Activation of telomerase reverse transcriptase (hTERT) is essential for unlimited cell growth and plays a critical role in tumorigenesis. We investigated hTERT gene expression in 134 B-cell chronic lymphocytic leukemia (B-CLL) cases and evaluated its prognostic value with other prognostic markers (IgVH mutation status, CD38 and ZAP-70 expression). Real-time PCR assays to quantify either all hTERT transcripts (AT) or only the full length (FL) transcript encoding the functional protein were developed. hTERT-AT levels strongly correlated with hTERT-FT levels (r=0.743, P<0.0001); both inversely correlated with the percentage of IgVH mutation (P<0.005) and were significantly higher in unmutated than in mutated cases (P=0.004 and P=0.001, respectively). The hTERT values which best discriminated between the unmutated and mutated IgVH cases were 150 and 40 copies for hTERT-AT and hTERT-FL, respectively. Using these cut-off values, there was a significant difference in the survival of patients with high or low hTERT levels (P<0.0001). Unmutated cases with low hTERT levels had an overall survival close to mutated cases with high hTERT levels. Thus, this work identifies hTERT-RNA level as a new prognostic marker in B-CLL, and may be used to identify previously unrecognized patient groups with the same IgVH mutation status and different disease outcomes.
Telomerase, a ribonucleoprotein complex containing an internal RNA template (hTR) and a catalytic protein with a telomere-specific reverse transcriptase activity (hTERT), extends telomeres at the end of eukaryotic chromosomes.1 The progressive shortening of telomeres that occurs at each cell division cycle, owing to failure of DNA-dependent DNA polymerase to replicate fully DNA molecules, is a key mechanism in controlling cellular replicative potential; when telomere erosion reaches a critical point, cells cease to proliferate and undergo senescence or apoptosis. Telomerase activation, usually absent from normal somatic cells, is essential for unlimited cell growth and plays a critical role in tumorigenesis. Evidence that almost all human tumours have telomerase activity and that inhibition of telomerase activity in tumour cells prevents the formation of tumours in experimental animal models2 strongly supports the key role of telomerase in the neoplastic process. Although hTR is constitutively present in normal and tumour cells, hTERT, which de novo encodes the telomeric sequence using hTR as a template, is the rate-limiting component of the telomerase complex, and its expression is tightly controlled at both transcriptional and post-transcriptional levels.3
Recent studies have related the levels of telomerase activity and/or hTERT expression to clinical aggressiveness and prognosis in a variety of malignancies,4 but the prognostic role of telomerase activity (TA) or hTERT expression in B-cell chronic lymphocytic leukemia (B-CLL) is still unclear. B-CLL is the most common leukemia in adults. The identification of a reliable prognostic marker is essential in B-CLL, which is clinically highly heterogeneous; some patients rapidly progress and die from disease within a few months of diagnosis, whereas others live for several years with minimal or no treatment.5 Clinical staging systems may divide patients into risk groups; nevertheless, they do not predict disease evolution in low-risk patients, who make up the majority of B-CLL cases at presentation.6 Among molecular indicators, positivity for ZAP-70 kinase and the presence or absence of somatic mutations in the immunoglobulin V (IgVH) gene regions appear to be the best discriminators between stable and aggressive disease, with the unmutated IgVH profile (i.e., <2% difference from germ-line) being associated with an aggressive clinical course.7, 8, 9, 10 A previous study from our group using a small cohort of B-CLL patients demonstrated that TA was significantly higher in patients with progressive disease than in patients with stable disease.11 Furthermore, it has recently been reported that B-CLL with an unmutated IgVH profile and poor prognosis expressed more TA than B-CLL with mutated IgVH genes and better prognosis; however, in this study TA was not significantly related to patient outcome.12 No correlation between TA and survival was reported by Versothech et al.;13 however, 59% of the samples included in this study had no detectable TA. By contrast, a recent study demonstrated the prognostic value of hTERT expression in a series of 90 B-CLL cases.14 However, the hTERT transcript has been shown to contain alternate splicing sites, and mRNA variants lacking α and/or β regions resulted in truncated and dysfunctional protein products.15
The aim of this study was to determine hTERT gene expression in B-CLL and to evaluate its prognostic value. To this end, we set up methods able to quantify all hTERT transcripts and selectively quantify hTERT transcripts encoding for the functional protein. Levels of hTERT expression were then compared with IgVH mutation status and patient outcome. As recent findings suggest that hTERT expression is not only required for telomere maintenance, but may also influence cellular response to apoptotic stimuli,16 the assessment of hTERT expression in B-CLL may have importance for setting up therapeutic strategies as well.
