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

Abstract

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.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

S.R. Mallery is a member of NanoMed’s Pharmaceutical’s Scientific Advisory Board and is the principal investigator on NanoMed’s STTR grant on the topic of gel technology. R.J. Mumper is the founder of NanoMed Pharmaceuticals. All of the data and studies describes in the current article were planned, conducted, and funded independently of NanoMed Pharmaceuticals.

Figures

Fig. 1

Oral IEN tissue biopsy protocol. , dysplastic lesion; □, adjacent normalmucosa; , healing mucosa; ---, biopsy incision; –■–, tissue sectioning. A, pretreatment dysplastic lesion with adjacent normal mucosa. B, pretreatment hemisection with removal of half of the lesion with additional tissue sectioning for baseline analyses. C, residual dysplastic lesion and healing biopsy site treated with FBR gel for 6 wk. D, posttreatment excision of remaining dysplastic lesion and healed mucosa with tissue sectioning for final analyses. By creating a wound, this protocol activated the epithelial transient amplifying and stem cell pools. RT-PCR, reverse transcription-PCR.

Fig. 2

A subset of oral IEN participants showed marked clinical and histologic improvements following a 6-wk application of the 10% berry gel. The pretreatment clinical appearance of the left ventral tongue lesion depicted in A (patient #10) shows a lesion showing marked hyperkeratosis with crisp demarcation of lesional tissue. The corresponding pretreatment biopsy (B, 100× image scale, diagnosed as mild tomo derate dysplasia) shows characteristic dysplastic features, including basilar hyperplasia, increased nuclear to cytoplasmic ratios, and bulbous epithelial rete ridges. C, clinical regression, as evidenced by overall loss in lesional tissue delineation and reduction in hyperkeratosis, is evident in the posttreatment photograph. The corresponding posttreatment histopathology (D, 100× image scale, diagnosed as hyperkeratosis and atypia) reveals a more appropriate maturational pattern characterized by a reduction in basilar hyperplasia and a reduction in the nuclear toc ytoplasmic ratios.

Fig. 3

Pretreatment topo sttreatment comparisons of changes in LOH prevalence. A, changes in all events of LOH combined for the eight markers examined at three loci (3p14, 9p21-22, and 17p3). The initial biopsy site and residual lesion site are depicted together and separately. B, a subset of data from A, restricted to the changes in LOH observed for the four markers targeting locus 9p21-22. Small sample size precluded evaluation of the other two loci in this manner. C, individual patient changes in LOH prevalence. Each patient has three potential outcomes (decrease, no change, and increase) for eachmeasurement of LOH. There are 16 total (8 from the biopsy site and 8 from the residual lesion site) measurements for each patient. Participants with no data entry did not exhibit LOH for any of the evaluated markers either before or after berry gel application.