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Targeted RNA-sequencing for MRD in AML
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Figure 3. Comparison of the per- formance of the acute myeloid leukemia measurable residual disease targeted RNA-sequenc- ing assay with that of current gold-standard, single-target methodologies. Gold-standard quantitative real-time poly- merase chain reaction (qPCR) or digital droplet polymerase chain reaction (ddPCR) (right) tech- niques performed on the serial dilution samples for (A) NPM1 mutA, (B) RUNX1-RUNX1T1, and (C) CBFB-MYH11 exhibited com- parable performance to that of the acute myeloid leukemia (AML) measurable residual dis- ease (MRD) targeted RNA- sequencing (RNA-seq) tech- nique (left). The data are pre- sented as a ratio of the number of target copies/ABL1 copies (log10) on the y-axis and the cell dilution (-log10) on the x-axis. A linear regression was calculated for each dilution series (exclud- ing the 100% leukemia samples and any samples for which the target was not detected) and the coefficient of determination (r2) indicated in the graph area.
as used in the AML MRD RNA-sequencing assay. We con- firmed that the Myeloid FusionPlex assay can detect these mutations, but at varying frequencies (Online Supplementary Table S2). Compared to the Myeloid FusionPlex assay, our AML MRD RNA-sequencing assay exhibits a 1,000-fold increased limit of detection for NPM1 mutA and 100-fold increased limit of detection for CBFB-MYH11 (Figure 4), indicating that our assay out- performs a commercially available targeted RNA-sequenc- ing assay for MRD-level detection in AML.
Validation of assay performance in patient specimens
Finally, we examined the performance of the AML MRD RNA-sequencing assay applied to patient samples. RNA was isolated from diagnostic peripheral blood or bone marrow samples of patients clinically annotated to harbor mutations included in our assay and assessed the samples by AML MRD targeted RNA-sequencing. In all cases, the annotated mutation was detectable using our assay (Table 1). For NPM1 mutA and RUNX1-RUNX1T1-
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geted RNA-sequencing (left graph), thus confirming that the AML MRD RNA-sequencing assay on its own serves as an adequate replacement for current, single-target MRD detection assays.
Comparison of the performance of the study assay with that of a commercially available myeloid targeted RNA-sequencing assay
The Myeloid FusionPlex assay from ArcherDx is a com- mercially available targeted RNA-sequenced assay which also utilizes molecular barcodes and is designed to detect and identify fusions, point mutations, and expression level changes in a panel of 84 genes associated with malignan- cies of myeloid origin. In contrast to the assay presented here, the FusionPlex technology utilizes an anchored mul- tiplex-PCR enrichment (AMP-E) approach, which is intended for fusion discovery in a bulk tumor setting.
The performance of the Myeloid FusionPlex assay for the detection of MRD was assessed for NPM1 mutA and CBFB-MYH11 mutations using 250 ng of the same RNA
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