The Combination of BRAFV600E mutation and Chinese Thyroid Imaging Reporting and Data System is Helpful in the Management of AUS/FLUS Thyroid Nodules

Abstract

Purpose To explore the utility of the BRAF^V600E mutation in combination with Chinese Thyroid Imaging Reporting and Data System (C-TIRADS) in the management of atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) thyroid nodule (TN).

Methods 138 AUS/FLUS TNs in 129 patients were included. Each TN underwent preoperative BRAF^V600E mutation analysis and was classified using the C-TIRADS. Histopathologic diagnosis served as reference standard.

Results 46 benign TNs and 92 malignant TNs were identified. Malignant TNs were more commonly observed in TNs with a size less than 10mm (P = 0.033). There were significant differences in malignancy rate among the different C-TIRADS TNs (P = 0.000), and these TNs with higher C-TIRADS were associated with increased malignancy rate (P for trend = 0.000). The rate of nodule with BRAF^V600E mutation increased with the increase of C-TIRADS (P for trend = 0.001). For AUS/FLUS TNs without BRAF^V600E mutation, the malignancy rates of the C-TIRADS 3, 4A, 4B, 4C, and 5 were 0%, 21.4%, 20.8%, 70.8%, and 100%, respectively (P = 0.000), and the malignancy rate increased from C-TIRADS 3 to C-TIRADS 5 (P for trend = 0.000). The diagnostic performance of C-TIRADS was similar to the BRAF^V600E mutation (P > 0.05), and the sensitivity, negative predictive value, and accuracy of the combination were significantly higher than BRAF^V600E gene or C-TIRADS alone (P < 0.05).

Conclusions C-TIRADS can effectively provide risk stratification for AUS/FLUS TNs. The combination can be helpful in selecting appropriate management for AUS/FLUS patients.

Introduction

Ultrasound (US)-guided fine-needle aspiration (FNA) cytology is a reliable and effective diagnostic approach in patients with thyroid nodules (TNs) [1]. FNA can determine up to 70% of benign and 50% of malignant TNs without subjecting them to surgical resection [2, 3]. Nonetheless, 10–27% of the FNAs present the “atypical” category [2, 4]: either “atypia of undetermined significance” (AUS) or “follicular lesion of undetermined significance” (FLUS). The malignancy rate was assessed to be 5–15% by current Bethesda system [2, 5, 6], yet 6–48% of these nodules finally were confirmed to be malignant when underwent surgery [2, 7]. Current guidelines advise performing repeat FNA to guide clinical decision-making in these nodules [1]. However, repeat FNA is not an adequate solution due to a significant portion of again AUS/FLUS results and a high false-negative rate [8].

Molecular testing has been proposed in an attempt to improve preoperative risk evaluation of AUS/FLUS nodules [6, 9]. BRAF^V600E mutation is proved as the most widespread gene mutation in thyroid cancer, especially in papillary thyroid cancer (PTC), with high specificity and positive predictive value (PPV) [9, 10]. When BRAF^V600E mutation occurs in AUS/FLUS nodules, guidelines recommend surgical procedure rather than observed management [1]. Unfortunately, BRAF^V600E gene has limited sensitivity and negative predictive value (NPV) [11], resulting in approximately 33% of cancer undetected. The rate is too high to be beneficial in making decisions between active surveillance and surgery. Meanwhile, the incidence of BRAF^V600E mutation in PTC patients was only 35–65% [12], and has not been discovered in follicular thyroid carcinoma (FTC) [13]. Therefore, a challenge remains on the AUS/FLUS nodules tested negative for BRAF^V600E mutation in which malignancy fails to be excluded.

US is an effective tool to distinguish malignant from benign AUS/FLUS nodules [14]. Several suspicious US characteristics, such as markedly hypoechoic, infiltrative margin, and microcalcification, can be regarded as predictors of malignancy. Unfortunately, none of these features are sensitive or specific sufficient to be used in isolation. For this reason, several thyroid imaging reporting and data systems (TIRADS) have been formulated to classify TNs and stratify the risk of malignancy. Among them, Zhou et al. established the Chinese-TIRADS (C-TIRADS) suitable for Chinese clinical practice based on the Chinese TNs US database [15]. C-TIRADS had a sensitivity exceeding 90% and an NPV greater than 95% in a multicentric retrospective study [16]. In addition, compared to other systems, C-TIRADS was simpler and easier to apply [16, 17]. To date, there has been no report to evaluate the value of C-TIRADS for AUS/FLUS nodules.

