virus and host factors are both important determinants of response to interferon treatment among...

j o i i r t i d i f V i r d Hrpntitis. 1996. 3 , 85-96

Virus and host factors are both important determinants of response to interferon treatment among patients with chronic hepatitis C R. Lin, C. Liddle, K. Byth and G. C. Farrell Storr I . i w Utiit. Drpnrltwnl ojMrdiciw Utiiwrsit!j ojS!jdnr!j nt Wrslrnmd Hospital.

Wrs1twnd. Austrnlin

SUMMARY. Virus and host factors have both been linked to the response to interferon treatment among patients with chronic hepatitis C but their relative importance and potential interactions are unclear. Hepatitis C virus genotype and level of viraemia were determined in pretreatment sera from 65 Australian patients treated with interferon-a2b (IFN-a2b). 3 MU tiw for 6 months. Hepatitis C viraemia was quantitated by a competitive reverse transcription-polymerase chain reaction (RT-PCR) method and genotype was determined by a line probe assay. By univariate analysis, there were positive associations between initial (short-term) responses to IFN treatment and younger age (P=O.O04). absence of cirrhosis ( P = 0.01). and injecting drug use as risk factor for infection ( P = 0.05) but not gender, duration of infection, or level of viraemia. Genotype appeared to be important ( P = 0.06) but failed to reach statistical significance. By multivariate analysis, absence of cirrhosis was the only

significant independent predictor of treatment response ( P = 0.01). Among initial responders, the factors associated with long-term response were the pretreatment HCV RNA titre and the duration of infection. There was a close association between viral genotype. but not viral load, and the severity of liver disease. An interplay of factors determines the outcome of a 6-month course ofinterferon treatment for hepatitis C. Severity of liver disease, but not the viral load, is the most crucial determinant of initial response to interferon, and histological severity appeared to be influenced by the viral genotype. The level of hepatitis C virus (HCV) viraemia and the duration of infection are independent determinants of long-term response by affecting the relapse rate after interferon treatment.

Key words: chronic HCV. host determinants, response; virus.

INTRODUCTION

Since identification of the hepatitis C virus (HCV) genome in 1989 [l]. it has been shown that HCV is a common cause of chronic liver disease in all parts of the world [2]. Among infected individuals, chronicity devel- ops in 50-70% and, in at least 20% of these, chronic

Abbreviations: ALT. alanine aminotransferase: Kl', blood product transfusion: EI.ISA. enzyme-linked immunosorbent assay: HCV. hepatitis C virus: IDU. intravenous drug use: IFN. interferon: LTR. long-term response; NR. non-response: RT-PCR. reverse transcription-PCR: SP. sporadic infection; STR. short-term response: S'UTR. 5' untranslated region.

Correspondence: Professor GeolTrey C. Farrell. Storr Liver Unit. Westmead Hospital. Westmead. NSW 2 145. Australia.

hepatitis results in the development of cirrhosis within 20 years. Effective treatment of HCV infection is the only known way to prevent the progression of chronic hepatitis C. Administration of interferons (IFN) of the a@ subtypes normalizes serum aminotransferase levels and clears serum HCV RNA in about 50% of cases [3-81. The short-term response rate has been higher ( 6 5%) among Australian patients [ 7,9], and the reasons for this are not clear. A major problem with IFN treatment continues to be the high frequency of relapse upon drug withdrawal. Thus, long-term responses have been achieved in only 1 5-30'% of cases overall [ 10-1 I ] .

In order to improve the cost efficacy of IFN treatment for hepatitis C, it is desirable to identify factors that are predictive of a good outcome, especially long-

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86 R. Lin et al.

term response with eradication of HCV viraemia. Several investigators have noted that host factors, such as age [6,13,14], gender [6], risk factor for acquisition of HCV infection [ 7,131, duration of infection [ 151 and histological severity of liver disease [6,7,15-181 are associated with response to IFN treatment. More recently, it has been suggested that viral determinants may also play a major role in determining the long- term response [ 19-26], but the respective importance of viral and host factors and the ways in which they are inter-related have not been clearly characterized.

Assessment of viral genotype in relation to outcome of IFN treatment of HCV has been complicated by the number of genotypes, including a high proportion of mixed infections, in isolates from Japan, North America and Western Europe [27.28]. We postulated that the comparatively high response rate to IFN treatment among Australian patients with chronic HCV might be due to an unusual frequency of IFN-responsive HCV genotypes, such as type 2 or type 3 (using the classification of Simmonds [29]). To test this, the frequency of HCV genotypes was determined in stored sera from carefully studied patients who had been treated with IFN and whose initial (short-term) and long-term treatment outcomes had been documented, virologically as well as biochemically and clinically.

