J Clin Pathol 1997;50:735-740

Papers

Hepatic histology of patients with HIV infectionand chronic hepatitis C treated with interferon

Renzo Boldorini, Paolo Vigano, Guido Monga, Manuela Nebuloni, Antonietta Cargnel,Guido Gubertini, Giuseppe Migliaretti, Giulio Costanzi

AbstractAims-To evaluate the histologicalchanges seen in liver biopsies after inter-feron (IFN) treatment in patients withchronic hepatitis C and human immuno-deficiency virus (HIV) infection.Methods-Twenty four intravenous drugusers with chronic hepatitis C were inves-tigated histologically before beginning a12 month course of IFN treatment and 18months later. Twelve were HIV positive,without opportunistic or other viral infec-tions (group A), and 12 were HIV negative(group B).Results-According to alanine amino-transferase concentrations, four sustainedresponders and eight non-responderswere found in group A; six sustainedresponders, five relapsers, and one non-responder were found in group B. HCVRNA became negative in one sustainedresponder of group A and in the sixsustained responders of group B. Whenhistological findings of biopsies per-formed before therapy and 18 monthslater were compared, no significantchanges in the mean value of Knodell'sindex and subindices were found in groupA, whereas in group B Knodell's index,piecemeal necrosis, and focal hepatocellu-lar necrosis decreased significantly.Conclusions-In chronic hepatitis C, co-infection with HIV showed a tendencytowards a lower response to IFN, althoughthis did not reach statistical significance;however, none of the HIV positive patientsdeveloped cirrhosis during the follow upand this should be considered in clinicalmanagement of such patients.(7 Clin Pathol 1997;50:735-740)

Keywords: chronic hepatitis C; human immunodefi-ciency virus; liver biopsy

Hepatitis C virus (HCV) infection is frequentin patients with acquired immunodeficiencysyndrome (AIDS),' especially in intravenousdrug users or subjects who have received bloodtransfusions.2 Its clinical outcome is reportedto be worse than in patients without human

immunodeficiency virus (HIV) infection. Berket a13 described a case of chronic hepatitis Cinfection in an HIV positive patient with severeliver cell damage resembling that of autoim-mune type, and Martin et al4 found a rapidprogression to liver cirrhosis in three HIV posi-tive patients co-infected with HCV.The unfavourable evolution of chronic

hepatitis C in HIV positive subjects could becaused solely by the severe impairment of theimmunological system, or by the simultaneouspresence of opportunistic agents or neoplasms,which are common in HIV positive patients.'

Several studies have shown that interferon(IFN) therapy is of potential benefit to patientswith chronic hepatitis C, and both clinical andmorphological effects in treated patients havebeen reported.6"9 On the contrary, only a fewstudies have evaluated its effects in chronichepatitis C in HIV positive patients, and theyhave been mainly from the clinical point ofview.""'2 To our knowledge, no study has thor-oughly examined the histological changes inliver biopsies induced by IFN in HIV positivepatients.The aim of this study was to describe the

effects on liver histology of a 12 month courseofIFN in a group ofHIV positive patients, andto compare the results with those from a groupof HIV negative patients selected for similarage, sex, risk factors, and duration ofintravenous drug addiction, all with chronichepatitis C.

Materials and methodsPATIENT SELECTIONTwenty four patients, all intravenous drugusers with chronic hepatitis C, were selectedfor the study and divided into two groups: 12HIV positive patients (group A), and 12 HIVnegative patients (group B). The mean age forboth groups was 27 years (range 21-37 forgroup A and 23-35 for group B); bothcomprised seven men and five women; theduration of intravenous drug addiction wassimilar in the two groups (mean seven years forgroup A and six years for group B).The patients had been referred to the 2nd

Division of the Infectious Disease Unit of L.Sacco Hospital, Milan, from 1991 to 1994, and

Dipartimento diScienze Mediche,Facolta di Medicina eChirurgia di Torino,sede di NovaraR BoldoriniG MongaG Migliaretti

Unita Operativa diMalattie Infettive,Ospedale di LegnanoP Vigan6

I1 Divisione di MalattieInfettive, Ospedale L.Sacco, MilanoA CargnelG Gubertini

V Cattedra diAnatomia Patologica,Istituto di ScienzeBiomedicheM NebuloniG Costanzi

Correspondence to:Dr Boldorini, Servizio diAnatomia Patologica,Ospedale Maggiore dellaCarita, Corso Mazzini 18,28100 Novara, Italy.

