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Purpose: To analyze the impact of demographic, clinical, and management variables on time to treatment initiation (TTI) and overall survival (OS)., Study Design: Retrospective chart review., Materials and Methods: Medical records of patients diagnosed with head and neck cancer from 2018 to 2020 were reviewed. Univariate linear and Cox-regressions identified predictors of TTI and OS. Kaplan Meier (KM) curves assessed the difference in survival by diagnostic year and TTI., Results: 381 patients met eligibility criteria. Median TTI was 35.0 days (IQR: 25.0-49.0). Only 10.8 % of all patients reported any treatment delay, with TTI exceeding 90 days found in 3.7 % of patients. TTI increased with African American race (p = 0.02), ED referrals (p = 0.02), and direct admission status (p = 0.01). When compared to treatment with surgery alone, TTI was shorter in patients undergoing surgery with adjuvant radiation (p = 0.02), adjuvant chemoradiation (p = 0.04), and salvage surgery (p = 0.04). Univariate Cox-regressions found smoking (p = 0.01), direct admission status (p = 0.02), increased duration of symptoms (p = 0.02), placement of PEG tubes (p < 0.01) and tracheostomies (p < 0.01), combination treatment (p < 0.01), and surgery with adjuvant chemoradiation treatment (p = 0.01) to increase mortality risk. Disease characteristics, including tumor size (p < 0.01), presence of nodal disease (p = 0.02), and late-stage disease (p < 0.01), increased mortality risk. TTI and diagnostic year did not impact survival., Conclusions: Our analysis determined several demographic, referral, and treatment factors impacted TTI. However, increased TTI did not impact survival. Characteristics consistent with advanced disease worsened OS. Despite the pandemic burden, patients diagnosed in 2020 showed no difference in short-term survival compared to prior years., Competing Interests: Declaration of competing interest The Authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Understanding biological differences between different racial groups of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) patients, who have differences in terms of incidence, survival, and tumor morphology, can facilitate accurate prognostic biomarkers, which can help develop personalized treatment strategies., Methods: This study evaluated whether there were morphologic differences between HPV-associated tumors from Black and White patients in terms of multinucleation index (MuNI), an image analysis-derived metric that measures density of multinucleated tumor cells within epithelial regions on hematoxylin-eosin images and previously has been prognostic in HPV-associated OPSCC patients. In this study, the authors specifically evaluated whether the same MuNI cutoff that was prognostic of overall survival (OS) and disease-free survival in their previous study, T TR , is valid for Black and White patients, separately. We also evaluated population-specific cutoffs, T B for Blacks and T W for Whites, for risk stratification., Results: MuNI was statistically significantly different between Black (mean, 3.88e-4; median, 3.67e-04) and White patients (mean, 3.36e-04; median, 2.99e-04), with p = .0078. Using T TR , MuNI was prognostic of OS in the entire population with hazard ratio (HR) of 1.71 (p = .002; 95% confidence interval [CI], 1.21-2.43) and in White patients with HR of 1.72 (p = .005; 95% CI, 1.18-2.51). Population-specific cutoff, T W , yielded improved HR of 1.77 (p = .003; 95% CI, 1.21-2.58) for White patients, whereas T B did not improve risk-stratification in Black patients with HR of 0.6 (p = .3; HR, 0.6; 95% CI, 0.2-1.80)., Conclusions: Histological difference between White and Black patient tumors in terms of multinucleated tumor cells suggests the need for considering population-specific prognostic biomarkers for personalized risk stratification strategies for HPV-associated OPSCC patients., (© 2022 The Authors. Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Objective: To analyze the prognostic significance of microscopic vs macroscopic extranodal extension and to assess the impact of chemoradiation on overall survival among patients with oral cavity squamous cell carcinoma and varying degrees of extranodal extension., Methods: Utilizing the National Cancer Database, we performed a retrospective cohort study of 7975 patients with oral cavity squamous cell carcinoma and varying degrees of extranodal extension who underwent primary surgical intervention. Propensity-score matched models following Cox regression analyses allowed us to assess the impact of adjuvant radiation alone vs adjuvant chemoradiation on overall survival in patients with microscopic extranodal extension and macroscopic extranodal extension., Results: 7975 patients with oral cavity squamous cell carcinoma were included in the final analysis. Within this cohort, 25.4% had microscopic extranodal extension and 5.2% had macroscopic extranodal extension. On univariate analysis, we found that microscopic and macroscopic extranodal extension were associated with decreased overall survival when compared to those with positive nodes without extranodal extension (HR = 1.67; 95% CI 1.56, 1.79 and HR = 1.88; 95% CI 1.66, 2.14, respectively). On multivariate analysis after propensity-score matching, we found no significant difference in overall survival in patients who received adjuvant radiation alone vs. adjuvant chemoradiation for both microscopic and macroscopic extranodal extension., Conclusion: Our data suggest that microscopic extranodal extension in oral cavity squamous cell carcinoma is associated with worse overall survival than patients without extranodal extension following primary surgical intervention with neck dissection. The results of this study also suggest that the addition of chemotherapy to adjuvant radiation may not provide a significant survival benefit in patients with oral cavity squamous cell carcinoma with microscopic and macroscopic extranodal extension. Comprehensive assessment of the benefits of adjuvant chemoradiation in the setting of microscopic vs macroscopic extranodal extension would need to be studied in a randomized controlled trial., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Purpose: VSIR is a novel immune checkpoint protein whose expression on tumor cells across cancers remains largely uncharacterized. Here we purposed to decode the pan-cancer biologic and clinical significance of VSIR overexpression in the tumor compartment., Experimental Design: We performed multi-omics integrative analyses of 9,735 tumor samples to identify cancers with non-leukocytic expression of VSIR (VSIR High), followed by association with overall survival and immune cell infiltration levels. Orthogonal assessments of VSIR protein expression and lymphocytic infiltration were performed using quantitative immunofluorescence (QIF)., Results: Integrative modeling identified a subset of cancer types as being enriched for VSIR High tumors. VSIR High tumors were associated with significantly poorer overall survival in immunogenic ovarian serous adenocarcinoma (SA) and oral cavity squamous cell carcinoma (SCC). QIF assessments in an independent validation cohort confirmed overexpression of VSIR as being associated with poorer overall survival within immunogenic oral cavity SCC. VSIR overexpression was associated with lower CD4 helper T-cell infiltration in both ovarian SA and oral cavity SCC, but did not impact CD8 T-cell infiltration. VSIR overexpressing tumors in both cancer types exhibited significantly higher STAT3 signaling activity. Pharmacologic inhibition of STAT3 signaling resulted in dose-dependent reduction of VSIR expression in ovarian SA and oral cavity SCC cells., Conclusions: The STAT3-VSIR axis is a potentially significant immunomodulatory mechanism in oral cavity and ovarian cancers, whose activation is associated with poorer survival and an immune microenvironment marked by decreased CD4 helper T-cell activity. The role of VSIR as a tumor-intrinsic modulator of resistance to immunotherapy warrants further exploration., (©2021 American Association for Cancer Research.)