Peripheral blood lymphocytes were collected from 1990 to 2003 from 134 patients (65 females and 69 males) with B-CLL who attended two different institutions (Department of Clinical and Experimental Medicine, University of Padova, and Centro Riferimento Oncologico, Aviano). Most of the samples (82%) were collected at the time of diagnosis, and no patient underwent therapy at the time of sampling. The median age was 61 years (range, 32 to 90), and the median follow-up time (from the blood sample) was 70 months. Peripheral-blood mononuclear cells were isolated by density-gradient centrifugation with the use of Ficoll-Paque Plus (Amersham Biosciences). Cell samples were further enriched in B lymphocytes by rosetting with neuroaminidase (Sigma)-treated SRBC and by removing residual CD3+, CD16+, CD56+ and CD14+ cells using magnetic separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany), as described previously.17 Following this multistep negative selection procedure, more than 98% of the resulting cell population was CD19+. All studied samples contained more than 90% tumour cells.
RNA extraction and cDNA synthesis
Total cellular RNA was isolated from cells (1–5 million) with the TRizol reagent (Invitrogen, Gaithersburg, MD, USA). Integrity of RNA was then evaluated by visualizing the 18S and 28S RNAs by agarose gel electrophoresis, and the amount was quantified by spectrophotometer. One microgram of RNA was retro-transcribed into cDNA using the SuperScript TM III RNase Reverse Trancriptase assay (Invitrogen) according to manufacturer's instructions.
Real-time PCR for quantification of hTERT transcripts
Real-time PCR-assays were developed to quantify either all hTERT transcripts (hTERT-AT) or the full-length hTERT transcript (hTERT-FL), which encode for the functional protein,15 by using AT1/AT2 and FL1/FL2 primer pairs, respectively. Details of primers, probes, and real-time PCR conditions are described in the Supplementary Section.
Telomerase activity detection assay
IgVH gene status assessment
RNA was extracted from 2 × 106 B cells using the RNeasy™ Total RNA kit (Qiagen) and reverse transcribed using the SuperScript™ Preamplification System for first-strand cDNA synthesis (Life Technologies, Inc.). The B-CLL cells VH gene family was assigned as previously described19 using a sense VH family-specific framework region (FR) primer in conjunction with the appropriate antisense CH primer. VH gene DNA sequences were determined by reamplifying 5 μl of the original cDNA using the appropriate VH leader and CH primers. PCR products were sequenced directly after purification with Wizard PCR Preps (Promega, Madison, WI) using an automated genetic analyser (Applied Biosystems, Foster City, CA, USA). Sequences were compared with those in the V BASE sequence directory.
Flow-cytometry analysis of ZAP-70 and CD38 protein expression
Cytoplasmic ZAP-70 expression was determined by flow cytometry. Permeabilized cells were analysed with the anti-ZAP-70 antibody Alexa Fuor488 (Caltag), CD3-phycoerythrin (PE), CD56-PE (BD Biosciences), CD 19-peridinin chlorophyll protein-cytochrome 5,5 (Caltag) and CD 5 APC (BD Biosciences). After appropriate lymphocyte gating, cytoplasmic ZAP-70 expression was determined in CD19+ CD5+ B-CLL cells. The threshold level for ZAP-70 was set at 20%.8
Analysis of CD38 expression on B-CLL cells was carried out by incubating whole blood with 5 μl of the following antibodies: anti-CD5 FITC, anti-CD-38-PE, and anti-CD19-RPECy5 for 20 min and at least 1 × 105 cells were counted. Each sample was run with the appropriate isotype control antibodies to define the negatively stained cells. The percentage of CD38+ cells was defined as the percentage of CD19+CD5+ that were CD38+. The threshold for CD38 expression was set at 30%; <30% were defined as CD38- and >30% as CD38+.7, 10
Associations between categorical variables were analysed by Fisher's exact tests. The central tendency differences between groups were compared with the Mann-Whitney test, because of non-normal distribution. Non-parametric linear correlations between characteristics were analysed by the Spearman rank test. To identify the level of hTERT expression that could best be used to discriminate between IgVH mutated and unmutated cases, we used a receiver-operating-characteristic (ROC) method.20, 21 Survival was estimated according to the method of Kaplan and Meier and compared between groups by the log-rank test. The hazard ratios of various risk factors for survival were estimated by Cox's proportional hazard method with stepwise forward variable selection option, after checking for proportional assumptions. As both hTERT AT and FL are highly correlated and as because they measure similar parameters, they were put into 2 distinct models. All P values were two-sided. Statistical analyses were performed with use of SPSS software (SPSS advanced statistics, version 13, Chicago, IL, USA, SPSS Inc).
