DIAGNOSIS OF ENDOCRINE DISEASE: Usefulness of genetic testing of fine-needle aspirations for diagnosis of thyroid cancer

in European Journal of Endocrinology
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  • 1 Arnie Charbonneau Cancer Institute, University of Calgary Cumming School of Medicine, Calgary, Alberta, Canada
  • | 2 Departments of Oncology, Pathology and Laboratory Medicine, Biochemistry and Molecular Biology, University of Calgary Cumming School of Medicine, Universitätsklinikum Halle, Institute of Pathology
  • | 3 Departments of Medicine, Oncology, Pathology and Laboratory Medicine, Biochemistry and Molecular Biology, and Arnie Charbonneau Cancer Institute, University of Calgary Cumming School of Medicine, Calgary, Alberta, Canada

Contributor Notes

Correspondence should be addressed to R Paschke; Email: ralf.paschke@ucalgary.ca
Free access

Objective

Genetic testing is increasingly used to diagnose or rule out thyroid cancer in indeterminate fine-needle aspirations. This review evaluates the usefulness of these methods with considerations of advantages and limitations.

Design

Given the diagnostic problem associated with the increasing incidental detection of indeterminate thyroid nodules in the context of thyroid cancer overtreatment, we consider the conditions and respective necessary settings for the role of genetic testing to improve presurgical malignancy risk stratification.

Methods

We review diagnostic pathway requirements and commercially available molecular tests with their respective advantages and disadvantages and discuss the prerequisites required for local application and implementation including quality assurance for local ultrasound and cytopathology practices.

Results

Recent improvements in available molecular diagnostic tests have brought high sensitivity and specificity in initial validation studies, but whether these promising results translate to other clinical settings depends on the quality of the local thyroid nodule diagnostic pathway.

Conclusions

Genetic testing can meaningfully improve presurgical malignancy risk assessment, but more work is needed to implement and use genetic testing effectively in local settings.

Abstract

Objective

Genetic testing is increasingly used to diagnose or rule out thyroid cancer in indeterminate fine-needle aspirations. This review evaluates the usefulness of these methods with considerations of advantages and limitations.

Design

Given the diagnostic problem associated with the increasing incidental detection of indeterminate thyroid nodules in the context of thyroid cancer overtreatment, we consider the conditions and respective necessary settings for the role of genetic testing to improve presurgical malignancy risk stratification.

Methods

We review diagnostic pathway requirements and commercially available molecular tests with their respective advantages and disadvantages and discuss the prerequisites required for local application and implementation including quality assurance for local ultrasound and cytopathology practices.

Results

Recent improvements in available molecular diagnostic tests have brought high sensitivity and specificity in initial validation studies, but whether these promising results translate to other clinical settings depends on the quality of the local thyroid nodule diagnostic pathway.

Conclusions

Genetic testing can meaningfully improve presurgical malignancy risk assessment, but more work is needed to implement and use genetic testing effectively in local settings.

Invited Author’s profile

Paul Stewardson is currently a PhD Candidate in Medical Science with specialization in Cancer Biology at the University of Calgary under the supervision of Dr Markus Eszlinger and Dr Ralf Paschke. Paul has performed molecular biology research for several years in government and academic settings as well as in the biotechnology sector. His technical expertise includes genetics, genomics, virology, fermentation, food microbiology, cancer biology, molecular testing, and he is currently focused on integrated diagnostics. In the course of his doctoral research, Paul applied these technical skills to develop a molecular diagnostic test, ThyroSPEC, from a prototype to a validated test routinely used in the province of Alberta and available to patients across Canada.

