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Familial Medullary Thyroid Carcinoma: Clinical Variability and Low Aggressiveness Associated with RET Mutation at Codon 804

Départment de Biologie Clinique and Service de Médecine Nucléaire, Institut Gustave Roussy (F.L., E.B., B.C., J.-M.B., M.S.), 94805 Villejuif, France; Dipartimento di Medicina Sperimentale e Clinica G. Salvatore, Università di Catanzaro (E.C., F.A., S.F.), 88100 Catanzaro, Italy; Istituto CSS-Mendel (C.C., B.D.), 00161 Rome, Italy; Service de Médecine Nucléaire, Centre François Baclesse (S.B.), 14076 Caen, France; Division of Medical Oncology, Ospedale San Luigi (D.G.), 95123 Catania, Italy; and Dipartimento di Scienze Cliniche, Università di Roma La Sapienza (S.F.), 00161 Rome, Italy

Address all correspondence and requests for reprints to: Sebastiano Filetti, M.D., Dipartimento Scienze Cliniche, Clinica Medica 2, Policlinico Umberto I Viale del Policlinico, 155, 00161 Rome, Italy. E-mail: . filetti{at}tin.it

Abstract

Sixty-one heterozygotes harboring the germline V804L mutation of the RET protooncogene were identified in five independent families. A total of 31 subjects underwent surgery. Histology identified C cell hyperplasia in 30 cases, isolated in 12 and associated with medullary thyroid carcinoma (MTC) in 18. Six patients with MTC had lymph node metastases. Among the 14 patients with basal detectable calcitonin (CT) level, 12 had MTC and 2 had isolated C cell hyperplasia. In most individuals carrying 804 RET mutation, C cell disease displayed late onset and an indolent course; a pentagastrin test was negative in the majority of heterozygotes during the first 2 decades and was positive in only half of them during the third and fourth decades of life. Interestingly, concomitant somatic M918T was detected in a 12-yr-old girl with MTC and was likely to be responsible for both the early clinical appearance and the aggressiveness of the disease.

Our data show that in these gene carriers, surgery may be postponed to the fourth decade of life or until the pentagastrin stimulation test becomes positive. Indeed, our data should be confirmed on a larger series of V804L carriers, but may offer a balanced strategy to keep under control and prevent development of the full disease phenotype.

GERMLINE MUTATIONS of the RET protooncogene predispose to hereditary medullary thyroid carcinoma (MTC). This autosomal dominant cancer syndrome can manifest either as a single disorder [familial MTC (FMTC)] or as MEN syndrome type 2A or 2B.

Mutations in one of six cysteine codons clustered in the extracellular domain (at codons 609, 611, 618, 620-exon 10, and 630, 634-exon 11) are found in the majority of MEN 2A families and in half of FMTC families (1, 2, 3). In recent years genotype analysis has consistently changed the clinical management of this disease by separating the hereditary from the far more common sporadic cases. In addition, in the hereditary forms, half of the first degree relatives can be excluded from the risk, and gene carriers can be identified at a presymptomatic stage. In these presymptomatic gene carriers, prophylactic thyroidectomy is advocated before age of 5–6 yr based on complete penetrance of MTC, early age at onset, and the potential for aggressive MTC (4, 5).

International RET mutation consortium analysis reported that only 10% of FMTC families showed germline mutation in the intracellular domain of the RET gene (1). Vice versa, the French Calcitonin Tumors Study Group found that a mutation located within the intracellular domain of the RET protooncogene (at codons 768, 790, 791-exon 13, or codon 804-exon 14) is present in about half of FMTC families (6). A codon 804 mutation, leading to substitution of valine with leucine was first identified in two unrelated French FMTC families, including family F426 (7, 8). In vitro studies have shown that this mutation is more weakly activating than mutations in exon 10 or 11 (9). Some observations suggested that the V804L RET mutation in codon 804 is causing low penetrant disease, with late onset and relatively indolent course (7, 8, 10), whereas other researchers reported that V804L mutation or other mutations in the same codon (substitution of valine with methionine) have an aggressive potential (11, 12, 13). These data argue for wide clinical variability, and there is not yet agreement as to the therapeutic strategies for gene carriers.

