HPV-associated oropharyngeal cancer: in search of surrogate biomarkers for early lesions.

The incidence of oropharyngeal cancer (OPSCC) has escalated in the past few decades; this has largely been triggered by high-risk human papillomavirus (HPV). Early cancer screening is needed for timely clinical intervention and may reduce mortality and morbidity, but the lack of knowledge about premalignant lesions for OPSCC poses a significant challenge to early detection. Biomarkers that identify individuals at high risk for OPSCC may act as surrogate markers for precancer but these are limited as only a few studies decipher the multistep progression from HPV infection to OPSCC development. Here, we summarize the current literature describing the multistep progression from oral HPV infection, persistence, and tumor development in the oropharynx. We also examine key challenges that hinder the identification of premalignant lesions in the oropharynx and discuss potential biomarkers for oropharyngeal precancer. Finally, we evaluate novel strategies to improve investigations of the biological process that drives oral HPV persistence and OPSCC, highlighting new developments in the establishment of a genetic progression model for HPV + OPSCC and in vivo models that mimic HPV + OPSCC pathogenesis.

Salivary microbiome changes distinguish response to chemoradiotherapy in patients with oral cancer.

BACKGROUND: Oral squamous cell carcinoma (SCC) is associated with oral microbial dysbiosis. In this unique study, we compared pre- to post-treatment salivary microbiome in patients with SCC by 16S rRNA gene sequencing and examined how microbiome changes correlated with the expression of an anti-microbial protein. RESULTS: Treatment of SCC was associated with a reduction in overall bacterial richness and diversity. There were significant changes in the microbial community structure, including a decrease in the abundance of Porphyromonaceae and Prevotellaceae and an increase in Lactobacillaceae. There were also significant changes in the microbial community structure before and after treatment with chemoradiotherapy, but not with surgery alone. In patients treated with chemoradiotherapy alone, several bacterial populations were differentially abundant between responders and non-responders before and after therapy. Microbiome changes were associated with a change in the expression of DMBT1, an anti-microbial protein in human saliva. Additionally, we found that salivary DMBT1, which increases after treatment, could serve as a post-treatment salivary biomarker that links to microbial changes. Specifically, post-treatment increases in human salivary DMBT1 correlated with increased abundance of Gemella spp., Pasteurellaceae spp., Lactobacillus spp., and Oribacterium spp. This is the first longitudinal study to investigate treatment-associated changes (chemoradiotherapy and surgery) in the oral microbiome in patients with SCC along with changes in expression of an anti-microbial protein in saliva. CONCLUSIONS: The composition of the oral microbiota may predict treatment responses; salivary DMBT1 may have a role in modulating the oral microbiome in patients with SCC. After completion of treatment, 6 months after diagnosis, patients had a less diverse and less rich oral microbiome. Leptotrichia was a highly prevalent bacteria genus associated with disease. Expression of DMBT1 was higher after treatment and associated with microbiome changes, the most prominent genus being Gemella Video Abstract.

Clinical, morphologic and molecular heterogeneity of HPV-associated oropharyngeal cancer.

The incidence of human papillomavirus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) is rising rapidly and has exceeded cervical cancer to become the most common HPV-induced cancer in developed countries. Since patients with HPV + OPSCC respond very favorably to standard aggressive treatment, the emphasis has changed to reducing treatment intensity. However, recent multi-center clinical trials failed to show non-inferiority of de-escalation strategies on a population basis, highlighting the need to select low-risk patients likely to respond to de-intensified treatments. In contrast, there is a substantial proportion of patients who develop recurrent disease despite aggressive therapy. This supports that HPV + OPSCC is not a homogeneous disease, but comprises distinct subtypes with clinical and biological variations. The overall goal for this review is to identify biomarkers for HPV + OPSCC that may be relevant for patient stratification for personalized treatment. We discuss HPV + OPSCC as a heterogeneous disease from multifaceted perspectives including clinical behavior, tumor morphology, and molecular phenotype. Molecular profiling from bulk tumors as well as single-cell sequencing data are discussed as potential driving factors of heterogeneity between tumor subgroups. Finally, we evaluate key challenges that may impede in-depth investigations of HPV + OPSCC heterogeneity and outline potential future directions, including a section on racial and ethnic differences.

