Stromal Changes in Early Invasive Breast Carcinoma

Path. Res. Pract. 186, 70-79 (1990)

Stromal Changes in Early Invasive Breast Carcinoma An Immunohistochemical, Histoenzymological and Ultrastructural Studyl,2 G. Chomette, M. Auriol, P. Tranbaloc and J. Blandon Department of Pathology (Prof. G. Chomette), Hopital de la Pitie, Paris, France; Department of Gynecological Surgery (Prof. J. Blondon), Hopital de la Salpetriere, Paris, France

SUMMARY The purpose ofthis work was to detect in periductal connective tissue ofbreast carcinoma in situ changes induced by intraductal tumor cells before any dehiscence in basement membrane. Histological, electron microscopic, immunohistochemical and histoenzymological methods were used in 3 carcinomas in situ, 4 microinvasive carcinomas and 13 control invasive carcinomas. We could demonstrate a high functional activity of fibroblasts with secretion of mucopolysaccharides and type III collagen around intraductal carcinomas. These changes occurred simultaneously to those ofthe basement membrane which was either thinning or thickening. Any dehiscence in basement membrane secondarily induced in this periductal stroma the usual changes seen in invasive stroma, peculiarly numerous vascular pedicles, myofibroblasts, elastic material. These periductal stromal changes are interpreted according to recent concepts about the possible influence of tumor cells upon their environment: stimulation of mitotic and metabolic activity of fibroblasts: stimulation of angiogenesis by means of an angiogenic factor.

Introduction Recently, the stroma of invasive breast carcinomas was studied by many authors. In order to improve histoprognostic criteria, several methods such as electron microscopy and immunohistochemistry were used to detect the possible influence of invasive tumor cells on the composition of this stroma, especially on the abundance of myofibroblasts 30- 32 and of some interstitial substances (elastin, fibronectin) 10-12, 20, 34.

1 2

Dedicated to Prof. h.c. Franz Buchner on the occasion of his 95th birthday, January 20, 1990. This work was supported by a grant from "Ie comite de Paris de la ligue nationale fran~aise contre Ie cancer".

0344-0338/90/0186-0070$3.50/0

Ductal carcinomas in situ or early invasive carcinomas were also studied by means of immunohistochemist ry2,8,21,25 and electron microscopy23. The main purpose of these studies was to detect the distortion and loss of continuity of periductal basement membranes and to discuss their prognostic and therapeutic incidences5. As a matter of fact, these anomalies were associated with changes in periductal conjunctive tissue 4,24,29. The aim of the present investigation was to further analyse the general features of periductal connective tissue and its vascular component in intraductal carcinoma in situ and microinvasive carcinoma compared with invasive carCInoma. Besides, we tried to correlate these stromal changes with some characteristics of the epitheliomatous component, peculiarly with its evolutive potential. ©

1990 by Gustav Fischer Verlag, Stuttgart

Early Invasive Breast Carcinoma . 71

Material and Methods a) Material The material used in this study consisted of 3 breast carcinomas in situ, 4 microinvasive carcinomas and 13 invasive breast carCInomas. b)

Methods

Histological examination was made on sections cut from paraffin blocks and stained with haematoxylin and eosin, alcian blue and P.A.S. (for mucines), Weigert's methods (for elastin) and Masson's trichromic stain. Specimens were also taken for histoenzymological investigation. They were immersed in liquid nitrogen and sections were obtained using a cryostat. According to Pearse's methods 26 , enzyme activities of alkaline phosphatase were tested on thick sections (30 microns) for the demonstration of vessels. Leucine aminopeptidase was also studied for the appreciation of functional activity of fibroblasts. Immunohistochemical examination was performed on fresh frozen cryostat sections. We used the following tests: - anti-laminin serum (Gibco BRL), diluted 1:100. Indirect immunofluorescence technique. - anti-type I collagen serum diluted 1: 100 anti-type III collagen serum diluted 1: 1000 anti-type IV collagen serum diluted 1:5 anti-fibronectin serum diluted 1:20 All these sera were obtained from the Institut Pasteur Lyon (Docteur Grimaud) and were used according to indirect immunofluorescence technique. anti-transferrin receptor (Becton-Dickinson), direct fluorescence technique. Expression of this receptor on the tumor cell surface correlates with cellular proliferation, being highest in rapidly dividing cells and much lower in resting cells. - antibody Ki67 (Transbio) diluted 1:10, immunoperoxidase technique. This new antibody labels the nuclei of proliferating cells. It specifically recognizes a human nuclear epitope present in cells in the S,G2,M and postmitotic G1 phases of the cell cycle. It is not expressed in GO (resting) cells. Ultrastructural examination was carried out on tissue fixed immediately after surgical excision in 4 per cent glutaraldehyde cacodylate solution, post-fixed in osmium tetroxide and embedded in epon. Thin sections, cut on LKB ultramicrotome, stained with uranyl acetate and lead citrate, were examined with a Hitachi H 300 microscope.

