Controversy on the management of patients carrying RET p.V804M mutation

RET p.V804M is classified as a moderate risk mutation for familial medullary thyroid cancer (FMTC). There is a significant controversy on the management of patients carrying this mutation. We describe a family incidentally discovered to have this mutation and review the literature on RET p.V804M mutation. The proband was born to first-degree relative parents. He was noticed to have hypertrophy of some parts of the body and vascular skin changes. Whole-exome sequencing of DNA extracted from a skin biopsy showed a mutation in the PIK3CA (c.3132T>G, p.ASN1044LYS). This variant was not found in DNA extracted from blood. This confirmed the diagnosis of CLOVES syndrome (Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevi and Scoliosis, skeletal or spinal anomalies). Another incidentally found mutation in the skin biopsy and blood sample was RET p.V804M. Although there was no family history of MTC or MEN 2 syndromes, family screening revealed RET p.V804M mutation and FMTC in the proband’s father, paternal grandmother, one sister, and one aunt. There was significant interfamilial heterogeneity in the age of presentation and pathology. A review of literature showed that RET p.V804M mutation is a moderate risk mutation associated with late-onset FMTC, usually at middle to old age. Despite the controversy and the heterogeneous presentation of patients with RET p.V804M mutation, our study and review of the literature suggest that this seemingly “low” risk mutation is associated with late-onset but potentially aggressive MTC. This indicates the need for follow-up and timely intervention based on calcitonin level elevation.

The RET mutation c.2410G>A, p.V804M is one of the most commonly reported mutations [6,9,10]. It was described for the first time in 1995 [11] and has been widely reported in several studies since then [6,9,[12][13][14][15][16][17][18]. However, there is some controversy about its pathogenicity, age of penetrance, and phenotypes [6,12,13,17]. It is classified as a moderate risk mutation by the American Thyroid Association (ATA) guidelines and as such, it is recommended that children with moderate risk RET mutations should have an annual physical examination, neck ultrasonography, and serum calcitonin measurement starting at age 5 years. Thyroidectomy should be performed when serum calcitonin rises unless parents are concerned about long-term evaluation program and opt for prophylactic thyroidectomy at around age 5 years [5]. In moderate risk RET mutations including V804M, preoperative serum calcitonin has a high predictive value for cure after thyroidectomy. A large retrospective analysis of 170 patients with moderate risk RET mutations showed that no patient with a preoperative basal CT<31 ng/ml had persistent or recurrent disease or N1 disease status after thyroidectomy [15].
A recent analysis of population data based on non-cancer Exome Aggregation Consortium (ExAC) Databases found a high frequency of RET p.V804M mutation with a low lifetime penetrance of MTC of only 4% (95% CI, 0.9-8%) [6]. RET p.V804M mutation is mostly associated with FMTC without other components of MEN 2 syndromes but there are some reports of pheochromocytoma and primary hyperparathyroidism in some families carrying this mutation [12,[17][18][19]. It is possible that other genetic or environmental modifiers operate in those cases with more phenotypic manifestations.
CLOVES syndrome (OMIM 612918) was described for the first time in 2007 and is characterized by tissue overgrowth and complex vascular anomalies [20]. CLOVES is an acronym for Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevi and Scoliosis, skeletal or spinal anomalies [21]. It is caused by a somatic postzygotic mosaic activating PIK3CA mutations [22]. It is part of a complex group of diseases called PIK3CA-related overgrowth spectrum [23]. This includes overlapping clinical syndromes such as Fibroadipose Overgrowth/Hemihyperplasia-Multiple Lipomatosis, Isolated Large Lymphatic Malformation, Epidermal Nevi, Seborrheic Keratoses, Benign Lichenoid Keratoses, Megalencephaly-Capillary Malformation, Hemimegalencephaly and Dysplastic Megalencephaly [23].
In this report, we describe a family with FMTC due to the RET p.V804M mutation, which was discovered incidentally when a newborn (index case) was found to have significant dysmorphic features of unclear cause. Wholeexome sequencing (WES) was performed to understand the reason for these dysmorphia. An underlying PIK3CA mutation was found in DNA extracted from skin biopsy but not from blood sample indicating a postzygotic mosaic mutation of CLOVES syndrome. In addition, RET p.V840M was discovered incidentally in his WES of the skin and blood samples. The family screening revealed that the child's father has the same RET mutation and a 5-cm thyroid mass that turned out to be MTC. Further family screening revealed the presence of the RET p.V804M mutation in a number of family members and MTC and C-cell hyperplasia (CCH) in some of them. The findings in this family suggest that this mutation is of moderate risk and tends to affect patients at an older age than usual for other RET mutations. There was also significant intrafamilial heterogeneity of p.V804M-related FMTC/CCH. In this report, we describe all of these aspects and provide a literature review on this common RET mutation.

