背景及目的

甲状腺癌是最常见的内分泌肿瘤，其全球癌症发病率排行第七，在我国位列第三。快速且准确地诊断甲状腺癌至关重要。此外，随着人们对甲状腺癌惰性特征的认识不断深入，过度诊疗的问题日益受到关注。新背景下，甲状腺癌诊断效度的提高及高风险肿瘤的早期识别，对甲状腺癌患者个体化诊疗方案的制定具有重要意义。

超声是评估甲状腺癌首选的影像学方法，目前以灰阶超声为主，以此形成了多个版本的甲状腺结节超声风险分层系统，但存在良恶性结节灰阶特征重叠、各系统对甲状腺癌诊断特异度相对较低的问题。超微血流成像（Superb Microvascular Imaging, SMI）凭借其对微小血管及低速血流的高敏感性，可为甲状腺癌的无创评估提供新的指标。然而目前相关研究使用的SMI血流评价指标不同、诊断标准各异、血流特征形成原因不明，阻碍了SMI在甲状腺癌诊断方面的临床应用。

基于此，本研究拟应用SMI，通过评估甲状腺良恶性结节的血流特征，构建甲状腺癌诊断新指标；通过与灰阶超声联合，探究SMI对甲状腺癌超声诊断效度提升及穿刺活检推荐的临床价值；通过影像基因组学的方法，探索基于SMI的甲状腺癌相关微血流特征的形成原因，解析该特征所反映的肿瘤生物学行为，最终实现辅助甲状腺癌临床决策的目的。

方法

第一部分：前瞻性纳入2021年10月至2022年6月于我院行甲状腺切除术的患者，所有患者行术前甲状腺结节灰阶超声及SMI检查。对比分析SMI模式下甲状腺良恶性结节在微血流的丰富程度、大小、走行、分布及架构方面的血流特征，筛选出诊断效度高且观察者一致性较强的微血流指标。

第二部分：前瞻性纳入2023年5月至2024年2月于我院行甲状腺切除术或甲状腺结节穿刺活检术的患者，所有患者行甲状腺结节灰阶超声及SMI检查。参照五个临床常用的甲状腺结节超声风险分层系统对结节进行评级，根据SMI模式下微血流架构对结节进行级别调整，“蟹足征”及“残根征”为升级征象，“轮辐征”及“树枝征”为降级征象。对比与SMI联合前后各系统对甲状腺癌的诊断效度及不必要活检率。

第三部分：前瞻性纳入2024年9月至2024年12月于我院行甲状腺结节穿刺活检术的甲状腺乳头状癌（Papillary Thyroid Carcinoma, PTC）患者，所有患者行甲状腺结节灰阶超声及SMI检查。所有结节灰阶超声特征一致，以SMI模式下显示“蟹足征”的结节为A组，不显示“蟹足征”或“残根征”的结节为B组。在穿刺活检过程中留取肿瘤标本并进行RNA测序，对比两组的基因表达情况。以P ＜ 0.05且以2 为底的对数倍数变化（Log₂FC）的绝对值≥2为标准筛选显著差异表达基因，并进行生物信息分析，筛选与PTC“蟹足征”形成相关的差异表达基因。

结果

第一部分：共177名患者248个甲状腺结节被纳入研究（125个恶性结节，123个良性结节）。SMI模式下“穿支血流≥1” （恶性组82.4% vs 良性组30.9%，P < 0.001）、“蟹足征” （64.0% vs 10.6%，P < 0.001）及“残根征” （8.0% vs 2.4%，P = 0.049）是甲状腺恶性结节相对常见的微血流特征，其中“蟹足征”是甲状腺癌的独立危险因素；“轮辐征” （1.6% vs 33.3%，P < 0.001）及“树枝征” （0% vs 19.5%，P < 0.001）是甲状腺良性结节相对常见的微血流特征。微血流架构“蟹足征”、“残根征”诊断甲状腺癌的特异度分别为0.894、0.976，阳性预测值分别为 0.860、0.769；“轮辐征”、“树枝征”诊断甲状腺良性结节的特异度为0.984、1.000，阳性预测值为0.953、1.000。

