The Extent in Telomere Attrition and the Number of Affected Leukocyte Subsets Distinguishes Patients with Different Myeloproliferative Neoplasms (MPN).

Myeloproliferative neoplasms (MPN) are clonal disorders with an origin of the disease in a hematopoietic stem or progenitor cell. Except for chronic myeloid leukemia (CML), the diagnosis for Philadelphia-chromosome negative (Ph-neg.) MPN is less straightforward. Although many patients with a Ph-neg. MPN can be identified by mutations in JAK2 and/or TET2, the categorization into primary or secondary cythosis or specifically into polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) can be difficult. Since telomeres can be used to estimate the mitotic history of cells, our aims were to evaluate 1) whether the telomere length can be used to distinguish clonal from polyclonal hematopoiesis, 2) whether telomere attrition correlates to the mutational status of JAK2 and 3) whether the extent of telomere shortening in different subsets of leukocytes can point to the originating level of the hematopoietic stem or progenitor cell.

So far, 32 patients with Ph-neg. MPN diagnosed according to WHO criteria (range: 32 – 85 years; PV: n=19, ET n=5, PMF n=8; JAK2V617F positive n=22, JAK2V617F negative n=16) and 11 patients with secondary erythrocythosis (range 39 – 61 years) were included in this study. 400 healthy individuals (range 0-102 years) served as controls. After informed consent peripheral blood was taken from the patients to measure the telomere length in subsets of leukocytes by automated multicolor flow-FISH. In order to correct for the age-dependent decline in telomere length, telomere length differences to the 50^th percentile of the healthy cohort (DeltaTel) were calculated. Telomere length values below the 10^th percentile of those from healthy donors were considered as substantially affected by telomere attrition. The mutational status of the JAK2V617F was assessed by allele-specific real time quantitative PCR.

The mean telomere length in granulocytes from patients with MPN was considerably shorter compared to healthy controls (mean ± STD DeltaTel: 2.73kb ± 1.20kb), whereas there was no remarkable difference in lymphocytes (0.70kb ± 0.81kb). Furthermore, we found significant DeltaTel between granulocytes from patients with MPN and with secondary erythrocythosis (mean ± STD: 2.73kb ± 1.20kb vs. 1.66kb ± 0.894kb, p<0.0001). Regarding the JAK2V617F mutational status we found no difference for the average DeltaTel (mean ± STD: 2.35kb ± 1.25kb (JAKV617F+), 2.28kb ± 1.12kb (JAKV617F-), p=0.84). Most interestingly, there was a clear difference in the average DeltaTel in granulocytes from patients with different types of MPN (ANOVA p=0.017). The most striking DeltaTel was seen between patients with PMF and patients with PV and ET (PMF: 3.72kb ± 0.30kb, PV and ET: 2.40kb ± 1.18kb, p=0.005). In addition, in patients with PMF most subsets of leukocytes demonstrated substantial telomere length differences compared to healthy controls (Granulocytes: 2.06kb ± 0.55kb, p=0.0001, T-cells: 0.84kb ± 1.07kb, p=0.016, B-cells: 1.62kb ± 1.29kb, p=0.0001, NK-cells: 0.95kb ± 1.49kb, p=0.039), whereas in patients with ET, except for the granulocyte subset, no significant DeltaTel values were detected (Granulocytes: 1.01kb ± 0.856kb, p=0.014, T-cells: -0.435kb ± 0.723, p=0.34, B-cells: 0.26kb ± 1.00kb, p=0.65, NK-cells: 0.30kb ± 0.86kb, p=0.58). In patients with PV one to three leukocyte subsets showed substantial DeltaTel, but in varying combinations.

In our ongoing study we were able to confirm shorter telomeres in granulocytes of MPN patients compared to telomeres in granulocytes of healthy controls and of patients with secondary erythrocythosis. Based on the telomere length attrition patients with a clonal cythosis can be distinguished from such patients with a secondary cythosis, which could help diagnostically in uncertain cases. No correlation was found between the extent of telomere attrition and the JAK2V617F mutational status. The extremely short telomeres found in most subsets of leukocytes from patients with PMF could point to a very early hematopoietic stem cell as the cell of origin, whereas for ET and PV with only one or a few subsets of leukocytes affected by telomere attrition the cell of origin could be a hematopoietic stem cell at a somewhat later stage.

No relevant conflicts of interest to declare.