JOURNAL OF PATHOLOGY, VOL. 150: 43-49 (1986)

STROMAL CHANGES IN EARLY INVASIVE AND NON-INVASIVE BREAST CARCINOMA

A N ULTRASTRUCTURAL STUDY SALAH 0. TAMIMI A N D ALI AHMED

Department of Pathology, University Medical School, Manchester, MI3 9PT, U.K.

SUMMARY

Received 1 April 1986 Accepted3 June 1986

Six examples of histologically diagnosed, non-invasive breast carcinomas were studied by electron microscopy to elucidate the ultrastructural features for an accurate diagnosis of in situ carcinoma. The results obtained revealed two patterns of basal lamina/stromal cells relationship. One pattern showed intact basal lamina with associated periductal stromal cells consisting entirely of fibroblasts, the other pattern showed disruption of basal lamina by gaps and malignant cell protrusions with associated stromal cells consisting of both fibroblasts and myofibroblasts. As myofibroblasts are not a component of normal breast stroma but are known to be a prominent feature in the stroma of infiltrating breast carcinoma, the present observations suggest that myofibroblastic proliferation around in situ carcinoma represents an early sign of carcinomatous infiltration. Hence the definitive diagnosis of non-invasive carcinoma of the breast requires an intact basal lamina and a complete absence of a myofibroblastic reaction.

KEY woms-Carcinoma of the breast, early infiltrating carcinoma, myofibroblasts, ultrastructure of breast carcinomas.

INTRODUCTION

In situ breast carcinoma refers to the pathological condition in which the neoplastic epithelial cells are confined to the pre-existing parenchymatous struc- ture without evidence of penetration of the base- ment membrane. Detecting the disease in the in situ phase is very important since the method of treat- ment is less aggressive and the cure rate is high at this stage.' Unfortunately, in the diagnosis of in situ carcinoma it is difficult to be certain that all the malignant cells are confined within the paren- chymatous structure. Even after a thorough examination of the whole radical mastectomy speci- men, histological evidence of stromal invasion by carcinoma cells may not be demonstrated, yet metastases are found in regional lymph nodes.z

As the early neoplastic cell invasion may not be easily detected on histological examination, efforts

Addressee for correspondence: Dr A. Ahmed, Department of Pathology, The Medical School, University of Manchester, Manchester M13 9PT, U.K.

0022-341 7/86/090043-07$05.00 0 1986 by John Wiley & Sons Ltd.

have been directed to the identification of changes that indicate early invasion. Four changes have been described, namely inflammation, elastosis, col- lagenosis3 and basement membrane/basal lamina alteration^.^,^,^ However, according to some authors these changes have been shown to be of relatively limited diagnostic value.1,6,7

The purpose of the present study is to elucidate the ultrastructural features that accurately identify non-invasive breast carcinoma.

MATERIALS AND METHODS

Six examples of histologically diagnosed in situ ductal carcinomas were studied by electron microscopy. Suitable tissues were selected, cut to 1 mm cubes and fixed in glutaraldehyde buffered with sodium cacodylate, then post-fixed in osmium tetroxide, dehydrated in graded alcohol and embed- ded in Araldite. Thick sections were cut from multiple blocks and stained with toluidine blue for light microscopic examination. Ultrathin sections were cut from each block, double stained with

44 S. 0. TAMIMI AND A. AHMED

uranyl acetate and lead citrate. Sections were examined on a Philips 301 transmission electron microscope operating at 60KV.

tumour cells. The walls were lined by variable amounts of fibrous tissue as well as spindle cells and aggregates of lymphocytes.

Ultrastructurally the tumour cells contained pale cytoplasm and few cytoplasmic organelles. The nuclei were large with prominent nucleoli and smooth nuclear membranes. Flattened myoepithelial cells were observed forming a discon- tinuous layer at the periphery of the ducts. The

RESULTS

At light microscopy, the in situ carcinomas were formed of several dilated ducts filled with large

Fig. 1 -Electron micrograph of part of a duct containing malignant cells. The myoepithelial (My) basal plasma membrane is flat- tened. The basal lamina shows multiple defective areas (arrows). The periductal stromal cells are myofibroblasts (Mf) forming multilayering adjacent to the disrupted basal lamina. x 4600