Accepted for publication12 June 1997

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Table 1 Changes in serum ALT with IFN treatment and HCVgenotypes in 12 HIVpositive patients

ALT concentration (IUII)Response to

Case Time 0 Time 12 Time 18 HCVgenotype IFN

1 63 119 97 3a NR2 91 94 300 3a NR3 81 46 108 lb NR4 208 28 37 lb SR5 104 80 57 3a NR6 131 21 26 2a SR7 182 57 32 la SR8 71 38 36 3a SR9 154 153 196 lb NR10 94 79 84 3a NR11 75 54 69 3a NR12 116 146 228 la NR

Time 0, before treatment; time 12, at the end of 12 months' treatment; time 18, after a further sixmonths of follow up. HCV genotype according to Simmond's classification.'7NR, non-responder; SR, sustained responder.

Table 2 Changes in serum ALT with IFN treatment and HCVgenotypes in 12 HIVnegative patients

ALT concentration (IUII)Response to

Case Time 0 Time 12 Time 18 HCV genotype IFN

1 147 21 17 3a SR2 274 16 109 2a Relapser3 244 12 10 lb SR4 197 14 121 2a Relapser5 68 17 17 3a SR6 123 28 97 2a Relapser7 108 23 152 1 a Relapser8 205 36 19 3a SR9 105 16 17 3a SR10 75 62 120 lb NR11 82 16 87 3a Relapser12 87 24 29 3a SR

Time 0, before treatment; time 12, at the end of 12 months' treatment; time 18, after a further sixmonths of follow up. HCV genotype according to Simmond's classification. 17NR, non-responder; SR, sustained responder.

fulfilled the following criteria: (1) elevation ofserum alanine aminotransferase (ALT) levelsfor longer than six months (more than twicethe upper limit of normal values, 10-40 IU/1);(2) no evidence of hepatitis B virus infection(absence of detectable hepatitis B surface anti-gens and HBV DNA in serum) or other causesof chronic viral hepatitis by appropriate labora-tory tests, and (for patients ofgroup A) absenceof opportunistic infections; (3) CD4 cell countmore than 200 cells/mm'; (4) no zidovudine orother antiviral therapy before or during thestudy period.

In HIV positive patients, mean CD4 countwas 465 cells/mm3 (range 231-819) before thetreatment, and 414 cells/mm3 (range 234-950)18 months later.

SEROLOGYAnti-HCV antibodies were detected by thesecond generation enzyme linked immuno-sorbent assay (ELISA) (Ortho Diagnostic Sys-tems, Raritan, New Jersey, USA); HCV RNAwas detected in serum with a nested polymer-ase chain reaction (nPCR) and the amplifica-tion products were detected by DNA enzymeimmunoassay (Sorin Biomedica, Saluggia,Italy). HCV genotyping was carried out using atype specific detection system of the PCRamplified 5' non-coding region of the HCVgenome on the serum collected at the begin-ning of treatment."

Group A had anti-HIV antibodies detectedby ELISA (Pasteur, Paris, France) and con-firmed by western blot assay (Pasteur). Thep24 antigen of HIV was detected by an immu-noenzyme assay (Abbott Laboratories, NorthChicago, Illinois, USA).Written informed consent was obtained

from all patients and the trial was approved bythe local ethics committee.

