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Telomerase Expression Predicts Unfavorable Outcome in Osteosarcoma

From the St Jude Children's Research Hospital, Memphis, TN

Address reprint requests to Jeffrey S. Dome, MD, Department of Hematology/Oncology, St Jude Children's Research Hospital, 332 N Lauderdale St, Memphis, TN 38105; e-mail: jeff.dome{at}stjude.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: Osteosarcoma is distinct from most cancers in that the majority of osteosarcomas lack telomerase expression and use the alternative lengthening of telomeres (ALT) mechanism to maintain telomeres. Laboratory studies suggest that compared with ALT, telomerase expression is associated with increased tumor aggressiveness. We evaluated the clinical significance of telomerase expression in human osteosarcoma.

PATIENTS AND METHODS: Fifty-six osteosarcomas from 51 patients treated at St Jude Children's Research Hospital between 1982 and 2003 were evaluated for telomerase enzyme activity, mRNA expression of the catalytic component of telomerase (TERT), and presence of the ALT pathway.

RESULTS: Outcome analysis was based on TERT mRNA expression in the primary tumor samples from 44 patients. Fourteen primary tumors expressed TERT mRNA (32%; eight TERT only, six TERT and ALT) and 30 did not express TERT mRNA (68%; 29 ALT, one no ALT). Progression-free survival (PFS) was inferior in the TERT-positive group compared with the TERT-negative group (3-year estimates, 21.4% ± 9.5% v 63.7% ± 11.1%; P = .014). Likewise, overall survival was inferior in the TERT-positive group compared with the TERT-negative group (3-year estimates, 42.9% ± 12.2% v 70.0% ± 9.9%; P = .031). Among 31 patients with nonmetastatic disease at diagnosis, PFS was lower in the TERT-positive group compared with the TERT-negative group (3-year estimates, 33.3% ± 13.6% v 72.0% ± 11.5%; P = .092).

CONCLUSION: Telomerase expression in primary tumor samples is associated with decreased PFS and OS in patients with osteosarcoma. Additional studies are warranted to better define the clinical utility of this molecular marker.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Osteosarcoma is the most common primary bone tumor of childhood.^1,2 Despite significant treatment advances that occurred in the 1970s and 1980s, approximately 30% of patients with localized disease and 60% of patients with metastatic disease do not survive.^3-7 A greater understanding of osteosarcoma biology is critical for further strides to be made in osteosarcoma treatment.⁸ The telomere maintenance mechanism (TMM) has recently emerged as a promising prognostic marker and therapeutic target for osteosarcoma.^9,10

Telomeres are nucleoprotein structures that cap chromosome ends, protecting cells from inducing inappropriate DNA damage responses and from chromosomal loss and recombination.¹¹ With each round of replication, telomeres erode because of the inability of DNA polymerases to replicate the very ends of DNA. When telomeres become critically short, senescence or apoptosis is induced.¹² A hallmark of cancer cells is that they have unlimited proliferative potential. To achieve this, they must develop mechanisms to counteract telomere shortening.¹³ Most human cancers activate the enzyme telomerase to maintain telomeres, whereas a minority of cancers use a poorly defined recombination-based mechanism called alternative lengthening of telomeres (ALT). ^14-16

Osteosarcoma is distinct from most human cancers in that many osteosarcomas are telomerase-negative and use the ALT mechanism.^9,16,17 Although telomerase and ALT both maintain telomeres, they are not biologically equivalent. From the standpoint of telomere maintenance, ALT is characterized by telomeres that are both longer and more heterogeneous in size than are those in telomerase-expressing cells. From the standpoint of biologic behavior, laboratory studies indicate that compared with ALT, telomerase expression is associated with increased tumorigenicity and metastatic potential. In one study, ALT-dependent, ras-transformed GM847 human fibroblast cells were unable to form tumors when injected into immunodeficient mice unless the telomerase catalytic component (TERT) was ectopically expressed.¹⁸ In another study, ALT-dependent, sarcomatous cell lines derived from telomerase-null mice failed to yield macroscopic distant metastases after tail vein injection unless telomerase activity was reconstituted.¹⁹ Together, these studies provide a strong rationale to evaluate the effect of TMM on the biologic behavior of human tumors. In this report, we present the results of an analysis of the clinical significance of telomerase expression in human osteosarcoma.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
We conducted a retrospective study involving patients with osteosarcoma who were treated at St Jude Children's Research Hospital (Memphis, TN) between February 1982 and September 2003, and who had frozen tumors available in the St Jude tissue bank. This study was approved by the Institutional Review Board. A total of 64 tumors from 59 patients were available for analysis, including 54 primary and 10 relapsed tumors from various sites. Of the 54 primary tumors, 36 were from prechemotherapy biopsies or resections and 18 were from postchemotherapy resections. Two patients had primary tumor samples available from both the prechemotherapy biopsy and the postchemotherapy resection, two patients had samples available from diagnosis and recurrence, and one patient had tumor samples available from two different recurrences.

