Laboratory of Molecular and Tumour Virology
RNDr. Ruth Tachezy, Ph.D.
Phone: +420 325 873 922, +420 221 951 724
The Laboratory of Molecular and Tumour Virology was established in 2015 by a group of researchers from the Institute of Hematology and Blood Transfusion who have joined the programme Cell Biology and Virology in the newly created research centre BIOCEV at Vestec. The Laboratory maintains close cooperation with the National Reference Laboratory (NRL) for Papillomaviruses and Polyomaviruses based in the Institute of Hematology and Blood Transfusion and is a platform which groups together researchers from the Faculty of Science. The projects are realized in close cooperation with the Laboratory of Immunotherapy and Laboratory of Virology of our Department.
The Laboratory of Molecular and Tumour Virology has long been involved in molecular epidemiology of papillomaviruses and polyomaviruses as well as in the study of newly discovered small DNA viruses. To detect the viral etiology in some diseases, both the direct detection and indirect detection by serology and immunohistochemistry have been used. The Laboratory team also conducts surveillance of some viral diseases which is of relevance to the implementation or modification of countrywide prevention screening and vaccination programmes. Some diseases associated with viral infection serve as model systems to identify diagnostic and therapeutic targets.
The focus is also on the study of animal viruses. In the past, the Laboratory team contributed to the identification of a range of novel animal papillomaviruses, and the experience gained in this field has been helpful in the diagnosis of animal infections which clinically manifest themselves as papillomavirus-associated diseases, i.e. papillomas, fibromas, fibropapillomas, or sarcomas (Rector et al. 2007, 2005, 2004; Tachezy et al. 2002a, 2002b; Jelínek et Tachezy 2005, Tan et al. 1994).
The surveillance of human papillomaviruses (HPV) was conducted for the purposes of the implementation of the screening programme for cervical cancer which is almost 100% associated with infection by high-risk (HR) types of HPV and implementation of countrywide vaccination against HPV. The type-specific prevalence of HPV infection in HPV-associated pre-malignancies and malignancies was analyzed in the population of the Czech Republic (CR) (Tachezy et al. 1999, 2011), the type-specific prevalence of HPV infection was monitored in the screening population of females of the CR (Tachezy et al. 2013), and the seroprevalence of HPV-specific antibodies was detected in both the healthy population (Hamšíková et al. 2013) and patients (Tachezy et al. 1999, 2003, 2006) in the CR. The analysis of the prevalence of HPV infection among the vaccinated females showed that many young women in the CR carry a wide range of HPV types. Eleven percent of the female population of the CR are incidentally and/or persistently infected with HR vaccine HPV types. Therefore, HPV vaccines are likely to be less effective in this population. HPV-specific antibodies were only detected in half of females, which indicates that HPV vaccination will also be highly beneficial to the women who are already sexually active (Hamšíková et al. 2017).
In cooperation with the Prague Voice Centre a study of the effect of HPV vaccine has been conducted in patients with recurrent laryngeal papillomatosis (RLP), a benign disease, caused by low-risk (LR) types of HPV, in particular by type 6. Although benign, this disease causes considerable morbidity and has the potential for conversion to malignancy. The currently available therapeutic options are limited. The tetravalent HPV vaccine comprising also the HPV6 and HPV11 antigens might offer hope for improved quality of life. Our previous studies have shown that patients with RLP often do not have antibodies against the causative types of HPV. These antibodies appear after several surgical attempts to remove papillomas (Tachezy et al. 1994). The vaccine is expected to trigger a robust antibody response and to induce neutralisation antibodies on the surface of the mucous membrane of the upper airways, which should slow down/inhibit the growth of HPV-associated lesions. Our phase IIIb clinical trial analyzes the effect of the tetravalent vaccine on recurrences in patients with RLP.
The team has also been involved in intensive study of possible involvement of HPV in vulvar, anal, bladder, and prostate cancer (Švec et al. 1999, 2003; Škapa et al. 2007; Tachezy et al. 2007, 2012; Hrbáček et al. 2011, 2013). At present, it has been participating in a project to optimize the diagnostic/therapeutic management of condylomata and vulvar precancerosis and cancer with the Department of Obstetrics and Gynaecology and the Institute of Pathology and Molecular Medicine of the 2nd Faculty of Medicine, Charles University and Motol University Hospital. Apart from viral infection markers, the association between the expression of the P16, P53, and SOX2 proteins, type of lesion and HPV presence has been studied.
Since 2001, a study of the etiology of HPV-associated head and neck tumours has been conducted in cooperation with the Department of Otorhinolaryngology and Head and Neck Surgery, 1st Faculty of Medicine, Charles University and Motol University Hospital, Prague. This study has already shown that oropharyngeal tumours in particular are up to 69%-associated with active infection by HR HPV. Our other studies have also identified risk factors for HPV-associated and non-HPV-associated tumours. The traditional prognostic markers, used so far in clinical practice, turned out to be less predictive in HPV-associated tumours (Tachezy et al. 2005, 2009; Rotnáglová et al. 2011; Košlabová et al. 2013; Vojtěchová et al. 2016). As the patients with HPV-associated tumours have a better response to therapy and better survival, the diagnosis of viral infection and consequent modification of the therapy need to be considered. A study, the results of which have been in progress for publication, focuses on the correlation between the etiology of oropharyngeal tumours and outcomes of therapeutic modalities. The outcomes suggest the possibilities for using modified therapeutic protocols in patients with HPV-associated tumours and will provide a basis for formulating therapeutic guidelines for these patients in the CR. The diagnosis of HPV-associated tumours needs to be made by a combination of two methods, PCR detection of HPV DNA and detection of P16 protein expression, to be conclusive of the viral etiology. Our studies will result in the recommendation for use of a combination of methods in a particular sequence in clinical practice.
