Radiol Oncol 2003; 37(2): 109-13. Telomerase in lung cancer diagnostics Elizabeta Kovkarova1, Tome Stefanovski1, Aleksandar Dimov2, John Naumovski3 1Pulmology and Allergology Clinic, Clinical Center Skopje, 2Macedonian Academy of Science, 3Urgent Internal Medicine Clinic, Skopje, Macedonia Background. Telomerase is a ribonucleoprotein that looks after the telomeric cap of the linear chromosomes maintaining its length. It is over expressed in tumour tissues, but not in normal somatic cells. Therefore the aim of this study was to determine the telomerase activity in lung cancer patients as novel marker for lung cancer detection evaluating the influence of tissue/cell obtaining technique. Material and methods. Using the TRAP (telomeric repeat amplification protocol), telomerase activity was determined in material obtained from bronchobiopsy (60 lung cancer patients compared with 20 controls) and washings from transthoracic fine needle aspiration biopsy performed in 10 patients with peripheral lung tumours. Results. Telomerase activity was detected in 75% of the lung cancer bronchobyopsies, and in 100% in transthoracic needle washings. Conclusions. Measurement of telomerase activity can contribute in fulfilling the diagnosis of lung masses and nodules suspected for lung cancer. Key words: lung neoplasms - diagnosis; telomerase; bronchoscopy Introduction Lung malignancies represent a repeated problem in the respiratory clinics constantly arousing attention due to the rapid evolution, extremely bad prognosis and frightening number of new patients every following year. Lung cancer is the most frequent pathology among all malignancies with more than Received 22 April 2003 Accepted 2 May 2003 Correspondence to: Elizabeta Kovkarova, M.D., Pulmology and Allergology Clinic Skopje, Vodnjanska 17, 1000 Skopje, Macedonia; Phone: +389 2 113 302; Fax: +389 2 237058; E-mail: naumovskie@yahoo.com 1000 000/year, and the main cause of death in 29% in this pathology. The distribution shows dominance in the developing countries with 61%, mainly due to the early presence of smoking habits.1,2 The 5-year survival is the lowest one, compared to the most frequent cancers: (colon 63%, breast 83%, prostate can-cer 93%).2,3 It remains alarmatic low: from 8% in 1960 to 14% 2001. On the other hand there is a dramatic difference in the 5-year survival, related to the stadium of the disease. The lo-calized form of the disease shows survival up to 40% (IA=67% IB=57%, IIA=55%, IIB=39%, II-IA=23%), compared to the extensive form of the disease -only 14%. The emergency need to deal with this highly fatal disease pointed out 110 Kovkarova E et al. / Telomerase and lung cancer that the main interest in this field has to be: developing and using methods for early de-tection of lung cancer such as sputum cytol-ogy, native chest radiogram, spiral chest CT, fluorescence bronchoscopy and molecular markers for malignancy.2,3 One of the most important factors in es-tablishing the cellular integrity are the telom-eres, specialized structures of the chromosomal end in all eucariotic cells build of repeti-tive short DNA sequences (TTAGGG) and associated combining proteins. They serve as puffer zones against the chromosomal spend-ing in the aging process and protectors in the degradation and recombinant process during the chromosomal junctions in the mitosis. The shortening of the telomere is the signal for stopping the cell division.4-6 The enzyme telomerase is a ribonucleic protein with func-tion of resyntetising the telomeric DNA of the chromosomal ends. It maintains the telomer-ic length giving the cell the opportunity for uncontrolled cellular division. Its quantity is carefully regulated, but genetic mutation and DNA damage can cause its activation or de-activation. The main characteristics of the tu-mour growth are avoidance of normal prolif-erative control so the renewal of telomeric re-peats by activating the enzyme telomerase may be a path for the tumour cells to avoid senescence and death. This enzyme is nor-mally detected only in reproductive cells and cells with self renewal capacity (bone mar-row, lymphocytes, intestinal crypt cells, epidermal basal cells), but it is undetectable in normal somatic cell. The development of highly sensitive PCR-based commercial kit (TRAP) by Kim et al. in 1995 allows telom-erase detection in various biosamples.7 The analysis of the telomerase activity in lung tumours was evaluated predominantly in surgical specimens (frozen samples of proven tumour tissues), after the diagnosis of lung cancer was already established. Therefore this study was designed to evaluate the role of telomerase in lung cancer diagnos-Radiol Oncol 2003; 37(2): 109-13. tics6,8 in fresh specimens obtained by routine lung cancer diagnostic sampling: bronchosco-biopsy and transthoracic fine needle aspiration biopsy. Material and methods The study involved 60 patients with central lung tumour, and 20 pts with pneumonia as a control group. All of the pts underwent bron-choscopy and in cases of endoscopic lesion, bronchobiopsy (1 mm) was obtained for TRAP analysis. The same was performed in the control group taking 1mm sample from bronchial mucosa. Another 10 patients with peripheral lung lesion were included, and transtoracic fine needle aspiration biopsy (FNAB) was performed. The mean smear was sent to citopathology lab and the needle washings were analysed for telomerase. Analysis of telomerase was performed in total of 80 samples of bronchial mucosa, 10 sam-ples of FNAB. Telomerase assay Telomerase activity was qualitatively evaluat-ed using the TRAP (Telomerase Repeat Amplification Protocol) of Boehringer Mannheim. Practically it is a four step process: Telomerase, if present, adds multiple 6-nucleotide telomeric repeats to a bi-otinylated synthetic