Our study aimed to assess the ability of C-TIRADS to stratify the malignancy risk for AUS/FLUS nodules and to evaluate the diagnostic performance of C-TIRADS, BRAF^V600E mutation and their combination when applied to AUS/FLUS nodules.

Materials And Methods

Thyroid Ultrasound Examination and C-TIRADS Classification

All US examinations were performed by one of five US experts with more than 6 years of experience in thyroid US, using Philips IU22, EPIQ5 or GE Logiq 9 devices equipped with a 5–12 MHz linear-array transducer. US images were independently reviewed by two US experts with 10 and 14 years of experience in the thyroid US and blinded to the cytological, BRAF^V600E gene testing, and histological findings.

Composition, echogenic foci, echogenicity, margin, and orientation were evaluated in all TNs [15]. The composition was classified into five categories: solid, predominately solid, predominately cystic, cystic, and spongiform. The echogenic foci were classified as microcalcification, comet-tail artifact, punctate echogenic foci of undetermined significance, macrocalcification, peripheral calcification, and no echogenic foci. When a nodule had both microcalcification and other types of echogenic foci, we regarded it as having microcalcification. The TN was considered hyperechoic, isoechoic, or hypoechoic with respect to the thyroid parenchyma or as showing marked hypoechoic when compared with strap muscle. The margin was classified into four categories: circumscribed, irregular margin, ill-defined, or extrathyroidal extension. Orientation was classified as vertical (taller-than-wide) or horizontal (wider-than-tall) on transverse or longitudinal sections. Solid composition; microcalcification; markedly hypoechoic; irregular margin, ill-defined, or extrathyroidal extension; and vertical orientation were considered the positive US features, while comet-tail artifact showed benign status. Thus, the interpretation of these US features was the same for all US experts.

All TNs were assessed according to C-TIRADS proposed by Zhou [15]. The nodule’s C-TIRADS ranging from C-TIRADS 1 to C-TIRADS 6 was determined by the counting method, being classified as C-TIRADS 1 (no nodule); C-TIRADS 2 (− 1 point); C-TIRADS 3 (0 point); C-TIRADS 4A (1 point); C-TIRADS 4B (2 points); C-TIRADS 4C (3–4 points); C-TIRADS 5 (5 points); and C-TIRADS 6 (biopsy proved malignant). When there was no consensus through the independent assessment, a third US expert with 20 years of experience in the thyroid US reviewed the images independently and made a final decision (Table 1).

US-guided FNA was executed with 23- to 25-gauge needles by one of seven US experts with more than 6 years of experience in FNA. Each nodule was aspirated at least three passes. FNA samples were smeared onto frosted-end glass slides and immediately fixed in 95% alcohol for hematoxylin and eosin (H&E) staining. Evaluation of FNA samples was executed by a pathologist with more than 10 years of experience in thyroid cytology using Bethesda system criteria [6].

One of the FNA specimens was added to the gene kit (ADx-BR02, Amoy Diagnostics Co. Ltd) followed by DNA extraction and polymerase chain reaction (PCR) amplification (real-time fluorescence quantitative PCR-ARMS was used for detection). The results were analyzed based on the instructions, and mutations in the BRAF^V600E gene were obtained.

Results

Among 138 AUS/FLUS nodules, BRAF^V600E mutation was observed in 67 nodules, including 64 malignant nodules (64 PTCs) and 3 benign nodules (2 atypical hyperplasia nodules and 1 adenomatous goiter). Negative BRAF^V600E mutation was identified in other 71 AUS/FLUS nodules, including 28 malignant nodules and 43 benign nodules (Table 2).

Correlation Between the C-TIRADS and Pathological Findings

The overall distribution of the C-TIRADS for all AUS/FLUS nodules was as follows: C-TIRADS 3 in 6, C-TIRADS 4A in 18, C-TIRADS 4B in 47, C-TIRADS 4C in 65, and C-TIRADS 5 in 2. The malignancy rates in C-TIRADS 3, 4A, 4B, 4C, and 5 were 0%, 33.3%, 57.4%, 87.7%, and 100%, respectively (P = 0.000), and the malignancy rate increased from C-TIRADS 3 nodules to C-TIRADS 5 nodules (P for trend = 0.000) (Table 4).

For AUS/FLUS nodules without BRAF^V600E mutation, C-TIRADS was classified as C-TIRADS 3 in 6, C-TIRADS 4A in 14, C-TIRADS 4B in 24, C-TIRADS 4C in 25, and C-TIRADS 5 in 2, respectively. The malignant rates of C-TIRADS 3, 4A, 4B, 4C, and 5 were 0%, 21.4%, 20.8%, 70.8%, and 100%, respectively (P = 0.000), and the malignancy rate increased from C-TIRADS 3 nodules to C-TIRADS 5 nodules (P for trend = 0.000) (Table 4).

