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L.W. Dillon et al. et al.
Figure 4. Comparison of the performance of the acute myeloid leukemia measurable residual disease targeted RNA-sequencing assay to that of the commercially available Myeloid FusionPlex assay. NPM1 mutA and CBFB-MYH11 serial dilution samples were analyzed using the ArcherDx Myeloid FusionPlex assay. The ratio of target copies/ABL1 copies (log10) is plotted on the y-axis and the cell dilution (-log10) on the x-axis for both the National Institutes of Health acute myeloid leukemia measurable residual disease targeted RNA-sequencing assay (NIH AML MRD RNA-Seq) (blue) and the Myeloid FusionPlex assay (red). The shaded areas indicate the cell dilution frequencies in which the mutation was detectable by both methodologies.
Table 1. Validation of mutation detection in diagnostic patient samples by acute myeloid leukemia measurable residual disease targeted RNA- sequencing.
Patient
A
B
C
D
E
F
G
H
I
J
Sample type
PB
BM
PB
PB
BM
BM
BM
BM
BM
BM
Annotated mutation
NPM1 mut NPM1 mut NPM1 mut RUNX1-RUNX1T1 PML-RARA PML-RARA PML-RARA BCR-ABL1 p210 BCR-ABL1 p210 CBFB-MYH11
Mutation detected by RNA-seq
NPM1 mutA NPM1 mutA NPM1 mutA RUNX1-RUNX1T1 PML-RARA bcr1 PML-RARA bcr1 PML-RARA bcr3 BCR-ABL1 p210 BCR-ABL1 p210 CBFB-MYH11 type D
Gene expression detected by RNA-seq
WT1, PRAME
WT1 WT1, PRAME WT1, PRAME WT1, PRAME WT1, PRAME WT1 WT1, PRAME WT1 WT1, PRAME
RNA-seq: RNA-sequencing; PB: peripheral blood; BM: bone marrow.
positive patient samples, we also confirmed the detectability of these mutations by qPCR (Online Supplementary Table S3). Furthermore, using our AML MRD RNA-sequencing assay we were able to specifically define the isoform present in each sample, including the rarer CBFB-MYH11 type D isoform (Online Supplementary Figure S5), and to identify the overexpression of WT1 or PRAME as potential secondary tracking targets (Table 1).
To test the ability of this assay to track disease burden in a patient and detect relapse relative to standard clinical assessments, we performed AML MRD targeted RNA- sequencing on RNA isolated from peripheral blood mononuclear cells of an NPM1 mutA-positive AML patient at various time-points during treatment. NPM1 mutA transcripts were detected at the time of pathological cytomorphological complete remission prior to allogenic stem cell transplantation. While NPM1 mutA transcript expression was not detected immediately after transplan- tation, an increase in NPM1 mutA transcript was detected at two subsequent time-points, up to 3 months prior to clinical relapse (Figure 5).
Collectively, these findings confirm that the AML MRD targeted RNA-sequencing assay can identify all of the included targets in both peripheral blood and bone mar-
row from patients harboring these mutations and is capa- ble of detecting MRD in patient samples during clinical complete remission.
Discussion
Here we present the development of a targeted RNA- sequencing method for the detection and measurement of MRD in AML. This assay, which can cover over two- thirds of patients in a single standardized assay (Figure 1), is highly specific, sensitive, and resilient to variations in sequencing depth and platform used during data collec- tion. MRD status is now integrated into response criteria for AML,4 with ELN consensus guidelines for measure- ment now available.6 The assay presented here detects all of the molecular targets included in these guidelines.
To date, molecular MRD detection in AML has primari- ly focused on single target qPCR assays.18 The use of next- generation sequencing for AML MRD detection has begun to emerge but has primarily focused on DNA as the start- ing material.21-27 Several features of the AML MRD RNA- sequencing assay presented here in theory make it ideal for MRD detection, including: (i) the use of next-genera-
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haematologica | 2019; 104(2)