Large chromosomal rearrangements resulting in fusion gene formation are implicated in tumorigenesis and cancer progression in multiple hematologic malignancies.
While karyotyping, FISH, RT-PCR and microarrays are routine research techniques to detect fusion genes, they all have limitations. With improvements in NGS-based methods, DNA- and RNA sequencing are rapidly becoming established as the method of choice. NGS panels facilitate the simultaneous discovery of novel alterations alongside known mutations and structural alterations in genomic research. The rapid growth in the development of this mutation detection capability has moved beyond SNVs and indels to now include translocations.
Acute myeloid leukemia (AML), characterized by myeloid cell overproliferation, is typically underpinned by a range of fusion genes. In this study, we tested OGT’s SureSeq™ Myeloid Fusion Complete NGS Workflow Solution V2 to detect known fusions in AML research samples.
100ng - 500ng RNA was converted to cDNA followed by library generation and hybridization enrichment, using SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 (Figure 1).
Figure 1: Universal NGS Workflow RNA to sequencer in 3 days with minimal handling time.
The SureSeq Myeloid Fusion panel allows the detection of 30 common myeloid fusions in addition to novel fusion partners. Specific gene targeting (bold text) allows accurate partner gene agnostic identification of fusion genes (Table 1). Moreover, inv(3)/t(3;3)(q21.3q26) detection is achieved through analysis of MECOM overexpression.
Sequencing was conducted using 2 x 150 bp reads on an Illumina MiSeq® V2 300.
We tested 69 RNA samples (20 commercially available cell lines and 49 research samples) previously characterized by FISH and/ or qPCR, including 52 samples with known fusion events: BCR-ABL1 (n=8), ETV6-RUNX1 (n=10), RUNX1-RUNX1T1 (n=7), PML-RARA (n=11), CBFB-MYH11 (n=5), KMT2A-partner (n=8), inv(3)/t(3;3) (q21.3q26) (n=3) as well as 17 fusion-negative samples.
Sequencing data analysis including detection of fusions as well as relative expression of MECOM was performed using OGT’s Interpret NGS Analysis Software.
Table 1: SureSeq Myeloid Fusion panel content. Gene targets are highlighted in bold text.
We achieved 100% concordant detection for all 69 samples tested (Table 2-3). Sample-cohort comprised 52 fusion-positive and 17 fusion-negative samples previously characterized via FISH and/or qPCR. Reciprocal transcripts are detected for a number of fusion-positive samples.
Table 2: Characterization of commercial samples using SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 and OGT’s Interpret NGS Analysis Software.
Table 3: Characterization of research samples using SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 and OGT’s Interpret NGS Analysis Software.
OGT’s SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 successfully detects MECOM overexpression, which is especially important for atypical translocations involving MECOM inv(3)(q21q26) and t(3;3)(q21;q26) that result in MECOM overexpression rather than fusion gene formation (Figure 2).
Figure 2: MECOM expression detection in research and commercial samples.
SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 provides exon-level resolution of chromosomal breakpoints for fusions and identifies multiple breakpoints within the same experiment. BCR-ABL1 (e14-a2, e13-a2 and e1-a2), CBFB-MYH11 (exon5-exon34), PML-RARA (typical isoforms: bcr1 (exon6-exon3), bcr3 (exon3-exon3) and atypical isoforms: bcr3 (exon4-exon2).
Table 4: Breakpoint detection by SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 and OGT’s Interpret NGS Analysis Software.
OGT’s SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 is capable of partner-agnostic fusion detection. In this study, we detected 8 KMT2A fusions involving 4 gene partners: MLLT3, MLLT4 (AFDN), MLLT10 and AFF1 (Tables 2-3). Examples of these fusions are presented in Figures 3-4.
Figure 3: SureSeq Interpret NGS Analysis Software displaying KMT2A-MLLT3 fusion in 2 research samples
Figure 4: SureSeq Interpret NGS Analysis Software displaying KMT2A-MLLT10 fusion in 1 research sample
SureSeq: For Research Use Only; Not for Diagnostic Procedures
Call +44 (0)1865 856800 Email contact@ogt.com
Send us a message and we will get back to you
Visit International site
Visit USA site