Projects

HPV is known for causing cervical cancer, but it also causes large proportions of other anogenital cancers (anal, vaginal, vulva, penis) and head and neck, especially oropharyngeal cancer (OPC) comprising mostly cancer of the palatine tonsils and the base of tongue.

OPC is rare, but shows strongly increasing incidence rates in many countries. The "classical" risk factors tobacco and alcohol have been superseded by HPV in the US and some European countries (e.g. UK, Sweden), where more than 70% of OPC are attributable to HPV, predominantly type 16 (HPV16). In these countries, the number of men with HPV-driven OPC (HPV-OPC) already exceeds the number of women with cervical cancer. This is a paradigm shift for HPV, and requires entirely new public health interventions, given low HPV vaccination rates, especially among boys. Although HPV-OPC is characterized by favorable survival, diagnosis typically occurs after the primary tumor has metastasized to the neck, often necessitating the use of multimodality treatment which may result in severe treatment-related morbidity, such as speech and swallowing dysfunction. To date, no precursor lesion for OPC has been identified making early detection challenging.

Serum antibodies against HPV16 proteins, especially the oncoprotein E6, are promising biomarkers for identifying individuals at risk for HPV-OPC development. HPV16 E6 antibodies have been shown to be present in approximately 90% of HPV-OPC patients at the time of diagnosis, with a specificity of 99%, and are detectable more than ten years prior to diagnosis. HPV16 E6 seroprevalence in the general population and in cancer-free controls is about 0.5%, making HPV16 E6 seropositivity a promising biomarker to develop an efficient screening strategy for early detection of HPV-OPC. The 5-year risk of a 60-year old HPV16 E6 seropositive male to develop HPV-OPC has been estimated at >14% (>27% after 10 years), reflecting a positive predictive value (PPV) exceeding that of established screening methods for e.g. cervical, breast or colorectal cancer.

 

<sup>1. Kreimer AR, Johansson M, Waterboer T, Kaaks R,…, 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.

2. Kreimer AR, Johansson M, Yanik EL, Katki HA,…, Michel A, Pawlita M, Brennan P, Waterboer T. Kinetics of the Human Papillomavirus Type 16 E6 Antibody Response Prior to Oropharyngeal Cancer. J Natl Cancer Inst. 2017 Aug 1;109(8):djx005.

3. Kreimer AR, Ferreiro-Iglesias A, Nygard M, Bender N,…, Freedman ND, Brennan P, Waterboer T, Johansson M. Timing of HPV16-E6 antibody seroconversion before OPSCC: findings from the HPVC3 consortium. Ann Oncol. 2019 Aug 1;30(8):1335-1343.

4. Hibbert J, Halec G, Baaken D, Waterboer T, Brenner N. Sensitivity and Specificity of Human Papillomavirus (HPV) 16 Early Antigen Serology for HPV-Driven Oropharyngeal Cancer: A Systematic Literature Review and Meta-Analysis. Cancers (Basel). 2021 Jun 16;13(12):3010.

5. Robbins HA, Ferreiro-Iglesias A, Waterboer T, Brenner N,…, Freedman ND, Kreimer AR, Johansson M, Brennan P. Absolute Risk of Oropharyngeal Cancer After an HPV16-E6 Serology Test and Potential Implications for Screening: Results From the Human Papillomavirus Cancer Cohort Consortium. J Clin Oncol. 2022 Nov 1;40(31):3613-3622.</sup>

The incidence of oropharyngeal cancer attributable to human papillomaviruses (HPV-OPC) has been rising constantly over the last decades. How oral HPV infections progress into cancer is poorly understood, and the lack of detectable precancerous lesions makes early detection of HPV-OPC difficult. Antibodies to HPV16 early proteins, especially E6, are strongly associated with HPV-OPC at the time of, but also prior to diagnosis^1,2. 

