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Insights into the diagnosis of hepatocellular carcinomas with hepatobiliary MRI

  • Valérie Vilgrain
    Affiliations
    Department of Radiology, University Hospitals Paris Nord Val-de-Seine, Beaujon, 100 Boulevard du Général Leclerc, 92118 Clichy, France

    University Paris Diderot, Sorbonne Paris Cité, Paris, France
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  • Bernard E. Van Beers
    Affiliations
    Department of Radiology, University Hospitals Paris Nord Val-de-Seine, Beaujon, 100 Boulevard du Général Leclerc, 92118 Clichy, France

    University Paris Diderot, Sorbonne Paris Cité, Paris, France

    Inserm U1149, Centre de Recherche sur l’Inflammation, Paris, France
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  • Catherine M. Pastor
    Correspondence
    Corresponding author. Address: Département d’imagerie et des sciences de l’information médicale, Hôpitaux Universitaires de Genève, Rue Gabrielle-Perret-Gentil, 4, 1205 Geneva, Switzerland. Tel.: +41 22 372 38 36.
    Affiliations
    University Paris Diderot, Sorbonne Paris Cité, Paris, France

    Département d’imagerie et des sciences de l’information médicale, Hôpitaux Universitaires de Genève, Geneva, Switzerland
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Published:November 26, 2015DOI:https://doi.org/10.1016/j.jhep.2015.11.016

      Summary

      The incidence of hepatocellular carcinomas (HCCs) has increased worldwide in line with an improved screening by high-resolution imaging of cirrhotic livers. Besides abdominal ultrasonography and computerised tomography, magnetic resonance imaging (MRI) is an important tool to detect HCCs. With commercialisation of MR hepatobiliary contrast agents that cross membrane transporters in hepatocytes or tumour cells, MRI adds new information to detect and characterise HCCs. When tumour cells lose organic anion transporting polypeptides (OATP1B1/B3) in cell membranes facing sinusoidal blood, tumours appear hypointense (decreased contrast agent concentrations) in comparison to surrounding normal or cirrhotic liver that retains OATP1B1/B3 expression. However, expression, regulation, and prognostic significance of transporter evolution along carcinogenesis are not completely known. Moreover, understanding signal intensities in focal lesions also relies on transport functions of cellular efflux transporters. This manuscript reviews all the publications that associate liver imaging with hepatobiliary contrast agents and expression of transporters. The regulation of transporters along carcinogenesis to anticipate the prognosis of focal lesions is also included.

      Abbreviations:

      HCCs (hepatocellular carcinomas), MRI (magnetic resonance imaging), HB-MRI (Hepatobiliary MRI, images acquired during the hepatobiliary phase), DN (dysplastic nodule), LGDN (low grade dysplastic nodule), HGDN (high grade dysplastic nodule), BOPTA (gadobenate dimeglumine, MultiHance®, Bracco Imaging SpA, Milan, Italy), EOB-DTPA (gadoxetate dimeglumine, Primovist® or Eovist®, Bayer Health Care Pharmaceuticals, Berlin, Germany), Hypointense HCCs (lower signal intensities (darker) than surrounding parenchyma at HB-MRI), Hyperintense HCCs (higher signal intensities (brighter) than surrounding parenchyma at HB-MRI), OATP (Organic Anion Transporting Polypeptide), MRP (Multiple Resistance-associated Protein)

      Keywords

      Introduction

      Over the past decade, the incidence of hepatocellular carcinomas (HCCs) has increased worldwide in line with an improved screening by high-resolution imaging of cirrhotic livers [
      • Forner A.
      • Gilabert M.
      • Bruix J.
      • Raoul J.-L.
      Treatment of intermediate-stage hepatocellular carcinoma.
      ,
      • Forner A.
      • Llovet J.M.
      • Bruix J.
      Hepatocellular carcinoma.
      ,
      • El-Serag H.B.
      Hepatocellular carcinoma.
      ]. Besides abdominal ultrasonography and computerised tomography, magnetic resonance imaging (MRI) is an important tool to detect HCC [
      • Choi J.Y.
      • Lee J.M.
      • Sirlin C.B.
      CT and MR imaging diagnosis and staging of hepatocellular carcinoma: part II. Extracellular agents, hepatobiliary agents, and ancillary imaging features.
      ,
      • Choi J.Y.
      • Lee J.M.
      • Sirlin C.B.
      CT and MR imaging diagnosis and staging of hepatocellular carcinoma: Part I. Development, growth, and spread: key pathologic and imaging aspects.
      ,
      • Kudo M.
      Diagnostic imaging of hepatocellular carcinoma: recent progress.
      ], while the use of positron emission tomography is increasing [
      • Tsurusaki M.
      • Okada M.
      • Kuroda H.
      • Matsuki M.
      • Ishii K.
      • Murakami T.
      Clinical application of 18F-fluorodeoxyglucose positron emission tomography for assessment and evaluation after therapy for malignant hepatic tumor.
      ].
      Figure thumbnail fx1
      Figure thumbnail gr1
      Fig. 1Carcinogenesis evolution from dysplasic nodules to hepatocellular carcinomas with corresponding signal intensities at liver magnetic resonance imaging (MRI) following hepatobiliary contrast agents. (A) Macroscopic evolution of focal lesions, presence of pseudoglands and sinusoidal capillarisation are illustrated. (B) Evolution of arterioles and venules in focal lesions with corresponding signal intensities at MRI following injection of extracellular contrast agents in comparison to surrounding tissues. Hyperintense lesion during the hepatic artery phase (MRIHA) reflects the presence of numerous isolated arterioles. Hypointense lesion during the portal vein phase (MRIPV) reflects the disappearance of portal triads. (C) Putative evolution of transporter expression from hepatocyte to tumour cell and corresponding signal intensities during the hepatobiliary phase (MRIHB). Adapted from
      [
      • Van Beers B.E.
      • Pastor C.M.
      • Hussain H.K.
      Primovist, Eovist: what to expect?.
      ]
      . LGDN, low grade dysplastic nodule; HGDN, high grade dysplastic nodule; OATP, organic anion transporting polypeptide; MRP, multiple resistance-associated protein.

      Pathology: from dysplastic nodules to HCCs

      Progression of tissue injury from dysplastic nodules (DN) to HCCs is summarised because histological structures such as pseudoglands interfere with signal intensities at HB-MRI (Fig. 1). Most HCCs develop from clonal cells and expand into foci or DN, before evolving into a true carcinoma with exclusive arterial blood supply as well as stromal and vascular invasion [
      • Roskams T.
      • Kojiro M.
      Pathology of early hepatocellular carcinoma: conventional and molecular diagnosis.
      ,
      • Efremidis S.C.
      • Hytiroglou P.
      • Matsui O.
      Enhancement patterns and signal-intensity characteristics of small hepatocellular carcinoma in cirrhosis: pathologic basis and diagnostic challenges.
      ]. Classification of these lesions was rationalised during several international meetings and is briefly summarised [
      • Roskams T.
      • Kojiro M.
      Pathology of early hepatocellular carcinoma: conventional and molecular diagnosis.
      ,
      Pathologic diagnosis of early hepatocellular carcinoma: a report of the international consensus group for hepatocellular neoplasia.
      ].