Purpose: Human papilloma virus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC), diagnosed with p16 immunohistochemistry, is associated with favorable prognosis; however, this connection was established using European American (EA)-skewed populations. The impact of p16/human papillomavirus status on outcomes in African American (AA) OPSCC patients remains to be settled. In this study, we determine the association between cancer disparity and p16 status in an OPSCC cohort controlling for time to treatment initiation (TTI), a surrogate for medical care access., Materials and Methods: We analyzed data from all patients diagnosed with OPSCC (N = 440) between 2010 and 2017, who received treatment at our academic medical center. Associations between age, disease stage, sex, p16 status, race, TTI, and overall survival (OS) were investigated., Results: TTI was similar between AA and EA OPSCC patients in our p16+ ( P = .291) or p16- ( P = .715) cohorts. Among p16+ OPSCC patients, the median OS was > 8.65 years for EA patients compared with 5.038 years (95% CI, 2.019 to 5.30; P = .003, log-rank) for AA patients. For p16- patients, the median OS was 5.74 years (95% CI, 3.32 to 6.99) for EA patients and 1.85 years (95% CI, 0.978 to 4.50; P = .03, log-rank) for AA patients. Multivariate Cox regression analysis showed that race was an independent prognostic biomarker and the most impactful co-variate for OS (hazard ratio, 0.40; 95% CI, 0.00 to 0.69; P = .001)., Conclusion: Our work showed that AAs with p16+ OPSCC have surprisingly poor clinical outcomes and are thus poor candidates for treatment de-escalation regimens. Caution should be exercised when extending clinical guidelines based on EA-majority studies to non-EA populations., Competing Interests: Rod RezaeeHonoraria: Zimmer BiometSpeakers' Bureau: Zimmer BiometPatents, Royalties, Other Intellectual Property: Zimmer/Biomet trauma fixation deviceTravel, Accommodations, Expenses: Zimmer BioMet Quintin PanResearch Funding: Bristol Myers Squibb FoundationPatents, Royalties, Other Intellectual Property: I have patents on p53 reactivation therapeutics for HPV+ HNSCCNo other potential conflicts of interest were reported.

