Telomerase expression and telomere length in breast cancer and their associations with adjuvant treatment and disease outcome.

Abstract

Telomere length plays important roles in maintaining genome stability and regulating cell replication and death. Telomerase has functions not only to extend telomere length but also to repair DNA damage. Studies have shown that telomerase may increase cancer cell resistance to DNA-damaging anticancer agents; tamoxifen may suppress telomerase expression in breast cancer cells. This study aimed to investigate the role of telomere length and telomerase activity in breast cancer prognosis.
qPCR and qRT-PCR were used to analyze telomere length and telomerase expression, respectively, in tumor samples of 348 breast cancer patients. Cox regression analysis was performed to examine telomere length and telomerase expression in association with disease-free survival and cause-specific mortality.
Telomere length had no relation to tumor features or disease outcomes. Telomerase expression was detected in 53% of tumors. Larger tumors or aggressive disease were more likely to have telomerase expression. Among patients treated with chemotherapy, high telomerase was found to be associated with increased risk of death (hazard ratio (HR) = 3.15; 95% CI: 1.34 to 7.40) and disease recurrence (HR = 2.04; 95% CI: 0.96 to 4.30) regardless of patient age, disease stage, tumor grade, histological type or hormone receptor status. Patients treated with endocrine therapy had different results regarding telomerase: high telomerase appeared to be associated with better survival outcomes. Telomerase expression made no survival difference in patients who received both chemotherapy and endocrine therapy.
Overall, telomerase expression was not associated with disease outcome, but this finding may be masked by adjuvant treatment. Patients with high telomerase expression responded poorly to chemotherapy in terms of disease-free and overall survival, but fared better if treated with endocrine therapy.

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RESEARCH ARTICLEOpen Access
Telomerase expression and telomere length in
breast cancer and their associations with
adjuvant treatment and disease outcome
Lingeng Lu1, Chong Zhang1,2, Gongjian Zhu1,3, Melinda Irwin1, Harvey Risch1, Guido Menato4, Marco Mitidieri4,
Dionyssios Katsaros4and Herbert Yu1*
Abstract
Introduction: Telomere length plays important roles in maintaining genome stability and regulating cell
replication and death. Telomerase has functions not only to extend telomere length but also to repair DNA
damage. Studies have shown that telomerase may increase cancer cell resistance to DNA-damaging anticancer
agents; tamoxifen may suppress telomerase expression in breast cancer cells. This study aimed to investigate the
role of telomere length and telomerase activity in breast cancer prognosis.
Methods: qPCR and qRT-PCR were used to analyze telomere length and telomerase expression, respectively, in
tumor samples of 348 breast cancer patients. Cox regression analysis was performed to examine telomere length
and telomerase expression in association with disease-free survival and cause-specific mortality.
Results: Telomere length had no relation to tumor features or disease outcomes. Telomerase expression was
detected in 53% of tumors. Larger tumors or aggressive disease were more likely to have telomerase expression.
Among patients treated with chemotherapy, high telomerase was found to be associated with increased risk of
death (hazard ratio (HR) = 3.15; 95% CI: 1.34 to 7.40) and disease recurrence (HR = 2.04; 95% CI: 0.96 to 4.30)
regardless of patient age, disease stage, tumor grade, histological type or hormone receptor status. Patients treated
with endocrine therapy had different results regarding telomerase: high telomerase appeared to be associated with
better survival outcomes. Telomerase expression made no survival difference in patients who received both
chemotherapy and endocrine therapy.
Conclusions: Overall, telomerase expression was not associated with disease outcome, but this finding may be
masked by adjuvant treatment. Patients with high telomerase expression responded poorly to chemotherapy in
terms of disease-free and overall survival, but fared better if treated with endocrine therapy.
