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HCV-coinfected patients
Journal of Clinical Virology 61 (2014) 423–429
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CXCL9, CXCL10 and CXCL11 polymorphisms are associated with sustained virologic response in HIV/HCV-coinfected patients Daniel Pineda-Tenor a , Juan Berenguer b,c , María A. Jiménez-Sousa a , María Guzmán-Fulgencio a , Teresa Aldámiz-Echevarria b,c , Ana Carrero b,c , Mónica García-Álvarez a , Cristina Diez b,c , Francisco Tejerina b,c , Verónica Briz a , Salvador Resino a,∗ a
Unidad de Infección Viral e Inmunidad, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain Unidad de Enfermedades Infecciosas/VIH, Hospital General Universitario “Gregorio Mara˜ nón”, Madrid, Spain c Instituto de Investigación Sanitaria Gregorio Mara˜ nón (IiSGM), Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 22 April 2014 Received in revised form 20 August 2014 Accepted 24 August 2014 Keywords: HIV/AIDS Chronic hepatitis C CXCL HCV therapy SNPs
a b s t r a c t Background: The CXCL9, CXCL10 and CXCL11 (CXCL9–11) chemokines play a critical role in eradication of hepatitis C virus (HCV), although HCV-specific immunity often fails to eradicate the HCV, allowing the chronicity of hepatitis C. Objective: To examine the association between CXCL9–11 polymorphisms and the sustained virological response (SVR) following hepatitis C virus (HCV) therapy with pegylated-interferon-alpha plus ribavirin in HIV/HCV-coinfected patients. Study design: We performed a retrospective study in 176 naïve patients who started HCV treatment. The CXCL9 rs10336, CXCL10 rs3921 and CXCL11 rs4619915 polymorphisms were genotyped by GoldenGate® assay. Genetic data were analyzed under recessive inheritance model. The SVR was defined as undetectable HCV viremia through 24 weeks after the end of HCV treatment. Results: In the intention-to-treat analysis, the SVR rate was higher in HCV genotype 1/4 (GT1/4) patients carrying rs10336 TT (p = 0.042), rs3921 GG (p = 0.021), and rs4619915 AA (p = 0.024) genotypes; and they had higher likelihood of achieving SVR (adjusted odds ratio (aOR) = 3.26 (p = 0.038), aOR = 4.21 (p = 0.019), and aOR = 4.08 (p = 0.022), respectively). For CXCL haplotype analysis (CXCL9/rs10336, CXCL10/rs3921, and CXCL11/rs4619915), the TGA haplotype (favorable alleles) had better odds of achieving SVR than the CCG haplotype (unfavorable alleles) in GT1/4patients (OR = 2.69; p = 0.003). No significant results were found in GT2/3 patients. Moreover, similar results were obtained in the on-treatment analysis. Conclusions: The presence of homozygous for the minor allele of CXCL9 rs10336, CXCL10 rs3921 and CXCL11 rs4619915 was related to higher likelihoods of achieving the HCV clearance after pegIFN␣/ribavirin therapy in HIV infected patients coinfected with HCV GT1/4. © 2014 Elsevier B.V. All rights reserved.
1. Background During the last decade, dual therapy with pegylated-interferonalpha plus ribavirin (pegIFN␣/ribavirin) has been administrated for the treatment of hepatitis C virus (HCV) infection in patients co-infected with HIV but the effectiveness of this treatment has been low [1]. The rate of sustained virological response
∗ Corresponding author at: Centro Nacional de Microbiología, Instituto de Salud Carlos III (Campus Majadahonda), Carretera Majadahonda-Pozuelo, Km 2.2, 28220 Majadahonda, Madrid, Spain. Tel.: +34 918 223 266; fax: +34 918 223 269. E-mail addresses: [email protected], [email protected] (S. Resino). http://dx.doi.org/10.1016/j.jcv.2014.08.020 1386-6532/© 2014 Elsevier B.V. All rights reserved.
(SVR) of pegIFN␣/ribavirin therapy is around 20–40% for patients infected with HCV genotype 1 and 4 (GT1/4), and 50–60% in HCV genotype 2 and 3 (GT2/3) patients [2,3]. Nowadays, the new directacting antivirals (DAAs) are generally administered in combination with pegIFN␣/ribavirin, particularly in difficult-to-treat patients infected with GT1/4 [1]. Despite the emergence of new DAAs, the potential use of these drugs in HIV/HCV coinfected patients is complicated due to choice of patients to treat, potential for drug–drug interactions and overlapping toxicities between HIV and HCV therapy [1]. To date, the best baseline predictors for HCV therapy are age, sex, liver fibrosis, HCV genotype, HCV viral load, obesity, and singlenucleotide polymorphisms (SNPs) around interleukin 28B (IL28B)
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gene [4]. Moreover, CXC chemokine ligand 10 [CXCL10, also known as interferon-␥-inducible protein-10 (IP-10)] has been proposed as predictive biomarker of virologic response to pegIFN␣/ribavirin therapy in GT1/4 patients [5–7], whereas the combination of CXCL10 levels and IL28B polymorphisms improves the predictive performance for achieving SVR in both HIV/HCV coinfected and HCV monoinfected patients [8,9]. The CXCL10, together with CXCL9 (monokine induced by interferon ␥; Mig) and CXCL11 (interferon-inducible T-cell ␣ chemoattractant; I-TAC), are produced in the liver during chronic hepatitis C (CHC), inducing migration of activated T cells (T-helper/T-cytotoxic type-1 cell (Th1/Tc1) response) from the periphery to infected liver parenchyma via CCR5 or CXCR3 chemokine receptors [10]. These CXCL9–11 chemokines play a critical role in eradication of HCV, although HCV-specific immunity often fails to eradicate the HCV, allowing the chronicity of hepatitis C [10]. 2. Objective The aim of the study was to evaluate the association between CXCL9–11 polymorphisms and SVR in HIV/HCV-coinfected patients on pegIFN␣/ribavirin therapy. 3. Study design 3.1. Patients We carried out a retrospective study in 176 European HIV/HCV˜ coinfected naïve patients from Hospital Gregorio Maranón (Madrid, Spain) who started treatment with pegIFN␣/ribavirin on regular follow-up from October 2000 to June 2010. The study was conducted in accordance with the Declaration of Helsinki. Patients gave their written consent before the start of the study. The Institutional Review Board and the Research Ethic Committee of the Instituto de Salud Carlos III (ISCIII) approved the study. The criteria for starting HCV antiviral treatment were: (A) inclusion criteria: chronic hepatitis C (presence of detectable HCV replication for at least six months after HCV infection), negative hepatitis B surface antigen, availability of DNA sample, no clinical evidence of hepatic decompensation, detectable HCV RNA by polymerase chain reaction at baseline, CD4+ count higher than 200 cells/mm3 , and stable cART for at least 6 months before study entry or no need for cART according to treatment guidelines used in the study period [11,12]. (B) Exclusion criteria: active opportunistic infections, active drug or alcohol addiction, and other concomitant diseases or conditions such as diabetes, nephropathies, autoimmune diseases, haemochromatosis, primary biliary cirrhosis, Wilson’s disease, a1-antitrypsin deficiency and neoplasia. 3.2. Epidemiological and clinical data Clinical and epidemiological data (including sex, age, HIV acquired by IVDU, years since HCV infection, prior AIDS, HIV markers and HCV markers) were obtained from medical records in order to obtain a complete data set before starting hepatitis C therapy. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. The degree of insulin resistance (IR) was estimated for each patient using the homeostatic model assessment (HOMA) [13]: fasting plasma glucose (mmol/L) times fasting serum insulin (mU/L) divided by 22.5. 3.3. Liver biopsy Liver biopsies were performed before starting hepatitis C therapy as part of usual clinical care as we described previously [14].
