Lung Cancer (2008) 61, 97—103

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate / lungcan

Telomerase activity in transthoracic fine-needlebiopsy aspirates from non-small cell lung canceras prognostic factor of patients’ survival

Tomasz Targowskia,∗, Karina Jahnz-Rozyka, Tomasz Szkodab,Tadeusz Płusaa, Sławomir Froma

a Department of Internal Medicine, Pneumonology and Allergology, Military Institute of Health Service, Warsaw, Polandb Department of Virology, National Institute of Hygiene, Warsaw, Poland

Received 9 October 2007; accepted 11 October 2007

KEYWORDSTelomerase;NSCLC;Prognostic factors;Lung cancer;Lung malignanttumours;Fine-needle biopsy

SummaryPurpose: Evaluation of the relationship between telomerase activity in transthoracic fine-needlebiopsy (TFNB) aspirates collected from peripheral non-small cell lung cancer (NSCLC), canceradvancement, risk of death and free of cancer recurrence survival.Material and methods: The study group consisted of 93 patients with peripheral infiltrationof the lung. All of them had TFNB of the focal pulmonary lesion performed. The aspirateswere subjected to standard cytological evaluation. Telomerase activity in the specimens wasdetermined with the PCR—ELISA PLUS method. Cancer advancement, manner of treatment andsurvival were assessed in patients with NSCLC.Results: A benign lesion of the lung was recognised in 14 cases. NSCLC was newly diagnosedin 79 subjects. Nobody with benign infiltration had a detectable level of telomerase in lunginfiltration. Increased telomerase activity was observed in 56 (70.9%) patients with lung cancer.It was significantly more often detected in patients with non-operable NSCLC (clinical stage IIIBplus IV) and patients with distant metastases (stage IV alone). Cox hazard analysis revealedthat presence of telomerase activity in primary NSCLC is an independent prognostic factor ofsurvival. Increased telomerase activity in TFNB aspirates was related to 7 times higher relative

risk of death during the study [RR = 6.9 (CI: 1.8—26.8); p < 0.05] and 2.5 times higher risk of

icalivity

cancer recurrence after radConclusion: Telomerase act

could be a helpful independentcancer recurrence.© 2007 Elsevier Ireland Ltd. All

∗ Corresponding author at: Military Institute of Health Service, DepartmSzaserow Street 126, 00-909 Warsaw, Poland. Tel.: +48 22 6818537; fax:

E-mail address: targowski.tomasz xl@wp.pl (T. Targowski).

0169-5002/$ — see front matter © 2007 Elsevier Ireland Ltd. All rights redoi:10.1016/j.lungcan.2007.10.017

treatment [RR = 2.5 (CI: 0.3—9.3); p < 0.05].in aspirates collected through TFNB of primary peripheral NSCLC

prognostic factor of distant metastases and risk of death or

rights reserved.

ent of Internal Medicine, Pneumonology and Allergology,+48 22 6818537.

served.

9

oncotapo6daitniafmaciTrts

1

Nin

leraffsasTi−Moil2ocAOatwa(sl

wiawtrwcdgsupw(

ttuwac

1

TrtawttKhrcs1(sppndvbasaftispn

8

Lung cancer is the most common malignant tumour allver the world and is the main cause of deaths related toeoplastic diseases. More than 80% of cases are non-smallell lung cancer (NSCLC). Due to a nearly asymptomaticnset of the disease and quite frequent distant dissemina-ion of locally non-advanced NSCLC, results of its treatmentre highly unsatisfactory. It is estimated that 10—15% ofatients with NSCLC achieve 5-year survival [1]. Even in casef non-advanced operable NSCLC (stage I) only from 38% to7% of patients survive 5 years after treatment [1]. NSCLCevelops in most cases peripherally in the lung parenchyma,nd for that reason transthoracic fine-needle biopsy (TFNB)s one of basic methods of cytological diagnosis of suchumours. Unfortunately aspirates collected through fine-eedle biopsy contain too few cells to provide sufficientnformation on cancer malignancy. The determination of thectivity of telomerase, a polymerase enzyme characteristicor the majority of immortalised cells of malignant tumours,ay be a helpful prognostic marker of NSCLC advancement

nd patients’ survival [2—4]. Up till now, no study has beenarried out dealing with the assessment of telomerase activ-ty in aspirates obtained from focal lesions in lungs throughFNB. The aim of the study performed was to assess theelationship between telomerase activity in TFNB aspiratesaken from peripheral NSCLC, and cancer advancement andurvival of patients.

