Jing-Yun Hung , , Zhi-Ling Hung , Zhe Yng , Xio-Ping Zheng
a Department of Ultrasound, Shulan (Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College, Hangzhou 310022,China
b Department of Hepatobiliary and Pancreatic Surgery, Shulan (Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College, Hangzhou 310022, China
c Department of Pathology, Shulan (Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College, Hangzhou 310022,China
The presence of microvascular invasion (MVI) in hepatocellular carcinoma (HCC) is an important indicator for HCC recurrence and poor prognosis  . Determining the preoperative MVI status is helpful for guiding clinical treatment and improving prognosis. Currently, MVI diagnosis is achieved primarily by postoperative histopathology, which makes it difficult to provide a definite diagnosis before surgery. However, significant advancements in imaging technology have begun to allow clinicians to make a diagnosis of HCC MVI prior to surgery. In this study, we used contrastenhanced ultrasound (CEUS) to observe the characteristics of HCC enhancement patterns and quantitatively analyze the changes in HCC blood perfusion parameters to explore the value of CEUS in evaluating HCC MVI.
This study enrolled HCC patients who underwent surgical resection in our hospital from February 2019 to February 2021. The inclusion criteria were as follows: (i) a single lesion, (ii) no prior treatment before the operation, iii) surgical resection, (iv) definite pathological results after the operation, and (v) a Child-Turcotte-Pugh score A or B. The exclusion criteria were as follows: (i)venous tumor thrombus observed on imaging examination, (ii)naked eye intravascular tumor thrombus observed in postoperative pathology, or (iii) the presence of metastasis. A total of 175 patients were newly diagnosed with HCC at our facility during the recruitment period. Ultimately, 63 HCC patients (38 males and 25 females with 63 lesions) were enrolled. The mean age was 57.36 ±8.36 years. The patients were divided into the MVI group (n= 35)and the non-MVI group (n= 28), based on postoperative pathological results. All patients underwent CEUS within 1 week before surgery.
The CEUS (HITACHI-Aloka, ARIETTA 70) procedure was carried out as follows: first, conventional two-dimensional ultrasound was performed. The tumor location, shape, size, boundary, blood flow,and other basic information were observed and recorded. For the acoustic contrast agent, 84 mg of SonoVue was dissolved in 5.0 mL of saline. The agent was suspended and rapidly injected into the median cubital vein. An estimate of the typical time required to administer the 2.4 mL injection was 1 s, which was followed by a 5.0-mL saline flush. The injection rate of the saline flush was 2 mL/s. A 6-minute process was recorded. After CEUS, the images were replayed, and the enhancement patterns of the lesions were observed and recorded. The most obvious and uniform part of the tumor lesion was selected as the region of interest. The time-intensity curve was obtained, and the enhancement intensity,initial increase time, time to peak, and washout time were calculated and recorded  .
Tumor tissues were obtained during surgery. Postoperative pathological specimens were determined according to the Hyung criteria for MVI  . Positive MVI was defined as follows: tumor thrombus seen microscopically in the small veins (capsular venule,branches of the portal vein and central vein, etc.) in the hepatic parenchyma around the tumor and cancer cells seen in the vascular endothelium and smooth muscle layer; otherwise, MVI status was considered negative.
Data analyses were performed using SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). Numerical variables were presented as mean ± standard deviation (SD) and compared using the Student’st-test. Categorical variables were compared using the Chi-square test. Postoperative pathological diagnosis was used as the “gold standard”. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic accuracy of CEUS parameters to predict HCC MVI. AP< 0.05 was considered statistically significant.
Age, sex, Child-Turcotte-Pugh score, tumor location, maximum tumor diameter, and alpha-fetoprotein were not different between the two groups (P> 0.05). Irregular tumor shape was more frequent in the MVI group than in the non-MVI group (65.7% vs.32.1%,P< 0.05). The postoperative pathological results showed that 22 cases (22/63, 34.9%) were poorly differentiated, 16 (16/63,25.4%) were moderately differentiated, and 25 (25/63, 39.7%) were well differentiated. The MVI group mainly had moderately or poorly differentiated tumors (26/35, 74.3%), while the non-MVIgroup mainly had well-differentiated tumors (16/28, 57.1%). This difference between the two groups was significant (P< 0.05)( Table 1 ).
Table 1 Comparison of the general data between the two groups.
Fig. 1. CEUS in patients with MVI. Patient 1 underwent CEUS and pathological examination. The enhancement pattern of CEUS was “high enhancement - low enhancement -low enhancement”, and the pathological examination showed MVI-positive HCC. A: Enhancement pattern in the arterial phase; B: enhancement pattern in the portal phase;C: enhancement pattern in the delayed phase; D: postoperative pathological pictures (HE staining, original magnification × 100). Tumor embolus can be seen in the vascular cavity. The red arrows indicate the location of the tumor/tumor embolus. CEUS: contrast-enhanced ultrasound; MVI: microvascular invasion; HCC: hepatocellular carcinoma.
Fig. 2. CEUS in patients without MVI. Patient 2 underwent CEUS and pathological examination. The enhancement pattern of CEUS was “high enhancement - equal enhancement - equal enhancement”, and the pathological examination showed MVI-negative HCC. A: Enhancement pattern in the arterial phase; B: enhancement pattern in the portal phase; C: enhancement pattern in the delayed phase; D: postoperative pathological pictures (HE staining, original magnification × 100). No tumor embolus was found in the vascular lumen. The red arrows indicate the location of the tumor. CEUS: contrast-enhanced ultrasound; MVI: microvascular invasion; HCC: hepatocellular carcinoma.
Table 2 Comparison of CEUS parameters between the two groups.
