Cytological Findings in Medullary Carcinoma of the Thyroid

Path. Res. Praet. 178,461-466 (1984)

Cytological Findings in Medullary Carcinoma of the Thyroid R. Schaffer, H.-A. MOiler, U. Pfeifer and W. Ormanns Pathologisches Institut der Universitiit Wiirzburg, Wiirzburg, FRG

SUMMARY In six cases of medullary carcinoma of the thyroidrather characteristic findings wereseen in cytological specimens obtained by aspiration or imprint. The roundish-polygonal or spindle-shaped cells were predominantly disseminated, but occasionally arranged in small groups. The nuclei were frequently found in an excentric position; binuclear cells were sometimes encountered. In addition, five of the six cases showedintranuclear cytoplasmic inclusions whichwerealso investigated by electron microscopy in specimens of two cases. Such inclusions in aspiration biopsies of the thyroid have so far been regarded, as characteristic for papillary carcinoma. In smears aswellasin histological sections amyloid and calcitonin could be demonstrated by congo red staining and by immunocytochemistry, respectively. The cytomorphology of the tumor cells, their pattern of arrangement, and the peculiar nuclear inclusions make up a combination of findings that may justify the diagnosis of medullary carcinoma in aspiration biopsies. In smears the diagnosis can already be confirmed when (immuno-) cytochemical methods are applied to smear preparations.

Among malignant epithelial tumors of the thyroid, papillary and follicular carcinomas and, to a lesser degree, anaplastic carcinomas are the most frequent types, as observed in clinical and patho-histological studies. In fine needle biopsy these tumors can be identified by their characteristic morphology and a more or less typical arrangement of their cells. The medullary carcinoma (C-cell carcinoma) of the thyroid, however, which is not derived from thyrocytes but from Earafollicular cells, i.e. C-cells, is a rather rare tumor 4,22. It contains amyloid and in addition to calcitonin can produce a number of other polypeptides" 8. As will be shown in the following report on aspiration and imprint cytological investigations, medullary carcinomas can be identified in smear preparations by a characteristic cell morphology and cell arrangement, and by additional properties to be detected by cytochemical methods. © 1984byGustavFischer Verlag, Stuttgart

Material and Methods Between 1960 and 1982 20 cases of C-cell carcinoma were seen in the Institute of Pathology of the University of Wiirzburg. Fine needle biopsies were obtained in six of these cases, additional smears by imprint from surgical specimens in two cases. All smears were wet-fixed with a cytospray (Merckofix's) and routinely stained with hematoxilin-eosin; in three cases staining withcongo red wasperformed afterremoval of thehematoxilineosin byacid alcohol. In twocases May-Griinwald-Giemsa stainingwas used in addition. Calcitonin wasdemonstrated by the avidin-biotin-peroxidasecomplex method? in three cases after removal of the HE-stain. Rabbit antihuman calcitonin (Immunonuclear Corp., USA) used in a dilution of 1: 1000served asprimary antibody. Diaminobenzidine (Sigma) was used as substrate for visualization of peroxidase linked to the second antibody via an avidin-biotin complex (Tago, USA). Three smears from follicular adenomas of the thyroidserved as controls. In two cases which had been examined at 0344-0338/84/0178-0461$3.50/0

462 . R. Schaffer, H.-A. Muller, U. Pfeifer and W. Ormanns first by aspiration and/or Imprint cytology, specimens of the tumor obtained during surgery were prepared for electron microscopy. Small tissue cubes of 1 mm diameter were fixed in 1,5% phosphate- buffered OS04 and embedded in Epon. Thin sections stained with lead citrate were examined with the Zeiss EM 10.

Results The smears of all C-cell carcinomas examined, whether obtained by aspiration biopsy or by imprint, showed a similar cytological appearance. They were rather rich in cells and in this regard similar to the smears of follicular and papillary carcinomas. However, while in the latter the presence of large cell complexes, which recall histological

features of the tumor allows the detection of characteristic patterns of cell arrangement, no such cell complexes are preserved in smears from C-cell carcinomas but only small aggregates and disseminated single cells. Three characteristic cell types could be identified in all smears, although their size may differ from one case to the other. The first cell type showed a roundish, polygonal cytoplasma with an excentrically placed round nucleus; small forms of this cell type sometimes resembled plasma cells. The second cell type showed a prismatic, more spindle like, sometimes elongated cellular body and an oval nucleus (Fig. 1). Type one or type two could predominate within different areas of the same preparation. As a third type "triangular" elements were frequently observed, which probably represent transitional forms between the

Fig. 1. Cytological appearance of medullary carcinoma of the thyroid in smear preparation: Roundish-polygonal tumor cells with round, frequently excentrically positioned nucleus (~) and spindle-like prismatic cellswith oval nucleus (_); nuclear chromatin pattern of moderate coarseness. Female, 52 years, aspiration biopsy. HE, X 1500.

