Quantitation of hTERT Gene Expression in Sporadic Breast Tumors with a Real-Time Reverse Transcription-Polymerase Chain Reaction Assay¹

Telomeres are structures located on the ends of eukaryotic chromosomes. In humans, they consist of several kilobases of a simple 5′-(TTAGGG)_n-3′ repeat. Telomeres protect the ends of chromosomes against degradation by exonucleases and ligases and against end-to-end fusion, rearrangements, and the loss of terminal DNA segments that occurs when linear DNA replicates (1). Telomeres in human somatic cells gradually shorten with each successive cell division, through replication-dependent sequence loss at DNA termini; this results in chromosome instability, leading to cellular senescence (2). A possible cause of human telomere shortening is repression of telomerase, a specialized ribonucleoprotein that consists of multiple protein subunits and a structural RNA component that contains a template sequence for the telomeric repeat (3). Telomerase activity is inactivated or repressed in the majority of normal somatic tissues but is activated in germ cells and in most malignant tumors. Telomerase reactivation may thus be a major step in human carcinogenesis (4).

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 hTERTexpression 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 hTERTresults 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 hTERTexpression levels and altered mRNA expression of MYC, RB1,and CCND1 genes.

We analyzed tissue from surgically removed primary breast tumors of 134 women followed at the Center René Huguenin from 1977 to 1989. The samples were examined histologically for the presence of tumor cells. A tumor sample was considered suitable for this study if the proportion of tumor cells was >60%. Immediately after surgery,the tumor samples were stored in liquid nitrogen until RNA extraction.

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).

To help validate the kinetic quantitative RT-PCR method, we also analyzed three breast tumor cell lines obtained from the American Tissue Type Culture Collection (SK-BR-3, BT-20, and MCF7). Ten specimens of adjacent normal breast tissue from breast cancers and normal breast tissue from 10 women undergoing cosmetic breast surgery were used to assess basal level of hTERT mRNA in normal breast tissue.

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 C_t (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 C_t 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 1× 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 hTERTC_t 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 N_hTERT, were determined as follows:

Primers and probes for the RPLP0 and hTERTgenes 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.

Total RNA was extracted from breast specimens by using the acid-phenol-guanidinium method. The quality of the RNA samples was determined by electrophoresis through agarose gels and staining with ethidium bromide, and the 18S and 28S RNA bands were visualized under UV light.

Reverse transcription of RNA was done in a final volume of 20 μl containing 1× 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 mmDTT, 50 units of Superscript II RNase H⁻ reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD), 1.5μ m random hexamers (Pharmacia, Uppsala, Sweden), and 1μg 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.

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 1× 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).

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 1× TaqMan buffer, 5 mmMgCl₂, 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.

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 hTERTmRNA copy numbers >10% were also retested.

RFS was determined as the interval between diagnosis and detection of the first relapse (local and/or regional recurrences and/or metastases). Clinical, histological, and biological parameters were compared using the χ² 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.

hTERT mRNA detection was assayed in 20 normal breast tissue RNAs. We observed no normal breast samples in which real-time PCR showed total absence of hTERT mRNA copies (hTERTC_t, 50). Indeed, a very faint positive signal was observed in all normal breast samples, with high hTERT C_t values. Because the hTERT C_t values consistently fell between 37 and 45, values of 35 or less were considered to represent overexpression of the hTERT gene in tumor RNA samples, which were thus scored hTERT-positive.

Among the 134 breast tumor RNA samples tested, 33 (24.6%) were scored hTERT negative (hTERTC_t, >35) and 101 (75.4%) were scored hTERT positive (hTERTC_t, ≤35) with the possibility of N_hTERT value determination, calculated as described in “Patients and Methods.” Among these 101 hTERT-positive tumors, major differences of N_hTERT 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 N_hTERT 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(N_hTERT = 8.2) and on two hTERT-positive tumors with low (TERT22, N_hTERT = 1.2) and high (TERT57, N_hTERT = 64.7) hTERT mRNA levels.

The N_hTERT value was based on the amount of the hTERT target message relative to the RPLP0endogenous control to normalize the amount and quality of total RNA;similar results were obtained by using a second endogenous RNA control,the gene TBP coding for the TATA box-binding protein (a component of the DNA-binding protein complex TFIID; data not shown).

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 (χ2 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)].

Using a Cox proportional hazards model, we also assessed the prognostic value for RFS of parameters that were significant in univariate analysis, i.e., lymph-node status and hTERTstatus (Table 1). 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).

To better analyze the hTERT mRNA level as a quantitative variable, patients with hTERT-positive tumors(n = 101) were subdivided into three equal groups (34,34, and 33 patients, respectively), with tumors with low (1.00–1.6),intermediate (1.6–3.5), and high (3.6–64.7) N_hTERT values. We observed statistical links between high hTERT mRNA levels and negative estrogen(P = 0.002) and progesterone (P =0.048) receptor status (Table 5). 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).

We tested the possible link between hTERT expression levels and altered mRNA expression of MYC, RB1, and CCND1 genes, tested previously for same tumor RNA series.⁴ We observed a statistical link between high hTERT mRNA levels and MYC overexpression (P = 0.007) but not between high hTERT mRNA expression and altered RB1 or CCND1 mRNA expression (Table 5).

Numerous published studies based on the TRAP assay have demonstrated that telomerase is activated in the vast majority of tumor types, including breast tumors, and also in certain normal tissues. Telomerase activity has been detected in 75–95% of breast tumor samples (20, 21, 22, 23, 24), and its up-regulation appears to be an early event in breast carcinogenesis (25). Studies that have correlated telomerase activity with clinical and pathological parameters have given conflicting results for traditional prognostic indicators, disease outcome, and telomerase activity (20, 21, 22, 23). This suggests that telomerase activity may be influenced by parameters other than clinical/pathological features alone, such as sensitivity, telomerase inhibitors, and tissue viability with regard to the different TRAP protocols used. Overall, however, the results suggest that telomerase reactivation may be an important step in the progression of normal epithelial tissue to breast cancer.

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 C_t 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 hTERTmRNA 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 hTERTmRNA 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 hTERTtranscription by c-myc transcriptor factor (15). Conversely, no correlation was observed between high hTERTexpression 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.

We thank the Center René Huguenin staff for assistance in specimen collection and patient care.