Pretreatment hepatitis C virus dynamics for predicting virological response to interferon-α2b monotherapy in patients with chronic hepatitis C virus infection

Hepatology Research 27 (2003) 181–191

Pretreatment hepatitis C virus dynamics for predicting virological response to interferon-␣2b monotherapy in patients with chronic hepatitis C virus infection Shigehiko Sainokami a,∗ , Kouichi Abe a , Kazuyuki Suzuki a , Kazuyoshi Ishikawa b a

The First Department of Internal Medicine, Iwate Medical University, 19-1 Uchimaru, Morioka, Iwate 020-8505, Japan b Department of Nursing, Iwate Prefectural University, 152-52 Sugo, Takizawa, Iwate 020-0173, Japan Received 22 January 2003; received in revised form 19 May 2003; accepted 3 June 2003

Abstract One hundred and forty-one patients with chronic hepatitis C virus (HCV) infection treated with 6 MIU of interferon (IFN)-␣2b for 24 weeks were studied to compare pretreatment viral dynamics during 1 month before the initiation of treatment (HCV) with other predictive factors. The patients were classified into three groups according to HCV: the Increase group (HCV > 0.20 log copies/ml/month), the Stable group (−0.20 ≤ HCV ≤ 0.20 log copies/ml/month) and the Decrease group (HCV < −0.20 log copies/ml/month). The sustained response (SR) rates were 0/40 (0%) in the Increase group, 6/54 (11.1%) in the Stable group and 25/47 (53.2%) in the Decrease group (P < 0.0001). Based on the HCV RNA level at baseline, the SR rates were lower in the Increase group than that in the Decrease group in less than 5.0 log copies/ml (0% vs. 100.0%, P = 0.0132) and at 5.00–5.49 log copies/ml (0% vs. 81.8%, P = 0.0270), respectively. The pretreatment HCV RNA decrease was determined as independent predictors of SR by multivariable logistic regression analysis (P < 0.0001). For serial assays of HCV RNA during treatment, there was not significant difference in mean HCV RNA level at baseline but was significantly different on day 14 between the Decrease and the Nondecrease groups (1.15 vs. 2.21 log copies/ml, P = 0.0417). These results suggest that HCV is independently associated with the IFN response and is useful for determining the indication of IFN treatment. © 2003 Elsevier B.V. All rights reserved. Keywords: Antiviral therapy; Hepatitis C virus; Viral dynamics; Sustained response

1. Introduction Treatment with pegylated interferon (Peg-IFN) [1,2] and a combination therapy with interferon (IFN) and ribavirin [3,4] have been attempted as a therapy for chronic hepatitis C and have been recently established as the most effective and available therapy, compared with IFN monotherapy. However, the percentage of patients who eliminated hepatitis C virus (HCV) is considered to be approximately 35– 40% after a 24- or 48-week course of the combination therapy [4]. In addition, it is considered that the main agent in the combination therapy is IFN. In combination with IFN, ribavirin increases the initial response and raises the ratio of

∗ Corresponding author. Present address: Mizusawa City Hospital, 3-1 Ootemachi, Mizusawa, Iwate 023-0808, Japan. Tel.: +81-197-25-3838; fax: +81-197-25-3834. E-mail address: [email protected] (S. Sainokami).

1386-6346/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1386-6346(03)00240-7

HCV elimination from infected hepatocytes, and markedly reduces relapse rates in these patients [4–6]. When administered alone, ribabirin was not proved to be efficient for patients with chronic HCV infection [7]. Therefore, it is necessary to develop a more effective therapy and investigate optimal factors to raise the availability of IFN therapy as the main agent. Several virus- and host-related factors affecting response to IFN have been defined to date. HCV genotypes 1a, 1b and 4a, and a higher baseline HCV RNA as virus-related factors [4,6,8–12] as well as male gender, an older age and histologically advanced case as host-related factors [3,4,6] have been shown to be resistant to IFN therapy. Furthermore, numerous studies have showed that daily administration of high-dose IFN is more effective than intermittent administration of low-dose IFN [13–15] and a long-term treatment significantly decreases the incidence of relapse [3,4,16,17]. However, the antiviral effect of IFN is still limited even if patients are treated considering the above-mentioned factors. In addition,

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IFN therapy is costly and accompanied by serious adverse effects during a long-term treatment. While it is necessary to investigate other useful factors to increase the efficacy of IFN, there are no reports on correlation between pretreatment HCV dynamics immediately before IFN treatment and the efficacy of IFN. The applicability of pretreatment HCV dynamics as a predictive factor of IFN therapy has also been unclear. Therefore, the aim of this study is to determine whether pretreatment HCV dynamics immediately before treatment is useful as a predictive factor of IFN treatment by investigating the changes in the level of serum HCV RNA during 1 month before treatment. In addition, we also studied the influence of pretreatment HCV dynamics on the early decrease in serum HCV RNA level during IFN therapy.

