Hepatocyte Expression of MinichromosomeMaintenance Protein-2 Predicts Fibrosis ProgressionAfter Transplantation for Chronic Hepatitis C Virus:

A Pilot StudyAileen Marshall,1 Simon Rushbrook,1 Lesley S. Morris,4 Ian S. Scott,4

Sarah L. Vowler,3 Susan E. Davies,2 Nicholas Coleman,4 and Graeme Alexander1

Although graft infection with hepatitis C virus (HCV)occurs in virtually all patients transplanted for HCV-re-lated liver disease, the outcome ranges from minimal dis-ease to the rapid development of cirrhosis. Induction ofhepatocyte cell cycle entry followed by inhibition of cellcycle progression has been proposed as a potential mech-anism whereby HCV may cause hepatocyte dysfunctionand may promote fibrogenesis. The aim of this study wasto assess whether early hepatocyte cell cycle entry mightpredict subsequent fibrosis progression in patients withgraft HCV infection after liver transplantation. Liverbiopsies from 21 liver transplant recipients diagnostic ofgraft HCV infection but before development of significantfibrosis were studied. Patients were classed as nonprogres-sors, intermediate progressors, or rapid progressorsaccording to the rate of fibrosis progression calculatedfrom the most recent biopsy. Minichromosome mainte-nance protein 2 (Mcm-2), a highly sensitive and specificmarker of cell cycle entry, and cyclin-dependent kinaseinhibitor p21 were detected by immunohistochemistry.Hepatocyte Mcm-2 expression increased significantlyaccording to rate of fibrosis. For nonprogressors, themedian percentage of positive hepatocytes was 5.3%(range, 0.92%-11.2%) compared with 20.7% (4.6%-43.7%) in intermediate progressors and 23.7% (11.6%-55.2%) in rapid progressors (P � 0.002). By contrast,there was no evidence of a difference in hepatocyte p21expression. Median values and ranges were 3.4% (range,1.1%-30%), 13.3% (range, 1.4%-42.3%), and 11.8%(range, 7.6%-52.3%) for nonprogressors, intermediate pro-gressors, and rapid progressors, respectively (P � 0.11). Inconclusion, hepatocyte cell cycle entry may be important inthe pathogenesis of posttransplant HCV hepatitis. Earlyassessment of hepatocyte Mcm-2 expression could help iden-tify patients at high risk for progressive fibrosis before itoccurs. (Liver Transpl 2005;11:427-433.)

Liver transplantation is an effective treatment forend-stage liver disease caused by chronic hepatitis

C virus (HCV) infection, with short- and medium-term survival rates similar to patients transplanted forother indications.1,2 However, graft infection by HCVoccurs in almost all cases, which causes a spectrum ofdisease. Some patients experience rapidly progressivefibrosis, whereas others have mild and apparently stableor more slowly progressive disease. The prevalence ofcirrhosis has been estimated at 10–30% at 5 years,3 but

Berenguer et al. found an apparent increase in the rateof fibrosis progression in HCV transplant patients.4,5

Although the short-term outcome of transplantation issimilar for HCV-positive and HCV-negative patients,reduced long-term patient and graft survival has beenreported.6 The apparent increase in rate of fibrosis pro-gression in HCV-positive transplant recipients may bebecause of the increased use of older donors4,7,8 and useof different immunosuppressive agents. Other factorsthought to contribute to the risk of rapidly progressivefibrosis include early and more severe histologic recur-rence,3 longer warm ischemic time,7 pretransplant viralload,9 more acute rejection episodes, and multiple treat-ments for acute rejection.10,11

Once cirrhosis occurs, the interval to decompensa-tion is shorter than for nontransplant patients—up to42% by 1 year.12,13 United Network for Organ Sharing(UNOS) data for patients transplanted between 1988and 2001 show that retransplantation for graft cirrhosiscaused by HCV is associated with reduced patient andgraft survival compared with other indications.14

One strategy to limit the adverse effect of HCV graft

Abbreviations: HCV, hepatitis C virus; Mcm-2, minichromo-some maintenance protein 2; LI, labeling index; OLT, orthotopicliver transplant.

