Intermediate Hepatobiliary Cells Predict an Increased Risk of Hepatocarcinogenesis in Patients With Hepatitis C Virus-Related Cirrhosis

GASTROENTEROLOGY 2010;139:335 343 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, and GISELE N KONTCHOU *Pathology Department, and Hepatology Unit, CHU Jean Verdier, Assistance Publique, Hôpitaux de Paris (AP-HP), Bondy and Paris 13 University, Bobigny, France; Clinical Research Unit Lariboisière/St Louis, Hôpital Fernand Widal, AP-HP, Paris, France; and Pathology Department, CHU Avicenne, AP-HP, Université Paris 13, Bobigny, France BACKGROUND & AIMS: The expression of biliary lineage markers such as cytokeratin (K) 7 by hepatocytes is thought to reflect an altered regeneration pathway recruiting a stem cell compartment, more prone to carcinogenesis. We aimed to investigate the presence of these so-called intermediate hepatobiliary cells (IHC) in liver biopsies of patients with hepatitis C related cirrhosis and their potential influence on the subsequent occurrence of hepatocellular carcinoma (HCC). METHODS: From a cohort of patients with hepatitis C related cirrhosis, prospectively screened for HCC, we retrospectively selected those with a liver biopsy performed for the initial diagnosis of cirrhosis. Presence of IHC was recorded when foci of K7-positive, intermediate-sized hepatocytes were detected. RESULTS: A total of 150 patients were included (87 men; mean age, 57 y; range, 19 84 y; body mass index, 25 kg/m 2 ). After a median follow-up period of 4.85 years, HCC was diagnosed in 36 patients (24%). Baseline liver biopsy showed intermediate hepatobiliary cell foci in 61 patients (41%). Intermediate cells co-expressed both hepatocytes markers and the progenitor cell markers Ep-CAM and K19. The presence of intermediate hepatobiliary cells was associated independently with HCC occurrence (Fine and Gray model; hazard ratio, 2.48; 95% confidence interval, 1.24 4.96; P.01). Other predictors of HCC were diabetes and low platelet count. The HCC annual incidence rate was significantly higher in patients with IHC compared with patients without (8.14% vs 3.12%, Gray s test, P.003). CONCLUSIONS: The aberrant expression of biliary K by hepatocytes in patients with hepatitis C virus related cirrhosis is related independently to HCC occurrence. Keywords: Hepatocellular Carcinoma; HCV-Related Cirrhosis; Intermediate Hepatobiliary Cells; Cancer Stem Cells. Chronic hepatitis C remains a major cause of cirrhosis and hepatocellular carcinoma (HCC) in Western countries, despite the decreasing incidence of carcinoma in patients with sustained response to antiviral therapy. 1 The detection of very early and early HCC (lesion diameter, 3 cm) is of high priority because of the development of curative treatments. 2 The identification of criteria associated with a high risk of HCC occurrence could select a group that could benefit from an enhanced surveillance with improvement in mortality and cost efficiency. The currently established risk factors for HCC in compensated hepatitis C virus (HCV)-related cirrhosis include age, male sex, 3 being overweight, diabetes mellitus, 4 high -fetoprotein (AFP) serum levels, and low platelet counts. 5 Regarding histologic factors, activity, and steatosis, 6 small-cell dysplasia (SCD) and large-cell dysplasia (LCD), 7 along with hepatocyte proliferative index, 8 have shown an independent influence on HCC occurrence. In the normal adult liver, hepatocytes display a cytokeratin (K)8- and K18-positive and a K7- and K19-negative profile whereas biliary cells have a K8/18-, K7-, and K19-positive profile. In diseased liver, a ductular reaction often takes place and some intermediate hepatobiliary cells (IHC) have been described. 9 Both types of cells observed in reactive lesions, resulting from an acute or chronic liver injury, are characterized by the simultaneous expression of biliary (K7, 19, Ovalbumin-6) and hepatocyte antigens (Anti-Human Hepatocyte, K8/18, albumin). This expression of both hepatocyte and biliary lineage markers is thought to reflect an altered hepatocyte regeneration pathway recruiting a stem cell compartment, more prone to carcinogenesis as suggested by experimental models 10,11 and in vitro studies. 12,13 Although a ductular reaction is observed in almost all chronic and acute liver disease, IHC, defined as larger than 6 m and smaller than 40 m, with a dual hepatocyte and biliary Abbreviations used in this paper: AFP, -fetoprotein; Ep-CAM, epithelial cell adhesion molecular; HBV, hepatitis B virus; HCC, hepatocellular carcinama; HCV, hepatitis C virus; IHC, intermediate hepatobiliary cells; K, cytokeratin; LB, liver biopsy; LCD, large-cell dysplasia; NCAM, neural cell adhension molecule; SCD, small-cell dysplasia. 2010 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2010.04.012

