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Second primary tumors in initially cured patients remain the greatest challenge in therapy for oral squamous cell carcinomas. The concept of field concerization is the most accepted hypothesis for the cellular and subcellular damages resulting in neoplastic transformation in the upper aerodigestive tract by risk factors such as chronic alcohol and tobacco abuse. Recent studies investigated several agents and regimens with benefit for chemoprevention of oral squamous cell carcinomas. In our review, the results of the most promising agents were studied. Despite discouraging results from recent intervention trials, studies in oral squamous cell carcinomas reported response rates up to 92%. In our investigation, two groups of patients were treated daily with 100 mg alpha-tocopherol, 75 mg beta-carotene, and 1000 mg ascorbic acid per os. The first group consisted of 24 patients with leukoplakia of the oral cavity. The second group included 24 patients with premalignant lesions after R0-resection of primary oral squamous cell carcinoma. Biopsies were taken from both groups prior to therapy and after 12 weeks follow-up. Flow cytometry analyses were performed and nucleolar organizing regions (NOR) were examined. An overall histological response rate of nearly 98% was noticed. Additionally, the pretherapeutic regimen increased cell kinetic parameters, such as the S-phase portion, and the average number of NOR per cell nucleus decreased. These results indicate that the chosen combination has substantial activity in oral premalignant lesions. Nevertheless, basic research is required in investigating valid biomarkers for chemoprevention studies.

The Bowman-Birk inhibitor (BBI) is a soybean-derived serine protease inhibitor and a potential cancer chemopreventive agent for humans. In this Phase I clinical trial, BBI concentrate was administered as a single oral dose to 24 subjects with oral leukoplakia. Pharmacokinetics of BBI was analyzed, and subjects were monitored clinically for toxic effects. Subjects received between 25 and 800 chymotrypsin inhibitor units (CIU) of the compound in a dose escalation trial. BBI was taken up rapidly, and a metabolic product of BBI was excreted in the urine within 24-48 h. No clinical or laboratory evidence of toxicity was observed in the study. Protease activity was also measured in buccal cells to evaluate usefulness as a biomarker. Single-dose BBI concentrate administered up to 800 CIU was well tolerated and appeared to be nontoxic. Further investigation in Phase II clinical trials is being done.

The aim of photodynamic diagnostics is complete visualization of all neoplastic lesions in a tumorous organ after the application of a tumor-selective photosensitizer. We explored the potential benefit of a combination of retinyl palmitate for the treatment of oral leukoplakias and follow-up diagnosis of malignant tissue by using 5-aminolevulinic acid (5-ALA) as a fluorescence marker. Semiquantitative measurements were performed following the topical application of 5-ALA in six patients. After biopsy and histological evaluation of tissue specimens the patients were treated with escalating doses of retinyl palmitate. After treatment periods ranging from 4 to 8 weeks 5-ALA was again applied for photodetection of persisting fluorescence. In all cases prior to retinyl palmitate treatment a prominent red fluorescence indicated tissue dysplasia. Eight weeks after treatment with retinyl palmitate decreased red fluorescence intensities were found, and repeated histological evaluations showed no persistent signs of dysplastic lesions. Our initial experience shows that high doses of retinyl palmitate can be useful for treating dysplastic lesions in the upper aerodigestive tract and can be monitored with 5-ALA induced protoporphyrin IX fluorescence.

Background: Beta-Carotene has been reported to produce regressions in patients with oral leukoplakia, a premalignant lesion. However, previous studies have all been of short duration, with clinical response as the end point., Objective: To evaluate the duration of response and the need for maintenance therapy in subjects who respond to beta-carotene., Methods: In this multicenter, double-blind, placebo-controlled trial, subjects were given beta-carotene, 60 mg/d, for 6 months. At 6 months, responders were randomized to continue beta-carotene or placebo therapy for 12 additional months., Results: Fifty-four subjects were enrolled in the trial, with 50 being evaluable. At 6 months, 26 subjects (52%) had a clinical response. Twenty-three of the 26 responders completed the second, randomized phase. Only 2 (18%) of 11 in the beta-carotene arm and 2 (17%) of 12 in the placebo arm relapsed. Baseline biopsies were performed in all patients, with dysplasia being present in 19 (38%) of the 50 evaluable patients. A second biopsy was obtained at 6 months in 23 subjects who consented to this procedure. There was improvement of at least 1 grade of dysplasia in 9 (39%), with no change in 14 (61%). Nutritional intake was assessed using food frequency questionnaires. There was no change in carotenoid intake during the trial. Responders had a lower intake of dietary fiber, fruits, folate, and vitamin E supplements than did nonresponders. Beta-carotene levels were measured in plasma and oral cavity cells. Marked increases occurred during the 6-month induction. However, baseline levels were not restored in subjects taking placebo for 6 to 9 months after discontinuation of beta-carotene therapy., Conclusions: The activity of beta-carotene in patients with oral leukoplakia was confirmed. The responses produced were durable for 1 year.

Background: Due to the possibility of malignant transformation of oral leukoplakia, these lesions must be assessed and managed closely and, if not resolved, must be reassessed on a regular basis., Methods: This study evaluated the use of topical 0.05% vitamin A (tretinoin) acid gel for the treatment of oral leukoplakia. Tretinoin was applied topically 4 times a day for the management of nonmalignant oral white lesions in 26 patients. The clinical response was evaluated in all patients and posttreatment biopsies were performed in ten patients., Results: The mean age of the patients at the time of diagnosis was 62 years. Of the 26 patients, 50% were tobacco users. Patients were followed for a mean of 23 months. Approximately 27% of the patients had a complete clinical remission. Recurrence of leukoplakia was observed in approximately 40% of patients in whom complete clinical remission occurred if topical applications were discontinued. A 50% reduction in the clinical grade of leukoplakia from a mean of 2.8 to 1.4 on a scale ranging from 0 (no leukoplakia) to 4 (speckled leukoplakia) was observed. When the pretreatment and posttreatment biopsies from 10 patients were evaluated, no change in the mean histologic grade (between mild and moderate dysplasia) was noted; however, some reduction in the histologic grade was noted in 3 of these patients (30%)., Conclusions: The use of topical vitamin A acid showed a limited effect in controlling oral leukoplakia. Further studies are needed to establish the appropriate indication, efficacy, and best choice for chemoprevention agents. Close follow-up of all patients with oral leukoplakia is required., (Copyright 1999 American Cancer Society.)

