Thyroid carcinoma associated with familial adenomatous polyposis

Histopathology 1997, 31, 231–236

Thyroid carcinoma associated with familial adenomatous polyposis F.CETTA, P.TOTI*, M.PETRACCI†, G.MONTALTO, A.DISANTO*, F.LORE` ‡ & A.FUSCO§ Institutes of Surgical Clinics, *Pathology, †Ophthalmology and ‡Endocrinology, University of Siena, and §Dipartimento di Medicina Sperimentale e Clinica, University of Reggio Calabria, Italy Date of submission 8 January 1997 Accepted for publication 23 April 1997

C E T TA F. , T O T I P. , P E T R A C C I M . , M O N TA LT O G . , D I S A N T O A . , L O R E` F & F U S C O A .

(1997) Histopathology 31, 231–236

Thyroid carcinoma associated with familial adenomatous polyposis Aims: Thyroid carcinoma is an extracolonic manifestation that is present in about 1% to 2% of patients with familial adenomatous polyposis (FAP). Less than 100 cases have been reported in detail. We have investigated the suggestion that FAP associated thyroid carcinoma is significantly different morphologically from both papillary and follicular types and can be considered as a separate entity. Methods and results: Specimens from three patients with FAP associated thyroid tumours, all but one having single nodules, have been analysed. All three patients belonged to an extended kindred (23 siblings in four generations) who had genetic analysis and intensive screening for thyroid nodules. Seven patients had the

same APC mutation at codon 1061. Pathological examination revealed a typical papillary carcinoma, encapsulated variant, in all patients, with follicular areas in one case. All thyroid specimens, in addition to histological and immunohistological examinations, were also specifically studied for activation of the RETPTC oncogene, that seems to be restricted to papillary thyroid carcinoma. Two of the three patients had RETPTC activation (PTC1 isoform). Conclusions: The findings suggest that the tumours were certainly papillary, at least in the present kindred. Further studies in different families are required for a better understanding of this peculiar tumour and of its biological behaviour.

Keywords: thyroid carcinoma, familial adenomatous polyposis, RET-PTC mutation

Introduction Thyroid carcinomas of follicular cell origin are usually sporadic tumours. However, inherited factors may be implicated in a minority of these cancers. Familial papillary thyroid carcinoma has been described as an isolated defect, but has also been associated with non polyposis colon cancer syndrome (HNPCC), Peutz– Jeghers’ syndrome, Cowden’s disease and ataxiatelangectasia1–4. The best-recognized association is with familial adenomatous polyposis (FAP). It is currently believed that FAP is caused by a single defective gene and that extracolonic lesions, including thyroid tumours, are probably the phenotypic Correspondence to: Dr F. Cetta, Professor of Surgery, Institute of Surgical Clinic, University of Siena, Nuova Policlinico, viale Bracci, 53100 Siena, Italy. q 1997 Blackwell Science Limited.

expression of a higher penetrance of the gene5. In fact, most of the patients have other extracolonic manifestations in addition to thyroid cancer, if specifically screened for them5,6. The current view is that the symptoms previously known as Gardner’s syndrome are due to a generalized disturbance of proliferative activity in tissues of endodermal, mesodermal and ectodermal origin5–15. The first documented case of thyroid cancer in a patient with FAP was reported in 19497, but the importance of this association was not fully appreciated until 1968, when Camiel et al. reported two sisters with papillary thyroid carcinoma and FAP, suggesting that this association was not fortuitous8. In a review of world literature by Bell and Mazzaferri in 199314, 49 cases with such association were found. A year later, a total of 63 patients were reported by Harach