The rate of nodule with BRAF^V600E mutation in each C-TIRADS was as follows: C-TIRADS 3 in 0, C-TIRADS 4A in 4, C-TIRADS 4B in 23, C-TIRADS 4C in 40, and C-TIRADS 5 in 0, respectively, and the rate of nodule with BRAF^V600E mutation increased from C-TIRADS 3 nodules to C-TIRADS 4C nodules (P for trend = 0.001).

Diagnostic Performance of the BRAF^V600E Mutation, C-TIRADS, and the Combination

The sensitivity, specificity, PPV, NPV, accuracy, and AUC of BRAF^V600E mutation were 69.6%, 93.5%, 95.5%, 60.6%, 77.5%, and 0.815, respectively. The best cut-off of C-TIRADS was C-TIRADS 4C according to the ROC curve. The sensitivity, specificity, PPV, NPV, accuracy, and AUC of C-TIRADS were 64.1%, 82.6%, 88.1%, 53.5%, 70.3%, and 0.783, respectively, and the diagnose performance was similar to the BRAF^V600E mutation (P > 0.05). The sensitivity, specificity, PPV, NPV, accuracy, and AUC of the combination were 91.3%, 78.3%, 89.4%, 81.8%, 87.0%, and 0.870, respectively. The sensitivity, NPV, and accuracy of the combination were significantly higher than BRAF^V600E gene or C-TIRADS alone (all P < 0.05) (Table 5, Fig. 2). Among them, a total of 18 AUS/FLUS nodules were misdiagnosed, including 10 false-positive nodules (1 benign nodule showed BRAF^V600E mutation and C-TIRADS 4C, 2 showed BRAF^V600E mutation, and 7 showed C-TIRADS 4C) and 8 false-negative nodules (3 malignant nodules showed C-TIRADS 4A, and 5 showed C-TIRADS 4B) (Table 6).

Discussion

AUS/FLUS is considered the “gray zone” of cytopathology, including atypia that cannot be readily assigned to a definitive cytological category due to architectural and/or nuclear atypia and sample preparation artifact. Previous studies indicated significant differences in the prevalence and malignancy in surgical samples for AUS/FLUS nodules [2, 4–7]. With these heterogeneous findings, the optimal approach to the management of these nodules remains controversial.

In our study, which included a larger surgically confirmed population, the malignancy rate was 66.7%, which is higher than previous reports [5–7], but lower than the other studies [18], and similar to some other studies [19]. This variability was caused by differences in inclusion criteria, modalities of preoperative assessment, and follow-up strategy. Thus, it is necessary to manage AUS/FLUS nodules with prudence. The size of benign and malignant AUS/FLUS nodules was different (P = 0.014), and malignant nodules were more commonly detected in nodules with a size less than 10mm (P = 0.033). These results were consistent with Suh et al. [20]. The difference in the size of benign and malignant nodules might be based on the fact that the US had high sensitivity and could detect many malignant nodules smaller than 10mm. In addition, a proportion of benign nodules performed surgery due to tracheal compression or cosmetic reasons.

Currently, BRAF^V600E gene has been confirmed as a highly specific marker of thyroid cancer. Compared with multi-gene panel or next-generation sequencing, BRAF^V600E test was already quite established in clinical application and more cost-effective. In our study, BRAF^V600E mutation was identified in 48.6% of AUS/FLUS nodules and 69.6% of malignant AUS/FLUS nodules, which were higher than that described by Xing [10], but approached the described by Kim et al. [21]. The possible reasons for the differences were diverse races, geographic area, methods of detecting gene mutation, and iodine intake. The specificity and PPV of the BRAF^V600E mutation were 93.5% and 95.5%, respectively, which is consistent with the previous report [10]. However, BRAF^V600E gene has limited sensitivity and NPV [11]. In our study, BRAF^V600E mutation demonstrated a sensitivity and NPV of 69.6% and 60.6%, respectively, which was similar to the reports.

Coincidentally and interestingly, 3 surgery-proven benign AUS/FLUS nodules harbored BRAF^V600E mutation. Two AUS/FLUS nodules were confirmed as atypical hyperplasia, which embraced some cytological characteristics of PTC. We believed that these nodules were identical to the atypical hyperplastic nodule reported by Chung et al. and it might be the precursor of PTC [22]. The other was proven to be an adenomatoid nodule. The highly sensitive of BRAF^V600E test might explain the false-positive result [23].

