VOLUME

22

NUMBER

10

MAY

15

2004

JOURNAL OF CLINICAL ONCOLOGY

O R I G I N A L

R E P O R T

Telomere Length and Human Telomerase Reverse Transcriptase Expression As Markers for Progression and Prognosis of Colorectal Carcinoma
Ralf Gertler, Robert Rosenberg, Dominik Stricker, Jan Friederichs, Axel Hoos, Martin Werner, Kurt Ulm, Bernhard Holzmann, Hjalmar Nekarda, and Joerg-Ruediger Siewert
From the Chirurgische Klinik und Poliklinik, Institut fu r Pathologie und Pathologische Anatomie, and Institut fu r Medizinische Statistik und Epidemiologie, Klinikum rechts der Isar der Technischen Universita t Mu nchen, Munich, Germany. Submitted September 26, 2003; accepted February 27, 2004. Authors disclosures of potential conicts of interest are found at the end of this article. Address reprint requests to Ralf Gertler, MD, Chirurgische Klinik und Poliklinik, Klinikum rechts der Isar der Technischen Universita t Mu nchen, Ismaninger Str 22, 81675 Mu nchen, Germany; e-mail: gertler@ nt1.chir.med.tu-muenchen.de. 2004 by American Society of Clinical Oncology 0732-183X/04/2210-1807/$20.00 DOI: 10.1200/JCO.2004.09.160

Purpose Maintenance of telomeres through reactivation of telomerase is a prerequisite for tumors to preserve their ability to proliferate. The purpose of this study was to evaluate telomere length and human telomerase reverse transcriptase (hTERT) expression as markers for progression and prognosis of colorectal carcinoma. Patients and Methods Telomere length and hTERT expression were analyzed in matched cancer and adjacent noncancer mucosa samples from 57 patients with R0-resected colorectal carcinoma. The median follow-up time was 76 months. Results Telomere length and hTERT expression correlated signicantly in cancer tissues and adjacent mucosa samples (r 0.52, P .001; and r 0.54, P .001, respectively). Overall, cancer tissue had shorter telomeres than adjacent mucosa (P .001). Only in noncancer tissue did telomere length decrease with age (r 0.36; P .01). Telomere length in cancer tissue was signicantly correlated with tumor stage (P .01), with longer telomeres in advanced tumors. Patients with ratios of telomere length in cancer to noncancer tissue greater than 0.90 had a signicantly poorer overall survival compared with patients with smaller telomere length ratios (P .002). In multivariate analysis, the telomere length ratio proved to be of independent prognostic value (P .03). Conclusion Telomeres in colorectal carcinoma tissue were signicantly shorter compared with adjacent normal mucosa as an indication for extensive cell proliferation. The correlation with tumor stage and patient survival suggest that hTERT-mediated telomere stabilization may be critical for progression and prognosis of colorectal carcinoma. J Clin Oncol 22:1807-1814. 2004 by American Society of Clinical Oncology

INTRODUCTION

Telomeres constitute the ends of eukaryotic chromosomes.1 In somatic cells, they progressively shorten during each cell cycle by replication-dependent loss of DNA termini. Ongoing shortening nally prevents telomeres from adequately protecting chromosome ends from further degradation, resulting in chromosomal instability.2,3 Cells with shortened telomeres eventually succumb to proliferative senescence and crisis (mitotic clock).4 Consequently, tumor cells need to compensate for replicative telomere losses

to preserve their ability to proliferate indefinitely.5 In 90% of human tumors, maintenance of telomeres is achieved by human telomerase reverse transcriptase (hTERT) expression and activation of telomerase.3,6-8 The key role of telomere maintenance by hTERT expression for human carcinogenesis was rst described by Hahn et al in 1999 and reassessed in 2002.8,9 They identied hTERT expression as one of three fundamental genetic changes for human tumorigenesis by showing that the expression of hTERT together with the two oncogenes large-T and H-ras resulted in direct malig1807