Molecular Studies
Tumors were flash frozen at the time of surgery and stored in liquid nitrogen or at –80°C until ready for analysis. Tumor samples were analyzed for TERT mRNA expression, telomerase enzyme activity, and ALT expression. Experiments were conducted in a blinded manner; data on clinical characteristics and outcome of patients were not revealed until after the molecular assays were completed.

TERT mRNA Expression
Total RNA was extracted by Tri-reagent (Molecular Research Center, Cincinnati, OH) and cDNA was synthesized using 2 µg of total RNA, random hexamer, and Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). Analysis of TERT mRNA expression was performed by quantitative real-time polymerase chain reaction (PCR), using primers that detect all TERT alternative splice forms.²⁰ Standard curves were generated from serial dilutions of cDNA derived from the TERT-positive neuroblastoma cell line SY5Y. mRNA expression of the gene GAPDH was also analyzed for each tumor as an indicator of RNA quality—tumors with inadequate GAPDH expression compared with the standard curve were excluded from the study. Tumors were considered to have detectable TERT mRNA if the C_t value (the number of PCR cycles required for the fluorescence level to exceed a predefined threshold level) was less than 39 cycles. This cutoff was based on TERT mRNA expression data obtained in our laboratory from normal human fibroblasts and kidney tissue, which are considered to be TERT-negative.²¹

Telomerase Enzyme Activity
Total protein was extracted with 0.5% 3-[3-cholamidopropyl-dimethylammonio]-1-propane-sulfonate lysis buffer. The telomeric repeat amplification protocol (TRAP) assay was used to detect telomerase enzyme activity and was performed as described previously.²¹ Tumors were considered to have detectable telomerase activity if the TTAGGG telomere amplification products were detected by enzyme-linked immunosorbent assay (TeloTAGGG PCR enzyme-linked immunosorbent assay kit; Roche Diagnostics, Indianapolis, IN) or by gel electrophoresis (TRAPeze kit; Chemicon, Temecula, CA). A range of 125 ng to 2 µg total protein was used for initial assays; lysates without detectable enzyme activity were reanalyzed at higher and lower protein concentrations to exclude the possibilities of low enzymatic activity level and tissue inhibitors of telomerase, respectively.

ALT Expression
Southern blot analysis was performed to assess telomere restriction fragment length. DNA was extracted using the DNeasy tissue purification kit (Qiagen, Valencia, CA), and telomere restriction fragment length analysis was performed using standard techniques.¹⁷ Wilms tumor samples were used as telomerase-positive and ALT-negative controls,²¹ and the osteosarcoma cell line Saos2 was used as an ALT-positive and telomerase-negative control.¹⁶ Samples were considered to have ALT if they exhibited high molecular weight telomeres ( 23.1 kb) with significant heterogeneity in length.

