High-Dose Fenretinide in Oral Leukoplakia

Cancer Prevention Research

William N. William, Jr.,1 J. Jack Lee,2 Scott M. Lippman,1,3 Jack W. Martin,4 Nitin Chakravarti,1 Hai T. Tran,1 Anita L. Sabichi,1,3 Edward S. Kim,1 Lei Feng,2 Reuben Lotan1 and Vassiliki A. Papadimitrakopoulou1

Abstract We previously showed that low-dose fenretinide (200 mg/d) had limited activity in retinoid- resistant oral leukoplakia (34% response rate) possibly because serum drug levels were insufficient to induce retinoid receptor–independent apoptosis. Therefore, we designed the single-arm phase II trial reported here to investigate whether higher-dose fenretinide would improve leukoplakia response over that of our previous study. Leukoplakia patients received fenretinide (900 mg/m2 twice daily) in four 3-week cycles (1 week on drug followed by 2 weeks off). At week 12, clinical responses were determined and blood samples were collected for serum drug level assessments. A planned interim futility analysis led to early trial closure after the initial 15 (of 25 planned) patients because only 3 (20%) had a partial response (stopping rule: ≤4 responses in first 16 patients). Fenretinide was well tolerated— only one grade 3 adverse event (diarrhea) occurred. Serum fenretinide levels changed from 0 (baseline) to 0.122 ± 0.093 μmol/L (week 12). In correlative in vitro studies, high-dose fenretinide inhibited the growth of head and neck cancer cells more and oral leukoplakia cells less than did lower doses of fenretinide. This result is consistent with our clinical finding that high-dose fenretinide did not improve on the historical response rate of lower-dose fenretinide in our previous oral leukoplakia trial.

Fenretinide is a synthetic retinoid [N-(4-hydroxyphenyl)reti- apoptosis is independent of retinoid receptors, whereas fenretinide- namide] with clinical activity in patients with the prema- induced differentiation is not. lignant lesion oral leukoplakia (1, 2).It is postulated that In a previous phase II trial, we found that low-dose fenreti- fenretinide is active in oral premalignant lesions via modulat- nide (200 mg/d) for 12 weeks produced a 34% response rate in ing cell growth and/or differentiation or inducing apoptosis. patients with retinoid-resistant leukoplakia (never responded A dual, dose-dependant mechanism of action mediates the or responded and then relapsed during treatment; ref.1).The antineoplastic effects of fenretinide.Fenretinide induced cell responses, however, were partial in all cases, often short lived, differentiation at a low concentration (1 μmol/L) and apop- and correlated with previous response to natural retinoids (1). tosis at higher concentrations (3-10 μmol/L) in cancer cells Because fenretinide serum levels (mean, 0.230 μmol/L) were in vitro – (3 6).Of note, the induction of differentiation was at least 10-fold lower than the concentrations required in vitro not observed in mutant F9 murine embryonal carcinoma cells for induction of apoptosis (>3 μmol/L; refs.6 –8), we hypothe- γ lacking expression of retinoic acid receptor and retinoid X sized that the limited clinical activity of low-dose fenretinide α receptor , whereas apoptosis was shown following exposure in this trial likely was due to retinoid receptor–mediated me- to fenretinide in both wild-type and receptor-deleted cells chanisms and did not involve retinoid receptor–independent (3).These collective results indicate that fenretinide-induced apoptotic effects. A previous phase I trial showed that fenretinide at 900 mg/ m2 twice daily in 21-day cycles (7 days on drug followed by 14 days off) was tolerable and produced peak serum drug levels of 3 to 5 μmol/L (9).Based on the preclinical data – Authors' Affiliations: Departments of 1Thoracic/Head and Neck Medical (3 6), fenretinide at this dose theoretically would produce Oncology, 2Biostatistics, 3Clinical Cancer Prevention, and 4Head and Neck high serum levels capable of inducing retinoid receptor– Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, independent apoptosis, eliminating premalignant clones, Texas Received 05/15/2008; revised 07/25/2008; accepted 07/28/2008. and thus producing sustained clinical responses in oral leu- Grant support: National Cancer Institute, Department of Health and Human koplakia.We hypothesized that fenretinide would have this Services grants P01 CA052051 and P30 CA016672 (The University of Texas cytotoxic-like effect of a rapid burst of apoptotic activity and M. D. Anderson Cancer Center support grant). Requests for reprints: Vassiliki A. Papadimitrakopoulou, Department of that the 14-day drug holiday would minimize potential Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. adverse effects, paralleling the rationale behind cytotoxic Anderson Cancer Center, 1515 Holcombe Boulevard, Box 432, Houston, TX drug regimens.Therefore, we assessed the effects of high- 77030. Phone: 713-792-6363; Fax: 713-792-1220; E-mail: [email protected]. ©2009 American Association for Cancer Research. dose fenretinide on oral leukoplakia in a phase II trial and doi:10.1158/1940-6207.CAPR-08-0100 correlative in vitro studies.

