The Influence of Fluticasone Inhalation on Markers of Carcinogenesis in Bronchial Epithelium Remco M. van den Berg1, Harm van Tinteren2, Nico van Zandwijk3, Christine Visser3, Arifa Pasic1, Clarissa Kooi1, Thomas G. Sutedja1, Paul Baas3, Katrien Gru ¨ nberg4, Wolter J. Mooi4, Peter J. F. Snijders4, Pieter E. Postmus1, and Egbert F. Smit1 1
Department of Pulmonology, VU University Medical Center (VUMC), Amsterdam, The Netherlands; 2Departments of Biometrics and Thoracic Oncology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital (NKI/AvL), Amsterdam, The Netherlands; and 4Department of Pathology, VU University Medical Center (VUMC), Amsterdam, The Netherlands
3
Rationale: Bronchial epithelium exposed to cigarette smoke undergoes a series of histologic changes that may ultimately lead to invasive cancer. Inhaled corticosteroids reduce the number of lung tumors developing in rats exposed to cigarette smoke. Objectives: We studied the effect of inhaled fluticasone on premalignant lesions in smokers and patients curatively treated for head and neck cancer or lung cancer. Methods: Participants were screened for premalignant lesions by bronchoscopy. Biopsies were taken from three to five locations and classified using WHO criteria. In case of a metaplasia index of ⬎ 15%, participants were randomized to receive a powder inhalation device containing either fluticasone 500 g or a placebo, to be used twice a day. After 6 months, biopsies were obtained from the same locations as previously sampled. Efficacy of treatment was assessed by reversal of metaplasia/dysplasia; secondary endpoints were reversal of increased p53 and KI-67 immunoreactivity and expression of human telomerase reverse transcriptase. Measurements and Main Results: Of the 201 subjects that were screened, 108 were included. Mean age was 53 yr (35–71), mean number of pack-years 48 (18–99), mean metaplasia index 48%, and 32% had some degree of dysplasia at baseline. The two treatment arms did not differ with respect to response or change in either metaplasia index or the expression of the markers p53, KI-67, or human telomerase reverse transcriptase. Conclusions: Inhaled fluticasone in a dose of 500 g twice a day does not affect the natural course of premalignant lesions in the central airways. Keywords: cancer chemoprevention; hTERT; KI-67; p53; inhalational corticosteroid
Despite advances in treatment, lung cancer kills more patients than any other malignancy (1). Its main cause is tobacco smoking (2), which is widely recognized as a danger to public health. In spite of anti-smoking campaigns and legislation intended to reduce the number of smokers, lung cancer will remain an important cause of cancer mortality in the foreseeable future. The process of carcinogenesis takes many years, and although lung cancer risk declines in the years after quitting, many former smokers develop lung cancer. This is illustrated by a study in which 42% of 1,039 patients with lung cancer were former smok-
(Received in original form December 6, 2006; accepted in final form February 8, 2007 ) Supported by a grant from GlaxoSmithKline, Zeist, The Netherlands. Correspondence and requests for reprints should be addressed to E. F. Smit, M.D., Ph.D., Department of Pulmonology, VU Medical Centre, PO Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail:
[email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 175. pp 1061–1065, 2007 Originally Published in Press as DOI: 10.1164/rccm.200612-1770OC on February 8, 2007 Internet address: www.atsjournals.org
AT A GLANCE COMMENTARY Scientific Knowledge on the Subject
Animal studies show a chemopreventive effect of inhalational corticosteroids on lung carcinogenesis. A trial of budesonide did not find an effect on histology of bronchial premalignant lesions in humans, but did find an effect on p53 expression. What This Study Adds to the Field
Inhalational corticosteroids have no effect on the natural course of premalignant bronchial lesions.
