Epithelial markers in thyroid carcinoma: an immunoperoxidase study

Histopathology 1986, 10, 815-829

Epithelial markers in thyroid carcinoma: an immunoperoxidase study

N.W.WILSON*, H.PAMBAKIAN*, T.C.RICHARDSON*, M.R.STOKOE*, C.A.MAKINt & E.HEYDERMAN* Department of * Histopathology, St Thomas’ Hospital, London and tDepartment of Surgery, Westminster Hospital, London, UK

Accepted for publication 20 January 1986

WILSON N.W., PAMBAUAN H., RICHARDSON T.C., STOKOE M.R., MAKIN C.A. & HEYDERMAN E. (1986) Histoparhology 10, 815-829

Epithelial markers in thyroid carcinoma: an immunoperoxidase study

Ten cases each of papillary, follicular, anaplastic and medullary carcinoma of the thyroid were stained for thyroglobulin, calcitonin, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and cytokeratin (CAM 5.2). Monoclonal or affinity purified polyclonal antibodies, and an indirect immunoperoxidase technique were used. All the papillary and follicular tumours, 5/10 anaplastic and 3/10 medullary carcinomas contained thyroglobulin. Only the 10 medullary carcinomas stained positively for calcitonin. Three out of 10 papillary, 1/10 follicular, 0110 anaplastic and 10/10 medullary carcinomas were positive for CEA. Nine out of ten papillary, 7/10 follicular, 2/10 anaplastic and 3/10 medullary carcinomas were positive for EMA. Ten out of 10 papillary, 10/10 follicular, 5/10 anaplastic and 10/10 medullary carcinomas were positive for cytokeratin. The presence of calcitonin and CEA is of value in the diagnosis of medullary carcinoma, and enable its distinction from anaplastic thyroid carcinoma. Thyroglobulin is a useful marker in thyroid carcinomas.

Keywords: calcitonin, carcinoembryonic antigen, immunoperoxidase, medullary thyroid carcinoma, thyroglobulin, thyroid neoplasms, tumour markers

Introduction

The purpose of this study was to investigate the distribution in thyroid carcinomas of five epithelial markers, thyroglobulin (LoGerfo et al. 1978), calcitonin (Busso- lati, Van Noorden & Bordi 1973), carcinoembryonic antigen (CEA) (Gold & Freedman 1965), epithelial membrane antigen (EMA) (Heyderman, Steele & Ormerod 1979) and cytokeratin (CAM 5.2) (Makin, Bobrow & Bodmer 1984). Ten

Address for correspondence: Dr E.Heyderman, Department of Histopathology, St Thomas’ Hospital, London SE1 7EH, UK.

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816 N . W. Wilson et al.

cases each of follicular, papillary, anaplastic and medullary carcinoma were studied.

In thyroid tumours, immunohistochemical methods have been used to localize both thyroglobulin (LoGerfo et al. 1978, Burt & Goudie 1979, Bocker, Dralle & Dorn 1980a, Bocker et al. 1980b, Kawaoi et al. 1982, Permanetter, Nathrath & Lohrs 1982, Franklin et al. 1982, Albores-Saavedra et al. 1983, Longmans & Jobsis 1984, Krisch et al. 1985) and calcitonin (Bussolati et al. 1973, Arnal-Monreal et al. 1977, Kameya et al. 1977, DeLellis et al. 1978, Mendelsohn et al. 1978, Talerman et al. 1979, Kameda et al. 1979, Cox et at. 1979, Deftos et al. 1980, Lloyd, Sisson & Marangos 1983, Logmans & Jobsis 1984, Sikri et al. 1985, Krisch et al. 1985). Calcitonin secretion has also been reported in non-thyroid tumours (Silva et al. 1973, Coombes et al. 1974, Milhaud et al. 1974, Hillyard et al. 1976, Lee, Ray & Sharifi 1982). Epithelial membrane antigen and CEA are more widely distributed. Epithelial membrane antigen, a large glycoprotein (Ormerod et al. 1983)- has been demonstrated on secretory and other epithelial surfaces (Heyderman, Steele & Ormerod 1979), and has been shown to be of value in histopathological diagnosis (Heyderman et al. 1979, Sloane et al. 1980, Sloane & Ormerod 1981, Dearnaley et al. 1981, Sloane et al. 1982, Sloane, Hughes & Ormerod 1983, Dearnaley et al. 1983, Bamford et al. 1983). Carcinoembryonic antigen, which was originally thought to be a gastrointestinal specific marker (Gold & Freedman 1965, Gold, Gold & Freedman 1968), has subsequently been demonstrated in a variety of non-gastrointestinal tumours (Goldenberg, Sharkey & Primus 1978), including medullary thyroid carcinoma (Isaacson & Judd 1976, Hamada & Hamada 1977, DeLellis et al. 1978, Cox et al. 1979, Talerman et al. 1979, Lloyd er al. 1983, Logmans & Jobsis 1984, Krisch et al. 1985). Cytokeratin polypeptides have been recognized in a wide variety of tissues, including thyroid (Miettinen et al. 1984). In this study an antibody to low molecular weight cytokeratins (CAM 5.2) was used (Makin, Bobrow & Bodmer 1984).

