Abstract
Graphical abstract
Abstract
RET is a key oncogene in neuroendocrine cancer. Pathogenic germline variants lead to multiple different phenotypes, including multiple endocrine neoplasia type 2, medullary thyroid cancer (MTC), Hirschsprung disease and kidney malformations. Pathogenic somatic variants are also associated with MTC, and RET rearrangements are observed in papillary thyroid cancer, non-small cell lung cancer and pan-cancer syndromes. Testing for both germline and somatic variants is now feasible in everyday clinical practice, and their identification has important clinical consequences, both for affected individuals and their families. This mini-review will discuss current germline and somatic testing strategies in the UK and worldwide, as well as reporting and test outcomes (including variants of uncertain significance or incidental findings). It will explore actions following identification of a pathogenic germline variant, including predictive, reproductive and childhood testing, and somatic testing of RET variants in solid tumours informing personalised cancer treatment. Finally, it will discuss the challenge of delivering rapid and equitable access to genomic testing to ensure that all individuals can benefit promptly and appropriately to improve clinical outcomes.
Introduction
Genomic testing has expanded greatly in the last decade, shifting from limited access within clinical genetics services into mainstream care. Specialists from multiple areas now routinely test for germline and/or somatic variants early in their patients’ pathways to determine whether there is a heritable cause and to inform mutation-specific clinical care (Eggermann et al. 2020). This transformation was possible due to technical advances in DNA sequencing and computational biology, reduced sequencing costs, more efficient variant interpretation, improved test turnaround times and upskilling in workforce understanding and utilisation – all of which have contributed towards earlier genomic diagnoses with prognostic, therapeutic and management implications for personalised care.
In 2019, the UK leveraged the 100,000 Genomes Project to commission a national Genomic Medicine Service (GMS) (Brittain et al. 2017), which now provides the entire English population with access to genomic tests for inherited rare diseases and cancer (similar services exist in Scotland, Wales and Northern Ireland). All clinicians can access the National Genomic Test Directory (NGTD) (https://www.england.nhs.uk/publication/national-genomic-test-directories/; accessed 26/08/2024), which lists available genomic tests (from whole genome sequencing (WGS) to single-gene testing), describes the technology used to deliver testing, and details patient eligibility criteria. Somatic testing is available to aid diagnosis and management of solid tumours and give prognostic information, identifying oncogenic drivers for targeted therapies. Testing is delivered through core and specialised arrangements within seven networked Genomic Laboratory Hubs, working to common national standards, specifications and ISO accreditation (ISO 151989:2012). All genomic tests are funded by NHS England at no direct cost to the patient or health provider. Globally, access to RET genomic testing is performed in multiple laboratories (https://www.orpha.net/en/diagnostic-tests/diagnostics/; accessed 12/01/2025) or (https://www.ncbi.nlm.nih.gov/gtr/; accessed 12/01/2025). Access to genomic testing, the extent of testing, techniques employed and reimbursement of costs will be country and healthcare system-dependent, guided by national guidelines, for example, https://www.nccn.org/professionals/physician_gls/pdf/thyroid.pdf; accessed 12/01/2025. Provided criteria for testing are fulfilled (for example, a patient presenting with medullary thyroid cancer (MTC)), insurers will often cover the cost of both germline and somatic RET testing in the USA and Europe.
RET: a key endocrine oncogene
Gene structure
The gene RET (rearranged during transfection) was identified in 1985 as an oncogene by its ability to transform NIH3T3 cells by DNA rearrangement (Takahashi et al. 1985). RET encodes a transmembrane RET that regulates cell migration, proliferation, survival and differentiation (Fig. 1). Ret also has a key role in the development of the renal tract, enteric nervous system and other neuroendocrine pathways; it is therefore unsurprising that RET germline mutations result in clinical phenotypes affecting these systems (Mulligan 2014).
Schematic representation of the RET gene, protein and phenotypes associated with common RET germline pathogenic variants. Representation of the RET gene (middle), RET protein (left) and phenotypes associated with LoF or GoF germline pathogenic variants (pv) (listed on right). The RET gene is located on 10q11.2, containing 20 exons. It encodes a transmembrane receptor which has a signal peptide, four cadherin-like domains and a cysteine-rich domain sited extracellularly. The intracellular domain contains a tyrosine kinase domain. The two main alternatively spliced isoforms of RET protein are RET9 and RET51. Hirschsprung disease (HSCR) and congenital anomalies of the kidney and urinary tract (CAKUT) occur with LoF pv across the entire coding sequence of the RET gene. In MEN2, GoF pv, listed as amino acid changes, are clustered in specific exons 8, 10, 11 and 13–16, as shown. aThe most frequent RET pvs and MEN2 phenotypes are shown in black, purple and red to represent the moderate (MOD), high and highest risk of MTC according to the Revised ATA 2015 guidelines (Wells et al. 2015). The MEN2 phenotypes associated with various common RET germline pvs are listed in the table on the right. +HSCR, +CLA, cutaneous lichen amyloidosis. Data from Waguespack et al. (2011).
