Leukemic Stem Cells Lscs in Aml Review Paper

• Loading metrics

Open Access

Peer-reviewed

Research Article

Leukemic Stalk Prison cell Frequency: A Potent Biomarker for Clinical Outcome in Astute Myeloid Leukemia

• Wendelien Zeijlemaker,
• Angèle Kelder,
• Arjo P. Rutten,
• Alexander N. Snel,
• Willemijn J. Scholten,
• Thomas Pabst,
• Gregor Verhoef,
• Bob Löwenberg,
• Sonja Zweegman,
• Gert J. Ossenkoppele,
• Gerrit J. Schuurhuis

Leukemic Stalk Cell Frequency: A Strong Biomarker for Clinical Outcome in Acute Myeloid Leukemia

• Monique Terwijn,
• Wendelien Zeijlemaker,
• Angèle Kelder,
• Arjo P. Rutten,
• Alexander Due north. Snel,
• Willemijn J. Scholten,
• Thomas Pabst,
• Gregor Verhoef,
• Bob Löwenberg,
• Sonja Zweegman

x

• Published: September 22, 2014
• https://doi.org/10.1371/journal.pone.0107587

Figures

Abstruse

Introduction

Treatment failure in acute myeloid leukemia is probably acquired by the presence of leukemia initiating cells, also referred to as leukemic stalk cells, at diagnosis and their persistence subsequently therapy. Specific identification of leukemia stalk cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses.

Results

For identification of leukemic stem cells, we developed menstruum cytometric methods using leukemic stem cell associated markers and newly-defined (low-cal scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, nowadays in the same patient'due south bone marrow, was demonstrated in eight patients by the presence or absenteeism of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n = 88), the frequency of the thus divers neoplastic role of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stalk cell compartments had no prognostic impact at all. Afterwards different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in consummate remission strongly correlated with shorter patient survival (n = 91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residue disease cells (n = 91), which reflect the total neoplastic brunt, revealed four patient groups with dissimilar survival.

Conclusion and Perspective

Discrimination between putative leukemia stalk cells and normal hematopoietic stem cells in this large-scale study immune to demonstrate the clinical importance of putative CD34+CD38- leukemia stalk cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.

Citation: Terwijn G, Zeijlemaker Due west, Kelder A, Rutten AP, Snel AN, Scholten WJ, et al. (2014) Leukemic Stalk Cell Frequency: A Potent Biomarker for Clinical Outcome in Astute Myeloid Leukemia. PLoS ONE 9(9): e107587. https://doi.org/10.1371/journal.pone.0107587

Editor: Kevin D. Bunting, Emory University, United States of America

Received: May 15, 2014; Accepted: August xi, 2014; Published: September 22, 2014

Copyright: © 2014 Terwijn et al. This is an open-admission article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original writer and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant information are within the paper and its Supporting Data files.

Funding: This work was supported past Netherlands Cancer Foundation KWF grant 2006-3695. The funders had no role in study design, information collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors accept declared that no competing interests exist.

Introduction

There is increasing evidence that the evolution of solid and hematological tumors depends on the presence of small-scale populations of cells known as tumor-initiating cells or tumor stalk cells [1]. The first proof of the stem cell concept came from studies by John Dick and colleagues in astute myeloid leukemia (AML) [2], [3]. In AML, these cells are referred to equally leukemia-initiating cells or leukemic stem cells (LSCs) [4]. Since the first studies on LSCs, jail cell compartments defined by immunophenotype (CD34/CD38 expression) and function (side population, SP, and aldehyde dehydrogenase [ALDH] activity) take been reported to contain LSCs [5]–[9]. The first LSC compartment that was described had the CD34+CD38- immunophenotype [ii], [iii]. Although allowed reactivity of the CD38 antibody used in before studies likely caused the lack of engraftment of CD38+ cells [10], the CD34+CD38- compartment yet seemed to be the most robust compartment in CD34-positive (CD34+) patients, since information technology was found to be the predominant compartment containing leukemia-initiating cells in less immunocompromised mouse models [3]. On the other hand, in more severely immune-compromised mouse models, CD34+CD38+ and CD34-negative compartments were too constitute to contain leukemia initiating cells [5], [vii]–[9], [11].

In os marrow (BM) of AML patients, leukemic and normal cells are present within one compartment. The CD34+CD38- compartment in particular, was shown to contain both CD34+CD38- LSCs and normal hematopoietic stalk cells (HSCs) [10], [12]. Previous papers reported merely on the role of the size of the total CD34+CD38-stem cell compartment and only at diagnosis [13]–[17]. Nevertheless, for proper identification of LSCs, with the aim to establish the prognostic affect of their frequencies at diagnosis and to follow their fate during and after therapy, it is of import to identify distinguishing features betwixt LSCs and HSCs. Nosotros previously accept constitute that in a substantial number of AML cases, CD34+CD38- LSCs are characterized by the expression of C-type lectin-like molecule-1 (CLL-i) and aberrant expression of several lineage markers [18], [19]. Since these markers are absent on HSCs in regenerating BM afterward chemotherapy [eighteen], [nineteen], for the current study nosotros chose to use these markers to distinguish between LSCs and HSCs in both diagnosis and post-diagnosis samples. Other LSC markers accept also been described for AML diagnosis (reviewed in ref [20]), but since little is known near their behavior during and after therapy, their suitability for LSC tracking remains to be established. Still, despite the usefulness of CLL-1 and lineage markers, in at least 25% of the AML patients, aberrant marker expression on CD34+/CD38- cells is absent or as well weak, and therefore there is a need to identify other discriminative parameters [6], [nineteen]. In the beginning part of this newspaper, nosotros volition describe such additional parameters for CD34+CD38- compartment. Now beingness able to discriminate neoplastic from normal CD34+CD38- cells, in the 2d function nosotros volition focus on the prognostic role of the CD34+CD38- stalk jail cell compartment. This will be compared with the other CD34/CD38 divers compartments.

Moreover, another important aspect of cancer stem cells is their putative therapy resistance [21], with a subsequent power to crusade re-growth. The nowadays report is the start to address the resistance to therapy by showing the prognostic impact of AML stem cells postal service-therapy in a large patient group. The results demonstrate that defining residuum LSCs post-therapy has important prognostic impact. Moreover, nosotros evidence that information technology adds important prognostic touch on to a well-established immunophenotypical MRD approach, known to place and quantify the majority of neoplastic cells.

Patients, Fabric and Methods

More detailed information tin be found in the supporting text (Text S1).

Patient treatment and sampling

Patients between 18 and threescore years of age with AML, except those with FAB M3 and previously untreated RAEB and RAEB-t patients (with IPSS ≥ 1.5), were included in this study. Detailed data regarding treatment can exist found at http://www.hovon.nl. The HOVON/SAKK 42a and 92 studies were reviewed and canonical past an institutional review board (METc) of the Erasmus MC Rotterdam for the full written report (number 2000-220 for Hovon 42a) and 2008/216 for Hovon 92). In addition, the VU Amsterdam review board approved both studies with METc number 2001/50 (LUV) and 2008/292 (LUV), respectively. Patients provided their written informed consent to participate in this written report. In 250 CD34-positive patients, we used a specific gating strategy to place normal and neoplastic cells in the CD34+CD38-, CD34+CD38+ and the CD34- compartment at diagnosis. Patient details are in Table S1 (patient groups ane and two). Additionally, in the last part of the results, an extra patient group (n = 23) was included (details are in Table S1, patient grouping 3) to written report combination of LSC and minimal residuum disease (MRD) information after 2nd cycle of therapy.

Flow cytometry

Methods.

