Molecular diagnosis and comprehensive treatment of multiple endocrine neoplasia type 2 in Southeastern Chinese

Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant inherited endocrine malignancy syndrome. Early and normative surgery is the only curative method for MEN 2-related medullary thyroid carcinoma (MTC). In patients with adrenal pheochromocytoma, cortical-sparing adrenalectomy (CSA) can be utilized to preserve adrenocortical function. We present twenty-six of 33 MEN2 patients underwent prophylactic thyroidectomy with varying neck dissection and eight of 24 MEN2A patients with PHEO underwent adrenal-sparing surgery. Direct sequencing of entire RET exons was performed in all participants. The RET mutations (p.C634Y [n = 10], p.C634R [n = 9], p.C634F [n = 2], p.C618Y [n = 8], p.C618R [n = 3], and p.M918T [n = 1]) were confirmed in 20 symptomatic patients and identified in 13 at-risk relatives (RET carriers). Twenty-six of 33 MEN2 patients underwent thyroidectomies with neck dissections; the mean age at the time of the first thyroid surgery and the tumor diameter of the 6 RET carriers was decreased compared with 20 symptomatic patients (P < 0.001 and P = 0.007, respectively), while the disease-free survival was increased (80% vs.10%, P = 0.0001). Seven RET carriers who were declined surgery. One of 20 symptomatic patients with MTC bone metastases after surgery received vandetanib therapy for 20 months and responded well. Additionally, 8 of 24 MEN2A patients who initially had unilateral pheochromocytomas underwent CSA, 1 developed contralateral pheochromo cytomas 10 years later, then also accepted and also agreed to a CSA. None of the patients required steroid replacement therapy. Based on our results, integrated RET screening and the pre-operative calcitonin level is an excellent strategy to ensure earlier diagnosis and standard thyroidectomy. CSA can be utilized to preserve adrenocortical function in patients with pheochromocytomas.

MEN2 patients can be diagnosed early through systematic pedigree investigation and RET proto-oncogene screening [3][4][5]. In the vast majority of cases, > 70 RET mutations accumulate that are related to MEN2, and > 98% of RET mutations are located in exons 5, 8, 10, 11, and 13-16 [4]. In 2009, the American Thyroid Association (ATA) performed a timing prophylactic total thyroidectomy (TT) procedure based on a model that utilized the following genotype-phenotype correlations to stratify mutations into four risk levels: ATA-A → D, A < B < C < D. It was recommended that ATA-B and -C level mutation carriers should be considered for prophylactic TT, which should be performed before 5 years of age. However, it has been shown that family members with the same RET mutation may have different clinical phenotypes or offspring might exhibit earlier MTC phenotypes [4,5].
In recent years, combined detection of RET mutations and pre-operative calcitonin (pre-Ct) levels have been considered for comprehensive decision-making regarding the timing or surgical extent of prophylactic TT [8][9][10][11]. Moreover, phase III clinical trials have demonstrated that one oral drug (such as vandetanib), which is a small molecular multi-targeted tyrosine kinase inhibitor, provides a new therapeutic choice for advanced hereditary MTC patients whose disease cannot be resected or who present with metastatic lesions [12]. Because most of the MEN2Arelated PHEO presentations are benign with bilateral multiple onsets, it is currently preferred to perform laparoscopic cortical-sparing adrenalectomy (CSA) [5,13,14].
In the present study, we report 33 MEN2 cases derived from 9 unrelated MEN2 families residing in southeastern China. Our aim was to analyze the feasibility of prophylactic TT based on predictive integrated screening of RET and pre-Ct levels. We also observed and evaluated the efficacy and safety of vandetanib in the treatment of one advanced case of MEN2A-MTC, and determined the clinical significance of managing unilateral CSA for MEN2A-related PHEO.

Patients
Nine MEN2 families from Zhejiang Province of southeastern China were recruited. All patients were diagnosed and treated at the Zhejiang Cancer Hospital or the 117th PLA Hospital between August 1989 and January 2013. Systematic family screening was performed between 2005 and 2013, and comprehensive medical data, including clinical profiles, surgical procedures, pre-and post-operative biochemical data, and follow-up records were studied. The patients diagnosed before 1993 were submitted to RET genetic analysis at the time of family screening. All participants and/or their legal guardians provided written informed consent, as required by the institution's Ethical Committee.

