An economically efficient strategy for diagnosing atypia of undetermined significance or follicular lesion of undetermined significance thyroid nodules with ultrasound-based risk stratification systems and BRAF^V600E testing

Introduction

Fine needle aspiration (FNA) biopsy is the most accurate and cost-effective method for the preoperative evaluation of thyroid nodule properties (1). However, although FNA can obtain satisfactory specimens, there are still cases where nodules cannot be definitively diagnosed (2). The Bethesda System for Reporting Thyroid Cytopathology (BSRTC) refers to atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS), and this phenomenon is challenging for both pathologists and surgeons (3). The incidence and malignancy rates of AUS/FLUS vary widely in recent studies, ranging from 0.8% to 28% and from 6% to 48%, respectively (2,4,5). Currently, there is no consensus on a standardized clinical approach for the treatment of these nodules (6). The 2015 American Thyroid Association (ATA) guidelines recommend repeat FNA (7). However, rediagnosis of AUS/FLUS nodules occurs in 20% to 32% of patients (8), and repeated FNA does not necessarily improve the malignancy rate of these nodules (8,9). This has led to clinical controversy regarding the management of AUS/FLUS nodules.

Ultrasound (US)-based risk stratification systems (RSSs) have played a crucial role in standardizing the treatment of thyroid nodules and are widely recognized and applied in clinical practice. However, there is a lack of agreement regarding the significance of AUS/FLUS nodules (10,11). The BRAF^V600E gene mutation is commonly found in papillary thyroid carcinoma (PTC) and serves as a widely used molecular marker for detecting PTC in clinical practice (12). Studies have shown that the BRAF^V600E gene mutation exhibits high specificity and general sensitivity for diagnosing PTC (13,14). In some studies, the combination of BRAF^V600E and RSSs was used for the diagnosis of thyroid nodules (15,16). The general approach involved performing a BRAF^V600E test on all thyroid nodules and diagnosing those with positive BRAF^V600E or RSSs results as positive while diagnosing those with negative results for both markers as negative. However, since BRAF^V600E testing can be costly, it may impose a financial burden on patients, especially those from economically disadvantaged backgrounds. Hence, is not clear whether it is necessary to conduct BRAF^V600E testing on all nodules. To clarify this issue, we examined a substantial sample size to investigate the most effective approach for managing patients with AUS/FLUS nodules. We present this article in accordance with the STARD reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-23-1066/rc).

Methods

Patients

The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the Scientific Research and Clinical Trials Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No. 2022-KY-0974-001). The requirement for informed consent was waived due to the retrospective nature of the study, specifically with the sole purpose of conducting secondary analysis on existing data.

A total of 1,408 consecutive patients with 2,097 thyroid nodules initially diagnosed as AUS/FLUS after US-FNA from February 2019 to May 2023 at the First Affiliated Hospital of Zhengzhou University were enrolled in this study. After the exclusion criteria were applied, 762 nodules in 551 patients were examined in the final analysis (Figure 1). The exclusion criteria were as follows: (I) incomplete preoperative US images; (II) unclear results of BRAF^V600E gene testing; (III) inconclusive postoperative histopathology, including noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP); and (IV) cases with multiple nodules in one gland, where the consistency of US images, preoperative FNA, and postoperative pathological results could not be determined.

Figure 1 Flowchart of participant inclusion. AUS/FLUS, atypia of undetermined significance or follicular lesion of undetermined significance; US-FNA, ultrasound fine needle aspiration.

US examination and image interpretation

Each examination was conducted by one of three highly experienced US specialists in thyroid imaging, each of whom had more than 10 years of expertise. Each US examination was conducted within half a month before the surgical procedure. The examinations were performed using Aplio 300 or 500 (Toshiba Corporation, Tokyo, Japan) US machines equipped with a 5 to 12-MHz linear array transducer. To assess the US features of the nodules, two additional sonographers with 8 and 11 years of expertise in analyzing thyroid US images were recruited. Their responsibilities included evaluating the nodules’ characteristics, such as location (left, right, isthmus region), size, composition (solid or almost solid, mixed cystic and solid, cystic, spongiform), echogenicity (hyperechoic, isoechoic, hypoechoic, markedly hypoechoic), shape (taller-than-wide, wider-than-tall), margin (smooth, ill-defined, irregular or lobulated, extrathyroidal extension), and echogenic foci (punctate echogenic foci, macrocalcification, peripheral calcifications, comet-tail artifacts).