LSC characterisation at diagnosis and LSC monitoring during follow up was performed on fresh patient samples. Purified white blood cells were obtained from BM or Atomic number 82 using lysing solution (Pharm lyse, Becton Dickinson, BD, San Jose, CA, UsA.) to eradicate red blood cells. After washing with PBS containing 0.1% human serum albumin (HSA), cells were re-suspended in PBS containing 0.1% HSA, incubated with monoclonal antibody combinations (mAbs) for fifteen minutes at room temperature and washed with PBS containing 0.one% HSA. Details on antibodies (sources, clones, fluorochromes) are written in the supporting text (Text S1). Samples were analysed using a 4-color approach on a FACSCalibur from Becton Dickinson (BD, San Jose, CA, USA) using CellQuest and Infinicyte software. Cell sorting was performed using FACSAria (BD) with FACSDiva analysis software. More details are in the supporting text (Text S1).

Marker selection for the CD34+CD38- LSC.

In a previous paper [nineteen], we have shown that particular lineage markers, i.e. CD2, CD7, CD11b, CD19, CD22, CD56, can be positive on CD34+CD38- LSC, while e'er negative on CD34+CD38- HSC. This is true for CLL-1 too [18]. All markers have been shown to be negative on HSCs in normal bone marrow, but also on normal stem cells nowadays at diagnosis and at follow up [eighteen], [xix]. In add-on, the absence of CD13, CD33 and HLA-DR was considered aberrant, since these markers are always expressed on normal HSCs [19], [22]. For each new AML case one or more of these markers may be aberrantly expressed; in a next case this may be completely different. Therefore, for each new AML case, the choice for a mark was based on screening for presence (>10% positivity) of all markers. Fifty-fifty more heterogeneity was seen, since part of the CD34+CD38- population might be negative for these markers, and all or not covered by another marker.

Marker selection for the more than mature CD34+CD38+ and CD34- populations.

For CD34+CD38+ and CD34- leukemic cells, identification had to be done using lineage markers: it is but lineage markers that are absent (or nowadays at very low frequencies) on these more mature populations [23], [24]. This property offers the basis for detection of residual leukemic cells (all leukemic cells) in MRD approaches. CLL-ane, withal, cannot be used equally a neoplastic mark on CD34+CD38+ and CD34- cells since it is present on role of these populations in normal bone marrow [25]. For LSC identification/quantification at diagnosis and follow up, but AML samples were used that, at diagnosis, showed>1% CD34 expression relative to the total WBC count. This was done because we have previously shown that the CD34+ population in AML with <1% CD34 is of normal origin [26]. CD34 negative AML samples, together with normal bone marrow, were used as a command for FSC/SSC values in HSC (run across results section I.A). For details on the gating strategy to define the CD34+CD38- compartment, see Figure 1, part I. The details how to define scatter characteristics of marker positive and mark negative populations are outlined under Results. Stalk jail cell numbers (LSC and HSC) were divers every bit a per centum of total white blood cells (WBC).

Figure 1. Gating strategy for the CD34+CD38- compartment and identification of pLSCs and HSCs in this compartment. I. Gating of CD34+CD38- AML cells.

Cells were labeled with antibiotic-fluorochrome combinations every bit described in Patients, Materials and Methods. Remaining erythrocytes, debris and dead cells are largely excluded in an FSC/SSC plot (A). CD45^dim/SSC^dim blast cells (B) were gated to homogeneity in FSC/SSC plot (C). CD34 positive cells are gated (D) and the CD38- stalk cells are gated within this fraction (E). The CD38-negative fraction in D may contain two stem jail cell populations differing in CD34 expression (details in text). F. Inside the CD34+CD38- gate, CD38 is plotted against an abnormal marker (in this case CD19) to indicate presence of putative LSCs (pLSCs) and HSCs. II. Identification of pLSCs and HSCs. This patient (nr 317) was diagnosed with t(8;21). Main gating was as in I. Sorted CD34+CD38-/CD19+ cells (A, in scarlet) were t(eight;21) positive; sorted CD34+CD38-/CD19- cells (A, in green) were t(eight;21) negative. These two populations were backgated in FSC/SSC (B,E), CD34/SSC (C,F) and CD45/SSC (D,G) plots. The CD19- cells are shown in the upper panels and the CD19+ cells in the lower panels. Dotted vertical lines (B–D) bear witness that normal CD19- cells are FSC^low (B), CD34^low (C) and slightly lower in CD45 (D), compared to CD19+ cells (E–G). The dotted horizontal line shows that SSC of the normal stem cells (in green) was slightly lower than that of neoplastic stem cells (in ruddy). FISH data are from an example published previously [19]. Similar results were found in an boosted serial of seven patients (Tables 1 and 2). FSC and SSC of CD19+ pLSC were factor i.71 and 1.77 higher than lymphocyte present in the same samples. FSC and SSC of the CD19 negative cells were but 1.08 and i.20 times lower than lymphocytes.

https://doi.org/x.1371/periodical.pone.0107587.g001

Engrafting studies

Animal experiments were performed afterward approval of the animal ethical committee of the VU University, Amsterdam, The Netherlands under Dec-number: KNO06-02. NOD/SCID IL-2Rγ -/- mice were obtained from the Jackson laboratory (Bar Harbor, ME, USA). At the age of 8-ten weeks, the mice were irradiated sub-lethally with a dose of 350 cGy, 24 hours prior to transplantation of the homo AML cells. Details of irradiation, anesthesia, intravenous and intra-femoral injection are outlined in the supporting text (Text S1) under "Patients". Human being leukemic and multi-lineage engraftment was adamant based on positivity for CD45-PercP, the presence/absence of CD19-positive B-cells, CD13 and/or CD33-positive cells and, in 3 cases, past the presence/absence of abnormal marker expression.

Engraftment was defined as a clear clustered population in CD45 expression in a minimum of 200,000 acquired mice marrow cells. The minimum pct of human engraftment that was detected was 0.i%, which shows up equally a cluster of 200 cells on the scatterplot. Human leukemic engraftment was determined based on positivity for CD45-PercP, the absence of CD19-positive B cells and the presence of CD13 and/or CD33 positive cells: when CD13 was aberrantly absent-minded on the AML that was injected, CD33 was used and when CD33 was absent, CD13 was used. Homo multi-lineage engraftment was identified when CD45 positive cells consisted of both CD19 positive B-cells and myeloid cells with both CD13 and CD33 present (identified as monocytes and/or granulocytes in a FSC/SSC plot), and with absenteeism of aberrant markers.

FISH, FLT3-ITD and NPM1 assay

For the FISH assay, cytospins were prepared with FACS-sorted cells. LSI AML1/ETO dual color for t(eight;21) probe (Vysis, Abbott molecular, Illinois, UsA.) was practical to the denatured cells and incubated as previously described [17]. Genomic Dna from sorted jail cell populations was analyzed for the presence of an FLT3-ITD equally described before [27]. Mutations in NPM1 exon 12 were analyzed by PCR using genomic Deoxyribonucleic acid that had been isolated from sorted cell fractions (meet Text S1).

Survival analysis

Statistical analysis of the stalk cell information at follow-up was carried out using the SPSS twenty.0 software plan. The supplement provides details regarding the definition of overall survival (OS), effect-free survival (EFS), relapse-gratis survival (RFS), Kaplan-Meier analyses, and Cox regression analyses for both univariate and multivariate analyses. P-values beneath 0.05 were considered significant.

Results

In the absenteeism of formal proof of leukemia initiating ability in the stem cell compartments of about of the samples studied, the different stem jail cell compartments are referred to every bit putative LSCs (abbreviated as pLSC).

Since the aim of the study was to define the prognostic bear on of the neoplastic CD34+CD38- compartment, and to compare with the neoplastic CD34+CD38+ and CD34 negative compartments, starting time, backdrop will exist described that allow to discriminate between the neoplastic and the normal compartment within the CD34+CD38- compartment (Section I.A–D) and the CD34+CD38+ and the CD34- compartments (Department I.East). Proof of the resulting concept was obtained using either molecular biological and/or murine engraftment experiments.

In Section Two. the findings were used to assess the prognostic touch of the number of pLSCs at diagnosis and postal service-therapy for the CD34+CD38- compartment (II.A,B) and the CD34+CD38+ and CD34- compartment (Ii.C,D). Finally, the prognostic impact of the combination of follow up CD34+CD38- pLSC frequency, with MRD data volition be described (Two.Due east).