Materials and methods

The 10 primary follicular, papillary, medullary and anaplastic carcinomas of the thyroid were selected either from the routine surgical files of St Thomas’ Hospital, or were generously provided by outside pathologists. All tumours in this study were histologically reviewed to ensure an unequivocal diagnosis. The blocks were of formalin fixed paraffin embedded tissue, and sections were cut at 3 pm for immunohistological staining.

Staining was carried out by a modification of the indirect immunoperoxidase technique (Heyderman 1979, Heyderman et al. 1984). Positive control tissues were used for each first (specific) antibody. For thyroglobulin the positive control was papillary thyroid carcinoma; for calcitonin, rat thyroid; for CEA, carcinoma of the colon; for EMA, carcinoma of the breast, and for cytokeratin, carcinoma of the colon. Negative controls were carried out for each tumour which exhibited any positive staining with any of the specific antibodies. Negative controls using

Epithelial markers in thyroid carcinoma 817

specifically absorbed antibodies were carried out for each of the two polyclonal antibodies, thyroglobulin and calcitonin (Heyderman, Gibbons & Rosen 1981). The negative control for the monoclonal antibodies to EMA, CEA and CAM 5.2 was a mouse immunoglobulin supernatant fluid (P3 x63 Ag4 from D r J-P.Mach, LICR, Lausanne) which does not stain sections of fixed human tissues. A panel of 16 non-thyroid carcinomas (lung, breast, gastrointestinal, skin and urogenital) was also stained for thyroglobulin.

ANTIBODIES

Two different monoclonal antibodies to CEA were used (Accola ef al. 1979). These were a generous gift from Dr Mach (LICR, Lausanne). One of these antibodies (LICR 202), in addition to specificity for the CEA glycoprotein, appears to cross-react with non-specific cross-reacting antigen (NCA; CEX) (Mach & Putztas- zeri 1972, Von Kleist, Chavanel & Burtin 1972), a similar but smaller glycoprotein found in myeloid and monocytic cells. Monoclonal anti-EMA (E29) was produced in a collaborative study with Dr Cordell (Heyderman et al. 1985, Cordell et al. 1985). Monoclonal anti-cytokeratin (CAM 5.2) was raised against the colonic carcinoma cell line H T 29 (Makin et a f . 1984). The rabbit antiserum to calcitonin was obtained from Dr M.Ellison (Ludwig Institute for Cancer Research, Sutton). A crude lyophilized preparation of human thyroglobulin was obtained from Wellcome Diagnostics (Dartford, Kent) and further purified by gel filtration chromatography using Sephadex G200 (Pharmacia GB Ltd, Milton Keynes, Bucks). Antibodies were produced by emulsification of the thyroglobulin im- munogen in a non-ulcerogenic adjuvant (Guildhay Antisera, University of Surrey), and subcutaneous injection into New Zealand white rabbits. Both polyclonal antibodies to thyroglobulin and calcitonin were affinity purified on an agarose column (Affi-Gel 10, Bio-Rad Laboratories Ltd, Watford, Herts) to which purified thyroglobulin or synthetic human calcitonin had been attached. The specifically bound antibodies were eluted from the column using guanidine (pH 3; 3 mmol/l).

Results

The results are summarized in Table 1. All of the papillary and follicular carcinomas were positive for thyroglobulin. All of the medullary carcinomas contained calcitonin and CEA. All of the papillary, follicular and medullary carcinomas were positive for cytokeratin. Staining of the tumours was otherwise variably positive. In general, there was patchy staining of variable intensity.

