Agnieszka Kotecka-Blicharz M.D., Department of Nuclear Medicine and Endocrine Oncology, Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology, Gliwice Branch, Wybrzeża Armii Krajowej 15, Gliwice, Poland, phone. 32 278 99 32, e-mail: agnbli@wp.pl

This research was supported by Polish National Science Centre grant no. NN401410639.

Introduction

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominant genetic syndrome, caused by germline mutation in RET proto-oncogene, which encodes a tyrosine kinase receptor. The RET proto-oncogene is located on chromosome 10 and consists of 21 exons. RET receptor forms together with the co-receptor, which is glial cell-derived neurotrophic factor family receptor-α-1(GFRα1), the binary complex. The RET/GFRα1 complex is activated by the ligand called glial cell-derived neurotrophic factor (GDNF) [1].

RET activation is required in the early embryos for the development of neural crest-derived cell types and the kidneys [2]. It also plays a role in growth stimulatory and anti-apoptotic functions. Single amino acid substitutions in the extracellular or intracellular domains of the receptor, which are the result of germline point mutations of RET, cause ligand-independent activation as a dimer or homodimer [3]. The most common mutations are present in a cysteine-rich domain [1].

Different RET mutations are associated with different clinical phenotype. MEN 2 syndromes may be classified into three subtypes based on their occurrence. Medullary thyroid cancer (MTC) is characteristic for familiar medullary thyroid cancer (FMTC), MEN 2A and MEN 2B syndromes, while hyperparathyroidism occurs only in MEN 2A subtype, with penetrance of 15–20% [1]. Phaeochromocytoma is observed in 50% of MEN 2A and MEN 2B cases [1]. In patients with MEN 2, phaeochromocytomas are usually benign, but the consequences of abrupt secretion of catecholamines from the tumour may be lethal [4]. Taking in to account the latter risk and nonspecific clinical manifestation of the neoplasm and the fact that it develops usually after the diagnosis of medullary thyroid cancer, it is mandatory to screen all RET proto-oncogene mutations carriers for phaeochromocytoma.

Material and methods

We studied 228 patients with proto-oncogene RET mutations, registered in the Department of Nuclear Medicine and Endocrine Oncology, Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology, Gliwice Branch.

Genetic analysis of RET proto-oncogene was performed in each patient with confirmed medullary thyroid cancer, phaeochromocytoma, and positive family history of MEN 2.

The monitoring for the diagnosis of phaeochromocytoma was carried out in all patients with established genetic status. Mean time of follow up was 138 months. Surveillance consisted of periodically performed clinical evaluation, 24-hour urinary determinations of total metanephrines complementary with imaging (CT, MR, MIBG scintigraphy). To locate phaeochromocytoma, CT and MR were used without distinguishing between the effectiveness of these modalities. The size of the tumour was assessed using imaging modalities. The diagnosis of phaeochromocytoma each time was confirmed by histopathological analysis after adrenalectomy. All patients after surgery were under surveillance toward the recurrence or contralateral phaeochromocytoma.

Results

The studied population consisted of 228 RET protooncogene mutation carriers. The following distribution of RET mutations were observed in the cohort: 84 patients with codon 634 mutation (37%), 48 patients with codon 791 mutation (21%), 22 patients with codon 918 mutation (10%), 19 patients with codon 618 mutation (8%), 15 patients with codon 620 mutation (6%), and the remaining 18% consisted of patients with mutations in codon 611, 768, 790, 804, 844, and 891.

Phaeochromocytoma developed in 41 patients (18% of all RET proto-oncogene mutation carriers), in 26 patients with codon 634 mutation (31%), in 6 patients with codon 918 mutation (27%), in 4 patients with codon 620 mutation (27%), in 2 patients with codon 618 mutation (10.5%), in 2 patients with codon 791 mutation (4%), in 1 patient with codon 611 mutation (25%).

The codon-specific penetrance of phaeochromocytoma is illustrated in Table I.