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nant transformation of normal human epithelial and broblast cells. Accordingly, introduction of hTERT cDNA into telomerase-negative cells was shown to reconstitute telomerase activity10,11 and to extend the life span of these otherwise mortal cells.12 Moreover, inhibition of hTERT led to telomere loss and limited the growth of human tumor cell lines in vitro and their tumorigenetic capacity in vivo.13 Therefore, the telomere maintenance pathway seems to contribute directly to human oncogenesis. Previous studies demonstrated increased telomerase activity in colorectal cancer tissue14,15 and even suggested a prognostic value for patients with colorectal carcinoma.16 However, only few studies have addressed the link of telomere length and hTERT expression with histopathologic tumor parameters and patient survival. The previously published study on hTERT expression in colorectal carcinoma and corresponding normal mucosa was the rst report on the prognostic potential of hTERT expression in patients with colorectal carcinoma.17 In the present study, we analyzed telomere length by Southern blot and, as described earlier, hTERT-mRNA by real-time polymerase chain reaction (PCR) in cancer tissue and adjacent noncancer mucosa of 57 R0-resected patients with colorectal carcinoma to further understand the mechanisms of telomere regulation and to validate telomere length and hTERT expression as markers for progression and prognosis of colorectal carcinoma.
PATIENTS AND METHODS Patients and Tumor Specimens Our study group consisted of 57 patients with colorectal carcinoma with a mean age of 64.6 13.6 (standard deviation) years. All patients underwent primary R0 resection without neoadjuvant chemotherapy or neoadjuvant radiation therapy between 1993 and 1996. Two stage II patients with rectal carcinoma received adjuvant radiochemotherapy (uorouracil [FU]/folic acid, 50 Gy; in one case in combination with intraoperative radiation with 15 Gy). For the 20 stage III tumors, eight patients received adjuvant chemotherapy (FU/folic acid), four patients received adjuvant radiochemotherapy (FU/folic acid, 50 Gy), and eight patients did not get adjuvant therapy because of reduced state of health or patient refusal. The single stage IV patient was operated for stenosing rectal carcinoma and resectable solitary lung metastasis and refused adjuvant therapy. Statistical analysis showed no inuence of adjuvant therapy on patient survival in all stage groups. The resection procedures for the 30 rectal carcinomas (53%) included 10 abdominoperineal extirpations and 20 anterior resections. For the 27 colon carcinomas (47%), one anterior resection, six sigmoid resections, two left hemicolectomies, 15 right hemicolectomies, and three subtotal colectomies were performed. The absence of residual tumor after resection (R0 resection) and the tumor stages were classied according to the International Union Against Cancer (UICC; Table 1).18 From the resected specimen of each patient, samples from both the cancer tissue and the adjacent noncancer mucosa were
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obtained, immediately shock-frozen in liquid nitrogen, and stored at 80C within 1 hour after resection until use. Clinical Follow-Up Clinical follow-up was performed according to a standardized protocol for all 57 patients with a median follow-up time of 75.5 months (range, 52 to 87 months). One patient (2%) was lost for follow-up after 54 months. Recurrent disease was found in 19 patients (33%), with distant metastases in 12 patients (10 patients with liver, one with lung, and one with brain metastases) and local recurrence in seven patients (12%). Seventeen of these 19 patients with tumor recurrence died of recurrent disease during the follow-up period. Three patients (5%) died of postoperative complications within 3 months after surgery, and eight patients (14%) who did not develop tumor recurrence died of cancer-unrelated causes during follow-up. The latter two groups were considered censored by statistical survival analysis. DNA and RNA Extraction Genomic DNA and total RNA were extracted from 20 16-m cryostat sections of all samples, corresponding to 20 to 25 mg of tissue, using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and the High Pure RNA Tissue Kit (Roche Diagnostics, Mannheim, Germany), respectively. Before and after each 20 sections, a 7-m cryostat section from each tissue sample was stained with hematoxylin and eosin for histopathologic analysis. Histology-guided sample selection was performed to rule out any contamination of the adjacent mucosa samples with carcinoma cells and to identify a tumor content of greater than 80% in the cancer samples. A 1:100 diluted aliquot of the extracted RNA and DNA yield was quantied spectrophotometrically at 260- and 280-nm wavelength for RNA and DNA concentration. Integrity of the extracted RNA was determined by electrophoresis through agarose gels with ethidium bromide and visualization of typical 18S and 28S RNA bands under ultraviolet light. Telomere Length Measurement Telomere lengths were determined by a modied TeloQuant Telomere Length Assay Kit (Pharmingen, San Diego, CA) protocol. Briey, extracted DNA samples (4.0 g of DNA for noncancer tissue and 7.2 g of DNA for cancer tissue) were digested with the restriction enzymes RsaI and HinfI (Roche Diagnostics; 4 U/g DNA) at 37C for 12 hours and run on 0.6% agarose gels at 50 V for 13 hours. A biotinylized gamma DNA Molecular Weight Marker (Vector Laboratories, Burlingame, CA) was used as DNA length standard. TeloHi DNA, TeloLow DNA (Pharmingen), the colorectal carcinoma cell line SW480, and the pancreatic adenocarcinoma cell line PA-TU8902 were run as positive controls. The DNA samples were depurinated in 0.25 mol/L of HCl, denatured in 0.4 mol/L of NaOH/3 mol/L of NaCl, and transferred to a positively charged nylon membrane Hybond-N (Amersham Pharmacia Biotech, Little Chalfont, England) by capillary blotting over 12 hours. The membrane was washed in 2 saline-sodium citrate buffer (SSC; Sigma Chemical, St Louis, MO) and dried at 30C for 30 minutes. The blot was hybridized with 3 L of biotinylized (TTAGGG)3 telomere probe (Pharmingen) at 65C for 12 hours and washed in 2 SSC/0.1% sodium dodecyl sulfate. Chemiluminescent detection was performed according to the Biotin Luminescent Detection Kit (Roche Diagnostics). The blocked membrane was incubated with 4 L of Streptavidin-alkalinephosphatase complex (200 mU/mL) at room temperature for 30 minutes, washed, equilibrated in 0.1 mol/L of Tris HCl/0.1 mol/L of NaCl (pH 9.5), and incubated with the chemiluminescent subJOURNAL OF CLINICAL ONCOLOGY

