Predicting Response to Immunosuppressive Therapy By the Combination of Minor Paroxysmal Nocturnal Hemoglobinuria Clones and Lymphocyte Telomere Length in Children with Aplastic Anemia

Introduction: Acquired aplastic anemia (AA) is an immune mediated disease characterized by severe quantitative defects in stem cell number resulting in a hypocellular marrow and peripheral blood cytopenias. Immunosuppressive therapy (IST) using anti-thymocyte globulin (ATG) and cyclosporine A (CyA) is the standard therapy for patients with AA who lack a human leukocyte antigen (HLA)-matched sibling donor. Previous studies identified minor paroxysmal nocturnal hemoglobinuria (PNH) clones and short telomere length (TL) as predictive markers of the response to IST. However, the likelihood ratio of these markers was insufficient to influence clinical decisions. Here we assessed whether the combination of these markers could more accurately predict the response to IST.

Methods: Between July 2001 and November 2013, 116 patients (65 boys and 51 girls) diagnosed with AA were examined to identify a minor population of PNH-type cells and TL of peripheral blood lymphocytes. Fifty-three patients received horse ATG (Lymphoglobulin, 15mg/kg/day for 5 days) and 63 received rabbit ATG (Thymoglobulin, 2.5−3.75 mg/kg/day for 5 days). CyA (6mg/kg/day) was started on day 1 and continued to at least day 180. The dose of CyA was adjusted to maintain trough levels between 100 and 200 ng/ml. Written informed consent was obtained from all patients’ parents. The ethics committee of each participating hospital approved the study. We used a flow cytometric assay to detect PNH-type granulocytes and red blood cells (RBCs). Presence of >0.007% CD13⁺ CD55⁻ CD59⁻ granulocytes and/or >0.009% glycophorin A⁺ CD55⁻ CD59⁻RBCs was defined as PNH positive (PNH+), based on the results from healthy individuals. The average relative TL of peripheral lymphocytes was measured by flow-fluorescence in situ hybridization (flow-FISH) using a Telomere PNA Kit (Dako Cytomation). Based on our previous study, we defined −1.0 SD of age-adjusted controls as the cut-off value.

Results: The median age at IST was 9.8 years (0.9−16.0). Disease severity of patients was assessed as moderate in 39, severe in 37, and very severe in 40, respectively. The causes of AA were idiopathic in 104 patients and hepatitis-associated in 12 patients. The median follow-up period from the time of IST was 36 months (range 1−134). Thirty-nine patients (34%) carried a minor PNH clone at diagnosis. Compared with healthy individuals, their median of TL was −1.08 SD (−4.01 to +3.01 SD). Overall, 60 of 116 patients (51.7%) responded to IST at 6 months after administration of ATG. In univariate analysis, the response rate at 6 months was higher in PNH+ patients than in PNH- patients (74.4% vs. 40.3%, respectively, p = 0.001). Longer TLs (≥−1.0 SD of age-adjusted control) were also associated with a favorable response to IST at 6 months (73.2% vs. 31.7%, p<0.001). In multivariate logistic regression analysis, PNH clone presence [odds ratio (OR), 4.41; 95% CI, 1.68−11.60; p = 0.003], longer TLs (OR, 5.59; 95% CI, 2.33−13.40; p < 0.001), and male gender (OR, 2.72; 95% CI, 1.12−6.62; p = 0.028) were found to be independent favorable predictors of response to IST at 6 months. Different types of ATG (horse vs. rabbit) did not affect response rate (OR, 1.94; 95% CI, 0.92−4.10; p = 0.084). According to these findings, the cohort was stratified into two groups: possible poor responders [PPR; PNH− and shorter TLs (<−1.0 SD), 45 patients] and possible good responders (PGR; PNH+ and/or longer TLs, 71 patients). The response rate at 6 months in the PPR group was as low as 24.4%, which was significantly lower than that in the PGR group (69.0%, p < 0.001). There was no significant difference in the incidence of relapse (0.0% vs. 20.8%, p=0.190) and clonal evolution (4.5% vs. 5.7%, p=1.000) between two groups. Five-year probabilities of transplant-free survival (TFS) and failure-free survival (FFS) were significantly lower in the PPR group than in the PGR group (TFS, 49.1% vs. 71.4%, p = 0.008; FFS, 42.6% vs. 57.3%, p = 0.020). However, 5-year overall survival showed no difference between the two groups (PPR, 97.7% vs. PGR, 95.5%, p = 0.910) because of the effective salvage therapies.

Conclusion: Combination of the absence of a minor PNH clone and a short TL was an efficient predictor of poor response to IST. These patients had low chances to respond to IST, which challenges the current standard of treatment. Prospective clinical trials are required to confirm these findings.