The nodules were then classified into different risk categories based on the ATA guidelines (Figure 2) and American College of Radiology (ACR) Thyroid Imaging Reporting and Data System (TIRADS) (Figure 3) (17). The ACR-TIRADS system encompasses categories 1 through 5, while the ATA guidelines, while not explicitly employing the term “TIRADS”, classifies thyroid nodules into five groups: benign, very-low suspicion, low suspicion, intermediate suspicion, and high suspicion. For ease of expression, we refer to them as categories 1 to 5.

Figure 2 American Thyroid Association guidelines. *, suspicious features including irregular margins (infiltrative, microlobulated), microcalcifications, taller-than-wide shape, rim calcifications with small extrusive soft tissue component, and evidence of extrathyroidal extension. ATA, American Thyroid Association.

Figure 3 American College of Radiology Thyroid Imaging Reporting and Data System. *, cystic and spongiform nodules receive 0 points in total without addition of points for other categories. ACR, American College of Radiology; TIRADS, Thyroid Imaging Reporting and Data System; TR, Thyroid Imaging Reporting and Data System category.

The hired sonographers were blinded to the cytological results, postoperative pathology, and BRAF^V600E gene results of the nodules. In cases where there were differences of opinion between the two sonographers, the final decision was made by the third specialist, who had 33 years of experience in thyroid imaging. Prior to evaluating the US features, a training session involving 20 representative thyroid nodules was conducted to improve expertise and ensure consistent and dependable classification by the participating sonographers. The unclassified (NC) nodules that did not fit into any of the categories outlined in the ATA guidelines were categorized as having intermediate suspicion (18,19).

US-guided FNA and BRAF^V600E mutation analysis

US-FNA was performed by one of three physicians with more than 11 years of work experience using a EPIQ 7 (Philips, Amsterdam, The Netherlands) system equipped with a 5 to 12-MHz line array transducer. The procedure began with disinfection the neck area, which was then covered with a sterile towel to maintain a sterile field. The physician used US guidance to carefully insert a 23 G disposable puncture needle into the nodules and collected the specimens. The collected material was then injected into a specimen tube with a liquid-based cell preservation solution. The needle was rinsed in the preservation solution to ensure all residual cells were transferred. To ensure accuracy and consistency, the diagnosis of nodules classified as AUS/FLUS was confirmed by two independent pathologists following the criteria outlined in the 2017 Bethesda system (3).

DNA samples were extracted from FNA samples using a DNA Kit (ADX-FF01 DNA Kit, AmoyDx, Xiamen, China). The BRAF^V600E mutation was assessed using the Human BRAF^V600E Gene Mutation Detection Kit (ADx-BR02, AmoyDx) according to the manufacturer’s instructions. Real-time fluorescence polymerase chain reaction (PCR) was used as the detection method, and the ABI StepOne or ABI Sequence Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was employed as the detection instrument.

Combined approaches

In method 1, all nodules underwent BRAF^V600E testing, and those found positive in the BRAF^V600E gene testing or RSSs were diagnosed as malignant. If both were negative, the diagnosis was benign.

In method 2, according to the malignancy rate of each category, the RSSs were reclassified into a low-risk group (category 2 and 3 in the ATA guidelines and ACR-TIRADS), medium-risk group (category 4), or high-risk group (category 5). BRAF^V600E gene testing was applied only in the medium-risk group. Nodules with positive BRAF^V600E mutation were upgraded to the high-risk group, while the negative ones in the medium-risk group remained unchanged (Figure 4).

Figure 4 The new combined RSS and BRAF^V600E strategy for AUS/FLUS nodules. AUS/FLUS, atypia of undetermined significance or follicular lesion of undetermined significance; RSS, risk stratification system; ATA, American Thyroid Association; ACR, American College of Radiology; FNA, fine needle aspiration.

