Interobserver variability between expert urologic pathologists for extraprostatic extension and surgical margin status in radical prostatectomy specimens.

Accurate Gleason score, pathologic stage, and surgical margin (SM) information is critical for the planning of post-radical prostatectomy management in patients with prostate cancer. Although interobserver variability for Gleason score among urologic pathologists has been well documented, such data for pathologic stage and SM assessment are limited. We report the first study to address interobserver variability in a group of expert pathologists concerning extraprostatic soft tissue (EPE) and SM interpretation for radical prostatectomy specimens. A panel of 3 urologic pathologists selected 6 groups of 10 slides designated as being positive, negative, or equivocal for either EPE or SM based on unanimous agreement. Twelve expert urologic pathologists, who were blinded to the panel diagnoses, reviewed 40x whole-slide scans and provided diagnoses for EPE and SM on each slide. On the basis of panel diagnoses, as the gold standard, specificity, sensitivity, and accuracy values were high for both EPE (87.5%, 95.0%, and 91.2%) and SM (97.5%, 83.3%, and 90.4%). Overall kappa values for all 60 slides were 0.74 for SM and 0.63 for EPE. The kappa values were higher for slides with definitive gold standard EPE (kappa=0.81) and SM (kappa=0.73) diagnoses when compared with the EPE (kappa=0.29) and SM (kappa=0.62) equivocal slides. This difference was markedly pronounced for EPE. Urologic pathologists show good to excellent agreement when evaluating EPE and SM. Interobserver variability for EPE and SM interpretation was principally related to the lack of a clearly definable prostatic capsule and crush/thermal artifact along the edge of the gland, respectively.

Nuclear E-cadherin and VHL immunoreactivity are prognostic indicators of clear-cell renal cell carcinoma.

The loss of functional von Hippel-Lindau (VHL) tumor suppressor gene is associated with the development of clear-cell renal cell carcinoma (CC-RCC). Recently, VHL was shown to promote the transcription of E-cadherin, an adhesion molecule whose expression is inversely correlated with the aggressive phenotype of numerous epithelial cancers. Here, we performed immunohistochemistry on CC-RCC tissue microarrays to determine the prognostic value of E-cadherin and VHL with respect to Fuhrman grade and clinical prognosis. Low Fuhrman grade and good prognosis associated with positive VHL and E-cadherin immunoreactivity, whereas poor prognosis and high-grade tumors associated with a lack of E-cadherin and lower frequency of VHL staining. A significant portion of CC-RCC with positive VHL immunostaining correlated with nuclear localization of C-terminally cleaved E-cadherin. DNA sequencing revealed in a majority of nuclear E-cadherin-positive CC-RCC, subtle point mutations, deletions and insertions in VHL. Furthermore, nuclear E-cadherin was not observed in chromophobe or papillary RCC, as well as matched normal kidney tissue. In addition, nuclear E-cadherin localization was recapitulated in CC-RCC xenografts devoid of functional VHL or reconstituted with synthetic mutant VHL grown in SCID mice. These findings provide the first evidence of aberrant nuclear localization of E-cadherin in CC-RCC harboring VHL mutations, and suggest potential prognostic value of VHL and E-cadherin in CC-RCC.

Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel.

The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression by Nedd4 proteins.