Citation: Endocrine-Related Cancer 32, 5; 10.1530/ERC-24-0230
RET pathogenic variants
Dysregulation of RET via loss-of-function (LoF) or gain-of-function (GoF) mechanisms results in multiple autosomal dominant conditions.
Germline LoF pathogenic variants can cause Hirschsprung disease (Edery et al. 1994) or kidney malformations (Pini Prato et al. 2021). Germline GoF pathogenic variants cause MEN2, where the hallmark of the disease is MTC (Mulligan 2014). MEN2-associated variants cluster in exons 8, 10, 11 and 13–16 and are mostly single-nucleotide variants, although deletions, duplications, insertions, homozygotes or tandem variants in a cis configuration also occur (Fig. 1) (Elisei et al. 2019, Salvatore et al. 2021).
Considering somatic variants, RET GoF pathogenic variants are found in approximately half of sporadic MTC cases (Ciampi et al. 2019), and activating RET chromosomal rearrangements or gene fusions constitute oncogenic drivers in multiple solid tumours, including 10–20% papillary thyroid cancers (PTCs) (Agrawal et al. 2014) and 1–2% of NSCLC (Takeuchi et al. 2012). Somatic or germline RET alterations are druggable targets, which enables personalised cancer treatments (Filetti et al. 2022).
Clinical disease associated with germline RET pathogenic variants
MTC and MEN2
MTC (OMIM #155240) arises from the parafollicular C-cells of the thyroid gland. MTC is rare, accounting for ∼2–3% of thyroid cancers (TCs), with 107 cases reported in England in 2021 (National Disease Registration Service). 75% of MTC cases are sporadic; however, 25% are syndromic, occurring in patients with germline RET variants and MEN2 (Fig. 2).
Recommended RET genomic testing for patients presenting with MTC and personalised care. 75% of MTC cases are sporadic (sMTC) and 25% are syndromic, described as hereditary (hMTC). Testing of specific exons of RET exons 5, 8, 10, 11 and 13–16 is recommended, as the known GoF pathogenic variants leading to hMTC or sMTC are concentrated in these exons. MEN2, multiple endocrine neoplasia type 2; PCC, phaeochromocytoma; HPTH, hyperparathyroidism; PND, prenatal diagnosis; PGT-M, pre-implantation genetic testing for monogenic disorders; ATA guidelines, revised American Thyroid Association Guidelines (Wells et al. 2015); bilateral CND and LND, bilateral central neck dissection and lymph node dissection; NGS, next-generation sequencing. FFPE (formaldehyde-fixed-paraffin-embedded tissue) is required for somatic analysis, or ctDNA (circulating tumour DNA). RET testing algorithm data adapted from Belli et al. (2021).
Citation: Endocrine-Related Cancer 32, 5; 10.1530/ERC-24-0230
Ninety-five percent of MEN2 cases have MEN2A (OMIM #171400), characterised by developing MTC with a variable risk of phaeochromocytoma and hyperparathyroidism. MTC is usually the presenting tumour in MEN2A, but in 30% of cases, phaeochromocytoma develops first, whereas hyperparathyroidism is an uncommon presentation (Rodriguez et al. 2008, Larsen et al. 2020). Some MEN2A cases additionally present with Hirschsprung disease (codons 609, 611, 618 or 620) or cutaneous lichen amyloidosis (codons 634 or 804) (Fig. 1) (Verga et al. 2003, Coyle et al. 2014).
MEN2B (OMIM #162300) (5% of MEN2 cases) arises in individuals with a pathogenic variant M918T or, infrequently, A883F (Mathiesen et al. 2017). M918T patients present with early-onset, aggressive MTC and 50% develop phaeochromocytoma, but hyperparathyroidism is not described. Other pathognomonic non-endocrine features include intestinal ganglioneuromatosis, marfanoid habitus, mucosal neuromas of the lips and tongue, and corneal nerve thickening (Castinetti et al. 2018). Furthermore, >90% M918T cases are de novo (Brauckhoff et al. 2014).
Strong genotype–phenotype correlations are described in MEN2 (Eng et al. 1996). The American Thyroid Association (ATA) classifies RET variants into three risk levels for the age of onset/aggressiveness of MTC (highest, high and moderate) (Wells et al. 2015). Patients with RET M918T represent the highest risk category, with the earliest age of MTC onset, whereas C634X or A883F variants are categorised as high risk. In contrast, all other MEN2A variants are categorised as moderate risk (Machens & Dralle 2024).
Up to 7% cases of apparently sporadic isolated MTC have germline MEN2A RET pathogenic variants, predominantly non-cysteine variants, e.g., V804M, with a moderate MTC risk (Romei et al. 2011), whereas ∼55% of sporadic MTC cases have a somatic RET variant (Fig. 2) (Ciampi et al. 2019).