. Materials and methods

inety-three patients with suspicious peripheral lungnfiltrations, who had been qualified for transthoracic fine-eedle biopsy (TFNB), were enrolled to the study.

Transthoracic fine-needle aspiration biopsy of the focalesion was done (Becton Dickinson needle 0.7 mm in diam-ter) under fluoroscopy control in each patient. Afteradiologically proven placement of the needle in the tumour,n aspirate was taken and divided in two equal specimensor cytological and molecular examinations. The aspirateor the cytological examination was smeared on defattedlides fixed in 95% ethyl alcohol, and stained with eosinnd haematoxylin. The cytological examination of eachample was carried out by two independent pathologists.he aspirates for the telomerase activity assessment were

mmediately placed in 1 ml probes and deeply frozen (at70 ◦C). PCR—ELISA PLUS (ROCHE Molecular Biochemicals,annheim, Germany) method was used for the assessmentf telomerase activity, as described below [5]. After defrost-ng, the aspirates were homogenised in 200 �l ice-cooledysis reagent. The lysate was centrifuged at 16,000 × g for0 min at 4 ◦C. One hundred and seventy five microlittresf the supernatant was gently removed and its proteinoncentration was measured with the Bio Rad Proteinssay Kit. Each supernatant was divided into two aliquots.ne, inactivated at 85 ◦C for 10 min, was used as a neg-tive control, while the other one was used to evaluateelomerase activity. For each assay 3 �g of protein extract

as used. The telomerase activity assessment was doneccording to the Telomeric Repeat Amplification protocolTRAP) method, consisting in amplification of telomericequences added by telomerase to the 3′ end of biotin-abelled synthetic primer. These elongation products, as

1

T2C

T. Targowski et al.

ell as the internal standard (IS), which constitutes a pos-tive control, included in the same reaction vessel, weremplified using the appropriate primers. The PCR productsere split into two aliquots, denaturated and hybridised

o digoxigenin-labelled probes, specific for the telomericepeats and for the IS, respectively. The resulting productsere immobilised, via the biotin label to a streptavidin-oated microtitre plate. Immobilised amplicons were thenetected with an anti-digoxigenin antibody that is conju-ated to horseradish peroxidase and the sensitive peroxidaseubstrate. The absorbance of the samples was measuredsing an ELISA reader with 450 nm of wavelength. The sam-le was considered as telomerase positive if its absorbanceas higher than the absorbance of heat-treated aspirate

negative control) [5].Tumour size, lymph node invasion and distant metas-

ases were determined in all cancer subjects according tohe World Health Organization criteria [6]. In patients whonderwent surgery, the final stage of cancer advancementas determined by a combination of pathomorphologicalnd clinical findings according to the current TNM classifi-ation for lung cancer staging [1].

.1. Statistical analysis

elomerase activity in TFNB specimens was compared inelation to the clinical advancement (i.e. TNM classification,umour size, lymph nodes infiltration, distant metastases,nd surgical treatment) of NSCLC. The chi-square testas used for the evaluation of significant differences in

elomerase activity. The survival of patients in respect toelomerase activity in TFNB aspirates was analysed by theaplan—Meier method. Patients with the status which couldave a significant influence on survival, i.e.: significantespiratory or blood circulation failure, haemoglobin con-entration less than 11 g/dl, hypoalbuminemia (<3 g/dl),erum lactate dehydrogenase concentration higher than50 U/l, platelet count higher than 600 g/l, hypercalcemia>2.75 mmol/l) or bad general status (≥3 points of Zubrod’scale) were excluded [7—12]. Patients with malignant neo-lastic diseases during the previous 5 years, as well asatients with NSCLC recognised before TFNB were alsoot included. Deaths because of cancer or its recurrenceuring the study period were qualified as complete obser-ation and they were analysed separately. The relationshipetween age, sex, telomerase activity in primary tumournd clinical advancement of NSCLC, on the one hand, andurvival, on the other, was assessed by the Cox hazardnalysis. Relative risk coefficients of overall survival andree-of-recurrence survival were calculated in relation toelomerase activity in TFNB specimens. The 95% confidencenterval (CI) was set, and p value ≤0.05 was considered astatistically significant. All of the statistical analyses wereerformed with the STATISTICA PL 7.0 programme (serialo. AAAP510C860213FA).

