Full text of DOI:10.1097/01.LAB.0000017165.26718.60

0023-6837/02/8206-729$03.00/0
LABORATORY INVESTIGATION
Vol. 82, No. 6, p. 729, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Prediction of Survival in Stage I Lung Carcinoma
Patients by Telomerase Function Evaluation
Antonio Marchetti, Caterina Pellegrini, Fiamma Buttitta, Monica Falleni,
Solange Romagnoli, Lara Felicioni, Fabio Barassi, Simona Salvatore,
Antonio Chella, Carlo Alberto Angeletti, Massimo Roncalli, Guido Coggi, and
Silvano Bosari
Department of Oncology and Neurosciences (AM, FB, LF, FB, SS), University of Chieti, Chieti; Department of
Medicine, Surgery, and Dental Sciences (CP, MF, SR, GC, SB), University of Milan, A.O.S. Paolo and IRCCS
Ospedale Maggiore, Milan; Department of Surgery (AC, CAA), University of Pisa, Pisa; and Department of Medicine,
Surgery, and Dental Sciences (MR), University of Milan, Istituto Clinico Humanitas, Rozzano (Milan), Italy
SUMMARY: Telomerase activity and telomerase reverse transcriptase (hTERT) expression are elevated in human malignancies.
We have investigated telomerase activity measured by the telomeric repeat amplification protocol (TRAP) assay and hTERT levels
by real-time RT-PCR in stage I non-small-cell lung carcinomas. The purposes of our study included the comparison of these two
techniques in the assessment of telomerase function and the evaluation of their prognostic significance. Telomerase activity and
hTERT levels were determined in 90 stage I non-small-cell lung carcinoma patients, using TRAP assay and real-time RT-PCR,
2
respectively. Variables were analyzed by the ␹ and Fisher exact tests. Survival was analyzed by the Kaplan-Meier method.
Multivariate analysis was performed with the Cox’s proportional hazards model. Telomerase activity was elevated in 60 (67%)
carcinomas. hTERT was elevated in 43 (48%) carcinomas. Only 21 (23%) tumors had low telomerase function by both TRAP and
hTERT expression levels. Telomerase activity and hTERT were significantly correlated (p ϭ 0.017), although 35 cases displayed
discordant results. Both telomerase activity and hTERT levels were significantly associated with poor patient overall and
disease-free survival (p ϭ 0.019 and p ϭ 0.018 for TRAP, and p ϭ 0.011 and p ϭ 0.012 for hTERT, respectively). Among the 21
patients with tumors displaying low telomerase function, defined by both TRAP and hTERT expression levels, only one
succumbed to the disease (p ϭ 0.0053). Our results suggest that the two techniques used in this study evaluate separate aspects
of telomerase function and their combination provides powerful prognostic information in lung cancer patients. (Lab Invest 2002,
82:729–736).
ung carcinoma is the first cause of death by
be used to accurately predict clinical outcome, there
Lcancer in developed countries, and its incidence is exists a great need to identify molecular markers with
which to define a subset of stage I NSCLC patients as
candidates for new investigational adjuvant therapies,
eventually leading to an improvement in patient
survival.
steadily increasing in the world (Greenlee et al, 2001).
Patients’ prognosis is largely dependent on the dis-
ease stage. The standard treatment regimen for pa-
tients with a traditionally better outcome, those
Much interest has recently focused on telomerase, a
ribonucleoprotein enzyme that uses its own integral
RNA as a template for synthesis of telomeric repeats
to compensate for the normal loss of terminal DNA
sequences during cell divisions (Greider and Black-
burn, 1985; Morin, 1989). After the introduction of the
PCR-based assay, called the telomeric repeat ampli-
fication protocol (TRAP), telomerase activity was
found to be present in most human tumors (Piatyszek
et al, 1995; Shay and Wright, 1996). Furthermore, a
correlation between telomerase activity and clinical
outcome in different malignancies has been reported
(Clark et al, 1997; Hiyama et al, 1995a, 1995b). We
have previously shown that telomerase activity, eval-
uated by the TRAP assay, is an independent prognos-
tic marker in stage I NSCLC patients (Marchetti et al,
1999).
grouped as stage
I non-small-cell lung cancer
(NSCLC), is resection of the primary tumor without
adjuvant therapy. This treatment can cure about two-
thirds of the patients with stage I disease, although the
remaining 30% to 40% of the patients will have tumor
recurrence and will die despite complete resection
(Bunn et al, 1994; Strauss et al, 1995). Because careful
pathologic examination of the primary lesions cannot
DOI: 10.1097/01.LAB.0000017165.26718.60
Received December 29, 2001.
