IL-7 producing immunotherapy improves ex vivo T cell functions of immunosenescent patients, especially post hip fracture.

Following acute stress such as trauma or sepsis, most of critically ill elderly patients become immunosuppressed and susceptible to secondary infections and enhanced mortality. We have developed a virus-based immunotherapy encoding human interleukin-7 (hIL-7) aiming at restoring both innate an adaptative immune homeostasis in these patients. We assessed the impact of this encoded hIL-7 on the ex vivo immune functions of T cells from PBMC of immunosenescent patients with or without hip fracture. T-cell ex vivo phenotyping was characterized in terms of senescence (CD57), IL-7 receptor (CD127) expression, and T cell differentiation profile. Then, post stimulation, activation status, and functionality (STAT5/STAT1 phosphorylation and T cell proliferation assays) were evaluated by flow cytometry. Our data show that T cells from both groups display immunosenescence features, express CD127 and are activated after stimulation by virotherapy-produced hIL-7-Fc. Interestingly, hip fracture patients exhibit a unique functional ability: An important T cell proliferation occurred compared to controls following stimulation with hIL-7-Fc. In addition, stimulation led to an increased naïve T cell as well as a decreased effector memory T cell proportions compared to controls. This preliminary study indicates that the produced hIL-7-Fc is well recognized by T cells and initiates IL-7 signaling through STAT5 and STAT1 phosphorylation. This signaling efficiently leads to T cell proliferation and activation and enables a T cell "rejuvenation." These results are in favor of the clinical development of the hIL-7-Fc expressing virotherapy to restore or induce immune T cell responses in immunosenescent hip fracture patients.

Elevated IL-6/IL-1Ra ratio as a prognostic biomarker of poor chemotherapy efficacy in Chinese patients with metastatic NSCLC, validated in a Caucasian patient cohort.

The treatment options for advanced (stage IV) non-small cell lung cancer (NSCLC) at diagnosis remain disappointing. The development of immunotherapeutic drugs may represent a promising alternative approach to the treatment of late-stage cancer at diagnosis. These current paradigms in cancer treatment highlight the need for new biomarkers related to the immune status of the patients and/or the tumor microenvironment, for immune as well as chemotherapeutic treatment options. The aim of the present study was to analyze soluble immune factors in patients with lung cancer treated with chemotherapy to identify prognostic biomarkers. For this purpose, the data obtained from two cohorts of patients from different clinical trials were analyzed: A Chinese patient cohort to identify potential prognostic biomarkers, and a validation cohort comprising patients with a similar clinical stage from a clinical trial in Europe. Analyses of soluble markers for inflammation and immune status were performed by standard assays and multiplex Luminex assays. Differences in overall survival (OS) and progression-free survival (PFS) were evaluated with the log-rank test and robustness was evaluated with the resampling approach. In the Chinese cohort, four prognostic biomarkers of poor response to chemotherapy were identified, which had a significant impact on OS and PFS. It was confirmed in the Caucasian validation cohort that an increased value of the interleukin (IL)-6/IL-1Ra cytokine ratio at inclusion was correlated with significantly shorter OS and PFS, whereas no other biomarkers were found to be significant. The IL-6/IL-1Ra ratio reflects the imbalance between pro- and anti-inflammatory status in the plasma of patients and may be associated with tumor inflammatory status and the therapeutic outcome. The present study highlights the identification of the IL-6/IL-1Ra ratio as a biomarker of poor prognosis in terms of response to chemotherapy in two independent clinical studies.

Viral based vaccine TG4010 induces broadening of specific immune response and improves outcome in advanced NSCLC.

BACKGROUND: Advanced non-small cell lung cancer patients receiving TG4010, a therapeutic viral vaccine encoding human Mucin 1 and interleukin-2 in addition to standard chemotherapy, displayed longer overall survival in comparison to that of patients treated with standard chemotherapy alone. Our study intended to establish the association between overall survival and vaccine-induced T cell responses against tumor associated antigens (TAA) targeted by the vaccine. METHOD: The TIME trial was a placebo-controlled, randomized phase II study aimed at assessing efficacy of TG4010 with chemotherapy in NSCLC. 78 patients from the TIME study carrying the HLA-A02*01 haplotype were analyzed using combinatorial encoding of MHC multimers to detect low frequencies of cellular immune responses to TG4010 and other unrelated TAA. RESULTS: We report that improvement of survival under TG4010 treatment correlated with development of T cell responses against MUC1. Interestingly, responses against MUC1 were associated with broadening of CD8 responses against non-targeted TAA, thus demonstrating induction of epitope spreading. CONCLUSION: Our results support the causality of specific T-cell response in improved survival in NSCLC. Additionally, vaccine induced epitope spreading to other TAA participates to the enrichment of the diversity of the anti-tumor response. Hence, TG4010 appears as a useful therapeutic option to maximize response rate and clinical benefit in association with other targeted immuno-modulators. TRIAL REGISTRATION: Registered on ClinicalTrials.gov under identifier NCT01383148 on June 23rd, 2011.