.2. Ethics committee

he study was conducted from February 2004 to January006. The study protocol was approved by the Ethicsommittee of the Military Medical Chamber (no. 59/2002).

(conawf

doawdol3ciImhn

Telomerase activity in as prognostic factor

The study was financially supported by the Polish Ministryof Science.

2. Results

Benign lung lesions were finally recognised in 14 out of 93patients (9 cases of non-specific inflammatory infiltrationand 5 cases of tuberculous tumours). The analysed groupcomprised 79 patients with newly diagnosed non-small celllung cancer, and included 20 patients (25.3%) with stage INSCLC, 15 (19.0%) with stage II, 22 (27.85%) with stage III,and 22 (27.85%) with stage IV. Cytological diagnosis of theperipheral lung tumour was established on the basis of theresult of TFNB in 62 (78.5%) patients in 52 (65.8%) during thefirst TFNB, and in the remaining 10 during subsequent aspira-tions. In 15 out of 17 patients with negative results of TFNB,histological diagnosis of lung tumour was done after openlung biopsy and in 2 out of 17 after another procedure (1—–resection of brain metastases, 1—–bronchial brushing duringbronchofibroscopy).

Elevated telomerase activity was found in 56 (70.9%)

aspirates obtained during the first TFNB (including 18 caseswithout cancer cells in specimens from the first TFNB).Telomerase activity was found in 32 (82.0%) of 39 histo-logically unspecified non-small cell cancers, 9 (56.3%) of 16squamous cell cancers, 8 (57.1%) of 14 adenocarcinomas, 3

(e(

t

Table 1 Telomerase activity in aspirates from primary tumours in

Number ofpatients

Telomerase activ

Telomerase posit

StageI 20 10 (50)II 15 8 (53.3)

22 14 (63.6)III 22 22 (100)

IV

T classificationT1 11 5 (45.5)T2 41 28 (68.3)T3 14 10 (71.4)T4 13 11 (84.6)

N classificationN0 35 24 (68.6)N1 19 12 (63.2)N2 13 8 (61.5)N3 12 10 (83.3)

M classificationM0 57 32 (56.1)M1 22 22 (100)

(1) Operable (stage I-IIIA) 44 22 (50)(2) Non-operable (stage IIIB-IV) 35 32 (91.4)

*Significant p value ≤ 0.05

99

60%) of 5 broncho-alveolar cancers and 3 (75%) of 4 largeell cancers. Telomerase activity was also found in one casef mixed lung cancer. In 9 TFNB aspirates neither cancer cellsor telomerase activity were found. Results of telomerasectivity assessment in relation to clinical data of patientsith NSCLC are set in Table 1. Telomerase activity was not

ound in any of 14 cases of benign nodules.Increased telomerase activity in TFNB specimens was

etected significantly more often in patients with non-perable cancer (stage IIIB and IV) than in subjects with lessdvanced stages (p = 0.0001) (Table 1). Among 22 patientsith stage IV NSCLC, 12 patients had metastases to oneistant organ, 9 subjects to two organs and 1 to threergans. Eleven subjects had distant metastases to the otherobe of the lung, 7 to brain, 5 to bones, 4 to liver, and

to pleura. Suprarenal glands invasion was observed in 2ases and in 1 case skin was involved. Telomerase activ-ty was found in primary tumours of all patients with stageV of NSCLC and only in 56.1% cancers without distantetastases (p = 0.00001). Moreover patients with T4 feature

ad detectable telomerase activity in primary tumour sig-ificantly more frequently than patients with T1 feature

p = 0.05). There was not any relationship between telom-rase activity and infiltration of lymph nodules by cancerTable 1).

