Interferon Alpha Resistance Research Articles

Limited Sequence Variation and Similar Phenotypic Characteristics of HIV-1 Subtype C Gag Variants Derived From the Reservoir and Pre-Therapy Plasma

HIV variants present in the reservoir, particularly in tissues, may differ from those present in peripheral blood prior to therapy initiation, and characterisation of these reservoir variants could better inform immune-based interventions for HIV cure. In the present study, Gag sequence differences between variants derived from the lymph node and peripheral blood mononuclear cell (PBMC) reservoirs as well as those derived from pre-therapy plasma, were investigated in 24 HIV-1 subtype C-infected individuals. HIVgagamplification was successful for 20 individuals, where 4 were controls including one untreated individual and 3 early treated individuals with LN collection within 2 weeks of treatment initiation. The remaining 16 individuals with LN and PBMC collection > 3 months after treatment initiation (median = 665 days), were further characterised. Recombinant viruses encoding patient-derived Gag-protease sequences from the pre-therapy plasma, LN reservoir, and PBMC reservoir, were constructed and the replication-competent viruses that grewin vitrowere used to further investigate whether there are specific features of Gag reservoir variants that may have relevance for strategies to cure HIV. Virus characteristics measured included replication capacity, interferon-alpha resistance, cell-to-cell spread ability, and induction of antiviral cytokines. A limited number of novel Gag mutations (median = 4) in the reservoir of 3/7 early treated participants and 9/9 late treated participants were observed, where the majority of these mutations were likely cytotoxic T lymphocyte (CTL)-driven and 48% were represented in the replication-competent viruses. The reservoir variants had very few unique potential CTL escape mutations (median = 3) in Gag compared to the number of these Gag mutations that were already present in the plasma-derived virus (median = 23) at the time of treatment initiation, which was similar whether treatment was initiated late or early. The data suggest that the extent of CTL escape in Gag overall is likely similar between early and late treated individuals as well as between the reservoir and pre-therapy variants. The sequence differences in Gag that were unique to the reservoir viruses did not result in significantly altered virus characteristics overall, and are therefore unlikely to affect effectiveness of immune-based interventions for virus eradication.

Monitoring Molecular Response, Minimal Residual Disease and Clonal Structures in Polycythemia Vera Patients Treated with Interferon Alpha

Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) primarily characterized by an elevated red blood cell count, consequently to clonal expansion of myeloid progenitor cells driven almost exclusively by a mutation of a specific single nucleotide in JAK2 exon 14 (JAK2-V617F). JAK2-V617F mutational burden therefore represents a surrogate measure for the size of the "liquid tumor" underlying PV, i.e. the amount of malignant cells in the peripheral blood. We previously reported on sustained molecular responses (MR) in PV patients on interferon alpha (IFNa) based therapies. Monitoring MR is crucial for understanding the therapeutic potential of IFNa and can be an important parameter in future PV patient care. Thus, highly sensitive laboratory techniques providing most accurate quantification at lowest limit of detection (LOD) are required. Here we evaluate four different methods for JAK2-V617F mutational burden monitoring, which are digital droplet PCR (ddPCR), next-generation sequencing (NGS), quantitative PCR (qPCR) and allele-specific PCR (AS-PCR). Moreover, using an NGS-based approach we investigate the patients' clonal composition by targeted re-sequencing involving 54 genes in longitudinal patient samples.To determine the characteristics of the different methods for burden quantification, we benchmarked a ddPCR assay (Assay ID dHsaCP2000061/dHsaCP2000062, Bio-Rad, Hercules, CA) using serial dilutions of JAK2-V617F positive in JAK2-V617F negative gDNA. The assay showed an LOD of 0.01% JAK2-V617F with a false positivity rate of 0 mutation-positive droplets in 8 independent measurements of healthy donor gDNA. Next we quantified the JAK2-V617F burden in baseline and follow-up (FU) gDNAs from a PV patient cohort on IFNa-based therapy (n=51) using ddPCR, a qPCR-based assay (ipsogen MutaQuant, Qiagen, Hilden, Germany), an NGS-based approach (TruSight Myeloid Sequencing Panel, Illumina, San Diego, CA) and an AS-PCR assay described previously (Kralovics et al, Blood, 2006). Covering the full spectrum of allelic burden values, correlations with ddPCR-derived values were best for NGS (R2=0.998) followed by qPCR (R2=0.976) and AS-PCR (R2=0.951). While mutational burden values >10% JAK2-V617F showed high concordance between methods, JAK2-V617F burdens <10% exhibited significant deviations from the benchmarked ddPCR assay, suggesting that ddPCR is most suitable for monitoring residual disease in PV. Therefore, applying ddPCR, we next re-evaluated two PV patients with baseline JAK2-V617F burdens of 46% and 28%, respectively, both previously classified as complete MR based on JAK2-V617F undetectable by AS-PCR. For those two patients, ddPCR revealed an actual residual disease of 3.02% and 2.39% mutant allele burden at 138 and 68 weeks post initiation of IFNa therapy, respectively. Further monitoring of these patients showed a sustained decrease in mutant allele burden to 1.53% and 0.20% at weeks 217 and 171, respectively.In addition to JAK2-V617F burden quantification, our NGS approach allowed for quantitative evaluation of mutations in 54 genes implicated in myeloid diseases. After somatic variant calling, paired analysis of baseline and FU samples (n=96) from 48 PV patients revealed an average of 2.8 variants per patient in a total of 17 genes. Besides JAK2, recurrently affected genes were TET2, PHF6, ASXL1 and CEBPA. The paired analysis allowed for tracking changes in clonal structures upon therapy. Most notably, TET2-JAK2 double-positive clones present in two patients showed mutant burden decreases of 13% and 59% respectively, arguing against an implication of TET2 mutations in IFNa resistance. Furthermore, PHF6 positive clones present in three patients showed ambiguity upon therapy, shrinking concordantly with the JAK2 positive clone in two patients while expanding as a JAK2-independent clone in the third patient.In summary, while larger studies will be required to assign statistical significance to mutations implicated in IFNa-based therapies, targeted re-sequencing of serial samples in MPN patient cohorts has the potential to deconvolute the impact of the patients' clonal architecture on therapeutic success. Moreover, as medications such as IFNa-based drugs have been shown to induce deep MR in a substantial fraction of PV patients, the use of highly accurate assays such as ddPCR will be crucial for monitoring minimal residual disease. DisclosuresKlade:AOP Orphan: Employment. Gisslinger:Baxalta: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria. Hoermann:Novartis: Honoraria; Ariad: Honoraria; Gilead: Research Funding; Amgen: Honoraria. Kralovics:AOP Orphan: Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees.

