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
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Abstract |
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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<107). 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
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Introduction |
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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 [11, 12
] 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 [13
15
] 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 [16]. Very recently, higher telomerase activity was reported in a poor-prognosis Ig-unmutated B-CLL subgroup, as compared with an Ig-mutated subgroup [17
]. 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.
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Patients and methods |
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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 [10]. 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 [15]. 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 KruskalWallis 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 [18]. Overall survival was calculated using the KaplanMeier 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.
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Results |
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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).
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Discussion |
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Bechter et al. [16] 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 [17
]. 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 [19
]. 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 [20
]. 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. [17]. 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 [21
]. 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 [17, 22
]. In addition, B-CLL cases with shorter telomeres were shown to exhibit higher levels of telomerase and hTERT mRNA [16
, 22
]. 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 [23], levels of ß2-microglobulin [24
] and soluble CD23 [25
], serum thymidine kinase levels [26
], genetic abnormalities [27
, 28
] and IgV mutations [13
15
].
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Acknowledgements |
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Received for publication November 23, 2003. Revision received May 16, 2004. Accepted for publication May 19, 2004.
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