Alternative Splicing of hTERT Exon 7 in AML: Biological Fuction and Prognostic Significance

hTERT, a telomere specific reverse transcriptase with a crucial role in telomerase activity, prevents telomeres shortening, a pro-apototic cellular event which occurs at every round of DNA replication. Its regulation is complex as alternative spliced variants which generate the full-length hTERT transcript (+α+β) and transcripts carrying α and/or β deletions (transdominant negative isoform -α+β, inactive products +α-β and -α-β) have been reported. In AML the prognostic significance of hTERT expression is still debated, even if its over-expression has been correlated with a poor clinical outcome. In contrast, up to now no study has dealt with the frequency and the prognostic significance of variant transcripts.

Thus, our study was aimed at estimating hTERT isoforms distribution in a series of de novo AML patients (pts), at establishing whether their expression varied between pts with a normal and a complex chromosome pattern, at estimating their prognostic significance. The ninety-seven de novo AML pts included in the present study came to our observation in the period January 2010 and January 2013. They were thirty-eight females and fifty-nine males; their median age was 59 years (range 18-84). On conventional cytogenetic studies fifty-two, whose median age was 56 years (range 24-84), presented a normal karyotype and forty-five, whose median age was 60 years (range 33-84), presented a complex karyotype (≥3 defects); the two pts groups were comparable by age. According to WHO classification 6 (6.1%) pts were classified as M0/M1, 47 (48.4%) as M2, 38 (39.1%) as M4, 5 (5.1%) as M5 and one as M6. An internal tandem duplication (ITD) of the FLT3 gene and a NPM1 mutation were revealed in 12 and 2 chromosomally normal pts. No CK presented a FLT3 ITD. All pts received standard induction chemotherapy followed by two courses of consolidation treatment. At the time of the study 38 pts achieved a complete remission (CR) and 36 died. Median follow-up was 22.7 months (range: 13.5-60.2).

hTERT isoforms expression was determined in bone marrow samples by real-time reverse transcriptase polymerase chain reaction, using SYBR Green I. hTERT transcript (+α+β) primers’ design was made using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/), while for the other primers we referred to Capraro et al. 2011. Specific amplifications were confirmed by sequences analysis. In order, to estimate hTERT isoforms expression levels in normal mononuclear cells, twenty-three umbilical cord bloods (UCB) were examined (control group).

When the Kruskal-Wallis rank test was applied to compare the expression of hTERT isoforms inAML pts with a normal and a complex karyotype no significant difference was observed (P=0.39, P=0.77; +α+β, -α+β respectively). Instead, independently from karyotype, in Cox univariate analysis, pts who over-expressed the trans-dominant negative isoform (-α+β) experienced a significant better response to treatment than the other pts (P=0.03, HR=2.11; 95% CI:1,13-3,93). In addition, pts who over-expressed inactive products (-α-β) (first group) presented a leukemia-free survival inferior to that of pts who presented a low expression of these products (second group) (P=0,003, HR=3,26; 95% CI:1,33-7,96). Moreover, pts who over-expressed the full-length hTERT transcript (+α+β) and the trans-dominant negative isoform (-α+β) presented only a trend towards significance. The first group of pts presented a worse overall survival (HR=1,66; 95% CI:0.94-2,91), whereas the second group a better overall survival (HR=0,58; 95% CI:0,33-1,02).

We conclude that the over-expression of the hTERT trans-dominant negative isoform identifies pts with low-risk AML, whereas the over-expression of the hTERT inactive product (-α-β) identifies pts with high-risk AML. In addition, this last hTERT isoform might control the expression of its trans-dominant negative isoform. Thus, our results suggest an intriguing link between the control of hTERT isoforms expression and AML outcome.