In this report, we describe a family with 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. 3132 T > GP), has never been reported to our knowledge. It is unlikely that the two mutations are related but it is tempting to speculate that the germline RET mutation has induced some chromosomal instability leading to the development of postzygotic mosaic PIK3CA mutation. 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, the pathogenesis of which is not fully understood. It is also 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.
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 work up for the leukemia and this revealed a p.V804M RET mutation [20]. Evaluation of his family led to detection of this mutation and MEN 2a in his father and 3 siblings [20]. These two cases reflect the relatively indolent nature of this 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 65-year old mother (paternal grandmother of the index case) had only a multifocal microMTC of 2 mm size and CCH. 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 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 his mother’s brother) and her daughter both had FMTC [21]. 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 are associated with more aggressive tumors and higher likelihood of MEN 2a syndromes rather than FMTC alone [21, 22]. No other genetic alteration in RET or other genes was found in our family to explain this intrafamilial heterogeneity.
A review of the literature since the initial description of this mutation in 1996 [8] shows that the p.V804M mutation is relatively indolent and associated with old age of development of MTC [6, 7, 9, 12, 14, 23]. FMTC and CCH are the most dominant presentations [6, 9, 14]. However, complete or partial MEN 2a syndromes also occur [9, 11, 22, 24]. Lymph node and especially distant metastases are rare in patients with p.V804M [12, 21, 25]. Pheochromocytoma appears to be rare as well [9, 15] as are other less classic manifestations of MEN 2a such as thickened corneal nerves [21] and cutaneous lichen amyloidosis [26]. These seemingly indolent features and the low lifetime risk of FMTC in patients with p.V804M mutations raised questions about the general ATA recommendations of prophylactic thyroidectomy for patients carrying the p.V804M mutation [6]. Some suggested that a “wait-and-see” approach guided by plasma calcitonin level monitoring is a better and safer strategy than routine prophylactic thyroidectomy for patients with this mutation [6, 12].
A number of reports have described the concomitant occurrence of PTC and FMTC in patients with p.V804M mutation [13, 14, 27, 28]. While this could be coincidental as PTC is very common, the several reports describing this combination in patients with p.V804M mutation suggest a possible association related to the transforming capacity of this mutation [13, 23, 27]. In our family, 3 out of 6 members (50%) who underwent thyroidectomy had micro-PTC (2–6 mm in size) in addition to MTC.
The prevalence of the RET p.V804M mutation varies between populations (Table 4) but overall, it is a common mutation [3, 6, 9, 24]. Two reports from Italy in 2010 and 2019 which included 250 and 195 families, respectively showed that the p.V804M mutation was the most common mutation occurring in 19.6% and 25.6% of families with RET mutations, respectively, suggesting that this variant might be a founder mutation in the Italian population [9, 24]. Similar prevalence has also been reported from a large study from France (RET Variants) which included 444 families with FMTC/MEN 2 syndromes and showed p.V804M mutations in 95 of them (21.2%)[10]. However, less prevalence rates were found in Germany (9.9%) and Austria (6.25%) and Brazil (8.5%) [7, 23, 29].
With the widespread use of next generation sequencing, large databases have now become available. These databases include some for patients with cancer and some non-cancer databases. Studies that have looked at the chance occurrence of p.V804M in these databases showed occasional occurrence of this mutation in patients with other diagnoses. In a study that included 1566 patients with different cancers who underwent tumor and normal tissue genomic sequencing for possible target therapies, 246 patients (15.7%) were found to have presumed pathogenic germline mutations including 198 subjects having mutations in cancer susceptibility genes [30]. Interestingly only around 41% of these germline mutations were concordant with the patient’s underlying cancer diagnosis. Six RET germline mutations were found in this series including p.V804M in a patient with colorectal cancer [30]. In another study that included 165 patients with metastatic lung and colorectal cancer who underwent WES on tumor tissue and matching blood samples, of 806 curated germline mutations, 5% were clinically relevant and 56% of unknown significance [31]. RET p.V804M mutation was found in 1 patient with metastatic lung adenocarcinoma [31]. In a large study of this type of analysis, Kuan-Lin Huang et al. analysed the pathogenic variants from 10,389 individuals across 33 cancer types in The Cancer Genome Atlas (TCGA) cohort [32]. They found 8% of cases carrying pathogenic or likely pathogenic germline variants, ranging in prevalence from 22.9% in pheochromocytoma and paraganglioma (PPGL) to a scarce 2.2% in cholangiocarcinoma [32]. RET p.V804M mutation was found in 3 cases [32]. However, this analysis included patients with MTC, MEN 2a and 2b and PPGL [32]. Therefore, it was not surprising to find this mutation in the 3 cases. In fact, the three cases had MEN 2a syndrome [32]. Loveday et al, evaluated 61 RET mutations in non-cancer ExAC database of around 51000 individuals [6]. They found only 10 of the 61 RET mutations present in this non-cancer database including p.V804M mutation [6]. However, they estimated the lifetime penetrance for MTC for this mutation to be quite low at about 4% (95% CI, 0.9-8%) and questioned the current ATA recommendations of prophylactic thyroidectomy for carriers of this mutation [6].
The age at which the risk of FMTC in p.V804M mutation starts to appear has been a subject of controversy. Although most studies show that the majority of patients carrying this mutation develop MTC in their 5th or 6th decade of life, some reported FMTC and MEN 2a syndrome in as early as second decade of life [33]. A comprehensive analysis of a large sample of patients with low risk mutations (class A and B according to the ATA old classification [34] and moderate risk according to the more updated classification in 2015 [5] included 160 patients with p.V804M mutations [33]. Their age ranged between 2–74 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) [33]. 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 70s 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. An 8 and 9-year old boy and girl had only CCH. The literature is conflicting on this subject with some recommending prophylactic thyroidectomy in childhood [25] and most recommending surveillance and thyroidectomy when serum calcitonin starts to rise [12, 25, 33]. Based on our family and the literature review, it seems reasonable to adopt a surveillance strategy for carriers of p.V804M mutation and proceeding with thyroidectomy only when serum calcitonin starts to rise.
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 post zygotic PIK3CA mutation consistent with his diagnosis of CLOVES syndrome but incidentally reported RET p.V804M mutation. Evaluation of this 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. Comprehensive 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. This is probably a reasonable approach since the disease associated with this mutation is generally indolent and small even when they develop early in life.