The influencing factors for tumor thrombus in patients with hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and has the second highest level of mortality rate among all cancers (1). Although surgery is considered to be the most effective treatment, HCC in the majority of patients is accompanied by intrahepatic metastasis, tumor thrombus, and ascites, leading to these patients being unable to undergo surgery (2). Tumor thrombus is one of key factors in determining suitability for surgery and includes microvascular invasion (MVI) and portal vein tumor thrombus (PVTT). Mähringer-Kunz et al. (3). reported that patients with HCC and tumor thrombus experience a very poor prognosis even when the tumor thrombus is in the early stage. Therefore, greater attention should be paid to the evolution and development of tumor thrombus. PVTT can be diagnosed by imaging methods, while the diagnosis of MVI is dependent upon pathology. MVI is considered to be an important factor in the early invasion and metastasis of liver cancer (4,5), with the incidence in liver cancer ranging from 15% to 57.1% (6-9), and the survival rate of HCC patients with MVI is much lower than that of patients without MVI. Therefore, it is crucial to accurately predict the independent predictors of tumor thrombus formation. The purpose of this study was thus to identify the influencing factors of tumor thrombus formation in patients with HCC. We present this article in accordance with the STROBE reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-983/rc).

Methods

Patients

From August 2011 to December 2022, 240 consecutive patients diagnosed with HCC in The First Hospital of Jiaxing were retrospectively enrolled in the study. The diagnostic criteria of HCC were based on the American Association for the Study of Liver Diseases (AASLD). Among them, nine patients were excluded due to preoperative treatment or incomplete imaging data, and a total of 231 patients were eventually enrolled in this study (Figure 1). This study was approved by the Ethics Committee of The First Hospital of Jiaxing (approval No. LS2021-KY-383). Informed consent of patients was not required for this study due to the retrospective nature of the design. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Figure 1 Flow diagram of patient selection for the study. HCC, hepatocellular carcinoma; MVI, microvascular invasion; PVTT, portal vein tumor thrombosis.

The inclusion criteria were as follows: (I) patients who underwent surgical treatment or needle biopsy in our hospital; (II) patients who were diagnosed with HCC as confirmed by pathology and immunohistochemistry; and (III) patients who underwent computed tomography (CT) or enhanced magnetic resonance (MR) imaging examination, with the images being usable for analysis. Meanwhile, the exclusion criteria were as follows: (I) incomplete clinical and examination data; (II) imaging examinations not performed in our hospital; and (III) patients who received transcatheter arterial chemoembolization (TACE) or radiofrequency ablation (RFA) before surgical treatment.

Definition

The diagnostic criterion of MVI was cancer cell nests with surrounding endothelial cells found in microvascular pathology (10).

The diagnostic criteria of PVTT included solid space-occupying lesions in the portal vein found in all phases on enhanced CT or MR images and partial enhancement and filling defects observed in the arterial phase and portal phase (Figure 2A-2C), respectively.

Figure 2 CT images of patients with HCC. (A-C) PVTT in a 65-year-old-man. A mass in the left lobe of liver with low density and an indistinct boundary was found (A). A solid space-occupying lesions was found in the portal vein on enhanced CT images (arrows in B and C) with mild enhancement. (D-F) CT images of peritumoral enhancement in a 73-year-old-man. The patchy enhancement in lesion was found at the edge of the tumor (arrow in D), showing isodensity in the portal vein phase and delayed phase (arrows in E and F). CT, computed tomography; HCC, hepatocellular carcinoma; PVTT, portal vein tumor thrombosis.

Peritumoral enhancement was defined as the focal patch of the lesion enhanced in the arterial phase, showing isointensity or isodensity in the portal vein phase and the delayed phase (Figure 2D-2F).

The Ki-67 index is determined by the proportion of Ki-67 positive cells in the total number of cells, when the nuclei and/or cytoplasm of Ki-67 positive cells are tan or brownish yellow. Ki-67 specific classification is: <10% as negative (−), 10% to 19% as weak positive (+), 20% to 49% as positive (++), and more than 50% as strong positive (+++).

Statistical analysis

Statistical analysis was performed using SPSS 26.0 software (IBM Corp). MVI-negative status, MVI-positive status, and PVTT were considered to be the dependent variables; gender, age, tumor diameter, tumor quantity, Ki-67, AFP and alanine aminotransferase levels, among others, were considered to be the independent variables. The data of patients with HCC were analyzed with univariate analysis. (I) The measurement data were analyzed with the Kolmogorov-Smirnov (K-S) test to determine whether they conformed to a normal distribution. If the data followed a normal distribution, a t-test or F test was used to determine whether there were differences between the three independent samples, with these data being expressed as the mean ± standard deviation. If the data did not follow a normal distribution, the nonparametric rank-sum test was used, with these data being expressed as the median and range. (II) The categorical data were tested with the χ² or Fisher exact test and are expressed as frequency or percentage. (III) Factors with P<0.05 in the univariate analysis were included in the multiple logistic regression model to further analyze the independent risk factors affecting the formation of tumor thrombus. (IV) A receiver operating characteristic (ROC) curve was drawn to predict the threshold of tumor thrombus formation. P<0.05 was considered statistically significant.

