Human Papillomavirus antibodies and risk of subsequent head and neck cancer

Dr. Tim Waterboer
© T. Waterboer

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 OPC1(Figure). 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, ... , 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.

© A.R. Kreimer et. al., Kinetics of the Human Papillomavirus Type 16 E6 Antibody Response Prior to Oropharyngeal Cancer; J Natl Cancer Inst. 2017

Figure HPV-16 E6 anti-bodies as predictive bio-marker for oropharyngeal cancer (OPC). Top panel: Multiplex serology enables early OPC detection. Left panel: HPV-16 E6 antibody levels (median fluorescence intensities, MFI) by years from blood draw (left) to diagnosis (right) among OPC cases. (Kreimer et al., J Natl Cancer Inst. 2017)

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 (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.

© Dr. Daniele Viarisio

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 16 weeks of storage, the stabilized plates are comparable to freshly prepared ones. Ongoing experiments suggest that the storage time can be further extended.

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 (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.


1. 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

© K. Hufnagel

Figure 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

Serology of gastrointestinal bacteria and colorectal cancer risk

Dr. Julia Butt
© T. Waterboer

Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth leading cause of cancer death. Apart from well-known risk factors like age, male sex, family history of CRC or obesity, research of the recent years focused on the relation of CRC and the bacterial community residing in the gut. Studies addressing more specifically individual bacterial species identified Streptococcus gallolyticus subsp. gallolyticus (SGG) and Fusobacterium nucleatum (FN) of being associated with CRC. Several open questions remain, i.e. whether the bacterial infection is a consequence or even a cause of CRC development and consequently whether infection might be an indicator for presence of/risk of developing CRC.

The aim of this project is to evaluate the association of SGG and FN infection with CRC using multiplex serology (Figure) in retrospective, as well as prospective sero-epidemiological studies.

Multiplex serology assays measuring antibody responses against SGG and FN have been developed to analyze large sero-epidemiological studies in collaboration with various partners in- and outside DKFZ. While there was no association of antibody responses to FN with CRC, we found an association of antibody responses to SGG in a German case-control study. This finding was reproduced in a multicenter prospective study across Europe (European Prospective Investigation into Nutrition and Cancer (EPIC)). The prospective nature of the EPIC study showed that antibody responses to SGG were present up to 8 years prior to diagnosis indicating that these antibody responses might serve as early marker for risk of developing CRC.

Currently our ongoing research focusses on prospective CRC studies covering a time span of even more than 10 years before diagnosis. This analysis could help answer the question whether the bacterial infections are potential cause or consequence of tumor development. Furthermore, it is attempted to detect specifically immunoglobulin A against SGG and FN as a mucosal infection marker as this might be of importance in assessing CRC risk.


1. Butt J, Romero-Hernández B, Pérez-Gómez B et al.: „Association of Streptococcus gallolyticus subspecies gallolyticus with colorectal cancer: Serological evidence.“, Int J Cancer. 138(7):1670-9., 2016, doi: 10.1002/ijc.29914.

2. Butt J, Werner S, Willhauck-Fleckenstein M et al.: „Serology of Streptococcus gallolyticus subspecies gallolyticus and its association with colorectal cancer and precursors.“, Int J Cancer, 2017, 6. doi: 10.1002/ijc.30765. [Epub ahead of print].

3. Butt J, Jenab M, Willhauck-Fleckenstein M, et al.: “Prospective evaluation of antibody response to Streptococcus gallolyticus and risk of colorectal cancer”, submitted

© J. Butt

Figure Multiplex serology of gastrointestinal bacteria to assess association with CRC risk. Antibodies to SGG and FN are detected using multiplex serology in case-control, as well as prospective studies to analyze their possible association with risk of developing CRC.

Determination of epidemiological and genetic risk factors for HPV16 E6 seropositivity in the healthy population

Nicole Brenner
© T. Waterboer

Antibodies against the oncoprotein E6 of human papillomavirus type 16 (HPV16) are considered highly sensitive and specific biomarkers for HPV16-driven oropharyngeal cancer1,2. However, approximately 0.6 % of healthy controls in previous nested case-control studies were also observed to have antibodies against HPV16 E61,2,3. To evaluate a potential application of HPV16 E6 antibodies in a future serology-based screening setting for HPV16-driven oropharyngeal cancer, presence of antibodies against HPV16 E6 in the healthy population needs further investigation. Thus, the project aims at determining epidemiological risk factors for HPV16 E6 seropositivity in the healthy population.

In multiple large European studies HPV16 E6 antibodies were measured by Multiplex Serology4. Risk factors for HPV16 E6 seropositivity are assessed by classical sero-epidemiology and genetic epidemiology (HLA allele analysis, GWAS see Figure). The majority of the analyzed studies are population-based and will help to determine HPV16 E6 seroprevalence estimates stratified by gender and age in different European countries. In addition, HPV16 E6 antibodies will be further characterized comparing for example immunogenic epitopes in OPC patients vs. seemingly healthy adults or children. This analysis will provide insight into antibody characteristics and might be a potential additional criterion for OPC risk stratification.

1. A. Kreimer et al., Evaluation of human papillomavirus antibodies and risk of subsequent head and neck cancer, J. Clin. Oncol. 2013

2. A. Kreimer et al., Kinetics of the Human Papillomavirus Type 16 E6 Antibody Response Prior to Oropharyngeal Cancer, J. Natl. Cancer Inst. 2017

3. K. Lang Kuhs et al., Human Papillomavirus 16 E6 Antibodies in Individuals without Diagnosed Cancer: A Pooled Analysis, Cancer Epidemiol. Biomarkers Prev. 2015

4. Waterboer et al., Multiplex human papillomavirus serology based on in situ-purified glutathione s-transferase fusion proteins, Clin. Chem. 2005


© D. Chen et al., Genome-wide association study of HPV seropositivity; Hum. Mol. Genet. 2011

Figure In genome-wide association analyses (GWAS), manhattan plots are used to illustrate the significance of all tested SNPs in association with the trait of interest. For each SNP, the negative logarithm of the p-value (y-axis) is plotted by chromosome and position (x-axis). Chen et al. showed a genome-wide significant association (indicated by the black line) of SNP rs9357152 (chr. 6) with seropositivity against HPV type 8 L1 antigen.

to top