      Dysplastic nodules

      DNs range between 1 and 3 cm and are either low (LGDNs) or high (HGDNs) grade dysplastic nodules (Fig. 1A). Hepatocytes in LGDNs show minimal nuclear atypia. Cytoplasm is normal but clonal cells (dysplastic foci) may accumulate fat, hemosiderin, or copper. DNs are often identified by the presence of a peripheral fibrous scar, accumulation of fat, and increased cell density. The vascular supply is identical to surrounding tissues. LGDNs do not contain pseudoglands, which are rosettes of hepatocytes that secrete bile into a closed space. Unpaired (isolated or not triadal) arteries are sometimes present, spreading outside portal tracts. They are not associated with portal vein or bile ductules and reflect lesion neovascularisation. Distinction between LGDNs and large regenerative nodules may be difficult.
      Cells of HGDNs have mild nuclear irregularities, basophilic cytoplasm, high nuclear-cytoplasmic ratio, and occasional mitosis but atypia is insufficient to diagnose malignancy. Architectural modifications include thickening of cell plates, pseudoglands, and may contain a malignant HCC referred to as ‘‘nodule-in-nodule’’. The subnodule may possess higher proliferation indexes than the parent nodule with abundant atypia and fatty change or can be a well differentiated HCC. Vascular supply is abnormal and HGDNs contain unpaired arteries and shows intermediate degree of sinusoidal capillarisation. HGDNs may be distinctly or vaguely nodular lacking a true capsule, similar to LGDNs. A considerable proportion of HGDNs progresses to HCCs within a few years, the risk of malignant transformation of LGDN being much lower [
      • Roskams T.
      • Kojiro M.
      Pathology of early hepatocellular carcinoma: conventional and molecular diagnosis.
      ].

      Early small HCCs

      Early small HCCs (up to 2 cm in diameter) are classified into distinctly nodular or vaguely (indistinctly) nodular types. Distinctly nodular small HCCs (dN-sHCCs) are delineated by a capsule and resemble large HCCs. Tumour invasion into portal tracts and metastases in the vicinity are frequent. Most of these dN-sHCCs have numerous unpaired arteries, complete sinusoid capillarisation, and a capsule that encircles the lesion. Pseudoglands and stroma invasion (or tumour cell invasion into portal tracts or fibrous septa) are present. Vaguely nodular small HCCs (vN-sHCCs) are not well delineated and the lesion grows at the boundary of surrounding tissue. They are mostly well differentiated with few atypia. vN-sHCCs have increased cell density and nuclear-cytoplasmic ratio and contain irregular thin trabecular structures. They possess pseudoglands. Diffused fat accumulations are frequent. The number of unpaired arteries associated with normal portal tracts is variable. At the tumour boundary, portal vessels of surrounding tissues may be connected with sinusoids of vN-sHCCs, explaining why lesions may be partially perfused when contrast agents arrive in portal vessels. vN-sHCCs and HGDNs may be difficult to differentiate, but stromal invasion is the proof of malignancy [
      Pathologic diagnosis of early hepatocellular carcinoma: a report of the international consensus group for hepatocellular neoplasia.
      ].

      Large HCCs

      Finally, large HCCs (>2 cm) have numerous unpaired arteries, complete sinusoid capillarisation, and a capsule that encircles the lesion. Pseudoglands and stroma invasion are present. Tumour invasion into portal veins and metastases at the vicinity are frequent. In routine practice, HCC is graded as; well, moderate, or poorly differentiated types [
      • Jain D.
      Tissue diagnosis of hepatocellular carcinoma.
      ,
      • Kim H.
      • Park Y.N.
      Role of biopsy sampling for diagnosis of early and progressed hepatocellular carcinoma.
      ]. The pattern of growth is trabecular, pseudoacinar (pseudoglands), or diffuse. Tumour cells are polygonal with granular eosinophilic cytoplasm, nuclear pleiomorphism and increased nuclear-cytoplasmic ratio. Cells may secrete bile and contain fat, glycogen, hyaline globules, or fibrinogen. There is no intercellular stroma except in rare scirrhous (fibrous) and fibrolamellar HCCs. In most HCCs, endothelial cells line tumour cells. Unpaired arteries are identified. Portal tracts are not present except at tumour boundary where entrapped portal tracts may be present with invading neoplastic cells.

      Transporter expression in HCCs and surrounding liver tissues

      As morphologic criteria alone are insufficient to differentiate HGDN from early HCC, additional markers, such as glypican-3, heat shock protein 70, and glutamine synthetase are helpful to stain on biopsies [
      • Roskams T.
      • Kojiro M.
      Pathology of early hepatocellular carcinoma: conventional and molecular diagnosis.
      ,
      • Schütte K.
      • Schulz C.
      • Link A.
      • Malfertheiner P.
      Current biomarkers for hepatocellular carcinoma: surveillance, diagnosis and prediction of prognosis.
      ,
      • Pinato D.J.
      • Pirisi M.
      • Maslen L.
      • Sharma R.
      Tissue biomarkers of prognostic significance in hepatocellular carcinoma.
      ]. Drug transporters present on hepatocyte membranes modify their expression along carcinogenesis with important implications for liver imaging (Fig. 1C).

      Expression of OATP1B1 and OATP1B3

      Hepatobiliary contrast agents (BOPTA and EOB-DTPA) are taken up from sinusoids and interstitium into hepatocytes by OATP1B1 and OATP1B3 in normal hepatocytes (Fig. 2). In normal livers, the expression of OATP1B1/B3 highly increases from portal (no transporters) to perivenous areas (maximal expression) along sinusoids [
      • Obaidat A.
      • Roth M.
      • Hagenbuch B.
      The expression and function of organic anion transporting polypeptides in normal tissues and in cancer.
      ]. Early studies measured OATP1B1/B3 expression in HCCs in comparison to surrounding liver parenchyma without imaging. The sinusoidal expression of OATP1B1 is found either decreased [
      • Kinoshita M.
      • Miyata M.
      Underexpression of mRNA in human hepatocellular carcinoma focusing on eight loci.
      ,
      • Zollner G.
      • Wagner M.
      • Fickert P.
      • Silbert D.
      • Fuchsbichler A.
      • Zatloukal K.
      • et al.
      Hepatobiliary transporter expression in human hepatocellular carcinoma.
      ] or unchanged [
      • Vavricka S.R.
      • Jung D.
      • Fried M.
      • Grützner U.
      • Meier P.J.
      • Kullak-Ublick G.A.
      The human organic anion transporting polypeptide 8 (SLCO1B3) gene is transcriptionally repressed by hepatocyte nuclear factor 3beta in hepatocellular carcinoma.
      ] while OATB1B3 is decreased by 60% in one study [
      • Vavricka S.R.
      • Jung D.
      • Fried M.
      • Grützner U.
      • Meier P.J.
      • Kullak-Ublick G.A.
      The human organic anion transporting polypeptide 8 (SLCO1B3) gene is transcriptionally repressed by hepatocyte nuclear factor 3beta in hepatocellular carcinoma.
      ]. Little information is available on a gradual decrease of OATP1B1/B3 expression between HGDN, early HCCs, and poorly differentiated HCCs (Fig. 1). Some moderately differentiated HCCs may have a strong expression of OATP1B3 [
      • Ueno A.
      • Masugi Y.
      • Yamazaki K.
      • Komuta M.
      • Effendi K.
      • Tanami Y.
      • et al.
      OATP1B3 expression is strongly associated with Wnt/β-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma.
      ]. Moreover, transporter expression correlates with tumour differentiation [
      • Ueno A.
      • Masugi Y.
      • Yamazaki K.
      • Komuta M.
      • Effendi K.
      • Tanami Y.
      • et al.
      OATP1B3 expression is strongly associated with Wnt/β-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma.
      ] or not [
      • Narita M.
      • Hatano E.
      • Arizono S.
      • Miyagawa-Hayashino A.
      • Isoda H.
      • Kitamura K.
      • et al.
      Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma.
      ,
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ].
      Figure thumbnail gr2
      Fig. 2In normal hepatocytes, the hepatobiliary contrast agent EOB-DTPA distributes from sinusoids (SIN) into interstitium (INT) and hepatocytes. EOB-DTPA sinusoidal clearance into liver (SINCLLIVER) and EOB-DTPA extraction ratio (SINERLIVER) evaluates EOB-DTPA transport through organic anion transporting polypeptides (OATPs). EOB-DTPA uptake clearance (SINUPTCLHC) from INT into hepatocytes (HC) is estimated by liver enhancement at MRI (hepatobiliary phase). When inside hepatocytes, EOB-DTPA exits into bile canaliculus (hepatocyte clearance to bile canaliculus, HCCLBC) and returns back to INT (hepatocyte clearance to INT, HCCLINT). These transfer rates between compartments generate concentrations (CEOB-DTPA) inside sinusoids, interstitium, hepatocytes, and bile canaliculi estimated by a single value at liver imaging. CEOB-DTPA in bile canaliculus is much higher than that measured in hepatocytes and MRP2 functions against a concentration gradient with the help of ATP. In tumour cells, MRP2, MRP3, MRP4 expression is often preserved while OATP1B1/B3 expression is more likely to decrease along hepatocarcinogenesis. Two structures trap EOB-DTPA in tumour: closed canaliculus and pseudogland. Tumour cells in pseudoglands can conserve transporters but once inside the glands, EOB-DTPA cannot exit from the lumen. Cell uptake transporters (pink circles); cell efflux transporters (blue circles).

