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RET Genetic Screening in Patients with Medullary Thyroid Cancer and Their Relatives: Experience with 807 Individuals at One Center

Departments of Endocrinology and Metabolism (R.E., C.R., B.C., L.A., V.B., E.M., M.S., L.G., A.P.), of Surgery (P.M.), and of Oncology (F.B.), University of Pisa, 56100 Pisa, Italy; Department of Internal Medicine, Endocrinology, and Metabolism, and Biochemistry (F.P.), University of Siena, 53100 Siena, Italy; and AMBISEN Center, High Technology Center for the Study of the Environmental Damage of the Endocrine and Nervous Systems (A.P.), University of Pisa, 56124 Pisa, Italy

Address all correspondence and requests for reprints to: R. Elisei, M.D., Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy. E-mail: relisei{at}endoc.med.unipi.it.

	Abstract

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Background: Germline RET gene mutations are causative of multiple endocrine neoplasia (MEN) 2 and may be identified by genetic screening. Three different syndromes are distinguished: MEN 2A, when medullary thyroid carcinoma (MTC) is associated with pheochromocytoma and/or parathyroid adenomas; MEN 2B, when accompanied by a marfanoid habitus and/or pheochromocytoma; and familial medullary thyroid carcinoma (FMTC), when only MTC is present.

Patients and Methods: During the last 13 yr, we performed RET genetic screening in 807 subjects: 481 with apparently sporadic MTC, 37 with clinical evidence of MEN 2, and 289 relatives. Genomic DNA was extracted from the blood of all subjects, and exons 10, 11, 13, 14, 15, and 16 were analyzed by direct sequencing after PCR.

Results: We unexpectedly discovered a germline RET mutation in 35 of 481 (7.3%) apparently sporadic MTC patients. A germline RET mutation was also found in 36 of 37 patients with clinical evidence of hereditary MTC. The distribution of RET mutations in cysteine and noncysteine encoding codons was significantly different in the two groups of patients, with the prevalence of RET mutations in noncysteine codons being higher in MTC that presented as apparently sporadic (P < 0.0001). A total of 34 FMTCs (75.5% of all FMTC) arrived with apparent sporadic MTC, with no familial history of other MTC cases. According to genetic screening and clinical data, our 72 families were classified as follows: 45 FMTC (62.5%), 22 MEN 2A (30.5%), and five MEN 2B (7%).

Conclusions: In this large series of MTC, hereditary forms, mainly FMTC, were clinically unsuspected in 7.3% of apparently sporadic cases. As a consequence, the prevalence of FMTC in our series is higher than that previously reported (60 vs. 10%). In these cases, RET mutations were more prevalently located in noncysteine codons. Data derived from our series helped elucidate the role of RET genetic screening for the identification of all forms of MEN 2, and especially for FMTC, which are frequently clinically misdiagnosed as nonheritable, sporadic cases.

	Introduction

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MEDULLARY THYROID carcinoma (MTC) originates from parafollicular C cells and represents 5–10% of well-differentiated thyroid tumors (1, 2, 3). In about 25% of cases, MTC is inherited as an autosomal dominant trait with a variable degree of expressivity and an age-related penetrance. In these cases, other organs (i.e. parathyroid and adrenal glands) may be involved, and three different syndromes, multiple endocrine neoplasia (MEN) 2A, MEN 2B, and familial medullary thyroid carcinoma (FMTC), can be distinguished (4, 5, 6).

In 1987, genetic linkage analysis localized the MEN 2 gene to the centromeric region of chromosome 10 (7). In 1993 and 1994, three independent groups reported that activating germline point mutations of the RET protooncogene are causative events in MEN 2A, MEN 2B, and FMTC (8, 9, 10).

The number and type of recognized RET mutations have grown over the last 10 yr, especially after the introduction of RET genetic screening in the workup of all patients with MTC, both hereditary and apparently sporadic types. As a consequence of this more careful research, RET mutations have been widely distributed, not only among the five cysteine codons 609, 611, 618, 620, and 634, but also in other noncysteine codons, such as codon 804 in exon 14, codon 883 in exon 15, and others (11, 12). These widely spread mutations are mainly associated with the FMTC phenotype (11).

In the present study, we report the results of 13-yr experience of RET genetic screening performed at one single Italian center, in 807 subjects who arrived for observation because of either sporadic or hereditary MTC, or because they were relatives of familial cases. In particular, we correlated the RET gene mutations and clinical features of positive cases.

	Subjects and Methods

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Subjects

Over the last 13 yr (1993–2006), we performed RET genetic screening in 807 Italian subjects, 481 of whom [289 female (F), 192 male (M); median age 51 yr, range 15–87] arrived for first clinical observation affected by a sporadic form of MTC (no familial history of MTC, no other endocrine diseases) and 37 (22 F and 15M; median age 31 yr, range 9–65) clearly affected by one of the familial forms of MTC. The remaining 289 subjects (145 F and 144 M; median age 31 yr; range 3–93) were first-degree relatives of patients affected by familial MTC.

