Christian Huebbers - Academia.edu (original) (raw)

Papers by Christian Huebbers

Research paper thumbnail of HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer

International Journal of Cancer, May 7, 2018

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-ce... more Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV 1 OPSCC metastasize faster than HPV 2 tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV 1 OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44 high /EpCAM high) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44 high /EpCAM low) CSC pool. This increase in migratory CSCs could also be confirmed in HPV 1 OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44 high /EpCAM low CSCs were validated by RT-qPCR and in situ hybridization on HPV16 1 OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16 1 OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV 1 and HPV 2 metastasis. As inhibition of this miRNA led to a markedly reduction of CD44 high /EpCAM low cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.

Research paper thumbnail of FGFR1 amplification and co-overexpression of c-MYC in oropharyngeal squamous cell carcinoma

Oral Oncology, Mar 1, 2016

Research paper thumbnail of Analysis of CpG promotor methylation status of the AKR1C3 gene in HPV16 positive and negative oropharyngeal squamous cell carcinoma

Abstract- und Posterband – 90. Jahresversammlung der Deutschen Gesellschaft für HNO-Heilkunde, Kopf- und Hals-Chirurgie e.V., Bonn – Digitalisierung in der HNO-Heilkunde, 2019

Research paper thumbnail of Aktuelles zu HPV-induzierten Oropharynxkarzinomen

Hno, Sep 30, 2011

Teilnahmemöglichkeiten-kostenfrei im Rahmen des jeweiligen Zeitschriftenabonnements-individuelle ... more Teilnahmemöglichkeiten-kostenfrei im Rahmen des jeweiligen Zeitschriftenabonnements-individuelle Teilnahme durch den Erwerb von CME.Tickets auf CME.springer.de Zertifizierung Diese Fortbildungseinheit ist mit 3 CME-Punkten zertifiziert von der Landesärztekammer Hessen und der Nord rheinischen Akademie für Ärztliche Fort-und Weiterbildung und damit auch für andere Ärzte kammern anerkennungsfähig. Hinweis für Leser aus Österreich Gemäß dem Diplom-Fortbildungs-Programm (DFP) der Österreichischen Ärztekammer werden die auf CME.springer.de erworbenen CME-Punkte hierfür 1:1 als fachspezifische Fortbildung anerkannt.

Research paper thumbnail of Abstract 2229: Targeted locus amplification based sequencing for mapping viral integration sites in human papillomavirus positive head and neck squamous cell carcinomas

Cancer Research

Background: Human papillomavirus (HPV) infections are the principal cause of cervical cancers, su... more Background: Human papillomavirus (HPV) infections are the principal cause of cervical cancers, subsets of anogenital and head and neck cancers (HNC). During persistent infection, viral DNA integration into the host genome may occur, which is suggested to affect carcinogenesis. One of the most critical limitations of currently used HPV integration detection techniques (PCR-based or NGS-based) is their application to FFPE material, because of DNA fragmentation. The aim of this study was to assess HPV integration in HPV-positive HNSCC cell lines and FFPE tissue comparing the new Targeted Locus Amplification (TLA) technology with previously used PCR technology (APOT/DIPS). Methods: Seven HPV-positive cell lines and FFPE material of 10 HPV-positive HNSCC were used for HPV integration detection by TLA, a proximity ligation-based next-generation sequencing technique. Crosslinked DNA is digested with restriction enzymes, and re-ligated into chimeric DNA molecules. For cell lines, a PCR base...

Research paper thumbnail of P16INK4A immunostaining patterns differentiate between the presence of high and low risk human papillomavirus types in head and neck dysplasias and papillomas

Introduction: Human papillomavirus (HPV) is a risk factor for head and neck mucosa lesions, inclu... more Introduction: Human papillomavirus (HPV) is a risk factor for head and neck mucosa lesions, including HPV-16 in tonsillar squamous cell carcinomas (TSCC) and HPV-6/11 in laryngeal papillomas. The aim of this study was to determine whether expression of the HPV surrogate marker p16INK4A could predict[for full text, please go to the a.m. URL]

Research paper thumbnail of Der Einfluss der viralen Onkoproteine E6 und E7 auf die miRNA-Expression in Keratinozyten

Einleitung: MiRNAs werden gewebespezifisch exprimiert und weisen zudem häufig im Tumorgewebe eine... more Einleitung: MiRNAs werden gewebespezifisch exprimiert und weisen zudem häufig im Tumorgewebe eine veränderte Expression im Vergleich zum Normalgewebe auf. MiRNAs fungieren als Modulatoren der Translation, indem gezielt 3'-untranslatierte mRNA-Abschnitte blockiert werden. In Voruntersuchungen[for full text, please go to the a.m. URL]

Research paper thumbnail of Die Bedeutung der Valosin-Containing Protein (VCP/p97)-Expression in Oropharynxkarzinomen

