ex-vivo aml-culture initiating cells reflect patients...

GRIESSINGER et al. L-LTC-IC dynamics stratify AML risk group of 11

Priority Reports

Frequency and dynamics of ex-vivo AML-culture initiating cells reflect patients’ outcome.

Emmanuel Griessinger1,2,*, Fernando Anjos-Afonso3,4, Jacques Vargaftig2, David C. Taussig5, François Lassailly2, Thomas Prebet6, Véronique Imbert1, Marielle Nebout1, Norbert Vey6, Christian Chabannon6, Andrew Filby7, Frederic Bollet-Quivogne8, John Gribben5, Jean-François Peyron1 and

Dominique Bonnet2,3*. AUTHORS AFFILIATION:

1Present address INSERM U1065, C3M, Team 4, Inflammation, Cancer, Cancer Stem Cells, Nice, France; 2 Cancer Research UK, London Research Institute, 3Francis Crick Institute, Haematopoeitic Stem Cell Laboratory, London, UK; 4Present address: Haematopoietic Signalling Group, European Cancer Stem Cell Research Institute, Cardiff University 5Centre of Haemato-Oncology, Cancer Research UK Clinical Centre, Barts Cancer Institute, St Bartholomew’s Hospital, Queen Mary University of London, London, UK; 6Haemato-Oncology Division, Paoli-Calmette Institute, Marseille, France; 7The Francis Crick Institute, FACS services; 8Neurophysiology Laboratory, Faculty of Medicine, Brussels University, Belgium. RUNNING TITLE:

L-LTC-IC dynamics stratify AML risk group

* CORRESPONDING AUTHORS:

Dr Emmanuel GRIESSINGER, INFLAMMATION, CANCER & CANCER STEM CELLS, INSERM U1065, C3M, Bâtiment Universitaire Archimed, 151 Route de Ginestière, BP 2 3194, 06204 NICE Cedex 3, France; Phone: +33(0) 4 8906 4318; Fax: +33(0)4 8906 4221 - e-mail: [email protected] - Website: http://www.unice.fr/c3m/EN/Equipe4.html Dr Dominique Bonnet, The Francis Crick Institute, Haematopoietic Stem Cell Laboratory, 44 Lincoln’s Inn Fields, London, UK; Phone: +44 (0) 20 7269 3282 – email: [email protected] - Website: http://www.crick.ac.uk/dominique-bonnet

Key terms: AML, LICs, L-LTC-ICs, co-culture, proliferation, CFSE, xenograft, prognosis.

Financial Supports: E. Griessinger was supported by Cancer Research UK internal fellowship and

by a grant from the Fondation de France. T. Prebet, N. Vey and C. Chabannon were supported by

grant INCa-DGOS-Inserm 6038 to the SIRIC PACA-Ouest. D Bonnet was funded by Cancer

Research UK, and by European grant (contract No:037632), J.F Peyron was supported by INSERM

and by a grant from the Cancéropôle PACA.

The authors have no conflict of interest to report.

Research. on February 15, 2020. © 2016 American Association for Cancercancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 9, 2016; DOI: 10.1158/0008-5472.CAN-15-2063

http://cancerres.aacrjournals.org/


Abstract: (188 out of 250)

Acute Myeloid Leukemia (AML) is sustained by a sub-population of rare Leukemia initiating cells

(LICs) detected in the xenograft assay by their capacity to self-renew and to generate non-LICs in

vivo. The xenotransplantation model captures functional properties of LICs that have clinical

prognostic value. However, the long duration of this in vivo assay has hampered its use as a

prognostic tool. Here, we show using an ex vivo co-culture system, that intermediate and poor risk

AML patient samples at diagnosis have a 5 to 7 times higher frequency of Leukemic-Long Term

Culture-Initiating cells (L-LTC-ICs) compared to the good risk group. We defined a Fluorescence

Dilution Factor (FDF) parameter that monitors sample proliferation over 1 week and established a

strong correlation of this parameter with the L-LTC-IC frequency. A higher FDF was found for poor

prognostic AMLs or for samples capable of engrafting NSG mice compared to good risk AMLs or

non-engrafters. Importantly, FDF could classify normal karyotype-intermediate risk patients into 2

groups with a significant difference in their overall survival, thus making this non-genetic and non-

in vivo approach a new clinically relevant tool for a better diagnosis of AML patients.