PAPILLARY CARCINOMA

All were positive for thyroglobulin (Figure l ) , which stained predominantly the surfaces of papillae, with focal staining of cytoplasm and colloid; 8/10 blocks contained adjacent thyroglobulin positive thyroid follicles. None was positive for


Table 1. Positive immunoperoxidase staining of thyroid carcinomas (number positive out of 10)

Papillary Follicular Anaplastic Medullary

Thyroglobulin (STH 934) 10 10 5 3 Calcitonin (STH 878) Nil Nil Nil I0 CEA (LICR, Lausanne 73) Nil 1 Nil 10 CEA (LICR, Lausanne 202) 3 1 Nil 10 EMA (STH E29168) 9 7 2 3 Cytokeratin (ICRF CAM 5.2) 10 10 5 10

CEA=Carcinoembryonic antigen; EMA=epithelial membrane antigen.

calcitonin. Nine out of 10 were focally and mostly rather weakly positive for EMA (Figure 2). Epithelial membrane antigen stained the surfaces of papillae and in one tumour there was also cytoplasmic staining. In four blocks, the adjacent non- neoplastic follicles were EMA positive. None was positive for CEA LICR 73, but 3/10 were focally and very faintly positive for CEA LICR 202, which showed variable cytoplasmic, cell membrane and extracellular material staining. All were positive for cytokeratin CAM 5.2, which stained cytoplasm with a moderate to strong intensity over much of the tumour area (Figure 3).

Figure 1. Papillary thyroid carcinoma showing positive staining for thyroglobulin of cell surfaces and cytoplasm. Immunoperoxidase. x240.


Figure 2. Papillary thyroid carcinoma showing positive staining for epithelial membrane antigen of cell surfaces. Immunoperoxidase. ~ 2 4 0 .

Figure 3. Papillary thyroid carcinoma showing a cytoplasmic pattern of staining for cytokeratin CAM 5.2. lmmunoperoxidase. x 240.


FOLLICULAR CARCINOMA

All were positive for thyroglobulin, with staining of cytoplasm, membrane and colloid. In most of the tumours, all of the tumour cells were positive. None was positive for calcitonin. Seven out of 10 were weakly and focally positive for EMA, which stained cell and follicle edges. The adjacent thyroid was also patchily positive. One was positive with both CEA antibodies, though there was staining of only cell margins in a few isolated cells, together with staining of colloid (Figure 4). All were positive for cytokeratin CAM 5.2, with cytoplasmic staining of variable intensity and extent.

ANAPLASTIC CARCINOMA

Five were thyroglobulin positive, with variable distribution and intensity of cytoplasmic staining (Figure 5) . Some tumours had only a few positive cells, but in others staining was diffuse. One of the thyroglobulin positive tumours had a distinctive spindle cell pattern. None was positive for calcitonin. Two out of 10 were EMA positive, with staining of some of the tumour cell edges in both, and cytoplasm in one of them. Six out of 10 had EMA positive entrapped follicles or adjacent thyroid. All of them were negative for CEA. Five out of 10 were positive for cytokeratin CAM 5.2, with patchy cytoplasmic staining of moderate intensity (Figure 6). CAM 5.2 also stained entrapped follicles and adjacent thyroid in four of

Figure 4. Follicular carcinoma showing positive staining for carcinoembryonic antigen in a few isolated cells, follicles and colloid. Immunoperoxidase. X240.


Figure 5. Anaplastic thyroid carcinoma showing patchy staining of cytoplasm for thyroglobulin. Immunoperoxidase. X280.

Figure 6. Anaplastic thyroid carcinoma showing patchy staining for cytokeratin CAM 5.2. Im- munoperoxidase. X280.


the tumours. In the case of some of the follicular structures which were stained with CAM 5.2, it was uncertain whether these were degenerate entrapped follicles, or structures representing follicular differentiation within an anaplastic tumour. Overall, 8/10 were positive with at least one of the five epithelial markers. The histology of the two entirely negative tumours was reviewed, and it was felt that the diagnosis of large cell anaplastic carcinoma was correct.

MEDULLARY CARCINOMA

In three of the tumours, there was positive staining for thyroglobulin in cell cytoplasm and in lumina of entrapped follicles. Some of the thyroglobulin positive follicular structures had an abnormal appearance which was difficult to interpret with certainty as either degenerative or neoplastic. Around some of the entrapped follicles there was a limited zone of strongly thyroglobulin positive tumour cells (Figure 7). Two of the tumours which contained thyroglobulin positive entrapped follicles also contained sheets of diffusely and strongly thyroglobulin positive tumour cells well away from entrapped follicles. One further tumour, which did not contain entrapped follicles, was also diffusely thyroglobulin positive. This tumour also stained diffusely for calcitonin. All medullary carcinomas were calcitonin positive (Figure S), with a very variable pattern and intensity of staining. In some, only isolated tumour cells were positive, but in others sheets of tumour cells were

Figure 7. Medullary carcinoma stained for thyroglobulin, showing a positive entrapped follicle, with a surrounding zone of positive tumour cells. Immunoperoxidase. X320.


