Frank G. Ondrey

Identification of a gene expression signature associated with recurrent disease in squamous cell carcinoma of the head and neck.

Molecular studies of squamous cell carcinoma of the head and neck (HNSCC) have demonstrated multiple genetic abnormalities such as activation of various oncogenes (Ras, Myc, epidermal growth factor receptor, and cyclin D1), tumor suppressor gene inactivation (TP53 and p16), and loss of heterozygosity at numerous chromosomal locations. Despite these observations, accurate and reliable biomarkers that predict patients at highest risk for local recurrence have yet to be defined. In an effort to identify gene expression signatures that may serve as biomarkers, we studied 41 squamous cell carcinoma tumors (25 primary and 16 locally recurrent) from various anatomical sites and 13 normal oral mucosal biopsy samples from healthy volunteers with microarray analysis using Affymetrix U133A GeneChip arrays. Differentially expressed genes were identified by calculating generalized t tests (P < 0.001) and applying a series of filtering criteria to yield a highly discriminant list of 2890 genes. Hierarchical clustering and image generation using standard software were used to visualize gene expression signatures. Several gene expression signatures were readily identifiable in the HNSCC tumors, including signatures associated with proliferation, extracellular matrix production, cytokine/chemokine expression, and immune response. Of particular interest was the association of a gene expression signature enriched for genes involved in tumor invasion and metastasis with patients experiencing locally recurrent disease. Notably, these tumors also demonstrated a marked absence of an immune response signature suggesting that modulation of tumor-specific immune responses may play a role in local treatment failure. These data provide evidence for a new gene expression-based biomarker of local treatment failure in HNSCC.

Constitutive activation of transcription factors NF-?B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines

We previously reported that human head and neck squamous cell carcinomas (HNSCCs) express the pro‐inflammatory and pro‐angiogenic cytokines interleukin (IL)‐1α, IL‐6, IL‐8, and granulocyte‐macrophage colony‐stimulating factor in vitro and in vivo. The promoter region of the genes encoding these cytokines include binding sites for the transcription factors nuclear factor (NF) κB/Rel A, activator protein‐1 (AP‐1), and CCAAT enhancer‐binding protein β (C/EBPβ, or NF‐IL6), which have been reported to contribute to activation of these cytokine genes. In the study presented here, we examined the activation, composition, and function of these transcription factors in HNSCC cell lines that express pro‐inflammatory cytokines, by using electrophoretic mobility shift and reporter‐gene assays. Constitutive activation of NF‐κB, AP‐1, and NF‐IL6 DNA‐binding proteins was detected. Supershift analysis with antibodies specific for NF‐κB, AP‐1, and NF‐IL6 binding proteins showed that the NF‐κB–binding protein included p65/Rel A and p50; AP‐1 activity included c‐jun, junB, junD, and Fra‐1; and NF‐IL6 included C/EBPβ. Mutational analysis of the NF‐κB, AP‐1, and NF‐IL6 sites in the IL‐8 promoter region showed that NF‐κB and AP‐1 sites contributed to constitutive IL‐8 reporter activity in HNSCC. HNSCC lines that exhibited increased IL‐8 secretion relative to simian virus 40–immortalized and primary keratinocyte cell lines also demonstrated a concordant increase in NF‐κB reporter activity relative to nonmalignant keratinocytes. We concluded that the early transcription factors NF‐κB, AP‐1, and NF‐IL6 are constitutively activated in human HNSCC cell lines and that NF‐κB and AP‐1 promote expression of the pro‐inflammatory and pro‐angiogenic cytokine IL‐8 in HNSCC. The demonstration of the activation of these transcription factors will be helpful in defining the identity and role of these and other early gene products that contribute to pathogenesis of the malignant phenotype in HNSCC and in defining potential targets for pharmacologic and molecular therapy of HNSCC. Mol. Carcinog. 26:119–129, 1999. Published 1999 Wiley‐Liss, Inc.

Phase III Randomized Trial of Induction Chemotherapy in Patients With N2 or N3 Locally Advanced Head and Neck Cancer

PURPOSE Induction chemotherapy (IC) before radiotherapy lowers distant failure (DF) rates in locally advanced squamous cell carcinoma of the head and neck (SCCHN). The goal of this phase III trial was to determine whether IC before chemoradiotherapy (CRT) further improves survival compared with CRT alone in patients with N2 or N3 disease. PATIENTS AND METHODS Treatment-naive patients with nonmetastatic N2 or N3 SCCHN were randomly assigned to CRT alone (CRT arm; docetaxel, fluorouracil, and hydroxyurea plus radiotherapy 0.15 Gy twice per day every other week) versus two 21-day cycles of IC (docetaxel 75 mg/m(2) on day 1, cisplatin 75 mg/m(2) on day 1, and fluorouracil 750 mg/m(2) on days 1 to 5) followed by the same CRT regimen (IC + CRT arm). The primary end point was overall survival (OS). Secondary end points included DF-free survival, failure pattern, and recurrence-free survival (RFS). RESULTS A total of 285 patients were randomly assigned. The most common grade 3 to 4 toxicities during IC were febrile neutropenia (11%) and mucositis (9%); during CRT (both arms combined), they were mucositis (49%), dermatitis (21%), and leukopenia (18%). Serious adverse events were more common in the IC arm (47% v 28%; P = .002). With a minimum follow-up of 30 months, there were no statistically significant differences in OS (hazard ratio, 0.91; 95% CI, 0.59 to 1.41), RFS, or DF-free survival. CONCLUSION IC did not translate into improved OS compared with CRT alone. However, the study was underpowered because it did not meet the planned accrual target, and OS was higher than predicted in both arms. IC cannot be recommended routinely in patients with N2 or N3 locally advanced SCCHN.

