MEDULLARY thyroid carcinoma (MTC) may arise as

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1 X/97/$03.00/0 Vol. 82, No. 10 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1997 by The Endocrine Society A Case of Metastatic Medullary Thyroid Carcinoma: Early Identification Before Surgery of an RET Proto-Oncogene Somatic Mutation in Fine-Needle Aspirate Specimens* DIEGO RUSSO, FRANCO ARTURI, EUSEBIO CHIEFARI, DOMENICO MERINGOLO, DAVIDE BIANCHI, BARTOLOMEO BELLANOVA, AND SEBASTIANO FILETTI Cattedra di Endocrinologia, Dipartimento di Medicina Sperimentale e Clinica (F.A., S.F., E.C.), Cattedra di Farmacologia, Facoltà di Farmacia (D.R.), Università di Reggio Calabria, Via T. Campanella, Catanzaro, Italy; Unità Operativa di Endocrinologia (D.M., D.B.), Ospedale Bentivoglio; and Servizio di Medicina Nucleare Policlinico S. Orsola-Malpighi (B.B.), Bologna, Italy ABSTRACT Medullary thyroid carcinoma (MTC) management requires determination of the sporadic or familial nature of the disease. RET protooncogene mutation analysis in the tumor tissue obtained at surgery and in the peripheral blood identifies somatic vs. germinal mutations. We now report a case of MTC in which a RET somatic mutation at codon 918 was detected in fine-needle aspiration specimens obtained from both the thyroid nodule and two enlarged neck lymph nodes but Received March 21, Revision received June 3, Accepted June 19, Address all correspondence and requests for reprints to: Sebastiano Filetti, Cattedra di Endocrinologia, Dipartimento di Medicina Sperimentale e Clinica, Via T. Campanella, Catanzaro, Italy. filetti@mbox.vol.it. * This work was supported by a Ministero dell Università e delle Ricerca Scientifica e Tecnologica grant (40%) and by a grant of Associazione Italiana per la Ricerca sul Cancro (AIRC) (to S.F.). not in peripheral blood. Therefore, a diagnosis of sporadic MTC was made before surgery. Thus, this approach, by excluding preoperatively multiple endocrine neoplasia disease, permitted immediate thyroidectomy without search for pheochromocytoma. PCR-based genetic analysis in fine-needle aspiration biopsy specimens, therefore, preoperatively identifies genetic abnormalities at an early and easily manageable stage and may well contribute to the management strategy of MTC. (J Clin Endocrinol Metab 82: , 1997) MEDULLARY thyroid carcinoma (MTC) may arise as sporadic or familial disease, eventually occurring in association with other endocrine tumors in multiple endocrine neoplasia (MEN) syndrome 2A and 2B. In these inherited disorders, germline abnormalities (mostly point mutations) in the RET proto-oncogene have been identified in the majority of patients affected (1). Somatic RET mutations occur as well in sporadic MTC with a frequency variable between 25 and 70% in different studies (1, 2). Identification and assessment of the nature of the RET mutation (somatic or germinal) in all of the MTC is, therefore, a critical tool for discriminating sporadic cases from unrecognized familial disease and could replace biochemical screening based on the calcitonin (CT) serum level at basal concentrations and after pentagastrin stimulation (3, 4). This investigation was undertaken to assess the possibility of preoperative detection of genetic abnormalities in fine-needle aspiration specimens from a patient with MTC. A major problem in the management of these patients is the necessity of excluding the presence of a pheochromocytoma. In fact, the asymptomatic normotensive patient with MEN2 syndrome is at risk of dangerous and fatal paroxysms during surgery if an unsuspected pheochromocytoma is present (5). Thus, we report a case of a patient with a MTC presenting with enlarged neck lymph nodes. In this patient, we performed the RET proto-oncogene mutation analysis in the complementary DNA (cdna) from the fine-needle aspiration biopsy (FNAB) of the thyroid nodule and the lymph nodes, as well in the peripheral blood DNA. Case Report and Methods A 47-yr-old male was referred to us in April 1996 because of a right neck mass. Physical examination revealed several palpable enlarged lymph nodes in the right cervical and submandibular region and a 2-cm diameter nodule in the right thyroid lobe. Ultrasound evaluation confirmed the presence of a solid right thyroid mass (cold at 99 Tc scintigraphy) and of multiple enlarged lymph nodes in the right cervical region. Serum-free T 3 and TSH were normal. Subsequently, FNAB (under US guidance) on both the thyroid nodule and the enlarged lymph nodes was performed. An aspirate aliquot was smeared for cytological examination, and another was utilized for messenger RNA extraction. At cytology, diagnosis of suspected medullary carcinoma and