A clinical laboratory-developed LSC17 stemness score assay for rapid risk assessment of patients with acute myeloid leukemia

Abstract

Leukemia stem cells (LSCs) are linked to relapse in acute myeloid leukemia (AML). The LSC17 gene expression score robustly captures LSC stemness properties in AML and can be used to predict survival outcomes and response to therapy, enabling risk-adapted, upfront treatment approaches. The LSC17 score was developed and validated in a research setting. To enable widespread use of the LSC17 score in clinical decision making, we established a laboratory-developed test (LDT) for the LSC17 score that can be deployed broadly in clinical molecular diagnostic laboratories. We extensively validated the LSC17 LDT in a College of American Pathologists/Clinical Laboratory Improvements Act (CAP/CLIA)-certified laboratory, determining specimen requirements, a synthetic control, and performance parameters for the assay. Importantly, we correlated values from the LSC17 LDT to clinical outcome in a reference cohort of patients with AML, establishing a median assay value that can be used for clinical risk stratification of individual patients with newly diagnosed AML. The assay was established in a second independent CAP/CLIA-certified laboratory, and its technical performance was validated using an independent cohort of patient samples, demonstrating that the LSC17 LDT can be readily implemented in other settings. This study enables the clinical use of the LSC17 score for upfront risk-adapted management of patients with AML.

Graphical abstract

Figure 1.

A NanoString-based laboratory-developed test reproducibly measures LSC17 score. (A) Workflow in the clinical laboratory for the LSC17 LDT. Samples received by the middle of a workday can be reported by the end of the next workday. (B-C) Plots showing within-run (B) and between-run (C) correlation in replicate measurements of the LSC17 score. (D) Box-and-whisker plots showing difference (Δ) in within-run and between-run replicate measurements of the LSC17 score. Boxes represent interquartile range (IQR), with median indicated. Whiskers represent 10th and 90th percentiles. The dotted line (Δ0.1) indicates 2 standard deviations of the technical variation of the assay. (E) Plot showing correlation between LSC17 scores measured using Elements and Elements XT reagents. In panels B-D, each dot represents an independent patient sample; R, Spearman correlation coefficient.

Figure 2.

LSC17 score measured by the LDT is strongly associated with survival outcomes. (A) Plot showing LSC17 scores of 306 patients with AML from PM measured by the original research assay and by the LSC17 LDT. Each dot represents 1 patient sample. R, Spearman correlation coefficient. (B-C) Kaplan-Meier estimates of overall (B) and relapse-free (C) survival of 306 patients in the PM AML cohort according to LSC17 scores measured by the LDT and classified as high (above median) or low (below median). The median LSC17 score for the cohort was 0.51.

Figure 3.

Measurement of LSC17 score under various sample collection and processing conditions. (A) Plot showing LSC17 scores measured in 4 PB samples processed either fresh (blue) or after being viably frozen and thawed (red). The dotted line represents the median LSC17 score (0.51). (B) Plot showing correlation between LSC17 score measured in Ficoll-separated and unseparated (non-Ficoll) BM samples from 41 patients with AML. (C) Plot showing LSC17 scores measured by the LDT in PB (blue) and BM (red) samples collected from 10 patients with AML at diagnosis. The dotted line represents the median LSC17 score. Patient samples are shown in order left to right by increasing PB blast percentage. Triangles, Ficoll-separated samples; circles, unseparated (non-Ficoll) samples. (D) Plot showing the difference (Δ) between paired PB and BM LSC17 scores compared with PB blast percentage. The horizontal dotted line indicates the extent of technical variation (Δ0.1 = 2 standard deviations); the vertical red line indicates the threshold (20% PB blasts) below which BM should be used as the sample source for the assay. (E) Plot showing LSC17 scores measured in 13 BM samples processed 8, 24, and/or 48 hours after collection. (F) Plot showing correlation between LSC17 scores measured in BM samples collected in EDTA tubes processed 8 hours after collection vs samples collected in PAXgene tubes and processed up to 5 days after collection. R, Spearman correlation coefficient.

Figure 4.

A synthetic plasmid control for the LSC17 LDT performs equivalently to patient RNA samples. (A) Box-and-whisker plots showing log₂-transformed read counts for each of the 17 score genes (filled boxes) and the 12 housekeeping genes (white boxes) on 9 consecutive runs of the LSC17 LDT measured in a patient sample with high LSC17 score (red boxes), a patient sample with low LSC17 score (blue boxes), and the synthetic plasmid control (green boxes). Each box represents the replicate read counts from a single probe. (B) Plot showing the LSC17 scores calculated from the data shown in panel A (high LSC17 score, red; low LSC17 score, blue; synthetic control, green.

Figure 5.

Establishment of the LSC17 LDT in a second clinical diagnostic laboratory. (A) Plot showing correlation between LSC17 scores measured in 24 patient samples collected and processed at PM (red dots) and 36 patient samples collected and processed at Mayo Clinic (blue dots). Each sample was tested independently in the laboratories at both sites. The dotted line represents no difference between the paired score measurements. R, Spearman correlation coefficient. (B) Kaplan-Meier estimates of overall survival of 26 patients with AML from Mayo Clinic treated with intensive induction therapy, according to LSC17 score classified as high (above median) or low (below median) using the PM reference median score of 0.51.