Thyroid Molecular Testing
Join us for our 31st episode on the latest advances in molecular testing, written in collaboration with Dr. Richard Payne, an otolaryngologist specializing in head and neck and thyroid cancer and a nationally recognized leader in endocrine surgery. You won’t want to miss this insightful overview.
Show Notes
Hey everyone and welcome to the OtoApproach, a podcast created by medical students for medical students to teach you about all things otolaryngology. I’m your host Mely and today we are discussing thyroid nodules and the very interesting novelties of molecular testing. This is one of those topics that shows up everywhere, from exams to real-life clinical decision-making.
Let’s start with a short scenario; You’re on ENT clinic. A 32-year-old patient with a 1.8 cm thyroid nodule… FNA comes back Bethesda III. Now what? If that question makes you pause, you’re not alone. This is where thyroid molecular testing enters the picture.
Let’s first discuss thyroid nodules.
We’ll keep this part high-yield and clinically focused.
Thyroid nodules are extremely common, with prevalence estimates ranging from 19% to 68% in the general population when assessed by high-resolution ultrasound. The prevalence is higher in women and increases with age. However, most thyroid nodules are benign, with only about 5% harboring malignancy. The majority are clinically insignificant and safely managed with surveillance rather than intervention(1–3). So right away, this sets up the core challenge: common problem, rare cancer.
Fine-needle aspiration (FNA) is the first-line diagnostic tool for evaluating thyroid nodules with concerning sonographic features or size criteria. FNA is simple, safe, and provides the most definitive diagnostic information, especially when performed under ultrasound guidance. The cytology results are classified using the Bethesda System for Reporting Thyroid Cytopathology, which stratifies nodules into six categories based on implied risk of malignancy and guides management (1,4,5). This system is something you’ll hear about constantly in clinic and on exams.
Bethesda category III (Atypia of Undetermined Significance/Follicular Lesion of Undetermined Significance) and category IV (Follicular Neoplasm/Suspicious for Follicular Neoplasm) represent the main areas of diagnostic uncertainty. These indeterminate categories account for 20–30% of all biopsies and carry a malignancy risk of approximately 10–30% (category III) and 25–40% (category IV). Most nodules in these categories are ultimately benign, and when cancer is present, it is usually nonaggressive (1,6).
The management dilemma for indeterminate thyroid nodules lies in balancing the risks of surgery against active surveillance. Historically, surgery was often pursued to obtain a definitive diagnosis; however, given the high prevalence of benign disease, this approach exposed many patients to unnecessary intervention and associated morbidity. Molecular testing has become crucial in this context, as negative molecular testing results support surveillance rather than surgery for Bethesda category III and IV nodules. Nevertheless, optimal surveillance intervals and clear criteria for repeat FNA or surgical intervention remain debated, and patient preferences play an important role in decision-making (1,3,4,6–8). And this is the exact gap molecular testing is trying to fill.
Before diving into the benefits of molecular testing in thyroid cancer, it’s helpful to briefly review the most common thyroid cancers we encounter in clinical practice. Think of this as a quick mental refresher.
The vast majority, about 80 to 85 percent, are papillary thyroid carcinomas. These tumors are most often associated with specific genetic alterations, particularly the BRAF V600E mutation, which is present in roughly 60 percent of cases, as well as RAS mutations and RET or NTRK gene fusions. Papillary thyroid cancer really dominates the landscape here.
The second most common type is follicular thyroid carcinoma. Here, the genetic landscape is different, with RAS mutations and PAX8–PPAR gamma rearrangements being most frequently observed.
Poorly differentiated thyroid carcinoma is much less common, accounting for about 5 percent of thyroid cancers, and is typically associated with more aggressive mutations such as TP53 and TERT promoter mutations.These mutations often raise red flags clinically.
Medullary thyroid carcinoma represents around 4 percent of cases and is most often driven by RET mutations, which may be either germline or somatic.
Finally, oncocytic, or Hürthle cell carcinoma and anaplastic thyroid carcinoma are rare entities and won’t be discussed further today. We’ll keep our focus on what you’re most likely to encounter.
With that overview in mind, it’s important to recognize that while many genetic alterations exist, two mutation pathways are particularly important in thyroid cancer: BRAF and RAS.
BRAF mutations, especially BRAF V600E, are most commonly seen in papillary thyroid carcinoma and are associated with the classic papillary cancer pathway; starting as papillary microcarcinoma and evolving into classic Papillary Thryoid Carcinoma. RAS mutations can also be seen in classic papillary thyroid carcinoma, as follicular variants of PTC.
RAS mutations, are also best understood as part of a biological spectrum. Lesions driven by RAS may initially appear histologically benign, but over time can progress toward malignancy. For example, RAS mutations are found in noninvasive follicular thyroid neoplasms with papillary-like nuclear features, often called NIFTP, which are slow-growing lesions typically removed surgically to prevent progression to invasive disease. RAS mutations are also seen in follicular adenomas, some of which may eventually evolve into follicular thyroid carcinoma if left untreated. In practical terms, this means that some RAS-positive tumors are surgically removed while they are still benign, but remember thay they may have progressed to cancer over time. So as you can see, a positive RAS result doesn’t give you a simple yes-or-no answer.
