RET Proto-Oncogene Genetic Screening of Families with Multiple Endocrine Neoplasia Type 2 Optimizes Diagnostic and Clinical Management in China

Abstract

Background:

Genetic screening for germline mutations in the RET proto-oncogene has been extensively exploited worldwide to optimize the diagnostic and clinical management of multiple endocrine neoplasia type 2 (MEN2) patients and their relatives. However, a distinct lag period exists not only in the recognition but also in the medical treatment of patients with MEN2. Here we present a comprehensive genetic and clinical analysis of MEN2 among Chinese families followed from 1975 to 2011. Our series comprises 36 index cases and 134 relatives from 11 independent families.

Methods:

Genetic diagnosis was performed in all participants by direct sequencing all relevant RET exons. Thyroidectomy was performed in 50 patients with varying cervical neck dissection procedures. Patients with pheochromocytoma (PHEO) underwent specific surgery. Demographic, clinical profiles, mutation types, tumor histopathologic features, and follow-up records were systematically analyzed.

Results:

The RET mutations p.C634Y (n=34), p.C634R (n=6), p.C618S (n=13), p.V292M/R67H/R982C (n=7), p.L790F (n=2), and p.C634Y/V292M/R67H/R982C (n=1) were confirmed in 31 index cases and then identified in 32 at-risk relatives (mutation carriers), with MEN2A as the most common clinical subtype. The overall penetrance of PHEO in patients with MEN2A was 46.7%. A total of 50 patients underwent thyroidectomy, and there was a significant lowering of their mean age at thyroidectomy and the tumor diameter of the mutation carriers that were detected and operated on compared with the index cases (age at first surgery: 29.3 vs. 39.3 years, p<0.05; maximum size: 1.1 vs. 3.3 cm, p<0.001). There was also a decrease in the TNM staging and the proportion of patients who underwent inappropriate initial thyroid surgery (pN1: 31.6% vs. 100%, p<0.001; inappropriate surgery: 0% vs. 29%). Meanwhile, disease-free survival (DFS) increased (DFS: 100% vs. 58.1%, p<0.05). Both medullary thyroid carcinoma-specific (n=1) and PHEO-specific (n=5) deaths were reported during the study period.

Conclusions:

Our results further substantiate that gene scanning of all relevant RET exons is a powerful tool in the management of MEN2 patients, especially in asymptomatic carriers, and has led to earlier diagnosis and more complete initial treatment of patients with MEN2 in China.

Introduction

M ultiple endocrine neoplasia type 2 (MEN2; OMIM 171400) is an autosomal dominant inherited multiglandular cancer syndrome with an estimated prevalence of 1 per 30,000 in the general population (1,2). It is primarily caused by germline-activating mutations of the rearranged during transfection (RET) proto-oncogene (OMIM 164761), which encodes a tyrosine kinase receptor expressed in a variety of neural crest-derived cells (1,2). Three distinct subtypes of MEN2 have been characterized, (i) MEN2A, by the presence of medullary thyroid carcinoma (MTC, >95% of cases), pheochromocytoma (PHEO, ∼50%), and primary hyperparathyroidism (HPT, ∼25%) in a single patient or family; (ii) MEN2B, by the association between highly aggressive MTC, PHEO, ganglioneuromatosis, and mucosal neuroma; and (iii) familial MTC (FMTC), by isolated MTC without other endocrine neoplasms (3,4). Almost always, MTC is the first manifestation to develop in these MEN2 subtypes, and the most common cause of death is due to extensive regional and/or distal metastases. Once established, it spreads early and is resistant to both chemotherapy and radiotherapy. Therefore, the only therapeutic method is total thyroidectomy, ideally before the development of MTC, or at least when the MTC is still localized within the thyroid capsule (5 –7).

The American Thyroid Association (ATA) categorizes almost all known RET variants into four levels (ATA-A, -B, -C, and -D) based on the different risks of development and aggressiveness of MTC. The majority of the reported mutations occur both in MEN2A and FMTC, except for the most aggressive and MEN2B-specific mutations of codons 883 and 918 (level D) (8). Mutations detected in one of the six cysteine codons (634 [level C]; 609, 611, 618, 620, 630 [level B]) account for 98% of classic MEN2A cases, with the most frequent mutations clustering to codon 634. Noncysteine codons (768, 790, 791, 804, 891; level A) are more frequently reported in FMTC. Rare mutations such as p.V292M are not yet included in the guidelines due to their recent identification (9). An excellent prognosis is associated with the early identification of MTC (8). All patients with a clinical suspicion of MTC should be screened for the presence of a RET mutation, and if positive, molecular screening of the family members should be performed. This offers the chance of codon-directed timing of prophylactic surgery and cure of MTC (8). As the second most common neoplasm to develop in MEN2, the majority of MEN2-related PHEOs are intra-adrenal and benign (10,11). Cortical-sparing adrenalectomy to preserve adrenocortical function is the first choice of approach in surgical treatment (12). Considering the risk of a catecholamine crisis, the coexisting PHEO should be removed before thyroidectomy (10,11).

Before the present work, the available published data on the rate, distribution, mutation analysis, and clinicopathological features of MEN2 among the Chinese population were very limited (13 –15). This relatively uncommon thyroid malignancy remains underdiagnosed and inadequately managed in China. Unlike previous publications, since most patients from emerging countries present with the MEN2A phenotype and have an RET gene test for confirmation rather than for screening, our study focused on systematic RET screening in patients with MTC and their at-risk relatives. In the present study, we analyzed a cohort of 68 patients from 11 independent MEN2 families, and investigated the prevalence of RET mutations and the genotype–phenotype correlation.