Introduction
Telomeres are repeated sequences of oligonucleotides
(TTAGGG) located at the ends of chromosomes, and
have important functions in regulating cell replication
and maintaining genome integrity [1-3]. The length of
telomeres decreases with the number of cell divisions;
reduced telomere length carries functional cellular sig-
nals. Cells enter senescence or initiate apoptosis when
telomere length is reduced to a critical level. Shortened
telomere length often leads to genome instability, result-
ing in loss of cell-cycle control, a hallmark of cancer. Tel-
omere shortening has been found to be associated with
increased risk of several human cancers, including blad-
der, lung, esophagus, pancreas, ovary and thyroid [4-8].
Telomerase, a ribonucleoprotein complex composed of
telomerase reverse transcriptase (TERT), telomerase
RNA (TERC) and dyskerin [9], is a nuclear enzyme that
increases the length of telomeres. Using TERC as a tem-
plate, TERT adds telomeric repeats to the ends of chro-
mosomes [10,11]. While it is important to maintain
appropriate telomere length for genome stability, dysre-
gulated telomere elongation also makes cells immortal,
* Correspondence: herbert.yu@yale.edu
1Department of Epidemiology and Public Health, Yale Cancer Center, Yale
University School of Medicine, 60 College Street, New Haven, CT 06520-8034,
USA
Full list of author information is available at the end of the article
Lu et al. Breast Cancer Research 2011, 13:R56
http://breast-cancer-research.com/content/13/3/R56
© 2011 Lu et al.; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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which is a feature of cancer cells. Telomerase expression
is low during early carcinogenesis, but markedly
increases in tumor invasion, preventing cancer cells
from entering senescence or apoptosis [12-14]. Com-
pared to that in adjacent normal tissues, the activity of
telomerase is up-regulated in many types of cancer,
including breast [15-17]. Several studies have shown
high telomerase activity to be linked to poor prognosis
of breast cancer [18-20]. Based on the experimental
observations of in vitro cell cultures and in vivo animal
models, telomerase inhibitors can reduce tumorigenicity
and suppress breast cancer growth and metastasis
[21-23]. Up-regulated telomerase activity and telomere
elongation may also increase drug resistance in breast
and colorectal cancer cell lines [17,24]. Telomerase inhi-
bitors can increase cancer cell death when used in com-
bination with DNA-damaging anticancer drugs [25].
However, our understanding of telomerase activity in
breast cancer remains limited. The purpose of this study
was to investigate the possible effects of telomerase
expression and telomere length on breast cancer treat-
ment outcomes.
Materials and methods
Study patients
Patients undergoing surgery for primary breast cancer
between January 1998 and July 1999 in the University
Hospital at University of Turin were recruited to a
clinical follow-up study. The study was approved by
the university’s ethical review committee, and a written
consent form was obtained from each study partici-
pant. In total, 348 consecutive patients were enrolled
in the study, with the average age of patients at sur-
gery being 57 years (range: 23 to 84). Of the patients,
302 were followed from surgery through February
2007. The median follow-up time was 86 months
(range: 8 to 108). Clinical and pathological information
on these patients was collected from the hospital
records and pathology reports (Table 1). The majority
of patients in the study were diagnosed with early
stage cancer (36.4% stage I and 53.4% stage II).
Patients with late stage diseases (stages III and IV)
accounted for only 10.3%. Grades 1 (well differen-
tiated), 2 and 3 tumors were seen in 16.6%, 41.1% and
42.3% of the patients, respectively. The major histolo-
gical tumor type was ductal carcinoma (63.1%), fol-
lowed by lobular carcinoma (16.1%). Other histological
types were ductal-lobular or tubular-lobular mixed
histologies which accounted for 10.7% and 10.1%,
respectively. Two hundred one patients (58.1%) had
tumors smaller than 2 cm, 120 patients (34.7%) had
tumors between 2 cm and 5 cm, and 25 (7.2%) had
tumors greater than 5 cm. One hundred sixty patients
(46.8%) had lymph node-positive tumors. According to
the 10% assay cutoff, 64.9% of the patients were ER
positive, and 52.2% were PR positive.