TREATMENT SCHEDULE

Lymphoblastoid a 2n IFN (Wellcome Re-search Laboratories, Kent, UK) was injectedsubcutaneously according to the followingschedule: 6 million units three times a week forsix months (induction phase), 3 million unitsthree times a week for a further six months(maintenance phase).Serum ALT concentrations were assessed

before treatment (time 0), at the end oftreatment (time 12), and six months later (time18). According to the outcome of serum ALT,the patients were defined as: sustained re-sponders (normal serum ALT achieved at time12 and lasting throughout follow up) and non-responders (no significative changes in ALTduring the therapy). Post-treatment relapserswere defined as the cases in which ALT levelswere normalised at the end of therapy butincreased above the normal range six monthsafter discontinuation of therapy.

EVALUATION OF LIVER HISTOLOGYLiver needle biopsies were obtained with amodified Menghini technique, at times 0 and18. The samples were fixed in formalin andembedded in paraffin; 5 mm thick sectionswere stained with haematoxylin and eosin,Masson's trichrome, periodic acid-Schiff afterdiastase digestion, Gomori's method for reticu-lin, and Perls method for evaluation of ironstores.The morphological changes were evaluated

by the histological activity index, according toKnodell et all4 and by the recently proposedDesmet's classification of chronic hepatitis. 15

Histological assessment of hepatic ironstores was performed using a score system pro-posed by Searle et al.'6

STATISTICAL ANALYSISData from Knodell's index and subindices wereanalysed by the Wilcoxon matched pairsnon-parametric test. The changes in serumALT levels were analysed by the parametric ttest for independent samples to evaluate theirchanges from time 0 to times 12 and 18between the two groups. Serological andbiochemical responses between the two groupswere compared by Fisher's exact test. Signifi-cance was established at p < 0.05.

ResultsSerum ALT values are summarised in tables 1and 2. Thei mean values at the beginning oftherapy were higher in group B than in group A(142 and 114, respectively; not significant),decreased more considerably at the end of thetherapy in group B than in group A (24 and 76,

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737Hepatic histology of interferon treated HIVpatients with chronic hepatitis C

Table 3 Histological changes induced by IFN treatment in 12 HIVpositive patients

FocalPiecemeal hepatocellular Portal Score Histological

Case Time necrosis necrosis inflammation Fibrosis (HAI) diagnosis ofCH

1 0 1 1 3 1 6 Mild18 3 1 3 1 8 Mild

2 0 1 1 1 3 6 Mild18 3 3 3 3 12 Moderate

3 0 3 3 3 1 10 Moderate18 1 3 3 3 10 Moderate

4 0 3 3 3 1 10 Moderate18 1 1 3 1 6 Mild

5 0 3 3 3 3 12 Moderate18 5 3 4 3 15 Severe

6 0 5 3 3 3 14 Severe18 3 1 3 3 10 Moderate

7 0 1 3 1 1 6 Mild18 3 3 3 1 10 Moderate

8 0 3 1 3 1 10 Moderate18 1 1 3 3 8 Mild

9 0 1 3 1 1 6 Mild18 3 3 3 1 10 Moderate

10 0 3 3 3 1 10 Moderate18 3 3 3 3 12 Moderate

1 1 0 5 4 4 3 16 Severe18 4 1 3 3 11 Moderate

12 0 1 3 3 3 10 Moderate18 1 3 3 3 10 Moderate

Time 0, before treatment; time 18, six months after treatment stopped.HAI, histological activity index according to Knodell's criteria'4; CH, chronic hepatitis classifiedaccording to Desmet et al."