Word count: 1981 /2500 words.

INTRODUCTION

Leukemia initiating cells (LICs) are functionally defined as SCID Leukemia-initiating cells

(SL-ICs) (1) in the xenograft assay by their capacity to initiate, propagate and maintain bulk

leukemia in vivo (2). SL-ICs functional studies showed a correlation between the xenograft capacity

of a sample as well as “stem cell gene signature” with poorer overall survival of the respective

patient (3, 4).

Additionally, an AML mathematical modelling of LICs proliferation was also separately shown to

correlate with the clinical outcome of the patients (5). Thus, LICs quantification and their monitoring

could have strong clinical applications especially for intermediate risk-normal karyotype AMLs that

account for approximately 60% of all AML patients. However, the recently described heterogeneous





SL-IC phenotypes (4, 6-8) combined with the long-duration of the in vivo assay have prevented the

use of the xenograft assay as a prognostic tool. We have recently optimized a niche-like co-culture

system capable of maintaining SL-IC ex vivo and demonstrated that the frequency of leukemic

long-term culture initiating cells (L-LTC-ICs) is a reliable functional read-out for monitoring the

activity of LICs (9). Here we combine this assay with a cell proliferation analysis to demonstrate that

the expansion rate of L-LTC-ICs in this culture system strongly correlates with patient clinical

outcome.

MATERIALS AND METHODS

Cells

AML cells were obtained at St Bartholomew’s Hospital (UK) and from the Institutional Tumor Bank

at Institut Paoli-Calmettes (Comprehensive Cancer Centre in Marseille, France). For both sources,

ethical approvals have been granted (via the East London Ethical community or under authorization

#AC-2013-1905 granted by the French Ministry of Research respectively). Details of patient

samples are listed in Table S1. Co-culture experiments were performed as previously described (9)

on confluent MS-5 monolayers. The stromal cell line MS-5 was purchased from German Collection

of Microorganisms and Cell Cultures (DSMZ, http://www.dsmz.de; Braunschweig, Germany) in

2012 and were maintained in IMDM 10% FCS + 2 mM L-glutamine and used between passage 3 to

5.

Fluorescence Dilution Factor (FDF)

AML cells were stained with 0.8µM carboxyfluorescein diacetate, succinimidyl ester (CFSE)

(Invitrogen, UK). Cells were washed and incubated on pre-established confluent MS-5. CFSE

median fluorescence (MFI) was measured by FACS at 18h and day 7 on viable (Annexin V and

DAPI negative) human hematopoietic cells (CD45 positive and Sca-1 negative). FDF was defined





as the ratio of the 18h CFSE MFI divided by the 1-week CFSE MFI (See supplemental information

for more details).

Statistics

Data were analyzed for statistical significance using the Mann-Whitney unpaired two tail test or the

one-way Anova test. Linear or non-linear regression trend lines were performed with GraphPad

Prism software. A non-parametric spearman test was applied for correlation. Spearman's rank

correlation coefficient ( ) is shown. Observed differences were regarded as statistically significant if

the calculated two-sided P value was below 0.05.

Supplementary information are included in supplemental Methods, found on the CR website.

RESULTS AND DISCUSSION

We analyzed 92 de novo AML patients classified as favorable (n=22), intermediate (n=54),

and poor (n=16) prognostic groups according to the British MRC and French BGMT classifications

(Supplemental Table S1). We performed ex vivo limiting dilution analyses to determine the initial

frequencies of leukemic long-term culture-initiating cells (L-LTC-IC 1° frequency)(see schematic

