Histopathologic and Clinical Features of Medullary Microcarcinoma and C-Cell Hyperplasia in Prophylactic Thyroidectomies for Medullary Carcinoma

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1 Histopathologic and Clinical Features of Medullary Microcarcinoma and C-Cell Hyperplasia in Prophylactic Thyroidectomies for Medullary Carcinoma A Study of 42 Cases Demet Etit, MD; William C. Faquin, MD, PhD; Randall Gaz, MD; Gregory Randolph, MD; Ronald A. DeLellis, MD; Ben Z. Pilch, MD Context. Prophylactic thyroidectomies are increasingly performed on patients at risk for developing medullary thyroid carcinoma (MTC); consequently, pathologists are more commonly encountering these specimens in routine practice. Objective. To describe the detailed clinicopathologic features of prophylactic thyroidectomies for medullary carcinoma. Design. We present a retrospective series of 42 prophylactic thyroidectomies for MTC performed for one or more of the following: family history of multiple endocrine neoplasia (MEN) or MTC, elevated serum calcitonin level, or detection of a RET proto-oncogene mutation. Results. Patients included 22 men and 20 women (mean age, 26.2 years). Among those with known RET proto-oncogene mutations, affected sites included exons 10, 11, 14, and 16. In 93% (n 39) of cases, either C-cell hyperplasia (n 36), medullary microcarcinoma (MMC; n 29), or medullary macrocarcinoma (n 1) was found. C-cell hyperplasia was often multifocal (n 30) and bilateral (n 23) and included both nonnodular and nodular patterns. A total of 94% of C-cell hyperplasia cases and all MMC cases were microscopically detectable using hematoxylin-eosin stains. The MMCs were characterized by a complex microarchitectural pattern with a desmoplastic stromal response (n 29) and focal amyloid deposition (n 12). Most MMCs exhibited a solid pattern (n 24) of round, polygonal, spindled, or plasmacytoid-shaped cells. Only 1 case of MMC showed evidence of metastatic disease to a pretracheal lymph node. Conclusions. Based upon our clinicopathologic findings and review of the literature, we conclude that thyroidectomies in at-risk patients are very frequently associated with C-cell hyperplasia and/or MMC; however, the clinical prognosis for these patients is very good. (Arch Pathol Lab Med. 2008;132: ) Medullary thyroid carcinoma (MTC) is an uncommon thyroid malignancy of C-cell derivation representing approximately 3% to 12% of all thyroid cancers. 1 3 It has a propensity for metastasis to regional lymph nodes, and the average 10-year survival for all types of MTC ranges from 61% to 75%. 3,4 Although a majority of MTCs are acquired as sporadic tumors, 25% to 30% of cases are heritable, being associated with multiple endocrine neoplasia (MEN) 2A, MEN 2B, or with the familial medullary thyroid carcinoma syndrome. 1,5,6 Inherited forms of MTC are due to autosomal dominant mutations of the RET pro- Accepted for publication April 8, From the Departments of Pathology (Drs Etit, Faquin, and Pilch) and Surgery (Drs Gaz and Randolph), Massachusetts General Hospital, and the Massachusetts Eye and Ear Infirmary, Harvard Medical School (Drs Faquin, Gaz, Randolph, and Pilch), Boston; and the Department of Pathology, Rhode Island Hospital, Brown University Medical School, Providence (Dr DeLellis). Dr Etit is now with the Department of Pathology, Ataturk Research and Training Hospital, Izmir, Turkey. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Ben Z. Pilch, MD, Department of Pathology, Warren 219, Massachusetts General Hospital, 55 Fruit St, Boston, MA ( bpilch@partners.org). to-oncogene with incomplete penetrance, often presenting as multifocal disease in a background of C-cell hyperplasia (CCH). With routine testing of family members of affected patients for RET proto-oncogene mutations coupled with screening of serum calcitonin levels in affected individuals, patients with inherited forms of MTC are being detected at earlier stages, often before the development of clinically detectable MTC. 7 Prophylactic thyroidectomies are increasingly being performed on patients at risk for MTC, and as a consequence, pathologists are more commonly encountering these specimens in routine practice. 