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Combined hepatocellular-cholangiocarcinoma: An update

Published:February 02, 2021DOI:https://doi.org/10.1016/j.jhep.2021.01.035

      Summary

      Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a tumour that exhibits both hepatocytic and biliary differentiation. Classical risk factors for hepatocellular carcinoma (HCC) seem to also predispose patients to the development of cHCC-CCA. The pathological definition of cHCC-CCA has significantly evolved over time. The last 2019 WHO classification highlighted that the diagnosis of cHCC-CCA should be primarily based on morphology using routine stainings, with additional immunostaining used to refine the identification of subtypes. Among them, “intermediate cell carcinoma” is recognised as a specific subtype, while “cholangiolocellular carcinoma” is now considered a subtype of iCCA. Increasing molecular evidence supports the clonal nature of cHCC-CCA and parallels its biphenotypic histological appearance, with genetic alterations that are classically observed in HCC and/or iCCA. That said, the morphological diagnosis of cHCC-CCA is still challenging for radiologists and pathologists, especially on biopsy specimens. Identification of cHCC-CCA’s cell of origin remains an area of active research. Its prognosis is generally worse than that of HCC, and similar to that of iCCA. Resection with lymph node dissection is unfortunately the only curative option for patients with cHCC-CCA. Thus, there remains an urgent need to develop specific therapeutic strategies for this unique clinical entity.

      Keywords

      Introduction

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      It is a heterogeneous group of tumours associated with distinct risk factors, clinical outcomes and histological and molecular features. Among these malignancies, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) are by far the most common, and represent both ends of the spectrum of primary malignant tumours.
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      Fig. 1Macroscopic and microscopic features of iCCA and HCC.
      (A-C) Macroscopic and microscopic features of iCCA: (A) Macroscopic features: white fibrous tumour developed in a non-cirrhotic liver, (B) Microscopic features: tumour glands embedded in a fibrous stroma, (C) Expression of cytokeratin 7 by tumour cells, (D) Masson’s trichrome staining: discrete portal fibrosis. (E-H) Macroscopic and microscopic features of HCC: (E) Macroscopic features: well-delimitated encapsulated tumour developed in a cirrhotic liver, (F) Microscopic features: neoplastic cells organised in cords (trabeculae) of separated by sinusoidal spaces, (G) expression of Hepar-1 by tumour cells in HCC, (H) Masson’s trichrome staining: cirrhotic liver parenchyma. HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma.
      iCCA is thought to originate from cholangiocytes and unlike HCC, is not normally associated with classical risk factors of chronic liver disease, except for primary sclerosing cholangitis.
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      WHO classification of Tumours Editorial Board
      Importantly, the definition of this entity (and its related terminology) have continuously evolved since its first description by H Gideon Wells in 1903.
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      This review will provide an overview of the current knowledge about the clinical, biological, histological and molecular features of cHCC-CCA.

      Epidemiology: a rare tumour, increasingly recognised

      The incidence of cHCC-CCA among the other primary liver cancers ranges from 0.4% to 14.2%.
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      This large range of incidence is probably related to the evolving definition of cHCC-CCA over time in highly heterogeneous studies. In a large series of over 60,000 patients with liver cancer captured within the SEER (Surveillance, Epidemiology, and End Results Program of the National Cancer Institute) database, cHCC-CCA accounted for 0.77% of cases. However, the true incidence of cHCC-CCA is likely to be underestimated, as most patients do not undergo surgical resection and may have thus been misdiagnosed with HCC or iCCA.
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      Classical risk factors for HCC seem to also predispose to the development of cHCC-CCA. The likelihood of viral hepatitis or cirrhosis in patients with cHCC-CC has been reported to be intermediate between the high proportion seen in patients with HCC and the lower prevalence in patients with iCCA.
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      Due to the rarity of the disease, the existence of specific risk factors related to the development of cHCC-CCA remains unknown.

      Clinical features: a poorly specific presentation

      Like other malignant liver malignancies, cHCC-CCA are predominantly observed in male patients.
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      As observed for HCC and iCCA, clinical symptoms are most often associated with advanced tumoural disease. They may include fatigue, obstructive jaundice, weight loss, or abdominal discomfort. Pre-operative diagnosis of cHCC-CCA is unfortunately very complex with the absence of specific non-invasive imaging features.
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      Given that neoplastic cells of cHCC-CCA may produce alpha-fetoprotein (AFP) and carbohydrate antigen 19.9 (CA 19-9), a simultaneous increase in serum levels of both markers strongly suggests the diagnosis of cHCC-CCA. Nevertheless, such increases in both serum markers are only detected in a minority of patients.
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      Combined hepatocellular and cholangiocarcinoma: demographic, clinical, and prognostic factors.
      ,
      • Li R.
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      Pathological features: an evolving heterogeneous entity

