HPV in Head and Neck Squamous Cell Carcinomas

Dana Holzinger, PhD
© M. Pawlita, dkfz.de

A subset of head and neck squamous cell carcinoma (HNSCC) is causally associated with human papilloma virus (HPV), notably HPV-type 16. The detection of the viral DNA in tumor tissue is not sufficient to determine truly HPV-driven HNSCC.(1) Therefore, we use different marker panels for the detection of HPV-driven HNSCC in large epidemiological studies, i.e. viral DNA (by Multiplex Papillomavirus Genotyping) (2), followed by HPV type-specific E6*I transcripts (by RT-PCR) and/or in combination with the cellular markers p16INK4a, pRb, Cyclin D1 and p53 (by immunohistochemistry).(3) If fresh-frozen tumor tissues are available, we use our HPV16-transformation specific RNA patterns for the detection of HPV16-driven HNSCC (Daniela Höfler).

Patients with HPV-driven HNSCC develop antibodies to the viral proteins, notably to the oncoproteins E6 and E7, but also to E1, E2 and E4. If serum of the patients is available, our HPV multiplex serology represents another powerful tool (Tim Waterboer) besides the molecular markers to determine HPV-driven HNSCC with very high sensitivity and specificity. (4, 5) By HPV serology we also determine HPV antibody kinetics during follow-up of the HPV-driven HNSCC patients and evaluate their prognostic value.

The HPV prevalence in HNSCC varies in populations of different geographical regions as well as it is dependent on the localization in the head and neck region, e.g. in oropharyngeal tumors (OPSCC) the HPV16 prevalence is highest and it is increasing since the last decades. To analyze these geographical as well as the temporal and anatomical differences, and the role of the other high-risk HPV-types in detail, we work closely together with national and international collaboration partners (e.g. HPV Ahead, Head and Neck 5000).(6) To analyze the HPV prevalence and the risk factors of HPV infections among the healthy population, we collected oral gargle samples within two large epidemiological cohorts from a younger population (18-35 years old), one from Heidelberg and one from Italy. (7)

In addition, we search for genome-wide epigenetic alterations (8, 9) and we study HPV integration among HPV16-driven OPSCC in detail (Elisabeth Schwarz), both might be associated with the uniform biological and clinical behavior of the truly HPV-driven OPSCC.

References

  1. Holzinger D, ... , Pawlita M, Bosch FX. Viral RNA patterns and high viral load reliably define oropharynx carcinomas with active HPV16 involvement. Cancer Res 2012;72: 4993-5003.
  2. Schmitt M, Dondog B, Waterboer T, Pawlita M. Homogeneous amplification of genital human alpha papillomaviruses by PCR using novel broad-spectrum GP5+ and GP6+ primers. J Clin Microbiol. 2008 Mar;46(3):1050-9. 
  3. Holzinger D, ..., Schmitt M, Hess J, Pawlita M, Bosch FX. Identification of oropharyngeal squamous cell carcinomas with active HPV16 involvement by immunohistochemical analysis of the retinoblastoma protein pathway. Int J Cancer 2013;133: 1389-99.
  4. Holzinger D, ..., Dyckhoff G, Boehm A, Del Mistro A, et al. Sensitivity and specificity of antibodies against HPV16 E6 and other early proteins for the detection of HPV16-driven oropharyngeal squamous cell carcinoma. Int J Cancer 2017;140: 2748-57.
  5. Broglie MA, ..., Pawlita M, Holzinger D. Evaluation of type-specific antibodies to high risk-human papillomavirus (HPV) proteins in patients with oropharyngeal cancer. Oral Oncol 2017;70: 43-50.
  6. Gheit T, Anantharaman D, Holzinger D, ..., Pawlita M, Ridder R, Rehm S, Bogers J, Maffini F, et al. Role of mucosal high-risk human papillomavirus types in head and neck cancers in central India. Int J Cancer 2017;141: 143-51.
  7. Holzinger D, ..., Pawlita M, Boscolo-Rizzo P. Prevalence and Determinants of Oral Human Papillomavirus Infection in 500 Young Adults from Italy. PLoS One 2017;12: e0170091.
  8. Kostareli E, ..., Holzinger D, Mucke O, Pawlita M, et al. Gene promoter methylation signature predicts survival of head and neck squamous cell carcinoma patients. Epigenetics 2016: 0.
  9. Kostareli E, Holzinger D, ..., Schmidt CR, Seiwert T, Dyckhoff G, et al. HPV-related methylation signature predicts survival in oropharyngeal squamous cell carcinomas. J Clin Invest 2013;123: 2488-501.