2) By immunohistochemistry we could study ductal basement membranes, periductal connective tissue and vessels (Table 1). Basement membrane, well-demonstrated by anti-type IV collagen and anti-Iaminin sera, showed several features: .It was continuous in 3 cases. In case 2, it remained normal, thin and regular (Fig. 2). In cases 1 and 3, it was thickened. Either this thickening was intense but regular or it was moniliform with succession of thick and thin areas. In the four cases of microinvasive carcinoma, dehiscences were detected (Fig. 3) (small clefts or large gaps). Between these dehiscences basement membrane was usually irregularly thickened, moniliform (3/4 cases). The periductal connective tissue was studied using anti-fibronectin and anti-collagen sera type I and III. In all cases, type I collagen was not quantitatively modified near ducts compared with that of normal breast. On the other hand, in the 3 intraductal carcinomas, type III collagen fibres were increased in number. They were regularly arranged around carcinomatous ducts. Fibronectin was present in the whole periductal connective tissue as a bright fibrillar fluorescence and obscured the adjacent ductal basement membrane (Fig. 4a). In the 4 microinvasive carcinomas, the feature was quite similar. But, in front of dehiscences of the basement membrane, collagen fibres were irregularly arranged, sometimes perpendicularly to the ductal lumen and thus broke the continuity of circular fibres. Fibronectin was also irregularly arranged (Fig. 4b). Vessels were obvious after anti-Iaminin and anti-collagen sera type IV. They were increased in number but regularly located in periductal connective tissue. They often were close to the basement membrane with which they seemed to fuse (cases 2 and 3). In microinvasive carcinomas, vessels were irregularly scattered with interruptions by perpendicular collagen fibres. Some of them had thick walls (3 cases).

Results We report here stromal changes firstly in intraductal and microinvasive carcinomas, secondly in invasive carcinomas.

I Intraductal Carcinoma in Situ and Microinvasive Carcinoma 1) By histological examination there were 3 intraductal carcinomas (1 comedocarcinoma, 1 papillary and 1 tubuloglandular carcinoma) and 4 microinvasive carcinomas (Fig. 1) (among them 1 comedocarcinoma). The periductal sheat, rarely thickened, was almost always alcianophilic (with a high intensity in 3 cases). Elastic fibers were lacking except for 2 microinvasive carcinomas which showed large deposits of elastin against basement membrane and in adventitia of arteriolae and veinulae.

Fig. 1. Microinvasive carcinoma (case 4). Dense epitheliomatous structures with small microinvasive evaginations (-.) in periductal stroma. Haematoxylin and eosin x 160.

72 . G. Chomette et al. Table 1. Study of 7 in situ or microinvasive breast carCInomas. Aspect of connective periductal tissue. Histological and immunohistochemical results Type of carcinoma

Histology Alcian blue Elastin

Immunohistochemistry Basement membrane Type III collagen and fibronectin

Vessels

Thickened

Concentric

Concentric

Thin

Concentric

Concentric

Thickened

Concentric

Concentric

4 - Microinvasive carcinoma

± ++ ++ ++

Thickened with dehiscences

Concentric with focal distortion

Irregular repartition

5 - Microinvasive carcinoma

+

Concentric + focal distortion

Irregular repartition

6 - Microinvasive carcinoma

±

Thickened + dehiscences

Concentric + focal distortion

Irregular repartition

7 - Microinvasive carcinoma

+

Thickened + dehiscences

Concentric focal distortion

Irregular repartition

1 - Carcinoma in situ 2 - Carcinoma in situ 3 - Carcinoma in situ

+

+

Thin

+ dehiscences

i

Fig. 2. Intraductal carcinoma (case 1). Periductal thin and continuous basement membrane. Fluorescence after anti-laminin serum. Immunohistochemistry X 250.