Statistical Analyses
Fisher's exact test was used to assess the relationship of telomerase status with patient sex and stage of disease at presentation. An exact Wilcoxon rank sum test was used to examine the relationship between patient age at diagnosis and telomerase status. Progression-free survival (PFS) was defined as the time interval from date of diagnosis to date of relapsed or progressive disease. Patients who did not experience relapse or disease progression were considered censored at their last follow-up date, with the exception of one patient who had a second malignancy before relapse; this patient was censored at the date of second malignancy. PFS was estimated using the method of Kaplan and Meier²²; PFS distributions were compared using the exact log-rank test, as were the overall survival distributions. In an effort to take into account the competing nature of the second malignancy as an event, we also estimated the cumulative incidence of progression/relapse (data not shown).²³ Second malignancy was considered a competing event in this analysis. Gray's test was used to compare the cumulative incidence curves by telomerase status.²⁴ Results using both methods were similar.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Characteristics of the Study Population
A total of 64 frozen tumors were available for analysis. Eight tumors were excluded because of inadequate quality or insufficient quantity of RNA. The remaining 56 tumors (46 primary and 10 relapsed) from 51 patients were evaluated in this study. Patients were treated according to the Pediatric Oncology Group 8107 study (n = 7; upfront surgery and adjuvant bleomycin, dactinomycin, cyclophosphamide, high-dose methotrexate [HDMTX], doxorubicin, and cisplatin—although this protocol included a nonchemotherapy arm, all seven patients included in the current study received chemotherapy),²⁵ the St Jude OS-86 study (n = 13; neoadjuvant ifosfamide, HDMTX, and doxorubicin, and the same drugs plus cisplatin postoperatively), the St Jude OS-91 study (n = 4; neoadjuvant ifosfamide and carboplatin, and the same drugs plus doxorubicin and HDMTX postoperatively),²⁶ the St Jude OS-99 study (n = 19; neoadjuvant and adjuvant ifosfamide, carboplatin, and doxorubicin for patients with localized disease, and neoadjuvant irinotecan, ifosfamide, and doxorubicin plus adjuvant ifosfamide, carboplatin, and doxorubicin for patients with metastatic disease at diagnosis), with surgery only (n = 1), or according to best clinical management (n = 7). The median duration of follow-up in patients without events (n = 21) was 46.8 months (range, 4.5 to 252.3 months). All survivors were seen or contacted within the last 15 months.

Distribution of TMMs
Representative real-time PCR TERT mRNA expression analyses, telomerase activity (TRAP) assays, and Southern blots are depicted in Figure 1. Table 1 illustrates the concordance between the TERT mRNA and TRAP assays. Twelve samples had detectable TERT mRNA expression but undetectable telomerase activity. One sample had detectable telomerase activity without TERT mRNA expression, which was most consistent with degradation of the TERT transcripts.

View larger version (56K): [in this window] [in a new window]   Fig 1. Representative telomere maintenance mechanism assays. (A) TERT mRNA expression by quantitative real-time polymerase chain reaction. OS 14 was TERT-positive, OS 9 to 13 were TERT-negative. (B) Telomeric repeat amplification protocol (TRAP) assay for telomerase enzyme activity. Only tumor OS 14 demonstrated telomerase activity in this example, as manifested by the 6-bp telomeric repeat ladder. (C) Southern blot of telomere restriction fragment length. ALT, alternative lengthening of telomeres; pos, positive; neg, negative. OS, osteosarcoma.

On the basis of the TERT expression analysis and Southern blot data, each of the 46 primary tumors from 44 patients was placed into one of four categories: TERT only (n = 8), ALT only (n = 31), both TERT and ALT (n = 6), and neither TERT nor ALT (n = 1). Two patients had primary tumor samples available from both the prechemotherapy biopsy and the postchemotherapy resection. One of these patients had both TERT and ALT in the biopsy sample and only ALT in the postchemotherapy resection. The other patient had only ALT in both samples. For the subsequent analyses, only the prechemotherapy biopsy samples for these two patients were considered. The patient characteristics according to TMM for the 44 patients are summarized in Table 2.

Ten samples taken at relapse were analyzed for TMM. Although only 14 of 44 primary osteosarcoma samples (31.8%) were TERT-positive, six of 10 relapse samples (60%) were telomerase-positive (P = .147). Two patients had tumor material from both diagnosis and recurrence; in both patients, both the diagnostic samples and samples from patients who had experienced relapse were TERT-negative and ALT-positive. In one patient who had two samples available after relapse, both showed evidence of telomerase expression and ALT activation.

TMMs and Patient Outcome
Analysis of patient outcome in relation to TMM was performed on the 44 patients whose primary tumors were evaluated. First events included progressive or relapsed disease in 29 patients and second malignancy (acute myelogenous leukemia) in one patient. PFS was inferior in the TERT-positive group compared with the TERT-negative group (P = .014); 3-year PFS estimates were 21.4% ± 9.5% and 63.7% ± 11.1%, respectively (Fig 2A). Similar results were found when overall survival was analyzed for the 44 patients whose primary tumors were evaluated for TERT expression (P = .031). Three-year overall survival estimates were 42.9% ± 12.2% for patients with TERT-positive tumors and 70.0% ± 9.9% for patients with TERT-negative tumors.

