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Human Telomerase Reverse Transcriptase mRNA Expression Assessed by Real-Time Reverse Transcription Polymerase Chain Reaction Predicts Chemosensitivity in Patients With Ovarian Carcinoma

Fiamma Buttitta, Caterina Pellegrini, Antonio Marchetti, Angiolo Gadducci, Stefania Cosio, Lara Felicioni, Fabio Barassi, Simona Salvatore, Carla Martella, Guido Coggi, Silvano Bosari

From the Department of Oncology and Neurosciences, University of Chieti, Chieti; Department of Medicine, Surgery and Dental Sciences, Division of Pathology, University of Milan, Milan; A.O. S. Paolo, IRCCS Ospedale Maggiore and Interuniversity Center of Oncological Research, Milan; and Department of Procreative Medicine, Division of Gynecology and Obstetrics, University of Pisa, Italy.

Address reprint requests to Fiamma Buttitta MD, PhD, Molecular Pathology Section, Department of Oncology and Neurosciences, Anatomia Patologica, Ospedale Clinicizzato, via dei Vestini, 66013 Chieti, Italy; email: fbuttitta{at}unich.it.

	ABSTRACT

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Purpose: To evaluate in vivo whether the expression of the human telomerase reverse transcriptase (hTERT) gene, the catalytic subunit of the telomerase complex, is predictive of response to chemotherapy in ovarian cancer patients.

Patients and Methods: Fifty-nine advanced-stage ovarian cancer patients who were treated with platinum-based chemotherapy were studied. hTERT levels were evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR) on tumor specimens obtained before the treatment. Variables were analyzed by the ² and Fisher’s exact tests. Logistic regression analysis was also performed to account for the effects of all the covariates investigated (residual disease, stage, histotype, and grade).

Results: Twenty-eight (47%) of the 59 tumors showed low hTERT levels, whereas 31 (53%) tumors displayed high hTERT levels. Seventy-five percent of complete responders showed high levels of hTERT expression, whereas 66% of partial responders or nonresponders exhibited low hTERT levels (P = .002). Only residual disease and hTERT expression were independent predictors of response (odds ratios, 13.455 and 7.586, respectively). The combination of these two parameters provides powerful predictive information: 18 of the 20 patients with residual disease more than 2 cm and low hTERT levels were partial responders or nonresponders, whereas 11 of the 12 patients with residual disease less than 2 cm and high hTERT levels showed a complete response (² = 21,416; P < .00001).

Conclusion: Our data indicate that hTERT expression, measured by real-time RT-PCR, is a possible independent marker of response to platinum-based therapy in advanced stage ovarian cancer patients. Prospective validation of this marker will be required to further define its predictive value.

	INTRODUCTION

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OVARIAN CANCER patients show a great variability in the response to chemotherapy, even when they present with similar histological tumor type, tumor grade, and disease stage. Because conventional clinical and pathologic parameters cannot be used to accurately predict the response to pharmacological treatment, there exists a great need to identify new markers with which to define the subset of patients who will respond to therapy.

It is now becoming evident that conventional chemotherapy exerts its function via the cellular pathways that regulate the cell cycle and apoptosis.^1–4 Therefore, genetic alterations can have a profound effect on individual patient response to cytotoxic drugs, as we and others have previously shown.^5–7 The identification of genes as targets for specific chemotherapeutic agents is becoming a crucial issue for the management of neoplastic patients.

Recently, growing attention has been focused on telomerase, a ribonucleoprotein enzyme complex 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 division.⁸ Several components of human telomerase, encoded by distinct genes, have been cloned, including the telomerase RNA component (hTR)⁹ and the telomerase catalytic subunit (hTERT).^10–12 The expression of hTERT is thought to have important prognostic significance in different forms of human malignancies.^13–18 In addition, the upregulated expression of telomerase activity in tumors has made this enzyme a potential new target for cancer treatment.^19–21 Recent in vitro studies suggest a link between telomere loss or telomerase activity and sensitivity to DNA-damaging agents.^22–24 However, the role that telomerase plays in chemoresponse in vivo is still unknown.