Table 4 Histological changes induced by IFN treatment in 12 HIV negative patients

FocalPiecemeal hepatocellular Portal Score Histological

Case Time necrosis necrosis inflammation Fibrosis (HAI) diagnosis ofCH

1 0 1 3 3 1 8 Mild18 0 0 0 1 1 Minimal

2 0 1 3 1 3 8 Mild18 1 0 1 1 3 Minimal

3 0 1 1 1 1 4 Mild18 0 0 1 1 2 Minimal

4 0 1 3 3 1 8 Mild18 1 1 3 1 6 Mild

5 0 3 1 3 1 8 Mild18 0 0 1 1 2 Minimal

6 0 1 1 1 1 4 Mild18 0 1 0 1 2 Minimal

7 0 1 0 1 1 3 Minimal18 0 1 1 1 3 Minimal

8 0 3 3 1 1 8 Mild18 1 1 1 1 4 Mild

9 0 1 1 1 1 4 Mild18 0 0 1 1 2 Minimal

10 0 1 1 1 1 4 Mild18 0 0 3 1 4 Mild

11 0 1 3 1 3 8 Mild18 0 0 1 1 2 Minimal

12 0 1 1 1 1 4 Mild18 0 0 1 1 2 Minimal

Time 0, before treatment; time 18, six months after treatment stopped.HAI, histological activity index according to Knodell's criteria'4; CH, chronic hepatitis classifiedaccording to Desmet et al."

respectively; p < 0.002), and remained lowersix months later in group B than in group A (66and 105, respectively; not significant).According to the above reported criteria, in

group A there were four sustained respondersand eight non-responders, while in group Bthere were six sustained responders, five re-lapsers, and one non-responder (not signifi-cant). In case 7 of group A a marked decrease ofthe serum ALT was found at time 12, althoughconcentrations were above normal; at time 18ALT levels were normal and the patient wasclassified as a sustained responder. At the end ofthe treatment, HCV RNA was negative in onepatient from group A (case 4, a sustainedresponder), and in six sustained responders ofgroup B (cases 1, 3, 5, 8, 9, and 12) (p < 0.05).

HCV genotypes are compared with theresponse to IFN in tables 1 and 2; the changesin the histological picture induced by IFN attime 0 and 18 are reported in tables 3 and 4.IFN therapy had different histological effects

in the two groups of patients. The mean valueof the Knodell's index increased from 9.6 to10.2 (not significant) in the HIV positivegroup, and decreased from 5.9 to 2.7(p < 0.005) in the HIV negative group.The response to IFN is better defined by

evaluating the single parameters of Knodell'sindex. In group A, the mean values ofpiecemeal necrosis and focal hepatocellularnecrosis did not change significantly (fig 1). Ingroup B, these subindices decreased signifi-cantly (piecemeal necrosis, p < 0.005; focalhepatocellular necrosis, p < 0.008) (fig 2). Inboth groups, the mean value of fibrosis andportal inflammation did not change signifi-cantly.Using the classification of Desmet et al,15 a

worsening of the the histological picture wasfound in four patients in group A (cases 2, 5, 7,and 9) (fig 1), while four patients (4, 6, 8, and1 1) improved, and four patients (1, 3, 10, and12) did not change their histological gradingafter therapy. On the contrary, no patient fromgroup B worsened, eight improved (1, 2, 3, 5, 6,9, 11, and 12) (fig 2), and in four patients theliver damage did not change significantly (4, 7,8, and 10).The evaluation of liver iron deposition

showed fine granules, barely discernible at amagnification of x250 and x400 (grades 0 and1); these were found mostly in the cytoplasm ofhepatocytes and sinusoidal cells, and occasion-ally in the portal spaces, in all the cases.

