Investigation of Clinically Significant Molecular Aberrations in Patients with Prostate Cancer: Implications for Personalized Treatment, Prognosis and Genetic Testing.

The data on tumor molecular profiling of European patients with prostate cancer is limited. Our aim was to evaluate the prevalence and prognostic and predictive values of gene alterations in unselected patients with prostate cancer. The presence of gene alterations was assessed in patients with histologically confirmed prostate cancer using the ForeSENTIA® Prostate panel (Medicover Genetics), targeting 36 clinically relevant genes and microsatellite instability testing. The primary endpoint was the prevalence of gene alterations in homologous recombination repair (HRR) genes. Overall, 196 patients with prostate cancer were evaluated (median age 72.2 years, metastatic disease in 141 (71.9%) patients). Gene alterations were identified in 120 (61%) patients, while alteration in HRR genes were identified in 34 (17.3%) patients. The most commonly mutated HRR genes were ATM (17, 8.7%), BRCA2 (9, 4.6%) and BRCA1 (4, 2%). The presence of HRR gene alterations was not associated with advanced stage (p = 0.21), age at diagnosis (p = 0.28), Gleason score (p = 0.17) or overall survival (HR 0.72; 95% CI: 0.41-1.26; p = 0.251). We identified clinically relevant somatic gene alterations in European patients with prostate cancer. These molecular alterations have prognostic significance and therapeutic implications and/or may trigger genetic testing in selected patients. In the era of precision medicine, prospective research on the predictive role of these alterations for innovative treatments or their combinations is warranted.

In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides.

BACKGROUND & AIMS: Current treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting. METHODS: A lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively. RESULTS: ASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro. CONCLUSION: Our results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.

Decreased infectivity of nucleoside analogs-resistant hepatitis B virus mutants.

BACKGROUND & AIMS: To understand the mechanisms of emergence and selection of HBV polymerase variants, which may also harbor mutations in the overlapping envelope protein, we analyzed the in vitro virus production and infectivity of the main viral mutants resistant to lamivudine and adefovir. METHODS: HBV-resistant mutants (rtL180M+M204V, rtV173L+L180M+M204V, rtM204I, rtL180M+M204I, rtN236T, rtA181V, rtA181V+rtN236T, rtA181T+N236T, and rtA181T) were produced in HepG2 cells permanently expressing the respective viral genomes. Viral protein expression, secretion, and viral particle production were studied by ELISA, Western blot, and transmission electron microscopy. To study only the effect of surface gene mutants on virus infectivity, HepaRG cells were inoculated with HDV pseudo-particles coated with the mutant HBV envelopes. To evaluate infectivity and replication in a global fashion, HepaRG cells were inoculated with HBV mutants. RESULTS: HBeAg was expressed and secreted in cell supernatants in all mutant-expressing cell lines. As expected, mutants harboring a sW196Stop mutation in the surface gene did not express small envelope proteins. All mutants expressing HBsAg were able to produce viral particles. HDV particles coated with mutant envelopes were less infectious than WT in HepaRG cells. Finally, we found that resistant mutants exhibit lower infectivity and replication ability than WT virus. CONCLUSIONS: Based on this study, we found that envelope substitutions modulate viral protein expression, HDV coating, and viral infectivity. These envelope modifications provide novel insights into the features of emerging HBV variants during antiviral therapies and suggest that such mutants are less prone to transmission than their WT counterpart.

Preclinical and Phase 1 Assessment of Antisense Oligonucleotide Bepirovirsen in Hepatitis B Virus-Transgenic Mice and Healthy Human Volunteers: Support for Clinical Dose Selection and Evaluation of Safety, Tolerability, and Pharmacokinetics of Single and Multiple Doses.

Dose-dependent reductions in hepatitis B virus (HBV) RNA, DNA, and viral proteins following bepirovirsen administration were observed in HepG2.2.15 cells. In HBV-transgenic mice treated at 50 mg/kg/wk, hepatic HBV RNA and DNA were reduced by 90% and 99%, respectively. Subsequently, a phase 1 first-in-human study assessed pharmacokinetics and tolerability of single (75-450 mg) and multiple (150-450 mg on days 1, 4, 8, 11, 15, and 22) subcutaneous bepirovirsen doses in 96 healthy volunteers. Bepirovirsen at all dose levels was rapidly absorbed (maximum plasma concentration 3-8 hours after dosing), rapidly distributed to peripheral tissues, and slowly eliminated (median plasma terminal half-life: 22.5-24.6 days across cohorts). Plasma exposure (dose-proportional at 150-450 mg) and concentration-time profiles were similar following the first and sixth doses, suggesting little to no plasma accumulation (steady state achieved by day 22). Renal elimination of full-length bepirovirsen accounted for <2% of the total dose. Across the single and multiple dose cohorts, 197 treatment-emergent adverse events were reported, with 99% and 65% classified as mild in severity and local injection site reactions, respectively. In conclusion, bepirovirsen showed an acceptable safety profile in humans with observed pharmacokinetics consistent with the chemical class, warranting further evaluation of bepirovirsen in chronic HBV infection.

