Serum Transaminases as a Predictor of Response to Combined Interferon and Ribavirin Therapy for Chronic HCV Infection Patients

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1 Med. J. Cairo Univ., Vol. 84, No. 2, June: 43-48, Serum Transaminases as a Predictor of Response to Combined Interferon and Ribavirin Therapy for Chronic HCV Infection Patients REDA M. AMER, M.D.*; MONA A. AMIN, M.D.*; DINA SABRY, M.D.** and AMRO ABD EL-AZIZ, M.Sc.* The Departments of Internal Medicine* and Medical Biochemistry**, Faculty of Medicine, Cairo University, Egypt Abstract Introduction: Chronic HCV infection is a major health problem both globally and at the national level. Combined pegylated interferon and ribavirin was considered the standard of care treatment for chronic HCV infection. Inspite of its significant side effects, response rate has been around 50%. Several factors including virus and host factors are reported to be correlated to therapeutic effects. Aim of Work: To determine contribution of serum transaminases in predicting early virological response to interferonribavirin therapy in patients with chronic HCV. Methods: 89 patients with chronic HCV receiving interferon-ribavirin therapy. All were subjected to thorough clinical, laboratory evaluation, liver biopsy. Comparing serum level of serum transaminases (ALT and AST) between responder and non-responders to therapy. Results: The responders to therapy had lower baseline of transaminases (ALT and AST) with statistically significant difference compared to the non-responders. Conclusion: Chronic HCV patients with lower levels of pretreatment serum transaminases have better response to combined interferon-ribavirin therapy. Key Words: Transaminases HCV Interferon. Introduction HEPATITIS C Virus (HCV) is one of the most important infections in humans and is one of the most common causes of hepatitis [1]. The World Health Organization (WHO) estimates that about 3% of the world's population has been infected with HCV and that there are more than 170 million chronic carriers who are at risk of developing liver cirrhosis and/or liver cancer [2]. Egypt contains a high prevalence of hepatitis C in the world about 12% with high HCV related morbidity and mortality [3]. Correspondence to: Dr. Reda M. Amer, The Department of Internal Medicine, Faculty of Medicine, Cairo University, Egypt The combination of pegylated interferon (peg- IFN)-α and ribavirin has been considered the standard of care for chronic HCV infection, with Sustained Virological Response (SVR) rates varying from 50% to 90%, depending on genotype. However, approximately 50% of the patients do not clear the infection during treatment, or relapse after the end of it [4]. Predictors of response to therapy serve as decision tools for physicians to help identify patients who are likely or unlikely to achieve an SVR, and to consider pre-treatment counseling in those patients with a reduced likelihood of successful therapy, perhaps sparing them the side effects and cost of therapy. Therefore, knowledge of predictors to these therapies is extremely valuable [5]. In the study presented here, we investigated the potential effect of serum transaminases level on the response to combined interferon and ribavirin therapy in patients with chronic HCV infection. Patients and Methods Between October 2013 and October 2014, we prospectively enrolled 89 patients with chronic HCV infection at Internal Medicine Department Kasr El-Aini Hospital. Ethical committee approval and written informed consents from the patients were obtained. The patients' age ranged from years eligible for combined therapy of Pegylated interferon (PEG-IFN) and Ribavirin. All patients were subjected to: 1- Clinical evaluation; (history and physical examination) assessing history of smoking, alcohol consumption, presence of chronic diseases (DM, hypertension...), and past history of dental intervention, surgery or blood transfusion. 2- Laboratory investigations; including, ALT, AST, ALP, CBC, Albumin, Bilirubin (Total and Di- 43