ers (3). Of the male patients, 15% had stopped smoking over 15 years before diagnosis. Successes in the preventive treatment of risk factors in cardiovascular medicine suggest that a similar course of action may be helpful in the fight against lung cancer. In theory, cancer chemoprevention is feasible. Certain drugs or (dietary) supplements may counteract the effects of carcinogenic substances on healthy subjects at risk of developing cancer. Many mechanisms of carcinogenesis have been elucidated over the past decades, and drugs affecting this process have proven their benefit in in vitro and animal studies. In large clinical trials tamoxifen reduced the risk of recurrence of breast cancer (4), which is a proof of principle for cancer chemoprevention. Inhalational corticosteroids (ICS) are promising for lung cancer chemoprevention. Corticosteroids inhibit tumor growth in vitro (5), and both ingested and inhaled, this class of drugs reduces the number of lung tumors developing in rats exposed to cigarette smoke (6) and in a transgenic (p53/p16 null) mouse model treated with benzo[a]pyrene (7). ICS have been used in the treatment of asthma for years and were proven safe in long-term use, which is important if a substance is to be used for large-scale chemoprevention. Bronchial epithelium exposed to cigarette smoke will often undergo a sequence of histologic changes, from hyperplasia through squamous metaplasia and different grades of dysplasia. These changes may eventually lead to invasive squamous cell carcinoma. This sequential morphologic progression is thought to be driven by an accumulation of genetic and epigenetic changes. In this trial we examined the effect of fluticasone on premalignant epithelial lesions of the bronchus: reversal of histologic abnormalities of the bronchial epithelium was the primary endpoint. As a secondary endpoint we chose the expression of three biomarkers: p53, KI-67, and hTERT (human telomerase
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reverse transcriptase, a subunit of the enzyme telomerase, important for tumor cell immortalization). These markers are indicative of changes on a molecular level and may add to the predictive value of histology (8–11). Some of the results of this study have been previously reported in the form of an abstract (12).
METHODS Subjects and Screening Volunteers with a smoking history of over 20 pack years (one pack of cigarettes a day for over 20 yr or equivalent) were recruited by advertisements in local and national newspapers. Patients who had undergone treatment with curative intent of head and neck or lung cancer of any stage and did not show signs of recurrence on computed tomography (CT) were also asked to participate. Subjects with serious comorbid disease, FEV1 values below 1,000 ml, or use of systemic or inhaled corticosteroids in the year before enrollment were excluded. Participants were screened with autofluorescence- (VUMC) or white light bronchoscopy (NKI). Biopsies were taken from three to five locations. Standard locations (main carina, and carinas of right and left upper lobes) were biopsied when no suspicious lesions could be detected. From each location, one biopsy was snap-frozen in liquid nitrogen, and another was formalin fixed and paraffin embedded for immunohistochemistry and histologic assessment on a series of hematoxylin and eosin–stained sections by a staff pathologist according to WHO guidelines. At least one biopsy was to contain squamous metaplasia or dysplasia for the subject to be included in the study. Protocols were approved by the institutional review boards, and written informed consent was obtained from all subjects.
Randomization and Treatment A centralized trial service assigned subjects to the treatment or placebo arm by blocked stratified randomization. The institution where screening and follow up took place was used as stratification factor. Subjects received a powder inhalation device (Diskus inhaler) containing sixty doses of either 500 g fluticasone or a placebo to be used twice daily. All inhalers were kindly provided free of charge by GlaxoSmithKline (Zeist, The Netherlands). Each month, subjects received a new inhaler; inhalation technique was evaluated; and compliance, smoking behavior, and adverse events were monitored (NCI common toxicity criteria). After 6 months a second bronchoscopy was performed. Those who had used a placebo and had persistent bronchial premalignant lesions were offered treatment for another 6 months with fluticasone.
Evaluation of Efficacy Histology. The effect of fluticasone on the histology of bronchial biopsies was analyzed by comparing the average change in metaplasia index (the number of squamous lesions divided by the number of locations biopsied) of the two arms. Also a modified version of the method of Lam and coworkers (13) was used (see Table 1).
Markers. Twenty-five 10-m sections were cut from the snap-frozen biopsies and lysed in RNAzol Bee (Campro Scientific, Veenendaal, The Netherlands). RNA was isolated according to the manufacturer’s instructions. Levels of hTERT mRNA expression were determined with a quantitative one-step real-time reverse transcriptase PCR, using the light cycler instrument and the light cycler TeloTAGGG hTERT quantification kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The housekeeping gene porphobilinogen deaminase was amplified and detected in the same reaction, serving as a reference as well as internal control for RNA quality. Experiments were performed in duplicate. Normalized mean hTERT level was used for analysis. p53 and KI-67 immunostaining was performed as previously described (9). Antibodies (DO-7 and MIB-1) were obtained from Dako (Glostrup, Denmark).
