Cytogenetic and Molecular Genetic Shifts in 27 Genes Investigated By NGS Depict Specific Routes from MDS to s-AML in 38 Patients with Paired Samples

Abstract

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological disorders that can progress to secondary acute myeloid leukemia (s-AML). We performed a comprehensive approach based on cytomorphology, cytogenetics and amplicon deep sequencing using a newly developed pan-myeloid gene panel in order to better clarify this process.Methods: Paired samples from 38 patients at initial diagnosis of MDS and at progression to s-AML were analyzed for the following genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3TKD,FLT3-ITD, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1,WT1 and ZRSR2. With exception of RUNX1 and ZRSR2 (sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT)) and MLL-PTD and FLT3-ITD (analysed by PCR), all remainder genes were sequenced using a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA). A median coverage of 7,626 reads (range 174-12,256) per gene was achieved. The lower limit of detection was set at 3%.Results: At MDS diagnosis, patients were classified according to WHO: RARS (n=2), RCMD (n=3), RCMD-RS (n=8), RAEB-1 (n=12), RAEB-2 (n=12), and MDS with isolated 5q deletion (n=1). Progression to s-AML was defined by blasts >20% in bone marrow or peripheral blood. Transformation to AML occurred at a median time of 18 months (range 2 – 73) after MDS diagnosis. According to chromosome banding analysis and FISH, 14 (36.8%) MDS and 22 (57.9%) s-AML patients had an aberrant karyotype. Thirteen patients (34.2%; 8 with normal and 5 with already aberrant karyotype) gained chromosome abnormalities during progression to s-AML. All samples presented at least one mutation. The most frequent mutated genes were identical for MDS and s-AML: ASXL1 (36.8% / 42.1%); RUNX1 (36.8% / 39.5%); SF3B1 (23.7% / 23.7%); SRSF2 (47.4% / 47.4%) and TET2 (34.2% / 34.2%). Mutations in GATA1 and MPL were not found. Only one KIT-mutation was detected in a patient at MDS stage, while FLT3-TKD mutations were identified in s-AML samples only (7.9%; 3/38 patients). Results for MLL-PTD and FLT3-ITD were available for 58 samples (76.3%). The incidence of MLL-PTD was low (4.5%, 1/22 for MDS and 8.3%; 3/36 in s-AML). FLT3-ITD mutations were exclusively detected in 5 patients (14.3%) at s-AML stage. A total of 21 (55.3%) patients won at least 1 mutation during progression to s-AML. Surprisingly none of the gene mutation frequencies differed significantly between the MDS and s-AML stages. Therefore, we further compared this data set to an independent cohort of 944 newly diagnosed MDS patients (Haferlach T et al, Leukemia 2014). WHO diagnosis categories were comparable between both cohorts. However, mutation frequencies differed: in the present cohort the following mutations were more frequent than in the published cohort: GATA2 (5.3% vs 0.7%; P=0.044), IDH2 (18.4% vs 3.9%; P=0.001), RUNX1 (36.8% vs 10.6%; P=<0.001) and SRSF2 (47.4% vs 17.5%; P=<0.001), for ASXL1 (P=0.078), ETV6 (P=0.068), NRAS (P=0.064), suggesting a role of these genes as s-AML drivers.MDS patients with DNMT3Amut transformed faster to AML than DNMT3A wild-type (wt) cases (median of 8 vs 19 months; P= 0.022). Intriguingly, also SF3B1mut were associated with a faster s-AML progression compared to SF3B1wt (median 8 vs 19 months; P= 0.008). All mutations that were lost at the time of s-AML transformation were small clones (mutation load ≤10%), which hypothetically could have succumbed to more aggressive clones leading to transformation to AML. In detail, in 4 patients with mutation loss there was an increase in the loads of the remaining mutations, whereas their karyotype remained stable during s-AML transformation. On the other hand, the remaining 3 patients with mutation loss at the time of s-AML diagnosis had stable mutation loads in the remaining mutated genes but had a cytogenetic progression.Conclusions: 1) The molecular mutation profile remains stable between MDS and s-AML state in most of the cases and mutations were gained or lost in only a few cases. 2) FLT3-ITD was only detected at AML stage. 3) Mutations in GATA2, IDH2, RUNX1 and SRSF2 seem to predispose to s-AML transformation and mutations in DNMT3A and SF3B1 lead to a significantly faster s-AML progression. 4) Shifts in the karyotype were detected in more than one third of patients underlining its impact on s-AML transformation. DisclosuresDe Albuquerque:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o.: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.