Hirschsprung disease
Hirschsprung disease (OMIM#142623) is characterised by the congenital absence of myenteric and submucosal plexuses in the distal colon and rectum. Isolated Hirschsprung disease is most frequently caused by LoF RET variants (Fig. 1). Hirschsprung disease can also occur in other genetic syndromes (Karim et al. 2021), including MEN2A, where co-segregation occurs in individuals with RET codon 609, 611, 618 and 620 pathogenic variants (Arighi et al. 2004, Moore & Zaahl 2012).
CAKUT abnormalities
LoF RET variants are described in congenital absence of the kidney and urinary tract (CAKUT) cases. Some patients have both CAKUT and Hirschsprung disease (Fig. 1) (Davis et al. 2014).
Testing approach for RET (MEN2) germline variants
Who to test
RET genetic testing is recommended for all patients suspected of having an inherited RET pathogenic variant to inform medical management and targeted treatment in MEN2 and to determine familial risk. Early thyroidectomy, before MTC develops, is recommended in MEN2 families to reduce MTC mortality (Wells et al. 2015).
Pre-test evaluation of the affected patient includes personal and family history, clinical phenotyping and collation of biochemical, histological, imaging and prior personal or familial genomic test results to confirm eligibility and inform the genomic testing strategy (Izatt et al. 2022). Table 1 lists eligibility criteria for RET germline testing.
Eligibility criteria for RET germline testing.
| Patients who are eligible for RET germline testing include* | |
|---|---|
| 1 | An individual diagnosed with MTC (any age) |
| 2 | An individual with ≥2 MEN2-related endocrine abnormalities (any age) |
| 3 | An individual with ≥1 MEN2-related endocrine abnormality and a first-degree relative with ≥1 MEN2-related endocrine abnormality (any age). MEN2 abnormalities include MTC, phaeochromocytoma/paraganglioma, parathyroid adenoma/hyperplasia or Hirschsprung disease |
| 4 | An individual with Hirschsprung disease (any age) |
| 5 | An individual with a RET pathogenic germline variant identified in a close relative |
| 6 | An individual with a potential RET pathogenic germline variant identified on somatic testing † |
The clinical indication for genomic testing and the eligibility for accessing the test are listed in the National Genomic Test Directory (NGTD) for rare and inherited diseases (https://www.england.nhs.uk/wp-content/uploads/2024/07/national-genomic-test-directory-rare-and-inherited-disease-eligibility-criteria-v7.pdf; accessed 26/08/2024).
A potential RET germline pathogenic variant might be present if insertions/deletions <50 bp >20%, or if a single nucleotide variant >30% variant allele frequency is detected on somatic tumour testing, regardless of tumour type.
Informed written consent before testing should be obtained. This will include discussion of the benefits and limitations of testing (including the possibility of uncertain or unexpected results), the implications of results for the patient’s own care and for their family (including cascade testing and reproductive decision-making), storage of DNA and sequencing data, and recording of the result in the individual’s medical record.
Test choice
In individuals with MTC, MEN2 or Hirschsprung disease, only RET gene testing is recommended (Rare and inherited disease eligibility criteria; https://www.england.nhs.uk/publication/national-genomic-test-directories/; version 7:2024; accessed 26/08/2024). However, in other laboratories, a next-generation sequencing (NGS) panel of endocrine genes, including RET, is employed because it is more efficient and cost-effective. In the UK, this is the testing strategy for individuals with phaeochromocytoma (<60 years), bilateral phaeochromocytoma or hyperparathyroidism (<50 years), which includes RET in the multigene NGS panel. The probability of detecting RET variants by clinical presentation and genetic test choice is detailed in Table 2. As the criteria for testing, choice of genes on an endocrine panel and process for testing will vary in different healthcare settings, the clinician is advised to confirm local provision.
Probability of detecting a RET pathogenic variant by clinical indication.