Telomere Length and hTERT in Colorectal Cancer

Table 1. Telomere Length in Cancer Tissue and Adjacent Noncancer Mucosa and the Ratios of Cancer to Noncancer Tissue of 57 Patients With Colorectal Carcinoma Noncancer (kb) No. of Patients Patients Sex Female Male P Tumor site Colon Rectum P Tumor invasion pT 1 pT 2 pT 3 pT 4 P Lymph node status pN 0 pN P UICC stage I II III IV P Grade G1 G2 G3 P Lymphatic invasion Absent Present P 57 27 30 % 100 47 53 Median 6.8 6.9 6.7 NS 27 30 47 53 6.9 6.8 NS 2 17 31 7 4 30 54 12 6.7 6.9 6.9 6.6 NS 37 20 65 35 6.8 6.9 NS 16 20 20 1 28 35 35 2 6.7 7.0 6.9 6.1 NS 2 30 25 3 53 44 6.7 6.8 6.9 NS 43 14 75 25 6.8 6.9 NS 5.5-8.6 5.6-8.3 5.5 6.0 NS 6.6-6.8 5.5-7.9 5.7-8.6 5.6 5.5 6.1 .06 4.1-7.6 4.2-7.1 0.84 0.90 NS 5.5-7.5 6.0-8.6 5.6-8.3 5.2 6.3 6.0 5.5 .01 4.5-6.7 4.1-7.1 4.5-7.6 0.84 0.83 0.86 NS 0.61-1.17 0.53-1.06 5.5-8.6 5.6-8.3 5.5 6.0 NS 4.1-6.7 4.2-7.6 4.4-7.1 0.77 0.86 0.85 0.90 .06 0.66-1.02 0.53-1.06 0.61-1.17 6.6-6.8 5.5-7.7 5.6-8.6 6.1-8.3 5.6 5.3 6.0 6.0 .08 4.1-7.6 4.4-7.1 0.84 0.85 NS 0.63-1.02 0.53-1.17 0.70-1.06 5.9-8.6 5.5-8.1 5.7 5.7 NS 4.5-6.7 4.1-6.5 4.2-7.5 4.5-7.6 0.84 0.81 0.86 0.77 NS 0.53-1.17 0.70-1.06 Range 5.5-8.6 5.7-8.6 5.5-8.6 Cancer (kb) Median 5.7 6.0 5.4 .05 4.1-7.6 4.3-7.1 0.82 0.85 NS 0.66-1.02 0.63-0.97 0.53-1.13 0.61-1.17 Range 4.1-7.6 4.3-7.6 4.1-7.4 Median 0.84 0.84 0.83 NS 0.53-1.17 0.61-1.13 Ratio Range 0.53-1.17 0.61-1.17 0.53-1.13