Prognostic impact of the expression of NCR1 and NCR3 NK cell receptors and PD-L1 on advanced non-small cell lung cancer.

The putative contribution of natural killer (NK) cells to immunosurveillance in non-small cell lung cancer (NSCLC) has been an ongoing conundrum. Here, we used a readily standardizable quantitative real time polymerase chain reaction (qRT-PCR) to measure the expression of NK cell receptors in total peripheral blood mononuclear cells (PBMC) from healthy volunteers (HV), patients with gastrointestinal stromal tumors (GIST), neuroblastoma (NB), melanoma or NSCLC. We quantified NCR1 (which codes for NKp46) and NCR3 (which codes for NKp30), as well as that of three NCR3 splice variants (which give rise to immunostimulatory NKp30A and NKp30B, as well as to immunosuppressive NKp30C). NSCLC patients expressed lower levels of NCR1 than did HV. Remarkably, NCR3 was lower in NSCLC patients than in HV as well as in all other malignancies. Moreover, a discrete proportion of NSCLC patients exhibited a particular low ratio between NKp30B and NKp30C (ΔBC). In the overall cohort, low expression of NCR3 correlated with poor overall and progression-free survival (PFS). When patients were stratified according to the level of PD-L1 expression by NSCLC cells, within the PD-L1high category (>5% positive tumors), the sole parameter that affected prognosis was the expression of NCR1. However, in patients bearing tumors with negative PD-L1 expression on tumor or tumor-infiltrating stromal cells, the ΔBClow patients exhibited a dismal prognosis. Altogether, these results strongly suggest that NK cells mediate immunosurveillance against NSCLC and that measuring NK cell receptor expression by blood cells can yield useful biomarkers for patient stratification.

Oncolytic virotherapy with an armed vaccinia virus in an orthotopic model of renal carcinoma is associated with modification of the tumor microenvironment.

Oncolytic virotherapy is an emergent promising therapeutic approach for the treatment of cancer. We have constructed a vaccinia virus (WR strain) deleted for thymidine kinase (TK) and ribonucleotide reductase (RR) genes that expressed the fusion suicide gene FCU1 derived from the yeast cytosine deaminase and uracil phosphoribosyltransferase genes. We evaluated this construct (VV-FCU1) in the orthotopic model of renal carcinoma (RenCa). Systemic administration of VV-FCU1 resulted in orthotopic tumor growth inhibition, despite temporary expression of viral proteins. VV-FCU1 treatment was associated with an infiltration of tumors by CD8+ T lymphocytes and a decrease in the proportion of infiltrating Tregs, thus modifying the ratio of CD8+/CD4+ Treg in favor of CD8+cytotoxic T cells. We demonstrated that VV-FCU1 treatment prolonged survival of animals implanted with RenCa cells in kidney. Depletion of CD8+ T cells abolished the therapeutic effect of VV-FCU1 while depletion of CD4+ T cells enhanced its protective activity. Administration of the prodrug 5-fluorocytosine (5-FC) resulted in a sustained control of tumor growth but did not extend survival. This study shows the importance of CD4+ and CD8+ T cells in vaccinia virus-mediated oncolytic virotherapy and suggests that this approach may be evaluated for the treatment of human renal cell carcinoma.

Adefovir dipivoxil resistance patterns in patients with lamivudine-resistant chronic hepatitis B.