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et al.15. Recent consecutive series from the largest FAP register suggest that about 1% of FAP patients have thyroid carcinoma9. However, little attention had been paid to the morphological features of thyroid tumours before a recent paper by Harach et al. suggesting that FAP associated thyroid carcinoma may represent a new morphological entity, different from both typical papillary and follicular carcinomas15. It has recently been shown that 20% of papillary thyroid carcinomas and up to 50% of occult thyroid carcinomas harbour the RET-PTC oncogene, an activated form of the RET proto-oncogene. RET activation is restricted to carcinomas of the papillary type and has never been found in non-thyroid neoplasms16–20. Here we report the histological appearance of thyroid carcinomas from three patients with FAP. The present patients have the following peculiarities: (1) they all belong to the same family; (2) they have been prospectively studied with an intensive screening for thyroid lesions; (3) the specific mutation of the APC gene in these patients has been identified; and (4) genetic analysis has been performed in thyroid tumoral tissue, aimed at detecting the activation of RET-PTC proto-oncogene.

Patients and methods The three patients with thyroid carcinoma were members of an extended kindred consisting of 23 members, spanning four generations. The extended pedigree of the kindred has been reported elsewhere, along with a detailed list of other extracolonic manifestations21. This kindred belonged to a series of 13 FAP kindreds that were intensive screened for FAP and associated extracolonic manifestations. All living patients except three who refused screening underwent colonoscopy, upper gastrointestinal (GI) endoscopy (supplemented by X-ray examination of the GI tract in selected cases), and multiple biopsies. In addition, patients were screened for osteomas, dental abnormalities and desmoid tumours. Fundus oculi was examined in all patients. All patients underwent ultrasound (US) examination of the thyroid gland. Fine needle aspiration biopsy (FNAB) of nodules larger than 5 mm was performed. Some patients underwent multiple US and FNAB procedures. Cytological examination of the FNAB specimens was performed according to standard methods.

HISTOLOGICAL TECHNIQUES

All grossly identifiable nodules, as well as normal thyroid areas, were extensively sampled. Sections were

routinely stained with haematoxylin and eosin (H & E). Immunohistochemistry was carried out using the following monoclonal antibodies: thyroglobulin (Biogenex, San Ramon, California, diluted 1 : 500), chromogranin A (Dakopatts, Glostrup, Denmark, diluted 1 : 200), carcinoembryonic antigen (Immunotech, Marseille, France, diluted 1 : 10), and cytokeratin AE1/AE3 (Boehringer–Mannheim, Mannheim, Germany, diluted 1 : 1000). Colour was developed using the APAAP method. A polyclonal antibody against calcitonin (Biogenex, diluted 1 : 200) was also used, and the colour was developed with 3,30 -diaminobenzidine tetrahydrochloride.

S T U DY O F R E T / P T C A C T I VAT I O N

Research for activation of RET-PTC was performed using immunohistochemistry. This method is based on the fact that the RET proto-oncogene is not expressed in thyroid follicular cells unless its expression is driven by activating sequences replacing its 50 portion. Therefore the detection of the RET protein, by immunohistochemical analysis, will indicate an activation of the RET. In addition, after RNA extraction from paraffin-embedded samples, RT-PCR was used according to a previously published procedure22 for subsequent identification of RET-PTC expression as RET-PTC1, 2 or 3. In fact, the RET-PTC oncogene derives from the fusion of the tyrosine–kinase domain of the RET proto-oncogene with the 50 terminal region of other genes. RET-PTC1 is fused with another gene, named H4, also located on the long arm of chromosome 10, while the 50 portions of the RET-PTC2 and RET-PTC3 are represented, respectively, by the regulatory subunit RIa of the cAMP-dependent protein kinase A and the RFG/ELE1 gene.

S E A RC H F O R A P C M U TAT I O N S I N G E R M - L I N E C E L L S

DNA was isolated from lymphocytes in 10 ml of EDTA anticoagulated venous blood by means of proteinase K digestion and phenol–chloroform extraction. The entire coding region (8532 bp) of the APC gene was analysed by the PCR-SSCP method23 in all seven patients positive for CHRPE. The APC gene coding region was divided into five overlapping amplification segments, varying in length from 1.8 to 2.5 kDa. The primers used for PCR included signals for transcription by T7 polymerase and in vitro translation at their 50 end. Thus, PCR products could be transcribed and translated in vitro to seek the presence of mutations which resulted in an altered size of the encoded polypeptide. Direct sequencing was performed to identify the nature of mutations. q 1997 Blackwell Science Ltd, Histopathology, 31, 231–236.