Figure 1A). We observed a high variability ranging from 1 L-LTC-IC in 10 to 105 bulk AML plated

cells and noticed that the intermediate and poor risk AMLs had a 5 to 7 times higher frequency as

compared to the favorable group (Figure 1B, left panel). Cell counts at 5-weeks were found to be

different depending on AML risk groups (Figure 1B, right panel). Importantly, for all samples we

also correlated the L-LTC-IC 1° frequency (Figure 1C) and the 5-week fold expansion (Figure 1D)

with the patients’ overall survival (OS). By plotting the L-LTC-IC 1° frequencies against the 5-week

fold expansion, we further confirmed that the very modest ex vivo proliferation of AMLs depends on

the L-LTC-IC compartment size of the sample (Figure 2A). This correlation was not seen after a 1-

week culture period (Figure 2B). This suggests either that L-LTC-ICs did not sustain the leukemic





expansion during the first week or more likely that cell death exceeded the L-LTC-IC-driven

leukemic expansion. To address this question we quantified secondary L-LTC-ICs (LTC-IC 2°) after

replating cells that have been cultured for one week (see schematic Figure 1A). We observed that

the median proportion of L-LTC-ICs increased on average 7.8 times (Figure 2C, left panel; n=42,

P<0.001). In parallel, we quantified the loss of cellularity during the first week to be 60% suggesting

that the 7.8 times increase in L-LTC-ICs proportion seen was due to the ongoing proliferation of

some LICs. This was confirmed by calculating the L-LTC-IC absolute count, which increases by a

factor 4.25 (n=16, P<0.05) (Figure 2C right panel). Consistently, the intermediate and poor risk

samples maintained their higher proportion of L-LTC-ICs as compared to the favorable risk group

after replating (Figure 2D). Thus L-LTC-IC self-renewal capacity is an intrinsic biological feature of

samples that can be monitored over a one-week culture period.

We next wondered whether a shorter and simpler test, using CFSE staining to track cell

division, could be implemented to stratify sample risk groups. However, a high-resolution of division

peaks could not be achieved for the majority of samples due to intra-sample morphological

heterogeneity, as the incorporation of CFSE depends on cell size (For more details see

Supplementary Material and Method and Supplemental Figure 1A to C). Moreover, we could

not use cell sorting to reduce biological heterogeneity without the possibility of biasing the cell

population studied (10). Since AML samples usually have a poor viability at thawing and at later

points (See details of influence of cell viability in Supplementary Fig. 2A- 4C) we measured the

median dye dilution of the non-apoptotic leukemic cell population (non-DAPI, Non Annexin positive

fraction, See Supplemental Fig 2B and C). We then defined the Fluorescence Dilution Factor

(FDF) parameter as the ratio of the median fluorescence intensity (MFI) at the start of the analysis

by the MFI measured after a 1-week of co-culture (Figure 3A and Supplemental Figure 3).

Heterogeneous FDF values ranging from 1 to 10.2 with a mean of 3.0 were determined for 80 AML

samples. We observed a strong correlation between FDF and the L-LTC-IC frequency (Figure 3B)

or the proliferation index (Supplemental Figure 4A), strongly supporting the notion that the FDF





reports leukemic stem/progenitors dynamics ex vivo. The FDF did not correlate with bulk leukemic

expansion or bulk sample viability (Supplemental Figure 4B and C). Thus, the FDF parameter is

informative of sub-population dynamics even when cell death over exceeds proliferation. Of note,

the FDF parameter cannot distinguish between a high-proportion of slowly dividing cells versus a

small fraction of highly dividing ones.

Most good risk AML samples are devoid of xenograft potential (4). Among our cohort, 32

samples were tested for their ability to propagate AML in a xenograft assay in NSG mice. We

compared the FDF values of NSG mice Engrafter (E) and Non-Engrafter (NE) AML samples. A

higher FDF was found for 18 E compared to 14 NE samples (Figure 3C), which further suggests

that the FDF index reports on the actual LICs dynamics ex vivo. On the other hand, no significant

differences were seen between the E/NE groups simply using fold cell expansion as parameter

(Figure 3D). 14 of the 19 good risk samples had a FDF below the mean value of 3.0 of this cohort

while 11 of the 15 poor risk AMLs tested were above the mean (Figure 4A). Intermediate risk-

normal karyotype AML samples were stratified across the whole range. Poor prognostic AMLs had

a significantly higher FDF values compared to good risk and intermediate risk group AMLs whereas

no statistical differences were seen between good risk and intermediate risk groups (Figure 4B).