Clinical approach
All RET gene mutation carriers underwent biochemical and imaging examinations to ascertain thyroid, parathyroid, and adrenal gland involvement. The biochemical examination included Ct by chemiluminescence assay (normal male, <8.4 ng/L; and female, <5.0 ng/L), parathyroid hormone (PTH) by electrochemical immunoassay, carcinoembryonic antigen by chemiluminescence assay, catecholamines, 3-methyl-4-hydroxy mandelic acid by radioimmunoassay, serum calcium by the arsenazo III method, and serum phosphate by UV molybdate. The imaging examinations included thyroid ultrasound/CT, adrenal gland and parathyroid gland nuclear magnetic resonance (MRI), emission computed tomography (ECT), and/or PET-CT scan assessments, if indicated.

Genetic testing
Genomic DNA was isolated from the peripheral blood of 86 family members. The coding region of RET exons 5, 8, 10, 11, and 13-16 was amplified and sequenced in sense and antisense directions with an ABI Prism 3730 automatic sequencer (Perkin-Elmer, USA).

Follow-up management
All patients were followed for evaluation of tumor recurrence and metastasis after surgery. Since 2005, the standard follow-up for MTC has consisted of determining the Ct level, plasma calcium level (every 6 months), and during the post-operative period under conditions in which the Ct level is abnormal, a detailed imaging examination may be considered (yearly). For MEN2Arelated PHEO patients, biochemical and imaging examinations were repeated every 6-12 months, and life-time follow-up was then required. For RET mutation carriers without PHEOs, after initial screening at 20 years of age, repeat biochemical and imaging examinations were performed every year to screen for PHEOs and HPT [5].

Statistical analysis
All data were analyzed with SPSS (version 12.0) software. We carried out univariate analysis using the Fisher's exact test. Student's t-test was used to analyze differences between mean values.
Twenty symptomatic patients were admitted to the hospital for neck masses or neck pain and diagnosed with MTC before clinical screening. Thirteen of 20 (65%) patients were subjected to sub-standard thyroid surgery: 8 patients underwent interval (at least 2 times) TT and bilateral VI region lymph node dissection [B-LND (VI)] or modified bilateral cervical lymph node dissection (MBND); 3 patients underwent bilateral sub-TT and/or MBND; and 2 patients underwent unilateral TT and B-LND (VI) with MBND. The other 7 patients were accepted for standard thyroid surgery, as follows: 5 patients underwent TT plus B-LND (VI) plus MBND; and 2 patients underwent TT plus B-LND (VI). The histopathologic results showed that 20 patients had bilateral MTC (1 patient with T2N0M0 and 19 patients with T1N1bM0~T3N1bM0; Table 2). The median time of follow-up thus far is 115.6 ± 90.4 months (range, 21-384 months). The results of post-Ct levels were normal in 2 patients (10% [T2N0M0 and T1N1bM0]) and elevated in 18 patients (90%). Moreover, 2 of 20 patients (F6-II5 and F4-II1) had elevated Ct levels > 2000 ng/L. One patient (F6-II5), a 39-year-male-old with T3N1bM0, underwent surgery successively 4 times and eventually exhibited multiple bone metastases based on ECT and MRI examinations 11 years after the initial surgery. Vandetanib (ZD6474, Zactima™) was administered orally at an initial dose of 300 mg/d; bone pain resolved and the Ct levels decreased from 8755.3 ng/L to 873.6 ng/L after 2 months. Eight months later, the patient appeared uncomfortable and distressed. An electrocardiogram showed Q-T interval prolongation and incomplete right bundle branch block. Subsequently, the dose of vandetanib was decreased from 300 mg to 200 mg once a day for 20 months (until the present). The Ct level increased to 5869.2 ng/L during therapy, while repeated PET-CT examinations failed to show any obvious advance bone metastatic lesions. The patient developed erythema after vandetanib therapy, but had no pruritus, headaches, nausea, or reduction in the number of white blood cells. The other patients (F4-II1), a 50-year-male-old with T2N1bM0, declined further therapy and has survived with the tumor in situ for 19 years (until the present; Table 2).
Thirteen asymptomatic RET carriers were confirmed by clinical family screening, 12 of whom had elevated pre-Ct levels; 6 patients underwent prophylactic TT (n = 2), prophylactic TT plus B-LND (VI; n = 3), or prophylactic TT plus MBND (n = 1). The histopathologic examination revealed MTC in 5 patients (4 patients with T1N0M0 and 1 patient with T1N1bM0) and 1 26-yearold patient with CCH (F8-IV5; Table 3). The median time of follow-up thus far has been 19.6 ± 3.2 months (range, 17-26 months) and the results of post-Ct levels were normal in 5 patients. Unfortunately, a 21-year-old male (F6-III4) with T1N1bM0 still has an elevated Ct level. In addition, the remaining 7 patients declined/ awaited surgery, 6 of whom had progressively increased Ct levels, and only a 53-year-old male (F8-III5) has a normal Ct level and no thyroid nodules based on imaging ( Table 3). The asymptomatic RET carriers who underwent surgery benefited greatly from genetic screening and were a significantly younger age at the time of surgery and had a smaller tumor diameter compared with symptomatic patients (P < 0.001 and P = 0.007, respectively). One RET carrier had lymph node metastasis compared with 19 of 20 symptomatic patients (P = 0.0001). The disease-free survival (DFS) increased in the asymptomatic RET carriers who underwent surgery compared to the symptomatic patients (80% vs. 10%, P = 0.0001; Table 2).