The study has been supported by grants from Ministero dell’Istruzione,
dell’Università e della Ricerca (MIUR-cofin 1999), and Associazione
Italiana per la Ricerca sul Cancro (AIRC).
Address reprint requests to: Dr. Silvano Bosari, Division of Anatomic
Pathology, A.O.S. Paolo, Via A. Di Rudinì 8, 20142 Milano, Italy.
E-mail: silvano.bosari@unimi.it
The constituents of the telomerase complex have
recently been identified, and human telomerase re-
Laboratory Investigation • June 2002 • Volume 82 • Number 6 729

Marchetti et al
verse transcriptase (hTERT) has been found to be
responsible for the enzymatic activity of telomerase
(Kilian et al, 1997; Nakamura et al, 1997; Nakayama et
al, 1998). The expression of hTERT is thought to have
important prognostic significance in different forms of
human malignancies. In particular, hTERT mRNA
overexpression evaluated by real-time PCR has been
correlated with the histopathologic grade and receptor
status in breast cancer, therefore identifying the tu-
mors with more aggressive behavior (Bieche et al,
2000). An increase in hTERT mRNA expression was
also correlated with the pathologic grade and clinical
stage in bladder carcinomas, discriminating between
low- and high-grade tumors and between superficial
and muscle-invasive cancers (de Kok et al, 2000). A
significant correlation between hTERT mRNA levels
and telomerase activity was established in hepatocel-
lular carcinomas (Hisatomi et al, 1999). Interestingly,
these authors found a significant difference between
tumor and non-neoplastic liver tissues, suggesting a
possible application of hTERT quantification in the
follow-up evaluation of patients at high risk to develop
hepatocellular carcinomas. In prostate cancer, ele-
vated hTERT levels were significant associated with
myc overexpression, although both parameters were
not correlated with tumor stage or Gleason grade (Latil
et al, 2000).
In lung cancer, hTERT expression has been associ-
ated with telomerase activity, but no relationship with
clinical and pathologic features has been reported
(Arinaga et al, 2000). Recently, poor survival of lung
cancer patients has been linked to high telomerase
expression levels evaluated by in situ hybridization
and immunohistochemistry, together with a semi-
quantitative TRAP assay (Kumaki et al, 2001). A
shorter survival was observed in patients with hTERT
expression, evaluated by nonquantitative RT-PCR,
indicating that hTERT expression might be an inde-
pendent negative prognostic factor (Komiya et al,
2000). The prognostic significance of hTERT mRNA
levels, assessed by current quantitative techniques, in
lung carcinomas has yet to be elucidated.
Results
hTERT Expression in Normal Lung and in Tumors
Ten normal lung tissue samples were used to establish
a basal level of hTERT mRNA. All of the normal
samples expressed detectable, but very low levels of
hTERT mRNA, ranging from 0.01 n to 0.82 n.
Primary tumors from 90 stage I NSCLC patients
were examined. Twenty-one (23%) cases showed
very low levels of hTERT mRNA, comparable to the
normal counterparts, whereas 69 (77%) tumors dis-
played hTERT levels higher than the normal samples.
The range of hTERT levels varied greatly, ranging from
1 n to 939 n. Setting a cut off at 2.6 n, a value that
represents the median value of expression distribution
of the lung tumors, 47 (52%) cases had low hTERT
expression and 43 (48%) cases high hTERT expres-
sion. hTERT levels, excluding the highest one, which
greatly exceeded the range in tumor samples, are
shown in Figure 1. The differences between normal
lung hTERT levels and low and high hTERT expression
tumors are statistically significant (p ϭ 0.00065 and p
ϭ 0.00001).