NOTE. Signicance level: P .05. I v II, P .005; I v III, P .02; I v II to IV, P .002. Abbreviations: NS, not signicant; UICC, International Union Against Cancer.

strate for alkaline phosphatase for membrane blotting assays (CSPD; Applied Biosystems, Foster City, CA) for 5 minutes at room temperature. Enzymatic dephosphorylization for another 10 minutes at 37C produced chemoluminescence for detection on an x-ray Hyperlm ECL (Amersham Pharmacia Biotech; Fig 1). To address the issue of tissue heterogeneity, mean terminal restriction fragment lengths were calculated as (ODi)/(ODi/Li) using Totallab-Software (Amersham Pharmacia Biotech). Li represents the mean molecular size of 35 equal intervals of the telomeric smears in the range of 2 to 23 kb as dened by the DNA length standard. ODi reects the measured intensity of luminescence in each of the 35 intervals. As reported in the literature earlier, terminal restriction fragment lengths were recorded as telomere lengths.15,19,20 Quantication of hTERT Expression Kinetic PCR quantication of hTERT-encoding mRNA was performed in a real-time, one-step reverse transcriptase PCR using
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the LightCycler TeloTAGGG hTERT Quantication Kit (Roche Diagnostics) as described earlier.17 Statistical Analysis Statistical analysis was performed using the SPSS software package (SPSS Inc, Chicago, IL). Differences in telomere length and hTERT-mRNA expression between matched tissue samples were determined by the Wilcoxon test for matched pairs. Differences in telomere length and hTERT levels among various groups of patients discriminated for histopathologic parameters were analyzed by the Kruskal-Wallis test and the Mann-Whitney twosample test. All tests were performed at a signicance level of P .05. Group-oriented curves for overall survival were calculated according to the Kaplan-Meier model.21 To determine the relative prognostic impact of telomere length and hTERT expression compared with established prognostic factors, overall survival was analyzed according to Coxs proportional hazards model.22 For uni- and multivariate Cox regression analysis,
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Fig 1. Representative x-ray lm for telomere length analysis of ve colorectal cancer patients (Pt) in noncancer (N) and cancer (Ca) colorectal tissue using Southern blot analysis. C1, positive control (colon cancer cell line SW 480); C2, positive control (pancreatic adenocarcinoma cell line PA-TU 8902); M, DNA molecular weight marker.

continuous variables were recoded to binary variables. The classication and regression trees technique was used to determine optimal cutoff values.23 RESULTS

Fig 2. (A) Correlation of telomere length (TL) with hTERT expression in noncancer colorectal tissue. (B) Correlation of TL with hTERT expression in colorectal carcinoma tissue.