Statistics Power analysis indicated that inclusion of 90 subjects would give the study 80% statistical power to detect a difference of 30% in response rate (CR⫹PR; two-tailed test, 2⫽ 0.05). The SAS version 9 program (SAS Institute, Inc., Cary, NC) was used for statistical tests. Categorical variables were tested using Fisher exact test (location specific analysis) or Jonkheere Terpstra test when appropriate. Continuous variables were tested by t tests. Logistic regression analysis was performed to adjust for sex, institution where screening and follow up took place, number of pack-years, and history of cancer. For the lesion-specific analysis, patients were considered a random factor in a mixed-effect model to account for multiple observations per patient.
RESULTS Characteristics
Between February 2002 and October 2004, 201 subjects were screened. Two were excluded because of lung cancer detected during initial screening. Of the volunteers who were screened, 108 were included (Figure 1). Mean metaplasia index at baseline was 47% (median 33%); 35% had some degree of dysplasia (Table 2). Baseline characteristics were well balanced among the intervention and the placebo group, except for one: seven subjects with a history of lung cancer or head and neck cancer (of whom three were treated more than 7 yr before) were allocated to the placebo group, whereas two subjects with a history of head and neck cancer were allocated to the fluticasone group. Disease stage ranged from T1N0 to T2N1 in lung and T1N0 to T4N2 in head and neck cancer; median time since treatment was 33 months (range, 9 mo to 10 yr; see Table E1 in the online supplement). In one subject a carcinoid tumor was discovered at the time of the second bronchoscopy; another developed bronchioloalveolar carcinoma. Both were assigned to the placebo group and are included in the analysis. Seventeen subjects failed to complete the trial according to protocol. One subject, allocated
TABLE 1. DEFINITIONS OF RESPONSE CATEGORIES Classification
Definition
Lesion-specific Complete response: Regression of a metaplastic/dysplastic lesion to hyperplasia or normal. Progressive disease: Appearance of lesions classified as squamous metaplasia or worse, irrespective of whether the site was biopsied at baseline, or worsening of a metaplastic lesion present at baseline by two or more grades. Stable disease: Metaplastic/dysplastic lesions that were not classified as complete response or progressive disease. Subject-specific Complete response: Regression of all metaplastic/dysplastic lesions found at baseline to lesions that were no worse than hyperplasia and the appearance of no new metaplastic/dysplastic lesions. Partial response: Regression of some but not all of the metaplastic/dysplastic lesions with the appearance of no new lesions that were squamous metaplasia or worse. Progressive disease: Progression of one or more sites by two or more grades or the appearance of new metaplastic/dysplastic lesion. Stable disease: Subjects who did not have a complete response, partial response, or progressive disease.
van den Berg, van Tinteren, van Zandwijk, et al.: Fluticasone in Bronchial Premalignancy
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Figure 1. Flow chart of trial subjects.
to the fluticasone arm of the study, underwent the second bronchoscopy 1 month earlier than planned because of side effects (recurrent stomatitis). This person had a complete response and was included in the analysis. Of the 16 subjects who were not included in the analysis, 9 were in the placebo and 7 in the fluticasone arm (Figure 1). Subjects failed to complete the trial because of side effects, because they refused to undergo the second bronchoscopy, or were lost to follow up. One subject was excluded because an acute exacerbation of chronic obstructive pulmonary disease necessitated treatment with systemic corticosteroids. Data on five subjects were incomplete. The average number of doses taken per month was 56 (20–60; median 57, SD 5.0), without any difference between subjects assigned to the fluticasone or placebo arm. Data from 92 subjects were available for analysis. Six subjects out of 92 quit smoking during the study, of which 3 (6.8%) were allocated to the placebo group and 3 (6.4%) to the fluticasone group. Of the subjects allocated to the placebo arm, 21 were subsequently included in the cross-
