Pediatric medullary carcinoma of the thyroid with point mutation of RET proto-oncogene associated with multiple endocrine neoplasia and initially diagnosed by fine-needle aspiration biopsy. (Case Report).

Abstract: A 7-year-old girl presented with a thyroid mass, elevated serum levels of calcitonin and carcinoembryonic antigen, as well as multiple mucosal nodules in the upper lip and tongue. Cytologic material obtained by fine-needle aspiration biopsy from the thyroid mass was diagnosed as medullary carcinoma and confirmed by immunohistochemical studies in the cell-block sections. Subsequent histopathologic examination showed involvement of both thyroid lobes by medullary carcinoma, and electron microscopic studies further confirmed the diagnosis. Molecular studies showed a point mutation in amino acid 918 in exon 16 of the RET proto-oncogene. Biopsies from the upper lip and tongue showed mucosal neuromas. Fine-needle aspiration biopsy is frequently used in the initial evaluation of thyroid nodules. This case illustrates the value of fine-needle aspiration biopsy as a safe and accurate diagnostic modality in the workup of pediatric thyroid nodules. Fine-needle aspiration biopsy should always be considered for the investigation of thyroid nodules in pediatric patients.

Key Words: medullary carcinoma, multiple endocrine neoplasia, RET proto-oncogene, pediatric neoplasm, point mutation, thyroid neoplasms

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Medullary carcinoma of the thyroid (MCT) is an uncommon malignant neoplasm arising from the parafollicular cells that produce calcitonin and carcinoembryonic antigen (CEA). It accounts for up to 10% of thyroid malignancies. (1) It may occur sporadically (70-80% of cases) or as a familial form (20% of cases) associated with multiple endocrine neoplasia (MEN) type 2. Sporadic MCT occurs primarily in middle-aged adults (aged 36 to 51 years) as unilateral enlargement of the gland. The familial form is seen in a younger population (aged 15 to 20 years); it is often multicentric, involving both lobes, and coexists with T-cell hyperplasia. (1) The combination of elevated serum calcitonin level plus a thyroid nodule is virtually pathognomonic of MCT. (2) The biologic behavior of MCT is ranked between the well-differentiated thyroid neoplasms and aggressive anaplastic carcinoma. The treatment approach is also more aggressive, with radical neck dissection recommended in all cases.

MCT is also disproportionately important because of its strong association with other neuroendocrine tumors and familial occurrence. Recently, molecular studies have demonstrated that patients with MEN 2A, MEN 2B, and familial MCT have specific sequence changes within the coding region of the RET proto-oncogene. (3) It has been recommended that thyroidectomy be performed as early as possible in children with the clinical manifestations of MEN 2B, such as the mucosal neuroma syndrome. Analysis of RET proto-oncogene mutation at codon 918 should be performed in all children born to parents with MEN 2B, as well as in children with ambiguous clinical features suggestive of MEN 2B. (4)

In pediatric patients, a thyroid nodule should always arouse suspicion of carcinoma, and the mass must be promptly investigated. Although the risk of relapse exists for young patients with thyroid carcinomas, the prognosis is very good when the disease is diagnosed early and properly treated. (5) Fine-needle aspiration biopsy (FNAB) has become a mandatory diagnostic tool in the preoperative diagnosis of thyroid nodules. (2) This safe, economic, and highly accurate technique has not been used as widely in children as it has been in adults.

Discussion

Almost all cases of MCT in childhood are familial. Two of the inherited syndromes associated with MCT, namely MEN 2A and familial MCT, were found to have mutations in the extracellular domain of the RET protein. The third and most severe inherited syndrome, MEN 2B, was found to have the characteristic M918T mutation in the intracellular domain of the RET tyrosine kinase. (6,7) The substitution of methionine in the tyrosine kinase catalytic core is thought to alter the substrate specificity of this kinase both quantitatively and qualitatively. This mutation has been found in 30 to 40% of sporadic cases of MCT. (8) It has not been found in MEN 2A or familial MCT.

Familial and multifocal MCT are more aggressive tumors with early metastasis, particularly in young patients with MEN 2B. In sporadic cases, RET mutations are generally seen only in tumor DNA, although a de novo germ-line mutation will be present in all tissues. In hereditary cases, the RET mutations are always present in tumor and in constitutive DNA. Both sporadic and familial cases are more prevalent in females and are associated with elevated serum levels of calcitonin. (4)

This case illustrates the value of FNAB as a safe and rapid diagnostic tool in the initial outpatient evaluation of pediatric thyroid nodules. Two cases similar to ours were reported in adults with MCT diagnosed by FNAB of thyroid gland or node metastases. Tissues evaluated for DNA studies confirmed the mutation, which led to the final diagnosis of MEN type 2. (9,10) In one of these cases, MCT and MEN type 2 went undiagnosed for years, in spite of the fully developed clinical phenotype, until a thyroid FNAB and DNA studies revealed the diagnosis. (10)