In vitro characterization of viral fitness of therapy-resistant hepatitis B variants.

BACKGROUND & AIMS: Because of the overlapping of polymerase and envelope genes in the hepatitis B virus (HBV) genome, nucleoside analog therapy can lead to the emergence of complex HBV variants that harbor mutations in both the reverse transcriptase and the envelope proteins. To understand the selection process of HBV variants during antiviral therapy, we analyzed the in vitro fitness (the ability to produce infectious progeny) of 4 mutant viral genomes isolated from one patient who developed resistance to a triple therapy (lamivudine, adefovir, and anti-HBV immunoglobulins). METHODS: The 4 mutant and the wild-type forms of HBV were expressed from vectors in hepatoma cell lines; replication and viral particle secretion capacities then were analyzed. The impact of envelope gene mutations on infectivity was tested in HepaRG cells using the hepatitis delta virus (HDV) model as a reporter for infection. RESULTS: The dominant HBV variant characterized from the therapy-resistant patient was found to have the best replicative capacity in vitro in the presence of high concentrations of lamivudine and adefovir. The expression of envelope proteins and secretion of subviral and Dane particles by this mutant was comparable with that of wild-type HBV. HDV particles enveloped by surface proteins from the selected mutant had the highest rates of infection in HepaRG cells compared with other mutants. CONCLUSIONS: These results illustrate the importance of viral fitness and infectivity as a major determinant of antiviral therapy resistance in patients. Understanding HBV mutant selection in vivo will help to optimize new anti-HBV therapeutic strategies.

The Prevalence of Antibodies against Sandfly Fever Viruses and West Nile Virus in Cyprus.

BACKGROUND: Sandfly fever is an incapacitating disease caused by sandfly-borne Phleboviruses that can lead to meningitis, encephalitis or meningoencephalitis. West Nile virus (WNV), a mosquito-borne Flavivirus, can induce neuroinvasive disease manifested by meningitis, encephalitis or acute flaccid paralysis. Both vectors are endemic in Cyprus and very active during summer. The aims of this study were to determine first the prevalence of sandfly fever viruses (SFV) and WNV infections in Cyprus and second, to investigate their role in central nervous system (CNS) infections. METHODS: For the prevalence study, 327 sera collected in 2013 and 2014 were tested for anti-SFV and anti-WNV IgG using indirect immunofluorescence assay and ELISA, respectively. In order to investigate a possible role of SFV and WNV in CNS infections, 127 sera of patients presenting symptoms of SFV or WNV infections were screened for IgM specific to SFV and WNV. RESULTS: The overall anti-SFV IgG seroprevalence was 28% and was increasing with age (P< 0.01). The seroprevalence rate for anti-WNV IgG in Cyprus was 5%. Concerning the role of SFVs in CNS infections, anti-SFV IgM was detected in 8 out of 127 sera from selected patients presenting relevant symptoms of infections during vector's active period. In addition, anti-WNV IgM were detected in 17 out of the 127 patients with compatible symptoms. CONCLUSION: The findings confirm the presence of sandfly fever and WNV in Cyprus and should, therefore, be considered in the differential diagnosis of patients with febrile illness/meningitis.

The main hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to non-nucleoside inhibitors of HBV replication.

Long-term treatment of chronic hepatitis B with nucleos(t)ide analogs can lead to the emergence of HBV resistant mutants of the polymerase gene. The development of drugs with a different mode of action is warranted to prevent antiviral drug resistance. Only a few non-nucleosidic molecules belonging to the family of phenylpropenamides (AT-61 & AT-130) and heteroaryldihydropyrimidines (BAY41-4109) can prevent RNA encapsidation or destabilize nucleocapsids, respectively. The sensitivity of the main nucleos(t)ide analog- resistant mutants to these inhibitors was evaluated in vitro. HepG2 stable cell lines permanently expressing wild type (WT) HBV or the main HBV mutants resistant to lamivudine and/or adefovir (rtL180M+rtM204V, rtV173L+rtL180M+rtM204V, rtM204I, rtL180M+rtM204I, rtN236T, rtA181V, rtA181V+rtN236T, rtA181T, rtA181T+rtN236T) were treated with AT-61, AT-130 or BAY-41 4109. Analysis of intracellular encapsidated viral DNA showed that all mutants were almost as sensitive to these molecules as WT HBV; indeed, the fold-resistance ranged between 0.7 and 2.3. Furthermore, the effect of a combination of either AT-61 or AT-130 with BAY41-4109, and the combination of these compounds with tenofovir was studied on wild type HBV as well as on a lamivudine and an adefovir-resistant mutant (rtL180M+M204V and rtN236T, respectively). These combinations of compounds resulted in inhibition of viral replication but showed slight antagonistic effects on the three HBV species. Based on this in vitro study, BAY-41 4109, AT-61 and AT-130 molecules that interfere with capsid morphogenesis are active against the main lamivudine- and adefovir-resistant mutants. These results suggest that targeting nucleocapsid functions may represent an interesting approach to the development of novel HBV inhibitors to prevent and combat drug resistance.

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.