STROMAL CHANGES IN BREAST CANCER 45

Fig. 2-A tumour cells (TC) is seen protruding from the duct to the periductal stroma. Note the rnyofibroblasts (Mf) adjacent to the tumour cell. x 6100

myoepithelial basal membrane was straight and hemidesmosomes were few or absent. In four exam- ples of in situ carcinoma the basal lamina surround- ing the ducts was intact. However, in the other two examples the basal lamina was often intact but thin and indistinct and occasionally there were demon- strable defective areas which ranged from small gaps to focal absence of the basal lamina (Fig. 1). Rarely tumour cell processes were observed pro-

truding into the stroma through the defective basal lamina (Figs 2,3).

The stromal cell population around the ducts of in situ carcinoma showed variations in the cell type in different lesions and sometimes around different ducts of the same lesion. In four examples which showed no defects of the basal lamina the stromal cells were entirely composed of fibroblasts and des- pite careful search of multiple blocks and serial sec-

46 S. 0. TAMIMI AND A. AHMED

Fig. 3GHigher magnification of Fig. 2. The tumour cell process protruding between the myoepithelial cells (My) and a defective ‘gap’ in the basal lamina (arrows). Note the adjacent myofibroblasts with distended endoplasmic reticulum and myofilaments with dense bodies (short arrow). x 22 800

tions no myofibroblasts were demonstrated. layers around the ducts. The nearest cells often However, in the other two examples with basal occupied the position of the delimiting fibroblasts lamina defects, a spectrum of stromal cells with and were in close proximity to the basal lamina ultrastructural features of both fibroblasts and (Fig. 4). Focal concentrations of stromal cells were myofibroblasts were observed. Fibroblasts and frequently observed around the ducts which myofibroblasts were often arranged in concentric demonstrated defective basal laminae (Figs 1,3) .

STROMAL CHANGES IN BREAST CANCER 47

The fibrous stroma around in situ carcinoma was loosely formed of fine fibrillary material and sparse collagen and elastic fibres (Fig. 4).

DISCUSSION

The accurate diagnosis and the behaviour of in situ breast carcinoma still remain problematic. Pre- vious ultrastructural studies have noted defective basal laminae and protrusion of cancer cells as evi-

dence of early The present observations suggest an additional feature of the presence of myofibroblasts in relation to the invasive process.

Myofibroblasts originally describe in granulation tissueYJO are modified fibroblasts exhibiting morphological features of both fibroblasts and smooth muscle cells. Myofibroblasts have been ob- served in many non-neoplastic’ 1-13 and neoplastic 1esi0ns.l~ l 6 Myofibroblasts have also been noted in the stroma of infiltrating breast carcinoma^.'^-^^

Fig. 4-Periductal stromal cells. Both fibroblasts and myofibroblasts are concentrated in layers parallel to the duct wall. The stroma contains newly formed elastic fibres and scattered aggregates of collagen fibres. x 7600

48 S. 0. TAMIMI A N D A. AHMED

Since myofibroblasts are not normally present in the breast stroma, it can be concluded that these cells appear at some stage during the development of breast carcinoma.

The present study has shown the presence of myofibroblasts in two examples of histologically non-invasive carcinoma which at ultrastructural level showed disruption of basal lamina. In contrast no myofibroblasts or basal lamina defects were demonstrated in the other four cases of histological non-invasive carcinoma.

Previous ultrastructural studies have disagreed regarding the basal lamina around non-invasive breast carcinomas. Some authors noted an intact basal lamina,20.21 while others found basal lamina

