VWF/ADAMTS13 ratio as a potential biomarker for early detection of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents the sixth most common cancer worldwide [1] and is the second leading cause of cancer-related death [2, 3]. Japan has one of the highest incidences of HCC in the world [1, 4]. Therefore, the diagnosis of very early stage HCC (defined as a single nodule sized ≤2 cm) is a vital prerequisite for optimizing the treatment outcomes; however, until date, it remains challenging. The professional society guidelines from the European Association for the Study of the Liver [5] and American Association for the Study of Liver Diseases [6] encourage surveillance using ultrasonography at every 6 months in patients with cirrhosis. However, they do not agree with the addition of alpha fetoprotein (AFP) as an adjunct surveillance test. The Japan Society of Hepatology Consensus-Based Clinical Practice Guidelines for the Management of HCC recommends the use of three tumor markers AFP, des-γ-carboxy prothrombin (DCP), and alpha-fetoprotein-L3% (AFP-L3) in combination with ultrasonography [7]. This approach has contributed to increase the detection of small HCCs in Japan in comparison to that in Europe and America. Furthermore, AFP-L3% has been identified as a biomarker for the early detection of HCC due to its higher specificity in comparison with that of AFP [8]. The combination use of AFP-L3 and DCP plus ultrasonography achieved a sensitivity of approximately 85% and a specificity of approximately 95% [9], whereas AFP yielded no additional benefit to ultrasonography [10]. However, AFP-L3 is not routinely used outside of Japan. Therefore, universally available biological markers for the early diagnosis of HCC are urgently needed to improve the survival rate among patients with cirrhosis.

The tumor microenvironment, notably including the surrounding blood vessels; the hepatic non-parenchymal cells, such as Kupffer cells, hepatic stellate cells (HSCs), liver sinusoidal endothelial cells; and the diverse types of lymphocytes, plays a crucial role in tumor initiation and progression in HCC [11]. A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) is predominantly produced in HSCs and cleaves newly secreted, unusually large von Willebrand factor (VWF) multimers (UL-VWFM) on the endothelial surface under high shear stress [12‐14]. An imbalance between ADAMTS13 activity and UL-VWFM, formed by endothelial cell (EC) secretion of VWF from the endothelial surface, triggers thrombosis by inducing platelet adhesion and aggregation [15]. This marked imbalance between VWF antigen (VWF:Ag) and ADAMTS13 activity (ADAMTS13:AC) is related closely to impaired hepatic functional reserve in cirrhotic patients [16, 17]. The components of the coagulation cascade regulate various steps in tumor initiation, progression, and metastasis [18, 19]; therefore, coagulation-related factors could be molecular targets for the diagnosis of patients with HCC. The aim of this study was to investigate the VWF and ADAMTS13 as potential biomarkers for the early detection of HCC in cirrhotic patients.

In recent years, several promising candidate biomarkers for the early detection of HCC have been identified; however, most of these have not been applied for clinical diagnosis due to their high cost and limited practicality in clinical practice [35, 36]. Accordingly, there is a critical unmet medical need to identify novel specific biomarkers for the early detection of HCC. To the best of our knowledge, this was the first report demonstrating that the VWF:Ag/ADAMTS13:AC ratio could serve as a novel biomarker for the early diagnosis of HCC in cirrhotic patients. In the present study, the findings showed that imbalances in the ADAMTS13 enzyme–VWF substrate were associated with HCC development. Moreover, ADAMTS13:AC was reduced in cirrhotic patients with HCC, compared with those without HCC. In contrast, Ikeda et al. revealed that plasma ADAMTS13:AC was higher in patients who developed HCC than in those who did not and that ADAMTS13:AC was an independent risk factor for HCC development [37]. The reason for this different result between the two studies remains unclear. However, one possible explanation is the differences in the progression and the underlying cause of chronic liver disease between the studies. All the patients in our study developed cirrhosis, whereas in the study by Ikeda et al., 43% of the patients had chronic hepatitis [37]. The patients in the present study included those with alcoholic hepatitis, non-alcoholic hepatitis, and autoimmune hepatitis, as well as those with HBV- and HCV-related cirrhosis; whereas only patients with HBV- and HCV-related chronic hepatitis and cirrhosis were included in the study by Ikeda et al. [37]. Furthermore, cirrhotic patients frequently have hypercoagulability, resulting in markedly increased risk for thromboembolism [38, 39]. ADAMTS13 cleaves the VWF in plasma to generate smaller, less thrombogenic fragments. ADAMTS13:AC decreases with increasing severity of liver disease, leading the observed imbalance between the decreased ADAMTS13:AC and the increased VWF:Ag in cirrhotic patients [16, 17]. These results further supported the hypothesis that ADAMTS13 enzyme–VWF substrate imbalances could be related with the hypercoagulability associated with HCC development in cirrhotic patients.