336 ZIOL ET AL GASTROENTEROLOGY Vol. 139, No. 1 expression profile, 14 have not been described thoroughly. Thus, hypothesizing the role of hepatocyte phenotypic alteration in the early steps of carcinogenesis and the potential role of progenitor cell renewal in cirrhosis, we aimed to investigate whether the presence of IHC in liver biopsies (LBs) of patients with hepatitis C related cirrhosis had an influence on the subsequent occurrence of HCC. Materials and Methods Patient Selection From a cohort of patients referred to our liver unit between January 1, 1996, and June 31, 2003 with HCV-related cirrhosis and prospectively screened for HCC detection, we selected consecutive patients for this study according to the following inclusion criteria: (1) transparietal or transjugular LB proving cirrhosis available with remaining liver tissue sample sufficient to perform subsequent immunohistochemical studies; and, at the time of liver biopsy: (2) no presence or suspicion of HCC, as judged by AFP level and ultrasonographic findings; (3) criteria for compensated cirrhosis (Child Pugh A); (4) presence of anti-hcv antibodies detectable with positive serum HCV RNA by reverse-transcription polymerase chain reaction; (5) absence of hepatitis B virus (HBV) infection, hemochromatosis, diagnosis of biliary cirrhosis, Wilson disease, -1-antitrypsin deficiency, or human immunodeficiency virus infection; (6) absence of severe life-threatening diseases; and (7) acceptance of regular follow-up evaluations and periodic HCC screening. In addition, patients who cleared HCV infection from January 1996 up to the last recorded visit in January 2008 were excluded from the study because the occurrence of HCC in these patients was proven to be decreased. 1,15 For each patient, the date of inclusion was the date of the first LB showing cirrhosis. On the day of the biopsy, the following parameters were recorded: age, sex, body mass index, diabetes status, liver tests including prothrombin activity (%), albumin level (g/l), total bilirubin level (um/l), aspartate aminotransferase level (UI/L), alanine aminotransferase level (UI/L), alkaline phosphatase level (UI/L), -glutamyl transferase level (UI/L), platelet count (/mm 3 ), and AFP level (ng/ml). The cause of cirrhosis was considered as mixed (HCV and alcohol) when patients had past or ongoing daily alcohol intake greater than 30 g/day at the time of liver biopsy. Diabetes status was collected as a binary parameter (yes/no) and was defined by a fasting serum glucose level greater than 126 mg/dl or by the use of antidiabetic drugs. Follow-Up Evaluation and HCC Assessment Patients were followed up prospectively and HCC was screened by abdominal ultrasonography and AFP level performed at least every 6 months. The diagnosis of HCC was assessed through histology (with a guided liver biopsy) or noninvasive criteria according to the recommendation of the Barcelona Conference. 16 Patients were considered lost to follow-up evaluation when absent for more than one visit for HCC screening. The main end point of the study was the occurrence of HCC. The time frame was defined as the time from the date of biopsy until the HCC occurrence. Follow-up evaluation ended at the date of HCC diagnosis, at the date of liver transplantation, at the date of death without HCC, or at the last recorded visit until January 31, 2008. Histology and Immunohistochemistry LBs from patients with hepatitis C related cirrhosis were examined for the presence of LCD and SCD according to the previously described criteria, 17 for grading activity 18 and for semiquantitative assessment of macrovesicular steatosis. In addition, we searched for the association of ballooned hepatocytes with Mallory bodies and polymorphonuclear cells to define alcoholic hepatitis. Dewaxed and rehydrated paraffin sections, 3- m thick, were immunostained with antibodies to K7, to a mixture of K8 and K18, to K19, to neural cell adhesion molecule (NCAM), to epithelial cell adhesion molecule (Ep-CAM), CD117, CD34, N-cadherin, and chromogranin as detailed in the Supplementary Materials and Methods section and Supplementary Table 1. K7 immunostained sections were assessed independently by 2 observers (M.Z., N.B.) without knowledge of the patient s outcome. The observation of at least 2 foci of more than 5 polyg- 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 m, 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.