High-risk human papillomavirus (HPV) is an etiologic factor in a spectrum of squamous cell carcinomas including anal, cervical, and oropharyngeal. HPV cell free DNA (cfDNA) is shed from the primary tumor into systemic circulation and can be detected using several platforms including quantitative PCR, digital droplet PCR, or next generation sequencing. Levels of HPV cfDNA at time of initial presentation is associated with known poor prognostic clinicopathologic variables, such as advanced stage and, locoregional and distant metastases. Moreover, longitudinal sampling revealed that persistent or increasing HPV cfDNA levels are indicative of treatment relapse and, in some studies, HPV cfDNA detection predicted treatment failures prior to routine post-treatment clinical imaging. A liquid biopsy platform using HPV cfDNA offers unique advantages over traditional approaches and may have clinical utility for detection of minimum residual disease, treatment response, and disease progression in patients with HPV+ cancers., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: microRNAs negatively regulate gene expression at the post-transcriptional level. Mounting evidence shows that miR expression is deregulated in human cancers including head and neck squamous cell carcinoma (HNSCC). Epigenetically silenced tumor suppressor miRs may be re-expressed upon treatment with histone deacetylases inhibitors. Suberoylanilide Hydroxamic Acid (SAHA) is a histone deacetylase inhibitor that is currently being investigated in clinical trials for HNSCC. We hypothesized that SAHA will re-express a set of tumor suppressor miRs and enhance the efficacy of cisplatin and radiation in HNSCC., Results: In this study, miR expression profile was utilized to identify the tumor suppressor miRs that are re-expressed following SAHA treatment in HNSCC. Our data demonstrated that two tumor suppressor miRs, miR-107 and miR-138, were significantly up-regulated in CAL27 and SCC25 cell lines, following SAHA treatment. In addition to this, treatment with SAHA in a dose dependent manner significantly inhibited the cell proliferation, cell migration, and anchorage dependent clonogenic survival in CAL27 and SCC25 cell lines, respectively. Further, the expression of several oncogenes, PKCε, HIF1β, CDK6, and RhoC were down regulated in response to SAHA treatment. Additionally, we demonstrated that the combination treatment with SAHA and a chemotherapeutic drug cisplatin caused a significant reduction of cell growth compared to the single agent treatment., Conclusion: Our data indicate that SAHA treatment results in reactivation of the silenced tumor suppressor miRs. Furthermore, this study emphasizes the usefulness of this drug as a novel combination therapy for HNSCC patients., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: The purpose of this study was to further define the impact of primary surgery in the management of oropharyngeal squamous cell carcinoma (SCC)., Methods: Two hundred ninety-six patients with oropharyngeal SCC treated with primary surgery were included. Multivariable analysis and recursive partitioning analysis (RPA) identified predictors of survival and gastrostomy tube presence., Results: Multivariable analysis identified that HPV negativity (p = .0002), presence of extranodal extension (p = .0025), and advanced T classification (p = .0081) were independent predictors of survival. For HPV-positive patients, surgical approach (p = .0111) and margin status (p = .0287) were significant predictors of survival. For HPV-negative patients, extranodal extension (p = .0021) and advanced T classification (p = .0342) were significant predictors of survival. Smoking status and advanced neck disease did not impact survival, and the addition of adjuvant chemotherapy did not confer survival benefit in HPV-positive or HPV-negative subgroups., Conclusion: Independent predictors of survival are unique in patients with oropharyngeal SCC treated with primary surgery. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1794-E1802, 2016., (© 2015 Wiley Periodicals, Inc.)