2 44 Serum Transaminases as a Predictor of Response to Combined Interferon rect), Alpha Fetoprotein (AFP), Prothrombin Time (PT), Antinuclear Antibody (ANA), Antischistosomal antibody test. 3- Quantitative HCV RNA. Before starting therapy and after 12 weeks of treatment with combined PEG-IFN/Ribavirin therapy, to detect the Early Virologic Response (EVR) was done. Complete EVR is considered if the HCV RNA level is undetectable. Incomplete EVR is considered when a greater than 2-log-fold reduction in HCV RNA level is present. 4- Abdominal sonography. 5- Ultrasound guided percutaneous liver biopsy. 6- Histopathological examination of the liver biopsy with staging of fibrosis and grading of inflammation using metavair score. Inclusion criteria: HCV antibodies and HCV RNA positive by PCR, liver biopsy in the with METAVIR score over A2 and over or equal to F 1, or over or equal A 1 and over F2, patients never treated with ribavirin, IFN alpha or PEG-IFN alpha, AFP under or equal to 3 times the normal range for the laboratory, HBs antigen negative, Hb over or equal 1 1g/dl for male and, over or equal 10g/dl for female, TLC over or equal 3000/mm 3, neutrophils over or equal 1500/mm 3, platelets over or equal /mm 3, normal TSH, ANA under 1/160, fasting blood sugar between mg/dl (if glucose intolerance or diabetes: HbA1C <=8,5%), normal ophthalmologic examination, effective contraception and no breastfeeding during the treatment period during the study period. Exclusion criteria: Patients were excluded if co-infected with HBV, HIV, other causes for chronic liver disease were also ruled out. Other exclusion criteria included neutrophil count <1.5 x 10 3 /dl, platelet count <100 x 10 3 /dl or Hb <10mg/dL for female and <1 1g/dL for male, positive auto-antibodies, patients with a history of severe psychiatric disease, seizure disorder, organ transplantation, or severe cardiac or pulmonary disease. Patients were excluded if they had significant retinopathies, clinical gout, requiring systemic steroids, overt active autoimmune, thyroid disorders, decompensated cirrhosis, focal lesion on abdominal ultrasonography, elevated a -fetoprotein or were substance abuser. Pregnant female was excluded negative serum pregnancy test. Breast feeders were excluded. After an initial assessment, patients were treated with pegylated interferon alpha2a, at a dose of 180g g per week or alpha2b at a dose of 1.5 g g per kilogram body weight per week. In addition to ribavirin at a dose of mg daily as per body weight-1000mg if <_75kg and 1200mg if >_75kg for 48 weeks. Clinical, biochemical and virological parameters were collected pretreatment. HCV RNA was measured by quantitative PCR at end of 12 th week. So, patients were divided into 2 groups: 1- : Those are patients whose PCR results showed complete EVR. 2- Non-responders: Those are patients whose PCR results didnot show complete EVR. Methods: The serological and biochemical tests were done at Clinical Pathology Department of Cairo University Hospital. Quantitative HCV RNA was done using Real Time PCR technique. Histopathological examination for the liver biopsy for all patients was done by pathologists at Pathology Department of Cairo University Hospital. The liver histopathology was staged on using METAVIR scoring system. Statistical analysis: The data were coded, and processed on an IBM- PC compatible computer using Statistical Packages for Social Sciences (SPSS). Data were expressed as mean and Standard Deviation (SD) unless otherwise stated. Student- t-test was used to ascertain the significance of difference between mean s of two continuous variables and Mann-Whitney test was used for non-parametric distribution. Chi- Square analysis was performed to test for differences in proportions of categorical variables between 2 or more groups. In 2 x 2 tables, the Fisher exact test (two-tailed) was used instead of Chi- Square, in particular, when sample size was small. One-way analysis of variance (ANOVA) and nonparametric Kruskal Wallis one-way analysis of variance (ANOVA) was employed for comparison of several group means and to determine the presence of significant differences between group means. The Pearson's correlation coefficient was used to evaluate the strength association between two variables. The level p<0.05 was considered as the cut-off for significance. Multivariate logistic regression analysis was performed [6]. Results From eighty nine chronic HCV patients were included in our study, their age raged between (21 and 57 years) with mean age of 39 years. All patients received antiviral therapy Peg-interferon

