A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth.

Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knockdown of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.

Lysosomal inhibition sensitizes TMEM16A-expressing cancer cells to chemotherapy.

Squamous cell carcinoma of the head and neck (SCCHN) is a devastating disease that continues to have low cure rates despite the recent advances in therapies. Cisplatin is the most used chemotherapy agent, and treatment failure is largely driven by resistance to this drug. Amplification of chromosomal band 11q13 occurs in ∼30% of SCCHN tumors. This region harbors the ANO1 gene that encodes the TMEM16A ion channel, which is responsible for calcium-activated chloride transport in epithelial tissues. TMEM16A overexpression is associated with cisplatin resistance, and high TMEM16A levels correlate with decreased survival. However, the mechanistic underpinning of this effect remains unknown. Lysosomal biogenesis and exocytosis have been implicated in cancer because of their roles in the clearance of damaged organelles and exocytosis of chemotherapeutic drugs and toxins. Here, we show that TMEM16A overexpression promotes lysosomal biogenesis and exocytosis, which is consistent with the expulsion of intracellular cisplatin. Using a combination of genetic and pharmacologic approaches, we find that TMEM16A promotes lysosomal flux in a manner that requires reactive oxygen species, TRPML1, and the activation of the ß-catenin­melanocyte-inducing transcription factor pathway. The lysosomal inhibitor hydroxychloroquine (HCQ) synergizes with cisplatin in killing SCCHN cells in vitro. Using a murine model of SCCHN, we show that HCQ and cisplatin retard the growth of cisplatin-resistant patient-derived xenografts in vivo. We propose that TMEM16A enables cell survival by the up-regulation of lysosomal sequestration and exocytosis of the cytotoxic drugs. These results uncover a model of treatment for resistance in cancer, its reversal, and a role for TMEM16A.

Systematic Analysis of IL-1 Cytokine Signaling Suppression by HPV16 Oncoproteins.

The human papillomavirus (HPV) E6 and E7 oncogenes are expressed at all stages of HPV-mediated carcinogenesis and are essential drivers of cancers caused by high-risk HPV. Some of the activities of HPV E6 and E7, such as their interactions with host cellular tumor suppressors, have been characterized extensively. There is less information about how high-risk HPV E6 and E7 alter cellular responses to cytokines that are present in HPV-infected tissues and are an important component of the tumor microenvironment. We used several models of HPV oncoprotein activity to assess how HPV16 E6 and E7 alter the cellular response to the proinflammatory cytokine IL-1ß. Models of early stage HPV infection and of established HPV-positive head and neck cancers exhibited similar dysregulation of IL-1 pathway genes and suppressed transcriptional responses to IL-1ß treatment. Such overlap in cell responses supports that changes induced by HPV16 E6 and E7 early in infection could persist and contribute to a dysregulated immune environment throughout carcinogenesis. HPV16 E6 and E7 also drove the upregulation of several suppressors of IL-1 cytokine signaling, including SIGIRR, both in primary keratinocytes and in cancer cells. SIGIRR knockout was insufficient to increase IL-1ß-dependent gene expression in the presence of HPV16 E6 and E7, suggesting that multiple suppressors of IL-1 signaling contribute to dampened IL-1 responses in HPV16-positive cells. IMPORTANCE Human papillomavirus (HPV) infection is responsible for nearly 5% of the worldwide cancer burden. HPV-positive tumors develop over years to decades in tissues that are subject to frequent stimulation by proinflammatory cytokines. However, the effects of HPV oncoproteins on the cellular response to cytokine stimulation are not well defined. We analyzed IL-1 cytokine signaling in several models of HPV biology and disease. We found that HPV16 E6 and E7 oncoproteins mediate a broad and potent suppression of cellular responses to IL-1ß in models of both early and late stages of carcinogenesis. Our data provide a resource for future investigation of IL-1 signaling in HPV-positive cells and cancers.

Identifying predictors of HPV-related head and neck squamous cell carcinoma progression and survival through patient-derived models.

