The Journal of Pediatrics Gill et al. 459 Volume 129, Number 3

Early presentation of metastatic medullary carcinoma in multiple endocrine neoplasia, type IIA: Implications for therapy James R. Gill, MD, Miguel Reyes-Múgica, MD, Sudha Iyengar, PhD, Kenneth

K. Kidd, PhD, Robert J. Touloukian, MD; Cynthia Smith, RN, Marc S. Keller, MD, and Myron Genel, MD

From the Departments of Pathology, Human Genetics, Surgery, Pediatrics, and Diagnostic Radiology, Yale University School of Medicine, New Haven, Connecticut. A girl 5 years 11 months of age, belonging to an extensive kindred with multiple endocrine neoplasia, type IIA (MEN IIA), was found to have multifocal medullary thyroid carcinoma with metastasis in one paraglandular lymph node after positive findings on a calcium-pentagastrin stimulation test. Her sister, 3 years 8 months of age, also had an elevated calcitonin level, and thyroidectomy revealed C-cell hyperplasia and a focus of medullary thyroid carcinoma. These two cases underscore the need for prophylactic thyroidectomies in MEN IIA patients as young as 5 years of age and strict yearly provocative screening beginning at age 1 year. (J Pediatr 1996;129:459-64)

Multiple endocrine neoplasia is composed of three autosomal dominant syndromes, types I, II, and III (some forms of MEN I may be recessive), that consist of tumors or hyperplasias in several endocrine organs. MEN I (Wermer syndrome) is characterized by parathyroid, pancreatic, and pituitary lesions; MEN IIA (Sipple syndrome) involves pheochromocytomas, parathyroid hyperplasia, and medullary carcinoma of the thyroid; and MEN IIB is similar to MEN IIA with the addition of multiple systemic neuromas. Various mutations have been identified in these disorders. The MEN II syndromes have been linked to germ line mutations in the RET protooncogene on chromosome 10q11.2.1,2 It has been diagnosed from childhood to adulthood and has led to family screening once it is diagnosed. We present two sisters from a previously described MEN IIA kindred,3,4 in both of whom a diagnosis of medullary thyroid carcinoma has been made. One sister is the youngest known patient with MEN IIA who has received a diagnosis of metastatic medullary carcinoma. This case underscores the need for prophylactic thyroidectomies in patients with MEN IIA as young as 5 years of age. Supported in part by National Institutes of Health grant CA-32066. Submitted for publication Feb. 7, 1996; accepted May 24, 1996. Reprint requests: Miguel Reyes-Múgica, MD, Department of Pathology, Yale University School of Medicine, 310 Cedar St. LB20, New Haven, CT 06520-8023. Copyright © 1996 by Mosby-Year Book, Inc. 0022-3476/96/$5.00+0 9/22/75363

CASE REPORTS Patient 1. Patient 1, the older sister, was initially seen 3 weeks shy of her sixth birthday for initial screening with calcium-pentagastrin stimulation. She is part of an extensive kindred with MEN IIA through multiple generations that has previously been reported.3,4 Her mother had had a thyroidectomy and bilateral adrenalectomy for MTC and pheochromocytoma. Her maternal grandmother was similarly affected and succumbed to the disease. Pentagastrin challenge of patient 1 revealed an elevated baseline calcitonin concentration of 220 and 260 pg/ml, with a rise to 1500 pg/ml at 3 minutes, and these results were regarded

as indicative of underlying disease. A total thyroidectomy was performed 6 weeks later, at which time metastatic nodal disease was recognized adjacent to the right recurrent laryngeal nerve. Postoperatively the patient showed laryngoscopic evidence of right vocal cord paresis. The thyroidectomy specimen demonstrated two foci of medullary carcinoma, one in each lobe of the thyroid. Grossly, the nodules were tan, firm, and measured 1.0 and 0.7 cm in greatest dimensions. Microscopically, nests of small polygonal cells were seen that stained positively for an anticalcitonin antibody, which is considered a positive finding in approximately 80 percent of the cases of MTC. A background of C-cell hyperplasia was also noted. A right paraglandular lymph node showed evidence of metastatic MTC (Fig.1). A repeated calcium-pentagastrin stimulation test 2 months postoperatively showed normal results but again showed abnormalities 30 months postoperatively, with elevated baseline

MEN Multiple endocrine neoplasia MRI Magnetic resonance imaging MTC Medullary thyroid carcinoma PCR Polymerase chain reaction

460 Gill et al. The Journal of Pediatrics September 1996

Fig. 1. A, Microscopic section from the initial thyroidectomy of patient 1 shows the lymph node with metastatic MTC. A group of cells with ample cytoplasm is located in the subcapsular area of the lymph node. B, High-power view of the same lymph node. C, Anticalcitonin immunohistochemical stain of the new right paratracheal mass. Several nests of tumor cells are seen. Dark cytoplasms correspond to positive staining of calcitonin (arrows). (Magnification: A, X50; B, X100; C, X50.)