BACKGROUND: Lamivudine (3TC)-resistant chronic hepatitis B patients demonstrated a higher rate of adefovir dipivoxil (ADV) resistance compared with nucleoside-naive patients. This study describes ADV mutation patterns in 3TC-resistant patients treated with ADV+3TC or ADV monotherapy, investigating whether mutations selected during 3TC therapy predispose to ADV resistance. Risk factors for ADV resistance were also evaluated. METHODS: A total of 60 3TC-experienced patients were treated with (or switched to) ADV monotherapy (30 patients) or ADV+3TC combination therapy (30 patients), and followed for at least 12 months. In all patients the hepatitis B virus reverse transcriptase (RT) region was amplified and directly sequenced before initiating ADV. The RT sequence was reevaluated for virological breakthrough patients and phenotypic analysis was performed for several patients. RESULTS: In total, 14 (23%) patients showed virological breakthrough (10/30 on ADV monotherapy and 4/30 on ADV+3TC). ADV resistance mutations (rtA181V/T and rtN236T) were detected alone or in combination for 11/14 patients, whereas novel substitutions were present in 3 patients. Before ADV treatment, apart from 3TC resistance signature mutations, additional changes were found, including the rtA181T mutation, which was already present in 2/14 ADV-resistant patients. CONCLUSIONS: Although most patients showed virological breakthrough because of the well known rtA181V/T and rtN236T substitutions, more complex patterns were also found. ADV monotherapy, dose reduction and suboptimal virological response after 48 weeks of therapy were significantly associated with ADV resistance.

Rolling circle amplification, a powerful tool for genetic and functional studies of complete hepatitis B virus genomes from low-level infections and for directly probing covalently closed circular DNA.

Complete characterization of the biological properties of hepatitis B virus (HBV) variants requires the generation of full-length genomes. The aim of this study was to develop new tools for the efficient full-length genome amplification of virus from samples with low viral loads. Rolling circle amplification (RCA) was used to amplify full-length HBV genomes from both sera and liver biopsy samples from chronic HBV carriers. Serum-derived relaxed circular HBV DNA could be amplified only after completion and ligation of plus-strand DNA. Covalently closed circular DNA (cccDNA) from liver biopsies could be amplified directly from as few as 13 copies, using RCA, followed by a full-length HBV PCR. Three serial liver biopsy samples were obtained from a lamivudine-resistant patient who cleared detectable serum HBV after adefovir dipivoxil was added to the lamivudine therapy and then seroconverted to anti-HBs. Only the genomes from the last biopsy specimen obtained after the emergence of lamivudine resistance contained the lamivudine resistance-associated mutations rtL180M and rtM204V ("rt" indicates reverse transcriptase domain). Defective genomes were also found in this biopsy sample. Genomes cloned from the liver biopsy specimens were transfected into HuH7 cells to study their replication competence and their susceptibility to lamivudine. RCA is a powerful tool for amplifying full-length HBV genomes and will be especially useful for the study of occult or inactive HBV infections and patients undergoing antiviral treatment. It can also be used to probe HBV cccDNA, the crucial intermediate in viral persistence and the archive of resistance mutations.

Impact of hepatitis B virus rtA181V/T mutants on hepatitis B treatment failure.

BACKGROUND/AIMS: Recent clinical observations reported the occurrence of amino acid substitutions at position 181 of the HBV polymerase, associated with a viral breakthrough under lamivudine or adefovir therapy. In this study, we characterized the main variants harboring the rtA181T/V mutation isolated from 10 consecutive patients who developed lamivudine and/or adefovir resistance. METHODS: We performed a clonal analysis of the HBV polymerase gene amplified by PCR from serum samples during viral breakthrough. The main mutants were then tested after transfection of Huh7 cells for their resistance profile to nucleoside analogs. RESULTS: Clonal analysis revealed the co-localization on the same HBV genome of rtA181T/V with rtN236T, but not with rtM204V/I mutations following lamivudine, adefovir or lamivudine+adefovir breakthrough. In cell culture, the rtA181T/V mutation induced a decreased susceptibility to lamivudine (<10-fold), adefovir (2- to 8-fold) and tenofovir (2- to 3-fold). Interestingly, the association of rtA181T with rtN236T on one clinical isolate genome increased the resistance to these three drugs. All the tested mutants remained sensitive to entecavir. CONCLUSIONS: Our observations suggest that a single amino acid change at position rt181 may induce cross-resistance to lamivudine and adefovir. These data emphasize the clinical relevance of genotypic and phenotypic analysis in the management of antiviral drug resistance.