Fig. 2. Demonstration of fine and moderately coarse granules in the cytoplasma of a tumor cell by May-Gninwald-Giemsa staining. Female, 45 years, imprint. MGG, X

2400.

Thyroid Medullary Carcinoma . 463

cell types one and two described above; small examples of these triangular cells sometimes resembled respiratory epithelia with fringy apical cell poles. In the HE preparations the cytoplasma of the tumor cells showed a moderately eosinophilic staining in five cases, but was strongly eosinophilic in the remaining case. In the peripheral parts of the cytoplasma fine granular structures of yellowish intrinsic color were sometimes observed. Azurophilic cytoplasmic granules could frequently be identified in the Giemsa-stained smear preparation of one of the two cases examined by this method (Fig. 2), but only rarely in the cells of the other case. These granules corresponded well to the fine-granular structures seen in the HE-preparation. Amorphous reddish material could already be seen between the tumor cells after HE-staining. Following re-staining with Congo red, this material frequently showed the orange-red color typical for amyloid and also the green birefringence which is specific for the amyloid-dye complex (Fig. 3). While the cell nuclei were rather variable in size in all smears they did not show significant polymorphy, which might be due to irregular contours. Nuclear folds could only be identified occasionally. Frequently binuclear, and rarely multinuclear cells were encountered. The nuclear chromatin pattern was characterized by moderate coarseness. In most cells a nucleolus could easily be identified, but two nucleoli within one nucleus were rare. Cells with intranuclear cytoplasma inclusions of 2 to 10/-tm were identified in the smears of five of the six medullary carcinomas; these inclusions, however, were definitely less frequent than in the cells of papillary carcinoma of the thyroid. Besides solitary nuclear inclusions almost completely occupying the inner space of the nuc-

a

b

Fig. 3. Demonstration of amyloid by polarized light. a) Abundant amorphous amyloid deposits under crossed polarizationfilters. Male, 41 years, aspiration biopsy. Congo red, x 600. b) Two small roundish amyloid deposits in partially polarized light. Male, 17 years, aspiration biopsy, Congo red, x 600.

a

Fig. 4. Nuclear inclusions in medullary carcinoma of the thyroid. a) Large nuclear inclusion in a triangular carcinoma cell. Female, 52 years, imprint. HE, x 2400. b) Large nuclear inclusion accompanied by two adjacent small inclusions. Male, 17 years, aspiration biopsy. HE, x 2400. c) Apparently solitary large nuclear inclusion. Female, 24 years, aspiration biopsy. HE, x 2400.

c

464 . R. Schaffer, H.-A. Miiller, U. Pfeifer and W. Ormanns

leus, smaller, often multiple ones could also be observed. The large nuclear inclusions were sometimes surrounded by very small satellitelike ones (Fig. 4). The cytoplasmic origin of the nuclear inclusions could be especially well confirmed by electron microscopy. They were surrounded by a double membrane which sometimes was only partially preserved. The sequestrated cytoplasma occasionally showed condensations or vesicular transformation of cisternas (Fig. 5 a). Vesicular and granular components of the cytoplasma were in some instances also seen free in the nuclear space (Fig. 5 a). An additional finding already well known from EM-studies on medullary carcinoma'S!' were intracyto-

plasmic granules of 100 to 400 nm with a matrix of variable density (Fig. 5 b). In the extracellular space, densely but irregularly packed amyloid fibrils of 8 to 12 nm in diameter were seen (Fig. 5 c). Calcitonin was demonstrated immunocytochemically in many tumor cells (Fig. 6). The definite intracellular localisation of the polypeptide was sometimes difficult to establish because of a diffuse brownish dye precipitation which may be due to diffusion of calcitonin out of the cells during preparation, or to a rather high concentration of calcitonin in the extra-cellular space of the tumor tissue. The smears of the thyroid adenomas incubated as controls did not react with the calcitonin antibody.

