The Expert Panel had a high level of consensus throughout the process and in the final algorithms. Areas of discussion highlight the aspects of thyroid cancer molecular testing and care which need to be addressed and improved.

Actionable Biomarker Testing to Aid in the Management of Patients Diagnosed with Medullary Thyroid Cancer (MTC)

A stepwise approach to germline and tumor/somatic testing specifically for RET variants needs to be conducted for those diagnosed with MTC (Fig. 1).

Fig. 1

Actionable biomarker testing to aid in the management of patients diagnosed with medullary thyroid cancer (MTC). White/clear shading represents the patient’s clinical diagnosis/stage; orange shading represents molecular test results. Dark blue shading conveys a necessary action for molecular testing or clinical provision, while light blue shading conveys a recommended action. Solid black arrows indicate a necessary pathway; dashed black shading represents “if/when” pathways. Abbreviations: P/LP, pathogenic/Likely pathogenic; VUS, variant of unknown significance

Recommendation 1: Constitutional (germline) Testing for RET Variants Is Required for All Patients at the Initial Diagnosis of MTC

Constitutional (germline) testing for RET variants is necessary to identify patients with MTC who have a familial cancer syndrome [6, 28, 29]. Although most patients with RET germline variants have a pre-existing familial cancer history, a small number (up to 10%) of patients presenting with sporadic MTC have de novo germline variants, which can subsequently be passed down to family members [17, 28, 29]. Recommended testing methods for RET variants are next-generation sequencing (NGS) or polymerase chain reaction (PCR) (Table 1). Pre-test genetic counselling is required for patients receiving germline testing, to obtain appropriate informed consent. This can be facilitated by the treating physician, who can also request germline testing on a blood sample. This process is known as mainstreaming, which refers to the incorporation of genetic testing into the standard practices of clinical care [49]. Alternatively, the patient can be referred to a clinical genetics service for pre-test counselling and ordering of testing: in some jurisdictions, there can be a long wait to access these services, and treatment should not be delayed while waiting for test results [28].

Table 1 Methods for detection of actionable biomarkers in thyroid cancerRecommendation 2: Post-test Counselling by Clinical Genetics Is Necessary for All Patients with A Germline Pathogenic/Likely Pathogenic (P/LP) RET Variant and Recommended for All Those with a Germline Variant of Unknown Significance (VUS).

For those with a germline RET P/LP variant, post-test counselling by clinical genetics is necessary to explore the presence of MEN syndromes and to initiate cascade testing of family members, consistent with guidelines [17, 28, 29, 31]. Post-test counselling is also recommended for patients with a germline RET variant of unknown significance (VUS) as these patients may require additional follow-up. A more detailed explanation of variant classifications of P/LP/VUS (for germline variants) and Tier1/2/3 (for somatic variants) is included in Supplement 2.

Patients who have RET P/LP germline testing results do not require further tumor/somatic testing for RET variants. These patients can be considered for selective RET-targeted therapy based on germline testing results, since the germline variant will also be present in the tumor tissue.

Recommendation 3: Cascade Testing Must Be Offered to Family Members of Those Who Have a Constitutional (Germline) RET P/LP Variant

Cascade testing helps to identify those who have familial MTC syndromes, prior to the emergence of clinical symptoms of thyroid and other endocrine cancers. This aids in clinical management of these patients and also identifies those who may be candidates for targeted therapy, consistent with published guidelines [28, 31].

Recommendation 4: Somatic Tumor Tissue Testing for RET Variants Is Required for Those with Advanced Disease, with Unknown or Negative Germline RET Variant Status

Approximately 75–80% of all cases of MTC occur sporadically [6, 28], and of these, approximately 50% have somatic RET variants [6, 31], but this number may be even higher in patients with distant metastases [6]. Somatic tumor tissue testing for RET variants is required when patients who have germline RET VUS, or with no variants identified, progress to metastatic/recurrent/progressive/inoperable MTC, especially when targeted therapy is being considered. This is consistent with published guidelines [6, 17, 50]. Tumor/somatic testing is also required for patients with advanced disease whose germline status is pending, as the turnaround time for germline testing can be lengthy in some geographic regions, and the results of the testing are needed to inform treatment decisions. As RET variants are identifiable on testing of DNA (single nucleotide variants or small insertions/deletions) or RNA (fusions), the recommended testing method for RET variants in MTC is NGS or appropriately designed PCR assays (see Table 1 for details) [29]. Clinicians are advised to become familiar with the different testing modalities that are available in their local laboratories.