7,8 To add to our understanding of the range of histopathologic changes within these prophylactic thyroidectomies, we describe our experience based on the clinicopathologic features of a series of 42 cases and compare it with results in the literature. MATERIALS AND METHODS The database of the Department of Pathology at Massachusetts General Hospital, Boston, was searched to retrospectively identify specimens of thyroidectomies that were performed prophylactically for possible MTC. The search encompassed cases accessioned during a 30-year period ( ). To qualify for inclusion within the study, the thyroidectomy had to be performed for one or more of the following reasons: a positive family history Arch Pathol Lab Med Vol 132, November 2008 Prophylactic Thyroidectomy for Medullary Carcinoma Etit et al 1767

2 of MEN 2A, MEN 2B, or familial MTC, an elevated serum calcitonin level, or a RET proto-oncogene mutation. In addition, a member of an affected family was screened for an elevated serum calcitonin or a RET proto-oncogene mutation in order to have a prophylactic thyroidectomy. Using these criteria, 42 cases with available histology slides were identified for inclusion in the study group. For each case, clinical charts and records were reviewed to document patient demographics, ultrasound findings, family history, including MEN status or history of MTC, serum calcitonin levels, RET proto-oncogene mutation status, and clinical follow-up. Although the cases in this study came from a 30- year period, the most current presurgical workup of patients prior to prophylactic thyroidectomy for MTC at Massachusetts General Hospital includes the following: serum calcitonin and carcinoembryogenic antigen measurements, calcium and parathyroid hormone determinations to exclude parathyroid hyperplasia, 24- hour urine for catecholamines, vanillylmandelic acid, and metanephrines to exclude pheochromocytoma, and RET oncogene analysis to determine the need for family screening. Preoperative imaging included ultrasound evaluation and neck computed tomography for evaluation of cervical lymph nodes. Among the patients in our series where presurgical ultrasound records were available, 13 had ultrasonographically visualized nodules ranging from less than 0.5 cm to 2.2 cm; 7 were multifocal and 4 were bilateral. All of these patients received a prophylactic thyroidectomy for possible MTC or CCH. Although fine-needle aspiration is typically performed for any thyroid nodule larger than 1.0 cm, none of the patients in our series, based upon medical records available for review, had a presurgical fine-needle aspiration evaluation. Serum Calcitonin Measurements Serum calcitonin measurements were conducted using the DPC Immulite 2000 Chemiluminescent Method (Siemens Healthcare Diagnostics, Deerfield, Ill) in blood samples. RET Proto-oncogene Analysis A total of 32 of 42 patients within our study were evaluated for mutations of the RET proto-oncogene prior to thyroidectomy once this test became available for standard screening. Genomic DNA was obtained from submitted blood samples according to standard procedures. 9,10 Polymerase chain reaction was used to amplify exons 10, 11, 13, 14, 15, and 16 of the RET gene. Sequences of primers and polymerase chain reaction protocols were obtained from previously published sources. 11 Histochemical and Immunohistochemical Stains Immunohistochemical stains for calcitonin were performed on selected paraffin blocks using the avidin-biotin peroxidase method with an anti-calcitonin antibody at a 1:500 dilution (Dako Corporation, Carpinteria, Calif) and processed using automated immunostaining (Ventana Benchmark, Tucson, Ariz). The positive control tissue was an MTC sample that was not included in the study. For each case, 1 to 5 blocks were stained for calcitonin immunoreactivity. In 21 cases where focal amyloid was suspected based upon hematoxylin-eosin findings, a Congo red stain was performed using standard methods. Histopathologic Evaluation All specimens were inspected grossly, and multiple sections were taken. Thirty-four of the thyroidectomies were entirely submitted for histologic evaluation, and 8 cases had representative sections submitted. For these 8 cases, the median number of sections per centimeter of thyroid was 1.1 sections per cm length of thyroid (range, sections per cm length). Glass slides and special stains from each of the 42 cases were reviewed by 3 pathologists (B.Z.P., W.C.F., and D.E.), and a selected number