Endocrinol. Japon. 1990, 37 (2), 247-254

Tumor Markers and Oncogene Expression in Thyroid Cancer usingBiochemical and Immunohistochemical Studies

TAKUMA HASHIMOTO1, FUJITSUGU MATSUBARA1, YUJI MIZUKAMI2,

ITSUO MIYAZAKI3, TAKATOSHI MICHIGISHI4 AND

NOBORU YANAIHARA5

1Departments of Laboratory Medicine, 2Pathology, 3Surgery and 4Nuclear Medicine,Kanazawa University School of Medicine, Kanazawa 920, Japan,

5Shizuoka College of Pharmacy ,Shizuoka 422, Japan

Abstract

In 111 thyroid cancer patients consisting of 89 papillary carcinomas, 17follicular carcinomas, 2 medullary carcinomas, 1 squamous cell carcinomaand 2 malignant lymphomas, the levels of 12 tumor markers, including thyro-globulin (Tg), were measured in the serum by radioimmunoassay and radio-immunoassay related methods. Serum levels of Tg were elevated in 58.6%,those of CA-M26 in 15.7%, CA 19-9 in 5.3%, CT in 3.6%, NSE in 3.6%,CA 15-3 in 2.6%, CA 125 in 2.6%, CEA in 0.9%, CA-M 29 in 0%, ferritinin 0%, SCC in 0% and AFP in 0% of cases.

Among the patients, there was a case of thyroid carcinoma secretingthyroglobulin and CA 19-9, both of whose titer decreased after surgery.

Immunohistochemical studies were carried out on 57 of the above mentionedpatients plus 6 anaplastic carcinomas, 15 adenomas, 5 adenomatous goiters, 6Hashimoto's thyroiditis, 15 Graves' disease and 15 normal subjects. CA 19-9was positive in 58% of the papillary carcinomas, EGF in 73% of papillarycarcinomas, 67% of anaplastic carcinomas, and 33% of follicular carcinomas,while EGF-R was found in 73% of the papillary carcinomas, and 33% ofthe follicular carcinomas. Enhanced expression of ras p 21 oncogene and (c-myconcogene) was demonstrated in 100% (100%) of anaplastic carcinomas, in 100%(67%) of follicular carcinomas and in 63% (90%) of papillary carcinomas.

Our results indicate that a better tumor marker is required and moreextensive molecular oncology research should be pursued.

In the last 5 years, various tumor markers

have been developed and introduced into

the clinical field. However, little infor-

mation has been published on the valueof these markers in the detection of thyroidcancer. In order to acertain their re-liability or usefulness in the diagnosis ofthyroid cancer, we have evaluated 12 tumormarkers employing 10 commercially availablekits and 2 newly developed kits (CA-M 26,CA-M 29).

Receeived November 22, 1989

Address request for reprints to: TAKUMA

HASHIMOTO, M. D. Kanazawa University

School of Medicine 13-1, Takara-machi, Kana-

zawa, Ishikawa, 920 Japan.

248 HASHIMOTO et al.Endocrinol. Japon.April 1990

So far, thyroglobulin (Tg) measurementby radioimmunoassay (RIA) has been solelyemployed for the detection and post-operative monitoring of patients with thyroidcancer. However, increased concentrationsof Tg have also been seen in many thyroidaldiseases, other than differentiated thyroidcarcinoma (Botsch et al., 1983; Madedduet al., 1985). RIA of Tg has often been

jeopadized by the presence of and-thy-roglobulin antibody in the patient's serum.Thus, serum Tg is not a sufficiently reliableindicator in the diagnosis and follow-up ofdifferentiated thyroid cancer, particularlywhen the patients have anti-thyroglobulinantibodies in their serum as in Graves'disease or chronic thyroiditis (Uller andVan Herle, 1978; Schneider and Pervos,1978). A better tumor marker is urgentlyrequired.

In addition to the detection of tumormarkers in serum, we have examined thetissue expression of those tumor markers,as well as the expression of oncogenes,by immunohistochemical procedures. Ourreasons for this were that overexpressionof several oncogenes has been described invarious cancers (Channing, 1987) and thereare suggestions that oncogenes and their

products may be present in the early stagesof carcinogenesis (Czerniak et al., 1989) butare not detectable in the serum.