Several studies have examined HCV load as a determinant of the outcome of interferon treatment [ 19.22,2&26]. The most frequent finding has been that the pretreatment circulating level of HCV RNA does not correlate with response to IFN during treatment, but the likelihood of a long-term response is greater in those with lower concentrations of circulating virus particles. In the present study, we developed a competitive PCR method that incorporated an internal standard for all the steps, including reverse transcription. This assay allowed the viral load to be quantitated accurately in every pretreatment sample. The data were used to determine how viral load correlated with outcome of IFN treatment, the possi- ble inter-relationships between viral load and genotype, as well as to the other host and disease-related factors associated with treatment outcome.

SUBJECTS AND METHODS

Enrolment ojpatients

The 65 patients (41 males and 24 females) included in this study all had serologically, virologically and histo-

logically proven chronic HCV infection. Most cases (n = 40) were part of an Australian multicentre trial of IFN-a2b (Intron A@, Schering-Plough International, Kenilworth, NJ) for chronic hepatitis C, the results of which have been published in abstract form 191. The remainder were either from a smaller randomized controlled trial of IFN-a2b (n = 17). the results of which have also been reported [7] or were given IFN on a compassionate basis (n = 8). This study was approved by the Human Ethics Committee of the Western Sydney Area Health Service, conformed to accepted international standards of ethical propriety, and all patients gave written informed consent.

Subjects were all anti-HCV positive by a second- generation enzyme-linked immunosorbent assay (ELISA) (Ortho Diagnostics, Raritan. NJ). All were positive for HCV RNA on pretreatment serum by reverse transcription-polymerase chain reaction (RT-PCR), using primers derived from the S’-untranslated region of the HCV genome (5’-UTR) as described below. All patients had had a liver biopsy performed within 6 months prior to treatment which demonstrated chronic hepatitis with or without cirrhosis. Histological assessment was made before treatment by an experi- enced pathologist, and the diagnoses of chronic hepatitis and cirrhosis were made according to the criteria outlined by Scheuer [ 301. Other causes of chronic liver disease, including infection with other hepatitis viruses, were excluded as described previously [ 71. Patients with any of the following were excluded: anti- HIV positivity, pregnancy, presence of other serious medical or psychiatric illness, presence of leukopenia (white cell count < 3 x lo3 mm-’) or thrombocytopenia (< 7 0 x lo’mm-’).

32 (49%) patients had acquired chronic HCV infection through previous intravenous drug use (IDU). 2 (3%) were at risk through occupational exposure, 2 1 (32%) had received transfusions of blood or blood products (BT) and 10 (1 6%) had no obvious parenteral risks [designated as sporadic (SP) cases]. Chronic hepatitis alone was found in 45 (69%) cases, while the remaining 20 ( 3 1%) had chronic hepatitis with established cirrhosis. Duration of infection was estimated in cases with parenteral risk factors as the time from blood transfusion or the time from first IDU.

Patients were selected on the basis that they were PCR positive before treatment and had undergone treatment for 6 months. In order to determine the pretreatment factors associated with outcome of IFN treat-

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Response to IFN treatment among HCVputients 87

ment, cases were classified according to the treatment response, defined as follows. lnitial (short-term) response (STR) to IFN was defined as normalization of serum alanine aminotransferase level (ALT) and loss of PCR signal during and until the end of treatment irrespec- tive of the post-treatment course. Relapse was defined as elevation in ALT after drug withdrawal. Long-term response (LTR) was defined as normalization of ALT during treatment with continued normal values for at least 12 months after discontinuation of IFN, together with negativity of serum HCV RNA during and at 6 months after treatment. Non-response (NR) was failure to nor- malize ALT during treatment. Selection of cases for the study was designed to ensure similar numbers of non- responders, short-term responders who relapsed and long-term responders.

Treatment schedule and follow up

All patients received IFN-a2b at 3 MU administered subcutaneously thrice weekly for 6 months. Patients were reviewed at 4 -weekly intervals both during treatment and for 6 months thereafter. Subsequent assess- ments were conducted at 9 and 12 months after completion of IFN. Liver tests and peripheral blood count were performed during each visit. Sera for HCV RNA testing were collected before, during (at 3 months), at the completion of therapy (6 months), and at 6 months after treatment withdrawal, using a protocol that con- forms to accepted international standards to minimize RNA degradation [ 3 11. Thus, venous blood samples were spun within 30min of collection, aliquots (200 pl) of serum were placed under aseptic conditions in sterile storage tubes to which 500 pl of a 4 M guanidinium thiocyanate solution were added. Samples were frozen and stored at -70°C until the time of viral assays.