Within the Hamburg City Health Study (HCHS), a prospective, epidemiologic cohort study, we measured HPV serum antibodies in 5,000 participants enrolled 2016-2017 (men and women of the general population of the city of Hamburg aged 45-74 years) and identified eleven individuals with a high-risk antibody profile for the development of HPV-OPC. These individuals were invited to regular 6-monthly, non-invasive head and neck examinations in 2019 to diagnose HPV-OPC as early as possible. Within 1.3 years of clinical follow-up, three individuals were diagnosed with asymptomatic stage I HPV-OPC.³ The study is ongoing and since publication of the first three cases, two additional HPV-OPC cases were diagnosed resulting in a PPV of ~40% within 3.5 years of follow-up.

We are currently extending this screening approach to the next ~18,000 HCHS participants enrolled 2017-2025, with additional clinical examinations and a more comprehensive panel of biomarkers in order to describe the combination that maximizes the positive predictive value for HPV-OPC.⁴ HPV serology-based screening may facilitate early detection of HPV-OPC and thus reduce treatment-related morbidity. 

 

^{1. Kreimer AR, Johansson M, Waterboer T, Kaaks R,…, 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.}

^{2. Kreimer AR, Ferreiro-Iglesias A, Nygard M, Bender N,…, Freedman ND, Brennan P, Waterboer T, Johansson M. Timing of HPV16-E6 antibody seroconversion before OPSCC: findings from the HPVC3 consortium. Ann Oncol. 2019 Aug 1;30(8):1335-1343.}

^{3. Busch CJ, Hoffmann AS, Viarisio D, Becker BT…, Lang Kuhs K, Pawlita M, Waterboer T, Brenner N. Detection of stage I HPV-driven oropharyngeal cancer in asymptomatic individuals in the Hamburg City Health Study using HPV16 E6 serology - A proof-of-concept study. EClinicalMedicine. 2022 Sep 17;53:101659.}

^{4. Robbins HA, Ferreiro-Iglesias A, Waterboer T, Brenner N,…, Freedman ND, Kreimer AR, Johansson M, Brennan P. Absolute Risk of Oropharyngeal Cancer After an HPV16-E6 Serology Test and Potential Implications for Screening: Results From the Human Papillomavirus Cancer Cohort Consortium. J Clin Oncol. 2022 Nov 1;40(31):3613-3622.} 

Human papillomavirus (HPV) infections have in the past been mostly associated with cervical cancer. In recent years, however, many developed countries have seen a strong and steady increase in HPV-driven oropharyngeal cancer (OPC), now being the most common HPV-associated cancer in some countries. Even though HPV-driven OPC has a better prognosis than HPV-negative (i.e., mostly smoking-driven) OPC, treatment often comes with severe long-term side effects for the patient. This is in part explained by its tendency to form lymph node metastases early on, making aggressive treatment regimens necessary. To reduce treatment-related morbidity, early detection of HPV-OPC is of highest importance. Effective screening programs have shown a massive impact on cervical cancer incidence rates, but since, contrary to cervical cancer, no pre-cancerous lesions for HPV-driven OPC are known to date, screening is currently not feasible.

Previous work by our group has shown that antibodies against the HPV16 E6 oncoprotein are a very sensitive and specific biomarker for HPV-driven OPC, but since they can be present decades before diagnosis this is primarily useful to estimate the long-term risk of developing HPV-OPC. For personalized risk prediction, an additional marker that provides a more short-term indication of tumor growth is necessary. One candidate for this is circulating tumor DNA from blood plasma (ctDNA). Detection of HPV DNA in plasma has been utilized in therapy surveillance of HPV-driven OPC, but little is known about its characteristics before onset of disease. The goal of my project is therefore to detect HPV ctDNA and to assess its dynamics before onset of disease to assess its potential as a biomarker for use in HPV-OPC screening.