Therapeutic innovation for human papilloma virus-related (HPV+) head and neck squamous cell carcinomas (HNSCCs) is impaired by inadequate preclinical models and the absence of accurate biomarkers. Our study establishes the first well-characterized panel of patient-derived xenografts (PDXs) and organoids from HPV+ HNSCCs while determining fidelity of the models to the distinguishing genetic features of this cancer type. Despite low engraftment rates, whole exome sequencing showed that PDXs retain multiple distinguishing features of HPV+ HNSCC lost in existing cell lines, including PIK3CA mutations, TRAF3 deletion and the absence of EGFR amplifications. Engrafted HPV+ tumors frequently contained NOTCH1 mutations, thus providing new models for a negatively prognostic alteration in this disease. Genotype-phenotype associations in the models were then tested for prediction of tumor progression and survival in published clinical cohorts. Observation of high tumor mutational burdens (TMBs) in the faster-growing models facilitated identification of a novel association between TMB and local progression in both HPV+ and HPV- patients that was prognostic in HPV- cases. In addition, reduced E7 and p16INK4A levels found in a PDX from an outlier case with lethal outcome led to detection of similar profiles among recurrent HPV+ HNSCCs. Transcriptional data from the Cancer Genome Atlas was used to demonstrate that the lower E2F target gene expression predicted by reduced E7 levels has potential as a biomarker of disease recurrence risk. Our findings bridge a critical gap in preclinical models for HPV+ HNSCCs and simultaneously reveal novel potential applications of quantifying mutational burden and viral oncogene functions for biomarker development.

Comparative secretome analysis of epithelial and mesenchymal subpopulations of head and neck squamous cell carcinoma identifies S100A4 as a potential therapeutic target.

Epithelial-mesenchymal transition (EMT) is a key contributor in tumor progression and metastasis. EMT produces cellular heterogeneity within head and neck squamous cell carcinomas (HNSCC) by creating a phenotypically distinct mesenchymal subpopulation that is resistant to conventional therapies. In this study, we systematically characterized differences in the secretomes of E-cadherin high epithelial-like and E-cadherin low mesenchymal-like subpopulations using unbiased and targeted proteomics. A total 1765 proteins showed significant changes with 177 elevated in the epithelial subpopulation and 173 elevated in the mesenchymal cells. Key nodes in affected networks included NFκB, Akt, and ERK, and most implicated cellular components involved various aspects of the extracellular matrix. In particular, large changes were observed in multiple collagens with most affected collagens at much higher abundance levels in the mesenchymal subpopulation. These cells also exhibited a secretome profile resembling that of cancer-associated fibroblastic cells (CAF). S100A4, a commonly used marker for cancer-associated fibroblastic cells, was elevated more than 20-fold in the mesenchymal cells and this increase was further verified at the transcriptome level. S100A4 is a known mediator of EMT, leading to metastasis and EMT has been proposed as a potential source of cancer-associated fibroblastic cells in solid tumors. S100A4 knockdown by small interfering RNA led to decreased expression, secretion and activity of matrix metalloproteinase 2, as verified by quantitative PCR, multiple reaction monitoring and zymography analyses, and reduced invasion in collagen-embedded spheroids. Further confirmation in three-dimensional organotypic reconstructs showed less invasion and advanced differentiation in the S100A4 RNA interference samples. Orthotopic metastasis model, developed to validate the findings in vivo, demonstrated a decrease in spontaneous metastasis and augmented differentiation in the primary tumor in siS100A4 xenografts. These results demonstrate the value of secretome profiling to evaluate phenotypic conversion and identify potential novel therapeutic targets such as S100A4.

A novel pipeline for prioritizing cancer type-specific therapeutic vulnerabilities using DepMap identifies PAK2 as a target in head and neck squamous cell carcinomas.

There is limited guidance on exploiting the genome-wide loss-of-function CRISPR screens in cancer Dependency Map (DepMap) to identify new targets for individual cancer types. This study integrated multiple tools to filter these data in order to seek new therapeutic targets specific to head and neck squamous cell carcinoma (HNSCC). The resulting pipeline prioritized 143 targetable dependencies that represented both well-studied targets and emerging target classes like mitochondrial carriers and RNA-binding proteins. In total, 14 targets had clinical inhibitors used for other cancers or nonmalignant diseases that hold near-term potential to repurpose for HNSCC therapy. Comparing inhibitor response data that were publicly available for 13 prioritized targets between the cell lines with high vs. low dependency on each target uncovered novel therapeutic potential for the PAK2 serine/threonine kinase. PAK2 gene dependency was found to be associated with wild-type p53, low PAK2 mRNA, and diploid status of the 3q amplicon containing PAK2. These findings establish a generalizable pipeline to prioritize clinically relevant targets for individual cancer types using DepMap. Its application to HNSCC highlights novel relevance for PAK2 inhibition and identifies biomarkers of PAK2 inhibitor response.