To verify in vivo whether telomerase function is predictive of response to chemotherapeutic drugs, we analyzed a large series of advanced ovarian carcinoma patients in which a second-look laparotomy was performed in all patients with complete clinical response. In patients subjected to second-look laparotomy, the pathologic evaluation after first-line treatment gives additional, accurate information on response to chemotherapy. Because it has been reported that the evaluation of hTERT expression is a marker of telomerase function more sensitive than the assessment of telomerase activity by the telomerase repeat-amplification protocol (TRAP) assay,^18,25–27 and hTERT expression is a rate-limiting determinant of the enzymatic activity of human telomerase,²⁸ we analyzed the expression of hTERT mRNA to investigate the role of the telomerase complex in chemosensitivity. The quantification of hTERT expression was performed by a real-time reverse transcription polymerase chain reaction (RT-PCR) assay. This method, which is based on TaqMan methodology (Applied Biosystems, Foster City, CA), uses the 5'-3' exonuclease activity of Taq polymerase to cleave a dual-labeled probe annealed to a target sequence during the extension step of PCR.^29,30 This technique of nucleic acid quantification is significantly more sensitive and specific and shows some advantages compared with other RT-PCR–based quantitative procedures.^26,31–33 Our study revealed a link between high levels of hTERT expression in ovarian carcinoma and chemosensitivity to platinum-based chemotherapy.

	PATIENTS AND METHODS

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Patients and Treatment
Tumor samples were collected from ovarian carcinoma patients who consecutively underwent initial surgery at the Department of Gynecology and Obstetrics, University of Pisa, Italy, from October 1992 to September 1997. Normal tissue samples were obtained from patients who underwent surgery for nonneoplastic disease. Informed consent for the experimental use of surgical samples was obtained for all patients.

Neoplastic patients were subjected to staging laparotomy including total abdominal hysterectomy, bilateral salpingo-oophorectomy, omentectomy, peritoneal biopsies, and surgical cytoreduction with or without pelvic and periaortic lymph-node sampling. Tumor stage was established according to the criteria of International Federation of Obstetrics and Gynecology (FIGO).³⁴ Tumors were histologically classified according to the World Health Organization classification.³⁵ Grading was performed by evaluation of tumor architecture, the amount of solid neoplastic areas, nucleus-cytoplasm ratio, and nuclear pleomorphism.^36,37 The tumors were subdivided into three groups, well-differentiated (G1), moderately differentiated (G2), and poorly differentiated (G3), according to these criteria.

After initial debulking surgery, the patients were subjected to six cycles of platinum-based chemotherapy. Cisplatin (75 to 100 mg/m²) plus cyclophosphamide (600 mg/m²) was administered with or without epirubicin (60 mg/m²).

Tumor Response Criteria
At the end of treatment, all patients were evaluated for response to chemotherapy by physical examination, chest radiography, abdominal-pelvic ultrasound, and computerized tomography scan. Three to 5 weeks after the last course of chemotherapy, a second-look laparotomy was performed in all patients in complete clinical response (defined as the disappearance of all signs of tumor), according to a previously reported procedure,^38,39 for accurate assessment of response to chemotherapy by histopathologic examination. A complete pathologic response at second look was defined as the disappearance of all tumor deposits with negative peritoneal washing and negative multiple random biopsies. A partial pathologic response corresponded to a more than 50% tumor reduction in all measurable lesions, whereas stable disease corresponded to a less than 50% reduction or less than 25% increase in the neoplastic disease. Progressive disease refers to a more than 25% increase in the size of existing lesions or the appearance of new tumor lesions.

Patient Eligibility
Fifty-nine ovarian cancer patients were selected from a larger series of consecutive patients on the basis of the following criteria: histologically confirmed diagnosis of epithelial ovarian cancer, FIGO III-IV disease, no prior chemotherapy or radiotherapy, and treatment with platinum-based chemotherapy. The above-mentioned criteria were adopted to evaluate a homogeneous series of patients.