The degree of enhancement in the MVI group decreased rapidly with the prolongation of the CEUS phase, showing dynamic fast clearance ( Fig. 1 ). Conversely, the degree of enhancement in the non-MVI group decreased slowly, showing dynamic slow clearance( Fig. 2 ). The enhancement patterns in the arterial phase between the two groups were not different (P> 0.05), but the enhancement patterns in the portal phase and delayed phase between the two groups were significantly different (P< 0.05). The enhancement intensity in the MVI group was slightly higher than that in the non-MVI group, but this difference was not significant (67.85% ± 6.82%vs. 65.99% ± 7.39%,P> 0.05). The initial increase time and time to peak were not significantly different between the two groups(P> 0.05). The washout time in the MVI group was significantly shorter than that in the non-MVI group (72.05 ± 7.92 vs. 87.29 ±8.61 s,P< 0.05) ( Table 2 , Fig. 3 A). ROC analysis showed that when washout time of 75.50 s was used as the cut-off point, the area under the curve was 0.843 (95% confidence interval: 0.742-0.945);the diagnostic sensitivity and specificity values were 85.70% and 82.10%, respectively ( Fig. 3 B).
Fig. 3. The scatter plot ( A ) and ROC ( B ) of washout time for predicting MVI. ROC: Receiver operating characteristic; MVI: microvascular invasion.
The incidence of HCC remains high in China. At present, surgical resection is the main curative approach for HCC. However, the risk of recurrence after surgery remains high. Previous studies [4–6] have confirmed that MVI is not only a precursor of HCC invasion or distant metastasis but also a high-risk factor for postoperative recurrence. Therefore, accurate preoperative determination of MVI is of great importance for the selection of treatment plans and the accurate evaluation of patient prognosis.
Histopathological examination is still the “gold standard” for MVI diagnosis. Liver biopsy is a common method for obtaining preoperative histopathological results, but it can cause trauma and carries potential risks, such as bleeding and fine-needle aspirationrelated metastasis. In addition, its accuracy is not ideal  . Therefore, it is of great significance to develop a non-invasive method that can accurately evaluate MVI before surgery.
There are many reports on the use of CT, MRI, and nuclear medicine to predict HCC MVI, but there are few reports on ultrasound-based prediction of MVI. CEUS has the advantages of strong repeatability, low cost, and real-time observation. Recent studies [8–10] have shown that the acoustic findings of CEUS are closely related to the differentiation and malignant potential of HCC, especially when distinguishing poorly differentiated HCC from HCC with other differentiation patterns. Our study revealed that the MVI group mainly had moderately or poorly differentiated tumors, while the non-MVI group mainly had well-differentiated tumors, suggesting that the degree of differentiation of HCC is closely related to MVI. The poorer the differentiation of HCC, the more aggressive the biological behavior, the stronger the tumor invasion ability, and the higher the probability of MVI. This is consistent with previous research showing that HCC MVI corresponds to poorer differentiation  . The present study also revealed that the enhancement patterns in the portal phase and delayed phase between the two groups were different, suggesting that the enhancement patterns of CEUS were closely related to MVI. MVI lesions often show fast clearance, while non-MVI lesions often show slow clearance. On the one hand, the transition from hyperplastic nodules to poorly differentiated HCC is a gradual process; with the increase in the degree of malignancy, the degree of differentiation decreases, new abnormal arteries form, and the arterial blood supply increases. Due to the decrease of portal vein blood flow,the enhancement time in the portal phase and delayed phase is shortened, and the degree of enhancement in the portal phase and delayed phase is decreased compared to that of the surrounding normal hepatic parenchyma. On the other hand, the cells involved in MVI easily form an arteriovenous short circuit, which allows for more rapid clearance of the contrast agent in the portal phase or delayed phase. Therefore, regarding the overall enhancement performance, with the prolongation of the CEUS phase, the degree of enhancement of MVI lesions decreases rapidly, showing fast clearance.
As a new ultrasound technique, CEUS can depict the blood perfusion of the lesion in real time. It can also reveal the quantitative parameters of blood perfusion of the lesion through timeintensity curve and objectively reflect the hemodynamic characteristics of the microcirculation of the lesion  . This study showed that among the CEUS parameters, washout time in the MVI group was shorter than that in the non-MVI group, suggesting that the shorter the washout time of HCC is, the greater the possibility of MVI will be. The washout time analysis was helpful for indirectly evaluating MVI in HCC patients. The explanation may be as follows: when MVI in HCC occurs, a large number of arteriovenous short circuits are more likely to form in and around the tumor tissue which result in a faster clearance of the contrast agent , as reflected in the CEUS parameters, the washout time is significantly shortened.
The ROC curve results suggested that washout time has good diagnostic efficacy in evaluating HCC MVI and can provide hemodynamic reference information for clinical evaluation.
In conclusion, CEUS reflects the hemodynamic differences in HCC and quantitatively evaluate the MVI status of HCC patients,and washout time has high applicable value for evaluating MVI in these patients. CEUS is a simple imaging method for predicting the presence of HCC MVI before surgery.
Jing-Yuan Huang: Conceptualization, Data curation, Formal analysis, Funding acquisition, Writing – original draft, Writing –review & editing. Zhi-Liang Huang: Data curation, Formal analysis, Methodology, Writing – review & editing. Zhe Yang: Writing –review & editing. Xiao-Ping Zheng: Data curation.
This study was supported by a grant from the Medical Health Science and Technology Project of Zhejiang Provincial Health Commission ( 2020KY789 ).
This study was approved by the Ethics Committee of Shulan(Hangzhou) Hospital Affiliated to Zhejiang Shuren University Shulan International Medical College (KY2021016).
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.