Methods

Participants

This study was based on patients with a diagnosis of MTC managed in our institute between April 1975 and August 2011. The cohort included 36 index cases and 134 relatives from 11 unrelated families with MEN2 (Fig. 1). Of these, two pedigrees have been included in a previous study that described a p.C634Y/V292M/R67H/R982C compound mutation in a patient who, by chance, harbored mutations of both paternal (p.V292M/R67H/R982C) and maternal (p.C634Y) origin (16). Clinical profiles, surgical procedures, preoperative and postoperative biochemical data, and follow-up records were obtained. A total of 165 samples were available for genetic analysis, which began in 2010. All patients and/or their legal guardians gave written informed consent, as required by the institution's Ethics Committee.

FIG. 1.

Genealogy of seven MEN 2A families with RET codon 634 mutations. □, male; ◯, female; ■/●, MTC-affected patients; ▲, pheochromocytoma-affected patients; ⋆, patients with distant metastasis; /, deceased; ⊙, suspected mutation carrier; F1-III1 (), male patient with p.V292M. MTC, medullary thyroid carcinoma.

Clinical approach

MEN2A/FMTC was clinically evaluated following the criteria recommended by the Consensus of the International Multiple Endocrine Neoplasia Workshop (4). The following parameters were studied: clinical and diagnostic data (age, gender, and clinical features), biochemical markers (serum calcitonin [Ct], carcinoembryonic antigen, parathyroid hormone, serum, and/or 24-h urinary determination of catecholamines), metaiodobenzylguanidine, Doppler ultrasound (US), computerized tomography (CT), emission CT, and magnetic resonance imaging.

RET mutation analysis

Genomic DNA was isolated from the peripheral blood of all family members. The entire coding region of RET was amplified and sequenced in both sense and antisense directions with the ABI Prism 377 automatic sequencer (Perkin-Elmer) (16).

Surgical treatment

Our strategy since 2005 has been to perform total thyroidectomy and prophylactic level VI compartmental dissection for patients who were beyond the age recommended by the ATA guidelines (8). Total thyroidectomy with modified bilateral/unilateral neck dissection was performed when a suspicious lymph node was present during surgery or on preoperative imaging. However, almost all patients who underwent operations before 2005 received less-than optimal initial surgical treatment, and most did not undergo normative lymph node dissection. Surgery with unilateral/bilateral adrenal cortical sparing or total adrenalectomy was arranged to precede surgery for MTC. Before adrenalectomy, all patients received pharmacotherapy (α-blockers with or without β-blockers and adrenergic antagonists). The clinical features of HPT and other endocrine neoplasms were also evaluated. Diagnosis of MTC or C cell hyperplasia (CCH) was further confirmed by histopathology. Tumor staging was performed according to the current American Joint Committee on Cancer (AJCC) 6th edition TNM classification system (17).

Follow-up management

All patients were followed-up for evaluation of tumor recurrence and metastasis after surgery. Since 2005, the standard follow-up for MTC has consisted of determining basal Ct, plasma calcium (every 6 months), and abdominal and chest CT (yearly).

Statistical analysis

All data were analyzed with SPSS version 12.0. We carried out univariate analysis using the χ ² test or Fisher's exact test. Student's t-test was used to analyze differences in mean values.

Results

Clinical and diagnostic data

Seven families were found to have MEN2A (45 individuals), 2 with FMTC (20 individuals), and 2 as unclassified hereditary MTC because of the limited number (3 individuals) (Table 1). RET mutations were identified in 31 of 36 index cases and 32 of 134 relatives. Blood samples were not available for the remaining five cases (F2-II6, F3-III3, F4-II3, F4-II6, and F4-III2), who were obligate carriers, because they died before the introduction of genetic testing. The average age at first diagnosis in the 68 patients was 34 years (range, 4–78). The index patients with clinical disease at diagnosis were markedly older than the mutation carriers (39.6 [13–78] vs. 28.5 [4–70] years, p<0.05].

Table 1.

Clinical and Molecular Features of 68 MEN2 Patients

Family	Number of patients	Sex ratio (M:F)	Age range (years)	RET mutation	MTC, age at onset (mean years)	PHEO, n; age at diagnosis (mean years)
MEN2A
F1	7	4:3	24–56	p.C634Y	33	3; 45.7
F2	6	4:2	19–51	p.C634Y	34.3	4; 31.5
F3	9	6:3	34–65	p.C634Y	32	2; 54
F4	8	3:3	8–80	p.C634Y	35.4	6; 44.2
F5	5	3:2	6.5–64	p.C634Y	29.9	3; 43
F6	4	0:4	5.5–54	p.C634Y	24.1	2; 42.5
F7	6	4:2	19–65	p.C634R	33.5	1; 21
FMTC
F8	7	5:2	19–75	p.V292M	44.6	0; 0
F9	13	2:11	4–67	p.C618S	32.8	0; 0
Unclassified
F10	1	0:1	33	p.C634Y	33	0; 0
F11	2	0:2	27–52	p.L790F	39.5	0; 0

MEN2, multiple endocrine neoplasia type 2; MTC, medullary thyroid carcinoma; PHEO, pheochromocytoma; US, ultrasound.

Among the 32 carriers, 4 (12.5%) presented with an MTC phenotype (a palpable thyroid nodule and/or lymph node enlargement, neck pain, a palpable neck mass, and/or diarrhea resulting from hypercalcitoninemia), whereas 28 (87.5%) did not show overt symptoms (asymptomatic carriers).

The manifestations of PHEO were found in 21 individuals with diverse penetrance (including F2-II6, F3-III3, F4-II3, F4-II6, and F4-III2), distributed in all 7 MEN2A families (Fig. 1). Based on the presentation of hypertension and/or headache, palpitations, and sweating, PHEO was diagnosed in 13 patients, and physical examination or familial screening disclosed PHEO in 8 others (asymptomatic).