Introduction

Thyroid nodules are very common in the general population; their prevalence increases with age, female sex, and adiposity (1, 2, 3). With thyroid evaluation by neck palpation, the prevalence is about 5% in iodine-sufficient regions. The frequent use of neck sonography and other imaging techniques such as CT, MRI, and fluorodeoxyglucose PET-CT produced prevalence as high as 65% (4, 5). Most of these incidentally discovered thyroid nodules are asymptomatic and have a low risk of cancer, and thyroid cancer has exceptionally high survival compared to cancer in other primary sites. Thyroid nodules have a recognized problem of overdiagnosis and overtreatment in developed countries, which results in unnecessary morbidity and an inefficient use of healthcare resources (6). Unnecessary diagnostic surgery of asymptomatic benign nodules is a common clinical outcome that may cause risk of recurrent laryngeal nerve injury, hypocalcemia, hemorrhage/hematoma, and hypothyroidism (7). A variety of diagnostic modalities may be applied to clinically define and risk stratify the nodule starting with a history and family history and physical exam, checking thyroid-stimulating hormone (TSH) and sometimes calcitonin levels, and ultrasound and if indicated, Tc scintigraphy. The diagnostic workup should follow a well-defined pathway from clinical assessment and ultrasound to fine-needle aspiration (FNA) biopsy and finally, if indicated, molecular testing for indeterminate FNA cytologies (Fig. 1). Ultrasound malignancy risk stratification according to one of the available guidelines is highly efficient for the initial malignancy risk stratification (8, 9). The American Thyroid Association (ATA) guidelines recommend that a radionuclide thyroid scan should be performed if the serum TSH is subnormal (10) whereas the American Association of Clinical Endocrinologists (AACE)/Associazione Medici Endocrinologi (AME) thyroid nodule guidelines recommends performing thyroid scintigraphy in formerly iodine-deficient regions to exclude autonomy of a thyroid nodule or multinodular goiter even when the TSH level is low-normal (11). The latter is supported by a meta-analysis of 8 studies with 2761 hot thyroid nodules that reported a pooled prevalence of hot nodules with normal TSH levels of 50% (12). Most evaluations of thyroid nodule ultrasound malignancy risk stratification systems or determinations of malignancy rates for the different Bethesda thyroid nodule FNA cytology categories or applications of molecular FNA diagnostic were performed in select secondary or tertiary care referral settings which are not applicable to a non-specialized environment (13). The workup of thyroid nodules will not only be determined by the outcome of the clinical and laboratory investigations but also most importantly by local health care settings, the level of heath care (primary, secondary, or tertiary), the incentives and restrictions within the respective health care system, and most importantly by different degrees of integration or fragmentation of the respective healthcare system and the respective thyroid nodule diagnostic pathway. The quality of each step in this pathway will have impact on all subsequent steps. Deficiencies for one diagnostic step cannot be compensated by (over)emphasizing any of the subsequent steps and expertise. For example, insufficient cytology expertise or lack of local FNA cytology quality insurance or malignancy risk data cannot be compensated by molecular diagnostics. The respective clinical endocrine, ultrasound, cytology and surgical expertise, their extent of integration in a diagnostic pathway, and its integration with the primary care setting will determine the diagnostic outcomes and the quality of care for patients with thyroid nodules or thyroid cancer. Assessment of local process quality and local diagnostic outcomes based on local healthcare quality assurance data is therefore a key requirement for any thyroid nodule diagnostic pathway.

Pathway overview

A retrospective study in Australia identified suboptimal initial surgery (overtreatment or undertreatment) in 60% of a cohort of 1239 indeterminate FNAs without molecular diagnostics (14).

Ultrasound can rule out cancer in approximately 50% of nodules with a very low 0.3% false-negative rate, whereas the nodules that are suspicious by ultrasound are referred for an FNA biopsy (15). However, malignancy risk stratification of thyroid nodules with ultrasound requires a structured evaluation and consistent description of all pertinent characteristics using a common dictionary of adjectives followed by a stratified interpretation (16). This is essential for a meaningful use of any of the currently available Thyroid Imaging Reporting and Data Systems (TI-RADS) (5, 10, 17, 18). However, it remains to be determined to what extent the respective ultrasound malignancy risk guidelines and validation studies for the different classification systems are actually applied in general practice (16). In a prospective study, the American College of Radiology (ACR) TI-RADS outperformed four other stratification systems for the reduction of the number of biopsies performed on nodules later diagnosed as benign (9). The ACR system classified more than one-half of the FNAs as deferrable, with a false-negative rate of only 2.2%. All these evaluations were performed at referral centers. Therefore, the validity of these results in the primary care setting needs further validation. Most important, the thyroid nodule malignancy risk stratification systems were developed mainly for the detection of papillary thyroid cancers, whereas the ultrasound classification of follicular thyroid or medullary thyroid carcinomas requires further study. Current efforts focus on establishing an integrated international thyroid nodule ultrasound malignancy risk classification system based on a validated lexicon and by incorporating state-of-the-art techniques (19), as well as electronic algorithmic tools (20) and machine learning approaches (artificial intelligence) with the goal to reduce the number of unnecessary FNA evaluations.