All the patients were treated by oncologists accordingo the evidence-based medicine rules. Out of 44 patients

relation to advancement of NSCLC

ity p chi-square test

ive n (%) Telomerase negative n (%)

10 (50) I vs. II = 0.67 (46.7) I vs. III = 0.38 (36.3) I vs. IV = 0.0001*

0 (0) II vs. III = 0.4II vs. IV = 0.0006*

III vs. IV = 0.002*

T1 vs. T2 = 0.16 (54.5) T1 vs. T3 = 0.4

13 (31.7) T1 vs. T4 = 0.05*

4 (28.6) T2 vs. T3 = 0.52 (15.4) T2 vs. T4 = 0.2

T3 vs. T4 = 0.4

N0 vs. N1 = 0.511 (31.4) N0 vs. N2 = 0.47 (36.8) N0 vs. N3 = 0.35 (38.5) N1 vs. N2 = 0.62 (16.7) N1 vs. N3 = 0.2

N2 vs. N3 = 0.2

25 (43.9) M0 vs. M1 = 0.00001*

0 (0)

22 (50) 1 vs. 2 = 0. 0001*

3 (8.6)

100 T. Targowski et al.

Fig. 1 Relationship between probability of overall survival and telomerase activity in whole NSCLC group (n = 79) during observationperiod.

Fig. 2 Relationship between probability of free-of-recurrence survival and telomerase activity in radically treated NSCLC group(n = 36) during observation period.

Telomerase activity in as prognostic factor 101

Table 2 Overall survival of patients with NSCLC and telomerase activity in TFNB aspirates

Group Number of patients Observation period

Overall survival,mean (S.D.) (weeks)

Number of completeobservations (deaths), n (%)

All participants 79 47.0 (25.5) 32 out of 79 (40.5%)Telomerase positive 56 41.2 (24.3) 30 out of 56 (53.6%)Telomerase negative 23 61.3 (23.0) 2 out of 23 (8.7%)

Surgically treated 35 59.8 (21.0) 6 out of 35 (17.1%)Telomerase positive 18 55.9 (20.1) 6 out of 18 (33.3%)Telomerase negative 17 64.1 (21.7) 0 out of 17 (0.0%)

Non-surgically treated 44 36.9 (24.4) 26 out of 44 (59.0%)Telomerase positive 38 34.2 (23.2) 24 out of 38 (63.2%)Telomerase negative 6 53.6 (30.0) 2 out of 6 (33.3%)

Table 3 Free-of-recurrence survival of patients with NSCLC after radical treatment and telomerase activity in TFNB aspirates

Group Number of patients Mean time of survival during observation period

Free-of-recurrencesurvival, mean (S.D.)(weeks)

Number of recurrences, n (%)

Radically treated 36 61.3 (25.0) 7 out of 36 (19.4%)Telomerase positive 20 51.9 (25.9) 6 out of 20 (30%)Telomerase negative 16 61.3 (25.0) 1 out of 16 (6.2%)

Table 4 Cox hazard analysis of influence of selected factors on overall and free-of-recurrence survival in patients with NSCLC

Overall survival p Free-of-recurrence survival p

Whole group (n = 79) Non-radically treated group (n = 43) Radically treated group (n = 36)

Telomerase activity 0.02* 0.03* 0.04*

Age 0.5 0.3 0.8Sex 0.1 0.1 0.07Clinical stage 0.7 0.5 0.6T 0.001* 0.06 0.9N 0.9 0.3 0.6

d(aiN(

3

T

M 0.1 0.6

*Significant p value ≤ 0.05

with potentially operable NSCLC (stage ≤IIIA), 35 were sur-gically treated. Thirty-three patients were treated onlywith chemotherapy and/or radiotherapy. The best support-ive therapy was conducted in the remaining 11 patients.Treatment was radical in 36 patients (35 after surgi-cal treatment and 1 after chemoradiotherapy). Thirty-two(40.5%) participants (including 30 telomerase positive) diedduring the study (Table 2). Increased telomerase activ-ity in TFNB aspirates was related to 7 times higher riskof death during the observational period [RR = 6.9 (CI:1.8—26.8); p < 0.05] (Fig. 1). Among 35 patients, who had

been qualified for surgery, 6 (17.1%) died because of can-cer progression, all with increased telomerase activity inTFNB aspirates (Table 2). NSCLC recurrence after radicaltreatment appeared only in 1 (6.2%) case without increasedtelomerase activity in comparison to 6 (20%) cases with a

psct[

—

etectable telomerase level [relative risk factor—–RR = 2.5CI: 0.3—9.3); p < 0.05] (Table 3, Fig. 2). The Cox regressionnalysis revealed that telomerase activity in TFNB aspiratess a significant and independent of clinical advancement ofSCLC prognostic factor of death and cancer recurrenceTable 4).

