Black raspberries (BRBs) demonstrate potent inhibition of aerodigestive tract carcinogenesis in animal models. However, translational clinical trials evaluating the ability of BRB phytochemicals to impact molecular biomarkers in the oral mucosa remain limited. The present phase 0 study addresses a fundamental question for oral cancer food-based prevention: Do BRB phytochemicals successfully reach the targeted oral tissues and reduce pro-inflammatory and anti-apoptotic gene expression profiles? Patients with biopsy-confirmed oral squamous cell carcinomas (OSCCs) administered oral troches containing freeze-dried BRB powder from the time of enrollment to the date of curative intent surgery (13.9 ± 1.27 days). Transcriptional biomarkers were evaluated in patient-matched OSCCs and non-involved high at-risk mucosa (HARM) for BRB-associated changes. Significant expression differences between baseline OSCC and HARM tissues were confirmed using a panel of genes commonly deregulated during oral carcinogenesis. Following BRB troche administration, the expression of pro-survival genes (AURKA, BIRC5, EGFR) and pro-inflammatory genes (NFKB1, PTGS2) were significantly reduced. There were no BRB-associated Grade 3–4 toxicities or adverse events and 79.2% (N = 30) of patients successfully completed the study with high levels of compliance (97.2%). The BRB phytochemicals cyanidin-3-rutinoside and cyanidin-3-xylosylrutinoside were detected in all OSCC tissues analyzed, demonstrating that bioactive components were successfully reaching targeted OSCC tissues. We confirmed that hallmark anti-apoptotic and pro-inflammatory molecular biomarkers were over-expressed in OSCCs and that their gene expression was significantly reduced following BRB troche administration. Since these molecular biomarkers are fundamental to oral carcinogenesis and are modifiable, they may represent emerging biomarkers of molecular efficacy for BRB-mediated oral cancer chemoprevention.

The presence of RDINK4/ARF (RD) enhancer in the INK4-ARF locus provides a novel mechanism to simultaneously increase the transcription of p15INK4b (p15), p14ARF (p14), and p16INK4a (p16). While such up-regulation can be repressed through interactions between RD and oncoproteins CDC6 and BMI1, little is known about the involvement of RD in cancer. In this study we investigated RD deletions in 30 squamous cell carcinoma of the head and neck (SCCHN) and the patient-matched High At-Risk Mucosa specimens (HARM, “phenotypically normal” tissues neighboring SCCHN foci but beyond the surgical resection margin). RD was deleted (homozygously/heterozygously) in SCCHN and HARM at the incidence of 36.7% (11/30) and 13.3% (4/30), respectively. In comparison, no RD deletion was detected in 26 oral buccal brush biopsy specimens from healthy donors. Both p16 and p14 were lowly expressed in SCCHN and HARM, and their mRNA expression levels were positively associated with each other (p<0.01). Moreover, BMI1 was highly expressed in both SCCHN and HARM, and BMI1 over-expression was associated with p16 down-regulation in SCCHN (p<0.05). These results indicate that RD deletion and BMI1 overexpression frequently occur in the early stage of oral carcinogenesis and BMI1 overexpression may down-regulate the transcription of p16 and p14 through interfering with RD.