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Fig. 2. Serum calcitonin concentrations through time in patient 1. Dotted lines with upward arrows depict basal and peak stimulated calcitonin levels after combined calcium-pentagastrin provocation. Note normal baseline with minimal peak stimulated calcitonin levels immediately after surgery at age 6 years. Provocation stimulation was discontinued after age 12 years when baseline calcitonin levels exceeded 400 pg/ml. Normal calcitonin values for Bioscience Laboratories are less than 100 pg/ml; normal values for Nichols Institute are 2 to 27 pg/ml at baseline and less than 94 pg/ml at 1 or 2 minutes after stimulation with calcium and pentagastrin.

calcitonin levels that rose more than threefold with calcium-pentagastrin stimulation (Fig. 2). A whole-body thallium scan with 1 mCi of thallium-201 chloride showed no evidence of recurrence or metastases. A similar response to calcium-pentagastrin stimulation was observed 1 year later, with the peak calcitonin concentration rising to 692 pg/ml. A whole-body thallium scan again was normal. At age 11 years 5 months, 5 years after surgery, baseline calcitonin levels were further elevated and rose more than fourfold after calcium-pentagastrin stimulation. MRI of the neck demonstrated recurrent tumor in the right thyroid bed. Surgical exploration of the neck was performed when the patient was 12 years of age, with excision of cervical and cervical-mediastinal masses. The specimens, consisting of a histologically normal thymus and one parathyroid gland, showed no carcinoma. All tissue was submitted for microscopic examination. Postoperative MRIs during a 4-year period demonstrated persistence of a higher-density region in the right thyroid bed, thought to represent residual thyroid tissue, and a right peritracheal lymph node with a somewhat increased signal intensity but without significant progression. Baseline calcitonin levels continued to be grossly elevated. When the patient was 15 years 4 months of age, 9 years after her initial surgery, a fine needle aspiration of the right peritracheal structure identified on MRI was obtained by means of ultrasound guidance and was interpreted as a possible recurrent tumor. A repeated exploration of her neck was performed when she was 15 years 8 months of age, with complete resection of the mass seen on MRI (Fig. 3).

The right peritracheal mass measured 2 cm in greatest dimension and consisted of red, lobulated tissue with a firm, 1 cm tan nodule. Microscopic examination revealed thyroid tissue and two lymph nodes. Both lymph nodes were involved by metastatic medullary carcinoma (Fig. 1). The thyroid tissue was unremarkable, with no evidence of C-cell hyperplasia. Medullary carcinoma was also identified in the surrounding soft tissues. Postoperative MRI scans of the neck and CT scans of the chest, abdomen, and pelvis were interpreted as normal, though a calcitonin measurement obtained 3 and 6 months after surgery continued to be elevated at 516 pg/ml and 706 pg/ml, respectively. Fractionated catecholamine excretion has been normal. Patient 2. Patient 2, the younger sister of patient 1, was initially seen at 3 years 5 months of age for screening with calcium-pentagastrin stimulation after medullary carcinoma with nodal involvement was discovered in her older sister. Initial testing demonstrated a modestly elevated baseline of 53 pg/ml with a peak response of 144 pg/ml at 15 minutes. A study repeated 6 weeks later revealed baseline calcitonin levels of 33 and 36 pg/ml and a peak response of 200 pg/ml at 5 minutes. A total thyroidectomy, performed at 3 years 8 months of age, revealed C-cell hyperplasia and one focus with histologic features of medullary carcinoma. It was entirely submitted for histologic study. Aside from transient postoperative hypocalcemia, patient 2 had an uneventful postoperative course, with a normal calcitonin response to calcium-pentagastrin stimulation. Baseline and stimulated calcitonin levels have remained

