Cancer Research Targets Telomeres
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[Cancer Research 65, 9623-9627, November 1, 2005]
© 2005 American Association for Cancer Research

Priority Reports

Telomerase Expression in Noncancerous Bronchial Epithelia Is a Possible Marker of Early Development of Lung Cancer

Yuka Matsuoka Miyazu1,7, Teruomi Miyazawa1,7, Keiko Hiyama3, Noriaki Kurimoto4,8, Yasuo Iwamoto1, Hiroo Matsuura2, Koji Kanoh1, Nobuoki Kohno5, Masahiko Nishiyama3 and Eiso Hiyama6

Departments of 1 Pulmonary Medicine and 2 Pathology, Hiroshima City Hospital; 3 Department of Translational Cancer Research, Research Institute for Radiation Biology and Medicine Hiroshima University; 4 Department of Surgery, Hiroshima National Hospital; 5 Department of Molecular and Internal Medicine, Graduate School of Biomedical Sciences; and 6 Natural Science Center for Basic Research and Development, Hiroshima University, Hiroshima, Japan; and Departments of 7 Respiratory and Infectious Diseases and 8 Surgery, St. Marianna University School of Medicine, Kawasaki, Japan

Requests for reprints: Eiso Hiyama, Natural Science Center for Basic Research and Development, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8551 Japan. Phone: 81-82-257-5951; Fax: 81-82-257-5219; E-mail: eiso{at}hiroshima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Result
 Discussion
 References
 
Centrally located lung cancers in smokers frequently associated with subsequent primary tumors. We evaluated the telomerase expression chronologically in noncancerous epithelia as a risk factor of susceptibility to lung cancer development. Telomerase protein expression was examined in situ by immunohistochemistry in 26 noncancerous bronchial epithelia adjacent to centrally located early-stage lung cancers in sequential 23 patients treated by photodynamic therapy or surgery among 206 patients who underwent autofluorescence bronchoscopy from 1997 to 2003. Among the 15 lesions in 12 patients treated by photodynamic therapy alone, 11 lesions achieved complete remission after photodynamic therapy, and none of their noncancerous bronchial epithelia was telomerase positive. On the contrary, in the remaining four lesions, either recurrence or secondary lung cancer developed adjacent to the successfully treated primary cancer within 26 months, and the telomerase protein expression in noncancerous epithelia was detected before the secondary cancer development (P < 0.001). The overall relationship of human telomerase reverse transcriptase positivity in noncancerous epithelia and subsequent lung cancer development, including patients treated by radiation or surgery, showed higher significance (P < 0.0001). Histologically "normal" bronchial epithelia in smokers may unphysiologically express telomerase as a field, and such epithelia are likely susceptible to develop lung cancer. We propose that ectopic expression of telomerase in bronchial epithelia may precede transformation in human lung cancer development and that detection of telomerase protein in noncancerous bronchial epithelia will become a useful marker detecting high-risk patients for lung cancer development.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Result
 Discussion
 References
 
Most patients with centrally located lung cancers have been exposed to carcinogens in tobacco smoke and frequently develop subsequent primary and/or multicentric tumors (1, 2). Autofluorescence bronchoscopy has increased the detection rate of centrally located early-stage lung cancers and enhanced opportunities for curative endobronchial therapeutic strategies, such as photodynamic therapy (3, 4), as well as enabling chronological observation of lesion development. Although most of properly selected candidates for photodynamic therapy have a good therapeutic outcome, some cases, mainly heavy smokers, develop recurrence or multiple metachronous cancers shortly after a successful photodynamic therapy.

Telomerase is expressed in immortal cells and germ line cells maintaining chromosome termini called telomeres, so that these cells can proliferate indefinitely (5). Telomerase activity is detectable in 80% to 90% of human cancer tissues, whereas normal adjacent tissues usually lack measurable activity, indicating that telomere stability is required for immortalization of cancer cells (6). It is generally believed that activation of telomerase in cancer occurs after many cell divisions through several clonal selections (7). Despite these common understandings, we here propose with evidence that ectopic expression of telomerase in epithelia may precede transformation as a field and predispose the lesion to cancer development.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Result
 Discussion
 References
 
We did autofluorescence bronchoscopy in 206 patients at high risk for developing lung cancer from September 1997 to March 2003 at Hiroshima City Hospital. When suspicious lesions were detected, patients underwent autofluorescence bronchoscopy every 3 to 6 months. Written informed consent was obtained from all patients. In 12 patients, 15 lesions have been treated by photodynamic therapy with curative intent for biopsy-proven carcinoma in situ or squamous cell carcinomas, which satisfied the criteria of centrally located early-stage lung cancer and followed without any other intervention, such as chemotherapy, radiation, or operation (group P). We confirmed by endobronchial ultrasonography that the depth of cancer invasion did not extend to the cartilage in all group P cases (4). Among the remaining cases who had a centrally located early-stage lung cancers, three patients underwent radiation or chemotherapy after photodynamic therapy (group R) and nine patients underwent surgery (group O), since the cancer invaded beyond the cartilage, and were followed for >2 years.

