Blood cancer profiling challenges with NGS

Hematology oncology research has greatly benefited from next generation sequencing (NGS) methods to identify relevant cancer-driving biomarkers. Heme malignancies have a complex biomarker spectrum, but NGS methods to detect relative fusions, splicing, expression, copy number variants, single nucleotide variants, and complex alterations have been vital to labs.

However, traditional NGS methods may not provide all relevant information and can have difficult workflows and analysis. One specific challenge in blood cancer profiling is myeloid-relevant genes rich in GC-content, such as CEBPA, are difficult to amplify and sequence with NGS. Additionally, internal tandem duplications (ITDs), such as FLT3-ITD relevant in AML, can be difficult to detect with NGS due to variable locations and lengths along with random sequence insertions which can confound genome aligners. Due to this, most NGS methods have difficulty detecting ITDs of all sizes and insertion points, and sensitivity dramatically reduces above 100 bp ITDs. Finally, there are a multitude of relevant fusions in hematological neoplasms, including many novel fusions. In fact, one study found that 41% of acute myeloid leukemias and 88% of myelodysplastic syndromes had novel fusions [1]. However, since these are relatively rare, traditional opposing primer-based NGS methods may not be designed with primers to detect all of them, thus missing potentially relevant biomarkers (Figure 1).

Figure 1. Opposing primer amplicon assays lack detection of unknown fusions. In this example, a primer pair for ETV6::RUNX1 can only amplify that specific fusion since the ETV6 primer cannot bind to an unknown partner, therefore missing a novel RUNX1 fusion.
Figure 1. Opposing primer amplicon assays lack detection of unknown fusions. In this example, a primer pair for ETV6::RUNX1 can only amplify that specific fusion since the ETV6 primer cannot bind to an unknown partner, therefore missing a novel RUNX1 fusion.

Additionally, NGS library preparation workflows can be cumbersome, increasing the opportunity for manual errors. In-house bioinformatics pipelines can be expensive and time-consuming to build and maintain. They also may lack the QC and statistical tools necessary to reduce errors and guide calling, leaving it up to lab staff to make difficult calls.

Detect more with AMP chemistry

For blood cancer profiling, Anchored Multiplex PCR (AMP) is a target enrichment method that uses molecular barcoded adapters and single, nested, gene-specific primers for amplification, permitting open-ended capture of DNA, RNA, and cfDNA fragments. This approach enables the detection of novel fusions (Figure 2), allows for flexible primer design, and protects against dropout of primers. MBC adapter ligation prior to PCR combined with Archer Analysis software power the correction of systematic errors and quantitative analysis of the original unamplified template.

Figure 2. FUSIONPlex™ assays detect both known and novel fusions. FUSIONPlex assays include MBC adapters with a universal priming site. Since it is paired with the relevant gene-specific primer (in this case, RUNX1), both known and novel fusions are amplified.
Figure 2. FUSIONPlex™ assays detect both known and novel fusions. FUSIONPlex assays include MBC adapters with a universal priming site. Since it is paired with the relevant gene-specific primer (in this case, RUNX1), both known and novel fusions are amplified.

Watch video about AMP

Archer blood cancer assays amplify through GC-rich regions, such as those in CEBPA, with high target coverage. Statistically significant and confident variant detection is powered by Archer’s unique outlier identification algorithm informed by normalized data to characterize position specific noise stemming from PCR and sequencing.

Figure 3. Excellent coverage of CEBPA with VARIANTPlex™ Core Myeloid panel, with 95 MDAF plotted overall. Greater than 1000X unique molecule coverage depth from 50 ng DNA input and 2 M reads with the Core Myeloid panel (gray area), the minimal allele fraction that can be differentiated from background noise 95% of the time (95 MDAF) for the least sensitive base substitution, in blue line.
Figure 3. Excellent coverage of CEBPA with VARIANTPlex™ Core Myeloid panel, with 95 MDAF plotted overall. Greater than 1000X unique molecule coverage depth from 50 ng DNA input and 2 M reads with the Core Myeloid panel (gray area), the minimal allele fraction that can be differentiated from background noise 95% of the time (95 MDAF) for the least sensitive base substitution, in blue line.

VARIANTPlex blood cancer assays also provide comprehensive detection of ITDs. AMP chemistry enables tiling across both strands of DNA with primers that amplify independently of each. When reads from these primers are analyzed with the Archer Analysis pipeline, long consensus reads are generated based on sequence similarity and aligned against the genome to identify duplication events, allowing for confident detection of ITDs, including FLT3-ITD (Table 1).