      The first case of cHCC-CCA was reported in 1903.
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      Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.
      In 1949, Allen and Lisa suggested that single tumours showing features of both HCC and iCCA should be considered separately from HCC and iCCA arising at a distance in the same liver, or only intermingling at their borders.
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      Combined liver cell and bile duct carcinoma.
      In 1954, these tumours, called “hepatobiliary cancers”, were grouped with HCC by Edmondson and Steiner.
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      Primary carcinoma of the liver: a study of 100 cases among 48,900 necropsies.
      In 1959, Steiner and Higginson described the first cases of cholangiolocellular carcinoma (CLC), characterised by a proliferation of small ducts in a fibrous stroma and composed of relatively monotonous small cells resembling the epithelium of the Hering canal.
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      Cholangiolocellular carcinoma of the liver.
      In 1985, a classification of 3 types of cHCA-CCA (collision type, transitional type and fibrolamellar type [resembling the fibrolamellar variant of HCC with mucin-producing pseudoglands]) was proposed by Goodman et al.
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      Combined hepatocellular-cholangiocarcinoma. A histologic and immunohistochemical study.
      . Since 2000, the World Health Organization (WHO) classification defines cHCC-CCA as a tumour containing unequivocal elements of both HCC and CCA that are intimately admixed.
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      Then, several studies suggested the existence of cHCC-CCA with stem-cell features (recognised by the presence of clusters of small cells with scant cytoplasm and high nucleo/cytoplasmic ratio at the periphery of other tumour cells) and cHCC-CCA with intermediate morphology between HCC and iCCA (showing simultaneous expression of both hepatocytic and cholangiocytic markers).
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      The canals of Hering and hepatic stem cells in humans.
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      Primary liver carcinoma of intermediate (hepatocyte-cholangiocyte) phenotype.
      Accordingly, in 2010, the fourth WHO classification described different subtypes, including a classical type for tumours harbouring distinct areas with clear-cut hepatocytic and cholangiocytic differentiation, and 3 subtypes with stem/progenitor cell features including the typical one (clusters of small cytokeratin (CK)19-positive cells intermingled with other tumour cells), the intermediate cell type and CLC.
      • Bosman F.
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      Several studies have then challenged this classification, more particularly the 3 subtypes with stem/progenitor cell features. Indeed, several criticisms have arisen, such as i) the stem/progenitor cell features can also be observed in HCC or iCCA, ii) the 3 subtypes with stem/progenitor cell features often coexist, iii) CLC is not always associated with HCC.
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      Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.
      ,
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      • Theise N.D.
      Biphenotypic (hepatobiliary) primary liver carcinomas: the work in progress.
      cHCC-CCA tumours also exhibit a high degree of intratumour heterogeneity and features related to distinct subtypes frequently coexist within the same tumour.
      Finally, in 2018, an international group, composed of a community of pathologists, radiologists, and clinicians experienced in liver tumours, especially cHCC-CCA, proposed consensus terminology for these tumours.
      • Brunt E.
      • Aishima S.
      • Clavien P.-A.
      • Fowler K.
      • Goodman Z.
      • Gores G.
      • et al.
      cHCC-CCA: consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation.
      They recommended diagnosing cHCC-CCA based on routine histopathology with H&E, with immunostains being supportive but not essential for diagnosis. Stem-cell phenotypes/features may be present within cHCC-CCA, and if so, can be noted in the descriptive report without the need to be recognised as a specific subtype.
      • Brunt E.
      • Aishima S.
      • Clavien P.-A.
      • Fowler K.
      • Goodman Z.
      • Gores G.
      • et al.
      cHCC-CCA: consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation.
      Following these recommendations, significant changes were made in the fifth WHO classification of cHCC-CCA in 2019
      WHO classification of Tumours Editorial Board
      (Table 1).
      Combined hepatocellular-cholangiocarcinoma denotes a specific group of primary liver malignancies that are less frequent than hepatocellular carcinoma or intrahepatic cholangiocarcinoma.
      Table 1Evolution of the WHO classification of cHCC-CCA.
      2010 WHO classification (4th edition)2019 WHO classification (5th edition)
      cHCC-CCA, classical typecHCC-CCA, classical type
      cHCC-CCA with stem-cell features, intermediate cell subtypeIntermediate cell carcinoma
      cHCC-CCA with stem-cell features, typical-subtype
      cHCC-CCA with stem-cell features, cholangiolocellular typeIncluded in cholangiocarcinoma classification
      cHCC-CCA, combined hepatocellular-cholangiocarcinoma.

      2019 WHO classification of cHCC-CCA

      The WHO classification defines cHCC-CCA as a primary liver carcinoma with the unequivocal presence of both hepatocytic and cholangiocytic differentiation within the same tumour on routine histopathology with H&E (so-called “collision” cases are thus excluded). Importantly, diagnosis is made regardless of the percentage of each component (no cut-off values) (Fig. 2). Given the substantial intratumour heterogeneity of cHCC-CCA, extensive tumour sampling is highly recommended (sampling all different macroscopic areas with at least 1 block/cm collected at the macroscopic analysis) in order to best cover the various tumour areas throughout the nodule.
      Figure thumbnail gr2
      Fig. 2Macroscopic and microscopic features of cHCC-CCA.
      (A) Fibrous well-demarcated tumour developed in a cirrhotic liver in macroscopic examination; (B) Unequivocal cholangiocytic (arrow) and hepatocytic area (star) of cell differentiation (Hematein and eosin staining x100); (C) Hepatocytic area showing tumour cells organised in trabeculae (H&E staining x200); (D) Expression of HepPar-1 by tumour cells in hepatocytic area (x200); (E) No expression of CK7 by tumour cells in hepatocytic area (x200); (F) Cholangiocytic area showing tumour glands embedded in a fibrous stroma (H&E staining x200); (G) No expression of HepPar-1 by tumour cells in cholangiocytic area (x200); (H) Expression of CK7 by tumour cells in cholangiocytic area (x200). cHCC-CCA, combined hepatocellular carcinoma-cholangiocarcinoma; CK7, cytokeratin 7.
      The presence of typical stem-cell features is not sufficient to classify a tumour as cHCC-CCA, as this phenotype may be observed in classical HCC or iCCA. The terminology intermediate cell carcinoma can only be used in homogenous biphenotypic tumours cells (Fig. 3).
      WHO classification of Tumours Editorial Board
      The pathological features of cHCC-CCA are summarised in Table 2.
      Figure thumbnail gr3
      Fig. 3Microscopic features of intermediate cell carcinoma.
      (A) Monotonous tumour cells arranged in strands in an abundant fibrous stroma (H&E staining x100); (B) Tumour cells with scant cytoplasm and inconspicuous nucleoli showing intermediate morphological features between hepatocytes and cholangiocytes (H&E staining x100); immunostaining confirmed the biphenotypic nature of the tumour with expression by tumour cells of both (C) hepatocytic markers (HepPar-1) and (D) cholangiocytic markers (cytokeratin 7) (x200).
      Table 2Morphological and immunohistochemistry features of cHCC-CCA.
      Type of cHCC-CCAMicroscopic featuresImmunohistochemistry features
      ClassicalUnequivocal presence of both hepatocytic and cholangiocytic differentiation within the same tumour:
      • all architectural and cytological differentiation patterns described for HCC and Icca are possible
      • no minimum cut-off amounts of each component
      Possible identification of cancer stem cells in various proportions: small uniform cells with scant cytoplasm and inconspicuous nucleoli
      Confirmation of both differentiations by immunohistochemical markers:
      • Hepatocytic differentiation: Glypican, AFP, Hepar-1, Arginase-1, Polyclonal CEA and CD10
      • Cholangiocytic differentiation: CK7, CK19, EpCAM
      Identification of stem-cell features: CK19, EpCAM, CD56, CD117, CD133
      Intermediate cell carcinomaBiphenotypic differentiation:
      • Tumour cells have a morphology intermediate between a hepatocyte and a cholangiocyte
      • Monotonous tumour cells with scant cytoplasm arranged in strands in an abundant fibrous stroma
      Expression by tumour cells of both hepatocytic and cholangiocytic markers.
      cHCC-CCA: combined hepatocellular-cholangiocarcinoma, HCC: hepatocellular carcinoma, iCCA: intrahepatic cholangiocarcinoma.
      Tumours with histological features of CLC but without evidence of clear-cut hepatocellular differentiation should also be considered as a particular subtype of iCCA (Fig. 4). Indeed, several molecular studies demonstrated that CLC exhibit an overlapping gene expression profile with iCCA, with a downregulation of the hepatocyte differentiation programme and a commitment to the biliary lineage.
      • Moeini A.
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      • Zhang Z.
      • Camprecios G.
      • Stueck A.
      • Dong H.
      • et al.
      Mixed hepatocellular cholangiocarcinoma tumors: cholangiolocellular carcinoma is a distinct molecular entity.
      ,
      • Sasaki M.
      • Sato Y.
      • Nakanuma Y.
      Cholangiolocellular carcinoma with “ductal plate malformation” pattern may Be characterized by ARID1A genetic alterations.
      ,
      • Balitzer D.
      • Joseph N.M.
      • Ferrell L.
      • Shafizadeh N.
      • Jain D.
      • Zhang X.
      • et al.
      Immunohistochemical and molecular features of cholangiolocellular carcinoma are similar to well-differentiated intrahepatic cholangiocarcinoma.
      Additionally, immunophenotyping of CLC also reveals constant expression of markers of biliary lineage (CK7, CK19).
      • Balitzer D.
      • Joseph N.M.
      • Ferrell L.
      • Shafizadeh N.
      • Jain D.
      • Zhang X.
      • et al.
      Immunohistochemical and molecular features of cholangiolocellular carcinoma are similar to well-differentiated intrahepatic cholangiocarcinoma.
      Figure thumbnail gr4
      Fig. 4Microscopic features of cholangiolocellular carcinoma.
      (A) Fibrous well-demarcated tumour developed in a non-cirrhotic liver in macroscopic examination; (B) tubular, cord-like, anastomosing pattern (H&E staining x100); tumour expression of (C) CK7 and (D) CK19 (D x100). CK, cytokeratin.