Epithelial-restricted with serine box (ESX), a member of the ETS transcription factor family, is elevated and regulates EGFR in head and neck squamous cell carcinoma (HNSCC). However, the molecular mechanisms that contribute to ESX dysregulation remain to be elucidated. In this study, in silico analysis of the 3'-untranslated region (UTR) of ESX predicted two miR-124-binding sites. Delivery of miR-124 inhibited the 3'UTR ESX-driven reporter activity by 50% (P < 0.05) confirming ESX as a direct target of miR-124. Loss of miR-124 was found to be a frequent event in HNSCC. miR-124 expression was significantly depleted in the primary tumor compared with matched normal tissue in 100% (12/12) of HNSCC patients; relative mean miR-124 expression of 0.01197 and 0.00118 (P < 0.001, n = 12) in matched normal adjacent tissue and primary HNSCC tumor, respectively. Overexpression of miR-124 decreased ESX and EGFR levels in miR-124(low)/ESX(high)/EGFR(high) SCC15 HNSCC cells and reduced cell invasion, migration, proliferation, and colony formation. SCC15 cells with miR-124 restoration were less tumorigenic in vivo than miR-control SCC15 cells (70% inhibition, P < 0.01). Restoration of miR-124 in SCC15 cells enhanced the antiproliferative efficacy of the EGFR/Her2 tyrosine kinase inhibitors. Furthermore, recapitulation of EGFR in miR-124-overexpressing SCC15 cells was sufficient to completely block the antiproliferative effects of lapatinib and afatinib. Taken together, our work provides intriguing evidence that miR-124 is a novel therapeutic approach to reduce ESX/EGFR, and may be a tractable strategy to enhance the response rate of HNSCC patients to current anti-EGFR/Her2 therapies., (©2015 American Association for Cancer Research.)

Background: Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies, and it is postulated that they are responsible for disease recurrence and/or progression. Because the prognoses of patients with HPV16-positive and HPV-negative HNSCC are distinct, the authors sought to determine whether differences in the number of CSCs could account for this clinical observation., Methods: CSC populations in HPV16-positive and HPV-negative HNSCC were assessed using a proprietary assay based on expression of the enzyme aldehyde dehydrogenase (ALDH), an in vitro tumorsphere formation assay, and an in vivo limiting cell dilution in nonobese diabetic/severe combined immunodeficiency mice. A high-density tissue microarray was stained with ALDH1, a CSC marker, to determine the association between CSCs and HPV16-positive/HPV-negative OPSCC., Results: HPV16-positive HNSCC had a greater intrinsic CSC pool than HPV-negative HNSCC. Inactivation of p53 has been identified as a major mechanism for the elevated CSC population in HPV16-positive HNSCC. In vivo limiting cell dilution experiments using tumors from patients with HPV16-positive and HPV-negative OPSCC indicated that the CSC frequency was 62.5-fold greater in an HPV16-positive OPSCC tumor than in an HPV-negative OPSCC tumor. Primary tumors from patients with HPV16-positive OPSCC were associated with elevated tumor ALDH1 staining, further extending the association between HPV16 and CSCs., Conclusions: The current data and the clinical observation that patients with HPV16-positive HNSCC respond more favorably to current treatment paradigms than patients with HPV-negative HNSCC support the suggestion that CSC phenotype is not homogeneous. Therefore, the reliance on the CSC number may be insufficient to accurately assess the potential of a particular tumor for disease recurrence and/or progression., (© 2013 American Cancer Society.)