FNA biopsies are used to risk stratify thyroid nodules for risk of malignancy (ROM), with most classification systems using five or six diagnostic categories, including indeterminate categories such as follicular lesion of undetermined significance (FLUS) and follicular neoplasm (FN) in the Bethesda classification system, Thy3a and Thy3f in the British system, and TIR3A and TIR3B in the Italian system (21, 22). In practice, the published variability for the percentages of assignment of the Bethesda cytology categories vary greatly from 39% to 74% for cytologically benign results, 2–16% for malignant, 1–27% for atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS), 1–25% for follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), 1–6% for suspicious-for-malignancy, and 2–24% for a nondiagnostic result (23). Cytology is unable to detect the difference between follicular adenomas and adenomatous nodules as compared to follicular carcinomas and follicular variant of papillary carcinomas because the vascular and capsular invasion that distinguish these benign and malignant tumors cannot be determined by cytology (24). This inherent problem prevents cytology from accurately diagnosing the roughly 20% of thyroid nodules classified as indeterminate. Depending on upstream malignancy risk stratification, referral settings, etc., these indeterminate categories have an implied ROM of ~6–30% for AUS/FLUS and 10–40% for FN/SFN (10, 21) and a reported malignancy risk of 14–48% in resected indeterminate nodules (25). A large part of these malignancy risk variations are due to different referral and healthcare settings (13, 26). These data again emphasize the need for local data for the use of the respective cytology categories and their malignancy risks. Due to the high inter-observer variations in AUS/FLUS and FN/SFN diagnoses, consensus cytopathology is an effective way to reduce discrepant or indeterminate FNA cytology results (27).

To solve this diagnostic problem of cytologically indeterminate thyroid nodules, molecular diagnostic tools have been utilized for routine thyroid nodule diagnostics primarily in the United States for presurgical risk stratification, with varying levels of success. ThyroSeq v3, Afirma GSC, ThyGeNEXT + ThyraMIR, and ThyroidPrint are each tests that have published multicenter validation studies showing high sensitivity and specificity, providing rule-in and rule-out molecular diagnosis for indeterminate thyroid FNAs (28, 29, 30, 31, 32). However, even these sophisticated molecular tests have drawbacks such as dependency on quality metrics of local diagnostic pathways, insufficient long-term follow-up data, limited or conflicting independent validation, high cost, and the need for additional dedicated FNA passes (33, 34, 35, 36, 37). Despite these drawbacks, older European Thyroid Association and ATA guidelines as well as AACE (which need to be updated) supported the use of molecular testing for indeterminate cytologies (10, 11, 38). A recent 2020 guideline from the American Association of Endocrine Surgeons gave a strong recommendation based on moderate-quality evidence for consideration of molecular testing as a diagnostic adjunct for cytologically indeterminate nodules (39).

Comparison of available tests

The two molecular diagnostic tests included in Table 1 most commonly used in the United States are Afirma GSC and ThyroSeq v3, each of which reported high-quality evidence with high sensitivity and specificity in multicenter studies with ThyroSeq v3 in ten tertiary referral centers and Afirma GSC in 33% tertiary referral centers although all cytopathology diagnoses were centralized (28, 29). The Afirma GSC test uses a classifier to diagnose or rule-out malignancy according to gene expression based on RNA sequencing and utilizes the same RNA sequencing data to determine mutations and gene fusions with its accompanying Xpression Atlas Panel. The ThyroSeq v3 test uses targeted DNA and RNA sequencing to determine genetic alterations including mutations, gene fusions, gene expression, and copy number alterations to diagnose or rule out malignancy. Although the high accuracy originally reported by these tests has been confirmed in some independent validation studies, other independent validation studies have demonstrated reduced accuracy and limits to the generalizability of initial validation studies (41, 42, 43). A recent randomized controlled (albeit unblinded) trial found no significant difference in test performance between ThyroSeq v3 and Afirma GSC (44). ThyGeNEXT + ThyraMIR uses a mutation panel and a miRNA expression classifier, and ThyroidPrint uses an RT-qPCR mRNA classifier, both tests have reported similar high accuracy but lacked sufficient independent validation (30, 31, 32).

