Epstein-Barr virus (EBV) causes nasopharyngeal carcinoma (NPC) in endemic regions,where almost every tumor is EBV-positive. In Western populations, NPC is rare, and human papillomavirus infection (HPV) has been suggested as another viral cause. We validated multiplex serology with molecular tumor markers, to define EBV-positive, HPV-positive and EBV-/HPV-negative NPCs in the United Kingdom, and analyzed survival differences between those groups. Sera from NPC cases (n = 98) and ageand sex-matched controls (n = 142) from the Head and Neck 5000 clinical cohort study were analyzed. IgA and IgG serum antibodies against 13 EBV antigens were measured and compared with EBER in situ hybridization (EBER-ISH) data of 41 NPC tumors (29 EBER-ISH positive, 12 negative). IgG antibodies to EBV LF2 correctly diagnosed EBV-positive NPCs in 28 of 29 cases, while all EBER-ISH negative NPCs were seronegative to LF2 IgG (specificity = 100%, sensitivity = 97%). HPV early antigen serology was compared to HPV molecular markers (p16 expression, HPV DNA and RNA) available for 41 NPCs (13 positive, 28 negative). Serology matched molecular HPV markers in all but one case (specificity = 100%, sensitivity = 92%). EBV and HPV infections were mutually exclusive. Overall, 67% of the analyzed NPCs were defined as EBV-positive, 18% as HPV-positive and 14% as EBV/HPV-negative. There was no statistical evidence of a difference in survival between the three groups.These data provide evidence that both, EBV-positive and HPV-positive NPCs are present in a low incidence country, and that EBV and HPV serum antibodies correlate with the viral status of the tumor. K E Y W O R D S nasopharyngeal carcinoma, multiplex serology, Epstein-Barr virus, human papillomavirus Abbreviations: CI, confidence interval; EA-D, early antigen diffuse; EBER-1, Epstein-Barr virus small RNA; EBNA1, EBV nuclear antigen 1; EBV, Epstein-Barr virus; HPV, human papillomavirus; HPyV, human polyomavirus; IHC, immunohistochemistry; IMD, index of multiple deprivation; ISH, in situ hybridization; LSCC, laryngeal squamous cell carcinoma; MFI, median fluorescence intensity; MPG, multiplex papillomavirus genotyping; NCT,

The process of extracting comprehensive proteome representations is a crucial step for many proteomic studies. While antibody microarrays are an evolving and promising methodology in proteomics, the issue of protein extraction from tissues for this kind of analysis has never been addressed. Here, we describe a single-step extraction buffer for the isolation of proteins from mammalian tissues under native conditions in an effective and reproducible manner. Protein was extracted from cell lines BxPC-3 and SU.86.86, rat organs (pancreas, liver, heart and lung) and human pancreatic cancer tissues using several buffer systems that contained individual nonionic or zwitterionic detergents in comparison to commercial extraction buffers. Also, detergent combinations were used that included at least one polymeric phenylethylene glycol, a long-chain amidosulfobetaine, cholate and a zwitterionic detergent. Extracts were analyzed for protein quantity and quality. The detergent cocktails exhibited superior extraction capacity. Additionally, they demonstrated a substantially higher recovery of membrane and compartmental proteins as well as much better preservation of protein functionality. Also, they did not interfere with subsequent analysis steps such as labeling. In Western blot and antibody microarray assays, they outperformed the other buffer systems, indicating that they should also be useful for other types of proteomic studies.