At the time of this study, the clinical follow-up of our MTC patients ranged from 1–25 yr (1982–2007); all MTC patients diagnosed before 1993 were submitted to RET genetic analysis at the time of their first annual clinical control after the introduction of RET genetic screening in our center.

An informed consent for RET genetic screening and other clinical procedures was signed by all investigated subjects.

RET genetic analysis

Blood was collected in EDTA from all 807 subjects. Genomic DNA was purified from peripheral blood lymphocytes using the QIAMP DNA mini kit (QIAGEN, Hilden, Germany). The RET gene exons 10, 11, 13, 14, 15, and 16 were analyzed in all cases using PCR and DNA sequencing, as previously reported (13). An aliquot of DNA for each patient was stored at –20 C with the intent of searching for new RET mutations whenever described.

Clinical evaluation

MTC patients and RET mutated gene carriers were submitted to clinical and biochemical examination to ascertain thyroid, parathyroid, and adrenal gland involvement. Screening for pheochromocytoma (PHEO) and hyperparathyroidism (HyperPTH) was performed both at diagnosis and annually during the follow-up in both MTC affected patients and RET positive gene carriers.

Histopathology

MTC histological diagnosis was addressed by typical histological (i.e. tumoral cells arranged in trabecular, insular, or sheet-like growth patterns) and immunohistochemical (cells positive for calcitonin and chromogranin) findings.

	Results

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Germline RET mutations

We discovered an unsuspected germline RET mutation in 35 of 481 (7.3%) MTC patients that presented as sporadic cases according to their negative familial history and isolated MTC presentation. These patients were reclassified as having a hereditary form, and their relatives were invited to participate in the genetic screening. As shown in Table 1, these 35 RET mutations were distributed among several exons, and, in 23 of 35 (65.7%) cases (Table 1, cases 13–35), they affected noncysteine encoding codons. In one case (Table 1, case 6), a PHEO was diagnosed 6 yr after the MTC diagnosis. In all other cases, the MTC was the only endocrine neoplasia present both at diagnosis and during follow-up (range 3–25 yr, mean 8.1 yr, median 6.0 yr).

When considering the 27 hereditary cases with a RET mutation found in noncysteine coding codons, the most prevalent mutation was at codon 804 [14 of 27 (51.8%)] with a ValMet substitution in all cases. In particular, 12 of them were found in apparently sporadic MTC cases. The other 13 noncysteine mutations were widely distributed in different codons: three Glu768Asp, one Leu790Phe, six Ser 891Ala, one Ala883Thr, one Met848Thr, and one Ser904Phe. It is worth noting that Met848Thr and Ser904Phe germline mutations have never been described and that Ala883Thr has been reported for the first time by our group (14). RET germline mutations in our 72 families and their relationship with the extracellular and intracellular domains of the receptor are shown in Fig. 1.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Distribution of germline RET mutations found in our series of MEN 2 cases according to different RET gene domains, codons, and exons. One of 72 families was negative for germline RET mutations (not reported in this figure). Please note that mutations in codons 618 and 634 were associated with both FMTC and MEN 2A phenotypes. N, Number.

Clinical and biochemical examination of the 35 apparently sporadic index cases did not show any other associated endocrine neoplasia in all cases harboring the RET mutation in noncysteine coding codons in exons 13, 14, and 15 (Table 1, cases 13–35). After a mean follow-up of 8.1 yr, 11 of 12 cases with mutations in the cysteine coding codons did not develop any other endocrine disease: only one case with a RET Cys618Arg mutation (Table 1, case 6) developed a PHEO 6 yr after the MTC diagnosis.

Clinical examination and familial histories of the 37 cases with an evident diagnosis of a hereditary form of MTC showed that the four cases in this group that harbored the RET mutation in the noncysteine coding codons in exons 13, 14, and 15 (Table 2, cases 28–31) did not show any other associated endocrine neoplasia. All cases (21 of 21, 100%) with the classical MEN 2A mutation in exon 11 at codon 634 (Table 2, cases 1–21) showed the presence of a PHEO at the MTC diagnosis. At variance, the other six cases with RET mutations in cysteine coding codons other than Cys 634 (Table 2, cases 22–27) were negative for both PHEO and HyperPTH, not only at the diagnosis, but also at the time of the present study.

When the two groups of hereditary cases were analyzed together, we observed that among FMTC cases, RET mutations were mainly distributed in noncysteine coding codons in 27 of 45 (60%). The other 18 cases harbored RET mutations in cysteine coding codons distinct from Cys634 in 15 of 45 (33.3%) and in Cys634 in two of 45 (4.5%). In one of the 45 FMTC cases (2.2%), no RET mutation was found. Although we cannot completely exclude the possibility that these cases are indeed MEN 2A that have not yet developed other endocrine diseases, the quite long follow-up is suggestive of the isolated nature of their MTC. However, the periodic control of adrenal and parathyroid glands is mandatory for all of these cases. In our series, the genotype-phenotype correlation was more consistent for MEN 2B (100% with a Met918Thr substitution) and MEN 2A, which had a classical Cys634 RET mutation in 21 of 22 (95.5%) cases and Cys618 substitution in one of 22 (4.5%) cases.