Table 1

Summary of three molecular diagnostic tests for indeterminate FNAs according to their initial validation study results.

ThyroSeq v3Afirma GSCThyGeNEXT/ThyraMIR
TechnologyTargeted DNA and RNA NGSRNA NGS (mRNA expression)Targeted NGS + miRNA expression
Coverage112 genes + >120 fusions +10 CAN + 19 genes (expression)1115 genes (expression) + mutation hotspots + fusions + LOH10 genes + 28 fusions + 10 miRNA (expression)
NPV97%96%95%
PPV66%47 %74 %
Sensitivity94%91%93%
Specificity82%68%90 %
Sample size Bethesda III + IV (n)286190178
ROM Bethesda III + IV24%24%30%
ROM Bethesda III, IV23%, 25%25%, 22%36%, 24%
Sample size Bethesda III, IV154, 93114, 7692, 86
InputDedicated pass or 1–2 drops from first pass into proprietary sample collection kitTwo dedicated passes into proprietary sample collection kitDedicated pass into proprietary sample collection kit
Follicular cell content cassetteYesYesYes
Parathyroid cassetteYesYesYes
MTC cassetteYesYesYes
Validation studySteward et al. (29)Patel et al. (28)Lupo et al. (30)

There are several other thyroid cancer molecular diagnostic tests currently with less robust validation, including miR-THYpe, Nexthyro, ThyroSPEC, ThyroSure, PTC-MA, the test by Titov et al., the test by Sponziello et al., and a number of local generic mutation panels, each of which use various genetic markers including mutations, gene fusions, miRNA expression, and/or mRNA expression (45, 46, 47, 48, 49, 50, 51). Some of these tests offset their weaker validation with unique advantages over the leading diagnostic tests in the United States, such as low cost and/or the ability to use residual FNA material from air-dried smears or liquid cytology instead of a dedicated FNA pass in a proprietary preservative solution (45, 49, 51).

Limitations of molecular testing

The negative predictive value (NPV) and positive predictive value (PPV) of molecular tests achieved in validation are not equivalent across settings since NPV and PPV vary with pre-test ROM, albeit the specificity and particularly high sensitivity of tests primarily used in the United States are permissive of ruling out malignancy across a wide range of pre-test ROMs according to Bayes’ theorem (52). However, without knowledge of local ROM in each cytology category, the NPV and PPV of molecular testing is unknown for a given local application as emphasized in Fig. 2 due to the large variability in local ROM between cytology categories (53). Compounding this dilemma, currently available molecular tests lack long-term follow-up data which are necessary to clarify the malignancy risk of nodules diagnosed as benign without surgery, further limiting the generalizability of clinical validation data to local practice (24). The best available literature on long-term follow-up does not include specific ultrasound or specific clinical data for the 93% of initially unresected thyroid nodules in the study beyond the statement that they did not identify malignancy (54), and it is not yet possible to have long-term follow-up data for the newest versions of Afirma or ThyroSeq due to their recent release. The potential impact of this lack of long-term follow-up was seen with an earlier version of the Afirma classifier, which was found to have lower NVP in post-marketing findings compared to the data published in the original clinical validation, when excluding unresected nodules from the NPV calculation (35).