over part of the trial. Adverse events related to toxicity of study medication were reported on 68 occasions and were limited to thrush, cough, and hoarseness of the voice. All toxicity-related adverse events were below CTC grade 3. Histology
Lesion-specific analysis. At baseline a total of 437 locations were biopsied. Of these locations 320 yielded biopsies of sufficient quality from subjects who finished the trial according to protocol. After 6 months, 291 locations that were sampled at baseline were biopsied a second time. Fifty-three locations were sampled that were not biopsied at baseline. The complete response rate on a lesion level was 27% in the fluticasone group and 22% in the placebo group (p ⬎ 0.30; see Table 3). The progressive disease rate was 13% and 18% for fluticasone and placebo, respectively. For analysis of the crossover part of the trial, the response of 39 locations (21 subjects) after using open-label fluticasone for 6 months was compared with the response of all
TABLE 2. SUBJECT CHARACTERISTICS
Institute Sex Age, yr
Current smokers History of cancer
Pack-years
NKI/AvL VUMC Male Female Mean SD Range NSCLC Head&neck Both Other Mean Range
Placebo (n ⫽ 54)
Fluticasone (n ⫽ 54)
Total (n ⫽ 108)
27 (50%) 27 (50%) 37 (69%) 17 (31%) 54.31 8.34 38–71 49 (91%) 2 2 3 0 48.26 24–100
25 (46%) 29 (54%) 37 (69%) 17 (31%) 51.89 8.28 35–70 54 (100%) 0 2 0 2 47.85 18–125
52 (48%) 56 (52%) 74 (69%) 34 (31%) 53.10 8.36 35–71 103 (95%) 2 4 3 2 48.06 18–125
Definition of abbreviations: NKI/AvL ⫽ Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital; NSCLC, non–small cell lung cancer; VUMC, VU University Medical Center.
TABLE 3. LESION- AND SUBJECT-SPECIFIC ANALYSIS OF HISTOLOGY
Lesions Not evaluable Normal* Progressive disease Stable disease Complete response Total Subjects Progressive disease Stable disease Partial response Complete response Total
Placebo
Fluticasone
29 52 34 31 38 (22%) 184
31 55 28 32 47 (27%) 191
17 7 4 17
20 4 7 16
(38%) (16%) (9%) (38%) 47
(43%) (9%) (15%) (34%) 45
p Value
⬎ 0.30
0.75
Number of locations and subjects per treatment and response category (for categories see Table 1). * No histological abnormality at baseline and after 6 mo of treatment.
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locations in the placebo arm. Again, fluticasone did not influence the response (data not shown). Subject-specific analysis. Ninety-two subjects had results suitable for analysis. Complete response rate was 34% in the fluticasone group and 38% in the placebo group; progressive disease was found in 43% in the fluticasone and 38% in the placebo group (p ⫽ 0.75; see Table 3). Stratified analysis showed that institution, number of pack-years, sex, and history of cancer did not confound the main comparison. Before intervention mean metaplasia index was 50% and 51%, and after treatment 35% and 37% for fluticasone and placebo groups, respectively (p ⫽ 0.78; Wilcoxon 2-sided). Markers
p53. p53 Immunohistochemistry showed mainly basal staining concordant with physiologic stabilization (possibly as a consequence of cellular stress and DNA damage). In 60 biopsies suprabasal p53 staining was found in small numbers of cells in the superficial layers, without staining of the intermediate layers. Contiguous suprabasal staining of squamous lesions was found in three locations (one subject). Corresponding sections stained with hematoxylin and eosin were scored as squamous metaplasia without dysplasia. After 6 months one of these locations progressed to mild dysplasia, the others remained stable. There was no significant change in the percentage of cells with p53 expression after placebo or fluticasone treatment (Tables E3, E4, and E7). KI-67. The proportion of cells staining positive with KI-67 antibody (proliferation index) did not change significantly when comparing the two treatment arms (Tables E5–E7). hTERT. hTERT data before and after treatment could be compared for 245 locations. Mean hTERT was just below 6 per 1,000 copies of PBGD at baseline and equal for both treatment arms. The mean change of hTERT expression was equal in the placebo and fluticasone groups (p ⫽ 0.81; Table E8).