      Immunohistochemistry features

      The last 2019 WHO classification defines combined hepatocellular-cholangiocarcinoma as a primary liver carcinoma with the unequivocal presence of both hepatocytic and cholangiocytic differentiation within the same tumour on routine staining.
      The immunohistochemical (IHC) detection of markers of hepatocytic and cholangiocytic differentiation may help but is neither necessary nor sufficient
      WHO classification of Tumours Editorial Board
      ,
      • Sciarra A.
      • Park Y.N.
      • Sempoux C.
      Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.
      (Table 2). A set of markers is available to confirm a hepatocellular component, including Glypican-3, AFP, HepPar1, and Arginase-1.
      WHO classification of Tumours Editorial Board
      ,
      • Sciarra A.
      • Park Y.N.
      • Sempoux C.
      Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.
      ,
      • Di Tommaso L.
      • Franchi G.
      • Park Y.N.
      • Fiamengo B.
      • Destro A.
      • Morenghi E.
      • et al.
      Diagnostic value of HSP70, glypican 3, and glutamine synthetase in hepatocellular nodules in cirrhosis.
      Arginase-1 performs better in less-differentiated HCC than HepPar1, while AFP is only rarely positive. Polyclonal carcinoembryonic antigen and CD10 are the most specific IHC markers when a canalicular pattern is observed, but their sensitivity is low.
      • El Jabbour T.
      • Lagana S.M.
      • Lee H.
      Update on hepatocellular carcinoma: pathologists’ review.
      ,
      • Sciarra A.
      • Park Y.N.
      • Sempoux C.
      Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.
      To confirm a cholangiocytic differentiation, several markers can be used, including CK7 and CK19. Nevertheless, it is important to note that HCC can also express cholangiocellular markers, in particular HCC with atypical features such as fibrous stroma, more infiltrative growth and nodal metastasis.
      • Sciarra A.
      • Park Y.N.
      • Sempoux C.
      Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.
      ,
      • Kim H.
      • Choi G.H.
      • Na D.C.
      • Ahn E.Y.
      • Kim G.I.
      • Lee J.E.
      • et al.
      Human hepatocellular carcinomas with “Stemness”-related marker expression: keratin 19 expression and a poor prognosis.
      ,
      • Yokomichi N.
      • Nishida N.
      • Umeda Y.
      • Taniguchi F.
      • Yasui K.
      • Toshima T.
      • et al.
      Heterogeneity of epigenetic and epithelial mesenchymal transition marks in hepatocellular carcinoma with keratin 19 proficiency.
      EpCAM positivity is observed in >90% of cholangiocytic differentiation areas and in 10–20% of hepatocellular areas.
      • Sciarra A.
      • Park Y.N.
      • Sempoux C.
      Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.
      Finally, if IHC is discordant with the first H&E, it is advised to perform IHC on other slides and to take new tumour samples.

      Cell of origin: progenitor or not?

