Applicability and reproducibility of acute myeloid leukaemia stem cell assessment in a multi-centre setting

Abstract

Leukaemic stem cells (LSC) have been experimentally defined as the leukaemia-propagating population and are thought to be the cellular reservoir of relapse in acute myeloid leukaemia (AML). Therefore, LSC measurements are warranted to facilitate accurate risk stratification. Previously, we published the composition of a one-tube flow cytometric assay, characterised by the presence of 13 important membrane markers for LSC detection. Here we present the validation experiments of the assay in several large AML research centres, both in Europe and the United States. Variability within instruments and sample processing showed high correlations between different instruments (R_pearson > 0·91, P < 0·001). Multi-centre testing introduced variation in reported LSC percentages but was found to be below the clinical relevant threshold. Clear gating protocols resulted in all laboratories being able to perform LSC assessment of the validation set. Participating centres were nearly unanimously able to distinguish LSC^high (>0·03% LSC) from LSC^low (<0·03% LSC) despite inter-laboratory variation in reported LSC percentages. This study proves that the LSC assay is highly reproducible. These results together with the high prognostic impact of LSC load at diagnosis in AML patients render the one-tube LSC assessment a good marker for future risk classification.

Fig. 1

Schematic overview of study setup. The study can be divided into four parts: (A) pre‐analysis by the central site, (B) analysis of four initial samples by six researchers of the central site and participating centres (C) identification of critical gating steps and (D) the validation of both the coordinating centre and the participating centres.

Fig. 2

Central site inter‐instrument processing. Multiple samples with similar processing were measured across multiple flow cytometric instruments (A) or platforms (B). (A) Training sample 1 was measured on BD FACSCanto II (I) and BD LSRFortessa (II). FACS plots of complete blasts (top row) and CD34⁺ blasts (bottom row). CD33 and CD34 were exchanged in the channel (see Table S3) for standardisation within the coordinating institute. (B) A diagnostic AML sample was measured on BD FACSCanto II (I) and BC Gallios EX (II). FACS plots for complete blast population (top row) and CD34⁺ blast population (bottom row). (C) LSC percentages (dots) and lymphocyte percentages (diamonds) analysed in samples measured on BD LSRFortessa (black) and BC Gallios EX (red), compared to BD FACSCanto II. 0·5 log error is depicted as diagonal lines. 0·03% clinical stem cell cut‐off depicted as dotted lines. Pearson correlation coefficients between BC Gallios EX and BD FACSCanto II, and BD LSRFortessa and BD FACSCanto II were r = 1·00, P =< 0·001 and r = 0·91, P =< 0·0001 respectively. Grey: (CD34⁺) population; Red: LSC; Green: HSC.

Fig. 3

Results of central site and local sites on initial samples. Results of analysis of FCS files generated at central site by six researchers from central site (black), results of FCS files generated at local sites with analysis by one researcher from central site (grey) and results of FCS files generated at local sites with analysis from local site (blue) on samples T1–T4. All individual results are shown as the percentage of leukaemic stem cells of the complete white blood cell compartment. Laboratories are specified using different symbols, showing that differences in LSC percentages is not consistently explained by one laboratory. Axes run from 0·000% to 0·030% (clinical relevant cut‐off) for sample T1 and T2, and 0·000–0·003% for samples T3 and T4. CD44 is not depicted due to high variance (shown in Figure S3).

Fig. 4

Results of central site and local sites on validation FCS files. Ten representative diagnostic AML samples were selected and corresponding flow cytometry files were sent to six researchers from the central site and seven participating centres for analysis. Results were reported back as the leukaemic stem cell percentage analysed by the most reliable stem cell marker (or markers). Results reported by one researcher from the central site compared to LSC percentages reported by all other participants (researchers from the central site in black, participating laboratories in blue). Previously determined (and validated) cut‐off of 0·03% is shown as a dotted line. Results above the clinical validated cut‐off fall within ±0·5 log error, shown as grey diagonal lines. Lower percentages fall outside ±0·5 log error but are clinically irrelevant.