Einleitung: Die Typ II AAA-ATPase Valosin-Containing Protein (VCP/p97) wird ubiquitär im Kern und... more Einleitung: Die Typ II AAA-ATPase Valosin-Containing Protein (VCP/p97) wird ubiquitär im Kern und im Zytoplasma verschiedener Zelltypen exprimiert und trägt zu mehr als 1% der gesamten zellulären Proteine bei. VCP ist u.a. ein Schlüsselprotein der Proteindegradation, DNA-Reparatur und Autophagie. Studien an verschiedenen Malignomen haben gezeigt, dass eine VCP-Überexpression signifikant mit einer schlechteren Prognose korreliert. Ziel dieser Arbeit war es, die Bedeutung der VCP-Expression in Oropharynxkarzinomen zu untersuchen. Material und Methoden: 106 Patienten mit neu diagnostiziertem Oropharynxkarzinom wurden in die Studie eingeschlossen. Die Gewebeproben wurden im Rahmen einer Tumorbiopsie gewonnen. 87,1% der Patienten wiesen fortgeschrittene Tumorstadien auf (Stadium 3 und 4, AJCC). Für alle Patienten wurde zunächst der HPV-Status ermittelt (PCR-Nachweis sowie p16INK4a immunhistochemische Färbung). Die Expression von VCP wurde immunhistochemisch nachgewiesen. Ergebnisse: 47,2...

Research paper thumbnail of Vergleich genomischer Veränderungen zwischen HPV-positiven und HPV-negativen Oropharynxkarzinomen mittels Array-CGH

Einleitung: Patienten mit HPV-positiven Oropharynxkarzinomen haben eine bessere Prognose als Pati... more Einleitung: Patienten mit HPV-positiven Oropharynxkarzinomen haben eine bessere Prognose als Patienten mit HPV-negativen Karzinomen. Dieser Unterschied spiegelt unterschiedliche genetische Pathomechanismen bei den durch HPV verursachtne und den durch Noxen verursachten Karzinomen wieder. Die Methode der Array-CGH ermöglicht es, im Hochdurchsatz genomweite Veränderungen der DNA-Kopienzahl, entstanden durch den Zugewinn oder Verlust bestimmter Chromosomenregionen < 1 MB sensitiv zu erkennen. Die Kennzeichnung von systematisch deletierten oder amplifizierten Regionen in einer Gruppe von untersuchten Tumoren ermöglicht die Identifizierung von Genen, die bei der Tumorentstehung und -entwicklung eine Rolle spielen, sowie einen Vergleich der Unterschiede zwischen beiden Tumorentitäten. Methode: Wir untersuchten mittels Array-CGH (Comparative genomic hybridization) 4 HPV-positive und 4 HPV-negative Oropharynxkarzinome. Der HPV-Status wurde mittels PCR und FISH zum Nachweis von HPV-DNA un...

Research paper thumbnail of Comprehensive Analysis of VEGFR2 Expression in HPV-Positive and -Negative OPSCC Reveals Differing VEGFR2 Expression Patterns

Cancers, 2021

VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a de... more VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells (p = 0.012). However, with respect to blood vessel supply, the intensity of VEGF...

Research paper thumbnail of LAG-3, TIM-3 and VISTA Expression on Tumor-Infiltrating Lymphocytes in Oropharyngeal Squamous Cell Carcinoma—Potential Biomarkers for Targeted Therapy Concepts

International Journal of Molecular Sciences, 2020

Tumor growth and survival requires a particularly effective immunosuppressant tumor microenvironm... more Tumor growth and survival requires a particularly effective immunosuppressant tumor microenvironment (TME) to escape destruction by the immune system. While immunosuppressive checkpoint markers like programmed cell death 1 ligand (PD-L1) are already being targeted in clinical practice, lymphocyte-activation-protein 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA) inhibitors are currently under investigation in clinical trials. Reliable findings on the expression status of those immune checkpoint inhibitors on tumor-infiltrating lymphocytes (TILs) in the TME of oropharyngeal squamous cell carcinoma (OPSCC) are lacking. This work aims to describe the expression of LAG-3, TIM-3, and VISTA expression in the TME of OPSCC. We created a tissue microarray of paraffin-embedded tumor tissue of 241 OPSCC. Expression of the immune checkpoint protein LAG-3, TIM-3, and VISTA in OPSCC was evaluated using immunohistochemi...

Research paper thumbnail of PD-L1 Expression and a High Tumor Infiltrate of CD8+ Lymphocytes Predict Outcome in Patients with Oropharyngeal Squamous Cells Carcinoma

International Journal of Molecular Sciences, 2020

Carcinogenesis of human papillomavirus (HPV)-related (+) oropharyngeal squamous cell carcinoma (O... more Carcinogenesis of human papillomavirus (HPV)-related (+) oropharyngeal squamous cell carcinoma (OPSCC) differs from HPV-negative (–) OPSCC. HPV-related immune-escape-mechanism could be responsible for the development and progression of HPV+ tumors and an immunophenotype different from HPV– OPSCC is expected. The purpose of this study was to analyze the expression of programmed cell death protein 1 ligand 1 (PD-L1) and its prognostic relevance in relation to CD8+ tumor infiltrating lymphocytes (TILs) and the major histocompatibility complex (MHC) I expression in OPSCC. We quantified PD-L1 expression on tumor cells (TC) and macrophages and MHC I expression in association to CD8+ TILs by immunohistochemistry on tissue microarray derived from 171 HPV+/-OPSCC. HPV-status was determined by p16INK4a immunohistochemistry/HPV-DNA detection. Presence of CD8+ TILs, PD-L1 expression on TC, and a more frequent loss of MHC I in HPV+ compared to HPV- OPSCC was detected. A high amount of CD8+ TILs ...