Ozzello and Sanpitak4 suggested that such disagreements are more likely to be apparent than real, since great variation can be found in the behaviour of the basal lamina from one case to another and even within a single tumour when multiple blocks are examined. In the present study, careful examination of the basal lamina around non-invasive carcinoma revealed an interesting relationship to the stromal cells. In the non-invasive lesions which showed no associated myofibroblasts, the basal lamina was always intact and no gaps were detected. In the lesions where myofibroblasts were present, however, the ducts showed basal lamina gaps although occasionally the basal lamina appeared to be intact. These observations suggest a possible causal relationship between the basal lamina changes and the myofibroblastic reaction. V r a ~ k o ~ ~ in a detailed review of the basal lamina stated that the presence of a basal lamina defines the special relationship among similar and dis- similar cells and between these cells and the connec- tive and supportive tissues. Also the basal lamina was said to determine the final outcome of repair in injured tissue since if the basal lamina was intact, the replenishment of cells occurred in an orderly way along the framework of the basal lamina. How- ever, if the basal lamina was destroyed the healing process resulted in granulation tissue formation and eventual scar formation.23 MajnoZ4 also found that the form of wound healing depended upon the integrity of the basal lamina. In open wounds myo- fibroblasts were demonstrated and the wound healed with a scar while in linear wounds only fibroblasts were seen and the wound healed without formation of scar. In breast carcinomas the process of early carcinomatous infiltration appears to resemble wound healing. The basal lamina of the ducts containing in situ carcinoma is damaged or

destroyed. Such destruction of the basal lamina triggers the formation of myofibroblasts. In cases where the basal lamina appeared intact it is possible that changes produced by chemical mediators ~

altered the integrity of the basal lamina causing stimulation of myofibroblasts without morphologi- cal change. It is also possible that basal lamina gaps represent a late stage of basal lamina destruction.

The present observations clearly demonstrate that the myofibroblastic proliferation around in situ carcinomas represents an early sign of invasion. As myofibroblasts are more easily detectable than gaps in the basal lamina and malignant cell protrusions, their identification should be of value in the accu- rate diagnosis of non-invasive carcinomas. Moreover at light microscopy the presence of a rim of loose stroma with multilayered spindle cells around the ducts can be considered as suggestive of early infiltration and such lesions should not be designated as non-invasive without ultrastructural examination. At the ultrastructural level the diagnosis of a non-invasive breast carcinoma requires the presence of an intact basal lamina around the duct and a complete absence of myo- fibroblasts in the periductal stroma. It should, how- ever, be emphasized that, although myofibroblastic proliferation represents a significant differentiating feature between non-invasive and invasive breast carcinomas, it is not of value in distinguishing between all benign and malignant breast lesions since myofibroblastic proliferation has been sug- gested in infiltrating epi thel iosi~~~ and demonstra- ted in radial scars in which basal lamina defects were also observed.26 Myofibroblastic proliferation has also been observed in duct ectasia, fat necrosis and breast absce~s.~’ Accordingly, it is possible to sug- gest that the inductive factors for myofibroblastic proliferation are products of injury to connective tissue, a feature common to reactive and benign breast lesions, as well as infiltrating breast carcinoma.

Myofibroblasts are known to have well- developed synthetic and contractile proper tie^.^^,^^ The presence of myofibroblasts within the periduc- tal collagenosis and elastosis seen in non-invasive carcinoma suggests that these cells are responsible for the formation of the dense fibrous tissue, thereby attempting to heal the injury produced by the invading malignant cells and to localize the car- cinomatous process. The degree of such stromal reaction, as for example seen in so-called regressive in situ c a r ~ i n o m a , ~ ~ . ~ ~ may reflect an effective body response and therefore be of prognostic value.

STROMAL CHANGES IN BREAST CANCER 49

ACKNOWLEDGEMENTS

Dr S. 0. Tamimi is supported by a grant from the University of Jordan.

REFERENCES 1. Azzopardi JN. Problems in breast pathology. In :

Major Problems in Pathology. Vol. 11. London: W. B. Saunders Co., 1979; 266-273.

2. Ozzello L. The behaviour of basement membranes in intraductdi carcinoma of the breast. Am J Pathol

3. Bonser GM, Dossett JA, Jull JW. Human and Experimental Breast Cancer. London : Pitman Medi- cal Publication co . , 1961 ; 381-395.

4. Ozzello L, Sanpitdk P. Epithelial-stromal junction of intraductal carcinomas of the breast. Cancer 1970;

5. Tulusan AH, Grunsteidal W, Ramming I, Egger H. A contribution to the natural history of breast can- cer. JJI. Changes in the basement membranes in breast cancers with stromal microinvasion. Arch Gynecol1982 ; 231 : 209-21 8.

