The Telomere Length In Peripheral Blood Lymphocytes Is a Useful Predictor of the Response to Immunosuppressive Therapy In Patients with Acquired Aplastic Anemia

The ability to predict the response to immunosuppressive therapy (IST) in patients with aplastic anemia (AA) will avoid unnecessary and possibly harmful treatment. To this end, several in vitro tests have been proposed as useful predictors of response in adult patients. However, we previously reported that the presence of either human leukocyte antigen (HLA)-DR15, a minor population of paroxysmal nocturnal hemoglobinuria (PNH)-type cells, or of autoantibodies to postmeiotic segregation increased 1 (PMS1) in children with AA, were not useful predictors of the response to IST, suggesting a different etiology of AA between adults and children (Yoshida, N et al Br J Haematol. 2008). Telomeres in peripheral blood cells from AA patients are shorter than those from normal individuals in 30% of patients. This shorter telomere length in AA patients is thought to result from genetic abnormalities in the genes related to telomere maintenance, stressed hematopoiesis due to immunological attack, and increased cell division. An NIH group and we reported that cryptic forms of dyskeratosis congenita (DC) are present in just under 5% of patients with AA who do not have physical abnormalities, but who harbour the gene mutation associated with telomere maintenance (Yamaguchi, H et al. N Engl J Med. 2005, Liang, J et al. Haematologica 2006). To identify such patients is important because those who have these mutations might respond poorly to IST. However, the presence of cryptic forms of DC does not explain the causes of short telomeres in the other patients. We hypothesized that patients with short telomeres may respond poorly to IST, and thus evaluated the relationship between telomere length in hematopoietic cells before IST and the response to IST in patients with AA.

We enrolled 40 newly diagnosed children with AA (median age, 10 years; range, 1 to 16 years) who received IST. We obtained peripheral blood lymphocytes from the patients with AA at the time of diagnosis and prospectively measured the telomere length of lymphocytes prior to IST 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 from 71 normal age-matched individuals. Delta RTL of each patient with AA was calculated using the distance of the RTL from the line of age-adjusted normal telomere length.

Of the 40 patients, at diagnosis AA was very severe in 12, severe in 13, and non-severe in 15. All received IST with horse antithymocyte globulin (ATG) and cyclosporine. The causes of AA were idiopathic in 38 patients and hepatitis in 2. In total, 25 of the 40 patients (63%) responded to IST at 6 months after administration of ATG. Between the responder and non-responder groups, no significant differences in gender, age, etiology, disease severity or interval from diagnosis, and IST were observed. Univariate analysis for IST response did not find any significant differences in median WBC count, neutrophil count, Hb level, reticulocyte count, platelet count or minor PNH clone positivity between the groups. However, delta RTL was significantly lower in non-responders than in responders (p=0.035). Multivariate analysis for response to IST revealed that only delta RTL was significantly lower in non-responders than responders (p = 0.049).

The telomere length in lymphocytes is promising not only to identify cryptic DC but also to predict the response to IST of patients with AA. A further larger analysis is warranted to evaluate the correlation between telomere length and the response to IST in AA patients.

No relevant conflicts of interest to declare.