Combined hepatocellular and cholangiocarcinoma (CHC), which is characterized by hepatocellular and biliary epithelial differentiation within the same tumor [1–3], is a unique type of primary hepatic carcinoma that is distinct from hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). CHC has been reported to account for 0.4% to 14.2% of primary hepatic carcinoma cases [4]. Between January 1982 and December 2009, of 31,000 cases of primary hepatic carcinoma treated at the Eastern Hepatobiliary Surgery Hospital (EHBH) in Shanghai, China, 92.3%, 6.7%, and 1.9% were classified as HCC, ICC, and CHC, respectively [5].

Although some clinical features of CHC are similar to those of HCC and ICC [4], the biological behavior of CHC and the prognosis for patients with CHC are completely different. Because CHC has components of both HCC and ICC, the effects of its two different components on clonal origin and clinical outcomes have become a focus of attention. However, molecular alterations in CHC have not been extensively studied, and the clonal link between the HCC and ICC components of CHC remains unclear [4, 6–14].

An accurate method to identify the origin of CHC is to determine whether the tumor components (i.e., HCC and ICC) are from a single clonal origin or multiple clonal origins. For clonal analysis of CHC, the most precise techniques rely on the detection of common patterns in DNA aberrations within each tumor component (i.e., the hepatocyte- and the biliary tubular-differentiated zones). Previous studies have used the integration pattern of hepatitis B virus (HBV) DNA detection, the X-chromosome inactivation assay, and comparative genomic hybridization as methods of tumor clonal origin determination [6]. However, these methods have limitations, including only being suitable for female patients and requiring fresh tissue specimens and expensive equipment and reagents. In general, these methods are not appropriate for routine clinical pathologic analysis using paraffin-embedded blocks. Loss of heterozygosity (LOH) is a non-random allelic loss of specific chromosomal loci, and it is closely associated with silencing of known or unknown tumor suppressor genes. Our previous research indicated that LOH detection has technical advantages for clonal discrimination in recurrent HCC [15] and hepatic carcinosarcoma [16].

Although there have been several studies of CHC, systematic studies in large cohorts of CHC patients, with detailed information on diagnostic criteria, surgicopathologic features, clonal origin, and clinical prognosis, are lacking. In the present study, we analyzed the characteristics of molecular clones in 34 CHC cases and compared them with 29 cases of separated HCC and ICC (SHC) with their respective differentiations in the same liver. In this study, SHC refers to separated HCC and ICC nodules in the same liver. In addition, we retrospectively evaluated the clinicopathologic characteristics and prognostic factors related to overall survival (OS) and disease-free survival (DFS) in patients with CHC and compared them with those in patients with SHC, pure HCC, and pure ICC who underwent resection during the same period.

CHC is a rare form of primary liver cancer, showing a mixture of hepatocellular and biliary features. In the present study, we demonstrated that the HCC and ICC components of CHC may originate from the same clone compared with SHC. CHC tended to exhibit the biological behaviors of both HCC and ICC, which may lead to poor clinical outcomes for patients with CHC.

CHC was first described by Wells in 1903 [18], and, in 1985, Goodman et al. [19] reported criteria to classify CHC into three subtypes: type I, “collision tumors,” a coincidental occurrence of HCC and ICC within the same liver; type II, “transitional tumors,” with transition from HCC differentiation to typical ICC differentiation; and type III, “fibrolamellar tumors,” a unique variant of fibrolamellar HCC. However, the World Health Organization (WHO) classification defines CHC as a tumor with an intimate and unequivocal admixture of both hepatocellular carcinoma and cholangiocarcinoma cells [3]. The WHO classification further states that CHC should be distinguished from cases of (i) SHC in which HCC and ICC arise in the same liver and (ii) collision-type tumors in which HCC and ICC are present at adjacent sites. Because there were no typical collision tumors in the present study, SHC was used as a control group for evaluating clonal differences between collision tumors and CHC.

In our study, the male-to-female ratio for CHC was 7.5:1, which is similar to the 8:1 ratio reported by Zhan et al. [20]. The frequency of HBV infection in CHC group was 94.1%, which is higher than the 65% described by Yano et al. [21] and the 75.9% described by Kim et al. [22]. However, in our study, the median age of patients with CHC was 52 years, which is younger than the median ages of 57 years and 53 years in the above reports [21, 22]. These results strongly suggest that HBV infection is a key etiological mechanism in patients with CHC, and that Chinese CHC patients may be younger at diagnosis compared with patients in the above reports [21, 22]. More interestingly, we identified a concomitant increase in AFP and CA19-9 levels in CHC patients (29.4%, 10/34). This phenomenon indicates that the HCC and ICC components of CHC have individual biological functions, with expression of both cholangiocyte and hepatocyte markers. This is an important characteristic of CHC, which may help to distinguish CHC from pure HCC or ICC before surgery.

The cellular origin of CHC is a controversial issue, but three main theories have been proposed: (i) collision (double) tumor of HCC and ICC that coincidentally exist in the same liver; (ii) subsequent differentiation of HCC or ICC into the other component; and (iii) derivation from HPCs, which have the potential to differentiate into both HCC and ICC [21, 23–25]. Despite the above speculations on the cellular origin of CHC, extensive studies on genetic alterations in CHC and comparative analyses between CHC and SHC are lacking. No previous studies have investigated the clonal origin of CHC by microsatellite LOH analysis.

As a control group of multiple clonal origins, 10 cases of SHC (20 tumor nodules) were selected as collision tumors. The differences in LOH patterns of the 10 microsatellite markers in all 10 cases of SHC were greater than 30%, which was consistent with the HCC and ICC nodules having independent origins, or a tumor type of multiple clonal origins. In comparison, the LOH incidence of 10 microsatellite markers in all 16 cases of CHC was less than 30%, suggesting that each tumor component (i.e., hepatocellular- and biliary-differentiated zones) of CHC shares the same genetic alterations and may be derived from the proliferation of a single clone. Furthermore, 52.9% of CHC had cytoplasmic co-expression of Hep Par1, GPC3, and CK7 in both hepatic zones and biliary zones, indicating that the HCC component expresses biliary markers and the ICC component expresses hepatocyte markers. This dual-modality phenotype strongly suggests that CHC originates from HPCs with the ability for multipotent differentiation.

In our study, all the metastatic lymph nodes originated from the ICC component, whereas the HCC element was mainly identified in microvascular invasion, suggesting that ICC and HCC cells from CHC maintain their distinct metastatic pathways, resulting in CHC having a dual biological mechanism associated with high malignancy. Another interesting phenomenon was the tendency for the biological behavior of CHC to be influenced by the proportion of HCC and ICC components. In the case that the HCC component was dominant, CHC was likely to exhibit the characteristics of HCC, and vice versa for a dominance of ICC components.

Because of CHC’s dual biological mechanism, patients with CHC usually have a poor prognosis. It was reported that the postoperative median survival time of patients with CHC varied from 9 to 22 months and was substantially shorter than that of HCC patients [21, 26–29]. In our study, the mean OS of patients with CHC was 15.4 months, being shorter than that of the pure HCC group (29.2 months) but longer than that of the pure ICC group (10.1 months). Multivariate analysis revealed that vascular invasion, lymph node metastasis, and histologic differentiation of the ICC component were independent risk factors for OS. These findings suggest that ICC plays a more important role than HCC in influencing the prognosis of CHC, although this requires further confirmation.

In patients with CHC, the main etiological factor was HBV infection. The two components of CHC tumors (the hepatocellular- and biliary-differentiated zones) arose from a single clonal origin, which may be HPCs. CHC has a highly aggressive behavior with dual biological mechanisms.