      Expression of MRP2, MRP3, and MRP4

      Most HCCs conserve a high expression of MRP2 but the transporter may disappear in poorly differentiated lesions [
      • Zollner G.
      • Wagner M.
      • Fickert P.
      • Silbert D.
      • Fuchsbichler A.
      • Zatloukal K.
      • et al.
      Hepatobiliary transporter expression in human hepatocellular carcinoma.
      ,
      • Nies A.T.
      • König J.
      • Pfannschmidt M.
      • Klar E.
      • Hofmann W.J.
      • Keppler D.
      Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.
      ,
      • Richart J.
      • Brunt E.M.
      • Di Bisceglie A.M.
      Expression of P-glycoprotein and C-MOAT in human hepatocellular carcinoma: detection by immunostaining.
      ]. Consequently, MRP2 expression might be similar in HCCs and surrounding tissues [
      • Namisaki T.
      • Schaeffeler E.
      • Fukui H.
      • Yoshiji H.
      • Nakajima Y.
      • Fritz P.
      • et al.
      Differential expression of drug uptake and efflux transporters in Japanese patients with hepatocellular carcinoma.
      ]. MRP2 is localised in the canalicular membrane of hepatocytes as well as in the apical membrane of tumour cells arranged either in trabecular or pseudoglandular patterns (Fig. 2) [
      • Zollner G.
      • Wagner M.
      • Fickert P.
      • Silbert D.
      • Fuchsbichler A.
      • Zatloukal K.
      • et al.
      Hepatobiliary transporter expression in human hepatocellular carcinoma.
      ,
      • Nies A.T.
      • König J.
      • Pfannschmidt M.
      • Klar E.
      • Hofmann W.J.
      • Keppler D.
      Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.
      ,
      • Namisaki T.
      • Schaeffeler E.
      • Fukui H.
      • Yoshiji H.
      • Nakajima Y.
      • Fritz P.
      • et al.
      Differential expression of drug uptake and efflux transporters in Japanese patients with hepatocellular carcinoma.
      ,
      • Tsuboyama T.
      • Onishi H.
      • Kim T.
      • Akita H.
      • Hori M.
      • Tatsumi M.
      • et al.
      Hepatocellular carcinoma: hepatocyte-selective enhancement at gadoxetic acid–enhanced MR Imaging—Correlation with expression of sinusoidal and canalicular transporters and bile accumulation.
      ]. MRP2 staining can be higher in the luminal membrane of pseudoglands than in canalicular membrane of tumour cells arranged in trabecular structures [
      • Zollner G.
      • Wagner M.
      • Fickert P.
      • Silbert D.
      • Fuchsbichler A.
      • Zatloukal K.
      • et al.
      Hepatobiliary transporter expression in human hepatocellular carcinoma.
      ]. No detailed information is provided on the transporter expression in other hepatic lesions but DN probably retain a normal MRP2 expression [
      • Richart J.
      • Brunt E.M.
      • Di Bisceglie A.M.
      Expression of P-glycoprotein and C-MOAT in human hepatocellular carcinoma: detection by immunostaining.
      ].
      MRP2 is regulated by retrieval from canalicular membrane or insertion into it (Fig. 3). Insertion of MRP2 in the canalicular membrane relies on substrate concentrations to transport [
      • Shoda J.
      • Miura T.
      • Utsunomiya H.
      • Oda K.
      • Yamamoto M.
      • Kano M.
      • et al.
      Genipin enhances Mrp2 (Abcc2)-mediated bile formation and organic anion transport in rat liver.
      ]. In normal rat livers, we showed that phosphorylation of MRP2 retrieves the transporter from the membrane, preventing bile excretion of the hepatobiliary contrast agent BOPTA that remains trapped within hepatocytes [
      • Planchamp C.
      • Hadengue A.
      • Stieger B.
      • Bourquin J.
      • Vonlaufen A.
      • Frossard J.-L.
      • et al.
      Function of both sinusoidal and canalicular transporters controls the concentration of organic anions within hepatocytes.
      ]. OATPs are not regulated by such membrane insertion or retrieval.
      Figure thumbnail gr3
      Fig. 3Regulation of OATP1B3 and MRP2 transporters in hepatocellular carcinomas. β-catenin (β-CAT) encoded by CTNNB1 gene participates to hepatocarcinogenesis. When β-CAT cytoplasmic concentrations increase, proteins translocate into nuclei and bind to the transcription factors TCF/lymphoid enhancer family. In turn, newly formed β-CAT/TCF complexes trigger the activation of target genes. Pathway activation occurs when Wnt ligands bind to Frizzled (FZD) receptors on cell membranes. Newly formed β-CAT/TCF complex in nuclei is frequently associated with an upregulation of SLCO1B3, the gene encoding OATP1B3. A subtype of HCCs is described: HCCs associated with activating mutation of CTNNB1. Besides β-CAT, other nuclear factors have been investigated: hepatocyte nuclear factor (HNF1α, 3β, 4α) and farnesoid X receptor (FXR). Phosphorylation (P) of OATP1B3 by protein kinase C (PKC) decreases hepatocyte uptake of the hepatobiliary contrast agent BOPTA. PKC activation also decreases hepatocyte clearance into bile by Mrp2 retrieval from the canalicular membrane
      [
      • Planchamp C.
      • Hadengue A.
      • Stieger B.
      • Bourquin J.
      • Vonlaufen A.
      • Frossard J.-L.
      • et al.
      Function of both sinusoidal and canalicular transporters controls the concentration of organic anions within hepatocytes.
      ]
      .
      The efflux transporter MRP3 is also detected in HCC samples. However, MRP3 expression is usually weak in comparison to that of cholangiocytes [
      • Vander Borght S.
      • Libbrecht L.
      • Blokzijl H.
      • Faber K.N.
      • Moshage H.
      • Aerts R.
      • et al.
      Diagnostic and pathogenetic implications of the expression of hepatic transporters in focal lesions occurring in normal liver.
      ]. In HCCs, MRP3 expression is either similar [
      • Nies A.T.
      • König J.
      • Pfannschmidt M.
      • Klar E.
      • Hofmann W.J.
      • Keppler D.
      Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.
      ,
      • Vander Borght S.
      • Libbrecht L.
      • Blokzijl H.
      • Faber K.N.
      • Moshage H.
      • Aerts R.
      • et al.
      Diagnostic and pathogenetic implications of the expression of hepatic transporters in focal lesions occurring in normal liver.
      ], increased [
      • Kimura Y.
      • Sato S.
      • Hitomi E.
      • Ohyama M.
      • Adachi K.
      • Inagaki Y.
      • et al.
      Coexpression of organic anion-transporting polypeptides 1B3 and multidrug-resistant proteins 2 increases the enhancement effect of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid on hepatocellular carcinoma in magnetic resonance imaging.
      ], or decreased [
      • Zollner G.
      • Wagner M.
      • Fickert P.
      • Silbert D.
      • Fuchsbichler A.
      • Zatloukal K.
      • et al.
      Hepatobiliary transporter expression in human hepatocellular carcinoma.
      ] in comparison to surrounding tissues. In tumour cells, MRP3 is localised in sinusoidal membranes [
      • Zollner G.
      • Wagner M.
      • Fickert P.
      • Silbert D.
      • Fuchsbichler A.
      • Zatloukal K.
      • et al.
      Hepatobiliary transporter expression in human hepatocellular carcinoma.
      ,
      • Nies A.T.
      • König J.
      • Pfannschmidt M.
      • Klar E.
      • Hofmann W.J.
      • Keppler D.
      Expression of the multidrug resistance proteins MRP2 and MRP3 in human hepatocellular carcinoma.
      ,
      • Vander Borght S.
      • Libbrecht L.
      • Blokzijl H.
      • Faber K.N.
      • Moshage H.
      • Aerts R.
      • et al.
      Diagnostic and pathogenetic implications of the expression of hepatic transporters in focal lesions occurring in normal liver.
      ,
      • Kimura Y.
      • Sato S.
      • Hitomi E.
      • Ohyama M.
      • Adachi K.
      • Inagaki Y.
      • et al.
      Coexpression of organic anion-transporting polypeptides 1B3 and multidrug-resistant proteins 2 increases the enhancement effect of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid on hepatocellular carcinoma in magnetic resonance imaging.
      ] but positive staining in cytoplasm can be observed [
      • Zollner G.
      • Wagner M.
      • Fickert P.
      • Silbert D.
      • Fuchsbichler A.
      • Zatloukal K.
      • et al.
      Hepatobiliary transporter expression in human hepatocellular carcinoma.
      ]. Kitao et al. [
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ] reported that MRP3 expression was higher in isointense or hyperintense HCC than in hypointense HCC, raising the hypothesis than EOB-DTPA might return from tumour cells into sinusoids. However, efflux of EOB-DTPA from tumour cells back to blood spaces was not shown. In contrast, Kimura et al. [
      • Kimura Y.
      • Sato S.
      • Hitomi E.
      • Ohyama M.
      • Adachi K.
      • Inagaki Y.
      • et al.
      Coexpression of organic anion-transporting polypeptides 1B3 and multidrug-resistant proteins 2 increases the enhancement effect of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid on hepatocellular carcinoma in magnetic resonance imaging.
      ] did not find any correlation between tumour enhancement and MRP3 expression. Finally, in 44 HCCs, Sekine et al. [
      • Sekine S.
      • Ogawa R.
      • Ojima H.
      • Kanai Y.
      Expression of SLCO1B3 is associated with intratumoral cholestasis and CTNNB1 mutations in hepatocellular carcinoma.
      ] found only four positive staining for MRP4. The expression was diffused within one lesion, while 3 HCCs had focal staining. The transporter was not detectable in surrounding tissues.