Telomere Length and hTERT Expression hTERT-encoding mRNA was found in all 57 noncancer mucosa and colorectal carcinoma tissue samples, with expression levels as reported earlier.17 Median telomere lengths in noncancer mucosa and cancer tissue of all 57 patients were 6.8 kb (range, 5.5 to 8.6 kb) and 5.7 kb (range, 4.1 to 7.6 kb), respectively. Overall, cancer tissue had signicantly shorter telomeres than matched adjacent mucosa (P .001). Patient-by-patient comparison of matched tissue samples showed longer telomeres in the noncancer mucosa than in the cancer tissue in 49 patients (86%), with a median difference of 1.3 kb (range, 0.1 to 3.7 kb). The eight patients (14%) with longer telomeres in the cancer tissue showed a median difference of 0.4 kb (range, 0.1 to 1.1 kb). The ratio of telomere lengths in cancer tissue to corresponding noncancer mucosa showed a median of 0.84 (range, 0.53 to 1.17). Signicant positive correlations between telomere length and hTERT expression were found in both noncancer colorectal mucosa (r 0.54; P .001; Fig 2A) and colorectal carcinoma (r 0.52; P .001; Fig 2B). The ratios of cancer to noncancer tissue for telomere length and hTERT expression were signicantly correlated as well (r 0.47; P .001).
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Only in adjacent noncancer mucosa samples did both telomere length and hTERT expression decrease with aging (r 0.36, P .01; and r 0.25, P .06, respectively; Fig 3), with a telomere length reduction of 19 bp per year. In

Fig 3. Correlation of telomere length (TL) with age in noncancer colorectal tissue.

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Telomere Length and hTERT in Colorectal Cancer

colorectal carcinoma tissue, both parameters were independent of age. Correlation With Histopathologic Parameters As shown earlier, hTERT expression in both cancer and noncancer tissue increased signicantly with increasing tumor grade (P .04 and P .05, respectively).17 There was a trend of increasing hTERT levels in cancer tissue with increasing depth of tumor invasion (pT), which was statistically not signicant.17 hTERT expression was not correlated with any other histopathologic parameter.17 Overall, there was a statistically signicant correlation between telomere length in cancer tissue and UICC stage (P .01; Table 1). Stage I tumors (mean telomere length, 5.2 kb, n 16) had signicantly shorter telomeres than both stage II (mean telomere length, 6.3 kb, n 20; P .005) and stage III tumors (mean telomere length, 6.0 kb, n 20; P .02). The single stage IV tumor in our study had a mean telomere length of 5.5 kb. Telomeres of early-stage tumors (UICC stage I; n 16) were signicantly shorter than telomeres of advanced tumors (UICC stages II through IV; n 41; P .002). The telomere length ratio of cancer to noncancer tissue increased with higher stage groups, approaching statistical signicance (P .06; Table 1). The trend that locally advanced tumors (pT34) had longer telomeres in cancer tissue than pT12 tumors was statistically not signicant (Table 1). The trend of increasing telomere lengths with increasing tumor grade approached statistical signicance in cancer tissue (P .06) but was not signicant in normal mucosa (Table 1). For tumor site, lymph node involvement, or lymphatic vessel invasion, no correlation was found with telomere length (Table 1). Prognosis For survival analysis, optimal cut-offs for telomere length in cancer tissue (5.4 kb; P .03) and the telomere length ratio (0.9; P .001) were calculated for our study group of 57 patients by log-rank statistics using the classication and regression trees technique. Thirty-ve patients (61%) with telomeres 5.4 kb in the carcinoma tissue had a signicantly poorer overall survival, with a 5-year survival rate of 51.5% 9.4% compared with a 5-year survival rate of 85.7% 7.6% for 22 patients (39%) with telomere lengths 5.4 kb (P .03; Fig 4A). The Kaplan-Meier survival curve in Figure 4B illustrates the increased hazard rate of 14 patients (25%) with a telomere length ratio greater than 0.9 with a 5-year survival of 25.6% 13.8% compared with 43 patients (75%) with a ratio 0.9 with a 5-year overall survival rate of 78.2% 6.9% (P .002). Kaplan-Meier survival curves for hTERT expression in cancer tissue (P .05) and hTERT ratio (P .02) were published earlier. Besides the established prognostic factors, depth of tumor invasion (pT), lymph node status, and lymphatic
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Fig 4. (A) Kaplan-Meier survival curve for telomere length (TL) in cancer tissue of 57 patients with R0-resected colorectal cancer. A cut-off value of 5.4 kb was determined by log-rank statistics according to classication and regression trees (CART) technique. (B) Kaplan-Meier survival curve for TL ratio of cancer to noncancer tissue of 57 patients with R0-resected colorectal cancer. A cut-off value of 0.9 kb was determined by log-rank statistics according to CART technique.

