hTERT expression and prognosis in B-chronic lymphocytic leukemia

A. Tchirkov1,2,*, C. Chaleteix1, C. Magnac3, Y. Vasconcelos3,4, F. Davi5, A. Michel5, F. Kwiatkowski6, O. Tournilhac1, G. Dighiero3 and P. Travade1

1 Service d'Hématologie Clinique, CHU, Clermont-Ferrand; 2 Departement de Radiothérapie and 6 Service Statistiques et Communications Médicales, Centre Jean Perrin, Clermont-Ferrand; 3 Unité d'Immuno-Hématologie et d'Immunopathologie, Institut Pasteur, Paris; 5 Département d'Hématologie, Hôpital Pitié-Salpêtrière, Paris, France; 4 Division of Hematology, Universidade Federal de São Paulo, Sao Paulo, Brazil

* Correspondence to: Dr A. Tchirkov, Département de Radiothérapie, Centre Jean Perrin, 58, rue Montalembert, B.P. 392, 63011 Clermont-Ferrand Cedex 1, France. Tel: +33-4-73-27-83-75; Fax: +33-4-73-27-81-25; Email: andrei.tchirkov{at}cjp.fr


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background: In B-chronic lymphocytic leukemia (B-CLL), there is a need for molecular markers to predict the evolution of this heterogeneous disease in individual patients. The level of expression of the human telomerase reverse transcriptase (hTERT) gene has been associated with disease aggressiveness in human cancers. The purpose of the present study was to examine the prognostic significance of hTERT expression in B-CLL.

Patients and methods: We used real-time reverse transcription-PCR to quantitate the amount of hTERT transcripts in mononuclear blood cells from 90 B-CLL patients. In addition, samples were analyzed for somatic mutations in the immunoglobulin V (IgV) genes.

Results: The expression of hTERT gene was detected in 59% of patients. The level of expression increased with advancing B-CLL stage (P=0.0064). Patients expressing hTERT showed significantly shorter survival than hTERT-negative patients (P=0.000034), irrespective of the disease stage. On average, the level hTERT mRNA expression was seven-fold higher in the poor-prognosis B-CLL group with unmutated IgV than in the Ig-mutated group (P<10–7). The level of hTERT expression discriminated the Ig-unmutated from Ig-mutated B-CLL in 89% of cases.

Conclusion: Our data indicate that hTERT expression in B-CLL may serve as a molecular prognostic marker.

Key words: B-CLL, hTERT, prognosis, real-time reverse transcription-PCR


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Human cancer cells are believed to acquire immortality through the activation of telomerase that is repressed in the majority of normal somatic cells (reviewed in [1Go]). Telomerase is a ribonucleoprotein complex that maintains telomeres by adding hexameric TTAGGG repeats to the chromosomal ends, thus compensating for the continued replicative loss of telomeres [2Go]. Telomerase activity is regulated at the expression level of the human telomerase reverse transcriptase (hTERT) gene coding for the catalytic subunit of telomerase [3Go]. The acquisition of expression of hTERT seems to be an essential step in the development and progression of a majority of human tumors [4Go]. Recent studies have related the level of hTERT expression to clinical aggressiveness and poor prognosis in a variety of malignancies, including leukemia and lymphoma [5Go–10Go].

The identification of reliable prognostic markers is essential in B-cell chronic lymphocytic leukemia (B-CLL), which is clinically heterogeneous. Clinical staging systems enable physicians to divide patients into low-, intermediate- and high-risk groups, but they do not accurately predict disease evolution within the low-risk group, which contains 65% of B-CLL cases [11Go, 12Go] Among known biological indicators of prognosis, the presence or absence of somatic mutations in the immunoglobulin V (IgV) gene regions is considered to be the best discriminator between stable and progressive disease. Within all clinical B-CLL stages, an Ig-unmutated gene profile is associated with an aggressive clinical course [13Go–15Go] However, this analysis is difficult to perform.

In B-CLL, one study has shown that high telomerase activity may be a predictor of a shorter survival [16Go]. Very recently, higher telomerase activity was reported in a poor-prognosis Ig-unmutated B-CLL subgroup, as compared with an Ig-mutated subgroup [17Go]. However, in this study, telomerase activity evaluated using telomeric repeat amplification protocol (TRAP) technology was not significantly related to patient outcome. Thus, the prognostic value of telomerase analysis in B-CLL needs to be confirmed. Moreover, the prognostic significance of hTERT analysis at the gene expression level has not yet been investigated in B-CLL.