FAP associated thyroid carcinoma

Results DESCRIPTION OF THE KINDRED

The proband was a 22-year-old woman (patient 1) who underwent colonscopy because of anaemia and abdominal pain. Her kindred consisted of 22 additional members, spanning four generations. Five members of the third generation had colonic polyps and underwent total colectomy. In particular, one of them, a 42-yearold female, in addition to multiple polyps, already had cancer in the descending colon. A careful examination of the fundus oculi revealed that all affected patients showed congenital hypertrophy of the retinal pigment epithelium. Intensive screening of all siblings for extracolonic manifestations showed additional tumours in all patients except one (a 38year-old male). None of the patients had desmoid tumours. A detailed list of all extracolonic manifestations has been reported elsewhere21. In particular, three patients, i.e. the proband, her sister (patient 2), aged 20, and the maternal aunt (patient 3), aged 36, also had thyroid nodules which diagnosed malignant at US guided FNAB. Single nodules (8 and 11 mm in diameter, respectively) were found in the last two cases, while the proband presented three different nodules. In addition, a 15-year-old girl (patient 4), who had not yet developed colonic polyps, also had thyroid nodules. US guided biopsy showed the presence of epithelial cells without any malignant changes and numerous inflammatory mononuclear lymphoid cells with germinal centres, suggesting Hashimoto’s thyroiditis (Figure 1).

Figure 1. Patient 4 (15-year-old girl). FNAB: findings supporting Hashimoto’s thyroiditis; cytological appearance of a germinal centre, (May Grunwald-Griemsa × 400). q 1997 Blackwell Science Ltd, Histopathology, 31, 231–236.

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H I S T O L O G I C A L E X A M I NAT I O N

All tumours were encapsulated. The predominant architectural pattern of the tumours studied was papillary, showing complex and branching papillae with a central fibrovascular core (well-differentiated carcinoma, papillary type, encapsulated variant) (Figures 2–4). One case (aged 20) also had widespread follicular areas (well-differentiated carcinoma, papillary type, follicular encapsulated variant) with a cribriform pattern. No true laminated psammoma bodies were observed. The tumour cells most often showed eosinophilic or amphophilic cytoplasms. Cell nuclei were slightly irregular in shape, either hyperchromatic or with a ground glass appearance, with prominent nucleoli. They often had cytoplasmic inclusions or grooving (Figures 3 & 4). In particular, one nodule from the patient 1 showed just a small area (with a solid cribriform pattern) with capsular penetration (Figure 4). This patient had a micrometastasis in a neck lymph node with follicular architecture (Figure 5). Her thyroid also showed the presence of focal lymphocytic aggregates with no germinal centres, both within

Figure 2. Patient 2 (20-year-old woman). Papillary carcinoma of the thyroid, encapsulated variant. *Thick fibrotic capsule, which completely encircles the tumour (H & E, × 150).

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Figure 3. Patient 3 (36-year-old woman). Papillary neoplastic growth with characteristic nuclei (H & E, × 400).

Figure 4. Patient 1 (22-year-old woman). Papillary carcinoma: focus of solid growth with capsular infiltration (H & E, × 200).

malignant nodules and in surrounding tissue. Patient 3 had a typical papillary carcinoma, encapsulated variant, with no area showing a cribriform pattern (Figure 3). Immunohistochemistry revealed occasional foci of tumour cells positively stained for thyroglobulin and large areas of cytokeratin immunoreactivity. On the other hand, positive immunoreactivity was never observed with antibodies against chromogranin A, carcinoembryonic antigen and calcitonin. All three patients had no recurrence in the following 2 years.

deletion created a stop codon (TGA) at position 3189– 3191, which is a very frequent mutation in the APC gene of patients with FAP. More specifically, it is the second most frequent mutation following mutation at codon 1309.