A66 Background Black raspberries represent a food-based chemopreventive agent rich in polyphenolics compounds, including anthocyanins, that have been shown to reduce inflammation. Numerous studies have demonstrated that inflammation plays a prominent role in tumor growth, progression, and metastasis. Common risk factors for oral squamous cell carcinoma (OSCC), such as tobacco use, also contribute to the inflammatory process. Cell culture and animal studies have shown altered gene expression in tumor and diseased tissues following exposure to lyophilized black raspberries (LBR) or their extracts. Consistently, these chemopreventive studies show the ability of LBR to modulate the expression of genes prominently associated with inflammation, including members of the NFκB family. In OSCC cells exposed to LBR extract in culture, we have shown a down-regulation of inflammatory biomarkers, including NFkB1D, NFkB1 and NFkB2. To transition these in vitro findings into an in vivo setting, we have used the hamster cheek pouch (HCP) model of OSCC to demonstrate a striking suppression of oral tumor lesion incidence and multiplicity (Anticancer Res 22, 2002) following dietary LBR treatment. Ultimately, the role of these essential pre-clinical studies is to translate into a human patient-based clinical investigation. Consequently, the aim of our ongoing Phase 1 Clinical Trial is to evaluate the molecular changes in oral cavity tissues following short-term, locoregional, oral exposure to LBR troches. According to protocols approved by the IRB of The Ohio State University, biopsy-confirmed OSCC patients were administered three LBR troches 4x/day (4.3g cumulative dose) between pre-surgical enrollment and their normally scheduled surgery. Tissue biopsies were obtained from tumor and distant normal tissues at enrollment and during surgical resection, after an exposure range of 2.5-34 days. Using a partial interim cohort of patients, NFκB1 was used as a surrogate endpoint biomarker for inflammation in order to assess LBR-dependent gene expression changes. Methods TaqMan real-time PCR assays were used to examine NFkB1 gene expression in a partial interim cohort of 24 patients. Experimental gene expression levels were normalized to GAPDH, RPS18, B2M, HPRT, and ACTB “housekeeping” gene expression levels. The interim patient cohort was stratified into “young” (≤ 45 years of age) and “older” (&gt; 45 years of age) groups for the purpose of this analysis. Results When patients were grouped into young (n = 7) and older (n = 17) populations, statistically significant differences were seen between LBR-modulated NFκB1 expression in tumor tissue compared to pre-treated, patient matched tumor tissue, with regard to age. Patients in the older population showed a significant decrease in NFκB1 expression in tumor tissue following LBR exposure compared to the younger population (p = 0.003). Conclusions These results suggest that inflammation can play a prominent role in the development of OSCC in older patients, and that LBR treatment may help alleviate the pathological effects of the inflammatory process in this population of patients. The lack of significant NFκB1 LBR-dependent modulation in the young OSCC population may signify that NFκB-independent or other functional pathways play an important role in tumorigenesis. Support: NIH/NIDCR R21DE016361, ACS RSGT-06-126-01-CNE Citation Information: Cancer Prev Res 2008;1(7 Suppl):A66.

Background: Consumption of phytochemical-rich fruits and vegetables is associated with a lower risk of oral cancer and protection against oxidative stress-mediated damage from reactive oxygen species (ROS). We proposed that antioxidant components in black raspberries (BRBs) could “scavenge” ROS and diminish oxidative stress burden. We tested the hypothesis that a long-term, low-dose administration of BRBs to a population of disease-free oral cancer survivors is both (i) achievable and (ii) will result in the attenuation of oxidative DNA damage. Methods: Participants were assigned to consume 0, 4, or 8 gms of BRBs daily for 6 months and provided self-report logbooks of adherence. Mass spectrometry (MS) of urine for dimethyl ellagic acid (DMEA) at baseline, 10, and 20 wks was utilized as a biomarker of adherence. ELISA of urine was used to measure 8-hydroxy-2’-deoxyguanosine (8-OHdG). A logistic regression model was used to determine factors associated with willingness to continue on the regimen once enrolled. Results: 112 participants were enrolled and assigned to a BRB regimen. 