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Molecular Oncology, Markers, Clinical Correlates |
Laboratoire de Génétique Moléculaire [I. B., M. O., M. V.] and Centre National de la Recherche Scientifique UPRES-A 8067 [V. P., P. B.], Faculté des Sciences Pharmaceutiques et Biologiques de Paris, F-75006 Paris, and Laboratoire d’Oncogénétique [I. B., R. L.] and Département de Statistiques Médicales [C. N.], Centre René Huguenin, F-92211 St-Cloud, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONPATIENTS AND METHODSStatistical AnalysisRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSStatistical AnalysisRESULTSDISCUSSIONREFERENCES |
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Considerable interest is being focused on the potential use of telomerase-based assays as diagnostic and prognostic methods and for the development of telomerase-based therapies (5) . To date, most studies have used the TRAP3 to assay telomerase activity (6) . In the TRAP assay, telomerase is extracted and allowed to synthesize extension products, which then serve as the templates for PCR amplification. However, the TRAP assay is time consuming, not accurate enough to quantify the full range of telomerase activity, and inappropriate to carry out retrospective studies of clinical outcome from standard archival material (7) .
Recently, several components of human telomerase have been cloned, including the telomerase RNA component (hTERC; also termed hTR; Ref. 8 ) and the telomerase catalytic subunit (hTERT; also termed hTRT, hTCS1, or hEST2; Ref. 9 ). Telomerase activity correlates with the restricted expression pattern of the hTERT gene, whereas expression of the hTERC gene is widespread (9) . Ectopic hTERT expression is sufficient to confer enzymatic activity to telomerase-negative cells (10) , suggesting that hTERT mRNA may serve as a surrogate index for telomerase activity. Ectopic hTERT expression in combination with two oncogenes (the SV40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells (11) . Moreover, inhibition of hTERT results in telomere loss and limits the growth of human cancer cell lines in vitro and their tumorigenic capacity in vivo (12) .
We have developed a real-time quantitative RT-PCR assay based on TaqMan methodology to quantify hTERT mRNA in homogeneous total RNA solutions prepared from tumor samples (13) . This recently developed method is based on use of the 5'-3' exonuclease activity of Taq polymerase to cleave a dual-labeled probe annealed to a target sequence during the extension phase of PCR. This method of nucleic acid quantification in homogeneous solution may become a reference in terms of its performance, accuracy, sensitivity, wide dynamic range, and high throughput capacity, and it also eliminates the need for tedious post-PCR processing. Above all, this method is suited to the development of new target gene assays with a high level of interlaboratory standardization and yields statistical confidence values.
We used this technique to detect and to quantify hTERT gene expression in a series of 134 unilateral invasive primary breast tumor RNAs. We then compared hTERT gene expression with usual prognostic indicators and disease outcome.
In vitro studies suggest induction of telomerase activity by MYC overexpression (14 , 15) , and that both telomerase activity and Rb/CCND1/p16 pathway inactivation are necessary to immortalize human epithelial cells (16) . In consequence, we also tested the possible link between hTERT expression levels and altered mRNA expression of MYC, RB1, and CCND1 genes.
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PATIENTS AND METHODS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSStatistical AnalysisRESULTSDISCUSSIONREFERENCES |
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The patients (mean age, 58.3 years; range, 34–91 years) met the
following criteria: primary unilateral nonmetastatic breast carcinoma
on which complete clinical, histological, and biological data were
available; and no radiotherapy or chemotherapy before surgery. The main
prognostic factors are presented in Table 1
. The median follow-up was 8.9 years
(range, 0.3–15.9 years). Forty-eight patients relapsed (the
distribution of first relapse events was as follows: 14 local and/or
regional recurrences, 30 metastases, and 4 both).
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Real-Time RT-PCR
Theoretical Basis.
Reactions are characterized by the point during cycling when
amplification of the PCR product is first detected, rather than the
amount of PCR product accumulated after a fixed number of cycles. The
larger the starting quantity of the target molecule, the earlier a
significant increase in fluorescence is observed. The parameter
Ct (threshold cycle) is defined as the
fractional cycle number at which the fluorescence generated by cleavage
of the probe passes a fixed threshold above baseline. The
hTERT target message in unknown samples is quantified by
measuring Ct and by using a standard
curve to determine the starting target message quantity.
The precise amount of total RNA added to each reaction mix (based on absorbance) and its quality (i.e., lack of extensive degradation) are both difficult to assess. We therefore also quantified transcripts of the RPLP0 gene (also known as 36B4) encoding human acidic ribosomal phosphoprotein P0 as the endogenous RNA control, and each sample was normalized on the basis of its RPLP0 content.