| Clinical presentation* | Probability of detecting a germline RET pathogenic variant | Test required † |
|---|---|---|
| MTC, any age | 25% (Romei et al. 2011) | Sequencing of selected RET exons 5, 8, 10, 11, 13, 14, 15, 16 ‡ |
| MEN2A clinical diagnosis (with a confirmed family history) | 98% (Elisei et al. 2019) | Sequencing of selected RET exons 5, 8, 10, 11, 13, 14, 15, 16 ‡ |
| MEN2B clinical diagnosis | 100% (Elisei et al. 2019) | Sequencing of selected RET exons 5, 8, 10, 11, 13, 14, 15, 16 ‡ |
| Familial MTC | 98% (Elisei et al. 2019) | Sequencing of selected RET exons 5, 8, 10, 11, 13, 14, 15, 16 ‡ |
| Sporadic MTC | 7% (Romei et al. 2011) | Sequencing of selected RET exons 5, 8, 10, 11, 13, 14, 15, 16 ‡ |
| Sporadic phaeochromocytoma without syndromic features | 3.6% (Fussey et al. 2021) | Sequencing of a small panel of 11 phaeochromocytoma and paraganglioma genes including selected RET exons ‡ |
| Bilateral phaeochromocytoma | 50% (Neumann et al. 2019) | Sequencing of a small panel of 11 phaeochromocytoma and paraganglioma genes including selected RET exons ‡ |
| Primary hyperparathyroidism (HPTH) | <1% (Larsen et al. 2020) | Sequencing of a small panel of eight FHH (familial hypocalciuric hypercalcaemia) and HPTH genes, including selected RET exons ‡ |
| Isolated Hirschsprung disease-sporadic | Up to 20% (Attié et al. 1995) | Sequence entire RET gene (20 exons) |
| Familial Hirschsprung disease | 50% (Attié et al. 1995) | Sequence entire RET gene (20 exons) |
| Patient with a first degree relative with a RET pathogenic germline variant | Up to 50% | RET variant specific sequence test, targeting the known familial pathogenic variant |
| Patient with a potential RET pathogenic germline variant identified on somatic testing | 47.8% (Kuzbari et al. 2023) | RET variant specific sequence test, targeting the potential RET variant |
The clinical indication for genomic testing and the eligibility for accessing the test are listed in the National Genomic Test Directory (NGTD) for rare and inherited diseases (https://www.england.nhs.uk/publication/national-genomic-test-directories/; accessed 26/08/2024).
The type of test required is listed on the NGTD. The genes in the rare disease panel are listed on PanelApp (https://nhsgms-panelapp.genomicsengland.co.uk/; accessed 26/08/2024).
The list of selected RET exons is as recommended in the 2012 European Thyroid Guidelines (Elisei et al. 2012). However, the inclusion of exon 5 is under review by endocrine specialist genomic testing laboratories in England.
Genomic analysis and reporting
Identified variants are classified using American College of Medical Genetics (ACMG) and Association of Clinical Genomic Sciences guidelines (Richards et al. 2015, Loong et al. 2022). In addition, for MEN2-associated RET variants, modified ACMG guidelines are available (Margraf et al. 2022). These frameworks use multiple strands of evidence for variant classification, including the published literature, population data, in silico prediction data, functional data, clinical phenotype and family history, and public databases (e.g., ClinVar; https://www.ncbi.nlm.nih.gov/search/all/?term=clinvar; accessed 26/08/2024).
Variants are ‘scored’ from the combined evidence into five classes:
Class 5: pathogenic.
Class 4: likely pathogenic.
Class 3: variant of uncertain significance (VUS)
Class 2: likely benign.
Class 1: benign.
Results are reported in a consistent format, using standardised nomenclature to describe the gene variant (https://hgvs-nomenclature.org/stable/; accessed 26/08/024) (Deans et al. 2022).
This includes the approved gene name (RET), reference transcript (RET gene NM_020975.6) and human genome build used for sequence alignment and variant assessment (GRCh37/hg19) (den Dunnen et al. 2016).
Currently, only pathogenic/likely pathogenic variants are routinely reported.
Reporting of VUS is variable, with the approach differing between accredited laboratories. As RET is an autosomal dominant gene, most laboratories will report VUS in diagnostic tests for both oncology and non-oncology patients. However, in the UK, there is a more cautious approach. VUS are considered for reporting only where there is a high level of supporting evidence and if additional evidence might be obtained to allow reclassification as (likely) pathogenic (Loong et al. 2022).
Genomic test outcomes
The following outcomes are possible:
1) Confirmation of a genetic diagnosis: a RET pathogenic/likely pathogenic variant is identified, consistent with the patient’s condition, having been specifically solicited.
2) Incidental findings: a pathogenic/likely pathogenic RET variant is identified unrelated to the reason for requesting the test (e.g. after WGS analysis in a child with congenital malformation/dysmorphism). This is due to recommendations that common cancer-susceptibility genes should be assessed if/when the methodology allows, even if irrelevant to the clinical presentation (Miller et al. 2023).
3) Identification of a RET variant of uncertain significance (VUS).
4) No significant findings.
Clinical outcomes: how to action the test result
Care of the individual
All test outcomes should be discussed with the patient.
Confirmed genetic diagnosis, with a pathogenic/likely pathogenic variant
Onward referral to clinical genetics is advised to support individuals and their families in dealing with this new information (Izatt et al. 2022).
Management of pre-symptomatic MEN2 cases is based on the risk stratification of their genotype, as per ATA guidelines (Wells et al. 2015). These findings inform the timing of thyroidectomy and surveillance for phaeochromocytoma and hyperparathyroidism (Fig. 2, Table 3).
