Duane A. Sewell

Head and neck cancer in nonsmokers: A distinct clinical and molecular entity

Objectives/Hypothesis: Clinical and molecular patterns of head and neck squamous cell carcinoma (HNSCC) in nonsmokers and smokers may be different. Analysis of these patterns may improve understanding and management of this disease.

Recombinant Listeria Vaccines Containing PEST Sequences Are Potent Immune Adjuvants for the Tumor-Associated Antigen Human Papillomavirus-16 E7

Previous work in our laboratory has established that the fusion of tumor-associated antigens to a truncated form of the Listeria monocytogenes virulence factor listeriolysin O (LLO) enhances the immunogenicity and antitumor efficacy of the tumor antigen when delivered by Listeria or by vaccinia. LLO contains a PEST sequence at the NH2 terminus. These sequences, which are found in eukaryotic proteins with a short cellular half-life, target proteins for degradation in the ubiquitin-proteosome pathway. To investigate whether the enhanced immunogenicity conferred by LLO is due to the PEST sequence, we constructed new Listeria recombinants that expressed the HPV-16 E7 antigen fused to LLO, which either contained or had been deleted of this sequence. We then compared the antitumor efficacy of this set of vectors and found that Listeria expressing the fusion protein LLO-E7 or PEST-E7 were effective at regressing established macroscopic HPV-16 immortalized tumors in syngeneic mice. In contrast, Listeria recombinants expressing E7 alone or E7 fused to LLO from which the PEST sequence had been genetically removed could only slow tumor growth. Because CD8+ T cell epitopes are generated in the ubiquitin-proteosome pathway, we also investigated the ability of the vaccines to induce E7-specific CD8+ T cells in the spleen and to generate E7-specific tumor-infiltrating lymphocytes. A strong correlation was observed between CD8+ T-cell induction and tumor homing and the antitumor efficacy of the Listeria-E7 vaccines. These findings suggest a strategy for the augmentation of tumor antigen-based immunotherapeutic strategies that may be broadly applicable.

Genome-Wide Profiling of Oral Squamous Cell Carcinoma by Array-Based Comparative Genomic Hybridization†

Objectives: Array‐based comparative genomic hybridization (aCGH) was used to develop a genome‐wide molecular profile of oral squamous cell carcinoma (OSCC). Copy number alterations (CNAs) were identified by chromosomal region, mapped to specific genes, and compared with several previously documented CNAs associated with head and neck squamous cell carcinoma (HNSCC). The status of 512 commonly altered cancer genes was assessed and evaluated as potential correlates of tumor behavior.

Listeria-based HPV-16 E7 vaccines limit autochthonous tumor growth in a transgenic mouse model for HPV-16 transformed tumors

We have shown that Listeria-based cancer vaccines inhibit the growth of transplanted tumors in a transgenic mouse model of immune tolerance where HPV-16 E7 is expressed in the thyroid gland. In this study we determine whether these vaccines are able to inhibit autochthonous tumor growth in this animal model. Mice treated with Listeria vaccines expressing E7 had significantly smaller thyroid tumors than did mice treated with controls and possessed higher numbers of antigen-specific CD8(+) T cells within the spleens, tumors, and peripheral blood. This study shows that Listeria-based vaccines are able to slow autochthonous tumor growth and break immunological tolerance.

An Anti-Vascular Endothelial Growth Factor Receptor 2/Fetal Liver Kinase-1 Listeria monocytogenes Anti-Angiogenesis Cancer Vaccine for the Treatment of Primary and Metastatic Her-2/neu+ Breast Tumors in a Mouse Model

Thirty years after angiogenesis was shown to play an enabling role in cancer, modern medicine is still trying to develop novel compounds and therapeutics to target the tumor vasculature. However, most therapeutics require multiple rounds of administration and can have toxic side effects. In this study, we use anti-angiogenesis immunotherapy to target cells actively involved in forming new blood vessels that support the growth and spread of breast cancer. Targeting a central cell type involved in angiogenesis, endothelial cells, we immunized against host vascular endothelial growth factor receptor 2 to fight the growth of Her-2/neu+ breast tumors. Using the bacterial vector, Listeria monocytogenes (Lm), we fused polypeptides from the mouse vascular endothelial growth factor receptor 2 molecule (fetal liver kinase-1) to the microbial adjuvant, listeriolysin-O, and used Lm to deliver the Ags and elicit potent antitumor CTL responses. Lm-listeriolysin-O-fetal liver kinase-1 was able to eradicate some established breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumor-associated Ag Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to HER-2/neu and was not solely due to the reduction of tumor vasculature. However, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy. We show that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope spreading to an endogenous tumor protein and drive active tumor regression.

Optimizing Suicide Gene Therapy for Head and Neck Cancer

An effective “suicide gene” therapy strategy in experimental studies has been the use of the herpes simplex virus thymidine kinase gene(HSV‐tk) to sensitize tumors to the cytotoxic effects of ganciclovir administration. Previous studies using this model have focused on utilizing maximal viral titers and high levels of ganciclovir that are not compatible with human dosing. Because of the high ganciclovir doses and the maximal viral titers, this strategy has limited application to actual clinical scenarios. In the following studies the authors investigate tumor regression in an oral squamous cell carcinoma animal model as a function of variable adenoviral titers and more physiologic ganciclovir dosing. Using adenoviral titers ranging from 1 × 108 to 2 × 109 plaque forming units(pfu) to treat oral tumors, they found no statistical difference in tumor regression among the different viral doses, despite differences in mitotic activity. Each treatment group, however, demonstrated a significant effect on tumor regression when compared with controls. Furthermore, the authors were able to reduce the level of ganciclovir administration to 10 mg/kg twice daily from established levels of 100 to 150 mg/kg twice daily while maintaining significant tumor responses to the HSV‐tk therapy. Mean survival of animals treated with this lower ganciclovir dose was significantly higher than in controls and was equal to established means based on previous studies using higher ganciclovir doses. The optimization of this suicide gene therapy strategy is imperative in order to minimize theoretical and known viral and ganciclovir toxicities while establishing a foundation upon which to design appropriate and effective clinical trials.