Telomerase Activity
A sample was considered positive for telomerase
activity when the 36-bp internal control band (see
“Materials and Methods”) and a ladder of PCR prod-
ucts similar to that of the telomerase-positive control
lane were present. Extracts that showed a 36-bp band
but not ladders of PCR products were considered
negative (Fig. 2).
Telomerase activity was detected in 60 (67%) of the
90 tumors examined. There were no obvious differ-
ences in the ratio of tumor cells to stromal cells in the
carcinomas with and without detectable telomerase
activity, and the reproducibility of the TRAP assay was
confirmed by sampling multiple different sites of some
of the tumors.
Comparative Evaluation of Techniques with Pathologic
Correlation
In this study we report a comprehensive analysis of
the telomerase status, based on the evaluation of
hTERT mRNA expression and telomerase activity, in
stage I NSCLC. A large series of stage I NSCLC
patients was investigated for hTERT mRNA expres-
sion by real-time RT-PCR and for telomerase activity
by the TRAP assay. The purposes of our investigation
included: (a) the evaluation of real-time RT-PCR for the
quantitative assessment of hTERT; (b) the comparison
of hTERT expression and telomerase activity in lung
tumors; and (c) the assessment of the prognostic
utility of these two parameters, both individually and
combined.
No correlation was observed between telomerase
activity and/or hTERT expression and age, sex, smok-
ing habits, tumor size, and tumor histotype. A trend
suggesting an association between telomerase status
and histopathologic grade of the tumors was ob-
served, but data were not statistically significant. G1
tumors were scored positive in 20% of cases, whereas
G2 and G3 tumors were positive in 42% and 38% of
cases, respectively, p ϭ 0.11. Results are detailed in
Table 1. According to the telomerase activity results,
21 cases were devoid of telomerase activity and
hTERT expression, whereas 69 showed activity and/or
The results of our investigation suggest that these
two techniques evaluate separate aspects of telomer-
ase function. Their combination provides a better
overall assessment of telomerase function and pow-
erful prognostic information in lung cancer patients.
2
expression. Applying a ␹ test, we observed a statis-
tical link between hTERT expression and telomerase
activity (p ϭ 0.017), although 35 cases displayed
discordant results (Table 2).
730 Laboratory Investigation • June 2002 • Volume 82 • Number 6

Telomerase Function Evaluation in Lung Cancer
Figure 1.
Distribution of human telomerase reverse transcriptase (hTERT) expression levels in normal lung specimens, and in low- and high-expression tumors. Data are
expressed as mean and standard error of the mean for each group. The tumor sample with extremely high expression level (939 n) has been excluded from the
high-expression group.
patients with tumors showing low telomerase function
by both TRAP and hTERT expression levels, only one
succumbed to the disease (p ϭ 0.0053). Overall sur-
vival curves are shown in Figure 3.
A multivariate analysis, using Cox’s proportional
hazards regression model, was performed to investi-
gate the independent prognostic factors among these
markers: tumor stage, histology, histologic grade, and
telomerase function. As a results, the presence of
telomerase activity or hTERT levels were identified as
the only two independent predictor factors of disease-
free survival (p ϭ 0.0317, risk ratio, 3.36 [1.11–10.15];
and p ϭ 0.0449, risk ratio, 2.87 [0.91–8.76], respec-
tively) and overall survival (p ϭ 0.0318, risk ratio, 3.34
[1.09–10.08]; and p ϭ 0.0457, risk ratio, 2.84 [0.90–
8.69], respectively). A very strong association has
been documented when considering both telomerase
activity and hTERT expression and disease free sur-
vival (p ϭ 0.0092, risk ratio, 9.01 [1.16–71.43]) and
overall survival (p ϭ 0.0095, risk ratio, 8.62 [1.12–
66.67]; Table 3).
Figure 2.
Telomeric repeat amplification protocol analyses of non-small-cell lung cancer
specimens are shown. Tumors A, B, and C are examples of telomerase-positive
lung carcinomas, Tumor D represents a telomerase-negative case. The 36-bp
internal positive control band is present in every lane.