Whole-genome CpG-resolution DNA Methylation Profiling of HNSCC Reveals Distinct Mechanisms of Carcinogenesis for Fine-scale HPV+ Cancer Subtypes.

DNA methylation is a vital early step in carcinogenesis. Most findings of aberrant DNA methylation in head and neck squamous cell carcinomas (HNSCC) are array based with limited coverage and resolution, and mainly explored by human papillomavirus (HPV) status, ignoring the high heterogeneity of this disease. In this study, we performed whole-genome bisulfite sequencing on a well-studied HNSCC cohort (n = 36) and investigated the methylation changes between fine-scaled HNSCC subtypes in relation to genomic instability, repetitive elements, gene expression, and key carcinogenic pathways. The previously observed hypermethylation phenotype in HPV-positive (HPV+) tumors compared with HPV-negative tumors was robustly present in the immune-strong (IMU) HPV+ subtype but absent in the highly keratinized (KRT) HPV+ subtype. Methylation levels of IMU tumors were significantly higher in repetitive elements, and methylation showed a significant correlation with genomic stability, consistent with the IMU subtype having more genomic stability and better prognosis. Expression quantitative trait methylation (cis-eQTM) analysis revealed extensive functionally-relevant differences, and differential methylation pathway analysis recapitulated gene expression pathway differences between subtypes. Consistent with their characteristics, KRT and HPV-negative tumors had high regulatory potential for multiple regulators of keratinocyte differentiation, which positively correlated with an expression-based keratinization score. Together, our findings revealed distinct mechanisms of carcinogenesis between subtypes in HPV+ HNSCC and uncovered previously ignored epigenomic differences and clinical implications, illustrating the importance of fine-scale subtype analysis in cancer. Significance: This study revealed that the previously observed hypermethylation of HPV(+) HNSCC is due solely to the IMU subtype, illustrating the importance of fine-scale subtype analysis in such a heterogeneous disease. Particularly, IMU has significantly higher methylation of transposable elements, which can be tested as a prognosis biomarker in future translational studies.

Oral Microbiome Community Composition in Head and Neck Squamous Cell Carcinoma.

The impact of the oral microbiome on head and neck cancer pathogenesis and outcomes requires further study. 16s rRNA was isolated and amplified from pre-treatment oral wash samples for 52 cases and 102 controls. The sequences were binned into operational taxonomic units (OTUs) at the genus level. Diversity metrics and significant associations between OTUs and case status were assessed. The samples were binned into community types using Dirichlet multinomial models, and survival outcomes were assessed by community type. Twelve OTUs from the phyla Firmicutes, Proteobacteria, and Acinetobacter were found to differ significantly between the cases and the controls. Beta-diversity was significantly higher between the cases than between the controls (p < 0.01). Two community types were identified based on the predominant sets of OTUs within our study population. The community type with a higher abundance of periodontitis-associated bacteria was more likely to be present in the cases (p < 0.01), in older patients (p < 0.01), and in smokers (p < 0.01). Significant differences between the cases and the controls in community type, beta-diversity, and OTUs indicate that the oral microbiome may play a role in HNSCC.

Increased Nerve Density Adversely Affects Outcome in Oral Cancer.