462 Gill et al. The Journal of Pediatrics September 1996

Figure 3. Postoperative MRI scan of neck. An axial MRI scan of the base of the neck shows a medium-intensity signal from a nodule (arrow) situated between the trachea (t) and the right common carotid artery (c). This nodule was seen to be hypervascular by color Doppler imaging.

normal during the subsequent 9-year period. Fractionated catecholamine excretion has been normal. GENETICS

Genetic analysis of this family revealed that affected members had a T-to-C (cysteine-to-arginine) transition at codon 634 in exon 11 of the RET protooncogene; this T-to-C mutation is frequently observed in patients with MEN IIA.2,5 In an initial screening test for mutations of all 20 exons of the RET protooncogene, only a few affected members of the family were analyzed. Because this mutation is easily identified on digestion of polymerase chain reaction-amplified DNA with the restriction enzyme Cfo I, we subsequently screened all members of the family. The mutation was confirmed in all affected members of this family from whom DNA was available. The PCR was performed in a total volume of 50 microliter containing 100 ng of genomic DNA, 50 mmol of potassium chloride, 10 mmol Tris-hydrochloride (pH 8.3), 1.5 mmol magnesium chloride, 0.1 percent wt/vol gelatin, 0.5 U Taq polymerase (AmpliTaq, Perkin Elmer-Cetus, East Norwalk, Conn.), 200 micromole of each deoxyribonucleotide triphosphate per liter, and 2 micromole of each primer, with the use of previously published primers for exon 11.2 The PCR product size was 233 base pairs. Restriction digestion of each sample was performed in a total volume of 15 micron 1 of 10X buffer, 12.5 microliter of PCR product, and 1 microliter of restriction enzyme. The reaction was incubated at 37 degrees C for 1 hour, and the digested product was then analyzed on a 2 percent agarose gel. The restriction

enzyme Cfo I cleaves the PCR product only if the recognition sequence GCGC is present. The normal sequence at codon 634 in exon 11 of the RET protooncogene is CTG TGC CGC. The sequence of the chromosome with the mutation at the first position in codon 634 is CTG CGC CGC. Thus Cfo I cleaves only DNA of the mutant chromosomes, giving fragments of 173 and 60 base pairs. After digestion with Cfo I, a heterozygous pattern of fragments of size 233, 173, and 60 base pairs was observed for both sisters (patients 1 and 2) and their mother, showing that they had the T-to-C mutation in exon 11 of the RET protooncogene.

DISCUSSION

The known hereditary nature of these syndromes may result in the early detection of symptom-free individuals through family screening once a patient with MEN is identified. Patients have been screened by serial calcitonin levels and provocative tests (e.g., calcium-pentagastrin stimulations) that have bothersome side effects and result in decreased patient compliance.6 Genetic testing has been shown to be an accurate, rapid, and reproducible test for identification of these patients.7,8 Missense mutations in the RET protooncogene have been shown to be associated with MEN II inheritance.1 This findings has allowed direct testing of family members in MEN II kindreds. Because virtually all patients with mutations in the RET protooncogene will have MTC (the most common cause of death in patients with MEN II) by 31 years of age,9 it is imperative to identify them opportunely for total thyroidectomy. A timely total

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thyroidectomy offers a cure for these patients with few long-term side effects.10 The age to perform thyroidectomy, however, is far from agreed on.11 Wells et al.7 suggested operating at 5 years of age in patients whose cases have been diagnosed by molecular genetic testing, because none of their patients in this group had positive nodes.7 At the Fifth International Workshop on Multiple Endocrine Neoplasias, in Stockholm, Sweden, Learoyd et al.12 reported general agreement that surgery should be performed as early as 6 years of age. On the other hand, Lips et al.13 suggested postponing surgery until the stimulation test results become positive, or until 12 or 13 years of age. They offered the evidence that all their patients who underwent surgery after the results of their plasma calcitonin stimulation tests became positive have had no recurrence of disease. They also reported that the risk of surgical complications (recurrent nerve palsy and hypoparathyroidism) in young children is not counterbalanced by the gain of MTC prevention.13 Although injury to the right laryngeal nerve did occur in our patient, the presence of metastatic nodal disease adjacent to the nerve should be considered as an additional contributing factor. Lips et al.13 found that all eight patients with RET mutations and negative or equivocal results on calcitonin tests who underwent surgery had microscopic MTC. They ranged in age from 4 to 18 years. In this context, Utiger6 stated that to find children with negative stimulation test results and microscopic MTC is worrisome and recommended thyroidectomy at age 4 or 5 years regardless of the results of biochemical screening tests because complications of surgery are no more frequent in younger than in older children. Telander et al.14 followed 33 children with MTC or C-cell hyperplasia who underwent thyroidectomy. All seven patients with metastases were more than 11.9 years of age. The youngest patient with MTC was 2.8 years of age. One of our patients had a pathologically documented metastasis at age 5 years 11 months. Before genetic diagnosis, Telander et al. recommended initial screening for at-risk children beginning at 1 year of age and surgery once disease was detected. The case report on patient 1 illustrates how difficult it may be to deal with MTC once the disease becomes locally disseminated. Despite documented elevations of calcitonin levels, two thallium scans failed to detect any focus of potential tumor. Furthermore, an area suggestive of tumor by MRI 5 1/2 years after the original diagnosis was subsequently resected and corresponded only to thymus and parathyroid gland. This finding underscores the value and limitations of these procedures to detect tumor presence or recurrence. Another problem that complicated the clinical course of our patient was compliance, with long intervals between her scheduled appointments, which further delayed the management plan.

