Leukoplakia is a clinical diagnosis 1 that may represent histologic

629 Topical Bleomycin for the Treatment of Dysplastic Oral Leukoplakia Joel B. Epstein, D.M.D., M.S.D. 1 Meir Gorsky, D.M.D. 2 Frances L. W. Wong, M.D. Amelia Millner, M.D. 1 British Columbia Cancer Agency, Vancouver Hospital, University of British Columbia, Vancouver, British Columbia, Canada. 2 Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Tel Aviv, Israel. 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. Cancer 1998;83:629 34. 1998 American Cancer Society. KEYWORDS: bleomycin, oral leukoplakia, dysplasia, chemoprevention. Address for reprints: Joel B. Epstein, D.M.D., M.S.D., British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, B.C., Canada V5Z 4E6. Received January 23, 1998; accepted February 4, 1998. Leukoplakia is a clinical diagnosis 1 that may represent histologic findings of epithelial hyperplasia, hyperorthokeratosis and/or hyperparakeratosis with or without epithelial dysplasia, or carcinoma. Dysplasia may be present in an area of leukoplakia in up to 16% of lesions, and up to 10% may be malignant at the time of initial examination. 2 7 In dysplastic leukoplakia, the risk of malignant transformation has been reported to be as high as 43%. 8,9 Leukoplakia that represents carcinoma in situ (CIS) or invasive squamous cell carcinoma (SCC) must be treated by surgical excision, radiotherapy, or a combination of these treatments. Persistent leukoplakia and dysplastic mucosal lesions must be managed. Elimination or at least reduction in local irritants by modifying prostheses, use of tobacco products, and ethanol use is essential. Excision is indicated if the lesion is localized and accessible; however, recurrence of leukoplakia after local excision has been reported in 10 35% of cases. 8 10 Laser excision of oral leukoplakia is a common treatment of dysplastic lesions. 7,9 16 Improved healing and a low complication rate associated with laser excision has led to the increased use of this modality to treat oral leukoplakia. After laser excision, there is limited formation of scar tissue, which may result in better maintenance of function after treatment. Twenty-nine patients treated with extensive 1998 American Cancer Society

630 CANCER August 15, 1998 / Volume 83 / Number 4 leukoplakia measuring up to 6 cm 8 cm were followed for a mean of 5 years. 10 An initial recurrence rate of 10.8% was observed; a malignant transformation rate of 2.6% and a 3-year control rate of 97% were achieved after 1 2 procedures. 10 In another study, local control of leukoplakia was achieved in 17 of 20 patients (85%). 13 A cure rate of 90% was reported after laser excision of 70 patients, with a mean follow-up of 5.3 years. 15 In another report, after laser excision in 131 patients, there was a 19% probability of remaining lesion free after 3 years. 7 Laser excision requires access to equipment and additional expertise; it may require hospitalization and general anesthesia, and extensive lesions may be difficult to remove. Postoperative pain often is severe, and may persist several weeks. Tissue excised by laser evaporation cannot be evaluated histologically. Chemotherapy may provide an additional approach to the management of oral leukoplakia. Population studies and clinical trials have demonstrated the effects of vitamin A and retinoids in oral leukoplakia. 14,17 34 Topical application of vitamin A has been shown to cause regression of leukoplakia. 19,33 However, leukoplakia frequently recurs in patients treated with retinoids. The use of systemic bleomycin in treating oral squamous cell carcinoma reportedly yielded a response rate of approximately 25% 35 and response rates of 72.4% have been reported in patients with skin cancer. 36 When applied locally, the drug does not cause significant tissue irritation. 37 Bleomycin applied with iontophoresis was effective in treating nonmalignant lesions of the head and neck. 38 Resolution of dysplastic leukoplakia was reported after local injection of bleomycin weekly for 8 weeks. 39 Recently, topical bleomycin has been used for resolving Kaposi s sarcoma of the skin. 40 Topical application of bleomycin in dimethylsulfoxide (DMSO) has been evaluated in open clinical trials, resulting in regression of leukoplakia. 37,41,42 A randomized, double blind, placebocontrolled Phase III study using 1.0% bleomycin in DMSO demonstrated a significant effect of topical bleomycin on the resolution of leukoplakia. 