of cases were reviewed with a fourth pathologist (R.A.D.). A consensus opinion was reached for the presence of CCH and MTC. The criterion for CCH was the presence of 50 or more C cells per low-power field ( 100). For each case, at least 1 block was stained for calcitonin for confirmation. C-cell hyperplasias were histomorphologically separated into nodular CCH, that is, C cells filling a thyroid follicle, or nonnodular CCH, and solitary or multifocal. Relative to the classification scheme for CCH described by Perry et al, 12 the nodular CCH cases in our series are equivalent to their neoplastic nodular CCH. In addition, the nonnodular CCH category presented here encompasses both the focal and diffuse CCH as described by Perry et al. Medullary microcarcinomas (MMCs), as defined by the World Health Organization, were less than 1 cm in greatest dimension 13,14 and were evaluated for the implied invasion through the follicular basement membrane by assessing the presence of irregular outlines and sclerosis within solid foci of C cells. In 22 patients, regional lymph node samplings were evaluated histopathologically, and the parathyroid tissues of 12 patients were also examined. Follow-up Clinical follow-up information was available for 29 of the patients within our series. The range of follow-up was 1 month to 30 years (average follow-up, 4.7 years), and follow-up data included contents of clinical notes, radiologic imaging, and serum calcitonin levels. RESULTS There were 20 female and 22 male patients who ranged in age from 2 to 73 years (mean age, 26.2 years). The corresponding family history, MEN status, and results of RET proto-oncogene analysis for the 42 patients in our series are presented in Table 1. A total of 71.4% of patients (n 30) had MEN 2A, and 95% of patients (n 40) had a positive family history of MTC. Of 32 patients who had RET proto-oncogene analysis, 29 (90.6%) had a mutation detected, 14 in exon 14 codon 804 (V804M), 9 in exon 11 codon 634 (C634A), 5 in exon 10 codon 609 (C609S), and 1 in exon 16 codon 918 (M918T). In 31 (77.5%) of 40 patients where results of calcitonin screening were available, the basal and/or stimulated serum calcitonin level was high (range, pg/ml) relative to the reported ageand sex-specific normal reference range. Histomorphologic Findings All patients included in the series (n 42) had a prophylactic total thyroidectomy performed. A total of 93% of patients (n 39) had either CCH and/or MTC; 85.7% (n 36) had at least a single focus of CCH, and 71.4% of patients (n 29) had at least 1 focus of MMC. One patient had a medullary macrocarcinoma (1.1 cm). Among the MMCs, 62% (n 18) were multifocal, 48% (n 14) were bilateral, and 38% (n 11) were solitary. Only 3 cases (7%) showed neither MMC, MTC, nor CCH, and in all 3 cases, the entire thyroid glands had been submitted for evaluation. The results of the histomorphologic findings are summarized in Tables 1 and 2. Cases with CCH included solitary and multifocal CCH. In 83.3% of cases (n 30), the CCH found on the examined slides was multifocal, and 77% (n 23) were bilateral. The foci of CCH were nodular in 33% (n 10), nonnodular in 23% (n 7), and mixed nodular and nonnodular in 43% (n 13) of cases (Figure 1, A). Although not all foci of CCH were seen using hematoxylin-eosin staining alone, 94.4% (n 34) were detectable on hematoxylin-eosin. Foci of CCH were characterized histologically by cells that were slightly larger than adjacent follicular cells with granular cytoplasm, round nuclei, and coarse granular chromatin (Figure 1, B). The mean size of the MMCs was 0.3 cm, with a size range of less than 0.1 to 0.8 cm. In every case of MMC 1768 Arch Pathol Lab Med Vol 132, November 2008 Prophylactic Thyroidectomy for Medullary Carcinoma Etit et al

