Head and neck squamous cell carcinomas (HNSCCs) belong to a group of diverse tumors, which can be induced by infection with human papillomavirus (HPV) or tobacco and alcohol consumption. The viral etiology of HNSCC relates to better clinical outcomes reflecting a different immune system response. Here, we retrospectively analyzed 97 tissue samples from oral and oropharyngeal carcinomas associated and non-associated with HPV infection using multispectral fluorescent immunohistochemistry. To evaluate the immune cell infiltration in tumor and stroma compartments, we designed four panels of four to five antibodies. We detected more T lymphocytes in the stroma, compared to the tumor parenchyma. In HPV positive (HPV+) in comparison to HPV negative (HPV-) tumors, higher counts of CD3+CD4+, CD3+CD8+, PD1+CD4+, PD1+CD8+ T cells, and ICOS- Treg cells were detected while more ICOS+ Treg cells and CTLA4+CD4+ T cells were observed in HPV- than in HPV+ tumors. The results of the univariate and multivariate analyses confirmed the predominant impact of HPV status on prognosis. More importantly, the number of CD8+PD-1+ T cells was identified as an independent factor, influencing the overall and/or disease-specific survival of patients with oral cavity or oropharyngeal carcinomas.

• Keywords
• HPV, PD-1, head and neck cancer, survival,
• Publication type
• Journal Article MeSH

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV-) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV- tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers-cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)-was detected in HPV- patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.

• Keywords
• HPV, arginase 1, head and neck carcinoma, macrophages, prognosis,
• Publication type
• Journal Article MeSH

BACKGROUND: Better insights into the molecular changes involved in virus-associated and -independent head and neck cancer may advance our knowledge of HNC carcinogenesis and identify critical disease biomarkers. Here we aimed to characterize the expression profiles in a matched set of well-characterized HPV-dependent and HPV-independent tonsillar tumors and equivalent immortalized keratinocyte clones to define potential and clinically relevant biomarkers of HNC of different etiology. METHODS: Fresh frozen tonsillar cancer tissues were analyzed together with non-malignant tonsillar tissues and compared with cervical tumors and normal cervical tissues. Furthermore, relative miRNAs abundance levels of primary and immortalized human keratinocyte clones were evaluated. The global quantitation of miRNA gene abundance was performed using a TaqMan Low Density Array system. The confirmation of differentially expressed miRNAs was performed on a set of formalin-fixed paraffin-embedded tumor samples enriched for the tumor cell fraction by macrodissection. RESULTS: We defined 46 upregulated and 31 downregulated miRNAs characteristic for the HPV-positive tonsillar tumors and 42 upregulated miRNAs and 42 downregulated miRNAs characteristic for HPV-independent tumors. In comparison with the expression profiles in cervical tumors, we defined miR-141-3p, miR-15b-5p, miR-200a-3p, miR-302c-3p, and miR-9-5p as specific for HPV induced malignancies. MiR-335-5p, miR-579-3p, and miR-126-5p were shared by the expression profiles of HPV-positive tonsillar tumors and of the HPV immortalized keratinocyte clones, whereas miR-328-3p, miR-34c-3p, and miR-885-5p were shared by the miRNA profiles of HPV-negative tonsillar tumors and the HPV-negative keratinocytes. CONCLUSIONS: We identified the miRNAs characteristic for HPV-induced tumors and tonsillar tumors of different etiology, and the results were compared with those of the model system. Our report presents the basis for further investigations leading to the identification of clinically relevant diagnostic and/or therapeutic biomarkers for tumors of viral and non-viral etiology.