Sample Collection
To limit problems related to the heterogeneity of ovarian neoplasms, three to six tumor samples in nonnecrotic areas were obtained from each patient. Areas containing nonviable tissues were excluded. Normal tissue samples were obtained from eight patients undergoing surgery for nonneoplastic diseases. For each specimen, a portion was snap-frozen in liquid nitrogen within 10 minutes of excision and stored at -70°C, whereas the remaining part was formalin-fixed and processed for histologic examination. Histologic examination was performed to verify the proportion of tumor and stromal cells and to exclude the presence of inflammatory foci. Only tumor tissue samples making up more than 80% of the tumor cells were processed for molecular analysis.

RNA Extraction
Total RNA was extracted from frozen tumor and normal tissue specimens using a Trizol commercial kit (Life Technologies, GIBCO BRL, Carlsbad, CA) according to the manufacturer’s protocol. RNA was quantified spectrophotometrically, and its quality was checked by electrophoresis through agarose gels stained with ethidium bromide. Only samples that were not degraded and showed clear 18S and 28S bands under ultraviolet light were used for real-time RT-PCR.

cDNA Synthesis
Total RNA (200 ng) was reverse transcribed in a total volume of 50 µ containing 1x TaqMan buffer, 5.5 mmol/L MgCl₂, 1 mmol/L deoxynucleotides, 2.5 µmol/L random hexamers, 20 U RNase inhibitor, and 62.5 U MuLv reverse transcriptase. The samples were incubated at 25°C for 10 minutes, 48°C for 30 minutes, and 95°C for 5 minutes.

PCR Amplification
PCR was performed in a total volume of 50 µL containing 1x TaqMan buffer; 5.5 mmol/L MgCl₂; 200 µmol/L each of deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanosine triphosphate; 400 µmol/L deoxyuracil triphosphate; 300 nmol/L each primer; 100 nmol/L probe; 0.5 U of AmpErase uracil N-glycosilase; 1.25 U AmpliTaq Gold (Applied Biosystems, Foster City, CA); and 10 µL of cDNA. Both beta-actin and hTERT amplification were done in duplicate for each sample. The thermal cycling conditions included 2 minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All reagents used for RT-PCR were purchased from Applied Biosystems.

Primers and Probes
Primers and probes for beta-actin and hTERT mRNAs were chosen using the Primer Express (Applied Biosystems) computer program. The primers, placed in different exons, were tested to ensure that they did not amplify genomic DNA. Primers and probe nucleotide sequences for hTERT (GenBank accession number AF015950) were forward primer 5'-CACGCGAAAACCTTCCTCA-3' (placed in exon 10; nt 2,690 to 2,708), reverse primer 5'-CAAGTTCACCACGCAGCC-3', and TaqMan probe 5'(FAM)-CTCAGGGACACCTCGGACCAGGGT-(TAMRA)3' (both placed in exon 11; nt 2755 to 2738 and 2734 to 2711, respectively).

Primers and probe for beta-actin mRNA (GenBank accession number X00351) were forward primer 5'-TCCTTCCTGGGCATGGAG-3', reverse primer 5'- AGGAGGAGCAATGATCTTGATCTT-3', and TaqMan probe 5'(FAM)-CCTGTGGCATCCACGAAACTACCTTC-(TAMRA)3'. Probes were purchased from Applied Biosystems.

Real-Time RT-PCR
hTERT expression in tumors samples was measured by real-time quantitative RT-PCR, based on TaqMan methodology, using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). This technique allows, by means of fluorescence emission, identification of the cycling point when PCR product is detectable (threshold cycle or C_t value). As previously reported, the C_t value correlates to the starting quantity of target mRNA.²⁹

To normalize the amount of total RNA present in each reaction, we amplified the housekeeping gene beta-actin, which is assumed to be constant in both normal samples and ovarian carcinomas. Our results are expressed as relative levels of hTERT mRNA, referred to a sample called a calibrator, which is chosen to represent 1x expression of this gene. The calibrator used was an ovarian 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.

The amount of target, normalized to an endogenous reference (beta-actin) and relative to the calibrator is definite by the Ct method as described by Livak K (Sequence Detector User Bulletin 2, Applied Biosystems). Specifically, the formula is target amount = 2^-Ct, where Ct = {[Ct (hTERT sample) - Ct(beta-actin sample)] - [Ct(hTERT calibrator) - Ct(beta-actin calibrator)]}.

