- Email: [email protected]
Biological features of bronchial squamous dysplasia followed up by autofluorescence bronchoscopy
Lung Cancer (2004) 46, 187—196
Biological features of bronchial squamous dysplasia followed up by autofluorescence bronchoscopy Hidehisa Hoshino a , Kiyoshi Shibuya a , Masako Chiyo a , Akira Iyoda a , Shigetoshi Yoshida a , Yasuo Sekine a , Toshihiko Iizasa a , Yukio Saitoh a , Masayuki Baba a , Kenzo Hiroshima b , Hidemi Ohwada b , Takehiko Fujisawa a,* a
Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan b Department of Basic Pathology, Graduate School of Medicine, Chiba University, Chiba, Japan Received 30 December 2003 ; received in revised form 31 March 2004; accepted 15 April 2004
KEYWORDS Squamous dysplasia; Telomerase activity; Ki-67 labeling index; p53 immunoreactivity; Autofluorescence bronchoscopy
Summary Some dysplasias in the bronchial epithelium are thought to be precancerous lesions that can develop into squamous cell carcinomas. In this investigation, we assessed the biological behavior of bronchial squamous dysplasia in order to define which dysplasias have the potential to progress to squamous cell carcinoma. Using autofluorescence bronchoscopy, we followed up periodically localized dysplasias and examined for correlation between histological outcome and smoking status during the follow-up period, telomerase activity, Ki-67 labeling index, and p53 immunoreactivity of initial biopsy specimens. Ninety-nine dysplasias from 50 participants mainly with sputum cytology suspicious or positive for malignancy were followed up. Of 99 dysplasias, 3 dysplasias progressed to squamous cell carcinoma, 41 dysplasias remained as dysplasia, 6 dysplasias changed to metaplasia, 14 dysplasias changed to hyperplasia, and 35 dysplasias regressed to bronchitis or normal bronchial epithelium. There were no significant associations between histological outcome and smoking status. Mean initial telomerase activity and Ki-67 labeling index values in the dysplasias increased in proportion to the severity of the histological outcome at the second biopsy. There was also a significant difference between p53-positive and p53-negative dysplasia in terms of histological outcome at the second biopsy. Our results suggested that dysplasias with high telomerase activity, increased Ki-67 labeling index, and p53-positivity tended to remain as dysplasia and might have the potential to progress to squamous cell carcinoma. Patients with dysplastic lesions with these characteristics should be carefully followed up. © 2004 Elsevier Ireland Ltd. All rights reserved.
* Corresponding
author. Tel.: +81 43 222 7171x5464; fax: +81 43 226 2172. E-mail addresses: [email protected] (H. Hoshino), [email protected] (T. Fujisawa). 0169-5002/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2004.04.028
188
1. Introduction During the past decade, the incidence of lung cancer and its related death rate has dramatically increased in Japan. In 1999, lung cancer became the leading cause of death, exceeding that of gastric cancer. Significant reduction in lung cancer-related morbidity and mortality will depend not only on aggressive efforts at smoking cessation, but also on earlier detection and treatment. Early hilar lung cancers, especially carcinoma in situ and microinvasive carcinoma, detected from sputum cytological examination can be cured. However, patients with carcinoma in situ and microinvasive carcinoma may require repeated examinations over many months before the lesion is localized. Moreover, it is exceedingly difficult to detect bronchial squamous dysplasia, thought to be a precancerous lesion, by conventional white light bronchoscopy alone. The development of autofluorescence bronchoscopy has made it possible to localize dysplasias and carcinoma in situ, and the usefulness of autofluorescence bronchoscopy has been widely reported [1—5]. Centrally arising squamous cell carcinoma of the tracheobronchial tree is thought to develop in multiple stages; from normal bronchial epithelium to hyperplasia, metaplasia, dysplasia, followed by carcinoma in situ, and finally invasive cancer [6,7]. Alterations in gene expression and chromosome structure known to be associated with malignant transformation can be demonstrated in carcinoma in situ and to a lesser extent in dysplasia, but can also be sometimes detected in morphologically normal epithelium [8]. Such changes might be sequential, and their frequency and number increase with atypia [8,9]. It is thought that at early stages, telomerase dysregulation, high proliferative activity, and p53 overexpression occur as part of a multistage pathogenesis of lung cancer [9—15]. While some reports have followed up dysplastic lesions to elucidate the multistep carcinogenesis of bronchial epithelium, few reports have examined the use of biomarkers to determine the biological behavior of dysplastic lesions, that is, the probability that a dysplastic lesion will progress to squamous cell carcinoma. Since October 1997, autofluorescence bronchoscopy examination has been performed for the localization of hilar lung cancer and squamous dysplasia at our institute. Autofluorescence bronchoscopy has also been used to periodically follow-up localized dysplasias. The purpose of this investigation is to elucidate the biological behavior of bronchial squamous dysplasia, and to clarify whether telomerase activity, cell proliferative ac-
H. Hoshino et al. tivity, or p53 immunoreactivity could be used as biomarkers to predict which dysplasias will progress to squamous cell carcinoma.
2. Materials and methods 2.1. Autofluorescence bronchoscopy and follow-up system Bronchoscopic examination was performed by one of two investigators (H.H. and K.S.) trained in autofluorescence bronchoscopy using a LIFE-lung fluorescence endoscopy system (Xillix Technologies Corp., Richmond, British Columbia, Canada). Autofluorescence bronchoscopy examinations were performed following white-light bronchoscopy (BF-240, Olympus Optical Corp., Tokyo, Japan) under local anesthesia with sedation by intravenous injection (diazepam or mitazoram) and O2 inhalation. Biopsy specimens for pathological examination were obtained from all suspicious or abnormal areas identified by white-light bronchoscopy examination, autofluorescence bronchoscopy examination, or both. One section of each biopsy specimen was immediately snap-frozen in liquid nitrogen, and stored at −80 ◦ C until assay for telomerase activity. The other section was formalin fixed, paraffin embedded, mounted, stained with hematoxylin and eosin, and diagnosed blindly by two expert pulmonary pathologists at the Department of Basic Pathology, Graduate School of Medicine, Chiba University (K.H. and H.O.). Dysplasia was identified according to recent World Health Organization criteria [16] (Fig. 1). Autofluorescence bronchoscopy was also used to follow-up localized dysplasia approximately every 6 months, and the follow-up biopsies were taken from the same site as the baseline biopsies were taken. Results were used to test for correlation between histological outcome and smoking status during the follow-up period, telomerase activity of initial biopsy specimens, and Ki-67 labeling index and p53 immunoreactivity of the initial biopsy specimens. Informed consent was obtained from all participants prior to investigation.
2.2. Smoking status Smoking status was classified at the initial autofluorescence bronchoscopy examination into three categories. A current smoker was defined as a participant who smoked at the time of initial autofluorescence bronchoscopy examination or in the year preceding it. An ex-smoker was defined as a participant who had quit smoking more than a year before the time of initial autofluorescence
Biological features of bronchial squamous dysplasia
189
Fig. 1 Bronchial squamous dysplasias detected by autofluorescence bronchoscopy. A: mild dysplasia; B: moderate dysplasia; and C: severe dysplasia.
190 bronchoscopy examination. A non-smoker was defined as a participant who had never smoked. Smoking status was also classified during the follow-up period of this investigation into three categories: The ‘‘continue’’ group was defined as participants who continued smoking as before; the ‘‘cut-down’’ group was defined as participants who reduced smoking to approximately five cigarettes per day; and the ‘‘quit’’ group was defined as participants who had quit smoking after initial autofluorescence bronchoscopy examination. Smoking status data was obtained by asking each participant directly prior to investigation.
2.3. Study populations From October 1997 to October 2001, a total of 276 participants with mainly sputum cytology suspicious or positive for malignancy or with lung cancer were examined by autofluorescence bronchoscopy (LIFE) in the Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, and 209 dysplasias were found in 105 participants. Using autofluorescence bronchoscopy, 99 dysplasias were followed up in 50 participants approximately every 6 months, and were used in our study. Forty participants had sputum cytology suspicious or positive for malignancy, and 10 participants were being followed up after curative treatment for lung cancer. The study group consisted of 49 men and one woman, ranging from 46 to 76 years old with a mean of 67 years. Smoking history of the study group ranged from 11 to 153 pack-years with a mean of 60.0. Forty-four were current smokers, and six were ex-smokers.