July 2010 KERATIN 7 AND HEPATOCARCINOGENESIS 337 onal-shaped hepatocytes, measuring from 6 to 40 m large, with cytoplasmic positivity (positivity of either the whole cytoplasmic area or only the submembranous areas) defined the presence of IHC (Figure 1A). The same criteria were applied for K19 foci. The presence of small isolated positive cells distinct of cord-like structures also was reported as isolated cholangiocytes (Figure 1B). Cord-like structures were not considered (Figure 1C). In case of disagreement regarding IHC assessment, sections were re-examined using a multipipe microscope and a consensus was reached that was used for the analysis of HCC occurrence. We further investigated the expression profile of IHC on consecutive sections; K19 immunostaining was performed to investigate another biliary marker, K8 and K18, to check the conservation of hepatocyte markers, and Ep-CAM, NCAM, CD117, CD34, and chromogranin A to investigate other progenitor cell markers. Furthermore, when K7 immunostaining was positive, it was categorized as extensive staining when one or several cirrhotic nodules showed positive staining in more than 30% of the hepatocyte or categorized as focal staining in other cases. To investigate the within-biopsy reproducibility of the detection of IHC, we retrieved from our pathology files patients who had subsequent LB, either at the time of HCC diagnosis or during follow-up evaluation, and assessed the presence of IHC as described earlier without knowledge of the results of the first biopsy. Control Patients For control of cytokeratin 7 immunostaining, LBs of 28 patients with chronic hepatitis C with mild fibrosis (F1 or F2 according to the METAVIR scoring system), matched for age and sex, were selected consecutively from our pathology files. Statistical Analysis Continuous data were expressed as means standard deviation, and categoric data were reported as frequency and percentage. Continuous and qualitative data related to the presence of IHC were analyzed using the Mann Whitney test and the Fisher exact test, respectively. Because mortality related to the progression of liver disease without HCC is non-negligible in these cohorts of cirrhotic patients, we used the Fine and Gray 19 model, which extends the Cox model to competing risks data including those related to death, to determine independent factors associated with HCC occurrence. Variables Table 1. Clinical, Biological, and Histologic Characteristics of Patients With HCV-Related Cirrhosis at the Time of Inclusion According to the Presence of IHC Patient characteristics Patients selected for the study (n 150) Patients with IHC (n 61; 41%) Patients without IHC (n 89; 59%) P a Clinical characteristics Age at inclusion, y b 57 13 55 13 58 13.12 Sex, male c 87 (58) 38 (62) 49 (55).38 Alcohol, 30 g/day c 64 (42.7) 28 (46) 36 (40).51 Body mass index, kg/m 2b 25 4 25 4 25 4.31 Diabetes mellitus c 53 (36.3) 19 (31.7) 34 (39.5).33 Biological characteristics c Platelet count, 10 3 /mm 3 130 56 114 48 142 59.003 Prothrombin time, % 77 16 73 18 80 15.009 AFP level, ng/ml 13 19 14 18 13 21.13 Bilirubin level, mol/l 18 14 21 16 17 12.19 Albumin level, g/l 40 6 40 5.7 41.4 6.2.16 AST level, ULN 2.37 1.7 2.24 1.22 2.48 1.99.75 ALT level, ULN 2.99 2.81 2.8 1.88 3.13 3.32.97 -Glutamyl transferase level, 3.14 3.20 2.81 2.41 3.36 3.64.67 ULN ALP level, ULN 1.23 0.50 1.21 0.5 1.31 0.48.25 HCV genotype 1 104 38 (62.3) 66 (74.2).12 Histologic characteristics LB size, mm 12.48 5.53 12.78 5.78 12.27 5.4.6 LCD 12 (8) 5 (8.3) 7 (7.9) 1.00 Activity A2 or A3 93 (62) 41 (67.2) 52 (58.4).28 Macrovesicular steatosis, 30% 25 (17) 9 (14.8) 16 (18).6 Isolated cholangiocytes 125 (83) 60 (98.4) 65 (73) <10-4 Alcoholic hepatitis 17 (11) 8 (13) 9 (10).54 NOTE. Bold entries indicate significant differences between groups. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal. a The Fisher exact test was used for binary variables and the Mann Whitney test was used for continuous variables. b Mean value standard deviation. c Number (%).

