Clonal Changes Detected By Target Capture Sequencing and Molecular Cytogenetic Study in Patients with Aplastic Anemia

BACKGROUND: Aplastic anemia (AA) is a group of heterogenous disease. Aplastic anemia with somatic mutations or cytogenetic aberrations are considered as clonal disease either as pre-malignant stage or malignant disease and differential treatment approach from idiopathic AA need to set. The presence of clonality in aplastic anemia could be a determining point for the choice of treatment among bone marrow transplantation or immunosuppressive therapy, and the detection method of clonality could be conventional cytogenetics (CG), molecular cytogenetics or molecular genetic test. Detection sensitivity is 5% by CG, 20-30% by Sanger sequencing, and 0.5-1.0% by next generation sequencing (NGS). We aimed to investigate the frequencies of clonal changes including cytogenetic aberrations and somatic mutations and their correlation with clinical prognosis in aplastic anemia.

METHODS: We reviewed clinical data of the 440 patients (mean age 38 years, M:F ratio 1.1) who were diagnosed with AA from 1997 to 2013. Results of G-banding were available in 280 patients and in all them, fluorescent in situ hybridization (FISH) for -5/5q-, -7/7q-, +8, -20/20q-, and 1q gain was performed. Target capture sequencing for 357 hematopoiesis-related genes was performed in 77 patients. Mutations were selected by running algorithms including SIFT, Polyphen2, and CADD, and normal variant was filtered by 1000Gp, ESP6500, and the in-house Korean database (n=250). We assessed disease progression, survival and response to treatment in 248 patients.

RESULTS: Of 440 patients, 21(4.7%) showed disease progression: MDS in 14(66.7%), AML in 4(19.0%), PMF in 2(9.5%) and plasma cell myeloma in 1(4.8%). Mean duration of transformation to MDS was 59 months (1-151 month). Among 21, only 2 patients showed cytogenetic aberrations (CA) at initial diagnosis, but 9 patients showed cytogenetic evolution at the time of disease transformation: monosomy 7(50%), trisomy 8(25%), and 1q gain (17%). Among 280 patients in whom both G-banding and FISH panel were performed, 21 (7.5%) showed cytogenetic aberrations; numerical abnormality (13/21, 61.9%), structural abnormality (5/21, 23.8%), combined abnormality (3/21, 14.3%). FISH revealed trisomy 8 in 19.0% (7/21), -7/7q- in 9.5% (2/21), followed by 1q gain in 4.5% (1/21). Patients with aberrant FISH result for MDS was associated with disease progression (p=0.0160). Median percentage of FISH⁺ clonal cells were smaller (mean 3.0%), compared to those of MDS (27%) and AML (39%). In severe AA group (n=67), BMT treatment group showed better survival rate than other groups in patients with cytogenetic aberration (p=0.004), while BMT have no impact on survival rate among patients without cytogenetic aberrations (p=0.232). The average of survival duration is in BMT, immunosuppressive treatment (IST), combined BMT &IST group was 61, 153, 68 months. In normal karyotype (NK) group, survival was different depending on treatment regimen (p=0.043), but no different was noted in cytogenetic aberration group. Among 77 patients in whom target sequencing was performed, 20/77 (26%) presented at least 1 mutation. The number of mutated genes was 14 and frequently mutated genes was LRP1B (3/20, 15.0%), NOTCH1 (3/20, 15.0%), SCRIB (3/20, 15.0%), DNMT3A (2/20, 10.0%), BCOR (2/20, 10.0%), DIS3 (2/20, 10.0%), NF1 (2/20, 10.0%), ERCC6 (2/20, 10.0%), GATA2 (1/20, 5.0%), RUNX1 (1/20, 5.0%), RELN1 (1/20, 5.0%), U2AF1 (1/20, 5.0%), TCF12 (1/20, 5.0%), JAK2 (1/20, 5.0%). In patients with somatic mutations, 10% showed disease progression. But patients with somatic mutation did not show any difference in survival, treatment response and disease progression. When the patients were devided by clonal (cytogenetic aberration by G-banding or FISH or somatic mutation by NGS, 14.9%), and non-clonal (84.1%), IST in clonal group showed adverse response compared to non-clonal group (p<0.05), while BMT treatment showed similar response between 2 groups.

CONCLUSIONS: Five percent of among AA patients in Korea revealed disease progression and about half of AA patients harboring clonalities showed at the time of disease transformation. CA was strongly associated with adverse prognosis or treantment response, while somatic mutation was not. Increased sensitivity of detection for clonality could reveal the hidden clonality in AA, but significance of molecular clonal hematopoiesis in AA need to be further investigated.