HPV16 genome structure analysis in oropharyngeal cancer PDXs identifies tumors with integrated and episomal genomes.

HPV + oropharyngeal squamous cell carcinoma (OPC) incidence recently surpassed cervical cancer and is the most common HPV-related cancer in the developed world. HPV16 is in ∼90 % of HPV + OPCs, with episomal genomes in the majority of cases. Most existing HPV16+ cancer cell lines derive from outside the oropharynx and harbor integrated HPV genomes. Thus, there is need for OPC preclinical models to evaluate standard and experimental therapeutics in the presence of episomal HPV16 oncogenic drivers. Here we characterize HPV genome structures in eight HPV16+ OPC patient-derived xenografts (PDXs), and evaluate their responses to standard chemotherapy. HPV genome state was investigated by combining Southern blot, T5 exonuclease assay, whole genome sequencing, and RNAseq data. This analysis revealed complexity and variation in integrated vs. episomal HPV forms across PDXs and demonstrated that four PDXs predominantly contain episomal HPV16. Episomal status did not ensure favorable in vivo responses to cisplatin therapy, despite the more favorable prognosis previously attributed to episomal HPV + tumors; this could be due to the small number present in the dataset. Our analysis establishes PDX models as test platforms for novel therapies designed to target maintenance of the episomal forms of HPV16 that commonly appear in OPC.

Preoperative Circulating Tumor HPV DNA and Oropharyngeal Squamous Cell Disease.

Importance: The utility of preoperative circulating tumor tissue-modified viral human papillomavirus DNA (TTMV-HPV DNA) levels in predicting human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) disease burden is unknown. Objective: To determine if preoperative circulating tumor HPV DNA (ctHPVDNA) is associated with disease burden in patients with HPV+ OPSCC who have undergone transoral robotic surgery (TORS). Design, Setting, and Participants: This cross-sectional study comprised patients with HPV+ OPSCC who underwent primary TORS between September 2021 and April 2023 at one tertiary academic institution. Patients with treatment-naive HPV+ OPSCC (p16-positive) and preoperative ctHPVDNA levels were included, and those who underwent neck mass excision before ctHPVDNA collection were excluded. Main Outcomes and Measures: The main outcome was the association of increasing preoperative ctHPVDNA levels with tumor size and lymph node involvement in surgical pathology. The secondary outcome was the association between preoperative ctHPVDNA levels and adverse pathology, which included lymphovascular invasion, perineural invasion, or extranodal extension. Results: A total of 70 patients were included in the study (65 men [93%]; mean [SD] age, 61 [8] years). Baseline ctHPVDNA levels ranged from 0 fragments/milliliter of plasma (frag/mL) to 49 452 frag/mL (median [IQR], 272 [30-811] frag/mL). Overall, 58 patients (83%) had positive results for ctHPVDNA, 1 (1.4%) had indeterminate results, and 11 (15.6%) had negative results. The sensitivity of detectable ctHPVDNA for identifying patients with pathology-confirmed HPV+ OPSCC was 84%. Twenty-seven patients (39%) had pathologic tumor (pT) staging of pT0 or pT1, 34 (49%) had pT2 staging, and 9 patients (13%) had pT3 or pT4 staging. No clinically meaningful difference between detectable and undetectable preoperative ctHPVDNA cohorts was found for tumor size or adverse pathology. Although the median preoperative ctHPVDNA appeared to be higher in pT2 through pT4 stages and pN1 or pN2 stages, effect sizes were small (pT stage: η2, 0.002 [95% CI, -1.188 to 0.827]; pN stage: η2, 0.043 [95% CI, -0.188 to 2.600]). Median preoperative log(TTMV-HPV DNA) was higher in active smokers (8.79 [95% CI, 3.55-5.76]), compared with never smokers (5.92 [95% CI, -0.97 to 1.81]) and former smokers (4.99 [95% CI, 0.92-6.23]). Regression analysis did not show an association between tumor dimension or metastatic lymph node deposit size and preoperative log(TTMV-HPV DNA). After univariate analysis, no association was found between higher log(TTMV-HPV DNA) levels and adverse pathology. Conclusions and Relevance: In this cross-sectional study, preoperative ctHPVDNA levels were not associated with disease burden in patients with HPV+ OPSCC who underwent TORS.