Before photodynamic therapy or surgery, at 3-month intervals during the first year after the therapy, at 6-month intervals in the second year, and once a year thereafter, bronchoscopy, including autofluorescence bronchoscopy, was repeated. Biopsy specimens were taken from all areas suspicious for dysplasia or worse on bronchoscopic examination in addition to a random biopsy. Routine H&E staining was carried out for all specimens, and the preserved formalin-fixed, paraffin-embedded specimens available for this study were stained by immunohistochemistry using a polyclonal antibody against human telomerase reverse transcriptase (hTERT; EST21A, Alpha Diagnostic International, San Antonio TX) as previously described (8). Telomerase protein expression was evaluated as positive when nuclear staining was confirmed by two molecular oncologists, in a blinded manner by one and confirmed by another.

Statistical analysis on the relationship between hTERT positivity in noncancerous epithelia and subsequent lung cancer development was carried out using Fisher's exact test. This study was approved by our institutional ethics committee.


   Result
Top
 Abstract
 Introduction
 Patients and Methods
 Result
Discussion
 References
 
In group P, 11 of 15 lesions treated by photodynamic therapy in nine patients achieved complete remission (CR) with a median follow-up term of 52 months after photodynamic therapy. In the remaining four lesions, either recurrence (case 1) or secondary lung cancer (cases 2-4) developed adjacent to the successfully treated primary cancer within 26 months (Table 1). The immunohistochemistry of noncancerous bronchial biopsy specimens in cases 1 to 4 showed positive telomerase protein expression in the nuclei of adjacent bronchial epithelia diagnosed as "normal" or atypical epithelia by H&E staining before cancer development (Fig. 1F and Fig. 2A), and greater parts of the subsequently developed cancers were telomerase positive (Fig. 1I). However, all of the remaining 11 lesions repeatedly examined before photodynamic therapy and during the follow-up term in nine patients that achieved CR after photodynamic therapy showed no telomerase protein expression in noncancerous epithelia (Fig. 2B; Table 2, in brackets; P < 0.001).


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  		Table 1. Histology of lung cancer treated with photodynamic therapy or surgery, immunohistochemical staining of hTERT protein in adjacent noncancerous bronchial epithelia, and prognosis of the patients

 


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  		Figure 1. Case 1. Lower trachea examined by autofluorescence bronchoscopy (A, D, and G) and trachea epithelia with H&E staining (B, E, and H) and with immunohistochemistry using hTERT antibody (C, F, and I). Carcinoma in situ and adjacent atypical lesion before photodynamic therapy (B and C) with a wide area of decreased green autofluorescence by autofluorescence bronchoscopy (A), stratified ciliated columnar cells without atypia 4 weeks after successful photodynamic therapy (E and F) with normal green autofluorescence (D), and carcinoma in situ as well as the superficial normal ciliated columnar cells 10 weeks after photodynamic therapy (H and I) with marked dark red findings by autofluorescence bronchoscopy (G) were all hTERT positive. Bar, 50 µm.

 


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  		Figure 2. Noncancerous bronchial epithelia at Rt. B6 orifice in case 2 (A, pathologically normal epithelia) who developed squamous cell carcinoma in this area 2 years after photodynamic therapy, normal epithelia in case 9 (B) who achieved CR for >7 years, and squamous metaplasia (C) and germinal center of a lymph node (D) in the same section with C in case 24 who achieved CR for 4 years. Most of the bronchial epithelial cells in A and some possibly activated lymphocytes, as internal controls of the staining, in D revealed to be hTERT positive by immunohistochemistry, but all bronchial epithelia in B and C were negative. Bar, 50 µm.

 

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  		Table 2. Relationship between hTERT expression in noncancerous epithelia and outcomes in 23 patients with early-stage lung cancer whose follow-up terms are >2 y after judgment of hTERT expression

 
To investigate this relationship further, we additionally examined 11 patients who underwent radiation or chemotherapy after photodynamic therapy (group R) or operation (group O) and were followed for >2 years. Also in group R, case 13, whose adjacent noncancerous epithelia was positive for hTERT expression, developed secondary cancer after photodynamic therapy and radiation therapy, whereas case 14 without hTERT expression in noncancerous epithelia did not. In group O, case 16 developed the first lung cancer 4 months after the biopsy of normal epithelia with positive hTERT protein. In the remaining eight cases, we examined hTERT protein expression in noncancerous epithelia at surgical margins in the resected tissues, so that the hTERT protein expression status at the margins of noncancerous epithelia remained in patients after surgery could be inferred. The case 17 with positive hTERT expression in noncancerous epithelia developed secondary squamous cell carcinoma at this region, whereas the remaining seven cases without hTERT expression (Fig. 2C) remain disease free until now. Including these groups R and O, the overall relationship between hTERT positivity in noncancerous epithelia and subsequent lung cancer development was summarized in Table 2 and revealed to be highly significant (n = 26, P < 0.0001) by Fisher's exact test.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Result
 Discussion
References
 