3 Case No. MEN Status* Table 1. Clinicopathologic Features of Study Cases Family History Age, y/sex RET Proto-oncogene CCH Foci MTC Foci and Size in cm Specimen Sampling 1 MEN 2A Positive 37/F Positive (V804M) exon 14 MF nodular MF, up to 0.1 Entirely 2 MEN 2A Positive 19/F Positive (V804M) exon 14 MF nonnodular Absent Entirely 3 MEN 2A Positive 19/F Positive (V804M) exon 14 MF nonnodular Absent Entirely 4 MEN 2A Positive 20/M Positive (V804M) exon 14 MF nodular nonnodular Absent Entirely 5 Non-MEN Positive 9/M Positive (C609S) exon 10 MF nodular nonnodular S, 0.1 Entirely 6 MEN 2A Positive 20/F Positive (C620F) exon 10 S nodular (on recut gone) Absent Entirely 7 MEN 2A Positive 3/F Positive (C634A) exon 11 MF nonnodular nodular S, 0.1 Representative 8 MEN 2A Positive 11/F Positive (C634A) exon 11 MF nonnodular MF, up to 0.3 Entirely 9 MEN 2A Positive 70/F Positive (V804M) exon 14 MF nodular nonnodular MF, up to 0.4 Representative 10 MEN 2A Positive 45/F Positive (V804M) exon 14 Absent S, 0.1 Entirely 11 MEN 2A Positive 2/M Positive (C634A) exon 11 Absent MF, up to 0.2 Entirely 12 MEN 2A Positive 8/M Positive (C634A) exon 11 S nonnodular S, 0.2 Representative 13 MEN 2A Positive 7/M Positive (C634A) exon 11 MF nonnodular MF, up to 0.4 Entirely 14 MEN 2A Negative 30/M Positive (C634A) exon 11 MF nodular nonnodular MF, up to 0.2 Entirely 15 MEN 2A Positive 6/F No workup MF nodular S, 0.2 Entirely 16 Non-MEN Positive 73/F Negative MF nodular nonodular MF, up to 1.1 Entirely 17 MEN 2A Positive 19/M Positive (C609S) exon 10 S nonnodular Absent Entirely 18 MEN 2A Positive 40/M Positive (C609S) exon 10 MF nodular nonnodular MF, up to 0.2 Entirely 19 Non-MEN Positive 38/F Positive (V804M) exon 14 MF nodular MF, up to 0.4 Entirely 20 Non-MEN Positive 32/F Positive (V804M) exon 14 MF nodular MF, up to 0.6 Entirely 21 MEN 2A Positive 9/F No workup MF nodular nonnodular S, 0.2 Entirely (by report) 22 MEN 2A Positive 15/F No workup MF nonnodular MF, up to 0.6 Entirely 23 MEN 2A Positive 42/M No workup Absent S, 0.2 Entirely 24 Non-MEN Positive 48/M Negative MF nodular MF, up to 0.1 Representative 25 Non-MEN Positive 43/M Negative MF nodular nonnodular MF, up to 0.4 Representative 26 Non-MEN Positive 68/F No workup MF nodular nonnodular MF, up to 0.5 Representative 27 MEN 2A Positive 25/F No workup MF nodular S, 0.2 Entirely 28 Non-MEN Positive 7/M No workup MF nodular Absent Entirely 29 MEN 2B Positive 10/M Positive (M918T) exon 16 S nodular MF, up to 0.6 Entirely 30 MEN 2A Positive 3/M Positive (C634A) exon 11 MF nodular S, 0.3 Entirely 31 MEN 2A Positive 9/F Positive (C634A) exon 11 S nonnodular (on the report) Absent Entirely 32 MEN 2A Positive 11/M Positive (C634A) exon 11 Absent Absent Entirely 33 MEN 2A Positive 17/F No workup MF nonnodular MF, up to 0.5 Entirely 34 Non-MEN Positive 2/M Positive (V804M) exon 14 Absent Absent Entirely 35 Non-MEN Positive 2/M Positive (V804M) exon 14 Absent Absent Entirely 36 MEN 2A Positive 18/F No workup MF nodular MF, up to 0.8 Representative 37 Non-MEN Negative 68/M No workup S nonnodular S, 0.7 Representative 38 MEN 2A Positive 42/M Positive (V804M) exon 14 MF nonnodular nodular Absent Entirely 39 MEN 2A Positive 44/M Positive (V804M) exon 14 MF nodular nonnodular MF, up to 0.1 Entirely 40 MEN 2A Positive 39/F Positive (V804M) exon 14 MF nodular nonnodular S, less than 0.1 Entirely 41 MEN 2A Positive 52/M Positive (C620F) exon 10 MF nodular MF, up to 0.2 Entirely 42 MEN 2A Positive 19/M Positive (V804M) exon 14 MF nonnodular Absent Entirely * MEN indicates multiple endocrine neoplasia; Non-MEN, no evidence of MEN syndrome. V indicates valine; M, methionine; C, cysteine; S, serine; F, phenylalanine; A, arginine; and T, threonine. CCH indicates C-cell hyperplasia; MTC, medullary thyroid carcinoma; MF, multifocal; and S, solitary. Entirely indicates the entire thyroid was submitted for histologic evaluation; Representative, representative sections of the thyroid were submitted for histologic evaluation. except for one, the carcinoma was confirmed in at least 1 slide using immunohistochemical staining for calcitonin. In the 1 case where the calcitonin-stained slide was unavailable, it was reported as positive for this stain in the corresponding pathology report. For those cases where Table 2. Numbers of Cases With or Without C-Cell Hyperplasia (CCH) and Medullary Thyroid Carcinoma (MTC) MTC/CCH Combination No MTC/no CCH 3 No MTC/CCH 9 MTC/no CCH 3 MTC/CCH 27 Total 42 n immunohistochemistry for carcinoembryogenic antigen was performed, 100% of CCHs (n 23) and MMCs (n 20) were positive. All of the MMCs were recognizable microscopically using hematoxylin-eosin staining. Although most of the MMCs showed a rounded solid architectural growth pattern, lobular or irregular patterns were also seen (Figure 2, A and B). Although some were well circumscribed, most MMCs appeared histologically nonencapsulated. Unequivocal angiolymphatic invasion was not identified, and only 1 case (case 29) exhibited microscopic extrathyroidal extension. Most the MMCs were composed predominantly of round or polygonal cells, but occasional cases contained spindle-shaped or plasmacytoid cells (Figure 2, C). The cells were arranged in nests, sheetlike, and cordlike patterns. The cytoplasm was typically granular or amphophilic (Figure 2, D), and nuclei Arch Pathol Lab Med Vol 132, November 2008 Prophylactic Thyroidectomy for Medullary Carcinoma Etit et al 1769