© D. Holzinger, dkfz.de

Figure 1  Examples of typical protein expression patterns in standard IHC. Healthy oropharyngeal mucosa of a non-tumor patient with low p16INK4a, high pRb and low (i.e. normal) Cyclin D1 and p53 expression levels (left panel). HPV—tumor with low p16INK4a, high pRb, high Cyclin D1 and high p53 expression levels (middle panel). RNA+ tumor with highp16INK4a, low pRb, low Cyclin D1 and low p53 expression levels (right panel). Original magnification 500-times for the details of tissue cores, 100-times for the entire tissue cores (3).

© dkfz.de

Figure 2  Heat-map of HPV16 E antibodies, as well as DNA and RNA status in HNSCC patients. Patients were clustered by seropositivity (dark blue) to early antigens (E6, E7, E1, E2) and grouped according to their molecular HPV status (RNA+ and DNA+) in the tumor tissue. Seronegativity is displayed in black. Clustering of HPV16 E6 seropositive and/or HPV RNA+ patients is depicted in higher magnification. (Figure from (4))

 

RNA Patterns of high-risk HPV types: a new triage marker in HPV-based cervical cancer precursor screening

Daniela Höfler, PhD
© M. Pawlita, dkfz.de

Cervical cancer precursor screening can be done either by cytological Papanicolaou (Pap) test or by HPV DNA testing. Testing for HPV DNA has a higher sensitivity for detecting advanced cervical lesions and therefore is increasingly considered as primary screening tool. However, the poor clinical specificity of DNA tests require triage tests in order to reduce over-treatment, additional costs and appreciable anxiety for women concerned. A specific identification of advanced lesions is sufficient since those require therapy contrary to early lesions that are likely to regress spontaneously.

The aim of this project is to develop and validate multiplex high-throughput reverse quantitative PCR assays that can be used as triage tests of HPV DNA positive women to sensitively and specifically identify advanced lesions and to improve cervical cancer precursor screening.

References

  1. Schmitt M, Dalstein V, Waterboer T, Clavel C, Gissmann L, Pawlita M. Diagnosing cervical cancer and high-grade precursors by HPV16 transcription patterns. Cancer Res 2010;70:249-56.
  2. 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.

The Role of HPV in Neck Squamous Cell Carcinoma from Unknown Primary Tumor

Lea Schroeder, PhD
© M. Pawlita, dkfz.de

Neck squamous cell carcinoma from unknown primary tumor (NSCCUP) is probably the most challenging head and neck cancer subset with regard to diagnostics and treatment. NSCCUP patients present with lymph node metastases, but no primary tumor can be detected by extensive diagnostic work-up. Aggressive treatment often induces severe side effects and reduces quality of life. Primary tumors of NSCCUP patients might be hidden in the oropharynx. A subset of oropharyngeal SCC is caused by HPV and is associated with superior survival. Thus, HPV might serve as a diagnostic and therapeutic biomarker for NSCCUP patients.