2.4. Telomerase activity Telomerase activity was measured using the TRAP-ezeTM telomerase detection kit (Intergen Co., New York, NY) together with the fluorescencebased telomeric repeat amplification protocol (F-TRAP) assay. Biopsy specimens were homogenized in 300 L ice-cold CHAPS lysis buffer and incubated for 5 min at 4 ◦ C. After incubation, cell lysates were centrifuged at 12,000 × g for 20 min at 4 ◦ C. Supernatants were frozen rapidly and stored at −80 ◦ C prior to analysis. Protein concentration of the extracts was determined using Coomassie protein assay reagent (Pierce Chemical Co., Rockford, IL) and aliquots of extract containing 1 g protein used for each TRAP assay. Extract aliquots were incubated with 0.1 ng Cy-5-labeled TS primer (5 -AATCCGTCGAGCAGAGTT-3 ) in master mix (TRAP-ezeTM) for 30 min at 30 ◦ C. PCR was then performed for 30 cycles (94 ◦ C for 30 s, 60 ◦ C
H. Hoshino et al. for 30 s, and 72 ◦ C for 45 s). PCR products were applied to denaturing 10% polyacrylamide gels containing 6 M urea attached to an automated DNA sequencer. DNA sequence data was collected and automatically analyzed by the fragment Manager V1.1 system. To standardize telomerase activity, an internal standard for telomerase activity (TSR8) was used. Each peak was quantified in terms of size and peak area. The quantification of telomerase activity was determined by the formula: total product generated (TPG) (U/g protein) = (A/B)/(C/D) × 100, where A = measured total area of telomerase activity (50, 56, 62, 68 base pairs [bp] . . . ); B = measured area of internal control (36 bp); C = measured total area of telomerase activity (50, 56, 62, 68 bp . . . ) in the positive control; and D = measured area of internal control (36 bp) in the positive control.
2.5. Immunohistochemistry Proliferative activity was assessed using the Ki-67 labeling index, and p53 immunoreactivity determined using the monoclonal antibody DO-7 (DAKO, Glastrup, Denmark) that reacts with both wild type and mutant p53 proteins. Representative samples were obtained from paraffin embedded biopsy specimens fixed by neutral formalin immediately after biopsy. Biopsy specimens 3—4 m thick were cut and mounted onto silan-coated glass slides. Proliferative activity was determined using the MIB-1 monoclonal antibody (monoclonal antibody against the Ki-67 epitope, Immunotech, Marseilles, France), and p53 immunoreactivity using the DO-7 monoclonal antibody with slides stained according to the streptavidin—biotin staining procedure. Briefly, after deparaffinization, specimens were heated by microwave (four times at 100 ◦ C) in 0.01 M citrate buffer solution. After cooling gradually to room temperature, slides were incubated for 20 min in 0.3% hydrogen peroxide diluted in methanol. Slides were then washed with distilled water, incubated with normal rabbit serum to block nonspecific binding, and incubated with primary antibody (MIB-1 diluted to 1:100 or DO-7 diluted to 1:1600) in buffered solution overnight at 4 ◦ C. After incubation with bridging antibody for 30 min, slides then were incubated with avidin—biotin complex for 30 min. Immunostaining was visualized by diaminobenzine (stained for 8 min) and counterstained with hematoxylin. All slides were evaluated without any information with regard to clinical outcome or other clinicopathological data. Proliferative activity was expressed as the percentage of MIB-1-positive cells in dysplastic epithelium after counting at least 1000 nuclei of all layers of
Biological features of bronchial squamous dysplasia epithelial cells. Ki-67 labeling indices were counted twice and the reproducibility was within 5%, with the mean of the two values used for further analysis. p53 immunoreactivity was classified as positive if more than 10% of all dysplastic cells were stained with DO-7 antibody.
191
Table 1 Histological outcome of the followed-up
dysplasias
2.6. Statistical analysis Differences in the means of continuous measurements were tested by Student’s t-test, but if there is no normal distribution, we used the Mann—Whitney U-test or the Kruskal—Wallis rank test to compare among groups. Frequency analysis was performed with Fisher’s exact test. All reported P-values were two-sided, and those less than 0.05 were considered to be statistically significant.
3. Results 3.1. Histological outcome of the followed-up dysplasias Table 1 shows the histological outcome at second biopsy for the 99 dysplasias followed up. Of 99 dysplasias, 3 developed into squamous cell carcinoma, of which 2 were severe and 1 moderate. These lesions were curatively treated by laser irradiation. Overall, 41 dysplasias remained as dysplasia, 6 dysplasias had changed to metaplasia, 14 dysplasias had changed to hyperplasia, and 35 dysplasias had regressed to bronchitis or normal bronchial epithelium. Follow-up periods ranged from 5 to 17 months with a mean of 6.9 months. No significant associations between histological outcome and morphological severity of dysplasia (mild, moderate, or severe dysplasia) were observed by the Kruskal—Wallis rank test.
3.2. Smoking status during the follow-up period We assessed the correlation between histological outcome and smoking status during the follow-up period (Table 2). Twenty-one participants (54 dysplasias) belonged to the group that had continued or cut down smoking, and 29 participants (45 dysplasias) belonged to the group that had quit smoking. Two dysplasias had developed into squamous cell carcinoma in the group that had continued or cut down smoking, and one in the group that had quit smoking. The number of dysplasias that remained as dysplasia was 22 in the group that had continued or cut down smoking, and 19 in the
Table 2 Histological outcome of 99 dysplasias and smoking status Histological outcome
Smoking status Continue or cut down
Quit
Squamous cell (ca.) Dysplasia Metaplasia Hyperplasia Bronchitis or normal
2 22 4 11 15
1 19 2 3 20
Total
54 Pa = 0.3874
45
a
The Mann—Whitney U-test.
192
H. Hoshino et al.
Fig. 2 Initial telomerase activity in dysplasias according to histological outcome at the second biopsy. The mean value of initial telomerase activity in the dysplasias was found to increase in proportion to severity of histological outcome at the second biopsy. Values represent mean ± S.D. Kruskal—Wallis rank test was used to compare three groups (P = 0.0462).
group that had quit smoking. Lastly, the number of dysplasias that regressed to bronchitis or normal bronchial epithelium was 15 in the group that had continued or cut down smoking, and 20 in the group that had quit smoking. There were no significant associations between histological outcome and smoking status during the follow-up period (P = 0.3874, the Mann—Whitney U-test).
3.3. Initial telomerase activity in dysplasias in relation to histological outcome at second biopsy Because there were some inadequate samples to measure telomerase activity, of the 99 dysplasias, 46 were tested for telomerase activity at the initial examination. Results were classified into three groups according to the histological outcome at the second biopsy, as summarized in Fig. 2. In the 22 dysplasias that remained as dysplasia, mean telomerase activity was 19.5 ± 21.8 U/g protein (0—80.1) and in the seven dysplasias that changed to metaplasia or hyperplasia, the mean telomerase activity was 15.9 ± 14.5 U/g protein (0—36.3). In contrast, in the 17 dysplasias that regressed to bronchitis or normal bronchial epithelium, the mean telomerase activity was 5.2 ± 6.4 U/g protein (0—19.5). Dysplasias with high telomerase activity tended to remain as dysplasia (P = 0.0462, the Kruskal—Wallis rank test).
3.4. Initial Ki-67 labeling index in dysplasias in relation to histological outcome at second biopsy Because we could not use all of samples to measure Ki-67 labeling index, of the 99 dysplasias, 52
were tested for Ki-67 labeling index at the initial examination. Results were classified into four groups according to the histological outcome at the second biopsy as summarized in Fig. 3. For the three dysplasias that developed into squamous cell carcinoma, the mean Ki-67 labeling index was 13.0 ± 3.8% (8.7—15.8). For the 23 dysplasias that remained as dysplasia, the mean Ki-67 labeling index was 10.9 ± 6.2% (2.3—27.1). In contrast, for the six dysplasias that changed to metaplasia or hyperplasia, the mean Ki-67 labeling index was 5.4 ± 3.3% (1.0—10.1) and for the 20 dysplasias that regressed to bronchitis or normal bronchial epithelium, the mean Ki-67 labeling index was 6.8 ± 5.6% (0.7—19.2). Dysplasias with increased Ki-67 labeling index values tended to remain as dysplasia (P = 0.0407, the Kruskal—Wallis rank test).
3.5. Initial p53 immunoreactivity in dysplasias in relation to histological outcome at second biopsy Because we could not use all of samples to assessed for p53 immunoreactivity, of the 99 dysplasias, 55 were assessed for p53 immunoreactivity at the initial examination as summarized in Table 3. Results were classified into four groups according to the histological outcome at the second biopsy. A statistically significant difference in histological outcome was observed between the p53-positive and p53-negative groups (P = 0.0104, the Mann—Whitney U-test).
4. Discussion Centrally arising squamous cell carcinoma of the tracheobronchial tree is thought to develop via
Biological features of bronchial squamous dysplasia
193
Fig. 3 Initial Ki-67 labeling index in dysplasias according to histological outcome at the second biopsy. The mean value of initial Ki-67 labeling index in the dysplasias was found to increase in proportion to severity of histological outcome at the second biopsy. Values represent mean ± S.D. Kruskal—Wallis rank test was used to compare three groups (P = 0.0407).