Fig. 4 a. Intraductal carcinoma (case 2). After anti-fibroneetin serum, fibrillary fluorescent network is seen in periductal stroma and in basement membrane (....). Immunohistochemistry x250.

Fig. 3. Microinvasive carcinoma (case 5). Thickened and irregular basement membrane with a large dehiscence. Fluorescence after anti-laminin serum. Immunohistochemistry X 250.

Fig. 4 b. Microinvasive carcinoma (case 5). After anti-fibronectin serum, irregular fluorescence around ducts (0) and especially around vessels. Immunohistochemistry x 250.

Early Invasive Breast Carcinoma . 73

3) Histoenzymological methods were used to study vessels and functional activity of fibroblasts (Table 2): Alkaline phosphatase activity was higher in vessels surrounding carcinomatous ducts than in normal mammary parenchyma. In the 3 carcinomas in situ, numerous capillaries were detected around ducts (Fig. 5). In 2 cases, they were placed against basement membrane and in 1 case, alkaline phosphatase activity was diffusing to it. In microinvasive carcinoma, large sinuous pedicles were sometimes seen in front of dehiscences of the basement membrane (Fig. 6). They were located either near ducts or further away (cases 4 and 6). Leucine aminopeptidase activity was high in fibroblasts, especially in 2 carcinomas in situ (cases 2 and 3) and in Fig. 6. Microinvasive carcinoma (case 6). Alkaline phosphatase activity in irregularly arranged vessels around cancerous ducts (*) and true vascular pedicles. Histoenzymology X 250.

Fig. 5. Intraductal carcinoma (case 2). Cancerous duct (-+). Strong activity of alkaline phosphatase in vessels which are regularly concentrically arranged around duct. Histoenzymology x250.

front of basement membrane dehiscences of all microin. . vaSlve carCInomas. 4) Ultrastructural study (Table 2): In carcinomatous ducts, tumor cells showed various features according to their variable differentiation. Their proliferative activity was often expressed by nuclear anomalies, especially by large and numerous nucleoli. Myoepithelial cells characterized by myofilaments and desmosomal junctions were still present in 2 cases of intraductal carcinomas (Fig. 7) (cases 1 and 2). Basement membrane exhibited 3 different features: In case 1, it was regularly thickened, and highly electron-dense (Fig. 8). In 3 cases, it was thin (Fig. 9), similar to a normal lamina densa with 2 to 3 thin replicated lamellae. Besides, small dehiscences (100 to 300 nanometers) and cytoplasmic evaginations of tumor cells were detected (Fig. 10).

Table 2. Periductal connective tissue in 7 intraductal and microinvasive carcinomas - histoenzymological and ultrastructural results Type of carcinoma

Alkaline phosphatase (vessels)

1 - Carcinoma in situ

Small vessels

2 - Carcinoma in situ

Small vessels

+++ ++ +++

Basement membrane

Myofibroblasts

Myoepithelial cells

Linear membrane

+

Thinning duplicata

+

Irregular thickening granulo-filamentous deposits

3 - Carcinoma in situ

Small vessels

4 - Microinvasive carcinoma

small vessels pedicles

+

Irregular thickening granulo-filamentous deposits

5 - Microinvasive carcinoma

Samll vessels pedicles

++

Thinning duplication

6 - Microinvasive carcinoma

Small vessels ± pedicles

Irregular thickening granulo-filamentous deposits

7 - Microinvasive carcinoma

Small vessels ± pedicles

Thinning

+

+

Fig. 7. Intraductal carcinoma (case 1). Myoepithelial cell (ME) with microfihrils .... and hemidesmosomes in periphery of a cancerous duct. T.E.M. x 16000.

Fig. 8. Intraductal carcinoma (case 1). Homogeneous thickened electron opaque basement membrane (....) around a cancerous duct. T.E.M. x 6000.

Fig. 9. Intraductal carcinoma (case 2). Thin periductal basement membrane with reticulations (....). In periduetal stroma, globulous fibroblastic expansions (*), surrounded by granulo-filamentous material. T.E.M. x 18000.

