JOURNAL OF INTERFERON & CYTOKINE RESEARCH Volume 35, Number 8, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/jir.2014.0193
IP-10 Serum Level in Chronic Hepatitis C Virus Patients: Relation to Fibrosis and Response to Combined Interferon/Ribavirin Therapy Maissa El Raziky,1 Aisha Elsharkawy,1 Salma E. Said,2 Sahar Abdelatty,3 Wafaa El Akel,1 Omnia Tantawy,1 Hadeel Gamal Eldeen,1 and Mahasen Mabrouk1
Despite the appearance of the direct acting antiviral drugs, pegylated interferon/ribavirin (PEG-IFN/RBV) still has a place in the standard of care (SOC) therapy for chronic HCV4. Studies were conducted to find an accurate prediction in response to SOC therapy. Pretreatment serum interferon-g-inducible protein-10 (IP-10) is an independent predictive factor of sustained virological response (SVR) in HCV1-infected patients. To assess whether the pretreatment serum level of IP-10 influences hepatic fibrosis and PEG-IFN/RBV therapy response, a study was conducted on 88 chronic Hepatitis C virus (HCV) patients who received PEG-IFN/RBV. Patients were subjected to a pretreatment routine laboratory evaluation, liver biopsy, and serum IP-10 assessment. They were followed up for 6 months after cessation of therapy (week 72). Patients were classified into 3 groups according to their response; nonresponders, relapsers, or sustained virological responders. The relation of pretreatment IP-10 with fibrosis and response was assessed. The studied groups were matched regarding their demographic data. There was no statistically significant association between the pretreatment IP-10 level and fibrosis (P = 0.86) and no relation to response was found at week 12, 24, 48, and 72 (P = 0.58, 0.8, 0.47, and 0.43, respectively). Pretreatment IP-10 could not predict either fibrosis or response to PEG-IFN/RIB therapy in chronic HCV Egyptian patients.
Introduction
H
epatitis C virus (HCV) is highly endemic in Egypt, where nearly 9.8% of the population is chronically infected with HCV (Guerra and others 2012). The genotype 4 is predominant in 91% of these patients (Ray and others 2000). The sustained virological response (SVR) in genotype 4 Egyptian patients treated with pegylated interferon/ribavirin (PEG-IFN/RBV) was estimated around 60%, intermediate between the SVR observed in genotype 1 and genotypes 2 and 3 patients in western countries (El Makhzangy and others 2009). Despite the appearance of new oral antiviral drugs, PEGIFN/RBV may remain the standard-of-care (SOC) therapy for some time, especially with the expected high costs and the limited number of patients who will be eligible for the new treatment (Esmat and others 2015). Knowledge of predictors of response is extremely valuable, as this will lead to considering pretreatment counseling, sparing patients the side effects and cost of therapy (Abdo and Sanai 2009).
The interferon-g-inducible protein 10 (IP-10), also termed CXCL10, belongs to the CXC (or a) chemokine family of secreted 8–10 kD proteins (Murdoch and Finn 2000). IP-10 is an IFN-g- and TNF-a-inducible chemokine that can be highly expressed by fibroblasts, keratinocytes, mesangial cells, endothelial cells, monocytes, astrocytes, neutrophils, and hepatocytes (Casrouge and others 2011). The majority of liver-infiltrating T cells in patients with chronic HCV expressed CXCR3, and the amount of hepatic IP-10 mRNA and protein was markedly increased in these patients and correlated strongly with serum IP-10 concentrations (Apolinario and others 2004). Four independent studies demonstrated that the baseline levels of IP-10 are predictive of the failure of response to HCV treatment. This has been demonstrated in patients with genotype 1 and few cases of genotype 4 HCV (Butera and others 2005; Diago and others 2006; Lagging and others 2006; Romero and others 2006). The aim of this study was to assess whether the pretreatment serum level of IP-10 influences hepatic fibrosis and PEG-IFN/RBV therapy response in Egyptian patients predominantly infected with HCV4.
Departments of 1Endemic Hepatology and Gasteroenterology, 2Biochemistry, and 3Chemical and Clinical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt.
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Patients and Methods This is a prospective study conducted on 88 patients with chronic HCV who attended El- Fatemia hospital in Cairo and were naive to treatment with SOC therapy with PEGIFN/RBV.
Sample size The sample size was estimated by using a power calculation program (Roasoft program) based on the study by Diago and others (2006). With an error margin of 5%, confidence level of 85, and an expected population size of 95, the recommended sample size was at least 63 patients (www.raosoft.com/samplesize.html).
Subjects Patients were recruited according to the following inclusion criteria: positive HCV antibodies using a third-generation test and detectable HCV RNA by PCR (according to the Abbott RealTime HCV kit, with a detection limit of 12 IU/ mL), age between 18 and 65 years, body–mass index (BMI) < 30, and the following hematologic and biochemical criteria [WBC > 3,500/mm3, neutrophil count > 1,500/mm3, platelets > 75,000/mm3, International Normalized Ratio ( £ 1.5), total serum bilirubin < 1.5 mg/dl, albumin > 3.5 g/dl, serum creatinine within normal, thyroid stimulating hormone (TSH) within normal, and anti nuclear antibody < 1:16]. Patients with uncontrolled diabetes mellitus, severe psychiatric disease, decompensated liver disease, combined HBV (positive HBsAg) and HCV, and serious comorbid conditions were excluded from this study. The study was conducted according to the principles of the Declaration of Helsinki. The Institutional Review Board (IRB) study approval was obtained through the (NHTMRI) IRB office before commencement of the study.