Liver fibrosis and was estimated according to Metavir score as follows: F0, no fibrosis; F1, mild fibrosis; F2, significant fibrosis; F3, advanced fibrosis; and F4, definite cirrhosis. Activity grade was scored as follows: A0, no activity; A1, mild activity; A2, moderate activity; A3, severe activity. Liver steatosis was evaluated according to the existence of hepatocytes containing visible macrovesicular fat droplets. We considered hepatic steatosis to be clinically significant when fatty hepatocytes exceeded 10% of the hepatic parenchyma. 3.4. HCV assays HCV infection was documented in all patients by enzyme-linked immunosorbent assay (ELISA) and PCR test. HCV genotype was determined by hybridization of biotin-labeled PCR products to oligonucleotide probes bound to nitrocellulose membrane strips (INNO-LiPA HCV II, Innogenetics, Ghent, Belgium). Plasma HCVRNA viral load was measured by polymerase chain reaction (PCR) (Cobas Amplicor HCV Monitor Test, Branchburg, NJ, USA) and realtime PCR (COBAS AmpliPrep/COBAS TaqMan HCV test); and results were reported in terms of international units per milliliter (IU/mL), with a lower limit of detection of 10 IU/mL. 3.5. Hepatitis C therapy Following both international and national guidelines [11,12,15,16], HCV treatment regimens included pegIFN␣ 2a or 2b at standard doses (180 g/week or 1.5 g/kg/week, respectively) plus weight-adjusted ribavirin dosing (1000 mg/day for patients weighing <75 kg and 1200 mg/day for patients weighing ≥75 kg). Patients with HCV genotypes 1 or 4 received either 48 or 72 weeks of treatment, and patients with HCV genotype 2 or 3 were treated for 24 or 48 weeks. A SVR was defined as an undetectable serum HCV-RNA level (<10 IU/mL) at week 24 after the end of the treatment. 3.6. Genotyping of CXCL polymorphisms Genomic DNA was extracted from peripheral blood with Qiagen kit (QIAamp DNA Blood Midi/Maxi; Qiagen, Hilden, Germany). DNA samples were sent at the Spanish National Genotyping Center (CeGen; http://www.cegen.org/) for genotyping CXCL9 rs10336, CXCL10 rs3921, and CXCL11 rs4619915 polymorphisms, which are located in putatively regulatory regions such as 3 untranslated region (rs10336) or intronic regions (rs3921 and rs4619915). 3.7. Statistical analysis For the description of the study population, p-values were estimated with nonparametric tests: Mann–Whitney U test was used for continuous variable and Chi-square test for categorical variables. All SNPs were analyzed for Hardy–Weinberg equilibrium (HWE) by Chi-square test, considering equilibrium when p > 0.05. For association study, logistic regression analysis was used to investigate the relationship among CXCL polymorphisms and HCV-therapy response. These analyses were adjusted by the most important clinical and epidemiological characteristics, which were selected by a “Stepwise” algorithm (p-value for entry and exit of 0.15 and 0.20, respectively). The covariables used were gender, age, BMI, HOMA, nadir CD4+ T-cells, time on cART, HCV genotype, HCVRNA viral load, liver fibrosis, and IL28B rs12980275 polymorphism. Moreover, decision tree analyses were performed via classification and regression tree (CART) algorithm to classify GT1/4 patients according to SVR using IL28B and CXCL10 genotypes. These analyses were performed by using the Statistical Package for the Social Sciences (SPSS) 19.0 software (IBM Corp., Chicago,
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Table 1 Clinical and epidemiological characteristics of HIV/HCV-coinfected patients. Values expressed as absolute numbers (%) and median (percentile 25-percentile 75). Characteristics
All patients
HCV-GT 2/3
HCV-GT 1/4
No. (%) Male, n (%) Age, years BMI, kg/m2 BMI ≥ 25 kg/m2 HOMA HOMA ≥ 3 HIV acquired by IVDU, n (%) Years since HCV infection Prior AIDS, n (%) cART, n (%) Time on cART, years Current cART protocols, n (%) Any NRTIs + any PI Any NRTIs + PI + NNRTI Any NRTIs + any NNRTI Only NRTIs HIV markers Nadir CD4+, cells/L Nadir CD4+ <200 cells/L, n (%) CD4+ T, cells/L CD4+ ≥ 500 cells/L, n (%) HIV-RNA < 50 copies/mL, n (%) HCV markers, n (%) HCV-RNA ≥ 500,000 IU/mL HCV genotype HCV-GT1 HCV-GT2 HCV-GT3 HCV-GT4 Undefined Metavir score, n (%) Liver biopsy patients Significant fibrosis (F ≥ 2) Moderate or severe activity (A ≥ 2) Steatosis
176 (100%) 135 (76.7%) 40.6 (37.9–44.5) 22.5 (21–24.5) 40 (22.7%) 2.13 (1.44–3.85) 66 (37.5%) 153 (86.9%) 20.5 (15.4–23.7) 49 (27.8%) 145 (82.4%) 4.59 (2.58–7.53)
56 (100%) 42 (75%) 41 (38.5–44.5) 22.5 (20.8–24.5) 47 (83.9%) 1.98 (1.34–3.64) 18 (32.1%) 49 (87.5%) 19.5 (15–23.8) 11 (19.6%) 46 (82.1%) 3.94 (2.34–5.69)
115 (100%) 88 (76.5%) 40.2 (37.8–44.6) 22.5 (21–25.2) 86 (74.8%) 2.22 (1.59–4.07) 47 (40.9%) 99 (86.1%) 20.7 (16.1–23.5) 37 (32.2%) 96 (83.5%) 4.75 (3.09–7.65)
40 (22.7%) 1 (0.6%) 92 (52.3%) 12 (6.8%)
10 (17.9%) 0 (0%) 32 (57.1%) 4 (7.1%)
30 (26.1%) 1 (0.9%) 58 (50.4%) 7 (6.1%)
217 (96–356) 81 (46%) 455 (330–675) 76 (43.4%) 126 (72%)
230 (139–379) 24 (42.9%) 433 (374–682) 23 (41.1%) 72 (76.4%)
204 (72–314) 56 (48.7%) 465 (324–675) 51 (44.7%) 81 (70.4%)
117 (68.4%)
36 (65.5%)
78 (69%)
89 (50.6%) 3 (1.7%) 53 (30.1%) 26 (14.8%) 5 (2.8%)
– 3 (5.4%) 53 (94.6%) – –
89 (77.4%) – – 26 (22.6%) –
142 (80.7%) 91 (64.1%) 90 (64.7%) 75 (54%)
42 (75%) 27 (64.3%) 25 (61%) 29 (65.9%)
97 (84.3%) 62 (63.9%) 64 (67.4%) 46 (49.5%)
Abbreviations: AIDS, acquired immunodeficiency syndrome; BMI, body mass index; cART, combination antiretroviral therapy; GT, genotype; HCV, hepatitis C virus; HCV-RNA, HCV plasma viral load; HIV, human immunodeficiency; HIV-RNA, HIV plasma viral load; HOMA, homeostasis model assessment; IVDU, intravenous drug users; NNRTI, no nucleoside analog reverse-transcriptase inhibitors; NRTI, nucleoside analog reverse-transcriptase inhibitors; PI, protease inhibitors.
USA). In addition, pair-wise linkage disequilibrium (LD) analysis was computed by Haploview 4.2 software, and haplotype-based association testing was performed using the SNPstat software (http://bioinfo.iconcologia.net/SNPStats web). All p-values were two-tailed and statistical significance was defined as p < 0.05.
model that best fitted our data). We divided the analysis according to the course of the HCV treatment. On the one hand, the analysis was performed considering the patients that initiate the therapy (intention to treat), regardless of whether they finished it or not.
4. Results 4.1. Characteristics of patients The main characteristics of the study population are shown in Table 1. At baseline, 176 patients on HCV treatment were stratified by HCV genotype: 56 patients were infected with GT2/3 and 115 with GT1/4. 4.2. CXCL9–11 polymorphisms All SNPs had a minimum allele frequency (MAF) of 40.5% and displayed missing values lower than 5%. CXCL9–11 polymorphisms were in HWE [rs10336 (p = 0.221), rs3921 (p = 0.184), and rs10336 (p = 0.184), respectively]. In addition, a strong LD among SNPs was observed (Fig. 1). 4.3. CXCL9–11 polymorphism and virologic response to HCV therapy The relationship between the CXCL polymorphisms and SVR was performed under a model of recessive inheritance (the genetic
Fig. 1. Pairwise linkage disequilibrium (LD) patterns for the polymorphisms through CXCL9, CXCL10 and CXCL11 regions. Each diagonal represents a different SNP, with each square representing a pairwise comparison between two SNPs.