Table 3 Comparison of histological outcome with p53 immunoreactivity
p53-positive dysplasia p53-negative dysplasia a
Squamous cell (ca.)
Dysplasia
Metaplasia, hyperplasia
Bronchitis, normal
P-valuea
2 1 3
17 8 25
4 2 6
6 15 21
0.0104
The Mann—Whitney U-test.
multiple stages, from normal bronchial epithelium to hyperplasia, followed by metaplasia, dysplasia, carcinoma in situ, and finally invasive cancer [6—8]. However, few reports have actually followed dysplastic lesions to determine the squamous dysplasia—carcinoma sequence. For example, while it has been observed that dysplasias progress to invasive cancers during follow-up periods [7,12,17,18], experimental studies have suggested that all changes, up to and including carcinoma in situ, may reverse spontaneously or in response to removal of the stimulus/carcinogen [6,17—19]. Thus, questions remain as to the biological behavior of squamous dysplasia. In our study, we periodically followed up localized cases of dysplasia by autofluorescence bronchoscopy, and assessed the histological outcome of the dysplastic lesions. We also attempted to clarify which dysplasias had the potential to progress to squamous cell carcinoma by measuring telomerase activity, cell-proliferative activity, and p53 immunoreactivity. Studies have shown that 20—40% of subjects presenting with marked atypia in the sputum eventually develop invasive lung cancer over a 5—10-year follow-up [6,17,18,20]. However, these studies failed to show histologically that localized
dysplasias progressed to cancer, instead suggesting that dysplastic cells of unknown origin within the bronchial tree had progressed to lung cancer. In our investigation of 99 dysplasias, three dysplasias detected in 3 patients with sputum cytology suspicious or positive for malignancy, developed into squamous cell carcinoma and were curatively treated by laser irradiation. They did not have lung cancer at the baseline biopsy, and were diagnosed as lung cancer for the first time at this follow-up period. Forty-one dysplasias remained as dysplasia, supporting the idea of dysplasia as a precancerous lesion. However, 35 dysplasias regressed to bronchitis or normal bronchial epithelium. Thus, our investigation observed histologically that while some dysplasias progressed to cancer, others were reversible. But more attention needs to be paid to the fact that while some dysplasias were actually reversible, biopsy itself was concerned in the regression of dysplastic lesions as a surgical resection of the lesion. The spread of the lesion may be concerned in the outcome of dysplasias though we could not examine in this study. Most of significance, despite the fact of the short follow-up time still three lesions developed into squamous cell carcinoma and most of the dysplasias remained as
194 dysplasias. In a study by Bota et al. [21], precancerous bronchial areas were mapped in subjects and individual sites followed by repeated biopsy. Keith et al. [22] found that 9 of 20 angiogenic squamous dysplasias persisted at the time of re-biopsy 1 year after initial diagnosis. Venmans et al. [23] reported that of nine patients with carcinoma in situ followed with autofluorescence bronchoscopy at regular intervals, five progressed to invasive cancer despite endobronchial therapy. Our investigation is a short-term follow-up study with a mean follow-up time of 6.9 months, such that if the follow-up period was extended, it is likely that the proportion of cases showing progression of dysplasia to squamous cell carcinoma would increase. Experimental studies have suggested that squamous dysplasia might be reversible by smoking cessation [9,12,19]. In our study, there was no significant association between histological outcome and smoking status over the follow-up period. Bota et al. [21] also observed no differences in progression/regression rates of severe dysplasia or carcinoma in situ lesions between patients who continued to smoke as compared with those who quit smoking after the first endoscopy. Epidemiological studies have clearly shown the beneficial effect of smoking cessation on lung cancer risk development, with a four-fold drop in the prevalence of lung cancer 10 years after smoking cessation [24]. While a similar effect may apply to squamous dysplasia, a longer follow-up period may be necessary to reveal the correlation between dysplasia regression and smoking cessation. While some studies have followed up dysplasia histologically, the characteristics that identify dysplasias with the potential to progress to squamous cell carcinoma are still unknown. Although the progression of disease through squamous metaplasia and carcinoma in situ in the bronchi has long been recognized, it is only recently that some of the molecular and genetic changes associated with the morphological transformation have been elucidated [8,9,15,22]. In the present study, we examined the biological behavior of bronchial squamous dysplasia using the biomarkers of telomerase activity, cell-proliferative activity, and p53 immunoreactivity. Telomerase is a ribonucleoprotein enzyme that stabilizes telomere length by adding hexametric (TTAGGG) repeats to the telomeric ends of chromosomes [25]. Telomerase is activated and expressed in most human cancers and may be one of the mechanisms that confer immortality to tumor cell populations by preventing progressive telomere shortening, and therefore cellular senescence [9]. Yashima et al. [26] reported that preinvasive
H. Hoshino et al. lesions, including carcinoma in situ, had telomerase enzyme activity three- to four-fold higher than normal, whereas in invasive disease, telomerase activity was 40 times greater. We have previously reported that the progressive increase in telomerase activity along with histological appearance supports a multistage pathogenesis of squamous cell carcinoma of the lung, and demonstrated the importance of dysplasia as a precancerous lesion using biopsy samples obtained by autofluorescence bronchoscopy [27]. In the present investigation, dysplasias with high telomerase activity tended to remain as dysplasia. It is possible that in dysplasias with high telomerase activity, it is the dysplastic cells with the activated telomerase activity that proliferate and survive. Thus, our results suggest that telomerase activity may be a useful biomarker mat predict which dysplasias progress to squamous cell carcinoma. Dysregulated cell growth is a hallmark of epithelial carcinogenesis, and proliferating cell nuclear antigen (PCNA) is a marker of dysregulated proliferation that is highly expressed in non-small cell lung cancers [28]. In two studies of squamous metaplastic lesions from bronchial biopsies, a positive correlation was found between the degree of proliferation as assessed by PCNA, and the grade of dysplasia [10]. In smokers, PCNA-positive cells increased progressively and significantly in parallel with histological progression from normal to hyperplasia to metaplasia/dysplasia [28]. Ki-67 nuclear antigen immunostaining experiments have shown that Ki-67 is associated with cell proliferation and is present throughout most of the cell cycle (Gl, S, G2 and M stages), but is absent in resting (GO) cells [29,30]. Ki-67 is thought to be a more reliable indicator of cell-proliferative activity than PCNA [31]. We reported that biopsy samples containing areas of squamous metaplasia, dysplasia, or squamous cell carcinoma from smokers had even greater proliferative activity and suggested that increased cell proliferation may be one of the first steps in the multistep process of lung carcinogenesis [32]. In the present investigation, dysplasias with a raised Ki-67 labeling index tended to remain as dysplasia. Some Ki-67-positive dysplastic cells, that is, cells in the Gl, S, G2 or M phase, appear to have become continuously cycling cells and may be irreversibly dysplastic. In contrast, Ki-67-negative dysplastic cells, that is, cells that retain a GO phase, may remain reversible. This result suggests that the Ki-67 labeling index may be a useful biomarker for predicting which dysplasias will progress to squamous cell carcinoma. The p53 protein inhibits cell proliferation by arresting cells in the Gl phase of the cell cycle. Loss
Biological features of bronchial squamous dysplasia
195
Table 4 Telomerase activity, Ki-67 labeling index, and p53 immunoreactivity according to morphological severity of dysplasia
Severe dysplasia Moderate dysplasia Mild dysplasia
Telomerase activity (U/g protein)a
Ki-67 labeling indexb (%)
p53 immunoreactivity (positive/negative)c
18.1 ± 25.6 (n = 2) 16.8 ± 19.3 (n = 25) 9.2 ± 14.1 (n = 19)
11.1 ± 8.2 (n = 7) 8.9 ± 6.0 (n = 32) 7.4 ± 4.5 (n = 13)
3/4 20/14 6/8
a
Values represent mean ± S.D. The Student’s t-test was used to assess statistical differences. Differences were not statistically significant. b Values represent mean ± S.D. The Student’s t-test was used to assess statistical differences. Differences were not statistically significant. c The Fisher’s exact test was used to assess statistical differences. Differences were not statistically significant.