I. Bigotry between leukemic and normal stalk jail cell compartments

A. CD34+CD38-: discrimination of pLSCs and HSCs can be fabricated based on aberrant marker expression and besprinkle properties.

We have previously shown that the expression of abnormal markers on CD34+CD38- cells indicates the leukemic nature of stem cells [18], [19]. Figure 1.II illustrates the difference between CD34+CD38- pLSCs and HSCs based on aberrant marker expression and molecular aberrancies [in this example, t(8;21)]. In addition to Figure one, Table ane shows seven other patients with abnormal marker positive cells, all with molecular aberrancies indicating that these are in fact neoplastic cells. The figure likewise shows that marker-positive pLSCs, compared to HSCs, are further characterized by a tight clustered cell population with higher forwards scatter (FSC, reflecting jail cell size) and higher sideward scatter (SSC, reflecting granularity). This phenomenon was constitute in the other patients of Table i too (shown in Table 2). In Table 2, FSC and SSC of lymphocytes were used as internal controls to define the FSC/SSC position of pLSC and HSC. pLSC and HSC may also differ in CD34 and CD45 expression (in the case of Effigy 1, CD34 expression of pLSC was higher than HSC). Such differences were not consistent but helped to define clusters of cells particularly in cases with very low numbers of cells and/or small-scale differences in aberrant marker expression and/or FSC/SSC.

Tabular array S2 shows the FSC/SSC position relative to lymphocytes of CD34+CD38- HSCs nowadays in normal BM, besides equally CD34+CD38- nowadays in CD34 negative AML, which accept previously been shown to be normal [26]. FSC/SSC of HSCs present in these controls are all lower than pLSC nowadays in CD34+ AML (Table S2). Moreover, HSC nowadays in CD34+ AML also have low FSC/SSC and altogether there is not fifty-fifty overlap in FSC/SSC between pLSC in CD34+ AML and the HSCs present in these three os marrow sources. This allows accurate definition of the nature of a population peculiarly in those cases with merely a single population present.

B. CD34+CD38-: is there a role of FSC/SSC to ascertain pLSC and HSC in aberrant marker-negative office of CD34+CD38- compartment?

Aberrant markers may cover only part of neoplastic CD34+CD38- cells [19]. We therefore investigated whether scatter differences may aid to discriminate pLSCs from HSCs in aberrant marker-negative cells. This was indeed seen as illustrated for patient sample 456 in Effigy ii.I. The investigation of expression of only aberrant markers would take led to an over-interpretation of HSC numbers and an under-interpretation of pLSC numbers. Figure ii.Ii provides more examples.

Figure two. Marker-negative cells may contain leukemic cells defined past aberrant besprinkle. I. Gating/sorting strategy and molecular analysis for patient 456.

CD34+ CD38- cells (patient 456, figure 2) were gated equally in effigy i. CD34+CD38- cells were either CD7-negative (greenish in A and C, 25% of CD34+CD38-) or CD7 positive (red in A and D, 75% of CD34+CD38-). CD7+ cells were FSC/SSC^high (D) and of neoplastic origin (H). CD7-negative cells were farther subdivided into FSC/SSC^depression (left of the broken line in C), and FSC/SSC^loftier (right of the broken line in C). The CD7-negative, but FSC/SSC^high, cells were neoplastic (G), while the CD7-negative FSC/SSC^low cells were essentially normal (F). CD34+ cells was the positive control (E). 2. Molecular analysis of stem cell subpopulations in iv patients. Sorting and assay was done equally in figure two.I A for iii additional patients. ^# FLT3-ITD (% of total bespeak: FLT3-ITD + wt) adamant in cell populations sorted from CD34+ AML patients. * shown in figure ii.I A. ^§ FSC/SSC^depression and FSC/SSC^high defined as outlined in Tabular array 2.

https://doi.org/ten.1371/journal.pone.0107587.g002

In approximately 25% of AML cases, there is no clear aberrant marker expression in the CD34+CD38- compartment, and in fifty-fifty more cases, marking expression is weak and overlaps with the marker negative cells [19]. In social club to nevertheless discriminate betwixt pLSCs and HSCs, we investigated whether scatter might replace mark expression in this respect. Figure 3 (A–D) shows that, fifty-fifty without clear aberrant marking expression (CD19 covers but a very low frequency CD34+CD38- population), differences in scatter can be used. Since, in this particular AML case, aberrant expression of CD7 was found (Figure iii E–F), the validity of the scatter approach could be demonstrated (compare Figure 3 E–F with D). This shows that the scatter approach allows to discriminate pLSC and HSC in the absenteeism of aberrant markers.

Effigy iii. Marker negative pLSCs co-exist with marker positive pLSCs and are identified by scatter and CD34/CD45 expression patterns.

CD34+ CD38- cells (patient 372) were identified as described in figure one.I and gated for sub-compartments as described for figure 2.I. In the stem cell compartment of this AML case, only eleven% could be identified as CD19+ (A). When back-gated in FSC/SSC (similar equally performed in Figures ane and ii), two different populations were identified based on the position in FSC/SSC: the small CD19+ fraction (events in red in A–D) is characterized past FSC/SSC^high (B), low CD34 expression (C) and loftier CD45 expression (D). CD19 negative cells (events in green in A–D), autonomously from a small FSC/SSC^low/CD34^high/CD45^low population of putative HSCs (A–D), contained a large population of cells that, like to the CD19+ population in A–D, were FSC/SSC^loftier (B), CD34^low (C), and CD45^loftier (D). Apart from CD19, CD7 was an aberrant marking: 61% of the cells was CD7+ (E). Upon backgating, CD7+ cells (events in red in East–H), similar to CD19+ cells, were all FSC/SSC^high (F) CD34^low (G) and CD45^loftier (H). In dissimilarity to CD19, CD7 negative cells (events in green in E–H) now completely consisted of a small FSC/SSC^low (F), CD34^high(Grand), CD45^low (H) fraction. CD7 thus covered the whole neoplastic CD34+CD38- population and shows perfect discrimination between HSC and pLSC. CD19 expression in the absence of both CD7 expression and other scatter and CD34/CD45 expression parameters would accept nether-estimated the pLSC in the CD34+CD38- compartment by a factor 5.5 (61%/eleven%), while the HSCs would take been over-estimated by a cistron two.3 (89%/39%). In this case CD7 was a good marker to compare with the poor CD19 marker; it tin be seen, withal, that in the absence of CD7 expression, merely with the scatter and CD34/CD45 aberrancies nowadays, these would have enabled a complete discrimination between putative HSC and LSC compartments. This patient was identified as NPM1-positieve and FLT3-ITD positive. Other molecular aberrancies were not detected.

https://doi.org/10.1371/journal.pone.0107587.g003

C. CD34+CD38-: bigotry between HSCs and pLSCs in a big patient grouping using mark and besprinkle differences.

In cases with differences seen in FSC and SSC between pLSC and HSC, pLSC always had college FSC/SSC than HSC. Even so, at this point it should be emphasized that differences in scatter were found in part of the patients. In 250 diagnosis AML cases studied, the combination of marking expression and FSC/SSC differences allowed accurate identification of both pLSCs and HSCs in 117/250 cases (47%), (outlined in detail in Tabular array S3 and summarized in Table S4. For reasons mentioned earlier, differences in expression of CD34 and CD45 were merely used for fine-tuning. As might be expected, the pLSC compartment made upwardly the bulk of the total CD34+CD38- compartment (median of 82%, ranging from 0%–100%). Apart from these 117 cases, there was an extra group of 102 patients (41%) with only marker expression available, while 31 patients (12%) had no marker and scatter properties usable for pLSC detection (summarized in Table S4).

D. CD34+CD38-: multilineage and leukemic engraftment of CD34+CD38- HSCs and CD34+CD38- pLSCs in NOD/SCID IL-2R γ^-/- mice.