PHEO
Of 24 MEN2A patients, 8 presented with PHEOs, including 5 males and 3 females (7 p.C634Y/R/F and 1 p.C618R RET-mutations) ( Table 4). The mean age at the time of diagnosis was 35.8 ± 9.2 years (range, 21-47 years). Among 8 PHEO patients, 5 were symptomatic and 3 were asymptomatic. The asymptomatic patients were diagnosed during systematic pedigree analysis. In addition, all 8 PHEO patients initially presented with unilateral disease. Unilateral CSA was initially performed, and 4 of 8 patients underwent a laparoscopic technique. The average maximum diameter of the PHEOs was 3.9 ± 2.5 cm (range, 2.3-10.0 cm), and all of the PHEOs were located within the adrenal glands. One patient (F5-II1) relapsed after initially developing a contralateral tumor after 10 years, and underwent CSA on the opposite side (bilateral CSA). After surgery, none of the 8 patients required steroid replacement therapy.  symptom, but no evidence of a PHEO. Furthermore, in the F6 family, the first (I) and second (II) generation also had the only clinical manifestations of MTC, while only one patient in their offspring (III-2) had evidence of a PHEO.

Follow-up management
Seven asymptomatic RET carriers were strictly monitored and presented with gradually increasing Ct levels, but did not undergo surgery. The other 26 patients received thyroid hormone replacement therapy lifetime, and were given calcium and vitamin D3 as preventive supplements at least 1 year after surgery. Moreover, thyroid, adrenal, and parathyroid function was normal, with no evidence of complications, such as tetany.

