Tumor-associated lymphangiogenesis predicts unfavorable prognosis of intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer that accounts for approximately 10 to 15% of all primary liver malignancies [1, 2]. Radical resection of the tumor is the potential curative treatment that prolongs survival outcomes [3]. However, most patients lose the opportunity to undergo radical surgery due to advanced stage or extrahepatic metastasis at the time of diagnosis [4]. Moreover, application of adjuvant therapies, such as chemotherapy and radiotherapy, is yet to be conclusively confirmed whether these provide significantly better survival outcomes [5]. In consideration of increasing number of patients and limited therapeutic strategies, a better understanding of potential risk factors of ICC is essential to seek novel anti-tumor targets and to develop new treatment approaches.

Metastasis of tumor cells is one of the leading causes of mortality in ICC and occurs mainly through lymphatic vessels [6]. In recent years, increasing experimental and clinicopathological studies indicated that lymphatic vessels undergo dynamic changes within the tumor, including tumor-associated lymphangiogenesis, that facilitate metastasis [7]. Specific signaling pathways, such as vascular endothelial growth factor C (VEGF-C) and its cognate receptor VEGF receptor 3 (VEGFR-3) are confirmed to stimulate tumor-associated lymphangiogenesis, as well as lymph node metastasis in experimental tumor models [8‐10]. Apart from that, lymphangiogenesis was found to be associated with increased frequency of tumor cells in lymphatics and lymph nodes in hilar cholangiocarcinoma [11]. However, information regarding the role of tumor-associated lymphangiogenesis in progression and prognosis of ICC remains poor. In this study, we evaluated impact of tumor-associated lymphangiogenesis in patients with ICC, a highly malignant and aggressive primary liver cancer.

ICC is a highly malignant neoplasm with a dismal prognosis due to insufficient evident symptoms at the early stage [15]. Up to date, radical hepatic resection remains the only ideal curative treatment for ICC. However, only a minority of patients with ICC are candidates for radical surgical approaches, with a median survival of 6.3 to 16 months [16]. Lack of appropriate medical approaches for ICC calls for further studies on its clinical and biological characteristics. Frequent lymph node metastasis is one of the major hallmarks of ICC that has been identified as a significant prognostic predictor [17, 18], while tumor-associated lymphangiogenesis in prognosis of ICC remains further confirmation.

In the present study, quantitative analysis of MLVD was performed by counting lymphatic vessels stained with podoplanin. Previously, studies of lymphatic vessels and dissemination have been limited due to lack of specific lymphatic markers. Vascular endothelial growth factor receptor-3 (VEGFR-3) was formerly considered as a marker for lymphangiogenesis. However, VEGFR-3 has also been shown to be expressed in blood vessel endothelium and cancer cells [19, 20]. In recent years, some specific markers, including LYVE-1 and podoplanin, have been identified. However, LYVE-1 has also been found to express on the endothelium of blood vessels [21]. In contrast, podoplanin is expressed specifically in lymphatic endothelium and does not exist in blood vasculature [22]. Therefore, podoplanin was chosen to evaluate tumor-associated lymphangiogenesis in the ICC specimens. Moreover, although not described in the present study, lymphatic vessels in adjacent healthy tissues were also evaluated, which ranged from 0 to 5. These vessels seem to play normal biological roles in the propulsion of the lymph circulation considering its no association with metastasis and prognosis of ICC. In addition, high expression of tumor-associated lymphangiogenesis was found to be closely associated with lymph node metastasis and recurrence, suggesting that tumor cells of ICC are likely to migrate along tumor-associated lymphatic vessels into lymph nodes, thus facilitating recurrence of the tumor that leads to poor prognosis. These findings are in line with several previous studies that reported association between lymphatic vessels and lymph node metastasis in mass-forming tumors [23‐25].

In addition, it is noteworthy that patients with low expression of MLVD seemed to be more likely to be infected with HBV preoperatively (p = 0.007), which was identified as a prognostic factor for recurrence-free survival (p = 0.009). HBV infection has been considered associated with an increasing incidence of ICC in East Asia, where HBV is endemic [26, 27]. However, several studies found that HBV infection may be a favorable factor in patients with ICC. Zhou et al. [28] suggested that the survival of HBV-associated ICC patients was better due to inhibited tumor invasiveness. Wu et al. [29] regarded HBV infection as a favorable factor because of early detection of the unexpected tumor during chronic liver disease follow-up. On the contrary, Ahn et al. [30] reported that 37 HBV-positive and 255 HBV-negative ICC patients showed no significant difference in their outcomes. Since the distribution of HBV infection in ICC was found to vary worldwide, the different results may be attributed to patients enrolled from various regions and different stages of tumor upon diagnosis. Thus, our results require validations in populations from different regions.

Recently, prophylactic lymph node dissection has been considered as a benign factor that might improve the prognosis of the patient with ICC [31]. A study from Japan rendered that the wide dissection, including the regional and para-aortic lymph nodes, along with hemihepatectomy was found to be curative in the first 5-year survivor with periductal-infiltrating ICC [32]. Since tumor-associated lymphangiogenesis is an important independent prognostic factor that deteriorates survival by promoting lymph node metastasis, intraoperative biopsy to evaluate extent of tumor-associated lymphangiogenesis may support decision for performance lymphadenectomy.

Moreover, tumor-associated lymphangiogenesis may serve as one of the novel therapeutic targets of ICC, of which the VEGF-C/VEGF-D-VEGFR3 axis is considered a major driver of tumor-associated lymphangiogenesis [33]. In addition, other growth factors and signaling pathways are also involved in lymphangiogenesis. VEGF-A was found to promote lymphangiogenesis and lymph node metastasis in a mouse fibrosarcoma xenograft model [34]. Platelet-derived growth factor B also induced tumor lymphangiogenesis and promoted lymph node metastasis in mice, but this could not be prevented using a VEGFR3-targeted antibody [35]. While, transforming growth factor-β (TGF-β) has been reported to be a negative regulator of lymphangiogenesis in cancer. Inhibition of endogenous TGF-β signaling in mouse xenograft models of pancreatic adenocarcinoma induced lymphangiogenesis [36]. Based on these findings, various tyrosine kinase inhibitors targeting VEGF, EGRF or FGF receptors such as sorafenib, sunitinib, regorafenib, etc. are under clinical trials for different types of tumor [37‐39]. Since clinical trials and reports about antitumor therapy targeting lymphangiogenesis in ICC are still rare, the results of our study may provide a novel view of treatment for ICC.

This study provides a great deal of information about the critical influence of tumor-associated lymphangiogenesis on prognosis of hepatic resected patients with ICC. However, there are a few underlying limitations that remain to be confirmed. Firstly, limited number of patients may cause bias of the results, which could be improved by additional large-scaled studies. Secondly, the involved patients were local malignancies within the liver, patients with advanced and metastatic tumors may result in different outcomes. Lastly, all the involved patients were from Eastern countries, further confirmations by patients from other regions are needed.

High extent of tumor-associated lymphangiogenesis is significantly associated with lymph node metastasis and recurrence of the tumor in ICC. MLVD, a indicative factor for the extent of tumor-associated lymphangiogenesis, was found to be an independent prognostic factor for overall and recurrence-free survival in patients with ICC after resection. Evaluation of tumor-associated lymphangiogenesis by intraoperative biopsy seems promising to possess a great potential to improve survival outcomes by providing guidance to patient selection who may benefit from lymphadenectomy. We call for future large-scale prospective trials to verify our results for general application in real-world clinical medicine.