Fig. 10. Microinvasive carcinoma (case 5). Dehiscence of a thin basement membrane with passage of a tumor cell (TC). In periductal connective tissue, activated fibroblat (F). T.E.M. x 7000.

Fig. 11. Intraductal carcinoma (case 3). Irregularly thickened basement membrane with granulo-filamentous deposits (-.); activated fibroblasts (F) in periductal connective tissue. T.E.M. x 10000.

Fig. 12. Intraductal carcinoma (case 2). Vessels (V) in periductal connective tissue; one of them is against basement membrane of a cancerous duet. T.E.M. x 4000.

Early Invasive Breast Carcinoma . 77

In other cases, basement membrane was hardly seen among irregular granulo-filamentous deposits spreading like needles towards fibroblasts and surrounding vessels (Fig. 11). Dehiscences occurring in these cases were almost always large. In periductal connective tissue, numerous globular fibroblasts were found in the lining basement membrane. These cells seemed very active: they were filled with numerous organites (mitochondriae, ergastoplasmic cisternae) and their obvious cytoplasmic expansions were surrounded by granular-filamentous material. In addition, some myofibroblasts were detected in 3 microinvasive carcinomas. They were characterized by myofilaments, endocytotic vacuoles and focal densities. They were lined with discontinuous linear aggregates of thinly granular material. Beyond collagen fibres, intercellular tissue contained numerous microfibrilla. Some rare newly formed immature areas of elastic tissue were also seen. They were characterized by a high microfibril/elastic ratio. They were located either along fibroblasts or around vascular walls. Vessels were numerous, especially against ductal basement membranes (Fig. 12). They were lined by a turgescent endothelium and their subendothelial basement mambrane was often replicated. In microinvasive areas, connective tissue was quite different. Fibroblasts were more numerous and irregularly arranged. Myofibroblasts were also more abundant (Fig. 13). Besides, larger areas of elastic tissue were invaginated in cytoplasmic expansions of fibroblasts and myofibroblasts.

5) Immunohistochemistry of tumor cells (Table 3)

Proliferative activity of carcinomatous cells was studied by means of the monoclonal antibody Ki67. A positive

Fig. 13. Microinvasive carcinoma. In front of a dehiscence of basement membrane, active myofibroblasts with well-developed ergastoplasm and numerous intracytoplasmic myofibrils. T.E.M. x 7000.

Table 3. Expression of antitransferrin receptor and antibody Ki67 on tumoral cells (percentage of labeled cells). Comparison between intraductal, microinvasive and invasive brest carcinomas In . situ. and . mlcromvaslve carcinomas 7 cases

Invasive carcinomas

13 cases

Anti transferrin receptor

5 -15%

3 -10%

Antibody ki 67

10 - 40%

8 -34%

staining was found in all cases, but with wide variations from one carcinoma to another, with a range of 10-40% of tumor cells. This percentage was lower in intraductal carcinomas than in microinvasive carcinomas. Transferrin receptor expression was low (percentage of positive tumor cells variable with a range of 5-15%). There were nor correlations of this percentage with various types of carcinoma. However, the percentage of positive cells was higher in the periphery of carcinomatous ducts, near their basement membrane.

II Invasive Carcinoma By histological study, 3 of the 13 cases were welldifferentiated tumors. The others were of the trabecular type. In the periphery of these tumors, few ductal foci of cancerisation were seen. Stroma was highly a1cianophilic. Elastin was abundant around veins and ducts and sometimes around epitheliomatous trabeculae.