2. Patients and methods 2.1. Patients and treatment One hundred and forty-one patients with chronic HCV infection (male, 83; female, 58; mean age, 51.3 year) who had not received IFN treatment previously were enrolled in this study. All patients had undergone IFN-␣2b treatment from August 1991 to October 1998 (Table 1) at the National Defense Medical College Hospital. After informed consent had been obtained before IFN treatment, all procedures during the study were according to the Helsinki Declaration of 1975 (revision of 1983) and approved by the ethical comTable 1 Characteristics at baseline of 141 patients with chronic HCV infection Characteristics

Data

No. (M:F) Mean age (year)a AST (IU/l)a,b ALT (IU/l)a,b HBsAg ANA Anti-HCV

141 (83:58) 51.3 ± 10.2 (22–71) 88.1 ± 48.5 (23–283) 130.9 ± 83.0 (36–528) 0 (0%) 0 (0%) 141 (100%)

HCV genotype 1b 2a 2b 1b + 2b

118 (84%) 19 (13%) 3 (2%) 1 (1%)

Histological stage of fibrosisc Stage 1 (F1) Stage 2 (F2) Stage 3 (F3) Stage 4 (F4)

19 42 23 57

(14%) (30%) (16%) (40%)

AST, aspartate aminotransaminase; ALT, alanine aminotransaminase; HBsAg, hepatitis B surface antigen; ANA, anti-nuclear antibody. a Mean ± S.D. (range). b Normal reference ranges: less than 29 IU/l for AST; less than 34 IU/l for ALT. c Liver biopsy was performed within 6 months before initiation of treatment.

mittee of National Defense Medical College. The diagnosis of chronic HCV infection was based on elevated serum aminotransferase levels for more than 6 months, histological examination, consistent detection of anti-HCV antibodies (second generation enzyme immunoassay) and serum HCV RNA. Mean asparatate aminotransferase (AST) and alanine aminotransferase (ALT) levels (range) were 88.1 (23–283) and 130.9 (36–528), respectively. The causes of chronic liver diseases including hepatitis B virus infection, autoimmune hepatitis, primary biliary cirrhosis, alcohol abuse and druginduced liver disease were excluded from the study. Patients with decompensated cirrhosis, cancer, systemic illness, serious heart disease and renal disorders were also excluded. Patients were intramuscularly administered 6 MIU of recombinant IFN-␣2b, 6 days weekly for 2 weeks followed by three times/week for additional 22 weeks (480 MIU in total). All patients were followed up and the serum ALT level measured every 1 month from 1 month prior to IFN treatment to 12 months after cessation of IFN therapy. In all patients, liver biopsies were performed within 6 months prior to IFN therapy. The histological stage was determined on the basis of the degree of fibrosis as follows: Stage 0 (F0), no fibrosis; Stage 1 (F1), fibrous portal expansion; Stage 2 (F2), portoportal septa; Stage 3 (F3), portcentral linkage or bridging fibrosis with architectural distortion; and Stage 4 (F4), cirrhosis. The numbers of patients classified according to the histological stage were 19 in Stage 1, 42 in Stage 2, 23 in Stage 3 and 57 in Stage 4. 2.2. Determination of serum HCV RNA levels and HCV genotypes Serum samples were obtained 1 month (4–5 weeks) before initiation of treatment at baseline point and every 4 weeks during both the treatment and follow-up periods after the cessation of treatment. In 70 out of 141 patients, serum samples were also obtained on day 1, day 3, day 7 and day 14 of IFN treatment. All serum samples were stored by freezing at −80 ◦ C within 3 h of collecting until testing. The HCV RNA level in the sera was determined using 300 ␮l of serum sample by reverse transcription-nested polymerase chain reaction (RT-nested PCR) using the Amplicor HCV version 2.0 (Roche Japan, Tokyo, Japan), which had a lower detection limit of 50 copies/ml. HCV RNA in 100 ␮l of a serum sample was quantitated using the Amplicor HCV monitor version 2.0 (Roche Japan), which had a quantification range of 500–850 000 (2.7–5.9 log) copies/ml. The samples found to contain HCV RNA higher than the range of this assay were diluted accordingly with anti-HCV negative normal human serum. HCV genotyping was performed according to the method described by Okamoto et al. [18], which uses PCR amplification of core gene sequences with universal and five type-specific primers to generate DNA fragments with a size specific for five HCV types, and genotypes were classified according to Simmonds’ classification system [19].

