Everolimus downregulates STAT3/HIF-1α/VEGF pathway to inhibit angiogenesis and lymphangiogenesis in TP53 mutant head and neck squamous cell carcinoma (HNSCC).

TP53 mutant head and neck squamous cell carcinoma (HNSCC) patients exhibit poor clinical outcomes with 50-60% recurrence rates in advanced stage patients. In a recent phase II clinical trial, adjuvant therapy with everolimus (mTOR inhibitor) significantly increased 2-year progression-free survival in p53 mutated patients. TP53-driven mTOR activation in solid malignancies causes upregulation of HIF-1α and its target, downstream effector VEGF, by activating STAT3 cell signaling pathway. Here, we investigated the effects of everolimus on the STAT3/HIF-1α/VEGF pathway in TP53 mutant cell lines and xenograft models. Treatment with everolimus significantly inhibited cell growth in vitro and effectively reduced the growth of TP53 mutant xenografts in a minimal residual disease (MRD) model in nude mice. Everolimus treatment was associated with significant downregulation of STAT3/HIF-1α/VEGF pathway in both models. Further, treatment with everolimus was associated with attenuation in tumor angiogenesis and lymphangiogenesis as indicated by decreased microvessel density of vascular and lymphatic vessels in HN31 and FaDu xenografts. Everolimus downregulated the STAT3/HIF-1α/VEGF pathway to inhibit growth and in vitro tube formation of HMEC-1 (endothelial) and HMEC-1A (lymphatic endothelial) cell lines. Our studies demonstrated that everolimus inhibits the growth of TP53 mutant tumors by inhibiting angiogenesis and lymphangiogenesis through the downregulation of STAT3/HIF-1α/VEGF signaling.

A Randomized Multi-institutional Phase II Trial of Everolimus as Adjuvant Therapy in Patients with Locally Advanced Squamous Cell Cancer of the Head and Neck.

PURPOSE: Investigate whether adjuvant everolimus, an mTOR inhibitor, improves progression-free survival (PFS) in advanced-stage head and neck squamous cell carcinoma (HNSCC) and provide outcomes related to correlative biological factors associated with disease control. PATIENTS AND METHODS: This was a prospective, randomized, double-blind phase II trial of patients with advanced-stage HNSCC from 13 institutions who were confirmed disease-free post-definitive therapy and enrolled between December 2010 and March 2015. Patients received adjuvant everolimus or placebo daily (10 mg, oral) for a maximum of 1 year. p16 IHC as a surrogate marker for human papillomavirus infection and whole-exome sequencing were performed. Cox proportional hazard models estimated hazard rates. Log-rank tests evaluated differences in survival. The primary endpoint was PFS. Secondary endpoints and objectives included overall survival (OS) and toxicity assessment. RESULTS: 52 patients [median (range) age, 58 (37-76) years; 43 men (83%), 9 women (17%)] were randomized to placebo (n = 24) or everolimus (n = 28). PFS favored everolimus, but was not significant [log-rank P = 0.093; HR = 0.44; 95% confidence interval (CI), 0.17-1.17]. There was no difference in OS (P = 0.29; HR = 0.57; 95% CI, 0.20-16.2). Everolimus resulted in significant improvement in PFS for p16-negative patients (n = 31; P = 0.031; HR = 0.26; 95% CI, 0.07-0.97), although subgroup analysis showed no difference for p16-positive patients (n = 21; P = 0.93). Further, PFS was significantly higher in TP53-mutated (TP53mut) patients treated with everolimus compared with placebo (log-rank P = 0.027; HR = 0.24; 95% CI, 0.06-0.95). No treatment difference was seen in patients with TP53 wild-type tumors (P = 0.79). CONCLUSIONS: p16-negative and TP53mut patients may benefit from adjuvant treatment with everolimus.

Rapalogs induce non-apoptotic, autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma.

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Since mutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV-negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.

Impact of smoking on stage-specific survival in human papilloma virus-associated oropharyngeal squamous cell carcinoma.

