An Accumulation of Cytogenetic and Molecular Genetic Events Characterizes the Progression From MDS to Secondary AML: An Analysis of Paired Samples in 38 Patients Analyzed by Cytogenetics, Mutation Analysis and SNP Microarrays.
Authors: Flach Johanna , Schindela Sonja , Dicker Frank , Schnittger Susanne , Kazak Beray , Kohlmann Alexander , Kern Wolfgang , Haferlach Torsten , Haferlach Claudia 
Published: October 2019

Abstract
Abstract 1748
Poster Board I-774
A hallmark of myelodysplastic syndromes (MDS) is their progression to acute myeloid leukemia (AML). Even though the pathogenetic mechanisms underlying both MDS and de novo AML have broadly been studied on a genetic level, the molecular factors that determine the progression from MDS to secondary AML (s-AML) are largely unknown. In order to gain further insights into the underlying genetic events leading to progression from MDS to AML, we analyzed a total of 38 patients at both their MDS and s-AML disease state focusing on cytogenetics and mutations in NPM1, MLL, NRAS, RUNX1 and FLT3. Additionally, we performed genome-wide SNP microarray analyses (Affymetrix SNP Array 6.0) at both timepoints of investigation in a subset of 34/38 patients. The cohort comprised 38 MDS patients (male/female: 25/13; median age at diagnosis 69.5/range 29.2-82.9) representing WHO categories RARS (n=2), RCMD (n=3), RAEB-1 (n=11), RAEB-2 (n=8), MDS unclassifiable (n=9), CMML (n=3) and MDS/MPN overlap (n=2). Most patients presented in MDS phase with a normal karyotype (n=27; 71%) or showed a single cytogenetic abnormality (n=8). One patient had two alterations and two patients showed a complex karyotype. Molecular genetic analysis revealed the presence of MLL-PTD (n=2), NRAS (n=2) and RUNX1 (n=10, 26.3%) mutations, with mutations in RUNX1 being by far the most common molecular mutation detected in our MDS cohort. NPM1 and FLT3-ITD mutations were not seen at that stage. The median time between analysis of MDS and the diagnosis of s-AML was 269 days (range 40 to 1044). Progression to s-AML was defined by blasts in the bone marrow or peripheral blood >20% and was accompanied by accumulations of either cytogenetic or molecular genetic events. 10/38 patients showed increased cytogenetic complexity with gains of one to 10 aberrations in the course of disease evolution (median number of gained anomalies: 1). Three patients acquired trisomy 8 during disease progression. Moreover, individually different chromosomal losses (n=1), insertions (n=1), translocations (n=3) and further chromosomal gains (n=8) were detected. 16 additional molecular alterations were observed in 15/38 patients involving genes NPM1 (5/16), MLL (3/16), NRAS (3/16), RUNX1 (3/16) and FLT3-ITD (2/16). Of note, these patients exclusively showed either cytogenetic or molecular genetic progression. Only one patient acquired both cytogenetic abnormalities (del(5q) and del(7q)) and a new mutation in RUNX1. 12/38 patients did neither show cytogenetic progression nor did they gain mutations in the genes analyzed. The latter patients, however, showed a trend towards a better overall survival from time of diagnosis of s-AML when compared to the patients with progression markers (median overall survival: not reached vs 7.9 months, % surviving at 1 year: 87.5 vs. 49.7%, p=0.09). SNP microarray analyses revealed the presence of UPD (uniparental disomy) in 11/34 paired samples including chromosomal regions 2p, 4q (n=2), 7q, 11q, 12p, 13q, 17q, 19p, 19q and 21q. While 9/11 patients showed the same UPD at both their MDS and s-AML stages, UPD21q (upd(21)(q11.2-qter)) in one and UPD17q (upd(17)(q21.2-qter)) as well as UPD19q (upd(19q13.42-qter)) in another patient were acquired during disease progression. Interestingly, UPD21q acquired during disease progression coincided with a shift from heterozygous to a homozygous RUNX1 mutation suggesting a role for acquired UPD as a mechanism leading to copy-neutral loss of heterozygosity of RUNX1. UPD(4q) came along with a mutation in TET2, which is located on 4q. This mutation was already present at the MDS stage. In conclusion, our data provide evidence for a multistep pathogenetic mechanism in the progression from MDS to s-AML. Patients can be grouped in two categories; they either acquire cytogenetic aberrations or present a higher number of molecular genetic mutations. While RUNX1 mutations already occurred at a high frequency at the MDS stage, mutations in NPM1 and FLT3 were seen only after progression to s-AML.

Disclosures
Flach: MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kazak:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.