Inhibition of Epstein-Barr Virus Replication in Human Papillomavirus-Immortalized Keratinocytes.

Epstein-Barr virus (EBV) is implicated in the pathogenesis of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OSCC). EBV-associated cancers harbor a latent EBV infection characterized by a lack of viral replication and the expression of viral oncogenes. Cellular changes promoted by HPV are comparable to those shown to facilitate EBV latency, though whether HPV-positive cells support a latent EBV infection has not been demonstrated. Using a model of direct EBV infection into HPV16-immortalized tonsillar cells grown in organotypic raft culture, we showed robust EBV replication in HPV-negative rafts but little to no replication in HPV-immortalized rafts. The reduced EBV replication was independent of immortalization, as human telomerase-immortalized normal oral keratinocytes supported robust EBV replication. Furthermore, we observed reduced EBV lytic gene expression and increased expression of EBER1, a noncoding RNA highly expressed in latently infected cells, in the presence of HPV. The use of human foreskin keratinocyte rafts expressing the HPV16 E6 and/or E7 oncogene(s) (HPV E6 and E7 rafts) showed that E7 was sufficient to reduce EBV replication. EBV replication is dependent upon epithelial differentiation and the differentiation-dependent expression of the transcription factors KLF4 and PRDM1. While KLF4 and PRDM1 levels were unaltered, the expression levels of KLF4 transcriptional targets, including late differentiation markers, were reduced in HPV E6 and E7 rafts compared to their levels in parental rafts. However, the HPV E7-mediated block in EBV replication correlated with delayed expression of early differentiation markers. Overall, this study reveals an HPV16-mediated block in EBV replication, through E7, that may facilitate EBV latency and long-term persistence in the tumor context.IMPORTANCE Using a model examining the establishment of EBV infection in HPV-immortalized tissues, we showed an HPV-induced interruption of the normal EBV life cycle reminiscent of a latent EBV infection. Our data support the notion that a persistent EBV epithelial infection depends upon preexisting cellular alterations and suggest the ability of HPV to promote such changes. More importantly, these findings introduce a model for how EBV coinfection may influence HPV-positive (HPV-pos) OSCC pathogenesis. Latently EBV-infected epithelial cells, as well as other EBV-associated head-and-neck carcinomas, exhibit oncogenic phenotypes commonly seen in HPV-pos OSCC. Therefore, an HPV-induced shift in the EBV life cycle toward latency would not only facilitate EBV persistence but also provide additional viral oncogene expression, which can contribute to the rapid progression of HPV-pos OSCC. These findings provide a step toward defining a role for EBV as a cofactor in HPV-positive oropharyngeal tumors.

Curcumin inhibits carcinogen and nicotine-induced Mammalian target of rapamycin pathway activation in head and neck squamous cell carcinoma.

Curcumin appears to be a safe, bioactive food compound that is a potential chemopreventive for patients at a high risk for head and neck squamous cell carcinoma (HNSCC). Identification and validation of intermediate endpoints is an important step in evaluating chemopreventive agents. AKT/MTOR pathway biomarkers are intrinsic to the carcinogenic process as well as the mechanism of intervention with curcumin. Antiproliferative effects of curcumin were assayed in 9 HNSCC and a keratinocyte cell line. Nicotine, a genotoxic alkaloid involved in tobacco addiction, forms DNA adducts and has been implicated in upper aerodigestive tract cancer promotion. The antiproliferative effects of curcumin were associated with inhibition of the AKT/MTOR pathway in presence and absence of nicotine, which also induced this pathway. Curcumin was highly effective at suppressing growth of SCC40 xenografts and its activity is associated with modulation of MTOR's downstream target pS6. Curcumin at 15 mg significantly increased survival (286 ± 37 vs. 350 days) in the 4NQO carcinogenic model survival study. A major cause of lethal progression of HNSCC is local regional migration and invasion of malignant cells, and curcumin significantly inhibited cancer cell migration and invasion in vitro and in vivo where downregulation of pS6 was associated with a significant decrease in MMP-9. This is the first study to demonstrate that curcumin inhibits the adverse effects of nicotine by blocking nicotine-induced activation of the AKT/MTOR pathway in HNSCC, which retards cell migration. These studies indicate that inhibiting the AKT/MTOR pathway with curcumin may be useful as an oral chemopreventive agent.

Radical combined treatment of locally extensive head and neck cancer in the elderly.

PURPOSE: Few studies have described the effects of aggressive combined therapy for locally extensive head and neck cancer in the elderly. Our study evaluated the outcome of this particular cohort of patients after such treatments. METHODS: Survival, failure, morbidity, and complication rates were determined retrospectively in 43 elderly patients with stage III or IV head and neck cancer who underwent curative surgery and postoperative radiotherapy (n = 33) or neoadjuvant, 3-drug chemotherapy plus radiotherapy (n = 10) between the years 1977 and 1992. RESULTS: The crude survival rate at 3 years was 27% in patients managed by surgery plus radiotherapy, and 30% in individuals treated with chemoradiation; the corresponding locoregional failure rates were 23% and 30%; and the distant failure rates were 13% and 0%, respectively. The acute toxicity rate was 12% in the surgery plus radiotherapy group and 30% in the chemoradiation patients; the corresponding late complication rates were 0% and 10%. There were no toxic deaths. CONCLUSION: Radical combined treatments can be performed safely and achieve long-term, disease-free survival in selected elderly patients with locally extensive head and neck cancer.