Materials and Methods

One hundred eleven patients with variousthyroid cancers were studied: 89 papillarycarcinomas, 17 follicular carcinomas, 2 medul-lary carcinomas, 1 squamous cell carcinomaand 2 malignant lymphomas. None of the

patients had any other malignant or non-malignant diseases, Nor any clinical laboratoryevidence of hepaic, gastrointestinal or pancreaticdiseases.

Serum levels were measured for carci-noembryonic antigen (CEA), a-fetoprotein (AFP)and squamous cell carcinoma antigen (SCC) with

Dainabot kits (Dainabot Co. Japan); thy-

roglobulin (Tg), neuron specific enolase (NSE)

and calcitonin (CT) with EIKEN kits (EIKEN

ICL Japan); carbohydrate antigens (CA 19-9,

CA 15-3, CA 125, with Centocor kits (distributed

by Fujirebio INC. Japan); carcinoma-associated

mucin antigens (CA-M26, CA-M29 [generous

gifts from Dr. P. S. Linsley]): and ferritin using

a LPIA (DIA-IATRON Co. Japan) in patients

with thyroid carcinoma. These tumor markers

were measured by radioimmunoassay or enzyme

immunoassay. The normal ranges of these tumor

markers were described previously (Hashimoto

et al., 1986; Linsley et al., 1988; Hashimoto

et al., 1989). Serum was separated by centri-

fugation at 4°C and stored at-20•Ž until the

assay was undertaken. All the samples were

measured in the same assay run in order to

avoid interassay variations. The significance of

the differences between the mean values was

evaluated by Student t-test.

Immunohistochemical staining was carried

out on tissue obtained from 57 of the patients

previously mentioned: 39 papillary carcinomas,

16 follicular carcinomas, 1 medullary carcinoma

and 1 squamous cell carcinoma. In addition, 6

anaplastic carcinomas, 15 adenomas, 5 ade-

nomatous goiters, 6 Hashimoto's thyroiditis, 15

Graves' disease, and 15 normal thyroids obtained

from lobes opposite to thyroid adenoma, were

also studied. Routinely formalin-fixed and

paraffin-processed material and the avidin-

biotin-peroxidase complex (ABC) technique were

used as previously described (Mizukami et al.,

1988). Antisera to Tg, CT and CEA (Immulok,

Corp. U. S. A.), CA 19-9, and CA 125 Centocor

Co. U. S. A.) were purchased.

The expressions of the ras p21 and c-myc

oncogene were evaluated with the monoclonal

antibody RAP-5 (a generous gift from Dr. H.

Schlom) and the polyclonal antibody produced

by Dr. N. Yanaihara, respectively. The im-

munohistochemical staining of epidermal growth

factor (EGF: Wakunaga Pharmaceutical Co.

Japan) and its receptor (EGF-R: Transformation

Research Inc. Japan) were performed with

commercially available reagents.

Case Report

A 73-year-old female noticed a swelling

on the right side of her neck in Decemcer,

Vol.37, No.2 TUMOR MARKERS AND ONCOGENE EXPRESSION 249

1987. She did not seek medical help until

she came to our outpatient's clinic in

March, 1988, when we diagnosed thyroid

cancer. She was then admitted for surgery.

On examination prior to surgery, there wasno evidence of hepatic, pancreatic or gall

bladder disease. Her serum level of CA

19-9 was as high as 78.1 U/ml. After hertotal thyroidectomy, the subsequent CA 19-9levels dramatically decreased to within thenormal range (Table 1). In the immuno-histochemical study, intense CA 19-9 stain-ing was found in the cytoplasm of the

papillary adenocarcinoma of thyroid (Fig. 2).

Table 1. Changes in serum tumor marker levels in a patient with undifferentiated thyroid

carcinoma with squamous cell metaplasia before and after total thyroidectomy.

Note: the consistency within normal levels in the serum levels of SCC both pre- and post-

operation in the patient with squamous cells carcinoma while Tg levels demonstrated

dramatical changes after total thyroidectomy.

Fig. 1. In follicular cells of papillary carcinoma of the thyroid CA 19-9 is stained intensively.