The mean age of diagnosis for the whole cohort was 43 years. The age-related occurrence of phaeochromocytoma is illustrated in Figure 1. The lowest age of diagnosis was observed in a 15-year-old patient with codon 918 mutation. The lowest age of diagnosis for the remaining codons were respectively: 20 years for codon 634, 29 years for codon 620, 30 years for codon 791, 45 years for codon 618, and 54 years for codon 611. In eight cases phaeochromocytoma was the first manifestation of MEN 2 syndrome, in 4 patients with codon 634 mutation, in 2 patients with codon 791 mutation, in 1 patient with codon 918 mutation, and in 1 with codon 620 mutation. Codon-related occurrence of phaeochromocytoma and medullary thyroid cancer in MEN 2 syndrome is illustrated in Figure 2.

Only 8 (20%) patients were symptomatic at diagnosis of phaeochromocytoma. The mean size of the tumour was 4.3 cm. There was no extra-adrenal localisation. We observed one case of malignant phaeochromo – cytoma with metastases to lymph nodes in the female patient with mutation in codon 791. The 10-cm tumour was revealed at the age of 30 years, and the metastases occurred after nine years of observation.

Of the 41 patients with phaeochromocytoma, 11 (25%) had bilateral involvement at the time of diagnosis, and 14 (34%) revealed contralateral tumour throughout the follow-up period. From the whole group of patients with bilateral tumours, most (84%) had total bilateral adrenalectomy, which resulted in the development of adrenal insufficiency. Only four patients (16%) had adrenal-sparing surgery, of which only one did not require substitution of hydrocortisone.

The clinical traits of phaeochromocytoma in MEN 2 syndrome are shown in Table II.

Discussion

In our series phaeochromocytoma developed in patients with mutations at codons 611, 618, 620, 634, and 918 of RET gene. Other studies observed phaeochromocytoma in subjects with mutations at codon: 609, 618, 620, 634, 666, 791, 804, 883, and 918 of RET gene [7–11].

The expected frequency of phaeochromocytoma in MEN 2 syndrome is up to 50% [6]. But its penetrance is highly dependent on the RET codon mutations [7, 8].

In our study in the whole cohort of RET proto-oncogene mutation carriers the overall rate of phaeochromocytoma was 18%, which is similar to that reported by Nguyen et al. [9]. However, we observed higher frequency of the neoplasm (respectively, 31% and 27%) in patients with mutations at codon 634 and 918, which is very close to that reported by Machens et al. [8].

In the study by Rodriguez et al. they registered a 32% frequency of phaeochromocytoma in a cohort of 54 patients, among which 52 had mutation located in codon 634 [10].

Quayle at al. reported an overall rate of phaeochromocytoma of 32%, with the greatest expression in codon 634 (50% positive incidence of phaeochromocytoma) [7].

The low penetrance of phaeochromocytoma in our group of patients may be due to the fact that we still have under surveillance 66 patients (29%) below the age of thirty years. Taking in to account that the peak age for revealing the phaeochromocytoma in MEN 2 syndrome is estimated around 40 years [1], we expect that more cases of phaeochromocytoma will occur in our population.

The earliest manifestation of phaeochromocytoma in our study was in a 15-year-old patient with mutation in codon 918, while mean age of diagnosis was assessed as 43 years for the whole cohort of RET proto-oncogene mutations carriers. The youngest age at diagnosis of phaeochromocytoma reported in literature was 12 years, also in a codon 918 mutation carrier [9]. But others observed early manifestation at the age of 12 years in the case of a patient with mutation in codon 634 [12]. The youngest patient age ever reported with disclosed phaeochromocytoma determines the moment of implementation of screening protocol. According to the guidelines for diagnosis and therapy of MEN 2 published in 2001 the screening for phaeochromocytoma should be implemented by the age of 5–7 years in high- and highest-risk codon (630, 634, 883, 918 RET mutation carriers). In families with mutation in lowerrisk codons screening may be initiated at a later age. The familial pattern of phaeochromocytoma should be considered during the development of a screening plan [13]. The American Thyroid Association also stratifies the risk of phaeochromocytoma by RET genotype and recommends commencement of phaeochromocytoma screening by age of 8 years for mutated RET codons 630, 634, and 918 and by age 20 years for the remainder [14].

The oldest patient in whom phaeochromocytoma was detected was 61 years old, a RET proto-oncogene mutation carrier in codon 620. In other studies the oldest age was 73 years [7]. However, it must be emphasised that age at detection does not necessarily reflect the age of onset.