There was no clinical or biochemical evidence of HPT in any of the carriers. We also excluded the possibilities of both Hirschsprung disease and cutaneous lichen amyloidosis based on the absence of intestinal obstruction and pruritic lichenoid skin lesions located on the upper back, respectively.

RET germline variants

With one exception, each of 63 RET mutation-positive cases harbored one of 5 heterozygous mis-sense mutations within exons 5 (p.V292M, n=7), 10 (p.C618S, n=13), 11 (p.C634Y, n=34 and p.C634R, n=6), and 13 (p.L790F, n=2). The exception was a 23-year-old woman (F1-IV1) who had a compound mutation of p.C634Y/V292M/R67H/R982C inherited from her mother (p.C634Y) and p.V292M/R67H/R982C inherited from her father (16).

The most prevalent mutation, at codon 634, was seen in 41 cases (65.1%) (Table 2). As the major component of our cohort, patients with codon 634 mutation benefited greatly from genetic screening with significantly younger age at surgery and less-aggressive tumor behavior compared with the index cases (age: 23.5 vs. 38.8 years, p<0.05; size: 1.0 vs. 3.3 cm, p<0.05; pN0: 10/14 vs. 0/24, p<0.05). However, patients with p.V292M did not seem to benefit so much due to the uncommonly late age of onset and decades of indolent disease. The operated patients with p.C634R (n=6, mean age: 33.5 years) had no significantly more advanced disease (stage III+IV or IV) than those with p.C634Y (n=32, mean age: 33.9 years), and similar ages at diagnosis (4/6 vs. 24/32, p>0.05; 4/6 vs. 16/32, p>0.05).

Table 2.

Characteristics and Histopathological Findings of Medullary Thyroid Carcinoma Stratified by RET Genotype

	Level C			Level B	Level A	Unclassified
	p.C634Y+p.C634R (IC:MC)	p.C634Y(IC:MC)	p.C634R(IC:MC)	p.C618S(IC:MC)	p.L790F(IC:MC)	p.V292M(IC:MC)
Number of patients	40 (24:16)	34 (21:13)	6 (3:3)	13 (5:8)	2 (1:1)	7
Number of patients operated	38 (24:14)	32 (21:11)	6 (3:3)	7 (5:2)	2 (1:1)	2
Age at surgery (years, mean)	33.7 (42.8:22.3)	33.9 (38.9:24.4)	33.5 (46.7:20.3)	43.1 (43.2:26.8)	39.5 (52:27)	44.6
Tumor diameter (cm, mean)	2.4 (3.3:0.9)	2.4 (2.83:0.87)	2.4 (3.8:0.93)	3.0 (3.46:1.75)	1.5 (3.0:1.0)	CCH, 1
N0 (n)	10/38 (0/24:10/14)	8/32 (0:8/11)	2/6 (0:2/3)	0 (0:0)	1/2 (0:1/1)	2/2
N1 (n)	28/38 (24/24:4/14)	24/32 (21/21:3/11)	4/6 (3/3:1/3)	7/7 (5/5:2/2)	1/2 (1/1:0)	0
M1 (n)	4/38 (4/24:0/14)	2/32 (2/21:0)	1/6 (1/3:0)	1/7 (1/5:0)	0	0
Stage of disease
Stage I (n)	10/38	8/32	2/6	0	1/1	MTC, 1; CCH, 1
Stage II (n)	0	0	0	0	0	0
Stage III (n)	8/38	8/32	0	3/7	0	0
Stage IV (n)	20/38	16/32	4/6	4/7	1/1	0

IC, index case; MC, mutation carrier; CCH, C cell hyperplasia.

Surgical outcome and follow-up

A total of 50 MTC patients (31 index cases and 19 carriers) were managed during the study period. The median age at thyroidectomy was 35.9 years (range, 5.5–78). None had been operated on at an age recommended by the guidelines (8). Among these, 11 index patients (22%) had inadequate primary thyroid surgery that had been performed at least 8 years previously (range, 8–36), and were recently referred to our institute for recurrence of MTC (Table 3). Four of them underwent reoperation of the residual thyroid, except for F5-III1 who rejected lymph node dissection; the other seven individuals are still awaiting continued management. Notably, F1-III2 and F7-II6 had even gone through four thyroid operations, and both finally developed advanced disease. The remaining 20 index patients and 4 symptomatic carriers underwent a curative thyroidectomy with lymphadenectomy of the central and two lateral compartments (since 2005). The only exception was a patient with p.C618S who refused a further total left thyroidectomy and bilateral neck dissection. Fifteen of the 28 asymptomatic carriers underwent a prophylactic thyroidectomy, with at least level VI lymph node dissection performed in 13 individuals (one CCH patient was excluded for lymph node dissection). Modified left/right neck dissection was supplemented in F6-II4 and F7-III4 based on slightly enlarged cervical lymph nodes disclosed by US examination. Postoperative examination did not reveal any recurrent laryngeal nerve damage or hypocalcaemia.

Table 3.

