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© 2003 American Society for Clinical Oncology Association Between Telomerase Activity and Outcome in Patients With Nonmetastatic Ewing Family of Tumors
From the Department of Molecular Oncology, Felsenstein Medical Research Center, and the Department of Pediatric Hematology/Oncology, Schneider Children’s Medical Center of Israel, Petah Tikva; and Sourasky Medical Center, Sackler Faculty of Medicine, and Statistical Laboratory, School of Mathematics, Tel Aviv University, Tel Aviv, Israel. Address reprint requests to Smadar Avigad, PhD, Molecular Oncology, Felsenstein Medical Research Center, Rabin Medical Campus, Petah Tikva 49100, Israel; e-mail: savigad{at}post.tau.ac.il.
Purpose: Telomerase is considered a molecular marker for malignancy. The aim of this study was to determine telomerase activity (TA) as a prognostic factor at diagnosis and as a marker for minimal residual disease during therapy and follow-up in nonmetastatic Ewing family of tumors (EFT). Patients and Methods: Primary tumor specimens and 97 peripheral blood (PBL) samples from 31 EFT patients were analyzed for TA by the Telomeric Repeat Amplification Protocol (TRAP assay). The telomerase catalytic subunit (human telomerase reverse transcriptase [hTERT]) gene expression was evaluated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and telomere length was determined by Southern blotting. The presence of the EFT chimeric transcripts was analyzed by RT-PCR. Correlations with progression-free survival were evaluated. Results: At diagnosis, TA in primary tumors did not correlate with outcome. During therapy and follow-up, highly significant correlation was observed between high TA in PBL samples and adverse prognosis (P < .0001). None of the patients harboring low TA progressed, with a long follow-up (median, 60 months) and a progression-free survival (PFS) of 100%. In nine patients, high TA actually could predict relapse, long before overt clinical relapse. The group of patients with high TA and positive RT-PCR had the most adverse outcome; PFS of 20% (P = .0025). TA was found to be a better prognostic factor than RT-PCR and histopathologic response at surgery. Conclusion: The results suggest that TA is a significant prognostic variable, superior to the established clinical prognostic parameters during therapy and tumor surveillance. It could be used in combination with RT-PCR for a new risk classification.
EWING’S SARCOMA (ES) is the second most common primary malignant bone tumor in children and adolescents, and it belongs to a group of neuroectodermal tumors known as the ES family of tumors (EFT).1 All EFT share one of the following specific translocations: t(11;22), t(21;22), t(7;22), t(17;22), and t(2;22).2–7 These translocations result in the fusion of the EWS gene on 22q12 with different ETS oncogene superfamily-related genes, FLI-1 (85% to 90%), ERG (5% to 10%), ETV-1, E1AF (1%), and rarely, FEV. The various EWS rearrangements seem to be pivotal events in EFT tumorigenesis and these genetic alterations are considered distinct diagnostic features of these tumors.3 At diagnosis, approximately 25% of patients have detectable metastatic disease, but the majority of patients may have micrometastases, undetectable by conventional methods, as evidenced by a very low cure rate with local therapy. The standard care includes combination of aggressive chemotherapy, radiotherapy, and surgery. Initial response to therapy, assessed by histologic analysis of surgical specimens as the degree of tumor necrosis, has become an accepted valid prognostic factor, especially in localized tumors of the extremities. Despite advances in therapy, more than 50% eventually relapse, even after 5 years.8 Several groups have used reverse transcriptase polymerase chain reaction (RT-PCR) for the accurate diagnosis and for determining the presence of occult micrometastases in bone marrow (BM) or peripheral blood (PBL) at diagnosis and during therapy.9,10 Some studies have shown an association between outcome and the presence or absence of tumor cells in BM at diagnosis and in PBL during therapy,11,12 and recently in both at diagnosis.13 Telomeres are specialized structures at the ends of eukaryotic chromosomes, consisting of hundreds of repeated hexanucleotides (TTAGGG)n.14 Telomeres protect the chromosomes from DNA degradation, end-to-end fusions, rearrangements, and chromosome loss.15,16 Due to the inability of DNA polymerase to replicate the ends of double-stranded DNA, known as the "end replication problem," telomeres are progressively shortened with each round of cell division, leading to cellular senescence.17 A reduction in telomere lengths has been described in a wide range of human cancers,18–22 and progressive telomere shortening has been shown to contribute to genomic instability.23 Telomerase expression is