 

Table 1. FLT3-ITDs called at ultralow allele frequencies using the VARIANTPlex Core Myeloid panel. 
A FLT3-ITD-positive cell line was titrated into a background control sample. Triplicate libraries were prepared using 200 ng total input DNA and the VARIANTPlex Core Myeloid panel. ITD calling was performed using Archer Analysis.

FLT3-ITD fractionInput ITD molecules*Expected ITD called
0.000/3
0.75450003/3
0.07545003/3
0.00754503/3
0.00075453/3
0.00025152/3

*Assuming 300 haploid copies per ng.

Blood cancer panels and reagent formats fit for your lab’s needs

  Genes targetedGenomic alterations detectedRecommended reads
DNA inputVARIANTPlex
Myeloid75SNVs, indels, ITDs4 M
Lymphoma51SNVs, indels, CNVs5 M
Core Myeloid37SNVs, indels, ITDs3 M
MPN Focus12SNVs, indels, ITDs800 K
AML Focus11SNVs, indels, ITDs800 K
RNA inputFUSIONPlex
Pan-Heme199Fusions, SNVs, indels, expression levels4.5 M
Heme v287Fusions, SNVs, indels, expression levels1.5 M
Myeloid84Fusions, SNVs, indels, expression levels1.5 M
Lymphoma125Fusions, SNVs, indels, expression levels2 M
Acute Lymphoblastic Leukemia (ALL)81Fusions, SNVs, indels, expression levels1.5 M

In addition to detection of the above alterations, Archer IMMUNOVerse™ assays can provide T- and B-cell clonality, minimal residual disease (MRD), and somatic hypermutation information relevant for heme malignancies. IMMUNOVerse panels have parallel workflows with VARIANTPlex and FUSIONPlex assays, allowing for comprehensive, streamlined blood cancer profiling.

Pair DNA- and RNA-based assays to efficiently generate a complete biomarker profile

Paired panelsGenesReadsSamples† per NextSeq 500/550 high output kits v2.5
FUSIONPlex Pan-Heme1994.5 M47
VARIANTPlex Myeloid754 M
FUSIONPlex Lymphoma1252 M66
VARIANTPlex Lymphoma515 M
FUSIONPlex Myeloid841.5 M88
VARIANTPlex Core Myeloid373 M

Data assumes Archer libraries are prepared using the liquid reagent workflow.

†Sample = 1 VARIANTPlex library + 1 FUSIONPlex library

 

VARIANTPlex and FUSIONPlex panels can be paired for cost-effective solutions to provide SNVs, indels, ITDs, fusions, and expression alterations from a single sample.

Choice of assay reagent formats are available to suit your lab’s sample volumes.

  • For high-throughput labs, liquid reagents are ideal for automated liquid handling workflows. Liquid adapters are available in a 96‑well plate format and include an increased number of dual indexes (over 36,000 unique combinations of sequencing indices) to support high-throughput sequencing. 
  • For manual workflow labs, single‑use, reaction‑size lyophilized reagents allow your lab to avoid potential contamination during manual pipetting and provide extended shelf life.

Both reagent formats have demonstrated excellent performance, with detection of 100% of expected variants in reference materials‡. Regardless of reagent format, all reagents are color‑coded to match protocol steps, making benchwork easy (Figure 4).

‡Data available upon request.

 

Figure 4. Reagents are color-matched to protocol steps for easy benchwork. Each step of the protocol has a different color, which matches the packaging of the reagents (in this case, liquid reagents) for that step.
Figure 4. Reagents are color-matched to protocol steps for easy benchwork. Each step of the protocol has a different color, which matches the packaging of the reagents (in this case, liquid reagents) for that step.

Read the original article by IDT

References

  1. Stengel A, Shahswar R, Haferlach T, et al. Whole transcriptiome detects a large number of novel fusion transcripts in patients with AML and MDS. Blood Adv. 2020. 4(21): 5393-5401.

Supplier

IDT logo

IDT - Integrated DNA Technologies

With over 30 years experience as a manufacturer, IDT offers innovative tools for NGS, CRISPR, qPCR and PCR. IDT offers superior quality DNA and RNA oligos, genes, gene fragments, Cas nucleases and more, with fast turnaround times!

About IDT                   Shop for IDT products