Cancer Prev Res 2009;2(1) January 2009 22 www.aacrjournals.org

Materials and Methods Treatment plan Before study entry, patients were required to sign an informed Clinical trial consent statement.Baseline evaluation included a complete history This was an open-label, single-arm, phase II trial conducted at The and physical examination, serum chemistry and hematologic tests, University of Texas M.D.Anderson Cancer Center.The study was biopsy, and measurement of oral lesions.Patients were then treated approved by the Institutional Review Board and was conducted in with fenretinide (900 mg/m2 twice daily) in four 21-d cycles (7 d on accordance with the provisions of the Declaration of Helsinki and drug followed by 14 d off).Toxicity during treatment was regularly Good Clinical Practice guidelines. assessed using the National Cancer Institute Common Toxicity Criter- ia version 2.0 modified to include retinoid-specific toxicities (10). At Patient eligibility the end of the treatment period, oral lesions were measured and biop- The main eligibility criterion was the presence of bidimensionally sied again.Optional blood collection for pharmacokinetic analyses measurable or evaluable oral leukoplakia, which was required to have [i.e., measurements of serum levels of fenretinide and its major one or more of the following characteristics: presence of dysplasia metabolite, N-(4-methoxyphenyl)retinamide (4-MPR)] was done at (any grade), localization to the floor of the mouth, ventrolateral ton- baseline (before treatment) and at 14 d after the last dose of fenretinide gue, or soft palate complex, presence of symptoms, and/or extensive (week 12).Details of blood sample processing and analysis metho- area.Other criteria included adequate liver, kidney, and bone marrow dology were previously described (1). function; Zubrod performance status <2; no history of cancer within the preceding 6 mo (except for nonmelanoma skin cancer); and no Study end points and statistical analysis exposure to retinoid or carotenoid supplements within the preceding The primary end point of the study was determination of clinical 3mo. response rate (complete and partial response) at the end of the 3-mo treatment period.Response was evaluated as follows: complete response, gross inspection revealed no evidence of a lesion; partial response, the size of a lesion or of the sum of the measurements of Table 1. Baseline characteristics of eligible patients all lesions decreased by at least 50%; stable disease, lesion sizes increased by <25% or decreased by <50%; disease progression, in- Characteristic No. patients (%), N =15 crease of at least 25% in the sum of the measurements of all lesions or appearance of any new lesion (10). Median age, y (range) 59 (39-77) Simon's minimax two-stage design was applied, and we planned to accrue 16 patients in the first stage; if there were 4 or less responders, Gender the study would be terminated and the treatment would be consid- Female 7 (47) ered inefficacious.If there were 5 or more responders, additional Male 8 (53) patients would be added to the study in the second stage to achieve Race a total of 25 patients.At the end of the trial, the treatment was con- Asian 1 (7) sideredtobeefficaciousiftherewere11ormoreresponders.The Black 1 (7) study had 90% power with a one-sided 10% type I error rate, assum- White 13 (87) ing the null response rate would be 30%, and a response rate of 55% Smoking status or higher would be of interest (25% improvement over previously ∼ Never smoker 7 (47) reported of 30% with low-dose fenretinide; ref.1).Additional end Former smoker 6 (40) points included toxicity, pharmacokinetics assessments, and histologic response at month 3. Current smoker 2 (13) Histology In vitro studies Hyperplasia 4 (27) Leukoplakia cell lines MSK Leuk1 and MSK Leuk1s were obtained Mild dysplasia 6 (40) from Dr.P.G.Sacks(New York University College of Dentistry, New Moderate dysplasia 2 (13) York, NY).The MSK Leuk1 cell line was established from a moder- Severe dysplasia/carcinoma in situ 3 (20) ately dysplastic leukoplakia of the oral cavity.These cells are immortal Primary site but nontumorigenic (11, 12).MSK Leuk1 cells were exposed to high Oral tongue, lateral border 7 (47) concentrations of calcium and serum (10% FCS), and the more- Buccal mucosa 6 (40) advanced cells that had lost sensitivity to the growth-inhibitory and differentiation-inducing effects of calcium and serum were selected to Floor of the mouth 1 (7) become the MSK Leuk1s subline (11, 12).Both cell lines were main- Oral tongue, dorsum 1 (7) tained routinely in serum-free keratinocyte growth medium (KGM; Prior treatment for oral premalignancy Lonza, Inc.). The malignant cell line Ca9-22 was established from a No surgery 8 (53) human oral squamous cell carcinoma (13).These cells are tumorigenic Surgery 7 (47) in immunocompromised nude mice. No previous chemoprevention 6 (40) The cells were maintained in DMEM/F12 plus 10% FCS but were Previous chemoprevention* 9 (60) switched to serum-free KGM 24 h before growth inhibition experi- 13-cis retinoic acid 6 (40) ments.Fenretinide obtained from the National Cancer Institute was β-carotene + retinyl palmitate 1 (7) dissolved in DMSO.This stock solution was diluted into serum-free Fenretinide, low dose† 3 (20) KGM medium at the beginning of each experiment.Cells were plated in triplicate wells of a 96-well plate and treated with various doses of Celecoxib 5 (33) fenretinide; controls were treated with the carrier (DMSO) alone for up to 3 d.Cell numbers were estimated by staining the monolayer *Five patients received more than one chemoprevention regimen cultures with sulforhodamine B (14), and growth inhibition was in the past. Seven patients received prior retinoids. calculated from the equation (1 − At/Ac) × 100, where Ac and At † Fenretinide (200 mg/d). represent the absorbencies at 510 nm of control and treated cultures, respectively.

