Affiliations of authors: S. Lam (Department of Respiratory Medicine), C. MacAulay, M. Guillaud (Department of Cancer Imaging), J. C. le Riche (Department of Pathology), Y. Dyachkova, A. Coldman (Department of Population and Preventive Oncology), British Columbia Cancer Agency and the University of British Columbia, Vancouver, Canada; E. Hawk, Division of Cancer Prevention, Gastrointestinal and Other Cancers Research Group, National Cancer Institute, Bethesda, MD; M.-O. Christen, Solvay Pharma, Suresnes, France; A. F. Gazdar, Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center, Dallas.
Correspondence to: Stephen Lam, M.D., F.R.C.P.C., 2775 Heather St., Vancouver, British Columbia, Canada V5Z 3J5 (e-mail: sclam{at}interchange.ubc.ca).
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ABSTRACT |
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INTRODUCTION |
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One potential strategy to inhibit the development of invasive cancer in those who are at risk of developing lung cancer is to use chemopreventive agents that either block the DNA damage that initiates carcinogenesis or that arrest or reverse the progression of premalignant cells in which such damage has already occurred (4,5).
Anethole dithiolethione (ADT; 5-[p-methoxyphenyl]-1,2-dithiole-3-thione) and Oltipraz (5-[2-pyrazinyl]-4-methyl-1,2-dithiol-3-thione) belong to the dithiolethione class of organosulfur compounds, which have antioxidant, chemotherapeutic, radioprotective, and chemopreventive properties (6). These agents are a unique class of compounds for which in vivo anticarcinogenic activity was predicted on the basis of their abilities to induce expression of carcinogen detoxification enzymes. In animal models of carcinogenesis, dithiolethiones exert chemoprotective activity against the development of lung and other cancers (610). Although Oltipraz has been tested in humans, it is not an approved drug for humans. Recently, Oltipraz was found to be too toxic for chemoprevention (11). By contrast, ADT is an approved drug that has been used in Canada, Europe, and other countries to treat drug- and radiation-induced hyposalivation as well as xerostomia due to other causes (12,13). ADT is also marketed as a choleretic and hepatoprotective agent. ADT is safe and effective for these uses at the recommended therapeutic dose (25 mg three times daily).
We performed a randomized, double-blind, placebo-controlled, phase IIb clinical trial (14) to determine the efficacy and safety of ADT as a chemopreventive agent in smokers with premalignant lesions in their bronchial epithelia.
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METHODS |
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The flow diagram of study subjects as they progressed through the phases of the randomized trial is shown in Fig. 1.
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The biopsy samples were fixed in buffered formalin and embedded in paraffin, and serial sections were obtained. The first, sixth, and 13th sections were stained with hematoxylineosin and used to determine histopathologic classification of each biopsy sample. The seventh section of each biopsy sample was stained with Feulgen-thionin for quantitative nuclear morphometry, as described below. The hematoxylineosin-stained sections were systematically reviewed by two pathologists (J. C. le Riche, A. F. Gazdar). The pathologists were blinded to the intervention assignments. All biopsy samples were classified into one of the following seven groups according to World Health Organization (WHO) criteria (19): normal (as represented by pseudostratified ciliated columnar epithelium); basal cell hyperplasia (as represented by an increase in the number and stratification of normal-appearing basal cells still covered with normal ciliated or mucin-secreting cells); metaplasia (as represented by a stratified epithelium and cytoplasmic changes consistent with squamoid differentiation); dysplasia (mild, moderate, or severe); and carcinoma in situ.
The two pathologists resolved minor (i.e., one grade) differences in sample classification by telephone consultation. If the histopathology diagnosis differed by two or more grades, both pathologists reviewed the slides again and, if necessary, reached a consensus diagnosis after communication verbally by phone or when they met in person.
One hundred fifty-six subjects had one or more sites of bronchial dysplasia as determined by analysis of autofluorescence bronchoscopy-directed bronchial biopsy samples. Of those subjects, 112 agreed to participate in our study.
