Insights into the diagnosis of hepatocellular carcinomas with hepatobiliary MRI  Valérie Vilgrain, Bernard E. Van Beers, Catherine M. Pastor  Journal of Hepatology  Volume 64, Issue 3, Pages 708-716 (March 2016) DOI: 10.1016/j.jhep.2015.11.016 Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Carcinogenesis 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 [47]. LGDN, low grade dysplastic nodule; HGDN, high grade dysplastic nodule; OATP, organic anion transporting polypeptide; MRP, multiple resistance-associated protein. Journal of Hepatology 2016 64, 708-716DOI: (10.1016/j.jhep.2015.11.016) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 2 In 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). Journal of Hepatology 2016 64, 708-716DOI: (10.1016/j.jhep.2015.11.016) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Regulation 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 [29]. Journal of Hepatology 2016 64, 708-716DOI: (10.1016/j.jhep.2015.11.016) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Signal intensities in hepatocellular carcinomas (HCCs) according to expression and location of transporters (adapted from Tsuboyama et al. [27]). 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. Journal of Hepatology 2016 64, 708-716DOI: (10.1016/j.jhep.2015.11.016) Copyright © 2015 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2016 64, 708-716DOI: (10. 1016/j. jhep. 2015. 11 Copyright © 2015 European Association for the Study of the Liver Terms and Conditions