Clinical Profiles of Patients with Thyroid Surgery More Than 8 Years Previously

		Primary surgery				Re-operation (last time)
								Pre-US
Patient	RET mutation	Sex/age (years)	Tumor size (cm), L/R	Operative procedure	pTNM	Age	Pre-Ct (ng/L)	Tumor size (cm), L/R	LNM	Operative procedure	pTNM	Post-Ct (ng/L)	Interval time (years)
F1-III2	p.C634Y	F/28	3.5/1.1	S-TT+MLND	T₂N₁M₀	43	>2000	0.5/0.5	Yes	B-TT(residual)+MLND^a	T₂N₁M₁ ^b	1538	16
F1-IV1	p.C634Y	F/13	1.5/3.9	N-TT+MLND	T₂N₁M₀	22	143	No tumor	No	Awaiting re-operation	—	—	8
F2-III2	p.C634Y	F/37	3.0/1.3	N-TT+NLND	T₂N₁M₀	47	114	No tumor	Yes	Awaiting re-operation^a	—	—	10
F3-III9	p.C634Y	F/29	3.5/—	L-ST+NLND	T₂N₁M₀	65	890	No tumor	Yes	Awaiting re-operation	—	—	36
F3-III10	p.C634Y	M/40	3.7/0.3	L-TT+NLND	T₂N₁M₀	59	635	—/1.1	Yes	Awaiting re-operation^a	—	—	19
F3-IV9	p.C634Y	F/28	3.1/0.6	L-TT+NLND	T₂N₁M₀	39	478	—/1.3	Yes	Awaiting re-operation	—	—	11
F3-IV10	p.C634Y	M/30	0.3/2.1	R-TT+NLND	T₂N₁M₀	38	331	1.0/—	Yes	Awaiting re-operation	—	—	9
F3-IV11	p.C634Y	M/25	—/2.2	R-TT+NLND	T₂N₁M₀	34	623	1.5/—	Yes	Awaiting re-operation	—	—	9
F5-III1	p.C634Y	M/31	0.5/2.5	R-TT+NLND	T₂N₁M₀	44	—	1.7/—	Yes	L-TT^a	T₂N₁M₀	—	13
F6-I2	p.C634Y	F/32	0.5/2.5	R-TT+NLND	T₂N₁M₀	54	366	1.5/—	Yes	L-TT+L-MLND^a	T₂N₁M₀	3.4	22
F7-II6	p.C634R	M/28	1.2/5.2	R-TT+R-LND	T₃N₁M₀	40	1895	3.8/—	Yes	L-TT+L-LND	T₃N₁M₁ ^b	933	12

F, female; M, male; L, left; R, right; TT, total thyroidectomy; ST, subtotal thyroidectomy; MLND, modified lymph node dissection; NLND, nonstandard lymph node dissection; normal Ct (basal serum Ct), male <8.4 and female <5.0; LND, lymph node dissection; LNM, lymph node metastasis; Ct, calcitonin.

Patients with PHEO.

Patients with distant metastasis.

However, in 15 asymptomatic individuals who underwent prophylactic thyroidectomy, 12 developed node-negative MTC with sizes varying from 0.2 to 1.3 cm. The presentations of the other three cases were (i) Case 1 (F1-III1, 45 years, p.V292M/R67H/R982C) had only bilateral CCH at histology (16); (ii) Case 2 (F1-IV3, 25 years, p.C634Y, bilateral MTC: 0.8 and 1.1 cm, stage IVA) developed adjacent fibroadipose tissue metastasis, but no lymph node invasion was identified; and (iii) Case 3 (F7-III4, 21 years, p.C634R, stage IVA) developed fibroadipose tissue invasion with extensive lymph node metastases. Notably, a 1.2-cm multifocal MTC in F4-II8 (50 years, p.C634Y) was identified in the left thyroid lobe, but only CCH was noted in the right lobe. In addition, in F1-IV6 (24 years), a 0.2-cm papillary thyroid carcinoma (PTC) with positive immunostaining for thyroglobulin and negative for Ct was present in the right lobe, while an MTC (0.5 cm, pT1) with intense positive immunostaining for Ct and negative immunostaining for thyroglobulin was identified in the left lobe.

Statistically significant younger ages occurred with respect to the mean age at surgery and tumor diameter in mutation carriers, and there was a significant decrease in the proportions of class C genotype, node metastases, distant metastases, and inappropriate primary thyroid surgery, whereas disease-free survival increased (Table 4).

Table 4.

Characteristics of Index-Case and Mutation-Carrier Groups

	Total (n=63)	Index cases (n=31)	Mutation carriers (n=32)
	n (%)	n (%)	n (%)	p
Female patients	35 (55.6%)	17 (54.8%)	18 (56.3%)	0.091^a
Genotype risk level
Unclassified (p.V292M)	7 (11.1%)	0 (0)	7 (21.9%)
Class A (p.L790F)	2 (3.2%)	1 (3.2%)	1 (3.1%)
Class B (p.C618S)	13 (20.6%)	5 (16.1%)	8 (25%)
Class C (p.C634)	41 (65.1%)	25 (80.6%)	16 (50%)	0.011^a
Surgery
Numbers of patients operated (n)	50	31	19
Age at surgery, mean (years)	35.7	39.9	29.3	t=2.535, 0.015
TT with central and lateral LND	28 (56%)	22 (71%)	6 (31.6%)
TT with central LND	12 (24%)	0 (0)	12 (63.2%)
TT without LND	1 (2%)	0 (0)	1 (5.3%)
Inappropriate	9 (18%)	9 (29%)	0 (0)	0.008^b
Histology
Tumor diameter (cm, mean)	2.4	3.3	1.1	t=6.805, 0.000
CCH or μ-MTC	10 (20%)	0 (0)	10 (52.6%)	0.000^b
MTC	40 (80%)	31 (100%)	9 (47.4%)
Lymph node metastasis
N₀	13 (26%)	0 (0)	13 (68.4%)	0.000^b
N₁	37 (74%)	31 (100%)	6 (31.6%)
Distant metastasis
M₀	46 (92%)	27 (87.1%)	19 (100%)	0.284^b
M₁	4 (8%)	4 (12.9%)	0 (0)
Disease-free survival	37 (74%)	18 (58.1%)	19 (100%)	0.001^b

p-Value determined using ^aPearson χ ² or ^bFisher's exact test.