BACKGROUND: The American Joint Committee on Cancer (AJCC) released a new staging system for human papilloma virus (HPV) positive oropharyngeal cancer (OPC) in their eighth edition. The role of smoking in HPV positive oropharyngeal cancer remains controversial and is not factored into the updated staging system. METHODS: Single institutional, retrospective chart review of patients with HPV positive OPC from 2009 to 2017 was completed. Dichotomized smoking data were collected into 0-9 and ≥10 pack-year histories. Kaplan-Meier survival curves compared overall survival (OS) for smokers and nonsmokers. RESULTS: Five-year OS was not statistically different in stage I or stage II HPV positive OPC comparing nonsmokers versus smokers, but worse in stage III smokers (38% vs. 76%, p < 0.05). CONCLUSION: Greater than 10 pack-year smoking status may negatively affect survival in late stage HPV positive OPC but not in early stage disease. HPV positive smokers may require additional risk stratification.

Comparing cell-free circulating tumor DNA mutational profiles of disease-free and nonresponders patients with oropharyngeal squamous cell carcinoma.

OBJECTIVE: The purpose of this study was to investigate whether somatic nonsynonymous variants in tumor tissue can potentially be identified in circulating cell-free DNA (cfDNA) of head and neck oropharyngeal squamous cell carcinoma (OPSCC) patients using next-generation sequencing and can predict recurrence or persistence disease. METHODS: A total of 22 OPSCC patients with tumor tissue and respective plasma samples were included in this study. Matching cfDNA and tumor tissues were processed, and DNA sequencing was conducted using the MiSeq platform. Variants were identified using Biomedical Genomic Workbench and Genialis's online data analysis platform for Swift Biosciences' Accel-amplicon panels. RESULTS: Among 11 nonresponders, 6 matched mutations were detected in 5 patients suggesting a predictive factor for patients with likelihood of recurrence. The matched variants and their allele frequencies identified in the nonresponder group were (tumor DNA/cfDNA in %): TP53 G325fs (27/0.62), TP53 R282W (48/1.74), TP53 R273C (39/2.17), FBXW7 R505G (30/0.6), FBXW7 R505L (31/0.65), and TP53 Q331H (56.5/0.52). Interestingly, the matched somatic mutations were only detected in patients who did not respond to therapy or had persistent disease. CONCLUSIONS: Somatic nonsynonymous variants in tumor tissue can potentially be identified in cfDNA of OPSCC patients using NGS. The likelihood of variant detection in cfDNA is greater in nonresponders, especially in human papillomavirus-negative nonresponders, rendering it beneficial as a less invasive detection method for disease persistence/recurrence and prognosis. LEVEL OF EVIDENCE: Cohort study.

Curcumin gum formulation for prevention of oral cavity head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: Head and neck squamous cell carcinoma represents the sixth most common cancer. As a result of field cancerization, second primaries and recurrences are high. Hence, research has focused on chemoprevention. Curcumin, a polyphenol compound with anticarcinogenic properties, is one such promising nutraceutical. As poor bioavailability limits curcumin's use, a novel gum formulation was tested allowing for direct mucosal absorption into the bloodstream. This preliminary study validates curcumin gum efficacy by assessing release and transmucosal absorption, along with measuring its effects on serum cytokine levels. STUDY DESIGN: Clinical trial. METHODS: Protocols consisting of initial chew (chewing gum for 30 minutes) and revised chew (alternating chewing and parking gum against buccal mucosa for 30 minutes) were tested in healthy volunteers. High-performance liquid chromatography measured remnant curcumin in chewed gum, serum, and saliva. Serum levels were assayed for 15 proinflammatory cytokines via multiplex analysis. RESULTS: Revised chew samples demonstrated significantly higher curcumin release and absorption (P = .0078). Curcumin serum levels were significantly higher at 4 hours in samples > 2.0 g of curcumin release (P = .01). As saliva levels decreased, a concurrent increase in serum levels was observed, with no significance in the inverse relationship (P = .1423). When evaluating differences between gender, race, and age, the Asian population showed significantly lower curcumin release and serum levels (P = .009). CXCL1 (GRO-α) and TNF-α were significantly decreased in serum after chewing the gum (P = .036, P < .001, respectively). CONCLUSIONS: Enhanced mucosal contact appears critical in improving curcumin release and absorption. CXCL1 and TNF-α both represent potential biomarkers for the future study of curcumin chemoprevention. LEVEL OF EVIDENCE: 2b Laryngoscope, 129:1597-1603, 2019.

