The correlation between poor prognosis and increased yes-associated protein 1 expression in keratin 19 expressing hepatocellular carcinomas and cholangiocarcinomas

The two major histologic types of primary hepatic carcinomas are hepatocellular carcinomas (HCCs) and cholangiocarcinomas (CCAs). HCC is a malignancy with hepatocellular differentiation and CCA is a malignancy with biliary epithelial (cholangiocytic) differentiation. HCCs are the most common type of primary hepatic carcinomas, followed by CCAs. As the difference of histopathogensis, HCCs and CCAs have different clinical behaviors. HCCs can be treated by some loco-regional treatments, such as embolization, radiofrequency ablation, surgical resection, or liver transplantation. However, a high post-treatment recurrence rate remains a challenge for long-term survival [1]. On the other hand, CCAs have been known to have a poorer prognosis than HCCs [2]. Surgical resection is the first treatment of choice for limited stage CCAs, but more advanced stage CCAs are considered to be chemotherapy-resistant [3].

There are some intermediate entities between HCCs and CCAs. These tumors are characterized by their coexisting hepatocytic and cholangiocytic differentiation in the aspect of morphology and associated protein expression. Tumors typically included in this list are classical type combined hepatocellular-cholangiocarcinomas (cHC-CCAs), cHC-CCAs with stem cell features, HCCs expressing stemness markers, and the cholangiolocellular type of intrahepatic CCAs. Classical type cHC-CCAs are typical collision tumors of unequivocal HCC and CCA components. cHC-CCAs with stem cell features are a newly described subgroup, first categorized in 2010 in the 4th edition of the WHO classification of tumors of the digestive system [4]. cHC-CCAs with stem cell features are a collection of hepatic tumors that display morphological and biological evidence for hepatic stem cell or hepatic progenitor cell differentiation. Markers related to stemness are CD133, epithelial cell adhesion molecule (EpCAM), CD56, and cytokeratin 19 (CK19). These markers are normally expressed in the bile ducts in the terminal portal tracts or in the proliferative ductules, frequently identified in injured hepatic parenchyma, and act as a phenotypic marker for hepatic progenitor cells that play a role in liver regeneration and tumorigenesis [5, 6]. cHC-CCA with stem cell feature group has three subcategories: 1) the typical subtype, 2) the intermediate subtype, and 3) the cholangiolocellular subtype [4]. Differential points within these subcategories are morphologic differences, such as the histological pattern of tumor cells, or the distribution of stemness-expressing cells. The cholangiolocellular subtype was first described in the 2000 3rd edition of the WHO classification as a kind of histologic variant of intrahepatic CCAs [7]. The cholangiolocellular pattern has recently come into the spotlight, as intrahepatic CCAs have been classified into two groups, based on their cell of origin in the biliary system [8]. The first group is CCAs arising from the large bile duct with peribiliary glands and the second group is formed by CCAs arising from the canals of Hering, where the hepatic progenitor cells are located. These two groups have different morphological, etiological, clinicopathological, and molecular features [8]. Cholangiolocellular intrahepatic CCAs are considered the most characteristic type of CCAs arising from the canal of Hering. Therefore, cHC-CCAs with stem cell features and cholangiolocellular CCAs are suggested to share similar biological features and clinicopathological behaviors.

HCCs expressing stemness markers are a well-known HCC prognostic subgroup and are characterized by having a poor prognosis and aggressive tumor behavior [2, 9, 10]. This group shows both the typical histological features of HCCs, as well the expression of stemness markers (CD133, EpCAM, or CK19). Because the only major difference between cHC-CCAs with stem cell features and HCCs expressing stemness markers are their morphological features, a gray zone between two groups, based on the opinion of the pathologist in diagnosis, does exist.

YAP1 (Yes-associated protein 1), also known as YAP65, is the mediator of the mammalian Hippo pathway and is associated with development, organogenesis, and postnatal growth [11]. The physiological role of YAP1 in the liver is the commitment of the intrahepatic bile duct to the induction of the cholangiocyte phenotype in the embryonic period and the enlargement of the hepatic volume by expanding progenitor cells, such as oval cells in the adult liver of animal models [12, 13].

In tumorigenesis, YAP1 has been known as an oncogene, due to of its function in promoting cancer cell survival, proliferation, invasion, and migration in several types of malignancies. YAP1 has been associated with poor prognosis in gastric cancer, gallbladder cancer, and hepatocellular carcinoma [14‐16]. A meta-analysis on YAP1 expression in various cancers and its prognostic implication reported that upregulation or nuclear expression of YAP1 indicated a worse disease-free survival time and a reduction in the overall survival time (OS) [17]. Recently YAP1 has been reported to play a role as a tumor suppressor during intestinal regeneration in human colorectal cancer [18].

This study aimed to assess the role of the Hippo pathway in hepatic carcinogenesis and morphogenesis by comparing YAP1 expression in hepatic carcinomas, categorized into eight disease groups (including HCCs, CCAs, and intermediate hepatic carcinomas), and analyzing the clinicopathological characteristics of YAP1 expressing tumors in each disease group.