Study procedure After signing a written informed consent, the selected patients were subjected to the following: full history taking, clinical assessment, and laboratory investigations, including routine laboratory tests, pretreatment serum level of IP-10, and histopathological examination of a needle liver biopsy of a core of at least 15-mm length and containing 6 portal tracts. A single experienced pathologist analyzed all biopsy specimens and they were evaluated according to the METAVIR score (Bedossa and Poynard 1996). The Invitrogen Human IP-10 (Hu IP-10) kit used (Invitrogen corporation, Carlsbad, CA) is a solid-phase enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody specific for Hu IP-10 has been coated onto the wells of the microtiter strips provided. Samples, including standards of known Hu IP-10 content, control specimen, and unknowns, are pipetted into these wells followed by the addition of a second biotinylated monoclonal antibody (Farber 1997). The mean of 15 normal sera done was 105 – 35 pg/mL.
Follow-up of patients Patients with a complete early virological response (EVR) or partial EVR at week 12 continued treatment till week 24 (Ferenci and others 2005). Patients with a negative HCV RNA at week 24 continued their treatment for 48 weeks.
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Patients with an end-of-treatment response entered a followup period until week 72 (Ghany and others 2009). Patients who had negative HCV RNA at week 72 were considered to achieve an SVR (Sarrazin and others 2010). Patients were divided according to their response as follows: nonresponders; 39 patients (44.3%), relapsers; 14 patients (15.9%), and 35 patients (39.8%) who achieved an SVR according to AASLD (2009).
Statistical analysis Analysis of data was performed using SPSS 17 (Statistical Package for Scientific Studies) for Windows. Quantitative parametric variables were presented as mean and standard deviation. They were compared by the ANOVA test. They were correlated to IP-10 by Pearson correlation. Nonparametric variables were presented as the median and interquartile ratio. They were compared by the Kruskal– Wallis test. They were correlated by Spearman correlation. Qualitative variables were presented as numbers and percentages. They were compared by the chi-square or Fischer’s exact test, when appropriate. A receiver operator characteristic (ROC) curve was plotted to assess the relationship between IP-10 and response to treatment. Area under the curve (AUC) was considered reliable if more than 0.60. Uni- and multivariate regression analysis was done, in which the dependent factor is the failure of treatment. In all tests, P value was considered significant if less than 0.05.
Results The 3 studied groups were matched regarding the age, BMI, gender, and baseline laboratory data, apart from the TSH levels, which were found higher in nonresponders (NR) with P value of 0.03 (Table 1). There was no statistically significant difference in both the degree of necroinflammatory activity and fibrosis stage among the 3 studied groups with P values = 0.2 and 0.06, respectively. The pretreatment serum IP-10 levels were not significantly different in relation to different grades of necroinflammatory activity and fibrosis stages (Table 2 and Fig. 1). Verifying the predictive value of pretreatment serum IP10 levels, the study did not find a significant relation to response at week 12, 24, 48, and 72 in any of the mentioned groups (Table 3).
Table 1.
Demographic and Baseline Laboratory Data of the Studied Groups
Data Age, mean – SD BMI, mean – SD Male, n = 68 (%) Albumin, mean – SD TSH, mean – SD HCV_RNA_IU/ML AST, mean – SD ALT, mean – SD
NR
Relapsers
SVR
P value
44.9 – 9.8 45 – 11.6 28.4 – 4.1 25.6 – 4.6 26 (38.2) 12 (17.7) 4.1 – 0.5 4.1 – 0.4
42.9 – 9.1 28.7 – 3.4 30 (44.1) 4.3 – 0.6
0.694 0.06 0.08 0.194
1.3 – 1.6 5.0 – 1.5 58.1 – 41.4 60.9 – 57.2
1.0 – 0.8 5.0 – 1.2 49.0 – 30.9 59.6 – 43.2
0.03a 0.424 0.459 0.720
0.9 – 0.6 4.6 – 1.2 62.5 – 39.3 72.8 – 51.9
a P value £ 0.05. ALT, alanine transaminase; AST, aspartate transaminase; BMI, body–mass index; HCV, hepatitis C virus; NR, nonresponders; SD, standard deviation; SVR, sustained virological response; TSH, thyroid stimulating hormone.
RELATION OF IP-10 WITH FIBROSIS AND RESPONSE TO INTERFERON
Table 2.
Activity Fibrosis
Pretreatment IP-10 Level in Relation to Activity and Fibrosis
A1 A2-A3 F1 ‡ F2
IP-10 (mean – SD)
P value
221.52 – 177.44 224.2 – 156.4 219.43 – 168.68 226.03 – 160.65
0.94 0.86
IP-10, inducible protein 10.
When different biochemical parameters were correlated with the pretreatment IP-10 serum levels, there was no statistically significant correlation, apart from serum albumin in the nonresponders group (P value = 0.013, r = 0.373), which was found to be directly correlated with the pretreatment level of IP-10. Table 4 shows that patients were stratified into 3 categories, those with pretreatment serum IP-10 levels below 150 pg/mL, those between 150 and 600 pg/mL, and finally, those with levels above 600 pg/mL. All the patients with IP10 levels above 600 pg/mL failed to achieve an SVR. An ROC curve was designed to find a cutoff level for IP-10 for prediction of response, but the P value was = 0.43 and AUC = 0.55 as shown in Fig. 2. By the regression analysis, in which failure of response to treatment is the dependent factor, there was no statistically significant association between the pretreatment serum level of IP-10 and response to treatment (Table 5).