To study the role of HPV in NSCCUP, we performed a large retrospective NSCCUP study, comprising 180 patients from Germany, Spain and Italy. Major findings are an increase in the HPV prevalence with year of diagnosis and a strong survival benefit of patients with HPV-driven NSCCUP (see Figure). Detailed molecular analysis of HPV-driven NSCCUP revealed distinct DNA methylation patterns and absence of disruptive TP53 mutations, as described for HPV-driven oropharyngeal SCC.

Encouraged by these findings, we proceeded to assess the potential of HPV serology as a non-invasive tool for the detection of HPV-driven NSCCUP. We assessed HPV antibody patterns, which we had recently identified in patients with HPV-driven oropharyngeal SCC, in serum from NSCCUP patients from Leipzig in comparison with molecular HPV markers. These HPV antibody patterns are present in NSCCUP patients and are highly specific and sensitive for HPV-driven NSCCUP. Furthermore, HPV antibody patterns are associated with a better survival.

In conclusion, HPV appears to be a reliable diagnostic and prognostic biomarker in NSCCUP patients. Identification of HPV-driven NSCCUP can be of value for localization of the primary tumor and for therapeutic decision-making. Treatment de-intensification in patients with HPV-driven NSCCUP might hopefully increase treatment tolerability and quality of life.

 

References

  1. Boscolo-Rizzo P, Schroeder L, Romeo S, Pawlita M. The prevalence of human papillomavirus in squamous cell carcinoma of unknown primary site metastatic to neck lymph nodes: a systematic review. Clinical & experimental metastasis 2015
  2. Schroeder L, …, Pawlita M, Holzinger D. Human papillomavirus as prognostic marker with rising prevalence in neck squamous cell carcinoma of unknown primary: A retrospective multicentre study. European Journal of Cancer 2017;74: 73-81.
  3. Schroeder L, …, Pawlita M, Dietz A, Waterboer T, Holzinger D. Antibodies against human papillomaviruses as diagnostic and prognostic biomarker in patients with neck squamous cell carcinoma from unknown primary. International Journal of Cancer (under review).

© L. Schröder, dkfz.de

Figure Survival probability of patients included in the retrospective NSCCUP study compared for patients with HPV-driven (HPV+, blue) versus non-HPV-driven (HPV-, red) NSCCUP for a period of 10 years after diagnosis. Patients with HPV-driven NSCCUP show a significantly higher survival probability (p=0.002, log-rank test).

Characterization of Human Papillomavirus 16 (HPV16) E1C, a diagnostically highly relevant protein

Christy Susan Varghese
© M. Pawlita, dkfz.de

HPV16 E1C mRNA has been shown to be frequently expressed in high-grade precursor lesions of cervical cancer (1, 2). Therefore, HPV16 E1C mRNA can be a potential candidate for triage tests in cervical cancer screening program to distinguish CIN grades and cervical cancer in HPV16 DNA-positive women (1, 2).

However, the exact role of E1C in HPV16-transformed cells remains unclear. This project aims to elucidate the functions of E1C by identifying the postulated E1C protein in HPV16-transformed cells and its interaction partners, analyzing the functional effects of E1C overexpression and silencing.

To this end, E1C expression experiments have been performed in HEK and HeLa cell lines and E1C mRNA has been quantified by newly developed qPCR assays. In order to detect E1C protein, a monoclonal antibody capable of detecting recombinant E1C fusion proteins has been developed (Fig.1). With these newly generated tools, currently the effect of E1C expression on viral factors and cellular factors, for e.g. HPV16 oncogene transcripts is being analyzed.

References

  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, Dalstein V, Waterboer T, Clavel C, Gissmann L, Pawlita M. Diagnosing cervical cancer and high-grade precursors by HPV16 transcription patterns. Cancer Res 2010; 70:249-56.

© C. Varghese, dkfz.de

Figure Western blot depicting the detection of GST fused E1C protein (35kDa) and GFP fused E1C protein (36kDa) by E1C monoclonal antibody. GST-E1C and GFP-E1C proteins are recombinantly expressed in E.coli and HeLa cells respectively.

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