Characterization of humoral immune responses against p16, p53, HPV16 E6 and HPV16 E7 in patients with HPV-associated cancers Miriam Reuschenbach 1 , Tim Waterboer 2 , Keng-Ling Wallin 3 , Jens Einenkel 4 , Joakim Dillner 5 , Eva Hamsikova 6 , Denise Eschenbach 1 , Heike Zimmer 4 , Bernhard Heilig 1 ,J€ urgen Kopitz 1 , Michael Pawlita 2 , Magnus von Knebel Doeberitz 1 and Nicolas Wentzensen 1 * 1 Department of Applied Tumor Biology, University of Heidelberg, Heidelberg, Germany 2 Infection and Cancer Program, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Department of Molecular Medicine, Karolinska University Hospital Solna, Stockholm, Sweden 4 Department of Obstetrics and Gynecology, University of Leipzig, Leipzig, Germany 5 Department of Medical Microbiology, MAS University Hospital, Malmo, Sweden 6 Department of Experimental Virology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic The cellular tumor suppressor p16 is strongly overexpressed in cervical cancers and precancers. We have previously demon- strated that infiltrating T lymphocytes reactive against p16 can be found in cervical cancer patients. Here, we analyzed whether p16 induces humoral immune responses. Sera of patients with cervical cancer, oropharyngeal cancer, colorectal cancer and autoimmune disease were included. A total of 919 sera were analyzed, including 486 matched sera from a cervical cancer case control study. p16 antibodies were analyzed in Western blot and a newly developed peptide ELISA covering the complete p16 protein. In addition, a Luminex-based multiplex assay was used for simultaneous detec- tion of antibodies directed against p16, p53, HPV16 E6 and HPV16 E7. In all entities, only low p16 antibody reactivity was observed. Epitope mapping revealed 2 predominant epitope regions of the p16 protein. No significant difference in p16 anti- body frequency (OR 5 0.9; 95% CI 5 0.6–1.3) and p53 antibody frequency (OR 5 0.6; 95% CI 5 0.3–1.2) was found between patients and healthy controls in the cervical cancer case control study. Antibodies against the HPV16 oncoproteins E6 and E7 were detected more frequently in cervical cancer patients when compared with healthy controls (E6 OR 5 27.8; 95% CI 5 11.1– 69.7, E7 OR 5 5.7; 95% CI 5 2.9–11.1). In conclusion, despite the strong expression of p16 and the observed induction of cellular immune responses, antibody reactivity against p16 was observed only at very low levels independent of the disease background. ' 2008 Wiley-Liss, Inc. Key words: cervical cancer; tumor immunity; HPV; p16; humoral immune response The tumor suppressor protein p16 is strongly and consistently overexpressed in human papillomavirus (HPV)-induced cancers and precancers. 1 The irregular expression of p16 is caused by the viral oncogene E7 that releases E2F from its binding to pRb. 2 Because expression of p16 in normal cells leads to irreversible ter- minal differentiation and eventually cell death, p16 expression is barely seen in normal tissue. 3 Usually, p16 expression is only found in terminally differentiated or senescent cells. 4 In contrast, in HPV-transformed cells, strong accumulation of p16 is observed in both the nucleus and the cytoplasm of all cells constituting a high-grade lesion or an invasive cancer. Based on these character- istics, p16 has been successfully used as a biomarker for cervical cancer. 5–7 Several cancer entities express proteins that are not, or only at very low levels, expressed in the respective normal tissue. When cancer cells are destroyed, either by host reactions, cytotoxic therapies or due to deficient nutrient supply, these irregularly expressed proteins may be released, processed by antigen present- ing cells and elicit immune responses. 8 These mechanisms have been shown for a number of self-antigens, such as NY-ESO1, Melan-A, hTERT and p53. One reason to study immune reactions against tumor-associated antigens is to explore new therapy options against malignant tumors. Therapeutic vaccination studies designed to enhance the host’s immune reaction against some of these tumor antigens have already been initiated. 9 Apart from that immune responses against tumor-associated antigens may have diagnostic or prognostic relevance. About 50% of cervical cancer patients show a humoral immune response against the HPV oncoproteins E6 or E7. 10 The analysis of cellular immune responses against E6 and E7 in animal experi- ments and few cervical cancer patients has shown that E6 and E7 are only poorly immunogenic. 11 Many attempts have been made to develop a therapeutic vaccine directed against E6 and E7, but despite great technical progress, promising clinical results are still lacking. It is therefore warranted to explore alternative immuno- logical targets in cervical cancer. Because of its unique expression pattern, p16 represents an interesting candidate for cancer vaccina- tion strategies in patients with HPV-induced tumors. Recently we have demonstrated that T cells isolated from cervi- cal cancer tissue are reactive against p16 and that PBMCs from healthy donors can be activated by p16 peptides to lyse cervical cancer cell lines. 12 Considering these findings, we wanted to explore whether patients with HPV-induced cancers develop a hu- moral immune response against p16. In our study, we analyzed the frequency of antibodies directed against p16 in sera obtained from cervical cancer patients, patients with HPV16-related oropharyngeal cancer, colorectal cancer patients and patients with various autoimmune diseases. Sera of a cervical cancer case control study were analyzed to identify differ- ences in frequencies of antibodies directed against p16, p53 and the viral proteins E6 and E7. Antibodies were detected using Western blot, bead-based recombinant protein serology and a newly developed peptide ELISA. Material and methods Patients and healthy controls In total, 919 sera derived from 774 patients were analyzed, including 242 consecutive sera from 94 cervical cancer patients obtained over a 12-month period after initial diagnosis (University of Leipzig), 35 sera from patients with newly diagnosed HPV16- induced oropharyngeal carcinomas (University of Prague), 64 pre- operative sera from patients with colorectal adenocarcinoma and Abbreviations: cDNA, complementary DNA; ELISA, enzyme linked immunosorbent assay; HNSCC, head and neck squamous cell carcinoma; HPV, human papilloma virus; HRP, horse radish peroxidase; Ig, immune globuline; OD, optical density; p16, p16 INK4a ; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PVDF membrane, polyvinylidenedifloride membrane; TBS, tris buffered saline. *Correspondence to: Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 220, 69120 Heidelberg, Germany. Fax: 149-6221-56-5983. E-mail: nwentzensen@t-online.de Received 8 May 2008; Accepted after revision 13 June 2008 DOI 10.1002/ijc.23837 Published online 10 September 2008 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 123, 2626–2631 (2008) ' 2008 Wiley-Liss, Inc. Publication of the International Union Against Cancer