Multiple airway plasmacytomas: A rare cause of proximal airway obstruction requiring tumor debulking.

Extramedullary plasmacytomas (EMP) can present as airway lesions causing central airway obstruction. Though typically solitary, EMPs should be considered in the evaluation of multifocal tracheobronchial tumors. Bronchoscopic tumor debulking and radiation therapy can be used for symptomatic relief.

Functional Outcomes in Patients with Human Papillomavirus-Associated Oropharyngeal Squamous Cell Cancer Treated with Trimodality Therapy.

OBJECTIVES: To describe swallowing and feeding-tube outcomes in patients with high-risk oropharyngeal cancer treated with trimodality therapy (TMT), including transoral robotic surgery (TORS) and adjuvant chemoradiotherapy. METHODS: A chart review was conducted on patients with HPV+ OPSCC receiving TMT with TORS at an academic medical center from March 2010 to March 2021. Data collected included demographics, treatment, feeding tube placement, functional oral intake scale (FOIS) scores, and swallowing-language pathology (SLP) evaluations. RESULTS: A total of 255 patients met selection criteria (mean age 61 years, 88% male). Following intraoperative nasogastric tube (NG) placement, 31% remained NG tube dependent after 3 weeks. A gastrostomy tube was placed in 19% of patients, and at 1 year after end-of-treatment (EOT), 3.5% overall remained tube-dependent. Mean FOIS scores were 6.9 (SD = 0.3) at pre-operative visit, 2.6 (1.8) at first post-operative visit, and 5.5 (1.5) after EOT. In the subset of patients with follow-up longer than 2 years (n = 118), the mean FOIS was 6.1 (SD = 1.3) at most recent visit. Clinical signs of aspiration/penetration were suspected on SLP evaluation in 18% of patients. These patients were subsequently evaluated with fiberoptic endoscopic evaluation of swallowing (FEES) and/or barium swallow study, which confirmed signs of aspiration in 2.7% of patients overall. Delayed NG tube removal after 3 weeks was predictive of (1) gastrostomy tube requirement and (2) clinical signs of aspiration on an SLP visit after EOT. CONCLUSIONS: Favorable functional and feeding-tube outcomes are demonstrated in patients with HPV-associated OPSCC undergoing TMT. In this single-institution study, we found low rates of long-term feeding tube dependence and high median FOIS following treatment. Review of routine SLP visits provides a detailed and easily accessible means for assessing swallowing function in this cohort. LEVEL OF EVIDENCE: 4 Laryngoscope, 133:3013-3020, 2023.

YAP1 activation by human papillomavirus E7 promotes basal cell identity in squamous epithelia.

Persistent human papillomavirus (HPV) infection of stratified squamous epithelial cells causes nearly 5% of cancer cases worldwide. HPV-positive oropharyngeal cancers harbor few mutations in the Hippo signaling pathway compared to HPV-negative cancers at the same anatomical site, prompting the hypothesis that an HPV-encoded protein inactivates the Hippo pathway and activates the Hippo effector yes-associated protein (YAP1). The HPV E7 oncoprotein is required for HPV infection and for HPV-mediated oncogenic transformation. We investigated the effects of HPV oncoproteins on YAP1 and found that E7 activates YAP1, promoting YAP1 nuclear localization in basal epithelial cells. YAP1 activation by HPV E7 required that E7 binds and degrades the tumor suppressor protein tyrosine phosphatase non-receptor type 14 (PTPN14). E7 required YAP1 transcriptional activity to extend the lifespan of primary keratinocytes, indicating that YAP1 activation contributes to E7 carcinogenic activity. Maintaining infection in basal cells is critical for HPV persistence, and here we demonstrate that YAP1 activation causes HPV E7 expressing cells to be retained in the basal compartment of stratified epithelia. We propose that YAP1 activation resulting from PTPN14 inactivation is an essential, targetable activity of the HPV E7 oncoprotein relevant to HPV infection and carcinogenesis.