Also as outlined above, the proficiency and standardization of local ultrasound and cytology practices control which nodules receive molecular diagnostics and vary widely depending on the region, especially according to the structure and volume of the local healthcare system (24). Since the implementation of molecular diagnostics, a study showed a simultaneous increase in the percentage of FNAs classified as indeterminate, potentially because cytopathologists in the study preferred to rely on molecular testing rather than strictly adhere to cytology criteria; this increased overall cost and slowed the movement of patients through the diagnostic pathway (33). This study did not include any information on ultrasound malignancy risk stratification. To be clear, if molecular diagnostics is associated with a reduction in the quality of cytology or ultrasound then the net, incremental usefulness of genetic testing will be minimal. Each part of the diagnostic pathway must be assessed and optimized through quality assurance programs to optimize local care and to obtain local malignancy data in each cytology category, for molecular diagnostics to have a quality input which genetic testing can improve on (33) (see Fig. 1).

Figure 1
Figure 1

Illustration featuring an overview of the thyroid nodule diagnostic pathway (3, 24, 40). Suboptimal screening at any step will result in a worse output in each successive step both because the wrong nodules will be selected for molecular testing and because the upstream steps in the pathway affect molecular test diagnostic NPV and PPV.

Citation: European Journal of Endocrinology 187, 3; 10.1530/EJE-21-1293

Both the local cytopathology practice and the upstream local practices for detection and selection of thyroid nodules for FNA affects the NPV and PPV of each molecular test (56) (Figs. 1 and 2). Therefore, all components of a given local thyroid nodule diagnostic pathway must be considered to determine which commercially available test, if any, is ideally suited to that setting. Relatively high pre-test ROM would make sensitivity more important than specificity, and lower pre-test ROM would allow for prioritization of specificity while maintaining the rule out objective in tests with negative FNAs. Tests that require a dedicated pass are better suited for settings where indeterminate thyroid nodules are repeated before being submitted for molecular testing, since otherwise collection of the additional pass is inefficient given that only a fraction of total patients receiving an FNA will benefit from molecular testing. However, it has been shown that restricting molecular testing to repeat indeterminate FNAs, while increasing specificity and reducing unnecessary surgeries, does reduce sensitivity resulting in some missed cancers and NIFTPs (57).

Figure 2
Figure 2

(A) Negative and positive predictive values calculated according to the local pre-test risk of malignancy (ROM) and the sensitivity (47% assumed in A) and specificity (85% assumed in (A) of a hypothetical binary outcome molecular test according to Bayes’s theorem. The vertical black dotted line represents the 26% ROM of AUS/FLUS nodules in our setting (55), and the green box represents the implied 6–30% implied ROM for AUS/FLUS nodules according to the Bethesda guideline (21). (B) Factors in a heuristic summary of malignancy risk assessment.

Citation: European Journal of Endocrinology 187, 3; 10.1530/EJE-21-1293

A single-center study of surgical management before and after introduction of molecular diagnostics showed a decline in the aggressiveness of surgical treatment with a decrease in the rate of diagnostic thyroidectomy from 67% to 35% (P = 0.015) (58). However, this decrease in surgeries is not necessarily due to molecular diagnostics since clinical guidelines (esp. ATA) emphasizing avoiding overtreatment were introduced during the same period. Another study issued a cautionary tale of increased surgical rates following implementation of genetic testing (59). Although specificity of available molecular diagnostic tests has improved since then and the results might differ with the latest generation of molecular tests, this example of molecular diagnostics exacerbating the overtreatment problem rather than mitigating it reinforces the evidence that genetic testing does not exist in a vacuum but depends largely on the context in which is it introduced (60).

Conversely, a recently published study by Colombo et al. illustrates the virtue of applying a holistic approach to implementation of genetic testing. In this single-center study, a locally validated, limited but low-cost mutation panel was applied in the context of a larger scope evaluation of the ultrasound, cytology, and other clinical data for each thyroid nodule (50). These additional diagnostic data were combined with genetic results to create a thyroid risk score, which showed promising accuracy despite the limited sensitivity of the genetic test on its own (61). The success of this approach, although producing accuracy below the initial validation studies of molecular diagnostic tests used in the United States, opens the possibility of a standardized, integrated score combining genomic classifiers with ultrasound and cytology data such as nodule size and TI-RADS category compared to the standard of care which assesses nodule features with separate scoring systems.