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prompted early termination of the study due to lack of efficacy Table 2. Worst toxicities by grade (1-3) per patient of the investigational agent.

Grade No. patients (%), N =15 Fenretinide and 4-MPR serum levels 123Fourteen patients had both baseline and posttreatment blood samples collected for analysis of drug serum levels. Gastrointestinal (other) 9 (60) As expected, fenretinide was undetectable in all 14 samples Diarrhea 8 (53) 1 (7) at baseline.The mean (±SD) serum level of fenretinide in- Ocular visual (other) 8 (53) creased to 0.122 ± 0.093 μmol/L after treatment (i.e., 14 days Triglyceride, serum high 8 (53) after the last dose of fenretinide).Similarly, 4-MPR (a major Pain 6 (40) fenretinide metabolite) was undetectable in all 14 samples at Cholesterol, serum high 5 (33) baseline and increased to 0.643 ± 0.398 μmol/L (mean ± SD) Constipation 5 (33) after treatment.We extrapolated the mean trough level of Dermatology/skin (other) 4 (27) 0.122 μmol/L at day 21 (2 weeks after last dose) to time of Photosensitivity 4 (27) maximum concentration; this calculation showed that fenreti- Fatigue 3 (20) 1 (7) nide levels during the initial days of the cycle (which were not Glucose, serum high 3 (20) assessed directly) were comparable with the high concentra- Arthritis 2 (20) tion range reported previously for high-dose fenretinide Conjunctivitis 2 (20) (based on the 14- to 25-hour termination half-life of fenretinide Headache 2 (20) and linear pharmacokinetics; data not shown; refs.9, 15, 16). Hot flashes 2 (20) In vitro Nausea alone 2 (20) fenretinide effects on the growth of oral Nyctalopia 1 (7) 1 (7) leukoplakia and squamous cell carcinoma cells The immortalized less-progressed (MSK Leuk1) and more- NOTE: Only toxicities occurring in more than one patient (regard- progressed (MSK Leuk1s) leukoplakia cell lines and the less of grade) are included. malignant cell line (Ca9-22) were all sensitive to the growth- inhibitory effects of fenretinide.Growth inhibition was dose and time dependent (Fig.1).The least-progressed cell line Results (MSK Leuk1) was more sensitive to lower concentrations of fenretinide than were MSK Leuk1s and Ca9-22 cells and Patient characteristics and treatment showed inhibition effects below 1 μmol/L fenretinide.In con- Between February 2003 and April 2004, 21 patients were trast, MSK Leuk1s and Ca9-22 cells were more sensitive to the registered in this study.Six patients were found to be ineligible highest fenretinide concentration (versus MSK Leuk1; Fig.1). due to history of cancer within 6 months (3), nonmeasurable disease (2), or refusal of treatment (1).Therefore, 15 patients initiated and completed treatment with the study drug.Their Discussion baseline characteristics are described in Table 1.All 15 patients 2 received the four planned cycles of fenretinide with no dose Although well tolerated, high-dose fenretinide (900 mg/m reductions.Self-reported compliance (confirmed by capsule twice daily) produced objective responses in only 3 of the first count) was >90% in 13 of 15 patients. 