Sequence diversity of hepatitis C virus 6a within the extended interferon sensitivity-determining region correlates with interferon-alpha/ribavirin treatment outcomes

Studies on the association between sequence variability of the interferon sensitivity-determining region (ISDR) of hepatitis C virus and the outcome of treatment have reached conflicting results. In this study, 25 patients infected with HCV 6a who had received interferon-alpha/ribavirin combination treatment were analyzed for the sequence variations. 14 of them had the full genome sequences obtained from a previous study, whereas the other 11 samples were sequenced for the extended ISDR (eISDR). This eISDR fragment covers 192 bp (64 amino acids) upstream and 201 bp (67 amino acids) downstream from the ISDR previously defined for HCV 1b. The comparison between interferon-alpha resistance and response groups for the amino acid mutations located in the full genome (6 and 8 patients respectively) as well as the mutations located in the eISDR (10 and 15 patients respectively) showed that the mutations I2160V, I2256V, V2292I ( P < 0.05) within eISDR were significantly associated with resistance to treatment. However, the extent of amino acid variations within previously defined ISDR was not associated with resistance to treatment as previously reported. Four amino acid variations I248V ( P = 0.03–0.06) within E1, R445K ( P = 0.02–0.05) and S747T ( P = 0.03) within E2, I861V ( P = 0.01) within NS2 which located outside the eISDR may also associate with treatment outcome as identified by a prescreening of variations within 14 HCV 6a full genomes.

The hepatitis C virus NS5A protein and response to interferon α: mutational analyses in patients with chronic HCV genotype 3a infection from India

The hepatitis C virus (HCV) non-structural (NS)5A protein is linked to interferon alpha resistance in vitro and higher numbers of NS5A amino acid (aa) variations in HCV 1a/b isolates are associated with virologic response to interferon alpha-based therapy in vivo. Here, we aimed to study NS5A aa variations in Indian patients undergoing interferon alpha/ribavirin treatment infected with HCV 3a. The NS5A region [aa 2194-2401, comprising interferon sensitivity determining region, protein kinase resource (PKR) binding domain, V3 region] was sequenced from pre-treatment sera of 24 patients with HCV 3a infection. Mean number and physicochemical properties of aa variations (conserved vs. non-conserved) were assessed. Additionally, published NS5A sequences [NS5A region (n = 61), PKR binding domain (n = 111)] of characterized HCV 3a isolates were analyzed. The mean number of NS5A aa variations was not correlated with treatment response in our cohort. When all available NS5A sequences were included, a higher number of non-conserved aa variations within PKR binding domain and an extended V3 region of NS5A was associated with virologic response (P = 0.004 and 0.05, respectively). Mutational analyses of a large number of NS5A sequences suggest, that a higher number of non-conserved aa variations within the PKR binding domain and the extended V3 region is correlated with virologic response in HCV 3a infected patients.