43 The purpose of the current prospective study was to evaluate the effect of 1.0% topical bleomycin on dysplastic leukoplakia of the oral mucosa. MATERIALS AND METHODS Patients of the British Columbia Cancer Agency with dysplastic oral leukoplakia were eligible for the study. Institutional approved informed consent was obtained. Nineteen patients were identified with a diagnosis of dysplastic leukoplakia and were enrolled in the study. All patients had clinically visible leukoplakia. The duration of the lesion, smoking history and alcohol use, oral hygiene status, and dental status were recorded. All potential oral irritants were identified and eliminated when possible. Three-millimeter punch biopsies of identified lesions, guided by toluidine blue and Lugol s iodine application, were conducted as described previously. 44 Pathologic diagnoses of dysplasia and histologic grading of the dysplastic lesions were required before entering into the study. The pathology was interpreted by a single pathologist in a blinded fashion. Cases of CIS, invasive SCC, and lesions identified as inflammatory in nature (e.g., lichen planus) were excluded from the study. Patients were age 18 years. Exclusion criteria included pregnant women or women of childbearing age in whom contraception was not confirmed. The treatment solutions (1% weight/volume bleomycin in DMSO), prepared fresh by the Department of Pharmacy, were applied to the involved area by soaking pledgets of cotton in the solution with continuous application to the area for 5 minutes, once daily for 14 consecutive days. After application, the solution was stored in a refrigerator. The applications were applied in the Dental Department by clinic staff on weekdays, and on weekends by a family member of the patient who had received prior instruction in the method of application by clinic staff. Patients were assessed weekly during treatment and at least once in the 3 months after treatment. Biopsies, guided by toluidine blue and Lugol s iodine, were repeated at 4 weeks after treatment. Laboratory study was completed prior to treatment and 2 weeks after treatment, and included complete blood count, electrolytes, blood urea nitrogen, and serum creatinine. Complete response was defined as no clinical and histologic evidence of leukoplakia. Partial response was defined as a reduction in the severity of the clinical nature of the lesion and/or a reduction of the histologic grading of the dysplasia. RESULTS A total of 19 patients (10 men and 9 women) with oral dysplastic leukoplakia were included in the study (Table 1). The mean age at diagnosis was 54.9 years (53.9 years for men and 56 years for women) and ranged between 25 79 years. Fourteen patients (73.7%) were tobacco users and only 4 patients reported alcohol consumption more than on an occasional basis. Three of the alcohol users also were smokers. The mean follow-up of the 19 patients was 3.9 years (range, 0.5 10 years). When patients that were followed for

Topical Bleomycin in Dysplastic Oral Leukoplakia/Epstein et al. 631 TABLE 1 Patient Characteristics and Treatment Outcome Patient Age (yrs) Gender Tobacco Alcohol Site Pretreatment Immediate posttreatment Clinical Histology Clinical Histology Posttreatment follow-up (yrs) Management and follow-up Clinical end results 1 61 M Palate SPL 2 1 7.5 Remission of moderate atypical to hyperkeratosis by bleomycin 2 44 F Buccal SPL 2 1 3 Clinical follow-up 3 45 M Tongue 2 1 10 Is 8 yrs after 11 SCC of the tongue, laserleukoplakia recurred 4 60 M Buccal 2 1 7.5 Lichenoid lesions controlled with topical steroids WNL 5 54 M Tongue 4 ML 1 5.5 Dysplasia recurred after 4 yrs-laser excision, leukoplakia recurred 6 59 F Gingiva 2 ML 1 8 Clinical follow-up WNL 7 64 F Buccal 3 2 3 Leukoplakia partially controlled with topical vitamin A 8 32 M FOM 3 WNL 0 2 Clinical follow-up WNL 9 60 F Buccal SPL 2 WNL 0 6 Clinical follow-up WNL 10 52 M Tongue N 2 1 3 Repeated topical bleomycin or vitamin A reduced thickness of leukoplakia 11 63 F Tongue 4 ML 1 3 Clinical follow-up 12 25 F Tongue 4 ML 2 3 Is 5 yrs after T1 SCC of the tongue, not controlled with vitamin A (SCC recurred 4 yrs after follow-up discontinued) N 13 76 F Tongue 2 2 5 3 mos after T1 SCC of the tongue, vitamin A only partially maintained leukoplakia up to recurrence of SCC of the tongue SCC 14 38 M Tongue N 4 2 2.5 Bleomycin and vitamin A partially controlled clinical leukoplakia up to transformation to SCC SCC 15 64 M Lip SPL 3 1 0.5 13 yrs after SCC of the lip clinical follow-up 16 79 M Tongue 4 2 0.7 Clinical follow-up WNL 17 54 M FOM SPL 4 ND 1 Failed follow-up, transformed to SCC in 1 yr SCC 18 70 F FOM N 2 ML 1 2 Vitamin A periodically controlled leukoplakia ML 19 44 F FOM 3 ML 1 1.5 Clinical follow-up WNL M: male; F: female, : no history of use; : history of use; SPL: speckled leukoplakia; : homogenous leukoplakia (uniform, thin, flat); WNL: within normal limits; ML: minimal leukoplakia; SCC: squamous cell carcinoma; FOM: floor of mouth; N: non-homogenous leukoplakia or thick leukoplakia (irregular, thick, raised); ND: not done; 0: normal epithelium; 1: benign hyperkeratosis; 2: mild dysplasia; 3: moderate dysplasia; 4: severe dysplasia. 