The management of MEN2 patients according to the revised ATA guidelines for MTC (2015).
| ATA risk category (RET variants)* | Timing of genomic testing | Timing of thyroidectomy | Incidence of phaeochromocytoma (PCC) | Incidence of primary hyperparathyroidism (HPTH) | Age to start screening for PCC/HPTH † |
|---|---|---|---|---|---|
|
Highest M918T |
At birth-cord blood Ages 0–1 |
Before age 1 | 50% | Not applicable | 11 years Annual plasma metanephrines, calcium and parathyroid hormone (PTH) |
|
High C634S/G/R/Y/F/W A883F |
Ages 3–5 | Before age 5 Screen with annual calcitonin, thyroid ultrasound and neck examination from 3 years |
50% | 20–30% | 11 years Annual plasma metanephrines, calcium and parathyroid hormone (PTH) |
|
Moderate
G533C C609R/Y/F/S/W C611S/G/R/Y/F/W C618S/G/R/Y/F C620S/G/R/Y/F/W C630S/R/Y K666E/N E768D L790F V804L/M S891A S904F R912P |
Age 5 | From 5 years Screen with annual calcitonin, thyroid ultrasound and neck examination from 5 years. Thyroidectomy is recommended once serum calcitonin becomes elevated in childhood aged 5–10 years or adulthood |
10–20% | 10% | 16 years Annual plasma metanephrines, calcium and parathyroid hormone (PTH) |
The management of MEN2 patients according to the risk levels for the age of onset/aggressiveness of MTC, as recommended in the Revised American Thyroid Association guidelines (Wells et al. 2015). If genomic testing confirms hereditary MEN2, then early thyroidectomy is recommended. MEN2 care should be led by a specialist multidisciplinary team. The incidence of phaeochromocytoma and primary hyperparathyroidism is lower than that of MTC across all genotypes.
Germline RET variants are listed by affected codon and the predicted protein change, using the NM_020975.6 reference sequence.
Radiological surveillance is not routinely performed in the absence of symptoms or abnormal biochemical results suggestive of PCC.
However, risk management of MEN2 cases with moderate-risk MTC variants, when ascertained as incidental findings or via population screening, may require adjustment. The natural history of disease is not fully known outside of familial cases, and MTC penetrance may be reduced. Therefore, the appropriateness of preventative thyroidectomy, as reported for RET V804M, the most frequent moderate-risk variant, is unclear (Loveday et al. 2018).
Uncertain results
Variants of uncertain significance need careful consideration.
The laboratory report details investigations that could provide additional evidence to facilitate variant reclassification (e.g., further investigations in the patient, their family or additional laboratory studies) (Elisei et al. 2012). Clinical genetics can facilitate this process (e.g., assessing segregation in other affected family members) (Newey 2022). Submission of additional information to the genomic laboratory may provide sufficient evidence to reclassify the variant upwards to a (likely) pathogenic variant. An updated report is issued by the laboratory, confirming the genetic diagnosis, to enable MEN2-focused care.
However, variant reclassification is not always possible. Clinical management (including cascade testing of unaffected family members) should not be predicated on VUS, as variants may ultimately prove to be benign.
It is not standard practice for laboratories to systematically review previously classified variants. Variant reinterpretation is typically reactive (Loong et al. 2022). The clinician may consider asking the laboratory to reassess the variant in 3–5 years, or earlier if new information/evidence emerges (e.g., additional affected family members/new published knowledge). If variant reinterpretation leads to upward reclassification, the result is communicated nationally between laboratories for all cases to be notified (https://www.acgs.uk.com/media/12533/uk-practice-guidelines-for-variant-classification-v12-2024.pdf; accessed 26/08/2024). Reissued reports are relayed to clinicians to action, proactively re-contacting historic patients to manage them as MEN2.
Care of the family
Cascade testing
First-degree relatives should be offered a variant-specific gene test to determine their own genetic risk. If the RET variant is not detected, the individual can be reassured that they are not at risk of MEN2. They remain at population risk of MTC.
Reproductive options
Couples may want to explore options for having a child without inherited RET disease. These include adoption, gamete donation or not having children at all, as well as non-invasive and invasive testing.
Preimplantation genetic diagnosis uses IVF technology to create embryos that are screened for MEN2A/MEN2B or Hirschsprung disease. Only unaffected embryos are transferred to the mother. NHS funding is available for couples meeting certain eligibility criteria (e.g., the couple has no unaffected children or the female partner is <40 at the start of treatment), or couples may self-fund.
Non-invasive prenatal diagnosis (NIPD) is possible in couples where the father carries the RET variant, after developing a bespoke assay. NIPD tests for the paternal RET variant amplified from free foetal DNA, detected from 9 weeks in the mother’s blood (Macher et al. 2012).
Invasive prenatal diagnosis tests for the RET variant in a chorionic villus sample taken from the mother’s placenta from 11.5 weeks in pregnancy or in an amniotic fluid sample obtained by amniocentesis from 15 to 18 weeks in pregnancy.
Couples may decide to conceive naturally and instead seek childhood testing (including testing cord blood) to understand their child’s RET status.