      Identification of the cell of origin of liver cancers is a highly active research area. HCC and iCCA have long been considered to derive from mature hepatocytes and cholangiocytes, respectively. However, this is probably too simplistic, given the plasticity of cells that populate the adult liver. As far as cHCC-CCA is concerned, 3 different hypotheses have been postulated, i) incidental coexistence of HCC and iCCA within the same tumour, ii) malignant transformation of a hepatic progenitor cell, iii) dedifferentiation of an HCC or an iCCA.
      • Sia D.
      • Villanueva A.
      • Friedman S.L.
      • Llovet J.M.
      Liver cancer cell of origin, molecular class, and effects on patient prognosis.
      • Tanimizu N.
      • Nakamura Y.
      • Ichinohe N.
      • Mizuguchi T.
      • Hirata K.
      • Mitaka T.
      Hepatic biliary epithelial cells acquire epithelial integrity but lose plasticity to differentiate into hepatocytes in vitro during development.
      • Chen Y.
      • Wong P.P.
      • Sjeklocha L.
      • Steer C.J.
      • Sahin M.B.
      Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture.
      Most recent molecular studies do not support the first hypothesis, as HCC and iCCA components of cHCC-CCA most often share common key-genetic events, including a clonal pattern.
      • Moeini A.
      • Sia D.
      • Bardeesy N.
      • Mazzaferro V.
      • Llovet J.M.
      Molecular pathogenesis and targeted therapies for intrahepatic cholangiocarcinoma.
      ,
      • Xue R.
      • Chen L.
      • Zhang C.
      • Fujita M.
      • Li R.
      • Yan S.-M.
      • et al.
      Genomic and transcriptomic profiling of combined hepatocellular and intrahepatic cholangiocarcinoma reveals distinct molecular subtypes.
      The frequent existence of areas with mixed morphological features of both HCC and ICC is also not consistent with a simple collision of 2 independent tumours.
      A role for progenitor cells is more likely, given the morphological appearance of these tumours that exhibit both biliary and hepatocytic differentiation. In this line, hepatic progenitor or oval cells were shown to give rise to cHCC-CCA in different mouse models.
      • Chiba T.
      • Zheng Y.-W.
      • Kita K.
      • Yokosuka O.
      • Saisho H.
      • Onodera M.
      • et al.
      Enhanced self-renewal capability in hepatic stem/progenitor cells drives cancer initiation.
      • Lee K.-P.
      • Lee J.-H.
      • Kim T.-S.
      • Kim T.-H.
      • Park H.-D.
      • Byun J.-S.
      • et al.
      The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis.
      • Benhamouche S.
      • Curto M.
      • Saotome I.
      • Gladden A.B.
      • Liu C.-H.
      • Giovannini M.
      • et al.
      Nf2/Merlin controls progenitor homeostasis and tumorigenesis in the liver.
      However, other studies have shown that mature hepatocytes have the ability to dedifferentiate into progenitor cells; they may also further undergo full malignant transformation into cHCC-CCA.
      • Chen Y.
      • Wong P.P.
      • Sjeklocha L.
      • Steer C.J.
      • Sahin M.B.
      Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture.
      ,
      • Tarlow B.D.
      • Pelz C.
      • Naugler W.E.
      • Wakefield L.
      • Wilson E.M.
      • Finegold M.J.
      • et al.
      Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.
      We thus cannot exclude that cHCC-CCA may arise from a dedifferentiated transformed hepatocyte. Consistently, Holczbauer et al. transduced mouse hepatoblasts, adult hepatocytes and progenitor cells with driver oncogenes and showed that all 3 lineages acquired cancer stem cells/progenitor markers and gave rise to tumours with varying HCC and/or iCCA components.
      • Holczbauer Á.
      • Factor V.M.
      • Andersen J.B.
      • Marquardt J.U.
      • Kleiner D.E.
      • Raggi C.
      • et al.
      Modeling pathogenesis of primary liver cancer in lineage-specific mouse cell types.
      The presence of typical stem-cell features is no longer sufficient to classify a tumour as combined hepatocellular-cholangiocarcinoma.

      Molecular landscape: a distinct subtype of primary liver malignancies

      Due to their rare occurrence, the molecular alterations that drive the progression of cHCC-CCA have long remained unknown. Published studies have reported various and often conflicting conclusions, including some showing that cHCC-CCA was closely related to HCC, some to iCCA, and some reporting that it was a distinct entity.
      • Jarnagin W.R.
      • Weber S.
      • Tickoo S.K.
      • Koea J.B.
      • Obiekwe S.
      • Fong Y.
      • et al.
      Combined hepatocellular and cholangiocarcinoma: demographic, clinical, and prognostic factors.
      ,
      • Maeda T.
      • Adachi E.
      • Kajiyama K.
      • Sugimachi K.
      • Tsuneyoshi M.
      Combined hepatocellular and cholangiocarcinoma: proposed criteria according to cytokeratin expression and analysis of clinicopathologic features.
      • Joseph N.M.
      • Tsokos C.G.
      • Umetsu S.E.
      • Shain A.H.
      • Kelley R.K.
      • Onodera C.
      • et al.
      Genomic profiling of combined hepatocellular-cholangiocarcinoma reveals similar genetics to hepatocellular carcinoma.
      • Yano Y.
      • Yamamoto J.
      • Kosuge T.
      • Sakamoto Y.
      • Yamasaki S.
      • Shimada K.
      • et al.
      Combined hepatocellular and cholangiocarcinoma: a clinicopathologic study of 26 resected cases.
      • Cazals-Hatem D.
      • Rebouissou S.
      • Bioulac-Sage P.
      • Bluteau O.
      • Blanché H.
      • Franco D.
      • et al.
      Clinical and molecular analysis of combined hepatocellular-cholangiocarcinomas.
      • Zuo H.-Q.
      • Yan L.-N.
      • Zeng Y.
      • Yang J.-Y.
      • Luo H.-Z.
      • Liu J.-W.
      • et al.
      Clinicopathological characteristics of 15 patients with combined hepatocellular carcinoma and cholangiocarcinoma.
      As previously discussed, accurate diagnosis of cHCC-CCA is challenging and HCC or iCCA cannot be excluded from molecular studies with complete certainty.
      However, a better view of the oncogenic pathways and genetic alterations of cHCC-CCA (classified according to the Allen and Lisa criteria into separate, combined, and mixed subtypes) has recently been proposed by Xue et al., based on a large-scale integrative study of 133 cases (the largest series investigated so far) including whole-exome and whole-genome RNA sequencing and single-nucleus sequencing.
