Capturing fusions in haematological malignancies through targeted RNAseq

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Introduction

Large chromosomal rearrangements resulting in fusion gene formation are implicated in tumourigenesis and cancer progression in multiple haematologic 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 leukaemia (AML), characterised 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.

Methods

Workflow

100ng - 500ng RNA was converted to cDNA followed by library generation and hybridisation 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.

Panel

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

Sequencing was conducted using 2 x 150 bp reads on an Illumina MiSeq^® V2 300.

Samples

We tested 69 RNA samples (20 commercially available cell lines and 49 research samples) previously characterised 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.

Bioinformatic analysis

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.

Results I

A. Detection of fusions

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 characterised via FISH and/or qPCR. Reciprocal transcripts are detected for a number of fusion-positive samples.

Table 2: Characterisation of commercial samples using SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 and OGT’s Interpret NGS Analysis Software.

Table 3: Characterisation of research samples using SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 and OGT’s Interpret NGS Analysis Software.

B. Detection of MECOM overexpression

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.

C. Exon-level resolution of breakpoints

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.

D. Partner-agnostic fusion detection

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

Conclusions

• By achieving 100% concordance with qPCR and FISH for all samples tested, we have demonstrated the capability of the SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 to detect known rearrangements in AML
• SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 detects MECOM overexpression, which in turn, allows for the detection of translocation events such as inv(3)(q21q26); t(3;3)(q21;q26) that do not form fusion genes but rather result in MECOM overexpression
• The NGS data detected single-exon resolution of breakpoints, multiple breakpoints as well as reciprocal fusion transcripts that would have remained undetected with FISH. Thus, our NGS assay provides a more comprehensive transcriptomic landscape of fusions in Myeloid Leukaemias
• SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 allows partner-agnostic fusion detection, which is especially important for promiscuous driver genes like KMT2A that have multiple fusion partners