Chemopreventive effects of black raspberry gel on oral epithelial dysplasia.

1. Pre-Treatment Post-Treatment CD34 CD34 Chemopreventive effects of black raspberry gel on oral epithelial dysplasia. Jared C. Zwick, Brian S. Shumway, Henry W. Fields, Peter E. Larsen, Russell J. Mumper, Gary D. Stoner, Susan R. Mallery Colleges of Dentistry and Medicine, The Ohio State University, Columbus, OH 43210 The Ohio State University Comprehensive Cancer Center and Solove Research Institute, Columbus, OH 43210 Genes uniformly down-regulated by topical berry gelapplication. Abstract: Approximately 36% of untreated oral intraepithelial neoplasia (IEN) lesions undergo malignant transformation to oral squamous cell carcinoma (SCC). There is currently no means to accurately predict which IEN lesions will transform. Further, clinical management is complicated by recurrences of previously excised IEN tissues or development of new premalignant lesions. This study assessed the effects of topical application of a bioadhesive 10% (w/w) freeze dried black raspberry gel on oral IEN parameters that included histopathologic diagnosis, gene expression profiles, levels of cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS), and microvascular densities. Each participant with oral IEN lesions served as their own internal control. All lesions were initially photographed, and lesional tissue was hemisected to obtain a pre-treatment diagnosis and establish baseline biochemical and molecular parameters. Gel dosing to the remaining lesional tissue (0.5 grams applied four times daily for six weeks) was initiated one week after the initial biopsy. RNA was isolated from snap frozen tissues for microarray analyses, followed by QRT-PCR validation. Additional epithelial gene specific QRT-PCR analyses, and image analysis quantified immunohistochemistry (IHC) for lesional epithelial COX2 and iNOS were conducted. No participant (10 normal controls, 20 IEN) developed any adverse side effects during treatment. Histopathologic results demonstrate appreciable interpatient variability, as 35% of our participants showed a post-treatment decrease in lesional grade, 20% showed an increase in lesional grade, and 45% of the patients showed stable disease. Topical berry gel application uniformly suppressed genes associated with RNA processing, growth factor recycling, and inhibition of apoptosis. The relative fold changes obtained by QRT-PCR paralleled the trends observed in the gene chip. Although a majority of participants showed post-treatment decreases in epithelial iNOS and COX-2 proteins, only COX-2 reductions were statistically significant (Wilcoxon signed rank). Supplemental, epithelial targeted QRT-PCR analyses demonstrated berry gel treatment markedly upregulated genes associated with keratinocyte differentiation and tumor suppression in a subset of patients, as well as reducing vascular densities in the superficial connective tissues. These data, which show topical berry gel application modulated gene expression profiles towards a less biologically active, more terminally differentiated tissue, and reduced epithelial COX2 and iNOS proteins, imply that further evaluation of berry gels for oral IEN chemoprevention is warranted. Research supported by NIH R21 CA11121. Microarray analyses (Affymetrix Human Plus 133A_2 array) were conducted on 13 matched pre and post treated oral dysplasias. Our results indicate a pervasive inhibitory effect of berry gel treatment on genes associated with RNA processing, intracellular signaling and metabolism, inhibition of apoptosis, and protein translation. A subset of patients showed concurrent upregulation of gene pathways associated with induction of terminal differentiation and apoptosis, inhibition of intracellular signaling, and epithelial cell adhesion. While the relative fold changes observed by QRT-PCR generally paralleled the fold changes observed in the gene chip, genes that showed appreciable upregulation by microarray demonstrated even greater increases with QRT-PCR. Additional QRT-PCR analyses (on all 20 oral dysplastic lesions) demonstrated increases in expression of genes associated with keratinocyte terminal differentiation and epithelial cell adhesion (SPPR2, SPPR3, involucrin, loricrin, TGM1, KRT2B, KER13, DSC1), suppression of growth and intracellular signaling (PPP2C, UBD, KSR1), and reduction in expression of iNOS, COX2 and VEGF in a subset of patients. Fig 3. Berry gel application reduced microvascular density in 7 of the 11 evaluable patients’ tissues. Patient MLS 002 samples are depicted below. Berry Trial Patient Population and Analyses • A total of 30 participants [10 normal controls, 20 persons with oral intraepithelial dysplasia (IEN)] have completed the trial. No participant reported any adverse effects. • None of the 30 participants used any tobacco products for at least 6 weeks prior to, and during the trial. • Normal participant tissue analyses: histopathologic assessment of post treatment biopsies. These tissues were also used for baseline indices for microarray and LOH studies. • 4. Oral dysplasia analyses: histopathology and RT-PCR (n=20), LOH (n=17), microarray (n=13), image analyses of COX2 (n=20), iNOS (n=20) and microvascular density (n=11). • 5. As 10 of the 20 persons with oral dysplasia had at least 1 previous biopsy and lesional recurrence and 7 of these 10 patients had multiple recurrences, this was a challenging patient cohort. Pre-Treatment Fig 1. Clinical regression of lesional tissue was more readily apparent in participants with low grade lesions. Conclusions: 1) A 6 week four times daily topical application of a 10% FBR gel demonstrated therapeutic efficacy as determined by a statistically significant decreases in epithelial COX-2 proteins, a decrease in iNOS and modulation of pro-proliferative, pro-inflammatory and differentiation associated gene pathways. 2) A subset of patients also showed an improvement in histologic grade, reduction in microvascular densities and reduction in LOH indices. 3) Analyses are ongoing to determine parameters that modulate patient responsiveness. Post-Treatment iNOS Fig 2. Seventeen out of 20 treated oral dysplastic lesions showed a significant reduction in COX-2 (p<0.005), while 12 of 20 treated lesions showed a reduction in iNOS, these were not statistically significant.