invasion, telomere length and hTERT expression in cancer tissue as well as the telomere length ratio and the hTERT ratio were correlated signicantly with overall survival in univariate Cox regression analysis (Table 2). Tumor site, histologic grade, sex, and age had no prognostic relevance. In multivariate analysis, the telomere length ratio of cancer to noncancer tissue was shown to be an independent prognostic parameter for overall survival (P .03; Table 2). The relative risk of death for 14 patients (25%) with a telomere length ratio greater than 0.90 was 3.3 times higher compared with 43 patients (75%) who had telomere length ratios 0.90 (95% CI, 1.2 to 9.0). The only other independent prognostic factor for overall survival was lymphatic vessel invasion, with a relative risk of 4.1 and a CI of 1.5 to 11.6 (P .01). Forty-three patients (75%) without lymphatic vessel invasion showed a signicantly better overall survival rate, with a 5-year survival rate of 81.5% 6.31% compared with a 5-year survival rate of 20.5% 12.0% for 14 patients (25%) with lymphatic vessel invasion. When the hTERT ratio instead of the telomere length ratio was added to this model, it also proved to be of independent prognostic value for overall survival (P .05); however, this was at a lower signicance level than the telomere length ratio, as previously shown.17
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Table 2. Univariate and Multivariate Cox Regression Analysis for 57 Patients With R0 Resected Colorectal Carcinoma Multivariate Analysis Variable Tumor invasion, pT1-pT2 v pT3-pT4 Lymph node status, pN0 v pN Lymphatic invasion absent v present TL ratio, 0.9 v 0.9 TL cancer, 5.4 v 5.4 hTERT ratio, 0.57 v 0.57 hTERT cancer, 37 v 37 P (univariate analysis) .05 .01 .001 .001 .05 .02 .05 P NS NS .01 .03 NS NS NS Relative Risk 4.1 3.3 CI 1.5 to 11.6 1.2 to 9.0

Abbreviations: TL, telomere length; hTERT, human telomerase reverse transcriptase. Signicance level: P .05. Ratio of telomere length in cancer tissue to telomere length in noncancer mucosa. Cutoff values statistically determined by log-rank statistics using the Classication and Regression Trees technique. Telomere length in cancer tissue. Ratio of hTERT expression in cancer tissue to hTERT expression in noncancer mucosa. hTERT expression in cancer tissue.

