Projects

Human Papillomavirus antibodies and risk of subsequent head and neck cancer

Dr. Tim Waterboer

© dkfz.de

Human papillomavirus type 16 (HPV16) infection is causally associated with a subset of oropharyngeal cancers (OPC), mainly tonsillar cancer. Similar to cervical cancer, antibodies to the E6 oncoprotein are strongly associated with HPV-driven OPC in patients after diagnosis. We currently investigate whether HPV antibodies are associated with OPC risk when measured prior to tumor diagnosis. Pre-diagnostic plasma samples from patients and controls enrolled in the EPIC (European Prospective Investigation into Cancer and Nutrition) cohort were analyzed for antibodies against multiple proteins of HPV16 and other HPV types, and Odds Ratios (ORs) and 95% Confidence Intervals (CI) were calculated, adjusting for potential confounders.
HPV16 E6 seropositivity was present in pre-diagnostic samples for 34.8% of patients with OPC and 0.6% of controls (OR 274, 95% CI 110 to 681). The increased risk of OPC among HPV16 E6 seropositive participants was independent of time between blood collection and diagnosis and was observed more than 10 years before diagnosis of OPC [1]. We are currently replicating these findings in other international cohort studies and investigate the potential of E6 antibody assays to serve as screening tools.

1. Kreimer AR, Johansson M, Waterboer T, Kaaks R, Chang-Claude J, Drogen D, Tjønneland A, Overvad K, Quirós JR, González CA, Sánchez MJ, Larrañaga N, Navarro C, Barricarte A, Travis RC, Khaw KT, Wareham N, Trichopoulou A, Lagiou P, Trichopoulos D, Peeters PH, Panico S, Masala G, Grioni S, Tumino R, Vineis P, Bueno-de-Mesquita HB, Laurell G, Hallmans G, Manjer J, Ekström J, Skeie G, Lund E, Weiderpass E, Ferrari P, Byrnes G, Romieu I, Riboli E, Hildesheim A, Boeing H, Pawlita M, Brennan P. Evaluation of human papillomavirus antibodies and risk of subsequent head and neck cancer. J Clin Oncol. 2013 Jul 20;31(21):2708-15.

Development of a diagnostic tool for early detection of HPV-driven oropharyngeal cancer

Dr. Daniele Viarisio
© T. Waterboer

High risk human papillomaviruses (HR-HPV) cause carcinomas of the uterine cervix, the vulva, the vagina, the anus, the penis and the oropharynx. Patients with HPV-induced tumors frequently develop antibodies against the viral oncoprotein E6. Especially patients with HPV-induced oropharyngeal or anal carcinoma develop these antibodies years before tumor diagnosis1,2. In HPV infection and replication without malignant transformation, E6 is also expressed, but rarely induces a measurable antibody response. Thus, antibodies against HR-HPV E6 could be used as a prognostic diagnostic marker for HPV-induced oropharyngeal and anal carcinomas. We are currently developing an ELISA-based screening assay to detect the presence of specific HR-HPV E6 antibodies in human sera. The ELISA system offers a robust, cost-effective and easy to handle method that can be used in routine hospital-based laboratories. Since the conformation of purified HPV E6 proteins is highly unstable and quickly loses antigenic properties when immobilized for long periods, we are currently focusing on the improvement of assay stability over time (see Figure).

    1. Kreimer AR, Johansson M, Waterboer T, …, Pawlita M, Brennan P. Evaluation of human papillomavirus antibodies and risk of subsequent head and neck cancer. J Clin Oncol. 2013 Jul 20;31(21):2708-15.
    2. Kreimer AR, Brennan P, Lang Kuhs KA, Waterboer T, … , Pawlita M, Johansson M. Human papillomavirus antibodies and future risk of anogenital cancer: a nested case-control study inthe European prospective investigation into cancer and nutrition study. J ClinOncol. 2015 Mar 10;33(8):877-84.

Figure: When preserved in the storing buffer that we are currently developing, the immobilized HPV E6 proteins do not show a measurable loss in antigenic reactivity: after 9 weeks of storage, the stabilized plates are comparable to freshly prepared ones. Ongoing experiments suggest that the storage time can be further extended.
© Dr. Daniele Viarisio

Bacterial infections associated with cancer: Whole-proteome microarrays for antigenic target identification

Katrin Hufnagel
© T. Waterboer

Published whole-proteome microarrays to systematically investigate the humoral immune response to bacterial infectious agents were so far based on individual cloning, expression and purification of hundreds or thousands of bacterial open reading frames. To overcome this limitation, and to maintain at the same time the advantages of slide-based microarrays, we have developed a novel method to generate microarrays representing an entire bacterial proteome, using Chlamydia trachomatis (Ct) as a complex model organism. It is based on a combination of multiple spotting technique and on-chip in situ protein expression. Expression constructs for each individual bacterial protein were generated by two successive PCRs using bacterial genomic DNA as a template, and proteins were expressed directly on microarray slides. We performed proteome immunoassays by incubating clinically characterized sera of Ct infected patients onto the whole-proteome arrays, and were able to provide evidence of antibody reactivity patterns that represent either Ct exposure markers, markers associated with persistent infections or Ct associated cervical cancer markers (see Figure). De novo identified antigens were validated in a large population-based cervical cancer case control study using high-throughput suspension bead array serology. The combination of high-density, low-throughput proteome immunoassays and low-density, high-throughput suspension bead array serology is a powerful platform for the discovery of exposure and disease-specific biomarkers and can be easily adapted to other microorganisms in all areas of infection research as well as in e.g. autoantibody screening and epitope mapping. We have already initiated the generation of whole-proteome microarrays for Helicobacter pylori and plan corresponding analyses for all eight human herpes viruses to investigate the role of these pathogens in the development of different types of human diseases.

Hufnagel, K. et al. Immunoprofiling of Chlamydia trachomatis using whole-proteome microarrays generated by on-chip in situ expression (under revision)

Results of Proteome Immunoassays (PIA) using pools of five sera

In total 897 Ct proteins were spotted on one array. Sera from uninfected women did not show positive signals with any of the Ct proteins, while all serum pools of Ct-infected women revealed positive signals. Comparison of the antibody reactivity patterns identified antigens which reacted with all Ct seropositive pools but also antigens which reacted only with serum pools from cancer patients. Some antigens reacted only with pools of sera from women of higher age, possibly indicating persistent Ct infections. Examples for antigens associated with general Ct infections, potentially persistent infections and cervical cancer are highlighted in green, yellow, and blue boxes, respectively
© K. Hufnagel

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