Leukaemic stem cell load at diagnosis predicts the development of relapse in young acute myeloid leukaemia patients (2017)

Over the last decades, accumulating evidence suggests that acute myeloid leukaemia (AML) develops in a hierarchical structure, originating from haematopoietic stem cells (HSCs), which are transformed to leukaemia-initiating cells, referred to as leukaemic stem cells (LSCs) (Passegué et al, 2003; Corces-Zimmerman & Majeti, 2014). In many aspects, LSC and HSC are much alike, sharing features such as quiescence, self-renewal capacity and the expression of drug efflux proteins (Guzman & Allan, 2014; Terwijn et al, 2014). LSCs are of fundamental interest for AML patients at risk for relapse as these cells are thought to be resistant to current standard treatments and therefore believed to be responsible for initiation of disease relapse. In adult AML, the frequency of LSCs at diagnosis was shown to be of importance for clinical outcome (Terwijn et al, 2014). Although LSCs reside in different cell compartments, the CD34+/CD38− or CD34+/CD38dim fractions are considered to be most enriched for LSCs (Ng et al, 2016). The association between these fractions and risk of relapse and overall survival (OS) in paediatric AML was shown in one previous study with a limited number of patients (Witte et al, 2011). Given that CD34+/CD38− comprise both HSC and LSC, little is known about the prognostic impact of the LSC pool within this immature compartment. LSC can be distinguished from normal CD34+/CD38− cells on the basis of aberrant expression of cell surface molecules using multicolour flow cytometry (Van Rhenen et al, 2007; Terwijn et al, 2014; Zeijlemaker et al, 2016). In this study, we determined LSC burden at diagnosis of AML by defining aberrantly expressed cell surface markers in CD34+/CD38−/low cells using a single 8-colour LSC tube (Figures S1 and S2). Here, we show that high frequency of LSC at diagnosis in young AML patients can distinguish patients more likely to fail current treatment regimens, as these patients develop significantly more relapses. LSC measurements were implemented in routine diagnostics (see Data S1) using a minimal combination of markers in patients from the Dutch-Belgian AML-01 study (n = 86; Table S1). In this selected cohort, complete remission (CR) was achieved in 80 patients (93%) and 5-year relapse-free survival (RFS) was 54%. Second CR was achieved in 54% of relapsed patients resulting in a 5-year OS of 84%. Based on the CD34 expression of the blast cells, we identified 13 CD34-negative patients according to the definition of Zeijlemaker et al (2015) and 73 CD34-positive patients, of whom we could measure LSC in 68 patients. CD34 expression status showed enhanced RFS for the CD34-negative group (Figure S1B) although this did not reach statistical significance. As LSC have heterogeneous marker expressions, multiple LSC markers are necessary to discriminate LSC from normal HSC; Zeijlemaker et al (2016) concluded that 13 markers are needed for adequate identification. The present study used 4 markers (Table S2). As result, the sensitivity is still limited compared to the novel single tube LSC-assay (Zeijlemaker et al, 2016). In this study, 11% of all patients were classified as high risk for developing relapse based on an LSC load >17·2% of CD34+ blasts with high specificity determined by Receiver-operating characteristic analysis (96%, not shown). This is percentage is less than that reported for adult AML, for whom over 50% of patients are classified as LSChigh (Terwijn et al, 2014). Differences in patient characteristics between LSClow and LSChigh groups are summarized in Table 1. Kaplan-Meier survival analysis (Fig 1A) showed a significant association between high LSC-load and impaired RFS, and when the CD34-negative patient group was included, they showed a better RFS compared to both CD34-positive/LSClow and CD34-positive/LSChigh patients (Fig 1B). Univariate COX regression analysis showed a hazard ratio of 2·49 [95% confidence interval (CI): 1·08–5·73; P = 0·03; Table S3) for LSC load. Additional univariate analyses of prognostic factors for relapse (summarized in Table S3) showed that, in addition to LSC load, high white blood cell (WBC) count at diagnosis, core binding factor (CBF)-AML and blasts percentage had impact on RFS. Multivariate analysis identified LSC load, CBF-AML and high WBC count as having independent prognostic influence on RFS (Table S4). Due to relatively low patient numbers, previously defined risk classifiers, such as French-American-British classification, karyotype class and other molecular aberrations, were not significant. A single study has been published regarding the role of LSC in young AML patients. Witte et al (2011) showed that, in a small cohort of paediatric AML, higher numbers of CD34+/CD38−/CD45low cells corresponded with lower event-free survival. When applying this method to our cohort, we also observed that patients with a higher proportion of CD34+/CD38− cells have a worse OS, although this was not statistically significant for RFS (Figure S3). Seven of the nine patients (78%) with high LSC load relapsed, having an increased LSC load at time of relapse with the same aberrant marker expression (data not shown). Similar results have been reported in adult AML (Ho et al, 2016). Of LSClow patients, 39% still developed a relapse, suggesting that the current approach must be further improved. In addition to technical improvements of LSC measurements (i.e. addition of markers, higher cell numbers), LSC measurements during therapy (Zeijlemaker et al, 2015) could aid in the accurate assessment of patient prognosis, as outcome also greatly depends on many different factors during therapy. Next to increasing sensitivity of LSC identification, and thereby identifying patients at higher risk to relapse, defining a group of patients with very low risk to relapse could possibly spare these patients from undergoing more intensive treatment. A low CD34+ cell number is a prognostically favourable variable, similar to adult AML. These results need to be confirmed in a different cohort, taking into account the suggested improvements. Despite the fact that the number of patients included in this study is relatively small, our results confirm that LSC frequency at diagnosis in young AML patients can identify those patients more likely to fail current treatment regimens. The incorporation of CD34+/CD38− LSC frequency in conventional minimal residual disease measurements is warranted for future risk group stratification. Grant from the Clinical Research Fund (Ghent University Hospital) for B. Denys; Grant from the Foundation against Cancer, Belgium, for T. Lammens; grant from Cancer Plan, action 29 (KP_29_020) for J. Philippé. This study was partially financially supported by KIKA (grant 70, “Prognostic value of ‘Minimal Residual Disease’ detection in children with acute myeloid leukaemia”) (VHJvdV, VdH). The research for this manuscript was (in part) performed within the framework of the Erasmus Postgraduate School Molecular Medicine. Performed the research: DH, BDenys, JGtM, BDepreter, TL, AdJ, JP. Designed the research study: GJS, JC, VHJvdV. Contributed patient data: GJLK, VdH, BdM, ESdB, CMZ. Analysed the data: DH, BDenys, JC. Wrote the paper: DH, BDenys (all authors edited the manuscript). None Data S1. Patients and methods Fig S1. Diagrams showing median and variation in different population densities across total patient group using flow cytometry data. Fig S2. Examples of marker defined CD34+/CD38- leukaemic stem cells (LSC) in three patient samples at time of diagnosis. Fig S3. Kaplan-Meier analysis of impact of having high or low immature cell fraction. Table S1. Cohort statistics. Table S2. Antibody panel information. Table S3. Univariate statistics. Table S4. Multivariate regression model. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.