Rallón, Norma I. PhD*; Pineda, Juan A. MD, PhD†; Soriano, Vincent MD, PhD*; Neukam, Karin PhD†; Vispo, Eugenia MD*; Rivero, Antonio MD, PhD‡; Labarga, Pablo MD, PhD*; Caruz, Antonio PhD§; Restrepo, Clara MSc*; Camacho, Angela MD, PhD‡; Barreiro, Pablo MD, PhD*; Benito, Jose M. MD, PhD*
Hepatitis C virus (HCV) infects more than 175 million people worldwide.1 In western countries, HCV is the leading cause of end-stage liver disease and hepatocellular carcinoma and the main indication for liver transplantation.1 HCV and HIV-1 share routes of transmission and establish chronic infections; therefore, coinfection is relatively common (15%–40%).2 Until recently, therapy for chronic hepatitis C was based on a combination of peginterferon-α (pegIFNα) and ribavirin (RBV) given for 6–18 months, depending on early viral kinetics and HCV genotype.3 Unfortunately, these medications are poorly tolerated and overall less than a half of patients achieve sustained HCV clearance.3 This figure is lower in HIV/HCV-coinfected patients.2 Thus, identification of baseline predictors of treatment success is desirable for making adequate treatment decisions, encouraging therapy in those with a high likelihood of cure, and conversely deferring treatment when the chances of response are minimal and/or expected drug-related side effects may be troublesome.
Until recently, the best baseline predictors of sustained virological response (SVR) were HCV genotypes 2 or 3, low serum HCV RNA, null or minimal liver fibrosis, and younger age.4 More recently, different genome-wide association studies identified single nucleotide polymorphisms (SNPs) upstream of the IL28B gene coding for interferon (IFN)-λ3 as strongly associated with the chance of SVR in HCV-monoinfected individuals.5–7 Similar findings have been reproduced in HIV/HCV-coinfected patients.8 The SNP with the strongest association, rs12979860, confers more han 2-fold increase in SVR rates after adjusting for other baseline variables. The effect of this SNP can be recognized early on therapy strongly influencing early viral kinetics in both HCV-monoinfected and HIV/HCV-coinfected patients.9–11 Patients carrying CC alleles show faster reductions in serum HCV RNA during the first weeks of therapy, regardless of other baseline variables.11
The recent introduction of directly acting antivirals (DAA) is rapidly changing the management of chronic hepatitis C patients.12,13 HCV protease inhibitors telaprevir and boceprevir were the first DAA approved for the treatment of patients infected with HCV genotype 1. Their use in combination with pegIFNα/RBV increases the frequency of SVR by at least 25%.14,15 Given the high genetic diversity and rapid mutation rate of HCV, emergence of viral variants resistant to DAA has been observed in patients who did not achieve complete viral suppression very rapidly on therapy.16 Because adding pegIFNα/RBV to DAA greatly diminishes the risk of selecting drug-resistant variants, combination therapy will remain necessary in the near future.17 In this scenario, IL28B variants will continue to remain important as long as pegIFNα/RBV is kept as part of the hepatitis treatment regimen. Although IL28B allelic variants may not influence directly the efficacy of DAA, their effect modulating viral kinetics in response to pegIFNα/RBV may remain important on triple therapy.
PATIENTS AND METHODS
This is an observational retrospective study of 2 Spanish cohorts of HIV/HCV-coinfected patients recruited in Madrid11 and Andalucia,18 which were recently merged into a single database. All subjects completed a full course of pegIFNα/RBV therapy and had validated outcomes. All underwent liver fibrosis staging at baseline using elastography, and all had been tested for the IL28B rs12979860 genotype. Other relevant demographic, clinical, and laboratory parameters were recorded from medical records. The main exclusion criteria were baseline CD4 counts lower than 300 cells per microliter, positive serum HBsAg, current alcohol abuse (>60 grams per day), prior exposure to IFNα-based therapy, and prior episodes of hepatic decompensation.
