Response to L-Leucine Therapy in Patients with Daimond-Blakfan Anemia and Serum L-Leucine Concentrations

Background: Diamond-Blackfan anaemia (DBA) is a congenital bone marrow failure syndrome of childhood manifested as normochromic macrocytic anemia with absence or insufficient erythroid precursors in the bone marrow. The common basis for the anemia is haploinsufficiency of ribosomal proteins. The first line of therapy for patients with DBA is oral corticosteroids. Initially 80% of the patients respond and less than a half of the responders remain on therapy for extended periods. The majority of the DBA patients receive regular blood transfusions. Animal models showed efficacy of L-leucine, a translation enhancer, in the DBA and 5q- syndrome, which has similar altered ribosome function as the DBA. It was observed that treatment with L-leucine improves hemoglobin level and induce transfusion independence in patients with DBA and 5q- syndrome. L-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls cell growth and mRNA translation.

Aims: The aim of our study is to determine the correlation of serum L-leucine and molecular lesions with response to L-leucine in DBA patients.

Methods: After approval by Local Ethics Committee and signed informed consent, 8 DBA patients (aged 2 - 13 years, 5 girls and 3 boys) were enrolled in the study (NCT02386267). The diagnosis of DBA in all patients was established according to the criteria of the DBA Working Group of the European Society for Paediatric Haematology and Immunology. L-leucine (700 mg/ m2) was given orally 3 times a day during 9-12 months. The efficacy criteria for L-leucine therapy were haemoglobin (Hb) level, reticulocytes count (Retic) and serum level of soluble transferring receptor (sTFR). Complete response (CR) - Hb > 9 g/dL and transfusion-independence; partial response (PR) - Hb <9 gd/L and increased Retic >1% and increased interval between transfusions compare to baseline; no response (NR) - no change in transfusion requirements and no significant change in Hb or Retic. Baseline Retic range in all cases was 0.1-0.5%; transfusion's frequency - every 3 weeks. Serum L-leucine concentration was detected by LCMS-8030 Triple Quadrupole Liquid Chromatograph Mass Spectrometer (LC-/MS/MS).

Results: CR was achieved in 3 (37,5%) cases (all are girls, 2 and 13 years old). One of these patients has mutation in RPL11 ex.2 c.65delT (p.Cys21Ser_fs*33) hetero. In the other two cases the whole genome sequencing (WGS) is in progress. In one case (4-year old girl) PR was achieved, characterized by increased intervals between blood transfusions. No response was seen in 4 cases (50%); their genotype: RPS19 ex.2 c.3G>А (Met1Ile) hetero; RPL5 ex.3 c.187C>T (Gln63*); RPS19 ex.2 c.31 C>T (Gln11*) hetero. Increased level of serum sTfR was observed in 7 patients, however only 4 patients have shown respose. Significant increase of Retic count was noticed in 3 cases with CR. Growth velocity was increased in all cases. None of the patients showed any adverse events. The blood chemistry parameters were within reference range in all cases. The serum L-leucine level was 22±1,0 for the patients with CR/PR - ng/ml, for non-responders - 42±10,3 ng/ml, Mann-Whitney p = 0,053.

Conclusions: The L-leucine therapy induces hematological response and affects growth in a proportion of DBA patients.Our preliminary data show a trend of lower serum L-leucine concentartion among the responders. Further study is needed to make firm conclusions regarding the correlation of serum L-leucine and molecular lesions with response to L-leucine in DBA patients.