(•~200)

250 HASHIMOTO et al.Endocrinol. Japon.April 1990

Table 2. Serial analysis of Thyroglobulin (Tg) and CA 19-9 in a patient with CA19-9-producing thyroid cancer

Note: parallel patterns in serum concentrations of Tg and CA 19-9 both pre- and post-

operation

Because there were no other CA 19-9

producing diseases in evidence, weconcluded that this was a very rare

case of CA 19-9 producing thyroid

cancer.

Results

RESULT 1: BiochemicalResults

Serum levels of Tg were elevatedin 58.6%, CA-M26 in 15.7%, CA19-9 in 5.3%, CT in 3.6%, NSEin 3.6%, CA 15-3 in 2.6%, CA 125in 2.6%, CEA in 0.9%, CA-M29 in0%, ferritin in 0%, SCC in 0% andAFP in 0% of cases. One patientwith squamous cell carcinoma showednormal levels of SCC in the bio-chemical studies before and after totalthyroidectomy (Table 1).

RESULT 2: Immunohistochemical Results

In the immunohistochemical stu-dies, CA 19-9 positive staining wasobserved in 58% of papillary carci-nomas (Fig. 1, Table 2). No patientshowed positive CA 125 or CA 15-3staining.

On patient with a minute medul-lary carcinoma of the thyroid gland (0.5 cmin a diameter) showing negative serum CEAand CT levels (serum CEA level was <1.0ng/mg, and CT level was as low as 38pg/ml), demonstrated clear positive staining of

Fig. 2. Staining of EGF (bottom) and EGF-R (upper)

in papillary circinoma of the thyroid.(•~200)

CT and CEA in the immunohistochemicalstudy.

Of the patients studied, strong stainingof EGF in the cytoplasm of thyroid cancerswas found in 73% of paillary cancers

Vol.37, No.2 TUMOR MARKERS AND ONCOGENE EXPRESSION 251

Fig. 3. Intense staining with ras p 21 oncogene is apparent in most tumor cells, characterized

mainly by diffuse cytoplasmic staining and apical luminal surface staining. (•~200)

Fig. 4. Enhanced c-myc expression is observed in the cytoplasm of papillary thyroid

carcinoma. (•~200)

252 HASHIMOTO et al,Endocrinol. Japon.April 1990

(Fig. 2), 67% of anaplastic carcinomas and33% of follicular carcinomas. Intensestaining of EGF-R was found in 73% inthe papillary carcinomas of the thyroid

(Fig. 2), 67% of anaplastic carcinomas and33% of follicular carcinomas, althoughnegative staining was found in the normalthyroid glands or follicular cells of Graves'disease.

As shown in Fig. 3, intense expressionof the ras p21 oncogene was demonstratedin the apical cell surface and cytoplasm of

papillary carcinoma (Fig. 3). Frequency ofthe enhanced expression of ras oncogenewas demonstrated in 100% of follicullarcarcinomas, 100% of anaplastic carcinomasand 63% of palliary carcinomas.

Enhanced c-myc expression was observedin the cytoplasm in 90% of the papillarythyroid carcinomas (Fig. 4), 100% in ana-

plastic carcinomas and 67% in follicularcarcinomas. C-myc gene amplification wasalso observed 100% in Graves' disease.

Discussion

Our present results indicate that thetwelve commercially available RIA kitswhich we studied are not sensitive enoughfor the early detection of thyroid cancer.Only serum levels of Tg and CA-M26were elevated in 58.6% and 15.7% ofcases respectively. Since increased con-centrations of serum Tg have been observedin both thyroid diseases and in thyroidcarcinoma, recent investigations have focusedon exploring a cancer specific Tg structure,in order that an and Tg monoclonal anti-body can be prepared (Kim, Dunn andDunn, 1988; Kohno et al., 1985).

CA-M26 is known to be a reliablemarker for breast cancer (Linsley et al., 1988).We applied it to thyroid cancer and foundthat it is also a fairly good marker for this

purpose. If further studies were directedtowards producing monoclonal antibodies

to recognize different epitopes of mucinsaround CA-M26, successful results might beanticipated. Furtheremore, it is well knownthat about 30% (SD 10%) of all thyroidcancer patients have anti-Tg antibodies

(Bayer and Kriss, 1979) which falsely in-crease or depress Tg values (Schneider andPervos, 1978). Therefore, research into theuse of mucin glycoproteins such as CA-M26as a tumor marker for thyroid cancersshould be pursued.