78 . G. Chomette et al. By immunohistochemistry, laminin and type IV collagen were sometimes present around residual ducts with large dehiscences in their walls and around numerous thickened vascular walls. Some newly-formed basement membranes surrounded well-differentiated epitheliomatous structures. Type III collagen was irregularly distributed around neoplastic trabeculae. By histoenzymological methods, alkaline phosphatase activity was only found in scarce vessels in fibrous stroma. By electron microscopy, the feature was quite similar to that seen in microinvasive carcinoma. However, myofibroblasts and areas of elastic tissue were more numerous, Vessels showed a thickened wall and a replicated basement membrane. As to the proliferative potential of tumor cells, it seemed to be a bit lower than that noted in the first group of carcinomas (antibody Ki67:8 to 34%; anti-transferrin receptor: 3-10%). Table 3). Discussion 7 cases of intraductal in situ and microinvasive breast cancers (Fig. 1) were analysed. Since their in situ phase, they exhibited a true stroma circumscribing carcinomatous ducts (Fig. 16). This stroma, well demonstrated by means of histoenzymological, immunohistochemical and electron microscopic techniques was composed of numerous hyperactive fibroblasts with high activity of leucine aminopeptidase27 . These globulous cells were provided with many organites and cytoplasmic expansions (Figs. 9, 11)24. They secreted a large amount of alcianophilic proteoglycanes29 . Besides, they also produced granulofilamentous material seen by electron microscopy around cells and immunohistochemically corresponding to fibrils of type III collagen regularly arranged around ducts. It should be noted that fibronectin which maintains cohesion of collagen fibrilles 22 , peculiarly of type III collagen7, outlined the disposition of these fibrilles, scattering in the whole periductal connective tissue and also near ductal basement membranes (Fig. 4a). Vessels were also numerous in this stroma. They were lined with a turgescent endothelium and their functional activity was high, as demonstrated by the strong positivity of alkaline phosphatase28 . These vessels regularly circumscribing ducts (Fig. 5) were sometimes interwined to their wall. In apparently more advanced forms, these vessels, well outlined by antilaminin and anti type IV collagen sera, were limited by a thickened, inhomogeneous, often replicated basement membrane. This finding can prove a persistent regeneration of vascular endothelium after aggression by some agents secreted by tumor cells 8• The feature of epithelial-stromal junction, in other words of the basement membrane, well seen using antifibronectin serum, was variable in intraductal-like microinvasive areas. Except one case where persistent functional activity of myoepithelial cells (Fig. 7) could explain the thickening of basement membrane, changes of the basement membrane were always correlated with modifications of periductal connective tissue. In one case, vessels

were juxtaposed (Figs. 2, 12) or incorporated in the basement membrane and alkaline phosphatase activities were diffuse from these vessels towards basement membrane. In the other cases, changes were resulting in an abundant secretion of granulo-filamentous material (Fig. 11) by fibroblasts (perhaps precollagen or type III collagen). On the whole, before any dehiscence of basement membrane, intense changes of periductal connective tissue were obvious and probably influenced the behaviour of basement membrane towards cancerous cells still enclosed in ducts. Microinvasion, following rupture of the basement membrane, induces new changes in periductal connective tissue. According to the feature of that membrane, dehiscence had different aspects. In thin and possibly replicated membranes, dehiscences were usually small (100 to 300 nanometers) and cytoplasmic expansions of tumoral cells were passing through them (Fig. 10)23. In the thickened basement membranes, dehiscences were generally larger (Fig. 3). In front of them, periductal connective tissue exhibited obvious and early changes. Fibroblasts were irregularly oriented. The alcianophilic interstitial frame contained areas of immature, highly fibrillar elastic tissue34 scattered in periductal tissue or accumulated around vessels (principally veins)l. Near the dehiscences, beyond activated fibroblasts 24, we could see flexuous myofibroblasts by electron microscopy (Fig. 13). Vessels lost their regular arrangement around ducts and formed large pedicles often perpendicularly disposed towards the ducts (Fig. 4b, 6). Thus we discussed which factors may interfere in these periductal stromal changes and even in those of the basement membrane during the earliest phase of intraductal and microinvasive cancerous evolution. In spite of intact basement membrane, the influence of tumor cells upon adjacent connective tissue is admitted by several authors29 . Tumor cells could induce mitotic and functional activity of fibroblasts by means of cytokines 16, perhaps of estrogens 17,24 and may be a true molecular hybridization 15. The intense angiogenesis seen in peri-ductal stroma, without doubt, is also dependent upon factors secreted by tumor cells. The numerous vessels occurring in periductal areas are certainly consecutive to the secretion of an angiogenic factor 13 , whose role, proved in other types of carcinoma9, is demonstrated by experimentation 14. The progressive increasing influence of this factor results in the constitution of true vascular pedicles peculiar to the microinvasive stroma. The most curious morphological fact is the occurence, after dehiscence of the basement membrane, in periductal stroma of elastic lamellae4,5 and of myofibroblasts3, which are considered by many authors as a sign of early invasion. The meaning of those facts remains unclear. The occurrence of myofibroblasts is well known in tissue conditions of repair after destruction of subepithelial basement membrane 35 . In other words, changes of periductal connective tissue would be related to the general concept of conjunctive reparation already invoked for the stroma of invasive carcinomas 18 . As to the proliferative potential of tumor cells, hardly appreciated by the study of 2 factors of activation, the