There are some specific weaknesses in existing molecular tests, one such gap is in the diagnosis of Hurthle cell tumors (oncocytic nodules). Because genetic alterations in Hurthle cell nodules tend to be copy number variants or mutations in the mitochondrial DNA (62, 63, 64), biomarkers for this subset of thyroid nodules are less well characterized and evidence on classifier performance in this category has been limited (65). However, the latest versions of ThyroSeq and Afirma have been designed to detect Hurthle cell carcinoma, and early results from post-validation studies have shown an improvement in the classification accuracy for this thyroid cancer subtype (66). There may be similar gaps in rare variants of thyroid carcinoma where data on the genomic profile are incomplete, such as columnar cell or hobnail variants of papillary thyroid carcinoma (PTC) whose molecular tests have not been trained on. Although the possibility of molecular tests missing rare subtypes of thyroid carcinoma highlights the need for clinical judgment in interpreting molecular test results, it is no more an indictment of molecular testing at the population level than the rare false negatives of ultrasound or cytology. Precisely because these subtypes are so rare, especially in indeterminate categories, these gaps do not pose a major dilemma for population-level performance of molecular testing.

Finally, high prices at more than $3000 USD per molecular FNA analysis and the required extra dedicated FNA samples for molecular testing are considerable barriers to adoption under a single payer or European healthcare system (46, 56). Studies of cost-effectiveness have generated mixed results, depending on the assumptions (67, 68, 69, 70, 71, 72, 73, 74). If applied as part of an optimized pathway, genetic testing does have the potential to be cost-effective, but the cost-effectiveness studies that have been published to date appear to underweight or incompletely assess the significance of upstream and downstream aspects of the thyroid nodule diagnostic pathway. Most important, cost-effectiveness of molecular FNA tests needs to be evaluated for each specific interdisciplinary diagnostic pathway setting on the basis of local costs and demographics and ultrasound and FNA performance (24). This was recently demonstrated by the first large patient cohort study pre- and post-ThyroSeq implementation with 773 consecutive patients. It reported a doubling of AUS/FLUS and FN/SFN cytologies and an increase of the overall cost of care for patients with thyroid nodules (33). This emphasizes again the importance of local outcome evaluations covering all building blocks of the diagnostic pathway for the characterization of a nodule since a doubling in the number of FNAs referred to molecular testing will substantially further worsen the price threshold at which molecular diagnostics are cost-effective.

Some of the possible strategies to improve cost-effectiveness include using a highly accurate test, not referring <1 cm nodules for FNA, reflexively performing a repeat FNA for all Bethesda III FNAs (which often are clarified on repeat FNA), restricting molecular FNA to ATA intermediate suspicion (or equivalent) cytologically indeterminate nodules where molecular diagnosis is most differential (75), and identifying patient preferences based on local performance data – before submitting FNAs for molecular testing (57). These and other strategies may decrease gross spending on molecular diagnostics, focusing molecular testing utilization on cases where it will be most likely to alter treatment decision-making and thus outcomes.

Uses other than diagnosis

Reporting mutations and gene fusions provides more granular malignancy risk stratification, possible prognostic information (76), and early stratification for molecular targeted therapies (51) unlike the black box of a gene expression classifier (52), which explains why Afirma added the Xpression Atlas to its classifier to provide this information and why ThyraMIR is offered alongside a mutation panel, namely ThyGeNEXT. A recent publication (out of the lab that developed ThyroSeq) showed that RNA sequencing missed 52% of mutations detected with DNA sequencing, and a more recent publication (77) from the lab that developed Afirma showed that RNA sequencing detected mutations in 44% of Bethesda III–IV FNAs (only ‘GSC suspicious’, n  = 16 594 nodules) (78), which compares unfavourably to over 64% of all Bethesda III–IV nodules having alterations detected by ThyroSeq v3 (n = 50 734 nodules) (79). There are two major caveats to this head-to-head comparison, namely different cytology practices influencing the average genome of nodules classified as Bethesda III–IV and the enrichment for genetic alterations in the Afirma data because the data are limited to GSC suspicious nodules. Nonetheless, the large delta strongly suggest that some mutations are missed by RNA sequencing. On the other hand, missed mutations in the Kaya et al. study were heavily concentrated in low allelic frequency mutations which may be less clinically relevant. It should be observed that Xpression Atlas is not equipped to detect prognostically important TERT mutations since TERT mutations are located in the promoter region and therefore technically impossible to detect by RNA sequencing. For comparison, ThyGeNEXT detects all common mutations and gene fusions including TERT promoter mutations, whereas ThyroidPrint detects no mutations or gene fusions which is a substantial disadvantage.