DISCUSSION

Telomere length measurement has been widely used as a marker for cell proliferation.1,3 With a telomere length reduction of 19 bp per year in noncancer colorectal mucosa, our results are in line with earlier studies on telomere shortening.2,19 For the rst time, we could also demonstrate that telomere length and hTERT expression decrease in parallel with aging in normal mucosa. Considering the high proliferative activity of colorectal (stem) cells, the moderate telomere reduction rates and the age-dependent decrease of both telomere length and hTERT expression support the hypothesis that colorectal cells may indeed have some hTERT-mediated telomere regulation that compensates part of the replicative telomere losses.24-27 Because telomere length and hTERT-mRNA expression were independent of age in cancer tissue, colorectal carcinomas seem to escape age-related telomere regulation. The ability to compensate for replicative telomere losses (through hTERT expression) thus seems to be a specic characteristic of each individual tumor. To adjust this age-dependent variation of hTERT and telomere length values, the ratios of tumor tissue to adjacent normal mucosa for both parameters were calculated for each patient. These ratios also illustrated the individual differences between cancer tissue and adjacent mucosa that served as a representative from which carcinogenesis might have started. Given the difculties of longitudinal studies in a clinical setting, the comparison of cancer and adjacent noncancer tissue is a useful model to investigate carcinogenesis-related changes. Because no study has delivered complete data on hTERT expression and telomere length for both the primary tumor and corresponding nontumor tissue so far, our approach to compare hTERT expression and telomere length in colorectal carcinoma tissue and adjacent normal mucosa is unprecedented.
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In our study, most tumors (86%) had shorter telomeres compared with the adjacent normal mucosa, with a median difference of 1.3 kb. Engelhardt et al15 also reported on shorter telomeres in 90% of colon tumors compared with adjacent normal tissues, with a mean difference of 0.9 kb. Nakamura et al19 found shorter telomeres in cancer tissues than in normal mucosa in 96 (77%) of 124 colorectal cancer cases, with a mean difference of 3.1 kb. Two more studies also reported on mainly shortened telomeres in colorectal carcinoma.20,28 Shorter telomeres in cancer tissue compared with adjacent mucosa are indicative for extensive tumor cell proliferation. These data show that tumor cell proliferation exceeds telomere maintenance mechanisms for compensation of replicative telomere losses in most tumors. However, telomere stabilization is inevitable at a critical point of telomere shortening to prevent the onset of crisis and senescence.2,12 In this context, our study revealed a signicant correlation between telomere length in cancer tissue and tumor stage with shortest telomeres in stage I tumors. Engelhardt et al15 also reported on signicantly longer telomeres in late-stage Dukes C and D tumors compared with earlystage Dukes A and B tumors. We also found a trend of increasing telomere length and hTERT expression in colorectal carcinoma tissue with increasing depth of local tumor invasion (pT). To date, all other available studies have failed to correlate telomere length or hTERT expression with tumor stage or depth of tumor invasion in patients with colorectal carcinoma.20,28,29 Because telomere lengths are the result of the balance of proliferative telomere losses and de novo telomere synthesis, they serve as an indicator for the ability of each tumor to compensate for replicative telomere losses. Our ndings support the hypothesis that sufcient (hTERT-mediated) telomere stabilization is achieved late in tumorigenesis after extensive cell proliferaJOURNAL OF CLINICAL ONCOLOGY

Telomere Length and hTERT in Colorectal Cancer

tion and telomere shortening have already taken place.15 Nevertheless, telomere maintenance or even elongation (eight patients showed longer telomeres in the cancer tissue compared with the adjacent mucosa) seems to be essential for the tumor to maintain its (indenite) proliferate capacity and to continue further tumor invasion and progression.1,8 Effective (hTERT-mediated) telomere length stabilization might thus be a selection criterion for colorectal carcinoma to proceed from early to advanced tumor stages, illustrated by higher telomere length ratios in advanced tumors compared with early-stage tumors. On the basis of the correlation of hTERT expression with tumor grade in both normal mucosa and cancer tissue, it has been hypothesized earlier that colorectal cells might even be continuously selected for high hTERT levels as they acquire genetic changes associated with invasive cancer.17 Hahn et al8,9 identied hTERT-mediated telomere maintenance as a key step in cell immortalization and neoplastic transformation of human cells and also stated that cells are selected for reactivated telomerase. Our data suggest that the mere expression of hTERT is not only linked with the creation of malignant clones but that the level of hTERT expression, together with the resulting telomere length stabilization, might even determine the potential for invasion and progression of these clones. Quantication of hTERT expression and measurement of telomere length may thus be useful methods for additional biologic and prognostic staging of colorectal carcinoma. Moreover, the presented data do not only underline the importance of effective telomere stabilization for tumor development and progression but also reveal the prognostic impact of these molecular mechanisms. This study is the rst to show that both telomere length and hTERT expression are signicantly correlated with overall survival. So far, mainly telomerase activity has been measured to demonstrate the prognostic relevance of telomere regulation. Several studies found increased telomerase activity in colorectal carcinoma tissue,14,15 and Tatsumoto et al16 identied
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1. Blackburn EH: Structure and function of telomeres. Nature 350:569-573, 1991 2. Hastie ND, Dempster M, Dunlop MG, et al: Telomere reduction in human colorectal carcinoma and with ageing. Nature 346:866-868, 1990 3. Counter CM, Avilion AA, Le Feuvre CE, et al: Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 11: 1921-1929, 1992 4. Hoos A, Nekarda H: Telomerase: Potential und Grenzen der klinischen Anwendbarkeit. Dtsch Med Wschr 124:223-230, 1999 5. Lustig AJ: Crisis intervention: The role of telomerase. Proc Natl Acad Sci U S A 96:33393341, 1999