To address these issues, we measured the relative amount of hTERT mRNA using real-time reverse transcription (RT)-PCR in mononuclear blood cells obtained from 90 patients with B-CLL at various stages of the disease. The results were correlated to overall patient survival and IgV mutational status. We found that hTERT expression was significantly correlated to short patient survival and Ig-unmutated B-CLL subtype. These results suggest that hTERT expression may serve as a molecular prognostic marker in B-CLL.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and samples
Ninety B-CLL patients [60 men and 30 women with a mean age of 61.5 years (range 36–96)] were included in this study. Median follow-up was 6 years (range 1–22). Patients were seen at the Clermont-Ferrand Hospital, the Pasteur Institute and the Pitié Salpêtrière Hospital (France), and at the Division of Hematology, Universidade Federal de São Paulo (Brazil). After informed consent was given, peripheral blood samples were obtained for the study. Mononuclear cells were isolated from whole blood by means of a standard Ficoll procedure and cryopreserved until RNA extraction. At the time of sampling, 55 patients were in stage A, 22 in stage B and 13 in stage C according to the Binet classification, and 86% of them were untreated. In addition, mononuclear cells obtained from 15 age-matched healthy individuals were included as controls.

Real-time RT-PCR for hTERT mRNA
The amount of hTERT mRNA was assessed using real-time RT-PCR in the LightCycler system (Roche Diagnostics, Meylan, France) as reported previously, with minor modifications [10Go]. Total RNA was extracted, reverse transcribed with random hexamers and amplified using: hTERT-specific primers 5'-GGAGCAAGTTGCAAAGCATTG-3' (forward) and 5'-TCCCACGACGTAGTCCATGTT-3' (reverse); and probes 5'-CTGCGGGAGCTGTCGG-3'FITC (probe 1) and 5'LCRed 640-GCAGAGGTCAGGCAGCA-3'Ph (probe 2). In addition, the amount of ABL mRNA was quantified in all samples as an internal control using the forward primer 5'-GCCGCTCGTTGGAACTCCAAGG-3', reverse primer 5'-TGACTGGCGTGATGTAGTTTGCTT-3' and SYBRGreen I as a detection format. The results of real-time RT-PCR were given as normalized hTERT expression, i.e. the ratio between hTERT and ABL transcripts multiplied by 1000. All experiments were performed in triplicate, with good consistency of results (the mean coefficient of variation was 9.4%).

Analysis of IgV mutational status
A cDNA sample was amplified using primers for the VH gene, and cloned and sequenced as reported previously [15Go]. The sequence was aligned to the DDBJ/EMBL/GenBank and V-BASE databases. Homology of ≥98% to the germ line sequence was used to define the absence of IgV mutations.

Statistical analysis
The Kruskal–Wallis and Spearman rank tests were used to determine the significance of associations between characteristics. A receiver operating characteristic (ROC) analysis was used to determine which hTERT level best discriminated between unmutated and mutated B-CLL cases [18Go]. Overall survival was calculated using the Kaplan–Meier method and survival curves were compared using the log-rank test. Deaths not attributable to B-CLL were censored. Univariate and multivariate analyses were performed using Cox regression study.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Of 90 B-CLL cases, expression of the hTERT gene was detected using real-time RT-PCR in 53 (59%). In positive samples, the normalized amounts of hTERT mRNA transcripts varied between 0.9 and 155.8, with a median of 10.0 (interquartile range 3.7–20.6). The percentage of hTERT-positive cases was essentially identical in stage A (56%) and B/C patients (63%). In contrast, the level of hTERT expression significantly increased with advancing B-CLL stage. The mean normalized hTERT transcript numbers were 5.8 in stage A, 10.7 in stage B and 26.0 in stage C patients (Kruskal–Wallis test, P=0.0064).

Overall patient survival was analyzed as a function of hTERT expression. As shown in Figure 1A, hTERT-positive patients have significantly shorter survival than those with hTERT-negative B-CLL (log-rank test, P=0.000034), irrespective of the disease stage (Figure 1B and C).