From the 1University of Cambridge Department of Medicine, and2Department of Pathology, Addenbrooke’s Hospital, Cambridge, UK;3Centre for Applied Medical Statistics, Department of Public Health andPrimary Care, University Forvie Site, Cambridge, UK; and 4MRCCancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, UK.

Aileen Marshall is supported by the National Blood Service (UK) andthe Raymond and Beverly Sackler Fund. Simon Rushbrook holds a fel-lowship awarded by the Digestive Diseases Foundation. The MedicalResearch Council and Cancer Research UK support work in NicholasColeman’s laboratory.

Address reprint requests to Dr. Graeme Alexander, University ofCambridge Department of Medicine, Box 157, Addenbrooke’s Hospital,Hills Road, Cambridge, CB2 2QQ, UK. Telephone: 44 1223 686614;E-mail: gja1000@doctors.org.uk

Copyright © 2005 by the American Association for the Study ofLiver Diseases

Published online in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/lt.20347

427Liver Transplantation, Vol 11, No 4 (April), 2005: pp 427-433

infection would be to instigate antiviral therapy withinterferon and ribavirin in all patients undergoingtransplantation for HCV-related cirrhosis. However,because most patients will not experience rapidly pro-gressive fibrosis and because combination therapy is lesseffective and is associated with more side effects than innontransplant patients,15-20 treatment of all patientscould impose a greater risk for less benefit. Therefore, areliable method for early identification of patients athigh risk of rapidly progressive fibrosis would be animportant advance that would allow targeted interven-tion aimed to delay or prevent fibrosis.

Difficulties in accurately predicting the course ofgraft HCV infection reflect the limited understandingof pathogenesis of liver damage by HCV. Induction ofhepatocyte cell cycle entry by HCV followed by inhibi-tion of cell cycle progression has been proposed recentlyas a potential mechanism whereby HCV might impairhepatocyte function and promote fibrogenesis.21 Hepa-tocyte expression of minichromosome maintenanceprotein-2 (Mcm-2, a marker of cell cycle entry)21 andcyclin dependant kinase inhibitor p2122 have been asso-ciated significantly with fibrosis stage and serum HCVRNA status. This study was undertaken to determinewhether hepatocyte Mcm-2 or p21 expression in theearly posttransplant period, before the development offibrosis, could predict the subsequent rate of fibrosisprogression in patients transplanted for HCV-relatedliver disease.

Patients and Methods

Liver Biopsies

All patients receiving a first liver transplant for HCV-related cirrhosis (without hepatocellular carcinoma) inAddenbrooke’s Hospital, Cambridge, UK, between 1990and 1999 were considered for eligibility (n � 44). In thiscenter, protocol liver biopsies are performed yearly, as wellas according to clinical need. Twenty-three patients were

excluded for the following reasons: duration of histologicfollow-up less than 5 years (8), insufficient tissue availablefor study (11), chronic rejection (2), biliary disease (1), andautoimmune damage (1). Twenty-one patients wereincluded in the study. The diagnosis of posttransplant graftHCV infection was based on liver histology and detectionof HCV RNA in serum by polymerase chain reaction. Therate of fibrosis progression was calculated as follows: fibro-sis stage of the most recent liver biopsy was divided by thenumber of years since transplantation to give the numberof stages per year. Patients were grouped according to theposttransplant rate of fibrosis progression, which wasdefined as follows: (1) no or minimal progression—patientswhose liver biopsy did not progress beyond fibrosis stage 2(out of 6 stages)over at least 5 years of follow-up (n � 5),(2) intermediate progression—patients who progressedbeyond stage 2 fibrosis by 3 years but did not reach cirrho-sis by 5 years of follow up (n � 8), and (3) rapid progres-sion—patients whose liver biopsy demonstrated cirrhosisby 2 to 3 years of transplantation (n � 8). All biopsiesshowed histologic changes of HCV graft infection only.Liver specimens from patients who had undergone partialhepatectomy for colorectal cancer metastasis were used as acontrol (n � 10). These liver specimens were histologicallynormal liver distant to the metastasis, and there was noevidence of neoplastic tissue in the block studied. All tissuewas used in accordance with local research ethics commit-tee guidelines. Table 1 lists the demographic and clinicalfeatures for the patient groups.