Our recent research projects are dedicated to the study of miRNA expression profiles in head and neck cancer (HNC) in relation to the etiology and prognosis in an attempt to select the miRNAs suitable for use as diagnostic and treatment outcome markers in a specific group of HNC cases. Unlike cervical cancer which is almost always associated with HPV infection, HPV-positive and HPV-negative HNC provide a unique model for the study of cancer known to be triggered by different molecular mechanisms – either HPV dependent or HPV independent. Our recent studies compared the miRNA expression profiles in tonsillar tumours characterized in detail in terms of their etiology and tumoural HPV genome status. The miRNA expression profiles were compared with those in cervical cancer and a model system of human keratinocytes immortalized by HPV or human telomerase gene. The so-called HPV core miRNAs specific for tumours etiologically linked to HPV infection and miRNAs specific for HPV-associated tonsillar tumours and for tonsillar tumours of non-viral origin were identified. Further analyses are planned to detect HPV core miRNAs in other HPV-associated tumours of various anatomical locations and to perform function tests on selected miRNAs (Vojtěchová et al. 2016).
Another of our projects focuses on the elucidation of the mechanism of carcinogenesis of tumours associated with HPV infection where the virus is not present in an integrated but in an extrachromosomal form. Methylation and sequencing analyses have been performed of the regulatory region of the viral genome, in particular of the binding sites for the early E2 viral protein, which acts as a transcriptional regulation factor for viral oncoproteins E6 and E7.
The Laboratory research team has also been involved in immune profiling of peripheral blood immune cells and immune cells infiltrating HNC. Characterization of the immune response is an important prognostic tool in some carcinomas, which is even more relevant than the existing cancer classification system. The results of studies may be helpful in identifying novel targets for treatment strategies, including cancer immunotherapy, and in gaining a better understanding of the pathophysiology of virus-induced tumours. Our pilot results have shown that a lower ratio of CD8+/ CD4+ CD25+ T cells in the peripheral blood has a positive effect on the overall survival regardless of the HPV status. Patients with HPV-associated tumours have higher numbers of CD8+ T cells. In the tumour tissue of patients with tumours of viral etiology, higher numbers of IFN-gamma CD8+ lymphocytes, IL-17+ CD8+ lymphocytes, myeloid dendritic cells, and pro-inflammatory cytokines are present. HPV-positive tumours exhibit a lower expression of Cox-2 mRNA and a higher expression of PD1 mRNA (Lukešová et al. 2014; Partlová et al. 2015). At present, in cooperation with the teams of the Laboratory of Immunotherapy and of the Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, we intend to implement the methods for the detection, quantification, and phenotyping of immune cells in the peripheral blood and tumours using mass-flow cytometry CyTOF. We have also acquired a unique multimode multispectral PerkinElmer Mantra workstation which allows the detection, quantification, and phenotyping of cells in a fixed tissue (tissue cytometry or in situ cytometry).
Apart from clinical specimens /native tissues, we have also started the study of the HPV life cycle, including HPV infectivity, transmission, and ability to interact with the host cells and structures in vitro. As the HPV life cycle is tightly linked to the host cell differentiation, the virus is very difficult to grow in the cell culture. That is why systems were created for the production of recombinant papillomavirus particles such as virus-like particles (VLP) obtained by infecting insect cells with recombinant baculoviruses carrying genes for capsid proteins of the respective viruses or pseudovirions (PsVs) produced in HEK 293 cells containing viral DNA. Another model system suitable for use in the study of HPV are human keratinocytes immortalized by HPV DNA which mimics the early stage of viral infection.
Among the viruses studied by the Laboratory team is the newly discovered transfusion transmitted virus (TTV) linked to post-transfusion hepatitis of unknown origin. An epidemiological study has shown a considerable TTV infection rate in the population. TTV is a blood-borne virus which can also be transmitted sexually. However, its role in post-transfusion hepatitis has not been proven (Saláková et al. 2004, 2009). TTV infection is clearly more common and TTV viral load is higher in patients after allogeneic transplantation of hematopoietic cells than in the healthy population. Viral load kinetics corresponds to immune system reconstitution.
Last but not least, the Laboratory team has engaged in intensive study of human polyomaviruses, in particular in the epidemiology of human polyomaviruses HPyV6, HPyV7, TSPyV, HPyV9, MWPyV, KIPyV, and MCPyV discovered by molecular biological methods in 2007 through 2012. Apart from the ELISA test for the detection of antibodies to all known polyomaviruses, we have implemented viral DNA quantification tests. The team has also been involved in the study of MCPyV, which is the cause of Merkel cell skin cancer. MCPyV is the only human polyomavirus to be involved in cancer. Monoclonally integrated and replication defective form of the viral genome is found in up to 80-95% of Merkel cell skin cancer, a rare neuroendocrine tumour. TSPyV causes trichodysplasia spinulosa, an uncommon skin disease. The role of other polyomaviruses in human pathology is unknown (Šroller et al. 2014, 2016; Saláková et al. 2016). In cooperation with Dr Vojtěch Šroller from the Laboratory of Virology, the Laboratory team intends to continue the characterization of MCPyV from Merkel cell skin cancer and the study of its life cycle. Specific events in MCPyV life cycle probably play a crucial role in the pathogenesis of Merkel cell skin cancer, particularly the still little known events contributing to the development of tumours (site of productive virus replication, infection of permissive versus nonpermissive cells, sequence of events during the integration, host genome alterations, etc.).
In the polyomavirus group, more attention will be paid to BK virus. The planned study will focus on mapping the type specific prevalence of BK virus in the risk population of immunosuppressed and cancer patients in order to compare the risks from virus reactivation with new infection with another type of the virus in relation to hemorrhagic cystitis and nephropathy.