15 patients (20%).The trial was terminated early (after these 15 patients) by early stopping rule of the protocol (≤4re- Toxicity sponses in the initial 16 patients).Considering the hypothesis Treatment was well tolerated, with only one patient experi- that high-dose fenretinide would induce apoptosis in oral encing grade 3 toxicity (diarrhea lasting <24 hours).No grade leukoplakia tissue and thus eliminate premalignant clones, 4 toxicities were observed.The complete list of adverse events why did the response rate of the present trial not exceed that occurring in more than one patient (regardless of grade) is out- of the low-dose fenretinide trial we used for a historical con- lined in Table 2. trol? Two major considerations involved with this question are dose and schedule of fenretinide and mechanisms of fenreti- Efficacy nide actions in carcinogenesis. All 15 patients were evaluable for clinical response assess- About dose and schedule, prior phase I studies of high-dose ment at the end of 3 months.There were no complete res- fenretinide, including the same dose and schedule we used, ponses.Three patients (20%) had a partial response, whereas produced high mean peak serum levels of fenretinide of 3 to stable disease was observed in 11 patients (73%).Only one 10 μmol/L in cancer patients (9, 15, 16).It is possible, how- patient (7%) progressed during treatment in terms of clinical ever, that drug levels in leukoplakia tissue were inadequate response.Histologically, two patients (13%) had downgrading for response, although there are data showing that high tissue of the dysplasia, seven patients (47%) had no histologic levels occur in association with high serum fenretinide levels change, and six patients (40%) had worsening of the degree in other systems.A phase II study (17) of the same (as our) of dysplasia.None of the patients developed invasive cancer high dose and schedule in renal cancer patients showed that within the 3-month follow-up period.After the initial 15 pa- fenretinide concentrated at 3 to 10 μmol/L in serum and at 3 tients had been evaluated, we recognized that the prespecified to 8 μmol/L in kidney tumors.High ratios of tissue-to-serum minimum clinical response rate during the first stage of the fenretinide levels (associated with lower doses of fenretinide) trial (i.e., 5 of 16 patients) could not be achieved. This also have been documented in the bladder (18) and breast (19).

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Fig. 1. Effects of fenretinide on the growth of oral premalignant and malignant cells. Cells were seeded at a density of 20,000 per well in a 96-well plate; after 12 h, they were treated with DMSO control or fenretinide diluted in serum-free medium for 24, 48, and 72 h. Cell growth was determined via the sulforhodamine B assay. Points, mean (n = 3); bars, SD.