Cancer of the stomach accounted for almost 1 million new cancer cases in 2022 worldwide. The major risk factor for development of stomach cancer is an infection with the bacterium Helicobacter pylori (H. pylori), and antibiotic treatment of infected individuals was shown to be effective in gastric cancer prevention. Our research group has developed a multiplex serology assay¹ which is applied in multiple epidemiological studies with collaboration partners from all over the world. The results provide insights into the H. pylori prevalence in different populations/regions in the world,^2,3,4 its associations with cancer, ^5,6 also in other entities than the stomach,⁷ as well as biomarkers that may identify individuals with high risk of developing H. pylori-associated gastric cancer.⁸ The assay is under constant refinement to meet the current status of research, not only with respect to additional H. pylori antigens of interest,⁹ but also other biomarkers relevant for stomach cancer, including autoantibodies.⁶

 

^{1. Michel A, Waterboer T, Kist M, Pawlita M. Helicobacter multiplex serology. Helicobacter. 2009 Dec;14(6):525-35. doi: 10.1111/j.1523-5378.2009.00723.x.}

^{2. Yao P, Millwood I, Kartsonaki C, Mentzer AJ,…G, Li LM, Waterboer T, Yang L, Chen Z. Sero-prevalence of 19 infectious pathogens and associated factors among middle-aged and elderly Chinese adults: a cross-sectional study. BMJ Open. 2022 May 9;12(5):e058353. doi: 10.1136/bmjopen-2021-058353.}

^{3. Varga MG, Butt J, Blot WJ, Le Marchand L,…, Hildesheim A, Waterboer T, Pawlita M, Epplein M. Racial Differences in Helicobacter pylori CagA Sero-prevalence in a Consortium of Adult Cohorts in the United States. Cancer Epidemiology, Biomarkers, and Prevention, 2020;29(10):2084-92. doi: 10.1158/1055-9965.EPI-20-0525}

^{4. Wawro N, Amann U, Butt J, Meisinger C,…, Rathmann W, Kääb S, Waterboer T, Linseisen J. Helicobacter pylori Seropositivity: Prevalence, Associations, and the Impact on Incident Metabolic Diseases/Risk Factors in the Population-Based KORA Study. Frontiers in Public Health 7:96, 2019. doi: 10.3389/fpubh.2019.00096.}

^{5. Yao P, Kartsonaki C, Butt J, Jeske R,…, Waterboer T, Chen Z, Millwood IY, Yang L. Helicobacter pylori multiplex serology and risk of non-cardia and cardia gastric cancer: a case-cohort study and meta-analysis. Int J Epidemiol. 2023 Aug 2;52(4):1197-1208. doi: 10.1093/ije/dyad007.}

^{6. Butt J, Lehtinen M, Öhman H, Waterboer T, Epplein M. Association of Helicobacter pylori and Autoimmune Gastritis With Stomach Cancer in a Cohort of Young Finnish Women. Gastroenterology. 2022;163(1):305-307 e4.}

^{7. Butt J, Varga MG, Blot WJ, Teras L,…, Hildesheim A, Waterboer T, Pawlita M, Epplein M. Serologic Response to Helicobacter pylori Proteins Associated With Risk of Colorectal Cancer Among Diverse Populations in the United States. Gastroenterology. 2019;156(1):175–186.e2.} 

^{8. Epplein M, Butt J, Zhang Y, Hendrix LH,…, Waterboer T, Pawlita M, You WC, Pan KF. Validation of a blood biomarker for identification of individuals at high risk for gastric cancer. Cancer Epidemiology, Biomarkers, and Prevention 27(12):1472-79, 2018. doi: 10.1158/1055-9965.EPI-18-0582.}

^{9. Jeske R, Reininger D, Turgu B, Brauer A,…, Waterboer T, Butt J, Aragonés N, Hufnagel K. Development of Helicobacter pylori Whole-Proteome Arrays and Identification of Serologic Biomarkers for Noncardia Gastric Cancer in the MCC-Spain Study. Cancer Epidemiol Biomarkers Prev. 2020 Nov;29(11):2235-2242. doi: 10.1158/1055-9965.EPI-20-0348.}

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus infecting >90% of adults worldwide. While most carriers remain asymptomatic, EBV has been linked with the development of various malignancies, including nasopharyngeal carcinoma (NPC), Hodgkin lymphoma, Burkitt's lymphoma, and gastric cancer (GC). In addition, recent studies have strengthened the associations between EBV and neurodegenerative diseases, notably multiple sclerosis. 