338 ZIOL ET AL GASTROENTEROLOGY Vol. 139, No. 1 with a P value less than.10 in the univariate analysis were entered into a stepwise multivariate model. In addition, cumulative incidence function was computed and compared in terms of the HCC occurrence between individuals with and without IHC using the Gray 20 test. Interobserver and interbiopsy agreements were determined using the statistic. All analyses were 2-sided and differences with P values less than.05 were considered statistically significant. All statistical analyses were performed using SAS 9.2 and R 2.8 software (SAS Inc, Cary, NC). Results Baseline Patient Characteristics and HCC Occurrence A cohort of 290 patients with Child class A, HCVrelated cirrhosis was included in an HCC screening program from January 1996 to June 2003. The liver biopsy sample was available at our institution with sufficient remaining tissue for immunohistochemical analysis in 201 patients. Among them, 35 patients were excluded because of HCV infection clearing during the follow-up evaluation and 16 other patients were not studied because immunostaining was not obtained or not interpretable. Thus, 150 patients were included. Clinical, biochemical, and histologic characteristics are detailed in Table 1. The median duration of follow-up evaluation for HCC occurrence was 4.85 years. Thirty-six patients (24%) developed HCC and the incidence rate of HCC occurrence was 5% per year. Thirty-two patients were lost to follow-up evaluation before January 2008. The diagnosis of HCC was based on noninvasive criteria in 22 patients and on histology in 14 cases. During follow-up evaluation, 10 patients died of liver disease without HCC, 4 patients died of liver unrelated disease, and 3 patients were transplanted without HCC. Histology and Immunostaining Liver biopsy specimens consisted of 1 10 fragments and the sum of the length of fragments varied from 6 to 30 mm (mean standard deviation, 12 5 mm). The distribution of hepatitis activity grade and the percentage of steatosis are given in Table 1. Seventeen (11%) patients had alcoholic hepatitis. LCD was identified in 12 patients (8%), and no focus of SCD was observed. Two or more K7- or K19-positive intermediate hepatobiliary cell foci were observed in 61 cases (41%) and 43 cases (28%), respectively. The interobserver agreement for this parameter was 0.79 (confidence interval, 0.69 0.89). As shown in Figure 2, IHC could be observed either in periseptal areas and/or in the center of the cirrhotic nodules. The amount of IHC varied from 2 isolated foci of 3 5 cells distributed throughout the biopsy to the presence of cirrhotic nodules with extensive IHC areas. K7 staining was focal in 47 cases and extensive in 14 cases. The staining was cytoplasmic with a strong peripheral submembranous reinforcement or only submembranous with a clear perinuclear area giving a honeycomblike pattern. Isolated cholangiocytes were observed in 60 cases positive for intermediate cells and the 2 types of cells 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.

July 2010 KERATIN 7 AND HEPATOCARCINOGENESIS 339 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. colocalized as shown in Figure 2. In addition, 65 patients LBs showed isolated cholangiocytes without intermediate cells. LCD foci did not include any intermediate hepatocyte or isolated cholangiocyte. Examination of serial sections, as illustrated in Figure 3, disclosed that intermediate cells all were positive for K8/K18. Most of the cells positive for K7 also were positive for K19, but to a lesser extent with a few cells at the periphery of foci, being K7-positive and K19-negative. They also constantly were positive for Ep-CAM, known as a hepatocyte progenitor cell marker. However, membranous Ep-CAM staining generally encompassed the K7 stained areas and also was shown, albeit more slightly, in other areas negative for K7. IHC and isolated cholangiocytes were negative for the other progenitor cell markers such as CD34, CD117, NCAM, and chromogranin. With the exception of 2, all LBs from the 28 patients with chronic hepatitis C and low fibrosis (F1 or F2 according to METAVIR scoring system) were negative for either IHC or isolated cholangiocytes. One of the 2 positives cases showed obvious alcoholic hepatitis with Mallory bodies and polymorphonuclear infiltrate, a condition known to be associated with K7 positivity. 21 Thirty of the 150 patients have had a subsequent LB available for immunostaining (8 at the time of HCC diagnosis; 22 without evidence of HCC) with a mean delay between the 2 LBs of 2.4 years (6 mo to 7 y). In 25 cases (83%), the results of both biopsies were in agreement and the calculated coefficient showed a good agreement (, 0.67; confidence interval, 0.41 0.93) for the detection of IHC in sequential biopsies. Characteristics Associated With the Detection of IHC in Liver Biopsy The baseline characteristics are detailed in Table 1. Patients with IHC had a significantly lower platelet count and prothrombin time than patients without IHC. Among 36 patients who developed HCC, 22 had IHC (61%), whereas in the 114 patients without HCC, 39 had IHC (34%). In univariate analysis, clinical, biological, and histologic variables at inclusion that significantly affected the occurrence of HCC were as follows (Table 2): the presence of IHC (assessed by K7 or K19 immunostaining, either focal or extensive immunostaining), diabetes, platelet counts, and body mass index. Age, sex, histologic alcoholic hepatitis or alcohol consumption greater than 30 g/day, and LCD were not associated with HCC occurrence. Among these parameters, multivariate analysis using the Fine and Gray 19 model showed that the presence of IHC assessed by K7, diabetes, and a low platelet count remained independently related to the development of HCC. As shown in Figure 4, the HCC annual incidence rate was significantly higher in patients with K7 IHC compared with patients without IHC (8.14% vs 3.12%; Gray s test, P.003) as well as in patients with K19 IHC (10.1%