81 participants (72%) provided self-reported adherence logbooks. Adherence at wks 2, 10 and 20 was 92% (n=77), 85% (n=55), and 86% (n=44) respectively. The regimen was well tolerated with no significant adverse events. MS/MS measurements of DMEA was 10 fold higher (p&lt;0.0001; CI: 4-25) at 10 and 20 wks in participants treated with BRBs vs control. ELISA measurements of 8-OHdG in urine at 10 wks was 1.28 fold lower (p&lt;0.0183; CI: 1.04-1.58) in patients treated with BRBs vs control. Logistic regression analysis revealed that those having the least travel distance (OR: 0.98 [10 and 20 wks] p&lt;0.03) and those having government insurance (OR: 3.96 [10 wks]; 7.04 [20 wks]; p&lt;0.02) or private insurance (OR: 5.1 [20 wks]; p&lt;0.01) vs those with no insurance were more likely to complete the study. Also, the analysis showed that past (OR: 3.85; p&lt;0.02) and current (OR: 3.82; p&lt;0.03) tobacco users were more likely reach 20 wks than never smokers. Furthermore, those having had no adjuvant therapy (OR: 4.5; p&lt;0.004) vs those having had chemotherapy and/or radiation were more likely to complete 20 wks of intervention. Conclusions: A food-based prevention strategy utilizing BRBs in oral cancer survivors is safe and feasible. Compliance for those staying on regimen was high and factors identified that influenced continued participation included: having insurance, travel distance, having had adjuvant therapy, and tobacco use. While the number of active participants declined, those completing the intervention maintained outstanding levels of adherence (&gt;85%). Biomarkers of adherence were identified as an objective means of validating self-reported adherence. Moreover, 8-OHdG levels were reduced, providing a potentially useful biomarker of BRB efficacy. Citation Format: Lana K. Uhrig, Thomas J. Knobloch, Bruce C. Casto, Amit Agrawal, David E. Schuller, Theodoros N. Teknos, Enver Ozer, Matthew O. Old, Christine L. Sardo, Jeff Xueliang, Steven K. Clinton, Dennis K. Pearl, Christopher M. Weghorst. Biomarkers of adherence and efficacy: modulation of biomarkers of DNA damage in a Phase 1b trial of post-surgical oral cancer patients on a long-term food-based prevention study with black raspberries. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4680. doi:10.1158/1538-7445.AM2013-4680

Purpose: Cancer chemoprevention is the use of natural or synthetic agents, in combination or alone, to inhibit, delay or reverse the carcinogenic process, and is an active topic of cancer research. Numerous epidemiological studies have shown the strong correlation between the consumption of fresh fruits and vegetables and a decreased cancer risk. With a food-based approach to cancer prevention, emphasis is placed on the potential for complex mixtures of preventive agents in whole food to inhibit multiple process of carcinogenesis. In support of this approach, our laboratory conducted several preclinical animal studies that demonstrate the remarkable chemopreventive activities of black raspberries on chemically-induced oral squamous cell carcinoma (OSCC). We have shown that an extract of lyophilized black raspberries (LBR) dramatically inhibits the in vitro proliferation of cells derived from human OSCC, suggesting that the chemoprevention of oral cancer by LBR components in vivo is possible. Survivin (BIRC5) is a member of the Inhibitor of Apoptosis (IAP) family of caspase inhibitors that direct the negative regulation of apoptosis. BIRC5 is highly expressed in most human tumors, including oral squamous cell carcinoma (OSCC), but nearly undetectable in terminally differentiated normal adult tissues. BIRC5 acts to antagonize apoptosis in tumors and to promote resistance to common apoptotic-inducing therapies such as ionizing radiation. A series of studies examining BIRC5 in OSCC have associated increased BIRC5 expression levels with cancer progression and poor prognosis. The current study reports the LBR-dependent modulation of BIRC5 expression in OSCCs as part of an ongoing Phase I clinical trial. Methods: According to protocols approved by the Internal Review Board (IRB) of The Ohio State University, 24 biopsy-confirmed OSCC patients were administered three LBR troches 4x/day (4.3g cumulative dose) between presurgical enrollment and their normally scheduled surgery. Biopsies were obtained from distant high “at-risk normal” (N1) and tumor (T1) tissues at enrollment and following LBR exposure (N2, T2) acquired during surgical resection [exposure 2.5–34 days (mean exposure = 11.2±7.2 days)]. Pre-validated TaqMan gene expression assays were used to assess apoptosis-related genes for LBR-dependent expression changes. Results: OSCC tumor samples had significantly greater BIRC5 expression compared to the high “at-risk normal” oral samples, both prior to (T1 vs N1, p&lt;0.0001) and following (T2 vs N2, p=0.0115) LBR administration. In the high “at-risk normal” oral samples, there was no significant difference in BIRC5 expression between the pre- and post-LBR treatment groups (N2 vs N1, p=0.1425). There was a statistically significant decrease in tumor BIRC5 expression after LBR exposure (T2 vs T1, p=0.0199). Conclusions: BIRC5 expression was significantly decreased in OSCC tumor tissues following short-term exposure to LBR as part of an ongoing Phase 1 clinical trial. Consequently, down-regulation of BIRC5 expression following administration of LBR to OSCC patients may provide mechanistic insight for future food-based cancer risk reduction interventions. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A81.