PURPOSE: Perineural invasion (PNI) in oral cavity squamous cell carcinoma (OSCC) is associated with poor survival. Because of the risk of recurrence, patients with PNI receive additional therapies after surgical resection. Mechanistic studies have shown that nerves in the tumor microenvironment promote aggressive tumor growth. Therefore, in this study, we evaluated whether nerve density (ND) influences tumor growth and patient survival. Moreover, we assessed the reliability of artificial intelligence (AI) in evaluating ND. EXPERIMENTAL DESIGN: To investigate whether increased ND in OSCC influences patient outcome, we performed survival analyses. Tissue sections of OSCC from 142 patients were stained with hematoxylin and eosin and IHC stains to detect nerves and tumor. ND within the tumor bulk and in the adjacent 2 mm was quantified; normalized ND (NND; bulk ND/adjacent ND) was calculated. The impact of ND on tumor growth was evaluated in chick chorioallantoic-dorsal root ganglia (CAM-DRG) and murine surgical denervation models. Cancer cells were grafted and tumor size quantified. Automated nerve detection, applying the Halo AI platform, was compared with manual assessment. RESULTS: Disease-specific survival decreased with higher intratumoral ND and NND in tongue SCC. Moreover, NND was associated with worst pattern-of-invasion and PNI. Increasing the number of DRG, in the CAM-DRG model, increased tumor size. Reduction of ND by denervation in a murine model decreased tumor growth. Automated and manual detection of nerves showed high concordance, with an F1 score of 0.977. CONCLUSIONS: High ND enhances tumor growth, and NND is an important prognostic factor that could influence treatment selection for aggressive OSCC. See related commentary by Hondermarck and Jiang, p. 2342.

Cytokines secreted by inflamed oral mucosa: implications for oral cancer progression.

The oral mucosa has an essential role in protecting against physical, microbial, and chemical harm. Compromise of this barrier triggers a wound healing response. Key events in this response such as immune infiltration, re-epithelialization, and stroma remodeling are coordinated by cytokines that promote cellular migration, invasion, and proliferation. Cytokine-mediated cellular invasion and migration are also essential features in cancer dissemination. Therefore, exploration of cytokines that regulate each stage of oral wound healing will provide insights about cytokines that are exploited by oral squamous cell carcinoma (SCC) to promote tumor development and progression. This will aid in identifying potential therapeutic targets to constrain SCC recurrence and increase patient survival. In this review, we discuss cytokines that overlap in oral wounds and SCC, emphasizing how these cytokines promote cancer progression.

The 3D's of Neural Phenotypes in Oral Cancer: Distance, Diameter, and Density.

Squamous cell carcinoma of the oral cavity (OSCC) is the most common type of head and neck cancer; survival is poor, and response to treatment varies. Metastasis or recurrence in the regional lymph nodes is associated with poor survival. Consequently, overt or occult spread to the lymph nodes is used to identify patients who will receive adjuvant radiation therapy. Perineural invasion and the diameter of nerves exhibiting perineural invasion have also been suggested to be of prognostic significance. The explosion of interest in cancer neuroscience in the last two decades has led to novel biological insights into interactions between nerves and tumor cells. However, the criteria for defining perineural invasion have lagged behind current knowledge. It is important to re-evaluate the concept of perineural invasion and identify other neural phenotypes in OSCC that can impact treatment selection and prognosis. In addition to perineural invasion, neural phenotypes that are of potential relevance to tumor progression include nerve-tumor distance, nerve diameter, and nerve density. This manuscript discusses the translational significance of recent mechanistic studies on the progression of oral cancer.

Influence of tumor cell-derived TGF-ß on macrophage phenotype and macrophage-mediated tumor cell invasion.