Background: This study evaluates the role of bcl-2 protein, as well as, of apoptotic bodies in the topical treatment of oral leucoplakia with 13-cis-retinoic acid (isotretinoin). To test this role, a clinical, histological and immunohistochemical study of the treated lesions was carried out, so as to verify the role of apoptosis and its genetic control mechanism of the development, progression and therapy of preneoplastic lesions., Methods: A Double-blind study was carried out on 15 patients afflicted with oral leukoplakia. They were treated daily (3 topical applications) with 0.1% isotretinoin gel or a placebo for 4 months. Afterwards, the patients treated with placebo were administered the active medication for an additional 4 month period., Results: All the patients, with the exception of one patient who was lost to follow-up, who finished the treatment, showed a marked improvement of the dimension and the clinical aspect of the lesions (3 total remission, 11 improvement of the size and clinical appearance of the lesion of 50% or more). The immunohistochemical analysis for bcl-2 protein showed a weak positive reaction of the level in the basal membrane of the specimens collected before the pharmacological treatment; after the pharmacological treatment almost all the specimens with the exception of one, tested completely negative for bcl-2 protein. In the specimens collected before the pharmacological treatment only a few apoptotic bodies were observed, while after treatment the samples showed a noticeable increase of apoptotic bodies., Conclusions: A statistical analysis showed that the difference in the positive reaction to bcl-2 protein between the 2 groups is not statistically significant (p = 0.132). On the other hand, the difference in the count of apoptotic bodies between the 2 groups is statistically significant (p = 0.0193).

Background: Surrogate endpoint biomarkers (SEBs) are detectable molecular, cellular, and tissue changes that take place during tumorigenesis and can be modulated by a chemoprevention agent., Method: To identify candidate SEBs for invasive squamous cell carcinoma of the upper aerodigestive tract (SCC), we have studied the expression of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFr) in sequential biopsy specimens of dysplastic oral leukoplakia and adjacent normal-appearing mucosa. Biopsies were taken from patients before, during, and after treatment with 13-cis retinoic acid, a vitamin A derivative. Immunohistochemistry was performed using the Biogenex Super Sensitive Biotin-Streptavidin horseradish peroxidase detection system., Results: The pretreatment expression of TGF-alpha and EGFr in dysplastic oral leukoplakia was increased when compared with their expression in adjacent normal-appearing mucosa (p = 0.001 and p = 0.01, respectively). Eleven of 14 patients enrolled in the study (78.6%) completed 3 months of treatment with 13-cis retinoic acid (1. 0 mg/kg/day). TGF-alpha expression in dysplastic oral leukoplakia, but not in adjacent normal-appearing mucosa, decreased during treatment (p < 0.05)., Conclusions: TGF-alpha is a candidate SEB for future SCC chemoprevention trials., (Copyright 1999 John Wiley & Sons, Inc. Head Neck 21: 566-573, 1999.)

In a double-blind study 10 patients with oral leukoplakia were treated daily (three topical applications) with 0.1% isotretinoin gel or a placebo for 4 months. Nine patients completed the treatment, while one patient was lost to follow-up. Subsequently, the patients who had received the placebo used the active medication for an additional 4 months. All patients treated with the active medication showed a significant improvement of the oral lesions while, in the patients receiving only the placebo, the size of the lesions remained the same. Also the group of patients who received the active medication after the placebo showed an improvement in the size and clinical appearance of the lesions. A complete response was defined as the complete disappearance of the lesion as assessed by visual inspection, while a partial response was defined as a 50% or more reduction in the size of the lesions. In total we had a complete response and eight partial responses. No side-effects from the use of the gel were ever observed. The percentage of bcl-2-positive cells was evaluated in the basal layer from a minimum of 1000 cells in each case and the bcl-2 immunostaining was scored using three groups: - (< or = 10% cells); + (< or = 50% cells); +2 (> or = 50% cells). The presence of apoptotic bodies was evaluated in a random fashion in the parabasal layer in 20 HPF. Immunohistochemical analysis for bcl-2 protein showed that before treatment a weak positivity of the basal layers, with a focal positivity of some parabasal cells, was present: five out of nine specimens were positive. Only a few apoptotic bodies were observed. After treatment in almost all specimens it was possible to observe a complete bcl-2 negativity with a positivity in only one specimen out of nine. An increase in apoptotic bodies was observed. Statistical analysis showed that the difference between the bcl-2 positivity in the two groups was not statistically significant (P = 0.134) while, on the contrary, the difference in the count of the apoptotic bodies between the same two groups was statistically significant (P = 0.0193). In conclusion, the data obtained from this pilot study show that good results can be obtained with the topical use of 13-cis-retinoic acid.

A new therapy for the treatment of oral leukoplakia by 5-aminolevulinic acid (ALA) and photodynamic therapy (PDT) is presented. ALA, a precursor in the biosynthesis of haeme, induces the production of the endogenous photosensitizer protoporphyrin IX which can be used for PDT. Twelve patients, who had been suffering from leukoplakia of the oral mucosa for several years, were treated by ALA-mediated PDT. ALA was used as a topical photosensitizer and 20% ALA cream was applied to the leukoplakia lesion of the oral mucosa for two hours and then light activated at 630 nm, 100 mW/cm2 and 100 J/cm2. Five patients showed complete response to the treatment, four patients showed a partial response and in three patients treatment was unsuccessful. One patient with partial response was retreated, after which the lesion disappeared.