Inactivation of the tumor suppressor p53 is a pathogenetic event in the development of head and neck squamous cell carcinoma (HNSCC). In the absence of functional wildtype p53, HNSCC cells have increased resistance to standard chemotherapeutics and radiation. Numerous approaches to restore p53 function in cancer cells have been developed over the past several decades. This review article focuses on viral approaches to deliver wildtype p53 to HNSCC cells, a designer virus that selectively eliminates mutant p53 HNSCC cells, and chemical approaches to reactivate p53 function in HNSCC cells. These promising studies provide evidence that p53 therapeutics may prove useful alone or in combination with conventional chemotherapy and/or radiation for the management of HNSCC patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Epithelial to mesenchymal transition (EMT) is a dynamic cellular process that is essential for the development of metastatic disease. During EMT, a tumor cell with epithelial characteristics transitions to a tumor cell with mesenchymal characteristics through modulation of cell polarity and adhesion. Two hallmark EMT proteins, E-Cadherin and Vimentin, are tightly controlled during EMT through multiple signal transduction pathways. Epidermal growth factor (EGF) and transforming growth factorβ (TGFβ) promote EMT by regulating a distinct set of transcription factors, including Snail and Twist. Snail, Twist, and Slug are integral to the induction of EMT through direct regulation of genes involved in cellular adhesion, migration, and invasion. This review highlights the current literature on EMT in HNSCC. Understanding the role of EMT will provide insight to the pathogenesis of disease progression and may lead to the development of novel anti-cancer therapeutics for metastatic HNSCC., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600,000 new cases diagnosed in the last year. Our laboratory showed that miR-107 expression is reduced and functions as a tumor suppressor gene in HNSCC suggesting the potential application of miR-107 as a novel anticancer therapeutic. In this study, we determined the efficiency and efficacy of cationic lipid nanoparticles to deliver pre-miR-107 (NP/pre-miR-107) in HNSCC cells in vitro and in vivo. NP/pre-miR-107 increased delivery of miR-107 into HNSCC cells by greater than 80,000-fold compared to free pre-miR-107. Levels of known miR-107 targets, protein kinase Cε (PKCε), cyclin-dependent kinase 6 (CDK6), and hypoxia-inducible factor 1-β (HIF1-β), decreased following NP/pre-miR-107 treatment. Clonogenic survival, cell invasion, and cell migration of HNSCC cells was inhibited with NP/pre-miR-107. Moreover, NP/pre-miR-107 reduced the cancer-initiating cell (CIC) population and dampened the expression of the core embryonic stem cell transcription factors, Nanog, Oct3/4, and Sox2. In a preclinical mouse model of HNSCC, systemic administration of NP/pre-miR-107 significantly retarded tumor growth by 45.2% compared to NP/pre-miR-control (P < 0.005, n = 7). Kaplan-Meier analysis showed a survival advantage for the NP/pre-miR-107 treatment group (P = 0.017). Our results demonstrate that cationic lipid nanoparticles are an effective carrier approach to deliver therapeutic miRs to HNSCC.

Metastasis remains a major cause of mortality in patients with head and neck squamous cell carcinoma (HNSCC). HNSCC patients with metastatic disease have extremely poor prognoses, with an average survival rate of less than a year. Metastasis is an intricate sequential process that requires a discrete population of tumor cells to possess the capacity to intravasate from the primary tumor into systemic circulation, survive in circulation, extravasate at a distant site, and proliferate in a foreign, hostile environment. Literature has accumulated to provide mechanistic insight into several signal transduction pathways, receptor tyrosine kinases (RTKs), signal transducer and activator of transcription 3 (Stat3), Rho GTPases, protein kinase Cε (PKCsε), and nuclear factor-κB (NF-κB), that are involved in mediating a metastatic tumor cell phenotype in HN-SCC. Herein we highlight accrued information regarding the key molecular parameters of HNSCC metastasis.

Background: The metastatic spread of solid tumors is directly or indirectly responsible for most cancer-related deaths. Tumor metastasis is very complex and this process requires a tumor cell to acquire enhanced motility, invasiveness and anoikis resistance to successfully establish a tumor at a distal site. Metastatic potential of tumor cells is directly correlated with the expression levels of several angiogenic cytokines. Copper is a mandatory cofactor for the function of many of these angiogenic mediators as well as other proteins that play an important role in tumor cell motility and invasiveness. We have previously shown that tetrathiomolybdate (TM) is a potent chelator of copper and it mediates its anti-tumor effects by suppressing tumor angiogenesis. However, very little is known about the effect of TM on tumor cell function and tumor metastasis. In this study, we explored the mechanisms underlying TM-mediated inhibition of tumor metastasis., Results: We used two in vivo models to examine the effects of TM on tumor metastasis. Animals treated with TM showed a significant decrease in lung metastasis in both in vivo models as compared to the control group. In addition, tumor cells from the lungs of TM treated animals developed significantly smaller colonies and these colonies had significantly fewer tumor cells. TM treatment significantly decreased tumor cell motility and invasiveness by inhibiting lysyl oxidase (LOX) activity, FAK activation and MMP2 levels. Furthermore, TM treatment significantly enhanced tumor cell anoikis by activating p38 MAPK cell death pathway and by downregulating XIAP survival protein expression., Conclusions: Taken together, these results suggest that TM is a potent suppressor of head and neck tumor metastasis by modulating key regulators of tumor cell motility, invasiveness and anoikis resistance.