Randomization. Participants were randomly assigned to receive either ADT (Sialor®, Sulfarlem®; Solvay Pharma, Suresnes, France) at a dose of 25 mg three times daily by mouth for 6 months or a placebo. The placebo tablets were identical in size, shape, and color to the tablets containing active drug. Randomization codes were generated by a statistician at the Population and Prevention Oncology Division of the BCCA. Randomization was stratified according to smoking status (current or former smoker) and grade of dysplasia (mild, moderate, or severe) of the subject's bronchial biopsy sample. Two sets of random numbers were drawn. First, random numbers were used to generate a sequence of treatment assignments (ADT/placebo). Second, numbers were drawn randomly from the following set (6, 8, 10, and 12) to set up random block lengths within which treatment assignment was constrained to be balanced. The treatment assignments were contained in unmarked envelopes which were provided to the BCCA pharmacist who had no contact with the statistician who generated the randomization codes. When a participant met the eligibility criteria, the study nurse contacted the pharmacist, who then provided the study medication to the participant according to the randomization code. The randomization code remained with the generator until the trial was completed. The randomization plan was followed as judged by dates of allocation. All study personnel were blinded to the study codes as was confirmed by independent review. All data analyses used standard approaches and well established metrics for measuring outcome.
Follow-up. The participants were examined monthly for drug-related adverse events. Their liver enzymes were measured before starting the study drug (i.e., at baseline) and at months 3 and 6 on study medications to monitor for toxicity according to the National Cancer Institute's common toxicity criteria. For participants with grade 2 adverse reactions, the dose of the study medication was decreased to one tablet twice daily for 2 weeks. If symptoms persisted, the dose was decreased to one tablet once daily for 2 weeks. If symptoms still persisted, the drug was discontinued permanently. For participants with grade 3 or 4 adverse reactions, the study medication was discontinued permanently. The subjects were followed every 2 weeks or more frequently as indicated, until the symptoms or laboratory values had returned to normal. All participants underwent an additional fluorescence bronchoscopy after 6 months on study medication to identify the sites from which the original biopsy samples were obtained, and we took biopsy samples from those same sites. Biopsy samples were also taken from new areas of the bronchial tree that displayed abnormal fluorescence. The bronchoscopist was blinded to the intervention assignment.
During the study, current smokers were encouraged to stop smoking. They were invited to take part in the Fresh Start Program at the BCCA. Our study was approved by the Clinical Investigations Committees of the BCCA and the University of British Columbia. Written informed consent was obtained from all participants.
Nuclear Morphometry
Quantitative nuclear morphometry, which measures the size and shape of a cell nucleus, the amount of DNA, and the distribution of DNA within the nucleus, has been described in detail previously (16,20,21). To make these high-resolution quantitative morphometric measurements in bronchial biopsy samples that were stained with Feulgen-thionin, we used a Cyto-SavantTM system (Oncometrics, Inc.) (16,20,21) equipped with a x20 objective with a numerical aperture of 0.75 to collect digital images of cells from the biopsy. Analysis of the nuclei in each biopsy sample consisted of five steps: 1) focusing the field of view; 2) automatically segmenting the nuclei in the field; 3) interactively correcting segmentation errors; 4) selecting and classifying the cell nuclei into the categories of basal, intermediate, or superficial cells depending on their location within the biopsy sample, where possible; and 5) automatically collecting individually focused images of each selected cell. We analyzed at least 100 epithelial cell nuclei from each biopsy sample in the region where the pathologist made the histopathologic classification. To normalize the images for sample-to-sample variations in staining intensity, images of approximately 30 leukocytes in the subepithelial layer of each sample were collected and analyzed in a similar fashion. We used a continuous scale we had previously established that distinguishes between the nuclear features from normal biopsy samples and those from biopsy samples of invasive non-small-cell lung cancer (20) to calculate a morphometry index (MI) for each biopsy sample. An MI greater than 1.36 was classified as abnormal.
Outcomes
The primary outcome of this study was change in the histopathologic grade of the bronchial biopsy samples after 6 months of intervention. The secondary end point was change in the nuclear morphometry index, from less than or equal to 1.36 to greater than 1.36 and vice versa, of the biopsy samples after 6 months of intervention.