Objective: To evaluate the effect of gynostemma pentaphyllum mak (GP) on carcinomatous conversions of leukoplasia of the golden hamster cheek pouch induced by dimethylbenzanthracene (DMBA)., Methods: 284 golden hamsters, ranging from 4 to 8 weeks of age, 80 to 100 grams in weight were randomly divided into three groups: control, model and experimental groups. Control group (8 hamsters) were sacrificed at week 0 and week 12 for control. Model group (123 hamsters) were pasted in bilateral cheek pouches with 0.5% DMBA, 3 times a week for establishment of experimental leukoplasia, and sacrificed at week 2 to week 9 (12-22 hamsters were sacrificed a week). Experimental group (153 hamsters) were divided into three subgroups. Subgroup A underwent simultaneous DMBA pasting and oral taking of GP, and were sacrificed at week 6 and week 9. Subgroup B underwent oral taking of GP for 12 weeks after completion of DMBA pasting. Subgroup C underwent DMBA pasting after completion of oral taking of GP for 12 weeks., Results: The earliest epithelial dysplasia and carcinoma in situ occurred at week 3 and week 6 after DMBA pasting. There were significant differences between subgroup B and model group for 3 weeks' administration of DMBA (P < 0.05), and between subgroup C and model group (P < 0.01), while there was no difference between subgroup B and model group for 6 week's administration of DMBA (P > 0.05)., Conclusions: GP could inhibit and reverse the carcinomatous conversions of leukoplasia of golden hamster cheek pouches, indicating its positive anticarcinogenic effect.

Background: A complete in vitro multi-stage carcinogenesis model for oral cancer was developed to examine chemopreventive strategies. In the present study, the effects of EGCG [(-)-epigallocatechin-3-gallate], the major constituent of green tea, is being examined to understand mechanisms of action., Methods: Effects of EGCG on the cell populations were examined with growth assays, cell cycle analysis, and western blots for retinoblastoma protein (pRB)., Results: In each cell type, EGCG inhibited growth, with a decrease in efficacy as cells progressed from normal to cancer. A G1 block was induced with an increase in the underphosphorylated form of pRB; EGCG-induced inhibition was not permanent, cells recovered, and no resistance developed., Conclusions: Our multistage carcinogenesis model for chemoprevention was effective in defining the chemopreventive value of EGCG. The observation that cancerous oral epithelium was less responsive than normal or dysplastic tissues has implication in the use of this agent, and the mechanisms responsible for this result remain to be defined.

Background: Because malignant transformation of dysplastic oral leukoplakia has been reported in up to 43% of cases, these lesions must be managed., Methods: This study evaluated the use of topical 1% bleomycin in dimethylsulfoxide for the treatment of dysplastic oral lesions. Bleomycin was applied once daily for 14 consecutive days to lesions of the oral mucosa in 19 patients. Immediate posttreatment biopsies and the clinical response were evaluated and clinical follow-up was conducted for as long as possible., Results: The mean age of the patients at diagnosis was 59.4 years and 74% were tobacco users. Seventy-five percent of patients had resolution of dysplasia at follow-up biopsy, with a mean improvement of two histologic grades of dysplasia after topical chemotherapy. Ninety-four percent of the patients attained at least partial responses. After a mean follow-up period of 3.4 years, 31.6% of patients had no clinically visible lesions and 47.4% of patients had clinically benign lesions of homogeneous leukoplakia or minimal visible leukoplakia. In 2 patients (11%) malignant transformation occurred a mean of 1.75 years after bleomycin treatment., Conclusions: Topical bleomycin may prevent the potential progression of leukoplakia through dysplasia to carcinoma. Close follow-up of all patients with dysplasia is required.

Chronic graft-versus-host disease commonly appears with oral manifestations subsequent to allogeneic bone marrow transplantation. These manifestations include leukoplakia, mucosal atrophy, erythema, ulcers, and xerostomia. Some lesions are resistant to treatment with immunosuppressive medications. Ultraviolet A irradiation therapy with oral psoralen has been shown to be effective in treating these resistant lesions. This article presents a review of the literature and a case report.

Over the last few years, we have witnessed tremendous progress in both basic and clinical research on retinoids. Preclinical studies have indicated the potential of retinoids in cancer prevention and therapy, but the actual successful application of retinoids in clinical chemoprevention trials has been the recent and exciting development in the field of retinoid research. Our understanding of the role of retinoids in normal developmental processes and the differentiation of normal and malignant cells, and the fundamental discovery of the nuclear retinoid receptors that act as transcription modulating factors regulating specific gene expression have been major advances in the field of basic retinoid research. Chemoprevention is the newest research approach in our efforts to control upper-aerodigestive tract cancers, which have one of the lowest cure rates among epithelial malignancies, and in which the occurrence of second primary tumors further burdens the dismal prognosis of patients. The efficacy of retinoids in the reversal of oral premalignant lesions and the prevention of second primary tumors has generated tremendous enthusiasm among retinoid researchers, particularly those in the field of chemoprevention. Current explorations of combinations of retinoids with biologic response modifiers such as alpha-interferon, as well as new receptor-selective retinoids, hold promise for the future.

A major limiting factor in the successful implementation of chemoprevention for oral cancer has been the lack of suitable endpoints to measure efficacy and success of clinical trials. To depend on the measure of the ultimate goal of such trials, namely cancer incidence as an endpoint has serious feasibility problems including a need for large numbers of participants, long periods of follow up and high costs. The application of selected biological markers as intermediate endpoints to reveal responses to chemopreventive regimens within a limited time frame is therefore an attractive concept. By serving as surrogates for cancer they could become valuable tools for monitoring patient compliance, defining the process of carcinogenesis and evaluating effects of the chemopreventive agents on tumour progression. This paper examines various biological markers of oral carcinogenesis and their relevance to chemoprevention trials. If validated, these biological markers could take oral cancer chemoprevention to new frontiers and help fulfil the ultimate goal of disease prevention through intervention.