Early Invasive Breast Carcinoma . 79

monoclonal antibody Ki67 6, 19 and the transferrin receptor expression33 ,36, its role in the building of a periductal stroma is difficult to prove. At least, when very active, this proliferative potential could induce early rupture of the basement membrane and substitution of a usual invasive stroma to the preexistent periductal stroma. References 1 Adnet 11, Pinteaux A, Caulet T, Hibon G, Jetit J, Pluot M, Roth A (1976) L'elastine dans les cancers du sein. Etude anatomo-clinique, histochimique et ultrastructurale. Ann Med de Reims, 13: 147-153 2 Albrechtsen R, Nielsen M, Wewer U, Engvall E, Ruoslahti E (1981) Basement membrane changes in breast cancer detected by immunohistochemical staining for laminin. Cancer Res 41: 5076-5081 3 Alon Y, Horowitz AT, Biran S, Weiss OW, Doljanski F (1986) Immunofluorescent characterization of fibronectin, laminin, and keratin in normal and neoplastic human mammary epithelial cells in culture and in breast tissue sections. Int J Tiss React 8: 401-410 4 AzzopardiJG, Laurini RH (1974) Elastosis in breast cancer. Cancer 33: 174-183 5 Azzopardi JG (1979) Problems in Breast Pathology. WB Saunders, Philadelphia-London 6 Barnard NJ, Hall PA, Lemoine NR; Kadar N (1987) Ki67 determined growth factor in breast carcinoma: relation to clinical and pathological variables. J Path 152: 287-295 7 Birembaut P, Gaillard 0, Adnet 11, Dousset H, Kalis B, Leuteneccer M, Labat-RobertJ, Robert L (1981) La fibronectine. Ann Pathol 1: 140-146 8 Charpin C, Lissitzky JC,Jacquemier J, Lavaut MN, Kopp F, Pourreau-Schneider N, Martin PM, Toga M (1986) Immunohistochemical detection of laminin in 98 human breast carcinomas: a light and electron microscopic study. Hum Pathol 17: 355-365 9 Chomette G, Auriol M, Tranbaloc P, Blondon J (1989) Neoplasie intra-epitheliale du col uterin et angiogenese: etude morphologique. Arch Anat Cytol Path 37: 73-79 10 Clavel Ch, Birembaut Ph, Adnet 11, Foidart JM (1988) Carcinome mammaire et matrice extracellulaire. Ann Pathol 8: 107-113 11 Davies JD, Mera SL (1987) Elastosis in breast carcinoma. II Association with immature elastic fibres. J Path 153: 317-324 12 Fisher ER, Gregorio RM, Fisher BN (1975) The pathology of invasive breast cancer. Cancer 36: 1-85 13 Folkman J (1986) How is blood vessel growth regulated in normal and neoplastic tissue. G.H.A. Cloves Meorial Award Lecture. Cancer Res 46: 467-473 14 Gimbrone MA, Gullino PM (1976) Neovascularization induced by intraocular xenografts of normal, preneoplastic and neoplastic mouse mamary tissues. J National Cancer Inst 56: 305-310 15 Horak E, Mc Gee JOD (1987) Coordination of tumor and stromal cell growth. Interaction with malignant cells results in oncogene expression in host stromal fibroblast. J Pathol (Abstract) 151: 39 A 16 lozzo RV (1985) Proteoglycans: structure, function and role in neoplasia. Lab Invest 53: 373-396

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Received July 14, 1989 . Accepted August 1, 1989

Key words: Microinvasive and in situ breast carcinoma - Periductal stroma - Breast tumor - Connective tissue Prof. G. Chomette, Department of Pathology, H6pital de la Pitie, 83, Boulevard de I'H6pital, 75013 Paris, France