Analysis of surgical margins with the molecular marker eIF4E: a prognostic factor in patients with head and neck cancer.

PURPOSE: Complete excision of cancer is guided by histologic assessment of surgical margins. Molecular markers may be more sensitive in identifying malignant cells. eIF4E, a eukaryotic protein synthesis initiation factor, is found elevated in all head and neck squamous cell cancers (HNSCC). In a preliminary study using Western blots and a retrospective study using immunohistochemistry, eIF4E elevation in histologically tumor-free surgical margins correlated with a higher local-regional recurrence. We wanted to confirm this hypothesis in a prospective study. PATIENTS AND METHODS: Immunohistochemical analysis of surgical margins and tumors with an antibody to eIF4E was performed on all newly diagnosed HNSCC patients who underwent surgical resection for their disease between January 1996 and December 1997. RESULTS: All 65 patients had elevated levels of eIF4E in the tumors. Thirty-six patients (55%) had elevated eIF4E in histologically tumor-free margins, and 20 of these patients (56%) have had local-regional recu rrences. Twenty-nine patients (45%) had no elevation of eIF4E in the margins, and only two of these patients (6.9%) have had recurrences. Cox regression analysis showed that elevated eIF4E in the margins was an independent prognostic factor (P =.009) for recurrence. The Kaplan-Meier curves for the probability of nonrecurrence were significantly different for positive and negative eIF4E margins (P =. 0001, log-rank test). CONCLUSION: In histologically tumor-free surgical margins, elevated levels of eIF4E predict a significantly increased risk of recurrence. Elevated levels of eIF4E in tumor margins may identify patients who could benefit from additional therapy.

Expression of eIF4E during head and neck tumorigenesis: possible role in angiogenesis.

OBJECTIVE: The translation initiation factor eIF4E (4E) when overexpressed in mammalian cells results in their oncogenic transformation. 4E facilitates the synthesis of two powerful tumor angiogenic factors (VEGF and FGF-2) by selectively enhancing their translation. 4E is overexpressed not only in all head and neck squamous cell cancers but also in some dysplastic margins. Tumorigenesis in the head and neck is proposed to be a multistep process preceded by clinically evident precancerous lesions. Molecular events underlie the histological changes that herald transformation. We wanted to study the role of 4E in tumorigenesis and further elucidate its causal role in angiogenesis. METHODS: An immunohistochemical analysis with antibodies to 4E, VEGF, and basic (b)-FGF was performed on 115 specimens of the head and neck representing various stages of histological progression of malignancy. This was correlated with mean vessel density (MVD) using factor VIII. RESULTS: There were 41 cases of hyperplasia and low-grade dysplasia, 40 cases of high-grade dysplasia and 34 cases of cancer. There was a significant increase in the percent of cases expressing 4E from low-grade dysplasia through tumor. However, for VEGF and b-FGF the significant increase was only seen between the tumor group and dysplastic groups and no significant increase was noted between low-grade and high-grade dysplasia There was a significant increase in MVD from low- (10.7+/-1) to high-grade grade dysplasia (18.0+/-2.3). This increase was even more striking for the 4E positive cases. CONCLUSION: 4E elevation is correlated with progressive cell transformation in the head and neck. Its correlation with VEGF, b-FGF, and MVD potentiates its possible role in angiogenesis.

Botulinum toxin: adjunctive treatment for posterior glottic synechiae.

INTRODUCTION: Synechiae formation of the posterior glottis can result in tracheostomy dependence secondary to airway obstruction. Stenosis is caused by total or partial fixation of the vocal folds in adduction resulting from scar contracture. The treatment poses a management dilemma because of recurrent scar formation, made worse by mobility of the vocal folds. Although various treatment options from conservative endoscopic repair to open procedures have been proposed, the results are not satisfactory and patients often require multiple procedures. METHODS: We present the trial of a conservative approach that includes microscopic CO2 laser resection of the scar with concomitant botulinum toxin injection of the interarytenoid and thyroarytenoid muscles of the more mobile cord. This results in a temporary paresis of the adductor muscles and hence prevents overadduction in the posterior commissure during the postoperative healing period. STUDY DESIGN: We present the surgical technique and results in three patients who underwent the procedure. RESULTS: Treatment in all three patients was successful. CONCLUSIONS: The appropriate use of botulinum toxin may help improve the treatment outcome of posterior synechiae of the larynx without sacrificing any laryngeal components.

Neonatal rhinitis.

Neonatal rhinitis as a distinct disease entity has not been well-studied. The recognition and treatment of this condition is important since neonates are obligate nasal breathers, and mismanagement of this entity can result in poor feeding or even death from respiratory distress. We undertook a retrospective analysis of 20 patients seen at the Children's Hospital of San Diego over the period 1990-1991. Eighteen patients developed neonatal rhinitis in the months of August to January and only two between February and July. Clinical presentation and an effective management algorithm are discussed. Early recognition on the basis of clinical features followed by a two-step therapeutic trial consisting of conservative therapy and corticosteroid drops are advocated. Based on the above findings we have defined neonatal rhinitis as mucoid rhinorrhea with nasal mucosal edema in the afebrile newborn that results in stertor, poor feeding and respiratory distress which responds promptly to decadron 0.1% drops within a week. We recommend reserving diagnostic procedures for complicated cases that do not respond to the proposed regimen.