Recommendation 5: Tumor/Somatic Testing for Other Targetable Alterations Is Recommended

Gene variants other than RAS or RET make up only a very small proportion of those identified in sporadic MTC. Further tumor/somatic testing for other targetable alterations is recommended for all thyroid cancer patients with advanced disease who have no variants identified or Tier III results from tumor testing, including BRAF and ALK [6].

Table 1 compares testing methods for these biomarkers. IHC (when validated using rigorous protocols) and PCR are both recommended for BRAF p.V600E testing, with IHC providing the most rapid results. NGS is a preferred approach for simultaneous multigene testing such as for BRAF p.V600E, ALK and NTRK fusions. While DNA-based NGS is needed to detect variants such as BRAF p.V600E, RNA-based NGS has high sensitivity and specificity for fusions while also confirming presence of predicted fusion transcripts. DNA-based NGS has moderate sensitivity and moderate to high specificity for fusions due to difficulty of coverage of all intronic regions at the DNA level, and lack of fusion transcript confirmation [29]. Unfortunately, NGS is often not available or reimbursed in some jurisdictions and takes up to 2–3 weeks in Canadian centers. FISH or RNA-based NGS are also recommended for stand-alone ALK testing, with IHC and RT-PCR as alternatives for detecting these fusions. NTRK fusions are rare, consisting of only about 2% of gene variants in thyroid cancer [35], and a common approach to stand-alone NTRK fusion testing is an initial screen using pan-TRK IHC which is then followed by RT-PCR or RNA-based NGS [35, 36]. Where multi-gene NGS testing is already being done, consideration could be given to including NTRK gene fusion testing in the panel, which avoids the need for an initial screen by IHC.

Constitutional (Germline) Testing in Patients Diagnosed with Non-Anaplastic Follicular Cell-Derived Thyroid Cancer

Constitutional testing is necessary for patients diagnosed with non-anaplastic follicular cell-derived thyroid cancer who have a clinical history or specific pathological or biomarker features consistent with a potential germline pathogenesis. Pathological indicators may include the following: (1) multiple cellular follicular cell-derived thyroid nodules (benign and/or malignant) with microfollicular growth, in association with immunohistochemical global phosphatase and tensin homolog (PTEN) loss in all nodules, in PTEN-hamartoma syndrome; (2) PTEN-hamartoma syndrome-like histological features with global loss of succinate dehydrogenase complex subunit B (SDHB) immunohistochemistry in one or more than one nodule, in PTEN-like syndrome and SDH-deficiency syndrome; (3) follicular nodular disease including those with involuted areas or multiple follicular adenomas with papillary architecture and synchronous follicular patterned differentiated thyroid carcinomas and/or non-invasive follicular thyroid neoplasm with papillary-like nuclear features, and background thyroid parenchyma involutional changes in the setting of normal thyroid-stimulating hormone (TSH) levels, in DICER1 syndrome; and (4) multiple follicular adenomas with papillary architecture in association with differentiated thyroid carcinomas and suppressed TSH levels in Carney complex (due to germline PRKAR1A variant) and McCune-Albright syndrome (due to somatic GNAS mosaicism) [9, 60]. A pathologically distinct entity known as “cribriform-morular thyroid carcinoma” can be a harbinger of familial adenomatous polyposis (FAP) syndrome. Cribriform-morular thyroid carcinomas (formerly known as a subtype of papillary thyroid carcinoma) are no longer considered to be of follicular cell origin; thus, they are classified as primary thyroid carcinomas of uncertain cytogenesis [1, 2, 26, 60]. However, any patient with cribriform-morular thyroid carcinoma requires germline APC testing [1, 60]. Constitutional testing is also necessary for those with syndromic manifestations suspicious for other hereditary cancer syndromes not included above or in Table 1 [9, 32, 60, 61] (Fig. 2). The same holds true for those who have two or more first-degree relatives with papillary thyroid carcinoma or three or more first-degree relatives with follicular cell-derived thyroid cancer [28, 30, 60] (Fig. 2, Table 2).

Table 2 Syndromic and non-syndromic non-medullary thyroid carcinomas [9, 60, 62,63,64,65,66,67,68] Fig. 2

Constitutional (germline) testing in patients diagnosed with non-anaplastic follicular cell-derived thyroid cancer. White/clear shading represents the patient’s clinical diagnosis/stage; orange shading represents molecular test results. Dark blue shading conveys a necessary action for molecular testing or clinical provision, green shading represents an action to be considered for clinical provision, and grey shading represents no further action needs to be taken. Solid black arrows indicate a necessary pathway, a solid grey arrow indicates no further action, and a dashed grey arrow indicates a pathway to be considered. Abbreviations: P/LP, pathogenic/likely pathogenic; VUS, variant of unknown significance

Patients who meet the criteria for both germline testing and tumor testing (Fig. 3) should receive tumor testing first, with follow-up germline testing for those with P/LP results to determine whether the variant is present in the germline.