Fig. 5. Electron microscopic findings in medullary carcinoma. a) Intranuclear cytoplasma inclusion: Parts of the inclusion can hardly be delineated from the nuclear space; the border appears as a double membrane (~) or blurred zone (I»), Other derivates of the cytoplasma are seen freely in the nuclear space (-4). In contrast to the nuclear inclusion a section of a simple cytoplasma invagination (large arrow) shows a completely unchanged cytoplasma. Female, 24 years. x 20500. b) Secretory granules of variable size and electron density in the cytoplasma of a tumor cell. Same case as a. X 37400. c) Irregularly orientated extracellular amyloid fibrils. Same case as a. x c 31900.

Thyroid Medullary Carcinoma . 465

b Fig. 6. Immunocytochemical demonstration of calcitonin In medullary carcinoma. a) Numerous calcitonin -positive, mainly roundish tumor cells and single calcitonin-negative (~ ) cells. Male, 41 years, aspiration biopsy. AVIdin-biotin-complex, X 1500. b) Calcitonin-positive, triangular, binuclear tumor cell with dendrite-like process. Male, 17 years, aspiration biopsy. Avidin-biotin-complex, x 1500.

Discussion The cytological picture of medullary carcinomas is rather characteristic in smea r preparat ion s. In accordan ce with the find ings of other authors' t" 16-1 8,24 ,25 it is characterized by numerous pol ygon al, triangular, and spindlelike cellul ar elements frequently ha ving excentric and sometimes double nu clei. Ho wever , no such typical arrangement of the cells as in follicular or papillary ca rcinoma is present in medull ar y carcinomas. The nucl ear inclu sion s which - lik e th ose of papill ar y carcinomas/ represent intranuclea r cytopl asmic inclusions ha ve so far been described onl y occ asion ally in cells of medullary carcinoma s of th e thyroid. Kakudo et al. (197 8) were able to identify cytoplasmic inclusion s histologically and elect ron microscopically in a giant-cell metastasis of a C-cell carcinoma; Droese (1979) ha s published a microphotograph of a cell with a nuclear inclusion in a medullary carcinom a (Giemsa-stained smear). These cytoplasmic inclu sion s in medullary carcinomas - as in th e cells of numerous other organs, e.g. the liver or seminal vesicle - represent th e con sequence of a disturbance in the mitotic course! ' 1 ~,21.

In cytological preparations of primary th yroid tumors, nuclear inclusions have so far onl y been described for papillary carcinomas' r" although according to our own experience the y can also be ob served in anaplastic car cinomas. When smears are routinely stained with May-Griinwald-Giemsa, azurophilic granules in the cytoplasma can be helpful for the diagnosis of C-cell carcinoma. These granules, however, are pr esent in only 5% to 15 % of the tumor cells24 and are sometimes even completely missing', It must be emphasized that azurophilic granules have so metimes also been observed in follicular carcinomas of the thyroid and in metastases of breast carcinomas", This means that such granules are of diagnostic importance only in combination with additional characteristics of a Ccell carcinoma. The identification of amyloid in the smears of all three cases examined shows that Congo red staining can confirm the diagnosis of the tumor type, when C-cell carcinoma is suspected from the spectrum of the cytological findings described above. This is in accor dance with the earlier studies of Ljungberg (1972 b) in two carcinomas and of Soderstrom et al. (1975 ), who could demonstrate amyloid by Congo red staining of cytological smears in 9 of 14 medullary car cinomas. In the 20 cases of the pr esent series am yloid was absent in onl y one case whe re a lymph node met astasis had been exa mined. It is kno wn fro m the liter ature 13. 20 that some medullary car cinomas - even in numerous histological prepar ation s - contai n no or onl y littl e amyloid; th e safest technique for th e idenification of C-cell carcino ma therefo re seems to be the immunocytochemical dem on stration of calcitonin". Acco rding to ou r expe rience th is is even possible afte r remo val of the hem ato xylin-eo sin from routinely sta ined smears.

Addendum. While this work was in preparation Miller et a1. (Miller M J, Kini SR, Hamburger JI (1983) Needle biopsy of the thyroid. Praeger Pub, New York) had also demonstrated Intranuclear inclusions In smears of thyroid medullary carcinoma.