Immunosuppression

All patients had received initial triple immunosuppression,consisting of either cyclosporine or tacrolimus in combina-tion with azathioprine and prednisolone. Prednisolone wastapered with the aim of discontinuing within 3 months; aza-thioprine was discontinued within 6 months. There was nodifference between the number of patients receiving cyclo-sporine or tacrolimus in the 3 groups. Six patients (29%)experienced a single episode of acute rejection; no patient hadmore than 1 episode of acute rejection. All rejection episodeswere treated with 3 intravenous doses of 1 g methylpred-nisolone, and all 6 patients responded to this therapy.

Table 1. Demographic, Clinical, and Histologic Features of Patient Groups

GroupNonprogressors

(n � 5)

Intermediateprogressors

(n � 8)

Rapidprogressors

(n � 8)P

value

Median age (range), yrs 51 (34-60) 52 (40-67) 56 (46-62) 0.56Male, % 60 87.5 50 0.36Median donor age (range), yrs 43 (14-53) 38 (17-55) 45 (20-58) 0.47Median day of biopsy after OLT (range) 91 (19-2409) 274 (23-1030) 73 (38-678) 0.5Number with episodes of acute rejection 3 1 2 0.31Median fibrosis stage of biopsy studied (range) 1 (0-2) 1 (0-3) 2 (1-5) 0.3

428 Marshall et al.

Histology

Sections were assessed by a consultant liver histopathologist(S. Davies) who was unaware of the patients’ clinical out-comes. Histologic activity index and fibrosis were scoredusing criteria according to Ishak et al.,23 and steatosis wasrecorded. Histologic activity index represented the sum ofinterface hepatitis (0 -4), confluent necrosis (0 -6), lobularinflammation (0-4), and portal inflammation (0-4). Fibrosiswas scored from 0 (absent) to 6 (cirrhosis) and steatosis wasscored from 0 to 3, for none or a few vacuoles (0), less than25% (1), 25%-50% (2), and greater than 50% (3) of biopsyaffected.

Immunohistochemistry

Standard immunohistochemistry was used to detect Mcm-2and p21 in formalin-fixed, paraffin-embedded liver sections.Mouse monoclonal anti–Mcm-2 was generated as previouslyreported21 and was used at a dilution of 1:10. Mouse mono-clonal antibody to p21 (Dako, Ely, UK) was used at a dilutionof 1:25. Incubation with the primary antibody was carried outovernight at 4°C. The following day, secondary biotinylatedgoat antimouse antibody was applied (Dako), followed by astreptavidin-horseradish peroxidase system (Dako) with thesubstrate diaminobenzidine to develop the stain.

For assessment of immunohistochemistry, positive andnegative hepatocytes were counted in 4 random fields at 40�magnification. Approximately 1000 hepatocytes werecounted per case. Dark brown staining of hepatocyte nucleiwas considered positive. Two observers (A. Marshall and I.Scott) counted the slides and were unaware of the patients’subsequent clinical course. The difference in values obtainedby the 2 observers was less than 10%. The number of positivehepatocytes was expressed as a percentage of the total to give alabeling index (LI).

Statistical Analysis

Analysis of hepatocyte Mcm-2 or p21 expression according torate of fibrosis progression was performed using the Jonck-heere-Terpstra test. Association of Mcm-2 and p21 with his-

tologic variables was performed using the Kruskal-Wallis test.For comparison of demographic and clinical features, theKruskal-Wallis test was used for continuous variables, and theFisher exact test was used for categorical variables. A P valueless than 0.05 was regarded as significant.

