Comprehensive Analysis of VEGFR2 Expression in HPV-Positive and -Negative OPSCC Reveals Differing VEGFR2 Expression Patterns.

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 VEGFR2 staining differed only in HPV-positive OPSCC and was upregulated in the blood vessels of tumor-containing regions (p < 0.0001). These results may suggest different routes of VEGFR2 signaling depending on the HPV-status of the OPSCC. While in HPV-positive OPSCC, VEGFR2 might be associated with increased angiogenesis, in HPV-negative tumors, an autocrine loop might regulate tumor cell survival and invasion.

Tumor-associated B cells and humoral immune response in head and neck squamous cell carcinoma.

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.

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

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.

Valosin-containing protein (VCP/p97)-expression correlates with prognosis of HPV- negative oropharyngeal squamous cell carcinoma (OSCC).

Valosin-containing protein (VCP)/p97 has been shown to be associated with antiapoptotic function via activation of the nuclear factor-[Formula: see text]B (NF[Formula: see text]B) signaling pathway and with metastasizing of tumors in several studies. VCP is located on chromosome 9p13-p12, a region often deleted in oropharyngeal squamous cell carcinoma (OSCC). The clinical significance of VCP expression in OSCC however remains unclear. In this study, expression of VCP was determined in 106 patients (77 male (71.3%) and 31 female (28.7%); age-range: 34-79 years (mean age 57 years)) by immunohistochemistry and in a subset of 15 patients by quantitative PCR. HPV-DNA was detected by polymerase chain reaction and p16INK4a immunohistochemistry. The experimental findings were correlated with clinico-pathological data and survival parameters. 47.2% of all OSCC specimens were analyzed as negative or weak staining intensity for VCP. 52.8% of all specimens showed a high staining intensity for VCP. 73.1% of all patients were tested HPV-negative, 26.9% were HPV-positive. The 5-year disease-free and overall survival probabilities of all patients were 71.2% and 55.7%, respectively. No correlation could be found between HPV-status and VCP expression. VCP overexpression in HPV-negative patients was associated with significantly better 5-year disease-free survival (86.4% vs., 45.6%, p = 0.017). The level of VCP-intensity determined by immunohistochemistry could be an additional prognostic marker in HPV-negative OSCC. VCP expression seems not to correlate with the HPV-status.

Prevalence and risk factors for oral human papillomavirus infection in 129 women screened for cervical HPV infection.

BACKGROUND: Oncogenic human papillomaviruses (HPV) are known to be associated with carcinomas of the uterine cervix. Furthermore, current studies have shown that HPV-infection is also associated with a subtype of oropharyngeal cancers. In general, a sexual transmission of the viruses has been shown by numerous studies in the genital lesions. However, there are unknown factors regarding the prevalence and transmission of HPV in the oropharynx. The aim of this study was to evaluate HPV prevalence in the oropharynx in female participants with and without genital HPV infection. In addition, we analyzed risk factors for an oropharyngeal colonization with HPV in their sexual partners, too. METHODS: 129 Female participants were tested for presence of HPV-DNA by oral lavage, brush cytology of the tonsils and of the cervix. In addition, 15 male partners of these patients were included in the study. HPV-DNA was detected by PCR (polymerase chain reaction) amplification. For HPV-genotyping, PCR products were hybridized with type-specific digoxigenin-labeled oligonucleotide probes and discriminated into 14 high risk (HR) and 6 low risk (LR)-HPV types. The 129 female and 15 male participants were interviewed by a standardized questionnaire for socioeconomic details, drinking, smoking and sexual behaviours. RESULTS: 59 (45.7%) Female participants were negative for a genital HPV-infection. Of these women, 3 (5.1%) showed a positive HPV-PCR result (HR and LR) in the oropharynx. 70 (54.3%) Female participants were positive for a genital HPV infection. In this group, 4 (5.7%) had a positive HPV-detection (HR and LR) in the oral cavity and oropharynx. Female participants with cervical HPV-infection had no higher risk for HPV-detection in the oropharynx (not significant). The analysis of sexual risk factors revealed no specific risk factor for an oral HPV-infection. CONCLUSION: A correlation between cervical and oral colonization by HPV could not be demonstrated in our small cohort. Our limited data suggest that sexual transmission of HPV from the cervix uteri to the oropharynx is a rare and unlikely event.

p16 expression in carcinoma of unknown primary: diagnostic indicator and prognostic marker.

BACKGROUND: Carcinoma of unknown primary (CUP) of the neck are heterogeneous tumors in their clinical and biological characteristics, and a preoperative prognostic marker is desirable to optimize staging and therapy and to improve outcome and survival. For CUP syndrome, no optimized diagnostic and treatment strategy or biomarker have yet been determined. METHODS: Forty-seven patients presenting with CUP syndrome were analyzed after thorough standard diagnostic staging procedures. All patients were surgically treated with tonsillectomy, neck dissection of the diseased neck, as well as adjuvant chemoradiation. The tissue of lymph node metastases (and, if found, of the primary tumor) was analyzed regarding expression of p16, epidermal growth factor receptor (EGFR), and presence of human papillomavirus (HPV) DNA. RESULTS: In 39% of all cases (20 of 47), the primary cancer was found during diagnostic workup. If HPV DNA was detected in the neck lymph node metastasis, the primary cancer was significantly more frequently found in the oropharynx (p = .002). Patients with a p16-positive tumor had a significantly higher 5-year overall survival (OS; 33% vs 69%; p = .045, disease-free survival [DSF] 77% vs 89%; p = not significant [NS]). Patients with p16-positive neck metastasis and no detectable primary cancer had a better prognosis. Expression of EGFR in this series did not have a significant effect on prognosis. CONCLUSION: In patients presenting with CUP syndrome, p16 immunohistochemistry can serve to locate the primary cancer in the oropharynx. It is a positive prognostic indicator in patients with those heterogeneous cancers.

In vitro culturing of porcine tracheal mucosa as an ideal model for investigating the influence of drugs on human respiratory mucosa.

It has been previously shown that fresh mucosa from different mammals could serve as raw material for in vitro culturing with the differentiation of cilia, which are the most important morphological structures for the function of the mucociliary system. Increasing legal restrictions on the removal of human tissue and changing surgical techniques have led to a lack of fresh human mucosa for culturing. Most of the animals that have been used as donors up to now are genetically not very close to human beings and must all be sacrificed for such studies. We, therefore, established a modified system of culturing mucosa cells from the trachea of pigs, which is available as a regular by-product after slaughtering. With respect to the possibility of developing "beating" cilia, it could be shown that the speed of cell proliferation until adhesion to the coated culture dishes, the formation of conjunctions of cell clusters and the proliferation of cilia were comparable for porcine and human mucosa. Moreover, it could be demonstrated that the porcine cilia beat frequency of 7.57 +/- 1.39 Hz was comparable to the human mucosa cells beat frequency of 7.3 +/- 1.4 Hz and that this beat frequency was absolutely constant over the investigation time of 360 min. In order to prove whether the reaction to different drugs is comparable between the porcine and human cilia, we initially tested benzalkonium chloride, which is known to be toxic for human cells, followed by naphazoline, which we found in previous studies on human mucosa to be non-toxic. The results clearly showed that the functional and morphological reactions of the porcine ciliated cells to these substances were similar to the reaction we found in the in vitro cultured human mucosa.