Statistical analysis

The BRAF^V600E gene test rate (BGR) was considered to be the proportion of nodules that underwent BRAF^V600E gene testing. The false-negative rate (FNR) was considered to be the proportion of malignant nodules misdiagnosed as benign among all nodules. The false-positive rate (FPR) was considered to be the percentage of benign nodules incorrectly diagnosed as malignant among all the nodules.

The statistical analysis was conducted using SPSS 26.0 software (IBM Corp., Armonk, NY, USA) and MedCalc 18.2.1 software (MedCalc Software Ltd, Ostend, Belgium). Continuous data were presented as the mean ± standard deviation (SD) and were compared using the independent two-sample t-test. Categorical data were compared using the Chi-squared test or Fisher exact test. Receiver operating characteristic (ROC) curves were constructed, and the area under the curve (AUC) was compared with the z test or DeLong method. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, BGR, FNR, and FPR, along with their 95% confidence intervals, were calculated and compared using the McNemar or Pearson Chi-squared test. A two-sided P value of <0.05 was considered statistically significant.

Results

Basic characteristics of AUS/FLUS nodules

Of the 762 AUS/FLUS nodules in the 551 patients included in this study, 225 were benign and 537 were malignant. Of the 225 benign nodules, 144 were nodular goiters, 31 were adenomatous goiters, 24 were follicular adenomas, 11 were Hashimoto thyroiditis nodules, 6 were subacute thyroiditis nodules, and 9 were others. Of the 537 malignant nodules, 505 were PTCs, 11 were follicular thyroid carcinomas, 5 were medullary thyroid carcinomas, and 16 were others. Among the patients included, 135 were male and 416 were female. The age range of the patients was 7–84 years old, with an average age of 41.2±11.4 years old. No significant differences in sex, age, or location were found between those with benign and those with malignant nodules (P>0.05; Table 1). Notably, the size of the benign nodules group was significantly larger than that of the malignant nodules group (P<0.001).

Malignant nodule proportions and BRAF^V600E mutation rates in the RSS categories

Significant differences were observed in the malignancy rate across the ATA guidelines and ACR-TIRADS categories (P<0.001). Similarly, the BRAF^V600E mutation rate also exhibited significant differences among these categories (P<0.001, except for ATA category 2). Both malignancy rates and BRAF^V600E mutation rates increased with a higher category (Table 2).

Diagnostic value of BRAF^V600E and the two RSSs

The positive BRAF^V600E mutation rate was found to be 50.9% in all AUS/FLUS nodules and 70.9% in malignant nodules. BRAF^V600E exhibited high specificity (96.8%) with moderate sensitivity (70.9%).

According to the ROC curves, the best cutoff was category 5 for ATA guidelines and ACR-TIRADS (Figure 5). There were no statistically significant differences in sensitivity, specificity, PPV, NPV, or accuracy between the ATA guidelines and ACR-TIRADS (P>0.05). Additionally, although the AUC of ACR was higher than that of ATA, this difference did not reach statistical significance (P=0.069) (Table 3).

Figure 5 ROC curves of the two RSSs, BRAF^V600E mutation, and the combination approaches. ROC, receiver operating characteristic; RSS, risk stratification system; ATA, American Thyroid Association; ACR American College of Radiology; TIRADS, Thyroid Imaging Reporting and Data System.