The 'epithelial' cells that cover our bodies are in a constant state of turnover. Every few weeks, the outermost layers die and are replaced by new cells from the layers below. For scientists, this raises a difficult question. Cells infected by human papillomaviruses, often known as HPV, can become cancerous over years or even decades. How do infected cells survive while the healthy cells around them mature and get replaced? One clue could lie in PTPN14, a human protein which many papillomaviruses eliminate using their viral E7 protein; this mechanism could be essential for the virus to replicate and cause cancer. To find out the impact of losing PTPN14, Hatterschide et al. used human epithelial cells to make three-dimensional models of infected tissues. These experiments showed that, when papillomaviruses destroy PTPN14, a human protein called YAP1 turns on in the lowest, most long-lived layer of the tissue. Cells in which YAP1 is activated survive while those that carry the inactive version mature and die. This suggests that papillomaviruses turn on YAP1 to remain in tissues for long periods. Papillomaviruses cause about five percent of all human cancers. Finding ways to stop them from activating YAP1 has the potential to prevent disease. Overall, the research by Hatterschide et al. also sheds light on other epithelial cancers which are not caused by viruses.

Outcomes Among Patients With Mucosal Head and Neck Squamous Cell Carcinoma Treated With Checkpoint Inhibitors.

Importance: Immune checkpoint inhibitors (CPIs) are now part of standard therapy for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) because of improved outcomes compared with chemotherapy in clinical trials. However, data on outcomes in patients with HNSCC in the general population who are treated with CPIs remain limited. Objective: To assess response rates, survival outcomes, and associations with key clinical covariates in a large, contemporary cohort of patients with recurrent or metastatic mucosal HNSCC who were treated with CPIs with or without chemotherapy. Design, Setting, and Participants: This retrospective cohort study included patients older than 18 years who received CPI-based therapy for recurrent or metastatic HNSCC at the University of Pennsylvania from January 1, 2015, through August 15, 2021. Clinical and survival data were abstracted through medical record review. Exposures: Treatment with CPIs with or without chemotherapy for a diagnosis of HNSCC. Main Outcomes and Measures: The main outcomes were overall survival, progression-free survival, and response rates. Overall survival and progression-free survival were estimated by Kaplan-Meier methods. Multivariable Cox proportional hazards regression was used to examine associations of key clinical variables with survival; a χ2 test and logistic regression were used to assess associations with response rate. Results: The study cohort consisted of 212 patients, of whom 165 (77.8%) were male, 148 (69.8%) were former or current smokers, and 66 (31.1%) had an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or greater; median age was 63.2 years (IQR, 57.2-71.2 years). Primary tumor sites included the oropharynx (99 [46.7%]), oral cavity (61 [28.8%]), and larynx or hypopharynx (52 [24.5%]). Most (126 [59.4%]) received CPI as first-line systemic therapy, and 23 (10.8%) received combination CPI with chemotherapy. The overall response rate was 30.1%. Estimated 1-year overall survival was 51.8% (95% CI, 44.5%-58.8%), and estimated 1-year progression-free survival was 9.4% (95% CI, 5.0%-15.5%). Median overall survival was 12.9 months (IQR, 4.1-36.5 months), and median progression-free survival was 3.9 months (IQR, 1.9-17.8 months). Non-oral cavity primary site (vs oral cavity) was associated with improved overall survival (human papillomavirus-positive oropharynx: hazard ratio [HR], 0.567 [95% CI, 0.335-0.960]; all other sites: HR, 0.491 [95% CI, 0.298-0.810]), and T category of 4 at presentation (HR, 1.594; 95% CI, 1.062-2.394) and an ECOG performance status greater than 1 (HR, 2.720; 95% CI, 1.866-3.964) were associated with worse overall survival. Conclusions and Relevance: In this cohort study of patients with recurrent or metastatic HNSCC who received CPI therapy, the overall response rate was 30.1%. Patients with oral cavity cancer had worse overall survival compared with patients with HNSCC of other subsites. These findings support the use of CPI therapies for first- or second-line treatment of recurrent or metastatic HNSCC.

A Critical Role for p53 during the HPV16 Life Cycle.