Patients
The propositus is now a 2-year old boy who was born to consanguineous parents at full term after an uneventful pregnancy and without any history of maternal exposure to toxic substances, alcohol or drugs. His birth weight was below the Fig. 1 Photographs of the index case at age 2 years showing hypertrophic changes involving the right side of the skull and face, right arm, right hand fingers, right side of the trunk, right thigh, the left thumb, and index finger. It also shows extensive cutaneous vascular changes over the trunk, back, and left hand 3rd percentile and he was immediately observed to have gross hypertrophy of the right side of his body involving the skull, face, arms, trunk, and right leg (Fig. 1a). He also had some hypertrophy of the left index finger and thumb and cutaneous changes consistent with vascular abnormalities. He had a large vascular malformation (hemangioma) over the right buttock. This was surgically removed and confirmed to be a hemangioma. The diagnosis was unclear and for that reason, WES was carried out to look for any genetic cause for his congenital anomaly. This was performed in DNA extracted from skin biopsy and a blood sample. It revealed a PIK3CA mutation (NM_006218.2: c.3132T>G, p.ASN1044LYS) in the skin biopsy but not in the blood sample confirming that this mutation is a postzygotic mosaic mutation and that the patient has CLOVES syndrome. This mutation was classified as a variant of unknown significance but was predicted to be probably damaging by PolyPhen, deleterious by SIFT, diseasecausing by MutationTaster, Alig-GVGD: C65 (most likely to interfere with function) and the amino acid aspraginase is highly conserved. This variant was not reported in Genome Aggregation Database (GenomAD), 1000Genome project, and Exome Sequencing Project. In addition to the PIK3CA mutation, the RET p.V804M mutation was reported as an incidental finding from the skin and blood samples. Further evaluation of the newborn revealed normal thyroid gland on ultrasonography without nodules and normal calcitonin level (4.1 pg/dl, normal range 0-5.0 pg/dl). Magnetic Resonance Imaging (MRI) of the brain showed hemimegaloencephaly and thickened facial subcutaneous tissue, bulky right choroid plexus and prominent periventricular veins. The brain parenchyma was normal. An MRI of the lumbar spine was normal. Skeletal survey showed short big toes bilaterally and hypoplastic phalanges. The patient suffered from seizures that are currently controlled by three antiepileptic drugs. He also has a significant developmental delay in his milestones. After obtaining informed consents and an Institutional Review Board Approval from the Office of Research Affairs, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia, the family was screened for the RET p.V804M mutation. Following the discovery of RET p. V804M mutation in the newborn child (the index case), his father, two sisters, a brother, paternal grandmother, paternal aunt, and her daughter were screened and found to carry the RET p.V804M mutation (Fig. 2). Clinical evaluation showed that the father has an asymptomatic 5-cm thyroid mass and some pathological cervical lymph nodes. A fine-needle aspiration biopsy confirmed the diagnosis of MTC. Before referred to us, the father and several members of the family underwent total thyroidectomy either as a treatment of preexisting FMTC or as prophylactic thyroidectomy. The family pedigree is shown in Fig. 2 and the clinical, biochemical, and pathological features, management, and outcome of the family are shown in Table 1.

RET V804M mutation in the literature
We searched the literature for studies reporting the RET p.V804M mutation, its clinical presentations, age of presentation, and penetrance. We used the terms RET, FMTC, RET p.V804M. We found a number of case reports    (Table 3), and large case series (Table 4).