Results

According to the inclusion criteria, 178 males and 53 females with a mean age of 60 years (range, 26–88 years) were included in this study; the numbers of patients in the MVI-negative, MVI-positive, and PVTT groups were 194, 23, and 14, respectively (Table 1). Ki-67 expression level in the MVI-positive and PVTT groups was higher than that in the MVI-negative group (Figure 3), while tumor diameter in the PVTT group was higher than that in the MVI-negative and MVI-positive groups (Figure 4).

Figure 3 Ki-67 expression level in the MVI-negative, MVI-positive, and PVTT groups. MVI, microvascular invasion; PVTT, portal vein tumor thrombosis.

Figure 4 Tumor diameter in the MVI-negative, MVI-positive, and PVTT groups. MVI, microvascular invasion; PVTT, portal vein tumor thrombosis.

Univariate analysis of tumor thrombus grades in patients with HCC

Univariate analysis revealed statistically significant differences in eight characteristics [tumor diameter, alpha fetoprotein (AFP) level, Ki-67 expression level, gender, tumor quantity, arteriovenous fistula, peritumoral enhancement, and satellite nodules] among the three groups (P<0.05) (Tables 2,3).

Multiple logistic regression analysis of tumor thrombus grades in patients with HCC

Variables with a P value <0.05 in the univariate analysis were selected as candidate variables and subsequently analyzed via multiple logistic regression. Multiple logistic regression analysis revealed that tumor diameter, Ki-67 expression level, and peritumoral enhancement were independent risk factors for the formation of tumor thrombus (Table 4). The Ki-67 expression level in the PVTT group and MVI-positive group was higher than that in the MVI-negative group. The area under the curve (AUC) of Ki-67 expression level for predicting tumor thrombus occurrence was 0.713 (95% CI: 0.626–0.800), the cutoff was 17.5%, and the sensitivity and specificity were 61.3% and 75.7%, respectively (P<0.05). The AUC of tumor diameter for predicting tumor thrombus occurrence was 0.753 (95% CI: 0.669–0.837), the cutoff was 4.1 cm, and the sensitivity and specificity were 61.9% and 78.4%, respectively (P<0.05) (Figure 5, Table 5). The AUC of the combined factors was 0.805 (95% CI: 0.723–0.888), with a sensitivity and specificity of 65.5% and 86.5%, respectively (P<0.05).

Figure 5 ROC curves of Ki-67 expression level, tumor diameter, and their combination (Ki-67 expression level + tumor diameter) for identifying tumor thrombus occurrence in HCC. ROC, receiver operating characteristic; AUROC, area under the ROC curve; CI, confidence interval; HCC, hepatocellular carcinoma.

Discussion

Tumor thrombus is one of the most important aggressive biological behaviors in patients with HCC and also a critical factor affecting the prognosis of patients with HCC. MVI is an independent risk factor affecting the recurrence free survival time and the overall survival time of liver cancer, and is also an important clinicopathological index significantly related to the invasion and metastasis of liver cancer. If MVI can be predicted before operation, higher requirements should be put forward for the operation, emphasizing the tumor-free operation and radical resection. For postoperative liver cancer with microvascular infiltration, the commonly used and effective method is to transhepatic arterial chemoembolization to further kill residual microcarcinomas and improve postoperative survival rate. The diagnosis of MVI, also as known as microvascular carcinoma thrombosis, mainly depends on pathology (11,12). The incidence of MVI is high even in small HCCs (13,14). MVI tends to occur in small branches of the portal vein in cancer-adjacent tissues and can eventually develop into PVTT. Although PVTT can be well displayed on enhanced CT or MR images as a filling defect in the portal vein with slight enhancement, MVI is difficult to diagnose via imaging examination methods. At present, MVI is mainly diagnosed using a pathology-based 7-point sampling method, but this has certain limitations, and developing a means to predicting MVI in a noninvasive manner has become a goal of recent research. Previous studies (15-18) have shown that the occurrence of MVI is associated with many factors, such as age, cirrhosis, AFP level, tumor number, boundary and diameter of tumor, and abnormal enhancement around the tumor. Ki-67 is a sensitive and highly specific cell marker at proliferative stage, which is an important indicator reflecting the proliferation degree and biological behavior of tumor cells. The higher the positive rate of the marker, the faster the tumor growth, the worse the tissue differentiation, and the worse the prognosis. The expression level of Ki-67 can provide accurate, effective and objective technical means for evaluating the malignant degree of tumor and the efficacy of anti-tumor drugs. However, few studies (19,20) have been conducted concerning the relationship between tumor thrombus and Ki-67 expression level, other tumor indicators, and liver function indicators, and none has been conducted on the correlation between tumor thrombus and Ki-67 expression level. Ki-67, a type of monoclonal antibody, is mainly present in the nucleus, and as it is related to cell mitosis and cell cycle, it can reflect the proliferative ability of tumor cells. A previous study (21) confirmed that the Ki-67 expression level varies according to the degree of malignancy and pathological type of HCC, with the Ki-67 expression level being positively correlated with degree of HCC malignancy and negatively correlated with the prognosis and survival rate.