Hepatitis C viral markers

The presence of serum HCV RNA was determined by single-round RT-PCR. The specificity of this assay was confirmed by Southern hybridization utilizing a probe that lies internal to the amplified product. Absence of contamination was checked routinely, as described below. RNA was extracted from 200pl of serum by addition of 500 pl of a 4 M guanidinium thiocyanate solution, followed by addition of 50pl of 3 M sodium acetate (pH 5.2), 500pl of phenol (Sigma, MO), and l O O p l of ch1oroform:isoamyl alcohol (49:l). The mix-

ture was vortexed for 10s and chilled on ice before cen- trifugation (13000g for 20m at 4°C). The aqueous phase was transferred to a fresh tube and tRNA (Sigma, MO) was added. An equal volume of propan-2-01 was added and the solution was mixed well before storage at -20°C overnight. RNA was then pelleted by centrifuga- tion (as above), washed with 70% ethanol, dried and dissolved in l O p l of RNAase-free water.

To synthesize cDNA, the RNA solution was made up to 20 pl with the buffer recommended by the manufacturer and reaction solution: the latter contained 20 pmol of anti-sense primer (S'-GGTGCACGGTCTAC- GAGACCT-3' as described by Okamoto et al. [32]), 0.2 mM of each dNTP, 15 U of ribonuclease inhibitor (Promega Corp., Madison, WI) and 12U of Avian myeloblastosis virus reverse transcriptase (Promega Corp, Madison, WI). Reverse transcription was carried out at 42°C for 60rnin. The resultant cDNA was added to 30pl of PCR solution containing the recommended buffer along with 20pmol of sense primer (5'-GGCGA- CACTCCACCATAGAT-3'), 0.1 mM of each dNTP, 1 . 5 m ~ of MgC12, and 2.5Uof Taq polymerase (Promega Corp, Madison, WI) in a final volume of 50 pl. The mixture was covered with mineral oil and amplified in a DNA thermal cycler (Perkin Elmer Cetus Corp.. Norwalk, CT). Initial denaturation of DNA products was accomplished by incubation at 94°C for 5min, followed by 3 5 cycles of 94°C for 1 min, 5 5°C for 1 min, 72°C for 1 min and final extension at 72°C for 5 min.

The resultant PCR products consisted of 32 3 bp, as demonstrated by electrophoresis on 2% agarose gel and staining with ethidium bromide. Stringent precautions to prevent contamination were undertaken as outlined by Kwok & Higuchi [ 3 31. All samples were assayed twice and results were reproducible. Positive and negative controls were included in all runs. Following electrophoresis, the PCR products on the agarose gel were blotted on to a nitrocellulose membrane. A 5'-end [ "P]labelled probe ( 5'-CAA'ITCCGGTGTACTCACCGG'ITCCGC-3'), the sequence of which lay between the RT-PCR primers, was used for hybridization overnight at 3 7°C. The blot was then washed in 6 xSSC four times at room tempera- ture followed by four 20-min washes at 60°C. The blot was autoradiographed overnight at -70°C.

Quantitative RT-PCR for HCV RAJA

To quantify the level of HCV viraernia in pretreatment sera, a competitive RT-PCR method was used whereby

8 1996 Blackwell Science Ltd. Iournal ol Viral Hepntitis. 3 ,8 5-96

88 R. Lin et al.

a synthetic RNA template was introduced as the internal standard: this template was identical to the target sequence except for a deletion of 70 bp. To produce the template ('mutant HCV RNA fragment'), cDNA containing nucleotides 1-324 from the 5'UTR of the HCV genome was ligated into the plasmid pBlue Script Ks at the SnraI site, This plasmid, pllKS.D8, was obtained courtesy of Dr Eric Gowans (Sir Albert Sakzewski Virus Research Centre, Royal Childrens Hospital, Brisbane, Queensland. Australia). The plasmid was digested with two restriction enzymes, Bsgl and Bbsl (Promega, Madison, WI) that have unique restriction sites respectively at nucleotides 115 and 45. This resulted in a 70 bp deletion. The two ends were then filled using Klenow DNA polymerase (Promega, Madison, WI) and a blunt-ended self-ligation of the plasmid was performed. The pllKS.D8 plasmid was linearized with BnmHI (Promega, Madison, WI), followed by in vitro transcription with T3 RNA polymerase (Promega. Madison, WI) performed according to the manufacturer's instructions. Following transcription, residual DNA template was removed with RNAase free DNase RQ-I (Promega, Madison, WI). The resultant RNA was purified by phenol-chloroform extraction and precipi- tated with ethanol. The molar RNA concentration was determined spectrophotometrically by absorbance at 2 60 nm.