DiscussionA large number of studies have found positiveeffects of IFN treatment in HIV negativepatients with chronic hepatitis C, with im-provement of biochemical6 10 15 9 and histologi-cal pictures.8 9 20 In detail, this therapy normal-ises ALT in about 25-45% of the patients,'819and variably decreases lobular, portal, orperiportal damage,8 and even fibrosis.9 Thereare only a few papers in the literature reportingthe effects of IFN on chronic hepatitis C inHIV positive patients, most of them beingevaluated on the basis ofbiochemical responsesonly.'°0 " In addition, when histological studieswere performed, only some of the cases seem tohave been investigated and no scoring systemswere used.'2 Indeed, Boyer et all' described theevolution of serum ALT after a six monthcourse of IFN in 12 HIV positive patients anddid not find any differences in rates of responsefrom those usually described in the literaturefor HIV negative patients. Liver biopsies wereperformed at the end of the therapy in only twocases and showed improvement in one patientand no response in the other. Dalton et alreported in an abstract," a normalisation ofserum ALT in one of five HIV positive subjectswith chronic hepatitis C after six months oftherapy, but they did not report any data aboutthe histological evolution. Marriott et al,'2described the effects of high dose IFN in 14

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..sL ..

Figure 1 Liver biopsy of moderate chronic hepatitis C in a patient with HIVin(case number 10) (A) before and (B) six months after the end of interferon trealthe persistence ofpiecemeal necrosis and portal inflammatory infiltrate. Haematceosin, original magnification x200.

*-.d;

Figure 2 Liver biopsy of mild chronic hepatitis C in an HIV negative patientnumber 2) (A) before and (B) six months after the end of interferon treatment. ipiecemeal necrosis and portal inflammatory infiltrate are considerably decreasedHaematoxylin and eosin, original magnification x200.

HIV positive patients with chronic heLiver biopsies were obtained frompatients before the treatment but onlysecond biopsy six months after thttherapy and in all of them the authorsimprovement of histology. However,not evaluate the changes in liver histoscoring systems; and in addition, no c

available about the evolution of fibrograde of inflammation during treatme

In the present study we considchanges in liver damage after a 1.

therapy with IFN, in 12 HIVintravenous drug users with chronicC. This group was compared with a

12 patients with chronic hepatitis C,the same risk factors, mean age, maratio, and duration of intravenous drition, but differing in the absence ofHtion.

The number of cases was relatively small, asit is difficult to find patients fulfilling the abovereported criteria in HIV positive subjects(especially the absence of other viral and oropportunistic infections). In addition, the poorcompliance ofHIV positive patients, especiallywhen drug addicts, is well known. On the otherhand, the relatively short period of the study isjustified by the lower expectation of life in HIVpositive subjects with respect to the HIV nega-tive ones.Our results point out that the response to

IFN in HIV positive patients with chronichepatitis C is different from that observed inthe HIV negative group.The histological out-come indicates an overall worsening of liverdamage in HIV positive patients comparedwith HIV negative ones.

Pretreatment histological parameters havebeen indicated in several papers as possibly

ifection conditioning the response to treatment in HIVtment. Note negative patients. High pretreatment scores ofxylin and periportal and lobular necrosis are considered

to be associated with high rates of response tothe therapy,8 20 21 because they reflect activity ofthe immune system against HCV, which couldfacilitate the immunomodulatory mechanismof IFN. On the contrary, the role of fibrosis iscontroversial: marked fibrosis may represent adisease of long duration that could lead todevelopment of HCV strains resistant to IFNaction," causing a poor response to thetherapy. However, other groups have not founda significantly decreased response to IFN inpatients with a high fibrosis score or evencirrhosis.9 24

In our study, the mean value of the scores forpiecemeal and focal hepatocellular necrosisbefore the treatment were markedly higher ingroup A than in group B and, therefore, poten-tially more favourable; nevertheless, the re-sponse to the therapy was significantly worsefor these subindices in group A than in groupB. The scores of portal inflammation worsenedin HIV positive patients and improved in theHIV negative patients but their changes were

~'cote that not statistically significant.in (B). Finally, liver iron deposition, sometimes

found in chronic hepatitis C, has been reported.atitis

to complicate the clinical course of hepatitispatltls C. and modify the response to IFN.25 26 For these