Thanks to the growth of availability of genetic screening, most patients under a follow-up regimen because of detected mutation in RET proto-oncogene are asymptomatic at the time of phaeochromocytoma diagnosis.

In this study only 20% of patients presented typical symptoms. In the literature it is also emphasised that patients with MEN 2-related phaeochromocytoma often lack sustained hypertension or other symptoms [1]. But the incidence of asymptomatic patients reported in other studies is lower than that observed in our cohort, and ranges between 36 and 52% [7–10, 15]. Nguyen et al. reported that none of the patients in his series expressed all of the classical symptoms (hypertension, postural hypotension, palpitations, headaches, excessive sweating) [9]. We took into consideration that the clinical evaluation in our study might be imperfect due to the retrospective character of analysis with a lack of some information in available medical records, and due to known difficulties in assessing symptoms of phaeochromocytoma sometimes mimicking other entities. Likewise, the extensive genetic screening in family members of RET mutations carriers may impact the low incidence of symptomatic cases acquired in this report.

In eight patients (20%) phaeochromocytoma was the first manifestation of multiple endocrine neoplasia type 2, which is comparable with data that phaeochromocytoma may be diagnosed before medullary thyroid cancer in 9–27% [16–18]. However, Nguyen et al. reported no phaeochromocytoma preceding MTC [9].

In our study all tumours were localised in adrenals. These results are in agreement with literature data. In a multicentre analysis of 563 MEN 2 patients with phaeochromocytoma extra-adrenal tumours occurred only in five patients [19]. Localisation of the tumour together with biochemical phenotype may be helpful in ordering proper genetic testing [20]. In our analysis results of 24-hour urinary fractioned metanephrines, unlike the total metanephrines, were available only in some cases due to limited availability of this modality in patients who were diagnosed a few years ago and outside the clinical centres. According to the algorithm proposed by the Endocrine Society, adrenal localisations of phaeochromocytoma together with significant productions of metanephrine relative to combined production of all three catecholamine metabolites, in the absence of a syndromic or familial presentation, guide selection of RET proto-oncogene as a first step in genetic analysis [20].

Bilateral phaeochromocytomas at the time of diagnosis were observed in 25% of patients in this cohort, and 34% revealed contralateral tumour throughout the follow-up period. The reported frequency of bilateralism is variable in published reports, ranging between 35% and 80% [7, 8, 21, 22] and is higher in comparison to sporadic cases, which is estimated at 3% [13]. According to that fact, patients with MEN 2 are at high risk for bilateral adrenalectomy and lifelong steroid dependence. In our data only four patients had adrenal-sparing surgery that enables assessment of the outcomes of such a procedure. Castinetti et al. in a multicentre study showed that adrenal insufficiency is greatly reduced in adrenal-sparing operations compared with total adrenalectomies [19]. It is known that one third to one half of an adrenal gland is needed to preserve cortical function [20]. On the other hand, leaving some adrenal medullary tissue during non-radical surgery may be the cause of phaeochromocytoma recurrence. The cumulative recurrence rate for MEN 2 patients after adrenalsparing surgery at 10 years is 38.5% [20]. Nonetheless, Castinetti et al. have shown no significant difference in recurrences between those operated on with adrenalsparing surgery or with adrenalectomy. In material from the Department of General, Vascular, and Transplant Surgery, Medical University of Warsaw, Poland, in the case of phaeochromocytoma as a part of MEN 2, the lesions in adrenals have always been multiple [23]. The authors of the analysis emphasise the importance of precise assessment of the lesions with use of 3D CT in patients qualified for a sparing surgery [23].

In our study there was only one case (2%) of malignant phaeochromocytoma, which corresponds with the opinion that MEN 2-associated phaeochromocytomas are almost exclusively benign [1, 19].

Conclusions

It is mandatory to screen all RET proto-oncogene mutations carriers for phaeochromocytoma.

In patients with MEN 2 syndrome phaeochromocytomas are usually benign adrenal tumours with high risk of bilateral development. Patients who present unilateral phaeochromocytoma should be monitored for early diagnosis of contralateral tumour.

Taking into account that medullary thyroid carcinoma is the first manifestation of MEN 2 syndrome in most cases, it is recommended mandatory genetic study of RET proto-oncogene after diagnosis of MTC. Patients with confirmed germline RET mutation need careful screening for phaeochromocytoma before thyroid resection.