View larger version (15K): [in this window] [in a new window]   Fig 2. Progression-free survival for osteosarcoma patients with evaluable primary tumor samples. (A) All patients; (B) patients with localized osteosarcoma; (C) all patients by TERT/alternative lengthening of telomeres (ALT) status; excludes one patient with a TERT-negative and ALT-negative tumor. Pos, positive; neg, negative.

Most studies of telomerase expression as a prognostic marker for human cancer evaluated telomerase expression, but not ALT. Because our data indicated that telomerase expression and ALT are not mutually exclusive, we evaluated the impact of coexpression of these two TMMs on patient outcome. When the entire group of primary tumors (n = 44) was analyzed according to both telomerase and ALT status, a significant difference in PFS was detected (P = .012). Estimates of 3-year PFS were 50.0% ± 17.7% for TERT-positive and ALT-positive patients and 62.3% ± 11.5% for TERT-negative and ALT-positive patients, whereas all TERT-positive and ALT-negative patients had experienced disease relapse within 3 years (Fig 2C). Interestingly, the single tumor with neither TERT nor ALT expression (not included in Fig 2C) was an intermediate-grade periosteal osteosarcoma. The corresponding patient was treated with surgery only and remains disease-free 13 years after diagnosis.

Although we elected to base our outcomes analyses on TERT mRNA expression, telomerase enzyme activity has historically been regarded as the gold standard measurement of telomerase expression. We therefore evaluated the prognostic significance of detectable telomerase enzyme activity in the primary osteosarcoma samples (n = 44). PFS was inferior in patients whose tumors had detectable telomerase activity compared with patients whose tumors lacked telomerase activity (P = .073); 3-year PFS estimates were 20.0% ± 12.6% and 53.4% ± 9.7%, respectively (Fig 3). Although both TERT mRNA expression and telomerase activity were predictive of recurrence, our results indicate that TERT mRNA expression was the stronger prognosticator.

View larger version (13K): [in this window] [in a new window]   Fig 3. Progression-free survival for osteosarcoma patients with evaluable primary tumor samples by telomerase enzyme activity (telomeric repeat amplification protocol [TRAP] assay).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

Our observation that telomerase expression was associated with increased risk of tumor recurrence recapitulates the findings in animal models that telomerase expression confers an aggressive clinical phenotype.^18,19 However, several important differences between the laboratory-induced tumors and naturally occurring human osteosarcomas are apparent. First, transformed GM847 fibroblasts with the ALT phenotype were unable to form tumors unless TERT was ectopically expressed.¹⁸ In contrast, the human data demonstrate that telomerase expression is not required to generate tumors, given that the majority of osteosarcomas in our series were telomerase-negative. Second, the ALT cells derived from telomerase-null mice were unable to form macroscopic distant metastases without reconstitution of telomerase activity.¹⁹ Consistent with this finding, our study and a previous report¹⁰ showed that the majority of metastatic osteosarcomas have detectable TERT expression or telomerase activity. However, in contrast to the mouse model, telomerase expression was not an absolute requirement for macroscopic metastasis; some of the metastatic tumors in our series were telomerase-negative.

An important determinant of clinical outcome that the animal models did not consider was the effect of telomerase expression on the chemotherapy responsiveness of the tumors. We observed a higher prevalence of TERT expression in postchemotherapy samples compared with prechemotherapy samples, which supports the idea that TERT expression may be a marker of resistance to chemotherapy. Consistent with this premise, a study of human breast epithelial cell lines showed that telomerase inhibition produced a modest (two-fold) increase in sensitivity to doxorubicin.³² Other studies demonstrated that telomerase inhibition sensitized human glioblastoma cell lines to cisplatin,³³ leukemia cell lines to cisplatin,³⁴ and lung carcinoma cell lines to cisplatin, docetaxel, and etoposide.³⁵ It is interesting that these in vitro studies showed an association between telomerase expression and resistance to agents commonly used in the treatment of osteosarcoma.