Notably, the four index cases had distant metastases: F1-III2 (43 years, bone metastasis) at diagnosis, F4-I1 (80 years, liver and lung metastasis), F7-II6 (40 years, bone metastasis), and a 58-year-old woman with p.C618S (bone metastasis). F4-I1 died 8 months later because of metastatic MTC.

All patients received lifelong L-thyroxine replacement after total thyroidectomy, and were followed regularly. The median follow-up period for MTC was 5.6 years (range, 0.5–36). At the time of the last visit, 13/50 (26%) had developed recurrent disease, including 9 inadequately managed patients who were awaiting or had rejected reoperation, as well as 4 with distant metastases. None who had normal preoperative Ct levels developed persistent or recurrent disease during the study period, according to the postoperative Ct levels and imaging examination.

PHEO was found in 21 MEN2A cases (46.7%; mean age 40.8 years; range, 18–78) (Table 5). Five PHEO-specific mortality cases were diagnosed only by clinical profiles and details of surgical procedures; F3-III3, F4-II3 F4-II6, and F4-III2 died of a hypertensive crisis during the thyroid surgery, and F2-II6 died during appendicitis surgery. A surgical approach was carried out in the remaining 16 cases, laparoscopy in 11 (8 bilateral and 3 unilateral), and open surgery in 5 (4 bilateral and 1 unilateral). Bilateral PHEOs were found at initial presentation in 12 of the 16 patients, and a total bilateral adrenalectomy was performed in 5 as a result of a large and/or multifocal PHEO. Two patients with unilateral resection who relapsed initially developed contralateral tumor recurrence after 5 and 17 years. F2-IV4 (man, 18 years) and F7-III2 (man, 21 years) did not show any clinical evidence of ipsilateral or contralateral tumor recurrence 2 and 15 months after the primary unilateral adrenalectomy, respectively. Notably, neither of their fathers (p.C634Y and p.C634R; 48 and 49 years) has presented evidence of PHEO to date. Bilateral multifocal PHEOs (2–5 tumors/unilateral) were found in 14 patients. The mean maximum size was 4.8 cm (range, 2.7–11). No extra-adrenal PHEOs were found. No operative or postoperative deaths occurred. Nine patients required steroid replacement therapy: five synchronous and one metachronous total bilateral adrenalectomy, as well as three bilateral adrenal cortical sparing. None of the 14 patients with bilateral adrenalectomy developed adrenocortical insufficiency or an Addisonian crisis during the observation period (mean time, 3.8 years; range, 0.3–17).

Table 5.

Clinical and Pathological Features of 21 MEN 2A Cases with Pheochromocytoma

Patient	Sex/age (years)	RET mutation	Age (years), at diagnosis, first PHEO	Temporal relation of PHEO to MTC	Tumor characteristics (L/R size, cm)	Lag-time (years), unilateral PHEO to contralateral PHEO	Operative procedure	Follow-up
F1-III2	F/43	p.C634Y	43	Succeeding	Bilateral 1.5/3.0	—	Synchronous, subtotal	Disease-free
F1-III4	M/41	p.C634Y	41	Concomitant	Bilateral 1.5/3.0	—	Synchronous, subtotal	Disease-free
F1-III10	M/51	p.C634Y	45	Preceding	Bilateral 1.5/3.5	5	Metachronous, subtotal	Disease-free
F2-II6	F/51	p.C634Y	51	No specimen	No specimen	—	Appendicitis surgery	Died
F2-III2	F/48	p.C634Y	28	Preceding	Bilateral 7.5/11	17	Metachronous, total	SR
F2-III5	M/38	p.C634Y	28	Preceding	Bilateral 3.2/4.6	—	Synchronous, subtotal	SR
F2-IV4	M/19	p.C634Y	18	Concomitant	Unilateral —/5.5	No contralateral PHEO	Subtotal	Disease-free
F3-III3	F/49	p.C634Y	49	No specimen	No specimen	—	Thyroid surgery	Died
F3-III10	M/59	p.C634Y	59	Succeeding	Bilateral 8.0/5.5	—	Synchronous, total	SR
F4-I1	M/80	p.C634Y	78	Concomitant	Bilateral 3.0/3.4	—	Synchronous, subtotal	Died of MTC
F4-II3	F/33	p.C634Y	33	No specimen	No specimen	—	Thyroid surgery	Died
F4-II4	F/33	p.C634Y	33	No specimen	No specimen	—	Thyroid surgery	Died
F4-II8	M/43	p.C634Y	42	Succeeding	Bilateral 2.2/2.8	—	Synchronous, subtotal	Disease-free
F4-III2	F/33	p.C634Y	33	No specimen	No specimen	—	Thyroid surgery	Died
F4-III3	M/36	p.C634Y	35	Concomitant	Bilateral 2.8/2.8	—	Synchronous, total	SR
F5-II5	M/64	p.C634Y	56	Preceding	Bilateral 6.0/5.5	—	Synchronous, total	SR
F5-III1	M/42	p.C634Y	38	Succeeding	Bilateral 2.0/6.0	—	Synchronous, subtotal	SR
F5-III4	M/36	p.C634Y	35	Concomitant	Bilateral 6.0/5.2	—	Synchronous, total	SR
F6-I2	F/54	p.C634Y	54	Succeeding	Bilateral 3.6/1.1	—	Synchronous, total	SR
F6-II2	F/33	p.C634Y	31	Concomitant	Bilateral 5.0/3.8	—	Synchronous, subtotal	SR
F7-III2	M/21	p.C634R	21	Concomitant	Unilateral 3.5/—	No contralateral PHEO	Subtotal	Disease-free

SR, steroid replacement.