In regions where EBV-driven cancers such as NPC are endemic, screening methods typically involve detection of antibodies against the viral capsid antigen (VCAp18) and Epstein-Barr nuclear antigen 1 (EBNA-1). To develop a better understanding of EBV antibody signatures in NPC, we have developed a panel of 23 EBV antigens, assessing both IgA and IgG antibodies. Notably, IgG antibodies targeting LF2 - a previously overlooked switch protein in the B95-8 strain of EBV - and BGLF2, a late lytic tegument protein, have demonstrated the ability to distinguish EBV-driven NPC cases from controls with >90% accuracy in both endemic and non-endemic regions (1, 2, 3). This panel is also used to identify distinct antibody signatures in EBV-associated gastric cancer which constitutes approximately 8% of all gastric cancer cases. Additionally, the panel will also be tested in patients with ocular cancers and multiple sclerosis to explore EBV involvement and secondary prevention options.

For causality assessment in etiological studies, identification of EBV-driven tumors relies on Epstein-Barr encoded RNA in situ hybridization (EBER-ISH), a technique that visualizes EBER1/2 RNA within the nuclei of cancerous cells. While EBER-ISH is crucial for confirming EBV presence, it does not provide quantitative data or prognostic information. To address this limitation, we are developing quantitative reverse transcription PCR assays (qRT-PCR) aimed at detecting and quantifying coding and non-coding EBV RNA transcripts which could provide etiological and prognostic information. We also target specific single nucleotide variants linked to higher-risk EBV subtypes in NPC, thereby complementing existing diagnostic methods and offering comprehensive insights into EBV-related oncogenesis.

 

^{1. Simon J, Brenner N, Reich S, Langseth H,…, Johansson M, Pring M, Nygard M, Waterboer T. Nasopharyngeal carcinoma patients from Norway show elevated Epstein-Barr virus IgA and IgG antibodies prior to diagnosis. Cancer Epidemiol. 2022 Apr;77:102117.}

^{2. Simon J, Schroeder L, Ingarfield K, Diehl S,…, Pring M, Butt J, Ness A, Waterboer T. Epstein-Barr virus and human papillomavirus serum antibodies define the viral status of nasopharyngeal carcinoma in a low endemic country. Int J Cancer. 2020 Jul 15;147(2):461-471.}

^{3. Simon J, Liu Z, Brenner N, Yu KJ,…, Proietti C, Doolan DL, Hildesheim A, Waterboer T. Validation of an Epstein-Barr virus antibody risk stratification signature for nasopharyngeal carcinoma by use of multiplex serology. J Clin Microbiol. 2020 Apr 23;58(5):e00077-20.}

The development of malignant lesions after exposure to carcinogenic pathogens typically requires incubation times between years and decades, opening a wide diagnostic window before clinical cancer diagnosis. The aim of secondary prevention of infection-associated cancers is to identify asymptomatic individuals with precancerous lesions or early stage cancer. Consequently, early detection can reduce cancer incidence and/or mortality, and post-treatment morbidity. Secondary prevention often entails screening for biomarkers in minimally invasive liquid biopsies such as oral gargle samples, swabs or blood. An ideal screening test does not result in overdiagnosis and overtreatment (high specificity), and makes at the same time sure that only very few people with the disease remain undetected (high sensitivity).

About 20% of all cancer cases worldwide are associated with infections. We develop quantitative assays detecting nucleic acids of different infectious agents, including HPV, EBV, STIs such as C. trachomatis, and H. pylori. This includes the establishment of automated nucleic acid extraction protocols from different liquid biopsies, and the amplification and detection by quantitative PCR, digital PCR or multiplex PCR combined with Luminex-based hybridization.