lymph node metastases was made. High serum CT, 2800 ng/l (normal 0 10 ng/l) and carcinoembryonic antigen, 70 ng/ml (normal 0 5 ng/ml) levels confirmed the diagnosis of MTC. An accurate family history, extended to second-degree relatives, was negative for endocrine or neoplastic diseases. The presence of other neoplasia associated with the MTC in the MEN syndromes was excluded. In fact, there was no evidence of pheochromocytoma (by assessment of plasma and urinary catecholamines, by computerized tomographic scan of the abdomen, and scintigraphy by 131 I-labeled metaiodobenzylguanidine) or hyperparathyroidism (serum calcium and PTH levels in the normal range). The patient underwent total thyroidectomy and central neck lymph node dissection. Histological examination (kindly provided by Dr. C. Gallo) confirmed the diagnosis of MTC and MTC metastatic right cervical and submandibular lymph nodes; neither multifocal diseases or C cell hyperplasia were detected; immunohistochemistry 3378

2 RET MUTATION IN FNAB showed neoplastic cell positivity for CT and negativity for thyroglobulin (Fig. 1). Presence of amyloid was evident after staining with Congo red (Fig. 2). Genetic analysis RT-PCR amplification were performed as previously described (6). PCR conditions were the following: cycle 1 at 42 C for 15 min and 95 C for 15 sec; 40 cycles at 95 C (1 min), 61 C (1 min) and 72 C (1 min); the last cycle at 72 C for 7 min (42 total cycles) The following 39 and 59 oligonucleotides (from Genosys, Cambridge, UK) were used according to the published sequence of RET cdna (7): A) 10F (59-ATTGTTGGGGGACACGAG-39) sense primer; 11R (59-GCAGTGGATGCAGAAGGC-39) antisense primer. B) 12F (59-GCCGTGAAGATGCTGAAAGAG-39) sense primer; 14R (59-AAATGAGATGAGGTCGCCCAT-39) antisense primer. C) 15F (59-CTCGTTCATCGGGACTTGGCA39) sense primer; 17R (59-CCATACATCACTTTGCGTGGT-39) antisense primer. The amplification products were purified and examined by re- FIG. 1. Immunohistochemical staining of tumoral tissue. A, Immunostaining with anti-ct antibodies (immunoperoxidase) shows high positivity of tumoral cells. B, Immunostaining with antithyroglobulin antibodies (immunoperoxidase) shows high positivity of follicular peritumoral cells whereas tumoral cells are negative.

3 3380 RUSSO ET AL. JCE&M 1997 Vol 82 No 10 FIG. 2. Staining by Congo red shows presence of amyloid depots into tumoral cells. striction enzyme analysis and/or direct sequencing. Genomic DNA was extracted by 2 ml peripheral blood of the same patient using a blood DNA extraction kit (Qiagen, M Medical Genenco, Firenze, Italy). A search for RET gene abnormalities in exons 10, 11, 13, 14, and 16 was performed as described (8). Results The purified 198-bp DNA fragment, obtained by PCR amplification of a region containing codon 918 of exon 16 of the RET gene, which is the more frequently altered site in sporadic MTC (1, 2), was digested with Fok1. Figure 3 shows the presence of a stronger undigested band together with weak bands (of 156 and 42 bp), a pattern consistent with the loss of the Fok1 site in one allele of the gene. The same pattern was detected in the amplified cdna from FNAB of both the tumor and the lymph nodes (Fig. 3B). The nucleotide sequence of the same fragment confirmed the presence of an ATG to ACG heterozygotic mutation at codon 918 (Fig. 4). Analysis of genomic DNA extracted from peripheral blood of the same patient showed maintenance of the Fok1 site, revealing the somatic nature of the mutation (Fig. 3C). The other areas of the RET gene known to host abnormalities in familial medullary thyroid carcinoma (FMTC) or MEN2 patients were also examined in the FNAB specimens and blood DNA of the patient, showing absence of alterations (data not shown). Discussion MTC has a malignant clinical behavior. Regional lymph node metastases are often present at the diagnosis and are considered the most important prognostic factors (9). In approximately 20% of the cases, MTC is the primary manifestation of a MEN2 disease. In patients affected by MTC, therefore, evaluation of medullary adrenal gland and parathyroid function, as well as the assessment of a heritable disease, is required. Even without a suspicious family history, in fact, an inherited disease has been demonstrated in some cases by biochemical (10) or genetic screening (11). Recently, an alteration of RET proto-oncogene has been identified as the genetic marker of MEN 2A and 2B and hypothesized to be the causative event in such diseases. Point mutations of the RET gene occur also in sporadic MTC. Thus, new protocols for management of this illness using genetic analysis have been proposed (12, 13). In all of these protocols, the search for mutations in RET oncogene