Discussion An accurate prediction of response to SOC therapy is still needed even with the emergence of the new oral antiviral drugs due to their high cost in the context of a large number of patients and limited resources in developing countries (Esmat and others 2015). The recommendations currently followed in
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Egypt are those of the EASL 2014 guidelines, where patients infected with HCV genotype 4 are treated with a combination of weekly pegylated IFN-a, daily weight-based ribavirin (1,000 or 1,200 mg in patients < 75 kg or ‡ 75 kg, respectively), and daily sofosbuvir (400 mg) for 12 weeks. In the current study, there was no statistically significant difference found between the pretreatment serum level of IP-10 and the degree of viral load, necroinflammatory activity, or fibrosis stage. These results were in disagreement with Reiberger and others (2008), who concluded that the pretreatment IP-10 levels correlated with the HCV viral load, ALT levels, hepatic inflammation, and fibrosis. In addition, our results disagree with Bonacchi and others (2001) and Diago and others (2006), who found that HCV patients with advanced fibrosis had significantly higher serum IP-10 levels than those with mild fibrosis, strongly suggesting that IP-10 could play an important role in the progression of hepatic fibrosis. This difference might be because we investigated the pretreatment level of IP-10 in the Egyptian population, where there is a predominance of genotype 4. In the currently discussed study, there was no statistically significant difference in the pretreatment serum level of IP10 in relation to response to SOC therapy at different time points at week 12, 24, 48, and 72 weeks. These results do not agree with at least 4 independent studies, which have demonstrated that the baseline levels of CXCL10 (IP-10) are predictive of the failure to response to HCV treatment (Butera and others 2005; Diago and others 2006; Lagging and others 2006; Romero and others 2006). Moreover, Apolinario and others (2004) were the first to point out the association between serum levels of CXCL10 and the therapeutic response. The accuracy of CXCL10 in predicting the lack of an SVR was 0.748. Diago and others (2006) reported the association of serum IP-10 levels with SVR to (PEG-IFN/RBV) therapy in patients with genotype 1 chronic HCV, showing that the levels
FIG. 1. Pretreatment inducible protein 10 (IP-10) level in relation to fibrosis.
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Table 3. Pretreatment Level of IP-10 in Relation to Response IP-10 Median (IQR)
Response Response at week 12 EVR NR Response at week 24 Responders Breakthrough at 24 weeks Response at week 48 Responders at 48 weeks NR Response at week 72 SVR NR
P value
172 (201) 174 (288)
0.58
186 (198) 164 (257)
0.80
181 (169) 168 (254)
0.47
163 (139) 179 (245)
0.43
EVR, early virological response; IQR, interquartile ratio.
of IP-10 were found to be higher in NRs and lower in SVR patients; concentrations lower than 594.1 pg/mL had a positive predictive value of 86.8% in this study population. Noteworthy, after successful antiviral therapy with an SVR, serum IP-10 concentrations decreased to levels lower than baseline, whereas they were unchanged in nonresponders, suggesting that HCV itself may be responsible for elevated serum IP-10 concentrations found in HCV-infected patients, and thus, pretreatment serum IP-10 is an independent predictive factor of SVR in HCV patients infected by genotype 1. As IP-10 may play a role in the mechanism of failure of antiviral therapy, interventions neutralizing endogenous IP10 or blocking the function of its receptor, CXCR3, may provide new strategies to improve the treatment outcome of these difficult-to-cure patients. This might be because most of these studies were conducted on patients with genotype 1. Despite not performing genotyping for the studied population, HCV genotype 4 (HCV4) is predominant in Egypt. A recent study by Al-Ashgar and others (2013) concluded that the pretreatment serum IP-10 level is a predictor for SVR in HCV4-infected patients. The baseline IP-10 level is significantly lower in responders among HCV genotype-4d patients compared with 4a patients (64 patients were enrolled in that study). It is noteworthy that the mean IP levels of patients in all groups of this study were considerably higher than the levels of our patients. On the other hand, our results are supported by Feld and others (2013), who concluded that the baseline IP-10 levels were associated with EVR, but not SVR to SOC therapy in acute or early chronic HCV infection. Lagging and others (2006) stratified patients into 3 groups according to pretreatment IP-10 levels (0–150 pg/mL; 150–
Table 4. Stratification of Patients According to IP-10 Levels IP-10 level < 150 pg/mL (n = 36) 150–600 pg/mL (n = 48) > 600 pg/mL (n = 4)
SVR, N (%)
Failure, N (%)
14 (38) 23 (48) 0 (0)
22 (62) 25 (52) 4 (100)
FIG. 2. Receiver operator characteristic (ROC) curve of IP-10 levels for prediction of response: area under ROC curve = 0.55, P value = 0.43. 600 pg/mL; > 600 pg/mL). Levels greater than 600 pg/mL had a negative predictive value of 79% for an SVR. In a subanalysis, including only difficult-to-treat patients (ie, elevated BMI or high viral load) with high CXCL10 levels, none of the 7 patients achieved an SVR. Similarly, in the current study, all the patients with serum IP-10 levels above 600 pg/mL failed to achieve an SVR. Limitations of this study were not performing genotyping assays, as it is not part of the routine evaluation done in Egypt before treatment for cost-effective reasons. Also, due to the small number of patients enrolled with high pretreatment IP-10 levels, we failed to set a cutoff value for prediction of response to treatment.