A dilution series (from 10 to 10' mol. ml-') was made of the internal standard RNA sequence, while the target RNA (sample HCV RNA) was added at a con- stant concentration. The standard and target RNA templates were then reverse transcribed together, so that competition for reverse transcription occurred. This manoeuvre provided an appropriate internal

reverse-transcription control, a reaction that has been reported to be only 40-50% efficient [34,3 51. Subsequent PCR was performed according to the conditions described above. The sensitivity of the competitive PCR assay was such that the lower limit for detection of the mutant HCV fragment by RT-PCR was 100 mol. ml-' .

After staining with ethidium bromide, the two PCR products were distinguished on a 2% agarose gel by virtue of the difference in their sizes (Fig. 1). As the equivalent point determined in this technique is depen- dent on the molecular mass as well as the concentration of each template, the fluorescence of the internal standard template (which consisted of 2 5 3 bp, 70 bp less than the natural HCV sequence) was corrected by a factor of 323/253 to allow direct comparison of the fluorescence intensities generated by the mutant and natural HCV RNA templates. To determine the equivalent point, the log,,, ratio of the fluorescence intensity of the 2 5 3 bp band to that of the 32 3 bp band was plotted against the log,,, titre of mutant template added to each reaction (Fig. 2). The data points were fitted using linear regression with the typical r value being 0.96 (range, 0.93-0.99). At the equivalent point, the ratio between the competing RNA and the target sequence of interest is one (i.e. log,,, is 0). Thus exploration from the plot where the Y value was zero provided an accurate estimate of the number of HCV RNA molecules present in the test sample (Fig. 2). The results were expressed as log,,, molecules HCV RNA/ml serum.

HCV genoQjping

The classification of HCV genotypes in this study was according to the nomenclature of Simmonds 1291.

Fig. 1 Example ofcompetitive PCK. Lane 1, molecular marker VI (Boehringer Mannheim. Mannheim. Germany). Lanes 2-3, positive control. Lanes 4-1 1, decreasing concentration of mutant HCV fragment by one log in each lane, from 10" mol. mlF' to 1 0 mol. ml-'. The lower band (2 5 3 bp) is the mutant fragment. while the upper band ( 32 3 bp) is the target HCV KNA.

1 2 3 4 5 6 7 8 9 10 11

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Response to ZFN treatment nrnong HCVpatients 89

RESULTS '1

f

2 3 4 5 6 7 8 Concentratlon of mutant HCV RNAfragment

(log moi. mi-')

Fig. 2 Determining the concentration of target HCV RNA. Log,,, of the ratio of mutant HCV RNA over target HCV RNA is plotted against the known concentration of mutant HCV RNA added to each reaction. Where the Y value equals 0. i.e. the equivalent point. the concentration of the mutant fragment is the same as that ofthe target HCV RNA.

which is based on nucleotide sequence variability in the 5'-UTR sequence of the virus. The line probe assay (LIPA, Innogenetics, Belgium) used is based on a reverse-hybridization technique and allows classification of the five most common types of HCV genotypes, types 1 ( l a and lb) . 2 (2a and 2b), 3 , 4 and 5 . The assay was performed according to the manufacturer's instructions.

Statisticnl annlyses

Data were analysed using logistic regression analysis. The outcome variable was response to IFN and the co- variables considered were age, gender, mode of transmission, presence or absence of cirrhosis, pretreatment viral titre, HCV genotype and duration of infection. Both univariate and multivariate analyses were performed. The latter identified the independent predictors of response. Analysis was performed on a NEC computer using the SPSS Release 4 package (SPSS Inc, Chicago. IL). The significance level for all tests was P< 0 . 0 5 .

To compare the viral load in the different genotypes, a one-way ANOVA and the least-significant difference method of adjusting for multiple comparisons were used [ 3 61.

Hostfattors arid initial response to ZFN

Among the 65 patients selected for this study who were treated with IFN for h months. 43 (66%) had responded to treatment, 17 ( 4 0 % ) had a long-term response following drug withdrawal, 26 (hO'%l) relapsed, and 22 (34%) did not respond.

