The puncture result is “ambiguous”? Genetic testing is the final word!

When thyroid nodules are detected during physical examinations, the initial reaction of many people is panic. After undergoing a fine needle aspiration (FNA), if the pathological report clearly indicates “benign” or “malignant,” everyone will feel more at ease. Benign nodules only require regular observation, while malignant ones will be treated with timely surgery.

However, what is most perplexing clinically is the “ambiguous” report: “atypical cellular lesions of unclear significance (AUS/FLUS)” or “follicular tumors/suspicious follicular tumors (FN/SFN).” Choosing surgical resection, one fears that the nodule is benign and that the surgery is unnecessary; if one doesn't opt for resection, there is a fear of delaying the treatment for a malignant tumor.

In the face of this “gray area,” genetic testing has become the “golden key” to break the deadlock and assist in decision - making. Just by analyzing the remaining cells, it can help doctors and patients see the “true nature” of the nodules.

PART1

Why is the puncture result “ambiguous”?

The morphology of thyroid cells is complex. Some benign nodules (such as nodular goiter) are highly similar in morphology to certain types of thyroid cancer (such as follicular cancer), and it is difficult to accurately distinguish them only by cell morphology. According to the Bethesda thyroid Cytopathology reporting system, such conditions are classified as Bethesda Category III/IV. Among them, Bethesda category III is defined as atypical cellular lesions (AUS/FLUS) of unclear significance, with a malignancy risk of about 10% - 30%; Bethesda category IV is defined as follicular tumors or suspicious follicular tumors (FN/SFN), with a malignancy risk of about 25% - 40% [1].

In the past, for the results of these two types of examinations, doctors often could only recommend that patients repeat the puncture after a period of time. This not only increases the physical and mental burden on the patient but may also cause problems such as over - treatment (such as removal of benign nodules) or delayed treatment.

PART2

Genetic testing: Create a “molecular ID card” for nodules

The core of genetic testing is to identify specific genetic variants unique to cancer cells. If these “abnormal molecules” are detected in nodular cells, there is a high probability that the nodule is malignant; conversely, it is very likely to be benign.

01.Core "marker”: BRAF gene mutation

This is the most common and specific marker of papillary thyroid cancer (PTC, the most common type of thyroid cancer). Among BRAF gene mutations, BRAF V600E mutations are the most prevalent, accounting for over 90% of all BRAF gene mutations. Once this mutation is detected, the risk of malignancy in thyroid nodules is as high as 99.8%, and papillary thyroid cancer can basically be diagnosed. For individuals with Bethesda Type III/IV nodules, if the mutation is detected, direct surgical treatment is usually recommended [2].

02.Other key clues

In addition to the BRAF gene, there are also some other genes that can provide important information:

① T gene fusion: It is also a common and highly specific marker of PTC. It is mutually exclusive with BRAF gene mutations and is highly prevalent in children or individuals affected by radiation. For people with Bethesda Type III/IV nodules, if the mutation is detected, direct surgical treatment is usually recommended [3].

② RAS gene mutation: This is relatively unique. It can be found in both benign follicular adenomas and malignant follicular carcinomas. For Bethesda Type III/IV nodules, although it cannot be directly characterized, mutations in the RAS gene suggest that the nodules have “tumor - like properties”, and even if they are benign, there is a potential risk of malignancy. Close follow - up or preventive resection is usually recommended, rather than simply leaving them untreated [4 - 5].

③ TERT gene mutation: This is a “danger signal”. For Bethesda type III/IV nodules, if the mutation is detected, direct surgical treatment is usually recommended. If it exists simultaneously with the BRAF gene (double mutation), it indicates that the tumor is more aggressive and prone to recurrence, and a more extensive surgical scope is required [6].

④ RET gene mutation: The most common and specific marker of medullary thyroid cancer (MTC). MTC has a certain family genetic predisposition. Almost all hereditary MTC cases carry RET germline mutations, while about half of sporadic MTC cases carry RET somatic mutations. For the population with Bethesda type III/IV nodules, if the mutation is detected, different prevention and treatment programs can be used according to the risk levels of different mutation sites [3, 7, 8].

In addition, there are TP53 gene mutations, PIK3CA gene mutations, PAX8 - PPARγ rearrangement, etc., which can also be used to assist in the assessment of the benign and malignant nature of nodules.

03. The value of “exclusivity”: the meaning of negative results

If the test results of the above common driver genes are all negative, it means that thyroid nodules are most likely benign! At this time, you can safely choose to continue observation and avoid unnecessary surgery.

PART 3

Conclusion

With the advancement of technology, genetic testing has provided new perspectives for the diagnosis of the benign and malignant nature of nodules. Genetic testing for thyroid nodules is not to cause anxiety but to eliminate uncertainty. At the crossroads of “to cut or not to cut”, it is like a bright light, illuminating the way forward with molecular - level evidence (positive: decisive operation, negative: safe observation). When choosing genetic testing, it is recommended to give priority to multi - gene NGS testing (at least covering the genes mentioned above), which can not only avoid the limitations and missed detections of single - gene testing but also provide a more comprehensive analysis to better guide the formulation of follow - up treatment plans.

References

[1] Genetic Testing and Clinical Application of thyroid Cancer Guangdong Expert Consensus (2020 Edition)

[2] Thyroid Carcinoma NCCN Guidelines 2025 V1

[3] Expert consensus on RET gene detection and Clinical Application of thyroid cancer (2021 Edition)

[4] Head & neck,2022,44(6):1277-1300.

[5] The Oncologist,2013,18(8):926-932.

[6] Nat Commun,2018;9:579.

[7] Chinese expert consensus on Diagnosis and treatment of medullary thyroid cancer (2020)

[8] CSCO Guidelines for the Diagnosis and Treatment of Medullary thyroid cancer (2022)

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