DISCUSSION We have performed a randomized, controlled trial of fluticasone, followed by a one-way crossover from placebo to active treatment, in a high-risk group of smokers. Lung cancer in smokers is a relatively rare occurrence, and when used as the endpoint in a chemoprevention trial, it would take a large cohort and a long follow-up time to obtain a number of cases large enough for sufficient statistical power. Therefore, we investigated the chemopreventive potential of fluticasone by examining its effect on squamous precursor lesions of the bronchus. No difference in change of histology between the fluticasone or placebo arms could be detected after a 6-month treatment period. The same result was found in the crossover part of the trial. However, because of the absence of a proper control group, the value of the crossover data is limited. A similar study has been performed by Lam and colleagues (13). Their study showed that inhalation of budesonide (like fluticasone and ICS) has no effect on squamous dysplasia in high-risk smokers, but does cause a reduction of the percentage of p53-positive cells as well as the number of CT-detected nodules. Metaplasia rather than dysplasia was used as inclusion criterion because dysplastic lesions are relatively rare in this population and we were unable to screen enough volunteers to obtain an equal number of subjects with dysplastic lesions. The metaplasia index has been used before as endpoint for chemoprevention studies (14), and although it has not been validated it is clearly related to smoking (15). Metaplastic changes are manifestations of the repair of epithelial damage (16), which may be caused by smoking-induced inflammation (17, 18). The effect of ICS on
carcinogenesis could stem from an influence on this inflammation. If we found a reaction of the metaplasia index to treatment in this study, this would have been indicative of an influence of ICS early on in the process of carcinogenesis. In a study by Lee and coworkers, the number of subjects who quit smoking was of greater influence on the metaplasia index than the intervention that was tested (19). The number of subjects that quit in the current study was small (just over 6% in both arms) and unlikely to affect the comparison between the treatment arms. Could results be different in former smokers? Retinoic acid, a substance that has a chemopreventive effect in oral squamous cell carcinoma, was found to have a lung cancer–promoting effect in subjects who continued to smoke (20). There were indications of a beneficial effect in former smokers. However, no effect was found on metaplasia in a trial of retinoic acid exclusively in former smokers (21). The number of former smokers in our study was too small to make any statement about a possible effect of fluticasone on this subgroup. To investigate a possible effect later on in the process of carcinogenesis, we also included (change of) dysplasia in our analysis as well as three biomarkers: KI-67, p53, and hTERT. KI-67 protein is a proliferation marker, which cells in any stage of proliferation will express. The ratio of KI-67–positive and –negative cells, the proliferation index, is used as a measure of the speed of turnover, which is higher in (pre)malignant lesions. Smoking causes an elevated proliferative index regardless of the presence of premalignant lesions (14). The change in KI-67 percentage did not show a significant difference between the two treatment arms. We have shown that suprabasal p53 staining has additional value as a marker in premalignant lesions of the lung, in a study of patients with a history of lung cancer or high-grade premalignancy, in which lung cancer was the primary outcome (9). In the current study there was convincing suprabasal staining in lesions of only one subject. Possibly p53 mutation does not occur as often in this population consisting mainly of healthy smokers. In addition to scoring the lesions with suprabasal staining, the samples were categorized according to the percentage of p53-positive cells (no staining; ⬍ 10% staining; between 10 and 50%; and ⬎ 50%). As opposed to the results of the study by Lam and colleagues (13), we found no significant difference between the placebo and fluticasone arms with respect to the percentage of p53-positive cells. The reason for the difference in findings with regard to p53 percentage may be that all study subjects in the budesonide study had bronchial dysplasia, whereas our main inclusion criterion was squamous metaplasia. Telomerase activation is a crucial step in development of most tumors, since it allows tumor cells to bypass senescence and divide indefinitely. hTERT, one