Univnrinte nnalysis

The characteristics of the 65 cases are summarized in Table 1. There was no gender difference between initial responders and non-responders, but responders were significantly younger (Pc0.01). The risk factor for HCV infection was also associated with response to IFN. Thus the initial response rates among the IDU. occupational, BT and sporadic groups were 7 5'%,, 1 00'%1, 5 7% and 50% respectively (Table 1). Patients from the sporadic group were less likely to respond to treatment compared with the IDU group (P< 0.05). The duration of infection in the 5 3 cases for whom this could be estimated was similar between the responders and non-responders.

The histological finding of chronic active hepatitis without cirrhosis was associated with a significantly better initial response to IFN compared with established cirrhosis (82% vs 30%, P< 0.01) (Table 1).

Multivariate nnnlysis

Multivariate analysis failed to confirm age or mode of disease acquisition as independent determinants of initial response to IFN. The best-fitting multivariate model of response included only liver histology (presence vs absence of cirrhosis, P = 0.01 ) (Table 2).

Host fnrtors nnd long-tiv-ni response to IFN

As determined by univariate analysis, there was no relationship between age, gender, risk factor, histological severity of liver disease or any other factor to long- term response following IFN therapy. Duration of infection among responders, however, was significantly associated with relapse after treatment withdrawal (P=O.O5) such that the risk of relapse increased by a factor of 1.2 with every year since the patient acquired HCV (Table 2). Thus relapse after an initial response to treatment was not influenced by the severity of liver disease but was affected by the duration of infection.

90 R. Lin et al.

Table 1 Clinical and virological characteristics of initial responders and Non-responders Characteristic (n) Responders

Age (mean f SD) Sex (F/M) 17/26 Mode of transmission

4 2 f 12 years'

IDU (32) 2 4 (75%)

BT(21) 12 (57%) Occupation related (2) 2 (100%)

S P ( 1 0 ) 5 (SO%)?

No cirrhosis (45) 37 (821Y")S Histology

Cirrhosis (20) 6 (30%) Genotype

Type l ( 2 4 ) 12 (50%) Type 3 (38) 2 8 (74%)) Mixed ( 3 ) 3 ( loo'%,)

(molecules/ml)

105.5i 1.1 Pretreatment viral titre

I)urationofinfection(n = 53)$(years) 15.1 + 4 . 3

non-responders 5 3 f 14 years

711 5

8 (24%) 0 9 (41%) 5 (50%)

8(18%1) 1 4 ( 70'%1)

12 (50%) 10 (26%) 0

1 [ ) 5 . 3 * 1.2

15.5 + 6.4

11, number of cases: IDU, injecting drug use: BT, blood transfusion: SP, sporadic. Tomparing age of responders and non-responders (P = 0.004). tComparing risk groups, IDU to SP (P < 0.05). $Comparing histologic groups, cirrhosis versus noncirrhosis (P < 0.00 1 ). Comparisons are by logistic regression analysis. No other differences are significant. $Duration of infection was estimated on the 5 3 cases with parenteral risk factors,

Viralfactors and initial response to IFN

Pretreatment HCV RNA titre. The pretreatment serum titres of HCV RNA among the cases grouped according to treatment responses are illustrated in Fig. 3. HCV RNA levels did not differ significantly between non- responders and all responders (Fig. 3a: Table 1).

HCV genotype. Among the 6 5 selected cases, the most common HCV genotype was type 3 (n=38) (Fig.4). Other isolates were of type 1 ( n = 24) and mixed infections ( n = 3) between these two genotypes. Of the 24 patients with type-1 infection, five were infected with type l a and the remainder with type lb . As the number of type l a was small, the two subtypes were considered together. Initial response to IFN was observed in all cases with mixed infection, 74% of those with type 3 and 50% of those with type 1. On univariate analysis, genotype appeared to be important in that patients with type 1 were less likely to respond to IFN treatment compared with type 3. This difference, however, did not reach statistical significance ( P = 0 . 0 6 ) (Table 3 ) .

Virulfuctors and long-term response to IFN

Pretreatment HCV RNA titre. In contrast to the similar- ity in HCV RNA titre between responders and non- responders, the mean pretreatment viral titre of patients who had initially responded to IFN but who subsequently relapsed was significantly higher than

mol. ml-' respectively ( P = 0.01) (Fig. 3b).

that of long-term responders 105.y*".y "s 104.Y* 1.'

