Diagnostic RET genetic testing in 1,058 index patients: A UK centre perspective

Diagnostic germline RET analysis is offered to all patients with a diagnosis of medullary thyroid carcinoma (MTC), or other conditions associated with multiple endocrine neoplasia type 2 (MEN2) in the United Kingdom. Here, we report the experience of a single centre's germline RET analysis over a 21‐year period.


| INTRODUC TI ON
Medullary thyroid carcinoma (MTC) is a neural crest-derived malignant tumour accounting for up to 2% of thyroid cancers. 1 Approximately 20%-25% of cases are hereditary (hMTC), either as part of the multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) or in isolation (FMTC), however in the majority of cases the disease is sporadic (sMTC). 2 Whilst the aetiology of sMTC is not fully understood, the underlying cause for hMTC is well established. The REarranged during Transfection (RET) gene on chromosome 10q11.2 was first described in 1985. 3 It encodes a tyrosine kinase involved in the development of the parathyroid glands, adrenal medulla, parafollicular C cells and enteric ganglia. It is primarily these structures that are affected in patients with MEN2, with MEN2A comprising hereditary medullary thyroid carcinoma with or without phaeochromocytoma and primary hyperparathyroidism, and MEN2B comprising hereditary MTC with or without phaeochromocytoma and other characteristic phenotypic features such as mucosal neuromata, marfanoid habitus, corneal nerve thickening and gastrointestinal ganglioneuromata. Shortly after its discovery, germline RET pathogenic variants were found to be present in almost all patients with hMTC. 4 Despite the lack of heritable germline RET pathogenic variants in the sporadic form of MTC, somatic RET variants are identified in up to 66% of cases. 5,6 A genetic diagnosis of MEN2 is important not only in order to direct investigation for other manifestations of the syndromes, but also to enable genetic counselling and predictive testing for family members. It is particularly important to offer RET testing to all patients with MTC regardless of family history, as around 7% of those with apparently sporadic MTC do indeed harbour germline pathogenic variants, 7 and 75% of MEN2B cases result from de novo RET pathogenic variants. 1 For these reasons, the American Thyroid

Association, British Thyroid Association and European Thyroid
Association guidelines all recommend RET analysis in any patient with confirmed MTC regardless of family history. 1,8,9 In a research setting, germline RET pathogenic variants have been identified in exons 5, 8, 10, 11 and 13-16 in over 95% of patients with MEN2A and FMTC, and a pathogenic variant in codon 918 (M918T) has been identified in over 95% of patients with MEN2B. 10 However, RET pathogenic variant detection rates in the routine clinical setting are less studied. Furthermore, the last two decades have seen an increase in reports of novel germline RET pathogenic variants in hMTC, and this has resulted in a shift in the extent of RET analysis in clinical practice. The objective of the present study is to report the results of diagnostic RET analysis performed in a clinical setting at a single centre over a 21-year period.

| Genetic analysis
Genomic DNA was analysed for variants in selected exons of the RET gene using sequence-specific primers (sequences available on request). PCR products were sequenced using Big Dye Terminator chemistry on an ABI DNA sequencer (Applied Biosystems, Warrington, UK), and the sequences were compared with the published sequence (NM_020975).
During the period of this study, the extent of RET sequencing has evolved in line with reports of new causative pathogenic variants.

| RE SULTS
The clinical indications for germline RET analysis in the 1,058 index patients are shown in Table 1

| Index patients with MTC
Of the 766 UK patients with MTC, 433 were female (56.5%). The

| Patients with isolated MTC and no known family history of endocrine tumours
Of the 657 patients with confirmed isolated MTC and no family history (therefore presumed to have sporadic disease), 56 were found to harbour germline RET pathogenic variants (8.5%, Table 1). The median age of these patients was 58 years (age range: 2-94 years), and there was a 1.3:1 female to male ratio. One patient in this group who was diagnosed with isolated MTC at the age of 30 was found to harbour a pathogenic variant at codon In vitro assays indicated a low-grade transforming potential, and the authors proposed that other genetic determinants may also have contributed to tumorigenesis.