4 Figure 1. C-cell hyperplasia. A, Both nodular and nonnodular forms of C-cell hyperplasia are present, as highlighted by immunohistochemical staining for calcitonin (calcitonin immunohistochemistry, original magnification 50). B, The C cells have delicate granular cytoplasm, round nuclei, and an even chromatin pattern (hematoxylin-eosin, original magnification 400). were round to oval with coarse granular ( salt and pepper ) chromatin. Binucleated cells were occasionally present. In those MMCs with spindle cells, the nuclei were elongated, but the chromatin pattern was still granular (Figure 2, E). Regarding other variations in cellular morphology, none of the MMCs in our series exhibited a predominant clear cell pattern or oncocytic change, and histologic evidence of mucin was not identified. Although some tumors showed mild nuclear pleomorphism or hyperchromasia, the majority were not significantly pleomorphic. In 96.5% of MMCs, there was at least focal stromal sclerosis. Even among the smallest recognizable MMCs that were slightly less than 0.1 cm, stromal sclerosis and single cells were present. Necrosis was absent, and mitotic figures were rare, being found in only 6.9% (n 2) of cases. A total of 5.7% of cases (n 12) that were stained using Congo red showed focal positivity for amyloid. Associated Lymph Node, Thyroid, and Parathyroid Findings A total of 22 cases in the series had at least a limited regional lymph node dissection accompanying the total thyroidectomy. Only case 21, from a 9-year-old girl with a single 0.2-cm focus of MMC, had a lymph node metastasis of MTC involving a pretracheal lymph node. The MMC in this case was histomorphologically similar to those from other cases. It lacked histologic evidence of angiolymphatic invasion, and it was neither extensively sclerotic nor amyloid rich. Additional morphologic findings in the thyroidectomy specimens included papillary thyroid carcinoma (n 3) ranging in size from less than 0.1 to 1.7 cm, the presence of thymic tissue (n 2), and follicular adenomas or adenomatous nodules (n 5). Interestingly, in only 5 cases were definite solid cell nests identified, and 4 of these were male patients. Parathyroid exploration was performed in 13 cases, and in 5 cases (38.5%), hypercellular parathyroids were encountered. Postoperative follow-up information was available in 29 cases, with a mean clinical follow-up of 4.7 years (range, 1 month to 13 years). In none of the cases was a documented recurrent MTC encountered. One case had a 3-mm lesion seen on ultrasound in the thyroid bed, with a serum calcitonin level below the normal reference level and no change in the lesion on subsequent follow-up. In the 29 cases with clinical follow-up, serum calcitonin level tests were performed at regular intervals and the levels seen were below the age- and sex-dependent normal reference ranges. Nineteen patients had additional clinical findings. Two patients had bilateral pheochromocytomas, and one of these patients developed renal failure and underwent bilateral nephrectomy. Two patients from the same family had myotonic dystrophy, and one of these developed renal tubular acidosis. One patient developed oral mucosal neuromas, and 1 patient, 13 years postoperatively, developed hyperparathyroidism, hypercalcemia, and hypophosphatemia, as well as elevated levels of blood norepinephrine, epinephrine, dopamine, and urine epinephrine. Two patients had an intestinal tubular adenoma, and 1 patient developed Alzheimer disease and cardiac disease. COMMENT During the past century, the MEN syndromes have emerged as an important group of diseases. Among these, MEN 2A, MEN 2B, and familial medullary thyroid carcinoma are associated with the development of MTC, a potentially lethal malignancy with frequent lymph node metastases. 