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2.3. Response criteria We defined sustained response (SR) as undetectable HCV RNA during the entire follow-up period of 12 months after the cessation of treatment, together with persistently normal ALT levels ( < 34 IU/l). All other patients were considered as nonresponders (NR). 2.4. Statistical analysis All data are expressed as mean ± S.D. All HCV RNA determinations were analyzed after log10 transformation. The difference in viral load between 1 month before the treatment [HCV (−1 M)] and baseline [HCV (0 M)] was defined as HCV. Therefore, the HCV value was calculated as follows: HCV(logcopies/ml/month) = HCV(0M)−HCV(−1M) Furthermore, we classified the patients into three groups according to the HCV value: Increase group (HCV>0.20), Stable group (−0.20 ≤ HCV ≤ 0.20), Decrease group (HCV < −0.20). The χ2 test or Fisher’s exact test for expected values < 5 was used to compare two group frequencies as appropriate. Differences between variables were assessed by unpaired Student’s t-test. Analysis of variance (ANOVA) was used to

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compare variables among the three groups and Scheffe’s test as the post hoc test was performed when a significant difference was recognized by ANOVA. Moreover, multivariate logistic regression analysis was performed for four factors, i.e., pretreatment HCV dynamics, HCV RNA level at baseline, HCV genotype and stages of fibrosis, in order to assess whether these variables independently predicted the likelihood of SR. All P values were two-tailed and a level of P < 0.05 was accepted as statistically significant. Statistical analysis was performed by using statview 5.0 for Macintosh (SAS Institute Inc., NC, USA).

3. Results 3.1. Relationship between HCV RNA at baseline and pretreatment HCV dynamics according to IFN response Thirty-one (22.0%) out of 141 patients with chronic HCV infection showed SR to therapy. Patients with SR were distributed mainly in the low-level area of HCV RNA at baseline and the negative area of HCV (Fig. 1). On the other hand, patients with NR were distributed mainly in both the areas of high-level HCV RNA at baseline and the positive area of HCV. There were few patients with SR in the positive area of HCV even when HCV RNA level at baseline

Fig. 1. Relationship between HCV RNA at baseline and pretreatment HCV dynamics (HCV). Serum HCV RNA levels at baseline and 1 month before IFN-␣2b treatment were assessed by quantitative RT-PCR and the difference was subsequently obtained (HCV = HCV RNA at baseline—HCV RNA 1 month before treatment). Open circles indicate virological SR and solid circles indicate virological NR).

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was low. Furthermore, as the HCV RNA level at baseline increased, the range of HCV tended to be narrow, and there were no patients with SR when the HCV RNA level at baseline was higher than 6.0 log copies/ml even if HCV was negative. However, when the HCV RNA level at baseline was less than 6.0 log copies/ml, it was possible to achieve SR in patients with negative HCV compared with those with positive HCV. In addition, as the HCV RNA at baseline was decreasing, the probability that HCV value would be negative was higher. Therefore, patients tended to achieve SR when the HCV RNA level at baseline was lower and HCV was negative. The relationship between HCV RNA level at baseline and HCV in patients with SR was positively correlated (r = 0.424, P = 0.0165). 3.2. Relationship between HCV RNA level at baseline and pretreatment HCV dynamics according to histological stage and IFN response In any stage, patients with SR were concentrated in the low-level area of HCV RNA at baseline and the negative area of HCV (Fig. 2). In more than 5.5 log copies/ml HCV RNA at baseline, there were some patients with SR in Stage 1 or 2

but there were no patients with SR in Stage 3 or 4. Furthermore, even in advanced Stage 4, there were many patients with SR when the HCV RNA level was less than 5.0 log copies/ml and HCV was negative. All patients in Stage 4 who had HCV of less than −0.50 log copies/ml/month achieved SR. 3.3. Relationship between HCV RNA level at baseline and pretreatment HCV dynamics according to HCV genotypes and IFN response In this study, we compared genotype 1b with genotypes 2a and 2b because there were a few cases of genotypes 2a and 2b. In addition, one case of mixed genotype 1b + 2b was excluded from this analysis. Regarding genotype 1b, the range of HCV became narrow as HCV RNA level at baseline increased (Fig. 3). The distribution of HCV was nearly in the range between −1.0 and 1.0 log copies/ml/month in more than 5.0 log copies/ml of HCV RNA at baseline. In these conditions, patients with SR were distributed only in the negative area of HCV. On the other hand, in genotypes 2a and 2b, there were no patients in whom HCV was negative when the HCV RNA level at baseline was higher than 5.5 log copies/ml. Patients with SR distributed in the