Research paper thumbnail of Viral Integration Analysis Reveals Likely Common Clonal Origin of Bilateral HPV16-Positive, p16-Positive Tonsil Tumors

Archives of Clinical and Medical Case Reports, 2020

Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number ... more Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number of oropharyngeal squamous cell carcinoma (OPSCC), with infection rates currently rising at epidemic rates in the western world. Synchronous bilateral HPV+ tumors of both tonsils are a very rare event whose understanding, however, could provide important insights into virus-driven tumor development and progression and whether such integration events are of clonal origin. In this study we analyzed a single case of a bilateral tonsillar p16+ HPV+OPSCC. The viral integration status of the various tumor samples was determined by integration-specific PCR methods and sequencing, which identified viral insertion sites and affected host genes. Integration events were further confirmed by transcript analysis. Analysis of the tumors revealed common viral integration events involving the CD36 gene, as well as a unique event in the LAMA3 gene which resulted in loss of LAMA3 exon one in both tissues that had lost the complex viral LAMA3 integration event. In addition, there were several integration events into intergenic regions. This suggests a common origin but individual evolution of the tumors, supporting the single-clone hypothesis of bilateral tumor development. This hypothesis is further supported by the fact that the two cellular genes LAMA3 and

Research paper thumbnail of Downregulation of the α1- and β1-subunit of sGC in Arterial Smooth Muscle Cells of OPSCC Is HPV-Independent

Journal of Dental Research, 2018

The nitric oxide (NO)–sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an ... more The nitric oxide (NO)–sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an α and β subunit. NO binds to heme of the β1-subunit of sGC, activates the enzyme in the reduced heme iron state in vascular smooth muscle cells (VSMCs), and generates cGMP-inducing vasodilatation and suppression of VSMC proliferation. In the complex tumor milieu with higher levels of reactive oxygen species (ROS), sGC heme iron may become oxidized and insensitive to NO. To change sGC from an NO-insensitive to NO-sensitive state or NO-independent manner, protein expression of sGC in VSMC is required. Whether sGCα1β1 exists at the protein level in arterial VSMCs of oropharyngeal squamous cell carcinoma (OPSCC) is unknown. In addition, whether differences in the genetic profile between human papillomavirus (HPV)–positive and HPV-negative OPSCC contributes to the regulation of sGCα1β1 is unclear. Therefore, we compared the effects of HPV-positive and HPV-negative OPSCC on the expression of s...

Research paper thumbnail of Tumor-associated B cells and humoral immune response in head and neck squamous cell carcinoma

OncoImmunology, 2019

B lymphocytes are important players in immune responses to cancer. However, their composition and... more B lymphocytes are important players in immune responses to cancer. However, their composition and function in head and neck squamous cell carcinoma (HNSCC) has not been well described. Here, we analyzed B cell subsets in HNSCC (n = 38), non-cancerous mucosa (n = 14) and peripheral blood from HNSCC patients (n = 38) and healthy controls (n = 20) by flow cytometry. Intratumoral B cells contained high percentages of activated (CD86 +), antigen-presenting (CD86 + /CD21 −) and memory B cells (IgD − / CD27 +). T follicular helper cells (CD4 + /CXCR5 + /CD45RA − /CCR7 −) as key components of tertiary lymphoid structures and plasma cells made up high percentages of the lymphocyte infiltrate. Percentages of regulatory B cell varied depending on the regulatory phenotype. Analysis of humoral immune responses against 23 tumor-associated antigens (TAA) showed reactivity against at least one antigen in 56% of HNSCC patients. Reactivity was less frequent in human papillomavirus associated (HPV +) patients and healthy controls compared to HPV negative (HPV −) HNSCC. Likewise, patients with early stage HNSCC or MHC-I loss on tumor cells had low TAA responses. Patients with TAA responses showed CD4 + dominated T cell infiltration compared to mainly CD8 + T cells in tumors without detected TAA response. To summarize, our data demonstrates different immune infiltration patterns in relation to serological TAA response detection and the presence of B cell subpopulations in HNSCC that can engage in tumor promoting and antitumor activity. In view of increasing use of immunotherapeutic approaches, it will be important to include B cells into comprehensive phenotypic and functional analyses of tumor-associated lymphocytes.