a characteristic feature of permanent cell lines and a vast majority of human malignant tumors, whereas most normal somatic tissues do not express telomerase.24 Recent findings support the concept that activation of telomerase may be a critical, if not obligatory step in the development of cancer.25 Telomerase, a multisubunit ribonucleoprotein, is capable of adding telomeric DNA to the ends of linear chromosomes using its RNA template.26 Previous studies have found telomerase activity (TA) to be a useful diagnostic and prognostic marker in various neoplasms.27–30 High levels of TA have been found in a variety of malignancies in adults, particularly in gastric, prostate, colon, ovarian cancer, and brain tumors.21,31–34 Among pediatric solid tumors, the most studied one is neuroblastoma, where TA has been found as an independent prognostic factor.35,36 Recently, the telomerase catalytic subunit (human telomerase reverse transcriptase [hTERT]) was cloned, and its expression revealed a strong correlation with TA.37–39 Early detection and treatment of relapse may prevent or delay tumor progression. The search for new molecular markers for assessing the individual patient’s prognosis is of vital importance. We postulated that telomerase might be a good candidate for these goals. In our study, 31 EFT patients were analyzed for TA, hTERT expression and telomere length in primary tumors, and in PBL at diagnosis, during therapy and during surveillance; the results were correlated with clinical parameters and outcome.
Patients Twenty-five primary tumor specimens, 97 PBL samples, and two BM samples were obtained from 31 EFT patients with nonmetastatic disease who were admitted to the Pediatric Hematology/Oncology Department at Schneider Children’s Medical Center (Petah Tikva, Israel). Informed consent was obtained from the patients or their guardians, and the local and national ethics committees approved the research project. All patients were treated according to the protocol for EFT, including a combination of aggressive chemotherapy, radiotherapy, and surgery. Median age at diagnosis was 14 years (range, 0.3 to 27 years). Twelve patients were females, and 19 were males. Primary sites were: nine pelvis, three sacrum, 14 limbs, and five others. Response to therapy was defined by histopathologic response and assessed by percentage of tumor necrosis at the time of surgery (limb salvage procedure) following neoadjuvant chemotherapy and radiotherapy. Nineteen patients were good responders (  90% tumor necrosis at the time of resection), nine were poor responders (< 90% of tumor necrosis) and three had an inoperable tumor (primary site: cranium, chest sacrum). Nine patients (29%) progressed, four locally, three in the lung, and two in distal bone. Median follow-up was 60 months (range, 7 to 153 months). All tissue samples were snap-frozen in liquid nitrogen immediately after surgery, and all samples were stored at -80°C until use. All patients were clinically well with no evidence of disease at the time before relapse. In addition, PBL samples from 20 age-matched healthy individuals were included as controls.
TA
Telomere Length Determination
Telomerase Catalytic Subunit (hTERT) Expression Analysis
RT-PCR Analysis
Statistical Analysis
At Diagnosis TA. TA was tested on 25 EFT primary tumors and nine PBL samples available at diagnosis. TA was detected in 84% (21 of 25) and in 67% (six of nine) of the tumor and PBL samples respectively. The telomerase positive tissue extracts produced a characteristic 6-bp ladder as shown in Figure 1  .
High TA (HTA) was observed in 71% (15 of 21) of the tumors. Of these patients, eight relapsed (53%), while in the 10 low-TA patients, four (40%) relapsed. Evaluation of progression-free survival (PFS) analysis did not reach a significant difference between high and low TA groups. Among the PBL samples, 56% (five of nine) showed high TA; of these patients, 40% (two of five) relapsed and as in the primary tumors, had no significant correlation with clinical outcome.
By Fisher’s exact test, TA was correlated to known clinical parameters such as age at diagnosis, primary site of tumor, sex, and response to therapy (Table 1
hTERT expression. Twenty tumor samples were tested for expression of hTERT mRNA (Fig 2 ). High expression of hTERT ( 100 copies) was identified in 55% (11 of 20), and it significantly correlated with high TA as nine of the 11 samples overexpressing hTERT mRNA, also presented high TA (P = .038, Table 1 ).
Telomere length. Twelve tumor DNA samples were analyzed using Southern blot for telomere length (Fig 3 ). Eight of 12 tumors exhibited changes in telomere lengths: five shorter and three longer than the median benign samples. Alteration in telomere length was associated with high TA in five (71%) of seven tumors. No significant correlation was found between telomere length and TA.