In this study, univariate analysis showed that there were differences in several aspects, including tumor diameter, AFP expression level, Ki-67 expression level, gender, tumor quantity, arteriovenous fistula, peritumoral enhancement, and satellite nodules, across the three different tumor thrombus grade groups. Of these factors, gender, tumor quantity, and satellite nodules were significantly different, and this result was similar to previous studies (22-24). There was no standard cutoff for tumor diameter used in a study (25), but it is generally accepted that the incidence of tumor thrombus increases with the increase of tumor diameter. The cutoff of tumor diameter in this study was 4.1 cm, which was similar to the results reporting by Liu et al. (22), who considered a tumor diameter greater than >3.9 cm to be an independent risk factor for tumor thrombus classification.

In terms of the correlation between Ki-67 expression level and tumor thrombus, one study (26) found that Ki-67 can synergistically enhance the vascular density in HCC cells to promote the occurrence of tumor thrombus, while another study (27) confirmed that a high expression of Ki-67 is linked to a greater likelihood of HCC recurrence and metastasis. Therefore, there is an urgent need to examine the correlation between Ki-67 expression level and the occurrence of tumor thrombus, as this may provide a key reference for clinical treatment, but no related studies regarding this correlation yet exist.

The results of our study indicated that the Ki-67 expression level of HCC in the MVI-positive and PVTT groups was significantly higher than that in the MVI-negative group. With the increase of Ki-67 expression level, the grade of tumor thrombus increased, revealing that high Ki-67 expression level is more likely to induce invasion and metastasis, leading to a worse prognosis. Logistic analysis further demonstrated that Ki-67 was an independent predictor of tumor thrombus, and its cutoff for predicting tumor thrombus was 17.5%. In addition, the combined diagnostic efficacy of tumor diameter and Ki-67 also was assessed, and the resulting AUC of the combination was 0.805, the sensitivity was 65.5%, and the specificity was 86.5%, indicating that a tumor diameter greater than 4.1 cm and a Ki-67 index value greater than 17.5% are highly suggestive of tumor thrombus. A consideration of these factors can improve the diagnostic accuracy for tumor thrombus after surgery and may constitute an important reference for planning the postoperative treatment of patients.

Peritumoral enhancement can be defined as the abnormal enhancement of the tumor edge in the arterial phase, which manifests as isodense lesions in the delayed and liver parenchyma phase. This area is rich in tumor cells and has a high incidence of tumor thrombus. The results of this study showed that peritumoral enhancement was an independent risk factor for predicting tumor thrombus, which was consistent with previous studies (28,29), and was also associated with the early postoperative recurrence of HCC.

Regarding the correlation between AFP expression level and tumor thrombus, the results of previous studies are generally inconsistent, but most (23,30,31) suggest that AFP expression level is correlated with tumor thrombus classification and is an independent predictor of tumor thrombus occurrence. However, in our study, there was no correlation between AFP expression level and tumor thrombus, and AFP was not an independent risk factor for tumor thrombus. The possible reason for this is that previous studies solely focused on the presence or absence of MVI or different risk levels of MVI, with a PVTT group not being included. As PVTT generally occurs at an advanced stage of tumor, some patients have significantly higher AFP levels, leading to there being no significant correlation between AFP expression level and the occurrence of tumor thrombus. In addition, the small number of patients with PVTT included in this study may also be an influencing factor. Therefore, we will further investigate the correlation between AFP expression level and tumor thrombus.

There are several limitations in this study that should be mentioned. First, a retrospective study design was used. Second, although the sample size of this study was large, the coherence between the three groups was poor. Third, although the radiographic diagnosis of PVTT was clear, the different types of PVTT were not distinguished. Additionally, most of the patients in this study had hepatitis B virus infection or a history of hepatitis B, so our findings may not be applicable to patients with nonviral cirrhosis, such as alcoholic cirrhosis.

Conclusions

Tumor diameter, Ki-67 expression level, and peritumoral enhancement were found to be independent predictors of tumor thrombus. The combination of tumor diameter and Ki-67 expression level demonstrated high diagnostic efficacy for tumor thrombus. We believe these factors can serve as a key index for predicting the formation of tumor thrombus and the prognosis of patients with HCC, thus better informing clinical decision-making.

Acknowledgments

Funding: This work was supported by the General Project of National Natural Science Foundation of China (No. 81771945) and the Jiaxing Key Discipline of Medical Imageology (No. 2023-ZC-015).

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-983/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-983/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-983/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-23-983/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). This study was approved by the Ethics Committee of the First Hospital of Jiaxing (approval No. LS2021-KY-383). The requirement for individual consent was waived by the committee 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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