The availability of precise, specific genetic testing for family-specific mutations permits management to be focused on symptom-free children found to carry the defect. We favor a more aggressive approach in the management of children with the mutation. The documentation that metastatic medullary carcinoma can arise before 6 years of age raises concern about waiting until age 10 or 12 years. The young age of our patient makes us uncomfortable with waiting until the calcitonin test shows positive results, especially because MTC has been documented in other patients with negative results on stimulation tests.13 Thus we are in accord with Utiger6 and Wells et al.7 and recommend total thyroidectomy before the age of 5 years for all patients with the mutation, with strict yearly provocative screening beginning at age 1 year.

REFERENCES

1. Mulligan LM, Kwok JBJ, Healey CS, et al. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993;363:458-60.

2. Donis-Keller H, Dou S, Chi D, et al. Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet 1993;2:851-6.

3. Lichter JB, Wu JS, Genel M, et al. Presymptomatic testing using DNA markers for individuals at risk for familial multiple endocrine neoplasia type 2a. J Clin Endocrinol Metab 1992;74:368-73.

4. Graham SM, Genel M, Touloukian RJ, Barwick KW, Gertner JM, Torony C. Provocative testing for occult medullary carcinoma of the thyroid: findings in seven children with multiple endocrine neoplasia type IIa. J Pediatr Surg 1987;22:501-3.

5. Goodfellow PJ, Peiffor S, Donis-Keller H, et al. Identification and characterization of mutations in the RET proto-oncogene associated with MEN 2A [abstract 167]. Am J Hum Genet 1993;53 Suppl (Sept).

6. Utiger RD. Medullary thyroid carcinoma, genes, and the prevention of cancer. N Engl J Med 1994:331:870-1.

7. Wells SA, Chi DD, Toshima K, et al. Predictive DNA testing and prophylactic thyroidectomy in patients at risk for multiple endocrine neoplasia type 2a. Ann Surg 1994;220:237-50.

8. Neumann HPH, Eng C, Mulligan LM, et al. Consequences of direct genetic testing for germline mutations in the clinical management of families with multiple endocrine neoplasia, type II. JAMA 1995;274:1149-51.

9. Easton DF, Ponder MA, Cummings T, et al. The clinical and screening age-at-onset distribution for the MEN-2 syndrome. Am J Hum Genet 1989;44:208-15.

10. Leape LL, Miller HH, Graze K, et al. Total thyroidectomy for occult familial medullary carcinoma of the thyroid in children. J Pediatr Surg 1976;11:831-7.

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11. Wohllk N, Cote G, Evans D, Goepfert H, Ordonez N, Gagel RF. Application of genetic screening information to the management of medullary thyroid carcinoma and multiple endocrine neoplasia type 2. Endocrinol Metal Clin North Am 1996;25:1-25.

12. Learoyd DL, Twigg SM, Marsh DJ, Robinson BG. The practical management of multiple endocrine neoplasia. Trends Endocrinol Metab 1995;6:273-8.

13. Lips CJM, Landsvater RM, Hoppener JWM, et al. Clinical screening as compared with DNA analysis in families with multiple endocrine neoplasia type 2A. N Engl J Med 1994;331:828-35.

14. Telander RL, Zimmerman D, Sizemore GW, et al. Medullary carcinoma in children: results of early detection and surgery. Arch Surg 1989;124:841-3.