. Discussion

elomerase is a specific DNA polymerase consisting of a

rotein component, comprising telomerase reverse tran-criptase and telomerase-associated protein 1, and an RNAomponent, which is a template for the elongation ofelomeric repeat sequences located at the ends of DNA13,14].

1

aclibflfuoio

aa[scutcdahrpaaao[hssrlrscTcNlaqwtTicasfprmonndi

iHttt

isrsaeAowTeoa(wtaeon(iNd

C

N

R

02

In healthy mature organisms, very weak telomerasectivity was described in fast-dividing cells, such as germells, epithelial cells, and lymphocytes or activated fibrob-asts [14—16]. Sporadic cases of high-telomerase expressionn a fatal course of pneumonia and cystic fibrosis have alsoeen reported [17]. In our study telomerase activity was notound in any of 14 cases of benign peripheral lesions of theung. High-telomerase activity is characteristic first of allor cells of malignant tumours and is responsible for theirncontrolled proliferation [14]. In the last decade numer-us studies have shown highly increased telomerase activityn more than 80% of different carcinomas and no expressionr very low expression in normal tissues [3,18,14,19,20].

In patients with lung carcinoma, high levels of telomerasectivity have been observed in sputum, bronchial washingnd brushing specimens obtained during bronchofibroscopy2,21]. It is thought that telomerase as an enzyme respon-ible for unlimited replication of genetic material insideancer cells and not finishing cells division could be a molec-lar prognostic factor. It has been shown that a high level ofelomerase activity in surgically resected stage I non-smallell lung cancer is associated with significantly diminishedisease-free survival and overall survival [22—24]. Fujita etl. [22] observed that strong and moderate expression ofuman telomerase reverse transcriptase mRNA (hTERT) inesected NSCLC is a prognostic factor of survival with similarower to lymph nodes status, pathomorphological TNM stagend age of patients. Some authors did not find any associ-tion between clinicopathological features of lung cancernd hTERT status, however they observed longer survivalf patients without telomerase activity in primary tumours25]. So far, telomerase prognostic value in lung canceras been studied on specimens derived from less advancedurgically resected tumours [4,22,26,27]. This is the firsttudy demonstrating that telomerase activity in transtho-acic fine-needle biopsy aspirates from primary peripheralung cancer is related to NSCLC advancement, risk of cancerecurrence and risk of death. Telomerase activity in TFNBpecimens was found in 70.9% of NSCLCs, including only 50%ases of stage I TNM and as many as 100% cases of stage IV.elomerase activity was most often observed in histologi-ally unspecified non-small cell cancer (82.0%). UnspecifiedSCLC is first of all recognised in patients with advanced

ung cancer disqualified from surgery and diagnosed withless invasive technique, so telomerase activity more fre-

uently observed in the subgroup with unspecified NSCLCas rather the emanation of the high advancement of cancer

han real differences among histological subtypes of NSCLC.elomerase activity was significantly more often detectedn TFNB aspirates obtained from patients with non-operableancer (stages IIIB plus IV) and distant metastases (stage IVlone) than in those from patients with less advanced stages,o assay of telomerase activity may be a helpful methodor estimation of probability of NSCLC non-resectability androbability of distant metastases (Table 1). In particular, theelationship between increased telomerase activity in pri-ary tumour and presence of distant metastases could be

f importance for clinical practice. For example, in case ofon-small cell lung cancer computed tomography or mag-etic resonance of brain are not routinely recommendeduring staging of the disease, because of the low sensitiv-ty of these diagnostic methods in detection of metastases

T. Targowski et al.

n patients without evident neurological symptoms [28—30].igh-telomerase activity in primary tumour could be a fac-or suggesting dissemination of cancer to other organs andhe necessity of additional diagnostic procedures, howeverhis hypothesis needs further studies.

