Supplementary Information Material
Supplementary Methods
Patients
Diagnostic and follow-up ALL bone marrow (BM) samples were obtained after informed
consent and approval of relevant research ethics committees from patients at the Paediatrics
Haematology Unit, Lund University Hospital, Sweden; Second Faculty of Medicine,
Department. of Paediatric Haematology/Oncology, Charles University, Prague, Czech
Republic; Centro Ricerca Tettamanti, Clinica Pediatrica Univ. Milano Bicocca, Ospedale San
Gerardo, Monza, Italy and the Paediatrics Haematology Unit, John Radcliffe Hospital,
Oxford, UK. Samples from 10 children with t(12;21), TEL/AML-1- positive ALLs and one
child with t(9;22) p190 BCR/ABL1– positive ALL were analyzed. For further patient
characteristics see Table 1.
Cell purification
Mononuclear cells were isolated by ficoll gradient centrifugation. In some cases CD34+ cells
were enriched by magnetic bead separation (StemCell Technologies or Miltenyi). CD34enriched cells or MNCs from BM were stained with anti-CD19-PE (BD-Pharmingen), CD34FITC (BD-Pharmingen) and CD38-APC (BD-Pharmingen). Cells were analyzed in staining
buffer containing DAPI at 0.1µg/ml, for live cell analysis. I.) HSC (34+38-/low19-); II.) Stem/B
(34+38-/low19+) and ProB III.) (34+38+19+) cells were purified by flow cytometry (FACS Aria,
BD-Pharmingen) as described previously(1). Data acquisition and analysis were done with
CellQuest (Beckon Dickinson) or FlowJo (Tree star) software.
‘Fluorescence minus one’ controls were used to determine positive and negative staining
boundaries for CD34, CD38 and CD19. For cell frequency calculations the 90% CD34+ cells
1
expressing the highest levels of CD38 were gated for CD34+CD38+CD19+ proB cells and the
10% CD34+ cells with no/lowest expression of CD38 were gated for calculations of
CD34+CD38-/lowCD19- HSCs and CD34+CD38-/lowCD19+ Stem/B cells. If more than 10% of
34+ cells were negative for 38, frequencies were calculated for 34+ cells with CD38 gates set
according to “fluorescence minus one” (FMO) controls. In order to obtain pure populations
sorting gates were set within the boundaries defined by FMO for CD34 and CD19. HSCs and
Stem/B cells were defined by 2 different approaches, either by CD34+CD19-/+ and no/lowest
expression of CD38 (patient1 d93, d121, d149; patient2 d0, 5months, 10months and patient5
d15, d33) or by CD34+CD19+/- and CD38 negativity defined by FMO in the remaining cases.
Cell-cycle analysis
Cells were stained with CD19-PECy5 (BioLegend), then fixed and permeabilized with 1.6%
paraformaldehyde and 90% ice-cold methanol. This was followed by staining with CD34APC (BD), CD38-PETxR (Invitrogen) and Ki67-FITC (Becton-Dickinson). For DNA
content analysis cells were incubated with 0.5µg/mL DAPI. Stained cells were analyzed by
excitation of DAPI with a violet laser on a FACS LSRII SORP (Becton-Dickinson) at the
University of Oxford (UK).
FISH analysis
Cells were fixed on slides in methanol/acetic acid fixative (3:1 vol/vol) and then hybridized
with BCR/ABL1 and TEL/AML1 FISH probes using the LSI BCR/ABL1 Dual Color Dual
Fusion Translocation and the LSI TEL/AML1 ES Dual Color Translocation probes
respectively (Abbot). Leukaemic sub-populations were FACS sorted and subsequently
analysed by FISH for the presence of the appropriate clonal marker.
2
The slides were analyzed with an Olympus BX51 microscope equipped with epi-fluorescence
and a triple band pass filter. Images were captured using a Sensys charge-coupled device
camera (Photometrics, Tucson, AZ) and MacProbe software (Applied Imaging, Newcastle
upon Tyne, U.K.).
The translocation of BCR/ABL1 was detected by one red, one green and two red-green
fusion signals and the translocation of TEL/AML1+ by one green (TEL), one large red
(AML1), one small red (residual AML1) and one red-green fusion signal (1, 2). When
possible, and unless otherwise specified at least 100-150 nuclei were analyzed in each
sample. Based on FISH analysis of sorted subpopulations of BM samples from 2 healthy
individuals, cut-off values for negativity were determined. For TEL/AML1 100-200
nuclei/slide were analysed on 6 slides (3x34+, 1x34+38-19-, 1x34+38+19- and 1x34+38+19+
cells) and for BCR/ABL1 100-217 nuclei/slide were analysed on 4 slides (3x34+ and
1x34+38-19- cells). Cut-off values were <2.69% for TEL/AML1 and <2.51% for BCR/ABL1
(Mean +2 SD).