SCC is reported to be a sensitive tumormarker for squamous cell carcinoma. Weencountered a typical case of squamous cellcarcinoma of the thyroid gland. Contraryto our expectations, we found SCC insensi-tive in our case. The difference betweenthese results may depend on the organdifference or on the stage of the carci-noma.

Although Ohtani et al. (Ohtani et al.,1988) reported NSE was a good tumormarker of medullary thyroid carcinoma,NSE and ferritin estimations have no valuein the diagnosis of thyroid cancer, asreported by Touitou (Touitou and Heshmati,1982).

According to our results then, since sofar as no single one, nor any combinationof the tested tumor markers could con-sistently diagnose thyroid cancer, furtherstudies directed towards producing a specificthyroid tumors marker with monoclonalantibodies should be pursued.

In the course of this study, a case ofCA 19-9-producing papillary carcinoma wasdiscovered (Fig. 1, Table 2). We found ahigh concentration of CA 19-9 in theserum (Table 2). This tumor marker wasalso detected in the cytoplasma membraneof the thyroid cancer by immunoperoxidasestaining (Fig. 1). Thus, we speculatedthat the high serum level of CA 19-9 wasdue to its leakage from the thyroid cancerinto the serum. This possibility was sup-

ported by the finding that the serum levelof CA 19-9 returned to within the normal

Vol.37, No.2 TUMOR MARKERS AND ONCOGENE EXPRESSION 253

range after her total thyroidectomy (Table2). Similar CA 19-9-producing lung cystshave been reported by Okubo et al.(Okuboet al., 1989). Routinely this tumor wouldhave been broadly defined as a follicular or

papillary carcinoma. This case indicatesthat other carbohydrate antigen or cartinomaassociated mucin antigen-producing tumors,could be discovered by combining RIA andimmunohistochemical studies in this way.

During the past decade, there has beena dramatic renewal of interest in growthfactors and oncogenes in the field of cancerresearch (Fusco et al., 1987; Franklyn andSheppard, 1988., Burman et al., 1987). Inour immunohistochemical study, strong stain-ing of EGF and EGF-R in papillary thyroidcancers was demonstrated (Fig. 2). Sincenegative stainings were found in the normalthyroids glands or follicular cells of Graves'disease, these findings support"the autocrinetheory" of thyroid cancer (Schlessinger,1986).

As shown in Fig. 3, intense expressionof the ras p 21 oncogene was demonstratedin the apical cell surface and cytoplasm of

papillary carcinoma (Mizukami et al., 1988).Furtheremore, we observed that ras oncogeneexpression was enhanced in papillary carci-noma and follicular carcinoma. Increasedras p 21 oncogene expression has been ob-served in a variety of human cancers, how-ever, it is postulated that ras p 21 expressioncannot be used as a diagnostic marker inhuman cancer (Czerniak et al., 1989). Inaddition, as shown in Fig. 4, enhanced c-myc expression was observed in the cyto-

plasm of the papillary thyroid carcinomas.C-myc oncogene expression has also beendemonstrated in anaplastic carcinoma andfollicular carcinoma as previously reported

(Burman et al., 1987; Terrier et al., 1985).However, c-myc gene amplification was alsoobserved in Graves' disease. This infersthat c-myc expression might be involved inthe proliferation and development of thyroidcells and is not cancer specific (Nunes et

al., 1987). On the other hand, Burman etal.(Burman et al., 1987) emphasizes that achange in genomic c-myc regulation may

play a role in tumor development in somepatients with thyroid cancer.

In conclusion, to clarify these conflictingreports and to detect early thyroid cancer,more extensive research, including molecularoncology and technology to develop a DNA

probe to aid the diagnosis, is warranted inthis area.

Acknowledgements

We are indebted to Dr. Ellen Ingham for

encouragement, helpful discussions, and com

ments. We also thank Miss Misako Yagi and

Miss Yasuko Kamei for their excellent secretarial

assistance and to Miss Hermione Elliot and

Mr. David Shinya Tano for their help in

English in the preparation of this manuscript.

This work was supported in part by the

Japan Clinical Pathology Foundation for Inter-

national Exchange and Clinical Pathology Foun-

dation of Japan.

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