Although commercial diagnostic tests merely scratch the surface of the complexity of the underlying tumor biology, molecular testing may nonetheless provide important accessible prognostic information. This includes several independently identified prognostic biomarkers such as a TERT promoter mutation co-occurring with RAS or BRAF mutations or an EIF1AX mutation co-occurring with a RAS mutation (80, 81). TERT promoter mutations have been correlated with tumor recurrence and mortality (82) and radioiodine resistance (83). The coexistence of mutations in thyroid carcinomas has been shown to predict poor prognosis (80). Therefore, particularly for aggressive cancers, these data may improve treatment decision-making by informing the triage and extent of surgery only for prognostic markers (39), radioactive iodine administration, and for setting the frequency of follow-up, according to recurrence risk stratification by current criteria most likely complemented by mutations or gene expression classifiers (84). This has been shown in practice, with a recent matched case–control study showing accurate risk stratification of distant metastasis risk by molecular risk groups (76). Simpler methods for identifying prognostic biomarkers may be more efficient than use of a commercially available gene panel, such as single-gene sequencing of TERT or similar targeted approaches, particularly when some genomic information is already available which lessens the incremental value of a large gene panel.

Another strength of molecular tests that detect mutations and gene fusions is that following diagnosis of malignancy, such genetic alterations can inform the extent of surgery and determine patient eligibility for targeted therapies such as RET or TRK inhibitors. These highly selective and effective targeted therapies are only applicable to the subset of recurrent/metastatic thyroid cancer patients with specific driver mutations. Targeted genetic testing with appropriate clinical selection allows clinicians to identify which therapies a patient is eligible for (51).

Also, due to known limitations of histologic classification of thyroid tumors such as high rates of discordant pathological diagnosis due to inter- and intra-observer variability (85), it is being increasingly argued that the diagnostic gold standard of morphology-based histologic classification could use improvement and might benefit from incorporating genetic data into neoplasm taxonomy as has been done in breast, colon, and pituitary cancers (86, 87). A blended morphologic and genetic classification of thyroid nodules may improve the notorious diagnostic inter- and intra-observer variability present even among experienced endocrine pathologists (49, 88). More fundamentally, histology is a problematic ‘gold standard’ for the evaluation of molecular diagnostic studies, given that re-review of histology by an experienced endocrine pathologist results in re-classification of the histopathologic diagnosis in many follicular tumours (89). Over time, integrating histopathologic and genetic features has the potential to improve classification criteria and benefit patient care by facilitating increased treatment options (51).

Mutation-specific malignancy risk

Consistent data across cohorts have elucidated the malignancy risk at the level of individual mutations and gene fusions for certain high-risk genetic alterations. For example, the >98% ROM for a nodule with a BRAFV600E mutation as well as for essentially all coexisting mutations (excluding TSHR or other mutations associated with benign disease) has been well established (61, 78, 90, 91). Moreover, data show worse disease-free survival in BRAFV600E mutant carcinomas than in carcinomas with RAS-like driver mutations such as BRAFK601E, RAS, or PAX8-PPARG (84).

However, ROM is far less certain for rare mutations or fusions, or even for the most common family of mutations in thyroid nodules, namely RAS mutations. The PPV of RAS mutations in the absence of coexisting mutations has been reported from 0% to 100% in Bethesda III (91, 92). Given these discrepancies, the ETA guidelines point out the need for determining the impact in each specific setting of the detection of RAS mutations (38). Recent data continue to confirm the variable risk associated with RAS mutations, with recent publications concluding that indeterminate FNAs with RAS mutations should assess imaging, cytologic, and other clinical indications for surgery; identifying RAS mutation-positive nodules is only productive for otherwise cytologically and radiographically indeterminate nodules (90, 93). Part of the discrepancy in ROM for RAS and other mutations or fusions is due to the high inter- and intra-observer variation for the differential diagnosis of adenomas and minimally invasive FTC due to the subtle morphologic discrepancy between these entities (89). This also questions the general and unreflected use of histology as the ‘gold standard’ for the evaluation of molecular FNA diagnostics.