      • Xue R.
      • Chen L.
      • Zhang C.
      • Fujita M.
      • Li R.
      • Yan S.-M.
      • et al.
      Genomic and transcriptomic profiling of combined hepatocellular and intrahepatic cholangiocarcinoma reveals distinct molecular subtypes.
      This study showed that the most frequently mutated driver genes were TP53 (49% of the cases), TERT promoter (23% of the cases), AXIN1 (10% of the cases), and KMT2D (9% of the cases), mutations that may be associated with either HCC or iCCA (Fig. 5). Xue et al. further compared the mutational frequencies according to the histological subtype, and except for an enrichment of AXIN1 mutations in mixed type cHCC-CCA (cases with intimately mixed components of HCC and iCCA in the same tumour without clear boundaries), they did not observe any significant difference. There has been a long-standing debate about whether cHCC-CCA has a monoclonal or multiclonal origin. Xue et al. showed that all cases of combined type (cases with clearly defined areas of HCC and iCCA components in the same tumour) and mixed type cHCC-CCA (according to Allen and Lisa’s classification)
      • Allen R.A.
      • Lisa J.R.
      Combined liver cell and bile duct carcinoma.
      were of monoclonal origin, although a certain degree of intratumor heterogeneity was observed. In this line, HCC and CCA components of combined tumours showed very similar global gene expression profiles. Multiclonality was observed only in a minority (2/6) of separate subtypes. The lesions may thus represent intrahepatic metastasis (with transdifferentiation from one component into another) or the concomitant occurrence of distinct cancers.
      • Xue R.
      • Chen L.
      • Zhang C.
      • Fujita M.
      • Li R.
      • Yan S.-M.
      • et al.
      Genomic and transcriptomic profiling of combined hepatocellular and intrahepatic cholangiocarcinoma reveals distinct molecular subtypes.
      Tumours with histological features of cholangiocellular carcinoma, but without evidence of clear-cut hepatocellular differentiation, should now be considered as a particular subtype of intrahepatic cholangiocarcinoma.
      Figure thumbnail gr5
      Fig. 5Main molecular features of combined hepatocellular-cholangiocarcinoma, hepatocellular carcinoma and cholangiocarcinoma.
      By performing genome-wide transcriptional profiling of 20 cases, Coulouarn et al. showed that transforming growth factor-β (TGFβ) and Wnt/β-catenin were the 2 major signalling pathways activated in cHCC-CCA. Interestingly, the spectrum of β-catenin target genes upregulated in cHCC-CCA was also completely different from that of classical HCC. Based on a previous report of a TGFβ-driven activation of β-catenin signalling in a particular subset of HCC (S1 molecular subclass), the authors hypothesised that it may also occur in cHCC-CCA.
      • Coulouarn C.
      • Cavard C.
      • Rubbia-Brandt L.
      • Audebourg A.
      • Dumont F.
      • Jacques S.
      • et al.
      Combined hepatocellular-cholangiocarcinomas exhibit progenitor features and activation of Wnt and TGFβ signaling pathways.
      cHCC-CCA with stem-cell features, previously recognised as "intermediate" and "typical" subtypes in the 2010 WHO classification, were characterised by poor prognostic signatures (proliferation, G3, S2), with activation of various oncogenic pathways (IG2, MYC, mTOR and NOTCH) and chromosomal instability.
      • Moeini A.
      • Sia D.
      • Zhang Z.
      • Camprecios G.
      • Stueck A.
      • Dong H.
      • et al.
      Mixed hepatocellular cholangiocarcinoma tumors: cholangiolocellular carcinoma is a distinct molecular entity.
      Nevertheless, the number of cases investigated was limited and validation in additional cases is mandatory.
      Classical cHCC-CCA may display features of both typical iCCA or HCC, such as TERT promoter, TP53 or KRAS mutations.
      • Moeini A.
      • Sia D.
      • Zhang Z.
      • Camprecios G.
      • Stueck A.
      • Dong H.
      • et al.
      Mixed hepatocellular cholangiocarcinoma tumors: cholangiolocellular carcinoma is a distinct molecular entity.
      ,
      • Sasaki M.
      • Sato Y.
      • Nakanuma Y.
      Mutational landscape of combined hepatocellular carcinoma and cholangiocarcinoma, and its clinicopathological significance.
      A challenge is to determine whether the hepatocytic and biliary components share the same molecular alterations, which would suggest a monoclonal origin. For that purpose, Fujii et al. analysed each tumoural component extracted from cHCC-CCA and showed that they harboured common allelic losses.
      • Fujii H.
      • Zhu X.G.
      • Matsumoto T.
      • Inagaki M.
      • Tokusashi Y.
      • Miyokawa N.
      • et al.
      Genetic classification of combined hepatocellular-cholangiocarcinoma.
      Clonality analysis using copy number variation also showed a significant resemblance in 3 out of the 4 cases analysed by Moeini and collaborators, supporting a common cell of origin.
      • Fujii H.
      • Zhu X.G.
      • Matsumoto T.
      • Inagaki M.
      • Tokusashi Y.
      • Miyokawa N.
      • et al.
      Genetic classification of combined hepatocellular-cholangiocarcinoma.
      ,
      • Moeini A.
      • Sia D.
      • Zhang Z.
      • Camprecios G.
      • Stueck A.
      • Dong H.
      • et al.
      Mixed hepatocellular cholangiocarcinoma tumors: cholangiolocellular carcinoma is a distinct molecular entity.
      This hypothesis is also supported by morphological features, as classical cHCC-CCA frequently shows transition areas between HCC and iCCA components. Further studies will have to better determine the divergent expansion that may be associated with the acquisition of distinct phenotypes. Although it is likely that genetic alterations may drive histological differentiation, a role for the microenvironment (inflammatory and immune cells, fibroblasts and vessels) cannot be ruled out.
      Altogether, current data show that the molecular profile of cHCC-CCA parallels its biphenotypic histological appearance, with genetic alterations that are classically observed in HCC and/or iCCA. However, the number of cases investigated remains low and validation is needed. Large-scale studies with integration of clinical, biological, histological and molecular features are mandatory to establish a robust classification of cHCC-CCA.