3 Reda M. Amer, et al. 45 and ribavirin. According to their response to therapy, we divided our patients into two groups: 1- (44 patients): Their age ranged between (23 and 57 years) with mean age of (41.6±9 years). They are 15 females representing 34% of responders and 29 males representing 66%. 2- Non-responders (35 patients): Their age ranged between (21 and 57 years) with mean age of (42.2± 10 years). They are 25 females representing 60% of non-responders and 20 males representing 40%. Table (1) shows demographic data of both groups. There were no statistically significant difference between both groups as regard age and sex distribution. BMI is below 30 in both groups and responders has lower BMI than non-responders with statistically significant difference. Also responders had a significantly lower rate of diabetes mellitus compared to non-responders (= 0.03). There was no statistically significant difference between both groups as regard the type of interferon used (Table 2). Regarding the laboratory data among the studied groups, the responders had lower baseline of transaminases (ALT and AST) with statistically significant difference compared to the non-responders. Pretreatment viral load (HCV RNA measured by PCR) is higher in non-responders than responders but the difference is statistically insignificant. Platelets and WBCs are also higher in responders but with no statistically significant difference. There was no statistically significant difference between both groups as regard bilirubin, alkaline phosphatase, INR, serum albumin levels, TSH and ANA levels (Table 3). Regarding pretreatment liver histopathology, both groups were similar in pre-treatment inflammation grade, fibrosis stage, with no statistically significant difference. Of note, none of our patients had liver cirrhosis (F 4), (Table 4). Also, correlation between fibrosis stage and different laboratory parameters showed positive correlation between the degree of fibrosis and serum bilirubin and transaminases (ALT & AST) levels. There was also negative correlation between the fibrosis stage and the platelets count but of less statistically significant (Table 5). Multivariate analysis showed that pretreatment ALT is independent predictors of virological response to Peg-interferon and ribavirin therapy, while other factors that showed significant difference in univariate analysis are statistically nonsignificant in stepwise multivariate logistic regression analysis (Table 6). Table (1): Demographic data are presented as mean ± SD, frequency (percent), and median (IQR). Patients (n=89) (n=44) (49.44%) Non-responders (n=45) (50.56%) Age 41.6±9 42.2± Gender: Male 29 (66%) 20 (40%) 0.08 Female 15 (34%) 25 (60%) BMI 27± ± Diabetes 1 (2%) 7 (16) 0.03 Table (2): Comparison between types of interferon received in both patients groups. Type of interferon 2a 2b 22 (50%) 22 (50%) Nonresponders 18 (40%) 27 (60%) Table (3): Baseline laboratory data of both groups. Data are apresented as mean ± SD. (n=44) Non-responders (n=45) 0.2 AST 28.9± ± ALT 28.7± ± Bil 0.77± ± Alb 4.3± ± Alk 110.7± ± PT 13.1 ± ± INR 1.1 ± ± TSH 3.5± ± HB 13.7± ± TLC 7.59± ± PLT 220x10 3 ± x10 3 ± ve ANA 0 (0%) 1 (2%) 0.5 Pretreatment ± ± 0.74 PCR Table (4): Comparison between the two groups regarding inflammation grade and fibrosis stage according to metavir scoring system. Nonresponders Fibrosis stage 1.5 (1,2) 2 (1,3) 0.5 Inflammation grade 1 (1,1) 1 (1,2) 0.8

4 46 Serum Transaminases as a Predictor of Response to Combined Interferon Table (5): Correlation between fibrosis stage and laboratory data. Pearson correlation coefficient AST ALT Bilirubin Albumin Alkaline phosphatase PT Platelets PCR Table (6): Multivariate stepwise logistic regression analysis of factors affecting patient response to interferonribavirin therapy. Odds 95% confidence interval ratio Lower Upper ALT Discussion Pegylated interferon and ribavirin therapy for chronic HCV has been associated with significant side effects with response rate around 50%. Predicting good response to pegylated interferon and ribavirin therapy before staring therapy may be very beneficial for the patients. This study demonstrates that lower baseline serum AST and ALT