Apart from the relationship between telomerase activ-ty and NSCLC advancement, the fact that telomerase is atrong independent prognostic factor of death and cancerecurrence after radical treatment was revealed. During thetudy 40.5% of patients with increased telomerase activitynd only 8.7% of patients with no detectable level of telom-rase in TFNB aspirates died because of cancer (Table 2).mong radically treated patients, cancer recurred in 30%f telomerase positive patients and only in 6.2% of patientsithout detected activity of telomerase in primary tumour.he Cox regression analysis revealed that presence of telom-rase activity in primary tumour influenced shortening of theverall survival more significantly (p = 0.02) than patients’ge and sex, clinical stage of cancer, lymph node statusN) and presence of metastases to distant organs. In thehole analysed group, only large tumour size (T) according

o the TNM classification had a stronger impact on the over-ll survival (p = 0.001) than telomerase activity, however theffect of the T feature on the overall survival and free-f-recurrence survival was insignificant when radically andon-radically treated groups were considered separatelyTable 4). In summary, the assessment of telomerase activ-ty could supplement prognosis of survival in the course ofSCLC and may be a promising molecular marker of cancerissemination to other organs.

onflict of interest

one.

eferences

[1] Mountain CF. Revisions in the international system for staginglung cancer. Chest 1997;111:1710—7.

[2] Hirashima T, Yoshitaka O, Nitta T, Sasada S, Kobayashi M,Masuda N, et al. Telomerase activity in endoscopically visiblelung cancer. Anticancer Res 2001;21(5):3685—9.

[3] Hiyama K, Hiyama E, Ishioka S, Yamakido M, Inai K, Gazdar F,et al. Telomerase activity in small-cell and non-small-cell lungcancers. J Natl Cancer Inst 1995;87:895—902.

[4] Marchetti A, Bertacca G, Buttitta F, Chella A, Quattrocolo G,Angeletti CA, et al. Telomerase activity as a prognostic indi-cator in stage I non-small cell lung cancer. Clin Cancer Res1999;5:2077—81.

[5] Telo TAGGG Telomerase PCR ELISA PLUS. Photometric enzymeimmunoassay for quantitative determination of telomeraseactivity, utilizing the telomeric repeat amplification protocol(TRAP). Instruction Manual 1999. Manheim, Germany: RocheDiagnostics; 1999.

[6] Travis WD, Brambilla E, Muller-Hermelink. Pathology andgenetics of tumours of the lung, pleura, thymus and heart.In: WHO classification of tumours. Lyon: IARC Press; 2004. p.

1—25.

[7] Albain KS, Crowley JJ, LeBlanc M, Livingston RB. Survivaldeterminants in extensive-stage non-small-cell lung cancer:the Southwest Oncology Group experience. J Clin Oncol1991;9(9):1618—26.

[

[

[

[

[

[

[

[

[

[

Telomerase activity in as prognostic factor

[8] Espinosa E, Feliu J, Zamora P, Gonzalez Baron M, Sanchez JJ,Ordon ez A, et al. Serum albumin and other prognostic factorsrelated to response and survival in patients with advanced non-small cell lung cancer. Lung Cancer 1995;12(1/2):67—76.

[9] Paesmans M, Sculier JP, Libert P, Bureau G, DabouisG, Thiriaux J, et al. Prognostic factors for survivalin advanced non-small-cell lung cancer: univariate andmultivariate analyses including recursive partitioning andamalgamation algorithms in 1052 patients. The EuropeanLung Cancer Working Party. J Clin Oncol 1995;13(5):1221—30.

[10] Pedersen LM, Milman N. Prognostic significance of thrombo-cytosis in patients with primary lung cancer. Eur Respir J1996;9(9):1826—30.

[11] Roszkowski K. Nowotwory płuc. In: Rowinska-Zakrzewska, Kus,editors. Choroby układu oddechowego. Warsaw: PZWL; 2004.p. 563—97.