      Imaging features: a combined tumour

      In line with its histological appearance, the imaging features of cHCC-CCA are heterogeneous and significantly overlap with those of HCC and iCCA. The amount of each component seems to be a key determinant of the radiological features of cHCC-CCA. cHCC-CCAs with a predominant HCC component are thus likely to display an intense contrast uptake during the arterial phase, with a washout in the delayed or venous phase, while tumours resembling conventional iCCA show progressive centripetal enhancement or stable persistent enhancement of the nodule and/or capsule retraction.
      • Li R.
      • Yang D.
      • Tang C.-L.
      • Cai P.
      • Ma K.-S.
      • Ding S.-Y.
      • et al.
      Combined hepatocellular carcinoma and cholangiocarcinoma (biphenotypic) tumors: clinical characteristics, imaging features of contrast-enhanced ultrasound and computed tomography.
      ,
      • Gigante E.
      • Ronot M.
      • Bertin C.
      • Ciolina M.
      • Bouattour M.
      • Dondero F.
      • et al.
      Combining imaging and tumour biopsy improves the diagnosis of combined hepatocellular-cholangiocarcinoma.
      Several studies have suggested that a majority of tumours share imaging features with mass-forming iCCA, especially peripheral rim arterial enhancement, possibly peripheral washout and central enhancement.
      • Gigante E.
      • Ronot M.
      • Bertin C.
      • Ciolina M.
      • Bouattour M.
      • Dondero F.
      • et al.
      Combining imaging and tumour biopsy improves the diagnosis of combined hepatocellular-cholangiocarcinoma.
      ,
      • Aoki A.
      • Shiozaki A.
      • Sameshima A.
      • Higashimoto K.
      • Soejima H.
      • Saito S.
      Beckwith-Wiedemann syndrome with placental chorangioma due to H19-differentially methylated region hypermethylation: a case report.
      • Fukukura Y.
      • Taguchi J.
      • Nakashima O.
      • Wada Y.
      • Kojiro M.
      Combined hepatocellular and cholangiocarcinoma: correlation between CT findings and clinicopathological features.
      • Hwang J.
      • Kim Y.K.
      • Park M.J.
      • Lee M.H.
      • Kim S.H.
      • Lee W.J.
      • et al.
      Differentiating combined hepatocellular and cholangiocarcinoma from mass-forming intrahepatic cholangiocarcinoma using gadoxetic acid-enhanced MRI.
      This has been more recently confirmed by studies using the Liver Imaging Reporting And Data System (LI-RADS) terminology of ‘targetoid appearance’.
      • Lee C.-C.
      • Wu C.-Y.
      • Chen J.-T.
      • Chen G.-H.
      Comparing combined hepatocellular-cholangiocarcinoma and cholangiocarcinoma: a clinicopathological study.
      ,
      • Gigante E.
      • Ronot M.
      • Bertin C.
      • Ciolina M.
      • Bouattour M.
      • Dondero F.
      • et al.
      Combining imaging and tumour biopsy improves the diagnosis of combined hepatocellular-cholangiocarcinoma.
      ,
      • Jeon S.K.
      • Joo I.
      • Lee D.H.
      • Lee S.M.
      • Kang H.-J.
      • Lee K.-B.
      • et al.
      Combined hepatocellular cholangiocarcinoma: LI-RADS v2017 categorisation for differential diagnosis and prognostication on gadoxetic acid-enhanced MR imaging.
      Moreover, Gigante et al. have shown that a mixed pattern at imaging (combination of progressive enhancement of the lesion, arterial enhancement with washout and an atypical pattern including areas of arterial enhancement without washout and/or hypovascular lesions) had a 48% sensitivity and a 81% specificity for the diagnosis of cHCC-CCA
      • Gigante E.
      • Ronot M.
      • Bertin C.
      • Ciolina M.
      • Bouattour M.
      • Dondero F.
      • et al.
      Combining imaging and tumour biopsy improves the diagnosis of combined hepatocellular-cholangiocarcinoma.
      (Fig. 6).
      Figure thumbnail gr6
      Fig. 6Example of mixed pattern on contrast-enhanced computed tomography (figure previously published by Gigante et al. in Liver International
      • Gigante E.
      • Ronot M.
      • Bertin C.
      • Ciolina M.
      • Bouattour M.
      • Dondero F.
      • et al.
      Combining imaging and tumour biopsy improves the diagnosis of combined hepatocellular-cholangiocarcinoma.
      ).
      Contrast-enhanced images at (A, C) arterial phase showed subcapsular lesions with mild and heterogeneous non-rim arterial phase hyperenhancement (arrows). (B, D) On delayed phase images, the lesion showed 2 different components, the upper one with washout (white arrows) and the larger and lower one with progressive contrast uptake (dashed arrows), corresponding to ‘hepatocellular carcinoma’ and ‘intrahepatic cholangiocarcinoma’ patterns, respectively. The hepatocellular carcinoma part is responsible for a subtle capsular bulging, while the intrahepatic cholangiocarcinoma part presents with a capsular retraction.

      Role of the biopsy: a diagnostic challenge

      It remains critical to determine whether i) the use of biopsy allows for a diagnosis of cHCC-CCA with a sufficient degree of confidence, ii) the different components of a radiologically heterogeneous tumour should be biopsied. Few data are available on the performance of biopsy for the diagnosis of cHCC-CCA. We can also speculate that, given the lack of systematic biopsy for liver cancers, a significant number of cHCC-CCA are currently misdiagnosed as HCC. Gigante et al. have shown in a retrospective series of 21 cHCC-CCA that a 2-step strategy, combining imaging followed by biopsy, improved diagnostic performance (with a sensitivity of 60% and a specificity of 82%) compared to imaging or histology alone.
      • Gigante E.
      • Ronot M.
      • Bertin C.
      • Ciolina M.
      • Bouattour M.
      • Dondero F.
      • et al.
      Combining imaging and tumour biopsy improves the diagnosis of combined hepatocellular-cholangiocarcinoma.
      Beyond tumour diagnosis, tumour biopsy may also provide further genetic characterisation to identify specific molecular pathways involved in tumour biology (i.e. aggressiveness) and/or potentially druggable targets. However, given the different genomic profiles of cHCC-CCA reported in the literature, potentially related to the substantial intratumour heterogeneity, further molecular studies based on multicentric cohorts of tumour biopsies are required to demonstrate their performance.
      • Xue R.
      • Chen L.
      • Zhang C.
      • Fujita M.
      • Li R.
      • Yan S.-M.
      • et al.
      Genomic and transcriptomic profiling of combined hepatocellular and intrahepatic cholangiocarcinoma reveals distinct molecular subtypes.
      ,
      • Moeini A.
      • Sia D.
      • Zhang Z.
      • Camprecios G.
      • Stueck A.
      • Dong H.
      • et al.
      Mixed hepatocellular cholangiocarcinoma tumors: cholangiolocellular carcinoma is a distinct molecular entity.
      ,
      • Maeda T.
      • Adachi E.
      • Kajiyama K.
      • Sugimachi K.
      • Tsuneyoshi M.
      Combined hepatocellular and cholangiocarcinoma: proposed criteria according to cytokeratin expression and analysis of clinicopathologic features.
      • Joseph N.M.
      • Tsokos C.G.
      • Umetsu S.E.
      • Shain A.H.
      • Kelley R.K.
      • Onodera C.
      • et al.
      Genomic profiling of combined hepatocellular-cholangiocarcinoma reveals similar genetics to hepatocellular carcinoma.
      • Yano Y.
      • Yamamoto J.
      • Kosuge T.
      • Sakamoto Y.
      • Yamasaki S.
      • Shimada K.
      • et al.
      Combined hepatocellular and cholangiocarcinoma: a clinicopathologic study of 26 resected cases.
      • Cazals-Hatem D.
      • Rebouissou S.
      • Bioulac-Sage P.
      • Bluteau O.
      • Blanché H.
      • Franco D.
      • et al.
      Clinical and molecular analysis of combined hepatocellular-cholangiocarcinomas.
      • Zuo H.-Q.
      • Yan L.-N.
      • Zeng Y.
      • Yang J.-Y.
      • Luo H.-Z.
      • Liu J.-W.
      • et al.
      Clinicopathological characteristics of 15 patients with combined hepatocellular carcinoma and cholangiocarcinoma.
      • Coulouarn C.
      • Cavard C.
      • Rubbia-Brandt L.
      • Audebourg A.
      • Dumont F.
      • Jacques S.
      • et al.
      Combined hepatocellular-cholangiocarcinomas exhibit progenitor features and activation of Wnt and TGFβ signaling pathways.
      Combined hepatocellular-cholangiocarcinoma seems to be of monoclonal origin.