Local and systemic Curcumin C3 complex inhibits 4NQO-induced oral tumorigenesis via modulating FGF-2/FGFR-2 activation.

Head and Neck Squamous cell carcinoma (HNSCC) can be characterized by synchronous tumors in the upper aerodigestive tract. Second primary tumors as a result of field cancerization are a significant problem amongst patients with risk factors for HNSCC, indicating a need for chemo preventive agents. We investigated the efficacy of local and systemic Curcumin C3 complex (C3); a purified mixture of Curcumin, bisdemethoxy Curcumin and demethoxy Curcumin as a chemo preventative agent in 4-nitroquinoline-1-oxide (4NQO)-induced tumorigenesis in mice. The effect of local C3 application was compared to C3 administered orally and in combination with systemic administration. C57Bl/6 mice were administered 4NQO (50 µg/ml) in the drinking water for 16 weeks. At 12 weeks, mice were subjected to daily treatment with either vehicle (control), or 15 mg C3 complex by local delivery, gavage, or combined local and gavage for 28 days (16 week time point), and followed up to 22 weeks. Compared to local and oral systemic C3 administration, combination of local and systemic application significantly decreased multiplicity of 4NQO-induced preneoplastic and neoplastic lesions (p<0.05). Treatment with C3 correlated with a decrease in cell proliferation compared to the 4NQO group. Further, pre-treatment with C3 complex significantly attenuated 4NQO induced expression of basic fibroblast growth factor (FGF-2) and its cognate receptor FGFR-2, suggesting an important role of FGF-2/FGFR-2 axis in chemoprevention of HNSCC (p<0.05). Our findings suggest that a combination of local and systemic C3 complex could effectively target proliferation and inhibit 4NQO-induced tumorigenesis via modulation of the FGF-2/FGFR-2 axis as a mechanism for its efficacy.

Survival outcomes based on systemic agent used concurrently with radiation in human-papillomavirus associated oropharyngeal cancer.

PURPOSE: To investigate survival outcomes of patients treated with concurrent cetuximab and radiotherapy for primary management of both HPV positive and negative OPSCC, and compare the results to traditional platinum-based therapy. We hypothesize that the use of cetuximab in the HPV positive OPSCC patients will result in inferior survival based on tumor biological differences. STUDY DESIGN: A single institution retrospective analysis of 304 patients. The primary outcomes of interest were 1) overall survival and 2) relapse free survival. Pearson Chi-square tests were used to compare proportions between subgroups. One-way analysis of variance was used to compare the continuous variable age between subgroups. Kaplan-Meier method was used to produce survival curves, and comparisons between survival curves were made using the log-rank test. The survival functions comparing subgroups of chemotherapy were analyzed using semi-parametric (i.e. Cox proportional hazards models) and fully parametric regression with Weibull distributions. Multivariable models were adjusted for age at diagnosis, gender, race, chemotherapy, radiotherapy, and cancer stage. RESULTS: In the multivariable analysis, the hazard ratio for cetuximab compared to cisplatin or carboplatin/paclitaxel was HR=0.77[95% CI = 0.67, 0.90] in the HPV - group, suggesting more favorable outcomes for the patients on cetuximab in this group. However, in the HPV + cohort, the hazard ratio was 1.88 [95% CI = 1.42, 2.50] for those patients treated with cetuximab vs platinum-based therapy. CONCLUSIONS: Our data suggest that cetuximab may have inferior outcomes in HPV-associated OPSCC compared to traditional platinum-based therapy.

