Screening of Telomere Length in Peripheral Blood Lymphocytes From Patients with Aplastic Anemia by Flow-Fluorescence in Situ Hybridization.

Abstract 3205

Poster Board III-142

Telomeres in peripheral blood cells from patients with aplastic anemia (AA) are shorter than those from normal individuals. One possible explanation is that telomere shortening reflects the extent and duration of damage to hematopoietic stem cells and the number of compensatory stem cell divisions. Patients with such stressed hematopoiesis probably do not respond to immunosuppressive therapy (IST). Cryptic forms of dyskeratosis congenita (DC) have been recognized in patients with AA who do not have physical abnormalities, but who harbor the gene mutation associated with telomere maintenance. To identify such patients is important because those who have these mutations might respond poorly to IST. The present study evaluates the usefulness of screening telomere length in peripheral blood lymphocytes from patients at the time of diagnosis with AA by flow-fluorescence in situ hybridization (flow-FISH) using Telomere PNA kits (Dako Cytomation, Glostrup, Denmark). After gating diploid cells based on propidium iodine staining, lymphocytes were isolated according to size and granularity. Relative telomere length (RTL) was calculated as the ratio between the telomere signal of each sample and the telomere signal of the control cell line. Because telomeres shorten with age, we obtained age-adjusted measurements for comparison. We used flow-FISH to measure the length of telomeres in peripheral blood lymphocytes from five and two patients with classical and cryptic DC who had the TERT mutation, respectively, to determine whether these two forms of DC can be screened. Significant telomere shortening was detected in all seven patients compared with age-matched normal individuals. To determine the association between telomere length and response to IST, we compared telomere lengths in peripheral blood lymphocytes from 61 normal controls and 27 patients with AA (very severe, severe and non-severe, n = 9 each) at diagnosis who received IST with antithymocyte globulin (ATG) and cyclosporine. The median age of the patients was 9.0 years (range, 1.5 to 14.8). The causes of AA were unknown etiology in 25 patients and hepatitis in 2. In total, 17 of the 27 patients (63%) responded to IST at 6 months after administration of ATG. Age-adjusted delta RTLs did not significantly differ between normal controls and responders (p = 0.181), but those in non-responders were significantly lower than those in normal individuals (p = 0.000). Our findings suggest that screening telomere length in patients with AA at diagnosis is useful for detecting cryptic DC and for predicting responses to IST.