At present, according to our clinical data and results from genetic screening, we can distinguish our 72 families as follows: 45 FMTC (62.5%), 22 MEN 2A (30.5%), and five MEN 2B (7%).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The hereditary form of MTC represents only 20–25% of all MTC whose prevalence of thyroid cancer varies from 5–10%, according to different series (3). With the introduction of RET genetic screening, the finding of a germline RET mutation became sufficient to define the "hereditary" nature of the syndrome. In our series, which is the result of 13-yr RET genetic screening at one single center, the analysis of RET mutations performed in all cases of MTC, regardless of their negative or positive familial history and the presence or absence of other clinical manifestations, allowed us to discover 35 new hereditary cases of MTC. The prevalence (7.3%) of our misdiagnosed hereditary forms of MTC that presented as sporadic is in keeping with those previously reported and ranging from 4–10% in different series (15, 16). This confirms the need to perform complete RET genetic screening in all MTCs. It is worth noting that the majority of misdiagnosed hereditary cases were FMTC, and their identification was due to systematic RET genetic screening. The relatively low aggressiveness of many of these FMTC forms, especially those caused by noncysteine RET mutations (11, 17), may explain the absence of a positive familial history and their clinical diagnosis as sporadic cases. It is also worth noting that all but two of our FMTCs harbor RET mutations in codons other than those typically correlated with the classical MEN 2A and 2B forms, which would not be found if there were not specifically analyzed.

Of 35 (51.4%) newly discovered hereditary cases, 18 were the index cases of families in which several unaware members were affected by MEN 2 syndrome (Table 1). It is very likely that without the identification of the index cases, these subjects would be discovered much later when the MTC clinically manifested, thus impairing the possibility of successful treatment (18, 19). This observation is in keeping with the important role of RET genetic screening for the early diagnosis of hereditary MTC cases.

Although a strong correlation of some RET mutations with specific phenotypes (i.e. Met918Thr/MEN 2B, Cys634Arg/MEN 2A, noncysteine coding codons/FMTC) has been previously documented (11, 20), the classification as MEN 2A, MEN 2B, or FMTC is still largely based on the evidence of familial history, and/or the simultaneous presence or absence of other endocrine neoplasia or physical manifestations that are typical of these syndromes. In our series the percentage of FMTCs, defined on the basis of the absence of any association with other endocrine neoplasia, not only at the diagnosis, but after a follow-up ranging from 1–25 yr, was significantly higher than expected (about 60 vs. 10%). In particular, FMTC showed a quite high percentage of cases with RET mutations in noncysteine coding codons (60% of all FMTC). There are at least two reasons that may explain this phenomenon: the systematic search for RET mutations in all six exons usually involved in the MTC pathogenesis; and the search performed by sequencing analysis, which allows for the occasional discovery of mutations in non "hot spot" regions.

In conclusion, on the basis of our genetic screening and clinical evaluation performed during a follow-up of 1–25 yr, we can assume that among 72 families affected by MEN 2 syndromes, 62.5% are represented by FMTC, 30.5% by MEN 2A, and 7% by MEN 2B. Such a high prevalence of FMTC has not been reported before, and it is higher than that expected. Furthermore, the observation that 75% of our FMTCs were initially diagnosed as apparently sporadic cases with no familial history of other MTC is in keeping with the clinical evidence that FMTC is usually less aggressive than the MTC of the other MEN 2 syndromes. The FMTC cases had RET mutations spread among several codons, especially in those not coding for cysteines, which should be included in routine screening. Unfortunately, 1.4% of MEN 2 syndromes did not show any RET mutations, thus highlighting that, although small, there is still a risk that an apparently sporadic MTC case with no identified RET germline mutation is a hereditary form. Finally, because PHEO and HyperPTH may always appear during follow-up, the screening for both endocrine pathologies should be performed in all gene carriers independent of their RET mutation and even if they are already cured for MTC.

Data derived from our series confirm that RET genetic screening is the most powerful tool for the identification of all forms of MEN 2 and, especially, for FMTC, which could be clinically misdiagnosed as nonheritable, sporadic cases.

	Acknowledgments

 
We thank all colleagues and friends who referred us to the patients included in this study, in particular, Professors Luigi Bartalena from Varese, Luca Chiovato from Pavia, Stefano Mariotti from Cagliari, and Enio Martino from Pisa, and Dr. Nadia Cremonini from Bologna.

	Footnotes

 
This work was supported in part by grants from "Associazione Italiana per la Ricerca sul Cancro."

The Department of Endocrinology and Metabolism of Pisa University is a World Health Organization Collaborating Center for the Diagnosis and Treatment of Thyroid Cancer and Other Thyroid Diseases.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 25, 2007

Abbreviations: F, Female; FMTC, familial medullary thyroid carcinoma; HyperPTH, hyperparathyroidism; M, male; MEN, multiple endocrine neoplasia; MTC, medullary thyroid carcinoma; PHEO, pheochromocytoma.

Received May 4, 2007.

Accepted September 14, 2007.

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