View larger version (17K):
[in this window]
[in a new window]
 
Figure 1. Comparison of patient survival in the human telomerase reverse transcriptase gene (hTERT)-positive and -negative B-chronic lymphocytic leukemia (B-CLL) groups. Patients expressing hTERT exhibited significantly reduced survival within the whole patient population (A, P=0.000034), and among stage A (B, P=0.0025) and stage B/C patients (C, P=0.0041).

 
In addition, we compared the levels of hTERT transcripts with unmutated and mutated IgV genes. Data concerning IgV mutation status were available in 83 patients. Of these, 42 (51%) showed unmutated IgV profiles. On average, the level of hTERT mRNA expression was seven-fold higher in Ig-unmutated than in Ig-mutated B-CLL (P<10–7; Figure 2). Significantly higher hTERT expression in IgV-unmutated B-CLL was also observed when analyzing separately stage A (Kruskal–Wallis test, P=0.0000003) and stage B/C (P=0.00027) patients. Sensitivity and specificity estimates, calculated as area under a ROC curve (AUC-ROC), showed that hTERT expression level discriminated Ig-unmutated from Ig-mutated B-CLL in 89% of cases. This degree of concordance was found within the whole population (applying the cut-off for normalized hTERT as 0.9), and among stage A (cut-off 0.9) and among B/C stages (cut-off 0.0).



View larger version (14K):
[in this window]
[in a new window]
 
Figure 2. Levels of hTERT expression were significantly higher in B-CLL patients with unmutated IgV genes than in those with mutated IgV genes (Kruskal–Wallis test, P <10–7). Mean hTERT values (horizontal bars) were 18.1 in the Ig-unmutated and 2.6 in the Ig-mutated groups.

 
We also found, as expected, a significant relationship between unmutated IgV genes and shorter survival among the whole patient population (log-rank test, P=0.00068), and patients with stages A (P=0.013) and B/C (P=0.028). Univariate Cox analysis confirmed prognostic significance of both parameters investigated in the present study: IgV gene profile and hTERT expression (Table 1). When two prognostic markers were analyzed simultaneously in a multivariate study, the Cox model appeared to be in favor of hTERT expression (P=0.043 for hTERT and P=0.51 for IgV gene profile), suggesting that hTERT might be a better prognostic indicator.


View this table:
[in this window]
[in a new window]
 
Table 1. Cox analysis for overall survival

 
To verify whether the presence of non-malignant cells in unselected B-CLL samples would influence the results of hTERT analysis, we tested mononuclear blood cells from an age-matched normal cohort for hTERT expression. All 15 normal specimens showed no detectable hTERT mRNA by our assay. In addition, among B-CLL patients, we did not observe a correlation between the hTERT level and lymphocyte counts (Figure 3).



View larger version (13K):
[in this window]
[in a new window]
 
Figure 3. Comparison between normalized hTERT mRNA values and lymphocyte counts in B-CLL patients. Level of hTERT expression was not correlated with lymphocyte counts (Spearman rank correlation, not significant).

 

    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
This study was performed to investigate the potential prognostic significance of hTERT expression in B-CLL. Using real-time RT-PCR, we quantified the levels of hTERT mRNA in mononuclear blood cells from 90 patients with B-CLL. The results were correlated with clinical and biological data. We found that patients expressing hTERT have significantly shorter survival than hTERT-negative patients, regardless of disease stage. In addition, the levels of hTERT expression were significantly increased in advanced B-CLL stages and, within all stages, in the clinically aggressive Ig-unmutated B-CLL subtype. Expression of hTERT was a significant prognostic factor when evaluated in a univariate analysis and, simultaneously with IgV mutations, in a multivariate analysis. These results suggest that hTERT expression in B-CLL may be a molecular marker of progressive clinical course and adverse prognosis.