The following assays are currently developed and validated:

 

• Quantification of human papillomavirus (HPV) DNA in plasma and oral gargles by digital PCR as potential molecular biomarkers for early detection of HPV-driven oropharyngeal cancer
• Quantification of spliced HPV RNA patterns in routine cervical swabs by multiplex RT-qPCR as triage marker for HPV DNA positive women to identify the subset of women that require treatment
• Quantification of HPV DNA and RNA in different liquid biopsies in order to improve the detection of high grade anal precursor lesions with increased likelihood to progress to cancer
• Quantification of Epstein-Barr virus (EBV) DNA and RNA by multiplex RT-qPCR to investigate tumor etiology and EBV attributable fractions of multiple cancers in large epidemiological studies
• Detection of sexually transmitted infections (STI)² in urine by multiplex PCR/Luminex hybridization in order to improve prostate cancer screening
• Identification of non-pylori Helicobacter DNA in the human gastro-intestinal tract by multiplex PCR/Luminex hybridization to estimate its relevance in carcinogenesis³

 

^{1. Höfler D, Böhmer G, von Wasielewski R, Neumann H, Halec G, Holzinger D, Dondog B, Gissmann L, Pawlita M, Schmitt M. HPV16 RNA patterns defined by novel high-throughput RT-qPCR as triage marker in HPV-based cervical cancer precursor screening. Gynecol Oncol. 2015; 183(3):676-82.}

^{2. Schmitt M, Depuydt C, Stalpaert M, Pawlita M. Bead-based multiplex sexually transmitted infection profiling. J Infect. 2014; 69(2):123-33.}

^{3. Butt J, Schmitz M, Berkus B, Schmidt K, Höfler D. Validation of multiplex PCR and serology detecting Helicobacter species in mice. Microorganism 2023, 11(2): 249.}

The first study that was realized with the semi-automated Multiplex Serology workflow included ~40,000 samples from the China Kadoorie Biobank (CKB). The Multiplex Serology panel contained antigens from 17 different pathogens, including Human Papillomaviruses, several Herpesviruses, Human Polyomaviruses, Helicobacter pylori, Chlamydia trachomatis and many more. 

Results have demonstrated the robustness and reproducibility of the system throughout the entire duration of the study (~6 months) with CVs in the 5% range and no noticeable batch effects. 

The next project in our pipeline that is carried out with the semi-automated Multiplex Serology workflow includes 50,000 samples from the UK Biobank (UKB).

We are only at the beginning of understanding the epidemiology of the worldwide COVID-19 pandemic. Assessing SARS-CoV-2 seroprevalence in large population-based studies, the evolution of antibody development over time after natural infection and/or vaccination, secondary attack rates after household infection, or the importance of antibodies to non-structural proteins, is of high importance to better understand outcomes such as acute disease severity, re-infection, or the occurrence of Long COVID.

We developed a multiplex serology assay covering almost the entire SARS-CoV-2 proteome (SARS-CoV-2 serolomics). Seropositivity to structural proteins N and S1-RBD yielded sensitivities and specificities of both 100% in detecting COVID-19 positive patients 14 days after symptom onset in a German hospital-based study.¹ This assay is currently applied in multiple studies of national and international collaborations to answer the above-mentioned open questions. The multiplex ability of this assay moreover allows including antigens of different SARS-CoV-2 variants of concern, human "common cold" coronaviruses, and other pathogens including human herpesviruses that have been hypothesized to affect long-term outcomes of COVID-19 disease.

To allow for efficient high-throughput analyses of COVID-19 epidemiological studies, the SARS-CoV-2 serolomics assay will be integrated into a recently established automated workflow.

 

^{1. Butt J, Murugan R, Hippchen T, Olberg S,…, Schöttker B, Müller B, Merle U, Waterboer T. From Multiplex Serology to Serolomics-A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome. Viruses. 2021 Apr 24;13(5):749.}