Background: The clinical management of locally advanced head and neck squamous cell carcinoma (HNSCC) is a challenging problem and requires a multidisciplinary approach. Historically, locally advanced HNSCC has been primarily managed with surgery and radiation (RT). The integration of pharmacotherapy has rapidly expanded over the years into the multimodality treatment paradigm of locally advanced HNSCC., Objective: The studies leading to the adoption of the current standard of care for locally advanced HNSCC are discussed. In addition, the limitations of these various treatment approaches are presented., Methods: An extensive literature search was conducted using the PubMed database for studies published before January 2009. The keywords used for this search were: head and neck neoplasms, chemoradiation, adjuvant chemotherapy, induction chemotherapy, EGFR inhibitor, cisplatin, carboplatin, paclitaxel, docetaxel, 5-fluorouracil, and cetuximab. Publications of randomized clinical trials and other supporting references leading to the current standard of care were particularly selected and discussed in this review., Conclusions: Various single-agent and multi-agent chemotherapeutic regimens have been examined in the context of randomized clinical trials in locally advanced HNSCC for definitive, induction and adjuvant settings. Results from these clinical trials support the use of cisplatin-based chemoradiation as the standard of care for the definitive and adjuvant settings. Recent evidence indicates that cetuximab, an epidermal growth factor receptor (EGFR) inhibitor, is highly active as a single agent and in combination with standard chemotherapy and/or RT. Future studies should focus to determine the optimal pharmacotherapeutic regimens for use in locally advanced HNSCC.

The objective of the current study was to investigate the effects of Rap1GAP on invasion and progression of head and neck squamous cell carcinoma (SCC) and the role of matrix metalloproteinase (MMP) 9 and MMP2 in this process. Rap1GAP functions by switching off Rap1, the Ras-like protein that has been associated with carcinogenesis. Previous findings suggest that Rap1GAP acts as a tumor suppressor protein in SCC by delaying the G(1)-S transition of the cell cycle. However, cells transfected with Rap1GAP exhibit a more invasive phenotype than corresponding vector-transfected control cells. MMP2 and MMP9 are enzymes that mediate SCC invasion via degradation of the extracellular matrix. Using SCC cells transfected with empty vector or Rap1GAP, cell invasion and MMP secretion were determined by Matrigel assays and gelatin zymography, respectively. Rap1GAP up-regulated transcription and secretion of MMP2 and MMP9, as assayed by quantitative reverse transcription-PCR and zymography. Furthermore, chemical and RNA interference blockade of MMP2/MMP9 inhibited invasion by Rap1GAP-transfected cells. Immunohistochemical staining of a human oropharyngeal SCC tissue microarray showed that Rap1GAP and MMP9 expression and staining intensity are correlated (P < 0.0001) and that, in early N-stage lesions of SCC, high MMP9 is prognostic of poor disease-specific survival (P < 0.05). Furthermore, Rap1GAP staining is correlated with MMP2 (P < 0.03). MMP2 in combination with N stage has a prognostic effect on time to indication of surgery at primary site. MMP2 intensity is also positively correlated with T stage (P < 0.015). In conclusion, Rap1GAP inhibits tumor growth but induces MMP2- and MMP9-mediated SCC invasion and tumor progression, suggesting a role for this protein as a biomarker for early N-stage, aggressive SCCs.

Angiogenesis is well recognized as an essential process that influences not only the growth of head and neck squamous cell carcinoma (HNSCC) but also promotes its invasive and metastatic behavior. The critical role of copper in multiple facets of angiogenesis makes it an important therapeutic target. Tetrathiomolybdate is a potent copper chelator, which has shown remarkable ability to suppress angiogenesis. Although this may involve multiple mechanisms, the effects on vascular endothelial growth factor (VEGF) are pivotal. In previous work, tetrathiomolybdate suppressed production of several proangiogenic cytokines by HNSCC cell lines. Given these results, we hypothesized that tetrathiomolybdate would impair tumor growth and metastasis by HNSCC. To test this concept, we evaluated the effects of long-term tetrathiomolybdate treatment on the growth and metastatic progression of HNSCC using a xenograft animal model. The results showed that tetrathiomolybdate treatment is able to maintain effective inhibition of angiogenesis. There was a significant reduction in the tumor size and vascularity with evident gross necrosis in the tetrathiomolybdate-treated animals. These effects were highly correlated with suppression of human VEGF expressed in the developing tumors as well as the mouse VEGF levels detected in the plasma. Moreover, tetrathiomolybdate treatment drastically suppressed the development of lung metastases. Taken together, these results show that tetrathiomolybdate can act long-term as a suppressor of vascularity and inhibit the growth of metastasis in this model of HNSCC.