Bechter et al. [16Go] showed previously that high telomerase activity evaluated using TRAP assay in bone marrow from B-CLL patients was associated with a shorter median survival. However, this relationship was not seen in a more recent study of telomeres and telomerase in blood-derived B-CLL cells, although high telomerase activity was found in the poor-outcome, Ig-unmutated B-CLL subgroup [17Go]. Here, we investigated telomerase by using real-time RT-PCR for hTERT mRNA, an approach offering enhanced sensitivity and more precise quantitation in comparison with the TRAP assay [19Go]. The possibility to study telomerase activation in B-CLL at the transcriptional level is supported by a recent report showing that B-CLL patients with high telomerase activity had low methylation of the hTERT gene promoter, which may contribute to up-regulation of hTERT transcription [20Go]. The significant relationship between hTERT expression and decreased patient survival may be viewed as confirmation of the prognostic implication of telomerase in B-CLL. The advantage of hTERT analysis is its capacity to detect clinically relevant differences in telomerase expression in blood-derived B-CLL cells.

The results of the present study indicated that the level of hTERT mRNA was significantly higher in Ig-unmutated than that in Ig-mutated B-CLL, which is in line with a recent report by Damle et al. [17Go]. This difference suggests that hTERT expression may also be useful as a simple surrogate for IgV mutations. As mutation analysis is technically difficult, many efforts have recently been made to identify markers that are easy to detect and are predictive of IgV gene status. The most sensitive (91%) and specific (100%) distinction was achieved by immunofluorescence detection of ZAP-70 expression, which is higher in Ig-unmutated than in Ig-mutated B-CLL [21Go]. Since ZAP-70 is normally expressed in T-lymphocytes, the analysis should be performed selectively on leukemic cells. In contrast, our results showed that the presence of non-malignant cells in unselected B-CLL blood samples does not significantly influence the specificity of hTERT analysis. Eighty-nine per cent of patients were correctly assigned an Ig-unmutated or Ig-mutated profile on the basis of hTERT expression level.

The difference in hTERT expression between Ig-unmutated and Ig-mutated B-CLL subtypes might be related to their telomere status. Telomeres were found to be significantly shorter in Ig-unmutated than in Ig-mutated B-CLL [17Go, 22Go]. In addition, B-CLL cases with shorter telomeres were shown to exhibit higher levels of telomerase and hTERT mRNA [16Go, 22Go]. This association may be the result of a greater need for telomeric end maintenance in B-CLL cells with shortened telomeres to extend their proliferative lifespan. To prevent critical telomere attrition during expansion, Ig-unmutated B-CLL cells with short telomeres are likely to up-regulate hTERT and telomerase. In contrast, Ig-mutated B-CLL cells are less limited in their expansion potential because of their increased telomere reserve, at least in the ‘early’ stage of the disease.

In summary, hTERT expression appears to be a novel molecular prognostic marker in B-CLL. Further assessment of this approach awaits validation in a larger series of patients in comparison with other known prognostic parameters such as lymphocyte doubling time [23Go], levels of ß2-microglobulin [24Go] and soluble CD23 [25Go], serum thymidine kinase levels [26Go], genetic abnormalities [27Go, 28Go] and IgV mutations [13Go–15Go].


    Acknowledgements
 
The authors are grateful to Dr J. Chassagne for providing B-CLL samples and to F. Aït-Ouaret for expert technical assistance.

Received for publication November 23, 2003. Revision received May 16, 2004. Accepted for publication May 19, 2004.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1. Hahn WC, Counter CM, Lundberg AS et al. Creation of human tumour cells with defined genetic elements. Nature 1999; 400: 464–468.[CrossRef][ISI][Medline]

2. Morin GB. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 1989; 59: 521–529.[ISI][Medline]

3. Pool JC, Andrews LG, Tollefsbol TO. Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT). Gene 2001; 269: 1–12.[CrossRef][ISI][Medline]

4. Stewart SA, Weinberg RA. Telomerase and human tumorigenesis. Semin Cancer Biol 2000; 10: 399–406.[CrossRef][ISI][Medline]

5. Wang L, Soria JC, Kemp BL et al. hTERT expression is a prognostic factor of survival in patients with stage I non-small cell lung cancer. Clin Cancer Res 2002; 8: 2883–2889.[Abstract/Free Full Text]

6. Tomoda R, Seto M, Tsumuki H et al. Telomerase activity and human telomerase reverse transcriptase mRNA expression are correlated with clinical aggressiveness in soft tissue tumors. Cancer 2002; 95: 1127–1133.[CrossRef][ISI][Medline]

7. Poremba C, Scheel C, Hero B et al. Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: a molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues. J Clin Oncol 2000; 18: 2582–2592.[Abstract/Free Full Text]

