Identification of oropharyngeal squamous cell carcinomas with active HPV16 involvement by immunohistochemical analysis of the retinoblastoma protein pathway.

Viral oncogene RNA expression is regarded as reliable biomarker to identify oropharyngeal squamous cell carcinomas (OPSCC) with active HPV16 involvement. This study addressed whether the expression profile of the cellular proteins p16(INK4a), pRb, Cyclin D1 and p53 provide surrogate marker combinations that identify OPSCC with active HPV16 in situations where only formalin-fixed biopsies are available. Protein expression was analyzed by immunohistochemistry on tissue microarrays created from 188 OPSCC of which the HPV16 DNA and RNA status had been established previously from snap-frozen biopsies. Associations of single markers and of marker combinations with HPV16 DNA, viral RNA expression patterns and overall survival as primary end point were evaluated by statistical analysis. Most tumors with active HPV16 involvement (RNA(+) group; n = 40) showed a specific protein pattern, that is, high p16(INK4a) (80%), low pRb (85%), low Cyclin D1 (95%) and normal p53 (73%). This pattern was significantly different from the pattern observed in HPV DNA-negative tumors (HPV(-) group) and in HPV16 DNA-positive tumors lacking viral RNA expression patterns (RNA(-) group). The combination of high p16(INK4a) and low pRb levels was distinctly superior to p16(INK4a) alone; it was strongly associated with RNA(+) tumors (OR 41.4, 95%CI 10.7-162.5), with improved survival (HR 0.37, 95%CI 0.2-0.8) and had best predictive values (78% sensitivity, 93% specificity, 78% PPV, 93% NPV). In conclusion, if only formalin-fixed biopsy material is available, the marker combination high p16(INK4a) /low pRb is well suited to identify OPSCC with biologically active HPV16 which represent a distinct OPSCC entity with improved prognosis.

Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells.

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.