Heterogeneity and chronology of PTEN deletion and ERG fusion in prostate cancer.

TMPRSS2:ERG fusions, in combination with deletion of the phosphatase and tensin homolog (PTEN) tumor suppressor, have been suggested to cooperatively drive tumor progression in prostate cancer. We utilized a novel heterogeneity tissue microarray containing samples from 10 different tumor blocks of 189 prostatectomy specimens to study heterogeneity of genomic PTEN alterations in individual foci. PTEN alterations were found in 48/123 (39%) analyzable individual tumor foci, including 40 foci with deletions, 7 with deletion and rearrangement, and 1 focus with rearrangement only. PTEN was homogeneously aberrant in only 4 (8%) and heterogeneously in 44 (92%) of the foci. We found a specific sequence of molecular events from PTEN breakage followed by deletion of DNA sequences flanking the breakpoint, resulting in homozygous deletion. The observation that 16 of 19 foci with homogeneous ERG positivity had focal PTEN alterations but none of 10 foci with PTEN alterations had focal ERG positivity (P<0.0001) suggests that PTEN alterations typically develop subsequent to ERG fusions. We demonstrate a high level of intratumoral heterogeneity of PTEN alterations with deletions and rearrangements that challenges potential PTEN routine diagnosis testing in biopsies. The observation that PTEN alterations develop subsequent to ERG fusion strongly suggests that ERG expression may directly drive development of PTEN aberrations.

Variant of hepatitis B virus with primary resistance to adefovir.

The reverse-transcriptase inhibitor lamivudine (Zeffix, GlaxoSmithKline) is often used to treat chronic infection with hepatitis B virus (HBV) until resistance develops. Treatment may then be switched to the reverse-transcriptase inhibitor adefovir (Hepsera, Gilead), which has a lower frequency of resistance. Here, we describe three cases of primary adefovir resistance that were sensitive to tenofovir (Viread, Gilead). All three cases involved a rare HBV variant with a valine at position 233 of the reverse-transcriptase domain instead of isoleucine (rtI233V), as in the wild-type virus. This HBV variant also displayed resistance to adefovir and sensitivity to tenofovir in vitro.

pH-independent entry and sequential endosomal sorting are major determinants of hepadnaviral infection in primary hepatocytes.

Entry and intracellular transport of hepatitis B viruses have several unusual, largely unknown aspects. In this study, we explored the mode of virus entry using the duck hepatitis B virus (DHBV) and the primary hepatocyte infection model. Upon internalization, viral particles were enriched in an endosomal compartment, as revealed by biochemical and ultrastructural analysis. Virus-containing vesicles harbored early endosome markers. Kinetic analysis revealed time-dependent partial translocation of viral DNA from endosomes into the cytosol. This was strongly reduced by inhibition of vacuolar ATPase; (vATPase) activity with bafilomycin A1 and resulted in abortive infection and prevention of cccDNA formation. Inactivation of vATPase induced accumulation and stabilization of incoming viral particles in endosomes, presumably by blocking endosomal carrier vesicle-mediated cargo transport and sorting. Although neutralization of the endomembrane organelles alone led to stabilization of incoming viral particles, it did not inhibit virus infection. In line with this, a pH-dependent ectopic virus fusion at the plasma membrane could not be artificially induced. This provided further evidence for a pH-neutral translocation mechanism. Endosomal membrane potential was required for viral infection because cotreatment of cells with monensin partially overcame the inhibitory effect of bafilomycin A1. In conclusion, hepatitis B viral infection is mediated by a novel cellular entry mechanism with features different from that of all other known viruses.