Peroxisome Proliferator-Activated Receptor γ Pathway Targeting in Carcinogenesis: Implications for Chemoprevention

The peroxisome proliferator-activated receptor (PPAR) γ is one member of the nuclear receptor superfamily that contains in excess of 80 described receptors. PPARγ activators are a diverse group of agents that range from endogenous fatty acids or derivatives (linolenic, linoleic, and 15-deoxy-Δ12,14-prostaglandin J2) to Food and Drug Administration-approved thiazolidinedione drugs [pioglitazone (Actos) and rosiglitazone (Avandia)] for the treatment of diabetes. Once activated, PPARγ will preferentially bind with retinoid X receptor α and signal antiproliferative, antiangiogenic, and prodifferentiation pathways in several tissue types, thus making it a highly useful target for down-regulation of carcinogenesis. Although PPAR-γ activators show many anticancer effects on cell lines, their advancement into human advanced cancer clinical trials has met with limited success. This article will review translational findings in PPARγ activation and targeting in carcinogenesis prevention as they relate to the potential use of PPARγ activators clinically as cancer chemoprevention strategies.

Cisplatin and radiation sensitivity in human head and neck squamous carcinomas are independently modulated by glutathione and transcription factor NF-κB

Response to neoadjuvant cisplatin‐based chemotherapy has been used to predict overall response to chemoradiation therapy and to select patients with head and neck squamous cell carcinoma (HNSCC) for organ preservation therapy in NCI and VA cooperative group trials. However, different molecular determinants have been reported to contribute to sensitivity of cells to cisplatin and radiation, including glutathione (GSH), and activation of nuclear factor‐κB (NF‐κB), a transcription factor that regulates cytoprotective genes. We have reported that NF‐κB is constitutively activated in HNSCC, but the relationship of NF‐κB to GSH and to cisplatin and radiation sensitivity in HNSCC is unknown.

The feasibility of monitoring NF‐κB associated cytokines: TNF‐α, IL‐1α, IL‐6, and IL‐8 in whole saliva for the malignant transformation of oral lichen planus

Previous investigations have demonstrated that immune activation and chronic inflammation may be one of the causes of oncogenesis. A previous study from our lab has shown significant increases of NF‐κB dependent cytokines, TNF‐α, IL‐1α, IL‐6, and IL‐8 in different oral fluids from oral lichen planus (OLP) patients. The aim of this analysis was to explore the potential of detecting these cytokines in whole unstimulated saliva (WUS) in monitoring the malignant transformation of OLP. Thirteen patients with OLP (with epithelial dysplasia), 13 cases with oral squamous cell carcinoma (OSCC), and 13 age‐sex matched controls were enrolled in the study. The WUS samples were collected and the level of TNF‐α, IL‐1α, IL‐6, and IL‐8 in WUS was determined by ELISA. In moderate and severe dysplasia, the level of each cytokine was significantly higher than in control. In moderate dysplasia, TNF‐α and IL‐1α were significantly increased at a level without difference from OSCC, but IL‐6 and IL‐8 was detected at a concentration significantly lower than OSCC. In severe dysplasia, the level of TNF‐α was also not significantly different from that of OSCC, and the level of IL‐1α, IL‐6, and IL‐8 was still significantly lower than that of OSCC. The level of four cytokines between smokers and non‐smokers in each group did not show a significant difference. These results indicate that the change of NF‐κB dependent cytokines in WUS may in part reflect the malignant transformation of OLP and the analysis of these cytokines and may provide a useful, non‐invasive surrogate endpoint for monitoring malignant transformation as well as the therapeutic response of OLP. This is the first in vivo study utilizing saliva to confirm preclinical data that NF‐κB is upregulated in oral carcinogenesis.

