—  SPECIALTY CONFERENCE  —

Liver Pathology

Case 4 - Combined Hepatocellular Cholangiocarcinoma

Matthew M Yeh
University of Washington School of Medicine
Seattle, Washington, USA




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Clinical History
A 59-year-old man with cirrhosis due to chronic hepatitis C had a 2 cm, solitary liver lesion in segment 2. His alpha-fetoprotein (AFP) was 21. Three phase CT scan showed a hypervascular lesion on arterial phase with rapid washout on delayed phase. Chest CT scan did not show any evidence of metastatic disease. Orthotopic liver transplantation was performed.


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Pathologic Findings
The liver explant showed a cirrhotic liver with a 1.8 cm white, soft and focally firm nodule in segment 2/3. Microscopic examination demonstrated a neoplasm with a component of the neoplastic cells forming trabecular and pseudoacinar architecture with endothelial cells wrapping around. There were also many unpaired arteries embedded within the neoplasm. Areas of canaliculi-like structure containing bile were present. The neoplastic cells had relatively abundant and eosinophilic cytoplasm with increased nucleus to cytoplasmic ratio. These features resembled a moderately differentiated hepatocellular carcinoma (HCC). There was also another component of the neoplasm forming glandular structures with cribriforming. Focally these glandular structures were embedded in a dense fibrous and desmoplastic stroma. The neoplastic cells demonstrated hyperchromatic nuclei with pleomorphism. Irregular and aborted glandular structures with occasional single cells were also present, mimicking an adenocarcinoma. There was also an interface between these two neoplastic components. At the interface of these two components, the neoplastic cells showed features that were intermediate between the HCC and adenocarcinoma. A mucicarmine stain showed mucin in the adenocarcinoma, sometimes merging with the HCC component. Immunohistochemical studies showed positive Hep Par 1 staining in the HCC component, and positive CK7 and CK19 stainings in the adenocarcinoma component.

Diagnosis
Combined Hepatocellular Cholangiocarcinoma

Discussion

Diagnostic Issues
Combined hepatocellular-cholangiocarcinoma (HCC-CC), a.k.a. mixed HCC-CC, is a rare (incidence among primary liver cancer ranges from 1.0% to 4.7%) but an increasingly recognized neoplasm in the liver [1, 2, 3, 4]. It shares unequivocal features of both HCC and CC as defined by the WHO classification that also emphasizes the diagnosis should not be used for tumors in which either form of growth is insufficiently differentiated for positive identification [5]. This neoplasm was first described and reviewed in detail by Allen and Lisa in 1949 [6] and it was the subtype with an intimate intermingling of hepatocellular and glandular elements that was subsequently defined by Goodman et al. [7] as type II (transitional) tumor that draws most interests in exploring its histogenesis, establishing the pathological diagnosis and characterizing the clinical outcome, whereas separate HCC and CC coincidentally found in the same liver is generally considered as collision tumor and is excluded by the WHO classification of combined HCC-CC. The concept of collision tumor occurring as separate HCC and CC in the same liver has been further supported by the genetic findings that two independent neoplastic clones develop at close proximity [8] and hence no histologic transitions exist.

Although a unified criteria for combined HCC-CC is still not available, it is generally accepted a firm diagnosis of combined HCC-CC requires evidence of HCC differentiation such as trabecular growth pattern, bile production, or bile canaliculi as well as clear evidence of CC such as true glandular structures formed by biliary-type epithelium, mucin production or prominent desmoplastic stoma [4, 9]. Combined HCC-CC will often express both biliary cytokeratins and markers of HCC, and is an important diagnosis to consider when there is an apparently conflicting or overlapping immunophenotype [10].

Histogenesis
The cell of origin of combined HCC-CC has been a matter of dispute. Overall three possibilities may be postulated regarding its cell of origin: (1) collision (double) tumor of HCC and CC that incidentally coexist in the same liver; (2) subsequent differentiation of HCC or CC into the other component; (3) the cancer derives from the hepatic progenitor cells. The fact that the HCC and CC elements intermingle with each other in a transitional area in most combined HCC-CC makes the first hypothesis less likely. Depending on various investigations, patients with combined HCC-CC share similar clinical and pathologic features with patients with HCC [4, 11] or CC [10, 12, 13] or their tumors are clinicopathologically different from those of CC [14] or HCC [10]. One needs to be cautious in interpreting the results of these studies regarding how combined HCC-CC was defined in the studies and there may have been population and etiology difference. Apparently some studies used looser criteria, e.g., any biliary differentiation by immunohistochemistry diagnosed as combined HCC-CC without the confirmation with a mucicarmine stain may have led to the conclusion that combined HCC-CC resembles HCC clinically and pathologically. Taken together, it appears combined HCC-CC lacks a specific clinical parameter and this may be explained by various studies across different geographic regions, etiologies, and populations.