Diagnostic value of the different combinations

Combining the RSSs with BRAF^V600E testing significantly improved the sensitivity, NPV, and accuracy compared to the RSSs or BRAF^V600E testing alone (all P values <0.05). However, the specificity and PPV were lower when compared to BRAF^V600E alone (all P values <0.05), but there were no significant differences compared to using a single RSS (all P>0.05). ACR combined with BRAF^V600E demonstrated a significantly higher AUC compared to using ACR or BRAF^V600E alone (the AUCs for ACR combined with BRAF^V600E, modified ACR combined with BRAF^V600E, ACR alone, and BRAF^V600E alone were 0.875, 0.878, 0.832, and 0.839, respectively; P<0.05 for both combinations vs. ACR or BRAF^V600Ealone). Meanwhile, ATA combined with BRAF^V600E had a significantly higher AUC compared to using ATA alone (the AUCs for ATA combined with BRAF^V600E, modified ATA combined with BRAF^V600E, and ATA alone were 0.851, 0.846, and 0.809, respectively; P<0.001 for both combinations vs. ATA), but no difference was observed compared to using BRAF^V600E alone (P=0.450 and P=0.680 for both combinations vs. BRAF^V600E). Additionally, the AUC of ACR combined with BRAF^V600E was significantly higher than that of ATA combined with BRAF^V600E (P=0.047 and P=0.007) (Table 3).

The two combined methods showed no statistically significant difference in diagnostic performance (P=0.428 for ACR combined with BRAF^V600E and P=0.314 for ATA combined with BRAF^V600E). Method 2 (modified combination method) significantly reduced the BGR compared to method 1 (both P values <0.001), but there was no statistically significant difference in FNR (P=0.818 and P=0.394 for ACR and ATA, respectively) or FPR (P=0.908 and P=0.916 for ACR and ATA, respectively) between the two methods (Table 4).

Discussion

Thyroid nodules classified as AUS/FLUS have historically presented a significant diagnostic and therapeutic challenge in clinical practice. Our findings revealed that the malignancy rates for category 2 nodules in the ATA guidelines and ACR-TIRADS ranged from 0% to 3.1%, suggesting that additional BRAF^V600E testing would be unnecessary. In fact, no positive BRAF^V600E mutations were detected in this category. For category 3 nodules in the ATA guidelines and ACR-TIRADS, the malignancy rates ranged from 22.2% to 22.8%, with positive mutation rates ranging only from 7.6% to 8.9%. It is worth noting that the majority of low-risk nodules underwent FNA due to their larger size. However, in cases where nodules lack suspicious US features, it is advisable to refrain from using BRAF^V600E testing, as it does not aid in identifying non-PTC malignancies, such as follicular thyroid carcinomas and poorly differentiated thyroid carcinoma (20). Therefore, we recommend a more cost-effective follow-up approach for managing low-risk nodules.

In the high-risk group, the malignancy rates were found to be as high as 90.2–92.0%. Due to the limited sensitivity of BRAF^V600E alone in AUS/FLUS nodules (21), the utility of BRAF^V600E was considered less valuable compared to that of the RSSs in such cases. It is worth mentioning that a negative BRAF^V600E result could not definitively exclude a malignant diagnosis. However, combining BRAF^V600E with the RSSs for the high-risk group yielded significant benefits. For these nodules, it is recommended to consider more aggressive treatments, such as surgical intervention.

The diagnostic capabilities of the RSSs have been found to be limited for medium-risk nodules. To address this, BRAF^V600E testing has been used to aid in the identification of malignant nodules. Given the high specificity of BRAF^V600E, the detection of a positive mutation indicates a high likelihood of PTC (21). Therefore, in this study, combining RSSs with BRAF^V600E allowed for the screening of malignant nodules. Nodules with a positive BRAF^V600E mutation were upgraded to the high-risk group, while those with a negative mutation remained in the medium-risk group. For this subset of nodules, follow-up or repeat FNA could be advised based on the patient’s preference. Compared to testing all nodules with BRAF^V600E, performing BRAF^V600E testing only on the medium-risk group resulted in a significant 69.2–72.8% reduction in BGR in AUS/FLUS nodules. Importantly, although there was a slight increase in FNR, it was not statistically significant (P=0.818 and P=0.394 for ACR and ATA, respectively). This reduction in BGR alleviated the financial burden on patients and avoided wasting medical resources. The study findings suggested that combining ACR with BRAF^V600E can provide superior diagnostic performance compared to the use of ATA alone. The observed superiority could be attributed to ACR’s higher specificity, which led to a lower FPR when there was a direct classification of category 5 in the ATA guidelines and ACR-TIRADS for the high-risk group. Therefore, combining ACR with BRAF^V600E appears to be a more appropriate approach for AUS/FLUS nodules.

