Dynamics of Telomere Length and Telomerase Activity in Ph¹-Negative Chronic Myeloproliferative Disorders

Evolving data demonstrate the pathogenetic significance of chromosomal ends telomeres and telomerase activity in the molecular pathogenesis of many hematological disorders. Furthermore, the presence of eroded telomeres and enhanced telomerase activity in hematopoietic cells has been associated with poor prognosis both in myeloid and lymphoid malignancies. The aim of the present study was to evaluate telomere length and telomerase activity in patients with Ph¹-negative Chronic Myeloproliferative Disorders (Ph⁻-CMPD) either at diagnosis or during the course of the disease and to assess their possible clinical utility. Sixty-six bone marrow and 60 peripheral blood samples were obtained from 80 Ph⁻-CMPD patients (aged 58.57±16.42 years) and 18 healthy age-matched controls (aged 53.94±15.16 years). Thirty-six patients diagnosed suffering from Polycythemia Vera, 36 from Essential Thombocythemia, 4 from Idiopathic Myelofibrosis and 4 from Unclassified CMPD. Twenty-six samples were studied at diagnosis, whereas 54 during the course of the disease. Telomere length analysis of individual chromosome ends was performed on bone marrow metaphases using Telomere/Centromere Quantitative-Fluorescence In Situ Hybridization (T/C Q-FISH) (Dako A/S, Denmark). Telomerase activity was determined in bone marrow purified CD34(+) and CD20(+) cells as well as in peripheral blood CD3(+) T-lymphocytes and granulocytes with the PCR-based Telomeric Repeat Amplification Protocol (TRAP) assay (Roche, Germany). Gene expression of telomerase-associated proteins (hTERT, hTER, TEP1, TRF-1 and TRF-2) was assayed by Real-Time Multiplex PCR (Maximbio, USA). Ph⁻-CMPD patients showed significantly more eroded telomeres (P=0.010) and increased telomerase activity in CD34(+) cells (P=0.005) compared to healthy age-matched individuals. However, there was no statistical difference in telomere length (P=0.451) and enzyme activity (P=0.538) among different groups of Ph⁻-CMPD. Telomerase activity was not detected in the remaining hematopoietic cells both in patients and healthy controls, which was closely correlated with downregulation in hTERT mRNA expression. hTER, TEP1, TRF-1 and TRF-2 showed no apparent differential expression of mRNA in all hematopoietic cell fractions. Chromosomal aberrations (+8, +9, del13q14, del20q12) were found by FISH in 37% Ph⁻-CMPD patients with reduced telomere lengths (P=0.001) and enhanced telomerase activity (P=0.014), especially during the course of the disease (P=0.028). The patients with shortened telomeres displayed a higher incidence of having thrombotic or hemorrhagic events during follow-up (P=0.011), treatment failure (P=0.024) and disease progression to myelofibrosis, myelodysplastic syndromes, secondary leukemia or death (P=0.137). Nevertheless, telomerase expression was not correlated with the above complications. The event free survival (survival without complications, e.g. myelofibrosis, myelodysplastic syndromes, secondary leukemia and death) was significantly shorter in patients with reduced telomere lengths (Log Rank P=0.033), who demonstrated a 7,71-fold higher probability of having complications within five years from the initial diagnosis (95% CI=2,04–31,49 P<0.001). In conclusion, accelerated telomere shortening may not be prevented or restored by telomerase activity in most of the Ph⁻-CMPD myeloid cells. Loss of telomere stability seems to predispose to further genetic events such as chromosomal rearrangement and consequently to trigger off a multistage neoplastic transformation of these diseases. Moreover, the negative correlation between telomere length and survival probability of Ph⁻-CMPD patients is indicative that telomere dynamics may serve as a useful prognostic tool for these patients.