Survival Analysis
Discussion
The Kaplan-Meier survival curves in all patients with
stage I NSCLC defined a significant association be-
tween telomerase activity and both disease-free (p ϭ
0.018) and overall survival (p ϭ 0.019). This correlation
is preserved considering hTERT levels alone (p ϭ
0.012 and p ϭ 0.011, respectively). Among the 21
In the present study we have carried out a compre-
hensive analysis of telomerase function in a series of
90 stage I NSCLC lung cancer patients. We evaluated
hTERT mRNA levels by real-time RT-PCR analysis and
telomerase activity by TRAP assay. The specific goals
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Marchetti et al
Table 1. Comparison of Clinical and Pathological
Variables with the Presence of Telomerase Activity
and/or hTERT Expression in Stage I NSCLC Patients
Telomerase activity
and/or hTERT
expression
Factor
Negative
Positive
p value
Age
(mean Ϯ SD, yr)
Sex
63.4 Ϯ 7.2 63.1 Ϯ 6.4 NS (0.7534)
Male
19 (90%) 65 (94%) NS (0.6209)
Female
2 (10%)
4 (6%)
Smoking habits
Smoker
Former smoker
Nonsmoker
Tumor stage
IA
13 (68%) 33 (57%)
6 (32%) 24 (41%) NS (0.6070)
0 (0%)
1 (2%)
6 (29%) 23 (33%)
IB
15 (71%) 46 (67%) NS (0.7932)
Histology
Squamous
carcinoma
Adenocarcinoma
Large cell
carcinoma
Histologic grade
G1
12 (57%) 37 (54%) NS (0.5288)
9 (43%) 28 (40%)
0 (0%)
4 (6%)
9 (43%) 14 (20%)
7 (33%) 29 (42%) NS (0.1101)
5 (24%) 26 (38%)
G2
G3
NS, not significant; NSCLC, non-small-cell lung cancer.
Table 2. hTERT Expression and Telomerase Activity in
Stage I NSCLC
Telomerase activity
n/(%)
HTERT
expression Positive Negative Total n/(%) p value
High
Low
34 (79)
26 (55) 21 (45)
9 (21)
43 (100)
47 (100)
0.017
–
NSCLC, non-small-cell lung cancer.
of the study included the evaluation of real time
RT-PCR for the quantitative assessment of hTERT, the
comparison of the two techniques, and the assess-
ment of their prognostic significance. We quantified
the expression of the hTERT gene in 10 normal lung
RNAs and 90 NSCLC RNAs. hTERT expression was
present in all of the normal and tumor samples tested.
However, all normal tissues and 21 (23%) lung tumors
showed very low hTERT expression levels by real-time
RT-PCR. When tumors were subdivided into two
groups with low and high hTERT expression, accord-
ing to the median value of expression distribution, a
significant association was observed between high
hTERT levels and shorter periods of disease-free and
overall survival. To the best of our knowledge, this is
the first report on the prognostic meaning of hTERT
expression evaluated by real-time RT-PCR in NSCLC
Figure 3.
Overall survival curves according to the telomerase activity status by telomeric
repeat amplification protocol (TRAP) assay (A), hTERT mRNA expression
levels (B) and telomerase function, combining both telomerase activity and
hTERT levels (C). All survival curve differences are statistically significant (see
text for details).
patients. Our data are in keeping with those recently
reported using the same methodologic approach on a
series of 134 breast cancer patients: high hTERT
levels in primary tumors were found to be significantly
associated with shorter relapse-free survival (Bieche
et al, 2000). A correlation between real-time quantita-
tive measurement of hTERT and clinicopathologic
parameters of poor prognosis, such as histologic
grade and muscle invasion, has also been observed in
bladder urothelial cell carcinomas (de Kok et al, 2000).
732 Laboratory Investigation • June 2002 • Volume 82 • Number 6

Telomerase Function Evaluation in Lung Cancer
Table 3. Statistical Analysis of Prognostic Markers and Clinical Outcome in Stage I NSCLC Patients
Disease-free survival Overall survival
Factor
Tumor stage (IA, IB)
Histologic grade
Histotype
p
Risk ratio
p
Risk ratio
NS (0.2347)
NS (0.7455)
NS (0.5368)
0.0092
1.70 (0.71–4.09)
1.19 (0.42–3.38)
1.27 (0.47–3.49)
9.01 (1.16–71.43)
NS (0.2204)
NS (0.8845)
NS (0.4339)
0.0095
1.73 (0.72–4.17)
1.08 (0.38–3.09)
1.32 (0.49–3.68)
8.62 (1.12–66.67)
Telomerase activity and/or hTERT expression
NSCLC, non-small-cell lung cancer.