The relative target gene expression level was also normalized to a calibrator, or 1x sample, consisting of a breast tumor tissue sample that contained the smallest accurately quantifiable amount of hTERT mRNA. The calibrator thus indicates the limit of assay quantitation of the target which corresponds to a hTERT Ct value of 35. Each sample normalized hTERT value is divided by the calibrator normalized hTERT value to give the final relative expression level.
Final results, expressed as N-fold differences in hTERT gene
expression relative to the RPLP0 gene and the calibrator,
termed NhTERT, were determined as follows:
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Primers, Probes, and PCR Consumables.
Primers and probes for the RPLP0 and hTERT
genes were chosen with the assistance of the computer programs Oligo
4.0 (National Biosciences, Plymouth, MN) and Primer Express
(Perkin-Elmer Applied Biosystems, Foster City, CA). We conducted BLASTN
searches against dbEST and nr (the non-redundant set of GenBank, EMBL,
and DDBJ database sequences) to confirm the total gene specificity of
the nucleotide sequences chosen for the primers and probes and the
absence of DNA polymorphisms. The nucleotide sequences of the
oligonucleotide hybridization probes and primers are shown in Table 2
. Primers and probes are designated by
the nucleotide position (relative to hTERT GenBank accession
number AF015950 and RPLP0 GenBank accession number M17885)
corresponding to the 5' position, followed by the letter U for upper
(sense strand) or L for lower (antisense strand). To avoid
amplification of contaminating genomic DNA, one of the two primers was
placed in a different exon. For example, the upper primer of
hTERT (2673U) was placed in exon 10, whereas the probe
(2711U) and the lower primer (2767 L) were placed in exon 11.
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cDNA Synthesis.
Reverse transcription of RNA was done in a final volume of 20 µl
containing 1x RT buffer (500 µM each deoxynucleotide
triphosphate, 3 mM MgCl2, 75
mM KCl, 50 mM Tris-HCl, pH 8.3), 10 units of
RNasin RNase inhibitor (Promega Corp., Madison, WI), 10 mM
DTT, 50 units of Superscript II RNase H- reverse
transcriptase (Life Technologies, Inc., Gaithersburg, MD), 1.5
µM random hexamers (Pharmacia, Uppsala, Sweden), and 1
µg of total RNA. The samples were incubated at 20°C for 10 min and
42°C for 30 min, and reverse transcriptase was inactivated by heating
at 99°C for 5 min and cooling at 5°C for 5 min.
Standard Curve Construction.
The relative kinetic method was applied using a standard curve
constructed with 4-fold serial dilutions of cDNA obtained from the MCF7
breast cell line known to strongly express the hTERT gene
(17)
; the cDNA was obtained by reverse transcription from
1 µg of total RNA and 5-fold dilution in 1x RT buffer. The standard
curve used for PCR is composed of 5 points (equivalent to 100, 25,
6.25, 1.6, and 0.4 ng of MCF7 total RNA).
PCR Amplification.
All PCR reactions were performed using a ABI Prism 7700 Sequence
Detection System (Perkin-Elmer Applied Biosystems). For each PCR run, a
master mix was prepared on ice with 1x TaqMan buffer, 5 mM
MgCl2, 200 µM dATP, dCTP, dGTP, and
400 µM dUTP, 300 nM each primer, 150
nM probe, and 1.25 units of AmpliTaq Gold DNA polymerase
(Perkin-Elmer Applied Biosystems). Ten µl of each appropriately
diluted RT samples (standard curve points and patients’ samples) were
added to 40 µl of the PCR master mix. The thermal cycling conditions
comprised an initial denaturation step at 95°C for 10 min and 50
cycles at 95°C for 15 s and 65°C for 1 min.
Experiments were performed with duplicates for each data point. Each
PCR run included the five points of the standard curve (4-fold serially
diluted MCF7 cell line cDNAs), a no-template control, the calibrator
cDNA, and 41 unknown patient cDNAs. The target gene mRNA copy value of
the 41 patients was obtained in 
2 h with this assay format.
Inclusion Criteria for hTERT Assay.
Breast tissue samples were considered eligible for study when the
RPLP0 Ct value was 
20, i.e., suggesting an
appropriate starting amount and quality of total RNA. All samples with
a coefficient of variation for RPLP0 and/or hTERT
mRNA copy numbers >10% were also retested.
