Telomere dynamics and telomerase expression in chronic myeloid leukaemia

INTRODUCTION: Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of the haemopoietic stem cell (HSC), with a variable clinical course. Chronic phase (CP) disease, typically of 4-5 years duration, progresses to accelerated (AP) and then blastic phase (BP), with the latter behaving as a particularly aggressive acute leukaemia of either myeloid (MBP) or lymphoid (LBP) lineage. Treatment is most successful when delivered in CP, and accurate prognostic indices are required to individualise treatment. Increased telomere shortening has been described during progression of CML, and may be of prognostic relevance. Paradoxically, telomerase activity (TA, as determined by the TRAP assay) has been shown to be elevated in the CP stem/progenitor cell (CD34+) compartment: however this may not accurately reflect telomere maintenance in-vivo. We sought to further define the prognostic significance of telomere shortening at diagnosis of CML, monitor the rate of telomere loss during the disease and characterise expression of the major telomerase components (hTR and hTERT) in CD34+ selected cells at diagnosis and during disease progression. METHODS: Peripheral blood leucocyte (PBL) telomere length measurement was performed by flow-FISH on cohorts of normal individuals, patients at diagnosis and all stages of CML. To define the degree of telomere shortening in individual patients at diagnosis ex-vivo expanded (BCR-ABL') T-cells were used as an internal control for 'normal' somatic cell telomere length. Expression of hTERT and hTR was quantified by Q RT-PCR and hTERT mRNA splice variants detected by RT-PCR. CD34+ selected cells from CML patients were confirmed as BCR-ABL+ by FISH. TA was determined by TRAP assay. RESULTS: Telomere shortening in CP and AP CIVIL patients progressed at 10-20 times the rate of age-related shortening observed in the normal control group. Furthermore, high-risk prognostic score patients at diagnosis had significantly shorter telomeres than low-risk patients. High purity CD34+ selected cells from CML, as compared to normal, demonstrated increased TRAP activity which correlated with the proportion of cycling cells. However, hTERT mRNA expression was not significantly elevated. Unexpectedly, Q-RT-PCR for hTR demonstrated a mean five-fold reduction in levels in the CML samples, raising the possibility that telomere homeostasis is disrupted in CML. In BP samples, hTERT expression was significantly lower in MBP than LBP and this was mirrored by a corresponding shift in hTERT splicing patterns. MBP hTERT expression correlated inversely with telomere length. CONCLUSIONS; In summary, increased TRAP activity is not synonymous with telomere maintenance in CML, and dysregulated expression of hTR may contribute to the telomere loss observed in these patients. Indeed TRAP activity appeared largely dependent on the proportion of cycling cells. In the context of progressive (i.e. BP) disease, hTERT expression is lineage and telomere length dependent, thus explaining inconsistent reports of TA levels in BP samples. We have also demonstrated that subtle shifts in splicing of hTERT mRNA is likely to have a regulatory role in primary HSC. In prognostic terms telomere shortening in CML is greatest in high-risk score patients at diagnosis, and occurs rapidly during disease progression. These data further emphasise the potential clinical utility of telomere length measurement for prognostic modelling and monitoring of disease progression.