R E T- P T C AC T I VAT I O N

Widespread RET-PTC activation was found in the tumoral tissue of the two young sisters (patients 1 and 2), respectively, but not in the thyroid specimen of their aunt (patient 3). RT-PCR documented that the specific isoform in these two patients was RET-PTC1. G E N E T I C A NA LYS I S O F G E R M - L I N E C E L L S

A 5 bp deletion of codon 1061 (A CAA A) (position 3183–3187) was observed in all affected patients. This

Discussion Sporadic papillary and follicular thyroid carcinoma seem to be related to different epidemiologic factors and also to different genetic alterations. In a recent review of the literature, a total of 63 cases of thyroid carcinoma associated with FAP were reported15. There have only been four cases of two siblings affected by thyroid carcinoma in a single family. To our knowledge, the present report is the first with three affected relatives in the same FAP kindred. Thyroid carcinoma usually occurs early, between 1 and 5 years after FAP detection. In particular, both the papillary and the follicular histotype were reported to occur in the thyroid of FAP patients. Since the present kindred was prospectively studied, all of the members had an intensive screening for thyroid nodules from youth. Therefore, nodules or other q 1997 Blackwell Science Ltd, Histopathology, 31, 231–236.

FAP associated thyroid carcinoma

Figure 5. Patient 1 (22-year-old woman). The neck lymph node micrometastasis shows a follicular architecture and immunohistochemical positivity for thyroglobulin (Thyroglobulin, × 400).

thyroid lesions could have an early detection. In particular, one of the three patients with thyroid carcinoma showed evidence of lymphocytic infiltration within the nodule and surrounding thyroid. Moreover, patient 4, who had not undergone thyroidectomy, showed multiple thyroid nodules, featuring abundant lymphoid infiltrate at FNAB examination (Figure 1). The probability that this inflammatory lesion could be a non-causal finding deserves further investigation. To our knowledge, there is only one other report describing non malignant or ‘premalignant’ thyroid lesions in two members of a FAP family, with relatives affected by thyroid carcinoma24. Both were females. Intralobular lymphoid infiltrate in the former patient (a 15-year-old girl) was reported as Hashimoto’s thyroiditis, while in the latter, the lymphocytic infiltrate was associated with a small follicular carcinoma24. APC gene mutations (at codon 1061, as in the present family, but also at codon 130925), or at other sites (848)14, may also cause predisposition to thyroid tumours. FAP associated thyroid tumours have been reported in most cases as papillary, yet in q 1997 Blackwell Science Ltd, Histopathology, 31, 231–236.

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some cases as follicular or mixed24. They are seldom associated with distant metastases and usually show an indolent behaviour11,26,27. The fact that the prognosis of papillary carcinoma is excellent, especially in young people26, could support the hypothesis by Harach suggesting that FAP associated thyroid tumour is a tumour with unique features and still unknown biological behaviour15. On the other hand, the observation of RET-PTC activation in two of the three patients, i.e. a finding that is unique to papillary tumours27, strongly suggests that these tumours are papillary, even in the patient with widespread follicular areas. In particular, a large cribriform area was observed in one of our three patients (patient 2) and, in a small area, in one of the three nodules of patient 1. Therefore, morphological findings are similar to those observed in most FAP associated thyroid tumours that have previously been described7–15,28. In any case, they are significantly different from typical ‘follicular’ tumours, that also have been reported, even if infrequently, in sporadic patients with FAP. Characteristics of these tumours seem to be papillary growth and encapsulated variant, with frequent, but not constant, cribriform areas (absent in patient 3, Figure 3). Since the causative role of inherited factors in thyroid tumours of the present kindred is beyond reasonable doubt, the question could be raised as to whether previous tumours reported as ‘follicular’ were (i) ‘follicular’ (re-examination of the specimens and/or search for RET-PTC activation could be of help); and (ii) a casual finding, instead of being determined by the APC gene mutation. Current opinion is that follicular and papillary types greatly differ among them for both risk factors and pathogenetic mechanisms. The former showed a frequent activation of ras oncogene, the latter of the chimeric gene RET-PTC. RET-PTC and ras activations seem to be mutually exclusive of each other. On the basis of the present findings, FAP associated thyroid tumours seem to suggest a cooperation between APC mutation, at least in some codons and in germ-line cells29, and the RET-PTC, but not ras, activation. More generally, RETPTC activation could detect a subgroup of papillary thyroid tumours, particularly frequent in young patients, possibly related to different aetiological factors (genetic predisposition, exposure to fall-out radiation during the first years of life30–32, etc.) with possible lymph nodal metastasis, but with infrequent poor outcome25. A definitive answer is still difficult. In particular, the following questions should be addressed: (i) Can initial papillary tumours in patients carrying APC and/or RET-PTC mutation turn into other types?