Rapamycin targets Interleukin 6 (IL-6) expression and suppresses endothelial cell invasion stimulated by tumor cells.

mTOR inhibitors have potent antiangiogenic and anti-lymphangiogenic effects in addition to their growth inhibitory effects in head and neck squamous cell carcinoma (HNSCC). Lymphatogenous spread is much more predominant in HNSCC than hematogenous spread and significantly decreases survival. In this study we evaluated the effects of rapamycin on targeting tumor-stroma crosstalk in HNSCC. HNSCC tumor cells (FaDu) and human lymphatic endothelial cells (HMEC-1A) were co-cultured in various combinations using transwell cell culture inserts to study tumor-stroma crosstalk and the effects of mTOR inhibitor rapamycin. Levels of growth factors and cytokines in cell culture media were measured using Milliplex bead immunoassay (EMD Millipore) and ELISA assay (R&D Systems). We found that conditioned media collected from tumor cells or co-culture with tumor cells significantly increased the invasiveness of lymphatic and blood vascular endothelial cells (P<0.05), while there was no effect of conditioned media collected from endothelial cell cultures or co-culture with endothelial cells on tumor cell invasiveness. There was a significant effect of rapamycin on both baseline and tumor cell stimulated invasiveness of endothelial cells (P<0.05). Importantly the level of IL-6 secreted in media increased significantly in tumor-endothelial cell co-culture compared to monocultures. Rapamycin significantly suppressed secretion of IL-6 by tumor cells (P<0.05). Thus, HNSCC cells produce chemotactic stimuli that promote endothelial cell invasion toward tumor cells that can stimulate lymphangiogenesis. Rapamycin effectively reverted the stimulatory effect of IL-6 secreted by tumor cells on endothelial cell invasiveness.

Biomarker and Pathologic Predictors of Cutaneous Squamous Cell Carcinoma Aggressiveness.

OBJECTIVE: Aggressive cutaneous squamous cell carcinoma (cSCC) patients are at increased risk of metastasis. Currently, there are no accepted criteria or biomarkers for reliably predicting individuals at risk for recurrence and metastasis. Our objective is to determine if pS6 and pERK can predict cSCC aggressiveness and to identify primary tumor characteristics that may predict parotid metastasis. STUDY DESIGN: Retrospective case series. SETTINGS: Tertiary care center. SUBJECTS AND METHODS: An Institutional Review Board-approved retrospective review was performed for patients with facial cSCC, with and without metastasis to the parotids. Subjects for the study were identified through the Louisiana Tumor Registry, Veterans Medical Records, and LSU Health-Shreveport pathology database. Tumor specimens from patients with cSCC and cSCC with parotid metastasis were analyzed for pERK and pS6 expression through immunohistochemistry. To identify risk factors for tumor aggressiveness, multiple logistic regression analysis was used to evaluate patients with cSCC that was metastatic to the parotid and managed surgically. RESULTS: cSCC with parotid metastasis specimens exhibited significantly higher average pS6 but not pERK positivity than those from cSCC without metastasis (P < .05). Primary lesion-positive margins (P < .01), size of the skin tumor (P < .01) and degree of tumor differentiation (P < .01) were significantly associated with parotid metastasis. CONCLUSION: Surgical history of cSCC, primary lesion-positive margins, degree of differentiation, and lesion size together with pS6 positivity appear to be predictors of cSCC aggressiveness and should prompt increased monitoring or elective parotidectomy.

Photopreventive Effect and Mechanism of AZD4547 and Curcumin C3 Complex on UVB-Induced Epidermal Hyperplasia.