The majority of patients (303 out of 348) received
adjuvant therapy. Of these patients, 119 (34.2%) received
chemotherapy, 77 (22.1%) had hormonal therapy, and
107 (30.8%) received both chemotherapy and endocrine
therapy. Forty-five patients did not receive any adjuvant
treatment. Chemotherapy protocols used for the patients
included CMF (cyclophosfamide, methotrexate, 5 fluoro-
uracil; dose: 600/60/600 mg/mq every three weeks for
six cycles), CEF (cyclophosfamide, epirubicin, 5 fluoro-
uracil; dose: 600/90/600 mg/mq every three weeks for
six cycles), EPI-TAX (epirubicin-paclitaxel; dose: 90/175
mg/mq every three weeks for six cycles), EPI-VNB (epir-
ubicin-vinorelbine; dose: 90/30 mg/mq every three
weeks for six cycles), DTX-EPI-VNB (doxetaxel-epirubi-
cin-vinorelbine; dose: 75/90/30 mg/mq every three
Table 1 Clinical and pathological features of breast
cancer patients in the study
Variable Number%
Stage (TMN) (n = 341)
I
II
III
IV
Grade (n = 343)
1
2
3
Histological type = 347)
Ductal
Lobular
Mix
Other
Tumor size (n = 346)
T1
T2
T3 and T4
Nodal status (n = 342)
Negative
Positive
ER status (n = 342)
Negative
Positive
PR status (n = 341)
Negative
Positive
Treatment (n = 348)
No adjuvant therapy
Chemotherapy
Endotherapy
Chemotherapy and endotherapy
124
182
30
5
36.4
53.4
8.8
1.5
57
141
145
16.6
41.1
42.3
219
56
35
37
63.1
16.1
10.1
10.7
201
120
25
58.1
34.7
7.2
182
160
53.2
46.8
120
222
35.1
64.9
163
178
47.8
52.2
45
119
77
107
12.9
34.2
22.1
30.8
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weeks for six cycles), and TXT-EPI-VNB (paclitaxel-
epirubicin-vinorelbine; dose: 175/90/30 mg/mq every
three weeks for six cycles). Tamoxifen was the only
endocrine therapy used at that time, and the dose was
one 20 mg tablet per day for five years or until disease
progression or unacceptable toxicity. By the end of fol-
low-up, 55 of the 81 patients who had had recurrences
had died. Among the 221 patients without disease pro-
gression, 5 had died.
Tissue analysis for telomere length and telomerase
expression
Fresh tumor samples were collected from the enrolled
patients during surgery. The specimens were snap-fro-
zen in liquid nitrogen immediately after resection and
stored at -80°C until analysis. All tissue samples were
examined by pathologists to confirm tumor content
which varied between 80% and 90%. The specimens
were pulverized manually in liquid nitrogen; aliquots
of the tissue powders (about 100 mg each) were pro-
cessed to extract DNA and total RNA separately, fol-
lowing conventional phenol-chloroform protocols.
RNA samples were quantified and treated with DNase
using a commercial kit (Ambion Inc, Austin, TX,
USA).