Hepatitis C virus-related resistance mechanisms to interferon α-based antiviral therapy

Only 50-60% of the patients chronically infected with the hepatitis C virus (HCV) achieve a sustained virologic response to the current standard antiviral therapy consisting of pegylated interferon alpha in combination with ribavirin. The definite reasons for virologic response or non-response to interferon alpha-based therapy are unknown. Besides host and treatment efficacy factors, it is presumable that HCV is able to antagonize the antiviral activity of interferon alpha. So far, among the different HCV proteins, the envelope (E)2 protein, the non-structural (NS)3/4A protein, and the NS5A protein have been associated with interferon alpha resistance mechanisms in vitro. The clinical significance of amino acid mutations within these HCV proteins in HCV isolates from patients who did or did not respond to interferon alpha-based therapy was investigated in multiple studies. Within the E2 (HVR2, CD81 binding sites, PePHD) and the NS3/4A proteins no specific mutations in correlation with virologic response to interferon alpha-based therapy were observed. For the NS5A protein, mutations within the interferon sensitivity determining region (ISDR) and the complete NS5A protein may be of importance for response to interferon alpha-based treatment in patients infected with HCV subtype 1a/b.

The nature of interferon-alpha resistance in hepatitis C virus infection.

HCV infection becomes chronic in 50-85% of cases. The treatment of chronic hepatitis C is currently based on a combination of pegylated interferon (IFN)-alpha and ribavirin. With this regimen, a failure to eradicate infection occurs in 18-24% of patients infected by genotype 2 or 3, and in 54-58% of patients infected by genotype 1. IFN resistance, i.e. the capacity of HCV strains to attenuate IFN antiviral responses in order to evade them, could play a role in the establishment of chronic infection at the acute stage of infection. IFN resistance could also play a role in the virological response to IFN therapy through similar or different mechanisms. The involved mechanisms however remain unclear. Several viral proteins were recently shown to mediate IFN resistance through inhibition of IFN antiviral effectors in vitro, but the relevance of such mechanisms in vivo is not proven. Whatever the mechanisms, IFN resistance could play a role at the early stages of infection, but a qualitative and quantitative defect of both CD4-positive and CD8-positive immune responses appears as the main determinant of viral persistence. IFN treatment failure to eradicate infection is multifactorial. IFN resistance could play a partial role through unclear mechanisms. However, immune clearance of infected cells appears to be the principal determinant of IFN treatment success. In spite of active research, the role and the mechanisms of IFN resistance in HCV persistence and IFN treatment failure remain partly unknown. A better understanding is needed in order to further improve IFN treatment strategies.

Identification of Genes Involved in Resistance to Interferon-α in Cutaneous T-Cell Lymphoma

Interferon-alpha therapy has been shown to be active in the treatment of mycosis fungoides although the individual response to this therapy is unpredictable and dependent on essentially unknown factors. In an effort to better understand the molecular mechanisms of interferon-alpha resistance we have developed an interferon-alpha resistant variant from a sensitive cutaneous T-cell lymphoma cell line. We have performed expression analysis to detect genes differentially expressed between both variants using a cDNA microarray including 6386 cancer-implicated genes. The experiments showed that resistance to interferon-alpha is consistently associated with changes in the expression of a set of 39 genes, involved in signal transduction, apoptosis, transcription regulation, and cell growth. Additional studies performed confirm that STAT1 and STAT3 expression and interferon-alpha induction and activation are not altered between both variants. The gene MAL, highly overexpressed by resistant cells, was also found to be expressed by tumoral cells in a series of cutaneous T-cell lymphoma patients treated with interferon-alpha and/or photochemotherapy. MAL expression was associated with longer time to complete remission. Time-course experiments of the sensitive and resistant cells showed a differential expression of a subset of genes involved in interferon-response (1 to 4 hours), cell growth and apoptosis (24 to 48 hours.), and signal transduction.

Interferon-alpha resistance in renal carcinoma cells is associated with defective induction of signal transducer and activator of transcription 1 which can be restored by a supernatant of phorbol 12-myristate 13-acetate stimulated peripheral blood mononuclear cells.

Therapy of selected human malignancies with interferon-α is widely accepted but often complicated by the emergence of interferon-α resistance. Interferon is a pleiotropic cytokine with antiproliferative, antitumour, antiviral and immunmodulatory effect; it signals through the Jak-STAT signal transduction pathway where signal transducer and activator of transcription 1 plays an important role. Here we report both, a lack of signal transducer and activator of transcription induction in interferon-α resistant renal cell carcinoma cells and signal transducer and activator of transcription 1 reinduction of phorbol 12-myristate 13-acetate-stimulated peripheral blood mononuclear cells supernatant. Preliminary experiments on the identification of the molecules that reinducing signal transducers and activators of transcription 1 indicate that interferon-γ may be the responsible candidate cytokine, but several others may be involved as well. This work provides the basis for therapeutic strategies directed at the molecular modulation of interferon-α resistance in human neoplasms.British Journal of Cancer (2002) 86, 449–455. DOI: 10.1038/sj/bjc/6600066 www.bjcancer.com© 2002 The Cancer Research Campaign