Childhood testing
Childhood predictive testing for RET pathogenic variants is appropriate because children diagnosed with MEN2 are recommended to undergo pre-emptive thyroidectomy by 1 year (ATA highest-risk category) or by 5 years (ATA high-risk). There is variability in the age of MTC onset in moderate-risk families, but the factors that modify penetrance are not fully known (Machens & Dralle 2024). Advanced MTC in V804M from age 5 is reported (Shankar et al. 2021); however, if calcitonin remains normal, thyroidectomy may be delayed beyond 5 years (Fig. 2, Table 3).
New genomic testing programmes: population screening
Genomic newborn screening (gNBS) for rare disease is being piloted globally, with the promise of increasing the number of treatable early conditions that could be detected and prevented (Stark & Scott 2023). Two studies, Early Check (https://earlycheck.org/assets/Uploads/Gene-Condition-Lists/14920_Early_Check_NC_Gene_List_group1v2.pdf; accessed 26/08/2024) and Baby Screen+ (https://babyscreen.mcri.edu.au/media/lh1duxez/babyscreen-gene-list-v1-108.pdf; accessed 19/05/2024), list RET as a target gene, with Early Check reporting only MEN2B cases. Over 90% of MEN2B cases are de novo, with the highest risk of developing MTC, often in infancy. The ability to measure calcitonin as a marker of MTC development serves as a confirmatory test of disease before early thyroidectomy with curative intent (Zenaty et al. 2009). Many gNBS programme, including the UK Newborn Genomes programme (https://www.genomicsengland.co.uk/initiatives/newborns; accessed 26/08/2024), are more cautious in their approach, choosing not to include RET as a target gene in their gNBS condition list due to uncertainties in MTC/MEN2 penetrance when ascertained at the population level. gNBS programmes continue to review and update target gene lists, and therefore, inclusion of specific RET variants may change as evidence emerges (https://www.genomicsengland.co.uk/initiatives/newborns/choosing-conditions; accessed 12/01/2025). Reporting the outcomes from these studies to understand the costs and long-term ethical, legal and psychosocial challenges will be fundamental to demonstrating lessons from this new approach.
RET-altered cancers
Activating RET mutations
Medullary thyroid cancer
Somatic RET point mutations, small deletions and/or insertions are identified in ∼55% of sporadic MTCs, with M918T being the most frequent oncogenic driver (Table 4) (Ciampi et al. 2019). Patients with somatic RET variants have more severe MTC, with an increased risk of lymph node and distant metastasis compared to those with wild-type RET (Salvatore et al. 2021).
Somatic RET mutations in sporadic MTC.
| RET mutation | Frequency among somatic RET mutations in sporadic MTC (%, n = 1,193)* |
|---|---|
| M918T | 68.2 |
| C634R | 5 |
| A883F | 2.2 |
| C634W | 1.9 |
| C634Y | 1.9 |
| D898_E901del | 1.9 |
| C630R | 1.5 |
| E632_L633del | 1.4 |
| C620R | 0.9 |
| S891A | 0.7 |
| C618S | 0.7 |
| C634S | 0.6 |
| C618R | 0.5 |
| Other | 12.6 |
Somatic RET mutation frequencies are derived from the COSMIC database (https://cancer.sanger.ac.uk/cosmic; COSMIC v99, released 28-NOV-23; accessed 19/05/2024).
A total of 1,193 medullary thyroid cancer samples with identified RET mutations are listed in the COSMIC database. The top 13 mutations are listed, with M918T being the most common. Somatic variants cluster in specific exons, as seen in germline MEN2 cases, exons 10, 11 and 13–16. Mutation frequencies less than 0.5% are not listed individually.
Phaeochromocytoma
Somatic point RET mutations are identified in ∼7% sporadic phaeochromocytomas (intra-adrenal paragangliomas) (https://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=RET; accessed 19/05/2024)
RET rearrangements
RET can combine with multiple other gene partners to create a somatically acquired oncogenic fusion protein. Over 61 different RET fusion genes have been reported, usually mutually exclusive to other oncogenic drivers (Parimi et al. 2023). RET chromosomal breakpoints are commonly in introns 7, 10 or 11. The chimeric protein created from RET breakpoints retains the intracellular kinase domain. The partner genes contribute protein dimerisation domains, which constitutively activate RET signalling via multiple mechanisms (Salvatore et al. 2021). The most common RET fusion genes in solid tumours are shown in Fig. 3.