To provide further proof of principle for the strategy based on marker expression, secondary gating and molecular profiles, murine engraftment experiments were performed. HSCs were sorted by marking negativity, and clustering as FSC/SSC^depression, and/or distinct CD34/CD45 clustering, injected intrafemorally and evaluated for engraftment. In 5/6 cases, multilineage engraftment was found with no signs of leukemia (Figure 4, Table S5). In accordance with our in vitro gating strategy, AML engraftment with low jail cell numbers was observed in two/6 cases for CD34+CD38- marker-positive samples and in i/6 cases for the CD34+CD38- marker-negative cells, the latter being identified as neoplastic based on high FSC and aberrant CD34 expression (run across legends of Table S5).

Figure iv. Multilineage engraftment of CD34/CD38 and scatter-divers putative HSCs.

Unsorted mononuclear cells (MNCs) were injected intravenously and resulted in leukemic engraftment: cells were CD45+ (A) and of myeloid origin (B). In this case, the myeloid cells were positive for the diagnosis of leukemia-associated phenotype (LAP): partly CD33+CD13- (C) and CD11b+ (D). Sorted putative HSCs were injected intrafemorally (details, see Table S5). Engrafted CD45+ cells (East), contained both B-cells and myeloid cells (F), and lacked LAP (G,H). Multilineage engraftment of the sorted subpopulations was seen for patient 598 (I), 661 (J), 423 (K), and 928 (L). B-cells and myeloid cells (percentage of CD45+ cells) are in the upper left and lower right corners of the plots, respectively. The AML cells of patients 598 (I) and 661 (J) had an aberrant phenotype at diagnosis that was nowadays in the neoplastic engrafted cells, but absent-minded in the normal cells (not shown).

https://doi.org/10.1371/journal.pone.0107587.g004

E. CD34+CD38+ and CD34-: bigotry between normal and leukemic CD34+CD38+ and CD34- cells and their leukemic engraftment in NOD/SCID IL-2R γ^-/- mice.

Although emphasis in this paper is on the CD34+CD38- compartment, results described later for prognosis of CD34+CD38- pLSC will be compared with the other CD34/CD38 defined compartments. To enable such, neoplastic CD34+CD38+ and CD34- cells were identified past aberrant expression of markers used to define and so-called Leukemia Associated (Immuno) Phenotypes established at diagnosis and used past others and united states of america [23], [24] for detection of minimal remainder disease. Cells were sorted equally described in Text S1. In understanding with recent reports, these cell compartments besides had leukemia initiating potency since AML engraftment was plant in four/6 cases for the CD34+CD38+ compartment and in two/6 cases using CD34- compartments (referred to in the legends of Tabular array S5). In all but ane case, this was accomplished with high cell numbers (100,000-1,000,000).

II. Prognostic office of putative stem cell compartments

To assess the clinical impact of our findings, we assessed the prognostic value of the size the three individual LSC compartments both at diagnosis and, where applicable, specially at clinical follow-up.

A. CD34+CD38- at diagnosis.

To appraise prognostic bear upon, different cut-off values were established to define patients with high pLSC count (above the cutting-off level: referred to as pLSC+) and patients with low pLSC count (below the cut-off level: pLSC-). Patients who afterward turned out not to have achieved consummate remission (not-CR patients) had median 6-fold college pLSC count than patients who had achieved CR (meet legends of Figure 5). Moreover, the CR patient group could be divided into ii groups with significantly dissimilar prognoses at multiple cut-off points ranging from 0.002% to i% (Figure 5A and Table S6A). Figure 5B shows that farther splitting up the CR grouping resulted in the definition of three groups with big differences in relapse-free survival (RFS). The latter result was also found upon inclusion of non-CR patients (not shown) and replacement of RFS by EFS (Figure 5C). Multivariate analysis showed pLSC frequency to exist an contained prognostic factor (Figure 5, legends).

Effigy 5. Prognostic value of frequencies of pLSC compartments at diagnosis.

This figure shows the Kaplan-Meier analyses at diagnosis for the three compartments putatively containing pLSCs: CD34+CD38- (A,B,C), CD34+CD38+ (D) and CD34- (E). Of the 117 patients shown in Table S3, for Figures five and vi, 88 patients were chosen who had at least 1 follow-up time point. Of these, 70 entered Complete Remission, of whom 53 after the first form, 13 afterward the 2nd course and 4 at after stages. Eighteen never reached CR. The size (median values) of the CD34+CD38- compartment at diagnosis was significantly (half dozen-fold) higher in patients who did non enter CR (n = eighteen) compared with patients who did (n = 70): 0.225% of WBC versus 0.036% of WBC (p = 0.041). For CD34+CD38+ and CD34-, there were no pregnant differences (see text). Cut-off levels were defined to divide the total population into loftier stalk cell frequencies (above cut-off) and low stem cell frequencies (beneath cut-off). A particular cut-off value was chosen (A, D, E) to ensure approximately equally numbers of patients in the resulting loftier and low stem cell frequency compartments. Results for other cut-offs for the iii pLSC compartments are in Table S6. A–C: CD34+CD38-; D: CD34+CD38+; Eastward: CD34-. A. RFS in remission patients (northward = 70) with diagnosis CD34+CD38- cut-off of 0.03%; B. RFS in the same patient grouping (north = 70), but at present with two cut-offs (0.005% and 0.1%); C. Outcome-free survival for all CR and non-CR patients (n = 88); D. RFS in remission patients (n = 70) with CD34+CD38+ cutting-off of 25%; E. RFS in remission patients (n = 70) with CD34- cut-off of 3%. All relevant prognostic variables with statistical significance were investigated in a multivariate model. In this multivariate analysis it was found that take chances grouping (according to the HOVON 102 trial) was an contained prognostic cistron for OS at diagnosis (p = 0.001). For RFS, both risk grouping and CD34+CD38- leukemic stem cell load (using a 0.03% cut-off point) were independent prognostic factors at diagnosis (p = 0.017 and p = 0.011, respectively).

https://doi.org/10.1371/journal.pone.0107587.g005

B. CD34+CD38- during follow-up in CR.

The gating approach shown in Figure 1 was used to follow the fate of pLSCs over time using sequential sampling (examples in Effigy S1). Cox regression assay showed a stiff significant inverse correlation between the pLSC percent later all therapy cycles and RFS and OS (for details, see legends to Table S7). Kaplan-Meier analyses showed markedly improved RFS and OS in pLSC- patients compared with pLSC+ patients (case in Figure 6, A–C), which applies for a big range of cutting-off values (Table S7). Multivariate analyses with cut-off values showed that pLSC frequency later on the first and second treatment cycles was an contained prognostic factor for RFS and OS (case with 0.0003% and 0.0001% shown in Table S8).

Figure 6. Prognostic value of frequencies of CD34+CD38- pLSC compartment at follow-upwardly.

This effigy shows the Kaplan-Meier analyses for RFS for the CD34+CD38- pLSC compartment at follow up for iii consecutive therapy cycles. The optimal cut-off levels were chosen to define pLSC + and pLSC- after 1^st induction cycle (0.0003%,which is 3 pLSCs in 1,000,000 WBC) and after two^nd induction bike and consolidation therapy 0.0001% (one pLSC in 1,000,000 WBC). Results for other cut-offs are in Table S7. Later on the start induction cycle (B, 71 patients), 2nd induction cycle (C, 77 patients), and after consolidation therapy (D, 48 patients), patients with loftier pLSC frequency (pLSC+) showed significantly more adverse performance compared with patients with low pLSC frequency (LSC-).

https://doi.org/10.1371/periodical.pone.0107587.g006

C. CD34+CD38+ and CD34- at diagnosis and at follow up.

The CD34+CD38+ and CD34- compartments have been shown to incorporate leukemia initiating cells [5], [11] (also in this paper). In social club to appraise whether these were clinically important, when present together with the leukemia initiating CD34+CD38- compartment, nosotros assessed their prognostic affect.