In the present study, we evaluated the expression of hTERT protein in the bronchial epithelia in situ. The hTERT protein, a catalytic reverse transcriptase, and a 451-bp template RNA (human telomerase RNA) are essential components of telomerase that endow human cells with immortalization (9, 10). Among these components, human telomerase RNA is known to be constitutively expressed even in cells without telomerase activity (10). Then, the detection of hTERT mRNA has been used as an indicator of positive telomerase activity, including in bronchial epithelia (11–14). However, it was proved that hTERT mRNA is often alternatively spliced, prohibiting the expression of hTERT protein in a dominant-negative manner (15). Moreover, detection of telomerase activity in clinical materials without in situ analysis may cause a false positive due to the infiltrated lymphocytes (16). Thus, detection of human telomerase RNA overexpression (17), hTERT mRNA expression (12–14), or telomerase activity without in situ evaluation (13, 17, 18) in noncancerous bronchial epithelia or lung tissues in smokers might have represented the expression of an alternatively spliced dominant-negative form of hTERT mRNA or lymphocyte inflammation rather than substantial telomerase expression in epithelia in some cases.

Telomerase activation in solid tumors has been considered to occur after many cell divisions through several clonal selections (7). However, cases 1 to 4, 13, 16, and 17, all heavy smokers, showed expression of telomerase protein in noncancerous epithelia as a field before the development of cancer. One possible explanation for this finding may be "field immortalization" in the bronchial epithelia in heavy smokers, in a manner similar to "field cancerization" as a step of carcinogenesis. The fact that majority of cancer cells in subsequently developed cancers showed positive telomerase protein even at an early stage, whereas it is uncommon in non–small cell lung cancers (7), supports our hypothesis that the second cancers have developed from the telomerase-positive epithelia. Recently, it was proved in vitro that once telomerase is activated, human cells are susceptible to transformation (19), and human bronchial epithelial cells can be immortalized by activation of telomerase with cyclin-dependent kinase 4 in the absence of viral oncoproteins (20). Considering these, telomerase-activated bronchial epithelia possibly through a field immortalization phenomenon might be susceptible to developing lung cancer in vivo.

Especially in group P or R cases, photodynamic therapy is considered to provide selective tumor necrosis. It may be natural that photodynamic therapy does not destroy morphologically normal but telomerase-positive epithelia. Consequently, such epithelia may survive after photodynamic therapy and be predisposed to cancer development, whereas telomerase-positive transformed cells be destroyed. We propose that telomerase expression may precede transformation in cancer development and that detection of hTERT protein in noncancerous bronchial epithelia in situ will become a useful indicator of a high-risk of developing lung cancer.


   Acknowledgments
 
Grant support: Grants-in-aid for Scientific Research A#13307050 and A#15209058 and Ministry of Education, Culture, Science, Sports and Technology of Japan grant-in-aid for Development of Highly Advanced Medical Technology B#K-500 (E. Hiyama).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Prof. M. Ohtaki (Department of Environmetrics and Biometrics) for statistical evaluation and I. Fukuba (Department of Surgery, Graduate School of Biomedical Sciences, Hiroshima University) for her excellent technical support.


   Footnotes
 
Note: Presented in part at the 13th World Congress for Bronchology, June 20-23, 2004, Barcelona, Spain.

Received 3/23/05. Revised 9/ 4/05. Accepted 9/16/05.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Result
 Discussion
 References
 

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  5. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–5.[Abstract/Free Full Text]
  6. Hiyama E, Hiyama K. Clinical utility of telomerase in cancer. Oncogene 2002;21:643–9.[CrossRef][Medline]
  7. Hiyama K, Hiyama E, Ishioka S, et al. Telomerase activity in small-cell and non-small-cell lung cancers. J Natl Cancer Inst 1995;87:895–902.[Abstract/Free Full Text]
  8. Hiyama E, Hiyama K, Yokoyama T, Shay JW. Immunohistochemical detection of telomerase (hTERT) protein in human cancer tissues and a subset of cells in normal tissues. Neoplasia 2001;3:17–26.[CrossRef][Medline]
  9. Bodnar AG, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998;279:349–52.[Abstract/Free Full Text]
  10. Weinrich SL, Pruzan R, Ma L, et al. Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 1997;17:498–502.[CrossRef][Medline]
  11. Kolquist KA, Ellisen LW, Counter CM, et al. Expression of TERT in early premalignant lesions and a subset of cells in normal tissues. Nat Genet 1998;19:182–6.[CrossRef][Medline]
  12. Soria JC, Moon C, Wang L, et al. Effects of N-(4-hydroxyphenyl)retinamide on hTERT expression in the bronchial epithelium of cigarette smokers. J Natl Cancer Inst 2001;93:1257–63.[Abstract/Free Full Text]
  13. 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.[CrossRef][Medline]
  14. Soria JC, Xu X, Liu DD, et al. Retinoic acid receptor ß and telomerase catalytic subunit expression in bronchial epithelium of heavy smokers. J Natl Cancer Inst 2003;95:165–8.[Abstract/Free Full Text]
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  16. Hiyama K, Hirai Y, Kyoizumi S, et al. Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immunol 1995;155:3711–5.[Abstract]
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