hTERT expression in B cells from B-CLL patients
hTERT transcripts were determined in a total of 134 samples; 122 (91. 0%) and 100 cases (74.6%) tested were positive for hTERT-AT and hTERT-FL transcripts, respectively. Overall, the mean level of hTERT-AT was 296 copies, with a median value of 96 copies (interquartile range (IQR) 37–357) and the mean value of hTERT-FL was 76 copies, with a median value of 20 copies (IQR 0–68). In the positive samples, the mean level of hTERT-AT was 325 copies, with a median value of 130 copies (IQR 49–385), and the mean level of hTERT-FL was 101 copies, with a median value of 44 copies (IQR 13–93) (Figure 1a). A significant linear correlation was found between hTERT-AT and hTERT-FL levels (r=0.743, P<0.0001) (Figure 1b). In some representative cases, positive or negative results obtained by real-time PCR were confirmed by qualitative analyses using agarose gel electrophoresis, and the correspondence between the presence of hTERT transcripts and telomerase activity was evaluated by TRAP assay. Samples positive for both hTERT-AT and hTERT-FL transcripts also tested positive for telomerase activity (nos.1, 3, 4, 6, 7, 9, Figure 1c and d), whereas samples positive only for hTERT AT transcripts (no. 2, Figure 1c and d) or negative for both (nos. 5 and 8, Figure 1c and d) were found to be negative for telomerase activity.
Correlation between hTERT expression and IgVH gene mutation
The hTERT transcript levels were compared with the IgVH mutational profile, which was available in 117 B-CLL cases. Values of IgVH mutation inversely correlated with both hTERT-AT levels (r=−0.279, P=0.002) and hTERT-FL levels (r=−0.345, P=0.0001). When the cut-off of 2% of IgVH mutations was used to label the B-CLL in unmutated (<2%) or mutated cases (>2%), the number of hTERT negative samples was higher in mutated than unmutated B-CLL (9 and 2, respectively for hTERT-AT, and 21 and 9, respectively for hTERT FL), and overall the levels of both hTERT-AT and hTERT-FL were significantly higher in unmutated than in mutated B-CLL (mean 455 vs 212 copies, P=0.004 for hTERT-AT and 117 vs 51 copies, P=0.001 for hTERT-FL) (Figure 2a). From ROC curve analyses, hTERT values which best discriminated between IgVH unmutated and mutated cases were 150 copies for hTERT-AT and 40 copies for hTERT-FL. By using these cut-off values, a high level of hTERT was associated with unmutated IgVH, and a low level of hTERT was associated with mutated IgVH. The overall association between levels of hTERT and IgVH status was 65% (hTERT-AT) and 68% (hTERT-FL) (Figure 2b).
Comparison of hTERT expression and CD38 and ZAP-70 expression
High expression of the surface membrane CD38 protein was found to be associated with the presence of unmutated IgVH and unfavourable prognosis.7 However, the relation between CD38 expression and mutational status and survival is still controversial.22, 23 When the cut-off of 30% positive cells for CD38 was employed,7, 10 we found that both hTERT-AT and hTERT-FL values were significantly higher (P=0.011 and P=0.017, respectively) in the CD38 high-positive samples rather than in the CD38 low-positive samples (Figure 3). The expression of the tyrosine kinase ZAP-70, which is essential for T-cell signalling and expresses in activated normal B cells,24 has recently been reported to be a reliable prognostic marker for B-CLL.25 When a cut-off of 20% was employed to discriminate between ZAP-70 positive and negative cases,25 hTERT levels were higher in the ZAP-70 high-positive cases than in the ZAP-70 negative cases, although the differences were not statistically significant (Figure 3). Furthermore, no correlation was observed between hTERT levels and sex or age (data not shown).