Table 2 Best-fitting multivariate models of initial and long- term response to interferon treatment

Odds Variable ratio 95%CI Pvalue

Initial response Histology (no cirrhosis 6.8 1 . h 2 9 . 4 0.01 vs cirrhosis)

Duration of infection (years) 1.2 1 .O. 1 . 5 0.0 3

Pretreatment viral titre 1 1 . 3 1 .4 .92 .1 0.02 (titre > 1 P molecules ml-' vs titre c 1 0 ~ . ~ molecules mI-'

Long-term response

~_______

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Response to ZFN treatment among HCVpatients 91

NS

8 "1 (*) 0 0

T E3

0

0

0

Y 8 Rmponders (n = 43) Non-responders (n = 22)

(b) P = 0.01 d 3 8

!2 7 -

3 .

2 Respondere who relapsed (n = 26) Long-term responders ( n = 17)

0

Fig. 3 Pretreatment HCV RNA titre in non-responders and short-term responders (a). and in long-term responders (b). Data are presented as box plots: the median values are indicated bythebarwithintheboxwhichcomprise the25thto 75th percentiles. Error bars denote 10th and 90th percentiles. NS. not significant. comparing viral titres of initial responders and non-responders. P = 0.01, comparing viral titres between responders who relapsed and long-term responders.

There was a statistically significant association between the risk of relapse and increasing viral load (P=O.Ol). To investigate this association further, patients were divided into three groups: those with a pretreatment load of 1104.5 rnol. ml-' (group 1). those whose load lay between 104.4-105.5 mol. ml-' (group 2) and those patients with a pretreatment load > 105.5 mol. ml-' (group 3). In group 1, long-term response was observed in four cases (67%). In group 2, it occurred in 10 patients (59%), while in group 3. only three patients (1 5%) achieved a long-term response. The odds of relapse in groups 1 and 2 were almost identical. However, the odds of relapse in group 3 patients were 11-fold higher (95% CI 1.4, 92) compared with those from group 1. The effect of pretreatment viral load on long-term response to IFN was independent of the duration of infection (Table 2).

HCV genotype. Among patients who had responded to IFN. long-term response was 25% in type 1, 50% in type 3 and 33% in cases with mixed infection (Table 3). None of the genotypes was found to have a significant influence on long-term response. Type 1 appeared to be more likely than the other genotypes to relapse after completion of treatment but this association was not significant (P = 0.09).

Inter-relationships between host and virus factors associated with short- and long-term responses to ZFN

HCV viral load and histology. Mean pretreatment serum HCV RNA levels were almost identical between cases

mol. ml-' respectively).

with and without cirrhosis ( 105.4* '.' and 1 0 5 . 5 * 1.1

HCV viral load and genotype. In Fig 4. the levels of HCV RNA for cases grouped by gemotype are presented. The

8 0 0

GI 4- 0

0

2 1 3 Mixed

(n = 24) (n = 38) (n = 3)

Fig. 4 Pretreatment HCV RNA titre according to HCV genotype. Format of box plot is as for Fig. 3

Genotype

Table 3 Hepatitis C viral genotype and initial and long-term responses to interferon treatment

Short-term Long-term response response n (%I of initial % of all

Genotype ( n ) ( n ) (%I) responders) treated

l ( 2 4 ) 12 ( 50%) 3 (2 5%) 12.5%

3 ( 3 8 ) 28 ( 7 4 % ~ ) ~ 14(50'%1) 3 7%)

Mixed ( 3 ) 3 ( 1 0 0 ' % 1 ) 1 ( 3 3%) 3 3%

No differences are significant. *Comparing initial response rate of genotype 3 to genotype 1 (P = 0.06).

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92 R . Lit1 et al.

mean HCV RNA titres for types 1. 3 and mixed infections were comparable.

HCV genotype and liver histology. The frequency of cirrhosis among cases classified according to viral genotype was 54% for type 1. 18% for type 3 and 0% for mixed infections (Table 4). Thus patients with type 1 were significantly more likely to have cirrhosis ( P = 0.02), while those with type 3 were significantly less likely to have cirrhosis (P=O.Ol). Patients with type-3 infection were found to be younger than those with type-1 infection (P=O.Ol). There were no other significant associations between HCV genotype and host factors in relation to treatment response including risk factor or duration of infection (Table 4).

DISCUSSION

The present data confirm that viral and host factors are both important determinants of response to IFN treatment in patients with chronic HCV infection, and that these factors are related. Earlier studies identified a variety of host and disease-related factors that were associated with a favourable response to IFN [6-7,141. The most consistent of these have been age and histological severity of liver disease: younger patients are

more likely to respond than older subjects. and patients without cirrhosis are considerably more likely to respond than those with cirrhosis [7,9,13-181. In addition, some, but not all, studies have found better responses in women than in men [ 6 ] . among those with injecting drug use as risk factor for infection [7.9,13]. and in patients with a shorter duration of infection [ 151.