We conducted a double-blind placebo-controlled trial to evaluate the chemopreventive potential of either vitamin A alone or beta carotene alone in subjects with oral leukoplakia in Kerala, India. We randomised 160 fishermen and women with oral precancerous lesions to receive oral vitamin A (retinyl acetate 300,000 IU/week x 12 months, n = 50), or beta carotene (360 mg/week x 12 months, n = 55), or placebo (n = 55). Blood, saliva and urine samples were collected at baseline and at exit to study serum micronutrients and mutagenicity assays. Biopsies of the mucosal lesions at entry were performed for histopathological exclusion of malignancy. The subjects were examined once every 2 months to establish clinical response of lesions and toxicity, if any. The results are based on 43 complaint subjects on placebo, 42 on vitamin A and 46 on beta carotene. The complete regression rates were: 10% in the placebo arm, 52% with vitamin A and 33% with beta carotene (P < 0.0001). Homogeneous leukoplakias and smaller lesions responded better than non-homogeneous and larger lesions. No major toxicities were observed. Half of the responders with beta carotene and two thirds with vitamin A relapsed after stopping supplementation. Serum beta carotene concentration increased substantially with beta carotene administration while with vitamin A supplementation there was no change in serum retinol levels. In the vitamin A treated group there was a significant decrease in serum alpha tocopherol. Vitamin A administration resulted in a significant remission of oral leukoplakia without any side effects of prolonged vitamin A supplementation. The results of this study, as well as those from previous studies, appear to provide strong supporting evidence to justify long term trials with vitamin A in subjects with high-risk leukoplakias with oral cancer as an endpoint.

Background: Retinoids are under intensive study for the treatment and prevention of cancer. Substantial dose-related toxicities of retinoids are a major obstacle to this work. In a recent retrospective analysis of combined 13-cis-retinoic acid (13cRA) and alpha-tocopherol (AT) in myelodysplasia, 13cRA toxicity was reduced significantly and 13cRA activity was enhanced. These results suggested the need for prospective testing of this new combination. This trial tested the hypotheses that At can reduce toxicity of high-dose 13cRA and does not interfere with 13cRA absorption/activity as reflected by reduced 13cRA serum levels., Patients and Methods: This was a phase I trial design in which patients received fixed-dose 13cRA (100 mg/m2/d) plus escalating-dose AT (beginning at 800 IU/d, increased 400 IU/d each month until 2000 IU/d). We collected toxicity data every four weeks from self-report forms, clinical examinations and laboratory studies. AT effects on 13cRA toxicity were determined by comparing maximum toxicity at lowest AT dose with that at highest AT dose. We also measured serum levels of both agents every four weeks., Results: Of the 45 patients registered, 36 had cancer (active or prior history), 9 had premalignant lesions. Thirty-nine patients could be evaluated for initial-course toxicity; 31 for final course toxicity. Median time on treatment (all patients) was four months (range, 1-9 months); a total of 223 month-long courses of treatment were given. Eighteen percent of patients (7/39) developed grade 3 or 4 toxicity in the initial course. The rates of increase and decrease in 13cRA toxicity associated with increasing AT doses were similar: 36% decreased (11/31), 32% increased (10/31) (P = 0.84). At did not reduce 13cRA serum levels. After initial increases of mean AT plasma levels (17.9 micrograms/ ml at baseline to 45.4 micrograms/ml after first four-week course), subsequent AT plasma increases (< 2-fold) did not keep pace with increased AT doses (2-3-fold). No major activity occurred in the 21 patients with active refractory cancer. The complete response rate in patients with premalignant head-and-neck or lung lesions was 77.8% (7/9), which included two patients previously refractory to 13cRA alone., Conclusion: Although escalating doses of AT did not reduce 13cRA toxicity, the rate of initial-course (including 800 IU/d of AT) high-grade toxicity was substantially lower than that typical of high-dose 13cRA-alone and similar to that typical of low-dose 13cRA-alone. Indeed, a trial of 13cRA-alone followed by 13cRA plus AT may have detected a significant toxicity difference. We did not design such a trial out of ethical concern for known side effects of high-dose 13cRA. The increase in AT serum levels was not proportional with increasing doses of AT, which may explain the lack of a dose-response effect of AT on 13cRA toxicity. Previous trials have established that 13cRA has an approximate 10% complete response rate in oral premalignancy. Our small trial's 77.8% complete response rate in premalignant lesions suggests that AT may enhance 13cRA clinical activity. Future trials of 13cRA plus AT are needed to define this combinations toxicity profile, clinical activity and pharmacokinetics.

A particularly aggressive form of oral leukoplakia that commences with a hyperkeratosis, spreads to become multifocal and verruciform in appearance, and later becomes malignant has been termed proliferative verrucous leukoplakia. Ten patients with persistent multifocal verruciform white patches were investigated. Lesions were often bilateral and affected predominantly mandibular alveolar and buccal mucosa. At first biopsy no lesion was graded higher than a verrucous hyperplasia, but subsequently all patients had squamous cell carcinoma, and two patients have died of their disease. Lesions were managed with surgery, carbon dioxide laser, and photodynamic therapy. The patients presented here confirm the existence of proliferative verrucous leukoplakia as a clinicopathologic entity. Careful examination of the whole mouth is essential when a hyperplastic white patch is seen to check for possible proliferative verrucous leukoplakia. Early aggressive treatment must then be started, and regular long-term review is crucial.

To better understand genetic alterations in oral premalignant lesions, we examined 84 oral leukoplakia samples from 37 patients who had been enrolled in a chemoprevention trial. The samples were analyzed for two microsatellite markers located at chromosomes 9p21 and 3p14. Loss of heterozygosity (LOH) at either or both loci was identified in 19 of the 37 (51%) patients. Of these 19 patients, seven (37%) have developed head and neck squamous cell carcinoma (HNSCC) while only one of 18 (6%) of patients without LOH developed HNSCC. Our data suggest that clonal genetic alterations are common in oral premalignant lesions; that multiple genetic alterations have already occurred in oral premalignant lesions, allowing at least a focal clonal expansion; and that losses of the 9p21 and 3p14 regions may be related to early processes of tumorigenesis in HNSCC. These genetic alterations in premalignant tissues may serve as markers for cancer risk assessment.

Inhibition of buccal pouch carcinogenesis induced by dimethyl-benzanthracene in hamsters with retinamide was studied. Group A received oral retinamide at a dosage of 20mg/kg, every day for eight weeks. Group B was treated by retinamide orally at the same dosage, plus topical treatment by retinamide. Group C was treated by retinamide topically for eight weeks. Experiments exhibited that under the action of retinamide the incidence of tumor was lowered significantly. All the Group A, B and C had a good response. Clinically 80 patients were randomly arranged into two groups. One group received placebo and served as controls and the other group was treated by retinamide. Results indicate that retinamide is effective against leukoplakia and the response rate is as high as 84.0%. By contrast, only 16.7% of patients receiving placebo showed some improvement.