Human papillomaviruses (HPV) are causative agents in ano-genital and oral cancers; HPV16 is the most prevalent type detected in human cancers. The HPV16 E6 protein targets p53 for proteasomal degradation to facilitate proliferation of the HPV16 infected cell. However, in HPV16 immortalized cells E6 is predominantly spliced (E6*) and unable to degrade p53. Here, we demonstrate that human foreskin keratinocytes immortalized by HPV16 (HFK+HPV16), and HPV16 positive oropharyngeal cancers, retain significant expression of p53. In addition, p53 levels increase in HPV16+ head and neck cancer cell lines following treatment with cisplatin. Introduction of full-length E6 into HFK+HPV16 resulted in attenuation of cellular growth (in hTERT immortalized HFK, E6 expression promoted enhanced proliferation). An understudied interaction is that between E2 and p53 and we investigated whether this was important for the viral life cycle. We generated mutant genomes with E2 unable to interact with p53 resulting in profound phenotypes in primary HFK. The mutant induced hyper-proliferation, but an ultimate arrest of cell growth; ß-galactosidase staining demonstrated increased senescence, and COMET assays showed increased DNA damage compared with HFK+HPV16 wild-type cells. There was failure of the viral life cycle in organotypic rafts with the mutant HFK resulting in premature differentiation and reduced proliferation. The results demonstrate that p53 expression is critical during the HPV16 life cycle, and that this may be due to a functional interaction between E2 and p53. Disruption of this interaction has antiviral potential. IMPORTANCE Human papillomaviruses are causative agents in around 5% of all cancers. There are currently no antivirals available to combat these infections and cancers, therefore it remains a priority to enhance our understanding of the HPV life cycle. Here, we demonstrate that an interaction between the viral replication/transcription/segregation factor E2 and the tumor suppressor p53 is critical for the HPV16 life cycle. HPV16 immortalized cells retain significant expression of p53, and the critical role for the E2-p53 interaction demonstrates why this is the case. If the E2-p53 interaction is disrupted then HPV16 immortalized cells fail to proliferate, have enhanced DNA damage and senescence, and there is premature differentiation during the viral life cycle. Results suggest that targeting the E2-p53 interaction would have therapeutic benefits, potentially attenuating the spread of HPV16.

T2R bitter taste receptors regulate apoptosis and may be associated with survival in head and neck squamous cell carcinoma.

Better management of head and neck squamous cell carcinomas (HNSCCs) requires a clearer understanding of tumor biology and disease risk. Bitter taste receptors (T2Rs) have been studied in several cancers, including thyroid, salivary, and GI, but their role in HNSCC has not been explored. We found that HNSCC patient samples and cell lines expressed functional T2Rs on both the cell and nuclear membranes. Bitter compounds, including bacterial metabolites, activated T2R-mediated nuclear Ca2+ responses leading to mitochondrial depolarization, caspase activation, and ultimately apoptosis. Buffering nuclear Ca2+ elevation blocked caspase activation. Furthermore, increased expression of T2Rs in HNSCCs from The Cancer Genome Atlas is associated with improved overall survival. This work suggests that T2Rs are potential biomarkers to predict outcomes and guide treatment selection, may be leveraged as therapeutic targets to stimulate tumor apoptosis, and may mediate tumor-microbiome crosstalk in HNSCC.

HPV E6 regulates therapy responses in oropharyngeal cancer by repressing the PGC-1α/ERRα axis.

Therapy with radiation plus cisplatin kills HPV+ oropharyngeal squamous cell carcinomas (OPSCCs) by increasing reactive oxygen species beyond cellular antioxidant capacity. To explore why these standard treatments fail for some patients, we evaluated whether the variation in HPV oncoprotein levels among HPV+ OPSCCs affects mitochondrial metabolism, a source of antioxidant capacity. In cell line and patient-derived xenograft models, levels of HPV full-length E6 (fl-E6) inversely correlated with oxidative phosphorylation, antioxidant capacity, and therapy resistance, and fl-E6 was the only HPV oncoprotein to display such correlations. Ectopically expressing fl-E6 in models with low baseline levels reduced mitochondrial mass, depleted antioxidant capacity, and sensitized to therapy. In this setting, fl-E6 repressed the peroxisome proliferator-activated receptor gamma co-activator 1α/estrogen-related receptor α (PGC-1α/ERRα) pathway for mitochondrial biogenesis by reducing p53-dependent PGC-1α transcription. Concordant observations were made in 3 clinical cohorts, where expression of mitochondrial components was higher in tumors of patients with reduced survival. These tumors contained the lowest fl-E6 levels, the highest p53 target gene expression, and an activated PGC-1α/ERRα pathway. Our findings demonstrate that E6 can potentiate treatment responses by depleting mitochondrial antioxidant capacity and provide evidence for low E6 negatively affecting patient survival. E6's interaction with the PGC-1α/ERRα axis has implications for predicting and targeting treatment resistance in OPSCC.