1 year (Patients 15, 16, and 17) were excluded, the mean follow-up time was 4.53 years (range, 1 10 years). Bleomycin treatment was well tolerated with minor mucosal reactions resulting in oral sensitivity occurring after approximately 1 week of treatment and resolving within 1 2 weeks of completion of treatment. Eighteen patients (94.7%) had at least a partial response to the application of topical bleomycin (Table 1). Two patients (Patients 8 and 9) had a complete response (10.5% complete response). In 94.7% of the study group (excluding Patient 13) histologic remission of the dysplasia was found. No posttreatment biopsy was performed in one patient (Patient 17) because the patient failed follow-up. In 14 patients (73.7%) a clinical improvement of the white lesions was observed after the bleomycin treatment. In five patients who had homogenous leukoplakia no immediate major clinical improvement was observed; however, in two of these patients (Patients 4 and 16) complete clinical remission was observed at last follow-up. In 2 patients (Patients 3 and 8) no change was noted in their lesions after treatment with bleomycin and a recurrence of the carcinoma occurred 5 years later in 1 patient (Patient 13). Transformation to carcinoma occurred in 15.8% of the group studied (Patients 13, 14, and 17). Of these, 1 transformation was a recurrence of a previously treated malignancy (Patient 13), and the other 2 transformations (11.1% of the remainder) transformed to SCC in a mean follow-up of 1.75 years. Two of these three patients were tobacco users

632 CANCER August 15, 1998 / Volume 83 / Number 4 and one also was a heavy alcohol user (Patient 17), and none had changed their habits after the first diagnosis of dysplasia. Approximately 78% (7 of 9) of the patients with an improvement in their leukoplakia stopped tobacco use after the first diagnosis of dysplasia; the clinical end result of 6 of these patients (75%) was homogenous leukoplakia or complete resolution of clinical lesions. A mean histologic grade of 2.84 was calculated (Table 1) prior to treatment with bleomycin, which is very close to a diagnosis of the moderate dysplasia. The posttreatment mean histologic grade was 1.16, which is very close to a nonmalignant hyperkeratosis stage. A mean improvement of nearly two histologic stages of dysplasia were recorded after chemotherapy with topical bleomycin. We compared the clinical description of the lesion and the histologic diagnosis. Homogenous leukoplakia was reported as mild dysplasia in four cases, moderate dysplasia in three cases, and severe dysplasia in four cases. In eight cases of nonhomogenous leukoplakia (including speckled leukoplakia), five cases were mild and three cases were either moderate or severe dysplasia. Clinical lesions were improved from the pretreatment description at last follow-up. Treated lesions were not detected clinically in six patients, and were described as homogenous leukoplakia in nine patients and nonhomogenous in one patient. However, three cases of SCC were diagnosed. Of the 16 patients who were followed for 1 year, no dysplasia was diagnosed in 12 patients (75%) after bleomycin treatment. Of the remaining 4 patients (25%), 2 had a previous history of carcinoma in the same site (Patients 12 and 13). In another patient (Patient 14), the lesion transformed into SCC 2.5 years later. In 1 patient (Patient 7), no clinical improvement was observed after bleomycin treatment; however, at 3 years of follow-up, homogenous leukoplakia was maintained clinically with topical vitamin A acid application (0.01%). Four patients had leukoplakia in a previous cancer site (three on the tongue and one on the lip). Two of these patients had a recurrence of the cancer (Patients 12 and 13). One patient was followed for 10 years with no recurrence (Patient 3). Although the remaining patient (Patient 15), who had history of carcinoma of the lip, responded well with remission of the lesion from severe dysplasia to nondysplastic hyperkeratosis with topical bleomycin, there was insufficient follow-up for any conclusion. DISCUSSION There has been increasing interest in preventing progression of leukoplakia to SCC. Because the risk of dysplastic leukoplakia to transform into SCC is much higher than that of hyperkeratotic leukoplakia, 9 and because transformation may occur close to the time of the diagnosis, an immediate clinical approach should be taken. Although surgical removal may be recommended, topical chemoprevention may represent an additional treatment option. Systemic chemoprevention may lead to toxic side effects, which may be reduced with topical therapy. Topical applications of vitamin A have been evaluated in small clinical trials. 19,33 Complete response was observed in 5 patients with snuff-induced leukoplakia, 33 and in a study of 11 patients an 82% response rate was reported. 