Determination of Sample Size
We used information obtained from the placebo arm of a previous trial of retinol versus placebo (National Cancer Institute U01CA68381) in a study population that had eligibility criteria similar to that for our study population to estimate the spontaneous regression rate of bronchial dysplasia. For individuals, the response rate was 24% (9 of 38). By assuming that this rate would increase to 54% in the ADT arm of our study and by specifying a power of 80% and a 5% level for the two-sided test of statistical significance, we determined that a sample size of 49 subjects per treatment arm would be required. We also assumed that each subject would present with an average of 2.4 dysplastic lesions and that, on the basis of data from the U01 study, this number would represent an equilibrium condition, with the number of new dysplastic sites being approximately equal to the number of regressing sites. We assumed that the number of lesions per subject was distributed as a Poisson distribution, and that distribution was used to estimate the sample size that was required to measure a change in the total number of dysplastic sites between the two intervention arms. At a 5% level for the test of two-sided statistical significance, 50 subjects per arm would permit the detection of a 50% reduction in the average number of lesions per subject (i.e., from 2.40 to 1.20) with a power of 97%, a 40% reduction in the average number of lesions per subject with a power of 87%, and a 30% reduction in the average number of lesions per subject with a power of 64%. To allow for a 10% dropout rate, we planned to randomly assign a total of 110 subjects to the two arms of this clinical trial.
Statistical Analyses
All subjects who had taken one or more doses of the intended 6-month course of treatment were included in the analyses. The primary end point of the study was change in the histopathologic grade of the bronchial biopsy samples; the end point was based on the risk of progression to invasive cancer that was reported in longitudinal studies that analyzed exfoliated cells in sputum and bronchial biopsy samples (2227).
For the lesion-specific analysis, complete response was defined as the regression of a dysplastic lesion to one classified as being either hyperplastic or normal. Progressive disease was defined as the appearance of lesions that were classified as mild dysplasia or worse, irrespective of whether the site was biopsied at baseline, or worsening of the dysplastic lesions present at baseline by two or more grades (e.g., mild dysplasia to severe dysplasia or worse). Dysplastic lesions that were not classified as complete response or progressive disease were referred to as stable disease. These stringent criteria to define regression or progression were based on our quantitative microscopy study (20) which used conventional histopathologic analysis and showed a statistically significant overlap in the classifications between metaplasia and mild dysplasia and between moderate and severe dysplasia.
For the subject-specific analysis, response was defined as follows: complete response referred to regression of all dysplastic lesions found at baseline to lesions that were no worse than hyperplasia, as defined by the site-by-site analysis at 6 months, and the appearance of no new dysplastic lesions that were mild dysplasia or worse. Progressive disease was defined as progression of one or more sites by two or more grades, as defined for the lesion-specific analysis above, or the appearance of new dysplastic lesions that were mild dysplasia or worse at 6 months. Partial response was defined as regression of some but not all of the dysplastic lesions with the appearance of no new lesions that were mild dysplasia or worse. Stable disease referred to subjects who did not have a complete response, partial response, or progressive disease.
For quantitative nuclear morphometry, the median MI for histologically normal biopsy samples was 1.25 (range = 1.0 to 2.2). The interobserver variation in MI measurements was such that a change in MI greater than 0.11 (i.e., the equivalent of 2 standard deviations) was considered a statistically significant change. For the lesion-specific analysis, complete response was defined as regression of a lesion with an MI of greater than 1.36 to one with an MI of 1.36 or less, and progressive disease was defined as an increase in the MI of a lesion from less than or equal to 1.36 to greater than 1.36. For the subject-specific analysis, complete response was defined as regression of all sites with an MI greater than 1.36 to an MI of 1.36 or less, and progressive disease was defined as the appearance of new lesions with an MI greater than 1.36.
Descriptive statistics were used to summarize subject characteristics and pathologic evaluations of the bronchial biopsy specimens. We used the MannWhitney U test for continuous variables (e.g., age, smoking intensity [in pack-years], and MI) to compare treatment arms. Pearson's chi-square test with continuity correction was used to compare categorical variables, such as sex, smoking status (current versus former smokers), and response rates (progression and regression), in the two arms. All P values are two-sided. A two-sided P value less than .05 was considered statistically significant.
We used multiple logistic regression analysis on a participant level to adjust for the effects of various pretreatment factors on the likelihood of regression or progression of dysplastic lesions. This analysis included the following variables: age, sex, smoking status, and smoking intensity (in pack-years). All analyses were unconditional, and tests of statistical significance and confidence intervals (CIs) for odds ratios (ORs) were based on the log-likelihood test.
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RESULTS |
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The characteristics of the 101 participants who completed the trial are shown in Table 1. There was no statistically significant difference in median age, sex, or smoking history between the ADT and placebo groups. Although there were fewer former smokers in the placebo group than in the ADT group (18% versus 34%), that difference was not statistically significant (P = .07).
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Lesion-specific analysis.