of p53 activity can lead to neoplastic transformation [33]. Accumulation of mutant p53 protein can occur after genetic alteration of the p53 gene, and can be detected immunohistochemically in the majority of tumor cells [34]. Various studies have found increased p53 protein staining with increasing areas of bronchial squamous dysplasia [14,35]. The general conclusion is that abnormal p53 expression occurs even in the very early stages of malignant transformation, as well as in the least dysplastic of the preinvasive lesions [9]. Our results indicated that p53-positive dysplasias tended to remain as dysplasia. Thus, some p53-positive dysplastic cells may have acquired the p53 mutant gene and become irreversibly dysplastic. Thus, p53 immunoreactivity may also be useful as a biomarker to predict which dysplasias progress to squamous cell carcinoma. While our study found no significant associations between histological outcome and morphological severity of the dysplasia, we did observe that mean values of telomerase activity and Ki-67 labeling index measured in our study increased in proportion to the morphological severity (Table 4). These results support the hypothesis that the morphological severity of dysplasia is sequential. Importantly, even when morphological severity was mild, dysplasias with high telomerase activity, increased Ki-67 labeling index, or showing p53-positivity tended to remain as dysplasia. Our study observed three dysplasias that progressed to squamous cell carcinoma. While it is possible that some cancer cells may have been in existence at the initial examination, because the biopsy specimens were obtained at areas of significantly diminished autofluorescence, and so diagnosed as dysplasia, we concluded that the cases of squamous cell carcinoma observed after follow-up had progressed from dysplasia. The initial Ki-67 labeling index values for the three dysplastic lesions were 15.8, 14.5, and 8.7%. Although the
sample size was too small to detect statistically significant differences, the results agreed with our findings that dysplasias with increased Ki-67 labeling index had the potential to progress to squamous cell carcinoma. Of the three dysplasias, two were p53-positive, and the other p53-negative. At the time of diagnosis of squamous cell carcinoma, the p53-positive dysplasias had progressed to p53-positive carcinomas, and the p53-negative dysplasia progressed to a p53-negative carcinoma. While we were unfortunately unable to measure the telomerase activity of these three dysplasias, we would expect that the telomerase activity in the three dysplasias would be high. The natural course of preinvasive change in the bronchi in high-risk subjects needs to be clarified through longitudinal prospective studies that investigate the histological changes in bronchi. Ongoing longitudinal studies with autofluorescence bronchoscopy, multiple biopsies with histology, and biomarkers will further refine the ability to assess risk [15]. In conclusion, our results suggested that dysplasia with high telomerase activity, increased Ki-67 labeling index, and p53-positivity tended to remain as dysplasia, and might have the potential to progress to squamous cell carcinoma. Patients with dysplastic lesions with these characteristics should be carefully followed up.
Acknowledgements The authors thank Dr. Hisashi Hisatomi and Dr. Hiroto Nakano (SRL Inc., Tokyo, Japan) for technical assistance.
References [1] Lam S, Kennedy T, Unger M, et al. Localization of bronchial intraepithelial neoplastic lesions by fluorescence bronchoscopy. Chest 1998;113:696—702.
196 [2] George PJM. Fluorescence bronchoscopy for the early detection of lung cancer. Thorax 1999;54:180—3. [3] Shibuya K, Fujisawa T, Hoshino H, et al. Fluorescence bronchoscopy in the detection of preinvasive bronchial lesions in patients with sputum cytology suspicious or positive for malignancy. Lung Cancer 2001;32:19—25. [4] Hirsch FR, Prindiville SA, Miller YE, et al. Fluorescence versus white-light bronchoscopy for detection of preneoplastic lesions: a randomized study. J Natl Cancer Inst 2001;93:1385—91. [5] Banerjee AK, Rabbitts PH, George J, et al. Lung cancer. 3: Fluorescescence bronchoscopy: clinical dilemmas and research opportunities. Thorax 2003;58:266—71. [6] Auerbach O, Gere JB, Forman JB, et al. Changes in bronchial epithelium in relation to smoking and cancer of the lung: a report of progress. N Engl J Med 1957;256:97— 104. [7] Saccomanno G, Archer VE, Auerbach O, et al. Development of carcinoma of the lung as reflected in exfoliated cells. Cancer 1974;33:256—70. [8] Wistuba II, Behrens C, Milchgrub S, et al. Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma. Oncogene 1999;18:643—50. [9] Kerr KM. Pulmonary preinvasive neoplasia. J Clin Pathol 2001;54:257—71. [10] Pendleton N, Dixon GR, Burnett HE, et al. Expression of proliferating cell nuclear antigen (PCNA) in dysplasia of the bronchial epithelium. J Pathol 1993;170:169—72. [11] Bennett WP, Colby TV, Travis WD, et al. p53 protein accumulates frequently in early bronchial neoplasia. Cancer Res 1993;53:4817—22. [12] Thiberville L, Payne P, Vielkinds J, et al. Evidence of cumulative gene losses with progression of premalignant epithelial lesions to carcinoma of the bronchus. Cancer Res 1995;55:5133—9. [13] Boers JE, ten Velde GP, Thunnissen FB. P53 in squamous metaplasia: a marker for risk of respiratory tract carcinoma. Am J Respir Crit Care Med 1996;153:411—6. [14] Brambilla E, Gazzeri S, Lantuejoul S, et al. p53 mutant immunophenotype and deregulation of p53 transcription pathway (bcl-2, bax, and wafl) in precursor bronchial lesions and lung cancer. Clin Cancer Res 1998;4:1609—18. [15] Hirsch FR, Franklin WA, Gazdar AF, et al. Early detection of lung cancer: clinical perspectives of recent advances in biology and radiology. Clin Cancer Res 2001;7:5—22. [16] World Health Organization. Histological typing of lung and pleural tumors. 3rd ed. Berlin: Springer-Verlag; 1999. p. 28—30. [17] Frost JK, Ball Jr WC, Levin ML. Sputum cytopathology: use and potential in monitoring the workplace environment by screening for biological effects of exposure. J Occup Med 1986;28:692—730. [18] Band PR, Feldstein M, Saccomanno G. Reversibility of bronchial marked atypia: implication for chemoprevention. Cancer Detect Prev 1986;9:157—60.
H. Hoshino et al. [19] Auer G, Ono J, Nasiell M, et al. Reversibility of bronchial cell atypia. Cancer Res 1982;42:4241—7. [20] Satoh Y, Ishikawa Y, Nakagawa K, et al. A follow-up study of progression from dysplasia to squamous cell carcinoma with immunohistochemical examination of p53 protein overexpression in the bronchi of ex-chromate workers. Br J Cancer 1997;75:678—83. [21] Bota S, Auliac JB, Paris C, et al. Follow-up of bronchial precancerous lesions and carcinoma in situ using fluorescence endoscopy. Am J Respir Crit Care Med 2001;164:1688— 93. [22] Keith RL, Miller YE, Gemmill RM, et al. Angiogenic squamous dysplasia in bronchi of individuals at high risk for lung cancer. Clin Cancer Res 2000;6:1616—25. [23] Venmans BJ, van Boxem TJ, Smit EF, et al. Outcome of bronchial carcinoma in situ. Chest 2000;117:1572—6. [24] Peto R, Darby S, Deo H, et al. Smoking, smoking cessation and lung cancer in the UK since 1950. BMJ 2000;321:323—9. [25] Shay JW, Gazdar AF. Telomerase in the early detection of cancer. J Clin Pathol 1997;50:106—9. [26] Yashima K, Litzky LA, Kaiser L, et al. Telomerase expression in respiratory epithelium during the multistage pathogenesis of lung carcinomas. Cancer Res 1997;57:2373—7. [27] Shibuya K, Fujisawa T, Hoshino H, et al. Increased telomerase activity and elevated hTERT mRNA expression during multistage carcinogenesis of squamous cell carcinoma of the lung. Cancer 2001;92:849—55. [28] Khuri FR, Lee JS, Lippman SM, et al. Modulation of proliferating cell nuclear antigen in the bronchial epithelium of smokers. Cancer Epidemiol Biomarkers Prev 2001;10:311— 8. [29] Gerdes J, Lemke H, Baisch H, et al. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984;133:1710—5. [30] Cattoretti G, Becker MH, Key G, et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB-1 and MIB-3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 1992;168:357—63. [31] Sabattini E, Gerdes J, Gherlinzoni F, et al. Comparison between the monoclonal antibodies Ki-67 and PC10 in 125 malignant lymphomas. J Pathol 1993;169:397—403. [32] Hiroshima K, Iyoda A, Shibuya K, et al. Evidence of neoangiogenesis and an increase in the number of proliferating cells within the bronchial epithelium of smokers. Cancer 2002;95:1539—45. [33] Levine AJ. P53, the cellular gate keeper for growth and division. Cell 1997;88:323—31. [34] Harris CC, Hollstein M. Clinical implications of the p53 tumor-suppressor gene. N Engl JMed 1993;329:1318—27. [35] Katabami M, Dosaka-Akita H, Honma K, et al. p53 and bcl-2 expression in pneumoconiosis-related pre-cancerous lesions and lung cancers: frequent and preferential p53 expression in pneumoconiotic bronchiolar dysplasias. Int J Cancer 1998;75:504—11.
