Role of contrast-enhanced ultrasound with time-intensity curve analysis about thyroid nodule and parenchyma for differentiating BRAF V600E mutation status

Introduction

Papillary thyroid carcinoma (PTC) is the most common endocrine tumor and accounts for approximately 80–85% of malignant tumors of thyroid (Gharib et al., 2013; Ali, 2011). The conventional PTC treatment was surgery, radioactive iodine, thyroid hormone suppression of TSH, external beam radiation (less commonly), and rarely, chemotherapy (Luster et al., 2019). While the majority of PTCs have a low death rate and respond well to radioiodine therapy, certain PTCs have worse prognosis and more aggressive clinicopathologic features (Feng et al., 2019; Yamada et al., 2014). Recently, biological differences between PTCs have been discovered from the identification of genetic alterations in various signaling pathways (Lee et al., 2017). One of the genetic mutations linked to the protein kinase pathway is the B-type Raf (BRAF) V600E kinase mutation on exon 15 (Riesco-Eizaguirre & Santisteban, 2016; Poulikakos, Sullivan & Yaeger, 2022). This mutation has been linked to thyroid cancer by upregulating cell division and proliferation, which results in the development of tumors (Chen et al., 2021; Fu et al., 2023). Additionally, numerous studies have demonstrated that the BRAF V600E mutation is linked to the invasive clinicopathological behavior of PTC as well as negative outcomes such distant metastases, lymph node metastases, loco-regional recurrences, and malignant recurrences (Haoran & Yongzhao, 2017; Yong-Heng et al., 2018; Jin et al., 2017; Baloch et al., 2022). Although the BRAF V600E mutant status is helpful for clinical decision-making, most papillary thyroid microcarcinoma are not recommended for its acquisition since it requires invasive diagnostic procedures such surgery and fine-needle aspiration biopsies (Haugen et al., 2015).

Contrast-enhanced ultrasound (CEUS) is a supplemental ultrasound imaging technique that is frequently employed in clinical settings to detect benign and malignant thyroid nodules. It is also capable of rapidly predicting the severity and prognosis of PTC (Kim et al., 2019; Ricarte-Filho et al., 2012). The advantage of CEUS is that it can measure more precisely and accurately in terms of shape. It can also evaluate the thyroid nodules’ hemodynamics and the sequence and intensity of vascular perfusion based on several parameters (washout, washout peak, ring sign, etc.) (Trimboli et al., 2020). According to certain research, CEUS has a strong predictive value for detecting PTC (Diao et al., 2017; Xi et al., 2020; Yan et al., 2018), but most studies were focused on the contrast enhancement patterns (Zhang et al., 2020; Fang et al., 2021; Wang et al., 2021). The BRAF V600E mutation was linked to certain CEUS quantitative characteristics in a number of investigations, although these research only examined the nodular parameters obtained by post-processing software, like arrival time (AT) and time to peak (TTP) (Chen et al., 2019). In this study, we aimed to compare both the nodular parameters and the quantitative parameters of the thyroid nodule and surrounding parenchyma in relation to BRAF status. This study is expected to provide additional evidence for differentiating aggressive PTC and to assist in personalized clinical treatment.

Materials and Methods

Image analysis

Two radiologists with over 5 years of CEUS expertise examined the quantitative CEUS parameters of PTC nodules. Both the nodules and the surrounding thyroid parenchyma were designated as regions of interest (ROI). The ultrasonic signal augmentation status was quantitatively examined using time-intensity curve (TIC) analysis. The following direct quantitative parameters were measured (Fig. 1): (1) The arrival time, AT; (2) time to peak, TTP; (3) basic intensity, BI; (4) peak intensity, PI. In addition, we measured more parameters which based on the direct quantitative parameters: (1) rise time, RT; (2) arrival time change between nodule and surrounding parenchyma, ATC; (3) time to peak change between nodule and surrounding parenchyma, TTPC; (4) rise time ratio, RTR; (5) time to peak ratio, TTPR; (6) basic intensity change between nodule and surrounding parenchyma, BIC; (7) peak intensity change between nodule and surrounding parenchyma, PIC; (8) rise intensity, RI; (9) peak intensity ratio, PIR; (10) rise intensity ratio, RIR.

Results

This study included 447 individuals (203 men and 244 women) who were enrolled between October 2021 and December 2023. Table 1 displays the clinical features of the tumors and patients. Multiple lesions, capsular invasion, and lymph node metastases did not significantly correlate with the presence of a BRAF mutation in our study.

The quantitative CEUS parameters were given using TIC curves. Direct measures showed that nodules in the BRAF-positive group had longer AT (7.68 (5.96, 8.99)) and TTP (15.58 (13.97, 17.42)) than nodules in the BRAF-negative group (6.82 (5.73, 8.48)) and TTP (14.62 (12.56, 17.52)) (p = 0.019 and p = 0.023, respectively). There was no correlation between the BI and PI and the state of mutations (Table 2).

In terms of indirect parameters, ATC (3.80 (2.33, 5.30)) and TTPC (4.07 (3.17, 4.61)) in the BRAF-positive group were longer than ATC (3.13 (2.36, 4.14)) and TTPC (3.85 (3.16, 4.31)) in the BRAF-negative group (p = 0.002 and p = 0.036 respectively), while other indirect parameters differences between the two groups were not statistically significant (Table 3).