In this series of 90 NSCLC patients, telomerase
activity, evaluated by an isotopic TRAP method, was
present in 67% of cases and was found to be a
predictor of clinical outcome, in keeping with our
previous data (Marchetti et al, 1999). Because we
found an expression of the hTERT gene in all of the
samples, whereas telomerase activity was evident in
67% of the tumors, our data indicate that the sensi-
tivity of the real-time RT-PCR method is much higher
than that of the TRAP assay. The relationship between
telomerase activity, detected by TRAP, and hTERT
expression, investigated by real-time RT-PCR, in hu-
man tumors in vivo has been studied only in a limited
number of cases. In 24 hepatocellular carcinoma
samples Hisatomi et al (1999) found a significant
correlation between the levels of hTERT mRNA and
that of telomerase activity (r ϭ 0.751). More recently,
in agreement with our data, Buchler et al (2001)
reported that in 29 pancreatic carcinomas the TRAP
assay was positive in 35% of cases, whereas hTERT
expression was present in 90% of cases. In our series
of tumors, a significant association between high
levels (above the median value) of hTERT expression
and telomerase activity was observed, however, in
39% of cases results were discordant. These discrep-
ancies may be explained by the following possibilities:
(1) A high expression of hTERT in the absence of
telomerase activity may be due to the presence of
telomerase inhibitors in the tissue extract that may
block the enzyme during the TRAP assay (Hohaus et
al, 1996; Lancelin et al, 2000). (2) Low levels of hTERT
gene expression in tumors with telomerase activity
could be a consequence of the fact that hTERT mRNA
and the catalytic subunit of the enzyme have different
half-life periods.
We decided to evaluate the combined results ob-
tained by TRAP and real-time RT-PCR: a stronger
association between telomerase status and disease-
free and overall survival has emerged. In particular,
among the 21 patients who were scored negative with
both methods, only 1 succumbed to the disease (p ϭ
0.0053). Therefore, taken in a broad sense, telomerase
alterations (overexpression and/or activity), which we
call here telomerase function, are frequent events in
NSCLC and strongly correlate with disease-free and
overall survival. The results of the present study indi-
cate that both techniques (real-time RT-PCR and
TRAP assay) are indicated to provide the most accu-
rate estimation of telomerase status in NSCLC. The
use of a single technique could underestimate the
actual number of telomerase abnormalities in lung
tumors. However, our data show that both methods
used separately can give useful prognostic
information.
The quantitative assessment of mRNA levels in
human tissues by real-time RT-PCR may be ham-
pered by the heterogeneity of samples, almost always
containing both neoplastic and non-neoplastic cell
populations. In our study we examined only samples
in which tumor cells comprised at least 80% of the
specimen, thus greatly reducing tissue heterogeneity.
Indeed our results document hTERT levels much
higher in tumor specimens than in non-neoplastic
ones. In future investigations, for cases in which tissue
heterogeneity is quantitatively more significant, the
cell populations to be studied may be enriched using
laser microdissection or flow cytometry and cell sort-
ing techniques.
Real-time RT-PCR provides several advantages to
quantify gene expression: (a) it does not require post-
PCR sample handling, thereby avoiding problems
related to carryover; (b) it has a high sample through-
put and possesses a wide dynamic range, so that
samples do not have to contain equal initial amounts
of total RNA; (c) it makes RNA quantitation more
precise and reproducible, because it is based on the
threshold cycle values established in the early expo-
nential phase of the PCR reaction (Gibson et al, 1996).
At the present time, few papers have been published
on hTERT expression evaluated by real-time RT-PCR
in human tumors, and no data are available for lung
cancer.
It is important to point out that the real-time RT-PCR
technique has several advantages over the TRAP
assay, in that it is faster, more reproducible, less
sensitive to RNase activity, proteases, and protein
inhibitors, and it can be applied to archival material for
retrospective analyses (Specht et al, 2001). The TRAP
assay may suffer some limitations primarily due to
inefficient extraction, PCR inhibitors, telomerase in-
hibitors, and telomerase degradation in clinical sam-
ples. In addition, the TRAP assay provides only semi-
quantitative data for telomerase activity evaluation
(Wu et al, 2000).