are significantly associated with response to interferon-ribavirin therapy in patients with chronic HCV infection. As regard AST, this is consistent with Al - Ashgar et al. [7], they reported that that lower baseline serum AST but not ALT is an independent predictor of response to interferon-ribavirin therapy. In our study, the mean AST level in responders is 28.9± 13U/l. This finding is in agreement with the finding of Pérez et al. [8], who demonstrated that serum AST levels <40U/l is correlated independently with complete response. Regarding ALT, our finding is consistent with Cupic et al. [9], they found that ALT level was significantly higher in NR than in SVR patients. Their patients (responders and non-responders) had serum ALT above the upper limit of normal. Ghweil et al. [10], noted that responders have significantly lower ALT levels than non-responders, but insignificant AST level. Derbala et al. [11], reported that there was a significant positive correlation between pre-treatment ALT and ETR in patients receiving Peg-IFN but not in patients receiving conventional IFN, all our patients received Peg-IFN. In contrast to our findings, it has been demonstrated that patients with normal ALT levels have a similar virologic response to interferon therapy as patients with elevated ALT levels [12]. Also, Pockros et al. [13], found no significant difference in ALT level between responders and nonresponders. Faisal et al. [14] noted that there is no significant difference in AST and ALT level between responders and non-responders. Moreover, our study showed that significant difference between increased serum aminotransferases (ALT & AST) and stage of liver fibrosis. We believe that these lower AST and ALT levels reflect less severe histological parameters in the sustained responders. A study by Zechini et al. [15], showed a statistically significant positive correlation of baseline aminotransferase s with the hepatitis activity index and fibrosis score. In support of our results, a study by Assy et al. [16], reported a significant positive correlation between AST s and the extent of hepatic fibrosis. Al-Ashgar et al. [7], showed that lower AST levels are independent predictor of fibrosis stage 0-2. On the other side, Cupic et al. [9], found that no significant difference between increased serum amintransferases and stage of liver fibrosis. We noted in this study that both responders and non-responders had low total billirubin serum level with no statistically significant difference. This is consistent with Faisal et al. [14] and Ghweil et al. [10]. Hosogaya et al. [17], reported that low total billirubin level is significantly associated with SVR. This study demonstrated that responders had lower fibrosis stage and inflammation grade in the pre-treatment liver biopsy than non-responders. However, neither the fibrosis stage nor the inflammation grade were found to be statistically different. Our findings are in agreement with Ćupic et al. [9], they found no correlation between the stage of liver fibrosis and response to therapy. This finding is also consistent with Kamal et al. [18], they demonstrated that pre-treatment liver pathology cannot predict the response to therapy. In addition, the study of Al-Ashgar et al. [7] did not find statistical difference between sustained responders and those who developed virological relapse after ETVR as regard fibrosis stage nor the inflammation grade in the pre-treatment liver pathology. This is contrary to what was previously reported by Hassan et al. [19] and Balart et al. [20]. They reported that patients with advanced liver necroinflamatory activity and fibrosis have poor response