During Therapy and Follow-Up TA. A retrospective analysis of TA was performed in 88 PBL and two BM samples from 26 patients at different time points during a long follow-up ranging from 7 to 153 months (median, 60 months). One to 10 samples were analyzed per patient (mean, five samples per patient). For the PFS and the prediction of relapse, the last available sample per patient was evaluated (24 PBL and two BM samples).
TA from the last sample analyzed was correlated with the same clinical parameters, as at diagnosis (Table 2
High TA was observed in 11 patients (42%). Nine (82%) of 11 patients with high TA relapsed, while none of the 15 patients with low TA did. Highly significant correlation was observed between TA in PBL samples and PFS. Low TA patients had 100% PFS, while high-TA patients had 18% PFS (P < .0001; Fig 4 ).
Since we analyzed many samples during a long follow-up, it was possible to observe the kinetics of TA. Low TA in all samples analyzed from the same patient was detected in seven patients (27%); high TA was detected in all samples from seven patients (27%). Samples from eight patients (31%) turned from high to low TA. In four patients, low TA turned to high TA. All seven patients that consistently exhibited high TA relapsed. Of the four patients who had a shift to high TA, two relapsed. None of the patients who consistently harbored low TA or turned to low TA relapsed.
In nine patients, high TA could predict relapse before overt clinical relapse (Table 3
Twenty-five samples of PBL were analyzed for both TA and the presence of the chimeric transcript EWS-FLI1 by RT-PCR (Table 4 ). Nineteen (76%) of 25 samples were in concordance, meaning they were RT-PCR positive with high TA, or RT-PCR negative with low TA. Of the six samples that were not in concordance, three were RT-PCR positive with low TA, of which no patient has progressed, and three were RT-PCR negative with high TA, of which one patient progressed. Of the 19 concordant results, nine relapsed, all with high TA and positive RT-PCR. A significant correlation between TA and RT-PCR was observed (P = .030), and their predictive value of relapse was similar.
Age older than 12 years at diagnosis (P = .01, data not shown), and a greater than 90% tumor necrosis at the time of tumor resection (P < .0001; Fig 5 ) were favorable prognostic factors for predicting PFS. We attempted to identify which independent factor had a significant influence on survival. It was not mathematically possible to establish the Cox multivariate study including TA, because one subset of events was empty (no cases of relapse within the group of low telomerase samples). Therefore, we performed Kaplan-Meier analysis of PFS with TA in the favorable group of patients who had more than 90% of tumor necrosis (Fig 6), and with both TA and positive/negative RT-PCR in this group (Fig 7).
Our data show that for patients with greater than 90% tumor necrosis, low TA was a significant favorable parameter and high TA was an adverse prognostic parameter regardless of their initial good histopathologic response (P = .0006), and therefore, TA is a better prognostic indicator. The same results were achieved when both TA and RT-PCR were analyzed. No relapses occurred in patients with low TA and negative or positive RT-PCR, with 100% PFS at a median follow-up of 60 months. High TA and negative RT-PCR determined an intermediate-risk group with 50% of 5-years PFS. The most adverse-risk group was high TA and positive RT-PCR with only 20% PFS (P = .0025). Thus, the combination of TA and RT-PCR results could identify three risk groups: good-, intermediate-, and high-risk. When we performed a multivariate analysis with high TA cases only, tumor necrosis was a significant protective parameter (P = .04) while RT-PCR lost its significance (P = .1; data not shown). According to our data, TA turned to be a significant prognostic factor in EFT patients during follow-up.
hTERT expression.
The expression of hTERT was tested in 22 PBL samples of EFT patients during follow-up. The samples of seven patients expressed high hTERT (>100 copies), four of them relapsed (57%), while of the 15 patients whose samples expressed low hTERT (<100 copies), only two relapsed (13%). PFS comparing patients with high and low hTERT expression was statistically significant (P = .038; Fig 8
Normal cohort. All 20 normal PBL samples studied showed no detectable TA by TRAP assay.
