Comprehensive Genomic Profiling of Chinese Acute Myeloid Leukemia (AML) with FLT3 ITD Using Ultra Deep Sequencing

Introduction: The FMS-like tyrosine (FLT3) gene encodes a class III receptor tyrosine kinase, belonging family members, including c-kit, c-FMS, FLT1, and PDGF-𝛽R. FLT3 plays a key role in the hematopoiesis. It was discovered that in acute myeloid leukemia (AML) FLT3 is frequently mutated. Furthermore, constitutive activation of FLT3 by internal tandem duplication (ITD) mutation is one of the most common molecular variants in AML, occurring in approximately 20% to 30% of AML patients who have a comparatively poor clinical outcome and increased relapse rate. In the last years, next generation sequencing technology has allowed a high- throughput, comprehensive profiling of cancer genomes that can aid in making clinical decisions in the process of diagnosis and treatment. A few reports included somatic genomic profiling in AML with FLT3-ITD. However, none comprehensive genomic profiling data shows in chinese population. In this study, we sequenced 26 FLT3-ITD AML samples to show genomic profiling with multiple mutations types and discover the mutations associated with FLT3-ITD based on in-house bioinformatics mutation calling pipelines.

Methods: DNA was extracted from blood or bone marrow of AML FLT3-ITD positive samples. Those DNA samples were subjected to comprehensive genomic profiling (CGP) assay consisting of whole exon coding region in 450 tumor actionable or cancer driver genes as well as selected introns (N=244) from 39 genes frequently involved in gene rearrangement using hybridization target capture and next generation sequencing (NGS) technologies and in house established bioinformatics pipelines. Somatic genomic alterations including SNV, short/long Indel, CNV and gene rearrangement were analyzed. Libraries were constructed with KAPA Hyper Prep kit( KAPA Biosystems), and hybridized to customized capture probes( Integrated DNA Technologies)and then sequenced on NovaSeq 6500 (Illumina). The mutation is detected with VAF of no less than 1% of point mutation, insertion and deletion, including long insertion and deletion( long indel). Long indel variants from 10bp-2kb were called with OriLindel algorithm, which is in silicon developed for structure variation calling, especially for FLT3-ITD variants calling.

Results: In this study, we sequenced 26 FLT3-ITD AML samples to show genomic profiling with multiple mutations types. The average sequencing depth of 26 samples is above 700X(720-2300X). The mutation is detected with VAF of no less than 1% of point mutation, insertion and deletion, including long insertion and deletion( long indel). Long indel variants from 10bp-2kb were called with OriLindel algorithm, which is in silicon developed for structure variation calling, especially for FLT3-ITD variants calling.

We discovered the previous reported high rate of mutations of NPM1(34.6%,9/26), DNMT3A(38.5%,10/26), IDH2(11.5%,3/26), NRAS(19.2%,5/26),RUNX1 (15.4%,4/26), WT1 (31%,8/26) and MLL3(KMT2C, 11.5%,3/26) which have consistent percentage with previous genomic profiling report of FLT3-ITD AML.

A total of 42 mutations were detected on the FLT3 gene of 26 samples (162%, 42/26). All 36 ITD sequences of 26 samples were distributed in the protein kinase domain with lengths ranging from 18 to 207bp. 5 samples have more than two ITD variants (19.2%,5/26)). 6 point mutation of FLT3 were detected, 5 of which were located in the 835th amino acid residue of tyrosine kinase domain and 1 in the protein kinase catalytic domain. One of the samples contained two mutations, D835H and V592G. Using NGS technology and in silico bioinformatics pipeline could call multiple type mutations including point mutation, insertion and deletion of FLT3 simultaneously.

We also found recurrent mutations found not often in AML, such as AXIN1 (7.7%,2/26) ,RIT1 (7.7%,2/26). AXIN1 belongs to Wnt signaling pathway. Wnt extracellular signaling pathway is one of the evolutionarily conserved pathways that regulate proliferation, polarity and cell migration. RIT1 belongs to Ras gene family. Somatic mutations of the Ras gene family are present in 20-30% of all human cancers,including15-20% of acute myeloid leukemia (AML). Ras-like-without-CAAX-1 (RIT1) gene is a new member of the family and has been found to be critical in noonan syndrome. One report shows the discovery of novel somatic mutations in the RIT1 gene in patients with myeloproliferative.