of its components, is the rate-limiting enzyme (22). Increased hTERT expression was found in 33 out of 38 squamous cell carcinomas in a recent study (23). Elevated expression in bronchial epithelium was found to be predictive of malignant transformation in several recent retrospective series including our own (10, 24, 25). We found no significant difference in the change of mean hTERT level between the placebo and fluticasone groups. Corticosteroids were previously proven effective in reducing the number of lung tumors in A/J mice and rats exposed to cigarette smoke. In a study by Yao and coworkers (26), the expression profile of mouse lung tumors treated with budesonide was analyzed by microarray. The authors concluded that budesonide affected mouse lung tumorigenesis through modulation of the Bcl-2– and caspase-2–regulated apoptosis pathways as well as the Mad2/3-regulated mitotic checkpoint. As Lam and colleagues (13) speculated, the explanation for the discrepancy between the promising results of trials of ICS in rodents and
van den Berg, van Tinteren, van Zandwijk, et al.: Fluticasone in Bronchial Premalignancy
the lack of effect in humans may lie in the fact that in the rodent lung carcinogenesis model the tumors that develop are all classified as adenocarcinomas and their precursor lesions as adenomas. Since the tumors that originate in epithelium of the large bronchi are squamous carcinomas, the mouse model may not be appropriate for investigating central bronchial carcinogenesis. The reduction in CT detected nodules in the budesonide study (13) could be indicative of an effect on adenomatous precursor lesions, similar to the lesions that develop in the mouse model. We conclude that ICS do not influence squamous bronchial lesions. A possible effect on peripheral adenomatous lesions found in a previous study warrants further investigation. Conflict of Interest Statement : R.M.v.d.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.v.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.v.Z. has received £2,500 for serving on an advisory board for Eli Lilly and Company and has received £14.000 during the last three years for speaking at conferences organized by Eli Lilly and Company and AstraZeneca. C.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. C.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.G.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. W.J.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.J.F.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.E.P. received €57,176 for the studies discussed in this manuscript in 2004/05; €164,000 was received for research in pulmonary hypertension. E.F.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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References 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global Cancer Statistics, 2002. CA Cancer J Clin 2005;55:74–108. 2. Hecht SS. Tobacco smoke carcinogens and lung cancer. J Natl Cancer Inst 1999;91:1194–1210. 3. Tong L, Spitz MR, Fueger JJ, Amos CA. Lung carcinoma in former smokers. Cancer 1996;78:1004–1010. 4. Fisher B, Costantino JP, Wickerham DL, Cecchini RS, Cronin WM, Robidoux A, Bevers TB, Kavanah MT, Atkins JN, Margolese RG, et al. Tamoxifen for the prevention of breast cancer: current status of the national surgical adjuvant breast and bowel project P-1 study. J Natl Cancer Inst 2005;97:1652–1662. 5. Greenberg AK, Hu J, Basu S, Hay J, Reibman J, Yie TA, Tchou-Wong KM, Rom WN, Lee TC. Glucocorticoids inhibit lung cancer cell growth through both the extracellular signal-related kinase pathway and cell cycle regulators. Am J Respir Cell Mol Biol 2002;27:320–328. 6. Wattenberg LW, Wiedmann TS, Estensen RD, Zimmerman CL, Galbraith AR, Steele VE, Kelloff GJ. Chemoprevention of pulmonary carcinogenesis by brief exposures to aerosolized budesonide or beclomethasone dipropionate and by the combination of aerosolized budesonide and dietary myo-inositol. Carcinogenesis 2000;21:179–182. 7. Wang Y, Zhang Z, Kastens E, Lubet RA, You M. Mice with alterations in both p53 and Ink4a/Arf display a striking increase in lung tumor multiplicity and progression: differential chemopreventive effect of budesonide in wild-type and mutant A/J mice. Cancer Res 2003;63: 4389–4395. 8. Hoshino H, Shibuya K, Chiyo M, Iyoda A, Yoshida S, Sekine Y, Iizasa T, Saitoh Y, Baba M, Hiroshima K. Biological features of bronchial squamous dysplasia followed up by autofluorescence bronchoscopy. Lung Cancer 2004;46:187–196. 9. Breuer RH, Snijders PJ, Sutedja TG, Linden H, Risse EK, Meijer CJ, Postmus PE, Smit EF. Suprabasal p53 immunostaining in premalignant
19.
20.
21.
22.
23.
24.
25.
26.