Research paper thumbnail of The relevance of the lymph node ratio as predictor of prognosis is higher in HPV-negative than in HPV-positive oropharyngeal squamous cell carcinoma

Clinical Otolaryngology, 2017

Lymph node ratio (LNR) is an established predictor in different entities of carcinoma, including ... more Lymph node ratio (LNR) is an established predictor in different entities of carcinoma, including head and neck malignancies. In oropharyngeal squamous cell carcinoma (OPSCC), lymph node involvement differs between Human Papilloma Virus (HPV)-positive and negative tumours. Herein, we evaluate the impact of HPV association on the concept of LNR. Methods 88 surgically treated patients were included in this retrospective chart review. HPV-positive and negative OPSCC were evaluated for prediction of outcome by LNR separately. The endpoints were five-year overall survival (OS) and recurrence free survival (RFS).

Research paper thumbnail of Hpv and cancer of the oral cavity

Virulence, Jan 5, 2015

Increased awareness of human papillomavirus (HPV) as an etiological cause of head and neck squamo... more Increased awareness of human papillomavirus (HPV) as an etiological cause of head and neck squamous cell carcinoma has increased the interest in analysis of distinct oral sub-sites. It is currently under debate, whether HPV plays a role in the development of squamous cell carcinoma of the oral cavity (OSCC). The weakness in most published studies is the lack of performing different HPV detection tests combined with analysis for biological activity of the virus. In addition, different sub-sites of the oral cavity had been combined to a single entity, which retrospectively leads to a highly heterogeneous basis of data. In this review we mainly discuss the unclear role of HPV in OSCC development.

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

PLoS ONE, 2014

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the... more 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 p16 INK4A 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.

Research paper thumbnail of Prognostic Importance of HPV Association in Locally Advanced Inoperable Oropharyngeal Cancer

International Journal of Radiation Oncology*Biology*Physics, 2012

Conclusions: MFO-IMPT is clinically feasible for head and neck cancer with low acute toxicity and... more Conclusions: MFO-IMPT is clinically feasible for head and neck cancer with low acute toxicity and excellent clinical response. Proton therapy may represent the next step in the technological advancement of radiation therapy for head and neck cancer. Author Disclosure: S.J. Frank: E. Research Grant; NIH/NCI PO1 Proton Therapy. F. Honoraria; Elekta. G. Consultant; MD Anderson Physician Network. H. Travel Expenses; Elekta. J.D. Cox: None. M. Gillin: None. D.I. Rosenthal: None. A.S. Garden: None. K. Ang: None. R. Mohan: E. Research Grant; NIH/NCI PO1 Proton Therapy. M.B. Palmer: E. Research Grant; Philips Medical System. M. Amin: None. X.R. Zhu: None.

Research paper thumbnail of Abstract 2255: Diagnostic and prognostic value of oncogenic human papillomavirus in patients with carcinoma of unknown primary of the neck

Cancer Research, 2011

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective. To date, ther... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective. To date, there is no consensus on the optimal diagnostic and therapeutic strategy for patients with carcinoma of unknown primary (CUP) of the neck. These tumors are heterogeneous in their clinical and biological characteristics, and a pre-operative prognostic marker is desirable to optimize therapy and improve outcome and survival. Human papillomavirus (HPV) is an etiologic factor in a subgroup of head and neck squamous cell carcinomas and has been identified as a significant prognostic biomarker. We sought to determine if HPV also may add relevant information on the origin and prognosis of these tumors. Material and method. 47 patients (mean age 58.7 years; 40 men, 7 women) presenting with CUP of the neck between 1994 and 2008 in Cologne were examined by standard diagnostic procedures including radiological imaging of the head, neck, thorax and abdomen, positron emission tomography (PET), panendoscopy, curettage of the nasopharynx, bilateral tonsillectomy and blind probes of the base of tongue. All patients were surgically treated with neck dissection of the diseased neck as well as adjuvant chemoradiation. The mean follow up time was 34 months. Formaldehyde-fixed, paraffin-embedded tissue specimens of the 47 metastases were examined retrospectively by means of p16INK4A immunohistochemistry, HPV-specific PCR and fluorescence in situ hybridization. Results were correlated with clinical follow-up data. Results. Oncogenic HPV was present in 12/47 (26%) metastases (10 with HPV 16), which showed also p16INK4A overexpression. In 42% of CUP patients the primary tumor was discovered during follow-up. A significant correlation between HPV positivity and later detection of the primary tumor in the oropharynx was found (p=0.038). Moreover, patients with a p16INK4A- (13/47) or HPV-positive tumor had a more favorable overall 5-year survival rate then p16INK4A- and HPV-negative tumors (69% vs 33%, p=0.05; 65% vs 37%, p=0.093; respectively). Conclusion. HPV is present in a quarter of neck metastases of CUP patients and the presence of oncogenic HPV and p16INK4A expression can serve to locate the primary tumor in the oropharynx. In addition, both biomarkers are indicators of a favorable prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2255. doi:10.1158/1538-7445.AM2011-2255

Research paper thumbnail of HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer

International Journal of Cancer, May 7, 2018

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-ce... more Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV 1 OPSCC metastasize faster than HPV 2 tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV 1 OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44 high /EpCAM high) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44 high /EpCAM low) CSC pool. This increase in migratory CSCs could also be confirmed in HPV 1 OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44 high /EpCAM low CSCs were validated by RT-qPCR and in situ hybridization on HPV16 1 OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16 1 OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV 1 and HPV 2 metastasis. As inhibition of this miRNA led to a markedly reduction of CD44 high /EpCAM low cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.