At diagnosis, neoplastic CD34+CD38+ and CD34- nail cells were identified equally described nether I.B. For CD34+CD38+, at that place was no single cut-off value (tested in the range 2%–60%) that resulted in bigotry of 2 patient groups with unlike RFS (Figure 5D; Table S6B), even when non-CR patients were included in the latter (data non shown). Although only borderline meaning, in contrast to CD34+CD38-, the neoplastic CD34+CD38+ compartment was even smaller in non-CR patients than in CR patients (10.viii% of WBC versus 23.vii% of WBC; p = 0.06). Similar results were obtained for the CD34- compartment with cutting-off points ranging from 0.1%–30% (Figure 5E; Table S6C), with the neoplastic compartment size, similar to CD34+CD38+, beingness even smaller (admitting not-significantly) in non-CR patients than in CR patients (0.87% of WBC versus 2.41% of WBC, p = 0.48).

At follow upward, the neoplastic component of the CD34+CD38+ and CD34- compartment represented a considerable portion of the total neoplastic blast compartment. This reflects the total leukemic burden, or MRD, which, in turn, has previously been shown by many authors, including ourselves, to have prognostic impact (come across also next paragraph) [23], [24].

D. Post-diagnosis prognostic impact of CD34+CD38- LSC combined with MRD.

The burden of leukemic stalk cells later on chemotherapy does not always reflect the full leukemic burden (known as MRD). It tin thus be argued that the combination of pLSC frequency and MRD frequency may ameliorate prognostic data at follow up. Because this combination would dissever the total patient group in four relatively small sub-groups defined by both a pLSC cutting-off (Figure half-dozen, Table 3) and a fixed MRD cut-off of 0.1 [24]), we increased the size of the nowadays patient remission grouping by including 23 similarly treated young adults in remission (<65 years) (details in Table S1 "Patients 3"). In the total grouping (n = 91) pLSC frequency over again had strong prognostic impact over a range of cutting-off points (from 0 in x^six WBC upwards to ten in 10^vi WBC. As an example, the cut-off signal of 0.0001% is shown in Figure 7A. For MRD, the cut-off signal of 0.1% had the expected prognostic touch on (Figure 7B) [24]. When combining MRD and pLSC in Figure 7C, iv groups were identified. The main conclusions from this effigy are: 1) within the full MRD- grouping (n = 64), pLSC+ patients (n = 31) have significantly poorer prognosis than pLSC- patients (due north = 33; p = 0.01); 2) within the pLSC+ grouping, MRD- patients, although having a relatively poor prognosis, may do better than MRD+ patients (p = 0.04); the pLSC-/MRD- group had relatively good prognosis, while the pLSC+/MRD+ grouping had very poor prognosis.

Figure 7. Prognostic value of combined p-LSC and MRD.

(A) Kaplan-Meier analyses after cycle II for RFS for the pLSC data as shown in Figure 6, with an additional 23 patients (Table S1). The pLSC cutting-off used is 0.0001%. (B) Kaplan Meier analysis of MRD information (cut-off 0.i%) obtained for the same patient grouping as in A (n = 91). (C) Combined pLSC and MRD (north = 91) data resulted in 4 patient groups: pLSC-/MRD-, pLSC-/MRD+, pLSC+/MRD- and pLSC+/MRD+.

https://doi.org/10.1371/journal.pone.0107587.g007

When including the distribution of cytogenetically/molecularly good, intermediate, poor and very poor patients, these were all represented in the 4 LSC/MRD divers subgroups (Table 3). This shows that, the pLSC/MRD prognostic touch is across cytogenetic risk groups, although in the LSC+/MRD+ group poor and very poor cytogenetic/molecular risk groups are prevalent.

When including the post-diagnosis prognostic parameter "cycle after which CR is reached" in Effigy 7, information technology turned out that there was no pregnant difference between the first iii pLSC/MRD defined patient groups in number of cycles needed to accomplish CR: in the kickoff group (pLSC-/MRD-) 29 after outset cycle versus iv afterwards second bike; in the tertiary grouping (pLSC+/MRD-) this was 25 versus half dozen. The second group (pLSC-/MRD+) was besides small-scale (all patients in CR after one bicycle). Still, in the fourth grouping (pLSC+/MRD+) for 9 patients ii cycles were needed, with one for the other nine patients.

These information show that combining cytogenetic/molecular defined risk groups together and clinical parameters like cycles to CR, together with pLSC/MRD defined risk cess may offer a very important new algorithm in risk cess.

Word

One of the major challenges in the design of new therapies to eradicate leukemia stem cells is to achieve high therapeutic specificity. To this end, it is important to distinguish LSCs from the concomitantly present HSCs, and to assess whether this distinction is of prognostic value, since it would underline the clinical importance of LSCs. Consequently, it was necessary to place parameters that immune bigotry betwixt these pLSCs and HSCs, preferably in all AML cases. Recent studies have shown that pLSCs may reside not only in CD34+CD38-, simply besides in CD34+CD38+ and CD34- compartments [v], [eleven]. In the present paper, we first present methods to discriminate between the neoplastic and normal portions of these compartments, and we subsequently assessed the prognostic value of these putative stalk cell compartments.

With regard to the prognostic impact, using a uniquely-designed multi-parameter catamenia cytometry protocol, we testify for the starting time fourth dimension that the CD34+CD38- pLSC load after different cycles of therapy was highly predictive of patient survival, independent of other prognostic parameters. In addition, post-obit our ain preliminary studies [thirteen], nosotros identified three patient groups at diagnosis defined by CD34+CD38- pLSC with very large differences in prognosis, again independent of other prognostic parameters. Past comparison with literature [thirteen]–[15], it can be appreciated that the prognostic impact using our new approach, in which the pLSC compartment within the total CD34+CD38- compartment was specifically used, is much college than using the full CD34+CD38- compartment as done in the previous studies. This is likely due to the "contagion" of the leukemic CD34+CD38- compartment with HSCs in the earlier studies (ranges of 0%–100% of pLSC, as seen in our report, also means that HSC range from 0%–100%), as seen in Tabular array S3. In contrast, the CD34+CD38+ and CD34- compartments at diagnosis completely lacked prognostic impact, which strongly suggests that CD34+CD38+ and CD34- pLSCs are of modest clinical importance, at least in AML cases where these compartments are accompanied by CD34+CD38- pLSCs (as past definition is the case in our current CD34 positive patient group). However, at follow-upwardly, leukemic CD34+CD38+ and CD34- compartments represent considerable portions of the total leukemic burden and thus reflect MRD cell frequency rather than pLSC frequency. Here, CD34+CD38+ and CD34- cells probably originate from (limited) differentiation of the CD34+CD38- pLSCs, a process that has been shown to occur in vivo by Goardon and colleagues [28].

The results are uniform with the following model: what the paper shows is that in CD34 positive AML cases, information technology is the percentage of the CD34+CD38- population at diagnosis that strongly correlates with clinical outcome and not the percentage of CD34+CD38+ or CD34- cells. This does not mean that CD34+CD38+ and CD34- cells do not contain leukemia initiating ability; it merely strongly suggests that, in the presence of CD34+CD38- cells, these CD34+CD38+ and CD34- leukemia initiating cells are either less therapy resistant and/or less malignant compared to CD34+CD38- cells. Likely, leukemia initiating ability in mouse models of CD34/CD38 defined sub-populations do not reflect clinical importance (see also next paragraph), since this ability is always assessed using purified populations, whereby the "contest" between these populations in outgrow and/or the relative therapy resistance cannot be taken into account. This model too implies that in CD34 negative AML (with only neoplastic CD34- populations present), it is the CD34- pLSC that takes over the leukemia initiating ability. Too in CD34 positive AML in the absenteeism of CD34+CD38- cells, but with neoplastic CD34+CD38+ and CD34- populations present, the latter 2 populations may take over the leukemia initiating power. Equally a logical consequence of the model these putatively less ambitious and/or less therapy resistant populations should define a ameliorate clinical upshot, which is indeed the case: in a separate cohort of 438 patients, survival of CD34 negative patients was significantly amend than survival of CD34 positive patients (unpublished results).

