Sequential Molecular Characterization Based Delineation of Potential Driver Aberrations in ACUTE Myeloid Leukemia Following Myelodysplastic Syndrome
Authors: Rücker Frank G. , Cocciardi Sibylle , Dolnik Anna , Blätte Tamara J. , Wüst Ulrike , Paschka Peter , Gaidzik Verena I. , Schlenk Richard F. , Döhner Hartmut , Bullinger Lars , Döhner Konstanze 
Published: October 2019

Abstract
Background: Myelodysplastic syndrome (MDS) patients exhibit an increased risk of progression towards secondary acute myeloid leukemia (sAML) with poor prognosis. The mechanisms underlying transformation from MDS to sAML are largely unknown.
Aims: To identify genetic lesions associated with transformation in AML [copy number alterations (CNA), chromothripsis, uniparental disomies (UPD), and gene mutations], we performed genome-wide SNP array 6.0 profiling and mutational screening of 9 AML associated genes (RUNX1, TP53, NPM1, NF1, FLT3, ASXL1, DNMT3A, IDH1 and IDH2) in 42 paired MDS/sAML samples including 5 relapsed AML samples (rAML) [WHO MDS categories: RA (n=5), RCMD (n=12), RAEB-I (n=10), RAEB-II (n=8), 5q- (n=1), CMML-1 (n=1), CMML-2 (n=1), unclassifiable (n=4)]. In addition, whole exome sequencing (WES) was performed in 10 selected cases.
Results: In total, 26 (62%) patients acquired genomic aberrations [CNAs and/or UPDs] at diagnosis of sAML as compared to the corresponding MDS sample. Most frequent numerical aberrations were trisomy 8 (n=7) and monosomy 7 (n=4); recurrent submicroscopic losses were identified at 17q11.2 (n=6, encompassing NF1), 17p13 (n=2, TP53), 12p13 (n=2, ETV6), and 21q22.12 (n=2, RUNX1); biallelic 17q11.2 and 21q22.12 losses were detected in one sAML each. Of note, all chromosome 5 alterations (n=11) were already present in MDS. Six (14%) cases acquired UPDs affecting the regions 1p, 11q, 13q, 20q, and 21q (n=2). Paired sequencing of candidate genes in 1p, 13q, and 21q delineated UPD related homozygous mutation patterns for pre-existing heterozygous NRAS, FLT3 -ITD, and RUNX1 mutations in sAML. Acquired AML associated gene mutations were identified in 11 (26%) patients with RUNX1 mutations being the most frequent. Sequential sequencing revealed mutation incidences in MDS and sAML as follows: RUNX1, 17% and 26%; TP53, 19% and 21%; ASXL1, 14% and 17%; DNMT3A, 12% and 14%; NF1, 10% and 14%; NPM1, 7% each; FLT3, 2% and 5%; IDH2, 2% and 5%; and IDH1, 2% each, respectively. While in the NPM1 mutated sAML cases the mutation was already present in the MDS sample (RCMD, n=2; RAEB-I, n=1), all cases transformed quickly within 5 to 6 months into AML compared to NPM1 wildtype MDS patients with a median time to transformation of 14 months. Other variables predicting inferior transformation free survival were biallelic TP53 alteration (n=5; p=.006), chromothripsis (n=4; p=.03), and presence of >4 CNAs (n=10; p=.04). Additional analysis of 5 rAML samples displayed aberration patterns closely related to the sAML in 4 cases; only one rAML seems to be evolved from an ancestral pre-MDS clone. In selected cases with available material (n=10) WES identified an average of 3.5 additional mutations per sAML. These included not only mutations in genes known to be associated with transformation (like e.g. NRAS, RUNX1, ETV6,and FLT3) but also in potential novel driver genes such as e.g. AFF1, NSD1, and STAG2. In accordance, the mutational pattern was not only significantly enriched for genes known to be associated with AML, but also for genes located in commonly gained regions of tumor genomes.
Summary/Conclusions: Over two thirds (71%) of MDS cases acquired additional genetic abnormalities during progression to sAML as detected by SNP profiling and conventional Sanger sequencing; WES suggests that in all cases additional mutations are acquired. The genetic spectrum of our cohort demonstrates a multistep process for leukemogenesis with mutations and genomic aberrations occurring over time. Deletion 5q and mutations in DNMT3A and ASXL1 seem to be early events usually already found in MDS, and UPDs and alterations of RUNX1 and NF1 rather late events acquired in sAML. Ongoing extensive molecular analyses will further unravel the genetic aberrations involved in this multistep process.
Supported by: FP7 NGS-PTL project.
LB and KD contributed equally.

Disclosures
Schlenk: Pfizer: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Teva: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Arog: Honoraria, Research Funding.