In oral squamous cell carcinoma (OSCC), macrophages are the most abundant immune cell type in the tumor microenvironment (TME). Macrophage infiltration is inversely proportional to prognosis and disease survival, particularly when these tumor-associated macrophages (TAM) assume an M2-like phenotype. This phenotype is determined by cues from the microenvironment, especially tumor cell-secreted molecules, and is associated with increased production of extracellular-matrix-degrading enzymes, angiogenic molecules and immunosuppressing cytokines. This study investigates, in vitro and in vivo, the relative contribution of OSCC cell-secreted transforming growth factor beta (TGF-ß) on the phenotype of macrophages and on macrophage-facilitated tumor invasion. TCGA database shows a positive correlation between high expression of TGFB1 and macrophage infiltrate in Head and neck squamous cell carcinoma (HNSCC). THP-1 derived-macrophages were exposed to the secretome of two OSCC cell lines using two strategies to block the effects of neoplastic cell-secreted TGF-ß: pre-treatment with a TGF-ß receptor type I kinase inhibitor (LY364947) and antibody-mediated depletion. RT-qPCR, ELISA and flow cytometry determined macrophage phenotype after exposure to conditioned medium (CM) from H-314 (TGF-ßhigh) or SCC-9 (TGF-ßlow) cell lines. The influence of TGF-ß on macrophage-mediated tumor cell invasion (myogel and CAM assays) and chemotaxis (Boyden chamber) was assessed using co-cultures of macrophages and OSCC cells in which macrophages were pre-conditioned with the secretome of OSCC cells in the presence and absence of LY364947. Blocking the effects of TGF-ß skewed macrophages to the M1 end of the phenotype by differential effects depending on the strategy for inhibiting the influence of TGF-ß and on the neoplastic cell secretome. In vitro and in vivo invasion of H-314 cell line was reduced by inhibiting TGFBR1 signaling in macrophages, whereas SCC-9 cell invasion was not affected. SCC-9/macrophage reciprocal chemotaxis were enhanced by inhibiting TGFBR1 signaling in macrophages, whereas only macrophage chemotaxis to H314 products was inhibited by inhibiting TGFBR1. In summary, blocking the effects of OSCC cell-secreted TGF-ß in macrophages attenuates M2-like phenotypical traits of macrophages and can impact invasion and chemotaxis of tumor cells differentially.

Spatial and Transcriptomic Analysis of Perineural Invasion in Oral Cancer.

PURPOSE: Perineural invasion (PNI), a common occurrence in oral squamous cell carcinomas, is associated with poor survival. Consequently, these tumors are treated aggressively. However, diagnostic criteria of PNI vary and its role as an independent predictor of prognosis has not been established. To address these knowledge gaps, we investigated spatial and transcriptomic profiles of PNI-positive and PNI-negative nerves. EXPERIMENTAL DESIGN: Tissue sections from 142 patients were stained with S100 and cytokeratin antibodies. Nerves were identified in two distinct areas: tumor bulk and margin. Nerve diameter and nerve-to-tumor distance were assessed; survival analyses were performed. Spatial transcriptomic analysis of nerves at varying distances from tumor was performed with NanoString GeoMx Digital Spatial Profiler Transcriptomic Atlas. RESULTS: PNI is an independent predictor of poor prognosis among patients with metastasis-free lymph nodes. Patients with close nerve-tumor distance have poor outcomes even if diagnosed as PNI negative using current criteria. Patients with large nerve(s) in the tumor bulk survive poorly, suggesting that even PNI-negative nerves facilitate tumor progression. Diagnostic criteria were supported by spatial transcriptomic analyses of >18,000 genes; nerves in proximity to cancer exhibit stress and growth response changes that diminish with increasing nerve-tumor distance. These findings were validated in vitro and in human tissue. CONCLUSIONS: This is the first study in human cancer with high-throughput gene expression analysis in nerves with striking correlations between transcriptomic profile and clinical outcomes. Our work illuminates nerve-cancer interactions suggesting that cancer-induced injury modulates neuritogenesis, and supports reclassification of PNI based on nerve-tumor distance rather than current subjective criteria.

Galanin mediates tumor-induced immunosuppression in head and neck squamous cell carcinoma.

PURPOSE: Galanin receptor 2 (GALR2) plays a significant role in the progression of head and neck squamous cell carcinomas (HNSCC). Since there is virtually no information on immunomodulation mediated by its ligand in the tumor microenvironment, we assessed the effects of galanin on peripheral blood mononuclear cells (PBMCs). METHODS: After verification of GALR2 expression and it activity in PBMCs we evaluated the effect of galanin and conditioned media from HNSCC cell lines silenced for galanin or antibody-depleted, on proliferation, apoptosis, cytokine expression and activation/differentiation of immune cells. RESULTS: We found that galanin alone and as a component of the HNSCC secretome decreased HNSCC cell proliferation and expression of pro-inflammatory cytokines (IFNγ, IL-12, IL-17A, IL-1α, IL-6 and TNF-α), whilst increasing apoptosis and expression of pro-tumoral cytokines/growth factors (IL-10, IL-4, PDGF and GM-CSF). T cell activation (using CD69 as activation marker) and anti-tumoral phenotypes in CD4+ T cells (Th1 and Th17) were found to be suppressed. In vivo, tumor growth was found to be increased in the presence of galanin-stimulated PBMCs. Data from The Cancer Genome Atlas (TCGA) revealed that high expression of galanin was associated with a reduced overall survival of patients with HNSCC. CONCLUSION: Our data indicate that galanin secreted by HNSCC cells exhibits immune-suppressive and pro-tumoral effects.