Squamous cell carcinomas of the oral cavity can be preceded by clinically obvious premalignant changes, and they have a high rate of incidence of development of second primary tumors. Recent studies suggest retinoids not only for the treatment of oral eukoplakia, but also for the prevention of second primaries. Although retinoids are promising therapeutic agents, their therapeutic potential has been limited by their undesirable side-effects. A complete network of nuclear receptors has now been identified that mediate the action of retinoids and can interfere directly with cell proliferation signals by interacting with transcription factors. It has recently been shown that retinoids with receptor-selective activities can be obtained that are likely to have fewer side-effects because of their restricted biologic activities.

An increasing public awareness of antioxidants may prompt a patient's request to be treated without surgery if a leukoplakic lesion is discovered. However, surgical excision remains the treatment of choice for oral leukoplakia. The use of antioxidant supplements has shown some promise, but the predictability of success remains uncertain and long-term results are unavailable. Before the decision to use any antioxidant is made, it is critical to obtain a histopathologic diagnosis of the lesion. When dealing with a lesion diagnosed as hyperkeratosis, it may be appropriate to choose an antioxidant that may take some time for clinical improvement to occur. However, as the grade of epithelial dysplasia becomes more severe, consideration must be given to the possibility of malignant transformation during antioxidant treatment. We do not recommend the use of antioxidant supplements in the treatment of any carcinoma. The therapeutic use of antioxidant supplements outside of clinical trials conducted at academic medical centers should be done with considerable caution by practitioners in private practice. It should be emphasized that in these clinical trial patients were seen at frequent intervals to monitor their progress and to intervene if there was a noticeable deterioration in the clinical appearance of the lesion. In spite of the uncertainty with respect to antioxidant treatment, there are circumstances in which it should be considered. Recurrence after surgical excision when there is little reason to believe that a second surgical excision would be any more successful is an ideal candidate. Also, patients with widespread leukoplakia that involves a large area of the oral mucosa might be suitable for treatment with antioxidants, as well as patients who have extensive medical problems that make them surgical risks. The choice of which antioxidant(s) to use is complex because thus far there is no combination that is superior to the others. Beta-carotene with ascorbic acid or alpha-tocopherol is attractive because of a lack of side effects, but the range in reported values for lesion improvement has been broad and the clinical improvement typically takes several months. Clinical response with 13-cRA is faster but requires baseline and periodic serologic testing, as well as close monitoring for side effects. In those circumstances in which time is an important consideration, 13-cRA might be useful because clinical improvement can be evaluated within a matter of weeks as compared with beta-carotene. The group from M.D. Anderson Hospital has shown the value of an induction dose of 13-cRA that is followed by a lower maintenance dose. Unfortunately, the problem of recurrence after discontinuation of 13-cRA is quite common. One aspect that has not been evaluated is the combination of conventional surgical excision and the administration of postoperative antioxidants. This would have the obvious advantage of conventional treatment of surgery together with the possible protective effect of the antioxidants. Although this is an attractive hypothesis, we do not know of any studies that have proven this to be beneficial.

The authors present methods of treatment of 90 patients with leukoplakia of oral mucosa. In 60 cases tissue material taken from focus of leukoplakia, has been tested in microscope. In 8 cases the cancer has been identified. Using method of Carr-Price, the level of vit. A and beta-karoten has been appointed.

Leukoplakia has evolved as a clinico-pathologic concept over many years, with the current clinical designation being accepted worldwide. Reflective of the biology of leukoplakia is the concept of cellular atypia and epithelial dysplasia. Adding to a better understanding of leukoplakia in general has been the definition of relevant clinical subsets which, in some cases, includes etiology (snuff), while in other cases a verrucous clinical appearance will suggest a more aggressive anticipated behavior pattern. Tobacco usage, in many of its forms, remains the prime etiologic factor; however, other considerations also apply. More recently, the potential etiologic role of Candida albicans has been stressed, as well as its possible role in carcinogenesis. So-called oral hairy leukoplakia has been defined in relation to a possible Epstein-Barr viral infection, usually in the immunosuppressed patient. Other viruses, human papilloma virus in particular, have been implicated in leukoplakia, while genetic alterations involving tumor suppressor elements (p53) have also been investigated. Finally, the management of this common condition remains a variable and includes local, topical, and systemic therapies such as anti-oxidants, carotenoids, and retinoids.

Biomarkers are being sought that could serve as surrogate end points for chemoprevention trials. Micronuclei, cytoplasmic fragments of DNA, have been proposed as a biomarker and studied in oral pre-malignancy. This study evaluated micronuclei frequency in a randomized chemoprevention trial of oral pre-malignancy. A recent clinical trial evaluated the responses of pre-malignant oral lesions to 3 months of therapy with isotretinoin followed by 9 months of either low-dose isotretinoin or beta-carotene. For 57 study participants, micronuclei were counted in mucosal scrapings of the lesion and in normal-appearing mucosa at baseline and following 3 months and 12 months of therapy. Micronuclei counts were higher in scrapings from the lesion than in the normal-appearing mucosa. Following 3 months of isotretinoin, the micronuclei counts in scrapings of the lesion were significantly reduced. With treatment, the mean micronuclei count declined at 3 months. In a randomized comparison, both isotretinoin and beta-carotene maintained the suppression of micronuclei. The change in micronuclei count was not associated with the clinical or histological response to treatment. Chemoprevention treatment with isotretinoin led to a reduction in frequency of micronuclei, a marker of recent DNA injury, which was then maintained by both isotretinoin and beta-carotene.