5 Reda M. Amer, et al. 47 to interferon therapy. The more advanced liver disease is more associated with poor response to IFN therapy. It should be noted that all our patients had fibrosis stage <_3, and none of them had fibrosis stage 4 (cirrhosis). We believe that the effect of pre-treatment fibrosis on the virological response to therapy becomes more obvious if comparisons between cohorts with predominantly stage 3-4 are compared with those with predominantly stage 1-2 are made. With respect to virus-related factors, we found that average viral loads were higher in non-responders, but differences were not significant, which is in accordance with Al-Ashgar et al. [7], and Derbala et al. [11]. In contrast to our findings, HCV pre-treatment viral load has been suggested as a predictor of SVR [18]. It is well-known that viral load fluctuates and a single reading of HCV quantification may not reflect the actual viral load at the time of treatment, especially if we know that viral load was assessed at varying intervals from the onset of treatment. It has also been reported that the differences in interferon response could be secondary to either a difference in the viral virulence and/or replication rate among different HCV genotypes and not the absolute viral load [21], which implies that HCV- RNA levels per se are less influential compared to the major impact of genotype that generally determines the rate of viral response. In addition, we found no statistically significant differences between pre-treatment viral load and stage of liver fibrosis, which confirms some previous results [22]. Conclusion: Chronic HCV patients with lower levels of pretreatment serum transaminases have better response to combined interferon-ribavirin therapy. Recommendations: Lower levels of transaminases should be used as a pre-treatment predictors of response of chronic HCV infection to combined interferon-ribavirin therapy, further studies are needed to prove this good prognostic in the setting of the new directly acting anti-hcv agents. References 1- LAUER G.M. and WALKER B.D.: Hepatitis C virus infection. N. Engl. J. Med., 345 (1): , ZEUZEM S.: Heterogeneous virologic response rates to interferon-based therapy in patients with chronic hepatitis C: Who respond less well? Ann. Intern. Med., 140: , KHATTAB M., ESLAM M., SHARWAE M.A., SHATAT M., ALI A. and HAMDY L.: Insulin resistance predicts rapid virologic response to peginterferon/ribavirin combination therapy in hepatitis C genotype 4 patients. Am. J. Gastroenterol., 105 (9): , HADZIYANNIS S.J., SETTE H J.R., MORGAN T.R., et al.: Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: A randomized study of treatment duration and ribavirin dose. Ann. Intern. Med., 140 (5): , KAMAL S. and NASSER I.: Hepatitis C genotype 4: What we know and we don't yet know. Hepatology, 47: , DAWSON B., TRAPP R.G., DAWSON B. and TRAPP R.G.: Basic and clinical biostatistics; Mcgraw-Hill Inc. Third edition, AL-ASHGAR H., KHAN M.Q., HELMY A., et al.: Predictors of sustained virological response to a 48-week course of pegylated interferon alfa-2a and ribavirin in patients infected with hepatitis C virus genotype 4. Ann. Saudi. Med., 29 (1): 4-14, PÉREZ ROLDÁN F., De DIEGO A., CASADO M., MA- TILLA A., BAÑARES R., GARCÍA-DURÁN F., SAL- CEDO M., COS E. and CLEMENTE G.: Treatment with interferon alfa-2b in patients with chronic hepatitis caused by hepatitis C virus: Predictive factors for the response, relapse and early development to cirrhosis after treatment. Rev. Esp. Enferm. Dig., 88 (9): , CUPIC S.J., GLISIC S., STANOJEVIC M., et al.: Response factors to pegylated interferon alpha/ribavirin treatment in chronic hepatitis C patient genotype1b. Arch. Biol. Sci., 66 (1): , GHWEIL A.A., ABD EL-MEGUID M.M., SHAMARDAN E.S., et al.: Serum Leptin, Ferritin and Uric Acid as Predictors of Fibrosis and Sustained Virological Response in Chronic Hepatitis C Patients. Life Sci. J., 11 (5): , DERBALA M.F., AL KAABI S.R., EL DWEIK N.Z., PASIC F., BUTT M.T., YAKOOB R., AL-MARRI A., AMER A.M., MORAD N. and BENER A.: Treatment of hepatitis C virus genotype 4 with peginterferon-2a : Impact of bilharziasis and fibrosis stage. World. J. Gastroenterol., 12 (35): , GORDON S.C., FANG J.W., SILVERMAN A.L., MCHUTCHISON J.G. and ALBRECHTJ.K.: The significance of baseline serum aminotransferase on pretreatment disease characteristics and response to antiviral therapy in chronic hepatitis C. Hepatology, 32: 400-4, POCKROS P.J., HAMZEH F.M., MARTIN P., LENTZ E., ZHOU X. and GOVINDARAJAN S.: Histologic Outcomes in Hepatitis C Infected Patients with Varying Degrees of Virologic Response to Interferon-Based Treatments. Hepatology, 52 (4): , FAISAL A., ALAA M., ASHRAF N., et al.: Predictors of Early Virological Response of Viral Hepatitis C to Combination Therapy with Pegylated Interferon Plus Ribavirin.

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