Positive expression of Midkine predicts early recurrence and poor prognosis of initially resectable combined hepatocellular cholangiocarcinoma

Hepatocellular carcinoma (HCC), the most common primary liver malignancy, originates from hepatocytes. Cholangiocarcinoma (CC), the second most common, originates from bile duct epithelial cells. Combined hepatocellular cholangiocarcinoma (CHCC-CC), a rare primary liver cancer consisting of two components, HCC and CC, accounts for 1% to 14.3% of all primary malignant liver tumors [1–9]. To date, complete tumor resection is its only possible cure. It is difficult to precisely diagnose this disease preoperatively. Although CHCC-CC is usually diagnosed post-operatively based on pathological findings, immunohistochemical stains have also been used to further confirm the presence of both components [6, 8, 10, 11].

The clinicopathological characteristics and prognosis of patients with CHCC-CC after surgery have not been reported in detail because the incidence of this disease is very low and studies on it are scarce. While these studies report conflicting outcomes, most conclude that CHCC-CC has a worse prognosis than either HCC or CC alone [12]. Only a few papers discuss the possible molecular markers that could be used to predict outcome in CHCC-CC [13, 14]. Because the molecules studied were not found to efficaciously predict likelihood of post-surgical CHCC-CC recurrence, there remains a need to identify biomarkers that can help assess its treatment response as well as predict recurrence and prognosis.

Midkine (MK) is a heparin-binding growth factor weakly expressed or undetectable in normal adult tissue but strongly expressed during embryogenesis [15]. It is expressed at abnormally high levels in several human cancers, including esophagus, gall bladder, pancreas, colorectal, breast, salivary gland and lung carcinomas [16–18]. It has been found to exacerbate disease by promoting many tumor specific functions, including cell growth, tumor cell survival, cell migrations, and carcinogenesis [19–21].

Some studies have investigated the possibility of using MK as a biomarker to predict prognosis and assess response to treatment in oral squamous cell carcinoma and report that its positive expression predicts poor prognosis in patients with various malignant tumors, including head and neck squamous cell carcinoma [22], esophageal squamous cell carcinoma [23] and non-small cell lung cancer [24]. Although MK is known to be overexpressed in hepatocellular carcinoma [25] and cholangiocarcinoma [26], little is known about its significance in CHCC-CC. Therefore, this study investigated the relationship between its expression and the pathogenesis of CHCC-CC as well as the disease’s clinicopathology and survival.

The incidence of CHCC-CC, a rare tumor, varies among different studies. The actual incidence may be higher than usually reported for two reasons. First, because most patients have either no symptoms or non-specific symptoms, clinical presentation does not generally contribute to diagnosis. Second, patients with CHCC-CC frequently present more advanced disease stages than those with typical HCC, and so a high percentage of patients with CHCC-CC do not receive surgical resection [28]. It is very difficult to make accurate diagnosis of CHCC-CC before surgery, because CT or MRI scans often do not show typical patterns of contrast uptake or washout. CHCC-CC may have CT features of both HCC and CC when a hepatic tumor contains an area of hyper-enhancement in the early phase and an area of delayed enhancement in the late phase on dynamics CT [29]. It may also have features of both when a hypovascular liver tumor is associated with significant elevation of a-fetoprotein levels and multiple regional lymph nodes metastases [30], or when a hepatic tumor has hypervascular expression and elevation of serum CEA and carbohydrate antigen 199. Although histological studies may be able to identify a dominant tumor type, they usually fail to detect the presence of both CHCC and CC. Therefore, precise diagnosis of CHCC-CC before surgery remains a challenge.

Some studies report CHCC-CC to have a worse prognosis than either HCC or CC alone [3, 9]. Similarly, our 52 CHCC-CC patients, who had received intensive curative tumor resection, had a two-year disease free survival of 42.1% (median 17.3 months) and a three-year overall survival of 44.6% (median 22.9 months). Because most of the CHCC-CC patients in this study were male patients with a high prevalence of HBV, chronic HBV infection may be a major cause for both HCC and CHCC-CC in Taiwan. The factors predicting survival also vary among different studies, possibly due to the limited number of patients [12]. Univariate analyses performed by prior studies found overall survival to be significantly related to microscopic vascular invasion, bilobar tumors and tumors > 6 cm [9], vascular invasion and satellite lesions [3], and lymph node metastases. Univariate analysis of our patients’ data found LVI, T stage III-IV, AJCC tumor stage III-IV and positive midkine expression to be associated with poorer disease free and overall survival. Our multivariate analysis revealed AJCC tumor stage III-IV to be the most important predictor of both survival rates. These findings are consistent with previous CHCC-CC studies [3, 31, 32].