6. Sommers SC. Histological changes in incipient car- cinoma of the breast. Cancer 1969; 23: 822-825.

7. Davies JD. Hyperelastosis, obliteration and fibrous plaques in major ducts of human breast. J Pathol

8 . Ozzello L. Breast. In: Johannessen JV, ed. Electron Microscopy in Human Medicine. Vol. 9 New York : McGraw-Hill International, 1979; 409450.

9. Majno G, Gabbiani G, Hirschel BJ, Ryan GB, Stat- kov PR. Contraction of granulation tissue in vitro: similarity to smooth muscle. Science 1971; 173:

10. Gabbiani G, Ryan GB, Majno G. Presence of modi- fied fibroblasts in granulation tissue and their poss- ible role in wound contraction. Experienta 1971 ; 27:

1 I . Gabbiani G, Majno G. Dupuytren’s contracture: fibroblast contraction? An ultrastructural study. Am

12. Feiner H, Kay GI. Ultrastructural evidence of myo- fibroblasts in circumscribed fibromatosis. Arch Pathol Lab Med 1976; 100: 265-268.

13. Lipper S, Kahn LB, Reddick RL. The myofibroblast. Pathol Ann 1980: 15:409441.

14. Churg AM, Kahn LB. Myofibroblasts and related cells in malignant fibrous and fibrohistiocytic tumors. Hum Pathol1977; 8: 205-218.

1959; 35: 887-899.

26: 1186-1 198.

1973; 110: 13-26.

548-550.

549-550.

JPatho11972;66: 131-146.

15. Seemayer TA, Schurch W, Lagace R, Tremblay G. Myofibroblasts in the stroma of invasive and metastatic carcinoma. A possible host response to neoplasia. Am JSurg Patholl979; 3: 525-533.

16. Seemayer TA, Schurch W, Lagace R, Thelmo WL. The myofibroblast, biologic, pathologic and theoreti- cal consideration. Puthol Ann 1980; 15: 443470.

17. Harris M, Ahmed A. The ultrastructure of tubular carcinoma of the breast. J Patholl977; 123: 79-83.

18. Tremblay G. Stromal aspects of breast carcinoma. Exp Molec Pathol1979; 31: 248-260.

19. Schurch W, Lagace R, Seemayer TA. Myo- fibroblastic stromal reaction in retracted scirrhous carcinoma of the breast. Surg Gynecol Obstet 1982;

20. Carter D, Yardley JH, Shelley, WM. Lobular car- cinoma of the breast. An ultrastructural comparison with certain duct carcinomas and benign lesions. Johns Hopkins M e d J 1969; 125: 25-43.

21. Goldenberg VE, Goldenberg NS. Sommers SC. Comparative ultrastructure of atypical ductal hyper- plasia, intraductal carcinoma and infiltrating ductal carcinomaofthe breast. Cancer 1969; 24: 1152-1 169.

22. Ahmed A. Atlas of Ultrastructure of Human Breast Diseases. Edinburgh : Churchill Livingstone, 1978 ; 1-23,7686.

23. Vracko R. Basal lamina scaffold-anatomy and sig- nificance for maintenance of orderly tissue structure. Am J Pathol1974; 77: 314-345.

24. Mdjno G. Thc story of myofibroblasts. Am J Surg

25. Azzopardi JN. Problems in Breast Pathology. In: Major Problems in Pathology, Vol. IJ. London: W. B Saunders Co, 1979; 181-182.

26. Battersby S, Anderson TJ. Myofibroblast activity of radial scars. J Patholl985; 147: 33-40.

27. Tamimi SO, Ahmed A. Stromal changes in invasive breast carcinoma: An ultrastructural study. In preparation.

28. Ryan GB, Cliff WJ, Gabbiani G , et al. Myo- fibroblasts in human granulation tissue. Hum PathoI

29. Rudolf R, Guber S , Suzuki M, Woodward M. The life cycle of the myofibroblast. Surg Gynecol Obstet

30. Muir R, Aitkenhead AC. The healing of intra-duct carcinoma of the mamma. J Pathol Bact 1934; 38: 117-127.

3 I . Linell F, Ljungberg 0. Regression of carcinoma. In : Atlas of Breast Pathology. Copenhagen: Munksgaard, 1984; 234-238.

154: 351-358.

Path01 1979; 3: 535-542.

1974; 5: 55-67.

1977; 145: 389-394.