A recent study using genome-wide allelotyping analysis has demonstrated recurrent LOH at 3p and 14q are common in both CC and combined HCC-CC, whereas no beta-catenin was observed in combined HCC-CC and CC, a common mutation in HCC, suggesting common carcinogenic pathways shared by combined HCC-CC and CC [15], however, the clinical and demographic features of the patients with combined HCC-CC in this study were intermediate to those patients with HCC and CC, and yet with a male predilection (M:F=14:1), closer to patients with HCC than CC. Using microdissection, Fujii et al. found single clonal tumor with homogeneous genetic background in both HCC and CC components of combined HCC-CC, suggesting that histologic diversity is a phenotypic expression of divergent differentiation potential of a single clone, or single clonal process in which genetic heterogeneity in the process of clonal evolution within the tumor parallel histologic phenotype and the neoplasm is composed of mosaics of closely related subclones [8]. Fujii's observations support the hypothesis the tumor is derived from a single clone which shows bidirectional phenotypic diversity and the tumor phenotype may also be altered by the divergent genetic changes in the process of tumor progression, given the mosaics of closely related subclones with varied phenotypic potential. An earlier experiment showing a primary cell line derived from resected combined HCC-CC differentiated to not only the characteristics of HCC but also those of CC also demonstrated that single clonally expanded tumor cell can give rise to both hepatocytic and biliary components [16]. So much so whether combined HCC-CC arises from malignant transformation of the hepatic progenitor cells or from de-differentiation of the malignant hepatocytes or cholangiocytes has remained a long debatable issue.

Some recent investigations, however, shed light on the hypothesis that at least a subset of the combined HCC-CC may derive from the hepatic progenitor cells [17, 18, 19]. For instance, Theise et al. [17] demonstrated cells morphologically and immunohistochemically resembling hepatic progenitor cells merged with the HCC and CC components and with mature appearing hepatocytes within some combined HCC-CC, supporting the notions that carcinogenesis of this unique neoplasm may be explained by the malignant transformation of the hepatic progenitor cells. A very recent study [20] nicely demonstrated the cell of origin of cholangiolocellular carcinoma, a very rare neoplasm accounting for less than 1% of the primary liver cancer [21, 22], may be the hepatic progenitor cells. Because the HCC and CC components altogether comprised less than 10% of the neoplasm and the cholangiolocellular carcinoma area (mixture of small monotonous glands, antler-like anastomosing pattern) occupied more than 90% of the neoplasm in this study, although these three histologic components showed transitions between each other, the relationship between this unique neoplasm and the typical combined HCC-CC remains to be clarified. However, they may overlap to some degree and belong to a spectrum of the primary liver neoplasm arising from the hepatic progenitor cells.

Differential Diagnosis and Diagnostic Pitfalls
Apparently combined HCC-CC needs to be distinguished from conventional HCC or CC. Pseudoglands are very common in HCC and they should not be confused with the true glandular formation in CC. Detection of mucin by a mucin stain in the CC component or the presence of bile in the HCC component can be very helpful. It has been recognized that the expression of CK7 and CK19 in HCC is not uncommon from several series and therefore a diagnosis solely based on immunohistochemistry may not be fully reliable [23, 24, 25]. Although rarely used nowadays, electron microscopy demonstrating dual differentiation in different cells within the same acini, such as bile canaliculi and abundant organelles (mitochondria, rough endoplasmic reticulum, lipid droplets, and abundant glycogen - characteristic of hepatocellular differentiation), and microvillous projections at the luminal surface or junctional complexes between the neoplastic cells, features of biliary differentiation, can be useful in some occasions when immunohistochemical and mucin stains are equivocal [26]. Despite intensive preoperative imaging studies, it is not uncommon many combined HCC-CC were misdiagnosed either as HCC or CC before surgery. Tissue sampling is always an issue and may hamper an accurate diagnosis in a core needle biopsy specimen.

Clinical Implication
Accurate preoperative diagnosis is important because the most appropriate treatment may depend on the major component of the tumor (HCC or CC), although patients with combined HCC-CC are seldom diagnosed before surgery. A high index of suspicion, imaging studies, levels of serum tumour markers (alpha-fetoprotein, carbohydrate antigen 19-9), and histopathology assist case detection and treatment option [27]. A recent study reported the survival rate of patients with combined HCC-CC after liver resection was poorer than that of patients with HCC or CC and pathologically combined HCC-CC showed more significantly vascular invasion and lymph node metastasis than HCC, with a similarity to CC [13]. This result is similar to that reported by Lee et al. [28], suggesting more aggressive treatment modality may have to be explored to improve the survival rate of these patients. There are very few outcome data on liver transplantation and the role of liver transplantation in combined HCC-CC needs to be defined. Further studies are also warranted to seek optimal therapeutic options in treating combined HCC-CC [29].

Conclusion
It has become clearer that hepatic progenitor cells are present in not only HCC [23, 24, 30] and CC [31] but also in combined HCC-CC [17, 18, 19, 20]. The role of hepatic progenitor cells as the cell of origin in combined HCC-CC is an interesting subject and ongoing studies may shade more light in the near future.

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