Wojtaszewska Marzena1, Szarawarska Marta1, Blajer-Olszewska Beata1, 2, Markiewicz Mirosław1, 2
1 Department of Hematology, Clinical University Hospital in Rzeszow, Poland
2 Department of Hematology, Institute of Medical Sciences, College of Medical Sciences, Rzeszow University, Poland
Recurrent gain-of-function translocations often result in gene fusion events, which are a hallmark of acute leukaemia, and many of these fusion events can significantly impact disease classification, prognosis and treatment approaches. Traditionally, fusion events are detected using fluorescence in situ hybridisation (FISH) and/or polymerase chain reaction (PCR)-based techniques, but these methods can be limited by their reliance on prior knowledge of what partner genes or gene breakpoints are involved and cannot be used to detect multiple and/or novel fusions. Conversely, next-generation sequencing (NGS) assays allow simultaneous detection of multiple fusion genes in a single assay, including multiple partners for the same genes and novel fusions.
Here we demonstrate the utility of a partner-agnostic targeted RNA sequencing workflow using OGT’s SureSeq™ Myeloid Fusion Complete NGS Workflow Solution V2 (890001-24) for functional detection of both canonical and rare fusions.
Bone marrow from 15 acute leukaemia patients (9 AML and 6 ALL), who had agreed to genetic testing, was collected for routine molecular and cytogenetic tests.
NGS fusion testing was performed using OGT’s SureSeq™ Myeloid Fusion Complete NGS Workflow Solution V2 as per manufacturers guidelines. All samples used in this workflow had undergone prior cytogenetic analysis using either qPCR and tagmentation-based sequencing, GTG and/or FISH. For OGT’s workflow, we used Trizol™ extraction to obtain 300ng total RNA per sample and were able to detect 14 previously characterised rearrangements with partner-agnostic fusion detection method by baiting for 18 clinically relevant target genes.
The median total read count across all samples was 5.9 M (2.5 – 9.3 M) with 67.4% median aligned reads (49.2- 82.6%). Duplicate rates ranged from 7.2 to 14%. The fusion-contributing read counts ranged from 10 to 4776.
Bone marrow from the patients was cultured for GTG-banding and FISH testing using routine diagnostic procedures. FISH analysis was performer using Metasystems™ probes. qPCR was used to confirm the expression of canonical fusions using standardized EAC methodology1 or Zheng et al. protocol2. After qPCR, products of amplification were processed using Illumina DNA prep™ and sequenced to confirm the exonic coordinates of breakpoints.
The SureSeq™ Myeloid Fusion Complete NGS Workflow Solution V2 allowed 100% concordant detection of clonal reciprocal translocations in all 14 samples, compared with either FISH or GTG-banding.
We successfully detected canonical gene fusions (RUNX1::RUNX1T1, CBFB::MYH11, PML::RARA and BCR::ABL1), known KMT2A rearrangements (KMT2A::ELL and KMT2A::ENL) and MECOM locus rearrangements (RUNX1::MECOM and ETV6::MECOM). In 3 cases, non-canonical translocations were detected: KMT2A::TET1 (known)3, MECOM::CBFA2T3 and NUP98::HOXD8 (novel)4, 5. No fusions were detected in negative samples or samples bearing non-targeted fusions.
Table 1: Fusions detected by SureSeq Myeloid Fusion Complete NGS Workflow Solution V2.
Figure 1: KMT2A::MLLT10 rearrangement excluded by qPCR (A and B), detected by OGT SureSeq™ Myeloid Fusion panel (C).
All fusion events identified with the SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 were in concordance with GTG and/or FISH results. Fusion partners orientation were compatible, and exons involved coherent with functional studies. qPCR reactions yielded positive results for 10 canonical fusions. Tagmentation-based sequencing or qPCR showed the involvement of the same exons described in the NGS panel (see table 1, labeled*). In two negative samples there were suspected non-canonical fusions of ETV6 and ABL1 genes identified by FISH, which were not targeted by the OGT workflow and therefore are considered outside of the test’s scope. The third negative sample was extracted from a healthy donor’s bone marrow and had no expected fusion events.
Figure 2: Unknown KMT2A rearrangement, described initially as 46,XY,t(10;11)(q22;q23) with KMT2A::MLLT10 rearrangement GTG (A), ISH (B) and nucISH(C).
In this study we demonstrated the capability of OGT’s SureSeq Myeloid Fusion Complete NGS Workflow Solution V2 to achieve 100% accurate detection for novel and canonical fusion events. By allowing concurrent detection of multiple known and novel rearrangements, NGS assays offer an economical and efficient alternative and addendum to routine cytogenetic approaches.
The corresponding author of the poster received research support in the form of reagents and travel reimbursement from Oxford Gene Technology Ltd., Unit 5, Oxford Technology Park, 4A Technology Drive, Kidlington, Oxfordshire, OX5 1GN, UK.
This presentation is intended for educational purposes only and does not replace independent professional judgment. Statements of fact and opinions expressed are those of the presenters individually and, unless expressly stated to the contrary, are not the opinion or position of the Oxford Gene Technology Group (OGT). OGT does not endorse or approve, and assumes no responsibility for, the content, accuracy or completeness of the information presented.
SureSeq™: For Research Use Only; Not for Use in Diagnostic Procedures.
Call +44 (0)1865 856800 Email contact@ogt.com
Send us a message and we will get back to you
Visit USA site
Visit Canada site