Biological features of bronchial squamous dysplasia followed up by autofluorescence bronchoscopy Hidehisa Hoshino a , Kiyoshi Shibuya a , Masako Chiyo a , Akira Iyoda a , Shigetoshi Yoshida a , Yasuo Sekine a , Toshihiko Iizasa a , Yukio Saitoh a , Masayuki Baba a , Kenzo Hiroshima b , Hidemi Ohwada b , Takehiko Fujisawa a,* a
Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan b Department of Basic Pathology, Graduate School of Medicine, Chiba University, Chiba, Japan Received 30 December 2003 ; received in revised form 31 March 2004; accepted 15 April 2004
KEYWORDS Squamous dysplasia; Telomerase activity; Ki-67 labeling index; p53 immunoreactivity; Autofluorescence bronchoscopy
Summary Some dysplasias in the bronchial epithelium are thought to be precancerous lesions that can develop into squamous cell carcinomas. In this investigation, we assessed the biological behavior of bronchial squamous dysplasia in order to define which dysplasias have the potential to progress to squamous cell carcinoma. Using autofluorescence bronchoscopy, we followed up periodically localized dysplasias and examined for correlation between histological outcome and smoking status during the follow-up period, telomerase activity, Ki-67 labeling index, and p53 immunoreactivity of initial biopsy specimens. Ninety-nine dysplasias from 50 participants mainly with sputum cytology suspicious or positive for malignancy were followed up. Of 99 dysplasias, 3 dysplasias progressed to squamous cell carcinoma, 41 dysplasias remained as dysplasia, 6 dysplasias changed to metaplasia, 14 dysplasias changed to hyperplasia, and 35 dysplasias regressed to bronchitis or normal bronchial epithelium. There were no significant associations between histological outcome and smoking status. Mean initial telomerase activity and Ki-67 labeling index values in the dysplasias increased in proportion to the severity of the histological outcome at the second biopsy. There was also a significant difference between p53-positive and p53-negative dysplasia in terms of histological outcome at the second biopsy. Our results suggested that dysplasias with high telomerase activity, increased Ki-67 labeling index, and p53-positivity tended to remain as dysplasia and might have the potential to progress to squamous cell carcinoma. Patients with dysplastic lesions with these characteristics should be carefully followed up. © 2004 Elsevier Ireland Ltd. All rights reserved.
* Corresponding
author. Tel.: +81 43 222 7171x5464; fax: +81 43 226 2172. E-mail addresses: [email protected] (H. Hoshino), [email protected] (T. Fujisawa). 0169-5002/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2004.04.028
188
1. Introduction During the past decade, the incidence of lung cancer and its related death rate has dramatically increased in Japan. In 1999, lung cancer became the leading cause of death, exceeding that of gastric cancer. Significant reduction in lung cancer-related morbidity and mortality will depend not only on aggressive efforts at smoking cessation, but also on earlier detection and treatment. Early hilar lung cancers, especially carcinoma in situ and microinvasive carcinoma, detected from sputum cytological examination can be cured. However, patients with carcinoma in situ and microinvasive carcinoma may require repeated examinations over many months before the lesion is localized. Moreover, it is exceedingly difficult to detect bronchial squamous dysplasia, thought to be a precancerous lesion, by conventional white light bronchoscopy alone. The development of autofluorescence bronchoscopy has made it possible to localize dysplasias and carcinoma in situ, and the usefulness of autofluorescence bronchoscopy has been widely reported [1—5]. Centrally arising squamous cell carcinoma of the tracheobronchial tree is thought to develop in multiple stages; from normal bronchial epithelium to hyperplasia, metaplasia, dysplasia, followed by carcinoma in situ, and finally invasive cancer [6,7]. Alterations in gene expression and chromosome structure known to be associated with malignant transformation can be demonstrated in carcinoma in situ and to a lesser extent in dysplasia, but can also be sometimes detected in morphologically normal epithelium [8]. Such changes might be sequential, and their frequency and number increase with atypia [8,9]. It is thought that at early stages, telomerase dysregulation, high proliferative activity, and p53 overexpression occur as part of a multistage pathogenesis of lung cancer [9—15]. While some reports have followed up dysplastic lesions to elucidate the multistep carcinogenesis of bronchial epithelium, few reports have examined the use of biomarkers to determine the biological behavior of dysplastic lesions, that is, the probability that a dysplastic lesion will progress to squamous cell carcinoma. Since October 1997, autofluorescence bronchoscopy examination has been performed for the localization of hilar lung cancer and squamous dysplasia at our institute. Autofluorescence bronchoscopy has also been used to periodically follow-up localized dysplasias. The purpose of this investigation is to elucidate the biological behavior of bronchial squamous dysplasia, and to clarify whether telomerase activity, cell proliferative ac-
H. Hoshino et al. tivity, or p53 immunoreactivity could be used as biomarkers to predict which dysplasias will progress to squamous cell carcinoma.
2. Materials and methods 2.1. Autofluorescence bronchoscopy and follow-up system Bronchoscopic examination was performed by one of two investigators (H.H. and K.S.) trained in autofluorescence bronchoscopy using a LIFE-lung fluorescence endoscopy system (Xillix Technologies Corp., Richmond, British Columbia, Canada). Autofluorescence bronchoscopy examinations were performed following white-light bronchoscopy (BF-240, Olympus Optical Corp., Tokyo, Japan) under local anesthesia with sedation by intravenous injection (diazepam or mitazoram) and O2 inhalation. Biopsy specimens for pathological examination were obtained from all suspicious or abnormal areas identified by white-light bronchoscopy examination, autofluorescence bronchoscopy examination, or both. One section of each biopsy specimen was immediately snap-frozen in liquid nitrogen, and stored at −80 ◦ C until assay for telomerase activity. The other section was formalin fixed, paraffin embedded, mounted, stained with hematoxylin and eosin, and diagnosed blindly by two expert pulmonary pathologists at the Department of Basic Pathology, Graduate School of Medicine, Chiba University (K.H. and H.O.). Dysplasia was identified according to recent World Health Organization criteria [16] (Fig. 1). Autofluorescence bronchoscopy was also used to follow-up localized dysplasia approximately every 6 months, and the follow-up biopsies were taken from the same site as the baseline biopsies were taken. Results were used to test for correlation between histological outcome and smoking status during the follow-up period, telomerase activity of initial biopsy specimens, and Ki-67 labeling index and p53 immunoreactivity of the initial biopsy specimens. Informed consent was obtained from all participants prior to investigation.
2.2. Smoking status Smoking status was classified at the initial autofluorescence bronchoscopy examination into three categories. A current smoker was defined as a participant who smoked at the time of initial autofluorescence bronchoscopy examination or in the year preceding it. An ex-smoker was defined as a participant who had quit smoking more than a year before the time of initial autofluorescence
Biological features of bronchial squamous dysplasia
189
Fig. 1 Bronchial squamous dysplasias detected by autofluorescence bronchoscopy. A: mild dysplasia; B: moderate dysplasia; and C: severe dysplasia.
190 bronchoscopy examination. A non-smoker was defined as a participant who had never smoked. Smoking status was also classified during the follow-up period of this investigation into three categories: The ‘‘continue’’ group was defined as participants who continued smoking as before; the ‘‘cut-down’’ group was defined as participants who reduced smoking to approximately five cigarettes per day; and the ‘‘quit’’ group was defined as participants who had quit smoking after initial autofluorescence bronchoscopy examination. Smoking status data was obtained by asking each participant directly prior to investigation.
2.3. Study populations From October 1997 to October 2001, a total of 276 participants with mainly sputum cytology suspicious or positive for malignancy or with lung cancer were examined by autofluorescence bronchoscopy (LIFE) in the Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, and 209 dysplasias were found in 105 participants. Using autofluorescence bronchoscopy, 99 dysplasias were followed up in 50 participants approximately every 6 months, and were used in our study. Forty participants had sputum cytology suspicious or positive for malignancy, and 10 participants were being followed up after curative treatment for lung cancer. The study group consisted of 49 men and one woman, ranging from 46 to 76 years old with a mean of 67 years. Smoking history of the study group ranged from 11 to 153 pack-years with a mean of 60.0. Forty-four were current smokers, and six were ex-smokers.