Results

Patient Demographic Details andPosttransplant Clinical Events

The no progression, intermediate progression, andrapid progression groups showed no evidence of a dif-ference in patient age at the time of transplantation,gender, donor age, bilirubin or alanine aminotransfer-ase level at the time of HCV recurrence, number ofpatients with an episode of acute rejection, or the fibro-sis stage of the biopsy included in the immunohisto-chemical study (Table 1).

Histologic Assessment of Posttransplant LiverBiopsies

Table 2 summarizes the results of the histologic assess-ment of the liver biopsies used to measure Mcm-2expression. Lobular inflammation was greater in therapid progression group (median 3, range 2-4) com-pared with the other 2 groups, but there was no evi-dence of a difference in fat, confluent necrosis, interfacehepatitis, portal inflammation, total histologic activityindex, or fibrosis stage.

Table 3 lists the timing of the early liver biopsiesused to measure Mcm-2 expression and the later biopsyused to calculate fibrosis progression. There was noevidence of a difference in the interval from transplan-tation to the liver biopsy used for measurement ofMcm-2 between the 3 groups. The median interval

Table 2. Histologic Activity Index of Posttransplant Liver Biopsies Showing Graft HCV Infection

Group NonprogressorsIntermediateprogressors

Rapidprogressors

Pvalue

Fat (0-3) 0 (0-1) 0 (0-2) 0 (0-1) 0.78Confluent necrosis (0-6) 0 (0) 0.5 (0-2) 0 (0-3) 0.13Interface hepatitis (0 -4) 2 (0-2) 1 (0-2) 1.5 (0-3) 0.66Lobular inflammation* (0-4) 2 (1-2) 2 (1-4) 3 (2-4) 0.026Portal inflammation (0-4) 2 (1-2) 1.5 (1-3) 2 (1-4) 0.85Histologic activity index (0-18) 6 (2-6) 5 (2-11) 6.5 (3-13) 0.47Fibrosis (0-6) 1 (0-2) 1 (0-3) 2 (1-5) 0.3

*There was no evidence of a difference between nonprogressors, intermediate progressors, and rapid progressors in individual featuresexcept in lobular inflammation.

429Mcm-2 Predicts Fibrosis in HCV Recurrence After OLT

from transplant to biopsy was 91 days for nonprogres-sors (range 19-2409), 274 days for intermediate pro-gressors (range 23-1030) and 73 days for rapid progres-sors (range 38-678), P � 0.5.

Hepatocyte Mcm-2 Expression in Normal Liver

In liver resected for colorectal cancer metastasis,hepatocyte expression of Mcm-2 and p21 was negligi-ble (� 0.01% of hepatocytes).

Hepatocyte Mcm-2 Expression inPosttransplant Liver Biopsies

Hepatocyte Mcm-2 expression rose significantly withincreasing rate of subsequent fibrosis progression (Figs.1-2). Median hepatocyte Mcm-2 LI for nonprogressorswas 5.3% (range 0.92%-11.2%), for intermediate pro-gressors 20.6% (range 7.2%-43.7%), and rapid pro-gressors 23.7% (range 11.6%-55.2%), P � 0.002, Jon-ckheere-Terpstra test. There was no evidence of adifference in interface hepatitis, confluent necrosis, por-

Table 3. Timing of Liver Biopsies (Days After Transplant) and Fibrosis Stage of Liver Biopsies Used to Measure Mcm-2 and Usedfor Calculation of Fibrosis Progression

Patients

Timing of biopsy used forassessment of Mcm-2

(days after OLT)

Fibrosis stage ofbiopsy used for

assessment of Mcm-2

Timing of biopsy used forcalculation of fibrosis

progression(days after OLT)

Fibrosis stage of biopsyused for calculation of

fibrosis progression

Fibrosisprogression

(stages per year)