Again, using cell expansion as parameter we were unable to detect any differences between the 3

groups (Figure 4C). Importantly, patients OS was significantly reduced in AML with high FDF (>3.0)

when compared with AML cases with low FDF (<3.0, Figure 4D, left panel). Since the

intermediate-risk group contains patients with variable outcomes we evaluated the usefulness of

the FDF index to correlate with their outcome. Using the same cut-off value we could divide the

patient cohort and show for 17 AML with high FDF a statistically significantly lower survival

compared to 27 AML with low FDF (Figure 4D, second panel from the left). Recently, the

combined mutational status of FLT3 and NPM1 has been found to stratify intermediate risk/normal

karyotype group in low molecular risk (NPM1mut FLT3wt) intermediate 1 and high molecular risk

(FLT3-ITD or NPM1wt FLT3wt) intermediate 2 groups (11). High FDF negatively correlated with a





lower overall survival in these two sub-groups (Figure 4D, first and second panel from the right).

FDF measurements could also refine good and poor risk groups, although the survival curve

between low and high FDF was not significant based on the small outlier number of patients in

these two groups (Supplemental Figure 5).

The advance of next generation sequencing for screening mutations in AML patients has

improved patients’ stratification, nevertheless this screening is not yet a bedside standardized

procedure. On the other hand, the quantification of LICs as well as the presence of a “stem cell

signature” have been shown to provide information on the clinical outcome of patients but are hard

to use in a routine setting. Here, our data demonstrate that the ex vivo frequency of L-LTC-IC and

its expansion dynamics reflect the intrinsic biology of the LICs. We further show that

monitoring AML-culture initiating cells expansion after 1 week could help predict the prognosis of

AML patients without the need of in vivo experiments. Here, our data demonstrate that the ex vivo

frequency of L-LTC-IC and its expansion dynamics reflects the intrinsic biology of LICs. We further

show that monitoring AML-culture initiating cell expansion after 1 week could help predict the

prognosis of AML patients without the need of in vivo experiments.

AKNOWLEDGEMENTS

Authors are indebted to patients who granted permission to use their samples for research. We

thank Finlay McDougall for providing diagnostic information, and all personnel at the Institut Paoli-

Calmettes Tumour Bank for the access of anonymized samples and clinical data. We are grateful to

Stuart Horswell for statistical analysis and Dr Katie Foster for proofreading the manuscript. This

work is dedicated in memory of Dr François Lassailly.

REFERENCES





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9. Griessinger E, Anjos-Afonso F, Pizzitola I, Rouault-Pierre K, Vargaftig J, Taussig D, et al. A

Niche-Like Culture System Allowing the Maintenance of Primary Human Acute Myeloid Leukemia-

Initiating Cells: A New Tool to Decipher Their Chemoresistance and Self-Renewal Mechanisms.

Stem cells translational medicine. 2014; 3: 520-9.

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and management of acute myeloid leukemia in adults: recommendations from an international

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FIGURE LEGENDS

Figure 1: AML long-term culture potential correlates with patient prognostic risk and clinical

outcome. A: Flow chart illustrating the experimental design for B–D. B (left panel): Frequency of

L-LTC-IC in primary plating (L-LTC-IC 1°) for good, intermediate/normal karyotype and poor risk

AML patients. B (right panel): Fold expansion of AML population after 5 weeks of co-culture with

MS-5 for good, intermediate/normal karyotype and poor risk AML patients. C, D: L-LTC-IC 1°

frequency (C) or 5-weeks AML fold expansion (D) as compared to patient’s Overall Survival (n=45).

Black line shows experimentally derived non-linear regression trend line. Mann-Whitney unpaired

two-tail test or the one-way Anova test was applied. * p< 0.05, **p<0.01, NS p>0.05.

Figure 2: L-LTC-IC self-renew during the first week of culture with amplitude related to

patient prognosis.





For schematic see Flow chart Fig 1A. A- B: 5-weeks or 1-week AML fold expansion plotting

against L-LTC-IC 1° frequency (n= 43 and 22 respectively). A.Thin black line shows experimental

derived non-linear regression trend line with 95% confidence band (dashed lines). For A, B a non-

parametric Spearman correlation test was applied. C: Frequency (left panel) (n=73) and absolute

count (right panel) (n=16) of L-LTC-IC in primary and secondary plating. D: Frequency of L-LTC-IC

in secondary plating (L-LTC-IC 2°) for good, intermediate/normal karyotype and poor risk AML

samples. Mann-Whitney unpaired or paired two-tail test (C) or the one-way Anova test (D) was

applied. * p< 0.05, **p<0.01, NS p>0.05.