Phosphorylation of TRIP13 at Y56 induces radiation resistance but sensitizes head and neck cancer to cetuximab.

Radiation therapy, a mainstay of treatment for head and neck cancer, is not always curative due to the development of treatment resistance; additionally, multi-institutional trials have questioned the efficacy of concurrent radiation with cetuximab, the epidermal growth factor receptor (EGFR) inhibitor. We unraveled a mechanism for radiation resistance; that is, radiation induces EGFR, which phosphorylates TRIP13 (thyroid hormone receptor interactor 13) on tyrosine 56. Phosphorylated (phospho-)TRIP13 promotes non-homologous end joining (NHEJ) repair to induce radiation resistance. NHEJ is the main repair pathway for radiation-induced DNA damage. Tumors expressing high TRIP13 do not respond to radiation but are sensitive to cetuximab or cetuximab combined with radiation. Suppression of phosphorylation of TRIP13 at Y56 abrogates these effects. These findings show that EGFR-mediated phosphorylation of TRIP13 at Y56 is a vital mechanism of radiation resistance. Notably, TRIP13-pY56 could be used to predict the response to radiation or cetuximab and could be explored as an actionable target.

Cancer-associated keratinocytes: new members of the microenvironment in head and neck cancer.

The tumor microenvironment is a complex ecosystem of malignant and nonmalignant cells and extracellular proteins that work together to enhance tumor progression. We identified a mechanism in which adjacent nonmalignant epithelium enhances invasion of squamous cell carcinoma, thereby expanding the tumor microenvironment to include cancer-associated keratinocytes.

Nerve density in cancer: Less is better.

The density of nerves in cancer is emerging as a relevant clinical parameter for patient survival. Nerves in the tumor microenvironment have been associated with poor survival and recurrence, particularly if involved in perineural invasion. However, usually only a few nerves inside a tumor are affected by perineural invasion, while most nerves are not. Mechanistic studies have shown nerve-secreted factors promote tumor growth and invasion thereby making tumors more aggressive. Therefore, the overall number of nerves in the tumor microenvironment should be more representative of the nerve-tumor biological interaction than perineural invasion. This review summarizes the available clinical information about nerve density as a measure of clinical outcome in cancer and explores the mechanisms underlying nerve density in cancer, specifically, neurogenesis, axonogenesis, and neurotropism.

Squamous cell carcinoma subverts adjacent histologically normal epithelium to promote lateral invasion.

Recurrent and new tumors, attributed in part to lateral invasion, are frequent in squamous cell carcinomas and lead to poor survival. We identified a mechanism by which cancer subverts adjacent histologically normal epithelium to enable small clusters of cancer cells to burrow undetected under adjacent histologically normal epithelium. We show that suppression of DMBT1 within cancer promotes aggressive invasion and metastasis in vivo and is associated with metastasis in patients. Cancer cells via TGFß1 and TNFα also suppress DMBT1 in adjacent histologically normal epithelium, thereby subverting it to promote invasion of a small population of tumor cells. The sufficiency of DMBT1 in this process is demonstrated by significantly higher satellite tumor nests in Dmbt1-/- compared with wild-type mice. Moreover, in patients, invasion of small tumor nests under adjacent histologically normal epithelium is associated with increased risk for recurrence and shorter disease-free survival. This study demonstrates a crucial role of adjacent histologically normal epithelium in invasion and its important role in the tumor microenvironment and opens new possibilities for therapeutic strategies that reduce tumor recurrence.