Figure 8. Medullary carcinoma showing positive staining for calcitonin in many of the tumour cells. I mmunoperoxidase. x 240.

Figure 9. Medullary carcinoma showing diffuse positive staining for carcinoembryonic antigen. Immunoperoxidase. X 320.


diffusely stained. Staining for calcitonin tended to be more patchy than staining for CEA. All of the medullary carcinomas contained amyloid, which in 7/10 stained positively for calcitonin. Ten out of 10 were positive for both CEAs, with a generally strong and diffuse pattern of staining (Figure 9). Carcinoembryonic antigen was located in cytoplasm, extracellular material and on cell margins. Staining for CEA LICR 202 was generally stronger and more widespread than that observed for CEA LICR 73. Three out of 10 had extremely localized areas in which cell edges were EMA positive. All were positive for cytokeratin CAM 5.2, with predominantly diffuse cytoplasmic staining of variable intensity. In one medullary carcinoma, however, positive staining for cytokeratin was very weak and isolated.

TUMOUR PANEL

All of the following tumours were negative for thyroglobulin: one each of carcinomas of lung (oat cell, squamous, adeno and large cell), breast (two tumours), pancreas, colon (two tumours), skin (squamous), sebaceous gland, prostate, kidney, ovary and carcinoma metastatic to the ovary. One case of testicular teratoma (MTI) was also negative.

CONTROLS

There was no staining with the absorbed antibodies or irrelevant immunoglobulin, confirming the specificity of the primary antibodies and the absence of anti-human activity in the second antibodies.

Discussion

Our results are in agreement with other reports that both papillary and follicular carcinomas of the thyroid are positive for thyroglobulin (LoGerfo et al. 1978, Burt & Goudie 1979, Bocker et al. 1980a, b, Kawaoi et al. 1982, Permanetter et al. 1982, Franklin et al. 1982, Albores-Saavedra et al. 1983, Logmans & Jobsis 1984, Krisch et al. 1985) and negative for calcitonin (Arnal-Monreal et al. 1977, Krisch et al. 1985). These are both useful markers in these thyroid tumours.

We found CEA in a minority of follicular and papillary carcinomas. In a recent study (Krisch et al. 1985) CEA was not detected in these tumours. However, weak CEA immunofluorescence has previously been demonstrated on the cell surface of one out of five papillary carcinomas (Hamada & Hamada 1977). In the thyroid, therefore, CEA is not confined exclusively to medullary carcinoma. We found EMA in the majority of follicular and papillary carcinomas, and it has also previously been noted in two papillary and two follicular carcinomas (Sloane & Ormerod 1981). Because neither EMA nor CEA are present in all cases of papillary and follicular carcinoma, and they are also found in other classes of thyroid tumour, their usefulness as markers in these two classes of thyroid tumour is limited.


Thyroglobulin was detected in half of the anaplastic carcinomas. In some previous studies, thyroglobulin was either not detected or only found in a small proportion of anaplastic carcinomas (Burt & Goudie 1979, Bocker et al. 1980a, Permanetter et al. 1982, Carcangiu et al. 1985). In other studies it has been reported in a larger proportion (LoGerfo et al. 1978, Bocker et al. 1980b, Kawaoi et al. 1982, Albores-Saavedra et al. 1983, Logmans & Jobsis 1984). Because thyroglobulin occurs in at least some anaplastic thyroid carcinomas, staining of undifferentiated carcinomas for thyroglobulin is worthwhile where the primary site is unknown, and clinical suspicion indicates thyroid as a possible source. It has been suggested that anaplastic carcinoma cells absorb thyroglobulin from invaded follicles rather than synthesizing it (Bocker et al. 1980, Kawaoi et al. 1982, Carcangiu et al. 1985). Demonstration of thyroglobulin in metastatic deposits would be necessary to resolve this question.