With this understanding of the main mutation pathways, and the idea that thyroid cancer exists along a molecular spectrum, let’s now dive into the role of molecular testing.
When we talk about molecular testing in thyroid nodules, the key question is: what is it actually trying to solve? At the end of the day, it’s about reducing uncertainty when cytology alone doesn’t give us a clear answer.
At its core, molecular testing helps refine risk stratification in indeterminate thyroid nodules, particularly when cytology alone is insufficient. Molecular testing for thyroid nodules evaluates both specific genetic mutations and gene expression profiles. Mutation analysis targets alterations such as BRAF (especially V600E), RAS, TERT promoter, RET/PTC fusions, PAX8/PPARG fusions, and TP53, among others. As mentioned earlier, these mutations are associated with different thyroid cancer subtypes and, in some cases, with benign lesions. For example, RAS mutations are found in both benign and malignant neoplasms as they are expected to develop into malignancy over time (1,4,6,9–12).
Gene expression profiling, as performed by platforms like Afirma GSC and ThyroSeq v3, evaluates the mRNA expression of a large panel of genes to classify nodules as benign or suspicious, without identifying specific mutations (1,4,6,9–12). Rather than asking “which mutation is present,” these tests ask “does this nodule behave more like cancer or not?”
An important gene expression profiling tool is next-generation sequencing (NGS), which enables simultaneous detection of point mutations, insertions/deletions, gene fusions, copy number alterations, and gene expression changes, increasing diagnostic yield (10,11,13). Both Afirma GSC and ThyroSeq v3 are examples of next-generation sequencing (NGS) for molecular testing of thyroid nodule. This ability to look at many alterations at once is what makes modern molecular testing so powerful.
Now that we understand better that molecular testing can identify oncogenic mutations, characterize gene expression patterns or combine both approaches, let’s discuss their main pros and cons.
The rule-out versus rule-in framework is central to the clinical use of molecular testing. Rule-out tests, such as NGS tools like Afirma GSC and ThyroSeq v3, are designed with high sensitivity and negative predictive value (NPV), allowing clinicians to confidently avoid surgery in patients with benign or negative molecular results. A benign Afirma GSC result or a negative ThyroSeq v3 result in a Bethesda III or IV nodule supports surveillance, with a very low risk of missed malignancy (1,4,6,7,9,10). For patients, this often means avoiding an operation they never needed.
On the other hand, rule-in tests focus on high specificity and PPV; the presence of certain mutations (e.g., BRAF V600E) is highly predictive of malignancy and can justify proceeding to surgery (1,4,13,14). These results tend to push clinicians toward more definitive intervention.
Despite RAS mutations being less specific for malignancy, as they are part of a spectrum and benign at first(1,6), management strategies are relatively well established, with conservative approaches, including active surveillance and radiofrequency ablation, being appropriate for select patients with RAS-positive tumors. This is where nuance and clinical judgment really matter.
Overall, molecular testing results directly influence the decision to operate, the extent of surgery, and patient counseling in the management of indeterminate thyroid nodules. A benign or negative molecular result supports active surveillance, reducing unnecessary diagnostic surgeries and associated morbidity (6,7,9,10). From a systems perspective, this also means fewer surgeries, fewer complications, and shorter wait times.
Conversely, positive or high-risk molecular result (e.g., BRAF, RET, TERT) favors surgical intervention and may influence the extent of surgery (lobectomy versus total thyroidectomy), especially if aggressive molecular features are present (1,4,11,12,14). So molecular results don’t just answer “operate or not,” but also “how much surgery is needed.”
Molecular findings also inform patient counseling by providing individualized risk estimates and, in advanced or refractory cases, can guide selection of targeted therapies. This is a clear example of precision medicine entering everyday clinical practice.
Despite its value, molecular testing has important limitations, including platform heterogeneity, cost, access, and challenges in interpreting RAS mutations and NIFTP, which may lead to overtreatment if not appropriately contextualized (1,6,9,11). So while the technology is impressive, it’s not foolproof.
Clinically, a benign molecular test result does not indicate zero risk; residual risk of malignancy remains (typically 3%), so ongoing surveillance is warranted (1,4,6). This is a key point to emphasize when speaking with patients.
Conversely, a positive test result does not mandate total thyroidectomy; management should integrate molecular findings with clinical, radiographic, and cytopathologic data. The American Thyroid Association recommends that molecular results may inform the extent of surgery, but do not supersede clinical judgment; lobectomy may be appropriate for low-risk profiles, while total thyroidectomy is reserved for high-risk molecular features or advanced disease (15). In other words, molecular testing informs decisions—it doesn’t replace them.