Clinical phenotype
The case reports, large families and case series show the different phenotypes and frequencies of the p.V804M mutation in different populations (Tables 2, 3, 4). Most cases carrying the RET p.V804M mutation present with FMTC or CCH (Table 2). MEN 2a, primary hyperparathyroidism, and pheochromocytomas occur rarely (Tables 2, 3, 4). These features are in agreement with the classification of the RET p.V804M mutation-related syndrome as MEN 2A with a low penetrance/late occurrence and low aggressiveness. Although lymph node metastases appear to be relatively uncommon, distant metastases are rare. We found only one report of bone metastasis. Interestingly, more than one report described the presence of papillary thyroid cancer (PTC) with MTC (Tables 2, 3, 4).

Frequency of RET p.V804M mutation
Although the frequency of the RET p.V804M mutation varies between different series, it is common and is the most common RET mutation in some populations (Table 4). There are a number of large series of patients with familial and/or sporadic MTC who were screened for various RET mutations (Table 4). In these series, the frequency of the RET p.V804M mutation varied between 6.25 and 25.6% (Table 4). This mutation was the most frequent RET mutation in two series conducted in Italy a decade apart, with rates of 19.6% and 25.6% (Table 4). It was also common in a large series from France (21.4%) but less common in Germany, Austria and Brazil with rates of 9.9%, 6.25%, and 8.5%, respectively ( Table 4). The high frequency of this low penetrant mutation is probably because most families with high penetrant mutations and MEN 2A have already been identified and this and other low penetrant mutations are currently the most frequent mutations found in "newly" identified families.