Discussion

MEN2 exhibits nearly 100% penetrance for MTC, which can be lethal if inappropriately treated (18 –20). In MEN2A, early thyroidectomy reduces the cancer mortality rate from 15% to 20% to <5% (4,18). Since 1993, with the discovery of the pathogenic role of RET in MEN2, DNA-based strategies have gained worldwide acceptance, leading to early diagnosis and timely surgical intervention in mutation carriers at risk of developing aggressive MTC, and eventually promoting survival (1,2). Numerous International Mutation Consortium analyses have been carried out in different ethnic lines by focusing on the genotype–phenotype correlation and management of MEN2 (21 –27).

Here, we found a significant inter- or intrafamilial heterogeneity in the disease spectrum, age at onset, and tumor behavior in Chinese patients with MEN2. The median age at diagnosis of MTC was 34 years, consistent with the global data (∼30 for hereditary MTC) (28). The most frequent phenotype was MEN2A with codon 634 mutations, accounting for 66.2% (45/68). Contrary to previous studies, patients with p.C634R did not seem to have a more aggressive tumor (development of tumor extending beyond the thyroid capsule or with distant metastases) than those with p.C634Y (29). Thirty-one index cases had clinically suspected and a heritable predisposition to MTC at the time of molecular diagnosis, and we confirmed the diagnosis of MEN2 by systematic gene screening of all relevant RET exons. This enabled us to unequivocally determine the carrier status of the rest of the families, and 32 at-risk relatives were documented, of whom most (87.5%, 28/32) were unaware of their condition. What is more is that the rigorous approach utilized here resulted in the discovery of infrequent mutations such as ATA-B (p.C618S), ATA-A (p.L790F), and p.V292M mutations in Chinese patients.

We also evaluated the histological and tumor stages of 50 patients who underwent total or partial thyroidectomy, including 11 who received inappropriate treatments for more than 8 years. Most patients having unilateral or less-than total thyroidectomy developed contralateral or bilateral recurrences soon after the primary surgery. Two patients even suffered from painful bone metastases. This highlights the importance of establishing consensus treatment guidelines and improving the management of MTC in China (8). The mean age at thyroidectomy was 35.9 years, ranging from 5.5 to 78. As expected, the thyroid tumor size and TNM stages in the group of carriers were significantly different from those in the index patients. In addition to four index patients who presented with distant metastases of MTC, nine index patients developed recurrent disease at follow-up. These results are most likely explained by the age differences between the groups (30). Our result is also concordant with the observation that older age at surgery is associated with an increased risk of advanced and persistent disease (4,31). Moreover, previous studies indicated that lymph node metastases are uncommon in young patients with MEN2A (32,33). Thus, delayed diagnosis could greatly jeopardize the final outcome in patients, especially those with high-level mutations.

Surprisingly, a rare occurrence of fibroadipose tissue invasion without lymph node involvement was identified in F1-IV3 (25 years, p.C634Y). An explanation for this phenomenon might be micrometastases of cervical lymph nodes, which were undetectable by conventional histology and immunohistochemistry (34). Lymph node micrometastases are detected frequently in PTC and MTC, but rarely in follicular thyroid carcinoma, and are associated with increased rates of locoregional recurrence and, in some reports, decreased survival (35). Moreover, the simultaneous occurrence of MTC and papillary thyroid microcarcinoma in the same thyroid gland was documented in F1-IV6 (24 years, p.C634Y). Although MTC with concurrent PTC was described in 9.1% of patients with mutations in exons 13/14, this has rarely been reported in patients with cysteine mutations (36,37). Recent reports suggested that the simultaneous occurrence of these distinct tumors in the same thyroid might be a fortuitous association (38).

MEN2A displays an age-related penetrance of PHEO with variable expressivity (1 –3). In our series, PHEO was diagnosed in 21 of 45 cases (46.7%) with MEN2A. Fourteen cases (66.7%) were bilateral, which is concordant with the reported frequency (35%–80%) (10,11,39). Two of four unilaterally treated cases developed another PHEO in the contralateral adrenal with lag times of 5 and 17 years; unilateral PHEO was diagnosed in the other two cases (≤25 years), who were relatively young for PHEO occurrence, while neither of their mutation-carrier fathers with MTC had clinical or biochemical evidence of PHEO. Genetic anticipation or clinical heterogeneity is suggested for this condition. Our finding also implied that PHEO may occur by the accumulation of genetic changes that have a dominant effect on the mutant allele, either through errors in chromosomal replication during cell division or other mechanisms such as a second somatic mutation or modifying factors (40). Lifelong steroid replacement therapy was needed in nine patients with unavoidable bilateral total or near-total adrenalectomy due to the multifocal distribution of tumors or large tumor size. By careful reviewing of patient records and family history, PHEO-specific death was noted in five family members, demonstrating that sudden death from PHEO was frequent before the recognition of MEN2, perhaps as frequent as death from progression of MTC. In fact, improved recognition and management of PHEO has decreased the rate of premature death in MEN2A even more than that of MTC (4).

In conclusion, our findings emphasize the importance of systematic molecular screening for RET in at-risk patients and their families due to the importance of early intervention in the management of MTC and screening for PHEO. The comprehensive care of identified families and patients should also include appropriate genetic counseling and long-term surveillance. Only by this, we can expect significant improvements in the treatment of MEN2 patients in China.

Footnotes

Acknowledgments

The authors thank all the patients and their families who agreed to participate in this study. This work was supported by the Key Scientific Research Project of Nanjing Military Command, China (09Z038 and 10Z036).

Disclosure Statement

The authors declare that no competing financial interests exist.