The specific PPV of mutations in genes including TP53, DICER1, RET, TERT, EIF1AX, PIK3CA, and CTNNB1 and gene fusions including BRAF, NTRK, RET, ALK, and PAX8/PPARG is summarized in Fig. 3, but it must be noted that a sample size of ~100 FNAs with a specific mutation is needed to refine the PPV CI to ±10%, which is only available for BRAFV600E, NRAS, and HRAS mutations. Given the lack of large-scale publications on indeterminate thyroid nodule mutations, less prevalent mutations have imprecise malignancy risk estimates (91). The inevitable imprecision of Fig. 3 points to the paucity of data and highlights the need for further research on the malignancy risk of individual mutations. There is especially a need for research on individual mutation malignancy risk stratified across variants within the same gene and stratified across geographic regions.

Figure 3
Figure 3

An approximate ROM for each mutation and fusion in cytologically indeterminate thyroid nodules from selected most informative publications (applicable sources listed next to each gene name). Sources: (29, 78, 91, 94, 95, 96, 97, 98, 99, 100, 101).

Citation: European Journal of Endocrinology 187, 3; 10.1530/EJE-21-1293

Conclusions

The clinical usefulness and cost-effectiveness of molecular diagnostic tests are dependent on the upstream and downstream diagnostic pathway and healthcare settings. A molecular test is only as good as the diagnostic pathway it is embedded within; a high-quality molecular diagnostic test depends on ultrasound and cytology malignancy risk stratification, among other clinical factors, to be properly assessed for the benefit of molecular testing to be maximized (24, 56). Guidelines-based ultrasound risk stratification for thyroid nodules and FNA cytology suffer from insufficient local data which are crucial to implement before or alongside rollout of a molecular diagnostic test in a new locale. Validation in the local setting is needed instead of importing a diagnostic test trained on a different population and different diagnostic pathway which may necessitate unique parameters for optimal benefit in the local population.

Scarcely more than 10 years since the first molecular diagnostic test became commercially available, genetic testing can undoubtedly be a significant improvement over previous standard of care for cytologically indeterminate thyroid nodules. However, to unlock the full capabilities of this technology, the current challenge is to reject the temptation of ‘plug and play’ molecular diagnostic tests. Rather, the aim must be to truly integrate molecular insights into optimized local pathways according to guidelines and best practices, with due consideration for every piece of the diagnostic puzzle, which is the only pathway to providing optimal patient care.

Declaration of interest

M Eszlinger and R Paschke receive licensing fees from ThyroSPEC. The authors have no other conflicts of interest.

Funding

This study did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

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  • View in gallery

    Illustration featuring an overview of the thyroid nodule diagnostic pathway (3, 24, 40). Suboptimal screening at any step will result in a worse output in each successive step both because the wrong nodules will be selected for molecular testing and because the upstream steps in the pathway affect molecular test diagnostic NPV and PPV.

  • View in gallery

    (A) Negative and positive predictive values calculated according to the local pre-test risk of malignancy (ROM) and the sensitivity (47% assumed in A) and specificity (85% assumed in (A) of a hypothetical binary outcome molecular test according to Bayes’s theorem. The vertical black dotted line represents the 26% ROM of AUS/FLUS nodules in our setting (55), and the green box represents the implied 6–30% implied ROM for AUS/FLUS nodules according to the Bethesda guideline (21). (B) Factors in a heuristic summary of malignancy risk assessment.

  • View in gallery

    An approximate ROM for each mutation and fusion in cytologically indeterminate thyroid nodules from selected most informative publications (applicable sources listed next to each gene name). Sources: (29, 78, 91, 94, 95, 96, 97, 98, 99, 100, 101).

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