Nucleid Acids


Viral load of high-risk human papillomaviruses as reliable clinical predictor for the presence of cervical lesions



Markus Schmitt



Introduction

Infections with high-risk human papillomaviruses can cause malignant transformation of the human cervical epithelium and the development of cervical cancer. A rapid and precise diagnosis by conventional methods of the precancerous lesions remains difficult. The high sensitivity of currently used HPV tests leads to the identification of transient HPV infections. As a consequence, they lead also to over-treatment of patients, additional costs and considerable anxiety for women concerned.

Objective
To improve cervical cancer precursor screening by increasing the clinical specificity applying viral load assessment.

Methods
After developing an assay for genotyping and quantification of all known mucosal HPV types (BSGP5+/6+-PCR/MPG), we are analyzing large screening populations presenting the disease continuum in order to determine the value of HPV quantification for primary screening of cervical cancer precursors.


References
Schmitt, M., I. G. Bravo, P. J. Snijders, L. Gissmann, M. Pawlita, and T. Waterboer. 2006. Bead-based multiplex genotyping of human papillomaviruses. J Clin Microbiol 44:504-12
Schmitt, M., B. Dondog, T. Waterboer, and M. Pawlita. 2008. Homogeneous amplification of genital human alpha papillomaviruses by PCR using novel broad-spectrum GP5+ and GP6+ primers. J Clin Microbiol 46:1050-9.


HPV type specific RNA analysis in cervical and head and neck cancers


Gordana Halec



Introduction
HPV is a main cause of cervical but also other anogenital cancers like head and neck cancers. Biological activity of HPV is represented via two main oncogenes, E6 and E7, transcribed from viral mRNA in full and/or truncated form. Although the efficiency of E6 and E7 oncoproteins of all high-risk (HR) HPV types is similar, the type specific prevalence varies dramatically in related cancers.

Aim and methods
To verify HPV mRNA presence as a proof of biological viral activity, this project involves development of type specific and highly sensitive RT PCR method. All PCR products are subsequently being analyzed by modern Luminex technique that involves hybridization of designed type-specific probes to the polystyrene beads. Additional verification of biological activity is done by detection of cellular proteins in fresh frozen and formalin-fixed paraffin-embedded (FFPE) tumour tissues biopsies by immuno-histochemistry.


RNA transcript patterns of high-risk HPV types: a new diagnostic marker for cervical lesions


Daniela Höfler



Introduction
Cervical smears can be screened both for abnormalities using the cytological Papanicolaou (Pap) test and for the presence of HPV DNA and HPV E6/E7 full-length mRNA. However, all of the available tests show considerable drawbacks exhibiting either poor clinical sensitivity or specificity. For example, reduced clinical specificity leads to an over-treatment, additional costs and considerable anxiety for women concerned. Indeed, a specific identification of high-grade lesions is sufficient since those require surgical therapy contrary to early lesions that are likely to regress spontaneously.

Objective
The aim of this project is to develop a high-throughput test distinguishing severe lesion (malignant and high-grade cervical lesions) from mild lesions (low-grade and no intraepithelial cervical lesions) with a high sensitivity and specificity to improve current screening programs.

References
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.


Novel multiplex assay for an improved health monitoring of laboratory rodents


Daniela Höfler




Introduction
Microorganisms including DNA-viruses, RNA-viruses, bacteria and fungi can infect laboratory rodents. Infections lead to physiological changes affecting behaviour, growth rate, relative organ weight and immune response, resulting in an increased inter-individual variability. Thus, health monitoring is essential for reproducible experiments and allows minimising the number of animals required per experiment. However, standard methods used to date are expensive, time- and animal-consuming.

Objective
The aim of this project is to develop multiplex high-throughput procedures to replace conventional testing and to simplify and improve routine health monitoring.

Methods
The method is based on the specific identification of nucleic acids from rodent pathogens. Therefore, DNA/RNA are amplified in multiplex (RT-) PCR reactions and biotinylated amplicons are detected via hybridisation to specific oligonucleotide probes coupled to spectrally distinct sets of fluorescent beads (Luminex).


HPV16 and Oropharyngeal Squamous Cell Carcinomas


Dana Holzinger


Background

Central European cohorts of OPSCC containing HPV16 DNA are heterogeneous regarding viral oncogenic activity and clinical behavior. Therefore, we analyse HPV16 DNA-positive tumors for viral RNA expression patterns as seen in cervical carcinogenesis, and we evaluate the association of viral DNA and RNA status with clinical parameters. Further, HPV16 DNA-positive tumors differ regarding the expression of cellular protein patterns. Therefore we examine the cellular proteins p16INK4a, p53, Cyclin D1 and pRb on tissue microarrays (TMA) by immunohistochemistry (IHC) and we evaluate their clinical utility as surrogate markers for viral oncogenic activity. In addition, we search for genome-wide epigenetic alterations and for the expression of transcriptional regulators which might be specifically induced by the viral oncogenes and might be associated with the uniform biological and clinical behavior of the truly HPV-driven OPSCC.


Objective
The aim of this project is to characterize in detail HPV16-positive oropharyngeal squamous cell carcinomas (OPSCC).

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