In this study, positive expression of MK in tumors was associated with poor prognosis and reduced survival in CHCC-CC patients, suggesting that MK could potentially be used as an independent post-surgical prognostic biomarker for CHCC-CC. MK expression predicted poor prognosis in disease free survival in both our univariate and multivariate analyses. Yoon et al. [33] attributed poor overall survival in this population to shorter survival after recurrence. Although our multivariate analysis did not find a significant association between MK expression and overall survival, our univariate analysis did, though the small number of cases may have affected this result.

MK, a heparin-binding growth factor, plays a central role in chemotaxis, angiogenesis, and the inhibition of apoptosis. The expression of MK is elevated in various tumors [34], and has been described as a potential prognostic marker in several malignancies, including esophageal cancer [35], endometrial carcinoma [36] and gastric cancer [37]. One study found breast cancer patients to have higher plasma levels of midkine than their healthy controls [38]. Another study also found a correlation between MK protein expression and malignant status and prognosis of breast cancer patients [39]. Zhu et al. found serum MK levels to be clearly increased in hepatocellular cancer patients and suggested they could be used to diagnose hepatocellular cancer with a high sensitivity. Moreover, serum MK levels, which were reported by one study to be markedly decreased in hepatocellular cancer patients after curative resection, were found by the same study to re-increased when tumors recurred [40]. Keto et al. also reported MK to be increased at messenger RNA and protein levels in patients with intrahepatic cholangiocarcinoma, although they stated that the ultimate biological significance and their possible relationship to tumor behavior had not been established. To the best of our knowledge, the current study is the first to report the results of MK immunohistochemical analysis of CHCC-CC tissue.

Cancer stem cell markers have been correlated with poor prognosis in primary liver malignancy and their presence has been associated with carcinogenesis, vascular invasion, and metastasis in this disease [41–43]. Therefore, the 2010 WHO classification divides CHCC-CC into two subtypes: the classical type and subtypes with stem cell features [44]. Classical type CHCC-CC includes HCC areas, CC areas and transitional zones, which comprise tumor cells with stem cell features. MK is known to have the ability to induce epithelial mesenchymal transition (EMT) in some types of cancer cells, the differentiation of lost polarity in epithelial cells and cell adhesion to contractile and motile mesenchymal cells [45]. In addition to angiogenesis, Takenaka et al. found that the growth of mouse embryonic stem cells could be induced while MK inhibits apoptosis through the PI3K/Akt signaling pathway [46]. Zhao et al. reported that mesenchymal stem cells overexpressing MK transplantation stimulate vasculogenesis effectively by increasing pro-angiogenesis factors (VEGF, TGF-β). These findings suggest that MK might be involved in the pathogenesis of liver cancers with stemness. Our study showed the patients with CHCC-CC who had a positive expression of MK to be at much higher risk of early recurrence and poor survival. However, currently MK is still not a suitable as therapeutic target and drugs need to be developed for this in the future.

This study has several limitations. First, it was based on a retrospective analysis of data collected from only 52 patients accumulated over a short period. Second, only three enrolled CHCC-CC patients had regional lymph node metastases. Preoperative diagnosis for patients with CHCC-CC is very difficult because there is no typical pattern of contrast uptake or washout in dynamic CT or MRI scans. In our series, most patients were diagnosed as having HCC before surgery. Patients who are found to have image study evidence of lymph node involvement before treatment often receive systemic treatment instead of hepatectomies, since HCC with lymph node metastases post curative resection has been reported to have poor disease free and overall survival [47]. Third, therapies used to treat recurrent CHCC-CC may affect overall survival. Some studies showed the recurrence rates following resections of these tumors were high and these recurrent tumors were commonly detected in the remnant liver [12]. Salvage treatment can include surgery, trans-arterial embolization, percutaneous ethanol injection, and radiofrequency ablation.

In conclusion, the findings of the present study suggest that heparin-binding growth factor MK contributes to the clinical outcome in patients with resectable CHCC-CC. It is significantly elevated in patients with advanced T-stage CHCC-CC. A high level of MK protein independently predicts a poor prognosis for patients receiving surgery for this disease.