Pharmacologic Inhibition of SHP2 Blocks Both PI3K and MEK Signaling in Low-epiregulin HNSCC via GAB1.

Preclinical and clinical studies have evidenced that effective targeted therapy treatment against receptor tyrosine kinases (RTKs) in different solid tumor paradigms is predicated on simultaneous inhibition of both the PI3K and MEK intracellular signaling pathways. Indeed, re-activation of either pathway results in resistance to these therapies. Recently, oncogenic phosphatase SHP2 inhibitors have been developed with some now reaching clinical trials. To expand on possible indications for SHP099, we screened over 800 cancer cell lines covering over 25 subsets of cancer. We found HNSCC was the most sensitive adult subtype of cancer to SHP099. We found that, in addition to the MEK pathway, SHP2 inhibition blocks the PI3K pathway in sensitive HNSCC, resulting in downregulation of mTORC signaling and anti-tumor effects across several HNSCC mouse models, including an HPV+ patient-derived xenograft (PDX). Importantly, we found low levels of the RTK ligand epiregulin identified HNSCCs that were sensitive to SHP2 inhibitor, and, adding exogenous epiregulin mitigated SHP099 efficacy. Mechanistically, epiregulin maintained SHP2-GAB1 complexes in the presence of SHP2 inhibition, preventing downregulation of the MEK and PI3K pathways. We demonstrate HNSCCs were highly dependent on GAB1 for their survival and knockdown of GAB1 is sufficient to block the ability of epiregulin to rescue MEK and PI3K signaling. These data connect the sensitivity of HNSCC to SHP2 inhibitors and to a broad reliance on GAB1-SHP2, revealing an important and druggable signaling axis. Overall, SHP2 inhibitors are being heavily developed and may have activity in HNSCCs, and in particular those with low levels of epiregulin.

A benchmark for oncologic outcomes and model for lethal recurrence risk after transoral robotic resection of HPV-related oropharyngeal cancers.

OBJECTIVES: Increasing use of transoral robotic surgery (TORS) is likely to impact outcomes for HPV+ oropharyngeal squamous cell carcinomas (OPSCCs). We aimed to describe oncologic outcomes for a large HPV+ OPSCC cohort after TORS and develop a risk prediction model for recurrence under this treatment paradigm. MATERIALS AND METHODS: 634 HPV+ OPSCC patients receiving TORS-based therapy at a single institution were reviewed retrospectively to describe survival across the entire cohort and for patients suffering recurrence. Risks for distant metastatic recurrence (DMR) and locoregional recurrence (LRR) were modeled using multivariate logistic regression analyses of case-control sub-cohorts. RESULTS: 5-year overall and recurrence-free survival were 91.2% and 86.1%, respectively. 5-year overall survival was 52.5% following DMR and 83.3% after isolated LRR (P = .01). In case-control analyses, positive surgical margins were associated with DMR (adjusted OR 5.8, CI 2.1-16.0, P = .001), but not isolated LRR, and increased DMR risk 4.2 fold in patients with early clinical stage disease. By contrast, LRR was associated with not receiving recommended adjuvant therapy (OR 13.4, CI 6.3-28.5, P < .001). CONCLUSIONS: This study sets a benchmark for oncologic outcomes from HPV+ OPSCC after TORS-based therapy. Under this treatment paradigm, margins are relevant for assessing lethal recurrence risk during clinical trial design and post-treatment surveillance.

Locoregional Recurrence in p16-Positive Oropharyngeal Squamous Cell Carcinoma After TORS.

