Measurable residual disease (MRD) — the presence of cancerous cells below the threshold of detection of conventional morphologic methods — is routinely investigated post treatment to research the risk of relapse and to explore potential therapeutic strategies.
Several methodologies are currently used to assess MRD in acute myeloid leukaemia (AML), including immunophenotypic multiparameter flow cytometry (MFC), real-time quantitative polymerase chain reaction (RT-PCR), digital droplet PCR (ddPCR) and next-generation sequencing (NGS).
MFC provides valuable information about antigen expression implicated in MRD but does not assess genetic biomarkers, while PCR-based techniques are highly sensitive but are limited by the number of targets that can be detected simultaneously.
In contrast next-generation sequencing (NGS) offers highly sensitive and specific analysis of multiple AML-associated genes, allowing comprehensive MRD analysis of a wide-range of AML subtypes, including the identification of sample-specific mutations.
* Exon examples not yet available
We have a regularly updated, expert-curated library of pre-optimised cancer panel content for you to select from. Simply mix and match the gene, exonic or intronic content you need to create an Myeloid MRD next generation sequencing cancer panel that meets your exact requirements.
The SureSeq™ Myeloid MRD Panel has been designed in collaboration with leading cancer experts and in accordance with the European LeukemiaNet (ELN) recommendations1 to offer a single, cost-effective NGS assay to investigate MRD in AML samples. The panel content incorporates key genes for assessing AML, including genes implicated in MPN and MDS to investigate secondary AML progression.
In addition, genes related to research on potential drug response are included, for truly comprehensive and informative sample analysis.
Utilising OGT’s intelligent panel design capabilities, the SureSeq Myeloid MRD Panel surpasses the typical sensitivities offered by alternative NGS panels to accurately detect SNVs, indels and internal tandem duplications (ITDs) down to 0.05% VAF.
NPM1 is the most commonly mutated gene in adult AML, present in approximately 25-35% of cases2, making it an essential marker for MRD monitoring. NPM1 mutations occur almost exclusively in exon 11; a difficult region to sequence due to multiple perfectly matched copies replicated across the genome. The sophisticated bait design used in the SureSeq Myeloid MRD Panel overcomes this issue to deliver exceptional coverage uniformity, enabling reliable detection of all target regions (Figure 1).
FLT3 internal tandem duplications (ITDs) are present in approximately 25% of AML cases and are an important negative prognostic marker3; however, their inherent repeat content and length (up to 300 bp4) make them challenging to target, and subsequently detect. As a result, they are masked in many gene panels, necessitating additional techniques to characterise these important mutations. The unique detection algorithms incorporated into the complimentary Interpret NGS Analysis Software enable accurate detection and quantification of FLT3-ITDs, including multiple and large ITDs (Figure 2).
Combining coverage uniformity with a focused, expert-led panel design allows greater sequencing depth, which further enhances sensitivity at lower sequencing cost. SNVs, indels and ITDs across all targeted genes and regions can be detected down to 0.05% VAF (Table 2).
Figure 1: IGV plot showing high uniformity of coverage of all target regions in the panel, including NPM1 exon 11.
Figure 2: FLT3-ITDs of various sizes and even regions containing multiple ITDs can be confidently detected. ITD sizes are [A] 174 bp, [B] 225 bp, [C] 195 bp with additional 6 bp, [D] 120 bp and [E] 168 bp with additional 69 bp.
Table 2: Detection of SNVs, indels and an ITD, with expected frequency ranges of 0.1%-0.05%. SNVs are filtered to remove unique molecular identifiers (UMI) with a read family size of one. The SureSeq Myeloid MRD Panel was used on a Myeloid Reference DNA Standard (Horizon Discovery) according to the standard protocol, with sequencing on a NextSeq® 500 (Illumina).
All SureSeq NGS panels combine the superior performance of hybridisation-based enrichment with the streamlined and automatable Universal NGS Complete Workflow Solution to deliver unparalleled results with minimal hands-on time (Figure 3). The incorporation of Unique Molecular Identifiers (UMIs) and Unique Dual Indexes (UDIs) prior to sample amplification, allows true variants to be distinguished from PCR artefacts, for highly sensitive and reliable results.
OGT’s powerful and easy-to-use Interpret NGS Analysis Software facilitates analysis and visualisation of a wide range of somatic variants including structural aberrations.
Designed to work seamlessly with all SureSeq NGS panels, the software delivers fast and accurate detection of all SNVs, indels and ITDs. Following detection, all variants are displayed in the user-friendly variant browser, for effortless translation of all your myeloid MRD data into meaningful results. The reporting tool also enables visualisation of changing MRD dynamics over time (Figure 4).
The software can be deployed locally or in the cloud to suit your analysis infrastructure.
Science doesn’t stand still, so we’ve made it easy for you to benefit from the latest scientific discoveries and guidance by customising the SureSeq Myeloid MRD Panel to meet your precise requirements. Choose from our regularly updated, expert-curated library of pre-optimised cancer content to create your ideal custom SureSeq myPanel™ assay.
Alternatively, browse our full range of myeloid panels, including the focused three-gene SureSeq Core MPN Panel and the SureSeq Pan-Myeloid Panel, incorporating key variants in 70 genes implicated in a wide range of myeloid disorders.
In addition, the RNA-based SureSeq Myeloid Fusion Panel enables detection of 30 common fusions and novel fusion partners for key myeloid cancer genes.
Overall, the promising beta test results with the SureSeq Myeloid MRD panel led us to consider implementing this panel for MRD analysis in our ongoing and future clinical trials. The detection of variants with the SureSeq Myeloid MRD panel was fully concordant with our laboratory developed test, generating no false positive calls. Importantly, several additional clinically relevant variants, including challenging ones such as FLT3-ITD were reliably detected. The high coverage uniformity and low background noise result in the high sensitivity of this panel, enabling the detection of variants down to 0.05% VAF for key genes implicated in AML. The streamlined workflow minimises hands-on time.
Dr. Klaus H. Metzeler, MD
Professor of Translational Hematology, Dept. of Hematology, Cell Therapy, Hemostaseology and Infectious Diseases, University of Leipzig, Germany