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(ii) Is RET-PTC activation a rare observation or a frequent finding in FAP associated thyroid cancer? (iii) Do APC mutations in FAP patients with thyroid carcinoma tend to cluster around some codons or are they scattered throughout the gene? Further studies are required for a deeper insight into FAP associated thyroid cancer. They could be of importance not only for nosologic, genetic and pathogenetic purposes, but also for early diagnosis and proper treatment.

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Acknowledgements The study has been supported in part by Regione Toscana Grant no. 358/C 1995, Murst 40%-Murst 60%, and National Research Institute (CNR) no. 9500897 CT04.

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References 1. Lote K, Andersen K, Nordal E, Brennhovd O. Familial occurrence of papillary thyroid carcinoma. Cancer 1980; 46; 1291–1297. 2. Stoffer SS, Van Dyke DL, Bach JV, Szpunar W, Weiss L. Familial papillary carcinoma of the thyroid. Am. J. Med. Genet 1986; 25; 775–782. 3. Phade VR, Lawrence WR, Max MH. Familial papillary carcinoma of the thyroid. Arch. Surg. 1981; 116; 836–837. 4. Farid NR, Shi Y, Zou M. Molecular basis of thyroid cancer. Endocrin. Rev. 1994; 15; 202–232. 5. Giardiello FM, Offerhaus GJ, Lee HD et al. Increased risk of thyroid and pancreatic carcinoma in familial adenomatous polyposis. Gut 1993; 34; 1394–1396. 6. Iwama T, Mishima Y, Utsonomiya J. The impact of familial adenomatous polyposis on the tumorigenesis and mortality at the several organs. Ann. Surg. 1993; 217; 101–108. 7. Crail HW. Multiple primary malignancy arising in the rectum, brain and thyroid. Report of a case. US Navy Med. Bull. 1949; 49; 123–128. 8. Camiel MR, Mule JE, Alexander LI, Benninghoff DL. Association of thyroid carcinoma with Gardner’s syndrome in siblings. New Engl. J. Med. 1968; 9; 1056–1058. 9. Bulow S. Papillary thyroid carcinoma in Danish patients with familial adenomatous polyposis. Int. J. Colorect. Dis. 1988; 3; 29– 31. 10. Delamarre J, Capron JP, Armand A, Dupas JL, Deschepper B, Davison T. Thyroid carcinoma in two sisters with familial adenomatous polyposis of the colon. J. Clin. Gastroenterol. 1988; 10; 659–662. 11. Plail RO, Bussey HJR, Glazer G, Thompson JPS. Adenomatous polyposis: an association with carcinoma of the thyroid. Br. J. Surg. 1987; 74; 377–380. 12. Thompson JS, Harned RK, Anderson JC, Hodgson PE. Papillary carcinoma of the thyroid and familial polyposis coli. Dis. Colon Rectum 1983; 26; 583–585. 13. Kashiwagi H, Konishi F, Kanazawa K et al. Sisters with familial adenomatous polyposis affected with thyroid carcinoma: a

21.