The present study is, to the best of our knowledge, the first report on TA and its possible prognostic significance in patients with EFT over a long period of follow-up (7 to 153 months; median, 60 months). Moreover, this is the first extensive study that attempted to evaluate TA in PBL as a marker for minimal residual disease (MRD). Many studies in various other malignancies showed that high TA in primary tumors at diagnosis was associated with unfavorable clinical features, and some of them revealed a significant correlation between TA and survival in cancer patients.21,22,27–36 In some of these studies, hTERT expression also correlated with TA. In contrast to these reports, we did not find in EFT primary tumors any correlation between TA and clinical outcome at diagnosis. Even so, there was a significant correlation between TA and hTERT expression in these tumors (P = .038). Numerous studies have shown that changes in telomere lengths are associated with unfavorable outcome. A reduction in telomere length has been reported in several malignancies: colorectal carcinoma, renal cell carcinoma, and childhood leukemia.18,19,42 A correlation between shortened telomeres and TA was also established for ovarian and gastric cancer.21,31 However, telomeres can be stabilized at virtually any length. Indeed some cancers, such as basal cell carcinomas, may have unchanged or elongated TRFs, whereas liposarcomas show a characteristically large variation in telomeric length, shortened or elongated in comparison with normal tissues.43,44 This could explain why, in contrast to enzyme activity, relatively few studies regard TRF measurements as a useful prognostic factor. TRFs in EFT revealed a heterogeneous pattern. In our small cohort study, we found changes in TRF in 69%; six had shortened TRFs, three had elongated ones, and the rest were unchanged. Seventy-five percent of the tumor samples with changes in TRF also expressed high TA. The three patients whose tumors showed elongated TRFs are well with no evidence of disease, while three of the six patients whose tumors exhibited shortened TRFs had relapsed. However, of the four patients with unchanged TRFs, three relapsed. Our preliminary data suggest that elongated TRFs may be associated with a more favorable outcome in EFT patients, but the study should be extended to a much larger cohort. In order to evaluate the potential use of TA as a marker for MRD, we analyzed PBL samples from 20 healthy donors by the TRAP assay and no TA could be detected in any of them, suggesting that TA determination could be used in PBL samples. Eighty-eight PBL and two BM samples were analyzed for TA during follow-up, many of which were also tested for hTERT expression. In the present study, a highly significant correlation between high TA and poor outcome was observed (P < .0001), and TA could distinguish between patients with a high- or low-risk of relapse. While 82% of the patients with high TA relapsed, none of the low TA group did. In nine patients, high TA actually could predict relapse long before overt clinical relapse (2 to 15 months). Tumor necrosis, which is considered a strong clinical prognostic factor, was also highly correlated with outcome. Five patients who were good responders still relapsed, and all of them harbored high TA. This suggests that high TA during follow-up significantly correlates with clinical outcome. The data that high TA in the PBL could be used as a reliable prognostic marker has additional added value for monitoring residual disease, particularly in children.
Based on the presence of unique chimeric transcripts in EFT cells, the feasibility of RT-PCR for follow-up of MRD in EFT patients has been demonstrated recently by several groups9–11 including ours (manuscript submitted for publication). In 25 patients, both TA and RT-PCR analysis of the chimeric transcripts were performed. A significant correlation between TA and RT-PCR was observed (P = .030). In the group of patients with over 90% tumor necrosis at surgery, TA turned to be a much stronger indicator when comparing the value of TA and RT-PCR in predicting the risk to relapse, as shown in Figure 7 Qualitative RT-PCR is known to be a very sensitive method which can detect as little as one tumor cell in 106 normal cells,45 therefore, positive RT-PCR samples may represent residual tumor cells which do not yet indicate evidence for early relapse. The sensitivity of detection TA by the TRAP assay is 1:104.24 Therefore, a sample harboring high TA may imply a higher number of tumor cells which by now already indicate relapse. This may explain the discrepancy between RT-PCR and TA results. A significant correlation between expression of hTERT and TA was observed during follow-up, and it was associated with adverse prognosis (P = .038). High TA during follow-up was a significant prognostic factor for PFS and was more predictive than the known clinical prognostic parameters. Low TA during follow-up may indicate a favorable localized group with low-risk of relapse. Although the number of patients in our study is small, our results suggest that TA could be used as a marker for monitoring EFT patients during follow-up. This study should be extended to a larger cohort of patients to further validate our observations. TA analysis, performed at precise time points, could be used for stratification of patients at risk of relapse.
The authors indicated no potential conflicts of interest.
This work was supported by the Josefina Maus and Gabriela Cesarman Maus Chair for Pediatric Hematology Oncology (R.Z.). This work was performed in partial fulfillment of the requirements for the PhD degree of Anat Ohali, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
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