Fig. 2. Scattered plots between HCV RNA at baseline and pretreatment HCV dynamics (HCV) according to histological stage ((a) Stage 1, (b) Stage 2, (c) Stage 3, (d) Stage 4) and IFN response in patients with SR (indicated by open circles) and with NR (indicated by solid circles). Viral load and HCV were lower in patients with SR as the histological stage advanced.

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However, HCV (−1 M) was 4.94 ± 0.86 (log copies/ml) in the Increase group, 5.55 ± 0.64 (log copies/ml) in the Stable group and 5.78 ± 0.64 (log copies/ml) in the Decrease group (P < 0.0001). In comparison among groups, HCV (−1 M) was significantly lower in the Increase group than that in the other groups (P < 0.01). In addition, HCV (0 M) were 5.57 ± 0.66 (log copies/ml) in the Increase group, 5.53 ± 0.64 (log copies /ml) in the Stable group and 5.14 ± 0.93 (log copies/ml) in the Decrease group (P = 0.0122). HCV (0 M) was significantly lower in the Decrease group than that in the other groups (P < 0.01). 3.5. SR rate according to HCV RNA level at baseline and pretreatment HCV dynamics

Fig. 3. Scattered plots between HCV RNA at baseline and pretreatment HCV dynamics (HCV) according to HCV genotype ((a) Genotype 1b, (b) Genotypes 2a and 2b) and IFN response in patients with SR (indicated by open circles) and with NR (indicated by solid circles).

negative area of HCV regardless of HCV RNA level at baseline. 3.4. Comparison of characteristics at baseline according to pretreatment HCV dynamics As for the pretreatment HCV dynamics, we classified the patients into three groups, i.e., the Increase group, the Stable group and the Decrease group; the mean HCV values for each group were 0.60 ± 0.55 (log copies/ml/month, −0.02 ± 0.11 (log copies/ml/month) and −0.66 ± 0.51 (log copies/ml/month), respectively (P < 0.0001). To estimate the influence of HCV on ALT level, we investigated ALT value at both 1 month before the initiation of treatment [ALT (−1 M)] and at the initiation of treatment [ALT (0 M)], and calculated the change in rate of ALT value during 1 month before treatment [ALT (OH)/ALT (−1 M)] (Table 2). Both ALT (−1 M) and ALT (0 M) levels were not significantly different among the three groups. ALT (0 M) of each group was lower than ALT (−1 M), but we noted no association between HCV and ALT (OH)/ALT (−1 M). Moreover, there were no significant differences in gender, age, histological stage and HCV genotype among the three groups (Table 2).

We divided the patients into four groups based on the HCV RNA level at baseline: < 5.00, 5.00–5.49, 5.50–5.99 and ≥ 6.00 log copies/ml, and investigated the SR rate in each group according to pretreatment HCV dynamics (Fig. 4). Among 31 patients with an HCV RNA level of < 5.00 at baseline, the proportions of patients with SR according to three groups of HCV were 0/8 (0.0%, the Increase group), 4/9 (44.4%, the Stable group) and 14/14 (100.0%, the Decrease group). The SR rate of the Decrease group was significantly higher than that of the Increase group (P = 0.0132). Among 27 patients with an HCV RNA level of 5.00–5.49 at baseline, the proportions of patients with SR according to three groups of HCV were 0/9 (0.0%, the Increase group), 2/7 (28.6%, the Stable group) and 9/11 (81.8%, the Decrease group). The SR rate of the Decrease group was also significantly higher than that of the Increase group (P = 0.0270). However, among 31 patients with an HCV RNA level of 5.50–5.99 at baseline, the SR rates of the Increase group, the Stable Group and the Decrease group were 0, 0 and 11.1%, respectively, which were not significantly different. Furthermore, among 56 patients with ≥6.00 HCV RNA at baseline, the SR rate was 0% in each group. Therefore, when HCV decreased < −0.20 log copies/ml/month, and the HCV RNA level at baseline decreased to less than 5.50 log copies/ml, we considered that we could expect a higher SR rate. Also, we considered that we could not expect an SR in patient with higher than 5.50 log copies/ml HCV RNA at baseline, even when HCV was negative. 3.6. Predictive variables of IFN response Of the various variables at baseline, we analyzed gender, age, histological stage, HCV genotype, HCV RNA level at baseline and pretreatment HCV dynamics according to the IFN response. There was no significant difference between SR and NR in gender, age and histological staging (Table 3). As for the HCV genotype, the proportions of patients with SR in genotype 1b including genotype 1b + 2b, and genotypes 2a and 2b were 20/119 (16.8%) and 11/22 (50.0%), respectively (P = 0.0014). As for HCV RNA at baseline, the proportions of patients showing SR with less

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Table 2 Characteristics at baseline according to pretreatment HCV dynamics Characteristics

Pretreatment HCV dynamics

P

Increasing group

Stable group

Decreasing group

No. Gender (M:F) Age (year)

40 20:20 52.5 ± 10.3

54 36:18 51.1 ± 10.1

47 27:20 50.6 ± 10.2

ALT (IU/l) ALT (−1 M)a Range ALT (0 M)a Range

123.4 ± 74.2 39–270 73.9 ± 11.7 38–339

166.8 ± 122.2 37–530 80.0 ± 10.9 38–355

168.8 ± 107.0 39–451 93.0 ± 13.6 36–528

0.3050

ALT (0 M)a /ALT (−1 M)a Range

0.99 ± 2.28 0.62–10.27

0.9 6 ± 0.83 0.18–3.89

0.90 ± 0.68 0.26–3.62

0.8162

Histology F1–F2 F3–F4

16 24

20 34

25 22

HCV genotype 1b 2a and 2b

31 9

50b 4

38 9

Viral load (log copies/ml) HCV (−1 M)c HCV (0 M)c HCV (log copies/ml)a

4.94 ± 0.86d 5.57 ± 0.66 0.60 ± 0.55

5.55 ± 0.64 5.53 ± 0.64 −0.02 ± 0.11

5.78 ± 0.64 5.14 ± 0.93e −0.66 ± 0.51

0.2584 0.6764

0.3488

0.2339

0.0807

< 0.0001 0.0122 < 0.0001

Note: Plus–minus values are mean ± S.D. a ALT (−1 M); ALT at 1 month before the initiation of treatment, ALT (0 M); ALT at the initiation of treatment. b Data includes genotype 1b + 2b. c HCV (−1 M); viral load at 1 month before the initiation of treatment, HCV (0 M); viral load at the initiation of treatment. d Increasing group vs. other groups, P < 0.01 (Scheffe’s test). e Decreasing group vs. other groups, P < 0.05 (Scheffe’s test).

than 5.5 log copies/ml and more than 5.5 log copies/ml were 29/58 (50.0%) and 2/83 (2.4%), respectively (P < 0.0001). Furthermore, as regards HCV, the proportions of patients with SR among the three groups were 25/47 (53.2%, the

Decrease group), 6/54 (11.1%, the Stable group) and 0/40 (0.0%, the Increase group) (P < 0.0001). Pretreatment HCV dynamics was included in a multivariate logistic regression analysis with other pretreatment

Fig. 4. Percentage of SR according to the serum HCV RNA levels measured at baseline in each group of pretreatment HCV dynamics. The number of patients studied in each subgroup is shown below the graph.

S. Sainokami et al. / Hepatology Research 27 (2003) 181–191 Table 3 Univariate analysis of predictive pretreatment variables on virological response to IFN-␣2b Pretreatment variables

Patients with SR

Patients with NR

No. Gender (M:F) Age (year)a

31 21:10 50.2 ± 11.4

110 62:48 51.6 ± 9.8

Histological stage of fibrosis Stage 1 (F1) Stage 2 (F2) Stage 3 (F3) Stage 4 (F4)

5 14 2 10

14 28 21 47

HCV genotype 1b 2a and 2b

20b 11

99 11

Viral load at baseline ≤ 5.5 log copies/ml >5.5 log copies/ml

29 2

29 81

25

22

6

48

0

40

Pretreatment HCV dynamics Decrease group (HCV < −0.20) Stable group (−0.20 ≤ HCV ≤ 0.20) Increase group (HCV>0.20)

P

0.5051 0.5519 0.0844

0.0014

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on day 0 (baseline), day 1, day 3, day 7 and day 14 during the early stage of IFN treatment. We compared the Decrease group with the Nondecrease group as the Increase Group and the Stable group and investigated the influence of pretreatment HCV dynamics on the early dynamics of viral load during IFN treatment. The serum HCV RNA levels on day 0 (baseline) were 5.27 ± 0.85 log copies/ml in the Decrease group and 5.63 ± 0.50 log copies/ml in the Nondecrease group, which were not significantly different. While it was noted that the difference between the two groups on day 1, day 3 and day 7 tended to increase, it was not significant. However, the serum HCV RNA levels on day 14 were 1.15 ± 1.92 log copies/ml in the Decrease group and 2.21 ± 2.30 log copies/ml in the Nondecrease group, and the serum HCV RNA level in Decrease group was significantly lower than that in the Nonincrease group (P = 0.0417).

< 0.0001

4. Discussion < 0.0001

Note: SR was defined as no detectable HCV RNA by qualitative RT-PCR at the end of follow up. a Mean ± S.D. b Data includes genotype 1b + 2b.

variables as associated with SR (Table 4). The four significant independent predictors of SR identified were pretreatment HCV RNA decrease (P < 0.0001), low HCV RNA level at baseline (P = 0.0003), decreased stage of fibrosis (P = 0.0122) and HCV genotypes 2a and 2b (P = 0.0406). 3.7. Serial early changes of serum HCV RNA level during IFN treatment Seventy out of 141 patients gave their informed consent for blood sampling. Serum HCV RNA level was measured

Table 4 Multivariable logistic regression analysis for predicting SR in patients with HCV Variable

Odds ratio

95% confidence interval

P

Pretreatment HCV RNA decreasea Lower HCV RNA level at baselineb Decreased stage of fibrosis HCV genotypes 2a and 2b

626.2

25.0–15706.7

< 0.0001

19.7 3.10 8.93

3.99–97.1

0.0003

1.28–7.50 1.10–71.4

0.0122 0.0406

a The odds ratios are those associated with a decrease of 1.0, in log copies/ml/month. b The odds ratios are those associated with a 1.0 lower baseline concentration, in log copies/ml.

To date it is reported that patients with HCV infection have no large changes in viral load during the long-term disease course [20–24], but there are no reports on the influence on IFN response with respect to short-term changes in viral load immediately before the IFN therapy. A prospective study by longitudinal follow up of HCV RNA levels for 2 months has revealed that natural fluctuations of viral load in six untreated patients with HCV ranged from 2.9 (0.46 log)- to 5.6 (0.75 log)-fold with Amplicor monitor assay [25]. However, the fluctuation range may be increased when levels are determined by a dilution assay if a serum sample is more than the quantification range. In a follow-up study of HCV RNA levels measured with the branched DNA assay for 2 years, it has been reported that the proportions of patients with 1–5 (0–0.70 log), 5–10 (0.70–1 log) and more than 10 (1 log)-fold fluctuation ranges (maximum/minimum) were 71.2, 21.7 and 7.1%, respectively [23]. In our study, the proportions of patients for each fluctuation range were 83.0, 7.1 and 9.9%, respectively. Therefore, it was considered that the fluctuation of viral load during 1 month immediately before the treatment was low compared with the natural fluctuation during a long-term observation. It was assumed, furthermore, that a fluctuation more than 1.6 (log 0.20)-fold was often observed in the natural course. In this dynamics of viral load, our results reveal that pretreatment dynamics of HCV RNA for 1 month before the treatment, as well as other factors such as viral load at baseline and HCV genotype, is an important virus-related factor based on which IFN therapy is decided. As the factor that influenced IFN response, host-related and therapy-related factors are important in addition to these virus-related factors. However, in this study we unified the therapy regimen such as pharmaceutical IFN and dosage, injection method and enrolled patients previously not treated with IFN. Thus, we were able to investigate predictive factors excluding therapy-related factors.

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In this study, age and gender as host-related factors had no influence on IFN response and there was no significant difference in the histological stage by univariable quantities analysis. However, it has been revealed that patients with the histologically advanced stage are resistant to IFN treatment [3,4,6]; thus our result is considered to contradict these reports. The reason for this contradiction was considered that many patients, i.e., 57 out of 141 patients (40.4%), with Stage 4 were enrolled in our study and the proportion of patients with SR in Stage 4 was as high as 10/57 (40.4%). Then, why do the patients with liver cirrhosis have a high rate of SR in this study? This was considered because there were more cases of both low viral load at baseline and negative of HCV in Stage 4 than the other three stages. Therefore, it is important to decide the indication of IFN therapy by observing the changes of viral load even in the histologically advanced stage. We investigated the relationship between HCV and HCV RNA at baseline according to the HCV genotype and identified important virological factors for IFN response. In genotype 1b, the viral load at baseline was important for IFN response in addition to HCV because there was no patient with SR even in the case of negative HCV for a case in whom the HCV RNA level at baseline was more than 6.0 log copies/ml. On the other hand, in genotypes 2a and 2b, almost all patients achieved SR in the case of negative HCV without a relation to viral load at baseline. In addition, all patients having HCV of less than −0.50 log copies/ml/month achieved SR. Taken together, it was reasonable to consider that in patients with genotypes 2a and 2b, HCV tended to decrease to become negative compared with patients with genotype 1b, and hence having genotype 2a or 2b was associated with better chance of achieving SR. Therefore, if HCV became negative regardless of the viral load at baseline in patients with genotypes 2a and 2b, antiviral therapy should be attempted aggressively. While in the case of positive HCV, particularly more than 0.20 log copies/ml/month, it is considered better that IFN monotherapy should not be given even in those with genotypes 2a and 2b. In this study, we analyzed pretreatment HCV dynamics among three groups: Increase group, Stable group and Decrease group based on HCV value in order to perform more detailed investigation. There were no significant differences in gender, age and histological stage among the three groups, and no significant difference in HCV genotype among virus-related factors was noted, but a significant difference only in viral load at 1 month before treatment and viral load at baseline was observed. Therefore, we investigated SR rate according to viral load at baseline and group of patients based on HCV. Even if HCV was less than 5.00 log copies/ml, the SR rate was 0% in the Increase group but 100% in the Decrease group. The SR rate in the Decrease group was significantly lower than that in the Increase group (P = 0.0132). Furthermore, in 5.00–5.49 log copies/ml of viral load at baseline, the SR rate was 0% in the

Increase group but 81.8% in the Decrease group. Therefore, even when viral load at baseline was relatively high, the SR rate was significantly higher in the Decrease group than in the Increase group (P = 0.027). Therefore, even when the viral load at baseline in patients with genotype 1b was relatively high at 5.00–5.49 log copies/ml, we considered that there was the indication for IFN monotherapy when HCV decreased to less than −2.0 log copies/ml/month. According to univariable analysis of predictive pretreatment variables on virological response to IFN-␣2b, significant pretreatment variables were HCV (P < 0.0001), HCV genotype (P = 0.0014) and the viral load at baseline (P < 0.0001). However, many patients with SR were distributed mainly in the low-level area of HCV RNA at baseline and in the negative area of HCV as shown in Table 1, and there was a significant difference in viral load at baseline among the three groups of HCV. Because viral load at baseline may affect IFN response as a confounding variable, we performed multivariable logistic regression analysis. As a result of the analysis, the independent factors that contributed to SR were HCV genotypes 2a and 2b (P = 0.0003), decreased stage of fibrosis (P = 0.0122), lower HCV RNA level at baseline (P = 0.0003) and pretreatment HCV decrease (P < 0.0001) with odds ratio of 626.2 (95% CI: 25.0–15705.7) with the decrease in 1.0 log copies/ml/month. Therefore, it was revealed that observing the changes in the viral load before IFN treatment once a month was a vital factor based on which the indication for IFN treatment is decided. Several studies have revealed that early kinetics of HCV RNA during IFN treatment was useful and reliable for prediction of IFN response [27–30]. Several papers have reported that during the early phase of IFN treatment, patients with early HCV RNA decline or clearance were more likely to have SR than those who responded later. Thus, we measured serum HCV RNA level on day 0, 1, 3, 5, 7 and 14 during IFN treatment in 70 out of 141 patients to study the influence of pretreatment HCV dynamics on the early kinetics of HCV RNA. From a report on kinetics of viral load during IFN-␣ therapy or acute phase of infection, it has been indicated that the production rate of HCV from hepatocytes was about 1012 viral particles/day and an estimated half-life of free HCV virion was estimated to be approximately 2.7 h [15]. It has been also indicated that daily IFN-␣ administration induced a biphasic decline of viral replication: the first rapid slope on day 1 was related to direct dose-dependent IFN inhibition of viral production, whereas the second slower slope of viral decrease that started on day 2 appeared to be related to infected cell death in the context of efficient inhibition of virus production [13,14,26,30,31]. On the other hand, it was also reported that early viral kinetics may predict IFN response after only 24 h of treatment initiation and indicate a strong link between the degree of viral load reduction during the first phase and the subsequent second phase of decline [32]. In this study, we compared the Decrease group with the Nondecrease group,

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which consisted of the Increase group and the Stable group because of low number of patients. The gradient of the first slope of viral decrease was −2.16 ± 1.63 log copies/ml/day in the Decrease group and −1.87 ± 1.61 log copies/ml/day in the Nondecrease group (P = 0.4590). Therefore, it was considered that pretreatment HCV dynamics had little influence on the first phase of decline and viral load after 24 h of treatment initiation. In addition, the gradient of the second slope of viral decrease from day 1 to day 14 was −0.15 ± 0.15 log copies/ml/day in the Decrease group and −0.12 ± 0.15 log copies/ml/day in the Nondecrease group (P = 0.4005). However, the gradient of the second slope in the Decrease group was estimated lower than that in the Nondecrease group, because the value was calculated as 0 when the viral load on day 1 was below the detection level and it often occurred in the Decrease group than in the Nondecrease group. According to the serial assay of serum HCV RNA levels from day 0 to day 7, there was no significant difference between the two groups, but on day 14 serum HCV RNA levels were 1.15 ± 1.92 log copies/ml in the Decrease group and 2.21 ± 2.30 log copies/ml in the Nondecrease group, which were significantly different (P = 0.0417) (Fig. 5). From the results, it was suggested that pretreatment HCV RNA dynamics exerted a great influence on the second phase of decline of HCV RNA level during the early phase of IFN treatment, particularly after 14 days of treatment initiation. Therefore, we considered that pretreatment HCV dynamics may be closely related to immunological mechanism eliminating HCV from infected hepatocytes and may influence IFN response. The host cellular immunity including the CD4 + and CD8 + T-cell response is activated in patients with HCV infection. In chronic viral infection, virus-specific CD8 + cytotoxic T

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lymphocytes (CTL) have been observed to be important in host defense and viral infection [33–37]. The presence of HCV-specific CTL activity in bulk-expanded CTL was found to be associated with a lower level of viremia and a greater inflammatory activity in the liver, suggesting that HCV-specific CTLs may assist in the control of HCV infection but contribute to the hepatocellular damage [37]. Moreover, several studies indicated that patients with higher levels of HCV-specific CTL activity have lower level of viremia [37–39]. Therefore, in this study, it was considered that patients in the Decrease group may have higher levels of HCV-specific CTL activity than patients in the Nondecrease group. However, according to the result showing no significant difference in changes in ALT level among the three groups during 1 month before the treatment, it was assumed that HCV-specific CTL activity was not always associated with ALT flare. This suggests a mechanism different from that in chronic hepatitis B patients who typically exhibit an immune activation and ALT flare before a decrease in virus load. Further studies are required to investigate the reason HCV RNA levels decrease without ALT flare. However, our study indicated that the change in the HCV RNA level during 1 month before the treatment was more closely associated with IFN response than with the ALT level. While we investigated by IFN monotherapy, antiviral therapies such as Peg-IFN and combination therapy did not always result in a high rate of SR, so that it was desirable to decide the indication for antiviral therapy by observing changes in the viral load. In conclusion, in this study of 24-week therapy with IFN-␣2b at 6 MIU/day, it was demonstrated that a decrease in viral load during 1 month before IFN treatment was a strong predictive factor for SR and would be useful for

Fig. 5. Serial changes in serum HCV RNA levels (mean ± S.E.) on day 0 (baseline), 1, 3, 7 and 14 after IFN treatment in patients with the Decrease group (open circles) and the Nondecrease group (solid squares) according to pretreatment HCV dynamics. Serum HCV RNA levels on day 14 was significantly lower in the Decrease group than that in the Nondecrease group (P = 0.0417). Values are mean ± S.E.

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deciding more accurate indications for IFN treatment. In addition, it has been considered that pretreatment HCV dynamics had a strong influence on early kinetics of HCV RNA concentration during IFN monotherapy. Therefore, we considered that it was extremely important to observe the changes in viral load monthly before IFN treatment or other antiviral therapy in order to decide the indication for the therapy.

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