References

Mulligan

, Kwok

, Healey

, Elsdon

, Eng

, Gardner

, Love

, Mole

, Moore

, Papi

, Ponder

, Telenius

, Tunnacliffe

, Ponder

BAJ

. 1993. Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature, 363:458–460.

Donis-Keller

, Dou

, Chi

, Carlson

, Toshima

, Lairmore

, Howe

, Moley

, Goodfellow

, Wells

Jr . 1993. Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC. Hum Mol Genet, 2:851–856.

Raue

, Frank-Raue

, Grauer

. 1994. Multiple endocrine neoplasia type 2. Clinical features and screening. Endocrinol Metab Clin North Am, 23:137–156.

Brandi

, Gagel

, Angeli

, Bilezikian

, Beck-Peccoz

, Bordi

, Conte-Devolx

, Falchetti

, Gheri

, Libroia

, Lips

, Lombardi

, Mannelli

, Pacini

, Ponder

, Raue

, Skogseid

, Tamburrano

, Thakker

, Thompson

, Tomassetti

, Tonelli

, Wells

Jr , Marx

. 2001. Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab, 86:5658–5671.

LiVolsi

. 1997. C cell hyperplasia/neoplasia. J Clin Endocrinol Metab, 82:39–41.

You

, Lakhani

, Wells

Jr . 2007. New directions in the treatment of thyroid cancer. J Am Coll Surg, 205:S45–S48.

Higgins

, Forastiere

, Marur

. 2009. New directions in the systemic treatment of metastatic thyroid cancer. Oncology (Williston Park), 23:768–775.

Kloos

, Eng

, Evans

, Francis

, Gagel

, Gharib

, Moley

, Pacini

, Ringel

, Schlumberger

, Wells

Jr . 2009. Medullary thyroid cancer: management guidelines of the American Thyroid Association. Thyroid, 19:565–612.

Castellone

, Verrienti

, Magendra Rao

, Sponziello

, Fabbro

, Muthu

, Durante

, Maranghi

, Damante

, Pizzolitto

, Costante

, Russo

, Santoro

, Filetti

. 2010. A novel de novo germ-line V292M mutation in the extracellular region of RET in a patient with phaeochromocytoma and medullary thyroid carcinoma: functional characterization. Clin Endocrinol (Oxf ), 73:529–534.

10.

Quayle

, Fialkowski

, Benveniste

, Moley

. 2007. Pheochromocytoma penetrance varies by RET mutation in MEN 2A. Surgery, 142:800–805discussion 805 e801.

11.

Ilias

, Pacak

. 2009. Diagnosis, localization and treatment of pheochromocytoma in MEN 2 syndrome. Endocr Regul, 43:89–93.

12.

Asari

, Scheuba

, Kaczirek

, Niederle

. 2006. Estimated risk of pheochromocytoma recurrence after adrenal-sparing surgery in patients with multiple endocrine neoplasia type 2A. Arch Surg, 141:1199–1205discussion 1205.

13.

, Wat

, Lam

, Tiu

, Chan

, Lam

. 2003. Multiple endocrine neoplasia type 2A in Chinese families. Clin Endocrinol (Oxf ), 58:528.

14.

Lau

, Lang

, Lo

, Tso

, Garcia-Barcelo

, Tam

, Lam

. 2009. Prophylactic thyroidectomy in ethnic Chinese patients with multiple endocrine neoplasia type 2A syndrome after the introduction of genetic testing. Hong Kong Med J, 15:326–331.

15.

Zhou

, Zhao

, Cui

, Gu

, Zhu

, Li

, Liu

, Yin

, Zhao

, Yin

, Yu

, Chen

, Wang

, Xiao

, Hong

, Zhang

, Tang

, Wang

, Li

, Ning

. 2007. RET proto-oncogene mutations are restricted to codons 634 and 918 in mainland Chinese families with MEN2A and MEN2B. Clin Endocrinol (Oxf ), 67:570–576.

16.

, Ma

, Du

, Ying

, Fei

, Jin

, Han

, Wang

, Chen

, Liu

, Lu

, Zhang

. 2011. RET germline mutations identified by exome sequencing in a Chinese multiple endocrine neoplasia type 2A/familial medullary thyroid carcinoma family. PLoS One, 6:e20353.

17.

Greene

, Page

, Fleming

, Fritz

, Balch

. 2003. AJCC Cancer Staging Manual, 6th. Springer Verlag: Chicago, IL.

18.

Kakudo

, Carney

, Sizemore

. 1985. Medullary carcinoma of thyroid. Biologic behavior of the sporadic and familial neoplasm. Cancer, 55:2818–2821.

19.

Pelizzo

, Boschin

, Bernante

, Toniato

, Piotto

, Pagetta

, Nibale

, Rampin

, Muzzio

, Rubello

. 2007. Natural history, diagnosis, treatment and outcome of medullary thyroid cancer: 37 years experience on 157 patients. Eur J Surg Oncol, 33:493–497.

20.

Paszko

, Sromek

, Czetwertynska

, Skasko

, Czapczak

, Wisniewska

, Prokurat

, Chrupek

, Jagielska

, Kozlowicz-Gudzinska

. 2007. The occurrence and the type of germline mutations in the RET gene in patients with medullary thyroid carcinoma and their unaffected kindred's from Central Poland. Cancer Invest, 25:742–749.

21.

Romei

, Mariotti

, Fugazzola

, Taccaliti

, Pacini

, Opocher

, Mian

, Castellano

, degli Uberti

, Ceccherini

, Cremonini

, Seregni

, Orlandi

, Ferolla

, Puxeddu

, Giorgino

, Colao

, Loli

, Bondi

, Cosci

, Bottici

, Cappai

, Pinna

, Persani

, Verga

, Boscaro

, Castagna

, Cappelli

, Zatelli

, Faggiano

, Francia

, Brandi

, Falchetti

, Pinchera

, Elisei

. 2010. Multiple endocrine neoplasia type 2 syndromes (MEN 2): results from the ItaMEN network analysis on the prevalence of different genotypes and phenotypes. Eur J Endocrinol, 163:301–308.