The early detection of the RET proto-oncogene mutation in children with MEN type 2 has become a crucial diagnostic tool to guide preventive thyroidectomy. (11) According to a consensus statement from an international group, RET testing should replace evaluation of calcitonin level to diagnose the MEN type 2 carrier state. (12)

The cytologic findings of MCT mirror the histologic findings. (1,2) Amyloid, demonstrated by Congo red stain, is present in 60 to 80% of the reported cases. (2) The amyloid is found more often on EM and is seen as webs of nonbranching fibrils (as in our case). (1)

The differential diagnosis of MCT on morphologic grounds includes papillary carcinoma of the thyroid (PCT), anaplastic carcinoma (AC), and small cell carcinoma. Among the benign lesions in the differential diagnosis are hyalinizing trabecular adenoma, follicular Hurthle cell neoplasms, hyperplastic nodules, and granulation tissue. (2,13) Pseudopapillae may be seen in MCT, but the tumor rarely has true papillae with fibrous cores, as seen in PCT. (2) In addition, both neoplasms may contain intranuclear pseudo-inclusions. (1,2,14) The spindle cells in MCT may be mimicked by fibroblasts from stroma, as well as granulation tissue and cellular repair in old hemorrhagic goiter. (13) Small cell MCT represents the poorly differentiated end of the spectrum of neuroendocrine carcinomas and can be identical to small cell carcinomas of the lung. Immunohistochemical testing of calcitonin level should be performed every time diagnostic doubts arise. (1,2)

Conclusion

Fine-needle aspiration biopsy of the thyroid can be an important diagnostic tool for thyroid nodules in pediatric patients. Vital information for the diagnosis, prognosis, and epidemiology of tumors can be gained from DNA studies of pathology specimens.

Accepted May 6, 2002.

References

(1.) Rosai J, Carcangiu ML, DeLcllis RA. Medullary carcinoma: Tumors of the thyroid gland, in Rosai J, DeLellis RA (eds): Atlas of Tumor Pathology: Thinors of the Thyroid Gland. Washington, DC, Armed Forces Institute of Pathology, 1994, 3rd series, fascicle 5, pp 207-241.

(2.) DeMay RM. Thyroid, in The Art and Science of Cyropothology: Vol 2. Aspiration Cytology. Chicago, American Society of Clinical Pathologists Press, 1996, pp 703-766.

(3.) Ferraris AM, Mangerini R, Gaetani GF, Romei C, Pinchera A, Pacini F. Polyclonal origin of medullary carcinoma of the thyroid in multiple endocrine neoplasia type 2. Hum Genet 1997;99:202-205.

(4.) Evans DB, Burgess MA, Goepfert H, Gagel RF. Medullary thyroid carcinoma. Curr Ther Endocrinol Metab 1997;6:127-132.

(5.) Fassina AS, Rupolo M, Pelizzo MR, Casara D. Thyroid cancer in children and adolescents. Tumor 1994;80:257-262.

(6.) Takahashi M. Oncogenic activation of the RET protooncogene in thyroid cancer. Grit Rev Oncog 1995;6:35-46.

(7.) Hofstra RM, Landsvater RM, Ceceherini I, Stulp RP, Stelwagen T, Luo Y, et al. A mutation in the RET proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thyroid carcinoma. Nature 1994;367:375-376.

(8.) Blaugrund JE, Johns MM Jr, Eby YJ, Ball DW, Baylin SB, Hruban RH, et al. RET proto-oncogene mutations in inherited and sporadic medullary thyroid cancer. Hum Mol Genet 1994;3:1895-1897.

(9.) Russo D, Arturi F, Chiefari E, Meringolo D, Bianchi D, Bellanova B, et al. A case of metastatic medullary thyroid carcinoma: Early identification before surgery of an RET proto-oncogene somatic mutation in fine-needle aspirate specimens. J Clin Endocrinol Metab 1997;82:3378-3332.

(10.) Ohyama T, Sato M, Murao K, Kittaka K, Namihira H, Matsuhara S, et al. A case of multiple endocrine neoplasia type 2B undiagnosed for many years despite its typical phenotype. Endocrine 2001;15:143-146.

(11.) Sanso GE, Domene HM, Garcia R, Pusiol E, De M, Roque M, et al. Very early detection of RET proto-oncogene mutation is crucial for preventive thyroidectomy in multiple endocrine neoplasia type 2 children: Presence of C-cell malignant disease in asymptomatic carriers. Cancer 2002;94:323-330.

(12.) Brandi ML, Gagel RF, Angeli A, Bilezikian JP, Beck-Peccoz P, Bordi C, et al. Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab 2001;86:5658-5671.

(13.) Kaur A, Jayaram G. Thyroid tumors: Cytomorphology of medullary, clinically anaplastic, and miscellaneous thyroid neoplasms. Diagn Cytopathol 1990;6:383-389.