Aggressive cutaneous squamous cell carcinoma (cSCC) of the skin is the second most common type of skin cancer in the United States due to high exposure to ultraviolet B (UVB) radiation. In our previous studies, Curcumin C3 complex (C3), a standardized preparation of three curcumonoids, delayed UVB-induced tumor incidence and inhibited multiplicity. Exposure to UVB activates mTOR and FGFR signaling that play a key role in skin tumorigenesis. The purpose of this study was to investigate the efficacy of C3 complex to afford protection against acute UVB-induced hyperproliferation by targeting the mTOR and FGFR signaling pathways. Pretreatment with C3 complex significantly inhibited UVB-induced FGF-2 induction, FGF-2-induced cell proliferation, progression and colony formation, mTORC1 and mTORC2 activation, and FGFR2 phosphorylation in the promotion-sensitive JB6 cells epithelial cells. Further, FGFR was critical for UVB-induced mTOR activation, suggesting an important role of FGFR2 in UVB-induced mTOR signaling. SKH-1 mice pretreated with C3 (15 mg/kg/b.w.) for 2 weeks followed by a single exposure to UVB (180 mj/cm(2)) significantly attenuated UVB-induced mTORC1, mTORC2, and FGFR2 activation. To further assess the role of FGFR in UVB-induced hyperproliferation, SKH-1 mice were pretreated with AZD4547 (5 mg/kg/b.w.); a selective pan-FGFR kinase inhibitor followed by single exposure to UVB (180 mj/cm(2)). AZD4547 significantly inhibited UVB-induced mTORC1 and mTORC2 activation, epidermal hyperplasia and hyperproliferation. Our studies underscore the importance of FGFR signaling in UVB-induced acute skin changes and the role of FGFR/mTOR signaling in mediating the effects of C3 complex in the pathogenesis of skin cancer.

Interobserver agreement of confocal laser endomicroscopy for detection of head and neck neoplasia.

OBJECTIVES/HYPOTHESIS: We have described the feasibility of using the probe-based confocal laser endomicroscopy (pCLE) in differentiating benign from malignant lesions of the head and neck. Therefore, we wanted to determine the interobserver agreement of pCLE offline images of noncancerous, precancerous, and cancerous lesions of the head and neck. STUDY DESIGN: Single tertiary referral center. METHODS: In the feasibility study, image criteria for nondysplasia, dysplasia, and cancer were defined. The pCLE was performed before lesions were biopsied. Fifty offline images and 10 videos of good quality were selected. Seven surgeons and one pathologist were asked to review and categorize the images into the three categories above. The overall accuracy of 29 offline pCLE images and six videos were compared with histopathology. Interobserver agreement and accuracy kappa (κ) scores were measured with 95% confidence intervals (CI). RESULTS: There were six nondysplasia, seven dysplasia, and 11 squamous cell carcinoma (SCCA) cases, each with multiple images. There was substantial agreement between the eight reviewers on the pCLE images and videos (κ = 0.66; 95% CI 0.51-0.82 and κ = 0.71; 95% CI 0.42-0.97, respectively). The overall agreement with the final histopathology was also substantial for both the images and video sequences (κ = 0.70; 95% CI 0.50-0.88 and κ = 0.73; 95% CI 0.39-1.00, respectively). CONCLUSION: The ability to differentiate normal mucosa, dysplasia, and invasive SCCA using pCLE with high accuracy and reliability was demonstrated. This technology has the potential to decrease sampling error of lesions in the head and neck. This is the first study to test the reliability of this technology in mucosal lesions of the head and neck. LEVEL OF EVIDENCE: NA. Laryngoscope, 126:632-637, 2016.

Enhanced Systemic Bioavailability of Curcumin Through Transmucosal Administration of a Novel Microgranular Formulation.