Research paper thumbnail of FGFR1 amplification and co-overexpression of c-MYC in oropharyngeal squamous cell carcinoma

Oral Oncology, Mar 1, 2016

Research paper thumbnail of Analysis of CpG promotor methylation status of the AKR1C3 gene in HPV16 positive and negative oropharyngeal squamous cell carcinoma

Abstract- und Posterband – 90. Jahresversammlung der Deutschen Gesellschaft für HNO-Heilkunde, Kopf- und Hals-Chirurgie e.V., Bonn – Digitalisierung in der HNO-Heilkunde, 2019

Research paper thumbnail of Aktuelles zu HPV-induzierten Oropharynxkarzinomen

Hno, Sep 30, 2011

Teilnahmemöglichkeiten-kostenfrei im Rahmen des jeweiligen Zeitschriftenabonnements-individuelle ... more Teilnahmemöglichkeiten-kostenfrei im Rahmen des jeweiligen Zeitschriftenabonnements-individuelle Teilnahme durch den Erwerb von CME.Tickets auf CME.springer.de Zertifizierung Diese Fortbildungseinheit ist mit 3 CME-Punkten zertifiziert von der Landesärztekammer Hessen und der Nord rheinischen Akademie für Ärztliche Fort-und Weiterbildung und damit auch für andere Ärzte kammern anerkennungsfähig. Hinweis für Leser aus Österreich Gemäß dem Diplom-Fortbildungs-Programm (DFP) der Österreichischen Ärztekammer werden die auf CME.springer.de erworbenen CME-Punkte hierfür 1:1 als fachspezifische Fortbildung anerkannt.

Research paper thumbnail of Abstract 2229: Targeted locus amplification based sequencing for mapping viral integration sites in human papillomavirus positive head and neck squamous cell carcinomas

Cancer Research

Background: Human papillomavirus (HPV) infections are the principal cause of cervical cancers, su... more Background: Human papillomavirus (HPV) infections are the principal cause of cervical cancers, subsets of anogenital and head and neck cancers (HNC). During persistent infection, viral DNA integration into the host genome may occur, which is suggested to affect carcinogenesis. One of the most critical limitations of currently used HPV integration detection techniques (PCR-based or NGS-based) is their application to FFPE material, because of DNA fragmentation. The aim of this study was to assess HPV integration in HPV-positive HNSCC cell lines and FFPE tissue comparing the new Targeted Locus Amplification (TLA) technology with previously used PCR technology (APOT/DIPS). Methods: Seven HPV-positive cell lines and FFPE material of 10 HPV-positive HNSCC were used for HPV integration detection by TLA, a proximity ligation-based next-generation sequencing technique. Crosslinked DNA is digested with restriction enzymes, and re-ligated into chimeric DNA molecules. For cell lines, a PCR base...

Research paper thumbnail of P16INK4A immunostaining patterns differentiate between the presence of high and low risk human papillomavirus types in head and neck dysplasias and papillomas

Introduction: Human papillomavirus (HPV) is a risk factor for head and neck mucosa lesions, inclu... more Introduction: Human papillomavirus (HPV) is a risk factor for head and neck mucosa lesions, including HPV-16 in tonsillar squamous cell carcinomas (TSCC) and HPV-6/11 in laryngeal papillomas. The aim of this study was to determine whether expression of the HPV surrogate marker p16INK4A could predict[for full text, please go to the a.m. URL]

Research paper thumbnail of Der Einfluss der viralen Onkoproteine E6 und E7 auf die miRNA-Expression in Keratinozyten

Einleitung: MiRNAs werden gewebespezifisch exprimiert und weisen zudem häufig im Tumorgewebe eine... more Einleitung: MiRNAs werden gewebespezifisch exprimiert und weisen zudem häufig im Tumorgewebe eine veränderte Expression im Vergleich zum Normalgewebe auf. MiRNAs fungieren als Modulatoren der Translation, indem gezielt 3'-untranslatierte mRNA-Abschnitte blockiert werden. In Voruntersuchungen[for full text, please go to the a.m. URL]

Research paper thumbnail of Die Bedeutung der Valosin-Containing Protein (VCP/p97)-Expression in Oropharynxkarzinomen

