Ultrastructural Characteristics and Variations in Human Mutinous Breast Carcinomas

Path. Res. Pract. 180, 28-35 (1985)

Ultrastructural Characteristics and Variations in Human Mucinous Breast Carcinomas Birgitte Bruun Rasmussen and Allan Henschel Department of Pathology, The Finsen Institute, Strandboulevarden 49, Copenhagen DK-2100

SUMMARY Nineteen human mucinous breast carcinomas have been studied by electron microscopy in order to investigate the variations in the fine structure of this type of tumor. All the investigated tumors are characterized by a well-developed, rough endoplasmic reticulum and prominent Colgi complexes. The majority of tumor cells contain secretory granules. Approximately 50% (9/19) of the tumors have cytoplasmic dense core granules that are morphological identical to the neurosecretory granules found in APUD-cell derived tumors. Six out of the nine tumors react positively in a Crime/ius staining for light microscopic argyrophilic granules. Two of the investigated breast carcinomas contain tonofilaments that are normally regarded as characteristic of squamous epithelium. It is concluded that mucinous breast carcinomas - that to this time have been regarded as a morphological homogenous group of tumors - are ultrastructurally characterized by heterogeneity.

Introduction Mucinous breast carcinomas (MC) are defined by

WH0 18 as tumors that contain vast amounts of extracellu-

lar mucin, as evaluated macroscopically and by the light microscope; the tumor cells are typically arranged in groups and are surrounded by mucin (Fig. 1). In larger materials of primary breast carcinomas, MC comprise only 2--4% of all tumors 8,14, and only a few studies based on a small number of tumors that have dealt with the fine structure have been published 1, 2, 9, 12, 16. Publications in recent years have shown that MC can contain intracytoplasmic "neurosecretory" granules s- 7,1O, that may be a sign of a hitherto unrecognized heterogeneity . In the present study of nineteen mucinous breast carcinomas the variations in the fine structure that characterizes this group of tumors are described. 0344-0338/85/0180-0028$3.50/0

Material and Methods The investigation is based on nineteen primary breast carcinomas, where at least 25% of the tumor is a mucinous carcinoma as defined by WH0 18 • All tumors are from patients registered in the Danish Breast Cancer Cooperative Group (DBCG). Fifteen of the tumors are primarily fixed for electron microscopy using Karnovsky's fixative 13. Three tumors are received fixed in formalin but have been refixed in Karnovsky's fixative. In one case the tissue is taken from a paraffin block, dewaxed in xylene, and refixed in Karnovsky's fixative. All biopsies are rinsed in cacodylate buffer, postfixed in osmium tetraoxyde in cacodylate buffer, stained with uranyl acetate and dehydrated in alcohol before embedding in epon. Sections are cut on a Reicherts Ultramicrotome (OM U3), and sections one micron thick are used for the selection of representative tumor areas after staining with toluidine blue. Ultrathin sections, ca. 800 A, are stained with uranyl acetate and lead citrate and studied in a Philips EM 301. © 1985 by Gustav Fischer Verlag, Stuttgart

Ultrastructure of Mucinous Breast Carcinomas . 29

Fig. 1. The light microscopy appearance of a typical mucinous breast carcinoma. HE staining. x 250.

Fig. 2. Mucinous breast carcinoma with a mixture of round and moderately folded nuclei. x 6800.

30 . B. Rasmussen and A. Henschel In one case, where material was available, tumor tissue were impregnated with the Grimelius technique before processing for electron microscopy. The method used was that of Vassallo et aJ1?, with the reservation that we had to make use of dewaxed material. Thus, a 100 my thick section was cut from the paraffin block, and dewaxed in xylene overnight. The tissue was then postfixed in Karnovsky's fixative for three hours, and subsequently treated according to the referred method. For the light microscopic demonstration of dense core granules, all tumors are stained according to the Grimelius method for the detection of argyrophilic granules and according to the Masson-Fontana method for the detection of argentaffin granules.

Results Ultrastructurally the nuclei generally appear round; however in ten tumors, a few moderately folded nuclei are also found (Fig. 2). The nuclear membrane appears to be a normal, double membrane. The heterochromatine is delicate, and there is a tendency toward peripheral condensation. The majority of nuclei contain one or two unconspicuous nucleoli.

The cytoplasm is abundant and rich in organelles. The mitochrondria appear round or elongated with normal cristae in an electron dense matrix. In a few cells the mitochondria are swollen and have incomplete cristaes, indicating degenerative changes. The number of mitochondria are unconspicuous. The rough endoplasmic reticulum is well developed and the Golgi complexes are prominent. Smooth endoplasmic reticulum has not been observed. Most cells contain membrane bound mucin granules that measure from 150-1200 nm (Fig. 3). The number of granules varies from one cell to another. In some cells only a few granules are found whereas in other cells the cytoplasm is totally occupied by secretory granules. In eight tumors, larger intracytoplasmic membrane bound mucin pools are formed as the result of the fusing of several mucin granules (Fig. 4). The mucin is homogenous in three tumors and made up of a fibrillar material in seven tumors. In nine cases both types of mucin are present. The extracellular mucin is always morphologically identical to the mucinous material in the secretory granules. Apart from the mucinous secretory granules, membrane bound electron dense granules that might represent lysosomes are often seen. Glycogen and lipid granules are rare.