Therefore, we do not believe that lack of efficacy in the present the angiogenic switch in more-advanced than less-advanced trial was due to low levels of fenretinide in target oral tissue. lesions (26).These findings highlight the importance of con- We further examined the dose issue in correlative in vitro ducting preclinical studies of agents in advanced versus non- studies in a cell model of human multistage oral carcino- advanced carcinogenesis before launching clinical trials. genesis (11): immortalized (MSK Leuk1; established from a Although limited in the past, preclinical technology and mod- clinical leukoplakia sample), progressed (MSK Leuk1s), and els for testing agents in premalignancy are rapidly emerging. malignant cells (Ca9-22).We found that sensitivity to lower- Fenretinide at the dose and schedule of the present study concentration fenretinide was higher in premalignant than was not an effective chemoprevention strategy in patients malignant cells and that sensitivity to higher-concentration with oral premalignant lesions.Randomized studies of this fenretinide was higher in malignant than premalignant cells. agent for chemoprevention of breast and bladder cancer also This high-dose in vitro result is consistent with our negative have failed to show efficacy (18, 27).It is important to note, high-dose clinical finding.The results of several other in vitro however, that the value of oral leukoplakia as a surrogate studies also bear on the question of whether premalignant end point for cancer development is not certain, notwithstand- cells are less sensitive to fenretinide than are malignant cells, ing substantial data indicating that it may be (28).A very where the vast majority of data on receptor-independent recently reported chemoprevention trial found that oral leuko- apoptosis have been generated (20, 21).Low-concentration plakia response did not correlate with oral cancer develop- fenretinide was less inhibitory of colony formation by prema- ment (29), and so it cannot be ruled out categorically that lignant SV40 T–immortalized lung cells than by malignant high-dose fenretinide would reduce oral cancer risk despite lung cells (22).Sensitivity to fenretinide at low or high doses its limited activity in oral premalignancy.Leukoplakia in this was similar, however, in human papillomavirus–immortalized prior and the present trial was not selected based on advanced premalignant skin cells and malignant skin cells of a human stage and so likely had a relatively moderate overall risk.Our skin carcinogenesis model (23).Different fenretinide effects current in vitro results showing that neoplastic oral cells pro- in the various in vitro systems could relate to differences be- gressed in association with higher sensitivity to high-dose tween organ systems and methods of immortalization [e.g., (retinoid receptor independent) fenretinide suggest that viral versus spontaneous (e.g., MSK Leuk1)]. high-dose fenretinide may be more clinically active in ad- What mechanism potentially underlies the ineffectiveness of vanced head and neck premalignancies such as that with se- high-dose fenretinide in both our in vitro and clinical studies? vere dysplasia, cyclin D1 aberrations (30, 31), or certain loss of High-dose fenretinide can induce apoptosis through genera- heterozygosity profiles (32, 33).A new formulation of fenreti- tion of reactive oxygen species (24, 25).Normal and premalig- nide incorporated into an organized lipid matrix called nant cells have more endogenous antioxidant capacity than LYM-X-SORB may improve the oral bioavailability, increase do cancer cells; therefore, normal and premalignant oral cells blood and tissue levels to high micromolar levels, and thus may tolerate reactive oxygen species and thus counteract improve clinical outcomes in advanced oral leukoplakia apoptosis induction better than do cancer cells. patients; several phase I clinical trials of this formulation are Our original hypothesis was that high-dose fenretinide under way (34). would be highly active in less-advanced leukoplakia if low- Given the limitations of retinoid-based therapy for oral can- dose fenretinide (as shown in our previous trial; ref.1) was cer chemoprevention illustrated by this and previous trials moderately active in more-advanced leukoplakia.This hypo- (1, 10, 29, 35–37), our group is now focusing on the develop- thesis is consistent with general prevention drug development ment of molecular-targeted agents for patients with oral (much of it in cancer), which hypothesizes that agents active in premalignant lesions.As an example, we are currently con- cancer should be more active in premalignancy.Our in vitro ducting a placebo-controlled randomized study of the epidermal findings contradicted this general hypothesis.Some angiogen- growth factor receptor tyrosine kinase inhibitor erlotinib, with esis inhibitors also reflect this contradiction, better preventing cancer development as the primary end point.Molecular-targeted

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Downloaded from cancerpreventionresearch.aacrjournals.org on September 25, 2021. © 2009 American Association for Cancer Research. Cancer Prevention Research agents may improve therapeutic index and the chances for Disclosure of Potential Conflicts of Interest establishing standard chemoprevention of oral cancer in the future (38). No potential conflicts of interest were disclosed.

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William N. William, Jr., J. Jack Lee, Scott M. Lippman, et al.

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