22.

Eng

, Clayton

, Schuffenecker

, Lenoir

, Cote

, Gagel

, van Amstel

, Lips

, Nishisho

, Takai

, Marsh

, Robinson

, Frank-Raue

, Raue

, Xue

, Noll

, Romei

, Pacini

, Fink

, Niederle

, Zedenius

, Nordenskjold

, Komminoth

, Hendy

, Mulligan

, Gharib

, Thibodean

, Lacroix

, Frilling

, Ponder

BAJ

, Mulligan

. 1996. The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2. International RET mutation consortium analysis. JAMA, 276:1575–1579.

23.

Fink

, Weinhusel

, Niederle

, Haas

. 1996. Distinction between sporadic and hereditary medullary thyroid carcinoma (MTC) by mutation analysis of the RET proto-oncogene. “Study Group Multiple Endocrine Neoplasia Austria (SMENA).”. Int J Cancer, 69:312–316.

24.

Bergant

, Hocevar

, Besic

, Glavac

, Korosec

, Caserman

. 2006. Hereditary medullary thyroid cancer in Slovenia—genotype-phenotype correlations. Wien Klin Wochenschr, 118:411–416.

25.

Machens

, Dralle

. 2008. Familial prevalence and age of RET germline mutations: implications for screening. Clin Endocrinol (Oxf ), 69:81–87.

26.

Chung

, Kim

, Min

, Lee

, Kim

, Ki

, Kim

, Chung

. 2004. RET proto-oncogene mutations are restricted to codon 634 and 618 in Korean families with multiple endocrine neoplasia 2A. Thyroid, 14:813–818.

27.

Alvandi

, Akrami

, Chiani

, Hedayati

, Nayer

, Tehrani

, Nakhjavani

, Pedram

. 2011. Molecular analysis of the RET proto-oncogene key exons in patients with medullary thyroid carcinoma: a comprehensive study of the Iranian population. Thyroid, 21:373–382.

28.

Machens

, Gimm

, Hinze

, Hoppner

, Boehm

, Dralle

. 2001. Genotype-phenotype correlations in hereditary medullary thyroid carcinoma: oncological features and biochemical properties. J Clin Endocrinol Metab, 86:1104–1109.

29.

Punales

, Graf

, Gross

, Maia

. 2003. RET codon 634 mutations in multiple endocrine neoplasia type 2: variable clinical features and clinical outcome. J Clin Endocrinol Metab, 88:2644–2649.

30.

Milos

, Frank-Raue

, Wohllk

, Maia

, Pusiol

, Patocs

, Robledo

, Biarnes

, Barontini

, Links

, de Groot

, Dvorakova

, Peczkowska

, Rybicki

, Sullivan

, Raue

, Zosin

, Eng

, Neumann

. 2008. Age-related neoplastic risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germ line RET Cys634Trp (TGC>TGG) mutation. Endocr Relat Cancer, 15:1035–1041.

31.

Schreinemakers

, Vriens

, Valk

, de Groot

, Plukker

, Bax

, Hamming

, van der Luijt

, Aronson

, Borel Rinkes

. 2010. Factors predicting outcome of total thyroidectomy in young patients with multiple endocrine neoplasia type 2: a nationwide long-term follow-up study. World J Surg, 34:852–860.

32.

Machens

, Niccoli-Sire

, Hoegel

, Frank-Raue

, van Vroonhoven

, Roeher

, Wahl

, Lamesch

, Raue

, Conte-Devolx

, Dralle

. 2003. Early malignant progression of hereditary medullary thyroid cancer. N Engl J Med, 349:1517–1525.

33.

Skinner

, Moley

, Dilley

, Owzar

, Debenedetti

, Wells

Jr . 2005. Prophylactic thyroidectomy in multiple endocrine neoplasia type 2A. N Engl J Med, 353:1105–1113.

34.

Weber

, Amann

, Weckauf

, Lacroix

, Weitz

, Schonfuss

, Holting

, Klar

, Herfarth

, von Knebel Doeberitz

. 2002. Detection of disseminated medullary thyroid carcinoma cells in cervical lymph nodes by cytokeratin 20 reverse transcription-polymerase chain reaction. World J Surg, 26:148–152.

35.

Cranshaw

, Carnaille

. 2008. Micrometastases in thyroid cancer. An important finding? Surg Oncol, 17:253–258.

36.

Brauckhoff

, Gimm

, Hinze

, Ukkat

, Brauckhoff

, Dralle

. 2002. Papillary thyroid carcinoma in patients with RET proto-oncogene germline mutation. Thyroid, 12:557–561.

37.

Machens

, Dralle

. 2012. Simultaneous medullary and papillary thyroid cancer: a novel entity? Ann Surg Oncol, 19:37–44.

38.

Kim

, Gong

, Kim

, Hong

, Bae Kim

, Shong

. 2010. Concurrent occurrence of medullary thyroid carcinoma and papillary thyroid carcinoma in the same thyroid should be considered as coincidental. Clin Endocrinol (Oxf ), 72:256–263.

39.

Rodriguez

, Balsalobre

, Ponce

, Rios

, Torregrosa

, Tebar

, Parrilla

. 2008. Pheochromocytoma in MEN 2A syndrome. Study of 54 patients. World J Surg, 32:2520–2526.

40.

Lengauer

, Kinzler

, Vogelstein

. 1998. Genetic instabilities in human cancers. Nature, 396:643–649.