high telomerase activity as an independent prognostic indicator of poor outcome in colorectal cancer. For hTERT, the previously published study was the rst report on the prognostic potential of hTERT expression in patients with colorectal carcinoma.17 In hTERT studies on other tumor entities, results are inconsistent.30-32 The coexistence of the two independent prognostic parameters identied in this study are consistent with the two main established prognostic aspects of malignant tumors, metastatic spread and invasive tumor growth. On one hand, metastatic tumor spread, represented by lymphatic vessel invasion in our study (and eventually lymph node involvement), is mainly determined by tumor-host interactions and independent of telomere regulation. On the other hand, a proliferative advantage for further tumor growth, nally resulting in poor prognosis, is provided by sufcient telomere maintenance, as indicated by longer telomeres and greater telomere length ratios. Despite these facts, no data have been published on the prognostic value of telomere length thus far, neither for colorectal carcinoma nor for other tumor entities, although the length of telomeres as the end point of telomere regulation is the crucial parameter for protecting chromosome ends. All other parameters of the telomere maintenance pathway, including hTERT expression and telomerase activity, might be bypassed by alternative lengthening of telomeres or inuenced by additional factors such as telomerase inhibitors, alternate splicing of hTERT transcripts,33-35 and changes of hTERT-mRNA at the posttranscriptional level.23 We therefore consider telomere length as the most reliable and most signicant parameter of telomere regulation with highest prognostic potential when calculated as the ratio of cancer to noncancer tissue.
I I I

Authors Disclosures of Potential Conicts of Interest The authors indicated no potential conicts of interest.
11. Weinrich SL, Pruzan R, Ma L, et al: Reconstitution of human telomerase with template RNA component hTERC and the catalytic protein subunit hTERT. Nat Genet 17:498-502, 1997 12. Bodnar AG, Ouelette M, Frolkins M, et al: Extension of life-span by introduction of telomerase into normal human cells. Science 279:349352, 1998 13. Hahn WC, Stewart SA, Brooks MW, et al: Inhibition of telomerase limits the growth of human cancer cells. Nat Med 5:1164-1170, 1999 14. Chadeneau C, Hay K, Hirte HW, et al: Telomerase activity associated with acquisition of malignancy in human colorectal cancer. Cancer Res 55:2533-2536, 1995 15. Engelhardt M, Drullinsky P, Guillem J, et al: Telomerase and telomere length in the development and progression of premalignant lesions

6. Morin GB: The human telomere transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59:521-529, 1989 7. Kim NW, Piatyszek MA, Prowse KR, et al: Specic association of human telomerase activity with immortal cells and cancer. Science 266: 2011-2015, 1994 8. Hahn WC, Weinberg RA: Rules for making human tumor cells. N Engl J Med 347:15931603, 2002 9. Hahn WC, Counter CM, Lundberg AS, et al: Creation of human tumor cells with dened genetic elements. Nature 400:464468, 1999 10. Nakayama Y, Tahara H, Tahara E, et al: Telomerase activation by hTERT in human normal broblasts and hepatocellular carcinomas. Nat Genet 18:65-68, 1998

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