Topical application of a mucoadhesive freeze-dried black raspberry gel induces clinical and histologic regression and reduces loss of heterozygosity events in premalignant oral intraepithelial lesions: results from a multicentered, placebo-controlled clinical trial.

PURPOSE: Approximately 30% higher grade premalignant oral intraepithelial neoplasia (OIN) lesions will progress to oral cancer. Although surgery is the OIN treatment mainstay, many OIN lesions recur, which is highly problematic for both surgeons and patients. This clinical trial assessed the chemopreventive efficacy of a natural product-based bioadhesive gel on OIN lesions. EXPERIMENTAL DESIGN: This placebo-controlled multicenter study investigated the effects of topical application of bioadhesive gels that contained either 10% w/w freeze-dried black raspberries (BRB) or an identical formulation devoid of BRB placebo to biopsy-confirmed OIN lesions (0.5 g × q.i.d., 12 weeks). Baseline evaluative parameters (size, histologic grade, LOH events) were comparable in the randomly assigned BRB (n = 22) and placebo (n = 18) gel cohorts. Evaluative parameters were: histologic grade, clinical size, and LOH. RESULTS: Topical application of the BRB gel to OIN lesions resulted in statistically significant reductions in lesional sizes, histologic grades, and LOH events. In contrast, placebo gel lesions demonstrated a significant increase in lesional size and no significant effects on histologic grade or LOH events. Collectively, these data strongly support BRB's chemopreventive impact. A cohort of very BRB-responsive patients, as demonstrated by high therapeutic efficacy, was identified. Corresponding protein profiling studies, which demonstrated higher pretreatment levels of BRB metabolic and keratinocyte differentiation enzymes in BRB-responsive lesions, reinforce the importance of local metabolism and differentiation competency. CONCLUSIONS: Results from this trial substantiate the LOH reductions identified in the pilot BRB gel study and extend therapeutic effects to significant improvements in histologic grade and lesional size.

Topical application of a bioadhesive black raspberry gel modulates gene expression and reduces cyclooxygenase 2 protein in human premalignant oral lesions.

Reduced expression of proapoptotic and terminal differentiation genes in conjunction with increased levels of the proinflammatory and angiogenesis-inducing enzymes, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), correlate with malignant transformation of oral intraepithelial neoplasia (IEN). Accordingly, this study investigated the effects of a 10% (w/w) freeze-dried black raspberry gel on oral IEN histopathology, gene expression profiles, intraepithelial COX-2 and iNOS proteins, and microvascular densities. Our laboratories have shown that freeze-dried black raspberries possess antioxidant properties and also induce keratinocyte apoptosis and terminal differentiation. Oral IEN tissues were hemisected to provide samples for pretreatment diagnoses and establish baseline biochemical and molecular variables. Treatment of the remaining lesional tissue (0.5 g gel applied four times daily for 6 weeks) began 1 week after the initial biopsy. RNA was isolated from snap-frozen IEN lesions for microarray analyses, followed by quantitative reverse transcription-PCR validation. Additional epithelial gene-specific quantitative reverse transcription-PCR analyses facilitated the assessment of target tissue treatment effects. Surface epithelial COX-2 and iNOS protein levels and microvascular densities were determined by image analysis quantified immunohistochemistry. Topical berry gel application uniformly suppressed genes associated with RNA processing, growth factor recycling, and inhibition of apoptosis. Although the majority of participants showed posttreatment decreases in epithelial iNOS and COX-2 proteins, only COX-2 reductions were statistically significant. These data show that berry gel application modulated oral IEN gene expression profiles, ultimately reducing epithelial COX-2 protein. In a patient subset, berry gel application also reduced vascular densities in the superficial connective tissues and induced genes associated with keratinocyte terminal differentiation.

Effects of a topically applied bioadhesive berry gel on loss of heterozygosity indices in premalignant oral lesions.

PURPOSE: The aim of this study was to assess the effects of topical application of a 10% (w/w) freeze-dried black raspberry (FBR) gel on oral intraepithelial neoplasia (IEN) variables that included histologic diagnoses and loss of heterozygosity (LOH) indices. Microsatellite instability and/or LOH at tumor suppressor gene-associated chromosomal loci have been associated with a higher risk for oral IEN progression to oral squamous cell carcinoma. Previously, our laboratories have shown that FBRs are well tolerated and possess potent antioxidant, apoptotic, and differentiation-inducing properties. EXPERIMENTAL DESIGN: Each participant with IEN served as their own internal control. Before treatment, all lesions were photographed, and lesional tissue was hemisected to obtain a pretreatment diagnosis and baseline biochemical and molecular variables. Gel dosing (0.5 g applied four times daily for 6 weeks) was initiated 1 week after the initial biopsy. Genomic DNA was isolated from laser-captured basilar and suprabasilar surface epithelial cells followed by PCR amplification using primer sets that targeted known and presumed tumor suppressor gene loci associated with INK4a/ARF, p53, and FHIT. Allelic imbalance was determined by sequence analysis using normal participant tissues to establish microsatellite marker peak patterns and allele sizes. RESULTS: Confirming earlier phase I data, none of the 27 participants developed FBR gel-associated toxicities. Furthermore, our results show histologic regression in a subset of patients as well as statistically significant reduction in LOH at tumor suppressor gene-associated loci. CONCLUSIONS: These preliminary data suggest that further evaluation of berry gels for oral IEN chemoprevention is warranted.