      Transporter expression and hepatocarcinogenesis

      Several reviews suggest that OATP1B1/B3 expression decreases from normal hepatocytes to tumour cells but few studies show the transporter expression along carcinogenesis. OATP1B3 expression gradually decreased from high grade DN, early HCCs, well differentiated HCCs, moderately differentiated, and poorly differentiated HCCs [
      • Ueno A.
      • Masugi Y.
      • Yamazaki K.
      • Komuta M.
      • Effendi K.
      • Tanami Y.
      • et al.
      OATP1B3 expression is strongly associated with Wnt/β-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma.
      ]. However, one HGDN (over 3) had no OATP1B3 while few moderately and poorly differentiated HCCs exhibit strong expression of OATP1B3. Some well and moderately differentiated HCCs may have strong expression of OATP1B3, but overall OATP1B3 expression and tumour differentiation grade correlated significantly. Kitao et al. [
      • Kitao A.
      • Matsui O.
      • Yoneda N.
      • Kozaka K.
      • Shinmura R.
      • Koda W.
      • et al.
      The uptake transporter OATP8 expression decreases during multistep hepatocarcinogenesis: correlation with gadoxetic acid enhanced MR imaging.
      ] found similar results but the study includes only 1 DN with OATP1B3 expression.

      Regulation of transporters

      β-catenin

      Some studies investigated the regulation of OATP1B1/B3, MRP2, MRP3, and MRP4 expression in HCCs, including β-catenin (Fig. 3). β-catenin encoded by CTNNB1 gene belongs to cellular adherens junctions and desmosomes [
      • Pez F.
      • Lopez A.
      • Kim M.
      • Wands J.R.
      • Caron de Fromentel C.
      • Merle P.
      Wnt signaling and hepatocarcinogenesis: molecular targets for the development of innovative anticancer drugs.
      ]. β-catenin also transmits proliferative signals and participates to early hepatocarcinogenesis. When β-catenin cytoplasmic concentrations increase, proteins translocate into the nuclei and bind to the transcription factors TCF/lymphoid enhancer family. In turn, newly formed β-catenin/TCF complexes trigger the activation of target genes involved in cell proliferation, survival, and migration. Pathway activation occurs when Wnt ligands bind to Frizzled (FZD) receptors on cell membranes (Fig. 3). CTNNB1-mutated HCCs lead to a newly formed β-catenin/TCF complex that triggers activation of target genes. These activating mutations of CTNNB1 are present in 8–44% of HCCs [
      • de La Coste A.
      • Romagnolo B.
      • Billuart P.
      • Renard C.A.
      • Buendia M.A.
      • Soubrane O.
      • et al.
      Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas.
      ,
      • Monga S.P.
      β-Catenin signaling and roles in liver homeostasis, injury and tumorigenesis.
      ].
      Interestingly, a newly formed β-catenin/TCF complex in the nuclei is frequently associated with an upregulation of OATP1B3 expression. In 2011, Sekine et al. [
      • Sekine S.
      • Ogawa R.
      • Ojima H.
      • Kanai Y.
      Expression of SLCO1B3 is associated with intratumoral cholestasis and CTNNB1 mutations in hepatocellular carcinoma.
      ] found that SLCO1B3 (gene encoding OATP1B3) expression was high in CTNNB1-mutated HCCs. Immunohistochemistry confirmed the frequent high expression of OATP1B3 in CTNNB1-mutated HCCs, but not in most HCCs with wild-type CTNNB1. However, OATP1B3 on tumour cell membranes is not always regulated by CTNNB1 mutation and 8% of HCCs without mutation possess OATP1B3 while 17% with mutations have no OATP1B3 expression [
      • Sekine S.
      • Ogawa R.
      • Ojima H.
      • Kanai Y.
      Expression of SLCO1B3 is associated with intratumoral cholestasis and CTNNB1 mutations in hepatocellular carcinoma.
      ]. A subtype of HCCs is now described, HCCs associated with activating mutation of CTNNB1. Glutamine synthetase detection on liver biopsy is a good marker of β-catenin activation. These HCCs develop more frequently in chronic HCV hepatitis and in non-cirrhotic livers [
      • Monga S.P.
      β-Catenin signaling and roles in liver homeostasis, injury and tumorigenesis.
      ]. CTNNB1-mutated HCCs are either differentiated lesions [
      • Ueno A.
      • Masugi Y.
      • Yamazaki K.
      • Komuta M.
      • Effendi K.
      • Tanami Y.
      • et al.
      OATP1B3 expression is strongly associated with Wnt/β-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma.
      ] or proliferating and poorly differentiated lesions [
      • Kitao A.
      • Matsui O.
      • Yoneda N.
      • Kozaka K.
      • Kobayashi S.
      • Sanada J.
      • et al.
      Hepatocellular carcinoma with β-catenin mutation: imaging and pathologic characteristics.
      ]. This HCC type has higher OATP1B3 expression and can be detected at HB-MRI by hyperintense signal intensities (see later section) [
      • Sekine S.
      • Ogawa R.
      • Ojima H.
      • Kanai Y.
      Expression of SLCO1B3 is associated with intratumoral cholestasis and CTNNB1 mutations in hepatocellular carcinoma.
      ,
      • Kitao A.
      • Matsui O.
      • Yoneda N.
      • Kozaka K.
      • Kobayashi S.
      • Sanada J.
      • et al.
      Hepatocellular carcinoma with β-catenin mutation: imaging and pathologic characteristics.
      ]. However, the OATP1B3 expression in mutated HCCs remains incompletely known. In several HCC cell lines, activation of β-catenin signalling does not induce SLCO1B3 expression [
      • Ueno A.
      • Masugi Y.
      • Yamazaki K.
      • Komuta M.
      • Effendi K.
      • Tanami Y.
      • et al.
      OATP1B3 expression is strongly associated with Wnt/β-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma.
      ]. In contrast, the β-catenin activator LiCl significantly induces OATP1B3 mRNA expression in KYN-2 cells [
      • Ueno A.
      • Masugi Y.
      • Yamazaki K.
      • Komuta M.
      • Effendi K.
      • Tanami Y.
      • et al.
      OATP1B3 expression is strongly associated with Wnt/β-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma.
      ]. Because HCCs may express high concentrations of OATP1B3 in the absence of CTNNB1 mutation, other factors are implicated in the regulation of uptake transporters.