This method is based on the assumption that the target (hTERT) and the reference (beta-actin) display equal amplification efficiencies. To verify this condition, we checked Ct (Ct hTERT - Ct beta-actin) variations according to template dilution. To this end, we prepared a standard curve composed of five different dilutions of MCF7 total RNA (100, 25, 6.25, 1.6, and 0.4 ng). The slope of this curve was 0.049. To ensure the appropriate amplification efficiency, the slope of the standard curve should be less than 0.1.

Statistical Procedure
The variables measured in the study were investigated for association by using the ² and Fisher’s exact tests. To this aim, the variables were categorized as follows: FIGO stage (III versus IV), histologic type (serous versus nonserous), histologic grade (G1-G2 versus G3), residual disease ( 2 cm v > 2 cm), and hTERT expression ( 2.76 v > 2.76, which was the median value). The association of response to chemotherapy as a dependent variable with residual disease, hTERT expression, tumor stage, histologic type, and histologic grade was also investigated by using logistic regression analysis to account for the effect of the different variables. P < 0.05 was considered to be a significant value. The statistical analysis was performed using the Statview 5 (SAS Institute Inc, Cary, NC) statistical software run on a PowerPC G4 Macintosh computer.

	RESULTS

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Clinical and Pathologic Features
Of the 59 patients considered eligible for this study, 47 were at FIGO stage III, and 12 were at FIGO stage IV (Table 1). Histopathologic evaluation of ovarian tumors according to the World Health Organization classification³³ revealed that 45 tumors were serous ovarian carcinoma and 14 tumors were nonserous carcinoma. Six (10%) tumors were G1, 18 (31%) tumors were G2, and 35 (59%) tumors were G3. Twenty patients had residual disease less than 2 cm in greatest dimension at the time of initial surgery; 39 patients had residual disease more than 2 cm. After the completion of first-line chemotherapy, 27 patients were found to be in pathologic complete response, whereas 32 patients showed a partial response, a stable disease, or a progressive disease (Table 2).

In the 59 ovarian carcinomas, the range of hTERT levels varied greatly, ranging from 0.01 to 90.82. The values of hTERT expression were categorized as low or high, using a cutoff at 2.76, which was the value that represented the median value of the expression distribution. Thus, 28 tumors (47%) showed low hTERT expression, whereas 31 tumors (53%) had high hTERT expression (Table 1)

A significant correlation (P = .002) between hTERT levels and response to chemotherapy was documented: 74% (20 of 27) of pathologically complete responders showed high levels of hTERT expression in the primary untreated tumor, whereas 66% (21 of 32) of patients with partial or no response exhibited low hTERT expression levels (Table 2).

The association of hTERT expression with response to platinum-based chemotherapy, as a dependent variable, was also evaluated by logistic regression analysis to take into consideration the effects of the other covariates investigated, including FIGO stage, histologic type, histologic grade, and residual disease. The results obtained are shown in Table 3. Only residual disease and hTERT expression reached statistical significance as independent markers of response to chemotherapy (odds ratios, 13.455 and 7.586, respectively). When the two independent predictors (residual disease and hTERT expression) were combined, a strong predictive value emerged (Table 4): 18 (90%) of the 20 patients with residual disease more than 2 cm and low hTERT levels were partial responders or nonresponders. Conversely, 11 (92%) of the 12 patients with residual disease less than 2 cm and high hTERT levels showed a complete pathological response (² = 21.416; P < .00001).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

Little is known about the value of telomerase activity in predicting tumor chemosensitivity. It has been reported that telomere length and telomerase activity in cisplatin-resistant human ovarian cancer cell lines were generally reduced compared with their parent cell lines.⁴⁰ In addition, esophageal cancer cell lines with high telomerase activity were found to be more sensitive to cisplatin.²³ However, in other studies, no significant correlation was observed in vitro between telomerase activity or telomere length and cellular sensitivity to different DNA-damaging agents including cisplatin and doxorubicin.²⁴ Takahashi et al⁴¹ investigated the relationship between telomerase activity and response to chemotherapy in epithelial ovarian cancer patients in vivo and reported no significant differences between responders and nonresponders. In all of these studies, telomerase activity was evaluated by the TRAP assay. The different results obtained may be ascribed, at least in part, to technical problems related to the TRAP methodology. In particular, the presence of telomerase inhibitors in the tissue extract may block the enzyme during the TRAP assay. Evidence exists that quantitative determination of hTERT mRNA by real time RT-PCR is a powerful method to investigate the telomerase status and is superior in specificity and sensitivity to the evaluation of telomerase activity by the TRAP assay.^26,31–33 In addition, it has emerged that the expression of hTERT may not always parallel telomerase activity. Lin et al⁴² recently reported a decline of telomerase activity and increasing levels of hTERT mRNA and protein in three lymphoma cell lines after exposure to cisplatin. Further studies are required to elucidate this point.