Schematic representation of RET fusions and partner genes at different solid tumour sites and recommended RET somatic testing algorithm for non-MTC solid tumours. (A) Schematic representation of somatic RET fusions and partner genes at different solid tumour sites. Frequencies of RET fusions are listed for PTC (10–20%) and non-small cell lung cancer (2%) (NSCLC), but in all other solid tumours, the frequency is very low (0–1%). RET gene breakpoints are mainly clustered in intron 11, with introns 7, 10 and exon 11 also observed. RET receptor activation occurs by aberrant expression or ligand-independent dimerisation with activation of RET. Red arrows indicate somatic RET fusions at that cancer site: skin (Spitz tumours), thyroid (papillary), NSCLC, breast and colon cancer. CCDC6, NCOA4 and KIFB are the most common fusion partners. KIFB is the most frequent fusion partner in NSCLC (83.6%) and CCDC6 in PTC (59%). Data from the COSMIC database (https://cancer.sanger.ac.uk/cosmic; COSMIC v99, released 28-NOV-23; accessed 19/05/2024). (B) Recommended RET somatic testing algorithm for NSCLC and non-MTC solid tumours. FFPE = formaldehyde-fixed-paraffin-embedded tissue. If a tissue sample is unavailable or exhausted, or if sufficient quality RNA cannot be extracted from an old sample, then a liquid biopsy can be performed to analyse circulating tumour DNA (ctDNA). NGS = next-generation sequencing. Data from Belli et al. (2021). aIf NGS is unavailable, FISH (fluorescence in situ hybridisation) or RT-PCR might also yield results, but they are less sensitive. bIf no RET fusion is detected on a liquid biopsy, then a new tissue biopsy is required to re-test for RET fusions to establish whether a RET fusion is present or not.
Citation: Endocrine-Related Cancer 32, 5; 10.1530/ERC-24-0230
Non-medullary thyroid cancer
RET fusions occur in 10–20% PTC cases, most commonly with CCDC6 or NCOA4 fusion partners (>90%) (Romei & Elisei 2012). RET fusions are more common in children with PTC, particularly after radiation exposure (Salvatore et al. 2021). The presence of RET fusions in PTC cases is associated with more aggressive tumour behaviour, with high rates of local lymph node and distant metastasis (Bulanova Pekova et al. 2023). RET fusions also occur infrequently in follicular, poorly differentiated, Hurtle cell or anaplastic TC. The identification of RET fusions in patients with advanced radioiodine-refractory TC is key to patient eligibility for targeted therapies and trials.
Lung cancer
RET fusions are found in 1–2% of NSCLC, with the KIF5B fusion partner found most frequently (Takeuchi et al. 2012).
Other solid tumours
RET fusions are detected at low frequency (0.05–1%) in multiple solid tumours (Fig. 3) (Desilets et al. 2023).
Somatic testing in cancers
Who to test
Tumour testing should be discussed with the patient, as genomic results may alter their clinical care (e.g., personalised cancer treatment). Informed consent should be sought for broad-based genomic testing, as potential pathogenic germline variants could be identified (Casolino et al. 2024).
Choosing the correct solid tumour test
The NGTD for cancer lists genomic tests commissioned for oncology management and test eligibility (https://www.england.nhs.uk/publication/national-genomic-test-directories/; version 9; accessed 26/08/2024); and iterates which individuals might benefit from testing after biopsy or surgical resection (e.g., patients with particular tumour types or with advanced/metastatic cancer) (see Supplementary Table 5 (see section on Supplementary materials given at the end of the article)). Molecular assessment is not usually required in well-differentiated TCs, as the treatment plan is based on histopathology results. However, in poorly differentiated TC, anaplastic TC and MTC, molecular testing is recommended to identify oncogenic drivers that can be targeted therapeutically, as discussed in these UK pathology guidelines (https://www.rcpath.org/static/920f6f31-f2ed-445c-a523e71c2002ff7f/67693af7-0b91-4a0a-b2a8c94150a830bd/G098-Dataset-for-the-histopathological-reporting-of-thyroid-cancerfor-publication.pdf; accessed 12/01/2025).
Testing approach for somatic RET alterations
RET alterations are best identified using massively parallel DNA and RNA sequencing from tumour samples, which can detect RET small variants or rearrangements in a histotype-agnostic manner (Belli et al. 2021). Currently, tumour-only sequencing is the standard approach, using a multigene panel to analyse multiple somatic drivers, including RET. As successful genomic analysis requires a minimum tumour cellularity of >20%, samples require pathology assessment before submission for testing. However, RNA assays, which detect RET rearrangements, may be limited by RNA quality. Analysis of circulating tumour DNA offers an alternative approach (Figs 2 and 3) (Rich et al. 2019).
Paired somatic-germline testing is currently reserved for specific indications, e.g., paediatric cancer WGS. WGS optimally requires fresh–frozen tissue (current NGTD requirements) and a paired blood sample for parallel analysis. WGS results take longer; therefore, in many cases, NGS-panel tumour testing may provide more timely results.
Somatic analysis and reporting
Somatic variant classification is a two-step process, determining both pathogenicity and actionability for each variant. Identified somatic variants are annotated as described in the ‘Genomic analysis and reporting’ section and are given an oncogenicity classification (Horak et al. 2022).
The clinical significance of the variant is assessed for actionability using established guidelines, AMP/ASCO/CAP joint consensus or ESCAT (https://www.esmo.org/scales-and-tools/esmo-scale-for-clinical-actionability-of-molecular-targets-escat; accessed 26/08/2024) (Li et al. 2017). The AMP guidelines categorise somatic variants as follows:
Tier I: variants of strong clinical significance.
