Page 333 - 2021_02-Haematologica-web

Case Reports
Detectable mutations precede late myeloid
neoplasia in aplastic anemia
Aplastic anemia (AA) is bone marrow failure character- ized by a hypocellular marrow and peripheral pancytope- nia. Immunosuppressive therapy (IST) results in hemato- logic recovery in the majority, decreased short term cytopenia related complications, and increased survival. However, clonal evolution to a secondary myeloid malig- nancy is a major complication in long-term survivors.1 Clonal evolution occurs in 10-15% of severe aplastic ane- mia (SAA) patients after IST.2 Most high risk clonal evo- lution is associated with complete or partial loss of chro- mosome 7, which is also frequent in other marrow failure syndromes.3 Previous studies have found older age, mul- tiple rounds of IST, and severity of cytopenia as risk fac- tors for clonal evolution.2,3
Shorter telomere length at diagnosis of AA and acceler- ated telomere attrition preceding the malignant transfor- mation are associated with chromosome 7 clonal evolu- tion, suggesting genomic instability as a possible mecha- nism.1 Frank myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) without chromosome 7 involvement is much less common.3-5 Mutations in myeloid neoplasia related genes are present in 20-30% of patients with SAA.6,7 Excluding PIGA mutations, the most frequently affected genes are epigenetic regulators: ASXL1, DNMT3A and BCOR. The presence of unfavor- able somatic mutations as a group is associated with a higher risk of clonal evolution and poorer survival.7,8 Surprisingly, somatic mutations were not frequently detected in clonal evolution to monosomy 7,9 including in patients treated with eltrombopag (EPAG).10
We retrospectively assessed all subjects with SAA treated at the NIH Clinical Center on Hematology Branch protocols, with either horse- anti thymocyte globulin (ATG), rabbit-ATG or alemtuzumab based regimens for treatment-naïve or relapsed/refractory disease from 1989 to 2019. A total of 666 subjects were identified, of whom 96 had clonal evolution. The definition of clonal evolu- tion was consistent across protocols. Clonal evolution was defined as an acquisition of new cytogenetic abnor- malities with or without morphologic evidence of a myeloid malignancy. It was considered a high-risk clonal evolution when there was a diagnosis of a myeloid malig- nancy, an isolated acquisition of chromosome 7 abnor- mality or complex karyotype. High-risk evolution was
noted in 58 of 96 subjects (Figure 1). Fifteen of these 58 subjects had evolved at 5 years or later after initial IST treatment. Among these 15 patients, eight subjects evolved with cytogenetic abnormalities and seven had MDS/AML with normal karyotype. We further investi- gated whether the late evolution to MDS/AML in the absence of cytogenetic changes was associated with somatic mutations in myeloid genes. Three subjects evolved at outside institutions and were excluded from the analysis due to insufficient data. One subject with loss of Y, a common age-related cytogenetic abnormality, was included.
In this case series, we report four cases of late occurring MDS/AML without chromosome 7 abnormalities. Patients were screened for mutations in genes recurrently mutated in myeloid malignancies by next generation sequencing (NGS) at the time of clonal evolution. Detected variants were confirmed and tracked back in earlier serial samples by digital droplet PCR (ddPCR).
All patients were evaluated with clinic visits, bone mar- row examinations, and cytogenetics, as dictated by treat- ment protocols No NCT number: Rosenfeld, Blood. 1995:NCT00001964 and NCT01623167. All patients gave written informed consent to inclusion in the respec- tive studies. Telomere length (TL) was measured by flow- fluorescence in situ hybridization (FISH) at a commercial laboratory (Repeat Diagnostics) or by Southern blotting at the time of SAA diagnosis. The details about NGS methods are included in the Online Supplementary Appendix.
The median age of SAA diagnosis in the four included subjects was 38 years (range: 29-59; Table 1). All four subjects had normal TL at SAA diagnosis. At 6 months, three subjects had achieved partial response and one a complete response (CR) to IST. Only unique patient number (UPN)-4 relapsed 7 months after treatment and required 2 years of additional cyclosporine and EPAG until CR was achieved. This subject also received eculizumab treatment for a large glycosylphosphatidyli- nositol (GPI) deficient granulocyte clone.
At evolution to myeloid malignancy, median time from initial IST was 5.7 years (range: 5-7.3 years). UPN-1 had pancytopenia and UPN-2 had progressive thrombocy- topenia at clonal evolution, but UPN-3 and UPN-4 were diagnosed on protocol-mandated bone marrow evalua- tions with minimal to no change in their blood counts. Cytogenetics were normal in all except UPN-4, who had loss of chromosome Y present a year prior to evolution.
Figure 1. Cytogenetic abnormalities in high-risk clonal evolution stratified by time to evolution from initial immunosuppressive treatment for severe aplastic anemia. There were 58 high-risk clonal evolutions in this large cohort of 666 subjects with severe aplastic anemia (SAA) diagnosis treated with immunosup- pressive treatment (IST). Majority occurred before 5 years and the most common cytogenetic abnormalities were in chromosome 7 including aneuploidy, partial loss/deletion, or inversion. Chr7: chromosome 7; NA: not available.
haematologica | 2021; 106(2)
647