To the best of our knowledge, no data are available on the relationship between hTERT mRNA expression and chemosensitivity to antineoplastic drugs. Our results represent the first evidence supporting the premise that hTERT expression is related to the response to chemotherapy in vivo. It is now well documented that TERT expression has profound and diverse effects on cellular molecular mechanisms. Forced expression of TERT in cardiac myocytes of mice promotes muscle cell proliferation and DNA synthesis, as evidenced by increased 5-bromo-2'-deoxyuridine incorporation, histone H3 phosphorylation, and prolongation of cyclin-dependent kinase 2 and cyclin-dependent kinase 6 activity.⁴³ Similarly, transgenic mice overexpressing mTERT in basal keratinocytes display increased incidence of epidermal tumors and increased skin wound healing potentials, suggesting that telomerase-transgenic skins have a proliferative advantage over controls.⁴⁴ In turn, it has been shown that TERT transcription is activated by cMYC,⁴⁵ whereas in TERT-driven immortalized human mammary epithelial cells, c-myc is expressed at increased levels, comparable to those seen in mammary breast cancer cell line HBL100.⁴⁶

Taken together, these data indicate that TERT expression plays a role in tumor cell proliferation and therefore may modulate the cellular response to chemotherapy, particularly for drugs that affect DNA synthesis and integrity.

It is generally recognized that an optimal cytoreductive surgery is the most important prognostic variable in advanced ovarian carcinoma.^47,48 In addition, several studies indicate that an optimal cytoreductive surgery is also significantly associated with a high probability that the patient will obtain a complete response to chemotherapy.⁴⁹ In our series of ovarian tumor patients, we have shown by logistic regression analysis that only residual disease and hTERT expression are independent predictors of response to chemotherapy (Table 3). This is particularly noteworthy in that never before in ovarian cancer patients has a biologic parameter been found to have a strong independent predictive value in a multivariate analysis including residual disease among the covariates. Because hTERT expression and residual disease were independent predictors of response to platinum-based therapy, we decided to evaluate the predictive value of a new parameter obtained by cumulating these variables. When these two independent variables were combined, they proved to have a strong predictive value: 90% of the patients with residual disease more than 2 cm and low hTERT levels were partial responders or nonresponders (P < .00001) (Table 4).

Many efforts have been devoted in recent years to the identification of molecular predictive markers in ovarian cancer. The evaluation of alterations of oncogenes and tumor-suppressor genes have had varying results relative to the prediction of the sensitivity or resistance of ovarian cancers to different chemotherapeutic drugs; currently, however, these molecular markers have no clinical utility. The primary purpose of surgery in patients with advanced ovarian cancer is cytoreduction. Indeed, patients with optimal cytoreductive surgery have an increased likelihood of achieving a complete clinical response to chemotherapy. Results of this study have shown that hTERT mRNA expression is a possible new predictive marker in ovarian cancer patients and that the level of hTERT mRNA expression combined with the residual disease could be an excellent parameter of response to platinum-based treatment. These combined parameters allowed us to identify more than 90% of the patients who responded to chemotherapy. Additional studies are required to validate these data and determine whether hTERT mRNA expression can predict the response to other antineoplastic drugs.

	NOTES

 
Supported by Consiglio Nazionale delle Ricerche (CNR), Progetto Strategico "Oncologia" (BRIDGE) CNR-MIUR, Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST 2000), and Associazione Italiana per la Ricerca su Cancro (AIRC).

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Submitted September 12, 2002; accepted December 27, 2002.

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