Discussion
In this report, we describe a family with RET p.V804M mutation with many interesting aspects. Initially, this was found incidentally while evaluating a newborn with congenital anomalies that eventually turned out to be part of CLOVES syndrome. The finding of two rare genetic alterations, one is germline (RET p.V804M) and the other is a mosaic somatic mutation (PIK3CA c. 3132T>GP), has never been reported to our knowledge. However, it is unlikely that the two mutations are related. Although distinct from the hypertrophy of CLOVES syndrome, RET mutations are sometimes associated with some forms of growth such as cutaneous lichen amyloidosis and thickened corneal nerves, and mucocutaneous ganglioneurmas, the pathogenesis of which is not fully understood. It is tempting to speculate that these are similar to hypertrophy in this newborn and are related to postzygotic mutations limited to the areas of growth but this has not been studied. Another possible explanation is that RET is expressed in a limited number of tissues and that activating RET mutations induce some abnormalities only in those tissues. It is also intriguing but unclear on how the two mutations interact in the tissue that harbor both (the hypertrophic and vascular tissue). It is also interesting how the congenital anomaly and its evaluation led to the discovery of FMTC in this family, which was not known to have any cancer syndromes. A similar situation was described in a 3-year-old boy with leukemia who had WES as part of workup for the leukemia and this revealed a p.V804M RET mutation [24]. Evaluation of his family led to detection of this mutation and MEN 2a in his father and 3 siblings [24]. These two cases reflect the relatively indolent nature of RET p.V804M mutation and the previously shown observation of late development of MTC and MEN 2a in carriers of this mutation. The other interesting aspect of the RET p.V804M mutation is the intrafamilial heterogeneity (Table 1). While the father of the index case had a large MTC of 5 cm size with lymph node metastases at his age of 43 years, his 65year old mother (the paternal grandmother of the index case) had only a multifocal micro-MTC of 2 mm size and CCH ( Table 1). The 13-year-old sister also had a 2-mm micro-MTC and CCH while a 46-year-old aunt had a 5-mm micro-MTC and CCH. Two younger siblings of 8 and 9 years had CCH only (Table 1). A previously reported family with RET p.V804M mutation showed a skipping of a 93-year-old mother (no disease) of a 53-year-old son who had FMTC and corneal nerve thickening and his cousin (daughter of a maternal uncle) and her daughter both had FMTC [25]. In our family and this reported family, males seem to be more affected but it is not clear whether these different phenotypic expressions are due to sex differences. Another possible explanation is the presence of other genetic modifiers that may lead to more manifestations in some family members. For example, it has been shown that the presence of tandem or double mutations in RET is associated with more aggressive tumors and higher likelihood of MEN 2a syndromes rather than FMTC alone [25,26]. However, no other genetic alteration was found in our family to explain this intrafamilial heterogeneity. We also looked for other somatic RET mutations in the MTC from the father who had the most aggressive disease among the family members and none was found including RET p. M918T mutation.
A review of the literature since the initial description of this mutation in 1995 [11] shows that the RET p.V804M mutation is associated with old age of development of MTC [6,7,9,13,18,27]. FMTC and CCH are the most dominant presentations [6,9,18]. However, complete or partial MEN 2a syndromes also occur [9,14,17,26]. The risk of lymph node metastases increases with age in carriers of this mutation [28] but distant metastases are rare [13,25,29]. Pheochromocytoma appears to be rare as well [9,19] as are other less classic manifestations of MEN 2a such as thickened corneal nerves [25] and cutaneous lichen amyloidosis [30]. These seemingly indolent features and the low lifetime risk of FMTC in patients with RET p.V804M mutation raised questions about the general ATA recommendations of prophylactic thyroidectomy for patients carrying the RET p.V804M mutation [6]. Some suggested that a "wait-andsee" approach guided by plasma calcitonin level monitoring is a better and safer strategy than routine prophylactic thyroidectomy for patients with this mutation [6,13]. However, Other data suggest that the RET p.V804M mutation is not indolent and is associated with late-onset but significant disease entailing a careful follow-up and a timely thyroidectomy. In a study of 53 carriers of RET p.V804M, 14 (26.4%) developed node-negative MTC at a median age of 42.9 years and 13 (24.5%) developed node-positive MTC at a median age of 51.3 years [28].
The age at which the risk of FMTC in p.V804M mutation starts to appear has also been a subject of controversy. Although most studies show that the majority of patients carrying this mutation develop MTC in their 4th to 6th decade of life, some reported FMTC and MEN 2a syndrome in as early as second decade of life [16]. A comprehensive analysis of a large sample of patients with low-risk mutations (class A and B according to the ATA old classification) [31] and moderate risk according to the more updated classification in 2015 [5] included 160 patients with RET p.V804M mutations [16] was undertaken. Their ages ranged between 2 and 74 years and 52% were females. No cases developed MTC at 5, 10, and 15 years but the probability was 0.02, 0.03, 0.17, 0.31, 0.67 and 0.87 at 20, 30, 40, 50, 60, and 70 years, respectively and the median age to develop MTC was 54 years (range 52-60) [16]. However, other studies of large families showed variable expression of the disease even within the same family with some carriers not developing MTC in their 70 and 80s and some developing metastatic disease in their 20s and 30s ( Table 2). This was also the case in our family where the youngest member with MTC was the 13-year old girl (patient number III.5). An 8-and 9-year old boy and girl had only CCH ( Table 1). The literature is conflicting on this subject with some recommending prophylactic thyroidectomy in childhood [29] and most recommending surveillance and thyroidectomy when serum calcitonin rises [13,16,29]. Based on our family and the literature review, it seems reasonable to adopt a surveillance strategy for carriers of p.V804M mutation using serum calcitonin as a guide for timing of thyroidectomy. This strategy involves annual serum calcitonin measurements starting at age 5 years and then every year as long as calcitonin remains low or undetectable and proceeding with thyroidectomy when calcitonin increases and before it reaches the level of 30 pg/ml.
A number of reports have described the concomitant occurrence of PTC and FMTC in patients with p.V804M mutation [12,18,32,33]. Several reports describing this combination in patients with p.V804M mutation suggest a possible association related to the transforming capacity of this mutation [12,27,32]. However, this is likely to be coincidental related to the high prevalence of micro-PTC and the scrutiny of histopathological examination of the resected thyroid tissue [34,35]. In our family, 3 out of 6 members (50%) who underwent thyroidectomy had micro-PTC (2-6 mm in size) in addition to MTC.
In summary, we have described an interesting patient born with congenital anomaly that turned out to be the rare and recently described CLOVES syndrome. WES to investigate for the congenital anomaly revealed a postzygotic PIK3CA mutation consistent with his diagnosis of CLOVES syndrome but also incidentally reported RET p.V804M mutation. Evaluation of his family found several members with this mutation and therapeutic or prophylactic thyroidectomy showed CCH or MTC at different ages with clear intrafamilial heterogeneity with respect to the features of MTC and the age of the patients. Literature review showed that p.V804M RET mutation is common especially in Europe and is associated mostly with FMTC/CCH and rarely with other components of MEN 2a syndromes. The age of MTC development is variable but generally is late (4th to 5th decade of life) suggesting that prophylactic thyroidectomy might be deferred until serum calcitonin starts to rise.