Treatment regimens included pegIFNα-2a or pegIFNα-2b at standard doses (180 μg/week or 1.5 μg/kg/week, respectively) plus weight-adjusted RBV (1000 mg/day for patients weighting <75 kg and 1200 mg/day for patients weighting >75 kg). Following international guidelines for coinfected individuals,2 patients with HCV genotypes 1 or 4 received either 48 or 72 weeks of treatment, and patients with HCV genotype 3 received 24 or 48 weeks of therapy, according to virological response at week 4. Early stopping rules were applied for subjects with suboptimal virological responses at weeks 12 or 24.2
The following on-treatment virological responses were assessed as follows: rapid virological response (RVR, HCV RNA <10 IU/mL at week 4), early virological response (EVR, >2 log IU/mL reduction in HCV RNA levels at week 12), complete early virological response (cEVR, HCV RNA <10 IU/mL at week 12), and end of treatment virological response (EOTVR, HCV RNA <10 IU/mL at the end of therapy). SVR was defined as undetectable serum HCV RNA 24 weeks after completion of therapy; otherwise patients were considered as relapsers.19 Moreover, HCV RNA declines at weeks 4 and 12 after initiating treatment were recorded for each patient. For the purpose of this study, relapsers were considered along with nonresponders, who were patients who experienced suboptimal virological responses during the treatment period, and accordingly did not complete the planned duration of therapy. Patients with poor drug compliance and/or who discontinued therapy due to side effects were excluded from this analysis.
To participate in the study, written informed consent for genetic testing was obtained from all individuals, and the study protocol was evaluated and approved by the hospital ethics committee. Both plasma and peripheral blood mononuclear cells were stored for all patients.
Plasma HCV RNA was measured using a real-time polymerase chain reaction assay (COBAS TaqMan, Roche, Barcelona, Spain), which has a lower limit of detection of 10 IU/mL. HCV genotyping and subtyping was performed using a commercial reverse transcriptase-polymerase chain reaction hybridization assay (Versant HCV Genotype v2.0 LiPA, Siemens, Barcelona, Spain), which maximally reduces the chances of HCV genotype misclassification.20 Plasma HIV RNA was measured using Versant HIV-1 RNA v3.0 (Siemens, Barcelona, Spain), which has a lower limit of detection of 50 copies per milliliter.
Liver Fibrosis Staging
The extent of liver fibrosis was measured using transient elastography by FibroScan (Echosens, Paris, France). Details about this noninvasive method, the examination procedure, and correlation of liver fibrosis estimates with liver biopsy have been reported elsewhere.21 The median value of all tests per patient is expressed in Kilopascal (kPa) units. Advanced liver fibrosis (severe fibrosis or cirrhosis, corresponding to METAVIR scores F3 and F4, respectively) was defined for liver stiffness values ≥9.5 kPa, based on results from studies conducted in both HCV-monoinfected and HIV/HCV-coinfected patients.22
The characterization of IL28 allelic variants was performed at Hospital Carlos III and Universidad de Jaen. It was conducted in a blinded fashion on DNA specimens collected from each individual. The SNP rs12979860 was assessed with a custom TaqMan assay designed by Ge et al5 using the 5' nuclease assay with allele specific TaqMan probes (ABI TaqMan allelic discrimination kit and the ABI7900HT Sequence Detection System, Applied Biosystems, Carlsbad, CA). Genotyping calls were manually inspected and verified before release.
The main characteristics of the study population, and the different parameters evaluated are expressed as median and interquartile ranges. Comparisons between groups were carried out using nonparametric Analysis of Variance. Associations between different qualitative parameters were explored using χ2 or Fisher exact tests, as appropiate. Univariable (χ2 test) and multivariable (logistic regression) analyses were used to assess the predictors of treatment outcome. Linear regression analysis was employed to assess the parameters associated with the decrease of HCV RNA level at weeks 4 and 12. All statistical analyses were performed using the SPSS software version 13 (SPSS Inc, Chicago, IL). All P values were 2-tailed and were considered as significant only when below 0.05.
Table 1 summarizes the main characteristics of the HIV/HCV-coinfected study population. The whole population comprised 331 patients, 178 recruited at Hospital Carlos III in Madrid, and 153 at 2 different hospitals in the south of Spain. HCV geno/subtypes distribution was as follows: 97 (29%) HCV-1a, 62 (19%) HCV-1b, 122 (37%) HCV-3, and 50 (15%) HCV-4. Of note, all patients were of European ancestry, being absent Asians or Africans.
Some baseline characteristics differed in subjects infected with distinct HCV geno/subtypes. Serum HCV RNA was higher in HCV-1 compared with HCV-3 or HCV-4 patients. Among HCV-1 patients, slightly greater levels were seen in HCV-1a than HCV-1b patients. Accordingly, the proportion of HCV-1a patients with low serum HCV RNA was significantly lower compared with the rest. The frequency of IL28B CC alleles was significantly higher in HCV-3 patients than in the rest, being similar when comparing HCV-1a and HCV-1b patients. Last, aspartate aminotransferases tended to be higher in HCV-3 patients, with no significant differences comparing HCV-1a and HCV-1b patients.
Influence of HCV Geno/Subtypes and IL28B Variants on Treatment Response
The proportion of patients achieving virological response at different time points is recorded in Table 2. As expected, it was lower in patients infected with HCV-1 or HCV-4 than in those infected with HCV-3. Interestingly, there were marked differences when comparing HCV-1a and HCV-1b patients, with significantly higher rates of treatment response at all time-points on therapy in HCV-1b patients (except for RVR that showed only a trend). Overall, rates of treatment responses in HCV-1b patients were intermediate between those seen in HCV-1a (the lowest) and those achieved by HCV-3 patients (the highest).
The effect of IL28B variants on treatment responses was assessed in patients infected with different HCV-1 subtypes. As expected, CC carriers showed a higher rate of RVR, EVR, cEVR, EOTR, and SVR than CT/TT patients, and this was true for both HCV-1a and HCV-1b (Fig. 1A). However, the beneficial effect of the IL28B CC variant was more pronounced in HCV-1a than HCV-1b patients at any given time point.
Using another approach, we compared treatment responses according to HCV-1 subtypes in IL28B CC and non-CC carriers. In non-CC carriers, treatment responses were higher in HCV-1b than HCV-1a patients; whereas in CC carriers, there were generally no significant differences according to HCV-1 subtype (Fig. 1B). Altogether, these data supports an independent effect of both HCV-1 subtypes and IL28B variants on treatment response. Accordingly, when patients were split out into 4 categories defined by HCV-1 subtype and IL28B variant, the lowest treatment response rates were seen in HCV-1a/IL28B non-CC patients, whereas the highest responses were seen in HCV-1b/IL28B CC patients (see Figure, Supplemental Digital Content 1, http://links.lww.com/QAI/A299).
The results of univariate analyses were confirmed using a multivariate logistic regression model, in which the independent influence of HCV-1 subtypes and IL28B variants on treatment responses was adjusted to baseline serum HCV RNA, liver fibrosis staging, gender, and age. As shown in Table 3, HCV-1b and lL28B CC remained as independent predictors of EVR, cEVR, and EOTVR. The same analysis applied separately to HCV-1a and HCV-1b patients confirmed the independent influence of IL28B variants on treatment outcomes in both groups of patients, with a more pronounced effect on HCV-1a than HCV-1b patients (Table 3).
Influence of HCV Geno/Subtypes and IL28B Variants on Early Treatment Viral Kinetics
The significant and independent association of IL28B variants and HCV-1 subtypes with treatment responses prompted us to explore the mechanism behind it. For this purpose, the variation in plasma HCV RNA levels at weeks 4 and 12 of therapy with respect to baseline was calculated for the subset of patients for whom these data were available (266 patients had HCV RNA values at week 4 and 313 patients at week 12). Then, the median variation in viremia at these 2 time points was compared in different groups of patients. First, we compared viral kinetics in patients according to HCV geno/subtype and found that patients infected with HCV-1b experienced significantly faster kinetics than those infected with HCV-1a (Fig. 2A). It must be highlighted that the fastest decay, however, was seen in HCV-3 patients. Overall, viral kinetics in HCV-1b patients were intermediate between those seen in HCV-3 and HCV-1a/HCV-4 patients.
Patients harboring the IL28B CC variant showed significantly faster viral decays than non-CC carriers regardless HCV-1 subtype (Fig. 2B), although the influence was more pronounced in HCV-1a patients. There was a difference of nearly 3 log IU/mL in the level of decrease of HCV RNA at week 12 when comparing CC and non-CC carriers infected with HCV-1a, whereas it was only of 1 log in HCV-1b patients (Fig. 2B). A multivariate linear regression analysis confirmed that both IL28B variants and HCV-1 subtypes independently predicted viral kinetics at weeks 4 and 12 of pegIFNα/RBV therapy. Moreover, IL28B variants remained as the only significant predictor of viral kinetics when considering separately HCV-1a and HCV-1b (see Table, Supplemental Digital Content 2, http://links.lww.com/QAI/A300).
The recognition of the strong impact of IL28B variants on response to pegIFNα/RBV has been a major breakthrough in hepatitis C therapeutics. Besides its influence on SVR in both HCV-monoinfected5–7 and HIV/HCV-coinfected8,18,23–25 patients, recent data in both groups of patients have shown that early HCV kinetics is also significantly modulated by IL28B polymorphisms,9–11,26,27 especially in patients infected with the most difficult-to-treat HCV genotypes 1 and 4. Our study is the first to extend these findings to HIV/HCV-coinfected patients carrying different HCV-1 subtypes. We noticed that on treatment responses and SVR rates were uniformly worse in HCV-1a as compared with HCV-1b patients, and statistically significant at all time points with the exception of RVR, most likely due to the small number of HCV-1 patients that attained undetectable HCV RNA at week 4. This striking observation has only occasionally been reported in HCV-monoinfected individuals28 and without deserving much attention.
Our study has shown that the effect of IL28B variants on HCV RNA kinetics during pegIFNα/RBV therapy in seen in both HCV-1 subtypes, although the beneficial effect is more noticeable in HCV-1a than HCV-1b patients. A multivariate analysis confirmed that both HCV subtype and IL28B variant were independent predictors of on-treatment responses. More importantly, this was independent of other well-known predictors of viral response, including baseline serum HCV RNA.
Another important finding of our study was that early viral kinetics in HCV-1 patients was significantly influenced by both IL28B alleles and HCV subtype. In fact, these 2 variables were the only ones significantly determining changes in serum HCV RNA during the first 12 weeks of therapy. As with treatment response rates at different time points, the effect of IL28B variants on early serum HCV RNA kinetics was more pronounced in HCV-1a than HCV-1b patients. Altogether, the fastest viral kinetics was seen in IL28B CC carriers infected with HCV-1b, whereas the slowest viral declines were seen in IL28B non-CC carriers infected with HCV-1a. Thus, the innate immunity mechanisms involved in treatment-induced viral clearance might be more efficiently operating in HCV-1b than HCV-1a.
The results obtained in our study may have important clinical implications in the new DAA era. Given that most DAA already approved or in advanced stage of clinical development are being used as part of triple combinations along with pegIFNα/RBV, an important question to address is the role of IL28B variants in this setting. Although a direct effect of IL28B alleles on DAA should be negligible if any at all, the global efficacy of triple combinations may still be dependent on the modulating effect of IL28B variants on the pegIFNα/RBV backbone. The strong influence of IL28B variants on early viral kinetics we recognized, with a differential impact on distinct HCV-1 subtypes, highlights that patients infected with distinct HCV-1 subtypes and harboring different IL28B variants may achieve different SVR rates using triple combinations.
There are scenarios in which our findings may be particularly relevant. First, boceprevir, one of the recently approved HCV protease inhibitors, is given as part of a triple combination including a lead-in phase of 4 weeks with only pegIFNα/RBV.17 In this way, the final efficacy of triple therapy is the best in the subset of patients that achieved maximal early viral load declines with pegIFNα/RBV alone. Selection of drug resistance to subsequently added boceprevir often compromises the achievement of SVR in initially poorly responders. Our findings suggest that this adversity should be particularly feared at baseline in the subset of patients harboring IL28B CT/TT and infected with HCV-1a. This population might require closer monitoring or deserve other treatment options.
Secondly, since most DAA exhibit a high antiviral effect but suffer from a low barrier to resistance, one of the most crucial effects of pegIFNα/RBV as part of triple combination therapy is to prevent selection and outgrowth of pre-existing DAA-resistant mutant viruses. In this way, the global efficacy of any triple combination regimen could vary according to pegIFNα/RBV-driven viral kinetics. Interestingly, an intriguing lower efficacy of triple therapy has been reported for HCV-1a compared with HCV-1b in several trials.15,29 Our results support that an intrinsic lower efficacy of pegIFNα/RBV on HCV-1a versus HCV-1b could contribute to this observation. Clearly, further studies analyzing the combined effect of HCV subtypes and IL28B variants on the efficacy of triple combination therapies are warranted.
Last, the results of our study highlight the continuous importance of IL28B testing in the new DAA era, at least whereas pegIFNα/RBV remains part of combination therapy. This test is cheap and has to be made only once in life. Therapeutic decision-making may be supported by baseline IL28B testing and be particularly helpful in patients infected with HCV-1a, in whom treatment failure and emergence of drug-resistant viruses may be more common.16 This subset of patients may require closer monitoring of early viral kinetics and/or plan in advance proper rescue interventions.
In summary, this is the first study demonstrating an important role of both IL28B variant and HCV-1 subtype in predicting on-treatment virological responses to pegIFNα/RBV therapy in HIV/HCV-coinfected individuals. This effect is largely mediated by an enhancement of viral kinetics during the first 12 weeks of therapy. Interestingly the protective effect of the IL28B CC variant is mainly seen in HCV-1a patients in whom responses are the poorest. Thus, baseline IL28B testing may helpfully assist treatment decisions in the new era of triple combination therapy including DAA.
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