Increased viral load correlates with improved survival in HPV-16-associated tonsil carcinoma patients.

Conclusion. Our results indicate that viral load may serve as an independent prognostic indicator for patients with HPV-16-associated squamous cell carcinoma of the tonsil. Objective. HPV-16 has gained increasing attention as a possible causative agent for squamous cell carcinoma of the tonsil. Recent reports have indicated that the viral load within the tumor, along with other factors, may be correlated to the patient survival. In this study, we sought to examine HPV-16 viral load as an independent prognostic indicator. Patients and methods. DNA was extracted from 35 tonsil carcinoma samples and the viral load was determined by real-time PCR. The patients were divided into four groups according to HPV-16 viral load. The correlation between viral load and recurrence, disease-free survival, and overall survival was assessed. Results. We found that HPV-positive patients with the highest viral loads had improved overall and disease-free survival. Recurrences of squamous cell carcinoma were significantly less likely to occur with increasing viral load.

The effects of exposure to intense sound on the DC endocochlear potential in the chick

Chicks were exposed to an intense pure tone (0.9 kHz, 120 dB SPL) for 48 h. The DC endocochlear potential was measured with a microelectrode inserted into scala media in six separate groups of animals between 0 and 12 days post exposure. Similar data were obtained from seven groups of unexposed control chicks between 2 and 15 days of age. One to nine animals were tested in each control or exposed group. The results in the control chicks revealed that the EP at 2 days of age (6.7 mV) was 36% of the mature value (15.2 mV) noted at 6 days of age. In the exposed animals, at 0-days recovery, the EP showed a 63% reduction relative to that measured in age matched control chicks. The level of EP in the exposed animals quickly recovered, and by 3 and 6 days post exposure exhibited a loss of only 7 and 3 percent relative to the age-matched controls. The recovery of EP was plotted against the recovery of evoked potential thresholds reported by others, and the time-course of the recovery functions were very similar. This relationship suggested that recovery of auditory function in the chick was highly correlated with the restoration of the EP. Damage to the tegmentum vasculosum and its capacity to secrete potassium, as well as the shunting of current through the damaged basilar papilla, are considered as mechanisms for the reduction of EP loss in the exposed ear.

Functional analysis of mild-type and malignant glioma derived CDKN2Aβ alleles: Evidence for an RB-independent growth suppressive pathway

The tumor suppressor gene CDKN2A (p16/MTS1/INK4A), which encodes the cyclin-dependent kinase inhibitor p16INK4a, is a target of 9p21 deletions during the malignant progression of human gliomas. This gene also encodes a second protein product (human p16β, murine p19ARF), which originates from an unrelated exon of CDKN2A (exon 1β) spliced onto exon 2 in an alternate reading frame. Cell cycle arrest by p16β is caused by an as yet unidentified pathway. In order to test the candidacy of p16β as a glioma suppressor, we replaced p16INK4a, p15INK4b and p16β wild-type as well as a series of seven glioma-derived p16β alleles (R87H, A112V, R120H, A121V, G125R, A128A and A128V), into glioma cell lines that had either CDKN2A−/RB+ (U-87MG and U-251MG) or CDKN2A+/RB− (LN-319) endogenous backgrounds and demonstrated that p16β can act as a functional glioma cell growth suppressor. Moreover, p16β, but not p16INK4a or p15INK4b inhibited the growth of RB-negative LN-319 cells, indicating that p16β likely exerts its effects through an RB-independent pathway. In vitro and in vivo assays of pRB phosphorylation were consistent with this interpretation. Since none of the glioma-derived p16β mutations inactivated their growth suppressive activities, it appears that mutations in CDKN2A exon 2 (which is shared in the coding sequences of p16INK4a and p16β) likely exclusively target p16INK4a.

Proteomic Signatures in Laryngeal Squamous Cell Carcinoma

Laryngeal cancer remains a worldwide health problem. The identification of biomarkers unique to laryngeal cancer may provide new insights into its pathogenesis, as well as provide potential targets for novel therapies and early detection. In order to identify potential biomarkers, we performed a proteomic analysis of laryngeal cancer specimens. Using two-dimensional differential in-gel electrophoresis and mass spectroscopy, protein expression profiles from two laryngeal carcinoma specimens and corresponding adjacent normal tissue were analyzed. The results of our analysis showed that the expression of a number of proteins was significantly altered in the tumor specimens when compared to matched normal controls. The differentially expressed proteins were identified, and they included stratifin, S100 calcium-binding protein A9, p21-ARC, stathmin, and enolase. With these findings, we have identified potential biomarkers which may contribute to the pathogenesis of laryngeal carcinoma, and which may be suitable as targets for novel therapeutic and/or diagnostic modalities.