Fig. 3. Carcinoma with numerous mucinous secretory granules (arrow). A neighboring cell is dominated by a well-developed, rough endoplasmic reticulum (double arrow). Microvilli are present at the cell surface and in an intracytoplasmic lumen (arrowheads). X 14800.

Fig. 4. Membrane bound mucin granules fusing into a larger pool lying close to the cell surface (arrow). x 8800.

Fig. 5. Dense core granules and mucin granules in the same cell (D and M). x 18500. Inset shows the dense core granules at higher magnification. x 39000.

32 . B. Rasmussen and A. Henschel

In thirteen tumors, intracytoplasmic lumina with microvilli are present, whereas in six tumors no such lumina can be found. a

In nine tumors, intracytoplasmic membrane bound granules with an electron dense center and a clear halo are found. The granules measure from 80-200 nm and are morphologically indistinguishable from neurosecretory granules of APUD-cell derived tumors (Fig. 5). The "neurosecretory" or dense core granules can be present as a small number of granules in just a few cells, but they can also be present in large amounts (up to 50) in many cells. In the same cell both dense core granules and mucinous secretory granules can be present. In six of the nine cases where dense core granules are found, the staining for argyrophilic granules in the light microscope is positive. In one of these cases, the Grimelius staining is further found to be positive ultrastructurally, with small grains of silver deposits in the dense core granules (Fig. 6). In the remaining three cases with dense core granules it is not possible to demonstrate either argyrophilic or argentaffin granules in the light microscope. Seventeen of the nineteen investigated tumors have cytoplasmic filaments that measure ca. 80 A. In two tumors, the filaments are arranged in bundles, that are morphologically identical with the tonofilaments typical for squamous epithelium. The tonofilaments are concentrated in the

b

Fig. 6. a + b. A mucinous breast carcinoma with dense core granules after impregnation with Grimelius' silver technique before processing for electron microscopy. Fig. 6 a illustrates the presence of argyrophilic cells (arrows) in 0.5 my thin sections stained with toluidineblue. X 1000. Fig. 6 b shows the same area ultrastructurally. The silver grains are seen localized to the dense core granules. X 46500.

Ultrastructure of Mucinous Breast Carcinomas . 33

Fig. 7. Tonofilaments around the nucleus and along the cell membrane (arrows). One of the filament bundles is in contact with a desmosome (double arrow). In the figure a granule that might represent a dense core granule is seen (arrowhead). x 21300. perinuclear area, but they can also be found in the peripheral part of the cytoplasm along the cell membrane. In a few instances they converge toward desmosomes (Fig. 7). Filaments with focal densities have not been noticed. The cells containing tonofilaments are otherwise indistinguishable from cells without tonofilaments, and even granules, maybe, representing dense core granules can be seen together with these filaments.

The cell membrane appears to be normal with both junctional complexes and desmosomes. The presence of microvilli varies from one tumor to another; three tumors are devoid of microvilli. Basement membranes have not been observed. The ultrastructural findings are summarized in Table 1 and 2. Table 1 describes the tumors without dense core granules while the tumors containing these granules are listed in Table 2. As can be seen, mucinous breast car-

Table 1. Ultrastructural characteristics in 10 mucinous breast carcinomas Case

.) 1 .) 2

3 4 5 6 7 8 9 10

Dense core granules

nuclei

Mucin granules

Mucin structure

Filaments Microvilli Desmosomes

folded round folded folded folded folded folded folded round folded

many + pools few + pools few many + pools few few + pools few many + pools few many

fibrillar fibrillar fibrillar homogenous + fibrillar homogenous + fibrillar homogenous + fibrillar fibrillar fibrillar homogenous homogenous

+ + + + +

*) Present intracytoplasmic tonofilaments

+ + +

+ + + + + + + + +

+ + + + + + + + + +

34 . B. Rasmussen and A. Henschel Table 2. Ultrastructural characteristics in nine mucinous breast carcinomas containing dense core granules Case

Dense core granules

1 2 3 4 5 6

*)

7 8 9

*) *) *) *) *)

+ + + + + + + +

+

nuclei

Mucin granules

Mucin structure

round round round round round round round folded folded

many many many few few few few many many

homogenous homogenous fibrillar homogenous homogenous homogenous homogenous homogenous fibrillar

+ pools

+ pools + pools

+ fibrillar + fibrillar + fibrillar + fibrillar + fibrillar + fibrillar

Filaments

+ + + + + + + + +

Microvilli Desmosomes

+ + + + + + +

+ +

+ + + + + +

*) Argyrophilic granules in light microscope

cinomas with and without dense core granules otherwise have the same characteristics. Tumors with and without tonofilaments also otherwise resemble each other.