A quantitative PCR (qPCR) developed by Cawthon
[26] was used to analyze telomere length. The method
compares the quantities of telomere and albumin in a
common PCR reaction assuming that both products
are amplified with similar efficiency. The primer
sequences used were 5’- ACA CTA AGG TTT GGG
TTT GGG TTT GGG TTT GGG TTA GTG T (for-
ward) and 5’- TGT TAG GTA TCC CTA TCC CTA
TCC CTA TCC CTA TCC CTA ACA (reverse) for tel-
omere, and 5’-CGG CGG CGG GCG GCG CGG GCT
GGG CGG AAA TGC TGC ACA GAA TCC TTG
(forward) and 5’- GCC CGG CCC GCC GCG CCC
GTC CCG CCG GAA AAG CAT GGT CGC CTG TT
(reverse) for albumin. Chromo4™ continuous fluores-
cence detector system (MJ Research Inc., Waltham,
MA, USA) was used to obtain Ct values for both telo-
mere and albumin in each sample. Based on the for-
mula 2-(ΔCt), where ΔCt = Cttelomere- Ctalbumin, the
relative quantity of telomere versus albumin (T/S) was
calculated. qPCR was done in 20 μl solution which
mixed 10 μl of 2 × Quantifast SYBR green (Qiagen,
Valencia, CA, USA) with telomere primers (900 nM),
albumin primers (900 nM), and approximately 20 ng
DNA template. The PCR conditions included initial
denaturing at 95°C for 15 minutes, followed by 2 cycles
of 15 sec at 94°C, and 15 sec at 49°C, and then 26
cycles of 15 sec at 94°C, 10 sec at 62°C, and 15 sec at
73°C with signal acquisition for telomere, 10 sec at 84°
C, and 15 sec at 87°C with signal acquisition for
albumin. After PCR amplification, melting curves were
analyzed to confirm the size of PCR products. Each
sample was run in duplicate. Samples with coefficient
of variation >15% were reanalyzed.
Levels of telomerase expression along with an inter-
nal control (GAPDH) were determined with real-time
reverse-transcriptase PCR (qRT-PCR). The forward
and reverse PCR primers for telomerase described by
Zhu et al. [27] were 5’- CGT CGA GCT GCT CAG
GTC TT and 5’- AGT GCT GTC TGA TTC CAA
TGC TT, respectively. The primer sequences for
GAPDH and the qRT-PCR methods are described else-
where [28-30]. Approximately 1.0 μg of DNase-treated
total RNA from each sample was used for reverse tran-
scription using the Cloned AMV First-Strand cDNA
Synthesis kit (Invitrogen™, Carlsbad, CA, USA). qRT-
PCR was performed using the Chromo4™ continuous
fluorescence detector system. The PCR reaction was
carried out in a 20 μl solution which contained 0.1 μg
of cDNA template plus 10 μl of 2 × Power®SYBR
Green PCR Master Mix (Applied Biosystems, Foster
City, CA, USA) and a pair of primers at a final con-
centration of 100 nM. PCR thermal cycle conditions
included initial incubation at 50°C for 2 minutes, dena-
turing at 95°C for 10 minutes, and 40 cycles of 95°C
for 15 sec and 60°C for 1 minute. Dissociation curves
were generated after PCR to determine the size of PCR
product. Each sample was tested in duplicate for each
gene, and the analysis was repeated for those samples
with large variation in their duplicates (coefficient of
variation >5%).
Statistical analysis
An expression index (EI) was calculated for telomerase
expression using the formula 1,000 × 2(-ΔCt), where
ΔCt = Ct(telomerase)- Ct(GAPDH). In data analysis, telo-
merase expression was analyzed as a categorical vari-
able. Patients with undetectable telomerase, Ct
(telomerase)>35, were grouped as low telomerase, and
those with detectable telomerase as high telomerase.
Telomere length was analyzed first as a continuous vari-
able for its correlation with other variables, and then
samples were classified into short and long telomere
groups using median length as cutoff. The Chi-square
test was used to compare differences in telomerase
expression and telomere length among patients with dif-
ferent clinical and pathological features of breast cancer.
Survival analysis was performed by proportional hazards
regression methods to assess the associations of telo-
merase expression and telomere length with the risk of
disease recurrence and death. SAS version 9.2 (SAS
Institute, Cary, NC, USA) was used for all statistical
analyses. P-values at 0.05 or smaller (two-sided) were
deemed statistically significant.
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Results
Telomerase expression and telomere length in breast
cancer
Telomerase expression was analyzed in the 336 tumor
samples that had sufficient quantities of RNA. Among
these, 159 samples had low (undetectable) and 177 had
high (detectable) telomerase expression. For the detect-
able samples, the average expression was 0.23 EI, and
the expression range varied widely from 0.001 to 2,266
EI. The average value of telomere length in 348 tumor
samples was 7.6, and the range was between 0.4 and
40.9. When analyzed as continuous variables, no correla-
tion was found between telomerase expression and telo-
mere length (r = 0.02, P = 0.746). This analysis was
repeated for the samples with detectable expression (n =
177) using the log transformed data, and the result
remained same. In categorical analysis, patients with
detectable telomerase expression seemed more likely to
have long telomeres compared to those with undetect-
able telomerase, but the difference was not significant
(53.1% versus 45.3%, P = 0.152).