In addition, angiogenesis plays an important role in hepatocarcinogenesis in the early stages. The development of HCC is dependent on the formation of new blood vessels, in which the role of VEGF is critical [40]. The binding of the VWF to integrin avβ3 represses the VEGFR-2 activity and the downstream pro-angiogenic signaling pathways [41, 42]. However, in the current study, VWF:Ag were higher in cirrhotic patients with HCC than in those without HCC. Furthermore, Liu et al. demonstrated that VWF:Ag increased with the progression of chronic hepatitis to HCC [43]. Recently, we found that VWF:Ag increases during the development of HCC [44]. The biological function of VWF depends largely on the size of VWF multimers [45]. Large VWF multimers have been reported to be deficient in malignant diseases [46], indicating a decline in the VWF function. Moreover, the loss of large VWF multimers has been shown to be compensated by an increase in the VWF antigen levels [47]. In addition, ADAM is a fascinating family of secreted transmembrane proteins that function to regulate cell phenotypes through the effects on cell adhesion, migration, proteolysis, and signaling [48]. The levels of ADAM domain-containing protein 28 (ADAM 28) are upregulated in malignant tumors [49], and ADAM 28 derived from cancer cells cleaves and inactivates the pro-apoptotic endogenous agent VWF [50]. These findings suggested that a reduction in the function of VWF results in elevated VWF:Ag in HCC.

ADAMTS13 promotes VEGFR-2 phosphorylation, leading to enhanced VEGF expression and improved angiogenic activity of ECs [51]. In contrast, an in vitro study revealed that when VEGF expression was abundant, ADAMTS13 exerted its anti-angiogenic effects on human ECs [52]. Other studies indicated that VEGF levels progressively increased during the successive stages of hepatocarcinogenesis [40] and that elevated VEGF expression was linked with early-stage HCC [53]. A recent study by Xu et al. revealed the critical role of a balance in the ADAMTS13 enzyme–VWF substrate in regulating blood vessel formation [42]. These findings reiterate the potential role of the VWF:Ag/ADAMTS13:AC ratio in HCC development, suggesting a potential new biomarker that may allow early detection of HCC.

The present study had several limitations, including the absence of clinicopathologic or prognostic data and the small sample size. Patients with liver cirrhosis sometimes develop thrombosis or inflammation, including portal thrombosis, bacterial overgrowth, and translocation. When the VWF:Ag/ADAMTS13:AC ratio is used as a biomarker for the early detection of HCC, thrombosis and inflammation might affect the value of the ratio. Only patients with hypervascular HCCs were analyzed in the present study; therefore, pathologically, early or hypovascular HCC should be examined in the future. Further studies need to be carried out to validate the findings of the present study.

The VWF:Ag/ADAMTS13:AC ratio probably contributes to HCC development identified small, early-stage, and AFP-negative HCC in cirrhotic patients. Although the use of either VWF:Ag or ADAMTS13:AC alone would be insufficient in aiding the early diagnosis of HCC, the ratio of the two biomarkers can greatly increase the accuracy. The diagnostic accuracy of the VWF:Ag/ADAMTS13:AC ratio was comparable with that of AFP-L3%; nonetheless, the VWF:Ag/ADAMTS13:AC ratio is a remarkably superior diagnostic biomarker of AFP-L3% in terms of universality and objectivity and could serve as a potentially favorable biomarker for the early diagnosis of HCC in cirrhotic patients.