BACKGROUND/AIM: Curcumin is a promising nutraceutical for chemoprevention of head and neck squamous cell carcinoma (HNSCC). Capsular formulations of curcumin demonstrate low systemic bioavailability. We aimed to determine if curcumin levels were higher in healthy volunteers and cancer patients with microgranular curcumin that allows for transmucosal absorption and identify a consistent biomarker. PATIENTS AND METHODS: Eight healthy volunteers and 15 HNSCC patients completed the trials. Serum levels of curcumin were measured by HPLC. Biological activity of curcumin was assessed with Multiplex Immunoassay and immunohistochemistry. RESULTS: We achieved higher serum levels of curcumin compared to trials using capsular formulation. In cancer patients a significant decrease in expression of fibroblast growth factor-2 (FGF-2) in post-biopsy samples and decreased serum levels of FGF-2, granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-17 (IL-17) (p<0.05) was observed. CONCLUSION: Transmucosal administration of microgranular curcumin leads to enhanced curcumin bioavailability that is associated with significant biological effects.

Association between human papilloma virus/Epstein-Barr virus coinfection and oral carcinogenesis.

BACKGROUND: The recent epidemic of head and neck squamous cell carcinomas associated with human papilloma virus (HPV) has not addressed its association with lymphoid tissue in the oropharynx or the potential role of Epstein-Barr virus (EBV)/HPV coinfection. METHODS: The prevalence of HPV and EBV infection/coinfection and CD21 mRNA expression were determined in normal and cancerous tissues from the oropharynx using in situ hybridization (ISH), p16, and quantitative reverse transcriptase PCR (qRT-PCR). The effects of coinfection on tumorigenicity were evaluated using proliferation and invasion assays. RESULTS: Normal oropharynx, tonsil, non-cancer base of tongue (BOT), and BOT from sleep apnea patients demonstrated EBV positivity ranging from 7% to 36% depending on the site and methods of detection used (qRT-PCR or ISH). Among non-malignant BOT samples, HPV positivity was noted only in 20%. The percent of tonsil and BOT cancers positive for HPV (up to 63% and 80%, respectively) or coinfected with HPV/EBV (up to 25% and 70%, respectively) were both significantly associated with cancer status. Notably, HPV/EBV coinfection was observed only in malignant tissue originating in lymphoid-rich oropharynx sites (tonsil, BOT). CD21 mRNA (the major EBV attachment receptor) was detected in tonsil and BOT epithelium, but not in soft-palate epithelium. Coinfected cell lines showed a significant increase in invasiveness (P < 0.01). CONCLUSIONS: There is a high prevalence of HPV/EBV infection and coinfection in BOT and tonsil cancers, possibly reflecting their origins in lymphoid-rich tissue. In vitro, cells modeling coinfection have an increased invasive potential.

Confocal Laser Endomicroscopy in the Detection of Head and Neck Precancerous Lesions.

OBJECTIVE: This study aimed to determine the feasibility of using probe-based confocal laser endomicroscopy (pCLE) in the diagnostic differentiation of non-neoplastic lesions from precancerous and cancerous lesions of head and neck patients. STUDY DESIGN: Diagnostic test evaluation. SETTING: Louisiana State University Health Shreveport. SUBJECTS AND METHODS: Intravenous injection of fluorescein was given to patients with precancerous and cancerous head and neck lesions (n = 21) followed by the use of a 1.8-mm GastroFlex probe in the oral cavity with subsequent biopsies of selected areas. Probe-based confocal laser endomicroscopy images were compared to histologic evaluation of visualized sites using sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV). RESULTS: The dorsal surface of the tongue was not well visualized. The remaining nonkeratinized subsites, including the buccal mucosa, floor of mouth, and ventral tongue, were well visualized. Diagnoses based on pCLE images correlated well with the gold standard diagnoses based on tissue histology. The overall sensitivity for diagnosis of dysplasia versus nondysplasia was 80.0% (95% confidence interval [CI], 62.0-98.0), specificity and PPV were 100%, and the NPV was 80.0% (95% CI, 60.0-100.0). The overall specificity, sensitivity, PPV, and NPV for pCLE diagnosis of carcinoma versus nondysplasia were 100%. The overall sensitivity for diagnosis of carcinoma versus dysplasia was 85.7% (95% CI, 73.0-99.0), specificity and PPV were 100%, and the NPV was 80.0% (95% CI, 60.0-100.0). CONCLUSION: The pCLE is a promising method for differentiating between nondysplastic, precancerous, and cancerous lesions of the head and neck.