In the present study, the age of the patient, the mode of disease acquisition and histologial severity of liver disease were associated, by logistic regression analysis. with the initial response to IFN treatment. However, it was revealed by multivariate analysis that the presence of cirrhosis was the only host factor that correlated independently with response. Several studies have employed multivariate analysis to examine associations between patient characteristics and outcome of IFN treatment for chronic HCV [7,13.14,22.37-391: cirrhosis was identified in each study as the most consistent independent predictor of treatment outcome. It would therefore seem likely that factors such as age. risk factor and duration of infection are identified in univariate analyses as they determine the likelihood of cirrhosis developing among patients with chronic HCV.

It should be noted that for a long-term response to IFN treatment to be achieved, two conditions must be

Table 4 Associations between host Type 1 Type 3 Mixed factors and hepatitis C virus genotype (ti = 24) (n = 38) (t1 = 3 )

Age (years. mean f SD) Risk for infection (n)

IDU (32) Occupation (2) BT(21) SP (10)

Duration ofinfection ( t i = 5 3 ) (years)

Histology (n) N o cirrhosis (45) Cirrhosis (20)

5 2 f 12

9 (28'%,) 0 8 (38%) 7 (70%)

16.3 f h.7

43f 1 3 *

20 (6 3%) 2 ( 100%)

1 3 (62%)

15.8* 5.4 3 ( 3 0 % )

3 2 f 3

3 ( 1 O O ' X r ) 0

*Cornparing the age of patients with type 1 and type 3 infection (P = (1.01). there were too few patients with mixed infection for comparison. tComparing the incidence of absence vs presence of cirrhosis in genotype 1 ( P = 0.02). $Comparing the incidence of absence vs presence of cirrhosis in genotype 3 ( P = 0.01). $Duration of infection was estimated in only one patient with mixed infection. No other differences are significant.

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Response to IFN treatment among HCVpatients 9 3

met. First, there must be a response during treatment, in this paper termed 'initial' or 'short-term' response as defined by normalization of ALT and loss of HCV viraemia. Second, relapse should not occur when therapy is withdrawn. In the present study, although cirrhosis was not associated with an increased relapse rate, the presence of cirrhosis still greatly influenced the chance of obtaining a long-term response to IFN treatment because it was the most important determinant of initial response. No other host factors in the present study population were associated independently with the relapse rate or the long-term response to IFN.

This study was designed to test the hypothesis that viral determinants are the most important factors that determine response to IFN treatment [ 19-26]. Specifically, we postulated that the relatively high response rates in Australian patients (65% short-term and 30% long-term response rates after 6 months treatment [7,9]) could be explained by a high local preva- lence of HCV genotypes that are responsive to IFN. Other authors have reported that different HCV genotypes are associated with variable treatment responses [20-2 31. Although response according to genotype did not quite reach statistical significance in this study ( P = 0.06). short-term response was more likely among patients infected with type 3 than in those with type 1. The failure to demonstrate significance may be related to the size of the study population. A strong association was also demonstrated between genotype and underly- ing histology: patients with type 1 were significantly more likely to have cirrhosis compared to patients with type 3. Thus it is likely that the influence of viral genotype on response to IFN may not be due to direct bio- logical effects related to viral replication or immune responses to it but may be an indirect effect consequent on the severity of the liver disease caused by infection with different viral strains.

The other determinants that could potentially be related to the outcome of IFN treatment are viral load, as determined by pretreatment titre of serum HCV RNA, and the duration of infection. Previous studies of viral load and IFN treatment have produced varying results, but most have not found a relationship between HCV RNA titre and short-term response [19.26,40]. The present results are in agreement with these findings. In contrast, the data clearly show that there was a direct relationship between viral titre and relapse after completion of IFN treatment. However,

unlike the other host and viral factors that determined treatment outcome, viral load affected long-term response by influencing relapse rather than by determining initial response. Other authors have reached similar conclusions regarding the role of viral load in determining long-term but not initial response to IFN [ 19,40,41].

The assays used to quantitate HCV RNA in some of the earlier studies were not truly quantitative for all patient samples. In particular, serial dilution studies are semi-quantitative while the branched-chain DNA (bDNA) assay fails to detect low viral titres in up to one- third of samples [26,40,42]. In the present study, a competitive PCR assay was developed that incorporated an internal standard for all steps of RT-PCR for HCV RNA. This assay was shown to be very sensitive and accurate. It allowed us to estimate the likelihood of post-treatment relapse according to the pretreatment HCV RNA titre. Thus, a viral load of > 105.5 mol. ml-' carried an 1 1-fold increase in the risk of relapse compared with a load of I 105.5 mol. ml-'.

