PS1008 A NEW NEXT‐GENERATION SEQUENCING STRATEGY FOR THE SIMULTANEOUS DETECTION OF GENE MUTATIONS AND COPY NUMBER VARIATIONS IN MYELOID MALIGNANCIES
Authors: M.I. Prieto‐Conde, I. Vázquez, M. Fernández‐Mercado, N.C. Gutiérrez, T. González‐Martínez, M.J. Larrayoz, M.E. Sarasquete, M. Alcoceba, V. González‐Calle, M. González‐Díaz, R. García‐Sanz, M.J. Calasanz, M.C. Chillón
Journal: HemaSphere (2019)

Background: The diagnosis of myeloid malignances includes multiple testing strategies for genetic abnormalities assessment, including gene mutations and copy number variations (CNVs), which is critical for therapeutic decision making. Although next‐generation sequencing (NGS) has been implemented in clinical diagnostic laboratories, it has focused on the detection of single nucleotide variants and small indels, while CNV detection still relies on karyotyping and fluorescence in situ hybridization (FISH). Aims: In the present study, a new strategy was developed and validated to simultaneously detect CNVs and somatic mutations in a series of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) by using a capture‐based NGS custom panel. Methods: We designed a NGS target‐DNA panel associated to a custom bioinformatic pipeline (Sophia Genetics) for one‐step identification of somatic and germline mutations in 48 genes, as well as CNV. The CNV algorithm was based on the coverage levels across samples within the same batch. We studied the complete CDS or targeted exons of 16 genes sited in chromosome regions 5q14‐q35 (5 genes), 7q22‐q36 (3 genes), 8q12‐q24 (4 genes), 20q11‐q13 (3 genes), 17p13 (1 gene, TP53 ), as well as other genes in 5p, 7p, 20p and 17q as control regions. We analyzed 45 diagnostic samples of myeloid malignances (39 AML and 6 MDS), that had previously been characterized by FISH and/or karyotyping, as well as by conventional molecular techniques ( FLT3‐ITD , NPM1 and CEBPA mutations). Paired‐end sequencing runs were performed on a MiSeq (Illumina) genome sequencer. Results: We detected 133 pathogenic mutations in 42 patients, including all 8 FLT3‐ITD (size 18–204 bp) and 4 mutations in CEBPA . We were also able to detect exon‐level duplications, such as KMT2A ( MLL ) partial tandem duplication, in 3 cases. Four different CNV types were distinguished by the algorithm: del(5q), del(7q), del(20q) and trisomy 8, showing a full concordance with those alterations identified by FISH probes. All 8 patients with 5q33 deletions (8/8, 100%) were detected by NGS, as well as 5 cases with 7q35 deletion (5/5, 100%), 3 patients with trisomy 8 (3/3, 100%) and 3 with 20q12 deletion (3/3, 100%). None of the patients presented 17p abnormalities by any technique. Moreover, we detected alterations in the copy number of KMT2A gene in 4 of 5 patients with 11q23 alterations (translocation or amplification) identified by FISH and cytogenetics. One case of t(11;19)(q23;p13) was not detected by NGS, which is explained because the breakpoint region of this translocation was not covered by our design. Next panel will overcome this caveat by including the complete CDS of the KMT2A gene. Finally, NGS estimated abnormal copy numbers in three cases of 7q deletions and one trisomy 8, where FISH and cytogenetics had failed to detect any change. Summary/Conclusion: Our study demonstrates that both somatic mutations and targeted CNVs can be accurately detected by NGS covering adequate exons on selected genes. Therefore, this strategy complements conventional methods to identify biomarkers for the diagnosis, prognosis and therapy of myeloid malignances.