Here we report our experience on 61 heterozygotes from 5 independent families harboring the V804L RET mutation. These observations have allowed study of the phenotypic heterogeneity associated with this mutation and the determination of penetrance, mean age at onset of the disease, clinical course, and thus the age at which surgery should be performed (1, 5, 6).

Subjects and Methods

Patients

A total of 61 heterozygotes have been identified in 5 unrelated families and form the basis of the present report (Table 1). Family 4 has been partly reported as family F426 (8). No evidence of hyperparathyroidism and pheochromocytoma was found in any of these subjects.

Calcitonin (CT) measurement

Plasma CT was measured by immunoradiometric assay, using the ELSA-hCT kit (Cis Bio International, Gif-sur-Yvette, France), with a normal range of less than 10 pg/ml (14).

The pentagastrin (Pg) stimulation test consisted of a slow iv injection of 0.5 µg/kg pentagastrin (Peptavlon, I.C.I. Pharma, Cergy, France) during 3 min. Blood samples were collected at 0, 3, and 5 min after starting Pg infusion (15).

Screening for RET mutation

DNA extraction and PCR amplification. Genomic DNA was extracted from peripheral leukocytes of family members on two separate blood samples using a blood DNA extraction kit (Amersham Pharmacia Biotech, Milan, Italy). Tumoral DNA was prepared from paraffin-embedded tissue from 12 tumor tissues obtained at surgery using an appropriate DNA extraction kit (Amersham Pharmacia Biotech, Milan, Italy). Genomic amplicons encompassing exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene were obtained by PCR amplification, using primers designed on the basis of the intronic sequence (16, 17).

Single strand conformation polymorphism (SSCP) analysis. Exons 10, 11, 13, 14, and 15 were screened for mutations by SSCP analysis, as previously reported (18). Briefly, an aliquot of each sample was incubated with a specific buffer (10 ml formamide, 0.5 M EDTA, 0.3% bromophenol blue, and 0.3% xylene cyanol) for 8 min at 100 C and run on 15% Tris-borate-EDTA gel using a Minigel apparatus (Bio-Rad Laboratories, Inc., Milan, Italy), at 100 V at 4 C. After electrophoresis, the gel was examined by a DNA silver staining kit (Amersham Pharmacia Biotech, Milan, Italy) according to the manufacturer’s instructions.

Sequence analysis. Sequence analysis was performed to identify the alteration found in the proband by SSCP analysis and to analyze other family members. Briefly, 100 ng double-stranded amplified DNA were sequenced with an ABI dideoxyterminator cycle sequencing kit (PE Applied Biosystems, Les Ulis, France). Thirty-five cycles of denaturation, annealing, and extension were performed at 94 C (15 sec), 60 C (15 sec), and 60 C (2 min), respectively, using an automatic thermocycler (model 9700, Perkin-Elmer Corp., Palo Alto, CA). Electrophoresis and sequence analysis were performed on a 377A DNA sequencer (PE Applied Biosystems).

Restriction analysis. Amplicons of exon 16 were directly screened for mutations by restriction enzyme analysis with FokI as previously described (19).

Surgical treatment and follow-up

Surgery consisted of total thyroidectomy in all patients. In the case of carcinoma, a bilateral dissection of the central neck compartment and of the lower third of the jugulo-carotid chains was also performed. Methylene blue staining of the parathyroid glands with autotransplantation was routinely performed.

All patients were followed postoperatively. In patients with undetectable basal CT, a Pg test was performed 2 months after and again 1 yr after surgery. Then patients were followed up annually with clinical examination, basal calcitonin determination, calcemia and PTH-(1–84) determinations, and urinary measurements of catecholamines and derivates.

Histopathology

Surgical specimens, including thyroid gland and lymph nodes, were studied according to standard histological. C Cell hyperplasia was defined as the presence of at least 50 C cells in 1 low power field (x100 magnification). Serial sections were examined for CT staining using an immunohistochemical method.

Results

Sixty-one individuals from five unrelated families (two from France and three from Italy) were found to harbor the germline RET V804L mutation (Figs. 1 and 2).

View larger version (17K): [in this window] [in a new window]   Figure 1. Pedigree of families 1 and 2. Squares and circles indicate males and females, respectively. In each family the arrow indicates the proband.