      Other nuclear factors

      Besides β-catenin, other factors have been investigated in the regulation of transporters (Fig. 3). In HepG2 and HuH7 cells, co-expression of hepatocyte nuclear factor 1α (HNF1α) stimulates OATP1B1 promoter activity while promoter mutations in HNF1α binding site abolishes the stimulation [
      • Jung D.
      • Hagenbuch B.
      • Gresh L.
      • Pontoglio M.
      • Meier P.J.
      • Kullak-Ublick G.A.
      Characterization of the human OATP-C (SLC21A6) gene promoter and regulation of liver-specific OATP genes by hepatocyte nuclear factor 1a.
      ]. The human OATP1B3 gene is also responsive to HNF1α in HepG2 cells [
      • Jung D.
      • Hagenbuch B.
      • Gresh L.
      • Pontoglio M.
      • Meier P.J.
      • Kullak-Ublick G.A.
      Characterization of the human OATP-C (SLC21A6) gene promoter and regulation of liver-specific OATP genes by hepatocyte nuclear factor 1a.
      ]. HNF1α expression is one of the major determinants of inter-individual variability of OATP1B1 mRNA expression [
      • Furihata T.
      • Satoh T.
      • Yamamoto N.
      • Kobayashi K.
      • Chiba K.
      Hepatocyte nuclear factor 1 alpha is a factor responsible for the interindividual variation of OATP1B1 mRNA levels in adult Japanese livers.
      ] while OATP1B3 mRNA is regulated by farnesoid X receptor (FXR), HNF1α, and HNF3β [
      • Ohtsuka H.
      • Abe T.
      • Onogawa T.
      • Kondo N.
      • Sato T.
      • Oshio H.
      • et al.
      Farnesoid X receptor, hepatocyte nuclear factors 1alpha and 3beta are essential for transcriptional activation of the liver-specific organic anion transporter-2 gene.
      ].
      In human HCC cells, activation of FXR induces OATP1B3 promoter activity and increases OATP1B3 mRNA expression [
      • Jung D.
      • Podvinec M.
      • Meyer U.A.
      • Mangelsdorf D.J.
      • Fried M.
      • Meier P.J.
      • et al.
      Human organic anion transporting polypeptide 8 promoter is transactivated by the farnesoid X receptor/bile acid receptor.
      ]. HNF3β increases in 70% of HCCs and is inversely correlated with OATP1B3 expression [
      • Vavricka S.R.
      • Jung D.
      • Fried M.
      • Grützner U.
      • Meier P.J.
      • Kullak-Ublick G.A.
      The human organic anion transporting polypeptide 8 (SLCO1B3) gene is transcriptionally repressed by hepatocyte nuclear factor 3beta in hepatocellular carcinoma.
      ]. In vitro, when HNF3β is co-transfected with OATP1B3, the promoter activity of OATP1B3 is inhibited by 70%. A HNF3β binding site is located on OATP1B3 promoter while no such binding site is found on OATP1B1, explaining the absence of OATP1B1 expression regulation by HNF3β. Along carcinogenesis, the downregulation of OATP1B3 expression from hepatocytes to tumour cells might be explained by a reduced transcription of SLCO1B3 by HNF3β [
      • Vavricka S.R.
      • Jung D.
      • Fried M.
      • Grützner U.
      • Meier P.J.
      • Kullak-Ublick G.A.
      The human organic anion transporting polypeptide 8 (SLCO1B3) gene is transcriptionally repressed by hepatocyte nuclear factor 3beta in hepatocellular carcinoma.
      ]. We will see later that HCCs have a good prognosis when both OATP1B3 and HNF4α are upregulated [
      • Yamashita T.
      • Kitao A.
      • Matsui O.
      • Hayashi T.
      • Nio K.
      • Kondo M.
      • et al.
      Gd-EOB-DTPA- enhanced magnetic resonance imaging and alpha-fetoprotein predict prognosis of early-stage hepatocellular carcinoma.
      ]. HNF1α and HNF4α also play an important role in the regulation of MRP2 but no data exist in HCCs [
      • Qadri I.
      • Hu L.J.
      • Iwahashi M.
      • Al-Zuabi S.
      • Quattrochi L.C.
      • Simon F.R.
      Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2).
      ].

      Post-translational regulation of OATP transporters

      Phosphorylation of transporters by the protein kinase C (PKC) pathway modifies the function of hepatocyte transporters [
      • Anwer M.S.
      Role of protein kinase C isoforms in bile formation and cholestasis.
      ]. We showed in rat livers that PKC activation decreases hepatocyte uptake of the hepatobiliary contrast agent BOPTA (see later section). PKC activation also decreases BOPTA hepatocyte clearance into bile, associated with decreased liver and bile concentrations [
      • Planchamp C.
      • Hadengue A.
      • Stieger B.
      • Bourquin J.
      • Vonlaufen A.
      • Frossard J.-L.
      • et al.
      Function of both sinusoidal and canalicular transporters controls the concentration of organic anions within hepatocytes.
      ]. Similar experiments were performed in human sandwich-cultured hepatocytes using cholecystokinin-8 (CCK-8) [
      • Powell J.
      • Farasyn T.
      • Kock K.
      • Meng X.
      • Pahwa S.
      • Brouwer K.L.
      • et al.
      Novel mechanism of impaired function of organic anion-transporting polypeptide 1B3 in human hepatocytes: post-translational regulation of OATP1B3 by protein kinase C activation.
      ]. Whether PKC activation occurs in HCCs is puzzling but it is described in other cancers [
      • Garg R.
      • Benedetti L.G.
      • Abera M.B.
      • Wang H.
      • Abba M.
      • Kazanietz M.G.
      Protein kinase C and cancer: what we know and what we do not.
      ,
      • Guo K.
      • Li Y.
      • Kang X.
      • Sun L.
      • Cui J.
      • Gao D.
      • et al.
      Role of PKCb in hepatocellular carcinoma cells migration and invasion in vitro: a potential therapeutic target.
      ].