1065
endobronchial lesions in combination with histology is associated with bronchial cancer. Lung Cancer 2003;40:165–172. Snijders PJ, Breuer RH, Sutedja TG, Egging M, Voorhorst FJ, Steenbergen RD, van der Linden HC, Risse EK, Berkhof J, de Vries EG, et al. Elevated hTERT mRNA levels: A potential determinant of bronchial squamous cell carcinoma (in situ). Int J Cancer 2004; 109:412–417. Lantuejoul S, Soria JC, Morat L, Lorimier P, Moro-Sibilot D, Sabatier L, Brambilla C, Brambilla E. Telomere shortening and telomerase reverse transcriptase expression in preinvasive bronchial lesions. Clin Cancer Res 2005;11:2074–2082. Van Den Berg RM, Van Tinteren H, Van Zandwijk N, Sutedja TG, Baas P, Visser C, Grunberg K, Mooi WJ, Postmus PE, Smit EF. The influence of fluticasone inhalation on premalignant lesions in the bronchial epithelium of a high risk population: a double blind placebocontrolled randomised phase II study. J Clin Oncol 2006;24:7200. (Meeting Abstracts). Lam S, leRiche JC, McWilliams A, Macaulay C, Dyachkova Y, Szabo E, Mayo J, Schellenberg R, Coldman A, Hawk E, et al. A randomized phase IIb trial of pulmicort turbuhaler (budesonide) in people with dysplasia of the bronchial epithelium. Clin Cancer Res 2004;10:6502– 6511. Lee JJ, Liu D, Lee JS, Kurie JM, Khuri FR, Ibarguen H, Morice RC, Walsh G, Ro JY, Broxson A, et al. Long-term impact of smoking on lung epithelial proliferation in current and former smokers. J Natl Cancer Inst 2001;93:1081–1088. Peters EJ, Morice R, Benner SE, Lippman S, Lukeman J, Lee JS, Ro JY, Hong WK. Squamous metaplasia of the bronchial mucosa and its relationship to smoking. Chest 1993;103:1429–1432. Park KS, Wells JM, Zorn AM, Wert SE, Laubach VE, Fernandez LG, Whitsett JA. Transdifferentiation of ciliated cells during repair of the respiratory epithelium. Am J Respir Cell Mol Biol 2006;34:151–157. Martey CA, Baglole CJ, Gasiewicz TA, Sime PJ, Phipps RP. The aryl hydrocarbon receptor is a regulator of cigarette smoke induction of the cyclooxygenase and prostaglandin pathways in human lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 2005;289:L391–L399. Martey CA, Pollock SJ, Turner CK, O’Reilly KMA, Baglole CJ, Phipps RP, Sime PJ. Cigarette smoke induces cyclooxygenase-2 and microsomal prostaglandin E2 synthase in human lung fibroblasts: implications for lung inflammation and cancer. Am J Physiol Lung Cell Mol Physiol 2004;287:L981–L991. Lee JS, Lippman SM, Benner SE, Lee JJ, Ro JY, Lukeman JM, Morice RC, Peters EJ, Pang AC, Fritsche HA Jr. Randomized placebocontrolled trial of isotretinoin in chemoprevention of bronchial squamous metaplasia. J Clin Oncol 1994;12:937–945. Lippman SM, Lee JJ, Karp DD, Vokes EE, Benner SE, Goodman GE, Khuri FR, Marks R, Winn RJ, Fry W, et al. Randomized phase III intergroup trial of isotretinoin to prevent second primary tumors in stage I non-small-cell lung cancer. J Natl Cancer Inst 2001;93:605–618. Kurie JM, Lotan R, Lee JJ, Lee JS, Morice RC, Liu DD, Xu XC, Khuri FR, Ro JY, Hittelman WN, et al. Treatment of former smokers with 9-cis-retinoic acid reverses loss of retinoic acid receptor-{beta} expression in the bronchial epithelium: results from a randomized placebocontrolled trial. J Natl Cancer Inst 2003;95:206–214. Weinrich SL. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 1997;17:498–502. Zhu CQ, Cutz JC, Liu N, Lau D, Shepherd FA, Squire JA, Tsao MS. Amplification of telomerase (hTERT) gene is a poor prognostic marker in non-small-cell lung cancer. Br J Cancer 2006;94:1452–1459. Shibuya K, Fujisawa T, Hoshino H, Baba M, Saitoh Y, Iizasa T, Sekine Y, Suzuki M, Hiroshima K, Ohwada H. Increased telomerase activity and elevated hTERT mRNA expression during multistage carcinogenesis of squamous cell carcinoma of the lung. Cancer 2001;92:849–855. Kolquist KA, Ellisen LW, Counter CM, Meyerson M, Tan LK, Weinberg RA, Haber DA, Gerald WL. Expression of TERT in early premalignant lesions and a subset of cells in normal tissues. Nat Genet 1998; 19:182–186. Yao R, Wang Y, Lemon WJ, Lubet RA, You M. Budesonide exerts its chemopreventive efficacy during mouse lung tumorigenesis by modulating gene expressions. Oncogene 2004;23:7746–7752.