View larger version (18K): [in this window] [in a new window]   Figure 2. Pedigree of families 3, 4, and 5. Squares and circles indicate males and females, respectively. In each family the arrow indicates the proband.

Isolated C cell hyperplasia was found in 2 patients with detectable basal CT levels and in 10 patients with undetectable basal CT levels and Pg-stimulated CT in the range of 14 pg/ml in 1 case, 50–100 pg/ml in 8 patients, and more than 100 pg/ml in 1 case (Tables 1 and 2).

Six patients with MTC had lymph node metastases. Patients with lymph node metastases had detectable basal CT levels ranging from 45–1750 pg/ml (Tables 1 and 2). One patient (family 4, patient III-1) underwent total thyroidectomy at the age of 34 yr for benign follicular adenoma; the preoperative Pg stimulation test was negative, and no C cell hyperplasia was found at immunohistological examination.

As a function of age, basal CT levels was undetectable in 37 of 38 gene carriers less than 40 yr of age (Table 3). Pg-stimulated CT levels were undetectable in 22 of the 32 subjects with basal CT levels and in whom a Pg stimulation test was performed before the age of 40 yr. In contrast, in the 21 subjects over 40 yr of age, basal CT levels were elevated in 12 patients. Seven of 8 patients with undetectable basal CT levels underwent a Pg stimulation test; pentagastrin stimulated CT levels were elevated in 5 patients and were less than 10 pg/ml in 2 patients. Basal and Pg-stimulated CT measurements were not available in 2 patients (Table 3).

Postoperatively, basal plasma CT levels were undetectable in 28 patients and remained undetectable after Pg stimulation in the 22 patients who underwent this stimulation test, even in those operated on after the age of 40 yr, suggesting the absence of residual disease (Table 3). With a median follow-up of 8.5 yr, no biochemical or clinical recurrence was observed.

Postoperatively, the basal CT level was elevated in 3 index cases (Table 3). A value of 5200 pg/ml was found in 1 patient (family 5, patient I-1). This index case was 70 yr old at diagnosis of disease and presented with a large thyroid tumor, neck lymph node metastases, and liver metastases. In 2 other patients, basal CT levels were 87 and 150 pg/ml, respectively (family 3, patient II-1; family 1, patient IV-4; Table 2). Both subjects presented with thyroid tumor, clinical lymph node involvement, and elevated basal CT levels. One patient (family 3, patient II-1) was 44 yr old at surgery, and the other was 12 yr old (family 1, index case IV-4). In the tumor of this latter patient, we detected a somatic missense RET mutation in codon 918, exon 16, leading to methionine substitution with threonine (Fig. 3). Thus, this patient displayed concomitant germline V804L and somatic M918T RET mutations that were located in different alleles (data not shown). No other RET gene mutation was observed in this tumor or in any of the other 11 tumors studied (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 3. Presence of double point mutation in the RET protooncogene. Left, Point mutation in codon 804 of the RET gene in genomic DNA (coexistence of T and G, as indicated by the point). Right, Point mutation in codon 918 of the RET gene in tumoral DNA (coexistence of T and C, as indicated by the point).

In the present five families, a conservative approach was used. RET protooncogene testing disclosed heterozygotes. The highly sensitive Pg stimulation test with CT measurement was performed on a yearly basis, starting during the second decade of life (14, 15). Surgery was performed in gene carriers presenting with detectable basal plasma CT levels or significant CT response to Pg stimulation. In most individuals with the 804 RET mutation, C cell disease was of late onset and had an indolent course. Different lines of evidence support this point.

First, the Pg test was negative in the majority of heterozygotes during the first 2 decades and was still negative in almost half of them during the third and fourth decades of life. This is in sharp contrast to studies of family members affected by RET mutation at codon 634, in whom the Pg test usually becomes positive during the first decade of life and is strongly positive at the age of 30 yr (20, 21, 22, 23). Similarly to heterozygotes for other RET gene mutations, the risk of C cell disease was higher in patients with higher CT levels. In addition, MTC and C cell hyperplasia were found in patients with undetectable basal CT levels and were moderately increased after Pg stimulation.

Second, most gene carriers were disease free in the first 2 decades of life; C cell hyperplasia developed during the third decade or even later, and C cell neoplasia developed during or after the fourth decade. Again, this pattern is different from that observed in MEN2A patients, in which C cell hyperplasia and MTC usually become manifest during the first 2 decades of life (20, 21, 22, 23).

