Intestinal Spirochaetosis

Path. Res. Pract. 182, 627-631 (1987)

Breast Carcinomas with Protein 5-100 Immunoreactivity An Immunocytochemical and Ultrastructural Study S. Lunde, Jahn M. Nesland and R. Holm Department of Pathology, The Norwegian Radium Hospital and Institute for Cancer Research, 0310 Oslo, Norway and The Norwegian Cancer Society, Oslo, Norway

Jan V. Johannessen Departments of Pathology, The Norwegian Radium Hospital, Institute for Cancer Research and Columbia University College of Physicians and Surgeons, New York, N. Y. and The Norwegian Cancer Society, Oslo, Norway

SUMMARY

Protein S-l'OO immunoreactivity was observed in 5 of 50 breast carcinomas (3 infiltrating lobular and 2 infiltrating ductal carcinomas). A diffuse cytoplasmic staining was present in single cells and groups of cells. The majority of normal myoepithelial cells in ducts of unremarkable appearance next to tumor areas were stained in all 50 breast carcinomas. The 5 protein S-lOO positive tumors all stained for prekeratin and 4 of them were vimentine-positive. No immunoreactivity for actin or NSE was observed in the 5 tumors. Electron microscopy did not distinguish the protein S-l 00 positive carcinomas from the 45 protein S-lOO negative tumdrs. The significance of protein SolDO immunostaining in breast carcinomas is discussed.

Introduction Protein S-100 is an acid Ca ++ -binding protein which is soluble in 100% ammonium sulfate. The protein was initially identified in the central nervous system, where it is mainly located in glial cells 17• Later, immunoreactivity for protein S-100 was reported in Schwann cells of peripheral nerves and sustentacular cells of sympathetic ganglia and adrenal medulla10, melanocytes and Langerhans cells of the skin3, interdigitating reticulum cells of lymph nodes 30 , certain subpopulations of lymphocytes32 , chondrocytes 28 , lipocytes l l and myoepithelial cells of sweat glands, salivary glands and the breast lO . The list of neoplasms with immunoreactivity for S-100 is extensive and includes tumors of the central and peripheral nervous system, granular cell tumors and paragangliomas 20 , malignant m~.1anomas25, mixed sweat gland tumors and pleomorphic adenomas of salivary glands, bronchio-alveolar carcinomas of the lung, teratomas of © 1987 by Gustav Fischer Verlag, Stuttgart

the ovarylO, tumors of cartilage and bone 16,2\ chordomas 20 , eosinophilic granulomas18,34, histiocytosis X8 and lymphomas 2. Because of the demonstration of protein S-100 immunoreactivity in nonneoplastic myoepithelial cells and the observation of protein S-100 staining of some breast carcinomas2o , we examined a series of breast carcinomas to identify tumors immunoreactive for S-100 protein. With the aid of electron microscopy and immunohistochemistry with a series of antibodies, we compared these tumors with those that lacked protein S-100 reactivity. Material and Methods 50 consecutive breast carcinomas from the files of the Laboratory for Ultrastructural Pathology, Department of Pathology, The Norwegian Radium Hospital, were included in this study. Sections from formalinfixed, paraffin-embedded material were 0344-0338/87/0182-0627$3.50/0

628 . S. Lunde, J. M. Nesland, R. Holm and J. V. Johannessen stained with haematoxylin and eosin and used for light microscopic classification according to WHO. Sections from the paraffin blocks were also used for immunocytochemical studies, applying the avidin-biotin peroxidase complex (ABC) method? The techni~ues have been described in more detail in an earlier publication1 . The number of positively stained cells was quantified from + to + + + (+ = scattered single cells positively stained; + + + = the majority of the neoplastic cells stained). All 50 breast carcinomas were tested for protein S-100. The 5 positively stained cases and 5 cases negatively stained for protein S-100 were then further analyzed for the presence of vimentin, keratin, actin and neurone specific enolase (NSE). The sources of the antibodies, concentrations, incubation times and temperatures are shown in Table 1. Fresh material from all 50 breast carcinomas was also fixed in a cacodylate buffered mixture of 4% formaldehyde and 1% glutaraldehyde l 4, postfixed in buffered 1% osmium tetroxide, dehydrated in graded ethanols and embedded in an Epon-Araldite mixture l5 • After polymerization at 60-80°C, semithin sections were cut with glass knives, stained with toluidine blue and used for light microscopic orientation. Ultrathin sections were cut with diamond knives, contrasted with uranyl acetate and lead citrate and examined in the electron microscope. Clinical data were taken from patients' records in the files of The Norwegian Radium Hospital.