2.4. Telomerase activity Telomerase activity was measured using the TRAP-ezeTM telomerase detection kit (Intergen Co., New York, NY) together with the fluorescencebased telomeric repeat amplification protocol (F-TRAP) assay. Biopsy specimens were homogenized in 300 L ice-cold CHAPS lysis buffer and incubated for 5 min at 4 ◦ C. After incubation, cell lysates were centrifuged at 12,000 × g for 20 min at 4 ◦ C. Supernatants were frozen rapidly and stored at −80 ◦ C prior to analysis. Protein concentration of the extracts was determined using Coomassie protein assay reagent (Pierce Chemical Co., Rockford, IL) and aliquots of extract containing 1 g protein used for each TRAP assay. Extract aliquots were incubated with 0.1 ng Cy-5-labeled TS primer (5 -AATCCGTCGAGCAGAGTT-3 ) in master mix (TRAP-ezeTM) for 30 min at 30 ◦ C. PCR was then performed for 30 cycles (94 ◦ C for 30 s, 60 ◦ C
H. Hoshino et al. for 30 s, and 72 ◦ C for 45 s). PCR products were applied to denaturing 10% polyacrylamide gels containing 6 M urea attached to an automated DNA sequencer. DNA sequence data was collected and automatically analyzed by the fragment Manager V1.1 system. To standardize telomerase activity, an internal standard for telomerase activity (TSR8) was used. Each peak was quantified in terms of size and peak area. The quantification of telomerase activity was determined by the formula: total product generated (TPG) (U/g protein) = (A/B)/(C/D) × 100, where A = measured total area of telomerase activity (50, 56, 62, 68 base pairs [bp] . . . ); B = measured area of internal control (36 bp); C = measured total area of telomerase activity (50, 56, 62, 68 bp . . . ) in the positive control; and D = measured area of internal control (36 bp) in the positive control.
2.5. Immunohistochemistry Proliferative activity was assessed using the Ki-67 labeling index, and p53 immunoreactivity determined using the monoclonal antibody DO-7 (DAKO, Glastrup, Denmark) that reacts with both wild type and mutant p53 proteins. Representative samples were obtained from paraffin embedded biopsy specimens fixed by neutral formalin immediately after biopsy. Biopsy specimens 3—4 m thick were cut and mounted onto silan-coated glass slides. Proliferative activity was determined using the MIB-1 monoclonal antibody (monoclonal antibody against the Ki-67 epitope, Immunotech, Marseilles, France), and p53 immunoreactivity using the DO-7 monoclonal antibody with slides stained according to the streptavidin—biotin staining procedure. Briefly, after deparaffinization, specimens were heated by microwave (four times at 100 ◦ C) in 0.01 M citrate buffer solution. After cooling gradually to room temperature, slides were incubated for 20 min in 0.3% hydrogen peroxide diluted in methanol. Slides were then washed with distilled water, incubated with normal rabbit serum to block nonspecific binding, and incubated with primary antibody (MIB-1 diluted to 1:100 or DO-7 diluted to 1:1600) in buffered solution overnight at 4 ◦ C. After incubation with bridging antibody for 30 min, slides then were incubated with avidin—biotin complex for 30 min. Immunostaining was visualized by diaminobenzine (stained for 8 min) and counterstained with hematoxylin. All slides were evaluated without any information with regard to clinical outcome or other clinicopathological data. Proliferative activity was expressed as the percentage of MIB-1-positive cells in dysplastic epithelium after counting at least 1000 nuclei of all layers of
Biological features of bronchial squamous dysplasia epithelial cells. Ki-67 labeling indices were counted twice and the reproducibility was within 5%, with the mean of the two values used for further analysis. p53 immunoreactivity was classified as positive if more than 10% of all dysplastic cells were stained with DO-7 antibody.
191
Table 1 Histological outcome of the followed-up
dysplasias
2.6. Statistical analysis Differences in the means of continuous measurements were tested by Student’s t-test, but if there is no normal distribution, we used the Mann—Whitney U-test or the Kruskal—Wallis rank test to compare among groups. Frequency analysis was performed with Fisher’s exact test. All reported P-values were two-sided, and those less than 0.05 were considered to be statistically significant.
3. Results 3.1. Histological outcome of the followed-up dysplasias Table 1 shows the histological outcome at second biopsy for the 99 dysplasias followed up. Of 99 dysplasias, 3 developed into squamous cell carcinoma, of which 2 were severe and 1 moderate. These lesions were curatively treated by laser irradiation. Overall, 41 dysplasias remained as dysplasia, 6 dysplasias had changed to metaplasia, 14 dysplasias had changed to hyperplasia, and 35 dysplasias had regressed to bronchitis or normal bronchial epithelium. Follow-up periods ranged from 5 to 17 months with a mean of 6.9 months. No significant associations between histological outcome and morphological severity of dysplasia (mild, moderate, or severe dysplasia) were observed by the Kruskal—Wallis rank test.
3.2. Smoking status during the follow-up period We assessed the correlation between histological outcome and smoking status during the follow-up period (Table 2). Twenty-one participants (54 dysplasias) belonged to the group that had continued or cut down smoking, and 29 participants (45 dysplasias) belonged to the group that had quit smoking. Two dysplasias had developed into squamous cell carcinoma in the group that had continued or cut down smoking, and one in the group that had quit smoking. The number of dysplasias that remained as dysplasia was 22 in the group that had continued or cut down smoking, and 19 in the
Table 2 Histological outcome of 99 dysplasias and smoking status Histological outcome
Smoking status Continue or cut down
Quit
Squamous cell (ca.) Dysplasia Metaplasia Hyperplasia Bronchitis or normal
2 22 4 11 15
1 19 2 3 20
Total
54 Pa = 0.3874
45
a
The Mann—Whitney U-test.
192
H. Hoshino et al.
Fig. 2 Initial telomerase activity in dysplasias according to histological outcome at the second biopsy. The mean value of initial telomerase activity in the dysplasias was found to increase in proportion to severity of histological outcome at the second biopsy. Values represent mean ± S.D. Kruskal—Wallis rank test was used to compare three groups (P = 0.0462).
group that had quit smoking. Lastly, the number of dysplasias that regressed to bronchitis or normal bronchial epithelium was 15 in the group that had continued or cut down smoking, and 20 in the group that had quit smoking. There were no significant associations between histological outcome and smoking status during the follow-up period (P = 0.3874, the Mann—Whitney U-test).
3.3. Initial telomerase activity in dysplasias in relation to histological outcome at second biopsy Because there were some inadequate samples to measure telomerase activity, of the 99 dysplasias, 46 were tested for telomerase activity at the initial examination. Results were classified into three groups according to the histological outcome at the second biopsy, as summarized in Fig. 2. In the 22 dysplasias that remained as dysplasia, mean telomerase activity was 19.5 ± 21.8 U/g protein (0—80.1) and in the seven dysplasias that changed to metaplasia or hyperplasia, the mean telomerase activity was 15.9 ± 14.5 U/g protein (0—36.3). In contrast, in the 17 dysplasias that regressed to bronchitis or normal bronchial epithelium, the mean telomerase activity was 5.2 ± 6.4 U/g protein (0—19.5). Dysplasias with high telomerase activity tended to remain as dysplasia (P = 0.0462, the Kruskal—Wallis rank test).
3.4. Initial Ki-67 labeling index in dysplasias in relation to histological outcome at second biopsy Because we could not use all of samples to measure Ki-67 labeling index, of the 99 dysplasias, 52
were tested for Ki-67 labeling index at the initial examination. Results were classified into four groups according to the histological outcome at the second biopsy as summarized in Fig. 3. For the three dysplasias that developed into squamous cell carcinoma, the mean Ki-67 labeling index was 13.0 ± 3.8% (8.7—15.8). For the 23 dysplasias that remained as dysplasia, the mean Ki-67 labeling index was 10.9 ± 6.2% (2.3—27.1). In contrast, for the six dysplasias that changed to metaplasia or hyperplasia, the mean Ki-67 labeling index was 5.4 ± 3.3% (1.0—10.1) and for the 20 dysplasias that regressed to bronchitis or normal bronchial epithelium, the mean Ki-67 labeling index was 6.8 ± 5.6% (0.7—19.2). Dysplasias with increased Ki-67 labeling index values tended to remain as dysplasia (P = 0.0407, the Kruskal—Wallis rank test).
3.5. Initial p53 immunoreactivity in dysplasias in relation to histological outcome at second biopsy Because we could not use all of samples to assessed for p53 immunoreactivity, of the 99 dysplasias, 55 were assessed for p53 immunoreactivity at the initial examination as summarized in Table 3. Results were classified into four groups according to the histological outcome at the second biopsy. A statistically significant difference in histological outcome was observed between the p53-positive and p53-negative groups (P = 0.0104, the Mann—Whitney U-test).
4. Discussion Centrally arising squamous cell carcinoma of the tracheobronchial tree is thought to develop via
Biological features of bronchial squamous dysplasia
193
Fig. 3 Initial Ki-67 labeling index in dysplasias according to histological outcome at the second biopsy. The mean value of initial Ki-67 labeling index in the dysplasias was found to increase in proportion to severity of histological outcome at the second biopsy. Values represent mean ± S.D. Kruskal—Wallis rank test was used to compare three groups (P = 0.0407).