There were 45 NC nodules according to the ATA guidelines. However, their malignancy rate of 51.1% was similar to that of the intermediate suspicion group (48.1%). As a result, we classified them as intermediate suspicion nodules. Subsequently, the sensitivity, specificity, PPV, NPV, accuracy, and AUC were determined to be 77.3%, 80.0%, 90.2%, 59.6%, 78.1%, and 0.809, respectively. When the NC nodules were excluded, the corresponding values became 80.7%, 77.8%, 90.2%, 61.5%, 75.2%, and 0.812, respectively. There was no significant difference observed between the two definition methods (P>0.05). This is consistent with the study by Kuru et al. (19), in which a similar risk of malignancy was found in the NC group (63%) compared to the intermediate suspicion group (57%). Consequently, they suggested that the NC group should be included in the intermediate category of the ATA guidelines.

The sensitivity and specificity of BRAF^V600E in this study were consistent with those reported previously (13,14). It should be pointed out that BRAF^V600E gene mutations were detected in seven benign AUS/FLUS nodules in our study. Among these, two nodules were diagnosed as atypical hyperplasia, while the remaining five were nodular goiters. Similar findings were reported by Chung et al. (22), who described a benign nodule with a positive BRAF^V600E mutation. This particular nodule was initially classified as indeterminate for PTC based on FNA, but subsequent postoperative pathology revealed atypical hyperplasia. This suggests that atypical hyperplasia may represent a precancerous thyroid lesion, potentially leading to false-positive BRAF^V600E results. Among the five nodular goiters in our study, two surgical specimens were negative for the BRAF^V600E gene mutation, while the remaining three cases were not evaluated for the BRAF^V600E mutation after surgery. A similar case was reported by Yin et al. (23), where cytology indicated a positive result, but histology did not detect the BRAF^V600E mutation. In such cases, it is speculated that there may be small and indolent papillary carcinomas present, which could be missed even with careful examination. Additionally, other studies have reported BRAF^V600E mutations in benign nodules, and the reason for this could be attributed to oversensitive detection methods or procedural errors (24).

In this study, benign nodules were generally larger than malignant ones. This observation can be explained by the fact that benign nodules are typically managed through follow-up, and surgery is only considered when they cause compression-related symptoms or aesthetic concerns. The combination of BRAF^V600E and RSS assessments outperformed individual evaluations, highlighting the improved diagnostic efficacy of incorporating BRAF^V600E gene testing as compared to using US alone. Of particular note is the combination of ACR with BRAF^V600E, which exhibited superior performance compared to ATA combined with BRAF^V600E. Furthermore, the restriction of BRAF^V600E gene testing to medium-risk nodules significantly reduced the rate of BGR while maintaining consistent FNR, supporting it as a preferable strategy over testing all nodules.

This study also had several limitations. First, the use of postoperative pathology as a reference led to a selection bias, which resulted in a relatively higher malignancy rate. Second, this study employed a retrospective design, thus limiting to the use of static images, which might have introduced some variability in the assessment of US features. Future prospective research is needed for further validation. Third, this study encountered a subset of nodules that were misdiagnosed as benign by all tests (both RSSs and BRAF^V600E), with the majority of these cases being non-PTC malignancies, primarily follicular-patterned lesions. This underscores the limited effectiveness of the methods employed in this study for non-PTC malignancies. To enhance diagnostic accuracy in such cases in the future, it is imperative to develop more suitable diagnostic approaches, such as elasticity imaging, contrast-enhanced US, RAS gene analysis, and other potential modalities.

In conclusion, the combination of RSSs with BRAF^V600E significantly enhances the efficacy of diagnosing AUS/FLUS nodules. Moreover, compared to testing all nodules with BRAF^V600E, only testing those nodules considered to be medium risk can significantly reduce the BGR without increasing the FNR.

Acknowledgments

Funding: None.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-23-1066/rc

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-23-1066/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) and was approved by the Scientific Research and Clinical Trials Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No. 2022-KY-0974-001). The requirement for informed consent was waived due to the retrospective nature of the study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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