The relationship between the amount of circulating virus particles and histological severity of chronic HCV has been controversial. Some authors have reported that higher HCV viral load is associated with more severe types of liver disease [43,44], while others have reported lower viral load among patients with mild chronic hepatitis (chronic persistent hepatitis) and similar loads in more severe chronic hepatitis or cirrhosis [45]. or even a fall in HCV RNA level with increasing severity of liver disease [40]. In the present study, the relationship between mean circulating viral titre and liver histology was examined by use of competitive PCR. It was evident that there was no difference in viral load between cases with and without established cirrhosis. Thus the influence of viral load on IFN treatment response is unrelated to histological severity of liver disease. The conflicting data of earlier studies may have resulted from methodological deficiencies of the assays used to quantify HCV RNA titre.

The effect of the duration of virus infection on treatment response in HCV has not been adequately defined. In a study by the US Hepatitis Interventional Therapy Group, the duration of infection did not affect short- or long-term response to IFN [46]. A Swedish study, however, has found that non-responders tended to present with a longer history of disease than responders, although this difference did not reach statistical significance [15]. A more recent meta-analysis of 361

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94 R. Lin et al.

patients concluded that a short duration of infection not only influenced short-term response to IFN but was also associated with a significantly lower incidence of relapse [47]. In chronic hepatitis B. patients with disease duration of more than 2 years have been found to respond less well to IFN treatment than those with a shorter history [48]. The same appears to be true for chronic HCV. It is often difficult to identify the time at which patients became infected, especially in the absence of parenteral risk factors or clinical features of acute hepatitis. This may partly explain why there is a lack of consensus data concerning the effect of duration on response to treatment. In this study, the duration of infection was calculated among 5 3 patients who had parenteral risk factors. As with pretreatment viral load, disease duration was found to influence outcome by virtue of its effect on relapse.

The present results have practical implications in considering case selection for IFN treatment. Knowledge about pretreatment histology remains the most important determinant of treatment outcome, even though viral genotype could be a critical determinant of histological severity. Quantitation of viral load is of no value in predicting the short-term response to IFN treatment, but among patients who have responded, pretreatment viral load becomes the critical determinant of relapse, and therefore of long-term response. Duration of infection, likewise, does not affect short-term response but is an important and independent determinant of long-term response among responders. The application of these findings in clinical prac- tice is that consideration of pretreatment histology (for short-term response) and pretreatment viral load, as well as the duration of infection (for relapse), should allow more accurate identification of cases that have a particularly high or low chance of a long-term response to IFN treatment. The predictive value of these factors should now be tested in other study populations. Larger studies are also required to determine whether knowledge of genotype adds predictive accuracy to pretreatment histology in determining short-term response to IFN treatment. Finally, the influence of the treatment regimen needs to be considered in relation to the long- term outcome. Several studies have failed to find an advantage in increasing dosage schedules to greater than 3MU thrice weekly [9,49,50], but others have indicated that a reduction in relapse rate can be achieved after a course of IFN treatment given for more than 6 months [9,51,52]. Whether prolonging IFN

treatment will overcome the disadvantageous effect of a high pretreatment viral load in predisposing to relapse is the subject of continuing research.

In conclusion, liver histology was the most important host-related factor associated with a short-term response to IFN among patients with chronic HCV treated for 6 months. Viral genotype was intimately associated with histological severity of liver disease, but was not an independent correlate of treatment response by multivariate analysis. Pretreatment viral load was not related to short-term response, but it was the only significant viral factor that determined the relapse rate, and therefore affected the frequency of sustained virological response. Similarly, the duration of infection affected long- term but not initial response to treatment. These findings support a complex interplay between host- related and virusdependent factors in pre-ordaining the response to IFN treatment of patients with chronic HCV infection. Application of these findings may assist in defining optimal case selection for IFN treatment.

ACKNOWLEDGEMENTS

We are indebted to Dr Eric Gowans for providing the plasmid pllKS.D8: to Associate-Professor Anthony Cunningham for technical support: to Ms Shirley Coverdale for serum collection: and to Schering-Plough Australia for sponsoring the two IFN trials. R.L. is an Australian National Health and Medical Research Council Postgraduate Medical Research Scholar. GCF is supported by the Robert W. Storr Endowment to the University of Sydney for Liver Research.

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