The lack of correlation between prognosis and the size of the different CD34+CD38+ and CD34- compartments, while all compartments in purified course do engraft in a mouse model, may be explained as follows: the overall immune condition of a patient grouping like that in our report, may best be represented past a less immune-restricted mouse model, where CD34+CD38- pLSCs are the predominant engrafting cells [29], [30]. In line with that, in our earlier engrafting experiments using CD34+ cells (i.e., containing CD34+CD38-, CD34+CD38+ and CD34- cells) in the less immune-restricted NOD/SCID mice, it was only the size of the CD34+CD38- compartment at diagnosis that correlated with levels of engraftment [13]. More immune-restricted mouse models are useful to study LSC engraftment of probably less aggressive pLSC sub-populations [five], [28]. In this respect, an important initial observation was made by Costello and co-workers, who found that, in vitro, CD34+CD38- cells were more therapy-resistant and less immunogenic than other compartments [21]. Moreover, farther compelling evidence that the CD34+CD38- compartment is most of import in the clinical setting comes from our clinical observations: in virtually cases which had depression frequency mutations at diagnosis, which became predominant at relapse, these mutations were nowadays and/or enriched in the neoplastic CD34+CD38- diagnosis compartment [31].

Survival and outgrowth of leukemia cells after therapy may depend on many factors and include the LSC load and likely specific LIC backdrop, but also the frequency of AML blast cells referred to as MRD. Many authors, including us, take shown that MRD is a strong independent prognostic gene [24], [32], [33]. In this newspaper we accept shown that both CD34+CD38- pLSC frequency and MRD jail cell frequency are complementary, thereby defining a new post-diagnosis combination factor that offers strong prognostic data, fifty-fifty beyond cytogenetics/molecular defined risk groups. This will prospectively be validated in a new patient accomplice of the HOVON/SAKK cooperative report grouping for which patient enrollment has already started.

With regard to our approach to distinguish between pLSCs and HSCs within the CD34+CD38- compartment, we have shown that aberrant expression of antigens (including CLL-1) and lineage markers, too every bit additional aberrancies (including differences in CD34 and CD45 expression and differences in light scatter) allow unequivocal discrimination between pLSCs and HSCs. Moreover, this approach even immune CD34+CD38- pLSC detection in patients lacking abnormal antigen expression. By applying the boosted parameters, underestimation of pLSC numbers or overestimation of HSC numbers (frequently seen when using marker expression alone) is avoided. The present study thereby confirms the findings of others [29] that different CD34+CD38- pLSC markers expressed in individual AML cases may miss substantial portions of LSCs present (Effigy three). Although secondary parameters are thus very useful, it requires experience.

The number of accurately-identified pLSCs would increment with the utilize of boosted AML markers. Such markers may include: CD123, CD96, CD44, CD47, CD25, CD32, CD33, TIM-3 [12], [xviii], [29], [34]–[38]. In the present study, both lineage markers and CLL-1 were used to place CD34+CD38- pLSCs and HSCs during follow-up, since these markers remained highly specific for these LSCs during and after therapy [xviii], [19]. To appointment, other markers have not yet been tested and considerable efforts volition exist necessary to demonstrate their applicability both for pLSC quantification at diagnosis and peculiarly at follow upward, and as putative targets in antibody therapies. In particular, undesirable post-chemotherapy up-regulation on HSCs may occur for some of these markers [xviii], [19]. Thus, our study implies that, for adequate pLSC (and HSC) tracking, a spectrum of markers and probably additional parameters should be screened for each individual AML example. Therefore, wide application of a unmarried target antigen for diagnostic purposes, likewise every bit for future LSC-directed antibiotic therapies, all the same seems unlikely.

Although FSC/SSC characteristics are often effective in discriminating betwixt CD34+CD38- pLSCs and HSCs, the underlying cause of this difference is not yet known. An attractive option is that, similar to normal stem cells, CD34+CD38- AML sub-populations may exist with slightly different levels of differentiation [28]. In fact, nosotros now have more formal proof for that: AML cases with high FSC and SSC of the CD34+CD38- cells all accept expression of the differentiation marker CD45RA. Information technology is known that this mark identifies GMP CD34+CD38+ progenitors in contrast to MEP and CMP CD34+CD38+ progenitors. We have establish that CD45RA is completely absent on the real CD34+CD38- HSCs, just marked corresponding CD34+CD38- pLSC populations in roughly half of the AML cases. In the other half, the pLSC had FSC and SSC close to those of respective HSCs; these pLSCs were CD45RA negative. The CD45RA positive cases may indeed reflect more progenitor-like pLSCs compared to the CD45RA negative pLSCs that resemble HSCs [28].

In AML cases with no malignant CD34+CD38- compartments, the pLSCs volition be located in the CD34+CD38+ and/or CD34- compartments. It is likely, withal, that inside these relatively large compartments, further compartmentalization will be necessary to identify the true LSC sub-compartment which likely occurs at low frequencies. Ane candidate fraction is the side population (SP), which does occur in low numbers (usually <ane% of the WBC population) and contains both normal and AML cells at diagnosis [8], [9]. Moreover, the SP has also been found to comprise different CD34/CD38-divers sub-fractions [6], which may represent CD34/CD38/SP pLSCs. In the future, it would be interesting to examine the relationships between the functional phenotypes (based on SP or aldehyde dehydrogenase activity) [vi]–[9], [39], [40] and the CD34/CD38 immunophenotype.

In decision, the present written report offers tools for detecting concomitantly present pLSCs and HSCs, both at diagnosis and at disease follow-up, and provides the first proof that CD34+CD38- pLSCs are non only clinically important at diagnosis, only also at follow-up. No evidence was found to suggest that the CD34+CD38+ and CD34- leukemic fractions contain clinically important LSCs, at least non in CD34 positive AML with leukemic CD34+CD38- nowadays. The combination with well-established MRD assessment likely opens a new field in prognostication in patients with AML. Ultimately, our findings may contribute to the development of new diagnostic tools and to novel, more selective therapies, including antibody-based therapies that would be highly effective against AML stem cells, while leaving the normal HSCs intact. Finally, these results may stimulate farther research into the role of cancer stalk cells in other cancers, such equally solid tumors.

Supporting Information

Figure S1.

pLSCs monitoring during sequential BM sampling. A patient (243) with AML positive for a t(viii;21) showed CLL-ane expression in the CD34+CD38- compartment (A), while normal BM misses CLL-ane+ CD34+CD38- cells [1]. In CR, CLL-ane+ CD34+CD38- cells (B) were sorted and assessed for t(viii;21): mainly neoplastic cells were nowadays (E). Normal CD34+CD38-CLL-1 negative cells were well-nigh completely absent here. Shown in F-H are examples of sequential monitoring in three cases with increasing periods of consummate remission until relapse (F, pt 253; G, pt 372) and in continuous remission (H, pt 572). Note the increase of pLSC frequency preceding relapse (F, Grand).

https://doi.org/ten.1371/journal.pone.0107587.s001

(PPTX)

Table S1.

Patient characteristics. *"Patients 1" are all CD34+ patients. "Patients two" are all patients were authentic discrimination betwixt HSC and pLSC was enabled using our extensive gating strategy. "Patients 3" are CR patients with MRD and pLSC data to enlarge the full patient group as shown in Figure 7.

https://doi.org/10.1371/periodical.pone.0107587.s002

(DOCX)

Table S2.

FSC and SSC position relative to lymphocytes. FSC, forrad besprinkle; SSC, side scatter; HSC hematopoietic stem cells; pLSC, putative leukemia stalk cell; NA, not applicable. * FSC and SSC values relative to those of lymphocytes present in the same sample.

https://doi.org/10.1371/periodical.pone.0107587.s003

(DOCX)

Table S4.