Table 5. Binary Logistic Regression, in Which Failure of Response to Treatment is the Dependent Factor
Age > 50 Female gender Fibrosis Activity Viremia > 600 · 103 AST ALT IP-10 results
Univariate
Multivariate
95% CI for OR
95% CI for OR
OR Lower Upper
P OR Lower Upper
1.9 4.6
0.11 0.9
8.5 24
0.38 2.2 0.08 3.5
1.1 1.4 0.6
0.53 0.4 0.2
2.3 4.8 2.3
0.78 0.56 0.46
1.0 1.0 1.0
1.0 1.0 1.0
1.0 1.0 1.0
0.46 0.47 0.05 1.0
CI, confidence interval; OR, odds ratio.
P
0.6 0.8
7.6 15.9
0.23 0.10
1.0
1.0
0.09
RELATION OF IP-10 WITH FIBROSIS AND RESPONSE TO INTERFERON
Conclusion The pretreatment serum level of IP-10 is not an accurate predictor of either the fibrosis stage or response to PEGIFN/RIB therapy in chronic HCV Egyptian patients.
Author Disclosure Statement No competing financial interests exist.
References Abdo AA, Sanai FM. 2009. Predictors of sustained virologic response in hepatitis C genotype 4: beyond the usual suspects. Ann Saudi Med 29(1):1–3. Al-Ashgar HI, Khan MQ, Helmy A, Al-Thawadi S, Al-Ahdal MN, Khalaf N, Al-Qahtani A, Sanai FM. 2013. Relationship of interferon-g-inducible protein-10 kDa with viral response in patients with various heterogeneities of hepatitis C virus genotype-4. Eur J Gastroenterol Hepatol 25(4):404–410. American Association for the Study of Liver Diseases (AASLD). 2009. Diagnosis, management and treatment of Hepatitis C. Hepatology 49(4):1335–1374. Apolinario A, Diago M, Lo Iacono O, Lorente R, Pe´rez C, Majano PL, Clemente G, Garcı´a-Monzo´n C. 2004. Increased circulating and intrahepatic T-cell-specific chemokines in chronic hepatitis C: relationship with the type of virological response to peginterferon plus ribavirin combination therapy. Aliment Pharmacol Ther 19(5):551–562. Bedossa P, Poynard T. 1996. An algorithm for the grading of activity in chronic hepatitis C. The METAVIR Cooperative Study Group. Hepatology 24(2):289–293. Bonacchi A, Romagnani P, Romanelli RG, Efsen E, Annunziato F, Lasagni L, Francalanci M, Serio M, Laffi G, Pinzani M, Gentilini P, Marra F. 2001. Signal transduction by the chemokine receptor CXCR3. Activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/AKT controls cell migration and proliferation in human vascular pericytes. J Biol Chem 276(13):9945–9954. Butera D, Marukian S, Iwamaye AE, Hembrador E, Chambers TJ, Di Bisceglie AM, Charles ED, Talal AH, Jacobson IM, Rice CM, Dustin LB. 2005. Plasma chemokine levels correlate with the outcome of antiviral therapy in patients with hepatitis C. Blood 106(4):1175–1182. Casrouge A, Decalf J, Ahloulay M, Lababidi C, Mansour H, Vallet-Pichard A, Mallet V, Mottez E, Mapes J, Fontanet A, Pol S, Albert ML. 2011. Evidence for an antagonist form of the chemokine CXCL10 in patients chronically infected with HCV. J Clin Invest 121(1):308–317. Diago M, Castellano G, Garcı´a-Samaniego J, Pe´rez C, Ferna´ndez I, Romero M, Iacono OL, Garcı´a-Monzo´n C. 2006. Association of pretreatment serum interferon gamma inducible protein 10 levels with sustained virological response to peginterferon plus ribavirin therapy in genotype 1 infected patients with chronic hepatitis C. Gut 55:374–379. El Makhzangy H, Esmat G, Said M, Elraziky M, Shouman S, Refai R, Rekacewicz C, Gad RR, Vignier N, Abdel-Hamid M, Zalata K, Bedossa P, Pol S, Fontanet A, Mohamed MK. 2009. Response to pegylated interferon alfa-2a and ribavirin in chronic hepatitis C genotype 4. J Med Virol 81(9):1576– 1583. Esmat G, El Raziky M, Elsharkawy A, Sabry D, Hassany M, Ahmed A, Assem N, El Kassas M, Doss W. 2015. Impact of vitamin D supplementation on sustained virological response in chronic hepatitis C genotype 4 patients treated by pegylated interferon/ribavirin. J Interferon Cytokine Res 35(1):49–54.