Einleitung: Die Typ II AAA-ATPase Valosin-Containing Protein (VCP/p97) wird ubiquitär im Kern und... more Einleitung: Die Typ II AAA-ATPase Valosin-Containing Protein (VCP/p97) wird ubiquitär im Kern und im Zytoplasma verschiedener Zelltypen exprimiert und trägt zu mehr als 1% der gesamten zellulären Proteine bei. VCP ist u.a. ein Schlüsselprotein der Proteindegradation, DNA-Reparatur und Autophagie. Studien an verschiedenen Malignomen haben gezeigt, dass eine VCP-Überexpression signifikant mit einer schlechteren Prognose korreliert. Ziel dieser Arbeit war es, die Bedeutung der VCP-Expression in Oropharynxkarzinomen zu untersuchen. Material und Methoden: 106 Patienten mit neu diagnostiziertem Oropharynxkarzinom wurden in die Studie eingeschlossen. Die Gewebeproben wurden im Rahmen einer Tumorbiopsie gewonnen. 87,1% der Patienten wiesen fortgeschrittene Tumorstadien auf (Stadium 3 und 4, AJCC). Für alle Patienten wurde zunächst der HPV-Status ermittelt (PCR-Nachweis sowie p16INK4a immunhistochemische Färbung). Die Expression von VCP wurde immunhistochemisch nachgewiesen. Ergebnisse: 47,2...

Research paper thumbnail of Vergleich genomischer Veränderungen zwischen HPV-positiven und HPV-negativen Oropharynxkarzinomen mittels Array-CGH

Einleitung: Patienten mit HPV-positiven Oropharynxkarzinomen haben eine bessere Prognose als Pati... more Einleitung: Patienten mit HPV-positiven Oropharynxkarzinomen haben eine bessere Prognose als Patienten mit HPV-negativen Karzinomen. Dieser Unterschied spiegelt unterschiedliche genetische Pathomechanismen bei den durch HPV verursachtne und den durch Noxen verursachten Karzinomen wieder. Die Methode der Array-CGH ermöglicht es, im Hochdurchsatz genomweite Veränderungen der DNA-Kopienzahl, entstanden durch den Zugewinn oder Verlust bestimmter Chromosomenregionen < 1 MB sensitiv zu erkennen. Die Kennzeichnung von systematisch deletierten oder amplifizierten Regionen in einer Gruppe von untersuchten Tumoren ermöglicht die Identifizierung von Genen, die bei der Tumorentstehung und -entwicklung eine Rolle spielen, sowie einen Vergleich der Unterschiede zwischen beiden Tumorentitäten. Methode: Wir untersuchten mittels Array-CGH (Comparative genomic hybridization) 4 HPV-positive und 4 HPV-negative Oropharynxkarzinome. Der HPV-Status wurde mittels PCR und FISH zum Nachweis von HPV-DNA un...

Research paper thumbnail of Comprehensive Analysis of VEGFR2 Expression in HPV-Positive and -Negative OPSCC Reveals Differing VEGFR2 Expression Patterns

Cancers, 2021

VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a de... more VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells (p = 0.012). However, with respect to blood vessel supply, the intensity of VEGF...

Research paper thumbnail of LAG-3, TIM-3 and VISTA Expression on Tumor-Infiltrating Lymphocytes in Oropharyngeal Squamous Cell Carcinoma—Potential Biomarkers for Targeted Therapy Concepts

International Journal of Molecular Sciences, 2020

Tumor growth and survival requires a particularly effective immunosuppressant tumor microenvironm... more Tumor growth and survival requires a particularly effective immunosuppressant tumor microenvironment (TME) to escape destruction by the immune system. While immunosuppressive checkpoint markers like programmed cell death 1 ligand (PD-L1) are already being targeted in clinical practice, lymphocyte-activation-protein 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA) inhibitors are currently under investigation in clinical trials. Reliable findings on the expression status of those immune checkpoint inhibitors on tumor-infiltrating lymphocytes (TILs) in the TME of oropharyngeal squamous cell carcinoma (OPSCC) are lacking. This work aims to describe the expression of LAG-3, TIM-3, and VISTA expression in the TME of OPSCC. We created a tissue microarray of paraffin-embedded tumor tissue of 241 OPSCC. Expression of the immune checkpoint protein LAG-3, TIM-3, and VISTA in OPSCC was evaluated using immunohistochemi...

Research paper thumbnail of PD-L1 Expression and a High Tumor Infiltrate of CD8+ Lymphocytes Predict Outcome in Patients with Oropharyngeal Squamous Cells Carcinoma

International Journal of Molecular Sciences, 2020

Carcinogenesis of human papillomavirus (HPV)-related (+) oropharyngeal squamous cell carcinoma (O... more Carcinogenesis of human papillomavirus (HPV)-related (+) oropharyngeal squamous cell carcinoma (OPSCC) differs from HPV-negative (–) OPSCC. HPV-related immune-escape-mechanism could be responsible for the development and progression of HPV+ tumors and an immunophenotype different from HPV– OPSCC is expected. The purpose of this study was to analyze the expression of programmed cell death protein 1 ligand 1 (PD-L1) and its prognostic relevance in relation to CD8+ tumor infiltrating lymphocytes (TILs) and the major histocompatibility complex (MHC) I expression in OPSCC. We quantified PD-L1 expression on tumor cells (TC) and macrophages and MHC I expression in association to CD8+ TILs by immunohistochemistry on tissue microarray derived from 171 HPV+/-OPSCC. HPV-status was determined by p16INK4a immunohistochemistry/HPV-DNA detection. Presence of CD8+ TILs, PD-L1 expression on TC, and a more frequent loss of MHC I in HPV+ compared to HPV- OPSCC was detected. A high amount of CD8+ TILs ...