Patient counseling should emphasize the probabilistic nature of molecular results and the need for individualized management. Framing results in terms of risk, rather than certainty, helps align expectations. Overall, molecular testing is a valuable adjunct in the management of indeterminate thyroid nodules, optimizing the balance between surgical intervention and surveillance. But its greatest strength lies in how thoughtfully it’s applied.
Now, let’s dive into some novelties in the field.
The most novel areas in molecular testing for thyroid nodules with indeterminate cytology (Bethesda III and IV) include the use of NGS platforms that simultaneously analyze multiple classes of genetic alterations: point mutations, gene fusions, copy number changes, and gene expression profiles. This enables more comprehensive risk stratification and diagnostic precision (9–11,13,14).
Innovations also include the integration of machine learning algorithms for gene expression classifiers, improved detection of oncocytic and Hürthle cell neoplasms, and the use of multi-omic approaches that combine DNA, RNA, and microRNA data for more nuanced classification (9,10,16).
Now, as with everything in medicine, there are some active debates in the molecular testing field.
Firstly, few studies, including a multicenter Canadian study highlight that molecular test performance is variable across institutions, with benign call rates and predictive values influenced by local cancer prevalence and patient selection (17). This variability means that a test’s negative or positive predictive value may not generalize across populations, and institutional validation is recommended before widespread adoption (18,19). B) In other words, cancer prevalence directly affects predictive values: in populations with lower malignancy rates, the negative predictive value of a “benign” molecular result is higher, but the positive predictive value of a “suspicious” result is lower, complicating risk stratification and management decisions (1,18).
Secondly, cost, access, and equity remain major concerns. Molecular testing is expensive and not universally available or covered, limiting its use in resource-constrained settings and raising questions about cost-effectiveness and equitable care (1,11). In Québec, the Régie de l’assurance maladie du Québec (RAMQ) covers molecular testing for many indeterminate thyroid nodules. Analyses have demonstrated that this policy reduces healthcare costs by avoiding unnecessary surgeries, while also improving patient care and decreasing surgical wait times. In Alberta, a limited program covers only a small number of mutations as part of a partial rule-in strategy, without rule-out capability, which is a key component of optimal patient management. In other Canadian provinces, molecular testing is generally paid for out of pocket, creating significant barriers for patients, particularly those with limited financial resources or health literacy, who may be unable to access or fully understand the potential benefits of testing.
Thirdly, while molecular tests have reduced the rate of diagnostic surgery for indeterminate nodules, there is debate as to whether they truly reduce overtreatment or simply shift the type of surgery performed (e.g., from total thyroidectomy to lobectomy), especially when positive results are interpreted aggressively (6,9,19,21).
Overall, ongoing debates should focus on whether molecular testing should be used universally or selectively. Current consensus favors selective use in indeterminate nodules with intermediate risk, rather than routine testing for all nodules (19,21,22).
Evidence suggest that surgeons should interpret molecular results as one component of a multidisciplinary risk assessment, not as a sole determinant of management, and should recognize that a “benign” molecular result does not equate to zero risk, nor does a “positive” result mandate total thyroidectomy (1,6,18,19).
So what would be the main take home points as a medical trainee?
1. Molecular testing is useful for indeterminate thyroid nodule (Bethesda III and IV)
2. There are 2 main types of molecular tests: mutation panels and gene expression classifiers.
§ Mutation panels provide rule-in information, with high specificity for certain mutations
§ Gene expression classifiers (such as Afirma GSC) offer rule-out utility, supporting surveillance when results are benign.
3. Interpreting results at a basic level
§ a benign molecular result (high NPV) supports surveillance, but does not mean zero risk of malignancy; ongoing follow-up is required (4,9,21,22)
§ A positive result (high PPV), especially for BRAF, RET, or TERT, increases the likelihood of cancer but does not mandate total thyroidectomy; the result must be integrated with clinical, radiologic, and patient factors (9,19,20).
§ RAS mutations require nuanced interpretation due to their presence in indolent and non-malignant lesions (1,9,21)
§ Cost, access, and equity concerns should be acknowledged, as molecular testing is expensive and not universally available (9,16,21)
4. Discussing uncertainty with patients
§ It is essential to communicate that molecular testing refines, but does not eliminate, diagnostic uncertainty. A benign result reduces the likelihood of cancer but does not exclude it; a positive result increases risk but does not dictate the extent of surgery.
§ Shared decision-making should incorporate patient values, preferences, and understanding of residual risk.
5. Multidisciplinary decision-making is critical for optimal management
§ Collaboration among otolaryngology, endocrinology, pathology, and radiology ensures that molecular results are interpreted in the context of clinical, cytologic, and imaging findings, and that management is tailored to the individual patient (9,19,20,22).
In summary, molecular testing is a powerful adjunct for indeterminate thyroid nodules, but results must be interpreted within a multidisciplinary framework, with careful attention to uncertainty, patient counseling, and individualized management.
Thank you for listening to this episode of the OtoApproach. The script was developed by myself, Melysiane Marcotte, and kindly reviewed by Dr Richard Payne. The script can be found at www.theotoapproach.com where you can also sign up for our newsletter and learn more about the team!
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