One of the anaplastic carcinomas, which had a spindle cell appearance, was diffusely stained for thyroglobulin. A previous study has not detected thyroglobulin in one thyroid angiosarcoma and three soft tissue sarcomas extending to the thyroid (Albores-Saavedra et al. 1983). Staining for thyroglobulin may therefore be useful in the distinction between spindle cell anaplastic thyroid carcinoma and sarcoma. Although we found EMA in only a minority of anaplastic carcinomas, an EMA positive anaplastic or spindle cell tumour in the thyroid would nevertheless favour the diagnosis of carcinoma rather than sarcoma (Heyderman et al. 1979, Sloane & Ormerod 1981, Sloane et al. 1983).

In medullary carcinomas, other workers have shown CEA in 75-100% (Isaacson & Judd 1976, Hamada & Harmada 1977, DeLellis et al. 1978, Cox et al. 1979, Talerman et al. 1979, Lloyd et al. 1983, Logmans & Jobsis 1984, Krisch et al. 1985), and calcitonin in 77-100% of tumours (Bussolati et al. 1973, Arnal-Monreal et al. 1977, Kameya et al. 1977, Mendelsohn et al. 1978, DeLellis et al. 1978, Kameda et al. 1979, Cox et al. 1979, Talerman et al. 1979, Deftos et at. 1980, Lloyd et al. 1983, Logmans & Jobsis 1984, Ghatei et al. 1985, Sikri et al. 1985, Krisch et -al. 1985). The presence of calcitonin in medullary carcinoma is consistent with its origin from C-cells (Williams 1966). Calcitonin has also been shown in 8-22% and CEA in 12-33% of undifferentiated carcinomas (Logmans & Jobsis 1984, Carcangiu et al. 1985).

All of the medullary carcinomas were positive for CEA and calcitonin, but these were not detected in any of the anaplastic carcinomas. Carcinoembryonic antigen may therefore be useful in addition to calcitonin in the distinction between anaplastic and medullary carcinomas. Although less specific for medullary carcinoma, CEA has the advantage over calcitonin in that it stains medullary carcinomas more diffusely, and the acquisition of CEA expression together with the loss of demonstrable calcitonin has been correlated with the more aggressive forms of medullary carcinoma (Mendelsohn, Wells & Baylin 1984).

Of the two antibodies to CEA, LICR 202 showed a more widespread and stronger staining reaction in medullary carcinomas. This antibody also stained positively in three papillary carcinomas which were negative with the other antibody to CEA (LICR 73). The greater intensity and more widespread

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distribution of staining for CEA observed with antibody LICR 202 is probably explained by its recognition of non-specific cross-reacting antigen (NCA; CEX) (Mach & Putztaszeri 1972, Von Kleist et al. 1972) in addition to CEA.

Cytokeratin CAM 5.2 was found in all of the medullary carcinomas. In situations where staining for calcitonin is weak or difficult to interpret, positive staining for CEA and CAM 5.2 may be useful in the diagnosis of medullary carcinoma. CAM 5.2 was detected in half of the anaplastic carcinomas, and is therefore less useful than calcitonin and CEA in the distinction between anaplastic and medullary carcinomas.

In the medullary carcinomas we observed thyroglobulin reactivity in tumour cells, in non-neoplastic residual follicles and in follicles with an abnormal appearance. Although some of the staining of medullary carcinoma cells may be due to uptake from residual follicles (LoGerfo et al. 1978), thyroglobulin positive medullary carcinoma cells were also found in areas well away from entrapped follicles. A thyroglobulin positive follicular variant of medullary carcinoma has been reported (Hales et al. 1982, Pfaltz, Hedinger & Muhlethaler 1983, Ljungberg et al. 1983). It has also been suggested that diffuse patches of thyroglobulin staining may be artifactually due to perfusion from blood vessels and lymphatics (Burt & Goudie 1979). We are not sure of the explanation of positive thyroglobulin staining seen in non-follicular areas of two of the medullary carcinomas.

From the results of this study three of the markers, thyroglobulin, calcitonin and CEA, appear to be useful in distinguishing between the different types of thyroid carcinoma. The other two markers, EMA and cytokeratin CAM 5.2, although less useful for this purpose, may have a role in distinguishing thyroid carcinomas from non-epithelium derived tumours.

Acknowledgements

We should like to thank Dr J-P.Mach, Dr M.Ellison and Messrs Hoffman- LaRoche for generous gifts of antisera. Several tumour blocks were kindly provided by pathologists from other hospitals. We thank Ms G.Powel1 for cutting the many tissue sections required for this study.

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Epithelial markers in thyroid carcinoma: an immunoperoxidase study

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