Research paper thumbnail of Viral Integration Analysis Reveals Likely Common Clonal Origin of Bilateral HPV16-Positive, p16-Positive Tonsil Tumors

Archives of Clinical and Medical Case Reports, 2020

Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number ... more Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number of oropharyngeal squamous cell carcinoma (OPSCC), with infection rates currently rising at epidemic rates in the western world. Synchronous bilateral HPV+ tumors of both tonsils are a very rare event whose understanding, however, could provide important insights into virus-driven tumor development and progression and whether such integration events are of clonal origin. In this study we analyzed a single case of a bilateral tonsillar p16+ HPV+OPSCC. The viral integration status of the various tumor samples was determined by integration-specific PCR methods and sequencing, which identified viral insertion sites and affected host genes. Integration events were further confirmed by transcript analysis. Analysis of the tumors revealed common viral integration events involving the CD36 gene, as well as a unique event in the LAMA3 gene which resulted in loss of LAMA3 exon one in both tissues that had lost the complex viral LAMA3 integration event. In addition, there were several integration events into intergenic regions. This suggests a common origin but individual evolution of the tumors, supporting the single-clone hypothesis of bilateral tumor development. This hypothesis is further supported by the fact that the two cellular genes LAMA3 and

Research paper thumbnail of Downregulation of the α1- and β1-subunit of sGC in Arterial Smooth Muscle Cells of OPSCC Is HPV-Independent

Journal of Dental Research, 2018

The nitric oxide (NO)–sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an ... more The nitric oxide (NO)–sensitive soluble guanylyl cyclase (sGC) is a heterodimeric enzyme with an α and β subunit. NO binds to heme of the β1-subunit of sGC, activates the enzyme in the reduced heme iron state in vascular smooth muscle cells (VSMCs), and generates cGMP-inducing vasodilatation and suppression of VSMC proliferation. In the complex tumor milieu with higher levels of reactive oxygen species (ROS), sGC heme iron may become oxidized and insensitive to NO. To change sGC from an NO-insensitive to NO-sensitive state or NO-independent manner, protein expression of sGC in VSMC is required. Whether sGCα1β1 exists at the protein level in arterial VSMCs of oropharyngeal squamous cell carcinoma (OPSCC) is unknown. In addition, whether differences in the genetic profile between human papillomavirus (HPV)–positive and HPV-negative OPSCC contributes to the regulation of sGCα1β1 is unclear. Therefore, we compared the effects of HPV-positive and HPV-negative OPSCC on the expression of s...

Research paper thumbnail of Tumor-associated B cells and humoral immune response in head and neck squamous cell carcinoma

OncoImmunology, 2019

B lymphocytes are important players in immune responses to cancer. However, their composition and... more B lymphocytes are important players in immune responses to cancer. However, their composition and function in head and neck squamous cell carcinoma (HNSCC) has not been well described. Here, we analyzed B cell subsets in HNSCC (n = 38), non-cancerous mucosa (n = 14) and peripheral blood from HNSCC patients (n = 38) and healthy controls (n = 20) by flow cytometry. Intratumoral B cells contained high percentages of activated (CD86 +), antigen-presenting (CD86 + /CD21 −) and memory B cells (IgD − / CD27 +). T follicular helper cells (CD4 + /CXCR5 + /CD45RA − /CCR7 −) as key components of tertiary lymphoid structures and plasma cells made up high percentages of the lymphocyte infiltrate. Percentages of regulatory B cell varied depending on the regulatory phenotype. Analysis of humoral immune responses against 23 tumor-associated antigens (TAA) showed reactivity against at least one antigen in 56% of HNSCC patients. Reactivity was less frequent in human papillomavirus associated (HPV +) patients and healthy controls compared to HPV negative (HPV −) HNSCC. Likewise, patients with early stage HNSCC or MHC-I loss on tumor cells had low TAA responses. Patients with TAA responses showed CD4 + dominated T cell infiltration compared to mainly CD8 + T cells in tumors without detected TAA response. To summarize, our data demonstrates different immune infiltration patterns in relation to serological TAA response detection and the presence of B cell subpopulations in HNSCC that can engage in tumor promoting and antitumor activity. In view of increasing use of immunotherapeutic approaches, it will be important to include B cells into comprehensive phenotypic and functional analyses of tumor-associated lymphocytes.

