Initiation, growth, recurrence, and metastasis of head and neck squamous cell carcinomas (HNSCC) have been related to the behavior of cancer stem cells (CSC) that can be identified by their aldehyde-dehydrogenase-isoform-1 (ALDH1) activity. We quantified and enriched ALDH1⁺ cells within HNSCC cell lines and subsequently characterized their phenotypical and functional properties like invasion capacity and epithelial-mesenchymal transition (EMT). Spheroid culture enriched CSC from five HNSCC cell lines by up to 5-fold. In spheroid-derived cells (SDC) and the parental monolayer-derived cell line ALDH1, CD44, CD24, E-Cadherin, α-SMA, and Vimentin expression was compared by flow-cytometry and immunofluorescence together with proliferation and cell cycle analysis. Invasion activity was evaluated by Matrigel assay and expression of stemness-related transcription factors (TF) Nanog, Oct3/4, Sox2 and EMT-related genes Snail1 and 2, and Twist by real-time PCR. All cell lines formed spheroids that could self-renew and be serially re-passaged. ALDH1 expression was significantly higher in SDC. ALDH1⁺ cells showed increased colony-formation. The proportion of cells with a putative CSC marker constellation of CD44⁺/CD24⁻ was highly variable (0.5% to 96%) in monolayer and spheroid cultures and overlapped in 0%–33% with the CD44⁺/CD24⁻/ALDH1⁺ cell subset. SDC had significantly higher invading activity. mRNA of the stemness-related genes Sox2, Nanog, and Oct3/4 was significantly increased in SDC of all cell lines. Twist was significantly increased in two while Snail2 showed a significant increase in one and a significant decrease in SDC of two cell lines. SDC had a higher G0 phase proportion, showed high-level expression of α-SMA and Vimentin, but significantly decreased E-Cadherin expression. HNSCC-lines harbor potential CSC, characterized by ALDH1 and stemness marker TF expression as well as properties like invasiveness, quiescence, and EMT. CSC can be enriched by anchorage-independent culture techniques, which may be important for the investigation of their contribution to therapy resistance, tumor recurrence and metastasis.

Human papillomavirus (HPV)–associated squamous cell carcinoma of the head and neck (SCCHN) seems to be a suitable target for cancer vaccination. HPV-encoded oncogenic proteins, such as E7, are promising tumor-specific antigens and are obligatory for tumor growth. Because few immunologic studies have analyzed the endogenous HPV-specific immune response in this subset of SCCHN patients, we studied T-cell frequencies against HPV-16 E7_11-20 or E7_86-93 in tumor-bearing, human leukocyte antigen (HLA)-A*0201⁺ SCCHN patients, whose tumors were either HPV-16⁺ or HPV-16⁻. In HPV-16⁺ SCCHN patients, frequencies of T cells against either peptide were significantly elevated (P < 0.005) compared with HPV-16⁻ patients or healthy volunteers. Tetramer⁺ T cells showed evidence of terminally differentiated phenotype (CD45RA⁺CCR7⁻) and an elevated level of CD107a staining for degranulation. Despite detectable expression of the restricting HLA class I allele, HLA-A*0201-E7_11-20– or HLA-A*0201-E7_86-93–specific CTL obtained by in vitro stimulation of healthy donor peripheral blood mononuclear cells only recognize a naturally HPV-16-transformed, HLA-A*0201⁺ SCCHN cell line after pretreatment with IFN-γ. This cell line had little or no expression of LMP2, TAP1, and tapasin, critical components of the HLA class I antigen-processing machinery, which were up-regulated by IFN-γ treatment. Immunohistochemistry of HPV-16⁺ SCCHN tumors showed that these antigen-processing machinery components are down-regulated in tumors in vivo compared with adjacent normal squamous epithelium. Thus, immunity to HPV-16 E7 is associated with the presence of HPV-16 infection and presentation of E7-derived peptides on SCCHN cells, which show evidence of immune escape. These findings support further development of E7-specific immunotherapy and strategies for up-regulation of antigen-processing machinery components in HPV-associated SCCHN.

Many environmental, host, and microbial characteristics have been recognized as risk factors for dissemination of extrapulmonary tuberculosis (EPTB). However, there are few population-based studies...

Consistent discrepancies in the p16/HPV-positivity have been observed in head and neck squamous cell carcinoma (HNSCC). It is therefore questionable, if all HPV⁺ and/or p16⁺ tested cancers are HPV-driven. Patients down-staged according to the HPV-dependant TNM are at risk for undertreatment and data in clinical trials may be skewed due to false patient inclusion. We performed a meta-analysis to classify clinical outcomes of the distinct subgroups with combined p16 and HPV detection. 25 out of 1677 publications fulfilled the inclusion criteria. The proportion of the subgroups was 35.6% for HPV⁺/p16⁺, 50.4% for HPV⁻/p16⁻, 6.7% for HPV⁻/p16⁺ and 7.3% for HPV⁺/P16⁻. The HPV⁺/p16⁺ subgroup had a significantly improved 5-year overall-survival (OS) and disease-free-survival in comparison to others both for HNSCC and oropharyngeal cancers. The 5-year OS of the HPV⁻/p16⁺ HNSCC was intermediate while HPV⁺/p16⁻ and HPV⁻/p16⁻ had the shortest survival outcomes. The clearly distinct survival of HPV⁻/p16⁺ cancers may characterize a new relevant HPV-independent subtype yet to be biologically characterized. The possibility also exists that in some HPV⁺/p16⁺ cancers HPV is an innocent bystander and p16 is independently positive. Therefore, in perspective, HPV-testing should distinguish between bystander HPV and truly HPV-driven cancers to avoid potential undertreatment in HPV⁺ but non-HPV-driven HNSCC.