Background: Oral leukoplakia and oral erythroplakia may be associated with benign and dysplastic cellular changes, and are at risk of malignant transformation. Additional means of management of these lesions is needed. The results of nonblinded trials using topical bleomycin in oral leukoplakia indicated the need for phase III study., Methods: A prospective, double-blind, randomized trial of topical bleomycin versus placebo was conducted. Bleomycin 1% in dimethylsulphoxide (DMSO) or the carrier was applied for 5 minutes for 14 consecutive days. Clinical assessment and pre-application and post-treatment biopsies were conducted., Results: Twenty-two patients were randomized. Of the patients who received bleomycin, decrease in clinical size of the lesion was achieved (p = 0.001), and histological reduction in dysplasia was seen (p = 0.094)., Conclusions: The topical application of bleomycin in DMSO may represent an additional approach to management of oral leukoplakia. The treatment is well-tolerated, and may be considered when the location or extent of the lesion may make surgical excision difficult.

Seventy-nine patients with oral leukoplakia that was histologically verified as either hyperkeratosis or epithelial dysplasia with hyperkeratosis were enrolled in an antioxidant supplementation program for the treatment of the oral lesions. The patients received 30 mg of beta-carotene, 1000 mg of ascorbic acid, and 800 IU of alpha-tocopherol per day for 9 months. Clinical improvement of the oral lesion was noted in 55.7% of the patients and was more likely to occur in patients who reduced their use of alcohol or tobacco (p = 0.0056). Although risk-factor reduction was important, approximately half of the patients who did not alter their exposure to either alcohol or tobacco showed clinical improvement. The antioxidant supplementation significantly increased serum and tissue levels of beta-carotene, ascorbic acid, and alpha-tocopherol, but these changes did not correlate strongly with clinical improvement.

Retinoids, natural or synthetic derivatives of vitamin A, have been studied as cancer chemopreventive agents and as therapeutic agents in the treatment of solid tumors. Intensive clinical research has focused on the role of retinoids in preventing second primary tumors following head and neck or lung cancer. The frequent occurrence of second primary tumors in these areas provides clinical support for the hypothesis of field carcinogenesis. Based on evidence of its efficacy in reversing oral premalignancy, the synthetic retinoid 13-cis-retinoic acid (13cRA) was studied in a 1-year trial to prevent the incidence of new cancers in patients who had been treated for squamous cell carcinoma (SCC) of the head and neck. Second primary tumors developed in only 4% of 49 patients treated with 13cRA, as compared with 24% of 51 patients treated with placebo (P = .005). These findings have led to two ongoing large-scale trials of 13cRA in North America. One study, performed through the M.D. Anderson Cancer Center and its affiliated Community Clinical Oncology Program and the institutions of the Radiation Therapy Oncology Group (RTOG), will determine whether long-term administration of low-dose 13cRA will prevent second primary tumors following an initial head and neck cancer. Another intergroup study using a similar randomized double-blind design is being performed among patients who have undergone resection of a stage I non-small-cell lung cancer. In Europe, a large chemoprevention study called Euroscan is currently examining the efficacy of another retinoid, retinyl palmitate, in preventing second primary tumors following head and neck or lung cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

In a prospective study we examined the effect of preoperative chemotherapy with cisplatinum and 5-fluorouracil (5-FU) in 40 patients with advanced carcinoma of the oral cavity. Histopathologic grading, cell kinetics and immunohistochemical parameters of the mucosa were determined at two different locations. Prior to therapy, histologic dysplasias in the mucosa "close" to the tumor were observed in 75 percent of the patients. Dysplastic changes of the mucosa "far" from tumor were also found in more than one-third of the patients. Our results show that more advanced dysplasias improve only temporarily under the influence of preoperative systemic chemotherapy. Moreover, new dysplasias appearing during chemotherapy and persisting after its termination are probably induced. The results point to a possible carcinogenic potential of the combined chemotherapy schema used.

Eight patients with diffuse (non-operable) pre-cancerous lesions (oral lichen or leukoplakias) were treated with fenretinidec (4-HPR) applied topically twice daily. After one month of therapy two patients had complete remission and the other six had a greater than 75% response. 4-HPR was well tolerated, and no local or distant side effects were observed. Topical treatment may be combined with oral administration at lower doses to obtain therapeutic results with less toxicity than observed with oral administration alone. A study to further evaluate the efficacy of topical treatment is in progress at our institute.

In earlier work, we demonstrated that 0.1 p. 100 topical tretinoin is clinically effective and well tolerated compared with placebo for the treatment of oral leukoplakia and oral keratosic or erythematous lichen planus. Here we aimed to complete this clinical protocol with histological and biochemical analyses comparing the biopsy specimens collected at inclusion and those collected after 4 months of treatment. Histological results were based on changes in keratinization observed between onset of treatment and 4 months treatment. Biochemical studies included the use of antibodies (anti-cytokeratins 10-11, anti-filaggrine) for the immunohistochemical evaluation of keratinization and 2-dimensional gel electrophoresis for measuring cytokeratins. In patients with lichen planus, histological changes during treatment showed that, in the 10 patients in the tretinoin group, keratinization disappeared in 6 and decreased significantly in 3. Immunohistochemistry revealed that cytokeratins 10-11 and filaggrin disappeared in 57 p. 100 of the patients treated with tretinoin versus 25 p. 100 in the patients given placebo. Bidimensional gel electrophoresis showed that cytokeratins 1, 2, 10 and 11 disappeared only in the tretinoin group (60 p. 100 of the cases). In patients with leukoplakia, histological changes during treatment showed that, in the tretinoin group, keratinization disappeared in 5 cases and decreased in 5 others. Immunohistochemistry revealed that cytokeratins 10-11 disappeared in 30 p. 100 of the patients treated with tretinoin versus 25 p. 100 in the placebo group. Bidimensional electrophoresis demonstrated that cytokeratins 1, 2, 10 and 11 disappeared in 43 p. 100 of the patients treated with tretinoin.