Research paper thumbnail of The relevance of the lymph node ratio as predictor of prognosis is higher in HPV-negative than in HPV-positive oropharyngeal squamous cell carcinoma

Clinical Otolaryngology, 2017

Lymph node ratio (LNR) is an established predictor in different entities of carcinoma, including ... more Lymph node ratio (LNR) is an established predictor in different entities of carcinoma, including head and neck malignancies. In oropharyngeal squamous cell carcinoma (OPSCC), lymph node involvement differs between Human Papilloma Virus (HPV)-positive and negative tumours. Herein, we evaluate the impact of HPV association on the concept of LNR. Methods 88 surgically treated patients were included in this retrospective chart review. HPV-positive and negative OPSCC were evaluated for prediction of outcome by LNR separately. The endpoints were five-year overall survival (OS) and recurrence free survival (RFS).

Research paper thumbnail of Hpv and cancer of the oral cavity

Virulence, Jan 5, 2015

Increased awareness of human papillomavirus (HPV) as an etiological cause of head and neck squamo... more Increased awareness of human papillomavirus (HPV) as an etiological cause of head and neck squamous cell carcinoma has increased the interest in analysis of distinct oral sub-sites. It is currently under debate, whether HPV plays a role in the development of squamous cell carcinoma of the oral cavity (OSCC). The weakness in most published studies is the lack of performing different HPV detection tests combined with analysis for biological activity of the virus. In addition, different sub-sites of the oral cavity had been combined to a single entity, which retrospectively leads to a highly heterogeneous basis of data. In this review we mainly discuss the unclear role of HPV in OSCC development.

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

PLoS ONE, 2014

Infection with high-risk human papillomavirus (HPV) type 16 is an independent risk factor for the... more 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 p16 INK4A 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.

Research paper thumbnail of Prognostic Importance of HPV Association in Locally Advanced Inoperable Oropharyngeal Cancer

International Journal of Radiation Oncology*Biology*Physics, 2012

Conclusions: MFO-IMPT is clinically feasible for head and neck cancer with low acute toxicity and... more Conclusions: MFO-IMPT is clinically feasible for head and neck cancer with low acute toxicity and excellent clinical response. Proton therapy may represent the next step in the technological advancement of radiation therapy for head and neck cancer. Author Disclosure: S.J. Frank: E. Research Grant; NIH/NCI PO1 Proton Therapy. F. Honoraria; Elekta. G. Consultant; MD Anderson Physician Network. H. Travel Expenses; Elekta. J.D. Cox: None. M. Gillin: None. D.I. Rosenthal: None. A.S. Garden: None. K. Ang: None. R. Mohan: E. Research Grant; NIH/NCI PO1 Proton Therapy. M.B. Palmer: E. Research Grant; Philips Medical System. M. Amin: None. X.R. Zhu: None.

Research paper thumbnail of Abstract 2255: Diagnostic and prognostic value of oncogenic human papillomavirus in patients with carcinoma of unknown primary of the neck

Cancer Research, 2011

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective. To date, ther... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective. To date, there is no consensus on the optimal diagnostic and therapeutic strategy for patients with carcinoma of unknown primary (CUP) of the neck. These tumors are heterogeneous in their clinical and biological characteristics, and a pre-operative prognostic marker is desirable to optimize therapy and improve outcome and survival. Human papillomavirus (HPV) is an etiologic factor in a subgroup of head and neck squamous cell carcinomas and has been identified as a significant prognostic biomarker. We sought to determine if HPV also may add relevant information on the origin and prognosis of these tumors. Material and method. 47 patients (mean age 58.7 years; 40 men, 7 women) presenting with CUP of the neck between 1994 and 2008 in Cologne were examined by standard diagnostic procedures including radiological imaging of the head, neck, thorax and abdomen, positron emission tomography (PET), panendoscopy, curettage of the nasopharynx, bilateral tonsillectomy and blind probes of the base of tongue. All patients were surgically treated with neck dissection of the diseased neck as well as adjuvant chemoradiation. The mean follow up time was 34 months. Formaldehyde-fixed, paraffin-embedded tissue specimens of the 47 metastases were examined retrospectively by means of p16INK4A immunohistochemistry, HPV-specific PCR and fluorescence in situ hybridization. Results were correlated with clinical follow-up data. Results. Oncogenic HPV was present in 12/47 (26%) metastases (10 with HPV 16), which showed also p16INK4A overexpression. In 42% of CUP patients the primary tumor was discovered during follow-up. A significant correlation between HPV positivity and later detection of the primary tumor in the oropharynx was found (p=0.038). Moreover, patients with a p16INK4A- (13/47) or HPV-positive tumor had a more favorable overall 5-year survival rate then p16INK4A- and HPV-negative tumors (69% vs 33%, p=0.05; 65% vs 37%, p=0.093; respectively). Conclusion. HPV is present in a quarter of neck metastases of CUP patients and the presence of oncogenic HPV and p16INK4A expression can serve to locate the primary tumor in the oropharynx. In addition, both biomarkers are indicators of a favorable prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2255. doi:10.1158/1538-7445.AM2011-2255