Fig. 3

Tumor testing of actionable biomarkers in patients diagnosed with non-anaplastic follicular cell-derived thyroid cancer. White/clear shading represents the patient’s clinical diagnosis/stage; orange shading represents molecular test results. Dark blue shading conveys a necessary action for molecular testing or clinical provision; green shading represents an action to be considered for clinical provision. Solid black arrows indicate a necessary pathway, and a dashed grey arrow indicates a pathway to be considered. Abbreviations: VUS, variant of unknown significance

Recommendation 6: Selection and Eligibility for Constitutional Germline Testing in Patients Diagnosed with Non-Anaplastic Follicular Cell-Derived Thyroid Cancer Will Most Likely be Determined by Multidisciplinary Team Members Including the Pathologist, the Treating Clinician, and Genetics Provider

While the majority of non-anaplastic follicular cell-derived thyroid cancers arise from sporadic mutations, about 3–9% result from familial non-medullary thyroid cancers (FNMTC) [9, 28, 61]. FNMTC can be more aggressive than the sporadic form [28]. Only 5% of FNMTC have well-characterized driver mutations, and the histological and molecular characteristics are still not as well defined as in hereditary C-cell neoplasia [9, 60]. Identification of patients with FNMTC is necessary to facilitate genetic testing of family members and to initiate surveillance for associated malignancies as appropriate [61].

In general, patients eligible for germline testing include those whose tumors have pathological and/or biomarker features indicating a need for germline testing or who have syndromic manifestations [1, 9, 60, 61] and/or two or more first-degree relatives with thyroid cancer [1, 9, 28, 30, 32, 60, 61]. For those with syndromic manifestations (where non-thyroid neoplasms dominate the clinical manifestations), clinical and pathological features often guide gene testing. As the histopathological findings of those without syndromic manifestations are usually non-specific [9, 60], important indicators for genetic screening and counselling include the 2022 WHO selection criteria which require exclusion of a non-syndromic non-medullary thyroid carcinoma (unassociated with ionizing radiation or hereditary cancer syndrome) when one of two rigid criteria are met: (a) at least three first-degree relatives with follicular cell-derived thyroid carcinoma or (b) the presence of papillary thyroid carcinoma in two or more first-degree relatives [1, 60]. In addition, the documentation of follicular nodular disease/multinodular goiter in at least three first- or second-degree kindreds of an index patient with a differentiated thyroid carcinoma (often PTC) is also considered among experts [9]. Table 2 summarizes key well-defined syndromic conditions (as recognized in the 2022 WHO classification) typically conferred by rare variants as well as susceptibility genes and loci that may be more common than in nature [9, 60, 62,63,64,65,66,67,68]. Apart from CHEK2 and POT1, most of the candidate genes and loci are not yet available or actionable for routine clinical use. Knowledge of these associations will be important with the increasing breadth of genetic testing modalities such as genome-wide sequencing approaches that may help to explain some portion of familial disease either independently or through use in polygenic risk scores. Although traditionally not being recognized as components of syndromic non-medullary thyroid carcinoma, recent evidence has raised the link between non-medullary follicular cell-derived thyroid carcinoma and other cancer syndromes (e.g., Li-Fraumeni syndrome, Lynch syndrome) [62,63,64,65,66,67,68]. Patients with germline variants may still develop neoplasms unrelated to their genetic predisposition or additional actionable molecular alterations; therefore, considerations for somatic testing algorithms are applicable regardless of germline status (see recommendation 9).

Recommendation 7: Post-test Counselling by Clinical Genetics Is Required for All Patients with a Pathogenic/Likely Pathogenic (P/LP) Constitutional (Germline) Variant and Can Be Considered for All Those with a Variant of Unknown Significance (VUS)

The standard practice for patients with P/LP variant results from germline testing is for them to receive post-test genetic counselling [17, 28, 29, 31]. Post-test genetic counselling can be considered for those with VUS gene alterations, particularly if the VUS is in a gene with known relevance to thyroid cancer management. For VUS occurring in genes without known relevance to targeted therapy, a consultation with clinical genetics may be less urgent, less necessary, or even less useful. A more detailed explanation of P/LP/VUS is included in Supplement 1.

Recommendation 8: Cascade Testing Must Be Offered to Family Members of Those Who Have a Germline P/LP Variant

Cascade testing is also recommended for family members of those who have germline P/LP variants, in accordance with standard practices [