Telomere Length In Ph - Negative Chronic Myeloproliferative Neoplasms: It Is Reduced According to JAK2 ^V617F Mutation Allele Burden and It Is Not Affected by Cytoreductive Treatment with Hydroxyurea

Telomeres are reliable indicators of previous cell proliferation and cell ageing. Moreover, a marked though variable loss of telomere length (TL) has been observed in several hematological malignancies. In particular, some recent studies have reported a marked TL reduction in patients with Ph-negative Chronic Myeloproliferative Neoplasms (Ph-neg-CMNs) (Ferraris AM et al, Br J Haematol 2005; Bernard L et al, Leukemia 2009). This supports the possible influence of TL in the development of CMNs. Moreover, TL might be of prognostic relevance in these disorders. The present study reports the analysis of TL in a large series of patients with Ph-neg-CMNs, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Myelofibrosis (MF), and in a few cases of Secondary Erythrocytosis (SE), as well. Aims of the study were: i. to verify the rate of TL reduction in Ph-neg-CMNs; ii. to compare TL in ET, PV and MF; iii. to investigate the role of TL as a marker of proliferation, evaluating differences in TL compared to healthy subjects and subjects with Secondary Erythrocytosis (SE); iv. to verify telomere dynamics according to the treatment received.

Peripheral blood (PB) samples were obtained from 239 Ph-neg-CMNs patients (median age 68 yrs, range 10–91): 78 had PV, 100 had ET and 61 MF. Most patients were analyzed for JAK2 mutations: among 72 evaluated PV patients, a JAK2^V617F mutation was found in 66 (92%) and an exon 12 mutation was identified in 2 patients; a JAK2 ^V617F mutation was detected in 50 (50%) ET patients and in 44 (73%) MF patients. Samples were obtained either at diagnosis or during follow-up. More than a half of CMNs patients received before TL analysis at least 1 year of cytoreduction (129 patients); 90% received Hydroxyurea (HU) and 26% other cytoreductive drugs with or without HU. As control, PB samples from 202 healthy age-matched subjects and from 14 SE subjects were analyzed. TL was assessed by Southern blot analysis, according to standard procedures (TeloTAGGG Telomere Length Assay Kit, Roche Diagnostic, Mannheim, Germany). JAK2^V617F mutation analysis was performed by ASO-PCR and digestion with BSAXI (Guerini et al, Leukemia 2008).

PV, ET and MF patients showed individual progressive TL shortening correlated with age as observed in the healthy population. However, CMNs patients had TL significantly shortened (5,890 bp, ± 1,305) compared to healthy age-matched individuals (median: 7,330 bp, ± 1574) (p<.0001). PV and MF showed the most pronounced TL loss among CMNs, significantly different from healthy controls, with TL values respectively of 5,621 bp (± 1,446) (p<.0001) and 5,361 bp (± 1,335 bp) (p<.0001). ET patients had a median TL of 6,422 bp, (± 1131bp). SE patients had a median TL of 6,972 bp (± 1355 bp) with no difference from healthy subjects (p= 1.00). At ANCOVA multivariate analysis of disease characteristics, short TL correlated significantly with JAK2^V617F mutation allele burden >50% (p=.0025), age (p=.0132) and diagnosis of PV (p=.0122). No significant correlation was found with disease duration, history of thrombosis and treatment with cytoreductive or anti-aggregants agents.

i. Ph-negative CMNs are confirmed to present with TL reduction compared to the age-matched healthy population; ii. PV and MF show higher degree of TL loss than ET; ii. high JAK2^V617F mutation burden is strongly correlated to TL loss; iii. SE patients and healthy subjects do not differ in TL, suggesting the possible use of TL as a parameter to distinguish SE from PV, particularly in the few cases lacking JAK2 mutations. iv. no evident influence on accelerated cell ageing is observed in patients receiving cytoreductive treatment with Hydroxyurea. The absence of a marked telomere loss in patients receiving Hydroxyurea, reduces the harm of using this drug when clinically indicated.

No relevant conflicts of interest to declare.