3,15 The clinical and pathologic features of these syndromes have been described, laboratory tests to detect them have been devised, and characteristic genetic abnormalities have been identified. As a result, the diagnosis of these syndromes in asymptomatic patients can today be accomplished by molecular genetic techniques. 16 In an attempt to identify patients with MTC or the precursor lesion CCH at a potentially curable stage of the disease, measurement of serum concentrations of calcitonin and, more recently, analysis of RET proto-oncogene mutations have increased in frequency. This has resulted in prophylactic thyroidectomies for such patients being performed at an early age, especially in patients with mutations in codons deemed to carry a high risk of development of MTC (eg, codons 609, 620, 804, 634, and 918). Consequently, thyroidectomy specimens with earlier and smaller lesions are being encountered in surgical pathology practices. Microcarcinomas of the thyroid are defined as tumors measuring less than 1.0 cm in greatest dimension. Most 1770 Arch Pathol Lab Med Vol 132, November 2008 Prophylactic Thyroidectomy for Medullary Carcinoma Etit et al

5 Figure 2. Medullary microcarcinoma. A, Some microcarcinomas are very well demarcated with a smooth interface between the outer thyroid parenchyma and tumor (hematoxylin-eosin, original magnification 50). B, Irregularly shaped focus of microcarcinoma with associated stromal fibrosis and nodular C-cell hyperplasia (hematoxylin-eosin, original magnification 100). C, A combination of spindled and round cells is present (hematoxylin-eosin, original magnification 100). D, The cells have amphophilic cytoplasm and round uniform nuclei with granular salt and pepper chromatin (hematoxylin-eosin, original magnification 400). E, Some microcarcinomas have a predominance of spindled cells (hematoxylin-eosin, original magnification 400). Arch Pathol Lab Med Vol 132, November 2008 Prophylactic Thyroidectomy for Medullary Carcinoma Etit et al 1771

6 of these tumors are papillary thyroid microcarcinomas, whereas MMCs are uncommonly encountered in thyroidectomy specimens. 13,14,17 In one study, MMCs have been described in autopsy findings with an incidence of 0.15%. 17 Medullary microcarcinomas are most commonly found in thyroids removed prophylactically, 14 and a number of recent studies have addressed the morphology of prophylactic thyroidectomy specimens. 2,5 8,18 22 We present our experience with a retrospective series of 42 patients who had prophylactic thyroidectomy specimens examined for the prevention or early cure of MTC. Currently, an assay for serum calcitonin is typically performed at Massachusetts General Hospital for patients presenting with nodular thyroid disease. 6,21 23 In a series of 667 cases reported by Kaserer et al, 6 routine screening of serum calcitonin levels in patients with nodular goiter demonstrated abnormal values in 4.5%, and in all of the abnormal cases, either MTC or CCH was found. In a study of patients with primary hereditary MTC, a serum calcitonin test was abnormal in 28 (87.5%) of 32 cases, 19 and in our series, preoperative serum calcitonin was abnormal in 31 patients (73.8%). The mean patient age in our study was 24.5 years: 18 patients (42.8%) were children or adolescents (2 18 years old), and 24 patients (57.2%) were adults (19 73 years old). This correlates well with the findings of a young patient age in other series of hereditary MTC. In a study by Kaserer et al, 5 the mean age of familial cases of MTC was 32 years, as opposed to 58 years for sporadic cases where MTC was found. In other series, the mean ages of patients with MTC following RET proto-oncogene screening were 18.9 years 19 and 16 years, 18 although the latter study involved only 14 patients from 2 families. The mean age at diagnosis for cases of sporadic MTC, by contrast, is reported as between 44 and 50 years. 24 Germline and somatic mutations in the RET proto-oncogene are associated with inherited and sporadic MTC, respectively. The various RET proto-oncogene mutations are reported to occur in different frequencies at various codons. 