Inhibitors of the Arachidonic Acid Pathway and Peroxisome Proliferator–Activated Receptor Ligands Have Superadditive Effects on Lung Cancer Growth Inhibition

Arachidonic acid (AA) metabolizing enzymes and peroxisome proliferator-activated receptors (PPARs) have been shown to regulate the growth of epithelial cells. We have previously reported that exposure to the 5-lipoxygenase activating protein-directed inhibitor MK886 but not the cyclooxygenase inhibitor, indomethacin, reduced growth, increased apoptosis, and up-regulated PPARalpha and gamma expression in breast cancer cell lines. In the present study, we explore approaches to maximizing the proapoptotic effects of PPARgamma on lung cancer cell lines. Non-small-cell cancer cell line A549 revealed dose-dependent PPARgamma reporter activity after treatment with MK886. The addition of indomethacin in combination with MK886 further increases reporter activity. We also show increased growth inhibition and up-regulation of apoptosis after exposure to MK886 alone, or in combination with indomethacin and the PPAR ligand, 15-deoxy-Delta12,14-prostaglandin J2 compared with single drug exposures on the adenocarcinoma cell line A549 and small-cell cancer cell lines H345, N417, and H510. Real-time PCR analyses showed increased PPAR mRNA and retinoid X receptor (RXR)alpha mRNA expression after exposure to MK886 and indomethacin in a time-dependent fashion. The results suggest that the principal proapoptotic effect of these drugs may be mediated through the known antiproliferative effects of the PPARgamma-RXR interaction. We therefore explored a three-drug approach to attempt to maximize this effect. The combination of low-dose MK886, ciglitazone, and 13-cis-retinoic acid interacted at least in a superadditive fashion to inhibit the growth of lung cancer cell lines A549 and H1299, suggesting that targeting PPARgamma and AA action is a promising approach to lung cancer growth with a favorable therapeutic index.

A pilot study of longitudinal serum cytokine and angiogenesis factor levels as markers of therapeutic response and survival in patients with head and neck squamous cell carcinoma

Head and neck squamous cell carcinomas (HNSCCs) were previously shown to express a repertoire of cytokines and angiogenesis factors that contribute to malignant pathogenesis and are detectable in serum. Pretreatment and posttreatment serum levels of cytokines and angiogenesis factors were evaluated as markers for outcome in patients with HNSCC.

Pilot Randomized Phase II Study of Celecoxib in Oral Premalignant Lesions

Purpose: Cyclooxygenase-2 (COX-2)–specific inhibition suppresses carcinogenesis in preclinical models and is a promising strategy for preventing oral cancer. In this pilot randomized phase II study, we evaluated the efficacy and safety of the COX-2 inhibitor celecoxib in patients with oral premalignant lesions (OPL). Experimental Design: Patients were randomly assigned to placebo (n = 18), celecoxib 100 mg twice daily (n = 17), or celecoxib 200 mg twice daily (n = 15) for 12 weeks. Six additional patients received celecoxib (400 mg twice daily) in an unblinded extension of the study. Biopsies were obtained at baseline and week 12. All patients entering the study were required to have at least one histologically confirmed early (atypical hyperplasia, atypical hyperkeratosis, or mild dysplasia) or advanced (moderate to severe dysplasia) OPL. Results: Forty-nine patients (46 of 50 randomized and 3 of 6 open label) were evaluable for efficacy analyses. There were no statistically significant differences between the response rates of the randomly assigned arms: placebo, 33.3% (6 of 18); celecoxib 100 mg twice daily, 41.2% (7 of 17); and celecoxib 200 mg twice daily, 20.0% (3 of 15). Two patients responded on celecoxib 400 mg twice daily. Celecoxib was generally well tolerated. Patients with higher baseline COX-2 mRNA levels had an increased risk of disease progression within 3 months. Conclusions: Celecoxib at 100 or 200 mg twice daily was ineffective in controlling OPLs in this randomized controlled trial. This result and cardiovascular toxicity results of other (large scale) randomized controlled trials of selective COX-2 inhibitors have discouraged the continued investigation of these agents in oral cancer chemoprevention. Better methods for identifying high-risk patients and more active interventions are needed for future oral cancer chemoprevention trials.

The role of inflammatory mediators in the pathogenesis of otitis media and sequelae.

This review deals with the characteristics of various inflammatory mediators identified in the middle ear during otitis media and in cholesteatoma. The role of each inflammatory mediator in the pathogenesis of otitis media and cholesteatoma has been discussed. Further, the relation of each inflammatory mediator to the pathophysiology of the middle and inner ear along with its mechanisms of pathological change has been described. The mechanisms of hearing loss including sensorineural hearing loss (SNHL) as a sequela of otitis media are also discussed. The passage of inflammatory mediators through the round window membrane into the scala tympani is indicated. In an experimental animal model, an application of cytokines and lipopolysaccharide (LPS), a bacterial toxin, on the round window membrane induced sensorineural hearing loss as identified through auditory brainstem response threshold shifts. An increase in permeability of the blood-labyrinth barrier (BLB) was observed following application of these inflammatory mediators and LPS. The leakage of the blood components into the lateral wall of the cochlea through an increase in BLB permeability appears to be related to the sensorineural hearing loss by hindering K+ recycling through the lateral wall disrupting the ion homeostasis of the endolymph. Further studies on the roles of various inflammatory mediators and bacterial toxins in inducing the sensorineumral hearing loss in otitis media should be pursued.