| Patients with isolated MTC and a family history of MTC or MEN2 components
This subgroup was formed of patients with a diagnosis of MTC and a positive family history of MTC, C-cell hyperplasia, phaeochromocytoma or primary hyperparathyroidism, and included 33 patients.
There were 12 pathogenic variants identified in this group (36.4%, Table S1). Amongst patients with multiple MEN2-associated conditions, 21 (23.1%) were found to harbour a germline RET pathogenic variant, and one was found to have a variant of uncertain significance (Table S1).

| Patients with MTC plus phaeochromocytoma or primary hyperparathyroidism
In those with MTC and Phaeochromocytoma, the pathogenic vari- Index patients with C-cell hyperplasia

| Index patients with phaeochromocytoma and primary hyperparathyroidism
Of the 22 index patients with a diagnosis of both phaeochromocytoma and primary hyperparathyroidism, only 1 (4.5%) was found to have a germline RET pathogenic variant (p.Cys634Arg). None of these patients had a family history of MEN2-related conditions.

| Index patients with isolated phaeochromocytoma
165 patients were referred for germline RET testing with a diagnosis of isolated phaeochromocytoma, with a median age of 39 years (range: 1-88 years), and only 6 were found to harbour pathogenic variants (3.6%). Of these, 4 were found to have pathogenic variants at codon 634, and one had a family history of MEN2-associated conditions.

| Index patients with isolated primary hyperparathyroidism
There were 56 patients with isolated primary hyperparathyroidism. RET mutations in presumed sporadic MTC in order to inform prognosis has been discussed extensively elsewhere, and there is evidence that certain somatic RET mutations predict a poorer outcome. [19][20][21][22][23] Despite this increasing body of evidence, guidelines on the manage- Overall, the most commonly affected codons amongst the UK patients with MTC were 634 in exon 11, followed by 804 in exon 14. The largest studies on RET analysis in MTC are from Italy, and they consistently find codon 804 to be most commonly mutated in MTC 16,26 ; however, codon 634 has been reported as the most commonly affected in both Germany and Brazil. 27,28 Interestingly, a large Danish study found that variants at codon 611 were most prevalent. 29 In keeping with reports from Italy, we also found that cysteine pathogenic variants at codon 634 were more common in those with MTC with a phaeochromocytoma. 16 The lowest pathogenic variant detection rates in the current series were in the subgroups of index patients with isolated primary hyperparathyroidism (0%) and isolated phaeochromocytoma (3.6%). can be the first presenting feature of MEN2B.

| Strengths and limitations
This study represents the largest UK series of germline RET analy-  37 and therefore, the ethnicity of patients in our series may have had an effect on the pathogenic variants identified. Finally, although samples from across the UK were tested, the applicability of our findings to other populations is limited, and we were unable to obtain follow-up data to identify metachronous MEN2-associated conditions diagnosed since referral for RET analysis.

| CON CLUS IONS
In a UK clinical setting as in other settings, the rate of germline RET pathogenic variants in presumed sporadic MTC is significant, and all patients with a diagnosis of MTC should be screened in order to identify familial cases. In patients with clinical manifestations of MEN2A, the majority did not receive a confirmatory genetic diagnosis. The rate of detection in patients with isolated phaeochromocytoma is not insignificant, and therefore, it seems this is an appropriate indication for germline RET analysis; however, the absence of a single positive result amongst patients with isolated primary hyperparathyroidism suggests that this may not be an effective use of resources.
Finally, the use of somatic RET analysis to confirm the diagnosis of sporadic MTC in patients with no identified germline RET variants may be a useful adjunct both in terms of reassuring family members about the lack of a heritable pathogenic germline variant, and riskstratifying sporadic tumours based on somatic variants.

CO N FLI C T O F I NTE R E S T
SE is a Wellcome senior investigator. No other author has any conflict of interest to declare.

AUTH O R CO NTR I B UTI O N S
JMF contributed to data collection, analysis and manuscript preparation; JAS and RC contributed to manuscript preparation; CB contributed to analysis and manuscript preparation; SE and BV contributed to conceptualization and manuscript preparation; MO contributed to conceptualization, data collection, analysis and manuscript preparation.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.