Previous studies have reached conflicting conclusions regarding the proportion of Merkel cell carcinomas (MCCs) that contain the Merkel cell polyomavirus (MCPyV) and the clinical significance of tumor viral status. To address these controversies, we detected MCPyV large T antigen using immunohistochemistry with two distinct antibodies and MCPyV DNA using quantitative PCR. Tumors were called MCPyV-positive if two or more of these three assays indicated presence of this virus. A total of 53 of 282 (19%) MCC tumors in this cohort were virus-negative using this multimodal system. Immunohistochemistry with the CM2B4 antibody had the best overall performance (sensitivity = 0.882, specificity = 0.943) compared with the multimodal classification. Multivariate analysis including age, sex, and immunosuppression showed that, relative to MCC patients with virus-positive tumors, virus-negative MCC patients had significantly increased risk of disease progression (hazard ratio = 1.77, 95% confidence interval = 1.20–2.62) and death from MCC (hazard ratio = 1.85, 95% confidence interval = 1.19–2.89). We confirm that approximately 20% of MCCs are not driven by MCPyV and that such virus-negative MCCs, which can be quite reliably identified by immunohistochemistry using the CM2B4 antibody alone, represent a more aggressive subtype that warrants closer clinical follow-up.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. Biochemical studies have demonstrated that NS5A interacts in vitro with and inhibits the IFN-induced, RNAdependent protein kinase, PKR, and that NS5A interacts with at least one other cellular kinase. The present study describes the establishment and characterization of various stable NS5A-expressing human cell lines, and the development of a cell culture-based assay for determining the inherent IFN resistance of clinical NS5A isolates. Human epithelioid (Hela) and osteosarcoma (U2-OS) cell lines were generated that express NS5A under tight regulation by the tetracycline-dependent promoter. Maximal expression of NS5A occurred at 48 hours following the removal of tetracycline from the culture medium. The half-life of NS5A in these cell lines was between 4 to 6 hours. NS5A protein expression was localized cytoplasmically, with a staining pattern consistent with the location of the Golgi apparatus and endoplasmic reticulum. In the majority of cell lines, no obvious phenotypic changes were observed. However, three genotype 1b NS5A-expressing osteosarcoma cell lines exhibited cytopathic effect and severely reduced proliferation as a result of high-level NS5A expression. Full-length NS5A protein isolated from a genotype 1b IFN-nonresponsive patient (NS5A-1b) was capable of rescuing encephalomyocardititis virus replication during IFN challenge up to 40-fold, whereas a full-length NS5A-1a and an interferon sensitivity determining region (ISDR) deletion mutant (NS5A-1a-⌬ISDR) isolated from a genotype 1a IFNnonresponsive patient showed no rescue activity. The NS5A-1b and NS5A-1a proteins also rescued vesicular stomatitis virus replication during IFN treatment by two-to threefold. These data cummulatively suggest that NS5A expression alone can render cells partially resistant to the effects of IFN against IFN-sensitive viruses, and that in some systems, these effects may be independent of the putative ISDR. A scenario is discussed in which the NS5A protein may employ multiple strategies contributing to IFN resistance during HCV infection. (HEPATOLOGY 1999;29: 1262-1271.)Hepatitis C virus (HCV), a hepacivirus in the Flaviviridae family, 1,2 is the etiological agent of chronic hepatitis C, a leading cause of liver disease worldwide. 3,4 Acute infection with HCV is associated with persistent viral replication in approximately 85% to 90% of cases, resulting in a spectrum of liver disease including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 5 Thus, HCV infection represents a serious global socioeconomic health burden.Interferon alfa (IFN-␣) is widely used to treat HCV infections, and although up to 50% of patients show an initial biochemical response (alanine transaminase normalization) to treatment, many patients relapse after therapy. 6,7 Subsequent studies revealed that virological end-of-treatment responses (HCV-RNA-negative) occurred in...

To study hepatitis C virus (HCV) genetic mutation during interferon (IFN) therapy, the temporal changes in HCV quasispecies heterogeneity were compared before and after treatment for nine patients infected with HCV genotype 1, including four nonresponders, four responders who relapsed after therapy, and one responder who experienced a breakthrough of viremia during therapy. Nine untreated patients with an average time between specimens of 8.4 years served as controls. Sequences from the second envelope glycoprotein gene hypervariable region 1 (HVR1) and the putative IFN sensitivity-determining region (ISDR) of the nonstructural NS5A gene were analyzed by heteroduplex mobility assays and nucleotide sequencing. A strong positive correlation was found between the percent change in a heteroduplex mobility ratio (HMR) and percent change in nucleotide sequence (r = 0.941,P < 0.001). The rate of fixation of mutations in the HVR1 was significantly higher for IFN-treated patients than for controls (6.97 versus 1.31% change in HMR/year; P = 0.02). Similarly, a higher rate of fixation of mutations was observed in the ISDR for IFN-treated patients than for untreated controls, although the result was not significant (1.45 versus 0.15 amino acid changes/year; P = 0.12). On an individual patient basis, IFN therapy was associated with measurable HVR1 and ISDR mutation in nine of nine (100%) and two of nine (22.2%) patients, respectively. Evolution to IFN-resistant ISDR sequences was observed in only one of nine IFN-treated patients. These data suggest that IFN therapy frequently exerts pressure on the HCV envelope region, while pressure on the ISDR was evident in only a subset of patients. Thus, the selection pressures evoked on HCV genotype 1 quasispecies during IFN therapy appear to differ among different patients.

Histological progression of hepatitis C is tightly associated with homogenization of HCV quasispecies, perhaps reflecting immune failure and/or selective outgrowth of aggressive viral variants.

Number of tables: 3 Number of figures: 3 Number of references: 38 Word count: 2936 Background Delirium is a common debilitating complication of advanced cancer. ObjectiveTo determine if a multicomponent non-pharmacological delirium prevention intervention was feasible for adult patients with advanced cancer, prior to a phase III (efficacy) trial. DesignPhase II (feasibility) cluster randomized controlled trial. All sites implemented delirium screening and diagnostic assessment. Strategies within sleep, vision and hearing, hydration, orientation, mobility and family domains were delivered to enrolled patients at intervention sites admission days 1-7.Control sites then implemented the intervention ('waitlist sites'). SettingFour Australian palliative care units MeasurementsThe primary outcome was adherence, with an a priori endpoint of at least 60% patients achieving full adherence. Secondary outcomes were interdisciplinary care delivery, delirium measures and adverse events, analyzed descriptively and inferentially. ResultsSixty-five enrolled patients (25 control, 20 intervention, 20 waitlist) had 98% delirium screens and 75% diagnostic assessments completed. Nurses (67%), physicians (16%), allied health (8.4%), family (7%), patients (1%) and volunteers (0.5%) delivered the intervention. There was full adherence for 5% patients at intervention sites, partial for 25%. Both full and partial adherence was higher at waitlist sites: 25% and 45%, respectively. One-third of control site patients (32%) became delirious within seven days of admission compared to one-fifth (20%) at both intervention and waitlist sites (p=0.5). Mean (SD) Delirium Rating Scale-Revised-1998 scores were 16.8 +12.0 control sites versus 18.4 +8.2 (p=0.6) intervention and 18.7 +7.8 (p=0.5) waitlist sites. The intervention caused no adverse events. ConclusionThe intervention requires modification for optimal adherence in a phase III trial.