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European Association for Study of the Liver. 2014. EASL recommendations on treatment of hepatitis C. J Hepatol 61(2): 373–395. Farber JM. 1997. Mig and IP-10: CXC chemokines that target lymphocytes. J Leukoc Biol 61(3):246–257. Feld JJ, Grebely J, Matthews GV, Applegate T, Hellard M, Sherker A, Cherepanov V, Petoumenos K, Yeung B, Kaldor JM, Lloyd AR, Dore GJ. 2013. Plasma interferon-gammainducible protein-10 levels are associated with early, but not sustained virological response during treatment of acute or early chronic HCV infection. PLoS One 8(11):e80003. Ferenci P, Fried MW, Shiffman ML, Smith CI, Marinos G, Gonc¸ales FL Jr, Ha¨ussinger D, Diago M, Carosi G, Dhumeaux D, Craxı` A, Chaneac M, Reddy KR. 2005. Predicting sustained virological responses in chronic hepatitis C patients treated with peginterferon alfa-2a (40 KD)/ribavirin. J Hepatol 43(3):425–433. Ghany MG, Strader DB, Thomas DL, Seeff LB. 2009. American Association for the Study of Liver Diseases (AASLD) practice guidelines diagnosis, management, and treatment of hepatitis C: an update. Hepatology 49(4):335–1374. Guerra J, Garenne M, Mohamed MK, Fontanet A. 2012. HCV burden of infection in Egypt: results from a nationwide survey. J Viral Hepat 19(8):560–567. Lagging M, Romero AI, Westin J, Norkrans G, Dhillon AP, Pawlotsky JM, Zeuzem S, von Wagner M, Negro F, Schalm SW, Haagmans BL, Ferrari C, Missale G, Neumann AU, Verheij-Hart E, Hellstrand K; DITTO-HCV Study Group 2006. IP-10 predicts viral response and therapeutic outcome in difficult to-treat patients with HCV genotype 1 infection. Hepatology 44(6):1617–1625. Murdoch C, Finn A. 2000. Chemokine receptors and their role in inflammation and infectious diseases. Blood 95:3032–3043. Ray SC, Arthur RR, Carella A, Bukh J, Thomas DL. 2000. Genetic epidemiology of hepatitis C virus throughout Egypt. J Infect Dis 182(3):698–707. Reiberger T, Aberle JH, Kundi M, Kohrgruber N, Rieger A, Gangl A, Holzmann H, Peck-Radosavljevic M. 2008. IP-10 correlates with hepatitis C viral load, hepatic inflammation and fibrosis and predicts hepatitis C virus relapse or non-response in HIV-HCV coinfection. Antivir Ther 13(8):969–976. Romero AI, Lagging M, Westin J, Dhillon AP, Dustin LB, Pawlotsky JM, Neumann AU, Ferrari C, Missale G, Haagmans BL, Schalm SW, Zeuzem S, Negro F, Verheij-Hart E, Hellstrand K; DITTO-HCV Study Group. 2006. Interferon (IFN)-gamma-inducible protein-10: association with histological results, viral kinetics, and outcome during treatment with pegylated IFN-alpha 2a and ribavirin for chronic hepatitis C virus infection. J Infect Dis 194(7):895–903. Sarrazin C, Shiffman ML, Hadziyannis SJ, Lin A, Colucci G, Ishida H, Zeuzem S. 2010. Definition of rapid virologic response with a highly sensitive real-time PCR-based HCV RNA assay in peginterferon alfa-2a plus ribavirin responseguided therapy. J Hepatol 52(6):832–838.
Address correspondence to: Dr. Hadeel Gamal Eldeen Department of Endemic Hepatology and Gasteroenterology Faculty of Medicine Cairo University Cairo 11562 Egypt E-mail: [email protected] Received 25 October 2014/Accepted 27 February 2015
IP-10 Serum Level in Chronic Hepatitis C Virus Patients: Relation to Fibrosis and Response to Combined Interferon/Ribavirin Therapy Maissa El Raziky,1 Aisha Elsharkawy,1 Salma E. Said,2 Sahar Abdelatty,3 Wafaa El Akel,1 Omnia Tantawy,1 Hadeel Gamal Eldeen,1 and Mahasen Mabrouk1
Despite the appearance of the direct acting antiviral drugs, pegylated interferon/ribavirin (PEG-IFN/RBV) still has a place in the standard of care (SOC) therapy for chronic HCV4. Studies were conducted to find an accurate prediction in response to SOC therapy. Pretreatment serum interferon-g-inducible protein-10 (IP-10) is an independent predictive factor of sustained virological response (SVR) in HCV1-infected patients. To assess whether the pretreatment serum level of IP-10 influences hepatic fibrosis and PEG-IFN/RBV therapy response, a study was conducted on 88 chronic Hepatitis C virus (HCV) patients who received PEG-IFN/RBV. Patients were subjected to a pretreatment routine laboratory evaluation, liver biopsy, and serum IP-10 assessment. They were followed up for 6 months after cessation of therapy (week 72). Patients were classified into 3 groups according to their response; nonresponders, relapsers, or sustained virological responders. The relation of pretreatment IP-10 with fibrosis and response was assessed. The studied groups were matched regarding their demographic data. There was no statistically significant association between the pretreatment IP-10 level and fibrosis (P = 0.86) and no relation to response was found at week 12, 24, 48, and 72 (P = 0.58, 0.8, 0.47, and 0.43, respectively). Pretreatment IP-10 could not predict either fibrosis or response to PEG-IFN/RIB therapy in chronic HCV Egyptian patients.
Introduction
H
epatitis C virus (HCV) is highly endemic in Egypt, where nearly 9.8% of the population is chronically infected with HCV (Guerra and others 2012). The genotype 4 is predominant in 91% of these patients (Ray and others 2000). The sustained virological response (SVR) in genotype 4 Egyptian patients treated with pegylated interferon/ribavirin (PEG-IFN/RBV) was estimated around 60%, intermediate between the SVR observed in genotype 1 and genotypes 2 and 3 patients in western countries (El Makhzangy and others 2009). Despite the appearance of new oral antiviral drugs, PEGIFN/RBV may remain the standard-of-care (SOC) therapy for some time, especially with the expected high costs and the limited number of patients who will be eligible for the new treatment (Esmat and others 2015). Knowledge of predictors of response is extremely valuable, as this will lead to considering pretreatment counseling, sparing patients the side effects and cost of therapy (Abdo and Sanai 2009).
The interferon-g-inducible protein 10 (IP-10), also termed CXCL10, belongs to the CXC (or a) chemokine family of secreted 8–10 kD proteins (Murdoch and Finn 2000). IP-10 is an IFN-g- and TNF-a-inducible chemokine that can be highly expressed by fibroblasts, keratinocytes, mesangial cells, endothelial cells, monocytes, astrocytes, neutrophils, and hepatocytes (Casrouge and others 2011). The majority of liver-infiltrating T cells in patients with chronic HCV expressed CXCR3, and the amount of hepatic IP-10 mRNA and protein was markedly increased in these patients and correlated strongly with serum IP-10 concentrations (Apolinario and others 2004). Four independent studies demonstrated that the baseline levels of IP-10 are predictive of the failure of response to HCV treatment. This has been demonstrated in patients with genotype 1 and few cases of genotype 4 HCV (Butera and others 2005; Diago and others 2006; Lagging and others 2006; Romero and others 2006). The aim of this study was to assess whether the pretreatment serum level of IP-10 influences hepatic fibrosis and PEG-IFN/RBV therapy response in Egyptian patients predominantly infected with HCV4.
Departments of 1Endemic Hepatology and Gasteroenterology, 2Biochemistry, and 3Chemical and Clinical Pathology, Faculty of Medicine, Cairo University, Cairo, Egypt.
649
650
Patients and Methods This is a prospective study conducted on 88 patients with chronic HCV who attended El- Fatemia hospital in Cairo and were naive to treatment with SOC therapy with PEGIFN/RBV.
Sample size The sample size was estimated by using a power calculation program (Roasoft program) based on the study by Diago and others (2006). With an error margin of 5%, confidence level of 85, and an expected population size of 95, the recommended sample size was at least 63 patients (www.raosoft.com/samplesize.html).
Subjects Patients were recruited according to the following inclusion criteria: positive HCV antibodies using a third-generation test and detectable HCV RNA by PCR (according to the Abbott RealTime HCV kit, with a detection limit of 12 IU/ mL), age between 18 and 65 years, body–mass index (BMI) < 30, and the following hematologic and biochemical criteria [WBC > 3,500/mm3, neutrophil count > 1,500/mm3, platelets > 75,000/mm3, International Normalized Ratio ( £ 1.5), total serum bilirubin < 1.5 mg/dl, albumin > 3.5 g/dl, serum creatinine within normal, thyroid stimulating hormone (TSH) within normal, and anti nuclear antibody < 1:16]. Patients with uncontrolled diabetes mellitus, severe psychiatric disease, decompensated liver disease, combined HBV (positive HBsAg) and HCV, and serious comorbid conditions were excluded from this study. The study was conducted according to the principles of the Declaration of Helsinki. The Institutional Review Board (IRB) study approval was obtained through the (NHTMRI) IRB office before commencement of the study.
Study procedure After signing a written informed consent, the selected patients were subjected to the following: full history taking, clinical assessment, and laboratory investigations, including routine laboratory tests, pretreatment serum level of IP-10, and histopathological examination of a needle liver biopsy of a core of at least 15-mm length and containing 6 portal tracts. A single experienced pathologist analyzed all biopsy specimens and they were evaluated according to the METAVIR score (Bedossa and Poynard 1996). The Invitrogen Human IP-10 (Hu IP-10) kit used (Invitrogen corporation, Carlsbad, CA) is a solid-phase enzyme-linked immunosorbent assay (ELISA). A monoclonal antibody specific for Hu IP-10 has been coated onto the wells of the microtiter strips provided. Samples, including standards of known Hu IP-10 content, control specimen, and unknowns, are pipetted into these wells followed by the addition of a second biotinylated monoclonal antibody (Farber 1997). The mean of 15 normal sera done was 105 – 35 pg/mL.
Follow-up of patients Patients with a complete early virological response (EVR) or partial EVR at week 12 continued treatment till week 24 (Ferenci and others 2005). Patients with a negative HCV RNA at week 24 continued their treatment for 48 weeks.
EL RAZIKY ET AL.
Patients with an end-of-treatment response entered a followup period until week 72 (Ghany and others 2009). Patients who had negative HCV RNA at week 72 were considered to achieve an SVR (Sarrazin and others 2010). Patients were divided according to their response as follows: nonresponders; 39 patients (44.3%), relapsers; 14 patients (15.9%), and 35 patients (39.8%) who achieved an SVR according to AASLD (2009).
Statistical analysis Analysis of data was performed using SPSS 17 (Statistical Package for Scientific Studies) for Windows. Quantitative parametric variables were presented as mean and standard deviation. They were compared by the ANOVA test. They were correlated to IP-10 by Pearson correlation. Nonparametric variables were presented as the median and interquartile ratio. They were compared by the Kruskal– Wallis test. They were correlated by Spearman correlation. Qualitative variables were presented as numbers and percentages. They were compared by the chi-square or Fischer’s exact test, when appropriate. A receiver operator characteristic (ROC) curve was plotted to assess the relationship between IP-10 and response to treatment. Area under the curve (AUC) was considered reliable if more than 0.60. Uni- and multivariate regression analysis was done, in which the dependent factor is the failure of treatment. In all tests, P value was considered significant if less than 0.05.
Results The 3 studied groups were matched regarding the age, BMI, gender, and baseline laboratory data, apart from the TSH levels, which were found higher in nonresponders (NR) with P value of 0.03 (Table 1). There was no statistically significant difference in both the degree of necroinflammatory activity and fibrosis stage among the 3 studied groups with P values = 0.2 and 0.06, respectively. The pretreatment serum IP-10 levels were not significantly different in relation to different grades of necroinflammatory activity and fibrosis stages (Table 2 and Fig. 1). Verifying the predictive value of pretreatment serum IP10 levels, the study did not find a significant relation to response at week 12, 24, 48, and 72 in any of the mentioned groups (Table 3).
Table 1.
Demographic and Baseline Laboratory Data of the Studied Groups
Data Age, mean – SD BMI, mean – SD Male, n = 68 (%) Albumin, mean – SD TSH, mean – SD HCV_RNA_IU/ML AST, mean – SD ALT, mean – SD
NR
Relapsers
SVR
P value
44.9 – 9.8 45 – 11.6 28.4 – 4.1 25.6 – 4.6 26 (38.2) 12 (17.7) 4.1 – 0.5 4.1 – 0.4
42.9 – 9.1 28.7 – 3.4 30 (44.1) 4.3 – 0.6
0.694 0.06 0.08 0.194
1.3 – 1.6 5.0 – 1.5 58.1 – 41.4 60.9 – 57.2
1.0 – 0.8 5.0 – 1.2 49.0 – 30.9 59.6 – 43.2
0.03a 0.424 0.459 0.720
0.9 – 0.6 4.6 – 1.2 62.5 – 39.3 72.8 – 51.9
a P value £ 0.05. ALT, alanine transaminase; AST, aspartate transaminase; BMI, body–mass index; HCV, hepatitis C virus; NR, nonresponders; SD, standard deviation; SVR, sustained virological response; TSH, thyroid stimulating hormone.
RELATION OF IP-10 WITH FIBROSIS AND RESPONSE TO INTERFERON
Table 2.
Activity Fibrosis
Pretreatment IP-10 Level in Relation to Activity and Fibrosis
A1 A2-A3 F1 ‡ F2
IP-10 (mean – SD)
P value
221.52 – 177.44 224.2 – 156.4 219.43 – 168.68 226.03 – 160.65
0.94 0.86
IP-10, inducible protein 10.
When different biochemical parameters were correlated with the pretreatment IP-10 serum levels, there was no statistically significant correlation, apart from serum albumin in the nonresponders group (P value = 0.013, r = 0.373), which was found to be directly correlated with the pretreatment level of IP-10. Table 4 shows that patients were stratified into 3 categories, those with pretreatment serum IP-10 levels below 150 pg/mL, those between 150 and 600 pg/mL, and finally, those with levels above 600 pg/mL. All the patients with IP10 levels above 600 pg/mL failed to achieve an SVR. An ROC curve was designed to find a cutoff level for IP-10 for prediction of response, but the P value was = 0.43 and AUC = 0.55 as shown in Fig. 2. By the regression analysis, in which failure of response to treatment is the dependent factor, there was no statistically significant association between the pretreatment serum level of IP-10 and response to treatment (Table 5).
Discussion An accurate prediction of response to SOC therapy is still needed even with the emergence of the new oral antiviral drugs due to their high cost in the context of a large number of patients and limited resources in developing countries (Esmat and others 2015). The recommendations currently followed in
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Egypt are those of the EASL 2014 guidelines, where patients infected with HCV genotype 4 are treated with a combination of weekly pegylated IFN-a, daily weight-based ribavirin (1,000 or 1,200 mg in patients < 75 kg or ‡ 75 kg, respectively), and daily sofosbuvir (400 mg) for 12 weeks. In the current study, there was no statistically significant difference found between the pretreatment serum level of IP-10 and the degree of viral load, necroinflammatory activity, or fibrosis stage. These results were in disagreement with Reiberger and others (2008), who concluded that the pretreatment IP-10 levels correlated with the HCV viral load, ALT levels, hepatic inflammation, and fibrosis. In addition, our results disagree with Bonacchi and others (2001) and Diago and others (2006), who found that HCV patients with advanced fibrosis had significantly higher serum IP-10 levels than those with mild fibrosis, strongly suggesting that IP-10 could play an important role in the progression of hepatic fibrosis. This difference might be because we investigated the pretreatment level of IP-10 in the Egyptian population, where there is a predominance of genotype 4. In the currently discussed study, there was no statistically significant difference in the pretreatment serum level of IP10 in relation to response to SOC therapy at different time points at week 12, 24, 48, and 72 weeks. These results do not agree with at least 4 independent studies, which have demonstrated that the baseline levels of CXCL10 (IP-10) are predictive of the failure to response to HCV treatment (Butera and others 2005; Diago and others 2006; Lagging and others 2006; Romero and others 2006). Moreover, Apolinario and others (2004) were the first to point out the association between serum levels of CXCL10 and the therapeutic response. The accuracy of CXCL10 in predicting the lack of an SVR was 0.748. Diago and others (2006) reported the association of serum IP-10 levels with SVR to (PEG-IFN/RBV) therapy in patients with genotype 1 chronic HCV, showing that the levels
FIG. 1. Pretreatment inducible protein 10 (IP-10) level in relation to fibrosis.
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Table 3. Pretreatment Level of IP-10 in Relation to Response IP-10 Median (IQR)
Response Response at week 12 EVR NR Response at week 24 Responders Breakthrough at 24 weeks Response at week 48 Responders at 48 weeks NR Response at week 72 SVR NR
P value
172 (201) 174 (288)
0.58
186 (198) 164 (257)
0.80
181 (169) 168 (254)
0.47
163 (139) 179 (245)
0.43
EVR, early virological response; IQR, interquartile ratio.
of IP-10 were found to be higher in NRs and lower in SVR patients; concentrations lower than 594.1 pg/mL had a positive predictive value of 86.8% in this study population. Noteworthy, after successful antiviral therapy with an SVR, serum IP-10 concentrations decreased to levels lower than baseline, whereas they were unchanged in nonresponders, suggesting that HCV itself may be responsible for elevated serum IP-10 concentrations found in HCV-infected patients, and thus, pretreatment serum IP-10 is an independent predictive factor of SVR in HCV patients infected by genotype 1. As IP-10 may play a role in the mechanism of failure of antiviral therapy, interventions neutralizing endogenous IP10 or blocking the function of its receptor, CXCR3, may provide new strategies to improve the treatment outcome of these difficult-to-cure patients. This might be because most of these studies were conducted on patients with genotype 1. Despite not performing genotyping for the studied population, HCV genotype 4 (HCV4) is predominant in Egypt. A recent study by Al-Ashgar and others (2013) concluded that the pretreatment serum IP-10 level is a predictor for SVR in HCV4-infected patients. The baseline IP-10 level is significantly lower in responders among HCV genotype-4d patients compared with 4a patients (64 patients were enrolled in that study). It is noteworthy that the mean IP levels of patients in all groups of this study were considerably higher than the levels of our patients. On the other hand, our results are supported by Feld and others (2013), who concluded that the baseline IP-10 levels were associated with EVR, but not SVR to SOC therapy in acute or early chronic HCV infection. Lagging and others (2006) stratified patients into 3 groups according to pretreatment IP-10 levels (0–150 pg/mL; 150–
Table 4. Stratification of Patients According to IP-10 Levels IP-10 level < 150 pg/mL (n = 36) 150–600 pg/mL (n = 48) > 600 pg/mL (n = 4)
SVR, N (%)
Failure, N (%)
14 (38) 23 (48) 0 (0)
22 (62) 25 (52) 4 (100)
FIG. 2. Receiver operator characteristic (ROC) curve of IP-10 levels for prediction of response: area under ROC curve = 0.55, P value = 0.43. 600 pg/mL; > 600 pg/mL). Levels greater than 600 pg/mL had a negative predictive value of 79% for an SVR. In a subanalysis, including only difficult-to-treat patients (ie, elevated BMI or high viral load) with high CXCL10 levels, none of the 7 patients achieved an SVR. Similarly, in the current study, all the patients with serum IP-10 levels above 600 pg/mL failed to achieve an SVR. Limitations of this study were not performing genotyping assays, as it is not part of the routine evaluation done in Egypt before treatment for cost-effective reasons. Also, due to the small number of patients enrolled with high pretreatment IP-10 levels, we failed to set a cutoff value for prediction of response to treatment.
Table 5. Binary Logistic Regression, in Which Failure of Response to Treatment is the Dependent Factor
Age > 50 Female gender Fibrosis Activity Viremia > 600 · 103 AST ALT IP-10 results
Univariate
Multivariate
95% CI for OR
95% CI for OR
OR Lower Upper
P OR Lower Upper
1.9 4.6
0.11 0.9
8.5 24
0.38 2.2 0.08 3.5
1.1 1.4 0.6
0.53 0.4 0.2
2.3 4.8 2.3
0.78 0.56 0.46
1.0 1.0 1.0
1.0 1.0 1.0
1.0 1.0 1.0
0.46 0.47 0.05 1.0
CI, confidence interval; OR, odds ratio.
P
0.6 0.8
7.6 15.9
0.23 0.10
1.0
1.0
0.09
RELATION OF IP-10 WITH FIBROSIS AND RESPONSE TO INTERFERON
Conclusion The pretreatment serum level of IP-10 is not an accurate predictor of either the fibrosis stage or response to PEGIFN/RIB therapy in chronic HCV Egyptian patients.
Author Disclosure Statement No competing financial interests exist.
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Address correspondence to: Dr. Hadeel Gamal Eldeen Department of Endemic Hepatology and Gasteroenterology Faculty of Medicine Cairo University Cairo 11562 Egypt E-mail: [email protected] Received 25 October 2014/Accepted 27 February 2015