      In vitro transport of hepatobiliary MR contrast agents

      Two hepatobiliary contrast agents for liver MRI are commercialised: gadobenate dimeglumine (BOPTA, MultiHance®, Bracco Imaging SpA, Milan, Italy) and gadoxetate dimeglumine (EOB-DTPA, Primovist® or Eovist®, Bayer Health Care Pharmaceuticals, Berlin, Germany). These agents are increasingly used to visualise liver morphology and detect and characterise focal lesions [
      • Choi J.Y.
      • Lee J.M.
      • Sirlin C.B.
      CT and MR imaging diagnosis and staging of hepatocellular carcinoma: part II. Extracellular agents, hepatobiliary agents, and ancillary imaging features.
      ,
      • Van Beers B.E.
      • Pastor C.M.
      • Hussain H.K.
      Primovist, Eovist: what to expect?.
      ,
      • Pastor C.M.
      • Müllhaupt B.
      • Stieger B.
      The role of organic anion transporters in diagnosing liver diseases by magnetic resonance imaging.
      ,
      • Lee D.H.
      • Lee J.M.
      • Baek J.H.
      • Shin C.I.
      • Han J.K.
      • Choi B.I.
      Diagnostic performance of gadoxetic acid-enhanced liver MR imaging in the detection of HCCs and allocation of transplant recipients on the basis of the Milan criteria and UNOS guidelines: correlation with histopathologic findings.
      ]. In HCCs, these agents image the vascularisation and transfer rates from sinusoids into interstitium, similarly to other extracellular contrast agents. Both agents also cross the sinusoidal and canalicular membrane through transporters described in the previous section: OATP1B1, OATP1B3, MRP2, and MRP3. Thus, imaging with these contrast agents shows hepatocyte transporter functions. We previously showed that in HCCs, signal intensities generated by contrast agents reflect their concentrations in tumour cells, interstitium, closed canaliculi and pseudoglands (Fig. 2).
      The in vitro transport of EOB-DTPA was studied in HEK293 cells infected with the human OATP1B1 and OATP1B3 [
      • Leonhardt M.
      • Keiser M.
      • Oswald S.
      • Kühn J.
      • Jia J.
      • Grube M.
      • et al.
      Hepatic uptake of the magnetic resonance imaging contrast agent Gd-EOB-DTPA: role of human organic anion transporters.
      ]. EOB-DTPA is taken up by both transporters after a 10-min incubation at concentrations close to those used in clinical imaging (Fig. 2) [
      • Leonhardt M.
      • Keiser M.
      • Oswald S.
      • Kühn J.
      • Jia J.
      • Grube M.
      • et al.
      Hepatic uptake of the magnetic resonance imaging contrast agent Gd-EOB-DTPA: role of human organic anion transporters.
      ,
      • Jia J.
      • Keiser M.
      • Nassif A.
      • Siegmund W.
      • Oswald S.
      A LC-MS/MS method to evaluate the hepatic uptake of the liver-specific magnetic resonance imaging contrast agent gadoxetate (Gd-EOB-DTPA) in vitro and in humans.
      ]. Canalicular transporter of EOB-DTPA was shown in vesicles isolated from MDCK cells overexpressing MRP2 [
      • Jia J.
      • Puls D.
      • Oswald S.
      • Jedlitschky G.
      • Kühn J.P.
      • Weitschies W.
      • et al.
      Characterization of the intestinal and hepatic uptake/efflux transport of the magnetic resonance imaging contrast agent gadolinium-ethoxylbenzyl-diethylenetriamine- pentaacetic acid.
      ]. A similar model shows that MRP3 transports EOB-DTPA from cells back to sinusoids [
      • Jia J.
      • Puls D.
      • Oswald S.
      • Jedlitschky G.
      • Kühn J.P.
      • Weitschies W.
      • et al.
      Characterization of the intestinal and hepatic uptake/efflux transport of the magnetic resonance imaging contrast agent gadolinium-ethoxylbenzyl-diethylenetriamine- pentaacetic acid.
      ].
      Finally, we showed that rat hepatocytes transport the second contrast agent BOPTA across Oatp1a1, Oatp1a4, and Oatp1b2. BOPTA is not excreted into bile when hepatocytes lack Mrp2 [
      • Millet P.
      • Moulin M.
      • Stieger B.
      • Daali Y.
      • Pastor C.M.
      How organic anions accumulate in hepatocytes lacking Mrp2: evidence in rat liver.
      ]. Human transporters of BOPTA were never confirmed. Moreover, EOB-DTPA is preferred to BOPTA in clinical liver imaging because its hepatocyte extraction ratio is higher [
      • Filippone A.
      • Blakeborough A.
      • Breuer J.
      • Grazioli L.
      • Gschwend S.
      • Hammerstingl R.
      • et al.
      Enhancement of liver parenchyma after injection of hepatocyte-specific MRI contrast media: a comparison of gadoxetic acid and gadobenate dimeglumine.
      ]. Both MR contrast agents are excreted into bile without metabolic transformation.

      Clinical imaging of EOB-DTPA transport across transporters

      Three factors can modify the membrane transport of hepatobiliary MR contrast agents: 1) inter-individual variability of transporter function; 2) drug-drug interactions or competition between drugs for the same transporter; and 3) modifications of transporter expression in injured livers and focal lesions including HCCs, as shown in previous sections [
      • Pastor C.M.
      • Müllhaupt B.
      • Stieger B.
      The role of organic anion transporters in diagnosing liver diseases by magnetic resonance imaging.
      ]. Several genetic variants of human OATP1B1/1B3 carry EOB-DTPA with various transport activities and accordingly, HB-MRI in volunteers are influenced by these genetic polymorphisms [
      • Nassif A.
      • Jia J.
      • Keiser M.
      • Oswald S.
      • Modess C.
      • Nagel S.
      • et al.
      Visualization of hepatic uptake transporter function in healthy subjects by using gadoxetic acid- enhanced MR imaging.
      ]. A single clinical study shows the absence of drug-drug interaction between erythromycin and EOB-DTPA at imaging, although both drugs enter by OATP1B1/B3 in human hepatocytes [
      • Huppertz A.
      • Breuer J.
      • Fels L.M.
      • Schultze-Mosgau M.
      • Sutter G.
      • Klein S.
      • et al.
      Evaluation of possible drug-drug interaction between gadoxetic acid and erythromycin as an inhibitor of organic anion transporting peptides (OATP).
      ]. In contrast, we showed in normal rat liver, that rifampicin decreases BOPTA entry into hepatocytes [
      • Daali Y.
      • Millet P.
      • Dayer P.
      • Pastor C.M.
      Evidence of drug-drug interactions through uptake and efflux transport systems in rat hepatocytes: implications for cellular concentrations of competing drugs.
      ]. Finally, EOB-MRI can visualise the expression of OATP1B1/B3 in normal hepatocytes as well as in tumour cells, a finding developed in the following sections.
      MRI is an important tool to detect HCC [
      • Choi J.Y.
      • Lee J.M.
      • Sirlin C.B.
      CT and MR imaging diagnosis and staging of hepatocellular carcinoma: part II. Extracellular agents, hepatobiliary agents, and ancillary imaging features.
      ,
      • Choi J.Y.
      • Lee J.M.
      • Sirlin C.B.
      CT and MR imaging diagnosis and staging of hepatocellular carcinoma: Part I. Development, growth, and spread: key pathologic and imaging aspects.
      ,
      • Kudo M.
      Diagnostic imaging of hepatocellular carcinoma: recent progress.
      ]. The technique offers a high contrast resolution to distinguish differences of signal intensities between focal lesions and surrounding parenchyma. In images acquired before injection of contrast agent, intra-tumour fat is suspicious for HCCs [
      • Min J.H.
      • Kim Y.K.
      • Lim S.
      • Jeong W.K.
      • Choi D.
      • Lee W.J.
      Prediction of microvascular invasion of hepatocellular carcinomas with gadoxetic acid-enhanced MR imaging: Impact of intra-tumoral fat detected on chemical-shift images.
      ]. Following injection, hepatobiliary contrast agents accumulate differently over time within surrounding tissue and lesions. Hyperintensity in HCCs during hepatic arterial phase (images acquired 25–30 s after contrast agent injection) characterises the development of isolated arteries associated with arterial angiogenesis (Fig. 1). Low signal intensities during the portal venous phase (images acquired 60–70 s after contrast agent injection) are related to the paucity of portal venules. The signal intensity during the delayed phase (3 to 5 min after injection) depends on the contrast agent used. BOPTA has a low hepatic extraction ratio (5%) and hypointensity during the delayed phase is mainly related to the paucity of portal venules. In contrast, EOB-DTPA has a high hepatic extraction ratio (50%) and besides the paucity of portal venules, a decreased contrast uptake in cells (associated with a decreased expression of transporters in HCCs) contributes to tumour hypointensity. The hepatobiliary phase is imaged 20 min after injection of EOB-DTPA and more than 1 h after injection of BOPTA. Evolution of transporter expression and signal intensities along with hepatocarcinogenesis is the subject of the following sections.