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Table 2 Relationship among CXCL polymorphisms and sustained virologic responses to HCV treatment in HIV/HCV-coinfected patients according to HCV genotypes. Statistically significant differences are shown in bold. (a) p-values were calculated Chi-squared test; (b) p-values were calculated by logistic regression adjusted by the most important clinical and epidemiological characteristics (see Section 3.7). SNP genotypes A) ITT analysis CXCL9/rs10336
p-value (a)
aOR (95% CI)
p-value (b)
All genotypes
CC/CT
TT
All patients HCV-GT2/3 HCV-GT1/4 CXCL10/rs3921
54.3% (95/175) 83.9% (47/56) 39.5% (45/114) All genotypes
50.7% (73/144) 86.0 (37/43) 35.7% (35/98) CC/CG
71.0% (22/31) 76.9% (10/13) 62.5% (10/16) GG
0.040 0.433 0.042
1.47 (0.55; 3.93) 0.26 (0.04; 1.84) 3.68 (1.07; 12.63)
0.436 0.178 0.038
All patients HCV-GT2/3 HCV-GT1/4 CXCL11/rs4619915
54.9% (95/173) 85.5% (47/55) 39.5% (45/114) All genotypes
50.3% (72/143) 86.0 (37/43) 35.1% (34/97) GG/GA
74.2% (23/31) 83.3% (10/12) 64.7% (11/17) AA
0.016 0.814 0.021
2.37 (0.86; 6.54) 0.52 (0.07; 3.99) 4.21 (1.27; 14.03)
0.095 0.528 0.019
All patients HCV-GT2/3 HCV-GT1/4 B) OT analysis CXCL9/rs10336
54.5% (96/176) 83.9% (47/56) 40.0% (46/115)
50.7% (73/144) 86.0% (37/43) 35.7% (35/98)
71.9% (23/32) 76.9% (10/13) 64.7% (11/17)
0.030 0.433 0.024
1.76 (0.66; 4.69) 0.26 (0.04; 1.84) 4.08 (1.22; 13.61)
0.259 0.178 0.022
All genotypes
CC/CT
TT
All patients HCV-GT2/3 HCV-GT1/4 CXCL10/rs3921
59.7% (95/159) 90.4% (47/52) 43.7% (45/103) All genotypes
55.7% (73/131) 90.2% (37/41) 39.8% (35/88) CC/CG
78.6% (22/28) 90.9% (10/11) 66.7% (10/15) GG
0.025 0.947 0.052
2.05 (0.67; 6.21) 1.08 (0.11; 10.78) 3.61 (0.99; 13.21)
0.205 0.947 0.052
All patients HCV-GT2/3 HCV-GT1/4 CXCL11/rs4619915
59.7% (95/159) 90.4% (47/52) 43.7% (45/103) All genotypes
55.4% (72/130) 90.2% (37/41) 39.1% (34/87) GG/GA
79.3% (23/29) 90.9% (10/11) 68.8% (11/16) AA
0.018 0.947 0.028
2.95 (0.98; 8.91) 0.97 (0.10; 9.79) 3.82 (1.08; 13.56)
0.055 0.981 0.037
60.0% (96/160) 90.4% (47/52) 44.2% (46/104)
55.7% (73/131) 90.2% (37/41) 39.8% (35/88)
79.3% (23/29) 90.9% (10/11) 68.8% (10/16)
0.019 0.433 0.032
2.88 (0.96; 8.68) 0.97 (0.09; 9.79) 3.65 (1.04; 12.92)
0.060 0.981 0.044
All patients HCV-GT2/3 HCV-GT1/4
Abbreviations: 95% CI, 95% of confidence interval; aOR, adjusted odds ratio; HCV-GT, hepatitis C virus genotype; ITT, intention-to-treat; OT, on-treatment.
On the other hand, we only analyzed the patients that completed the HCV treatment (on treatment). In the analysis by intention-to-treat (Table 2A), the SVR rate was higher in carriers of CXCL9 rs10336 TT (p = 0.040), CXCL10 rs3921 GG (p = 0.016), and CXCL11 rs4619915 AA (p = 0.030) genotypes. These trends continued for GT1/4 patients. In regards to the multivariate model, rs10336 TT (p = 0.042), rs3921 GG (p = 0.021) and rs4619915 AA (p = 0.024) genotypes had the highest likelihood of achieving SVR (adjusted odds ratio (aOR) = 3.26 (p = 0.038), aOR = 4.21 (p = 0.019) and aOR = 4.08 (p = 0.022); respectively). When analyzed IL28B rs12980275 and CXCL10 rs3921 polymorphisms in a decision tree, the SVR rate in GT1/4 patients increased from 40% to 52.2% in rs12980275 AA carriers and then to 66.7% in rs3921 GG carriers (Fig. 2A). Moreover, the SVR rate increased from 31.9% in rs12980275 AG/GG carriers to 62.3% in rs3921 GG carriers. The overall percentage of patients correctly classified (accuracy) was 64.3%. In the on-treatment analysis (Table 2B), 16 out of 176 patients were excluded by interruption of HCV therapy (12 adverse events and 4 abandonments) and 160 patients with a full course of HCV therapy were included. In this case, the SVR rate was also the highest in carriers of CXCL9 rs10336 TT (p = 0.025), CXCL10 rs3921 GG (p = 0.018) and CXCL11 rs4619915 AA (p = 0.019) genotypes. These trends continued for GT1/4 patients. Besides, rs10336 TT (p = 0.052), rs3921 GG (p = 0.028) and rs4619915 AA (p = 0.032) genotypes had the highest odds of achieving SVR (aOR = 3.61 (p = 0.052), aOR = 3.82 (p = 0.037) and aOR = 3.65 (p = 0.044); respectively). A decision tree was also performed using both rs12980275 and rs3921 polymorphisms in patients infected with GT1/4 (Fig. 2B) and the SVR rate increased from 44.2% to 63.2% in rs12980275 AA carriers and then to 75.2% in rs3921 GG carriers. Moreover, the SVR rate increased from 33.3% in rs12980275 AG/GG carriers to 62.5% in rs3921 GG carriers. The overall percentage of patients correctly classified (accuracy) was 67.3%.
4.4. CXCL9–11 haplotypes and virologic response to HCV therapy Finally, the relationship among CXCL haplotypes (comprised of rs10336, rs3921, and rs4619915) and SVR is shown in Table 3. Two haplotypes were found predominantly, which include the 99.7% of all patients: 58.3% for TGA haplotype (favorable alleles) and 41.4% for CCG haplotype (unfavorable alleles). The TGA haplotype had better odds of achieving SVR than the CCG haplotype both for intention-to-treat (aOR = 1.51; p = 0.099) and on-treatment approach (aOR = 1.89; p = 0.020). This trend improved for patients infected with GT1/4 (aOR = 2.69 (p = 0.003) and aOR = 2.62 (p = 0.008), respectively). 5. Discussion This study shows two major findings: (i) HIV infected patients coinfected with HCV GT1/4 and carriers of CXCL9 rs10336 TT, CXCL10 rs3921 GG and CXCL11 rs4619915 AA genotypes (homozygous for the minor allele) had the highest SVR rate to pegIFN␣/ribavirin therapy; (ii) only two main haplotypes were found, one of them with all favorable alleles (TGA haplotype) that showed the highest SVR rate, and another one with all unfavorable alleles (CCG haplotype) that had the lowest SVR rate. This is the first verification of the relation between the CXCL9–11 polymorphisms and SVR in HIV/HCV-coinfected patients on pegIFN␣/ribavirin therapy. The new IFN␣-free regimens with DAAs in combination with or without ribavirin are being developed for difficult-to-treat patients [17]. However, the new DAAs are extremely expensive and there are serious restrictions for its administration, and in many regions in the world these drugs are inaccessible. Also, not all patients have indications to be treated with these new antivirals, such as some patients coinfected with HIV and HCV. In fact, treatment with pegIFN␣/ribavirin remains the only option of therapy for many
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Fig. 2. Flowchart of the decision tree for sustained virologic responses (SVR) in HIV patients coinfected with HCV GT1/4 both by intention-to-treat as on-treatment.
patients in the world. Moreover, nowadays, pegIFN␣/ribavirin still remains as the backbone of some HCV treatment strategies and the new DAAs are generally being administered in combination with pegIFN␣/ribavirin [18]. Therefore, the search for predictors that may help select patient that will likely respond to HCV therapy, containing pegIFN␣/ribavirin, remains important today. The CXCL9–11 chemokines are expressed on many cell types, serving as critical mediators of the immune response to HCV infection. In our study, the protective genotypes of CXCL9 rs10336 TT, CXCL10 rs3921 GG and CXCL11 rs4619915 AA, and the TGA haplotype were associated with higher SVR rate in difficult-totreat patients (GT1/4). These CXCL9–11 polymorphisms are strongly linked in European whites in the HapMap and test populations [19]. Indeed, the CXCL9–11 genes are contained in a CXC chemokine “mini-cluster” at chromosome position 4q21.2, within a range of 29 kb on human DNA fiber [20]. Thus, the proximity and linkage of the loci makes difficult to determine which of the three chemokine
genes are actually related to successful virologic response to pegIFN␣/ribavirin therapy. Thus, these polymorphisms could be functional or could be closely linked to other functional polymorphism. Thus, three SNPs located in putatively regulatory sites were analyzed. These regions could affect the production levels of these CXCL chemokines in the liver of patients with HCV. Besides, there are a high number of additional SNPs tightly linked to CXCL9 rs10336, CXCL10 rs3921, and CXCL11 rs4619915 in the vicinity of the CXCL9–11 chemokine minicluster [19], some of which sited at regions with possible transcriptional regulatory activity and could be theoretically responsible for the association observed with SVR. The IL28B polymorphisms are strongly associated with SVR in both HCV monoinfected and HIV/HCV coinfected patients [21]. There is an apparent connection between IL28B and CXCL chemokines since patients with the unfavorable IL28B genotypes generally have high levels of the interferon stimulated genes, including CXCL family, suggesting that the pre-activation of these
Table 3 Relationship among CXCL haplotypes (rs10336, rs3921, and rs4619915) and sustained virologic responses to HCV treatment in HIV/HCV-coinfected patients stratified by HCV genotypes. p-values were calculated by multivariate logistic regression adjusted by the most important clinical and epidemiological characteristics. Statistically significant differences are shown in bold. CXCL Haplotype
All patients
HCV-GT 2/3
HCV-GT 1/4
rs10336
rs3921
rs4619915
Freq.