340 ZIOL ET AL GASTROENTEROLOGY Vol. 139, No. 1 Table 2. Clinical, Biological, and Histologic Characteristics Associated With the Risk of HCC Occurrence According to the Fine and Gray Model Univariate analysis Multivariate analysis HR (95%) P HR (95%) P Clinical data Male sex 1.174 (0.602 2.291).637 Alcohol, 30 g/day 0.897 (0.463 1.736).746 Age, y 1.018 (0.994 1.042).139 Diabetes mellitus 2.329 (1.217 4.457).011 3.195 (1.618 6.311).001 Body mass index, kg/m 2 1.078 (1.006 1.156).034 Biological data Platelet count, 10 3 /mm 3 0.908 (0.907 0.909).012 0.900 (0.899 0.900).014 Prothrombin time, % 0.999 (0.979 1.020).953 AFP level, ng/ml 1.010 (0.997 1.023).119 ALT level, ULN 1.068 (0.934 1.221).339 AST level, ULN 0.875 (0.714 1.073).201 Bilirubin level, mol/l 1.006 (0.986 1.027).540 ALP level, ULN 1.351 (0.786 2.323).276 GGT level, ULN 0.991 (0.864 1.135).892 Albumin level, g/l 0.983 (0.943 1.026)].440 HCV genotype 1 0.649 (0.337 1.253).198 Histologic data IHC (K7 extensive or focal) 2.697 (1.375 5.290).004 2.483 (1.242 4.963).010 Extensive staining 3.473 (1.211 9.963).02 Focal staining 2.531 (1.252 5.113).0097 IHC (K19) 2.642 (1.374 5.080).0036 Isolated cholangiocytes 0.842 (0.373 1.899).679 Activity A2/A3 1.443 (0.713 2.921).308 Macrovesicular steatosis, 30% 0.294 (0.070 1.244).096 LCD 1.067 (0.332 3.436).913 Alcoholic hepatitis 1.499 (0.665 3.379).329 ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, -glutamyl transferase; HR, hazard ratio; ULN, upper limit of normal. vs 3.6%; Gray s test, P.003). When only patients with extensive areas of IHC were considered (n 14), the HCC annual incidence rate was 10.6% vs 4.6% in patients without or with focal staining (Gray s test, P.091). Discussion In this cohort of patients with compensated HCVrelated cirrhosis prospectively screened for HCC, the detection of IHC in the baseline liver biopsy was a strong and independent risk factor for HCC occurrence (hazard ratio, 2.483; confidence interval, 1.242 4.963). We describe the distribution and the frequency of hepatocyte foci with an aberrant immunophenotype close to that observed in progenitor cells, in the biopsies of patients with HCV-related cirrhosis, and establish a link between its detection and the risk of developing HCC. This finding raises concerns regarding the early steps of carcinogenesis in cirrhosis and perspectives for focusing HCC screening in a high-risk population. The influence of K expression profile of nontumoral liver on subsequent HCC development could be supported indirectly by some previous studies. SCD is considered a preneoplastic lesion because of genetic alterations 22 and high proliferative rates. 23 However, the value of SCD in predicting HCC is impaired by its low frequency in LB 8,24,25 and conflicting results with no independent association with HCC in several studies, 7,24,25 although having independent influence in others. 26 Moreover, these foci are difficult to detect and the interobserver agreement for SCD has not been reported. We did not observe any SCD in our study, but foci of intermediate hepatocytobiliary cells showed small-sized hepatocytes with a slightly increased nuclear to cytoplasmic ratio. In addition, 55% of SCD foci have been shown to notably consist of intermediate cells expressing K7, K8, and K18. 27 Thus, IHC foci that we detected immunohistochemically with a very good interobserver agreement could represent early foci of SCD, more frequently sampled on LB and undetectable on routinely stained sections. A recent study 28 disclosed that gene expression profiles of HCC failed to yield a significant association with survival, whereas surrounding nontumoral profiles correlated with recurrence. K7 was one of the genes of this recurrence signature. Our study was not designed to investigate survival but, in line with these results, we found a relationship between altered K expression profile in nontumoral livers and subsequent HCC occurrence. In the normal human adult liver, mature hepatocytes proliferate whereas in chronic liver disease a reserve compartment of hepatic progenitor cells is activated. 29 These cells are identified morphologically as ductular reaction and

July 2010 KERATIN 7 AND HEPATOCARCINOGENESIS 341 Figure 4. Outcome of patients with HCVrelated 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) K7or(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). IHC, and they expressed markers of both hepatocytic and biliary lineage. 9 This altered type of renewal is observed in chronic hepatitis C 30 as a function of activity and fibrosis. Given the direct evidence for the involvement of progenitor cells in the genesis of HCC in experimental models 31 with the identification of human HCC expressing progenitor cell markers such as K7 and K19, 32 it is hypothesized that in human beings chronic hepatocellular injury favors the