After 6 months of intervention, the complete response rate was 53% in the ADT group and 41% in the placebo group (P = .14; difference in complete response rate = 12%, 95% CI = 3% to 26%). The progression rate was statistically significantly lower in the ADT group (8%) than it was in the placebo group (17%, P<.001, difference in progression rate = 9%, 95% CI = 4% to 15%) (Table 2). When new dysplastic lesions that were discovered during the follow-up bronchoscopy were excluded from the analysis, the difference in progression rate between the ADT group (2.7%) and the placebo group (7%) remained statistically significant (P = .046; difference = 4.3%, 95% CI = 0% to 9%).
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Effect of ADT Dose Reduction on the Histopathology of Bronchial Biopsy Samples
Thirty-one subjects in the ADT group were able to take the full dose of ADT (e.g., 25 mg thrice daily) for 6 months. For those who could not, the dose was reduced to 25 mg twice daily (17 subjects), 25 mg daily (four subjects), or was discontinued (four subjects). There was no statistically significant difference in the rates of complete response or progressive disease (all P values >.90) between the subjects who took the full dose of ADT and those who took a reduced dose or discontinued treatment before the intended end of the 6-month course (Table 5).
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Nuclear morphometry of bronchial biopsy specimens was used as a secondary end point biomarker. Although the eligibility criterion was bronchial dysplasia, not all of the dysplastic lesions had an abnormal MI (MI >1.36). Twenty-nine subjects (52%) in the ADT group and 25 subjects (56%) in the placebo group had at least one biopsy at baseline that had an MI greater than 1.36.
In the lesion-specific analysis, the complete response rate was 76% in the ADT group and 63% in the placebo group. The corresponding progressive disease rates were 24% and 33%, respectively. The difference between the two groups was not statistically significant for complete response (P = .37) or for progressive disease (P = .22) (Table 6).
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Subjects in both intervention groups frequently reported having excessive flatus, abdominal bloating, loose stools, and diarrhea (Table 8). Excessive flatus and abdominal bloating were statistically significantly more frequent in the ADT group than in the placebo group (P<.001 and P = .011, respectively). Grade 2 symptoms were observed in 51% of the participants taking ADT and in 20% of those on placebo. Grade 3 symptoms were observed in 11% of those taking ADT and in 6% of those on placebo. One of the participants taking ADT had a grade 3 elevation of liver enzymes. That subject's liver enzymes returned to normal levels after the subject stopped taking ADT. Dose reduction was required for 45% of the participants taking ADT and for 25% of those on placebo. The study medication had to be discontinued in four of the subjects in the ADT group and in one of the subjects in the placebo group because of complaints of intolerable abdominal bloating or flatulence.
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DISCUSSION |
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Bronchial dysplasia is one of the best surrogate end point biomarkers currently available to assess the effects of new chemopreventive agents, because the morphologic criteria for these pre-invasive lesions have been defined in the recent WHO classification (19) and grading of these lesions was found to be reproducible by a panel of experienced pulmonary pathologists (A. F. Gazdar: unpublished data). The precision of classifying dysplastic changes in the bronchial epithelium can be further improved by using quantitative image cytometry (28,29). The presence of these lesions also has prognostic significance. Cancer progression studies in animal models (30), as well as prospective studies in humans, have shown that the presence of dysplastic cells in sputum or bronchial biopsy samples is associated with the development of invasive lung cancer (2227). In animal models, reversal of dysplasia by treatment with a chemopreventive agent is associated with reduced cancer risk (28,29). We used nuclear morphometry to quantitate changes in dysplastic lesions and observed that subjects in the ADT group had an absolute increase in complete response rate of 21% and an absolute decrease in progressive disease rate of 19% compared with subjects in the placebo group. Although the differences between the two groups were not statistically significant, their magnitude was similar to that observed using histopathology criteria to classify changes in dysplastic lesions (17). Only 50% of the subjects in our study had at least one biopsy sample with an MI greater than 1.36 at baseline. A statistically significant difference might have been observed if the eligibility criteria for our study had included nuclear morphometry because the power of the study would be increased with a larger sample size. We did not use nuclear morphometry in the eligibility criteria in this clinical trial because that method was not developed until after the study began.