Telomerase expression and telomere length in relation
to clinical and pathological features of breast cancer are
shown in Table 2. A statistically significant association
was observed between telomerase expression and tumor
size (P = 0.035); patients with high telomerase expression
tended to have larger tumors. Patients with high telomer-
ase also seemed more likely to have stage III or IV dis-
ease (11.4% versus 9.1%) and grade 3 tumors (44.6%
versus 38.8%), but these differences were not statistically
significant. No significant associations were found
between telomerase expression and histological type,
lymph node involvement or hormone receptor status.
Telomere length, either analyzed as a continuous or cate-
gorical variable, was not associated with any of the clini-
cal or pathological features (categorical data in Table 2)
Telomerase expression and telomere length in relation to
disease outcomes
Table 3 presents telomerase expression and telomere
length in relation to disease outcomes. Patients with
high telomerase expression had higher risk of disease
recurrence and death than those with low expression,
but the elevated risks were not statistically significant.
Patients with long or short telomere lengths had quite
similar survival outcomes, either for disease recurrence
or death. Furthermore, these findings did not seem to
change substantially after the major clinical and patholo-
gical features of breast cancer were adjusted in the ana-
lysis, including age at surgery, disease stage, tumor
grade, histological type and receptor status.
However, since systemic adjuvant treatment may influ-
ence survival and interact with telomerase activity in its
therapeutic effects, we performed survival analyses
stratified by treatment modality (Table 4). Among
patients treated with adjuvant chemotherapy, high telo-
merase expression was associated with higher risk of
death and disease recurrence in comparison to low
expression: adjusted hazard ratios 3.15 (95% CI: 1.34 to
7.40) and 2.04 (95% CI: 0.96 to 4.30), respectively. Inter-
estingly, similar results were not observed in patients
treated with endocrine therapy. These patients, in con-
trast, appeared to have better survival outcomes with
high expression of telomerase, though none of the asso-
ciations were statistically significant. For patients who
received both chemotherapy and endocrine therapy, sur-
vival outcomes, either overall survival or disease-free
survival, were similar according to high or low telomer-
ase expression. No associations were found between tel-
omere length and patient survival when patients were
stratified by either adjuvant endocrine therapy or che-
motherapy in both univariate and multivariate analyses
(data not shown).
Discussion
In this study, we analyzed breast tumors for telomere
length and telomerase expression in order to assess
their possible effects on disease outcomes. We found no
evidence that telomere length was related to any of the
major clinical or pathological features of the disease. In
addition, we found no indication that telomere length
was associated with patient survival outcomes, either
disease-free survival or overall survival. We did notice,
however, that tumors with longer telomere lengths were
more likely to have detectable telomerase expression,
although statistically significant correlation between the
two was not observed. It has been reported that telo-
mere length can be maintained without telomerase
through an alternative mechanism, namely alternative
lengthening of telomeres (ALT) [31]. Although the
mechanisms by which ALT works remain unclear, it is
thought that homologous DNA recombination is
involved. About 10 to 15% of cancers including some
with extremely poor prognosis use ALT to elongate tel-
omere length [31]. Additionally, many environmental
and host factors including psychological stress, physical
activity, oxidative stress, hormones and growth factors
have been shown to affect telomere length [32-36].