NP1 2409 0 3699 0 0NP2 2007 2 3812 1 0.09NP3 91 2 1855 2 0.39NP4 19 1 2739 1 0.13NP5 21 1 1971 1 0.21IP1 87 2 1950 3 0.56IP2 241 1 1102 4 1.32IP3 755 0 2286 4 0.63IP4 509 1 2202 4 0.66IP5 97 2 1660 2 0.44IP6 1030 3 1527 5 1.2IP7 23 0 1201 2 0.61IP8 308 1 1133 2 0.65RP1 38 1 843 5 2.6RP2 56 1 526 5 3.47RP3 82 2 738 5/6 2.97RP4 54 1 225 5 8.06RP5 678 5 1044 6 2.1RP6 363 4 896 6 2.45RP7 53 2 220 6 10RP8 155 2 910 5/6 2.41

NOTE. Fibrosis progression (stages per year) was calculated by fibrosis stage of latest biopsy divided by number of years sincetransplantation. For fibrosis stage, 0 � no fibrosis and 6 � cirrhosis.Abbreviations: OLT, orthotopic liver transplantation; NP, nonprogressor; IP, intermediate progressor; RP, rapid progressor.

Figure 1. Hepatocyte minichromosome maintenanceprotein 2 (Mcm-2) expression according to rate of fibrosisprogression. Circles represent values for individualpatients, and black lines represent median values. P �0.002, Jonckheere-Terpstra test.

430 Marshall et al.

tal inflammation, fibrosis, or steatosis between the 3groups. Lobular inflammation was greater in the rapidprogressor group compared with the other two groups(Table 2, P � 0.026).

Hepatocyte p21 Expression in PosttransplantLiver Biopsies

There was no evidence of a difference in the hepatocytep21 LI between nonprogressors, intermediate progres-sors, or rapid progressors (Fig. 3). Median hepatocytep21 LI for nonprogressors was 3.4% (range 1.1%-30%),for intermediate progressors 13.3% (range 1.4%-42.3%),and rapid progressors 11.7% (range 7.6%-52.3%), P �0.11, Jonckheere-Terpstra test.

Discussion

To assess whether Mcm-2 or p21 expression in earlyposttransplant liver biopsies was related to subsequentfibrosis development, we analyzed Mcm-2 and p21expression according to the rate of subsequent fibrosisprogression in patients transplanted for chronic HCV.We found a significant association between hepatocyteMcm-2 expression and the subsequent development ofcirrhosis within 2 years after transplantation. There wasno evidence of a difference in p21 expression accordingto rate of fibrosis progression.

A reliable method of predicting which patients are athigh risk of rapidly progressive disease would be an

Figure 2. Sections of liver biopsies from 3 patients withgraft hepatitis C virus (HCV) infection and similar fibrosisstage but different posttransplant rate of fibrosis progressionshowing immunoperoxidase stain using monoclonal anti-body specific for minichromosome maintenance protein 2(Mcm-2) (brown staining indicates positive nucleus) (origi-nal magnification � 20). Hematoxylin and eosin stain andreticulin stain of the same biopsy are inset (original magnifi-cation � 10). (A) Immunoperoxidase stain for Mcm-2 fromindividual with no significant subsequent fibrosis. (B)Immunoperoxidase stain for Mcm-2 from individual withintermediate subsequent fibrosis progression, 0.66 stages peryear in this case. (C) Immunoperoxidase stain for Mcm-2from individual with rapid subsequent fibrosis progression,with cirrhosis by 2 years after transplantation.

Figure 3. Hepatocyte p21 expression according to rate offibrosis progression. Circles represent values for individ-ual patients and black lines median values. P � 0.11,Jonckheere-Terpstra test.

431Mcm-2 Predicts Fibrosis in HCV Recurrence After OLT

important advance in the management of posttrans-plant graft HCV infection. A recent study developed amodel for the prediction of progression to severe diseaseusing pretransplant and early posttransplant variables,including host, donor, viral factors, and type and dura-tion of immunosuppression.24 Significant factors asso-ciated with increased risk of severe disease in a multi-variate analysis of 285 patients were as follows:induction with mycophenolate mofetil, azathioprinefor less than 6 months, prednisone for less than 12months, and donor age older than 50 years. Althoughthis model may be helpful to define risk factors in apopulation, the complex nature of HCV infectionmeans that the accuracy was not sufficient to guidemanagement of individual patients.