Over 70% of patients with head and neck squamous cell carcinoma (HNSCC) present with locoregionally advanced stage III and IV disease. In spite of aggressive therapy, locoregional disease recurs in 60% and metastatic disease develops in 15% to 25% of patients causing a major decline in quality and length of life. Therefore, there is a need to identify and understand genes that are responsible for inducing an aggressive HNSCC phenotype. Evidence has shown that protein kinase C (PKC) epsilon is a transforming oncogene and may play a role in HNSCC progression. In this study, we determine the downstream signaling pathway mediated by PKC epsilon to promote an aggressive HNSCC phenotype. RNA interference knockdown of PKC epsilon in UMSCC11A and UMSCC36, two highly invasive and motile HNSCC cell lines with elevated endogenous PKC epsilon levels, resulted in cells that were significantly less invasive and motile than the small interfering RNA-scrambled control transfectants; 51 +/- 5% (P < 0.006) and 49 +/- 3% (P < 0.010) inhibition in invasion and 69 +/- 1% (P < 0.0005) and 66 +/- 3% (P < 0.0001) inhibition in motility, respectively. PKC epsilon-deficient UMSCC11A clones had reduced levels of active and serine-phosphorylated RhoA and RhoC. Moreover, constitutive active RhoA completely rescued the invasion and motility defect, whereas constitutive active RhoC completely rescued the invasion and partially rescued the motility defect of PKC epsilon-deficient UMSCC11A clones. These results indicate that RhoA and RhoC are downstream of PKC epsilon and critical for PKC epsilon-mediated cell invasion and motility. Our study shows, for the first time, that PKC epsilon is involved in a coordinated regulation of RhoA and RhoC activation, possibly through direct post-translational phosphorylation.

Purpose: Survival rates for squamous cell carcinoma of the head and neck (SCCHN) have remained unchanged for several decades due to local tumor recurrences as well as regional and distant metastases. Recent evidence has shown that RhoC GTPase is overexpressed in stages III and IV regionally metastatic SCCHN compared with stages I and II localized disease. This study evaluated the expression of RhoC in head and neck carcinoma and investigated the prognostic use of this marker on a large cohort of previously untreated patients with SCCHN., Experimental Design: Standard Western blot techniques were used to evaluate RhoC protein expression in nine established head and neck cancer cell lines and in normal oral epithelium. In vivo expression of RhoC in metastatic and nonmetastatic SCCHN was investigated using immunohistochemical analysis on a tissue microarray composed of 113 independent tumor samples. RhoC expression was analyzed as it related to clinical and pathologic variables of interest., Results: Levels of RhoC protein were increased in the SCCHN cell lines compared with normal oral epithelium. The in vivo expression of RhoC correlated with advanced clinical stage and lymph node metastases for the entire patient cohort as well as in small primary tumors (T(1) and T(2))., Conclusions: This study is the first to examine the expression of RhoC GTPase protein in SCCHN and normal squamous epithelium. It is clear from the results that RhoC is a specific marker of lymph node metastases in patients with this challenging form of carcinoma. RhoC levels seem to identify a subset of patients with early tumor stage primary tumors and high metastatic potential that might benefit from more aggressive therapy. Through continued investigation, blockade of RhoC activity may be a potential target in the development of novel strategies for treating metastases of head and neck cancer.

Objective: To assess the effect of tetrathiomolybdate on cytokine expression, angiogenesis, and tumor growth rate in human squamous cell carcinoma (SCC)., Design: Three human SCC cell lines were used in this study for both in vitro and in vivo investigations. Conditioned media from untreated and tetrathiomolybdate-treated cell lines were compared with regard to cytokine levels, endothelial cell chemotaxis, endothelial cell tubule formation, and migration and the ability to induce angiogenesis in a rat aortic ring array. In vivo UM-SCC-38 was seeded onto tissue-engineered scaffolds and surgically implanted into the flanks of immunodeficient mice. Tumor growth rates and the level of angiogenesis were compared after 2 weeks of therapy., Setting: A tertiary care facility., Results: In this study, we demonstrate that tetrathiomolybdate significantly decreases the secretion of interleukin 6 and basic fibroblast growth factor by head and neck SCC (HNSCC) cell lines in vitro. Furthermore, we demonstrate that tetrathiomolybdate significantly decreases the secretion of interleukin 6 and basic fibroblast growth factor by HNSCC cell lines in vitro. Furthermore, tetrathiomolybdate treatment of HNSCC cell lines results in significantly decreased endothelial cell chemotaxis, tubule formation, and neovascularization in a rat aortic ring assay. This in vitro evidence of decreased angiogenesis by tetrathiomolybdate is confirmed in vivo by using a severe combined immunodeficiency disorder mouse model in which tetrathiomolybdate therapy is shown to prevent human blood vessel formation. Finally, human HNSCC implanted into immunodeficient mice grow to a much larger size in untreated mice compared with those treated with 0.7 mL/kg per day of oral tetrathiomolybdate., Conclusions: These findings illustrate the ability of tetrathiomolybdate to down-regulate proinflammatory and proangiogenic cytokines in HNSCC. These observations are potentially exciting from a clinical perspective because a global decrease in these cytokines may decrease tumor aggressiveness and reverse the resistance to chemotherapy and radiation therapy seen in this tumor type.

