Nadine C. Olthof

Human papillomavirus reduces the prognostic value of nodal involvement in tonsillar squamous cell carcinomas.

Assessment of the prognostic value of nodal status in relation to human papillomavirus (HPV) status and the various treatment modalities in tonsillar squamous cell carcinomas (TSCC).

Radiosensitivity and effect of hypoxia in HPV positive head and neck cancer cells

BACKGROUND AND PURPOSE HPV associated Head and Neck Squamous Cell Carcinoma (HNSCC) represents a distinct subgroup of HNSCC characterized by a favorable prognosis and a distinct molecular biology. Previous data from the randomized DAHANCA 5 trial indicated that HPV positive tumors did not benefit from hypoxic modifications by Nimorazole during radiotherapy, whereas a significant benefit was observed in the HPV negative tumors. However, more studies have demonstrated equal frequencies of hypoxic tumors among HPV-positive and HPV-negative tumors. The aim of the present study was to determine radiosensitivity, the impact of hypoxia and the effect of Nimorazole in HPV positive and HPV negative cell lines. MATERIALS AND METHOD The used cell lines were: UDSCC2, UMSCC47 and UPCISCC90 (HPV positive) and FaDuDD, UTSCC33 and UTSCC5 (HPV negative). Cells were cultured under normoxic or hypoxic conditions, and gene expression levels of previously established hypoxia induced genes were assessed by qPCR. Cells were irradiated with various doses under normoxia, hypoxia or hypoxia +1mM Nimorazole, and the clonogenic survival was determined. RESULTS The HPV positive and HPV negative cell lines exhibited similar patterns of upregulation of hypoxia induced genes in response to hypoxia. The HPV positive cell lines were up to 2.4 times more radiation sensitive than HPV negative cell lines. However, all HPV positive cells displayed the same response to hypoxia in radiosensitivity, with an OER in the range 2.3-2.9, and a sensitizer effect of Nimorazole of 1.13-1.29, similar to HPV negative cells. CONCLUSIONS Although HPV positive cells had a markedly higher radiosensitivity compared to HPV negative cells, they displayed the same relative radioresistance under hypoxia and the same relative sensitizer effect of Nimorazole. The clinical observation that HPV positive patients do not seem to benefit from Nimorazole treatment is not due to inherent differences in hypoxia sensitivity or response to Nimorazole, but can be accounted for by the overall higher radiosensitivity of HPV positive cells.

Comprehensive Analysis of HPV16 Integration in OSCC Reveals No Significant Impact of Physical Status on Viral Oncogene and Virally Disrupted Human Gene Expression

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates with the level of viral gene transcription and influences the expression of disrupted host genes. We analyzed 75 patients with OSCC. HPV16-positivity was proven by p16INK4A immunohistochemistry, PCR and FISH. Viral integration was examined using DIPS- as well as APOT-PCR. Viral E2, E6 and E7 gene expression levels were quantified by quantitative reverse transcriptase (RT-q)PCR. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCCs. Viral copy numbers were assessed by qPCR in 73 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we did not find significant differences in the mean RNA expression of viral genes E2, E6 and E7 or the viral copy numbers per cell, nor did the RNA expression of the HPV-disrupted genes differ from either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC.

Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines.

HPV‐related HNSCC generally have a better prognosis than HPV‐negative HNSCC. However, a subgroup of HPV‐positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16‐positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD‐SCC‐2, UM‐SCC‐047, UM‐SCC‐104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT‐PCR and DIPS‐PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration‐specific staining patterns and signals indicating transcriptional activity using FISH. APOT‐ and DIPS‐PCR identified integration‐derived fusion products in six cell lines and only episomal products for UM‐SCC‐104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16‐positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

Next-generation treatment strategies for human papillomavirus-related head and neck squamous cell carcinoma: where do we go?

Oncogenic human papillomavirus (HPV) is currently recognised as a major risk factor for the development of head and neck squamous cell carcinomas (HNSCC). HPV is mostly detected in tumours arising from the oropharynx and more specifically from the tonsil. HPV‐related tumours display clinical and molecular characteristics that are distinct from HPV‐unrelated tumours, which are generally induced by alcohol and tobacco abuse. Detection of biologically active HPV in HNSCC has prognostic relevance, which warrants the separate classification of HPV‐induced tumours and is a prerequisite for further optimisation of treatment protocols for this distinct group. Current guidelines for the treatment of oropharyngeal squamous cell carcinoma (OPSCC) have not incorporated specific treatment modalities for HPV‐related tumours. The development of such treatment options is still in a preclinical phase or in early clinical trials. Recent data on treatment response of OPSCC have been obtained by retrospectively analysing HPV‐status and indicate that patients with HPV‐related tumours show a favourable prognosis, independent of the type of treatment. These patients may benefit from de‐intensified treatment, which should be assessed in prospective clinical trials. The development and future use of new antiviral and immunomodulatory therapeutics may be instrumental in this approach to improve survival rates and decrease disease‐and‐treatment‐related morbidity. In this review we will focus on present therapeutic HPV‐targeting strategies and discuss future directions for de‐intensified treatment of HPV‐positive HNSCC. Copyright

Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers

The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2‐binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)‐associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration.