Table 3 Comparison of histological outcome with p53 immunoreactivity
p53-positive dysplasia p53-negative dysplasia a
Squamous cell (ca.)
Dysplasia
Metaplasia, hyperplasia
Bronchitis, normal
P-valuea
2 1 3
17 8 25
4 2 6
6 15 21
0.0104
The Mann—Whitney U-test.
multiple stages, from normal bronchial epithelium to hyperplasia, followed by metaplasia, dysplasia, carcinoma in situ, and finally invasive cancer [6—8]. However, few reports have actually followed dysplastic lesions to determine the squamous dysplasia—carcinoma sequence. For example, while it has been observed that dysplasias progress to invasive cancers during follow-up periods [7,12,17,18], experimental studies have suggested that all changes, up to and including carcinoma in situ, may reverse spontaneously or in response to removal of the stimulus/carcinogen [6,17—19]. Thus, questions remain as to the biological behavior of squamous dysplasia. In our study, we periodically followed up localized cases of dysplasia by autofluorescence bronchoscopy, and assessed the histological outcome of the dysplastic lesions. We also attempted to clarify which dysplasias had the potential to progress to squamous cell carcinoma by measuring telomerase activity, cell-proliferative activity, and p53 immunoreactivity. Studies have shown that 20—40% of subjects presenting with marked atypia in the sputum eventually develop invasive lung cancer over a 5—10-year follow-up [6,17,18,20]. However, these studies failed to show histologically that localized
dysplasias progressed to cancer, instead suggesting that dysplastic cells of unknown origin within the bronchial tree had progressed to lung cancer. In our investigation of 99 dysplasias, three dysplasias detected in 3 patients with sputum cytology suspicious or positive for malignancy, developed into squamous cell carcinoma and were curatively treated by laser irradiation. They did not have lung cancer at the baseline biopsy, and were diagnosed as lung cancer for the first time at this follow-up period. Forty-one dysplasias remained as dysplasia, supporting the idea of dysplasia as a precancerous lesion. However, 35 dysplasias regressed to bronchitis or normal bronchial epithelium. Thus, our investigation observed histologically that while some dysplasias progressed to cancer, others were reversible. But more attention needs to be paid to the fact that while some dysplasias were actually reversible, biopsy itself was concerned in the regression of dysplastic lesions as a surgical resection of the lesion. The spread of the lesion may be concerned in the outcome of dysplasias though we could not examine in this study. Most of significance, despite the fact of the short follow-up time still three lesions developed into squamous cell carcinoma and most of the dysplasias remained as
194 dysplasias. In a study by Bota et al. [21], precancerous bronchial areas were mapped in subjects and individual sites followed by repeated biopsy. Keith et al. [22] found that 9 of 20 angiogenic squamous dysplasias persisted at the time of re-biopsy 1 year after initial diagnosis. Venmans et al. [23] reported that of nine patients with carcinoma in situ followed with autofluorescence bronchoscopy at regular intervals, five progressed to invasive cancer despite endobronchial therapy. Our investigation is a short-term follow-up study with a mean follow-up time of 6.9 months, such that if the follow-up period was extended, it is likely that the proportion of cases showing progression of dysplasia to squamous cell carcinoma would increase. Experimental studies have suggested that squamous dysplasia might be reversible by smoking cessation [9,12,19]. In our study, there was no significant association between histological outcome and smoking status over the follow-up period. Bota et al. [21] also observed no differences in progression/regression rates of severe dysplasia or carcinoma in situ lesions between patients who continued to smoke as compared with those who quit smoking after the first endoscopy. Epidemiological studies have clearly shown the beneficial effect of smoking cessation on lung cancer risk development, with a four-fold drop in the prevalence of lung cancer 10 years after smoking cessation [24]. While a similar effect may apply to squamous dysplasia, a longer follow-up period may be necessary to reveal the correlation between dysplasia regression and smoking cessation. While some studies have followed up dysplasia histologically, the characteristics that identify dysplasias with the potential to progress to squamous cell carcinoma are still unknown. Although the progression of disease through squamous metaplasia and carcinoma in situ in the bronchi has long been recognized, it is only recently that some of the molecular and genetic changes associated with the morphological transformation have been elucidated [8,9,15,22]. In the present study, we examined the biological behavior of bronchial squamous dysplasia using the biomarkers of telomerase activity, cell-proliferative activity, and p53 immunoreactivity. Telomerase is a ribonucleoprotein enzyme that stabilizes telomere length by adding hexametric (TTAGGG) repeats to the telomeric ends of chromosomes [25]. Telomerase is activated and expressed in most human cancers and may be one of the mechanisms that confer immortality to tumor cell populations by preventing progressive telomere shortening, and therefore cellular senescence [9]. Yashima et al. [26] reported that preinvasive
H. Hoshino et al. lesions, including carcinoma in situ, had telomerase enzyme activity three- to four-fold higher than normal, whereas in invasive disease, telomerase activity was 40 times greater. We have previously reported that the progressive increase in telomerase activity along with histological appearance supports a multistage pathogenesis of squamous cell carcinoma of the lung, and demonstrated the importance of dysplasia as a precancerous lesion using biopsy samples obtained by autofluorescence bronchoscopy [27]. In the present investigation, dysplasias with high telomerase activity tended to remain as dysplasia. It is possible that in dysplasias with high telomerase activity, it is the dysplastic cells with the activated telomerase activity that proliferate and survive. Thus, our results suggest that telomerase activity may be a useful biomarker mat predict which dysplasias progress to squamous cell carcinoma. Dysregulated cell growth is a hallmark of epithelial carcinogenesis, and proliferating cell nuclear antigen (PCNA) is a marker of dysregulated proliferation that is highly expressed in non-small cell lung cancers [28]. In two studies of squamous metaplastic lesions from bronchial biopsies, a positive correlation was found between the degree of proliferation as assessed by PCNA, and the grade of dysplasia [10]. In smokers, PCNA-positive cells increased progressively and significantly in parallel with histological progression from normal to hyperplasia to metaplasia/dysplasia [28]. Ki-67 nuclear antigen immunostaining experiments have shown that Ki-67 is associated with cell proliferation and is present throughout most of the cell cycle (Gl, S, G2 and M stages), but is absent in resting (GO) cells [29,30]. Ki-67 is thought to be a more reliable indicator of cell-proliferative activity than PCNA [31]. We reported that biopsy samples containing areas of squamous metaplasia, dysplasia, or squamous cell carcinoma from smokers had even greater proliferative activity and suggested that increased cell proliferation may be one of the first steps in the multistep process of lung carcinogenesis [32]. In the present investigation, dysplasias with a raised Ki-67 labeling index tended to remain as dysplasia. Some Ki-67-positive dysplastic cells, that is, cells in the Gl, S, G2 or M phase, appear to have become continuously cycling cells and may be irreversibly dysplastic. In contrast, Ki-67-negative dysplastic cells, that is, cells that retain a GO phase, may remain reversible. This result suggests that the Ki-67 labeling index may be a useful biomarker for predicting which dysplasias will progress to squamous cell carcinoma. The p53 protein inhibits cell proliferation by arresting cells in the Gl phase of the cell cycle. Loss
Biological features of bronchial squamous dysplasia
195
Table 4 Telomerase activity, Ki-67 labeling index, and p53 immunoreactivity according to morphological severity of dysplasia
Severe dysplasia Moderate dysplasia Mild dysplasia
Telomerase activity (U/g protein)a
Ki-67 labeling indexb (%)
p53 immunoreactivity (positive/negative)c
18.1 ± 25.6 (n = 2) 16.8 ± 19.3 (n = 25) 9.2 ± 14.1 (n = 19)
11.1 ± 8.2 (n = 7) 8.9 ± 6.0 (n = 32) 7.4 ± 4.5 (n = 13)
3/4 20/14 6/8
a
Values represent mean ± S.D. The Student’s t-test was used to assess statistical differences. Differences were not statistically significant. b Values represent mean ± S.D. The Student’s t-test was used to assess statistical differences. Differences were not statistically significant. c The Fisher’s exact test was used to assess statistical differences. Differences were not statistically significant.