Adoption of the Ishak scoring system for posttrans-plant HCV hepatitis has been proposed to aid predic-tion of early cirrhosis.25 In a study of 29 patients, his-tologic activity index greater than 9 at 6 months aftertransplantation was shown to predate early cirrhosis. Inour study, only 3 of the 8 patients who had rapidlyprogressive fibrosis had a histologic activity index above9, as did 3 of 8 patients with an intermediate rate offibrosis. However, because of the retrospective nature ofthe current study, the time points of the biopsies are notuniform and thus may not be comparable.

Another study found that high necroinflammatoryactivity at 12 months after transplant was associatedwith later development of severe fibrosis.26 By multivar-iate analysis, high necroinflammatory activity was itselfassociated with occurrence of acute lobular hepatitisand absence of pretransplant alcohol abuse. We foundno evidence of a difference in the overall necroinflam-matory activity, which may be because of the smallernumber of patients or the different time points at whichthe biopsies were taken. However, the severity of lobu-lar hepatitis was linked to rate of subsequent fibrosisprogression, and it may be that components of thehistologic classification have different prognostic valuefor nontransplant and transplant patients.

The pathogenesis of HCV-related liver disease is notwell understood. Immune-mediated damage is thoughtto be important, and viral cytopathic effects also mayplay a part. In particular, there is increasing evidence tosuggest that HCV-induced hepatocyte oxidative stressis important.27-29 In vitro, cell cycle arrest occurs inresponse to cellular oxidative stress.30 We have sug-gested previously that HCV-induced hepatocyte cellcycle arrest could be important in the pathogenesis ofchronic HCV infection. In liver biopsies from non-transplant patients with chronic HCV infection, hepa-tocyte expression of Mcm-221 and p2122 were elevated

in all biopsies. Mcm proteins 2-7 are essential in licens-ing DNA for replication and are highly sensitive andspecific markers of cell cycle entry. In the nontransplantcohort, the percentage of both Mcm-2– and p21-pos-itive hepatocytes rose significantly as fibrosis stageincreased and was significantly higher in liver biopsiesfrom serum HCV RNA–positive patients, supportinga direct viral effect on host cell cycle.

Risk factors for fibrosis progression in nontransplantHCV-infected patients include age at infection, heavyalcohol consumption and steatosis,31,32 and highernecroinflammatory activity33 in the liver biopsy; how-ever, the mechanisms of fibrosis remain unclear. Thehost immune response is believed to provide the stim-ulus for fibrosis, but this does not explain the morerapidly progressive disease seen in immunosuppressedpatients after liver transplant and those co-infected withHIV and HCV. Because high Mcm-2 expression pre-dated fibrosis development in our study, there could bea causative link between hepatocyte cell cycle entry andfibrogenesis. We have postulated that hepatocyteexpression of Mcm-2 does not necessarily denote aregenerating cell, but that in chronic HCV infection, aproportion of Mcm-2–positive hepatocytes might bearrested and unable to complete cell cycle.22 Arrestedhepatocytes could directly or indirectly promotehepatic stellate cell activation and hence induce fibro-genesis.

In addition to graft HCV infection, a number offactors could influence hepatocyte proliferation aftertransplantation, such as acute rejection, chronic rejec-tion, biliary complications, drugs, and infection. Weexcluded patients from this study who were known tohave any cause of graft dysfunction other than HCV tominimize potential confounding by these factors. How-ever, this means that the results do not necessarily applyto patients with other pathology in addition to graftHCV infection. This study is also limited by the varia-tion in time points of the biopsies because most weretaken according to clinical need. Prospective studiesusing liver biopsies taken according to clinical protocolat set time points are now required.

In conclusion, our results suggest that hepatocytecell cycle entry is likely to be important in the patho-genesis of posttransplant HCV hepatitis and that assess-ment of Mcm-2 expression could help identify patientsat high risk of early cirrhosis before it occurs.

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433Mcm-2 Predicts Fibrosis in HCV Recurrence After OLT