A Moderate Increase in the WT1 mRNA Copy Number in Peripheral Blood of Patients with Long-Standing Acquired Aplastic Anemia without Apparent Signs of Myelodysplastic Syndrome

Background: The copy number of Wilms tumor 1 mRNA (WT1) correlates well with the acute myeloid leukemia (AML) cell burden and is being used as a reliable marker of residual AML cells. WT1 has also been shown to be useful for distinguishing high-risk myelodysplastic syndrome from low-risk myelodysplastic syndrome (MDS) as well as diagnosing disease progression with time in patients with low-risk MDS. However, few studies have so far focused on the role of measuring WT1 in diagnosing clonal evolution in patients with acquired aplastic anemia (AA). Although normal values of WT1 are defined as <50 copies/µgRNA, the reference values in AA patients without a propensity toward progression to MDS also remain unclear. To address these issues, we retrospectively studied 36 patients with bone marrow failure whose WT1 copy numbers in peripheral blood (PB) were serially measured to determine their correlation with the clinical picture and prognosis of the patients. Patients & Methods: PB samples of 36 patients (male/female, 19/17; a median age, 53.5 [range, 23-85] years) were subjected to WT1 measurement using a quantitative PCR method. The diagnoses of the patients were acquired AA in 29, MDS-multilineage dysplasia (MLD) evolved from AA in 4, and hemolytic paroxysmal nocturnal hemoglobinuria (PNH) in 3. Among 29 AA patients, 2 was untreated, and 22 were in convalescence while 5 were refractory after 0.5-457 months of immunosuppressive therapy (IST) and/or androgen therapy. WT1 was serially measured over time for all the patients except 5 whose initial examination produced negative (<50 copies/µgRNA) results. Myeloid malignancy-related gene abnormalities were screened in 11 patients showing an increase in the WT1 copy number. Results: WT1 at the initial examination was negative in 12 and positive in 17 (59%) patients with AA ranging from 59 to 520 copies. The 17 patients with increased WT1 had a disease duration of 122 months (11-495) but did not shown any signs of progression to MDS. The WT1 levels in patients possessing PNH-type cells (<50-330, median 62) were comparable to those without PNH-type cells (<67-520, median 89. There was a significant difference in the WT1 level between the 10 patients with a disease duration <3 years (<50-190, median <50) and the 19 patients with a disease duration ≥3 years (<50-520, median, 110, P=0.032). All 6 patients with WT1>200 had a disease duration ≥3 years. In contrast, the WT1 levels in the 4 patients who relapsed with cytopenia due to progression to MDS were as high as 690-5,700 (median, 2,400). Deep sequencing revealed somatic mutations of myeloid malignancy-related genes in 4 of 11 patients studied, which included EZH2 in an MDS patient (WT1 2,000), BCOR and DNMT3A in another MDS patient (WT1 2,800), and ASXL1 in a PNH patients (WT1 96). Conclusions: The WT1 copy numbers in PB of AA patients were higher than in AML patients with minimal residual diseases, and the cut-off value that helps diagnose MDS evolving from AA was considered to be around 600 copies. The high WT1 levels in long-standing AA patients without any signs of progression to MDS may represent an increased proliferative potential of hematopoietic stem/progenitor cells that support sustained hematopoiesis with their limited numbers. An increase in the WT1 copy number was associated with somatic mutations in some MDS patients but not in AA patients. Although AA patients with increased WT1 copy numbers need to be carefully followed, a moderate increase in the WT1 copy number does not portend myeloid malignancies unless the number steadily increases.