第二部分：共203名患者253个甲状腺结节被纳入研究（136个恶性结节，117个良性结节）。与SMI联合后，各甲状腺结节超声风险分层系统对甲状腺癌诊断的特异度及受试者工作特征（Receiver Operator Characteristic, ROC）曲线下面积（Area Under the Curve, AUC）均明显升高（P < 0.05），其中诊断特异度以美国甲状腺协会的甲状腺结节风险分层系统（American Thyroid Association Risk Stratification System,  ATA RSS）（0.778 vs 0.863, P = 0.004）和韩国甲状腺放射学会的甲状腺影像报告及数据系统（Korean Society of Thyroid Radiology Thyroid Imaging Reporting and Data System, KSThR TIRADS）（0.795 vs 0.863, P = 0.013）最高，AUC以中国超声甲状腺影像报告和数据系统（Chinese Thyroid Imaging Reporting and Data System, C-TIRADS）最高（0.834 vs 0.900, P < 0.001）。此外，美国放射学会的甲状腺影像报告及数据系统（American College of Radiology Thyroid Imaging Reporting and Data System, ACR TI-RADS）（0.728 vs 0.860, P = 0.001）及KSThR TIRADS（0.801 vs 0.875, P = 0.034）对甲状腺癌的诊断敏感度也显著升高。与SMI联合后，各系统不必要活检率减少1.27% - 20.30%，以C-TIRADS下降最为显著（38.04% vs 17.74%）。

第三部分：共6名SMI模式下表现“蟹足征”的PTC患者纳入A组，7名SMI模式下既不表现“蟹足征”又不表现“残根征”的PTC患者纳入B组。通过RNA测序共获得基因58735个，其中显著差异表达基因174个。经筛选后获得与PTC“蟹足征”形成相关的表达上调基因BMP4（Log₂FC 2.085，P = 0.014）、MMP13（Log₂FC 3.625，P = 0.013）、COL11A1（Log₂FC 2.338，P = 0.015）和表达下调基因OGN（Log₂FC -2.704，P = 0.005）、PRELP（Log₂FC -2.095，P = 0.009），其功能主要富集于细胞外基质重塑、细胞增殖中。

结论

本研究通过应用SMI评估甲状腺结节微血流特征，提出了恶性结节微血流架构“蟹足征”、“残根征”，及良性结节微血流架构“轮辐征”、“树枝征”，建立了新的甲状腺结节评估指标；上述指标在与灰阶超声联合应用后可显著提高超声对甲状腺癌的诊断效度，并减少不必要活检率，体现出较好的临床应用价值；其中，“蟹足征”的形成与基因BMP4、MMP13、COL11A1表达上调及OGN、PRELP表达下调有关，这些基因在PTC肿瘤细胞外基质重塑、细胞增殖的生物进程中发挥重要作用，提示“蟹足征”或与PTC的侵袭性相关。

Background and Aims

Thyroid cancer is the most prevalent endocrine tumor, ranking seventh in global cancer incidence and third in China. It is crucial to diagnose thyroid cancer quickly and accurately. Moreover, recent years have witnessed increasing attention to the overdiagnosis and overtreatment of thyroid cancer with a deepening understanding of its indolent nature. In this context, improving the diagnostic validity of thyroid cancer while reducing the clinical intervention in benign thyroid nodules and indolent low-risk thyroid cancer is of great clinical value for individualized assessment.

Ultrasound is the preferred method for evaluating thyroid cancer, with grayscale ultrasound being the primary approach currently. Based on this, multiple versions of ultrasound risk stratification systems for thyroid nodules have been developed. However, grayscale features between benign and malignant thyroid nodules often overlap, leading to a relatively low diagnostic specificity of these risk stratification systems. Superb Microvascular Imaging (SMI) provides new indexes for non-invasive assessment of thyroid cancer owing to its high sensitivity for microvessels and low-speed flow. There are studies of SMI in diagnosing thyroid cancer, but problems of different microflow indexes, varying diagnostic criteria and unclear formation mechanisms do exist, which hinder the clinical utility of SMI in thyroid cancer evaluation.

On account of this, this study aims to construct a new ultrasound index for thyroid cancer by comparing the microflow characteristics of benign and malignant thyroid nodules via SMI; to explore the clinical value of SMI in the diagnostic validity of thyroid cancer and biopsy recommendation of thyroid nodules through the combination with grayscale ultrasound; to investigate the formation of the aforementioned microflow index and analyze the tumor biological behavior it reflects by means of radiogenomics, ultimately achieving the goal of optimizing clinical decision-making for thyroid cancer patients.

Methods

Part I: Patients who planned to undergo preoperative grayscale and SMI ultrasound examination as well as surgery for thyroid nodules in our center from October 2021 to June 2022 were prospectively recruited. The microflow features of benign and malignant nodules in terms of richness, size, morphology, distribution and patterns were compared to screen out the vascular indexes with great differentiation performance and acceptable observer agreement.

Part II: Patients who planned to undergo fine needle aspiration biopsy or surgery for thyroid nodules in our center from May 2023 to February 2024 were prospectively recruited. The risk categories of thyroid nodules were evaluated by grayscale ultrasound according to five widely used risk stratification systems of ultrasound for thyroid nodules and adjusted by microflow patterns on SMI. The crab claw-like and the root hair-like patterns were malignant signs for upgrading, with the wheel-like and the arborescent patterns for downgrading. The diagnostic validity for thyroid cancer and unnecessary biopsy rate of each system before and after the combination with SMI were compared.