Stepwise process for the development of entecavir resistance in a chronic hepatitis B virus infected patient.

BACKGROUND/AIMS: Complex mutants may be selected under sequential anti-VHB pressures. We analyzed the genotypic and phenotypic evolution of the viral quasi-species of a patient who developed resistance to entecavir following lamivudine breakthrough. METHODS: The polymerase gene was amplified, cloned and sequenced at different time points. Hepatoma cell lines were transfected to compare the replication capacity of HBV mutants and their drug susceptibility. RESULTS: A mixture of lamivudine-resistant HBV strains coexisted following viral breakthrough to lamivudine, all harboring the rtM204V mutation. The rtV173L+L180M+M204V dominant mutant displayed strong lamivudine-resistance and the highest replication capacity. Following the switch to entecavir, the viral load dropped but the lamivudine-resistant strains continued to be selected. Three years later, the viral load rose again, and a complex mixture of entecavir-resistant strains, all harboring the lamivudine-resistance signature rtL180M+M204V and the rtS202G mutation were observed. Although the rtL180M+S202G+M204V variant, that prevailed at the end of entecavir therapy, did not show the highest viral genome replication capacity, it conferred one of the strongest resistance levels to entecavir. CONCLUSIONS: We report the selection of complex HBV mutants that escaped lamivudine and entecavir antiviral pressures. Genotypic and phenotypic analysis provided additional information to understand the process of HBV variant selection.

Increase of doxorubicin sensitivity by doxorubicin-loading into nanoparticles for hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is known to be chemoresistant to anticancer drugs due to the multidrug resistant (MDR) transporters expression. Here, we compared in vitro and in vivo the anti-tumor efficacy of doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles (PIHCA-Dox) versus free doxorubicin (Dox). These nanoparticles are known to overcome the MDR phenotype. METHODS: We first determined in vitro the 50% inhibition concentration (IC(50)) of these drugs on different human hepatoma cell lines. Secondly, the efficacy of the drugs in vivo was determined on the X/myc transgenic murine model of HCC by histological counting of apoptotic tumorous hepatocytes and by TUNEL labeling. We characterized by semi-quantitative RT-PCR the MDR-related gene (mdr1, mdr3, mrp1) expression pattern in this model. RESULTS: In vitro, IC(50) was reduced with PIHCA-Dox versus Dox for Huh7 (1.7-fold reduction; P<0.001), HepaRG (4.5-fold reduction; P<0.01), HepG2 (1.5-fold reduction; P<0.001), and HepG2.2.15 (1.5-fold reduction; P=0.059). In vivo, HCC in transgenic mice overexpressed the mdr1 and mdr3 genes and the antitumor drugs efficacy was greatly enhanced after injection of PIHCA-Dox (9.0+/-5.0%; n=15) versus Dox (4.6+/-3.3%; n=13; P=0.01) for apoptotic bodies count. CONCLUSIONS: These promising data showing a higher anti-tumor efficacy on HCC of PIHCA-Dox versus Dox, warrant further studies in both animals and humans.

Long-term high-dose interferon-alpha therapy delays Hepadnavirus-related hepatocarcinogenesis in X/myc transgenic mice.

The role of interferon-alpha (IFN-alpha) remains unclear in prevention of virus-induced hepatocellular carcinoma in humans. We have investigated it herewith in the X/myc transgenic mouse model of Hepadnavirus-related hepatocarcinogenesis because of upregulation of c-myc oncogene in the liver. We have demonstrated that IFN-alpha can downregulate dose-dependently hepatocyte proliferation and c-myc overexpression at early premalignant stages, while it does not affect either hepatocyte apoptosis or telomerase activity at these steps. However, continuous and long-term administration of IFN-alpha dose-dependently delays tumor onset in dysplastic livers and increases overall survival of animals, more efficiently whether started before the onset of dysplasia. The present study therefore highlights that early preventive administration of IFN-alpha can slow down evolution towards hepatocellular carcinoma via repression of c-myc and hepatocyte proliferation at premalignant steps in experimental c-myc-induced hepatocarcinogenesis. However, the transient effect observed in this study emphasizes a need to clarify the possible mechanisms of acquired resistance and subsequent therapeutic escape. Our experimental model may be a pertinent tool to explore antioncogenic properties of IFN-alpha in human cirrhotic livers showing c-myc upregulation.