      Prognosis and treatment: still the grey zone

      Studies investigating the clinical outcome of cHCC-CCA have reported conflicting results. This is likely due to the small number of patients included and the discrepancies in the classification criteria used over time. The overall trend for its prognosis is to be worse than that of HCC, and similar to that of iCCA. Indeed, vascular invasion and lymph node metastasis seem to be more frequent in cHCC-CCA compared to HCC.
      • Wakizaka K.
      • Yokoo H.
      • Kamiyama T.
      • Ohira M.
      • Kato K.
      • Fujii Y.
      • et al.
      Clinical and pathological features of combined hepatocellular-cholangiocarcinoma compared with other liver cancers.
      The main prognostic factors identified were large tumour size (>5 cm), presence of satellite nodules, lymph node involvement, multifocality, vascular invasion, portal vein invasion, high tumour stage, high levels of CA19-9 (carbohydrate antigen 19-9), decreased capsule formation and free surgical margins <2 cm in cases of surgical resection.
      • Wakizaka K.
      • Yokoo H.
      • Kamiyama T.
      • Ohira M.
      • Kato K.
      • Fujii Y.
      • et al.
      Clinical and pathological features of combined hepatocellular-cholangiocarcinoma compared with other liver cancers.
      • Kim K.H.
      • Lee S.G.
      • Park E.H.
      • Hwang S.
      • Ahn C.S.
      • Moon D.B.
      • et al.
      Surgical treatments and prognoses of patients with combined hepatocellular carcinoma and cholangiocarcinoma.
      • Gera S.
      • Ettel M.
      • Acosta-Gonzalez G.
      • Xu R.
      Clinical features, histology, and histogenesis of combined hepatocellular-cholangiocarcinoma.
      • Chu K.
      • Lu C.
      • Dong H.
      • Fu X.
      • Zhang H.
      • Yao X.
      Hepatitis B virus-related combined hepatocellular-cholangiocarcinoma: clinicopathological and prognostic analysis of 390 cases.
      • Song S.
      • Moon H.H.
      • Lee S.
      • Kim T.-S.
      • Shin M.
      • Kim J.M.
      • et al.
      Comparison between resection and transplantation in combined hepatocellular and cholangiocarcinoma.
      Compared to HCC and CCA, management of cHCC-CCA is not yet standardised, and various therapeutic options have been proposed. Resection with lymph node dissection is unfortunately the only curative option for patients with cHCC-CCA.
      • Kim K.H.
      • Lee S.G.
      • Park E.H.
      • Hwang S.
      • Ahn C.S.
      • Moon D.B.
      • et al.
      Surgical treatments and prognoses of patients with combined hepatocellular carcinoma and cholangiocarcinoma.
      However, tumour recurrence is frequent (up to 80% at 5 years) and 5-year survival rates do not exceed 30%.
      • Yoon Y.-I.
      • Hwang S.
      • Lee Y.-J.
      • Kim K.-H.
      • Ahn C.-S.
      • Moon D.-B.
      • et al.
      Postresection outcomes of combined hepatocellular carcinoma-cholangiocarcinoma, hepatocellular carcinoma and intrahepatic cholangiocarcinoma.
      • Yin X.
      • Zhang B.-H.
      • Qiu S.-J.
      • Ren Z.-G.
      • Zhou J.
      • Chen X.-H.
      • et al.
      Combined hepatocellular carcinoma and cholangiocarcinoma: clinical features, treatment modalities, and prognosis.
      • Yamashita Y.-I.
      • Aishima S.
      • Nakao Y.
      • Yoshizumi T.
      • Nagano H.
      • Kuroki T.
      • et al.
      Clinicopathological characteristics of combined hepatocellular cholangiocarcinoma from the viewpoint of patient prognosis after hepatic resection: high rate of early recurrence and its predictors.
      The role of liver transplantation has been investigated in several retrospective studies that most often included patients for whom diagnosis was incidentally performed during the pathological examination of the explant. Garancini et al. analysed the outcomes of 465 patients with cHCC-CCA identified by interrogating the Surveillance Epidemiology and End Results (SEER) database and observed that liver transplantation did not provide any survival benefit compared to minor or major hepatectomy.
      • Garancini M.
      • Goffredo P.
      • Pagni F.
      • Romano F.
      • Roman S.
      • Sosa J.A.
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      Combined hepatocellular-cholangiocarcinoma: a population-level analysis of an uncommon primary liver tumor.
      Other authors reported higher rates of relapse after transplantation in patients with cHCC-CCA compared to patients with HCC; cHCC-CCA is now widely regarded as a contra-indication for liver transplantation. Further studies may in the future identify whether it may be considered for a subset of selected patients (with small and slowly growing tumours, for example).
      • Garancini M.
      • Goffredo P.
      • Pagni F.
      • Romano F.
      • Roman S.
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      • et al.
      Combined hepatocellular-cholangiocarcinoma: a population-level analysis of an uncommon primary liver tumor.
      ,
      • Magistri P.
      • Tarantino G.
      • Serra V.
      • Guidetti C.
      • Ballarin R.
      • Di Benedetto F.
      Liver transplantation and combined hepatocellular-cholangiocarcinoma: feasibility and outcomes.
      • Gupta R.
      • Togashi J.
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      • Sakamoto Y.
      • Kokudo N.
      Impact of incidental/misdiagnosed intrahepatic cholangiocarcinoma and combined hepatocellular cholangiocarcinoma on the outcomes of liver transplantation: an institutional case series and literature review.
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      • et al.
      The diagnostic conundrum and liver transplantation outcome for combined hepatocellular-cholangiocarcinoma.
      The treatment options for patients with advanced disease not amenable to resection include locoregional therapies (transarterial chemoembolisation, radioembolisation, ablative therapies) and systemic chemotherapy.
      • Stavraka C.
      • Rush H.
      • Ross P.
      Combined hepatocellular cholangiocarcinoma (cHCC-CC): an update of genetics, molecular biology, and therapeutic interventions.
      ,
      • Kassahun W.T.
      • Hauss J.
      Management of combined hepatocellular and cholangiocarcinoma.
      • Fowler K.
      • Saad N.E.
      • Brunt E.
      • Doyle M.B.M.
      • Amin M.
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      Biphenotypic primary liver carcinomas: assessing outcomes of hepatic directed therapy.
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      • An J.
      • Lee D.
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      The effectiveness of transarterial chemoembolization in recurrent hepatocellular-cholangiocarcinoma after resection.
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      • Rogers J.E.
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      • Elsayes K.M.
      • Elbanan M.G.
      • Law L.
      • et al.
      Systemic therapy for unresectable, mixed hepatocellular-cholangiocarcinoma: treatment of a rare malignancy.
      However, owing to the lack of randomised studies, there are no consensus guidelines and decisions are usually extrapolated from HCC and iCCA. Accordingly, patients with cHCC-CCA have been systematically excluded from recent clinical trials on immunotherapy (either as monotherapy or in combination). Liver-directed therapy seems to offer a superior objective response over systemic therapy, a survival advantage and may downstage patients for surgical resection.
      • Schizas D.
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      • Papapanou M.
      • Tsapralis D.
      • Vassiliu P.
      • et al.
      Combined hepatocellular-cholangiocarcinoma: an update on epidemiology, classification, diagnosis and management.
      ,
      • Na S.K.
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      The effectiveness of transarterial chemoembolization in recurrent hepatocellular-cholangiocarcinoma after resection.
      ,
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      Sorafenib, approved as the first-line treatment for advanced HCC, and gemcitabine and cisplatin, the standard of care for unresectable iCCA, do not seem to have significant antitumour efficacy.
      • Rogers J.E.
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      With the exception of 1 case report,
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      no data are available in the literature concerning immunotherapy. This clearly shows that cHCC-CCA should be considered a distinct entity that potentially requires specific therapeutic strategies.
      Current data show that the molecular profile of combined hepatocellular-cholangiocarcinoma parallels its biphenotypic histological appearance, with genetic alterations that are classically observed in hepatocellular carcinoma and/or intrahepatic cholangiocarcinoma.