References 1 Altmann HW, Muller HA (1973) Grundlagen der Karyologie Verh Dtsch Ges Path 57: 2-33 2 Beaumont A, Othman SB, Fragu P (1981) The fine structure of papillary carcinoma of the thyroid. Histopathology 5:

377-388

1 Blonk OI, Talermann A, Visser-van Dijk MN (1978) The cytology of medullary carcinoma of the thyroid with spindle cell pattern. Arch Geschwulstforsch 48: 313-3 17 4 Capella C, Bordi C, Monga G, Buffa R, Fontana P, Bonfanti S, Bussolati G, Solcia E (1978) Multiple endocrine cell types in thyroid medullary carcinoma. Virchows Arch A (path Anat) 377:

111-128 5 Christ ML, Haja J (1979) Intranuclear cytoplasmic inclu-

sions (invaginations) in thyroid aspirations. Frequency and specificity. Acta cytol (Baltimore) 23: 327-331

466 . R. Schaffer, H.-A. Muller , U. Pfeifer and W. Ormanns 6 De Lellis RA, Rule AH, Spiler I, Nathanson L, Tashjian AH, Wolfe HJ (1978) Calcitonin and carcinoembryonic antigen as tumour markers in medullary thyroid carcinoma. Amer J c1in Path 70: 587-594 7 Droese M (1979) Aspirationszytologie der Sclulddruse. Schattauer, Stuttgart - New York, 212- 229 8 Hazard JB (1977) The C Cells (parafolhcular cells) of the thyroid gland and medullar y thyroid carcinoma. Amer J Path 88: 214-249 9 Hsu SM, Raine L, Fanger H (198 1) Use of avidm-brotinperoxidase complex (ABC) in immunoperoxidase techmques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29: 577-580 10 Huang SN, Goltzman 0 (1978) Electron and immunoelectron microscopic study of thyroidal medullary carcmoma. Cancer 41: 2226-2235 11 JohannessenJV, Gould VE, jao W (1978) Thefine structure of human thyroid cancer. Human Path 9: 385-400 12 Kakudo K, Miyauchi A, Oginar a T, Takai SI, KItamura H, Kosaki G, Kumahara Y (1978) Medullary carcmoma of the thyroid. Giant cell type. Arch Path Lab Med 102: 445-447 13 Kracht J (1977) C-Zellen und C-Zellengeschwulste. Verh Drsch Ges Path 61: 235-264 14 Ljungberg 0 (1972 a) On medullary carcinoma of the thyroid. Acta path microbiol scand Sect A (Suppl) 231: 1-57 15 Ljungberg 0 (1972 b) Cytologic diagnosis of medullar y carcinoma of the thyroid gland with special regard to demonstration of amyloid in smears of fine needle aspirates. Acta cytol (Balnmore) 16: 253-255

16 Lowhagen T, Willems JS, Lundell G, Sundblad R, Granberg PO (1981) Aspiration biopsy cytology in diagnosis of thyroid cancer. World J Surg 5: 61-73 17 Lopes Cardazo P (1976) Atlas of clinical cytology. Lopes Cardazo, private publication, pp 566-585 18 Muller HA (1980) Die Feinnadelpunktion der Schilddriise aus der Sicht des Cytologen. Nuklearmediziner 3: 261-266 19 Muller HA, Diemer H, von Kietzell R (1973) Polymorphe GroBkeme im menschlichen Samenblasenepithel. Virchows Arch B (Cell Path.) 12: 281-284 20 Nieuwenhuijzen Kruseman AC, Bosman FT, van Bergen Henegouw JC, Cramer-Knijnenburg G, de la Riviere GB (1982) Medullary differentiation of anaplastic thyroid carcinoma. Amer J c1in Path 77: 541-547 21 Romen W, Bannasch P (1973) Karyokinese und Kernstruktur wahrend der Hepatocarcinogenese. Virchow Arch B (Cell Path.) 13: 267-296 22 Schaffer R, Muller HA (1983) Veranderungen im Spektrum maligner Schilddrusentumoren, Verh Dtsch Krebsges 4: 593-598 23 Soderstrom N, Biorklund A (1973) Intranuclear cytoplasmic inclusions in some types of thyroid cancer. Acta eytol (Baltimore) 17: 191-197 24 Soderstrom N, Telenius-Berg M, Akerman M (1975) Diagnosis of medullary carcinoma of the thyroid by fine needle aspiration biopsy. Acta med scand 197: 71-76 25 Tsuzaki K, Tanaka I, Kotoh K (1978) Cytologic appearances of pheochromocytoma and medullary thyroid carcinom a occurring within a family. Acta cyrol (Baltimore) 22: 321-326

Received July 12, 1983 . Accepted in revised form September 19, 1983

Key words: Thyroid - medullary carcinoma - C-cell - aspiration biopsy cytology - immunocytochemistry Dr. habil. R. Schaffer, Pathologische s Institut der Universitat, josef-Schneider-Strafie 2, 0-8 700 Wurzburg, FRG