We attempted to stratify the risk of AUS/FLUS nodules based on C-TIRADS guideline [15]. In our study, the malignancy rate of C-TIRADS 3 was 0%, and this value was consistent with the expected malignancy risk by the C-TIRADS guideline. This finding favors follow-up management instead of repeat FNA or surgery for these nodules. However, the number of C-TIRADS 3 nodules was small, and further researches with a large population are required. The malignancy rate of the C-TIRADS 4A and 4B nodules were 33.3% and 57.4%, respectively, and these values were higher than the described in the C-TIRADS guideline. This disparity might be due to selection bias since our study only contained AUS/FLUS nodules, while C-TIRADS guideline suggested a risk stratification for all TNs. However, the malignant rates of C-TIRADS 4A and 4B for AUS/FLUS nodules without BRAF^V600E mutation were 21.4% and 20.8%, respectively, which was relatively lower compared with all C-TIRADS 4A and 4B nodules. Thus, C-TIRADS 4A and 4B AUS/FLUS nodules with BRAF^V600E mutation can be surgical treatment, while the management of AUS/FLUS nodules without BRAF^V600E mutation will depend on the physician’s preference, including follow-up, repeat FNA, and surgery. For C-TIRADS 4C and C-TIRADS 5 nodules, the malignancy rates were very high (70.8–100%) regardless of the occurrence of BRAF^V600E mutation, which were consistent with the expected malignancy risk by the C-TIRADS guideline. In addition, there were significant differences in malignancy risk among the different C-TIRADS, and these nodules with higher C-TIRADS were associated with the increased malignancy rate. These findings suggested that C-TIRADS could provide risk stratification for benign and malignant AUS/FLUS nodules, especially in China where genetic test is not yet widely available.

It was important to note the association between C-TIRADS and BRAF^V600E mutation. Over two-thirds of C-TIRADS 4C nodules were detected to have BRAF^V600E mutation, and BRAF^V600E mutation nodules were more likely to be categorized into one of the higher C-TIRADS. In addition, the diagnostic performance of C-TIRADS was similar to BRAF^V600E mutation. Therefore, BRAF^V600E gene can offer a more cost-effective strategy in AUS/FLUS with C-TIRADS 4A or 4B. Using the combination of BRAF^V600E mutation with C-TIRADS 4C in AUS/FLUS nodules, the combination could get an increased sensitivity, NPV, and accuracy in comparison with BRAF^V600E test or C-TIRADS alone, which showed that the combination could promote the management of AUS/FLUS patients.

There are several limitations of this study. First, there was a selection bias because we did not include AUS/FLUS nodules that had no pathological findings. It might be a reason for the malignant rate of our study being higher than Bethesda system. However, this aspect is difficult to overcome, due to the fact that the reference criterion must be the postoperative pathology. Second, US features were categorized based on static images rather than actual images, which might influence the evaluation of US features. Third, the sub-classification of the atypia recommended by the 2017 revision was not considered in our study because this did not affect the management of patients. Fourth, our study focused on the BRAF^V600E mutation for the diagnosis of AUS/FLUS nodules and did not involve the detection of other genes. Finally, this study was a single-center retrospective study, with ensured consistency of diagnosis outcome.

In conclusion, this study demonstrated that C-TIRADS could effectively provide risk stratification for AUS/FLUS nodules, and the diagnostic performance of C-TIRADS is similar to BRAF^V600E mutation. C-TIRADS may be advised as an alternative method for providing information to manage patients with AUS/FLUS nodules, especially in circumstances in which molecular testing is not available. The combination can promote the diagnostic performance of AUS/FLUS nodules, and it is helpful in selecting appropriate clinical management in AUS/FLUS patients.

Declarations

Funding:

This study was supported by the Zhejiang Province Natural Science Foundation (grant no. LY20H180005) which is gratefully acknowledged.

Conflicts of interest:

The authors have no relevant financial or non-financial interests to disclose.

Availability of data:

All data generated or analyzed during this study are included in this article.

Code availability:

Not applicable.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Qiang Li, Lu Yang, Jianghong Lv, Lilong Xu, Murui Zhang. The first draft of the manuscript was written by Qiang Li and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics approval:

This study was approved by the board of medical ethics of Sir Run Run Shaw Hospital, Zhejiang University, School of Medicine.

Consent to participate:

Written informed consents were obtained from all participates included in the study.

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