Background: The survival of patients with head and neck squamous cell carcinoma (HNSCC) remains unaffected despite recent therapeutic advances. To reverse this trend, reliable and clinically applicable markers of tumor aggressiveness must be identified. One such marker may be the tumor-associated macrophage content. The authors hypothesized that tumor-associated macrophages contribute to HNSCC aggressiveness, and the objective of the current study was to prove this hypothesis using mRNA expression analysis and a large cohort of clinical specimens., Methods: Oligonucleotide microarray analysis was performed on a prospective cohort of 20 patients with previously untreated oral cavity or oropharynx squamous cell carcinoma (OC/OP SCCA) and on normal oropharyngeal mucosa from 4 patients. After determining whether macrophage chemoattractants were produced by tumors, conditioned media from three HNSCC cell lines were used to quantify macrophage migration in an in vitro assay. A high-density tissue microarray of 102 patients with previously untreated OC/OP SCCA was stained immunohistochemically for CD68 to identify tissue macrophages, and the results were correlated with clinicopathologic data and survival., Results: Monocyte chemoattractant protein 1 was up-regulated significantly in tumors compared with normal mucosa (P=0.0025; fold change=1.89). All University of Michigan SCC tumor cell line conditioned media caused a significant increase in macrophage migration (P <0.05). Tissue microarray data revealed that macrophage content of the primary tumor was associated strongly with lymph node metastasis (P <0.0001), extracapsular lymph node spread (P=0.0001), and advanced clinical disease stage (P=0.0002). When it was evaluated along with other clinicopathologic data, the macrophage content was found to be an independent predictor of lymph node metastasis (P <0.0001)., Conclusions: Primary tumor macrophage content is a strong predictor of tumor aggressiveness in HNSCC.

Objective To assess the effect of tetrathiomolybdate on cytokine expression, angiogenesis, and tumor growth rate in human squamous cell carcinoma (SCC). Design Three human SCC cell lines were used in this study for both in vitro and in vivo investigations. Conditioned media from untreated and tetrathiomolybdate-treated cell lines were compared with regard to cytokine levels, endothelial cell chemotaxis, endothelial cell tubule formation, and migration and the ability to induce angiogenesis in a rat aortic ring array. In vivo UM-SCC-38 was seeded onto tissue-engineered scaffolds and surgically implanted into the flanks of immunodeficient mice. Tumor growth rates and the level of angiogenesis were compared after 2 weeks of therapy. Setting A tertiary care facility. Results In this study, we demonstrate that tetrathiomolybdate significantly decreases the secretion of interleukin 6 and basic fibroblast growth factor by head and neck SCC (HNSCC) cell lines in vitro. Furthermore, we demonstrate that tetrathiomolybdate significantly decreases the secretion of interleukin 6 and basic fibroblast growth factor by HNSCC cell lines in vitro. Furthermore, tetrathiomolybdate treatment of HNSCC cell lines results in significantly decreased endothelial cell chemotaxis, tubule formation, and neovascularization in a rat aortic ring assay. This in vitro evidence of decreased angiogenesis by tetrathiomolybdate is confirmed in vivo by using a severe combined immunodeficiency disorder mouse model in which tetrathiomolybdate therapy is shown to prevent human blood vessel formation. Finally, human HNSCC implanted into immunodeficient mice grow to a much larger size in untreated mice compared with those treated with 0.7 mL/kg per day of oral tetrathiomolybdate. Conclusions These findings illustrate the ability of tetrathiomolybdate to down-regulate proinflammatory and proangiogenic cytokines in HNSCC. These observations are potentially exciting from a clinical perspective because a global decrease in these cytokines may decrease tumor aggressiveness and reverse the resistance to chemotherapy and radiation therapy seen in this tumor type. [ABSTRACT FROM AUTHOR]