342 ZIOL ET AL GASTROENTEROLOGY Vol. 139, No. 1 emergence of phenotypically altered cells resistant to cytotoxicity and growth arrest. 31 We provide here some basis to confirm this hypothesis by showing an independent relation between the presence of phenotypically altered cell foci and occurrence of HCC. Whether these intermediate cell foci represent true preneoplastic lesions with genetic alterations and clonality as it was shown for SCD 22,33 was not addressed in the present study. Further studies, which would be performed more easily on cirrhotic explanted livers with or without HCC, are needed to characterize these intermediate cells and identify potential markers that could be targeted specifically by therapy, especially including Ep-CAM. 34 We found that 41% of patients with HCV-related cirrhosis had IHC. Similar foci have been described in alcoholic and nonalcoholic fatty liver diseases, 29,35 in cirrhotic nodules adjacent to HCC in HBV patients, 36 and in cholestatic liver disease, 37 but its frequency in HCV-related cirrhosis and potential clinical significance has not been reported. The high frequency of IHC in our patients with HCV cirrhosis differed from its nearly complete absence in the large biopsy samples of patients with low fibrosis stages of chronic hepatitis C. This finding reinforces its potential value in helping to define high-risk patients. If some foci of ballooned hepatocytes were positive for K7, a significant relation was not found between the presence of alcoholic hepatitis, alcohol consumption, or steatosis and the finding of IHC. Whether these intermediate cells derive from progenitor cells or result from the transdifferentiation of mature hepatocyte into biliary cells as shown in animal models 38 and hepatoma cell lines 39 cannot be determined by this observational study. However, by using serial immunostaining, we showed that the intermediate cells expressed, as summarized recently, 40 some of the markers of progenitor cells such as Ep-CAM, K7, and K19, but did not express other potential stem cell markers such as NCAM, CD117, chromogranin, and CD34. Isolated cholangiocytes did not express NCAM, CD117, chromogranin, or CD34. Interestingly, IHC were observed in all cases in continuity with isolated cholangiocytes (Figure 3) that represent the progenitor cell compartment of normal liver. 9 This colocalization, along with the strong association between the detection of both types of cells, could favor the hypothesis that intermediate cells derive from isolated cholangiocytes. In contrast, intermediate cells were not observed in continuity with the ductular reaction that always was represented to a variable extent at the interface between fibrous septa and hepatocyte, and some large hepatocytes ( 40 m) also expressed K7. Thus, IHC do not appear to derive from the ductular reaction and also could represent a gradual phenotypic change from mature hepatocytes negative for biliary markers to hepatocytes expressing K7, then K19, and, finally, to progenitor cells. Identification of an immunohistochemical marker, associated with an increased risk of developing HCC, also could yield clinical applications because the relative risk was equal to 2 although no other clinical or biological parameter except diabetes mellitus and low platelet count was proved to have an independent influence on HCC development. In practice, this immunohistochemical characteristic can be determined retrospectively as part of a routine examination with low cost and a good interobserver agreement. Our findings need confirmation in other cohorts because this preliminary study was monocentric but the presence of intermediate cells on baseline liver biopsy, in combination with other clinical and biological criteria, efficiently could help to design a group of patients with a high risk of developing HCC. The univariate analysis did not identify the traditional risk factors such as age and male sex 41 for HCC occurrence in our 150 patients but it has been reported previously in studies including fewer than 200 patients. 42,43 We also found a higher incidence of HCC (5% per year) compared with that reported in other studies, 41 with frequencies ranging in Western patients from 1.5% to 4.6%. This is linked to the exclusion of cirrhotic patients who achieved HCV clearance during the follow-up evaluation for HCC, although most studies did not exclude these patients. 3,42,43 Patients who cleared HCV were excluded because we considered that it was an event occurring during the follow-up period, shown to significantly affect the risk of HCC. 15 In conclusion, we found a significant and independent association between the detection of IHC foci, identified with a good interobserver agreement using K7 immunostaining in nontumoral LB, and HCC occurrence in HCVrelated cirrhosis. These findings suggest that the acquisition of an aberrant cytokeratin expression profile by some hepatocytes could represent an early stage of carcinogenesis in cirrhosis that would be targeted and could help define patients with HCV-related cirrhosis at high risk for developing HCC. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2010.04.012. References 1. Camma C, Giunta M, Andreone P, et al. Interferon and prevention of hepatocellular carcinoma in viral cirrhosis: an evidence-based approach. J Hepatol 2001;34:593 602. 2. El-Serag HB, Marrero JA, Rudolph L, et al. Diagnosis and treatment of hepatocellular carcinoma. Gastroenterology 2008;134: 1752 1763. 3. Degos F, Christidis C, Ganne-Carrie N, et al. Hepatitis C virus related cirrhosis: time to occurrence of hepatocellular carcinoma and death. Gut 2000;47:131 136. 4. N Kontchou G, Paries J, Htar MT, et al. Risk factors for hepatocellular carcinoma in patients with alcoholic or viral C cirrhosis. Clin Gastroenterol Hepatol 2003;4:1062 1068.