      Hypointense or hyperintense lesions and transporter expression

      Narita et al. [
      • Narita M.
      • Hatano E.
      • Arizono S.
      • Miyagawa-Hayashino A.
      • Isoda H.
      • Kitamura K.
      • et al.
      Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma.
      ] published the first clinical study that correlates hepatocyte transporters and HB-MRI. This study differentiates hypointense from hyperintense HCCs. Most hypointense HCCs have a low expression of OATP1B3 that impedes EOB-DTPA entry into tumour cells. Overall, EOB-DTPA lesion accumulation correlates with OATP1B3 expression [
      • Narita M.
      • Hatano E.
      • Arizono S.
      • Miyagawa-Hayashino A.
      • Isoda H.
      • Kitamura K.
      • et al.
      Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma.
      ]. Moreover, in EOB-DTPA-positive HCCs, OATP1B3 expression is similar to that measured in surrounding tissues which contain either normal or cirrhotic hepatocytes. EOB-DTPA accumulation is not correlated to HCC differentiation and six moderately differentiated HCCs are EOB-DTPA positive while 11 similarly differentiated HCCs do not accumulate the agent. Kitao et al. [
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ] also find weak OATP1B3 expression in hypointense HCCs. This study also investigates efflux transporters and finds similar MRP2 mRNA expression in HCCs and surrounding livers. In contrast, MRP3 mRNA expression follows that of OATP1B3. Thus, hyperintense HCCs keep OATP1B3 and MRP3 expression while hypointense HCCs lose them. Another finding of hyperintense HCCs is a pseudoglandular proliferation pattern with bile plugs [
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ]. Similarly to the previous study, hypointense and hyperintense HCCs had similar tumour differentiation.
      These studies identify two types of HCCs, with hypointense lesions being more frequent than hyperintense lesions. Hypointense HCCs have less OATP1B3 [
      • Narita M.
      • Hatano E.
      • Arizono S.
      • Miyagawa-Hayashino A.
      • Isoda H.
      • Kitamura K.
      • et al.
      Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma.
      ,
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ] and MRP3 [
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ] expression on the sinusoidal membrane of tumour cells, while MRP2 is not modified in the canalicular membrane [
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ]. Differentiation is similar between both types of HCCs [
      • Narita M.
      • Hatano E.
      • Arizono S.
      • Miyagawa-Hayashino A.
      • Isoda H.
      • Kitamura K.
      • et al.
      Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma.
      ,
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ]. Hyperintense HCCs have a pseudoglandular proliferation pattern with bile plugs [
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ], a finding not found in the other study [
      • Narita M.
      • Hatano E.
      • Arizono S.
      • Miyagawa-Hayashino A.
      • Isoda H.
      • Kitamura K.
      • et al.
      Expression of OATP1B3 determines uptake of Gd-EOB-DTPA in hepatocellular carcinoma.
      ].

      Why are HCCs hyperintense?