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Statistical Analysis |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSStatistical AnalysisRESULTSDISCUSSIONREFERENCES |
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2 test. Differences between
the two populations were judged significant at confidence levels >95%
(P < 0.05). Survival distributions were estimated by
the Kaplan-Meier method (18)
, and the significance of
differences between survival rates was ascertained using the log-rank
test. Multivariate analysis using Cox’s proportional hazards model
(19)
was used to assess the independent contribution of
each variable to RFS. |
RESULTS |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSStatistical AnalysisRESULTSDISCUSSIONREFERENCES |
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hTERT mRNA Levels in Breast Tumor Tissues.
Among the 134 breast tumor RNA samples tested, 33 (24.6%) were scored
hTERT negative (hTERT
Ct, >35) and 101 (75.4%) were scored
hTERT positive (hTERT
Ct, 35) with the possibility of
NhTERT value determination, calculated as
described in "Patients and Methods." Among these 101
hTERT-positive tumors, major differences of
NhTERT values were observed, ranging from
1.0 to 64.7. hTERT gene expression was also investigated in
three breast tumor cell lines (SK-BR-3, BT-20, and MCF7), which were
scored hTERT-positive with
NhTERT values of 1.4 (BT-20), 3.6
(SK-BR-3), and 8.2 (MCF7). Fig. 1
gives
data on one hTERT-negative tumor (TERT91), on MCF7 cell line
(NhTERT = 8.2) and on two
hTERT-positive tumors with low (TERT22,
NhTERT = 1.2) and high (TERT57,
NhTERT = 64.7) hTERT mRNA
levels.
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Correlation between Qualitative hTERT mRNA Status
(hTERT negative/positive) and Clinical and Pathological
Parameters.
We sought links between qualitative hTERT mRNA status
(hTERT negative/positive) and standard clinical,
pathological, and biological factors in breast cancer (Table 3)
. hTERT-positive status was
not significantly associated (2 test) with menopausal status or
standard prognostic factors such as macroscopic tumor size,
histopathological grade, or lymph node or steroid receptor status.
Nevertheless, patients with hTERT-positive tumors had a
higher rate of relapse [42.6% (43 of 101) versus 15.2% (5
of 33)] than those with a hTERT-negative tumors
(P = 0.004; Table 3
). hTERT positivity was
associated with reduced RFS after surgery (log-rank test,
P = 0.017; Table 1
; Fig. 2
). The outcome for the 101 patients with
hTERT-positive tumors was significantly worse than that of
the 33 patients with a hTERT-negative tumors in term of RFS
[7-year RFS, 66.7% (57.1–76.3) versus 84.8%
(72.6–97.1); 10-year RFS, 57.3% (46.3–68.2) versus 84.8%
(72.6–97.1)].
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. The prognostic significance of these two parameters
persisted in Cox multivariate regression analysis (Table 4)
. Adjusted relative risk of these two
parameters, taking into account menopausal status, macroscopic tumor
size, histological grade, and steroid receptor status, did not change
their prognostic significance for RFS (data not shown).
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. A trend
toward a link between high hTERT mRNA levels and SBR
histopathological grade III was also observed (P =
0.066). The outcomes of the patients in the three groups did not differ
(Table 5
and log-rank test not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONPATIENTS AND METHODSStatistical AnalysisRESULTSDISCUSSIONREFERENCES |
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Recently, the gene encoding the catalytic subunit of human telomerase (hTERT) has been cloned (9) . Several studies have demonstrated that hTERT expression is a rate-limiting determinant of the enzymatic activity of human telomerase and that up-regulation of hTERT expression may play a critical role in human carcinogenesis (11) . In a recent report based on an RNA in situ hybridization assay, Kolquist et al. (26) showed that hTERT expression appeared early during breast tumorigenesis in vivo, beginning in normal epithelial cells with proliferative potential and increasing gradually during the neoplastic process.
In this study, we validated a recently developed RT-PCR method for the quantification of hTERT expression (13) . The method is based on real-time analysis of PCR amplification and TaqMan methodology and has several advantages over other RT-PCR-based quantitative methods, as well as over TRAP assay. The real-time PCR method does not require post-PCR sample handling, thereby avoiding problems related to carryover; it has a high sample throughput and possesses a wide dynamic range, meaning that samples do not have to contain equal starting amounts of total RNA. This technique should, therefore, be suitable for analyzing small early-stage tumors, fine needle aspiration specimens, or formalin-fixed, paraffin-embedded tissues. Real-time RT-PCR-based hTERT mRNA assay has also specific technical advantages over the TRAP assay: (a) because standard archival material (formalin-fixed, paraffin-embedded tissues) can be used to quantify hTERT mRNA by real-time RT-PCR, and retrospective studies of clinical outcome can be carried out; (b) real-time PCR reaction has endogenous control (RPLP0 gene in this study) for each sample, whereas the controls for TRAP assay are from separate samples and reactions.