Micronuclei frequency, a marker of genotoxicity, was studied within a trial of alpha-tocopherol for chemoprevention of oral leukoplakia. Oral swabs were obtained from two sites, the leukoplakia lesion and normal-appearing mucosa, at baseline and following 24 weeks of therapy with 400 international units of alpha-tocopherol twice daily. These specimens were analyzed for micronuclei frequency. The major risk factors for oral carcinogenesis in the group studied were cigarette smoking and alcohol consumption. alpha-tocopherol therapy produced a significant reduction in micronuclei frequencies in specimens from both the visible lesions (P < 0.01) and the normal-appearing mucosa (P < 0.01). The micronuclei frequencies, both at baseline and following therapy, were greater in specimens taken from the lesion than in those from the normal-appearing mucosa. Although these results indicate that alpha-tocopherol has a beneficial effect in oral carcinogenesis, there was no significant clinical or histological response associated with the change in micronuclei frequency. Micronuclei frequency has not yet been validated as a biomarker for cancer incidence, and consequently, its utility as an intermediate end point for chemoprevention trials is not known. Determining clinical significance of micronuclei frequency patterns in oral carcinogenesis and chemoprevention will require further study.

Several clinical trials have demonstrated the efficacy as chemopreventive agents on upper aerodigestive tract (UADT) cancer of vitamin A or its natural precursor, beta-carotene, or its synthetic analogues, retinoids. Particularly, 13-cis-retinoic acid (cRA) has been shown to reverse oral leukoplakia and to reduce the frequency of second primary tumours in patients treated for head and neck cancer. Since chemopreventive treatments must be of very long-term duration (even years), and because of apparent 'good health' of treated patients, the toxicity of used drugs and the compliance with treatment need to be carefully considered. Current clinical research has evaluated the efficacy of low doses of cRA in larger numbers of patients, and the increased effectiveness of cRA when used in combination with recombinant alpha-interferon 2a (r-alpha-IFN 2a) has also produced high objective response rate in patients affected with squamous cell carcinoma of the head and neck. In vitro and in vivo studies to verify the validity of several biomarkers (in particular micronuclei) as intermediate end-points in chemoprevention studies are continuing, to allow the short-term screening of promising chemopreventive drugs to be used in clinical trials. The published literature on this matter is reviewed, with special attention to retinoid treatment and toxicity/compliance-related problems, and compared with our own experience.

Among the tissue, cellular, and molecular changes which take place during the development of squamous cell carcinoma (SCC) of the upper aerodigestive tract, only a limited number can be used as surrogate endpoint biomarkers (SEBs) in cancer chemoprevention trials. Molecular SEBs will be genes or gene products which can be measured accurately and reliably, are altered in intraepithelial neoplasia (dysplasia), correlate strongly with the true outcome (invasive cancer), and are modulated by a chemoprevention agent(s). To identify and modulate molecular SEBs in intraepithelial neoplasia of the upper aerodigestive tract, we studied expression of the epidermal growth factor receptor (EGFR), transforming growth factor-alpha (TGF-alpha), and HER-2/neu genes in oral leukoplakia before, during, and after treatment with 13-cis-retinoic acid, a vitamin A derivative. Four of nine patients treated for 3 months with 1 mg/kg/day of 13-cis-retinoic acid had complete resolution of their leukoplakia. Biopsies were taken of leukoplakia and adjacent normal-appearing mucosa before, during, and after treatment. Immunohistochemistry was performed using the BioGenex Super Sensitive Biotin-Streptavidin horseradish peroxidase detection system. Pretreatment expression of EGFR, TGF-alpha, and HER-2/neu in leukoplakia was increased when compared to normal-appearing mucosa. TGF-alpha expression decreased during treatment in leukoplakia, but not in normal-appearing mucosa, suggesting that TGF-alpha may serve as an intermediate endpoint in cancer chemoprevention trials.

A randomized study was conducted to evaluate the effect of tretinoin and patient tolerance to treatment with topical applications in series of 20 cases of smoking-related or traumatic oral keratoses leukoplakia and of 20 cases of lichen planus. In each group, patients applied the topical ointment containing tretinoin (10 patients) or placebo (10 patients) twice daily. Clinical outcome was evaluated on the basis of the surface area of the lesion, measured monthly during treatment, as compared with the area observed at treatment onset. After 4 months treatment, there was a significant decrease in the surface area of the lesion in the patients with lichen planus (p < 0.02): 94 p. 100 in the tretinoin group versus 21.4 p. 100 in the placebo group. In patients with leukoplakias, there was also a very significant reduction in the surface area of the lesion after 4 months of treatment (p < 0.001): 80 p. 100 in the tretinoin group and 16 p. 100 in the placebo group. Tolerance to treatment was generally good despite a few complaints of quite temporary burning sensation at application rapidly resolutive.

The management of patients with 'field cancerization' of the oral mucosa, with multicentric foci of invasion, presents a considerable problem for the head and neck surgeon. Surgical resection of synchronous or metachronous primary squamous cell carcinomas, along with adjacent premalignant lesions, is likely to be associated with considerable mutilation. Photodynamic therapy (PDT) has been shown to be of value in the treatment of superficial tumours in the upper aerodigestive tract, with excellent healing of treated areas. This study reports the use of PDT to treat 11 patients with 'field cancerization' occurring in the oral cavity. Six patients had multiple primary cancers and five had single primary tumours. All had associated areas of leukoplakia. Each received Photofrin 2 mg/kg 48 hours prior to photoirradiation with 50-100 J/cm2 red laser light by surface illumination. Six to eight weeks later treated areas in 10 of the 11 patients showed a complete response to PDT; one patient had areas of residual leukoplakia. Two patients developed further areas of leukoplakia or erythroplakia within 12 months but no patient has had evidence of recurrent invasive carcinoma in the treated areas. Longer term follow-up will be necessary to exclude further recurrence. It is concluded that PDT offers an effective repeatable treatment option, whether on its own or as an adjunct to local excision, for patients with 'field cancerization' of the oral cavity.

Once considered a futuristic concept, chemoprevention for pre-malignant oral leukoplakia lesions is now a reality. Oral leukoplakia can proceed to invasive disease and expose the upper aerodigestive tract to carcinoma. Risk factors, diagnosis and research in chemoprevention are discussed.