• Keywords
• Head and neck cancer, Human papillomavirus, Tonsillar tumor, miRNA,
• MeSH
• Principal Component Analysis MeSH
• Gene Regulatory Networks MeSH
• Papillomavirus Infections genetics   MeSH
• Keratinocytes cytology   pathology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• MicroRNAs genetics   MeSH
• Uterine Cervical Neoplasms genetics   virology   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Gene Expression Profiling MeSH
• Tonsillar Neoplasms genetics   virology   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• MicroRNAs MeSH

Human papillomavirus (HPV) infection is one of the most important etiologic causes of oropharyngeal head and neck squamous cell carcinoma (HNSCC). Patients with HPV-positive HNSCC were reported to have a better clinical outcome than patients with HPV-negative cancers. However, little is known about the possible causes of different clinical outcomes. In this study, we analyzed a detailed immune profile of tumor samples from HNSCC patients with respect to their HPV status. We analyzed the characteristics of immune cell infiltrates, including the frequency and distribution of antigen-presenting cells and naïve, regulatory and effector T cells and the cytokine and chemokine levels in tumor tissue. There was a profound difference in the extent and characteristics of intratumoral immune cell infiltrates in HNSCC patients based on their HPV status. In contrast to HPV-negative tumor tissues, HPV-positive tumor samples showed significantly higher numbers of infiltrating IFNγ+ CD8+ T lymphocytes, IL-17+ CD8+ T lymphocytes, myeloid dendritic cells and proinflammatory chemokines. Furthermore, HPV-positive tumors had significantly lower expression of Cox-2 mRNA and higher expression of PD1 mRNA compared to HPV-negative tumors. The presence of a high level of intratumoral immune cell infiltrates might play a crucial role in the significantly better response of HPV-positive patients to standard therapy and their favorable clinical outcome. Furthermore, characterization of the HNSCC immune profile might be a valuable prognostic tool in addition to HPV status and might help identify novel targets for therapeutic strategies, including cancer immunotherapy.

• Keywords
• CD8+ T lymphocytes, Cox-2, cyclooxygenase 2, HNSCC, HNSCC, head and neck squamous cell carcinoma, HPV, HPV, human papillomavirus, PD-1, PD-1, programmed cell death 1, PD-L1, programmed cell death-ligand 1, Tim-3, Tim-3, T cell immunoglobulin and mucin protein 3, Treg, regulatory T cell, mDC, myeloid dendritic cell, pDC, plasmacytoic dendritic cell,
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Human papillomaviruses (HPVs) have been proved as one of the etiological factors of oropharyngeal squamous cell carcinoma (OPSCC). Patients with tumors of viral etiology have a lower recurrence rate and better prognosis. OPSCC is linked to an alteration in the immune system. Only a limited number of studies have correlated both the immunological parameters and HPV status with patient prognosis. The aim of this study was to determine whether HPV infection and the immunological status influence patient prognosis individually or in concurrence. MATERIAL AND METHODS: Sixty patients with oral and oropharyngeal carcinomas were enrolled. They were divided into HPV-positive and HPV-negative groups based on the expression of HPV 16 E6 mRNA. Basic lymphocyte subpopulations were determined in the peripheral blood by means of flow cytometry. RESULTS: Significantly better disease-specific survival (DSS) was observed in patients with HPV-positive tumors. Nodal status, tumor grade, recurrence, and CD8+/Tregs ratio were identified as factors influencing DSS. A higher level of Tregs and a lower ratio of CD8/Tregs influenced overall survival (OS) independently of HPV status and age. Patients with HPV-positive tumors and high levels of Tregs survived significantly better than patients from the other groups. CONCLUSION: Better survival is associated with HPV positivity and elevated Tregs levels. Our data suggest that HPV infection and Tregs do not influence patient prognosis in concurrence.

• MeSH
• Survival Analysis MeSH
• Biomarkers blood   MeSH
• Killer Cells, Natural immunology   MeSH
• Demography MeSH
• Cyclin-Dependent Kinase Inhibitor p16 metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Oropharyngeal Neoplasms blood   epidemiology   immunology   virology   MeSH
• Papillomaviridae physiology   MeSH
• Prognosis MeSH
• Proportional Hazards Models MeSH
• Regression Analysis MeSH
• T-Lymphocytes, Regulatory immunology   MeSH
• Carcinoma, Squamous Cell blood   epidemiology   immunology   virology   MeSH
• Neoplasm Grading MeSH
• Age Factors MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• Cyclin-Dependent Kinase Inhibitor p16 MeSH