aOR (95% CI)
p
Freq.
aOR (95% CI)
p
Freq.
aOR (95% CI)
ITT
C T
C G
G A
58.3% 41.7%
1 1.51 (0.90; 2.52)
– 0.099
56.3% 43.7%
1 0.61 (0.22; 1.66)
0.340
59.2% 40.8%
1 2.69 (1.41; 5.14)
– 0.003
OT
C T
C G
G A
58.2% 41.8%
1 1.89 (1.04; 3.44)
– 0.039
58.7% 41.3%
1 1.29 (0.33; 4.88)
– 0.740
57.9% 42.1%
1 2.62 (1.30; 5.27)
– 0.008
Abbreviations: 95% CI, 95% of confidence interval; aOR, adjusted odds ratio; HCV-GT, hepatitis C virus genotype; ITT, intention-to-treat; OT, on-treatment.
p
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genes may be related with IL28B expression [22]. Additionally, serum levels of CXCL10 have been related to HCV treatment response in patients infected with GT1/4, and the combination of CXCL10 levels and IL28B polymorphisms improves the prediction of SVR [9,23]. In contrast to CXCL10, the usefulness of serum CXCL9 and CXCL11 levels as predictive markers of response to HCV treatment are still unclear [10,24,25]. In our study, the association of CXCL9–11 polymorphisms was independently related to SVR since IL28B polymorphisms were included in the multivariate analysis. Moreover, in patients infected with GT1/4, the combination of both CXCL10 rs3921 and IL28B rs12980275 polymorphisms also improved the predictive performance to achieve SVR. Therefore, it is possible that both CXCL10 and IL28B polymorphisms are relevant to making individualized choices of HCV treatment. Finally, for the correct interpretation of the data, the following issues should be taken into account. This study design was retrospective and the number of patients was limited. This could limit the achievement of statistically significant values between CXCL9–11 polymorphisms and SVR. Secondly, HCV therapy regimens were not identical for all patients since they varied in some characteristics such as pegIFN␣ 2a or 2b and likely RBV dose. Thirdly, all selected patients met a set of criteria for starting HCV treatment and this may have introduced a selection bias. Fourthly, our study was carried out entirely on Caucasians, therefore since the frequency of these alleles varies among ethnicities, an independent study with different ethnic groups would clarify the current data. Finally, our study included only HIV/HCV co-infected patients, and may differ in HCV monoinfected patients. Further study of the role of CXCL9–11 polymorphism in this population is needed. In conclusion, the presence of homozygous for the minor allele of CXCL9 rs10336, CXCL10 rs3921 and CXCL11 rs4619915 was related to higher likelihoods of achieving the HCV clearance after pegIFN␣/ribavirin therapy in HIV infected patients coinfected with HCV GT1/4. Thus, CXCL polymorphisms might play a significant role in HCV virological response to pegIFN␣/ribavirin therapy although further analyses are needed to determine its potential use as a predictive marker.
Funding This work has been supported by grants from Fondo de Investigación de Sanidad (FIS) [PI08/0738, PI11/00245; PI08/0928, and PI11/01556], and Fundación para la Investigación y la Prevención ˜ (FIPSE) [361020/10]. Besides, this work has been del Sida en Espana (partially) funded by the RD12/0017/0024 and RD12/0017/0004 projects as part of the Plan Nacional R + D + I and cofinanced by ISCIII- Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). J.B. is an investigator from the Programa de Intensificación de la Actividad Investigadora en el Sistema Nacional de Salud (I3SNS) [INT10/009 and INT12/154]. D.P.T., M.G.F., M.A.J.S. and M.G.A. are supported by Instituto de Salud Carlos III [grant numbers CM12/00043, RD12/0017/0024, CD13/00013 and CD12/00442, respectively]. VB is supported by the Fondo de Investigación Sanitaria through the Miguel Servet program [grant number CP13/00098].
Competing interests The authors declare that they have no competing of interests.
Ethical approval The study was conducted in accordance with the Declaration of Helsinki and patients gave their written consent for the study. The
Institutional Review Board and the Research Ethic Committee of the Instituto de Salud Carlos III (ISCIII) approved the study. Author contributions DPT and SR performed all statistical analysis, interpretation of the data and wrote the manuscript. JB and SR participated in the study concept and design. JB, TAE, AC, CD, and FT, participated in patient selection, collection of samples and acquisition of data. MAJS, MGF, MGA, VB, and IM participated in sample preparation, DNA isolation and genotyping pre-procedure, and contributed with critical revision of the manuscript. SR supervised the study. All authors revised and approved the manuscript from a draft by SR. Acknowledgement The authors wish to thank the Spanish National Genotyping Center (CeGen) for providing the SNP genotyping services (http://www.cegen.org). References [1] Rockstroh JK, Bhagani S. Managing HIV/hepatitis C co-infection in the era of direct acting antivirals. BMC Med 2013;11:234 [Epub 2013/11/16]. [2] Moreno L, Quereda C, Moreno A, Perez-Elias MJ, Antela A, Casado JL, et al. Pegylated interferon alpha2b plus ribavirin for the treatment of chronic hepatitis C in HIV-infected patients. AIDS 2004;18(1):67–73 [Epub 2004/04/20]. [3] Laguno M, Murillas J, Blanco JL, Martinez E, Miquel R, Sanchez-Tapias JM, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for treatment of HIV/HCV co-infected patients. AIDS 2004;18(13):F27–36 [Epub 2004/08/19]. [4] Calvaruso V, Craxi A. 2011 European association of the study of the liver hepatitis C virus clinical practice guidelines. Liver Int 2012;32(Suppl. 1):2–8 [Epub 2012/01/11]. [5] Diago M, Castellano G, Garcia-Samaniego J, Perez C, Fernandez I, Romero M, et al. 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 2006;55(3):374–9 [Epub 2005/09/10]. [6] Lagging M, Romero AI, Westin J, Norkrans G, Dhillon AP, Pawlotsky JM, et al. IP-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with HCV genotype 1 infection. Hepatology 2006;44(6):1617–25 [Epub 2006/11/30]. [7] Romero AI, Lagging M, Westin J, Dhillon AP, Dustin LB, Pawlotsky JM, et al. Interferon (IFN)-gamma-inducible protein-10: association with histological results, viral kinetics, and outcome during treatment with pegylated IFNalpha 2a and ribavirin for chronic hepatitis C virus infection. J Infect Dis 2006;194(7):895–903 [Epub 2006/09/09]. [8] Zeremski M, Dimova RB, Makeyeva J, Sipley JD, Jacobson IM, Rennert H, et al. IL28B polymorphism, pretreatment CXCL10, and HCV RNA levels predict treatment response in racially diverse HIV/HCV coinfected and HCV monoinfected patients. J Acquir Immun Defic Syndr 2013;63(1):9–16 [Epub 2013/01/01]. [9] Darling JM, Aerssens J, Fanning G, McHutchison JG, Goldstein DB, Thompson AJ, et al. Quantitation of pretreatment serum interferon-gamma-inducible protein-10 improves the predictive value of an IL28B gene polymorphism for hepatitis C treatment response. Hepatology 2011;53(1):14–22 [Epub 2011/01/22]. [10] Zeremski M, Petrovic LM, Talal AH. The role of chemokines as inflammatory mediators in chronic hepatitis C virus infection. J Viral Hepat 2007;14(10):675–87 [Epub 2007/09/19]. ˜ [11] Panel de expertos de Gesida Plan Nacional sobre el Sida y Asociación Espanola para el Estudio del Hígado. Recomendaciones de Gesida/PNS/AEEHsobre tratamiento y manejo del paciente adulto coinfectado por VIH y virus de las hepatitis A, B y C [Recommendations of Gesida/PNS/AEEH for the management and treatment of the adult patient co-infected with HIV and hepatitis A, B and C virus]. Enferm Infecc Microbiol Clin 2010;28(1) [Epub 2010/02/02]. [12] Panel de expertos de Gesida y Plan Nacional sobre el Sida. Recomendaciones de Gesida/Plan Nacional sobre el Sida respecto al tratamiento antirretroviral en adultos infectados por el virus de la inmunodeficiencia humana (actualizacion febrero de 2009) [Recommendations from the GESIDA/Spanish AIDS Plan regarding antiretroviral treatment in adults with human immunodeficiency virus infection (update February 2009)]. Enferm Infecc Microbiol Clin 2009;27(4):222–35 [Epub 2009/02/28]. [13] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28(7):412–9 [Epub 1985/07/01]. [14] Resino S, Seoane JA, Bellon JM, Dorado J, Martin-Sanchez F, Alvarez E, et al. An artificial neural network improves the non-invasive diagnosis of
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Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
CXCL9, CXCL10 and CXCL11 polymorphisms are associated with sustained virologic response in HIV/HCV-coinfected patients Daniel Pineda-Tenor a , Juan Berenguer b,c , María A. Jiménez-Sousa a , María Guzmán-Fulgencio a , Teresa Aldámiz-Echevarria b,c , Ana Carrero b,c , Mónica García-Álvarez a , Cristina Diez b,c , Francisco Tejerina b,c , Verónica Briz a , Salvador Resino a,∗ a
Unidad de Infección Viral e Inmunidad, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain Unidad de Enfermedades Infecciosas/VIH, Hospital General Universitario “Gregorio Mara˜ nón”, Madrid, Spain c Instituto de Investigación Sanitaria Gregorio Mara˜ nón (IiSGM), Madrid, Spain b
a r t i c l e
i n f o
Article history: Received 22 April 2014 Received in revised form 20 August 2014 Accepted 24 August 2014 Keywords: HIV/AIDS Chronic hepatitis C CXCL HCV therapy SNPs
a b s t r a c t Background: The CXCL9, CXCL10 and CXCL11 (CXCL9–11) chemokines play a critical role in eradication of hepatitis C virus (HCV), although HCV-specific immunity often fails to eradicate the HCV, allowing the chronicity of hepatitis C. Objective: To examine the association between CXCL9–11 polymorphisms and the sustained virological response (SVR) following hepatitis C virus (HCV) therapy with pegylated-interferon-alpha plus ribavirin in HIV/HCV-coinfected patients. Study design: We performed a retrospective study in 176 naïve patients who started HCV treatment. The CXCL9 rs10336, CXCL10 rs3921 and CXCL11 rs4619915 polymorphisms were genotyped by GoldenGate® assay. Genetic data were analyzed under recessive inheritance model. The SVR was defined as undetectable HCV viremia through 24 weeks after the end of HCV treatment. Results: In the intention-to-treat analysis, the SVR rate was higher in HCV genotype 1/4 (GT1/4) patients carrying rs10336 TT (p = 0.042), rs3921 GG (p = 0.021), and rs4619915 AA (p = 0.024) genotypes; and they had higher likelihood of achieving SVR (adjusted odds ratio (aOR) = 3.26 (p = 0.038), aOR = 4.21 (p = 0.019), and aOR = 4.08 (p = 0.022), respectively). For CXCL haplotype analysis (CXCL9/rs10336, CXCL10/rs3921, and CXCL11/rs4619915), the TGA haplotype (favorable alleles) had better odds of achieving SVR than the CCG haplotype (unfavorable alleles) in GT1/4patients (OR = 2.69; p = 0.003). No significant results were found in GT2/3 patients. Moreover, similar results were obtained in the on-treatment analysis. Conclusions: The presence of homozygous for the minor allele of CXCL9 rs10336, CXCL10 rs3921 and CXCL11 rs4619915 was related to higher likelihoods of achieving the HCV clearance after pegIFN␣/ribavirin therapy in HIV infected patients coinfected with HCV GT1/4. © 2014 Elsevier B.V. All rights reserved.
1. Background During the last decade, dual therapy with pegylated-interferonalpha plus ribavirin (pegIFN␣/ribavirin) has been administrated for the treatment of hepatitis C virus (HCV) infection in patients co-infected with HIV but the effectiveness of this treatment has been low [1]. The rate of sustained virological response
∗ Corresponding author at: Centro Nacional de Microbiología, Instituto de Salud Carlos III (Campus Majadahonda), Carretera Majadahonda-Pozuelo, Km 2.2, 28220 Majadahonda, Madrid, Spain. Tel.: +34 918 223 266; fax: +34 918 223 269. E-mail addresses: [email protected], [email protected] (S. Resino). http://dx.doi.org/10.1016/j.jcv.2014.08.020 1386-6532/© 2014 Elsevier B.V. All rights reserved.
(SVR) of pegIFN␣/ribavirin therapy is around 20–40% for patients infected with HCV genotype 1 and 4 (GT1/4), and 50–60% in HCV genotype 2 and 3 (GT2/3) patients [2,3]. Nowadays, the new directacting antivirals (DAAs) are generally administered in combination with pegIFN␣/ribavirin, particularly in difficult-to-treat patients infected with GT1/4 [1]. Despite the emergence of new DAAs, the potential use of these drugs in HIV/HCV coinfected patients is complicated due to choice of patients to treat, potential for drug–drug interactions and overlapping toxicities between HIV and HCV therapy [1]. To date, the best baseline predictors for HCV therapy are age, sex, liver fibrosis, HCV genotype, HCV viral load, obesity, and singlenucleotide polymorphisms (SNPs) around interleukin 28B (IL28B)
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gene [4]. Moreover, CXC chemokine ligand 10 [CXCL10, also known as interferon-␥-inducible protein-10 (IP-10)] has been proposed as predictive biomarker of virologic response to pegIFN␣/ribavirin therapy in GT1/4 patients [5–7], whereas the combination of CXCL10 levels and IL28B polymorphisms improves the predictive performance for achieving SVR in both HIV/HCV coinfected and HCV monoinfected patients [8,9]. The CXCL10, together with CXCL9 (monokine induced by interferon ␥; Mig) and CXCL11 (interferon-inducible T-cell ␣ chemoattractant; I-TAC), are produced in the liver during chronic hepatitis C (CHC), inducing migration of activated T cells (T-helper/T-cytotoxic type-1 cell (Th1/Tc1) response) from the periphery to infected liver parenchyma via CCR5 or CXCR3 chemokine receptors [10]. These CXCL9–11 chemokines play a critical role in eradication of HCV, although HCV-specific immunity often fails to eradicate the HCV, allowing the chronicity of hepatitis C [10]. 2. Objective The aim of the study was to evaluate the association between CXCL9–11 polymorphisms and SVR in HIV/HCV-coinfected patients on pegIFN␣/ribavirin therapy. 3. Study design 3.1. Patients We carried out a retrospective study in 176 European HIV/HCV˜ coinfected naïve patients from Hospital Gregorio Maranón (Madrid, Spain) who started treatment with pegIFN␣/ribavirin on regular follow-up from October 2000 to June 2010. The study was conducted in accordance with the Declaration of Helsinki. Patients gave their written consent before the start of the study. The Institutional Review Board and the Research Ethic Committee of the Instituto de Salud Carlos III (ISCIII) approved the study. The criteria for starting HCV antiviral treatment were: (A) inclusion criteria: chronic hepatitis C (presence of detectable HCV replication for at least six months after HCV infection), negative hepatitis B surface antigen, availability of DNA sample, no clinical evidence of hepatic decompensation, detectable HCV RNA by polymerase chain reaction at baseline, CD4+ count higher than 200 cells/mm3 , and stable cART for at least 6 months before study entry or no need for cART according to treatment guidelines used in the study period [11,12]. (B) Exclusion criteria: active opportunistic infections, active drug or alcohol addiction, and other concomitant diseases or conditions such as diabetes, nephropathies, autoimmune diseases, haemochromatosis, primary biliary cirrhosis, Wilson’s disease, a1-antitrypsin deficiency and neoplasia. 3.2. Epidemiological and clinical data Clinical and epidemiological data (including sex, age, HIV acquired by IVDU, years since HCV infection, prior AIDS, HIV markers and HCV markers) were obtained from medical records in order to obtain a complete data set before starting hepatitis C therapy. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. The degree of insulin resistance (IR) was estimated for each patient using the homeostatic model assessment (HOMA) [13]: fasting plasma glucose (mmol/L) times fasting serum insulin (mU/L) divided by 22.5. 3.3. Liver biopsy Liver biopsies were performed before starting hepatitis C therapy as part of usual clinical care as we described previously [14].
Liver fibrosis and was estimated according to Metavir score as follows: F0, no fibrosis; F1, mild fibrosis; F2, significant fibrosis; F3, advanced fibrosis; and F4, definite cirrhosis. Activity grade was scored as follows: A0, no activity; A1, mild activity; A2, moderate activity; A3, severe activity. Liver steatosis was evaluated according to the existence of hepatocytes containing visible macrovesicular fat droplets. We considered hepatic steatosis to be clinically significant when fatty hepatocytes exceeded 10% of the hepatic parenchyma. 3.4. HCV assays HCV infection was documented in all patients by enzyme-linked immunosorbent assay (ELISA) and PCR test. HCV genotype was determined by hybridization of biotin-labeled PCR products to oligonucleotide probes bound to nitrocellulose membrane strips (INNO-LiPA HCV II, Innogenetics, Ghent, Belgium). Plasma HCVRNA viral load was measured by polymerase chain reaction (PCR) (Cobas Amplicor HCV Monitor Test, Branchburg, NJ, USA) and realtime PCR (COBAS AmpliPrep/COBAS TaqMan HCV test); and results were reported in terms of international units per milliliter (IU/mL), with a lower limit of detection of 10 IU/mL. 3.5. Hepatitis C therapy Following both international and national guidelines [11,12,15,16], HCV treatment regimens included pegIFN␣ 2a or 2b at standard doses (180 g/week or 1.5 g/kg/week, respectively) plus weight-adjusted ribavirin dosing (1000 mg/day for patients weighing <75 kg and 1200 mg/day for patients weighing ≥75 kg). Patients with HCV genotypes 1 or 4 received either 48 or 72 weeks of treatment, and patients with HCV genotype 2 or 3 were treated for 24 or 48 weeks. A SVR was defined as an undetectable serum HCV-RNA level (<10 IU/mL) at week 24 after the end of the treatment. 3.6. Genotyping of CXCL polymorphisms Genomic DNA was extracted from peripheral blood with Qiagen kit (QIAamp DNA Blood Midi/Maxi; Qiagen, Hilden, Germany). DNA samples were sent at the Spanish National Genotyping Center (CeGen; http://www.cegen.org/) for genotyping CXCL9 rs10336, CXCL10 rs3921, and CXCL11 rs4619915 polymorphisms, which are located in putatively regulatory regions such as 3 untranslated region (rs10336) or intronic regions (rs3921 and rs4619915). 3.7. Statistical analysis For the description of the study population, p-values were estimated with nonparametric tests: Mann–Whitney U test was used for continuous variable and Chi-square test for categorical variables. All SNPs were analyzed for Hardy–Weinberg equilibrium (HWE) by Chi-square test, considering equilibrium when p > 0.05. For association study, logistic regression analysis was used to investigate the relationship among CXCL polymorphisms and HCV-therapy response. These analyses were adjusted by the most important clinical and epidemiological characteristics, which were selected by a “Stepwise” algorithm (p-value for entry and exit of 0.15 and 0.20, respectively). The covariables used were gender, age, BMI, HOMA, nadir CD4+ T-cells, time on cART, HCV genotype, HCVRNA viral load, liver fibrosis, and IL28B rs12980275 polymorphism. Moreover, decision tree analyses were performed via classification and regression tree (CART) algorithm to classify GT1/4 patients according to SVR using IL28B and CXCL10 genotypes. These analyses were performed by using the Statistical Package for the Social Sciences (SPSS) 19.0 software (IBM Corp., Chicago,
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Table 1 Clinical and epidemiological characteristics of HIV/HCV-coinfected patients. Values expressed as absolute numbers (%) and median (percentile 25-percentile 75). Characteristics
All patients
HCV-GT 2/3
HCV-GT 1/4
No. (%) Male, n (%) Age, years BMI, kg/m2 BMI ≥ 25 kg/m2 HOMA HOMA ≥ 3 HIV acquired by IVDU, n (%) Years since HCV infection Prior AIDS, n (%) cART, n (%) Time on cART, years Current cART protocols, n (%) Any NRTIs + any PI Any NRTIs + PI + NNRTI Any NRTIs + any NNRTI Only NRTIs HIV markers Nadir CD4+, cells/L Nadir CD4+ <200 cells/L, n (%) CD4+ T, cells/L CD4+ ≥ 500 cells/L, n (%) HIV-RNA < 50 copies/mL, n (%) HCV markers, n (%) HCV-RNA ≥ 500,000 IU/mL HCV genotype HCV-GT1 HCV-GT2 HCV-GT3 HCV-GT4 Undefined Metavir score, n (%) Liver biopsy patients Significant fibrosis (F ≥ 2) Moderate or severe activity (A ≥ 2) Steatosis
176 (100%) 135 (76.7%) 40.6 (37.9–44.5) 22.5 (21–24.5) 40 (22.7%) 2.13 (1.44–3.85) 66 (37.5%) 153 (86.9%) 20.5 (15.4–23.7) 49 (27.8%) 145 (82.4%) 4.59 (2.58–7.53)
56 (100%) 42 (75%) 41 (38.5–44.5) 22.5 (20.8–24.5) 47 (83.9%) 1.98 (1.34–3.64) 18 (32.1%) 49 (87.5%) 19.5 (15–23.8) 11 (19.6%) 46 (82.1%) 3.94 (2.34–5.69)
115 (100%) 88 (76.5%) 40.2 (37.8–44.6) 22.5 (21–25.2) 86 (74.8%) 2.22 (1.59–4.07) 47 (40.9%) 99 (86.1%) 20.7 (16.1–23.5) 37 (32.2%) 96 (83.5%) 4.75 (3.09–7.65)
40 (22.7%) 1 (0.6%) 92 (52.3%) 12 (6.8%)
10 (17.9%) 0 (0%) 32 (57.1%) 4 (7.1%)
30 (26.1%) 1 (0.9%) 58 (50.4%) 7 (6.1%)
217 (96–356) 81 (46%) 455 (330–675) 76 (43.4%) 126 (72%)
230 (139–379) 24 (42.9%) 433 (374–682) 23 (41.1%) 72 (76.4%)
204 (72–314) 56 (48.7%) 465 (324–675) 51 (44.7%) 81 (70.4%)
117 (68.4%)
36 (65.5%)
78 (69%)
89 (50.6%) 3 (1.7%) 53 (30.1%) 26 (14.8%) 5 (2.8%)
– 3 (5.4%) 53 (94.6%) – –
89 (77.4%) – – 26 (22.6%) –
142 (80.7%) 91 (64.1%) 90 (64.7%) 75 (54%)
42 (75%) 27 (64.3%) 25 (61%) 29 (65.9%)
97 (84.3%) 62 (63.9%) 64 (67.4%) 46 (49.5%)
Abbreviations: AIDS, acquired immunodeficiency syndrome; BMI, body mass index; cART, combination antiretroviral therapy; GT, genotype; HCV, hepatitis C virus; HCV-RNA, HCV plasma viral load; HIV, human immunodeficiency; HIV-RNA, HIV plasma viral load; HOMA, homeostasis model assessment; IVDU, intravenous drug users; NNRTI, no nucleoside analog reverse-transcriptase inhibitors; NRTI, nucleoside analog reverse-transcriptase inhibitors; PI, protease inhibitors.
USA). In addition, pair-wise linkage disequilibrium (LD) analysis was computed by Haploview 4.2 software, and haplotype-based association testing was performed using the SNPstat software (http://bioinfo.iconcologia.net/SNPStats web). All p-values were two-tailed and statistical significance was defined as p < 0.05.
model that best fitted our data). We divided the analysis according to the course of the HCV treatment. On the one hand, the analysis was performed considering the patients that initiate the therapy (intention to treat), regardless of whether they finished it or not.
4. Results 4.1. Characteristics of patients The main characteristics of the study population are shown in Table 1. At baseline, 176 patients on HCV treatment were stratified by HCV genotype: 56 patients were infected with GT2/3 and 115 with GT1/4. 4.2. CXCL9–11 polymorphisms All SNPs had a minimum allele frequency (MAF) of 40.5% and displayed missing values lower than 5%. CXCL9–11 polymorphisms were in HWE [rs10336 (p = 0.221), rs3921 (p = 0.184), and rs10336 (p = 0.184), respectively]. In addition, a strong LD among SNPs was observed (Fig. 1). 4.3. CXCL9–11 polymorphism and virologic response to HCV therapy The relationship between the CXCL polymorphisms and SVR was performed under a model of recessive inheritance (the genetic
Fig. 1. Pairwise linkage disequilibrium (LD) patterns for the polymorphisms through CXCL9, CXCL10 and CXCL11 regions. Each diagonal represents a different SNP, with each square representing a pairwise comparison between two SNPs.
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Table 2 Relationship among CXCL polymorphisms and sustained virologic responses to HCV treatment in HIV/HCV-coinfected patients according to HCV genotypes. Statistically significant differences are shown in bold. (a) p-values were calculated Chi-squared test; (b) p-values were calculated by logistic regression adjusted by the most important clinical and epidemiological characteristics (see Section 3.7). SNP genotypes A) ITT analysis CXCL9/rs10336
p-value (a)
aOR (95% CI)
p-value (b)
All genotypes
CC/CT
TT
All patients HCV-GT2/3 HCV-GT1/4 CXCL10/rs3921
54.3% (95/175) 83.9% (47/56) 39.5% (45/114) All genotypes
50.7% (73/144) 86.0 (37/43) 35.7% (35/98) CC/CG
71.0% (22/31) 76.9% (10/13) 62.5% (10/16) GG
0.040 0.433 0.042
1.47 (0.55; 3.93) 0.26 (0.04; 1.84) 3.68 (1.07; 12.63)
0.436 0.178 0.038
All patients HCV-GT2/3 HCV-GT1/4 CXCL11/rs4619915
54.9% (95/173) 85.5% (47/55) 39.5% (45/114) All genotypes
50.3% (72/143) 86.0 (37/43) 35.1% (34/97) GG/GA
74.2% (23/31) 83.3% (10/12) 64.7% (11/17) AA
0.016 0.814 0.021
2.37 (0.86; 6.54) 0.52 (0.07; 3.99) 4.21 (1.27; 14.03)
0.095 0.528 0.019
All patients HCV-GT2/3 HCV-GT1/4 B) OT analysis CXCL9/rs10336
54.5% (96/176) 83.9% (47/56) 40.0% (46/115)
50.7% (73/144) 86.0% (37/43) 35.7% (35/98)
71.9% (23/32) 76.9% (10/13) 64.7% (11/17)
0.030 0.433 0.024
1.76 (0.66; 4.69) 0.26 (0.04; 1.84) 4.08 (1.22; 13.61)
0.259 0.178 0.022
All genotypes
CC/CT
TT
All patients HCV-GT2/3 HCV-GT1/4 CXCL10/rs3921
59.7% (95/159) 90.4% (47/52) 43.7% (45/103) All genotypes
55.7% (73/131) 90.2% (37/41) 39.8% (35/88) CC/CG
78.6% (22/28) 90.9% (10/11) 66.7% (10/15) GG
0.025 0.947 0.052
2.05 (0.67; 6.21) 1.08 (0.11; 10.78) 3.61 (0.99; 13.21)
0.205 0.947 0.052
All patients HCV-GT2/3 HCV-GT1/4 CXCL11/rs4619915
59.7% (95/159) 90.4% (47/52) 43.7% (45/103) All genotypes
55.4% (72/130) 90.2% (37/41) 39.1% (34/87) GG/GA
79.3% (23/29) 90.9% (10/11) 68.8% (11/16) AA
0.018 0.947 0.028
2.95 (0.98; 8.91) 0.97 (0.10; 9.79) 3.82 (1.08; 13.56)
0.055 0.981 0.037
60.0% (96/160) 90.4% (47/52) 44.2% (46/104)
55.7% (73/131) 90.2% (37/41) 39.8% (35/88)
79.3% (23/29) 90.9% (10/11) 68.8% (10/16)
0.019 0.433 0.032
2.88 (0.96; 8.68) 0.97 (0.09; 9.79) 3.65 (1.04; 12.92)
0.060 0.981 0.044
All patients HCV-GT2/3 HCV-GT1/4
Abbreviations: 95% CI, 95% of confidence interval; aOR, adjusted odds ratio; HCV-GT, hepatitis C virus genotype; ITT, intention-to-treat; OT, on-treatment.
On the other hand, we only analyzed the patients that completed the HCV treatment (on treatment). In the analysis by intention-to-treat (Table 2A), the SVR rate was higher in carriers of CXCL9 rs10336 TT (p = 0.040), CXCL10 rs3921 GG (p = 0.016), and CXCL11 rs4619915 AA (p = 0.030) genotypes. These trends continued for GT1/4 patients. In regards to the multivariate model, rs10336 TT (p = 0.042), rs3921 GG (p = 0.021) and rs4619915 AA (p = 0.024) genotypes had the highest likelihood of achieving SVR (adjusted odds ratio (aOR) = 3.26 (p = 0.038), aOR = 4.21 (p = 0.019) and aOR = 4.08 (p = 0.022); respectively). When analyzed IL28B rs12980275 and CXCL10 rs3921 polymorphisms in a decision tree, the SVR rate in GT1/4 patients increased from 40% to 52.2% in rs12980275 AA carriers and then to 66.7% in rs3921 GG carriers (Fig. 2A). Moreover, the SVR rate increased from 31.9% in rs12980275 AG/GG carriers to 62.3% in rs3921 GG carriers. The overall percentage of patients correctly classified (accuracy) was 64.3%. In the on-treatment analysis (Table 2B), 16 out of 176 patients were excluded by interruption of HCV therapy (12 adverse events and 4 abandonments) and 160 patients with a full course of HCV therapy were included. In this case, the SVR rate was also the highest in carriers of CXCL9 rs10336 TT (p = 0.025), CXCL10 rs3921 GG (p = 0.018) and CXCL11 rs4619915 AA (p = 0.019) genotypes. These trends continued for GT1/4 patients. Besides, rs10336 TT (p = 0.052), rs3921 GG (p = 0.028) and rs4619915 AA (p = 0.032) genotypes had the highest odds of achieving SVR (aOR = 3.61 (p = 0.052), aOR = 3.82 (p = 0.037) and aOR = 3.65 (p = 0.044); respectively). A decision tree was also performed using both rs12980275 and rs3921 polymorphisms in patients infected with GT1/4 (Fig. 2B) and the SVR rate increased from 44.2% to 63.2% in rs12980275 AA carriers and then to 75.2% in rs3921 GG carriers. Moreover, the SVR rate increased from 33.3% in rs12980275 AG/GG carriers to 62.5% in rs3921 GG carriers. The overall percentage of patients correctly classified (accuracy) was 67.3%.
4.4. CXCL9–11 haplotypes and virologic response to HCV therapy Finally, the relationship among CXCL haplotypes (comprised of rs10336, rs3921, and rs4619915) and SVR is shown in Table 3. Two haplotypes were found predominantly, which include the 99.7% of all patients: 58.3% for TGA haplotype (favorable alleles) and 41.4% for CCG haplotype (unfavorable alleles). The TGA haplotype had better odds of achieving SVR than the CCG haplotype both for intention-to-treat (aOR = 1.51; p = 0.099) and on-treatment approach (aOR = 1.89; p = 0.020). This trend improved for patients infected with GT1/4 (aOR = 2.69 (p = 0.003) and aOR = 2.62 (p = 0.008), respectively). 5. Discussion This study shows two major findings: (i) HIV infected patients coinfected with HCV GT1/4 and carriers of CXCL9 rs10336 TT, CXCL10 rs3921 GG and CXCL11 rs4619915 AA genotypes (homozygous for the minor allele) had the highest SVR rate to pegIFN␣/ribavirin therapy; (ii) only two main haplotypes were found, one of them with all favorable alleles (TGA haplotype) that showed the highest SVR rate, and another one with all unfavorable alleles (CCG haplotype) that had the lowest SVR rate. This is the first verification of the relation between the CXCL9–11 polymorphisms and SVR in HIV/HCV-coinfected patients on pegIFN␣/ribavirin therapy. The new IFN␣-free regimens with DAAs in combination with or without ribavirin are being developed for difficult-to-treat patients [17]. However, the new DAAs are extremely expensive and there are serious restrictions for its administration, and in many regions in the world these drugs are inaccessible. Also, not all patients have indications to be treated with these new antivirals, such as some patients coinfected with HIV and HCV. In fact, treatment with pegIFN␣/ribavirin remains the only option of therapy for many
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Fig. 2. Flowchart of the decision tree for sustained virologic responses (SVR) in HIV patients coinfected with HCV GT1/4 both by intention-to-treat as on-treatment.
patients in the world. Moreover, nowadays, pegIFN␣/ribavirin still remains as the backbone of some HCV treatment strategies and the new DAAs are generally being administered in combination with pegIFN␣/ribavirin [18]. Therefore, the search for predictors that may help select patient that will likely respond to HCV therapy, containing pegIFN␣/ribavirin, remains important today. The CXCL9–11 chemokines are expressed on many cell types, serving as critical mediators of the immune response to HCV infection. In our study, the protective genotypes of CXCL9 rs10336 TT, CXCL10 rs3921 GG and CXCL11 rs4619915 AA, and the TGA haplotype were associated with higher SVR rate in difficult-totreat patients (GT1/4). These CXCL9–11 polymorphisms are strongly linked in European whites in the HapMap and test populations [19]. Indeed, the CXCL9–11 genes are contained in a CXC chemokine “mini-cluster” at chromosome position 4q21.2, within a range of 29 kb on human DNA fiber [20]. Thus, the proximity and linkage of the loci makes difficult to determine which of the three chemokine
genes are actually related to successful virologic response to pegIFN␣/ribavirin therapy. Thus, these polymorphisms could be functional or could be closely linked to other functional polymorphism. Thus, three SNPs located in putatively regulatory sites were analyzed. These regions could affect the production levels of these CXCL chemokines in the liver of patients with HCV. Besides, there are a high number of additional SNPs tightly linked to CXCL9 rs10336, CXCL10 rs3921, and CXCL11 rs4619915 in the vicinity of the CXCL9–11 chemokine minicluster [19], some of which sited at regions with possible transcriptional regulatory activity and could be theoretically responsible for the association observed with SVR. The IL28B polymorphisms are strongly associated with SVR in both HCV monoinfected and HIV/HCV coinfected patients [21]. There is an apparent connection between IL28B and CXCL chemokines since patients with the unfavorable IL28B genotypes generally have high levels of the interferon stimulated genes, including CXCL family, suggesting that the pre-activation of these
Table 3 Relationship among CXCL haplotypes (rs10336, rs3921, and rs4619915) and sustained virologic responses to HCV treatment in HIV/HCV-coinfected patients stratified by HCV genotypes. p-values were calculated by multivariate logistic regression adjusted by the most important clinical and epidemiological characteristics. Statistically significant differences are shown in bold. CXCL Haplotype
All patients
HCV-GT 2/3
HCV-GT 1/4
rs10336
rs3921
rs4619915
Freq.
aOR (95% CI)
p
Freq.
aOR (95% CI)
p
Freq.
aOR (95% CI)
ITT
C T
C G
G A
58.3% 41.7%
1 1.51 (0.90; 2.52)
– 0.099
56.3% 43.7%
1 0.61 (0.22; 1.66)
0.340
59.2% 40.8%
1 2.69 (1.41; 5.14)
– 0.003
OT
C T
C G
G A
58.2% 41.8%
1 1.89 (1.04; 3.44)
– 0.039
58.7% 41.3%
1 1.29 (0.33; 4.88)
– 0.740
57.9% 42.1%
1 2.62 (1.30; 5.27)
– 0.008
Abbreviations: 95% CI, 95% of confidence interval; aOR, adjusted odds ratio; HCV-GT, hepatitis C virus genotype; ITT, intention-to-treat; OT, on-treatment.
p
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genes may be related with IL28B expression [22]. Additionally, serum levels of CXCL10 have been related to HCV treatment response in patients infected with GT1/4, and the combination of CXCL10 levels and IL28B polymorphisms improves the prediction of SVR [9,23]. In contrast to CXCL10, the usefulness of serum CXCL9 and CXCL11 levels as predictive markers of response to HCV treatment are still unclear [10,24,25]. In our study, the association of CXCL9–11 polymorphisms was independently related to SVR since IL28B polymorphisms were included in the multivariate analysis. Moreover, in patients infected with GT1/4, the combination of both CXCL10 rs3921 and IL28B rs12980275 polymorphisms also improved the predictive performance to achieve SVR. Therefore, it is possible that both CXCL10 and IL28B polymorphisms are relevant to making individualized choices of HCV treatment. Finally, for the correct interpretation of the data, the following issues should be taken into account. This study design was retrospective and the number of patients was limited. This could limit the achievement of statistically significant values between CXCL9–11 polymorphisms and SVR. Secondly, HCV therapy regimens were not identical for all patients since they varied in some characteristics such as pegIFN␣ 2a or 2b and likely RBV dose. Thirdly, all selected patients met a set of criteria for starting HCV treatment and this may have introduced a selection bias. Fourthly, our study was carried out entirely on Caucasians, therefore since the frequency of these alleles varies among ethnicities, an independent study with different ethnic groups would clarify the current data. Finally, our study included only HIV/HCV co-infected patients, and may differ in HCV monoinfected patients. Further study of the role of CXCL9–11 polymorphism in this population is needed. In conclusion, the presence of homozygous for the minor allele of CXCL9 rs10336, CXCL10 rs3921 and CXCL11 rs4619915 was related to higher likelihoods of achieving the HCV clearance after pegIFN␣/ribavirin therapy in HIV infected patients coinfected with HCV GT1/4. Thus, CXCL polymorphisms might play a significant role in HCV virological response to pegIFN␣/ribavirin therapy although further analyses are needed to determine its potential use as a predictive marker.
Funding This work has been supported by grants from Fondo de Investigación de Sanidad (FIS) [PI08/0738, PI11/00245; PI08/0928, and PI11/01556], and Fundación para la Investigación y la Prevención ˜ (FIPSE) [361020/10]. Besides, this work has been del Sida en Espana (partially) funded by the RD12/0017/0024 and RD12/0017/0004 projects as part of the Plan Nacional R + D + I and cofinanced by ISCIII- Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). J.B. is an investigator from the Programa de Intensificación de la Actividad Investigadora en el Sistema Nacional de Salud (I3SNS) [INT10/009 and INT12/154]. D.P.T., M.G.F., M.A.J.S. and M.G.A. are supported by Instituto de Salud Carlos III [grant numbers CM12/00043, RD12/0017/0024, CD13/00013 and CD12/00442, respectively]. VB is supported by the Fondo de Investigación Sanitaria through the Miguel Servet program [grant number CP13/00098].
Competing interests The authors declare that they have no competing of interests.
Ethical approval The study was conducted in accordance with the Declaration of Helsinki and patients gave their written consent for the study. The
Institutional Review Board and the Research Ethic Committee of the Instituto de Salud Carlos III (ISCIII) approved the study. Author contributions DPT and SR performed all statistical analysis, interpretation of the data and wrote the manuscript. JB and SR participated in the study concept and design. JB, TAE, AC, CD, and FT, participated in patient selection, collection of samples and acquisition of data. MAJS, MGF, MGA, VB, and IM participated in sample preparation, DNA isolation and genotyping pre-procedure, and contributed with critical revision of the manuscript. SR supervised the study. All authors revised and approved the manuscript from a draft by SR. Acknowledgement The authors wish to thank the Spanish National Genotyping Center (CeGen) for providing the SNP genotyping services (http://www.cegen.org). References [1] Rockstroh JK, Bhagani S. Managing HIV/hepatitis C co-infection in the era of direct acting antivirals. BMC Med 2013;11:234 [Epub 2013/11/16]. [2] Moreno L, Quereda C, Moreno A, Perez-Elias MJ, Antela A, Casado JL, et al. Pegylated interferon alpha2b plus ribavirin for the treatment of chronic hepatitis C in HIV-infected patients. AIDS 2004;18(1):67–73 [Epub 2004/04/20]. [3] Laguno M, Murillas J, Blanco JL, Martinez E, Miquel R, Sanchez-Tapias JM, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for treatment of HIV/HCV co-infected patients. AIDS 2004;18(13):F27–36 [Epub 2004/08/19]. [4] Calvaruso V, Craxi A. 2011 European association of the study of the liver hepatitis C virus clinical practice guidelines. Liver Int 2012;32(Suppl. 1):2–8 [Epub 2012/01/11]. [5] Diago M, Castellano G, Garcia-Samaniego J, Perez C, Fernandez I, Romero M, et al. 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 2006;55(3):374–9 [Epub 2005/09/10]. [6] Lagging M, Romero AI, Westin J, Norkrans G, Dhillon AP, Pawlotsky JM, et al. IP-10 predicts viral response and therapeutic outcome in difficult-to-treat patients with HCV genotype 1 infection. Hepatology 2006;44(6):1617–25 [Epub 2006/11/30]. [7] Romero AI, Lagging M, Westin J, Dhillon AP, Dustin LB, Pawlotsky JM, et al. Interferon (IFN)-gamma-inducible protein-10: association with histological results, viral kinetics, and outcome during treatment with pegylated IFNalpha 2a and ribavirin for chronic hepatitis C virus infection. J Infect Dis 2006;194(7):895–903 [Epub 2006/09/09]. [8] Zeremski M, Dimova RB, Makeyeva J, Sipley JD, Jacobson IM, Rennert H, et al. IL28B polymorphism, pretreatment CXCL10, and HCV RNA levels predict treatment response in racially diverse HIV/HCV coinfected and HCV monoinfected patients. J Acquir Immun Defic Syndr 2013;63(1):9–16 [Epub 2013/01/01]. [9] Darling JM, Aerssens J, Fanning G, McHutchison JG, Goldstein DB, Thompson AJ, et al. Quantitation of pretreatment serum interferon-gamma-inducible protein-10 improves the predictive value of an IL28B gene polymorphism for hepatitis C treatment response. Hepatology 2011;53(1):14–22 [Epub 2011/01/22]. [10] Zeremski M, Petrovic LM, Talal AH. The role of chemokines as inflammatory mediators in chronic hepatitis C virus infection. J Viral Hepat 2007;14(10):675–87 [Epub 2007/09/19]. ˜ [11] Panel de expertos de Gesida Plan Nacional sobre el Sida y Asociación Espanola para el Estudio del Hígado. Recomendaciones de Gesida/PNS/AEEHsobre tratamiento y manejo del paciente adulto coinfectado por VIH y virus de las hepatitis A, B y C [Recommendations of Gesida/PNS/AEEH for the management and treatment of the adult patient co-infected with HIV and hepatitis A, B and C virus]. Enferm Infecc Microbiol Clin 2010;28(1) [Epub 2010/02/02]. [12] Panel de expertos de Gesida y Plan Nacional sobre el Sida. Recomendaciones de Gesida/Plan Nacional sobre el Sida respecto al tratamiento antirretroviral en adultos infectados por el virus de la inmunodeficiencia humana (actualizacion febrero de 2009) [Recommendations from the GESIDA/Spanish AIDS Plan regarding antiretroviral treatment in adults with human immunodeficiency virus infection (update February 2009)]. Enferm Infecc Microbiol Clin 2009;27(4):222–35 [Epub 2009/02/28]. [13] Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985;28(7):412–9 [Epub 1985/07/01]. [14] Resino S, Seoane JA, Bellon JM, Dorado J, Martin-Sanchez F, Alvarez E, et al. An artificial neural network improves the non-invasive diagnosis of
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