Discussion

Over the past few decades, the prevalence of thyroid cancer, one of the most prevalent endocrine cancers, has steadily climbed globally (Miranda-Filho et al., 2021; Kitahara & Sosa, 2020). Although papillary thyroid carcinoma (PTC), the most prevalent histological form of differentiated thyroid cancer, is typically a slow-growing disease, the majority of PTC patients nonetheless underwent surgery in the clinic (Lee & Shin, 2014; Li, Dal Maso & Vaccarella, 2020). The potential complications from surgery including recurrent laryngeal nerve injury, hypoparathyroidism and lifelong thyroid hormone replacement. Considering the comparatively stable incidence-based mortality rates but the higher incidence rates of detection, customized treatments have been proposed (Xing, Haugen & Schlumberger, 2013; Miccoli, 2014; White et al., 2020). Previous studies have verified that patients with PTC frequently carry the BRAF mutation, with prevalence rates reaching 73.4% (Hiroyuki et al., 2003; Maaitah et al., 2019; Xing et al., 2015), while aggressive behaviors, such as capsule invasion, vessel invasion and lymph node metastasis are linked to the BRAF mutation (Conzo et al., 2012; Gambardella et al., 2019; Marotta et al., 2016). Thus, knowing the PTC mutate status offers crucial prognostic information for more individual treatment.

By replacing valine (V) at amino acid 600 with glutamic acid (E), the BRAF V600E mutation is created. This constitutively activates the MAPK pathway, which promotes carcinogenesis. Since its discovery, the BRAF V600E has emerged as a promising diagnostic and prognostic biomarker for PTC (Oler & Cerutti, 2010; Silver et al., 2021). High-risk clinicopathological features, tumor recurrence, metastasis, and decreased susceptibility to radioiodine therapy have all been linked to this mutation (Lee, Lee & Kim, 2007; Kim et al., 2012; Tufano et al., 2012). According to reports, the BRAF V600E mutation raises the aggressiveness of the tumor as well as the mortality and recurrence rates of PTC (Xing et al., 2013). According to studies, the BRAF V600E mutation in PTC has been linked to extra-thyroidal extension, multifocality, lymph node metastasis, and advanced TNM staging. Thus, it is crucial to include the BRAF V600E mutant state in the risk stratification management of PTC in order to lower the mortality and recurrence rates.

Although surgery is still the primary treatment for PTC patients, a number of recent studies have suggested that PTC may be eligible for active surveillance and avoidable surgery. Nevertheless, prior to histological evaluation, we were unable to determine whether these patients had any metastases. Noninvasive mutation status determination is crucial for stratified disease management since the BRAF V600E mutant state may be indicative of these possible malignant variables.

Ultrasound has been proved to be vital in evaluating thyroid diseases (Sui et al., 2016). With the advent and development of ultrasound contrast agents, it is now widely accepted that CEUS can provide more sensitive information of the microcirculation inside lesions and useful in differentiating benign and malignant nodules. The enhancement patterns of PTC on CEUS include centripetal mode of entrance, later arrival, heterogeneous hypo-enhancement, and earlier washout. These characters are associated with relevant pathologic mechanisms. Studies showed that tumor cells secrete large amounts of endothelial growth factor, resulting in intra-nodular vascularization. These new blood vessels of the marginal zone are relatively dense, while the center is sparse which might lead to the entrance mode. In addition, because of vascular stenosis or occlusion due to internal microthrombus, blood vessels with low efficacy and incompletely open state (Yuan et al., 2015), complicated collagen degeneration (Zhang et al., 2010), and the existence of arteriovenous fistula, this might lead to a later arrival but earlier washout enhancement mode in the CEUS process. BRAF V600E mutation is an important factor for the aggressive nature of PTC. The possible reason is that BRAF V600E might increase kinase activity and over-activate the mitogen-activated protein kinase pathway. Studies have shown that CEUS may offer qualitative traits associated with BRAF mutations (Ruan et al., 2022; Petrasova et al., 2022; Fan et al., 2024). However, the expertise of the interpreter determines these qualities accurately, therefore more objective quantitative measures are required. In this research, we have incorporated 10 indirect parameters from comparative studies in addition to quantitative data of the surrounding parenchyma that are readily derived from the TIC curve. Significant variations were seen in the time change parameters in this study, such as AT, TTP, ATC, and TTPC. The possible reason is that the BRAF V600E mutation promotes the formation of psammoma body in micro-calcification and associated with interstitial fibrosis which leads to the nodules showing a harder texture and more calcifications. This might result in a slow progression and slowly reaching a peak for the BRAF mutation-positive group, and could explain the results of our study.

Our study did have several drawbacks. First, this is a retrospective analysis. There may be selective bias in tumor selection. The tumor we choose might more readily available for collection and genetic analysis. A prospective well-designed study is needed for a more accurate assessment of the relation between the CEUS features and BRAF mutations. Second, choosing the point on the TIC to acquire quantitative parameters may result in random error. Third, in order to validate these results and assess the threshold of TIC characteristics in the future, a comprehensive prospective diagnostic investigation is required.

Conclusions

To sum up, our study’s findings showed that time-dependent measurements, such as arrival time, time to peak, arrival time change between the nodule and surrounding parenchyma, and time to peak change between the nodule and surrounding parenchyma, are crucial for differentiating between PTC that has a BRAF mutation and PTC that does not. Our research offers more proof that TIC-based quantitative metrics may help identify PTC patients’ BRAF V600E mutant status.

Supplemental Information