be a more constant finding than mucin production. Casein appoared free in the lumina and especially coating the cytoplasmic snouts which ultrastructurally showed a villous surface. These differences between mucin and casein synthesis are probably attributable not only to the highly sensitive method employed
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for casein detection but also to the excellent fixation essential for the optimal demonstration of mucin (Gad & Azzopardi 1975). In addition to the recognized distinctive biological, histological and ultrastructural features, tubular carcinoma appears to have an almost constant histochemical pattern which suggests a differentiation towards epithelial secretory cells engaged in intensive milk protein production. It has already been shown that in lobular carcinoma of the breast a large number of neoplastic cells produce mucosubstances (Gad & Azzopardi 1975) and, to an even greater extent, casein (Eusebi et al. 1977). Tubular carcinoma and lobular carcinoma share certain functional parameters of differentiation whilst infiltrating duct carcinoma shows a much more modest production of mucosubstances and casein (Gad & Azzopardi 1975, Pich, Bussolati & DiCarlo 1977). Lobular carcinoma in situ was present in six cases. This association is by no means unique to the present series because two of the 33 cases reported by Taylor & Norris (1970) and no fewer than five of the 35 cases reported by Carstens et al. (1972) showed in situ or infiltrating lobular carcinoma in association with the tubular carcinoma. This association between tubular and lobular carcinoma appears therefore to be more than a fortuitous one. In addition, one of our patients developed contralateral LCIS 6 months after the mastectomy for tubular carcinoma. Six of the 33 patients reported on by Taylor & Norris (1970) developed contralateral carcinomas, two of which were also tubular carcinomas. It appears, therefore, that tubular carcinoma has a high incidence of bilateral involvement which is also a well known feature of lobular carcinoma (Hutter & Foote 1969, Andersen 1974). Recently Haagensen et al. (1978) studied 38 carcinomas which developed subsequently to LCIS. Three cases were pure tubular carcinomas and in another two cases tubular carcinoma was associated with infiltrative lobular carcinoma. Furthermore, Martinez & Azzopardi (I979), in a study of 30 infiltrative lobular carcinomas have found, in seven cases, a further and separate invasive carcinoma in the same or the other breast. Four of these were tubular, two infiltrative lobular and one of uncertain classification. Lobular and tubular carcinoma are frequently associated together in the same or opposite breast; have a similarly high incidence of bilateral involvement; and in addition they share a similar functional differentiation. Both usually produce mucosubstances and casein and, at ultrastructural level, their constituent cells show an almost constant villous absorptive type of surface. Fisher et al. (1977) described the tubulo-lobular carcinoma as having histological and histochemical features of both infiltrative lobular and tubular carcinoma and they suggested that this entity could be regarded as an intermediate form of differentiation between the two structurally different tumours. The evidence presented by us points in the same direction and the concept that tubular carcinoma is necessarily a form of ductal carcinoma as currently believed needs reappraisal. Our findings suggest that both infiltrative lobular carcinoma and tubular carcinoma can be regarded either as divergent types of a specialized form of secretory breast carcinoma or, possibly inore likely, as two distinct variants of the same entity: the classical infiltrating lobular carcinoma representing the least differentiated variant
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whilst tubular carcinoma achieves a high degree of morphological as well as functional differentiation.
Acknowledgements The authors wish to thank Professor J.G.Azzopardi for critically reviewing this manuscript and Professor A.M.Mancini for assistance in its preparation. The authors are also grateful to Dr M.Monaco at the NCI, Bethesda, USA, for supplying the goat anti-human B-casein antisera and Dr G.F.Voglino at S . Anna Hospital, Torino for giving the guinea-pig anti-human B-casein antisera. Acknowledgement is also due to Dr Mary Osborn, at Max Plank Institute, Gottingen, West Germany, for supplying rabbit anti-actin antibodies. Mr M.Mariani has to be thanked for photographic assistance and Ms D.M.James for secretarial help. The work was supported by a CNR grant no. 79.00624.96, Ronie.
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