.all the reasons, liver iron deposition was assessed onsx had a liver biopsies, and only traces of iron wereefend of found in lobuli and portal spaces; this was con-found an sidered normal.they did The different rates of response to IFN in0logy with HIV positive subjects with chronic hepatitis Cdata were might be caused by several factors. Thesis or the impairment of the immune system caused bynt. HIV infection could induce greater replicationLered the of HCV and therefore more liver cell damage,2 month as suggested by Martin et al.4 In our study,positive, none of the HIV positive patients progressed tohepatitis AIDS and CD4 counts remained over 200group of cells/mm3, suggesting that other factors couldshowing be responsible for the lower response to IFN.

ile:female These could include a significantly lowerug addic- immunomodulatory activity of IFN in HIV[IV infec- positive patients or a promoter effect ofHIV on

HCV, as already demonstrated for other

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Hepatic histology of interferon treated HIVpatients with chronic hepatitis C

viruses.27 28 Another cause of liver damagecould be an indirect mechanism ofthe action ofHIV. Scoazek et al29 demonstrated the presenceof HIV receptors on Kupffer cells and hepaticsinusoidal cells; Housset et al'0 providedimmunohistochemical evidence of viral pro-teins in Kupffer cells and suggested that thesecells may constitute a reservoir for HIV in theliver. In in vitro studies, Lafon et al" observedthe presence of HIV particles in sinusoidalendothelium and Kupffer cells, in addition tovarious degrees of ultrastructural and func-tional damage. Damage to the endothelial bar-rier could modify the absorption of metabolitesand drugs and, therefore, impair IFN efficacy.

In our study, IFN therapy produced an over-all stabilisation of fibrosis in both groups ofpatients and no cases evolved to cirrhosis. Inthis respect, our results are similar to those ofMarriott et al.'2 In five out of the six patientswho underwent liver biopsy they did not findcirrhosis after the treatment; in the sixth casecirrhosis was present before the treatment butit improved after treatment. Martin et al'described three cases of chronic hepatitis C inHIV positive patients that progressed rapidly toliver cirrhosis, but the patients were elderly,infected by blood transfusion, and were nottreated with IFN.

Serological responses to the treatment weredifferent between the two groups of patients,although the difference was statistically signifi-cant only for the virological response (clear-ance of serum HCV RNA), and not for thebiochemical response.The percentage of sustained responders of

the non-HIV group was higher than thosereported in the literature.'8 This could be areflection of the different groups studied: theyoung age, infection by drug addiction, and theespecially high incidence of the HCV genotype3a in our patients have been reported as prog-nostically favourable conditions.32Our data for HIV positive patients are differ-

ent from those of Boyeret al' and Marriott etal."2 The former reported complete or nearlycomplete responses to IFN in 58% of HIVpositive patients with chronic hepatitis C; how-ever, these patients were treated for only four tosix months and did not undergo a comparablefollow up. In the work by Marriott et al,'2 onlynine out of 14 patients completed the therapyand four of them were sustained responders(45%) and were negative for HCV RNA sixmonths after the end of the therapy. These dif-ferences in results could be explained by thebetter immunological status of their patients(mean CD4 cell count more than 580 cells/mm') than ours (mean CD4 cell count 465cells/mm'), and by the different schedules oftreatment.The rate of response to IFN has been

reported to be connected with HCV genotypes;genotype 3a in particular is strongly associatedwith a high rate of response to IFN.6 32 33 In ourstudy, the outcome of chronic hepatitis C inHIV positive cases seems not to have beenrelated to the viral genotypes. In fact, one out ofsix patients from group A with genotype 3a hada sustained response; while five out of six cases

ofHIV negative patients with genotype 3a weresustained responders.Although no definitive conclusions can be

drawn, owing to the small number of cases andthe relatively short period studied, the treat-ment of chronic hepatitis C with IFN therapyproduces lesser rates of response in HIVinfected patients than in HIV negative patients,as judged by serological and histologicalparameters. Nevertheless, because IFNtherapy seems to prevent the progression offibrosis, HIV positive patients should still betreated in view of their more rapid progressionto liver cirrhosis as reported in the literature.4

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