This is the first phase IIb lung cancer chemoprevention trial to use bronchial dysplasia as the primary intermediate end point biomarker. Although previous lung cancer chemoprevention studies have used either metaplasia or a combination of metaplasia and dysplasia as primary intermediate end point biomarkers, those studies enrolled participants that had very few dysplastic lesions (31,32). Using metaplasia as the intermediate end point biomarker, isotretinoin and N-(4-hydroxyphenyl)retinamide were found to be ineffective chemopreventive agents (31,32). The effects of these agents on bronchial dysplasia are not known. In studies of retinoids (31,33), a difference in response was observed between former and current smokers. In our study, although ADT had a beneficial effect in preventing the development and progression of dysplastic lesions in both current and former smokers, the beneficial effect of ADT was also greater in former smokers than in current smokers. In addition, we observed that women had better responses to ADT than did men. The reason for this finding is not known and requires further investigation.
ADT has multiple mechanisms of action. For example, ADT exerts effects on glutathione. Animals pretreated with various toxins, as well as untreated animals, display an increase of intracellular glutathione after administration of ADT (34). This effect is produced by stimulation of glutathione synthesis via the glutamyl cysteine synthetase and is accompanied by an increase of glutathione-dependent enzyme activity (34). The activity of glutathione S-transferase (GST), a phase II enzyme implicated in chemoprevention against aflatoxin-induced hepatocarcinogenesis, is induced by ADT (68) and may explain the protective action of ADT against aflatoxin-induced tumorigenicity that was observed in rats by Kensler et al. (8). As was demonstrated in an in vitro rat liver microsome model, ADT displays free radical scavenger properties (35,36) that may protect the cellular membrane by inhibiting lipid peroxidation and diene formation. Moreover, ADT modulates the activation of the redox-sensitive cytolytic transcription factor nuclear factor-B (NF-
B) in human Jurkat cells (37). Pretreatment of Jurkat cells with ADT statistically significantly protected cells against oxidative stress-induced cytotoxicity (38).
Although the dithiolethione class of organosulfur compounds may, like ADT, act via several mechanisms including the inhibition of cell replication, they appear to act predominantly by increasing the expression or activity of phase II enzymes, such as GST. For example, in a chemoprevention study that used a rat model of hepatoma, the mean GST levels in liver were statistically significantly higher in animals treated with either ADT or Oltipraz than in untreated animals (P<.01) (8). Moreover, the GST levels increased by 3.2- to 4.5-fold as the concentration of Oltipraz in the animals' diets increased from 0.01% to 0.1% (8). A study from Qidong, China (39) showed that intermittent administration of high doses of Oltipraz in humans inhibited the activation of aflatoxins by phase I enzymes, whereas sustained administration of low doses of Oltipraz increased the conjugation of aflatoxin by phase II enzymes. These detoxification and antioxidant activities may explain why we observed that subjects in the ADT group had fewer new dysplastic lesions and less progression of existing dysplastic lesions to a higher grade but not more regression of existing dysplastic lesions than did subjects on placebo. Administration of ADT for longer than 6 months in a larger number of participants may be necessary to show the potential effects of ADT on regression of existing dysplastic lesions.
In this phase IIb study, we used the dose of ADT that was approved by Health Canada for the treatment of xerostomia to establish the potential efficacy of this agent in smokers with bronchial dysplasia. The only adverse events we observed were gastrointestinal. This finding is consistent with previous clinical data on the safety profile of ADT in the treatment of patients with dry mouth (12). Because we observed that ADT appeared to be active despite the fact that 45% of the participants in the ADT group took ADT at less than the full dose, the possibility that effective chemopreventive activity can be achieved in a once- or twice-daily dose needs to be investigated further.
The results of this study show, for the first time, that smokers with premalignant lesions who are treated with ADT have statistically significantly fewer new dysplastic lesions and less progression of pre-existing dysplastic lesions than do smokers with premalignant lesions who are treated with placebo. Given that more than half of all long-term smokers are unable to stop smoking despite aggressive behavioral and pharmacologic smoking cessation measures (40) and the persistent risk of lung cancer in former heavy smokers (2,3), our study suggests that the free radical scavenger and glutathione inducer ADT may represent a new chemopreventive strategy for lung cancer control.
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NOTES |
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We would like to thank the following people for technical assistance in subject recruitment, bronchoscopy, data management, and nuclear morphometry measurements: Suzan Ross, Carol Astrop, Jane Rozeluk-Gagnon, Myles McKinnon, Christen Oram, Chris Dawe, Sukhinder Khattra, and Lynne Ling. We would also like to thank Solvay Pharma for supplying anethole dithiolethione (Sialor®, Sulfarlem®) and its placebo for the clinical trial.
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Manuscript received September 17, 2001; revised April 24, 2002; accepted May 1, 2002.
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