Number of patients for dissimilar strategies in 250 CD34+ AML cases. *>xx% aberrant marker expression was considered substantial to place directly at least a substantial part of the pLSC population (179/250 patients; rows 3 and iv). In 102/179 patients (41% of all 250 CD34+ patients, row 3), pLSC frequencies may be under-estimated since additional gating strategy (with FSC/SSC etc, referred to in columns three–7) was not possible, probably leaving part of marking negative pLSCs unidentified. In 77 of these 179 patients, an additional gating step could be performed (FSC/SSC etc, come across row iv), assuasive a more accurate assessment of both pLSC and HSC frequencies. #: <twenty% aberrant marker expression (71/250 cases) is shown in rows five and 6. In 31 cases (12%) but inadequate LSC assessment was possible (row five). However, in 40 of these 71 cases HSCs could withal be distinguished from pLSCs with the use of secondary parameters (row 6). Highly adequate LSC assessment, using both aberrant marker expression and secondary parameters was thus possible in 77+twoscore cases (47%). Columns show parameters/plots used to distinguish HSCs from pLSCs.

https://doi.org/10.1371/journal.pone.0107587.s005

(DOCX)

Tabular array S5.

Multi-lineage engraftment of marking negative FSC/SSC^depression (CD34^high) CD34+CD38- cells present in AML. * in the missing mouse, engraftment could non exist assessed since this mouse died earlier examination was possible. # In the missing mouse, no human engraftment was detected. In terms of leukemic engraftment our results also confirmed the observation of Bonnet's group that purified CD34+CD38+ and CD34- were able to engraft be it in our case later injection of high cell numbers. CD34+CD38-/CLL-1+ in pts 1 and two (40,000 and 130,000 cells, respectively) CD34+CD38-/CLL-1-/FSC ^high CD34^depression in pt 1 (6,000 cells) CD34+CD38+ in pts ii, 4, v, 6 (high cell numbers, 100,000-ten⁶ injected in pts 2, 4, half-dozen and ane,000 in pt five) CD34- in pts two and v (high cell numbers injected:100,000-10⁶).

https://doi.org/ten.1371/periodical.pone.0107587.s006

(DOCX)

Tabular array S6.

Cut-off values in the CD34+CD38-, CD34+CD38+ and CD34- cell compartment at diagnosis to identify patient groups with dissimilar survival. *p-values refer to significance of differences in RFS between patients in a higher place and patients beneath the indicated cut-offs.

https://doi.org/10.1371/journal.pone.0107587.s007

(DOCX)

Table S7.

Relative run a risk of relapse determined for pLSC- and pLSC+ patients at follow upwardly defined by different cut-off points. Not shown in the Tabular array: for RFS and OS, without employ of cut-offs, Cox regression analysis showed a stiff significant inverse correlation between pLSC percentage and RFS subsequently one^st bicycle (n = 71, RR = ii.4, 95%CI:i.iii–iv.6, p = 0.008), two^nd wheel (n = 77, RR = 2.5, 95%CI:1.7–iii.7, p<0.001) and consolidation cycle (due north = 48, RR = iii.0, 95%CI:1.4–six.2, p = 0.004). For OS, these figures were RR = one.8 (p = 0.04), RR = 2.7 (p<0.001) and RR = 2.0 (p = 0.07). Futurity different cut offs were applied for risk on relapse. RR, relative risk of relapse using these different cut offs. Cut-offs of 0.0003% (3 in a million, 1^st wheel) and 0.0001% (2d and consolidation cycle) were used for relapse-complimentary survival (RFS) in Kaplan-Meier analyses shown in Figure 6. With these cut-offs median overall survival (OS, not shown in Effigy 6) was not reached (>42 months) for pLSC+ patients after 1^st cycle, but more patients survived in the pLSC- grouping (p = 0.002). After 2^nd bike median OS in pLSC- group was>45 months versus 35 months in pLSC+ grouping (p<0.001). After consolidation cycle these figures were both>37 months (p = 0.05).

https://doi.org/10.1371/periodical.pone.0107587.s008

(DOCX)

Tabular array S8.

Multivariate analysis^# for bear upon of pLSC frequency on RFS. ^# in univariate analyses performed on 162 CD34+ patients, cytogenetic/molecular risk (p = 0.001, n = 162), number of chemotherapy cycles needed to achieve CR (p<0.001, n = 162), and WBC count at diagnosis (p = 0.002, northward = 162) were meaning. Other factors showed a trend: NPM1 mutation (p = 0.093, n = 140) and EVI-1 (p = 0.18, n = 140). due north.r. not relevant since all evaluated patients achieved CR after commencement bike RR, relative adventure of relapse. * just the well-nigh optimal cut-offs (0.0003% after one^st cycle and 0.0001% after 2^nd and consolidation cycle) are shown.

https://doi.org/10.1371/journal.pone.0107587.s009

(DOCX)

Acknowledgments

The authors would like to thank Hans Meel and Rolf Wouters for their help with the sorting and PCR experiments.

Author Contributions

Conceived and designed the experiments: MT AK ANS WZ April SZ GJO GJS. Performed the experiments: MT AK ANS WZ Apr WJS. Analyzed the information: MT AK ANS WZ APR WJS SZ GJO GJS. Contributed reagents/materials/analysis tools: SZ GJO TP GV BL GJS. Wrote the paper: MT AK ANS WZ APR WJS TP GV BL SZ GJO GJS.

