Validation of automated semiconductor-based NGS in myeloid neoplasms

The detection of molecular alterations through approaches such as next-generation sequencing (NGS) can impact treatment decisions.¹ The 2022 European LeukemiaNet (ELN) recommendations identify 22 target genes or gene fusions that should be sequenced at diagnosis for optimal treatment decisions in acute myeloid leukemia (AML). An additional 20 optional genes should be sequenced at diagnosis for potential measurable residual disease monitoring.¹ Due to the high number of relevant genes, and with many of these genes requiring sequencing within 3–5 days, comprehensive molecular profiling with NGS has the potential to improve the diagnosis, risk stratification, and identification of therapeutic targets in patients with AML.¹

Here, we summarize a validation of the Oncomine Myeloid Assay GX v2 in combination with the Ion Torrent Genexus System using commercial controls in myeloid malignancies published by Zbieranski and Insuasti-Beltran.¹ in The Journal of Molecular Diagnostics.

Methods¹

• Pre-characterized samples negative for DNA mutations (n = 6) and for fusions (n = 10) were used to determine specificity.
• 130 clinical samples (bone marrow, n = 59; peripheral blood, n = 71) were included for validation.
  • Overall, 93.1% of samples were myeloid neoplasms, including AML (46.3%), chronic myeloid leukemia (23.1%), myeloproliferative neoplasms (15.7%), acute promyelocytic leukemia (5.8%), myelodysplastic syndromes (2.5%), myelodysplastic syndromes/myeloproliferative neoplasms (1.7%), and other myeloid neoplasms (5.0%).

• Sequencing was performed with the Oncomine Myeloid Assay GX v2 on the Ion Torrent Genexus Integrated Sequencer using the Ion Torrent GX5 semiconductor chip.
• The Oncomine Myeloid Assay GX v2 targets a panel of 17 complete genes, 28 hotspots, and 30 fusions (Figure 1).

ITD, internal tandem duplication; PTD, partial tandem duplication; TKD, tyrosine kinase domain.
*Adapted from Zbieranski and Insuasti-Beltran.¹ Created with BioRender.com.

 

Key findings¹

Target sequence metrics and control sample performance

• The raw read accuracy was >98.8% and the base call accuracy was >98.4% across all 11 lanes.
• Average turnaround time from nucleic acid extraction to results was 2 days.

• All 13 single nucleotide variants (SNVs) and 9 of 10 insertions/deletions (indels) from the synthetic DNA controls, and all nine fusion genes from the RNA controls, were successfully identified.

Analytical sensitivity and specificity

• Sensitivity for SNV and indel detection was 100% at >5% allele frequency (AF) (Table 1).
  • Although this sensitivity decreased below an expected AF of 5% (Table 1), detection down to 1.9% AF was verified.
• No SNVs, indels, or fusions were identified in the negative controls resulting in a specificity of 100%.

Table 1. Analytical sensitivity of the Oncomine Myeloid Assay GX v2 for SNVs, indels, and gene fusions*

Type of variant	Expected VAF	Expected variants/fusions	TP	TN	FP	FN	Sensitivity (PPA), %
SNVs/indels	>10%	7	7	0	0	0	100
5–10%	21	21	0	0	0	100
<5%	26	18	0	0	8	69
Fusions	95 copies/µL	7	7	0	0	0	100
32 copies/µL	7	7	0	0	0	100
11 copies/µL	7	7	0	0	0	100
FN, false negative; FP, false positive; indel, insertion or deletion; PPA, percent positive agreement; SNV, single nucleotide variant; TN, true negative; TP, true positive; VAF, variant allele frequency. *Adapted from Zbieranski and Insuasti-Beltran.1

Variant calling accuracy

• Results of variant identification from 145 samples were compared with orthogonal methodology results from the same samples to determine accuracy.
• The percent positive agreement was 99% and 97% for SNVs and indels, respectively.
• The positive predictive value was 100% for both SNVs and indels.
• The percent positive agreement and positive predictive value for the RNA samples were 100% for both.

Intra-assay and inter-assay reproducibility

• Six samples were used to measure intra- and inter-assay reproducibility, with an overall precision of 100% for SNVs, 96% for indels, and 100% for gene fusions.

Key learnings
The Oncomine Myeloid Assay GX v2 was successfully validated for the detection of myeloid neoplasms with high accuracy, sensitivity, specificity, and reproducibility. This NGS assay could provide clinically relevant molecular data for patients with AML in a relatively short turnaround time.