19 In a study of 6 patients treated with daily topical application of bleomycin in DMSO for 15 18 days, reductions in keratinization and dysplasia were observed. 41 In another study, ten patients were treated with topical bleomycin in DMSO. 42 Dysplasia was reversed in five of the ten patients, but was unchanged in the remaining patients. The thickness of the keratotic layer was decreased in five patients, increased in four patients, and unchanged in one patient. Recurrence appeared to be delayed compared with recurrence after surgery. 42 In a study in which bleomycin was applied topically once daily for 2 weeks, 12 patients were studied using 2 concentrations of bleomycin (0.5% and 1.0%). 37 Although partial responses were observed with the less concentrated application, complete remission was observed in 60% of those treated with the higher concentration, and 40% achieved a partial remission. We previously completed a prospective double blind study using topical 1% bleomycin compared with placebo. 43 A significant reduction in the size and the severity of dysplasia of the lesions was observed and the treatment was well tolerated. It is clear that bleomycin is an active drug for the treatment of leukoplakia and may be considered a treatment option for the management of oral leukoplakia. The clinical appearance of homogenous leukoplakia usually is considered a low risk lesion compared with nonhomogenous lesions, which may represent lesions at greater risk. However, in this series of patients the clinical descriptions of the lesions prior to treatment did not predict the histologic description. The histologic description of posttreatment biopsies improved or remained unchanged in all cases, and overall improvement in the histologic grade of dysplasia or reversal of dysplasia was observed. Only four patients with dysplastic leukoplakia were included in the previous randomized, double blind study and the follow-up was limited. 43 The current study, which was limited to dysplastic leukoplakias with extended follow-up, and previous study re-

Topical Bleomycin in Dysplastic Oral Leukoplakia/Epstein et al. 633 sults 43 demonstrate the potential role of topical bleomycin as a treatment option for dysplastic leukoplakia. In this study, we found that dysplasia reversed, at least partially, in 94% of our patients with a mean reversal of nearly 2 histologic stages. A previous study reported remission of the dysplasia in 50% of the patients and recurrence appeared to be slower than that after surgery. 42 In the previous study conducted at our center topical bleomycin was well tolerated and effective for the treatment of leukoplakia in general. 43 The current study demonstrates that topical bleomycin is a promising drug for controlling dysplasia in oral mucosal lesions. Although the lesions in three patients transformed into malignancies, it is important to note that in one patient the lesion may represent a recurrence rather than carcinoma de novo. The other two patients with transformation already were at high risk of transformation. At the initiation of the treatment, they were diagnosed with severe dysplasia, and it is possible that invasive SCC may have been present at the time of diagnosis and may represent a sampling error at the time of biopsy or pathologic review. There are no studies assessing the use of topical bleomycin for the treatment for oral SCC and this treatment should not be considered for SCC. Because an initial improvement was noted in the patients with subsequent transformation after the topical treatment, the possible transformation to SCC mandates regular, continuous follow-up of these patients. All patients who present with dysplastic mucosal lesions require regular and continuing clinical follow-up and histologic assessment when indicated. The number of cases progressing to SCC after topical bleomycin should be compared with the literature in which 16% in a study from a pathology laboratory 45 to 43% progress to SCC in a patient-based clinical follow-up study. 8 Due to the known recurrence of oral leukoplakia, additional therapeutic options are needed. Perhaps the most important aspect of the treatment of dysplastic leukoplakia is eliminating tobacco and alcohol use. To date excision has been the common method of management, and continues to be a choice in treatment, particularly with the use of lasers. However, there are sites in the oral cavity in which excision is difficult and may be associated with posttreatment complications and some lesions are extensive in their area of involvement, making excision difficult. There now are choices in the management of oral dysplastic leukoplakia. Topical application of bleomycin in DMSO represents an additional modality for the treatment of oral dysplastic leukoplakia. 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