July 2010 KERATIN 7 AND HEPATOCARCINOGENESIS 343 5. Rodríguez-Díaz JL, Rosas-Camargo V, Vega-Vega O, et al. Clinical and pathological factors associated with the development of hepatocellular carcinoma in patients with hepatitis virus-related cirrhosis: a long-term follow-up study. Clin Oncol 2007;19:197 203. 6. Pekow JR, Bhan AK, Zheng H, et a. Hepatic steatosis is associated with increased frequency of hepatocellular carcinoma in patients with hepatitis C-related cirrhosis. Cancer 2007;109: 2490 2496. 7. Ganne-Carrié N, Chastang C, Chapel F, et al. Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in Western patients with cirrhosis. Hepatology 1996;23:1112 1118. 8. Donato MF, Arosio E, Del Ninno E, et al. High rates of hepatocellular carcinoma in cirrhotic patients with high liver cell proliferative activity. Hepatology 2001;34:523 528. 9. Roskams TA, Theise ND, Balabaud C, et al. Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers. Hepatology 2004;39:1739 1745. 10. Thorgeirsson SS, Factor VM, Snyderwine EG. Transgenic mouse models in carcinogenesis research and testing. Toxicol Lett 2000;15:553 555. 11. Libbrecht L, Meerman L, Kuipers F, et al. Liver pathology and hepatocarcinogenesis in a long-term mouse model of erythropoietic protoporphyria. J Pathol 2003;199:191 200. 12. Cerec V, Glaise D, Garnier D, et al. Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor. Hepatology 2007;45:957 967. 13. Ma S, Chan KW, Hu L, et al. Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology 2007;132:2542 2556. 14. Roskams T. Different types of liver progenitor cells and their niches. J Hepatol 2006;45:1 4. 15. Mallet V, Gilgenkrantz H, Serpaggi J, et al. Brief communication: the relationship of regression of cirrhosis to outcome in chronic hepatitis C. Ann Intern Med 2008;149:399 403. 16. Bruix J, Sherman M, Llovet J, et al. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. J Hepatol 2001;35:421 430. 17. Watanabe S, Okita K, Harada T, et al. Morphologic studies of the liver cell dysplasia. Cancer 1983;51:2197 2205. 18. The French METAVIR Cooperative Study Group. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology 1994;20:15 20. 19. Fine JP, Gray RJ. A proportional hazards model for the subdistribution of a competing risk. J Am Stat Assoc 1999;94:496 509. 20. Gray RJ. A class of K-sample tests for comparing the cumulative incidence of a competing risk. Ann Stat 1988;16:1140 1154. 21. Crosby HA, Hubscher S, Fabris L, et al. Immunolocalization of putative human liver progenitor cells in livers from patients with endstage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6. Am J Pathol 1998;152:771 779. 22. Marchio A, Terris B, Meddeb M, et al. Chromosomal abnormalities in liver cell dysplasia detected by comparative genomic hybridisation. Mol Pathol 2001;54:270 274. 23. Adachi E, Hashimoto H, Tsuneyoshi M. Proliferating cell nuclear antigen in hepatocellular carcinoma and small cell liver dysplasia. Cancer 1993;72:2902 2909. 24. Libbrecht L, Craninx M, Nevens F, et al. Predictive value of liver cell dysplasia for development of hepatocellular carcinoma in patients with non-cirrhotic and cirrhotic chronic viral hepatitis. Histopathology 2001;39:66 73. 25. Brunt EM, Walsh SN, Hayashi PH, et al. Hepatocyte senescence in end-stage chronic liver disease: a study of cyclin-dependent kinase inhibitor p21 in liver biopsies as a marker for progression to hepatocellular carcinoma. Liver Int 2007;27:662 671. 26. Makino Y, Shiraki K, Sugimoto K, et al. Histological features of cirrhosis with hepatitis C virus for prediction of hepatocellular carcinoma development; a prospective study. Anticancer Res 2000;20:3709 3715. 27. Libbrecht L, Desmet V, Van Damme B, et al. The immunohistochemical phenotype of dysplastic foci in human liver: correlation with putative progenitor cells. J Hepatol 2000;33:76 84. 28. Hoshida Y, Villanueva A, Kobayashi M, et al. Gene expression in fixed tissues and outcome in hepatocellular carcinoma. N Engl J Med 2008;359:1995 2004. 29. Roskams T, Yang SQ, Koteish A, et al. Oxidative stress and oval cell accumulation in mice and humans with alcoholic and nonalcoholic fatty liver disease. Am J Pathol 2003;163:1301 1311. 30. Clouston AD, Powell EE, Walsh MJ, et al. Fibrosis correlates with a ductular reaction in hepatitis C: roles of impaired replication, progenitor cells and steatosis. Hepatology 2005;4:809 918. 