Discussion The nuclei in mucinous breast carcinomas have formerly been described by some authours as being generally regular with 1-2 nucleoles 2,16. Tellem l6 finds the mitochondria to be numerous and often abnormal. Since our material has not been fixed in the optimum way in all cases, the mitochondria are marked by degenerative changes in several cells. However, the well-preserved mitochondria resemble the ones described in normal breast epithelium 9• Most publications concerning the fine structure of MC agree on the presence of a prominent rough endoplasmic reticulum that is a sign of an active, secretory ceIU-3,9, 12, 15, 16. Tellem l6 describes a poorly developed Golgi complex whereas others 1-3,9, 12, 15, like us, find this organelle well developed, which is probably again a hint of the presence of a secretory cell type. This is in accord with the finding both by us and others l - 3 , 9, 2,15,16 that the tumors are dominated by mucin secretory granules. The presence of intracytoplasmic lumina in a substantial number is claimed by Battifora4 to be a strong indicator of a tumor of breast origin. In the present investigation only a few of these structures are generally seen, in accord with Gould et al. l1 • In a recent report, Yum et al. I9 have found intracytoplasmic lumina in a melanoma, thus indicating that the presence of these lumina is unspecific. They suggest that the lumina are formed by invagination of the cell membrane, a phenomenon that probably can be seen in tumors of varying origin. In nine tumors, dense core granules are detected. Such granules are described specifically in mucinous breast carcinoma by Capella et al) in 1980. Later others have confirmed the existence of "neurosecretory" granules ultrastructurally, both in MC and in infiltrating ductal carcinomas 7, 1O. In our material, the presence of granules is heterogenous, both intra- and intertumorally. The cells containing granules do not otherwise differ in their structure from the cells without granules. In the light micro-

scope it is possible to demonstrate argyrophilic granules in six of the nine tumors and in the one case where material is available for an ultrastructural detection of a positive Grimelius-reaction, silver deposits can be found in the dense core granules. Staining for argentaffin granules is negative in all cases. In the three cases where the ultrastructural granules cannot be detected in the light microscope, only a small number of granules are characteristically present in only a few cells. Approximately 50% of the MC examined contain ultrastructural dense core granules and about 30% contain argyrophilic granules. Using conventional techniques, it is impossible by either electron-or light microscopy to determine whether mucinous breast carcinomas demonstrating these granules are APUD-cell derived tumors. Until now only one study has been published where dense core granules in a breast carcinoma have been investigated by means of ultrastructural immunoperoxidase. It was found that the granules reacted positively for lactalbumin and negatively for a range of catecholamines6 • This finding is in favour of the hypothesis that dense core granules in the electronmicroscope and argyrophilic granules in the light microscope are not specific for APUD-cells as have previosly thought to be the case. The hypothesis is supported by the previously mentioned observation that a strikingly large number (30-50%) of mucinous breast carcinomas demonstrate these granules. Unexpectedly two of the investigated tumors contain structures that are identical to tonofilaments. Both Capella et al. 5 and Fetissof et aU describe the presence of bundles of filaments in the cytoplasm, while others I, 2, 9, IS have described filament bundles in cirrhous carcinomas and in infiltrating ductal carcinomas. Both Ahmed l and Sykes 15 have suggested that the bundles of filaments do in fact represent tonofilaments. In our material we have not found filaments with focal densities. The filament bundles are arranged around the nucleus and are in close approximation to desmosomes as is usually the case for tonofilaments. Sykes l5 has suggested that the presence of tonofilaments in breast carcinomas is a reflection of the fact that the breast epithelium is derived from ectoderm. Another possibility is that tonofilaments can be present in other epithelial cell types than the ectodermally derived cells, and thus is not specific for squamous epithelium.

Ultrastructure of Mucinous Breast Carcinomas . 35

It can be concluded that mucinous breast carcinomas that have previously been regarded as a morphologically and clinically well defined group of tumors are characterized by considerable ultrastructural heterogeneity. Whether the presence of ultrastructural organelles such as tonofilaments and dense core granules have any clinical relevance cannot be judged on this small material. However, the presence of argyrophilic granules in the light microscope seems to reflect the presence of dense core granules. The value of a positive reaction for argyrophilic granules as a marker of differentiation or as a prognostic factor is, therefore, under investigation in a larger patient population. Acknowledgements. This work has been supported by the Danish Cancer Society and by the Astrid Thaysen Fund. Material for the investigation has been contributed by Department of Electron Microscopy. The Herlev County Hospital, Departments of Pathology, The University Hospital, Copenhagen, Copenhagen Community Hospital Bispebjerg, Vejle County Hospital, Esbjerg County Hospital and The University Hospital, Odense. We are grateful for their cooperation. We wish to thank Dr. Klaus Hou-Jensen for having critically reviewed the manuscript, Miss Charlotte Laustsen and Mrs. Hanne Nissen for skilful technical assistance and Mrs. Ellen Margrethe Post for typing the manuscript.

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Received December 22, 1983 . Accepted June 19, 1984

Key words: Breast - Mucinous carcinomas - Neurosecretory granules - Dense core granules - Tonofilaments Birgitte Bruun Rasmussen, The Finsen Institute, Department of Pathology, Strandboulevarden 49, Copenhagen DK-l100