In conclusion, using real-time quantitative RT-PCR,
we have shown that the hTERT gene plays a major role
in lung cancer progression. In particular, we found
evidence that hTERT mRNA expression may serve as
a new prognostic tool for stage I NSCLC patients. In
addition, we demonstrated that telomerase activity is
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Marchetti et al
significantly associated with hTERT expression and
poor prognosis, but does not always reflect the status
of hTERT mRNA. Finally, we brought evidence that a
new parameter obtained by cumulating the results of
the real-time RT-PCR and the TRAP assay is a strong
indicator of clinical outcome. These new prognostic
markers should be considered in future prospective
trials to define high-risk stage I NSCLC patients who
could benefit from adjuvant therapy.
TRAP Assay
Frozen tissue samples (50 to 100 mg) were homoge-
nized with a pestle in 100 ␮l ice-cold CHAPS lysis
buffer (0.5% CHAPS, 0.1 mM benzamidine, 10 mM
Tris/HCl pH 7.5, 1 mM MgCl2, 1 mM EGTA, 10%
glycerol, 5 mM ␤-mercaptoethanol). After maintenance
at 4° C for 30 minutes, the lysate was centrifuged at
12,000 ϫg for 20 minutes at 4° C. The supernatant
was removed and its protein concentration was mea-
sured with the Biorad protein assay kit (Biorad Labo-
ratories, Munchen, Germany). The supernatant fluid
samples were diluted to a concentration of 0.3 ␮g/␮l
with lysis buffer and stored at Ϫ80° C. Telomerase
activity was assayed by using the Oncor TRAP-eze
telomerase detection kit (Oncor, Gaithersburg, Mary-
land; catalogue no. S7700). The presence of a primer
and a template for amplification in the TRAP-eze kit
resulted in the formation of a 36-bp band in every lane
and served as internal control to identify false nega-
tives due to the presence of Taq polymerase
inhibitors.
Materials and Methods
Study Population
The primary NSCLC specimens were obtained from
90 patients with pathologic stage I NSCLC surgically
treated at the Department of Surgery, University of
Pisa (Pisa, Italy) between 1993 and 1994. In each
case, tumor and macroscopically normal lung tissue
samples (taken as far as possible from the neoplastic
area) were snap-frozen in liquid nitrogen within 10
minutes of excision and stored at Ϫ80° C. Immedi-
ately adjacent pieces of tumor and normal tissue were
fixed and processed for light microscopy. In all tumor
specimens the amount of tumor cells equaled or
exceeded 80% of the overall sample, confirmed by
histopathologic examination. Similarly, all of the mac-
roscopically normal samples were judged to be
benign.
The study population consisted of 84 (93%) men
and 6 (7%) women, with a mean age of 63.3 years
(range, 43 to 74 years). Patients underwent lobectomy
(85% of cases) or pneumonectomy (15% of cases)
with hylar and mediastinal lymph node sampling.
Patient stage at the time of diagnosis was determined
according to the guidelines of the American Joint
Committee on Cancer (Fleming et al, 1997). Histologic
type and tumor cell differentiation were determined
according to the WHO criteria (Travis et al, 1999). The
most common histologic type was squamous cell
carcinoma (49 cases, 54%), followed by adenocarci-
noma (37 cases, 41%) and large cell carcinoma (4
cases, 5%). Twenty-three (26%) tumors were well
differentiated (G1), 36 (40%) moderately differentiated
(G2), and 31 (34%) poorly differentiated (G3). Smoking
history was available for 77 patients: 46 (60%) were
smokers, 30 (39%) were former smokers (stopped
smoking at least 1 year before the diagnosis of lung
cancer), and 1 (1%) was a nonsmoker.