primer. Then the telom-erase reaction product is amplified by PCR, using a biotinylated primer. Denaturation fol-lows and the PCR product hybridizes to a digoxigenin-labeled probe specific for the telomeric repeat. The last step is binding of the DNA hybrid to a streptavidin-coated mi-crotiter plate, and anti-digoxigenin-peroxi-dase so TMB substrate generates a coloured product measurable with a microplate read-er.9-11 Statistical analysis of the data included method of clinical test evaluation (Bayesian analysis). Kovkarova E et al. / Telomerase and lung cancer 111 Results Telomerase positively was detected in 45 out of 60 (75%) of the positive lung cancer biop-sies, compared with the controls of normal mucosa 2/20 (10%). Statistical analysis for telomerase in lung cancer biopsies showed accuracy of 80% (Ac), sensitivity of 75% (Son), specificity of 90% (Sp) positive predictive val-ue of 95.7% (PPV) and negative predictive val-ue of 40.4% (NPV). Histology analysis showed predominance of telomerase positivity in small cell lung can-cer (SCLC), and the lowest activity in metastatic deposits (Table 1). Telomerase ac-tivity was analyzed in needle washings after FNAB was performed in patients with pe-ripheral lung tumour. Telomerase was detect-ed in all 10 (100%) samples, compared to cy-tology where malignancy was confirmed in 8 out of 10 (80%) samples. Discussion This study design was performed in order to find out if the novel molecular marker of ma-lignancy telomerase can be used in diagnosis of lung cancer. This question revealed three new points: how can we use telomerase, in what way and what type of samples in lung cancer diagnostics. The routine lung cancer investigation usually starts with bron-Table 1. Histology of lung cancer and telomerase activity choscopy as a basic method for obtaining samples for histopathology. On the other hand up to 90% of the endobronhial lesions are confirmed by histopathological analysis of bronchobiopsy material, but sometimes we have to repeat the procedure in order to ob-tain sufficient sample for diagnosis. In our study telomerase positivity was detected in 75% in lung cancer bronchobiopsies com-pared to non-malignant tissue (10%). Statistical analysis showed highly significant value of c2=93.25 and p<0.0001 that proved this method to be significant in separating ma-lignant of non-malignant tissue. Several au-thors such as Hiyama et al.199512, Yang et al. 199813, Lee et al.9 1998 Shay et al.199914-16 etc reported very high telomerase activity in lung cancer tissue 75-85%.17 These results, includ-ing those from our study impose the conclu-sion that telomerase is one of the leading fac-tors in development of lung cancer. Most of the telomerase studies on lung cancer were conducted on material obtained by surgical re-section or lung cancer cell lines. Statistical analysis pointed out that telomerase activity is slightly higher in such material (increasing the Sn, Sp, Ac), but the PPV is the same (Table 2). This data suggests that in the clinical practice in cases where modest endobronchial changes lack histopathological conformation, and the telomerase activity is detected, the diagnostic procedure should be persistent and directed in establishing the malignant disease. Type (Total 60) Samples Telomerase/+/ Sn=TP/TP+FN NSCLC SCLS MS deposites 34 15 11 26 12 7 76.5% 80% 63.6% 76.5% 80% 63.6% Table 2. Telomerase activity in lung cancer: surgical sampling increases the Sn, Sp, and Ac of the method com-pared with bronchobiopsy sampling Sn Sp Ac PPV NPV Hiyama et al./199812 Sen et al./199918 Kovkarova et al./2002 80% 87% 75% 94% 100% 90% 96% 88% 81% 96% 96% 96% 89% 84% 40% Radiol Oncol 2003; 37(2): 109-13. 112 Kovkarova E et al. / Telomerase and lung cancer In the analysis of peripheral lung lesions, transthoracic fine needle aspiration biopsy is a basic diagnostic method for tissue sam-pling. Telomerase activity was determined in needle washing after the preparation of material for cytology analysis. Telomerase positiv-ity was found in all of the samples (10/10), compared to cytology were due to massive necrosis malignancy was confirmed in 8 out of 10 samples. Sen at al.18 and Naritoku et al.11 established telomerase sensitivity higher then the cytology (Table 3). Naritoku stresses the value of telomerase debating that this mo-lecular marker finally determines the blurre cytological report of rare atypical cells that of-ten confuses the pulmonologist.12,18 Second objective to the validation of the telomerase activity was to establish any con-nection with the histology type of the lung cancer (Table 1). This analysis showed no link to telomerase positivity in primary lung can-cers, but the lowest rate of telomerase activi-ty was detected in the metastatic type. Some authors like Strovel19 and Allbanell20 the telomerase activity can be associated with the tumour proliferation rate, response to therapy and final outcome. Quantification of these markers according to these authors can be used as valid prognostic marker of lung cancer. These results suggest that telomerase can be used as a complementary tool in lung can-cer diagnostics especially in cases where the first line diagnosis is confuse and unprecise. The studies that evaluate the telomerase ac-tivity in more simple sampling such as spu-tum or plasma can open a new field in early lung cancer detection especially in high risk population. On the other hand telomerase Table 3. Telomerase activity in FNAB washings of lung cancer: superior to cytology TTAP Telomerase/+/ Cytology Sen et al./199918 35/42 84% 68.4% Naritoku et al./199911 14/16 88% 68% Kovkarova et al./2002 10/10 100% 80% Radiol Oncol 2003; 37(2): 109-13. can be used as a valid target for lung cancer treatment. 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