Targeted Re-Sequencing Analysis of 31 Genes Commonly Mutated in Myeloid Disorders in Serial Samples from Myelodysplastic Syndrome Patients with Disease Progression
Authors: Pellagatti Andrea , Di Genua Cristina , Roy Swagata , Burns Adam , McGraw Kathy L , Larrayoz Maria José , Fernandez-Mercado Marta , Valletta Simona , Mason Joanne , Killick Sally , Mecucci Cristina , Calasanz Maria José , List Alan F. , Schuh Anna , Boultwood Jacqueline 
Published: October 2019

Abstract
Approximately 30-40% of patients with myelodysplastic syndromes (MDS) develop acute myeloid leukemia (AML). Several recurrent mutations have been identified in MDS using next-generation sequencing (NGS) technology and recent studies have greatly illuminated the molecular landscape of this disorder. However, the molecular events driving MDS progression to AML remain poorly understood.
In order to investigate the genetic basis of leukemic transformation in MDS during disease progression, we evaluated the frequency and chronology of the acquisition of mutations using a targeted NGS myeloid gene panel on serial (paired) samples from 41 MDS patients before (pre-progression) and after disease progression (post-progression) to a more advanced subtype (n=7) or to AML (n=34). The mutational profile was characterized using a TruSeq Custom Amplicon panel (Illumina) targeting the hotspots of 31 recurrently (>1%) mutated genes in myeloid malignancies. Samples were run on an Illumina MiSeq and variants were annotated and filtered using Illumina VariantStudio v2.1.36, and interpreted according to the ACMG recommendations. The proportion of sequencing reads reporting a given mutation (variant allele frequency, VAF) was used to estimate the fraction of tumor cells carrying that mutation, and to determine whether mutations are clonal (in all tumor cells) or subclonal (in a fraction of tumor cells).
A total of 100 and 123 mutations across 23 genes were identified in pre- and post-progression samples, respectively. The number of mutations was generally higher in the post-progression samples: the number of cases with one or two mutations was 24 in pre-progression samples and 16 in post-progression samples, while the number of cases with three or four mutations was 12 in pre-progression samples and 18 in post-progression samples. Several cases showed ≥5 mutations.
The most frequently mutated genes (in >15% of samples) were ASXL1, TET2, SRSF2, U2AF1, RUNX1 and TP53; ASXL1 was the top ranking mutated gene with a frequency of 44% in pre-progression samples and 46% in post-progression samples. SF3B1, the most frequently mutated gene in MDS, was mutated in only two cases in our cohort. This finding is consistent with the strong association of SF3B1 mutation with the low-risk MDS subtype RARS, and its status as a good prognostic marker. Our patient cohort is highly selected, comprised only of patients whose disease has progressed and our data thus indicate that ASXL1 mutations are strongly associated with MDS cases that show disease progression and conversely that SF3B1 mutations are rarely associated with MDS disease progression.
Mutations of genes involved in splicing (U2AF1, SRSF2), chromatin modification (EZH2, ASXL1) and DNA methylation (TET2, IDH1/2) were present in the pre-progression samples for almost all cases with mutations of these genes; these mutations often occur in founding clones (VAF >40%) and may play a role in disease initiation. Mutations of genes involved in transcriptional regulation (RUNX1, ETV6, PHF6) and signal transduction (NRAS, KRAS) were found in many cases in the post-progression sample only, suggesting that these are often late events that may co-operate with early events to drive disease progression. Interestingly, co-occurrence of NRAS and ASXL1 mutations was a frequent event in post-progression samples (n=5) in our cohort. It has been reported that NRas mutation and Asxl1 loss co-operate to drive myeloid proliferation and myeloid leukemia in mice, and our data support this observation.
The average VAF of some mutations changed markedly during disease progression, with NRAS showing the largest VAF fold increase among genes mutated in >5 cases. The majority of subclones containing mutations in RUNX1 or ASXL1 expanded with disease progression. EZH2 mutations were mainly in the founding clone, while TP53 mutations were mainly in a subclone.
For six cases we sequenced an additional serial sample, thus giving more precise information on the mutational profile evolution during disease progression.
This is the first study to investigate the mutation status of a large group of MDS patients showing disease progression by the study of serial samples using a NGS myeloid gene panel. This study informs the timing of mutation acquisition during disease initiation and progression in MDS and closely related conditions, illuminating the genetic basis of leukemic transformation in these disorders.

Disclosures
No relevant conflicts of interest to declare.