The optimal assay for telomerase expression remains an open question. We found that both TERT mRNA expression by real-time reverse transcriptase PCR analysis and telomerase activity by TRAP analysis were associated with unfavorable outcome in osteosarcoma, although TERT mRNA expression was the stronger of the two prognostic indicators. The TRAP assay is the most widely used measure of telomerase expression. Because it provides a functional readout of the enzyme, the telomerase activity assay is advantageous because it accounts for all levels of telomerase regulation (transcription, alternative splicing, and post-translational modifications). However, telomerase enzyme activity is subject to inactivation with heat and time, which is important to consider if this assay is to be validated in a multi-institutional setting and ultimately used in routine clinical practice, where procurement and processing of tumor may not be uniform. Unlike RNA degradation, which can be accounted for by assessing expression of housekeeping genes, there is no established method to correct for inactivation of telomerase activity. To circumvent the shortcomings of the TRAP assay, we were the first group to assess whether expression levels of TERT mRNA transcripts can serve as a prognostic indicator for human cancer.²¹ The primary advantages of measuring TERT mRNA expression are that this assay is amenable to the technique of real-time PCR and that RNA degradation can be detected with the amplification of housekeeping genes. Another advantage of assessing TERT mRNA expression is mounting evidence that TERT protein has tumorigenic effects beyond its role in lengthening telomeres. Some of these effects may be independent of telomerase activity, so measuring telomerase activity exclusively may miss an important biologic readout.¹⁸ Indeed, our study of several hundred Wilms tumor samples found that measurement of TERT mRNA expression was superior to telomerase enzyme activity in predicting tumor recurrence.^21,27 We recommend that future studies include both assays until this question is resolved.

Ulaner et al⁹ recently evaluated TMM in primary osteosarcoma samples from 60 patients treated at Memorial Sloan-Kettering Cancer Center. In this study, the telomerase and ALT status of the primary tumors were as follows: telomerase-positive and ALT-negative (n = 11); telomerase-positive and ALT-positive (n = 17); telomerase-negative and ALT-positive (n = 21); and telomerase-negative and ALT-negative (n = 11). The samples without evidence of telomere maintenance mechanism (neither telomerase nor ALT) were associated with better overall survival compared with those with either telomerase or ALT, but the presence of telomerase activity was not predictive of survival. Several factors may explain the discordance in findings between our study and the previous study. First, the median age at diagnosis in our study was 13 years, whereas that in the Ulaner study was 21 years. Biologic differences between adult and pediatric osteosarcomas, such as the increased prevalence of surface osteosarcomas in older patients, may have accounted for the high frequency of tumors without a TMM in the Ulaner study. Second, most of our samples were derived from prechemotherapy biopsies; it is unclear whether the samples in the Ulaner study were obtained pre- or postchemotherapy. As our results indicated, the timing of the surgical specimen may influence the readout of TMM. Finally, different laboratory methodologies were used in the two studies. For example, we used a real-time PCR-based TERT mRNA assay, whereas the Ulaner group used conventional PCR. A large confirmatory study of TMMs in human osteosarcoma is warranted to reconcile the differences between these studies.

The coexistence of ALT and telomerase is well documented in ALT cell lines that were genetically engineered to express telomerase,^36-39 but has only recently been demonstrated to occur naturally in human tumors.^9,40 Because most studies of telomerase expression in human cancer were not designed to evaluate ALT, the prevalence of ALT in human cancer may be underestimated. Our data indicated that patients with TERT-positive and ALT-positive tumors had superior outcomes compared with patients with TERT-positive and ALT-negative tumors (Fig 2C). It is possible that the ALT phenotype acts to mitigate the clinical aggressiveness conferred by TERT expression, although the small number and heterogeneity of tumor samples in our study precludes us from drawing a firm conclusion in this regard. It would be worthwhile to evaluate clinical outcome with respect to both telomerase expression and ALT activation in future studies of osteosarcoma and other tumor types.

In summary, our results indicate that telomerase expression is associated with unfavorable clinical outcome in patients with osteosarcoma. Our study was limited by its retrospective nature and relatively small sample size. Subgroup analyses of patients with nonmetastatic disease at initial presentation and patients with prechemotherapy primary tumor samples showed decreased PFS in the telomerase-positive group, but did not achieve statistical significance. Additional study of telomere maintenance mechanisms is warranted to better define the clinical utility of this molecular marker in human osteosarcoma.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The authors indicated no potential conflicts of interest.

	Acknowledgment

 
We thank Carol A. Bockhold and Jing Wu for their assistance with the laboratory assays and Mickey Cain for assistance with data collection.

	NOTES

 
Supported by grants from the National Institutes of Health (CA87903 and CA21765) and by the American Lebanese Syrian Associated Charities of St Jude Children's Research Hospital.

Presented in part at the American Association for Cancer Research Special Conference on the Role of Telomeres and Telomerase in Cancer, December 7-11, 2002, San Francisco, CA.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

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Submitted March 4, 2004; accepted June 19, 2004.

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