Despite documented success of immunosuppressive therapy (IST) in the treatment of aplastic anemia (AA), a significant minority of patients remain refractory, most responses are incomplete, and allogeneic stem cell transplantation is not available for older patients or those with significant comorbidities. Until introduction of the cMpl agonist eltrombopag, anabolic steroids were the most commonly used salvage drugs. At least theoretically, engaging growth factor receptors with eltrombopag has the potential to promote the evolution or expansion of mutant clones and thereby increase the rate of progression to secondary MDS, a feared complication of AA occurring in 10-20% of patients. Recently we and others reported detection of clonogenic somatic mutations typical of MDS in patients with AA and PNH. Subsequent study demonstrated that mutations characteristic of sMDS can be found in some patients at presentation of AA and may constitute risk for subsequent progression to MDS. As the risk of MDS evolution was a prominent concern when filgrastim was more widely used in management of AA, now similar questions have been raised regarding use of eltrombopag, be it as salvage therapy or to complement IST.

Recently, one of our primary refractory patients receiving eltrombopag progressed to AML. This clinical observation led to investigation of the impact of eltrombopag on evolution and clonal expansion using deep sequencing of a cohort of patients with AA. DNA from bone marrow cells was sequenced before and after initiation of eltrombopag to evaluate clonal expansion or evolution using a targeted multi-amplicon deep sequencing panel of the top 60 most commonly mutated genes in MDS.

Among 208 AA patients treated at Cleveland Clinic, we identified 13 patients (median age 68 yrs.) who were treated with eltrombopag for IST-refractory AA; median duration of treatment was 85 wks. The overall response rate, defined as sustained improvement in blood counts and transfusion independence after 12 weeks of therapy, was 46% (6/13), while 38% (5/13) of patients showed stable disease with intermittent transfusions (one of whom underwent HSCT). Among the two non-responders, one patient developed a PNH clone and another progressed to AML (see below). Expansion of PNH granulocytes after eltrombopag treatment was observed in 2 patients. Two patients had chromosomal abnormalities at initial diagnosis, one with t (10; 18) in 2 metaphases, and one with an extranumeral Y chromosome.

Use of next generation sequencing (NGS) allows for the quantitative detection of clonal events. We hypothesized that serial analysis by NGS before and after eltrombopag therapy may provide clues as to potential effects of this drug on clonal evolution. Sequencing analysis before eltrombopag treatment revealed the presence of a sole clonal mutational event in 3/13 cases, including CEBPA, EZH2, and BCOR. In the patient with a CEBPA mutation, the mutation persisted during treatment with minimal clonal expansion evidenced by a change in VAF from 53% to 65%. In the second patient, NGS results revealed the initial presence of an EZH2 mutation. A post eltrombopag sample clearly identified acquisition of additional clonal events in genes highly associated with advanced disease and clonal evolution (RUNX1 and U2AF1), as well as slight expansion of a persistent EZH2 clone from 2 to 8%. The third patient harbored a BCOR mutation which expanded markedly, increasing from 8% to 21%, and was accompanied by a hematological response. Sequencing results after eltrombopag treatment revealed the acquisition of new somatic mutations in 5/13 (38%) cases: 2 new CEBPA mutations, 1 new BCOR mutation, and, as discussed, one case with an initial EZH2 mutation in which RUNX1 and U2AF1 mutations were later discovered. In the 5th patient, evolution to AML was observed and accompanied by a large DNMT3A and U2AF1 clone that was absent on initial evaluation.

In conclusion, we did observe occasional expansion of clones with potentially leukemogenic mutations during treatment with eltrombopag. At our institution a case control study of patients with refractory aplastic anemia without treatment with eltrombopag is ongoing; ideally a prospective trial would be needed to confirm results. Our results suggest that the initial detection of certain somatic mutations (CBL, SETBP1 and RUNX1) associated with post-AA MDS may contraindicate use of eltrombopag in AA.


Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees.

Author notes


Asterisk with author names denotes non-ASH members.