22.

23.

24.

25.

26. 27.

28.

29.

30.

31.

32.

distinct type of follicular cell neoplasm. Br. J. Surg. 1996; 83; 228. Bell B, Mazzaferri EL. Familial adenomatous polyposis (Gardner’s syndrome) and thyroid carcinoma. A case report and review of the literature. Digest. Dis. Sci. 1993; 38; 185–190. Harach HR, Williams GT, Williams ED. Familial adenomatous polyposis associated thyroid carcinoma: a distinct type of follicular cell neoplasm. Histopathology 1994; 25; 549–561. Fusco A, Grieco M, Santoro M et al. A new oncogene in human papillary thyroid carcinomas and their lymph-nodal metastases. Nature 1987; 328; 170–172. Grieco M, Santoro M, Berlingieri MT et al. PTC is a novel rearranged form of the RET-proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell 1990; 60; 557–563. Santoro M, Carlomagno F, Hay ID et al. Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype. J. Clin. Invest. 1992; 89; 1517–1522. Santoro M, Grieco M, Melillo RM, Fusco A, Vecchio G. Molecular defects in thyroid carcinomas: role of the RET oncogene in thyroid neoplastic trasformation. Eur. J. Endocrinol. 1995; 133; 513–522. Molberg K, Albores-Saavedra J. Hyalinizing trabecular carcinoma of the thyroid gland. Hum. Pathol. 1994; 25; 192–197. Civitelli S, Tanzini G, Cetta F, Petracci M, Pacchiarotti MC, Civitelli B. Papillary thyroid carcinoma in three siblings with familial adenomatous polyposis. Int. J. Colorect. Dis. 1996; 11; 571–574. Viglietto G, Chiappetta G, Martinez-Tello FJ et al. RET-PTC oncogene activation is an early event in thyroid carcinogenesis. Oncogene 1995; 11; 1207–1210. Groden J, Thliveris A, Samowitz W et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell 1991; 66; 589–600. Herrera L, Carrel A, Rao U, Castilo N, Petrelli N. Familial adenomatous polyposis in association with thyroiditis. Dis. Colon Rectum 1989; 32; 893–896. Varesco L, Gismondi V, James R et al. Identification of the APC gene mutations in Italian adenomatous polyposis coli patients by PCR-SSCP analysis. Am J. Hum. Genet. 1993; 52; 280–285. Rosai J, Carcangiu ML, DeLellis RA. Tumours of the Thyroid Gland, third series. Washington DC: AFIP, 1992. Sugg SL, Zheng L, Rosen IB, Freeman JL, Ezzat S, Asa SL. Ret/PTC1, 2 and 3 oncogene rearrangements in human thyroid carcinomas: Implications for metastatic potential. J. Clin. Endocrinol. Metab. 1996; 81; 3360–3365. Hizawa K, Iida M, Yao T et al. Association between thyroid cancer of the cribriform variant and familial adenomatous polyposis. J. Clin. Pathol. 1996; 49; 611–613. Curtis L, Wyllie AH, Shaw JJ et al. Evidence against involvement of APC mutation in papillary thyroid carcinoma. Eur. J. Cancer 1994; 30; 984–987. Cetta F. Progression of tumorigenesis in patients with papillary carcinoma of the thyroid associated with familial adenomatous polyposis. Gastroenterology (Abstr.) 1996; 110; A502. Cetta F. Prevalence, significance and biological behaviour of retPTC1 associated papillary thyroid carcinoma. J. Clin. Endocrinol. Metab. 1997; 82; 1650. Cetta F, Petracci M, Montalto G, Fusco A. Thyroid cancer and the Chernobyl accident. Are long term and long distance side-effects of fall-out radiation greater than estimated? J. Clin. Endocrinol. Metab. 1997; 82; 2015–2017.

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