Tier II: variants of potential significance.
Tier III: variants of unknown significance.
Tier IV: variants of known insignificance (benign/likely benign).
The report details the actionability of identified variants and their impact on clinical care regarding diagnostic, prognostic or therapeutic implications.
Somatic test outcomes
The following outcomes are possible:
1) Clinically significant actionable somatic variant/s detected (Tiers I and II). Variants of strong and potential clinical significance are reported along with their therapeutic implication. Standard-of-care treatments are recommended based on therapies approved by the EMA (European Medicines Agency), FDA (Food and Drug Administration) or NICE (National Institute for Health and Care Excellence). If an oncogenic RET mutation in MTC or RET fusion in a solid tumour is detected, the patient may benefit from targeted tyrosine kinase inhibitor therapy.
2) Recommendation for germline testing. A potential RET germline pathogenic variant might be present if insertions/deletions <50 bp >20%, or if a single nucleotide variant >30% variant allele frequency is detected, regardless of tumour type (Kuzbari et al. 2023).
3) Complex, or uncertain results (Tier III). Discussion at a Molecular Tumour Board is advised before the final report is issued (Berner et al. 2019).
4) Information on suboptimal results, where testing has failed or is incomplete. Recommendations for repeat testing on new samples are given.
5) No clinically significant findings. Further investigations are listed for consideration.
How to action the somatic test result
Post-test management
Clinically actionable RET somatic variant
The clinician discusses the result at a multidisciplinary meeting to consider the therapeutic implications of the recommended targeted therapy relative to other treatments, and a precision oncological plan is devised. The options are discussed with the patient, and treatment is commenced (Filetti et al. 2022).
The RET-selective inhibitors selpercatinib or pralsetinib are the current targeted therapeutic choices, as they have greater efficacy and fewer adverse events compared to multikinase inhibitors (e.g., vandetanib) (Novello et al. 2023). In addition, selpercatinib has been shown to have greater efficacy in phase III trials over standard-of-care therapy, with an overall response rate of 69.4 versus 38.8% in controls for patients with RET-positive MTC (Hadoux et al. 2023).
Selpercatinib is approved for use in Europe, the US and the UK for RET-altered TC and NSCLC. Pralsetinib is approved in the US and Europe for specific indications but not in the UK; however, this is a constantly evolving landscape.
Potential germline pathogenic variants
A variant found on tumour testing may be somatic (i.e., only present in the tumour tissue) or germline (i.e., present in every cell of an individual, including their tumour cells). Thus, if a potential RET pathogenic variant is identified, confirmatory germline testing is recommended (Kuzbari et al. 2023). This involves testing a blood or saliva sample for the specific RET variant, noting that this will require specific consent.
If a germline MEN2 RET pathogenic variant is confirmed, management is as previously described (see the ‘Care of the individual’ section above), in addition to treatment of the primary tumour.
Conclusions
Genomic testing has revolutionised care of individuals with RET-associated disease – confirming diagnoses to guide disease management, pre-emptive thyroidectomy and personalised care. Families with inherited RET disease are supported by genetic teams to access predictive and childhood testing and consider reproductive options.
However, the majority of MTC cases are sporadic, and 35% present with advanced disease, with poorer outcomes compared to hereditary disease (Saltiki et al. 2019).
The development of RET-selective inhibitors provides an opportunity for precision therapy in those proven to have RET-driven tumours, particularly MTC, PTC and NSCLC. Timely and accurate cancer genomic profiling to understand oncogenic drivers is paramount, but implementation needs to be resourced equitably for patient benefit, and barriers in implementation exist (Segall et al. 2024, Taniere et al. 2024).
NGS has transformed diagnostic genomic testing in the last decade. In the future, long-read sequencing technology and integrating multimodal data are expected to accelerate precision oncology and rare disease discovery. Adoption of these new technologies holds much promise if they can become part of routine care.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ERC-24-0230.
Declaration of interest
The author declares that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review.
Funding
This work did not receive any specific grant from any agency in the public, commercial or not-for-profit sector.
Acknowledgements
Selected artwork (breast cancer/lung cancer/thyroid cancer/chromosome/blood sample/torso/DNA helix) shown in the graphical abstract or figures was reproduced from or adapted from pictures provided by Servier Medical Art (Servier; https://smart.servier.com/), licensed under a CC-BY Creative Commons Attribution 3.0 Unported Licence or CC0 https://creativecommons.org/publicdomain/zero/1.0/ Unported Licence. The DNA sequencer image was used without adaptation, provided by Servier Medical Art (Servier; https://smart.servier.com/) licensed under CC-BY 4.0 International Deed. This work uses data that have been provided by patients and collected by the NHS as part of their care and support. The data are collated, maintained and quality assured by the National Disease Registration Service, which is part of NHS England. The author thanks Emma Duncan for her review of the manuscript and helpful suggestions, and Martina Owens for her assistance.
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