31. Marongiu F, Doratiotto S, Montisci S, et al. Liver repopulation and carcinogenesis: two sides of the same coin? Am J Pathol 2008; 172:857 864. 32. Durnez A, Verslype C, Nevens F, et al. The clinicopathological and prognostic relevance of cytokeratin 7 and 19 expression in hepatocellular carcinoma. A possible progenitor cell origin. Histopathology 2006;49:138 151. 33. Terris B, Ingster O, Rubbia L, et al. Interphase cytogenetic analysis reveals numerical chromosome aberrations in large liver cell dysplasia. J Hepatol 1997;27:313 319. 34. Yamashita T, Ji J, Budhu A, et al. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology 2009;136:1012 1024. 35. Lackner C, Gogg-Kamerer M, Zatloukal K, et al. Ballooned hepatocytes in steatohepatitis: the value of keratin immunohistochemistry for diagnosis. J Hepatol 2008;48:821 828. 36. Xiao JC, Jin XL, Ruck P, et al. Hepatic progenitor cells in human liver cirrhosis: immunohistochemical, electron microscopic and immunofluorescence confocal microscopic findings. World J Gastroenterol 2004;10:1208 1211. 37. Crosby HA, Hubscher S, Fabris L, et al. Immunolocalization of putative human liver progenitor cells in livers from patients with endstage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6. Am J Pathol 1998;152:771 779. 38. Michalopoulos GK, Barua L, Bowen WC. Transdifferentiation of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary injury. Hepatology 2005;41:535 544. 39. Cerec V, Glaise D, Garnier D, et al. Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor. Hepatology 2007;45:957 967. 40. Mishra L, Banker T, Murray J, et al. Liver stem cells and hepatocellular carcinoma. Hepatology 2009;49:318 329. 41. Fattovich G, Stroffolini T, Zagni I, et al. Hepatocellular carcinoma in cirrhosis: incidence and risk factors. Gastroenterology 2004; 127:S35 S50. 42. Serfaty L, Aumaître H, Chazouillères O, et al. Determinants of outcome of compensated hepatitis C virus-related cirrhosis. Hepatology 1998;27:1435 1440. 43. Sangiovanni A, Prati GM, Fasani P, et al. The natural history of compensated cirrhosis due to hepatitis C virus: a 17-year cohort study of 214 patients. Hepatology 2006;43:1303 1310. Received July 15, 2009. Accepted April 12, 2010. Reprint requests Address requests for reprints to: Marianne Ziol, MD, PhD, Pathology Department, CHU Jean Verdier, Paris 13 University, Av du 14 Juillet, 93143 Bondy, France. e-mail: marianne.ziol@jvr.aphp.fr; fax: (33) 0148026877. Conflicts of interest The authors disclose no conflicts.

343.e1 ZIOL ET AL GASTROENTEROLOGY Vol. 139, No. 1 Supplementary Materials and Methods Immunohistochemistry Dewaxed and rehydrated paraffin sections, 3- m thick, were immunostained with antibodies to K7, to a mixture of K8 and K18, to K19, NCAM, Ep-CAM, CD117, CD34, N-cadherin, and chromogranin. The clone number, provider, antigen retrieval, and dilution used for these antibodies are detailed in Supplementary Table 1. All immunostaining was performed as previously described using an automated immunostaining device (Vision Biosystem Bond Max, Menarini, France). After heatinduced antigen retrieval (Vision BioSystems ER1) for 20 minutes at a ph of 8, sections were incubated with the primary antibody for 30 minutes and immunodetection was performed with a biotin-conjugated secondary antibody (Vision BioSystems DS9713) followed by peroxidase-labeled streptavidin and with 3,3=-diaminobenzidine tetrahydrochloride chromogen as the substrate (Vision BioSystems).

July 2010 KERATIN 7 AND HEPATOCARCINOGENESIS 343.e2 Supplementary Table 1. Summary of the Primary Antibodies Used for Immunohistochemistry Antibody directed against Used as a marker for Official gene symbol Identification Antibody clone Isotype Dilution Supplier K7 Intermediate hepatocytes KRT7 3855 OV-TL 12/30 IgG1 1:800 DAKO, Glastrup, Denmark K19 Intermediate hepatocytes KRT19 3880 BA17 IgG1 1/200 DAKO K8 Hepatocyte and biliary cells KRT8 16,691 35betaH11 IgGM Prediluted DAKO K18 Hepatocyte and biliary cells KRT18 3875 DC10 IgG1 1/25 DAKO NCAM Progenitor cells NCAM1 4684 CD564 IgG2b 1/200 Novocastra, Newcasatle, United Kingdom Ep-CAM Progenitor cells EPCAM 4072 BerEP4 IgG1 1/50 DAKO CD117 Progenitor cells KIT 3815 Polyclonal 1/200 DAKO CD34 Progenitor cells CD34 947 QBEND10 IgG1 1/200 DAKO Chromogranin Progenitor cells CHGA 1113 LK2H10 IgG1 1/20 Beckman, Marsceilles, France Ig, immunoglobulin.