5-Hydroxymethylation highlights the heterogeneity in keratinization and cell junctions in head and neck cancers.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide, with human papillomavirus (HPV)-related HNSCC rising to concerning levels. Extensive clinical, genetic and epigenetic differences exist between HPV-associated HNSCC and HPV-negative HNSCC, which is often linked to tobacco use. However, 5-hydroxymethylation (5hmC), an oxidative derivative of DNA methylation and its heterogeneity among HNSCC subtypes, has not been studied. RESULTS: We characterized genome-wide 5hmC profiles in HNSCC by HPV status and subtype in 18 HPV(+) and 18 HPV(-) well-characterized tumors. Results showed significant genome-wide hyper-5hmC in HPV(-) tumors, with both promoter and enhancer 5hmC able to distinguish meaningful tumor subgroups. We identified specific genes whose differential expression by HPV status is driven by differential hydroxymethylation. CDKN2A (p16), used as a key biomarker for HPV status, exhibited the most extensive hyper-5hmC in HPV(+) tumors, while HPV(-) tumors showed hyper-5hmC in CDH13, TIMP2, MMP2 and other cancer-related genes. Among the previously reported two HPV(+) subtypes, IMU (stronger immune response) and KRT (more keratinization), the IMU subtype revealed hyper-5hmC and up-regulation of genes in cell migration, and hypo-5hmC with down-regulation in keratinization and cell junctions. We experimentally validated our key prediction of higher secreted and intracellular protein levels of the invasion gene MMP2 in HPV(-) oral cavity cell lines. CONCLUSION: Our results implicate 5hmC in driving differences in keratinization, cell junctions and other cancer-related processes among tumor subtypes. We conclude that 5hmC levels are critical for defining tumor characteristics and potentially used to define clinically meaningful cancer patient subgroups.

Roadmap for the Emerging Field of Cancer Neuroscience.

Mounting evidence indicates that the nervous system plays a central role in cancer pathogenesis. In turn, cancers and cancer therapies can alter nervous system form and function. This Commentary seeks to describe the burgeoning field of "cancer neuroscience" and encourage multidisciplinary collaboration for the study of cancer-nervous system interactions.

Radiation resistance in head and neck squamous cell carcinoma: dire need for an appropriate sensitizer.

Radiation is a significant treatment for patients with head and neck cancer. Despite advances to improve treatment, many tumors acquire radiation resistance resulting in poor survival. Radiation kills cancer cells by inducing DNA double-strand breaks. Therefore, radiation resistance is enhanced by efficient repair of damaged DNA. Head and neck cancers overexpress EGFR and have a high frequency of p53 mutations, both of which enhance DNA repair. This review discusses the clinical criteria for radiation resistance in patients with head and neck cancer and summarizes how cancer cells evade radiation-mediated apoptosis by p53- and epidermal growth factor receptor (EGFR)-mediated DNA repair. In addition, we explore the role of cancer stem cells in promoting radiation resistance, and how the abscopal effect provides rationale for combination strategies with immunotherapy.

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.

Non-murine models to investigate tumor-immune interactions in head and neck cancer.

The immune response has important roles in the biology of solid tumors, including oncogenesis, tumor growth, invasion and metastasis, and response to treatment. Improved understanding of tumor-immune system interactions has provided promising therapeutic options that are based on the rescue and enhancement of the anti-tumoral host response. Immune-based treatments have been approved for clinical use in various types of cancer, including head and neck cancer (HNC); other strategies involving combination therapies are currently in development. These novel therapies were developed based on knowledge derived from in vitro, in silico, and in vivo pre-clinical studies. However, clinical trials seldom replicate the efficacy observed in pre-clinical animal studies. This lack of correlation between pre-clinical studies and clinical trials may be related to limitations of the models used; which highlights the relevance of considering immune-related aspects of different pre-clinical models. Murine models are the most frequently used pre-clinical models of HNC and are discussed elsewhere. Non-murine models have characteristics that offer unique opportunities for the study of HNC etiology, therapeutic strategies, and tumor-immune system interactions. The current review focuses on immune-related aspects of non-murine models, including dog, cat, pig, zebrafish, and frog, that could be used to investigate tumor-immune interactions in HNC.