Our study demonstrated that increased nuclear expression of YAP1 could be observed in cholangiocarcinomas (EHBCA and IHCCA) and a subset of hepatocellular carcinomas (CK19(+) HCC) and that nuclear YAP1 expression was correlated with poor overall survival in pT1 stage IHCCA patients. Interestingly, YAP1 expression rate in cHC-CCAs and cholangiolocellular IHCCAs was not so high as in EHBCAs, IHCCAs and CK19 (+) HCCs and was more similar to the expression levels seen in the CK19 (−) HCC group.

The Hippo pathway plays a major role in liver regeneration, development, and regulation. The determination of cellular lineage is of vital importance to this process. Activated YAP1 often induces hepatomegaly in animal models with mutated Hippo pathway genes, but the cellular components of the over-grown liver vary depending on the nature of the defective genes, e.g. hepatocyte proliferation, biliary hyperplasia, or abundant hepatic progenitor cells [21, 22] . Therefore, the determination of cell fate cannot be solely explained by activated YAP1. The level of activated YAP1 has also been associated with the determination of cell fates, differentiation to hepatocytes, and dedifferentiation to stem cells or progenitor cells [12]. In hepatic carcinogenesis, the genomic amplification of loci containing YAP1 in HCCs, suggests that YAP1 might possess an oncogenic effect [23]. As the determination of cell fate is extremely complicated, a simple inactivation of the Hippo pathway or the over-expression of YAP1 cannot induce liver cancer with an identical histological type [21]. Overexpression of YAP1 has been reported in the early phase of hepatocellular carcinomas, but the induction of hepatoblastoma required coactivation of β-catenin and interaction with PI3K has been reported to be involved in the induction of CCAs [24‐26].

Based on this function of YAP1, we expected a gradual increase of YAP1 expression from HCCs to CCAs and culminating in high levels of overexpression in cHC-CCAs, cholangiolocellular CCAs, and CK19(+) HCCs, which are known to have “stemness” traits. However, only CK19(+) HCCs showed increased levels of YAP1 expression, as the levels of YAP1 expression in non-cholangiolocellular IHCCAs. Regardless of subtype, the cHC-CCAs showed a low positive rate of YAP1 expression, as did the CK19 (−) HCC group. The cholangiolocellular type of IHCCAs, which was assumed to originate from the hepatic progenitor cells in the canal of Hering, also had low YAP1 expression. The results we obtained were different from those seen in a previous study, which reported heightened levels of YAP1 in both HCCs with stemness and in cHC-CCAs when compared to HCCs without stemness [27]. As CK19(+) HCCs in our study were similarly defined as “HCCs with stemness”, increased YAP1 expression in CK19(+) HCCs was a consistent finding between two studies, but a similar result for cHC-CCAs could not be reproduced. CK19(+) expression in HCCs has been generally considered an indicator of stemness and poor prognosis for CK19(+) HCC patients was thought to be due to this stemness trait [10]. However, hepatic carcinomas with stemness features showed heterogeneous clinical behavior in our study cohort. Among the 8 disease groups, CK19(+) HCCs, cHC-CCAs with stem cell features, classical cHC-CCAs and cholangiolocellular type IHCCAs could be classified into stem cell feature carcinomas and this 4 disease groups could be divided into two different survival groups (log rank p-value = 0.001, Additional file 1: Figure S1). cHC-CCAs with stem cell features and cholangiolocellular IHCCAs were grouped with CK19(−) HCCs in displaying similar or better PFS than CK19(−) HCCs (median, 103 vs. 23 months, log rank p-value within groups 0.123 within groups) (Additional file 1: Fig. S1). However, CK19(+) HCCs were included in the poor prognosis group, together with IHCCAs and classical cHC-CCAs (median, 18 vs. 11 vs. 12 months, log-rank p-value within groups 0.670) (Additional file 1: Figure S1). Although other prognostic factors such as stages, preoperative or postoperative treatment modalities and difference of group size should be considered on the interpretation of prognosis, this result suggests that hepatic carcinoma with stemness features may be a heterogeneous entity and is consistent with the short mention about the conflict evidences of prognosis of cHA-CCA with stem cell feature [4]. Furthermore, hepatic carcinomas with stemness features are extremely heterogeneous in regards to their morphology and clinicopathological behavior as described in Table 1 and result section. Therefore, the expression of stemness markers in hepatic carcinomas might be due to poor differentiation or the acquisition of invasiveness by the tumor cells, and not due to the acquisition of stemness. Although we could identify an association between poor prognosis and activated YAP1 in hepatic carcinomas, this was only confirmed in the pT1 stage IHCCA patients. Nevertheless, we did observe a similar trend in the tumor progression of pT1 stage HCC patients. These results are consistent with previous studies reporting on the prognostic value of activated YAP1 in CCAs and HCCs [28, 29]

Our study tried to elucidate the role of the Hippo pathway in the morphogenesis of the liver in regards to liver carcinomas and found that YAP1 activation was more commonly found in CCAs than pure HCCs. However, the heterogeneous pattern of YAP1 expression between cHC-CCAs and CK19(+) HCCs and the poor prognosis of YAP1 positive hepatic carcinomas suggests that YAP1 may have a preferential role in aggressive tumor behavior, rather than in the determination of cellular lineage in hepatic carcinomas.