is first performed either in specimen obtained at surgery or, after the histological diagnosis, by DNA extraction from paraffin-embedded tissue blocks. Subsequently, if genetic analysis is positive, the search is extended to peripheral blood samples from the patients and eventually to their close relatives. In consideration of the rare possibility of coexistence of somatic and germinal alterations in different sites (14), the search for RET alterations must include all of the sites known to host genetic anomalies. However, at present, even after such a complete investigation, a 100% exclusion diagnosis for familial disease may be not reached (4). Our study shows the possibility and the feasibility to search for RET mutations at a very early stage, even before performing surgery, by analyzing the aspirates obtained from the thyroid nodule and enlarged neck lymph nodes, when present. In our patient the genetic analysis established a diagnosis of sporadic MTC, making it unnecessary for either familial screening with the traditional, not well-tolerated pentagastrin test or for further investigation for other endocrine neoplasia as part of a MEN2. In this regard, it appears as an important feature of our approach to exclude preoperatively the presence of MEN diseases in our patient

4 RET MUTATION IN FNAB 3381 FIG. 3. A, Restriction analysis of cdna derived PCR products of RET exon 16 after FokI digestion (3 h at 37 C). Samples were run on 2% agarose-tris-borate-etilendiamminetetraacetate gel containing ethidium bromide. Lane 1, Undigested amplicon from medullary carcinoma TT cell line; lane 2, digested amplicon from TT cells; lane 3, digested amplicon from patient s tumor; point mutation at codon 918 determines loss of FokI site leading to a pathological pattern. B, Restriction analysis of cdna- derived PCR products of RET exon 16 after FokI digestion. Lane 1, DNA size marker; lane 2, undigested amplicon from medullary carcinoma TT cell line; lane 3, digested amplicon from TT cells; lanes 4 and 5, Digested amplicons from cervical lymph node metastases of patient; point mutation at codon 918 determines loss of FokI site leading to a pathological pattern. C, Restriction analysis of blood DNA-derived PCR products of RET exon 16 after FokI digestion. Lane 1, DNA size marker; lane 2, undigested amplicon from unaffected individual; lane 3, digested amplicon from unaffected individual; lane 4, digested amplicon from patient; presence of a normal restriction pattern indicates absence of germinal mutations. with MTC. In fact, pheochromocytoma, even if asymptomatic, should be removed first, because its presence greatly increases the surgical risk during thyroidectomy (5). On the other hand, it is certainly difficult to exclude the presence of an asymptomatic pheochromocytoma by both hormonal assay and different imaging modalities, whereas the detection of a germline RET mutation would demonstrate the presence of MEN2 disease. In addition, detection of a RET mutation suggests a more careful follow up of a patient with sporadic MTC, even if the role of RET alterations as poor prognostic marker is still controversial (8, 15, 16). Considering both the feasibility and the accuracy of the genetic analysis, this approach may be excellent for staging patients and for monitoring the disease before surgery, as well to achieve or confirm a definitive diagnosis of MTC in the presence of a neck mass of unclear histology/cytology. Thus, PCR-based genetic analysis in of FNAB aspirates may represent a feasible and reliable tool in the management strategy of medullary thyroid carcinoma. References 1. Eng C The RET proto-oncogene in multiple endocrine neoplasia type 2 and Hirschsprung s disease. N Engl J Med. 335: Lips CJM, Hoppener JWM Molecular genetics and clinical implications of medullary thyroid carcinoma and mutations of the RET proto-oncogene. Curr Opin Endocrinol Diabetes. 5: Wohllk N, Cote GJ, Bugalho MMJ, et al Relevance of RET protooncogene mutations in sporadic medullary thyroid carcinoma. J Clin Endocrinol Metab. 81: Eng C, Clayton D, Schuffenecker I, et al The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2: international RET mutation consortium analysis. JAMA. 276:19: Cervi-Skinner SJ Case record of the Massachusetts General Hospital. N Engl J Med. 289: Arturi F, Russo D, Giuffrida D, et al Early diagnosis by genetic analysis of differentiated thyroid cancer metastases in small lymph nodes. J Clin Endocrinol Metab. 82: Takahashi M, Buma Y, Hiai H Isolation of RET proto-oncogene cdna with an amino-terminal signal. Oncogene. 4: FIG. 4. Presence of a point mutation in codon 918 of RET gene in DNA of tumoral tissue (coexistence of C and T as indicated by arrow).