Real-Time RT-PCR
hTERT expression in these tumors was measured by
real-time quantitative RT-PCR, based on TaqMan
methodology, using the ABI PRISM 7700 Sequence
Detection System (Applied Biosystems, Foster City,
California). This technique allows, by means of fluo-
rescence emission, to find the cycling point when PCR
product is detectable (Ct value or threshold cycle). As
previously reported, the Ct value correlates to the
starting quantity of the target mRNA (Heid et al, 1994).
To normalize the amount of total RNA present in each
reaction, we amplified the housekeeping gene ␤-actin,
which is assumed to be constant in both normal
samples and lung tumors.
Our results are expressed as relative levels of
hTERT mRNA, referred to a sample, called calibrator,
chosen to represent 1X expression of this gene. The
calibrator used was a lung carcinoma of the tissue
collection under study, arbitrarily selected, that was
analyzed on every assay plate with the unknown
samples. All of the analyzed tumors express n-fold
hTERT mRNA relative to the calibrator. Furthermore,
one specific lung tumor sample was included in each
run as control of reproducibility among experiments
performed at different times.
Follow-up data of the study population were ob-
tained by direct patient contact. Follow-up occurred at
2-month intervals for the initial 2 years and at 4-month
intervals afterward. Recurrences were detected by
computed tomography scans or scintigrams and con-
firmed by pathologic examination, using biopsy spec-
imens. Patients were categorized as alive with evi-
dence of disease, alive without disease, and dead as a
result of lung carcinoma. No patient in this series died
of cancer-unrelated causes. Time in days from the
date of the operation to the date of follow-up or death
was recorded. The median follow-up in the series of
patients examined was 54 months (range, 7 to 94
months).
The amount of target, normalized to an endogenous
reference (␤-actin) and relative to the calibrator is
defined by the ⌬⌬Ct method as described by Livak K
(Sequence Detector User Bulletin 2; Applied Biosys-
tems). Specifically, the formula is applied as follows:
target amount ϭ 2Ϫ⌬⌬C
t
where ⌬⌬Ct ϭ {[Ct(hTERT sample) Ϫ Ct(␤-actin sam-
ple)] Ϫ [Ct(hTERT calibrator) Ϫ Ct(␤-actin calibrator)]}.
This method is based on the assumption that the
target (hTERT) and ␤-actin display equal amplification
efficiencies. To verify this condition, we checked ⌬Ct
(Ct hTERT Ϫ Ct ␤-actin) variations according to tem-
734 Laboratory Investigation • June 2002 • Volume 82 • Number 6

Telomerase Function Evaluation in Lung Cancer
plate dilution. To this end, we prepared a standard
curve, composed of five different dilutions of MCF-7
total RNA, corresponding to 100, 25, 6.25, 1.6, and 0.4
ng. The slope of this curve was 0.049. To assure the
appropriate amplification efficiency, the slope of the
standard curve should be Ͻ 0.1.
Statistical Analysis
The different variables of the tumors analyzed were
tested for association using the ␹ and Fisher exact
2
tests. Overall survival was estimated by the method of
Kaplan-Meier (Kaplan and Meier, 1958) and differ-
ences between curves were tested for statistical sig-
nificance with the log-rank test (Cox and Oakes,
1984). Cox’s proportional hazards regression models
(Cox, 1972) were used to assess the independent
prognostic contribution of clinicopathologic variables.
The statistical analysis was performed using the Stat-
View 5 statistical software run on a PowerPC G4
Macintosh computer.
RNA Extraction
Total RNA was extracted from frozen lung specimen
by using a commercial kit, Trizol (Life Technologies,
Gibco BRL), according to the manufacture’s protocol.
RNA was quantified spectrophotometrically, and its
quality was checked by electrophoresis through de-
naturing agarose gels. Only samples not degraded,
that showed clear 18S and 28S bands under ultravio-
let light, were used for real-time RT-PCR.
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Total RNA (200 ng) was reverse-transcribed in a total
volume of 50 ␮l containing 1X TaqMan buffer, 5.5 mM
MgCl2, 1 mM deoxynucleotides, 2.5 ␮M random hex-
amers, 20 units RNase inhibitor, 62.5 units MuLv
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primer, 100 nM probe, 0.5 units of AmpErase Uracil
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10 ␮l of cDNA. Both ␤-actin and hTERT amplification
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cycles of 95° C for 15 seconds and 60° C for 1 minute.
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