References

1. 1. Valent P, Bonnet D, De MR, Lapidot T, Copland Grand, et al. (2012) Cancer stem cell definitions and terminology: the devil is in the details. Nat Rev Cancer 12: 767–775.
   • View Article
   • Google Scholar
2. 2. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, et al. (1994) A cell initiating human acute myeloid leukaemia afterwards transplantation into SCID mice. Nature 367: 645–648.
   • View Article
   • Google Scholar
3. 3. Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic jail cell. Nature medicine 3: 730–737.
   • View Commodity
   • Google Scholar
4. 4. Dick JE (2008) Stem prison cell concepts renew cancer research. Blood 112: 4793–4807.
   • View Article
   • Google Scholar
5. 5. Taussig DC, Vargaftig J, Miraki-Moud F, Griessinger Eastward, Sharrock K, et al. (2010) Leukemia-initiating cells from some acute myeloid leukemia patients with mutated nucleophosmin reside in the CD34(-) fraction. Claret 115: 1976–1984.
   • View Commodity
   • Google Scholar
6. 6. Moshaver B, Van Rhenen A, Kelder A, Van der Pol M, Terwijn Grand, et al. (2008) Identification of a small subpopulation of candidate leukemia-initiating cells in the side population of patients with acute myeloid leukemia. Stem cells 26: 3059–3067.
   • View Commodity
   • Google Scholar
7. 7. Pearce DJ, Taussig D, Simpson C, Allen K, Rohatiner AZ, et al. (2012) Characterization of cells with a high aldehyde dehydrogenase activity from string blood and acute myeloid leukemia samples. Stalk cells 23: 752–760.
   • View Article
   • Google Scholar
8. viii. Wulf GG, Wang RY, Kuehnle I, Weidner D, Marini F, et al. (2001) A leukemic stem cell with intrinsic drug efflux chapters in acute myeloid leukemia. Blood 98: 1166–1173.
   • View Commodity
   • Google Scholar
9. 9. Feuring-Buske M, Hogge DE (2001) Hoechst 33342 efflux identifies a subpopulation of cytogenetically normal CD34(+)CD38(-) progenitor cells from patients with acute myeloid leukemia. Claret ix: 3882–3889.
   • View Article
   • Google Scholar
10. 10. Taussig DC, Miraki-Moud F, Anjos-Afonso F, Pearce DJ, Allen G, et al. (2008) Anti-CD38 antibody-mediated clearance of human being repopulating cells masks the heterogeneity of leukemia-initiating cells. Blood 112: 568–575.
    • View Article
    • Google Scholar
11. xi. Sarry JE, Spud K, Perry R, Sanchez P V, Secreto A, et al. (2011) Human being acute myelogenous leukemia stem cells are rare and heterogeneous when assayed in NOD/SCID/IL2Rgammac-deficient mice. J Clin Invest 121: 384–395.
    • View Article
    • Google Scholar
12. 12. Majeti R, Chao MP, Alizadeh AA, Pang WW, Jaiswal S, et al. (2009) CD47 is an agin prognostic factor and therapeutic antibody target on human astute myeloid leukemia stem cells. Cell 138: 286–299.
    • View Article
    • Google Scholar
13. xiii. Van Rhenen A, Feller Due north, Kelder A, Westra AH, Rombouts East, et al. (2005) High stem cell frequency in astute myeloid leukemia at diagnosis predicts high minimal rest disease and poor survival. Clinical cancer enquiry 11: 6520–6527.
    • View Article
    • Google Scholar
14. 14. Ran D, Schubert M, Taubert I, Eckstein Five, Bellos F, et al. (2012) Heterogeneity of leukemia stalk jail cell candidates at diagnosis of acute myeloid leukemia and their clinical significance. Experimental hematology 40: 155–165.
    • View Commodity
    • Google Scholar
15. fifteen. Witte KE, Ahlers J, Schafer I, Andre M, Kerst Chiliad, et al. (2011) High proportion of leukemic stem cells at diagnosis is correlated with unfavorable prognosis in childhood acute myeloid leukemia. Pediatr Hematol Oncol 28: 91–99.
    • View Article
    • Google Scholar
16. 16. Roshal Grand, Chien S, Othus M, Wood BL, Fang One thousand, et al. (2013) The proportion of CD34(+)CD38(low or neg) myeloblasts, but not side population frequency, predicts initial response to consecration therapy in patients with newly diagnosed astute myeloid leukemia. Leukemia 27: 728–731.
    • View Commodity
    • Google Scholar
17. 17. Vergez F, Green Equally, Tamburini J, Sarry JE, Gaillard B, et al. (2011) High levels of CD34+CD38low/-CD123+ blasts are predictive of an agin outcome in astute myeloid leukemia: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study. Haematologica 96: 1792–1798.
    • View Article
    • Google Scholar
18. 18. Van Rhenen A, Van Dongen GA, Kelder A, Rombouts EJ, Feller Northward, et al. (2007) The novel AML stem prison cell associated antigen CLL-1 aids in discrimination between normal and leukemic stem cells. Blood 110: 2659–2666.
    • View Article
    • Google Scholar
19. 19. Van Rhenen A, Moshaver B, Kelder A, Feller N, Nieuwint AW, et al. (2007) Aberrant marking expression patterns on the CD34+ CD38- stem cell compartment in acute myeloid leukemia allows to distinguish the cancerous from the normal stem jail cell compartment both at diagnosis and in remission. Leukemia 21: 1700–1707.
    • View Article
    • Google Scholar
20. 20. Becker MW, Jordan CT (2011) Leukemia stem cells in 2010: current understanding and future directions. Claret Rev25: 75–81.
    • View Commodity
    • Google Scholar
21. 21. Costello RT, Mallet F, Gaugler B, Sainty D, Arnoulet C, et al. (2000) Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and dumb dendritic jail cell transformation capacities. Cancer Res 60: 4403–4411.
    • View Commodity
    • Google Scholar
22. 22. Taussig DC, Pearce DJ, Simpson C, Rohatiner AZ, Lister TA, et al. (2005) Hematopoietic stalk cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia. Blood 106: 4086–4092.
    • View Commodity
    • Google Scholar
23. 23. Feller N, Van der Politico MA, van Stijn A, Weijers GW, Westra AH, et al. (2004) MRD parameters using immunophenotypic detection methods are highly reliable in predicting survival in astute myeloid leukaemia. Leukemia 18: 1380–1390.
    • View Article
    • Google Scholar
24. 24. Terwijn M, Van Putten WLJ, Kelder A, Van der Velden VHJ, Brooimans RA, et al. (2013) High Prognostic Impact of Period Cytometric Minimal Residual Affliction Detection in Astute Myeloid Leukemia: Information From the HOVON/SAKK AML 42A Study. Journal of clinical oncology 31: 3889–3897.
    • View Article
    • Google Scholar
25. 25. Bakker AB, Van den Oudenrijn South, Bakker AQ, Feller N, van Meijer Thousand, et al. (2004) C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia. Cancer Res 64: 8443–8450.
    • View Article
    • Google Scholar
26. 26. Van der Politician MA, Feller N, Roseboom M, Moshaver B, Westra G, et al. (2006) Assessment of the normal or leukemic nature of CD34+ cells in astute myeloid leukemia with low percentages of CD34 cells. Haematologica 88: 983–993.
    • View Commodity
    • Google Scholar
27. 27. Cloos J, Goemans BF, Hess CJ, Van Oostveen JW, Waisfisz Q, et al. (2006) Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples. Leukemia 20: 1217–1220.
    • View Article
    • Google Scholar
28. 28. Goardon N, Marchi E, Atzberger A, Quek 50, Schuh A, et al. (2011) Coexistence of LMPP-like and GMP-similar leukemia stem cells in acute myeloid leukemia. Cancer cell xix: 138–152.
    • View Article
    • Google Scholar
29. 29. Jan One thousand, Chao MP, Cha AC, Alizadeh AA, Gentles AJ, et al. (2011) Prospective separation of normal and leukemic stalk cells based on differential expression of TIM3, a human being astute myeloid leukemia stem cell marker. Proc Natl Acad Sci Usa 108: 5009–5014.
    • View Commodity
    • Google Scholar
30. 30. Ishikawa F, Yoshida Due south, Saito Y, Hijikata A, Kitamura H, et al. (2007) Chemotherapy-resistant man AML stem cells home to and engraft within the bone-marrow endosteal region. Nature biotechnology 25: 1315–1321.
    • View Article
    • Google Scholar
31. 31. Bachas C, Schuurhuis GJ, Assaraf YG, Kwidama ZJ, Kelder A, et al. (2012) The role of pocket-sized subpopulations within the leukemic nail compartment of AML patients at initial diagnosis in the evolution of relapse. Leukemia 26: 1313–1320.
    • View Article
    • Google Scholar
32. 32. Buccisano F, Maurillo L, Del Principe MI, Del PG, Sconocchia G, et al. (2012) Prognostic and therapeutic implications of minimal residual disease detection in acute myeloid leukemia. Claret 119: 332–341.
    • View Article
    • Google Scholar
33. 33. Freeman SD, Virgo P, Couzens Due south, Grimwade D, Russell N, et al. (2013) Prognostic relevance of handling response measured by period cytometric residual disease detection in older patients with acute myeloid leukemia. J Clin Oncol 31: 4123–4131.
    • View Article
    • Google Scholar
34. 34. Hosen N, Park CY, Tatsumi N, Oji Y, Sugiyama H, et al. (2007) CD96 is a leukemic stem jail cell-specific marker in man acute myeloid leukemia. Proc Natl Acad Sci Us 104: 11008–11013.
    • View Article
    • Google Scholar
35. 35. Hashemite kingdom of jordan CT (2002) Unique molecular and cellular features of acute myelogenous leukemia stem cells. Leukemia xvi: 559–562.
    • View Article
    • Google Scholar
36. 36. Jordan CT, Upchurch D, Szilvassy SJ, Guzman ML, Howard DS, et al. (2000) The interleukin-iii receptor alpha chain is a unique marker for homo acute myelogenous leukemia stalk cells. Leukemia xiv: 1777–1784.
    • View Article
    • Google Scholar
37. 37. Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE (2006) Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat med 12: 1167–1174.
    • View Article
    • Google Scholar
38. 38. Saito Y, Kitamura H, Hijikata A, Tomizawa-Murasawa Thousand, Tanaka Due south, et al. (2010) Identification of therapeutic targets for quiescent, chemotherapy-resistant human leukemia stalk cells. Sci Transl Med 2: 17ra9.
    • View Article
    • Google Scholar
39. 39. Ran D, Schubert One thousand, Pietsch L, Taubert I, Wuchter P, et al. (2009) Aldehyde dehydrogenase activity among primary leukemia cells is associated with stalk cell features and correlates with agin clinical outcomes. Exp Hematol 37: 1423–1434.
    • View Article
    • Google Scholar
40. 40. Cheung AMS, Wan TSK, Leung JCK, Chan LYY, Huang H, et al. (2007) Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential. Leukemia two: 1423–1430.
    • View Commodity
    • Google Scholar

gallegosqueng1989.blogspot.com

Source: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107587

Share this post