5 3382 RUSSO ET AL. JCE&M 1997 Vol 82 No Fink M, Weinhausel A, Niederle B, Haas OA Distinction between sporadic and hereditary medullary thyroid carcinoma (MTC) by mutation analysis of the RET proto-oncogene. Int J Cancer. 69: Duh QY, Sancho JJ, Greenspan FS, et al Medullary thyroid carcinoma. The need for early diagnosis and total thyroidectomy. Arch Surg. 124: Ponder BAJ, Finer N, Coffey R, et al Family screening in medullary thyroid carcinoma presenting without a family history. Q J Med. 67: Eng C, Mulligan LM, Smith DP, et al Low frequency of germline mutations in the RET proto-oncogene in patients with apparently sporadic medullary thyroid carcinoma. Clin Endocrinol (Oxf). 118: Ledger GA, Khosla S, Lindor NM, Thibodeau SN, Gharib H Genetic testing in the diagnosis and management of multiple endocrine neoplasia type II. Ann Intern Med. 122: Jhiang SM, Fithian L, Christopher M, et al RET mutation screening in MEN2 patients and discovery of a novel mutation in a sporadic medullary thyroid carcinoma. Thyroid. 6: Marsh DJ, Andrew SD, Eng C, et al Germline and somatic mutations in an oncogene: RET mutations in inherited medullary thyroid carcinoma. Cancer Res. 56: Marsh DJ, Learoyd DL, Andrew SD, et al Somatic mutations in the RET proto-oncogene in sporadic medullary thyroid carcinoma. Clin Endocrinol (Oxf). 44: Romei C, Elisei R, Pinchera A, et al Somatic mutations of the RET proto-oncogene in sporadic medullary thyroid carcinoma are not restricted to exon 16 and are associated with tumor recurrence. J Clin Endocrinol Metab. 81: The 25th Annual Meeting of the European Thyroid Association May 30 June 3, 1998 Athens, Greece For information, please contact: Prof. D.A. Koutras, Alexandra Hospital, 80 Vas. Sophias Avenue, Athens, Greece. Tel: ; Fax: A Local Meeting on Clinical Thyroidology with the participation of internationally esteemed scientists June 4, 1998 Athens, Greece For information, please contact: Prof. D. A. Koutras, Alexandra Hospital, 80 Vas. Sophias Avenue, Athens, Greece. Tel: ; Fax: Advances in Thyroid Eye Disease A Satallite Meeting of The 25th Annual Meeting of the European Thyroid Association May 28 29, 1998 Chalkidiki, Hellas For scientific organization and information; please contact: Dr. G.E. Krassas, MD, Associate Professor of Medicine, Endocrine Clinic Panagia Hospital, N. Plastira 22, N. Krini, Thessaloniki 55132, Hellas. Tel: ; Fax: Thyrotropin-Releasing-Hormone and Related Peptides on the Dawning of a New Millenium June 5 7, 1998 on the Island of Kos, Hellas For information contact: C/O Zita Congress, 46 Voulis Str., Athens, Hellas. Tel: ; Fax: Scientific Organization: Dr. L. Duntas, University of Athens, Evgenidion Hospital, 20 Papadiamantopoulou Str., Athens, Hellas. Tel: ; Fax: