Cyclic changes in gene expression induced by Peg-interferon alfa-2b plus ribavirin in peripheral blood monocytes (PBMC) of hepatitis C patients during the first 10 weeks of treatment

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Cyclic changes in gene expression induced by Peg-interferon alfa-2b plus ribavirin in peripheral blood monocytes (PBMC) of hepatitis C patients during the first 10 weeks of treatment
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  BioMed   Central Page 1 of 15 (page number not for citation purposes) Journal of Translational Medicine Open Access Research Cyclic changes in gene expression induced by Peg-interferon alfa-2b plus ribavirin in peripheral blood monocytes (PBMC) of hepatitis C patients during the first 10 weeks of treatment MiltonWTaylor* 1 , TakumaTsukahara 1 , JeanetteNMcClintick  2 , HowardJEdenberg  2  and PaulKwo 3  Address: 1 Department of Biology, Indiana University, Bloomington, IN. 47401, USA, 2 Department of Biochemistry and Molecular Biology and Center for Medical Genomics, Indiana University School of Medicine, Indianapolis, IN 46202, USA and 3 Department of Medicine, Hepatology Unit, Indiana University School of Medicine, Indianapolis, IN 46202, USA Email: MiltonWTaylor*;;;;* Corresponding author Abstract Background and Aims: This study determined the kinetics of gene expression during the first 10 weeksof therapy with Pegylated-interferon-alfa2b (PegIntron™) and ribavirin (administered by weight) in HCVpatients and compared it with the recently completed Virahep C study [1,2] in which Peginterferon-alfa2a(Pegasys™) and ribavirin were administered. Methods: RNA was isolated from peripheral blood monocytes (PBMC) from twenty treatment-naïvepatients just before treatment (day 1) and at days 3, 6, 10, 13, 27, 42 and 70 days after treatment. Geneexpression at each time was measured using Affymetrix microarrays and compared to that of day 1. Results: The expression of many genes differed significantly (p ≤  0.001 and changed at least 1.5-fold) atdays 3 (290 probes) and 10 (255 probes), but the number dropped at days 6 (165) and 13 (142). Mostgenes continued to be up regulated throughout the trial period. A second group of genes, including CXCL10 , CMKLR1 (chemokine receptor 1), TRAIL , IL1R α  and genes associated with complement and lipidmetabolism, was transiently induced early in treatment. CDKN1C (cyclin kinase inhibitor 1) was inducedearly but repressed at later times. Genes induced at later times were mostly related to blood chemistryand oxygen transport. By week 10, 11 of the patients demonstrated a positive response to therapy, andthe final sustained viral response (SVR) was 35%. The levels of gene induction or decrease was very similarto that previously reported with Pegasys/ribavirin treatment. Conclusion: The response to Pegintron/ribavirin was similar to that reported for Pegasys/ribavirindespite some differences in the amount administered. We did not detect major differences at the genomiclevel between patients responding to treatment or non-responders, perhaps because of limited power.Gene induction occurred in a cyclic fashion, peaking right after administration of interferon and decliningbetween administrations of the drug. Our data suggest that more than once a week dosing might bedesirable early during treatment to maintain high levels of response as measured by gene expression. Published: 5 November 2008  Journal of Translational Medicine  2008, 6 :66doi:10.1186/1479-5876-6-66Received: 16 September 2008Accepted: 5 November 2008This article is available from:© 2008 Taylor et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (   ), which permits unrestricted use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  Journal of Translational Medicine   2008, 6 :66 2 of 15 (page number not for citation purposes) Background Hepatitis C virus (HCV) infection is a significant globalpublic health problem, affecting approximately 200 mil-lion individuals in the world and over 4 million in theUnited States alone, where it is the most prevalent blood-borne infection [3]. It is currently the leading indicationfor a liver transplant and is responsible for 8,000–10,000deaths annually. Interferon (IFN) has formed the back-bone of therapy against HCV, first as monotherapy, thenin combination with the nucleoside analogue ribavirin[4]. Current standard of care for chronic HCV infectionconsists of a regimen of pegylated interferon- α  in combi-nation with ribavirin. The addition of the polyethyleneglycol (PEG) moiety (pegylation) increases the half-life of the IFN molecule and has facilitated once per week dosing instead of the two or three doses per week previously required with non-pegylated forms of IFN [5,6]. The com-bination of pegylated IFN- α  and ribavirin successfully eradicates the virus from 50–60% of those treated [7,8]. Two different pegylated molecules of IFN have beenapproved for clinical use in the U.S. The size and positionof the PEG moiety differs between pegylated-interferon- α -2a (Pegasys™) and Pegylated-interferon- α -2b (PegIn-tron™) [5,9,10]. Although pegylation improves the phar-macokinetic properties of the core IFN protein [11], it decreases the in vitro biological activity [12,13]. PegIn-tron™ has higher in vitro anti-viral activity than Pegasys™[11,14,15] (Taylor, unpublished data). Type I IFNs do not directly inactivate the virus, but exert their effects through binding to specific receptors on thecell surface, IFNAR1 and IFNAR2 [16]. This results in acascade of gene activation through the Jak-STAT pathway [17-19] and perhaps other transcription pathways[20,21]. Large number of genes are induced or down reg-ulated by non-pegylated IFN α   in vitro [22-25]. Previous work [15] has shown very similar in vitro profiles of geneinduction in monocytes (PBMC) treated with either pegylated or the non-pegylated version of IFN α . Virtually all of the changes in gene expression were due to the IFN,rather than the ribavirin [23]. We have recently reportedthat the expression of many hundreds of genes are signif-icantly modified, both up and down, in vivo following treatment of hepatitis C patients with pegylated-IFN α -2a(Pegasys™) and ribavirin [2]. Using a mathematical model we identified a core set of genes that appear to be relatedto the anti-viral effects. These include OAS2 ,  MX1 ,  MX2 , RIG1 , genes associated with ubiquitination, and many other genes previously shown to be associated with inter-feron treatment [26].In this report we analyze the response of patients to com-bination treatment with pegylated-interferon- α 2b (PegIn-tron™) and ribavirin during the first 10 weeks of treatment. Unlike the previous report from the Virahep Cstudy [2], which used a constant dose of 180 ug of pegylated-interferon- α -2a, in the present study the PegIn-tron™ was administered in an amount related to the body  weight of the patients. Blood samples were collectedbefore treatment initiation (day 1) and at days 3, 6, 10,13, 27, 42 and 70 after treatment. Interferon injections were weekly at day 1, 7, 14 etc. The selection of days wasbased on times just before interferon injection (days 6,13,27, 42 and 70) in order to analyze whether there was atrough in gene expression at the end of the weekly period. Affymetrix microarrays were used to detect genes up- or down- regulated during treatment. Viral assays for thepresence of HCV in serum were performed at the sametime points. In this study we report that changes in geneexpression levels are high 3 days after IFN injection andreturn toward baseline before the next injection; thereturn toward baseline is accompanied in many cases by aslight increase in virus titer. This pattern continues for thefirst few weeks. Genes induced by the treatment fall intothree classes, genes that are up regulated throughout thetreatment, immediate expressed genes with only transient expression, and late genes in which expression is elevatedonly after day 27. Fifty percent of the patients showed anantiviral response during the first 10 weeks, but the finalSVR was 35%. Materials and methods Subjects  Twenty (16 M, 4 F) genotype 1 hepatitis C patients whogave informed consent were entered into this trial. All sub-jects were previously untreated, and had no other cause of chronic liver disease, ALT levels above the upper limit of normal, compensated liver disease with minimal hemato-logical parameters including hemoglobin values of 12gm/dL for females and 13 gm/dL for males, WBC > 3,000/mm 3 , neutrophil count > 1,500/mm 3 , platelets > 70,000/mm 3 and no evidence of decompensation in those withcirrhosis. All patients had liver biopsies within 3 years of enrolling, with fibrosis graded by the Metavir scoring sys-tem. Patients were excluded if they had decompensatedcirrhosis, serum α -fetoprotein concentration above 50 ng/L, HIV infection, previous organ transplantation, other causes of liver disease, pre-existing psychiatric disease, sei-zure disorders, cardiovascular disease, haemoglobinopa-thies, haemophilia, poorly controlled diabetes, or autoimmune-type disease, or if they were unable to usecontraception. Table 1 presents the demographics of thepopulation used in this study. This study was approved by the institutional review board.Patients received PegIntron™ at 1.5 μ g/kg (based upon weight at initial visit) administered subcutaneously oncea week for 10 weeks (days 1, 7, 14, 21, 28 ...), plus ribavi-rin (13 ± 2 mg/kg/day). Patients had blood drawn for   Journal of Translational Medicine   2008, 6 :66 3 of 15 (page number not for citation purposes) analysis on day 1 prior to first injection of interferon (baseline) and at days 3, 6, 10, 13, 27, 42 and 70. HCV-RNA Serum Determinations Serum samples were collected before treatment initiation(day 1) and at days 3, 6, 10, 13, 28, 42, 70 and weeks 12,24, 48 and 72, for viral assays. HCV-RNA was determinedby qRT-PCR (TaqMan ® , Schering-Plough Research Insti-tute, Union, NJ) with a lower limit of detection of 29 IU/ml. Peripheral Blood Mononuclear Cell (PBMC) Preparation Blood was collected in sodium heparin-CPT tubes, diluted with an equal volume (8 ml) of phosphate buffered saline(PBS), carefully layered over a 10 ml Ficoll-Hypaque gra-dient (Amersham/Pharmacia, Piscataway, NJ) and centri-fuged at 800 rpm for 20 minutes at room temperature. The buffy coat layer was transferred to a 15 ml RNAse-freetube, diluted with PBS, and centrifuged at 100 × g for 15minutes at room temperature. The supernatants were dis-carded and the PBMC were retained. RNA Extraction  The PBMC were lysed in 1 ml of TRI reagent (Molecular Research Center Inc, Cincinnati, OH). The lysate wasmixed with 1-Bromo-3-chloropropane (BCP)-phase sepa-ration agent for 1 minute, and incubated at room temper-ature for 15 minutes. After centrifugation for 15 minutesat 12,000 rpm and 4°C, RNA was precipitated from thesupernatant overnight at -20°C with an equal volume of isopropanol and 1/10 volume of 7.5 M ammonium ace-tate. The precipitate was washed twice with 75% ethanol,and then with 95% ethanol. RNA was briefly air-dried andthen resuspended and further purified using RNeasy col-umns (Qiagen; Valencia, CA). The amount and quality of RNA were determined by spectrophotometry and by elec-trophoresis through 1% agarose with ethidium bromide.RNA was further analyzed by the Agilent Bioanalyzer;samples that did not show two clear bands of ribosomalRNA were discarded. RNA Labeling, Hybridization and Scanning  Preparation of cDNA, cRNA, and labeling were carried out according to the protocols recommended by Affymetrix inthe GeneChip ® Expression Analysis Technical Manual(Affymetrix, Santa Clara, CA), as previously described [2].Hybridization was to Affymetrix GeneChip ® HumanGenome U133A microarrays, which measure approxi-mately 22,000 genes. The microarrays were scanned using a dedicated Affymetrix Model 3000 7G scanner controlledby GCOS software. Statistical Analysis  The average intensity on each array was normalized by global scaling to a target intensity of 1000. Data wereextracted using the Affymetrix Microarray Suite 5 (MAS5)algorithm. To avoid analyzing genes that were not reliably detected, the MAS5 data were filtered to eliminate any gene that was not called present in at least 50% of thesamples in at least one group [27]. If a probeset was not reliably detected on day 1 but was later, it is noted as"turned on" and if it was detected on day 1 but not later it is noted as "turned off;" the exact fold change for suchgenes are not reliable because the signal for a gene that is Table 1: Pretreatment characteristics of the patients Patient IDAgeWeight (kg)GenotypeGenderFibrosis Score MetavirALT (IU/L)Day 1 HCV RNA level (IU/Ml)149761a14741.7E+06252921a00207.0E+05352921a14573.4E+06438901a10521.1E+07556801a12683.5E+066471061a131461.1E+07744651b121271.1E+078421121a11677.2E+06952781a14712.3E+061057761b02687.1E+061159701b03622.4E+0612561091a14745.8E+0613491161a12445.8E+061453901b14983.7E+051551611a03731.8E+061650781a13991.6E+0617601061a12374.1E+0518451141a141619.0E+051947741b131174.0E+052061831b121818.1E+05  Journal of Translational Medicine   2008, 6 :66 4 of 15 (page number not for citation purposes) not detected is largely background. Fold changes for eachgene were calculated using the ratio of the MAS5 signals of the post treatment time to the baseline (pre-treatment). If the signal for the post-treatment time point was greater than the baseline the fold change was calculated as +aver-age(post-treatment)/average(baseline), otherwise the foldchange was calculated as -average(baseline)/average(post-treatment). Genes were considered significant if thepaired t-test p-value of log(signal) ≤  0.001 and the foldchange was at least 1.5.Gene expression as a function of time was analyzed using Edge[28]; values are cal-culated on the log transform, but are plotted as percent of maximum signal values with gnuplot to show wider range of values. The 90 genesmost significant across all time points (by 1-way ANOVA) were clustered using Pearsons dissimilarity and averagelinkage, using Partek Genomics Suite (Partek Inc. St.Louis, MO); arrays were ordered by day to show the pat-tern of expression across time. Results Of the 20 patients enrolled, 19 were European Americanand one was African American. Sixteen were male. All were genotype 1, 14 with genotype 1A and 6 with geno-type 1B. The baseline features of the 20 patients in thisstudy are shown in Table 1. In this cohort, 11/20 hadadvanced hepatic fibrosis (Metavir stage 3–4), with 17/20having high viral load (> 600,000 IU/mL). The overallsustained viral response rate (SVR) at the end of treatment (72 weeks) was 35%; i.e. 7/20 individuals had undetecta-ble virus at 72 weeks. Table 2 presents virus titers withtime. By week 12 there were 11/20 patients who cleared virus, however one withdrew from treatment because of severe side effects, and 2 relapsed by the end of treatment. Changes in Global Gene Expression Gene expression in PBMC changed dramatically and rap-idly during PEG-interferon- α 2b (PegIntron™)/ribavirintherapy, with major changes being evident at all days after the initial administration of the drugs (Table 3, Figure 1). There was no significant difference in response betweenpatients with genotype 1A and 1B, nor between respond-ers and non-responders, so all patients were analyzedtogether. 973 genes were significantly (p ≤  0.001; FalseDiscovery Rate [29] 1.2%) induced or down regulated onday 3; the number induced was approximately the same asthe number down-regulated, as was seen in our earlier study [2]. The number of differentially expressed genes varied with time (Table 3, Figure 1); it was high on days 3and 10 (mid-way between injections) and much lower ondays 6, 13 and 42 (just before subsequent injections)(Table 3, Figure 1). The number of genes with alteredexpression was high again, particularly for down regulatedgenes, at day 70. Half of the up-regulated genes but only 16% of the down-regulated genes showed at least 1.5-foldchange (Table 3). For our subsequent analyses we focusedon the genes with more robust changes (p = 0.001 andabsolute fold-change ≥  1.5). There were 69 genes that showed at least 1.5-fold differ-ences in expression at either 6 or all 7 time points: 59 up-regulated and 10 down-regulated (Table 4). Many of theseup regulated genes have previously been shown to be reg-ulated by interferon [2,25,26]. A full list of all genesinduced or down regulated at p ≤  0.001 at any one day compared to day 1 is presented in Supplementary Table 1. There is a strong pattern of gene expression as a functionof time, as demonstrated by hierarchical clustering of the90 genes that differed most (Figure 2). There is a clearly  visible, alternating pattern of increases and decreases that decays over time. The patterns of gene expression can bedivided into four groups. The top cluster are genes that aredecreased as a result of treatment. These include genesassociated with protein synthesis including eukaryotic ini-tiation and elongation factors (EIF4B, EEI2, EIF3S5) andgenes involved in ribosomal proteins ( RPL3 ). The major-ity of genes fall into a second group, highly induced at days 3 and 10 but showing a decrease at day 6 and 13; thealternation decreases with time but is still high at day 70. This includes most of the well characterized IFN induciblegenes, including  MX1, MX2, OAS1, OAS2, OAL, RIG1(DDX58) and most interferon stimulated genes (ISGs). A third group are transiently induced genes, i.e. genesinduced at day 3 and then returning to base line at later times (Table 5); many have been described as important in the interferon antiviral response and include CXCL10,IL1RA (IL1RN), JAK2, TNFSF10 (TRAIL) , as well as CDKN1C, CXCL10, SMD4A . The last two genes at the bot-tom of the cluster array represent genes that are inducedlate. As is obvious for GYPA (glycophorin A), inductionfor such genes begins around day 27 and proceedsthrough day 70. Most of the genes in this latter group arerelated to blood chemistry, including hemoglobin com-plex formation, heme binding and oxygen transport (Table 6), which may reflect secondary response to long term treatment with ribavirin. A more complete list of genes in each category is presented in the accompanying  Tables 4, 5 and 6. To further examine the variation of gene expression withtime, we used Edge software [28], which tests for changesin gene expression over time vs. the null hypothesis that the gene was expressed at a constant level. Among the 518gene probes that were significantly modulated (absolutefold change ≥  1.5, p ≤  0.001) at any one time point in thestudy (Supplementary Table 1) 90% were shown to be dif-ferentially regulated over time (p ≤  0.001; False Discovery   Journal of Translational Medicine   2008, 6 :66 5 of 15 (page number not for citation purposes) Rate ≤  0.001) in a cyclic fashion. The ten most differen-tially expressed of these genes are plotted in Figure 3. These same genes were previously selected by an unbiasedmathematical model as being involved in interferon anti-HCV activity [26]. Comparison with previous studies  To compare the level of induction or down regulationbetween this study and a previous study (Virahep C; [2])performed with Peg-intron, we chose twenty patientsfrom the Virahep C study for whom we had data from day 3 (note that day 3 in Virahep C was the fourth day after interferon injection, which was day 0 in that study). Thetop 20 genes in terms of fold change are shown in Table7. All genes induced in both trials are presented in Supple-mentary Table 2. Note that in the Virahep C study the doseof Peginterferon-alfa2a (Pegasys™) was 180 ug; in thepresent study the dose of Pegylated-interferon-alfa2b(PegIntron™) was lower: 1.5 ug/kg, for an average of 133ug (standard deviation 25.6, maximum 174). Discussion  The aim of this study was to examine the effects of Peg interferon alfa-2b (PegIntron™, administered at 1.5 ug/kg) Table 2: Viral titer with time. Patient IDDay 0 HCV RNA level (IU/Ml)Day 3Day 7Day 10Day 13Day 27Week 6Week 10Viral Response*Week 12Week 24Week 48Week 72Final Response11.7E+063.5E+053.7E+051.0E+052.3E+059.6E+048.3E+045.3E+03NR2.0E+0300NRNR27.0E+051.3E+059.2E+041.5E+033.8E+03000R0000R33.4E+061.5E+062.8E+061.1E+061.9E+061.6E+061.9E+066.3E+05NR3.8E+05NRNR41.1E+073.5E+056.1E+053.6E+041.7E+059.0E+035.7E+020R0000R53.5E+062.9E+061.6E+064.9E+051.4E+066.9E+054.7E+052.0E+05NR1.7E+05NRNR61.1E+074.0E+065.6E+063.9E+066.3E+062.3E+061.0E+062.2E+05NR7.5E+05NRNR75.8E+061.3E+059.9E+041.4E+047.3E+041.3E+031.5E+020R0000R81.1E+071.9E+055.8E+052.2E+04no sample2.6E+038.7E+010R0000R97.2E+068.8E+032.2E+051.9E+034.4E+03000R0000R102.3E+061.7E+065.3E+066.1E+051.0E+065.5E+053.6E+058.0E+05NR3.1E+05NRNR117.1E+061.9E+062.7E+062.0E+065.8E+061.9E+061.9E+061.4E+06NR6.7E+05NRNR122.4E+061.7E+073.3E+061.3E+062.0E+067.3E+055.4E+051.0E+05NR6.6E+04NRNR135.8E+069.9E+059.2E+051.1E+051.9E+051.4E+057.4E+031.5E+02R0000R143.7E+051.0E+052.3E+057.0E+041.2E+057.0E+033.9E+021.6E+02R0001.2E+06NR151.8E+063.8E+051.0E+064.2E+058.0E+051.9E+052.3E+042.0E+03R3.6E+03NRNR161.6E+062.1E+063.9E+068.0E+055.8E+065.4E+062.1E+061.3E+06NR7.7E+04NRNR174.1E+051.2E+051.2E+061.3E+056.1E+053.1E+047.5E+034.5E+01R0000R189.0E+051.5E+066.2E+064.3E+051.4E+063.2E+053.3E+052.5E+05NR4.8E+04NRNR194.0E+058.7E+046.9E+053.1E+043.7E+041.5E+031.0E+030R0006.3E+06NR208.1E+053.3E+031.9E+033.8E+012.7E+02000R0WWWwithdrew*R = responder, NR = non-responder.W = withdrew
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