Expression of Human Telomerase Reverse Transcriptase (hTERT) Splice Variants In Chronic Lymphocytic Leukemia (CLL)

Abstract 2413

High telomerase activity and telomere length are prognostic markers in CLL. In a previous study we described hTERT as potential CLL-specific antigen. The rate-limiting component of telomerase is telomerase reverse transcriptase (hTERT), for which multiple transcripts exist. In particular two splicing sites named α and β have been described, which generate deleted transcripts. The existence of multiple hTERT transcripts suggests that telomerase activity may be regulated by transcriptional control mechanisms as well as alternative splicing.

In this study, we validated real-time RT-PCR assays for quantification of the hTERT transcripts with either α deletion (del-α, α-β+), or β deletion (del-β, α+β-) or both (del-αβ, α-β-). Only the full-length (FL, α+β+) transcript translates into a functional protein; the biological role of the other splice variants is still to be clarified. The aim of this work was to completely characterize hTERT splice variants in CLL, as well as study all hTERT splice variants in relation with disease activity.

The splice variant pattern was studied in 68 CLL cases, 6 healthy controls and two CD34+ cell samples. Fourteen of 68 pts (20%) did not express any of the splice variants. FL (α+β+) was five-fold more expressed in patients with unmutated than with mutated IgVH genes (mean normalized expression 1.68×10⁻⁰³ vs 3.36×10⁻⁰⁴, p=0.0004). FL levels correlated with the percentage of IgVH homology (r=0.36, p=0.005). The del-α and the del-β transcripts were also expressed at higher levels in IgVH unmutated patients than mutated patients (2.77×10⁻⁰³ vs 4.45×10⁻⁰⁴, p=0.0004; and 8.78×10⁻⁰⁴ vs 6.92×10⁻⁰⁴, p=0.002, respectively).

The expression of all four transcripts was higher in progressive compared to non-progressive patients. For FL average expression levels in progressive and non-progressive patients were 1.55×10⁻⁰³ vs 3.36×10⁻⁰⁴ (p<0.0001), respectively; for del-α 2.5×10⁻⁰³ vs 5.5×0⁻⁰⁴ (p=0.0003), respectively; for del-β 4.89×10⁻⁰⁴ vs 4.2×10⁻⁰⁴ (p= 0.01), respectively and for del-αβ 1.43×10⁻⁰³ vs 2.16×10⁻⁰³ (p<0.0001), respectively. FL expression was also significantly higher in progressive CLL patients compared to healthy controls (1.55×10⁻⁰³ vs 2.04×10⁻⁰⁴, p=0.03), while no difference was seen with regard to the other variants.

Subsequently, we analyzed IgVH mutated and unmutated patients separately. Notably, the difference in FL expression levels between progressive and non-progressive patients was found only in the unmutated subgroup (2.19×10⁻⁰³ and 4.85×10⁻⁰⁴, respectively, p=0.04), whereas in patients with mutated CLL no difference could be identified. Overall, the levels of expression of FL transcript significantly correlated with those of the other splice variants: del-α (r=0.52, p<0.0001), del-β (r=0.50, p<0.0001), del-αβ (r=0.47, p<0.0001). Two patients (both IgVH mutated) were tested repeatedly in different disease phases and at disease progression an increase in FL transcript levels was seen, while the del-α transcript decreased.

The levels of FL transcript detected in CD34+ cells and in the K562 cell line were 4.09×10⁻⁰⁴ and 1.85×10⁻⁰³, respectively, while for the del-α transcript they were 5.36×10⁻⁰⁴ and 1.73×10⁻⁰³, respectively and for the del-αβ transcript 1.67×10⁻⁰³ and 4.93×10⁻⁰³, respectively. The del-β transcript was not detected in CD34+ cells, while the expression in the K562 cell line was 2.7×10⁻⁰³.

This study provides a detailed insight into the hTERT transcript pattern in patients with CLL and shows that the expression of hTERT-FL is independent of disease phase in IgVH mutated but not in unmutated patients. Our results highlight the necessity of focusing on the functional transcript when analyzing hTERT expression in CLL patients and of interpreting the results in various phases of the disease in relation to the IgVH mutational status. The possibility that hTERT-FL mRNA gives rise to CLL-specific antigens that may be targeted with immunotherapy will be the object of further studies.