Upregulation of AKR1C1 and AKR1C3 expression in OPSCC with integrated HPV16 and HPV-negative tumors is an indicator of poor prognosis: AKR1C expression and prognosis in HPV16-positive and -negative OPSCC

Different studies have shown that HPV16‐positive OPSCC can be subdivided based on integration status (integrated, episomal and mixed forms). Because we showed that integration neither affects the levels of viral genes, nor those of virally disrupted human genes, a genome‐wide screen was performed to identify human genes which expression is influenced by viral integration and have clinical relevance. Thirty‐three fresh‐frozen HPV‐16 positive OPSCC samples with known integration status were analyzed by mRNA expression profiling. Among the genes of interest, Aldo‐keto‐reductases 1C1 and 1C3 (AKR1C1, AKR1C3) were upregulated in tumors with viral integration. Additionally, 141 OPSCC, including 48 HPV‐positive cases, were used to validate protein expression by immunohistochemistry. Results were correlated with clinical and histopathological data. Non‐hierarchical clustering resulted in two main groups differing in mRNA expression patterns, which interestingly corresponded with viral integration status. In OPSCC with integrated viral DNA, often metabolic pathways were deregulated with frequent upregulation of AKR1C1 and AKR1C3 transcripts. Survival analysis of 141 additionally immunostained OPSCC showed unfavorable survival rates for tumors with upregulation of AKR1C1 or AKR1C3 (both p <0.0001), both in HPV‐positive (p ≤0.001) and ‐negative (p ≤0.017) tumors. OPSCC with integrated HPV16 show upregulation of AKR1C1 and AKR1C3 expression, which strongly correlates with poor survival rates. Also in HPV‐negative tumors, upregulation of these proteins correlates with unfavorable outcome. Deregulated AKR1C expression has also been observed in other tumors, making these genes promising candidates to indicate prognosis. In addition, the availability of inhibitors of these gene products may be utilized for drug treatment.

Abstract 828: Methylation status of HPV16 E2-binding sites identifies subtypes of HPV-associated oropharyngeal squamous cell carcinomas

Background: The viral E2 protein is a transcriptional repressor of the HPV oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2 binding sites (E2BSs) in the HPV upstream regulatory region (URR) may interfere with transcriptional repression of E6 and E7 by the E2 protein. We hypothesized that CpG methylation of the E2BS is found in a proportion of HPV16-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. Methods: Methylation of 10 CpGs within the URR, encompassing E2BSs 1, 2, 3 and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC. E2 status was analyzed by gene amplification and quantitative real-time RT-PCR. Viral integration was determined by integration-specific PCR methods. Results: Three subgroups with differential E2BSs3 and 4 methylation were identified: a) complete methylation (>80%) associated with presence of integrated HPV genomes with intact E2 gene, b) intermediate methylation levels (20-80%) with predominantly episomal HPV genomes with intact E2, and c) no methylation ( Conclusions: E2BSs3 and 4 methylation in oropharyngeal cancers with episomal HPV and integrated HPV with intact E2 gene might explain deregulated viral oncogene expression in the presence of E2. The methylation level appears to be of prognostic significance for patients with HPV-associated OPSCC. Citation Format: Ernst-Jan M. Speel, Miriam Reuschenbach, Christian U. Huebbers, Elena-Sophie Prigge, Justo L. Bermejo, Simon Kalteis, Simon Preuss, Jutta Kolligs, Nadine C. Olthof, Bernd Kremer, Steffen Wagner, Jens P. Klussmann, Svetlana Vinokurova, Magnus von Knebel Doeberitz. Methylation status of HPV16 E2-binding sites identifies subtypes of HPV-associated oropharyngeal squamous cell carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 828. doi:10.1158/1538-7445.AM2015-828

Abstract 4775: HPV16-E2/E4, -E6 and -E7 genes and HPV-disrupted human genes show large variation in expression levels in OSCC independent of the viral physical status.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the development of oropharyngeal squamous cell carcinomas (OSCC). However, it is unclear whether viral integration is an essential hallmark in the carcinogenic process of OSCC and whether HPV integration correlates to the level of viral gene transcription and influences the expression of disrupted genes. HPV16 integration sites were assessed in 75 patients with HPV16-DNA- and p16INK4A-positive OSCC using Detection of Integrated Papillomavirus Sequences (DIPS)- as well as Amplification of Papillomavirus Oncogene Transcripts (APOT)-PCR. Quantitative RT-PCR assays were carried out to determine the viral E2/E4, E6 and E7 gene expression levels. Expression levels of 7 human genes disrupted by the virus were extracted from mRNA expression profiling data of 32 OSCC. Nuclear localization of HPV16 was visualized using Fluorescence In Situ Hybridization (FISH) in 59 OSCC, and corresponding viral copy numbers were assessed by quantitative RT-PCR in 61 tumors. We identified 37 HPV16-human fusion products indicating viral integration in 29 (39%) OSCC. In the remaining tumors (61%) only episome-derived PCR products were detected. When comparing OSCC with or without an integration-derived fusion product, we neither found significant differences in the mean expression of the viral genes E2/E4, E6 and E7, the nuclear FISH staining patters nor the viral copy numbers per cell. The expression of the HPV-invaded genes also did not significantly differ when comparing the expression in the affected tumor with the expression of that gene in either group of OSCC. In conclusion, our data do not support the hypothesis that integration affects the levels of viral and/or HPV-disrupted human gene transcripts. Thus constitutive, rather than a high level, of expression of oncogene transcripts appears to be required in HPV-related OSCC. Citation Format: Jens P. Klussmann, Nadine C. Olthof, Christian U. Huebbers, Jutta Kolligs, Frans CS Ramaekers, Simon Preuss, Uta Drebber, Emily Vucic, Wan Lam, Bernd Kremer, Ernst Jan Speel. HPV16-E2/E4, -E6 and -E7 genes and HPV-disrupted human genes show large variation in expression levels in OSCC independent of the viral physical status. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4775. doi:10.1158/1538-7445.AM2013-4775