Fig. 1. Infiltrating ductal carcinoma with immunostaining for protein S-100 in nearly" all tumor cells (Anti-protein S-100, X 140).

Table 1. Antiserum against

Dilution Incubation

Protein S-100 Prekeratin Vimentin Actin NSE

1: 700 1: 700 1:5 1:2000 1:200

18-22 18-22 18-22 18-22 18-22

h, h, h, h, h,

4°C 4°C 4°C 4°C 4°C

Source Dako Corporation, USA Dako Corporation, USA Amersham International Amersham International Dako Corporation, USA

Results

The 50 breast carcinomas were light microscopically classified as infiltrating ductal carcinoma (38 cases), infiltrating lobular carcinoma (9 cases), tubular carcinoma (2 cases) and papillary carcinoma (1 case). Immunoreactivity for protein 5-100 in single cells and groups of cells

Fig. 2. Higher magnification of protein S-100 pOSItive tumor cells. Immunoreactivity is present as a diffuse cytoplasmic staining and in a proportion of the nuclei (Anti-protein S-100, x 560).

Breast Carcinomas with Protein S-100 Immunoreactivity· 629 Table 2. Clinical Data Case Age

Localization

Tumor size (cm)

Oestrogen Receptor state

1

79

RR, LB

3

+

2

77

MUQ, LB

5

3

53

LUQ, RB

4

4

51

LUQ, RB

3.2

5

30

LUQ, RB

2.5

Progesteron Axillary receptor metastases state

+ +

+ +

+

and with a diffuse cytoplasmic staining was present in 5 cases (Fig. 1). Occasionally the nuclei were stained as well (Fig. 2). Immunostained tumor cells were present in both the periphery and center of tumor nests. The majority of normal myoepithelial cells in ducts of unremarkable appearance next to tumor areas were stained in all 50 breast carcinomas. The 5 breast carcinomas 'that were immunoreactive for protein 5-100 were light microscopically classified as infiltrating lobular carcinoma (3 cases) and infiltrating ductal carcinoma (2 cases). The ultrastructural features of the 5 breast carcinomas with immunoreactivity for protein 5100 were the same as in the other 45 tumors. In particular, there was no noteworthy amount of cytofilaments. Intracytoplasmic lumina were observed in 3 of the 5 cases, and in all cases exocrine granules were present in apical parts of the cells and in the proximity of intracytoplasmic lumina. The clinical data for the 5 patients with protein 5-100positive tumors are shown in Table 2. The age of the patients ranged from 30 to 79 years (mean age 58). The tumors measured from 2.5 to 5 em in largest diameter. Two tumors (cases 1 and 3) were oestrogen receptor positive and 1 tumor (case 4) was progesteron receptor positive. Axillary lymph node metastases were present in 3 cases (cases nos. 2, 3 and 5). All patients had been treated with mastectomy and axillary lymph node extirpation. Cases 2 and 3 had distant metastases a few months later Table 3. Immunohistochemistry Case Protein S-1 00 Actin 1 2 3 4 5

+ + + ++ +

NSE

-=

Vimentine

Prekeratin

++ ++

+++ ++ +++ +++ +++

++ ++

Primary treatment

Follow-up/survival

Mastectomy and axillary lymph node extirpation Mastectomy and axillary lymph node extirpation Mastectomy and axillary lymph node extirpation Mastectomy and axillary lymph node extirpation Mastectomy and axillary lymph node extirpation