Finally, and above all, real-time PCR makes RNA quantitation much more precise and reproducible, being based on Ct values established in the early exponential phase of the PCR reaction (when none of the reagents is rate-limiting) rather than end point measurement of the amount of accumulated PCR product. Real-time PCR has high intraassay and interassay reproducibility and gives statistical confidence values.
We validated the method on 20 normal breast tissue RNAs and on a large series (n = 134) of unilateral invasive primary breast tumor RNAs. hTERT mRNA was detected in 100% of breast tumor RNAs but also in all normal breast RNAs. These results reflect the higher sensibility of RT-PCR methods compared with the TRAP methods used, in agreement with Snijders et al. (27) . These latter authors also showed that the presence of hTERT mRNA itself was not indicative of telomerase activity, but that a certain threshold level of hTERT mRNA is required for real telomerase activity. In our series, all of the normal breast tissue RNAs (n = 20) and 33 (24.6%) of the human breast tumor RNAs showed very low levels of hTERT mRNA that were only detectable but not quantifiable by means of the real-time quantitative RT-PCR assay. An increase in hTERT mRNA levels compared with the normal breast tissues was observed clearly in 75.4% of breast tumors. This frequency of hTERT-positive breast tumors is in agreement with data reported by other teams using the TRAP assay (20, 21, 22, 23, 24) . Because the real-time quantitative RT-PCR assay is accurate enough to quantify the full range of expression values, the hTERT-positive group was subdivided into three equal subgroups, with tumors of low, intermediate, and high hTERT mRNA copy numbers. These additional cut points allowed to better study the possible correlations between hTERT gene expression levels and the usual prognostic indicators and disease outcome.
Overall, the results of this study agree with those reported in the literature: (a) We confirm, by quantitative evaluation of hTERT gene expression with a real-time RT-PCR assay, the association between telomerase activity in breast tumors and poor outcome reported by several previous studies based on the TRAP method (22 , 23) ; (b) we observed associations between high hTERT mRNA levels and SBR histopathological grade III and steroid receptor negativity, in agreement with Roos et al. (22) , who showed that high hTERT mRNA levels are associated with aggressiveness of breast tumors. These results suggest that tumor cells might be continuously selected for incrementally higher levels of telomerase activity as they proliferate and acquire genetic changes associated with invasive cancer (26) . In this regard, Hiyama et al. (20) showed that only tumors with high telomerase activity exhibited altered telomere lengths (33% of the breast tumors tested). This indicates that telomere alterations are linked to the multistep mutational events involved in tumor aggressiveness and occur a long time after reactivation of telomerase expression; (c) we observed a link between high hTERT expression levels and MYC gene overexpression. This in vivo study confirms the recently reported direct activation of hTERT transcription by c-myc transcriptor factor (15) . Conversely, no correlation was observed between high hTERT expression levels and altered expression of the RB1 and/or CCND1 genes. This is in disagreement with data from Kiyono et al. (16) , indicating that both telomerase activation and Rb/CCDN1/p16 pathway inactivation are required to immortalize primary epithelial cells.
In conclusion, this study points to a major role of the hTERT gene in breast tumorigenesis. In particular, we found evidence that hTERT mRNA status might serve as an exciting new prognostic tools in human breast cancer. These findings must now be confirmed in a larger series of breast cancer patients and in a large subpopulation of node-negative patients. Our rapid, highly sensitive, high-throughput RT-PCR-based hTERT mRNA assay should prove useful as a routine tool in hTERT-based clinical applications and therapeutic approaches to cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Comité Régional de
l’Essonne de la Ligue Nationale Contre le Cancer, the Association pour
la Recherche sur le Cancer, and the Ministère de l’Enseignement
Supérieur et de la Recherche. R. L. is a research director at
the Institut National de la Santé et de la Recherche
Médicale. 
2 To whom requests for reprints should be
addressed, at University of Paris V, Faculté des Sciences
Pharmaceutiques et Biologiques de Paris 4 Av. de
l’Observatoire, 75006, Paris France. Phone: (33)-1-53-73-97-25; Fax:
(33)-1-44-07-17-54; E-mail: mvidaud{at}teaser.fr)
3 The abbreviations used are: TRAP, telomeric
repeat amplification protocol; RFS, relapse-free survival; RT-PCR,
reverse transcription-PCR; SBR, Scarff-Bloom-Richardson.
Received 8/11/99; revised 11/15/99; accepted 11/17/99.
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