2,6,25 37 According to our study, the most commonly affected codon was V804M in exon 14, although 4 other codons were involved (Table 1). Eleven patients in our series were non-men cases. Ten of these had an associated family history of MTC and are considered examples of familial MTC. Three of these patients when tested in 1996 were negative for RET mutations, which were reported in up to 15% of familial MTC cases at that time. Only 1 non- MEN patient (case 37) had no family history but was found to have an elevated serum calcitonin level on routine testing. This case may represent a fortuitous early detection of a sporadic MMC. A total of 26 (89.6%) of 29 patients with a positive RET proto-oncogene mutation had either CCH, MMC, or both. In the 3 patients with negative pathology, surgery had been performed on the basis of a positive test for a RET proto-oncogene mutation. They were boys aged 2, 2, and 11 years. This is slightly less than the 100% of hereditary cases in other series that had CCH. 7,18,19 This small difference may possibly represent more extensive histopathologic sampling in the other series. None of our patients with MMC has died as a result of the tumor, and only 1 had a lymph node metastasis of MTC at the time of surgery. Interestingly, the single case in our series (a 9-yearold girl) with a metastasis to a pretracheal lymph node had a small, 0.2-cm primary tumor. Our follow-up results support the findings of Krueger et al, 8 who noted no mortality in patients with MMCs after an observation period of up to 70 months, and of Bergholm et al, 38 who found no excess in mortality compared with the general population of familial MTC patients detected by screening for a period of up to 20 years. It is noteworthy that 13 of our patients had mutations at codons considered to be at high risk for the development of MTC: 9 at codon 634, 3 at codon 609, and 1 at codon 918. In contrast to Krueger s series, 8 separation of MMCs from nodular CCHs was not easy in every case if there were no obvious invasion by tumor. In this situation, the most helpful finding to differentiate MMC from CCH was stromal sclerosis within the nodule of C cells. There was a high incidence (96.5%) of stromal sclerosis in the microcarcinoma group of patients in our series. CCH was easily identified in hematoxylin-eosin stained slides because of its similar cytomorphologic features to MMC. Most of the microcarcinomas had morphologic features of conventional medullary carcinoma: round and polygonal cells with granular to amphophyllic cytoplasm, and nuclei with salt and pepper chromatin. Most MMCs in our study were 0.1 to 0.2 cm in size (51.7%), and the presence of amyloid did not seem to be related to tumor size. Although the smallest MMC showed a positive reaction by Congo red stain, some larger MMCs were negative. Our findings in prophylactic thyroidectomies of small MMCs, often multiple and bilateral and often with multifocal CCH but only 1 macrocarcinoma, are in concert with the findings of others of small, often multifocal early C-cell proliferative lesions in these cases. 5,8 However, although the study of Kaserer et al 6 found that 11 males but no females had CCH only, in our study we found 9 patients with CCH but not MTC, of which 5 were male and 4 were female. In addition, we found very few solid cell nests associated with our C-cell lesions. It would be interesting to note the density of solid cell nests in association with C-cell lesions in other prophylactic thyroidectomies. In 3 cases from our series, there was concomitant papillary thyroid carcinoma present, and 1 was metastatic. This association has been reported previously and may be the result of oncogenic activation of RET in thyroid tumors From our study and review of the literature, it is clear that RET proto-oncogene analysis is a valuable test to detect early MTC. Our findings, together with those of others, support performing prophylactic thyroidectomy to give the best chance of cure to the family members of patients with MEN 2 or hereditary MTC with high calcitonin levels or RET proto-oncogene mutations. As MMC or CCH were found in almost every case in our series, prophylactic total thyroidectomy may indeed be a lifesaving procedure in many of these cases. This study was partially supported by a grant (B.02.1.TBT ) from The Scientific and Technological Research Council of Turkey (TUBITAK). The authors thank Lisa Gallagher, MD, for her valuable support collecting multiple previous cases. References 1. DeLellis R. Pathology and genetics of thyroid carcinoma. 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