Background: Oral leukoplakia is an important model for developing chemoprevention approaches for lesions in the upper aerodigestive tract. These lesions most often result from exposure to carcinogens such as tobacco and alcohol and may precede development of invasive cancer. The potent antioxidant alpha-tocopherol (vitamin E) has prevented the development of cancers of the oral cavities in animal models., Purpose: The objectives of this study were to evaluate the toxicity and efficacy of alpha-tocopherol in patients with oral leukoplakia and to assess the feasibility of performing chemoprevention trials through the network of the Community Clinical Oncology Program (CCOP)., Methods: A single-arm phase II study using the nontoxic agent alpha-tocopherol to treat oral premalignant leukoplakia was performed at seven institutions affiliated with the CCOP through The University of Texas M. D. Anderson Cancer Center. Patients with symptomatic leukoplakia or dysplasia were treated orally with alpha-tocopherol (400 IU) twice daily for 24 weeks. Follow-up was performed at 6, 12, and 24 weeks after the start of treatment to assess toxicity and response, and serum alpha-tocopherol levels were determined at baseline and at 6 and 24 weeks., Results: Of the 43 patients who have completed 24 weeks of treatment, 20 (46%) had clinical responses and nine (21%) had histologic responses. Mean serum alpha-tocopherol levels were 16.1 micrograms/mL at baseline and increased to 34.29 micrograms/mL after 24 weeks of treatment. Patient-recorded drug calendars, as well as serum drug levels, indicated excellent patient compliance; an average of 95% of the prescribed pills were taken. Treatment was extremely well tolerated; no grade 3 or 4 toxic effects were reported., Conclusions: Administration of alpha-tocopherol resulted in both clinical and histologic responses in premalignant leukoplakia lesions. The study also demonstrated that chemoprevention trials can be performed through the CCOP. The major problems were that a high percentage of patients were not assessable for response, some patients withdrew because expenses were not reimbursable, and there was limited participation within the CCOP network. These problems may reflect difficulties inherent in the implementation of multi-institutional chemoprevention trials., Implications: The efficacy of alpha-tocopherol alone and in combination with other chemopreventive agents for carcinogenesis in the upper aerodigestive tract should be explored in future trials.

A controlled clinical trial has been underway at the Istituto Nazionale Tumori (INT) of Milan since 1988. The goal of the trial is to evaluate the effectiveness of fenretinide (4-HPR) in preventing relapses, new localizations, and carcinomas in patients with benign postoperative diagnoses who have been surgically treated for oral leukoplakias. This paper presents the design and the preliminary results of this study. To date, 137 patients have been randomized, following surgical excision of oral leukoplakia, to receive either 200 mg 4-HPR daily for 52 weeks or no intervention. Twenty local relapses or new localizations have occurred so far in the control group and 9 in the 4-HPR group. Seven patients have interrupted the intervention because of toxicity. No impaired dark adaptation has been observed. We conclude that 4-HPR is well-tolerated and appears to be effective in preventing relapses and new localizations during the treatment period.

Over the past twenty years, research into the role of antioxidants in the prevention of cancer has increased dramatically. The use of antioxidant supplements to treat oral leukoplakia has gained acceptance due to the success demonstrated in several clinical trials. This review discusses the role of antioxidants in the development of cancer and their possible use in the treatment of oral leukoplakia.

Chemoprevention entails using specific agents to suppress carcinogenesis and thereby prevent the development of primary or second primary cancers. Because the concept of chemoprevention in patients with or at risk of lung cancer is new, ongoing clinical trials are based on data from epidemiologic and preclinical research, as well as on results of chemoprevention studies in head and neck cancer. The latter studies have provided a model for such studies in lung cancer, considering the two diseases have a similar etiology and biology of field carcinogenesis. Beta-carotene, natural vitamin A, and the retinoids may be effective chemopreventive agents. However, chronic administration of such agents may be required to prevent the development of cancer. Results of chemoprevention trials in head and neck cancer have demonstrated effective inhibition of the development of second primary tumors with the synthetic retinoid 13-cis-retinoic acid; investigators are hopeful this will be repeated in patients with lung cancer. Results of ongoing phase III trials and continued advances in the epidemiologic and biologic study of lung carcinogenesis should contribute to future research in this area.

16 patients with oral leukoplakia were treated with oral 13-cis-retinoic acid. The initial dose, given for 3 months, was 0.2 mg/kg/day, increasing by a further 0.2 mg/kg/day in successive 3 month cycles. The maximum dosage reached at 1.0 mg/kg/day, was given to only 2 patients (who received a total of 15 months treatment). However, 10 patients completed the cycle at 0.8 mg/kg/day (12 months treatment in total), but treatment could not continue due to toxicity (grade I and II), which was cutaneous, mucosal, and haematological (hypertriglyceridemia and hypercholesterolemia). There was grade III toxicity in the skin and mucosa in only 1 case, a patient treated at a dose of 1.0 mg/kg/day. The toxicity was reversible in all cases. 14 of the patients completed the trial. In 4 there were improvements graded as partial responses (PR) obtained at 0.2 mg/kg/day (3PR) and 0.6 (1PR) and there was one complete response (CR) obtained at 0.4 mg/kg/day. Overall there was thus an objective response rate of 36% who showed 50% or more reduction in lesion size. After the retinoic acid treatment was stopped, patients were followed-up for 12 months; 2 patients showed regression of the responses obtained after 6 and 9 months. This study shows that oral treatment with 13-cis-retinoic acid at low dosages is efficacious and with minimal toxicity. It also shows that it is not feasible to treat these patients at doses above 0.8 mg/kg/day for long periods--mainly due to poor compliance.

Chemoprevention involves efforts to block or reverse carcinogenesis before the development of invasive cancer. Natural agents, such as retinol and beta carotene, as well as synthetic retinoids have been studied as potential chemopreventive agents. In the head and neck, chemoprevention studies have included efforts both to reverse premalignant lesions such as oral leukoplakia and to prevent the development of second primary tumors. In one recent trial, high-dose 13-cis-retinoic acid treatment resulted in a dramatic reduction in the incidence of second primary tumors. However, significant toxicities were associated with the high dosage. This trial, as well as previous studies of oral leukoplakia, have led to the development of a chemoprevention trial using a low dose of 13-cis-retinoic acid to prevent second primary tumors following head and neck cancer. The rationale and design of this study are discussed in detail.