Discussion
In this study, the rate of occurrence of MEN2-related MTC/CCH, PHEO and the mean diagnostic age was similar to previously reported data [15,16]; however, MEN2-related HPT was not identified in the current study, which was different from Zhou et al. [17] reported that the prevalence of Chinese MEN2-related HPT was 10.8%, which is also lower compared with Western countries (20%-30%) [4]. The difference may be related to regional disparities, limited sample size and racial distribution.
Post-operatively, 17 of 20 (85%) symptomatic patients presented with residual MTC, metastases, and/or a recurrence. Five of 6 (83.3%) asymptomatic RET carriers underwent prophylactic TT had no abnormalities (P < 0.05). MEN2 had an age-related penetrance and metastasis of MTC with a variable intensity of expression. For the 10% of MEN2-related MTC patients with cervical LNM who were cured by TT and extensive lymph node excision, early standard surgery based on diagnostic RET testing might distinctly improve the cure rate of MEN2related MTC [3][4][5]18,19]. In addition, even the same family has significant variability in the clinical expression of disease and development of MTC [for example, F8-III5, F8-IV5 and F8 other 6 with p.C618Y]. This may occur by the accumulation of genetic changes that have a dominant effect on the mutant allele or other affected factors [20,21].
In 2009, at the 9th meeting of the European Thyroid Association and Cancer Research Network (ETA-CRN), there were comments reported to the 2009 ATA guidelines that pointed out that timing of prophylactic TT should be at best synthetically decided by combining the results of both RET gene testing and the ongoing pre-Ct level [9]. The timing of TT in RET carriers with a negative Ct level can be personalized and safely planned when the stimulated Ct becomes positive. Intrathyroid MTCs were found when the pre-Ct level was <60 ng/L, and individualization of surgery in RET carriers with normal pre-Ct levels may be avoided B-LND (VI) [8,10]. QI et al. [11] also reported that MTC remained located  [22] demonstrated that RET carriers with normal basal Ct levels undergoing prophylactic TT after testing positive for stimulated Ct could not always be cured, particularly in 12% of all the level ATA-B/C RET mutations cases. The threshold for a definitive cure of MEN2-related MTC still needs to be empirically determined. The 7 asymptomatic RET carriers declined surgery or chose a watchful waiting approach, which offered us the unique opportunity to observe the natural history of MEN2-related MTC. Thus, it is important to increase patient awareness to avoid treatment delay. Furthermore, our observation was also similar to reports detailing the use of vandetanib for advanced genetic MTC, and the Q-T interval prolongation of drug toxicity was remarkable [12,23,24]. Moreover, in the early stages of research, the MEN2A family (F6, p.C634R) was mistaken for the FMTC family; however, it was soon shown that the F6-III2 family accompanied PHEOs. FMTC is associated with mutations in codon 634 and is most commonly identified as p. C634Y, and almost never p.C634R [25]. It has been previously suggested that it is important to use clinical screening and deeper genotype-phenotype correlations in intact pedigree analysis including long-term followup, which may avoid missed diagnoses of PHEOs and/or HPT and other endocrine adenomas besides the diagnosis of MTC (FMTC). It has also been suggested that family members with the same RET mutation may display different clinical phenotypes, and the offspring may display earlier PHEO phenotypes [4]. Our findings also  implied that MEN2-related PHEO occurrence may be affected by modifying factors from parents or the environment, and even by other mechanisms, such as errors in chromosomal replication during cell division or a second somatic mutation [4,26]. Additionally, sufficient preoperative preparation before PHEO is necessary and preferential PHEO excision favors the avoidance of burst hypertensive crisis caused by PHEO during MTC or other non-PHEO surgical procedures or even death [4,5,26]. Unilateral MEN2A-related PHEO patients usually show contralateral PHEO over a period of 10 years and malignant MEN2A-related PHEO is rare (0.4%-5.0%) [4,27,28]. Based on a multi-institutional study, Castinetti et al. [28] recently reported 563 MEN2-related PHEO patients; recurrence of PHEOs in the same adrenal gland after CSA was only reported in 4 (3%) of 153 operated glands or in 4 (4%) of 114 patients after 6-13 years. Moreover, 47 of 82 (57%) patients with bilateral PHEO who underwent CSA did not become steroid-dependent. The CSA is a feasible surgical strategy for MEN2-related PHEOs, which may avoid or postpone the need for steroid replacement therapy and the associated risk of an Addisonian crisis [14,28]. The post-operative MEN2-related PHEOs should undergo follow-up, and monitored long-term for timely diagnosis and treatment options to prevent or reduce clinical risks [4,5]. In this study, our results also showed that initially unilateral CSA was technically safe and feasible, and may reduce steroid replacement therapy after a second/ contralateral CSA. The de novo occurrence rate of MEN2A/FMTC was 5.6%-9.0% [29], while in MEN2B > 90% of cases presented with de novo germline RET mutations [30]. In the current study, in 2 (F2 [p.C634Y] and F9 [p.M918T]) of 9 families (Figure 1), only 2 probands harbored mutations; all the other consanguineous family members had no p.C634Y or p.M918T mutations, which suggested that both patients had de novo mutations or that the mutations existed in a parental germ cell mosaicism [31]. The age at which the p.M918T mutation was diagnosed in our patient was advanced compared with the mean age (13 ± 9.3 years) reported by Brauckhoff et al. [30]. Thus, awareness of the clinical relevance of nonendocrine manifestations, timely detection of RET gene and pre-Ct facilitate an early diagnosis and normalize surgery to improve the long-term outcome of MEN 2B.

Conclusions
In summary, systematic evaluation of MEN2 patients by means of integrated screening of RET and pre-Ct levels and implementation of individualized prophylactic TT favors a reduction in surgical complications and improves DFS. For advanced MTC, vandetanib provides novel treatment options. In patients with PHEOs, CSA can be utilized to preserve adrenocortical function.