Distinct mutational pattern of myelodysplastic syndromes with and without 5q- treated with lenalidomide

Abstract

[EN]Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterised by ineffective haematopoiesis leading to peripheral blood cytopenias and an increased risk of transformation to acute myeloid leukaemia (AML) (Haferlach et al., 2014; Makishima et al., 2017). One of the most common cytogenetic alterations is the deletion of the long arm of chromosome 5q [del(5q)], which can be found isolated or with other alterations (10–30% of patients with MDS). Lenalidomide (LEN) has been approved for the treatment of patients with del(5q) low-risk MDS and transfusion dependence. Almost 50% of patients with del(5q) will show a complete cytogenetic remission and 70% of them will reach transfusion independence (List et al., 2006). LEN has also been approved for MDS non-del(5q) transfusion dependent and resistant to erythropoietin-stimulating agents (Santini et al., 2016), suggesting that other factors besides del(5q) modulate response to LEN (Negoro et al., 2016). Herein, we aimed to define the mutational spectrum of patients with MDS with and without del(5q) and define a signature of mutations influencing response to LEN.

We collected peripheral blood and/or bone marrow samples from patients with MDS treated with LEN from eight institutions at the Josep Carreras Leukaemia Research Institute (on behalf of the MDS Spanish Group and the MDS French Group) according to the institutional ethic committees and the revised Declaration of Helsinki. We collected 74 samples from patients with MDS at diagnosis or treatment- naïve with LEN follow-up treatment of two or more cycles; 32 patients presented with del(5q), while 42 patients did not have del(5q) in their karyotype (Table S1). The World Health Organization (WHO) classification (2017), Revised International Prognostic Scoring System (IPSS-R) and International Working Group response criteria (IWGc) (Cheson et al., 2006; Greenberg et al., 2012; Dolatshad et al., 2015) were used to classify patients. Responders to LEN included patients with complete and partial response, haematological response and cytogenetic response, while non-responders included patients with treatment failure, stable disease or relapse. We combined results of multi amplicon targeted sequencing with Ion Torrent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) (28 cases) and captured-based targeted sequencing with MiSeq (Illumina, San Diego, CA, USA) (46 cases). Amplicon and capture custom panels included 39 and 82 most recurrently mutated genes in MDS, respectively (Table S2). Capture libraries were generated using the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA, USA), enriched with the SeqCap EZ capture chemistry (Roche, Basel, Switzerland) and sequenced on MiSeq sequencers following a 150 base pairs (bp) paired-end reads Illumina standard protocol. Average coverage per gene was 777×. Reads were aligned against human genome build 19 (hg19) using Burrows-Wheeler Aligner (BWA) 0.7.12 and post-alignment was performed using Genome Analysis Toolkit (GATK) 3.4.46 software package. Libraries for the amplicon-based panel were prepared with Ampliseq (Thermo Fisher Scientific, Inc.) and sequenced in an ion torrent proton sequencer according to the manufacturer’s instructions. Average coverage per genes was 567×. Primary bioinformatic analysis [SAMtools 1.2 (http://www.htslib.org/), VarScan 2.4.0 (http://dkoboldt.github.io/varscan/), and ANNOtate VARiation (https://doc-openbio.readthedocs.io/projects/annovar/en/latest/)] was performed and followed by an in-house protocol (Ibáñez et al., 2016). Variants at highly variable regions, with low coverage (<100×), or a minor allele frequency >1% according to available population databases [Exome Aggregation Consortium (ExAC), Exome Variant Server, 1000 Genomes Project] were filtered out. Mutations were called when the variant allelic frequency (VAF) was >5%. Continuous variable comparisons were performed with Wilcoxon signed-rank tests, while Fisher’s exact test was used to compare variables. Survival curves were calculated using the Kaplan–Meier method and log-rank test were used for comparisons. Two-sided P values < 0·05 were considered as statistically significant.