of p53 activity can lead to neoplastic transformation [33]. Accumulation of mutant p53 protein can occur after genetic alteration of the p53 gene, and can be detected immunohistochemically in the majority of tumor cells [34]. Various studies have found increased p53 protein staining with increasing areas of bronchial squamous dysplasia [14,35]. The general conclusion is that abnormal p53 expression occurs even in the very early stages of malignant transformation, as well as in the least dysplastic of the preinvasive lesions [9]. Our results indicated that p53-positive dysplasias tended to remain as dysplasia. Thus, some p53-positive dysplastic cells may have acquired the p53 mutant gene and become irreversibly dysplastic. Thus, p53 immunoreactivity may also be useful as a biomarker to predict which dysplasias progress to squamous cell carcinoma. While our study found no significant associations between histological outcome and morphological severity of the dysplasia, we did observe that mean values of telomerase activity and Ki-67 labeling index measured in our study increased in proportion to the morphological severity (Table 4). These results support the hypothesis that the morphological severity of dysplasia is sequential. Importantly, even when morphological severity was mild, dysplasias with high telomerase activity, increased Ki-67 labeling index, or showing p53-positivity tended to remain as dysplasia. Our study observed three dysplasias that progressed to squamous cell carcinoma. While it is possible that some cancer cells may have been in existence at the initial examination, because the biopsy specimens were obtained at areas of significantly diminished autofluorescence, and so diagnosed as dysplasia, we concluded that the cases of squamous cell carcinoma observed after follow-up had progressed from dysplasia. The initial Ki-67 labeling index values for the three dysplastic lesions were 15.8, 14.5, and 8.7%. Although the
sample size was too small to detect statistically significant differences, the results agreed with our findings that dysplasias with increased Ki-67 labeling index had the potential to progress to squamous cell carcinoma. Of the three dysplasias, two were p53-positive, and the other p53-negative. At the time of diagnosis of squamous cell carcinoma, the p53-positive dysplasias had progressed to p53-positive carcinomas, and the p53-negative dysplasia progressed to a p53-negative carcinoma. While we were unfortunately unable to measure the telomerase activity of these three dysplasias, we would expect that the telomerase activity in the three dysplasias would be high. The natural course of preinvasive change in the bronchi in high-risk subjects needs to be clarified through longitudinal prospective studies that investigate the histological changes in bronchi. Ongoing longitudinal studies with autofluorescence bronchoscopy, multiple biopsies with histology, and biomarkers will further refine the ability to assess risk [15]. In conclusion, our results suggested that dysplasia with high telomerase activity, increased Ki-67 labeling index, and p53-positivity tended to remain as dysplasia, and might have the potential to progress to squamous cell carcinoma. Patients with dysplastic lesions with these characteristics should be carefully followed up.
Acknowledgements The authors thank Dr. Hisashi Hisatomi and Dr. Hiroto Nakano (SRL Inc., Tokyo, Japan) for technical assistance.
References [1] Lam S, Kennedy T, Unger M, et al. Localization of bronchial intraepithelial neoplastic lesions by fluorescence bronchoscopy. Chest 1998;113:696—702.
196 [2] George PJM. Fluorescence bronchoscopy for the early detection of lung cancer. Thorax 1999;54:180—3. [3] Shibuya K, Fujisawa T, Hoshino H, et al. Fluorescence bronchoscopy in the detection of preinvasive bronchial lesions in patients with sputum cytology suspicious or positive for malignancy. Lung Cancer 2001;32:19—25. [4] Hirsch FR, Prindiville SA, Miller YE, et al. Fluorescence versus white-light bronchoscopy for detection of preneoplastic lesions: a randomized study. J Natl Cancer Inst 2001;93:1385—91. [5] Banerjee AK, Rabbitts PH, George J, et al. Lung cancer. 3: Fluorescescence bronchoscopy: clinical dilemmas and research opportunities. Thorax 2003;58:266—71. [6] Auerbach O, Gere JB, Forman JB, et al. Changes in bronchial epithelium in relation to smoking and cancer of the lung: a report of progress. N Engl J Med 1957;256:97— 104. [7] Saccomanno G, Archer VE, Auerbach O, et al. Development of carcinoma of the lung as reflected in exfoliated cells. Cancer 1974;33:256—70. [8] Wistuba II, Behrens C, Milchgrub S, et al. Sequential molecular abnormalities are involved in the multistage development of squamous cell lung carcinoma. Oncogene 1999;18:643—50. [9] Kerr KM. Pulmonary preinvasive neoplasia. J Clin Pathol 2001;54:257—71. [10] Pendleton N, Dixon GR, Burnett HE, et al. Expression of proliferating cell nuclear antigen (PCNA) in dysplasia of the bronchial epithelium. J Pathol 1993;170:169—72. [11] Bennett WP, Colby TV, Travis WD, et al. p53 protein accumulates frequently in early bronchial neoplasia. Cancer Res 1993;53:4817—22. [12] Thiberville L, Payne P, Vielkinds J, et al. Evidence of cumulative gene losses with progression of premalignant epithelial lesions to carcinoma of the bronchus. Cancer Res 1995;55:5133—9. [13] Boers JE, ten Velde GP, Thunnissen FB. P53 in squamous metaplasia: a marker for risk of respiratory tract carcinoma. Am J Respir Crit Care Med 1996;153:411—6. [14] Brambilla E, Gazzeri S, Lantuejoul S, et al. p53 mutant immunophenotype and deregulation of p53 transcription pathway (bcl-2, bax, and wafl) in precursor bronchial lesions and lung cancer. Clin Cancer Res 1998;4:1609—18. [15] Hirsch FR, Franklin WA, Gazdar AF, et al. Early detection of lung cancer: clinical perspectives of recent advances in biology and radiology. Clin Cancer Res 2001;7:5—22. [16] World Health Organization. Histological typing of lung and pleural tumors. 3rd ed. Berlin: Springer-Verlag; 1999. p. 28—30. [17] Frost JK, Ball Jr WC, Levin ML. Sputum cytopathology: use and potential in monitoring the workplace environment by screening for biological effects of exposure. J Occup Med 1986;28:692—730. [18] Band PR, Feldstein M, Saccomanno G. Reversibility of bronchial marked atypia: implication for chemoprevention. Cancer Detect Prev 1986;9:157—60.
H. Hoshino et al. [19] Auer G, Ono J, Nasiell M, et al. Reversibility of bronchial cell atypia. Cancer Res 1982;42:4241—7. [20] Satoh Y, Ishikawa Y, Nakagawa K, et al. A follow-up study of progression from dysplasia to squamous cell carcinoma with immunohistochemical examination of p53 protein overexpression in the bronchi of ex-chromate workers. Br J Cancer 1997;75:678—83. [21] Bota S, Auliac JB, Paris C, et al. Follow-up of bronchial precancerous lesions and carcinoma in situ using fluorescence endoscopy. Am J Respir Crit Care Med 2001;164:1688— 93. [22] Keith RL, Miller YE, Gemmill RM, et al. Angiogenic squamous dysplasia in bronchi of individuals at high risk for lung cancer. Clin Cancer Res 2000;6:1616—25. [23] Venmans BJ, van Boxem TJ, Smit EF, et al. Outcome of bronchial carcinoma in situ. Chest 2000;117:1572—6. [24] Peto R, Darby S, Deo H, et al. Smoking, smoking cessation and lung cancer in the UK since 1950. BMJ 2000;321:323—9. [25] Shay JW, Gazdar AF. Telomerase in the early detection of cancer. J Clin Pathol 1997;50:106—9. [26] Yashima K, Litzky LA, Kaiser L, et al. Telomerase expression in respiratory epithelium during the multistage pathogenesis of lung carcinomas. Cancer Res 1997;57:2373—7. [27] Shibuya K, Fujisawa T, Hoshino H, et al. Increased telomerase activity and elevated hTERT mRNA expression during multistage carcinogenesis of squamous cell carcinoma of the lung. Cancer 2001;92:849—55. [28] Khuri FR, Lee JS, Lippman SM, et al. Modulation of proliferating cell nuclear antigen in the bronchial epithelium of smokers. Cancer Epidemiol Biomarkers Prev 2001;10:311— 8. [29] Gerdes J, Lemke H, Baisch H, et al. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984;133:1710—5. [30] Cattoretti G, Becker MH, Key G, et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB-1 and MIB-3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 1992;168:357—63. [31] Sabattini E, Gerdes J, Gherlinzoni F, et al. Comparison between the monoclonal antibodies Ki-67 and PC10 in 125 malignant lymphomas. J Pathol 1993;169:397—403. [32] Hiroshima K, Iyoda A, Shibuya K, et al. Evidence of neoangiogenesis and an increase in the number of proliferating cells within the bronchial epithelium of smokers. Cancer 2002;95:1539—45. [33] Levine AJ. P53, the cellular gate keeper for growth and division. Cell 1997;88:323—31. [34] Harris CC, Hollstein M. Clinical implications of the p53 tumor-suppressor gene. N Engl JMed 1993;329:1318—27. [35] Katabami M, Dosaka-Akita H, Honma K, et al. p53 and bcl-2 expression in pneumoconiosis-related pre-cancerous lesions and lung cancers: frequent and preferential p53 expression in pneumoconiotic bronchiolar dysplasias. Int J Cancer 1998;75:504—11.