8. Xu D, Gruber A, Peterson C, Pisa P. Telomerase activity and the expression of telomerase components in acute myelogenous leukaemia. Br J Haematol 1998; 102: 1367–1375.[CrossRef][ISI][Medline]

9. Harada K, Kurisu K, Arita K et al. Telomerase activity in central nervous system malignant lymphoma. Cancer 1999; 86: 1050–1055.[CrossRef][ISI][Medline]

10. Tchirkov A, Rolhion C, Kémény J-L et al. Clinical implication of quantitative real-time RT-PCR analysis of hTERT gene expression in human gliomas. Br J Cancer 2003; 88: 516–520.[CrossRef][ISI][Medline]

11. Rai KR, Sawitsky A, Cronkite EP et al. Clinical staging of chronic lymphocytic leukemia. Blood 1975; 46: 219–234.[Abstract]

12. Binet JL, Auquier A, Dighiero G et al. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer 1981; 48: 198–206.[ISI][Medline]

13. Damle RN, Wasil T, Fais F et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999; 94: 1840–1847.[Abstract/Free Full Text]

14. Hamblin TJ, Davis Z, Oscier DG et al. Unmutated immunoglobulin VH genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848–1855.[Abstract/Free Full Text]

15. Maloum K, Pritsch O, Magnac C et al. Expression of unmutated VH genes is a detrimental prognostic factor in chronic lymphocytic leukemia. Blood 2000; 96: 377–379.[Free Full Text]

16. Bechter OE, Eisterer W, Pall G et al. Telomere length and telomerase activity predict survival in patients with B-cell chronic lymphocytic leukemia. Cancer Res 1998; 58: 4918–4922.[Abstract]

17. Damle RN, Batliwalla FM, Ghiotto F et al. Telomere length and telomerase activity delineate distinctive replicative features of the B-CLL subgroups defined by Ig V gene mutations. Blood 2004; 103: 375–382.[Abstract/Free Full Text]

18. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 1982; 143: 29–36.[Abstract]

19. de Kok JB, Ruers TJ, van Muijen GN et al. Real-time quantification of human telomerase reverse transcriptase mRNA in tumors and healthy tissues. Clin Chem 2000; 46: 313–318.[Abstract/Free Full Text]

20. Bechter OE, Eisterer W, Dlaska M et al. CpG island methylation of the hTERT promoter is associated with lower telomerase activity in B-cell lymphocytic leukemia. Exp Hematol 2002; 30: 26–33.[CrossRef][ISI][Medline]

21. Crespo M, Bosch F, Villamor N et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003; 348: 1764–1775.[Abstract/Free Full Text]

22. Hultdin M, Rosenquist R, Thunberg U et al. Association between telomere length and V(H) gene mutation status in chronic lymphocytic leukaemia: clinical and biological implications. Br J Cancer 2003; 88: 593–598.[CrossRef][ISI][Medline]

23. Montserrat E, Sanchez-Bisono J, Vinolas N, Rozman C. Lymphocyte doubling time in chronic lymphocytic leukaemia: analysis of its prognostic significance. Br J Haematol 1986; 62: 567–575.[ISI][Medline]

24. Di Giovanni S, Valentini G, Carducc P, Giallonardo P. ß2 microglobuline is a reliable tumor marker in chronic lymphocytic leukemia. Acta Haematol 1989; 81: 181–185.[ISI][Medline]

25. Sarfati M, Chevret S, Chastang C et al. Prognostic importance of serum soluble CD23 level in chronic lymphocytic leukemia. Blood 1996; 88: 4259–4264.[Abstract/Free Full Text]

26. Hallek M, Langenmayer I, Nerl C et al. Elevated serum thymidine kinase levels identify a subgroup at high risk of disease progression in early, non smoldering chronic lymphocytic leukemia. Blood 1999; 95: 1732–1737.

27. Juliusson G, Oscier DG, Fitchett M et al. Prognostic subgroups in B-cell chronic lymphocytic leukemia defined by specific chromosomal abnormalities. N Engl J Med 1990; 323: 720–724.[Abstract]

28. Oscier DG, Gardiner AC, Mould SJ et al. Multivariate analysis of prognostic factors in CLL: clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 2002; 100: 1177–1184.[Abstract/Free Full Text]