Third, of the 18 patients with MTC, 6 (25%) had neck lymph node metastases, and 1 of them also presented with liver metastases. This is not the behavior expected in MTC, either in the sporadic form or in the classical MEN 2A disease, in which 10–30% of microcarcinomas and up to 90% of clinical cancers are associated with lymph node metastases (24).

Fourth, postoperative basal CT levels were undetectable in 28 of 31 patients, and stimulated CT levels remained undetectable in all 22 patients who underwent this stimulation test, even in those operated on after the age of 40 yr. Furthermore, none of these 28 patients relapsed, with a median follow-up of 8.5 yr, confirming the limited extent of the disease at the time of surgery. Again, this contrasts with sporadic MTC and with MEN2A operated on after the age of 40 yr, in which only 20–30% had undetectable CT levels postoperatively (24).

These data are in close agreement with in vitro studies showing that a codon 804 RET mutation has a weaker activating capability and a lesser transforming capacity compared with other RET mutations (9). Similarly, the rare and late occurrence of pheochromocytoma in patients with V804L mutation suggests that this disease should be regarded as a low penetrant and low aggressive form (25).

Other groups have reported an aggressive presentation of MTC at a young age in isolated cases with V804L mutation (11, 12, 13). Confirmatory evidence was obtained in 3 of our 61 heterozygotes who were not cured after initial surgery. All 3 of these patients were index cases. A 44-yr-old patient had lymph node involvement at initial surgery and, as expected, did not normalize CT level postoperatively. The second patient was 70 yr old at the time of surgery and presented with a clinical thyroid tumor, lymph nodes, and liver metastases. The third patient was a 12-yr-old girl exhibiting extensive and invasive MTC. However, in the same kindred the other gene carriers displayed low aggressive disease; indeed, the girl’s grandmother was a gene carrier, but at the age of 80 yr did not exhibit any evidence of C cell disease. The finding that identical RET mutation behaves differently, even in the same genetic background, suggests that other genetic or epigenetic or environmental factors play a role. Indeed, extensive gene analysis in the tumor tissue disclosed a second somatic RET mutation in this patient. It is likely that these 2 mutations were responsible for both the early clinical onset and the aggressiveness of the tumor. The fact that these 2 mutations were located on different alleles suggests that they are not related. Several studies demonstrated in some MTC from patients with MEN 2A/FMTC syndrome another somatic RET mutation (26, 27, 28), supporting the hypothesis that a somatic mutation, in addition to the preexisting germline mutation, may be responsible of the progression of C cell hyperplasia to MTC. This suggests that extensive genetic studies are needed to exactly predict the natural history of the C cell disease.

However, the discovery of a somatic 918 RET mutation in a single tumor will not modify our strategy in these family members with a germline RET V804L mutation. It consists firstly of identifying by genetic testing family members at risk and thereafter establishing in these gene carriers a surveillance program by using a sensitive biochemical test (i.e. Pg-stimulated CT). Surgery will be performed when Pg-stimulated CT becomes positive and in the other cases during the fourth decade of life. Indeed, our data should be confirmed on a larger series of V804L carriers, but may offer a balanced strategy able to keep these subjects’ disease under control and to prevent development of the full disease phenotype manifestation.

We are aware that Pg is no longer available to the medical community in the U.S.; our data will suggest a reconsideration of this choice. Alternatively, the use of calcium as a secretagogue (although the sensitivity of this test in the diagnosis of MTC is reduced) or the development of highly sensitive methods for CT measurement may overcome this difficulty.

Acknowledgments

Footnotes

This work was supported by a grant from Associazione Italiana per la Ricerca sul Cancro and MURST (to S.F.) and by a grant from the Italian Minister of Health (RC 1999; to B.D.).

¹ Recipient of Dottorato di Ricerca in Scienze Endocrine: Basi Molecolari dell’Azione Ormonale at University of Catania.

Abbreviations: CT, Calcitonin; FMTC, familial medullary thyroid carcinoma; MTC, medullary thyroid carcinoma; Pg, pentagastrin; SSCP, single strand conformation polymorphism.

Received September 21, 2001.

Accepted January 4, 2002.

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