Association between hTERT expression and survival
The median follow-up time of the patients studied was 70 months (range 2–172 months). Figure 4 shows the Kaplan-Meier survival curves, comparing patients with unmutated and mutated IgVH (Figure 4a) and those with hTERT-AT (Figure 4b) and hTERT-FL (Figure 4c) levels above or below the cut-off values (150 and 40 copies, respectively). All these systems showed strong predictive power for survival (P<0.00001). The median survival in IgVH unmutated cases was 76 months (95% Confidence Interval (CI) 47–105), and in patients with hTERT values above the cut-off was 71 months (95% 47–95 months) (hTERT-AT) and 76 months (95% CI 53–99 months) (hTERT-FL) (Figure 4). CD38 and ZAP-70 data were available from 111 and 77 patients, respectively. Survival curves obtained comparing CD38-positive with CD38-negative cases, and ZAP-70-positive with ZAP70-negative cases had a significant predictive value (P=0.0001, and P=0.0083, respectively) as reported in the literature7, 8, 9, 10 (data not shown).
hTERT levels discriminate IgVH mutated and unmutated B-CLL patients with different survival
Although hTERT levels showed a similar predictive value for IgVH mutational status, hTERT identified different groups of patients with poor and better outcome; indeed, although there is an overlap, about one third of the patients showed discordant hTERT high-positive/IgVH mutated and hTERT low-positive/IgVH unmutated cases, as also indicated by the correlation analysis (Figure 4). When patients were stratified according to both parameters we found that hTERT-AT high-positive/IgVH unmutated cases had a significantly poorer prognosis than hTERT-AT low-positive/IgVH unmutated cases (P=0.0020), with a median survival of 49 months (95% CI 18–90 months) in the former vs a median survival time of 101 months (95% 64–137 months) in the latter (Figure 4d). In agreement with the negative prognostic values of hTERT, hTERT-AT high-positive/IgVH mutated cases had a significantly worse prognosis than hTERT-AT low-positive IgVH mutated cases (P<0.05); though median survival is not reached for these curves given the high number of living patients, the median estimated survival was 135 months (95% CI 93–177 months) in the former and 171 months (95% 170–173 months) in the latter. Similar figures were obtained when patients were stratified for hTERT-FL and IgVH (Figure 4e). On the contrary, hTERT levels did not discriminate among CD38/ZAP-70 discordant patients, probably owing to the reduced number of samples investigated for these parameters (data not shown).
Using Cox proportional hazards regression analysis, we analysed IgVH status and hTERT-AT and hTERT-FL values with other prognostic factors available, that is CD38 expression, ZAP-70 expression, Rai stage, progressive or stable disease (progressive included lymphocyte doubling time of less than 12 months). In this analysis only the IgVH mutational status and hTERT were selected by the stepwise forward method as independent significant prognostic factors (Table 1).
The selection of patients who benefit from a precocious treatment instead of a wait and watch approach is a key decision in the clinical setting for patients with B-CLL. In recent years, several prognostic factors have been identified and proposed to distinguish groups of patients with different prognoses. The mutational status of the IgVH gene is one of the strongest prognostic indicators of survival in these patients. Nevertheless, all clinicians have experienced how some patients with unmutated IgVH status do not have progressive disease and how others with mutated IgVH experience diseases with a highly aggressive clinical course.26, 27 This observation implies that more prognostic factors are needed to identify groups of patients with different outcome and disease evolution.
Besides confirming that patients with mutated IgVH profiles had a better prognosis than those with unmutated IgVH status, our results demonstrate for the first time that the hTERT transcript level is a strong prognostic indicator for overall survival in B-CLL patients. Furthermore, the combination of IgVH status and hTERT levels allows us to identify previously unrecognized subgroups of patients with different outcomes.
Levels of telomerase activity were shown to have prognostic values in a variety of tumours, including B-CLL.11, 28 Nevertheless, the clinical application of measurements of telomerase activity has thus far been hampered by the fact that the telomeric repeat amplification protocol (TRAP) assay requires a relatively large amount of well-preserved fresh or frozen tumour cells, rarely available in a routine setting. It has, therefore, been proposed to assess the expression of hTERT, the catalytic rate-limiting component of the telomerase complex, as a substitute for measuring telomerase activity in archival and fresh materials.14 However, hTERT is alternatively spliced in specific patterns by different types of tissues, and such alternate transcripts may constitute a regulatory mechanism for telomerase activity.15 In particular, as α and β regions contain conserved reverse transcriptase domains of hTERT, spliced variants lacking these sites would not be expected to encode a catalytically active protein. Notably, the overall expression of hTERT mRNA in neuroblastomas showed only a limited correlation to telomerase activity, and did not have prognostic value.29 By contrast, detection of full-length hTERT mRNA, which contains the α and β regions and thus potentially encodes for the functional protein, was highly predictive of a poor outcome in the same tumors.30 This prompted us to quantify, in our B-CLL series, either the levels of all hTERT (hTERT-AT) transcripts or only the levels of full-length (hTERT-FL) transcript. Twenty-two of 134 (16%) B-CLL samples were found to be positive for hTERT-AT but negative for hTERT-FL; these discordant cases were also negative for telomerase activity, as expected. Nevertheless, there was a strong relationship between levels of hTERT-AT and hTERT-FL; moreover, when hTERT-AT levels above 150 copies was taken as the cut-off only 4 cases were found to be hTERT-FL negative. The overall results obtained using hTERT-AT levels were largely superimposable to those achieved by using hTERT-FL levels; this may imply that for routine investigations only one assay may be sufficient, and hTERT-FL may be selected as it potentially encodes for the functional protein.
The data we obtained point out that hTERT expression was a strong predictor for overall survival in the B-CLL patients, and that patients with high levels of hTERT had a worse prognosis than did those with undetectable or low levels of hTERT. This finding may be in keeping with the recent evidence that B-CLL cases with short telomeres had a poorer outcome than patients with longer telomeres.12, 31 Indeed, hTERT expression and telomerase activity may be activated by multiple cellular signalling pathways.32, 33 The selective pressure of hTERT activation by telomere erosion to a critical stage is a usual hallmark in tumorigenesis.34 Finding that B-CLL cases with short telomeres had more telomerase activity than those with longer telomeres12 supports this concept.
Levels of both hTERT-AT and hTERT-FL transcripts were significantly higher in patients with an unmutated rather than mutated IgVH profile, but there was no strong association between percentages of IgVH mutations and hTERT mRNA levels. It has been reported that a combination of telomere length and IgVH mutation status can be used to divide mutated B-CLL into two groups with different prognoses, one with longer telomeres and a favourable outcome and one with shorter telomeres and a poor prognosis.31 Of special interest, our results indicate that the combination of mutation status and hTERT levels could not only identify from among the mutated cases those with high levels of hTERT and an unfavourable outcome, but also recognize from among the IgVH unmutated cases those with low hTERT levels and a better outcome.
Finally, the identification of different groups of patients according to their hTERT expression and IgVH mutation status might be important not only for their management, but also for establishing new therapeutic strategies. Recent data suggest that, besides its ability to maintain telomere length, hTERT is also involved in several other cellular functions. In fact, hTERT was shown to promote cell survival35 and prevent apoptosis triggered by antiproliferative agents16 by mechanisms independent of its ability to prevent telomere erosion. Moreover, inhibition of hTERT induces rapid cell-growth decline and apoptosis in cancer cells in the absence of telomere shortening.36, 37, 38 To verify whether these effects take place in B-CLL, studies will be thoroughly performed in vivo in leukemic cells obtained from patients undergoing therapy and in vitro cultured neoplastic cells.
Therefore, because high levels of hTERT may result in a poor response to conventional therapies, it is conceivable that in the future therapies targeting hTERT may be developed and eventually find clinical application in B-CLL cases with high hTERT levels and a worse prognosis.
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This work was supported by the Italian Association for Cancer Research (AIRC, Milan), by the MURST (Rome) and by Fondazione Berlucchi (Brescia). The authors wish to thank M Donach for his help in the preparation of this paper.
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Terrin, L., Trentin, L., Degan, M. et al. Telomerase expression in B-cell chronic lymphocytic leukemia predicts survival and delineates subgroups of patients with the same igVH mutation status and different outcome. Leukemia 21, 965–972 (2007). https://doi.org/10.1038/sj.leu.2404607
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