In this study, telomerase expression was found more
often in larger tumors, and possibly in more aggressive
disease. Telomerase expression was not associated with
disease outcomes among patients when adjuvant treat-
ment was not considered. However, if the treatment
was considered, the associations between survival and
telomerase became quite different. Patients who
received adjuvant chemotherapy had worse survival out-
comes if they had high telomerase expression in their
tumors, but patients treated with endocrine therapy
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appeared to have better outcomes if their telomerase
was high in the tumors. This discrepancy suggests that
telomerase may interfere with adjuvant treatment. It is
known that telomerase can maintain genome stability
by elongating telomeres in normal cells, but evidence
also suggests that telomerase may play different roles
in tumors, and the effects are independent of telomere
length.The enzymemay facilitatemalignant
transformation and render cells more resistant to apop-
tosis [37-39]. It is found that when telomerase is sup-
pressed, short-term telomere integrity is not affected,
but the overall chromatin configuration undergoes sig-
nificant changes in terms of histone modification. These
epigenetic alterations can impair DNA repair capacity
and increase cell sensitivity to radiation [40]. Telomer-
ase not only promotes DNA damage repair and
Table 2 Telomerase expression and telomere length in association with clinical and pathological features of breast
cancer
VariableTelomerase expression
LowHigh
P* Telomere length
Short
P*
Long
Telomere length (n = 336)
Short
0.152
87 (54.7)83 (46.9)
Long
Disease stage (n = 329)
I-II
III-IV
Tumor grade (n = 332)
1-2
3
Tumor size (n = 334)
T1
T2
T3 and T4
Histological type (n = 335)
Ductal
Lobular
Mix
Other
Nodal status (n = 330)
Negative
Positive
ER status (n = 331)
Negative
Positive
PR status (n = 330)
Negative
Positive
72 (45.3)94 (53.1)
0.4870.788
140 (90.9)
14 (9.1)
155 (88.6)
20 (11.4)
149 (89.2)
18 (10.8)
146 (90.1)
16 (9.9)
0.2920.300
96 (61.2)
61 (38.8)
97 (55.4)
78 (44.6)
93 (55.4)
75 (44.6)
100 (61.0)
64 (39.0)
0.035
0.648
103 (65.6)
45 (28.7)
9 (5.7)
92 (52.0)
74 (41.8)
11 (6.2)
98 (58.3)
58 (34.5)
12 (7.1)
97 (58.4)
61 (36.8)
8 (4.8)
0.0940.252
98 (62.0)
31 (19.6)
19 (12.0)
10 (6.4)
114 (64.4)
25 (14.1)
15 (8.5)
23 (13.0)
101 (59.8)
35 (20.7)
17 (9.5)
17 (10.1)
111 (66.9)
21 (12.7)
18 (10.8)
16 (9.6)
0.6180.191
79 (51.0)
76 (49.0)
94 (53.7)
81 (46.3)
94 (56.0)
74 (44.0)
79 (48.8)
83 (51.2)
0.782 0.884
53 (34.0)
103 (66.0)
62 (35.4)
113 (64.6)
59 (35.1)
109 (64.9)
56 (34..4)
107 (65.6)
0.3520.181
70 (44.9)
86 (55.1)
87 (50.0)
87 (50.0)
86 (51.2)
82 (48.8)
71 (43.8)
91 (56.2)
Table 3 Survival outcomes in association with telomerase expression and telomere length in all patients
VariableOverall Survival
HR (95% CI)*
Disease-free Survival
HR (95% CI)* AHR (95% CI)**AHR (95% CI)**
Telomerase
Low telomerase
High telomerase
Telomere length
Short telomere
Long telomere
1.00 1.00 1.00 1.00
1.46 (0.87 to 2.45) 1.31 (0.78 to 2.22)1.25 (0.80 to 1.95)1.21 (0.79 to 1.94)
1.001.001.001.00
0.79 (0.47 to 1.31)0.83 (0.49 to 1.40)0.84 (0.55 to 1.31)0.83 (0.53 to 1.31)
*HR, hazard ratio; 95% CI, 95% confidence interval.
** AHR, adjusted hazard ratio; adjusted for patient age at surgery, disease stage, tumor grade, histological type, estrogen- and progesterone-receptor status.
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