OBJECTIVE: To analyze the patterns, risk factors, and salvage outcomes for locoregional recurrences (LRR) after treatment with transoral robotic surgery (TORS) for HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC). STUDY DESIGN: Retrospective analysis of HPV+ OPSCC patients completing primary TORS, neck dissection, and NCCN-guideline-compliant adjuvant therapy at a single institution from 2007 to 2017. METHODS: Features associated with LRR, detailed patterns of LRR, and outcomes of salvage therapy were analyzed. Disease-free survival (DFS) and overall survival (OS) were calculated for subgroups of patients receiving distinct adjuvant treatments. RESULTS: Of 541 patients who completed guideline-indicated therapy, the estimated 5-year LRR rate was 4.5%. There were no identifiable clinical or pathologic features associated with LRR. Compared to patients not receiving adjuvant therapy, those who received indicated adjuvant radiation alone had a lower risk of LRR (HR 0.28, 95% CI [0.09-0.83], P = .023), but there was no difference in DFS (P = .21) and OS (P = .86) between adjuvant therapy groups. The 5-year OS for patients who developed LRR was 67.1% vs. 93.9% for those without LRR (P < .001). Patients who initially received adjuvant chemoradiation and those suffering local, in-field, and/or retropharyngeal node recurrences had decreased disease control after salvage therapy. CONCLUSION: LRR rates are low for HPV+ OPSCCs completing TORS and guideline-compliant adjuvant therapy. Patients without indication for adjuvant therapy more often suffer LRR, but these recurrences are generally controllable by salvage therapy. Improved understanding of the patterns of recurrence most amenable to salvage therapy may guide treatment decisions, counseling, and adjuvant therapy de-escalation trials. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:E2865-E2873, 2021.

Retropharyngeal Internal Carotid Artery Management in TORS Using Microvascular Reconstruction.

OBJECTIVES: Guidelines for transoral robotic surgery (TORS) have generally regarded patients with retropharyngeal carotid arteries as contraindicated for surgery due to a theoretical risk of intraoperative vascular injury and/or perioperative cerebrovascular accident. We aimed to demonstrate that careful TORS-assisted resection and free flap coverage could not only avoid intraoperative injury and provide a physical barrier for vessel coverage but also achieve adequate margin control. STUDY DESIGN: Retrospective cohort analysis. METHODS: Retrospective review of patients with oropharyngeal malignancies and radiologically confirmed retropharyngeal carotid arteries who underwent TORS, concurrent neck dissection, and free flap reconstruction between 2015 and 2019. RESULTS: Twenty patients were included, 19 (95.0%) with tonsillar tumors and one (5.0%) with a tongue base tumor with significant tonsillar extension. Eighteen patients (90.0%) received a radial artery forearm flap, one (5.0%) an ulnar artery forearm flap, and one (5.0%) an anteromedial thigh flap. All 20 (100%) flaps were inset through combined transcervical and transoral approaches without mandibulotomy. There were no perioperative mortalities, carotid injuries, oropharyngeal bleeds, cervical hematomas, or cerebrovascular accidents. One patient (5.0%) had a free flap failure requiring explant. All patients underwent decannulation and resumed a full oral diet. The mean length of hospitalization was 6.8 (standard deviation 1.2) days. One (5.0%) patient had a positive margin. CONCLUSION: In this analysis, 20 patients with oropharyngeal malignancy and retropharyngeal carotid arteries underwent TORS, neck dissection, and microvascular reconstruction without serious complication (perioperative mortality, vascular injury, or neurologic sequalae) with an acceptable negative margin rate. These results may lead to a reconsideration of a commonly held contraindication to TORS. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:E821-E827, 2021.

Oncologic outcomes of transoral robotic surgery for HPV-negative oropharyngeal carcinomas.

BACKGROUND: Patients with human papillomavirus (HPV)-negative oropharyngeal squamous cell carcinoma (OPSCC) continue to experience disappointing outcomes following chemoradiotherapy (CRT) and appreciable morbidity following historical surgical approaches. We aimed to investigate the oncologic outcomes and perioperative morbidity of a transoral robotic surgery (TORS) approach to surgically resectable HPV-negative OPSCC. METHODS: Retrospective analysis HPV-negative OPSCC patients who underwent TORS, neck dissection and pathology-guided adjuvant therapy (2005-2017). RESULTS: Fifty-six patients (91.1% stage III/IV) were included. Three-year overall survival, locoregional control, and disease-free survival were 85.5%, 84.4%, and 73.6%, respectively (median follow-up 30.6 months, interquartile range 18.4-66.6). Eighteen (32.1%) patients underwent adjuvant radiotherapy and 20 (39.3%) underwent adjuvant CRT. Perioperative mortality occurred in one (1.8%) patient and hemorrhage occurred in two (3.6%) patients. Long-term gastrostomy and tracheostomy rates were 5.4% and 0.0%, respectively. CONCLUSION: The TORS approach for resectable HPV-negative OPSCC can achieve encouraging oncologic outcomes with infrequent morbidity.