Alive 17 months no metastases Dead, 22 months Widespread metastases Alive, 26 months Skeletal metastases Alive, 13 months no metastases Alive 15 months

and were treated with tamoxifen and irradiation. The short follow up period (maximum 26 months) does not permit further evaluation of clinical outcome for the patients with protein 5-100 positive tumors. The immunostaining results for the 5 cases positively stained for protein 5-100 are shown in Table 3. All tumors were prekeratin positive and actin and N5E negative. Four tumors were vimentin positive. Five protein 5-100 negative tumors (3 infiltrating lobular carcinomas and 2 infiltrating ductal carcinomas) used as controls, were all stained with antiprekeratin, but showed no immunoreactivity for vimentin, actin and N5E. Discussion Immunocytochemical methods have brought about a revolution in diagnostic tumor pathology. As in other revolutions, old, well-established systems have been toppled; in this case classification systems. The more experience we gain with immunocytochemical methods, the more we appreciate that they may bring as much diagnostic confusion as clarity. . Protein 5-100 was initially thought to be a specific marker of neurogenic cells and related tumors 17, but may be present only in sub-groups of certain tumors, and has now even been identified in a great number of non-neurally derived cells and tumors10. Protein 5-100 is composed of two subunits, an a- and~­ polypeptide chain9 • Human protein 5-100 positive cells are then divided into three subgroups31: cells containing only the a unit (aa), only the ~ unit (~~), or both units (a~). Polyclonal antibodies that are reactive with several epitop~s of the protein have been used to investigate the distribution of protein 5-100 in tissue and tumors10,20. There may also be a regional distribution of the subgroups of 5-100 protein in a specific organ or tissue, as demonstrated by Kimura et al. 12 in the bovine brain. However, the results of all these studies diverge to a certain extent,

630 . S. Lunde, J. M. Nesland, R. Holm and J. V. Johannessen probably due to differences in specificity and sensitivity of the various antisera applied, Recently, monoclonal antibodies reactive to antigenic determinants shared by both a and ~ units have been developed 13 ,33 as well as purified polyclonal antibodies that react more specifically with the subunits 31 , This has increased the specificity but probably reduced the sensitivity. The concentration of protein 5-100 in the cytosol seems to be regulated by ACTH and catecholamines29 • The protein may be depleted in the cells in various physiological conditions and thereby give confusing immunostaining results 6 , Immunoreactivity for protein 5-100 has repeatedly and consistently been identified in myoepithelial cells of the normal breast and its benign lesions 10, 23-27 and specific staining of ductal excretory epithelium has also been reported lO ,20. This fits with our findings. Nakajima and coworkers 20 reported positive protein 5100 immunostaining in 2 of 7 infiltrating ductal carcinomas, but we disagree with their conclusion that the protein S-100 positive cells "take part almost in the same fashion as myoepithelial cells in these lesions", since Fig. 20 of their report clearly shows immunoreactive tumor cells in an axillary l~mph node metastasis. Kahn and coworkers observed protein 5-100 immunoreactive neoplastic cells in medullary carcinomas of the breast, but not in ductal carcinoma or lobular carcinoma. No medullar,y carcinomas were included in our study, but we did find positive immunostaining for protein 5-100 in 3 infiltrating lobular carcinomas and in 2 infiltrating ductal carcinomas. The role of myoepithelial cells in breast carcinomas has been debated. Due to the abundance of cytofilaments, Murad and Scarpelli 19 suggested that some breast carcinomas were derived from myoepithelial cells. This was later refuted1,22 and Ahmed 1 expressed the firm view that myoepithelial cells do not become malignant, but only "participate secondarily" in infiltrating ductal carcinomas. Dairhee and coworkers 4 have isolated a monoclonal antibody claimed to be specific for myoepithelial cells. 5% of breast carcinomas stain with this antibody. The five breast carcinomas positive for protein 5-100 in our study did not reveal immunoreactivity for actin and N5E, both known as markers for myoepithelial cells, and the ultrastructural features were not indicative of myoepithelial origin. However, the existence of a subgroup of breast carcinomas sharing immunocytochemical features in common with myoepithelial cells should make us reluctant to exclude the possibility that myoepithelial cells and ductal epithelial cells can be derived from the same precursor cell.

°

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Received December 9, 1986 . Accepted in revised form March 14, 1987

Key words: Breast carcinomas - Protein 5-100 - Immunoreactivity - Electron microscopy Dr. S. Lunde, Department of Pathology, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway