LIUER, PANCREAS, AND BlllARY TRACT DIGEST LIVER OIS 2000;32:211-6

Tailoring interferon dose and monitoring viral load in hepatitis C virus genotype 1 b infected patients: a pilot study

l? Colombatto M. Baldi I F. Oliveri A. Randonel F. Bonino M. R. Brunetto

From: Gastroenterology and Hepatology Unit, Spedali Riuniti “Santa Chiara: Arienda Ospedaliera Pisena, Cisanello Hospital, Pisa; I Department of Digestive Diseases and Nutrition, Azienda Ospedaliera “S. Giovanni Battista” della Citta di Torino, Molinette Hospital, Turin, Italy.

Address for correspondence: Dr. F. Bonino, Divisione di Gastroenterologia ed Epatologia, Spedali Riuniti “Santa Chiare “, Azienda Ospedaliera Pisana. via Paradisa 2, Cisanello 56124, Pisa, Italy. Fax: +39-050-995457. E-mail: boninoamed-club. corn

Achnowi+dpenwntsi The study was supported in part by an educational grant from Bayer Diagnostics, Emeryville, CA, USA.

Submitted Decembar 28, 1999. Revised March 8, 2DOO. Accepted March 13, 2000.

Background and aims. Complete [biochemical and virological] pri- mary response remains the first goal of any antiviral therapy and its early assessment could be particularly useful in the management of the high viral load, genotype lb infected patients, who have the worst chance of response. We evaluated whether tailoring interfer- on dose according to pre-treatment viral load and early monitoring of quantitative HCV-RNA could either improve or predict the results of recombinant alpha-2a interferon treatment in these patients. Patients. Fifty-three consecutive genotype lb HCV-infected patients, stratified in two groups by viral load [cut off 6 MEq/ml], received randomly 6 or 9 MU of recombinant alpha-2a interferon thrice weekly for 6 months, followed by 6 MU for another 6 months. Methods. HCV-RNA was measured [b-DNA] assay] two months apart prior to therapy, at baseline, after 2 weeks of therapy and monthly thereafter Rasults. In the high viraemic group, complete primary response was observed in 80% of patients treated with high dose recombinant al- pha 2a interferon and only in 14.3% of low dose treated patients [p<O.O3]. In low viraemic patients, complete primary response was 53.8% in low dose patients and 80% 18 out of IO] in the high dose group. Sustained response was 60% in high viraemic patients treat- ed with high dose and absent in those treated with low dose [pcO.OS]. One log viral load decrease at 2 or 4 weeks showed 0.87 and 0.80 positive predictive values, 0.95 and I. 0 negative predictive values with 96% and Icoo/ sensitivities and 83% and 70% specificities. Conclusions. 6 MU recombinant alpha-2a interferon thrice weekly schedules were completely ineffective in the large majority [85.7%] of patients with viral load above 6 million HCV-RNA copies/ml and the treatment failure could be predicted by lack of one log viral load decrease after 2-4 weeks of treatment.

Digest Liver Dis 2000;32:211-6

Key words: alpha interferon; chronic hepatitis C; HCV-RNA; hepatitis C virus

Introduction

Chronic hepatitis C appears to have an indolent, slowly progressive course in the large majority of hepatitis C virus (HCV) infected patients I-4 and pro- gression to cirrhosis is related to sex, age, duration of HCV infection, cofac- tors of liver disease (for instance, hepatitis B virus infection and alcohol in-

lluantitative HEWRNA and interferon therapy

take 3 “) and viral characteristics (genotype and viral load 1-4. Randomized controlled trials of alpha-interfer- on (IFN) therapy have demonstrated that only 10% to 20% of patients have sustained biochemical (return to normal of serum alanine aminotransferase (ALT) lev- els) and/or virologic (undetectable circulating HCV- RNA) remissions when patients are followed up for at least 6 months after completing 6-12-month treatment courses I-5. Preliminary reports support the view that re- sponse to therapy can be significantly increased using more aggressive therapeutic schedules, namely higher IFN dose or combination with Ribavirin I-5. However, such a therapeutic strategy, if applied to every patient, may result in higher costs, less compliance and a larger number of side effects 5 6. In addition, a certain propor- tion of patients respond to low IFN dose schedules lm4. Most studies using multivariate analysis have identified several virus- and host-related factors which are predic- tive of a severe outcome of chronic hepatitis C both in untreated and treated patients 6-24. HCV genotype 1 and high pre-treatment HCV-RNA serum levels are the most consistently defined virus-related factors associat- ed with a poor response to IFN 9 I3 I4 l6 2’-23. The achieve- ment of both a biochemical and virologic response (complete primary response, CPR) at the end of treat- ment has been associated with a long-term sustained re- sponse in most studies lo ” 2o 2’ 23 24. The rapidity of the HCV decline in combination therapy has been shown to be dependent on the dose of IFN rather than the associ- ation of Ribavirin 24. In addition, the large majority of patients who experienced hepatitis relapses after either IFN alone or combination therapy had not achieved a CPR: they only had a biochemical, without a virologi- cal response 2s-27. Therefore, CPR remains the first goal of any IFN therapy and tools able to identify early those patients who are going to reach this goal might con- tribute to defining management of the single patient. On the basis of these observations, we tested, in a pilot study, the hypothesis of whether tailoring the IFN dose according to the baseline viral load in HCV genotype lb infected patients might improve the rate of CPR. We also monitored the early viraemia decrease during therapy in order to see whether it could be predictive or not of the achievement of CPR. The results of this pi- lot study are reported to prompt further trials on larger cohorts aimed at confirming the efficacy and verify the cost effectiveness of this approach.

Patients and methods

Patients Of 75 consecutive patients newly diagnosed as positive for the antibody to hepatitis C virus (anti-HCV) by 2nd generation enzyme-linked immunosorbent assay

(ELISA), immunoblotting assay and for serum HCV- RNA by reverse-transcriptase polymerase chain reac- tion (RT-PCR), after HCV genotyping, 53 patients (43 males and 10 females; median age 44 years, range 24- 61) were shown to be infected with genotype lb and 22 patients with other genotypes (2, genotype 1 a; 7, geno- types 2a/2c; 12, genotype 3a and one, genotype 4). All had biochemical evidence of liver disease for more than six months and histologically assessed chronic hepatitis without ultrasound signs of cirrhosis and without endoscopic or ultrasound signs of portal hy- pertension. None of them had been previously treated with antivirals or corticosteroids. In this group of 53 lb genotype infected patients, the viral load was deter- mined at monthly intervals in 3 serum samples prior to therapy and the mean of the results was used to classi- fy each patient as high or low viraemic, according to the cut-off value of 6~10~ HCV-RNA equivalent copies/ml. This value was selected on the basis of the mean baseline viraemia detected in 52 consecutive pa- tients infected with HCV genotype 1 b who did not re- spond to 6 million international units (MU) of IFN in our previously published trial 2s.

Treatment After the calculation of the mean pretreatment HCV- RNA level of the 3 monthly tests, 17 patients had vi- raemia levels above the cut-off value of 6x lo6 HCV- RNA equivalent copies/ml (median 8.6, range 5.4-39.5 MEq/ml). They were randomized as High viraemic pa- tients (Group H) into two groups of treatment: - Group Hl: 10 patients (8 males and 2 females, me-

dian age 44 years, range 24-59, median HCV-RNA level: 7.6, range 6.3-25.8) receiving 9 MU rIFN thrice weekly (tw) for 6 consecutive months fol- lowed by 6 MU rIFN for 6 additional months.

- Group H2: 7 patients (6 males and 1 female, medi- an age 43 years, range 25-61, median HCV-RNA level 7.4, range 6.1-39.6) receiving 6 MU rIFN tw for 12 consecutive months.

Thirty-six patients had viraemia levels below the cut-off value of 6~10~ HCV-RNA equivalent copies/ml (median 1.2 range 0.6-8.2). Twenty-three of them were random- ized as Low viraemic patients (Group L) into two groups: - Group Ll : 10 patients (8 males and 2 females, me-

dian age 45 years, range 26-60) receiving the same high dose schedule as Group Hl patients.

- Group L2: 13 patients (10 males and 3 females, me- dian age 48 years, range 3 l-60) receiving (as Group H2) 6 MU rIFN tw for 12 consecutive months.

The remaining 13 HCV 1 b infected patients (11 males and 2 females, median age 46 years, range 30-61), who also showed a mean viral load lower than 6x 1 O6 HCV- RNA equivalent copes/ml, were enrolled in another ongoing study testing the impact of therapy discontin-

212

1 1 - - - P. Colombatto et al.

uation guided by the HCV-RNA undetectability. They were treated with 6 MU rIFN tw and therapy was with- drawn only after 6 additional months since viraemia became undetectable at monthly test. These patients are, hereafter, referred to as Group L3. Once therapy was started, blood samples were ob- tained from all patients after 2 weeks, after 4 weeks and then monthly throughout the entire treatment peri- od and for 6 months after the end of treatment. Serum aliquots were stored a -20°C and thawed only at the time of HCV-RNA testing. The biochemical and virologic response to therapy was defined as follows: a) Complete Primary response (CPR) at the end of

treatment = return to normal of serum ALT levels and reduction of serum HCV-RNA levels to below 1,000 copies/ml;

b) No response (NR) = failure of serum ALT levels to return to normal or failure of serum HCV-RNA to drop to levels below 1,000 copies/ml during therapy;

c) Sustained response (SR) = persistence of the same features as CPR at the end of the post-treatment fol- low-up.

Methods Antibodies against the HCV proteins were detected by commercial immunoenzyme assay (2nd generation Or- tho HCV antibody Ortho Diagnostic System Milan, Italy). Antibodies against both structural and non struc- tural domains of the HCV polyprotein were deter- mined by recombinant immunoblot assay (RIBA 3, Ortho Diagnostic System, Milan, Italy). Geno/stubtyping of HCV (according to the classifica- tion of Simmonds et al.) was performed by commercial TNNO-Line Probe assay (Innogenetics, Nuclear Laser Medicine, Milan, Italy). Infections with other hepatotropic viruses (hepatitis A, hepatitis B, Epstein Barr virus and cytomegalovirus) were ruled out by serological testing with commercial- ly available kits (Abbott, Rome, Italy).

Detection of serum HCV-RNA Serum HCV-RNA was tested 3 times before starting therapy (at months -2 and -1 before therapy and at baseline), after 2 weeks, 4 weeks and monthly thereafter during therapy and af- ter the end of treatment. The qualitative assessment of serum HCV-RNA was performed by RT-PCR as pre- viously described 23 27 28. The sensitivity limit of our “in house” assay was 1,000 HCV-RNA copies per mil- liliter (ml) of serum 21 *’ 28. The quantitative analysis of viral load was performed by commercial Quantiplex HCV-RNA 2.0 assay (branched DNA, b-DNA-2) which was performed strictly according to the manufactures’ instructions (Bayer Diagnostics, Milan, Italy z9m33). Viraemia levels were expressed as equivalent copies (Equivalent, Eq) of HCV-RNA per milliliter (ml) of serum.

Statistical analysis Data are expressed as median or mean f SD. Categor- ical data were compared by chi-square analysis or Fisher’s exact test. All tests of significance were two- tailed, with p values of less than 0.05 considered to in- dicate statistical significance.

Results

Table I shows the demographic features of the patients, the therapy groups, the medians and ranges of baseline viraemia levels, the medians and ranges of baseline ALT levels, the percentages of the different types of re- sponse to IFN during therapy and at the sixth month of post-treatment follow-up. Overall, we observed a return to normal of ALT levels associated with undetectable serum HCV-RNA (CPR) in 31 out of 53 (58.5%) patients whereas 22 (41.5%) patients did not respond to therapy. In high viraemic patients, the CPR rate was 80% (8 out of 10) in Group Hl treated with high dose rIFN (9 MU tw for 6 months then 6 MU tw for 6 additional months) and 14.3% (1

TaLle 1. Demographic, clinical, virologic features at baseline, IFN regimens and outcome of treatment in 53 chronic hepatitis C patients infected with genotype 1 b.

Group N. Age (yearn1 median mnge

Male/ female

IFN dose

AU RI/l1 inediin range

HCU-RNA’ CPW NIP SW median range % % %

:: IO 7 z 24-60 25-61 ET 9 6 MU MU 108 120 48-229 62-175 7.9 7.4 6.3-25.8 6.1-39.6 80 14.3 20 85.7 60 0

:: :i 44 48 23-65 32-62 lW3 EV2 9 6 MU MU 113 118 49-275 54-188 2.0 2.2 0.7-5.2 0.5-5.0 80 53.8 46.2 20 50 38.5 L3 13 46 30-61 II/2 6 MU 125 58-203 2.3 0.6-4.5 45.2 54.8 NA

‘t-W-RNA: /eve/s are expressed as copies/m; ‘CPR: complete primary response; 3NR; non response; ?SR: sustained response; ‘NA: not avai/ab/e [ongoing study, see text). ALT: alanine aminotransferase.

~l-l----l-~----. 213

lluantitative HWRNA and interferon therapy -

out of 7) in Group H2 treated with low dose IFN (6 MU tw for 12 months) (95% confidence limits (CL) 0.175- 1.139; p=O.O29). In spite of comparable medians and ranges of baseline viraemia in Groups Hl and H2 (Table I), the decline of viral-load was earlier and more consistent in patients treated with the higher dose (Figs. 1, 2). In low viraemic patients, the CPR rate was not significantly different according to IFN dose: 80% (8 out of 10) of Group Ll patients treated with the higher dose (9 MU tw for 6 months, then 6 MU tw for 6 additional months) and 53.8% (7 out of 13) of Group L2 patients treated with the lower dose (6 MU tw for at least 6 months. High IFN doses induced identical CPR rates (80%) in both groups of high and low vi- raemic (Hl and Ll). Considering altogether the 33 pa- tients (7 in Group H2, 13 in L2 and 13 in L3) who re- ceived 6 MU tw for at least 6 months, CPR was achieved in 50% of the 26 patients with median viral load below 6 million HCV-RNA copies/ml, of serum, but only in 1 out of the 7 (14.2%) patients with higher viraemia. The large majority of those who achieved CPR (92.8%, 12 out of 13) were low viraemic. Fur- thermore, the analysis of variance for repeated mea-

bnekn WUkl wti2 Week-Y we464

during tr86tment

Fig. 1. Decline of viral load in 7 high viraemic patients treated with 6 MU IFN

so

5 26

.B

6

20

16

I Cig. 2. Decline of viral load in IO high viraemic patients treated with 9 MU IFN

surement in these low dose IFN-treated patients showed a significant difference (~~0.01) in baseline vi- ral load between patients who did not respond to ther- apy (median 6.1, range 1.1-39.5 M copies/ml) and those who achieved CPR (median 1.5, range 0.6-7.4 M copies/ml). Follow-up data showed that higher rates of CPR were associated with higher rates of SR (Table I). In high vi- raemic patients, the SR rate was 60% in patients treat- ed with the higher rIFN dose (6 out of 10 in Group Hl), whereas none of the patients treated with the low- er rIFN dose showed a sustained response (p=O.O42, 95% CL 0.130-1.062). The difference in the SR rates between Groups Ll and L2 did not reach a statistical- ly significant value (Table I).

Decline of viral load and prediction of response to therapy We tested the sera in all patients who achieved PR and in all non-responders after 2 and 4 weeks of treatment to establish how many patients showed normal serum ALT levels and undetectable or 1 log or 2 log decline

Table II. Predictive values, sensitivity and specificity of primary response prediction in 53 genotype 1 b HCV infected patients with chronic hepatitis C treated with interferon.

lbsts SNd Au AU llfiu-RMA lmf-RNA tlNlNNfblmd -Bea 2mkkIl 4u&rs 2Gmfu 4Ntebs

Predictive value Positive 0.87 0.80 Negative A.56 A.68 0.95 1

Sensitivity 35% 61% 96% 100%

Specificity 100% 100% 83% 70% '

Note: normal AL T [alanine aminotransfarasel and/or decline of viral load below 1 log of baseline levels.

AU and

ilt2weeke

A.53

29%

100%

ALTand llml-m

at 4 WNebs

A.68

62%

100%

214 - -

P. Colombatto et al.

of serum HCV-RNA levels (Eq of HCV-RNA per ml). Table II shows the positive and negative predictive val- ues, sensitivity and specificity for early ALT normal- ization and/or 1 log decline of viral load after 2 and 4 weeks of treatment. Undetectable viraemia and 2 log decline of viral load had worse predictive values, sen- sitivity and specificity than 1 log decline of viraemia (data not shown).

Discussion

A large series of published data support the view that more aggressive treatment schedules (high IFN dose and combination therapy) could improve the SR rate of chronic hepatitis C patients 1-S. The IFN dose rather than the addition of ribavirin appears to influence the rapidity of the HCV-RNA decline in the early phase of the treatment 24 and the achievement of both biochem- ical and virological response before therapy withdraw- al (CPR) appears to be associated with the SR 25 2h. In 53 consecutive genotype lb HCV infected patients, we tested the hypothesis of whether tailoring the IFN dose according to the pre-treatment viral load and its early monitoring during therapy can improve both rates and prediction of CPR in the individual patient. The mean baseline viraemia of a series of 52 consecutive geno- type lb infected patients who did not respond to a treatment course of 6 MU rIFN tw for at least 6 months 2s was used as the baseline viraemia cut-off (6 million HCV-RNA copies per milliliter of serum) to discriminate high viraemic from low viraemic patients who entered our pilot study. We confirmed that a high IFN dose significantly improved the CPR rate in high viraemic patients. In addition, the significantly higher CPR rate observed was associated with a significantly higher SR rate. This is consistent with a series of pre- viously reported studies 5 I8 I9 25. In low viraemic pa- tients, CPR and SR rates were only slightly better in those treated with the higher IFN dose. The lack of a statistically significant difference is probably due to the small sample size, adequate to assess only major differences in efficacy that could be helpful in deci- sion-making, at the individual patient level. Therefore, in order to confirm these findings, larger studies de- signed to deliver more statistical power are necessary. The results of our prospective pilot study, however, confirm other retrospective observations suggesting that tailoring the rIFN dose according to baseline vi- raemia is feasible and may be efficacious in clinical practice 20 ?I 33 24. It might be argued that a primary re- sponse rate of 80% in both high and low viraemic pa- tients treated with 9 MU IFN supports the indiscrimi- nate use of high dose regimens independently of base- line viral load measurement. This alternative approach,

whereas too aggressive in naive patients, could cer- tainly be attempted in previously treated patients who did not achieve CPR. In our study, we also addressed the issue of the significance of viral load monitoring during treatment and we found that a viral load decline of at least 1 log after 2 and 4 weeks of treatment has the highest specificity and sensitivity to identify the in- dividual patient whose treatment schedule is ineffec- tive to reach the normal levels of serum ALT and un- detectable HCV-RNA (below 1,000 Eq/ml) fulfilling our CPR criteria. Since sensitivity and negative predic- tive value are major factors for therapy decision-mak- ing, the measurement of viral load decline after 4 weeks of treatment appears more effective than earlier testing at 2 weeks. Using the delta negative variation of 1 log decline of viral load allows the evaluation of the response earlier than monitoring serum HCV-RNA negativization. Therefore, quantitative measurement is necessary. In treated patients where viral load does not decline by at least 1 log, within 4 weeks of treatment more aggressive therapeutic strategies could be at- tempted earlier, avoiding additional months of ineffec- tive IFN treatment. According to our data in naive pa- tients who are treated on the basis of currently avail- able consensus guidelines ’ 2, we hypothesize that the measurement of viraemia levels at baseline and at 4 weeks of treatment could be cost effective. This strate- gy provides a useful means to tailor and modify, as soon as possible, the inefftcacious treatment schedule in the individual patient. The result of our pilot study prompt larger prospective studies to validate such a therapeutic strategy in clinical practice.

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