Volume 139, Issue 1, Pages 335-343.e2 (July 2010) Intermediate Hepatobiliary Cells Predict an Increased Risk of Hepatocarcinogenesis in Patients With Hepatitis C Virus-Related Cirrhosis  Marianne Ziol, Jean–Charles Nault, Mounir Aout, Nathalie Barget, Maryline Tepper, Antoine Martin, Jean–Claude Trinchet, Nathalie Ganne–Carrié, Eric Vicaut, Michel Beaugrand, Gisele N'Kontchou  Gastroenterology  Volume 139, Issue 1, Pages 335-343.e2 (July 2010) DOI: 10.1053/j.gastro.2010.04.012 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Description of IHC, isolated cholangiocytes, and ductular reaction on K7 immunostained LBs of patients with HCV-related cirrhosis. (A) Groups of more than 5 K7-positive cells with a polygonal cytoplasmic shape that look like hepatocytes, measuring 6–40 μm, were identified as IHC, regardless of their localization (eg, close to or at a distance from fibrous septa and/or ductular reaction). (B) Isolated round small cells with high nuclear to cytoplasmic ratios were considered as isolated cholangiocytes. (C) Positive cord-like structures corresponding to ductular reaction, canal of Hering, or bile ductules were not taken into account. Gastroenterology 2010 139, 335-343.e2DOI: (10.1053/j.gastro.2010.04.012) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 (A–F) Representative samples of the distribution of IHC in HCV-related cirrhosis. (A and B) K7 decorated bile duct (black arrows) and (A–C) ductular reaction (white arrows) in all HCV-related cirrhosis and served as a positive internal control for this immunostaining. (A and D) In most cases, no IHC or isolated cholangiocyte was observed in cirrhotic nodules. When present, IHC foci were rare and barely detectable at (B) low-power field (black arrowheads) or (C) formed confluent zones (defined as an extensive staining) with a percentage of positive hepatocyte reaching 30% (black arrowheads). At a higher magnification (D–F are located in A–C, respectively), (E and F) IHC displayed a cytoplasmic positivity with a strong submembranous reinforcement (black arrows). (E and F) Isolated cholangiocytes frequently were observed in the vicinity of positive intermediate cells (white arrows) and showed a strong cytoplasmic staining without submembranous reinforcement. Gastroenterology 2010 139, 335-343.e2DOI: (10.1053/j.gastro.2010.04.012) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Complementary analysis of the staining pattern of IHC. At an intermediate (200×) and high magnification (1000×; inset), the immunostaining performed on consecutive sections showed that the foci of K7 IHC corresponded to foci positive for K19, another biliary marker, and Ep-CAM, a marker of progenitor cells. As indicated by black arrows, the foci of positive hepatocytes were detectable on these 3 different immunostainings. However, some differences were observed because membranous Ep–CAM staining generally encompassed the K7 stained areas and also was shown, albeit more slightly, in other areas negative for K7 (star). K19 immunostained the foci of K7 positive hepatocytes, but to a lesser extent, with a few cells at the periphery of foci being K7-positive and K19-negative. These K7/K19 foci retained a high expression of K8 and 18. NCAM was not expressed. No peculiar feature was observed on routine staining. Gastroenterology 2010 139, 335-343.e2DOI: (10.1053/j.gastro.2010.04.012) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Outcome of patients with HCV-related cirrhosis according to the presence of IHC on nontumoral liver biopsy. The cumulative curves of the incidence of HCC are adjusted for competing risk and compared according to the Gray test.35 The numbers of patients at risk are indicated under the x-axis. The HCC incidence rates were significantly higher in patients with (A) K7 or (B) K19 IHC compared with patients without (8.14% vs 3.12% per year, P = .003; and 10.1% vs 3.6%, P = .003, respectively). (C) When the extent of K7 hepatobiliary cells was taken into account, the incidence of HCC was higher in patients with extensive areas of IHC compared with other patients (10.6% vs 4.6%; P = .01). Gastroenterology 2010 139, 335-343.e2DOI: (10.1053/j.gastro.2010.04.012) Copyright © 2010 AGA Institute Terms and Conditions