      Future directions and perspectives

      The first step in improving clinical care of patients with cHCC-CCA is standardising its diagnosis. Although a recent consensus paper has recently provided simplified terminology and refined the diagnostic criteria for cHCC-CCA, inter-observer agreement remains suboptimal, even between experts.
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      Given the high intratumour heterogeneity, tumour sampling is a key issue for diagnostic purposes.
      The development of immunostains should thus be strongly encouraged. The study by Xue et al. suggested that the expression of Nestin, a marker of bipotent progenitor oval cells (expression of nestin-green fluorescent protein transgene marks oval cells in the adult) may serve as a diagnostic biomarker of cHCC-CCA. Nestin is a type VI intermediate filament that is thought to play an important role in cell proliferation and plasticity.
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      ,
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      Tschaharganeh et al. further showed that its downregulation by the tumour suppressor p53 restricts the reprogramming of differentiated hepatocytes into malignant progenitors.
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      • Xue W.
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      • Michurina T.V.
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      p53-dependent Nestin regulation links tumor suppression to cellular plasticity in liver cancer.
      However, nestin is expressed in a subset of HCC and iCCA so its value for cHCC-CCA remains to be assessed in large series.
      • Xue R.
      • Chen L.
      • Zhang C.
      • Fujita M.
      • Li R.
      • Yan S.-M.
      • et al.
      Genomic and transcriptomic profiling of combined hepatocellular and intrahepatic cholangiocarcinoma reveals distinct molecular subtypes.
      ,
      • Zhang Y.
      • Zeng S.
      • Ma J.
      • Deng G.
      • Qu Y.
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      Nestin overexpression in hepatocellular carcinoma associates with epithelial-mesenchymal transition and chemoresistance.
      ,
      • Malvi D.
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      The use of computational pathology approaches, such as automated image analysis or convolutional neural networks, may also provide an opportunity to standardise the diagnosis of cHCC-CCA.
      • Schmauch B.
      • Romagnoni A.
      • Pronier E.
      • Saillard C.
      • Maillé P.
      • Calderaro J.
      • et al.
      A deep learning model to predict RNA-Seq expression of tumours from whole slide images.
      For example, these approaches were recently used in a study by Calderaro et al. to refine the prediction of HCC prognosis.
      • Saillard C.
      • Schmauch B.
      • Laifa O.
      • Moarii M.
      • Toldo S.
      • Zaslavskiy M.
      • et al.
      Predicting survival after hepatocellular carcinoma resection using deep-learning on histological slides.
      Another approach to improve the diagnosis of cHCC-CCA could be the use of minimally invasive biomarkers such as circulating tumour cells. To the best of our knowledge, no data on this approach are available in cHCC-CCA, although it has been already used in HCC.
      • Cui K.
      • Ou Y.
      • Shen Y.
      • Li S.
      • Sun Z.
      Clinical value of circulating tumor cells for the diagnosis and prognosis of hepatocellular carcinoma (HCC): a systematic review and meta-analysis.
      ,
      • Trevisan França de Lima L.
      • Broszczak D.
      • Zhang X.
      • Bridle K.
      • Crawford D.
      • Punyadeera C.
      The use of minimally invasive biomarkers for the diagnosis and prognosis of hepatocellular carcinoma.
      In a recent meta-analysis of 20 studies including 1,191 patients, circulating tumour cell detection demonstrated good performance for the diagnosis of HCC with a sensitivity of 0.95 and a specificity of 0.60.
      • Trevisan França de Lima L.
      • Broszczak D.
      • Zhang X.
      • Bridle K.
      • Crawford D.
      • Punyadeera C.
      The use of minimally invasive biomarkers for the diagnosis and prognosis of hepatocellular carcinoma.
      Finally, the development of a dedicated staging system will also be important to adapt therapeutic strategies and stratify patients for upcoming trials. Indeed, it has been advised to apply the TNM classification of iCCA to cHCC-CCA.
      • Sciarra A.
      • Park Y.N.
      • Sempoux C.
      Updates in the diagnosis of combined hepatocellular-cholangiocarcinoma.
      Combined hepatocellular-cholangiocarcinomas are associated with a prognosis that is worse than that of hepatocellular carcinoma, but similar to that of intrahepatic cholangiocarcinoma.

      Conclusion

      cHCC-CCA represents a distinct group of primary liver malignancies. Despite significant recent advances in molecular and morphological characterisation, further studies are needed to increase our understanding of these tumours and to develop improved therapeutic options for patients with cHCC-CCA.

      Abbreviations

      AFP, alpha-fetoprotein; CA 19-9, carbohydrate antigen 19.9; cHCC-CCA, combined hepatocellular-cholangiocarcinoma; CK, cytokeratin; CLC, cholangiolocellular carcinoma; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma; IHC, immunohistochemical; LI-RADS, Liver Imaging Reporting And Data System; SEER, Surveillance, Epidemiology, and End Results Program of the National Cancer Institute; TGFβ, transforming growth factor-β; WHO, World Health Organization.

      Financial support

      The authors received no financial support to produce this manuscript.

      Authors’ contributions

      All authors (AB, JC, VP) equally contribute to bibliography research and manuscript redaction: A Beaufrère: Figure preparation; V Paradis: Coordination and final review.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Supplementary data

      The following is/are the supplementary data to this article:

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