[12] Takigawa N, Segawa Y, Okahara M, Maeda Y, Takata I, KataokaM, et al. Prognostic factors for patients with advanced non-small cell lung cancer: univariate and multivariate analysesincluding recursive partitioning and amalgamation. Lung Can-cer 1996;15(1):67—77.

[13] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell2000;100:57—70.

[14] Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL,et al. Specific association of human telomerase activity withimmortal cells and cancer. Science 1994;266:2011—5.

[15] Harley CB, Futcher AB, Greider CW. Telomeres shorten duringaging of human fibroblast. Nature 1990;345:458—60.

[16] Shammas MA, Koley H, Beer DG, Li C, Goyal RK, Munshi NC.Growth arrest, apoptosis, and telomere shortening of Barrett’s-associated adenocarcinoma cells by a telomerase inhibitor.Gastroenterology 2004;126(5):1337—46.

[17] Hiyama E, Ishioka S, Shay JW, Taooka Y, Maeda A, Isobe T, etal. Telomerase activity as a novel marker of lung cancer andimmune-associated lung diseases. Int J Mol Med 1998;1:545—9.

[18] Hiyama E, Saeki T, Hiyama K, Takashima S, Shay JW, MatsuuraY, et al. Telomerase activity as a marker of breast carcinomain fine-needle aspirated samples. Cancer 2000;90(4):235—8.

[19] Langford LA, Piatyszek MA, Xu R, Schold Jr SC, Shay JW. Telom-erase activity in human brain tumors. Lancet 1995;346:1267—8.

[

103

20] Tahara H, Nakanishi T, Kitamoto M, Nakashio R, Shay JW, TaharaE, et al. Telomerase activity in human liver tissues: comparisonbetween chronic liver disease and hepatocellular carcinoma.Cancer Res 1995;55:2734—6.

21] Xinarianos G, Scott FM, Liloglou T, Prime W, TurnbullL, Walshaw M, et al. Evaluation of telomerase activ-ity in bronchial lavage as a potential diagnostic markerfor malignant lung disease. Lung Cancer 2000;(28):37—42.

22] Fujita Y, Fujikane T, Fujiuchi S, Nishigaki Y, Yamazaki Y, NagaseA, et al. The diagnostic and prognostic relevance of humantelomerase reverse transcriptase mRNA expression detectedin situ in patients with non-small cell lung carcinoma. Cancer2003;98(5):1008—13.

23] Strauss GM, Kwiatkowski DJ, Harpole DH, Lynch TJ, Skarin AT,Sugarbaker DJ. Molecular and pathologic markers in stage I non-small cell carcinoma of the lung. J Clin Oncol 1995;43:1269—79.

24] Taga S, Osaki T, Oghami A, Imoto H, Yasumoto K. Prognosticimpact of telomerase activity in non-small cell lung cancers.Ann Surg 1999;230(5):715—20.

25] Wu TC, Lin P, Hsu CP, Huang YJ, Chen CY, Chung WC, et al. Lossof telomerase activity may be a potential favourable prognosticmarker in lung carcinomas. Lung cancer 2003;41(2):163—9.

26] Chen KY, Lee LN, Yu CJ, Lee YC, Kuo SH, Yang PC. Elevationof telomerase activity positively correlates to poor progno-sis of patients with non-small cell lung cancer. Cancer Lett2005;21:1—9.

27] Zhu CQ, Cutz JC, Liu N, Lau D, Shepherd FA, Squire JA, et al.Amplification of telomerase gene is a poor prognostic markerin non-small-cell lung cancer. Br J Cancer 2006;94(10):1452—9.

28] Ceresoli GL, Reni M, Chiesa G, Carretta A, Schipani S, Passoni P,et al. Brain metastases in locally advanced nonsmall cell lungcarcinoma after multimodality treatment: risk factors analysis.Cancer 2002;95(3):605—12.

29] Kormas P, Bradshaw JR, Jeyasingham K. Preoperative computed

tomography of the brain in non small cell bronchogenic carci-noma. Thorax 1992;47:106—8.

30] Shields TW. Screening, staging, and diagnostic investigation ofnon-small cell lung cancer patients. Curr Opin Oncol 1991;3(2):297—305.