Serum biomarkers in head and neck squamous cell cancer.

IMPORTANCE: Serum biomarkers may be useful in the evaluation of suspected head and neck squamous cell cancer (HNSCC) and as indicators of treatment success or failure in adjuvant and chemopreventive clinical trials. OBJECTIVE: To determine serum cytokine and chemokine concentrations altered in patients with HNSCC compared with healthy volunteers to identify potential biomarkers. DESIGN, SETTING, AND PARTICIPANTS: A retrospective experimental laboratory study at Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport. Serum samples from 50 patients with stages II, III, and IV HNSCC and 20 healthy volunteers were available for study. Primary tumor sites represented in the patient group included the nasal cavity, oral cavity, oropharynx, hypopharynx, and larynx. INTERVENTIONS: Following institutional review approval and written informed consent, blood samples were drawn from patients. No intervention, to include any kind of diagnostic workup or treatment, was provided to patients during the course of this study. MAIN OUTCOMES AND MEASURES: The main outcome measures were the quantification of cytokine and chemokine concentrations in serum samples. Luminex multiplex panel technology was used for simultaneous measurement of 18 analytes, including fibroblast growth factor 2, granulocyte-macrophage colony-stimulating factor, growth-related oncogene, interferon (IFN)-γ, interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, inducible protein (IP)-10, soluble CD40 ligand, tumor necrosis factor, and vascular endothelial growth factor. RESULTS: The serum samples of patients with HNSCC contained lower levels of IFN-γ (mean patient serum level, 6.08 pg/mL, compared with the mean control level, 26.20 pg/mL; P = .004), IL-13 (mean patient serum level, 2.85 pg/mL, compared with the control mean level, 7.23 pg/mL; P = .02), and macrophage inflammatory protein-1ß (MIP-1ß) (mean patient serum level, 14.91 pg/mL, compared with the mean control level, 28.98 pg/mL; P = .004), and elevated levels of IP-10 (mean patient serum level, 359.24 pg/mL, compared with mean control level, 216.40 pg/mL; P = .04). All other markers tested were not significantly different between patients with cancer and controls. CONCLUSIONS AND RELEVANCE: This pilot study demonstrated a significant decrease in serum IFN-γ, IL-13, and MIP-1ß levels and a significant elevation of serum IP-10 concentration in patients with HNSCC, irrespective of primary tumor site. If validated in larger, independent studies, these serum biomarkers may be useful in the diagnosis and treatment of HNSCC. In the future, a defined, multianalyte screening panel could facilitate early diagnosis of HNSCC, allowing for earlier treatment and thereby reducing patient mortality.

Anti-lymphangiogenic properties of mTOR inhibitors in head and neck squamous cell carcinoma experimental models.

BACKGROUND: Tumor dissemination to cervical lymph nodes via lymphatics represents the first step in the metastasis of head and neck squamous cell carcinoma (HNSCC) and is the most significant predictor of tumor recurrence decreasing survival by 50%. The lymphatic suppressing properties of mTOR inhibitors are not yet well understood. METHODS: Lymphatic inhibiting effects of rapamycin were evaluated in vitro using two lymphatic endothelial cell (LEC) lines. An orthotopic mouse model of HNSCC (OSC-19 cells) was used to evaluate anti-lymphangiogenic effects of rapamycin in vivo. The incidence of cervical lymph node metastases, numbers of tumor-free lymphatic vessels and those invaded by tumor cells in mouse lingual tissue, and expression of pro-lymphangiogenic markers were assessed. RESULTS: Rapamycin significantly decreased lymphatic vascular density (p = 0.027), reduced the fraction of lymphatic vessels invaded by tumor cells in tongue tissue (p = 0.013) and decreased metastasis-positive lymph nodes (p = 0.04). Rapamycin also significantly attenuated the extent of metastatic tumor cell spread within lymph nodes (p < 0.0001). We found that rapamycin significantly reduced LEC proliferation and was correlated with decreased VEGFR-3 expression in both LEC, and in some HNSCC cell lines. CONCLUSIONS: The results of this study demonstrate anti-lymphangiogenic properties of mTOR inhibitors in HNSCC. mTOR inhibitors suppress autocrine and paracrine growth stimulation of tumor and lymphatic endothelial cells by impairing VEGF-C/VEGFR-3 axis and release of soluble VEGFR-2. In a murine HNSCC orthotopic model rapamycin significantly suppressed lymphovascular invasion, decreased cervical lymph node metastasis and delayed the spread of metastatic tumor cells within the lymph nodes.