      EOB-DTPA concentrations in tumour cells result from two concomitant transporter functions: uptake rates from interstitium to hepatocytes and efflux rates from hepatocytes to bile canaliculi or interstitium (Fig. 2). Tsuboyama et al. [
      • Tsuboyama T.
      • Onishi H.
      • Kim T.
      • Akita H.
      • Hori M.
      • Tatsumi M.
      • et al.
      Hepatocellular carcinoma: hepatocyte-selective enhancement at gadoxetic acid–enhanced MR Imaging—Correlation with expression of sinusoidal and canalicular transporters and bile accumulation.
      ] carefully describe three compartments where EOB-DTPA can accumulate: cell cytoplasm, pseudogland lumens, and closed canaliculi, generating high signal intensities in comparison to surrounding livers [
      • Tsuboyama T.
      • Onishi H.
      • Kim T.
      • Akita H.
      • Hori M.
      • Tatsumi M.
      • et al.
      Hepatocellular carcinoma: hepatocyte-selective enhancement at gadoxetic acid–enhanced MR Imaging—Correlation with expression of sinusoidal and canalicular transporters and bile accumulation.
      ]. Over 27 HCCs vascularised predominantly by isolated arteries (hyperintense at arterial phase), the study identifies four types of HCCs at HB-MRI (Fig. 4): Type 1) OATP-positive HCCs with predominant MRP2 location on apical membrane of pseudoglands (n = 3); Type 2) OATP-positive HCCs with MRP2 location on canalicular membrane of tumour cells (n = 8); Type 3) OATP-negative HCCs (n = 13); and Type 4) OATP-positive HCCs with no expression of MRP2 (n = 3). Types 1 and 4 are hyperintense while types 2 and 3 are hypointense, showing that OATP-positive HCCs can be hypointense. OATP-positive HCCs represent 52% of all hypervascularised lesions. In contrast to normal or surrounding injured livers where OATP1B1/3 expression increased along sinusoids (from periportal to perivenous areas), the distribution of positive cells in HCCs were either broad and diffuse (n = 5) or focal and sparse (n = 9). A canalicular expression of MRP2 without zonal variation along sinusoids is observed in parenchyma surrounding HCCs and all HCCs are positive for MRP2 (74% with increased expression and 26% with unchanged or decreased expression). In lesions with increased expression, MRP2 is localised either in luminal membrane of pseudoglands (n = 6) or predominates in canalicular membrane (n = 14). As shown in previous studies, signal intensities are higher in OATP-positive than OATP-negative HCCs. Over 21 hypointense HCCs, 8 have a positive OATP expression, showing that OATP expression alone is insufficient to analyse signal intensities at HB-MRI (Fig. 4). MRP2 membrane location contributes to signal intensities in lesions. Following uptake, EOB-DTPA leaves tumour cells when MRP2 is present in canalicular membrane and the higher the MRP2 expression, the lower the signal intensities inside cells. In contrast, in the absence of canalicular MRP2, EOB-DTPA is trapped inside tumour cells with hyperintense images at HB-MRI. Similarly, we showed liver trapping of the hepatobiliary MR contrast agent BOPTA in hepatocytes lacking Mrp2 [
      • Millet P.
      • Moulin M.
      • Stieger B.
      • Daali Y.
      • Pastor C.M.
      How organic anions accumulate in hepatocytes lacking Mrp2: evidence in rat liver.
      ]. MRP2 retrieval from canalicular membrane might be another regulatory process [
      • Schonhoff C.M.
      • Webster C.R.
      • Anwer M.S.
      Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase C in HUH-NTCP Cells.
      ]. We showed MRP2 retrieval from canalicular membrane following vasopressin administration, with concomitant BOPTA accumulation inside hepatocytes [
      • Planchamp C.
      • Hadengue A.
      • Stieger B.
      • Bourquin J.
      • Vonlaufen A.
      • Frossard J.-L.
      • et al.
      Function of both sinusoidal and canalicular transporters controls the concentration of organic anions within hepatocytes.
      ]. Another source of signal intensities is EOB-DTPA trapping in pseudoglands that do not possess exit structure. Moreover, MRP3 expression interferes with EOB-DTPA accumulation in HCCs. Finally, Kimura et al. [
      • Kimura Y.
      • Sato S.
      • Hitomi E.
      • Ohyama M.
      • Adachi K.
      • Inagaki Y.
      • et al.
      Coexpression of organic anion-transporting polypeptides 1B3 and multidrug-resistant proteins 2 increases the enhancement effect of gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid on hepatocellular carcinoma in magnetic resonance imaging.
      ] also find that the co-expression of OATP1B3 and MRP2 influences the high contrast enhancement of HCCs at HB-MRI.
      Figure thumbnail gr4
      Fig. 4Signal intensities in hepatocellular carcinomas (HCCs) according to expression and location of transporters (adapted from Tsuboyama et al.
      [
      • Tsuboyama T.
      • Onishi H.
      • Kim T.
      • Akita H.
      • Hori M.
      • Tatsumi M.
      • et al.
      Hepatocellular carcinoma: hepatocyte-selective enhancement at gadoxetic acid–enhanced MR Imaging—Correlation with expression of sinusoidal and canalicular transporters and bile accumulation.
      ]
      ). Over 27 HCCs vascularised predominantly by isolated arteries (hyperintense at arterial phase of MRI), four types of HCCs at hepatobiliary phase of EOB-MRI are described: Type 1) OATP-positive HCCs with predominant MRP2 location on apical membrane of pseudoglands (n = 3); Type 2) OATP-positive HCCs with MRP2 location on canalicular membrane of tumour cells (n = 8); Type 3) OATP-negative HCCs (n = 13); and Type 4) OATP-positive HCCs with no expression of MRP2 (n = 3). Types 1 and 4 are hypertense at hepatobiliary phase of MRI (HB-MRI) while types 2 and 3 are hypointense. Over 21 hypointense HCCs, 8 have a positive OATP expression, showing that OATP expression alone is insufficient to analyse signal intensities at HB-MRI. MRP2 membrane location (canalicular membrane or pseudogland) contributes to signal intensities. EOB-DTPA leaves tumour cells when MRP2 is present in canalicular membrane and the higher the MRP2 expression, the lower the signal intensities into cells. In contrast, in the absence of canalicular MRP2, EOB-DTPA is trapped inside tumour cells with hyperintense images at HB-MRI. EOB-DTPA can also be trapped in pseudogland lumen.
      In summary, EOB-DTPA is localised in three compartments: tumour cells, pseudogland lumens, and closed bile canaliculi. To enter these structures, EOB-DTPA must cross OATP1B1/B3 localised in tumour cells and pseudoglands (Fig. 2). EOB-DTPA can be trapped into the cytoplasm of cancer cells (by MRP2 retrieval from canalicular membrane or by decreased expression of MRP2), in closed canaliculi (absence of flowing into bile ductules), and inside pseudogland lumens (absence of communication with bile ductules). Whether, EOB-DTPA might be trapped inside cells forming pseudoglands is not described but bilirubin that enters into hepatocytes across OATP1B1/B3 is identified in cells of pseudoglands [
      • Kitao A.
      • Zen Y.
      • Matsui O.
      • Gabata T.
      • Kobayashi S.
      • Koda W.
      • et al.
      Hepatocellular carcinoma: signal intensity at gadoxetic acid-enhanced MR Imaging–correlation with molecular transporters and histopathologic features.
      ].

      HCC imaging and β-catenin regulation

      Ueno et al. [
      • Ueno A.
      • Masugi Y.
      • Yamazaki K.
      • Komuta M.
      • Effendi K.
      • Tanami Y.
      • et al.
      OATP1B3 expression is strongly associated with Wnt/β-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma.
      ] found in hyperintense HCCs at EOB-MRI, a positive correlation between OATP1B3 expression and β-catenin activation, suggesting that β-catenin-activated HCCs might be detected by EOB-MRI. Very recently, Kitao et al. [
      • Kitao A.
      • Matsui O.
      • Yoneda N.
      • Kozaka K.
      • Kobayashi S.
      • Sanada J.
      • et al.
      Hepatocellular carcinoma with β-catenin mutation: imaging and pathologic characteristics.
      ] studied in 138 HCCs, the expression of β-catenin and its transcriptional product glutamine synthetase (GS). HCCs were divided into three groups, according to the presence or absence of β-catenin and GS: 1) HCCs with β-catenin mutations (β-catenin+/GS+); 2) intermediate HCCs (β-catenin−/GS+); and 3) HCCs without β-catenin mutation (β-catenin−/GS−). OATP1B3 expression was measured in the three groups. HCCs with β-catenin mutation (n = 27) had higher signal intensities at EOB-MRI than intermediate HCCs (n = 23) and HCCs without β-catenin mutation (n = 84). Positive correlations were found between the expressions of β-catenin, GS, and OATP1B3.
      In summary, most HCCs are hypointense at HB-MRI in comparison to surrounding parenchyma because OATP expression on the membrane of tumour cells is low and EOB-DTPA uptake into hepatocytes is decreased. However, OATP-positive HCCs may be hypointense if the expression of MRP2 is increased and located in the canalicular membrane. EOB-DTPA enters into tumour cells and its rapid exit impedes cellular accumulation of the contrast agent.
      Hyperintense HCCs have OATPs on tumour cells located in tissue or pseudogland. High signal intensities are generated by low canalicular MRP2 expression (EOB-DTPA cytosolic trapping), closed canaliculi (EOB-DTPA bile canaliculi trapping), or MRP2 expression in apical membrane of tumour cells inside pseudoglands (EOB-DTPA trapping in pseudogland lumen).

      Can HB-MRI predict HCC prognosis?

      Few studies have investigated the prognosis of HCCs by HB-MRI. Kitao et al. [
      • Kitao A.
      • Matsui O.
      • Yoneda N.
      • Kozaka K.
      • Kobayashi S.
      • Sanada J.
      • et al.
      Hepatocellular carcinoma with β-catenin mutation: imaging and pathologic characteristics.
      ] recently showed that HCCs with β-catenin mutation are associated with pseudoglandular proliferation, bile production, and a higher grade of differentiation. A new score of HCC prognosis that includes images at HB-MRI, α-fetoprotein serum concentrations, and tissue expression of OATP1B3 and HNF4α [
      • Yamashita T.
      • Kitao A.
      • Matsui O.
      • Hayashi T.
      • Nio K.
      • Kondo M.
      • et al.
      Gd-EOB-DTPA- enhanced magnetic resonance imaging and alpha-fetoprotein predict prognosis of early-stage hepatocellular carcinoma.
      ]. In this study, hyperintense HCCs at HB-MRI are observed in 15% of HCCs. Lesions that associate hyperintensity, low serum concentrations of α-fetoprotein, maintenance of hepatocyte function and increased tissue expression of OATP1B3 and HNF4α have a good prognosis. Other studies should now be performed to better define the early prognosis of HCCs by HB-MRI and other biomarkers. HB-MRI is an important non-invasive way to detect, characterise, and probably assess HCC prognosis in relation to the regulation of membrane transporters of hepatobiliary MR contrast agents. The review also highlights the various factors that contribute to generate high signal intensities in HCCs.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Authors’ contributions

      All author contributed to analysis of publications, drafting of the manuscript, and critical revision of the content

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