(14.) Uribe M, Fenoglio-Preiser CM, Grimes M, Feind C. Medullary carcinoma of the thyroid gland: Clinical,' pathological, and immunohisto-chemical features with review of the literature. Am J Surg Pathol 1985;9:577-594.

RELATED ARTICLE: Case Report

A 7-year-old white girl enjoyed general good health with steady growth and development, except for an intermittent, pruritic, circular rash about her face and trunk, which had been present for several years. Her mother had recently noticed a slow-growing mass in the patient's anterior neck. There was also a persistent swelling of the upper lip and tongue.

Physical examination revealed a normal prepubertal female with a thyroid mass left of midline, prominent upper lip (Fig. 1), and multiple submucosal nodules at the tip of her tongue. There was a circular, red skin rash distributed diffusely throughout the trunk. Family history was unremarkable. On admission, basal serum calcitonin level was 6,192 pg/mL (normal, 0-100 pg/mL) and serum CEA level was 36.2 ng/mL (normal, 0-10 ng/mL). Thyroid indices, including levels of triiodothyronine (T3) resin uptake, total thyroxine ([T.sub.4]), free [T.sub.4], and thyroid-stimulating hormone (TSH), were normal. Thyroid autoantibody test and biochemical screening for pheochromocytoma (vanillylmandelic acid, plasma metanephrine, and plasma free-catecholamine levels) showed no evidence of disease. Ultrasound examination of the neck revealed a 2.4-cm well-defined mass arising within the left thyroid lobe, and an additional 0.7-cm well-defined, solid mass in the center of the right lobe.

FNAB of the left thyroid mass showed hypercellular aspirates composed of loose clusters and scattered, single spindle cells with relatively uniform central nuclei, speckled chromatin pattern, occasional conspicuous nucleoli, and wispy cytoplasm (Fig. 2). Occasional polygonal cells were noted in the background. Colloid, amyloid, mitotic figures, and necrosis were all absent. Immunohistochemical tests performed on cell-block sections were strongly positive for calcitonin and chromogranin. The diagnosis of MCT was rendered based upon the morphology and immunohistochemical findings.

The patient underwent total thyroidectomy and bilateral neck dissection. The thyroid gland in the surgical specimen weighed 27.4 g. The right lobe had a well-demarcated, whitish tan, firm nodule that measured 0.7 X 0.4 )< 0.4 cm. The left lobe contained a similar firm nodule measuring 2.3 X 2.2 X 1.5 cm. The neck dissection specimen yielded 102 lymph nodes, all of which were negative for cancer. Microscopically, the tumor consisted of nests of neoplastic cells separated by thin strands of eosinophilic stroma, with a predominant pattern of spindle cells with mild to moderate nuclear pleomorphism and a trabecular pattern at the periphery. Mitoses were rare. Neither calcification nor amyloid was detected with hematoxylin and eosin staining. Congo red stain was negative for amyloid. Electron microscopy (EM), however, revealed a disordered meshwork of fibrils characterizing intracy-toplasmic amyloid; EM also showed numerous intracy-toplasmic, round, neurosecretory granules (150-300 nm in diameter) in the tumor cel ls. Repeated immunohisto-chemical studies of both nodules confirmed MCT. Biopsies from the nodules on both the upper lip and the tongue showed submucosal neuromas. One intrathymic parathyroid gland was histologically normal. Biopsies of the upper lip and the tongue revealed submucosal neuromas. DNA was isolated from a small fragment of tumor carved out from a paraffin block from the surgical specimen. Polymerase chain reaction (PCR) amplification of exon 16 of the RET proto-oncogene demonstrated loss of a normally present FokI restriction site. Electrophoretic separation of the digested PCR products in an agarose gel resulted in 3 bands, 2 of which had the expected sizes of the restricted normal allele, while 1 band represented the unrestricted, mutant allele (Fig. 3).

Postoperatively, the serum CEA and calcitonin levels returned to normal range. The skin rash, for which no etiology could be found, was never biopsied and persisted after surgery, but subsequently vanished. The dermatology consultation diagnosed an erythema multi-forme/marginatum type of lesion with pruritic scales. At last follow-up, 30 months after surgery, there was no evidence of recurrence of the thyroid tumor and no record of a skin rash. The patient's mother and father were evaluated for possible DNA mutations, and none were detected.

Key Points

* Fine-needle aspiration is important in the diagnosis of thyroid tumors in children.

* Molecular studies are fundamental in medullary carcinomas of the thyroid.

* Medullary carcinomas of the thyroid, particularly in children, can be a component of one of the multiple endocrine neoplasia syndromes.

From the Departments of Pathology and Preventive Medicine, University of Mississippi Medical Center, Jackson, MS.

Reprint requests to Luciano B. Lemos, MD, Department of' Pathology, Lyndon B. Johnson General Hospital, 5656 Kelley Street, Houston, TX 77026. Email: [e-mail]

Copyright [C] 2003 by The Southern Medical Association

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