Curcumin inhibits UV radiation-induced skin cancer in SKH-1 mice.

OBJECTIVE: As skin cancer incidence increases, research has focused on novel chemopreventive agents that inhibit tumor formation. In prior experimentation, curcumin, a naturally occurring food substance and anticarcinogenic agent, inhibited cutaneous squamous cell carcinoma xenograft growth. We hypothesize curcumin will inhibit UVB radiation-induced skin cancer growth in mice, approximating a human chemopreventive model. STUDY DESIGN: Randomized experimental animal and laboratory study. SETTING: Louisiana State University Health Sciences Center-Shreveport, Louisiana. SUBJECTS AND METHODS: SKH-1 mice were pretreated with oral or topical curcumin or oral or topical control (n = 11/group) for 14 days. Mice received UVB radiation 3 times weekly for 24 weeks or were not radiated. Number of tumors formed and time to tumor onset for each mouse were recorded through tumor harvest after week 24. Tumor multiplicity and time to tumor onset were compared. RESULTS: Time to tumor onset was significantly shorter in control mice compared to mice receiving either oral (P = .025) or topical (P = .015) curcumin. A significant difference in the average number of tumors formed per mouse was seen, as fewer tumors were formed in the oral curcumin (P = .01) and topical curcumin (P = .01) groups, compared with respective controls. No significant difference in average number of tumors per mouse was seen between oral and topical curcumin (P = .56), suggesting that both routes were equally effective. CONCLUSION: Curcumin appears to inhibit skin cancer formation and prolong time to tumor onset when administered by either an oral or topical route. These data suggest that curcumin may have chemopreventive potential against skin cancer, necessitating future experimentation with human subjects.

Oral dysplasia and squamous cell carcinoma: correlation between increased expression of CD21, Epstein-Barr virus and CK19.

OBJECTIVES: Epstein-Barr virus is an orally transmitted human gammaherpesvirus that infects B lymphocytes and epithelial cells. Although most primary infections are asymptomatic, long term carriage of the virus can be associated with either lymphoid or epithelial malignancies. The association of EBV with oral squamous cell carcinomas is sporadic and it is uncertain if the virus is involved in initiation of the tumor or, possibly, in its progression. Complement receptor type 2, CR2 or CD21, is a receptor for the major attachment protein of EBV, which significantly enhances epithelial cell infection, but its expression on normal tissues is restricted to tonsil and adenoid epithelium. As cells become dysplastic they are reported to express higher levels of CK19. We sought to evaluate whether CD21 and CK19 expression change as oral epithelial cells outside Waldeyer's ring become dysplastic. MATERIALS AND METHODS: Epithelial cells were isolated by laser capture microdissection and levels of CD21, CK19 and EBV RNA were measured by quantitative reverse transcriptase PCR. RESULTS: We report that expression of CD21 increases in frequency and intensity as oral epithelial cells become more dysplastic and that expression correlates with an increase in infection by EBV. Tumors or dysplastic lesions that carry EBV also generally express higher levels of CK19 than those that do not. CONCLUSION: The findings suggest that dysplasia may make cells more susceptible to infection by EBV and that infection by the virus may alter the phenotype of the infected cell in a manner which could affect prognosis.