Figure 3: One week Fluorescence Dilution Factor (FDF) correlates with L-LTC-IC and

predicts Engrafter versus Non-Engrafter in NSG mice.

A: Flow chart illustrates the experimental procedure to define the FDF values (top Panel) (See

supplemental Fig. 2 for FACS gating and cytometer calibration strategies) and (Bottom panel)

Representation of 18h and 1-week overlay CFSE Fluorescence profiles of one patient with high

FDF (left panel) and one patient with low FDF value (right panel). B: FDF plot against the L-LTC-IC

frequency (n=36). Thin black line shows experimental derived non-linear regression trend line. Non-

parametric Spearman correlation test was applied. C: FDF value between NSG mice engrafter (E)

and non-engrafter (NE) AML samples (E, n=18; NE, n=14). D: Fold expansion at 1-week for E and

NE samples.

Figure 4: Fluorescence Dilution Factor (FDF) predicts clinical outcome.

A: FDF for 80 AML samples. Poor risk (red bars) n=15; intermediate/normal karyotype (orange

bars) n=46; good risk (green bars) n=19. Black line shows the mean FDF value (=3.04). Dashed

line represents FDF=1, no dilution from the input fluorescence. B: FDF value comparison for

different risk group AML. C: 1-week fold expansion at 1-week for different risk group. D: Patients





with low FDF have better overall survival than high FDF patients. Kaplan-Meier 5 years survival

curves based on the mean FDF cut-off value determined in (E). Patients who underwent an

allograft were excluded from analysis. (Left Panel): All risk group; (Second Panel from the left):

Intermediate risk normal karyotype group. (Second Panel from the Right): Intermediate 1 group,

NPM1mut/FLT3wt cytogenetically normal AML; (First Panel from the Right) Intermediate 2,

NPM1wt or FLT3ITD cytogenetically normal AML. Mantel-Cox log rank test was applied for panel

D. One-way Anova test was applied for (B,C). *P<0.05, NS P>0.05.




AML

cells

L-LTC-IC 2° Freq

sorting

1st

plating 2nd

plating

2° LDA

1w

Fold expansion

L-LTC-IC 1° Freq

B A

Figure 1

5w

1° Limiting

Dilution

Assay

(1° LDA)

5w

Fold expansion

. . .

.

𝞀=-0.3664

P< 0.05 D 𝞀=-0.3776

P< 0.05 C




Figure 2

B A 𝞀=0,7073

P< 0.0001 𝞀=0,26

P=0.24

C D




AML

cells

CFSE

18h MFI

1w MFI CFSE MFI 1w

CFSE MFI 18h

Fluorescence

Dilution

Factor

(FDF) =

Gating:

mSCA-1-

/CD45+/Anne

xin-V-/DAPI-

Figure 3

𝞀=0.4597

P=0.0055

C B

A

D

1wk

FDF=7.2

#1056025

FDF=1.9

#1054491

18h

1w

18h

1w




Time (days)

Su

rviv

al (%

)

FDF>3.04 n=17

FDF<3.04 n=27

P<0.0001 P=0.0017

FDF>3.04 n=33

FDF<3.04 n=45

All Risk group Interm/NK Intermediate 1 D

Time (days)

Su

rviv

al (%

)

Time (days)

Su

rviv

al (%

)

Time (days)

Su

rviv

al (%

)

Intermediate 2

P=0.03

FDF>3.04 n=11

FDF<3.04 n=17

P=0.026

FDF>3.04 n=6

FDF<3.04 n=10

Mean FDF=3.04

(n=80)

B A C

Figure 4




Published OnlineFirst March 9, 2016.Cancer Res Emmanuel Griessinger, Fernando Anjos-Afonso, Jacques Vargaftig, et al. leukemia patientsshort-term ex vivo culture informs outcomes in acute myeloid Frequency and dynamics of leukemia-initiating cells during

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