Part III: This prospective study included patients who underwent fine needle aspiration biopsy for thyroid nodules which were confirmed as papillary thyroid carcinoma (PTC) later in our center from September 2024 to December 2024. All the nodules exhibited consistent grayscale features, but were divided into Group A for the crab claw-like pattern and Group B for other microflow patterns except for the crab claw-like or the root hair-like pattern on SMI. Tumor samples were collected during the biopsy process for RNA sequencing and the gene expression profiles of two groups were compared. Taking the P < 0.05 and |Log₂ (Fold Change) | ≥ 2 as standard for differentially expressed gene. Bioinformatics analysis was conducted to identify the differentially expressed genes related to the formation of the crab claw-like pattern in PTC.

Results

Part I: A total of 248 thyroid nodules (125 malignant, 123 benign) in 177 patients were included. Penetrating vessel ≥ 1 (82.4% in the malignant group vs 30.9% in the benign group, P < 0.001), the crab claw-like pattern (64.0% vs 10.6%, P < 0.001) and the root hair-like pattern (8.0% vs 2.4%, P = 0.049) on SMI were more common in malignant thyroid nodules, among which the crab claw-like pattern was the independent risk factor for thyroid cancer. The wheel-like pattern (1.6% vs 33.3%, P < 0.001) and the arborescent pattern (0% vs 19.5%, P < 0.001) were more likely to appear in benign nodules. The diagnostic specificities of the crab claw-like pattern and the root hair-like pattern for thyroid cancer were 0.894 and 0.976, and the positive predictive values were 0.860 and 0.769. The diagnostic specificities of the wheel-like pattern and the arborescent pattern for benign thyroid nodules were 0.984, 1.000, and the positive predictive values were 0.953, 1.000.

Part II: A total of 253 thyroid nodules (136 malignant, 117 benign) in 203 patients were included. The combination of SMI and grayscale ultrasound significantly enhanced the diagnostic specificity and the areas under the receiver operator characteristic curve (AUC) of all risk stratification systems of ultrasound for thyroid nodules (P < 0.05). Among these, American Thyroid Association Risk Stratification System (ATA RSS) (0.778 vs 0.863, P = 0.004) and Korean Society of Thyroid Radiology Thyroid Imaging Reporting and Data System (KSThR TIRADS) (0.795 vs 0.863, P = 0.013) shared the highest specificity, and Chinese Thyroid Imaging Reporting and Data System (C-TIRADS) owned the largest AUC (0.834 vs 0.900, P < 0.001). Moreover, the diagnostic sensitivities of American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS) (0.728 vs 0.860, P = 0.001) and KSThR TIRADS (0.801 vs 0.875, P = 0.034) improved evidently after the addition of SMI. The unnecessary biopsy rates of all systems were reduced by 1.27% - 20.30% according to microflow patterns on SMI, among which C-TIRADS had the largest reduction (38.04% vs 17.74%).

Part III: Six patients of PTC were included in Group A for nodules showing the crab claw-like pattern on SMI with 7 patients in Group B for other microflow patterns except for the crab claw-like or the root hair-like pattern on SMI. A total of 58735 genes were obtained through RNA sequencing, including 174 significantly differentially expressed genes. After bioinformatics analysis, the up-regulated genes BMP4 (Log₂FC 2.085, P = 0.014), MMP13 (Log₂FC 3.625, P = 0.013), COL11A1 (Log₂FC 2.338, P = 0.015) and down-regulated genes OGN (Log₂FC -2.704, P = 0.005), PRELP (Log₂FC -2.095, P = 0.009) were identified to be associated with the formation of the crab claw-like pattern in PTC. The functions of these genes are mainly enriched in extracellular matrix remodeling and cell proliferation.

Conclusions

This study established a novel ultrasound index microflow pattern for the assessment of thyroid nodules using SMI and identified the crab claw-like pattern and the root hair-like pattern as features of thyroid cancer, with the wheel-like pattern and the arborescent pattern for benign thyroid nodules. These microflow patterns on SMI substantially improved the diagnostic validity for thyroid cancer and reduced the unnecessary biopsy rate when combined with grayscale ultrasound features, demonstrating great clinical application value. Besides, the formation of crab claw-like pattern is correlated with the upregulation of BMP4, MMP13, COL11A1 expression and the downregulation of OGN, PRELP expression, which play vital roles in extracellular matrix remodeling and cell proliferation in tumors, suggesting that the crab claw-like pattern may be associated with the invasiveness of PTC.