RNA Extraction and Reverse Transcriptase
Total RNA was isolated from sorted cell populations using RNAeasy kits according to
manufacturer protocol (Qiagen, Hilden, Germany). Complementary DNA was prepared from
2-12µl RNA in a total volume of 15-20µl using SuperScript VILO kits according to
manufacturer protocol (Invitrogen, Carlsbad, CA, USA).
Real-time quantitative polymerase chain reaction (RQ-PCR) of sorted leukaemia
subpopulations
Absolute quantification was performed by real-time PCR using appropriate fusion transcript
quantification TaqMan assays (Ipsogen, Marseille, France), TEL-AML1 and BCR-ABL mbcr
3
FusionQuant® kits. PCR reactions were performed in 25 µL volume, containing 12.5 µL of
TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 µL of
the appropriate probe and primer mix (Ipsogen, Marseille, France) and 5 µL of cDNA.
Amplifications were performed in Qiagen Rotor-Gene 6000 under the following conditions:
50°C for 2 min, 95°C for 10 min followed by 50 cycles of amplification consisting of a
denaturation step at 95°C for 15 seconds and annealing and amplification at 60°C for 1 min.
Fluorescence acquisition was monitored to measure the accumulation of the reporter dye,
correlated to amplification of the target transcript, at every cycle, during the amplification
step. Simultaneous amplification of a control gene, c-abl, allowed normalization of variations
in the efficiencies of reverse transcription, while quantification was achieved by extrapolation
from standard curves, calculated from amplification of provided plasmid standards (5 for the
fusion gene of interest and 3 for the internal control, c-abl).(3) Samples were analysed in
duplicates for ABL and the respective fusion gene or in single reactions for ABL and the
respective fusion gene if cell number was limiting. Samples were counted as positive if >=5
copies of TEL/AML1 or BCR/ABL1 were detected. In order to assess negativity >50
cells/reaction had to be analysed or >50 ABL copies/reaction had to be detected.
Dilutions of BCR/ABL1+ (TOM-1) and TEL/AML1+ (REH) cells in negative cells (UOC-B6
for BCR/ABL1+ and Jurkat for TEL/AML1) were analysed to define the sensitivity of our
approach. We were able to detect BCR/ABL1 and TEL/AML1 cDNA copies in dilutions
down to 1/10000 cells. For BCR/ABL1 the lowest cell number tested was ~2.5 BCR/ABL1+
cells in 25000 cells. For TEL/AML1 even the detection of 6 copies in the cDNA equivalent
of 1250 cells of a 1/10000 dilution (TOM-1 in UOC-B6 cells) was possible.
4
Supplementary Figure
Supplementary Figure 1
A Pt 3
d0
d15
d33
D Pt 7
1
Stem/B
ProB
0.001
#
0.00001
d0
d15
Stem/B
Leukaemic cell
frequency
Leukaemic cell
frequency
*
d0
1
ProB
0.0001
0.00001
Leukaemic cell
frequency
0.0001
E Pt 8
0.001
d0
d15
days
Stem/B
0.01
ProB
0.001
#
0.00001
5
d15
d29
days
0.1
0.01
Stem/B
ProB
0.001
0.0001
0.00001
d29
0.1
0.0001
#
d36
0.01
1
ProB
0.001
days
0.1
C Pt 6
Stem/B
0.01
0.00001
1
d147
0.1
Leukaemia cell
frequency
Leukaemic cell
frequency
0.1
B Pt 5
d15
days
0.01
0.0001
d0
1
days
Supplementary Figure 1: Kinetics of leukaemic stem and progenitor cell elimination in
TEL/AML1+ cALL cases during chemotherapy. A. Frequencies of leukaemic ProB and
Stem/B cells, relative to total BM MNC (set as 1) at diagnosis and different time points after
initiation of chemotherapy in TEL/AML1 case which later relapsed (patient 3). B-E.
Frequencies of leukaemic proB and Stem/B cells, relative to total BM MNC (set as 1) at
diagnosis and different time points after initiation of chemotherapy of TEL/AML1 cALLs
which remain in long-term remission; one case in which leukaemic ProB and Stem/B cells
were eliminated with similar kinetics (B; patient 5), and 3 cases (C-E; patients 6-8) in which
leukaemic Stem/B cells were eliminated with slower kinetics than leukaemic proB cells.
Frequencies of leukaemic cells were determined by FISH and/or RQ-PCR, and in the case of
Stem/B cells also based on aberrant phenotype as described in the Supplemental Methods and
Supplemental Table 1. Cell populations with negative FISH/PCR results or with too few cells
to be sorted were scored as non-leukaemic. #Leukaemic cells scored based on aberrant
Stem/B phenotype. *ProB cells with unclear leukaemic status as PCR failed.
6
Supplementary Table 1
A
Patient 1
HSC
ProB
Stem/B
Diagnosis
Frequency
FISH
QPCR
Cells/reaction
0.15%
0/100
neg
(151)
31.31%
148/150
pos
(4491)
58.61%
140/146
pos
(6250)
Day93
Frequency
FISH
QPCR
Cells/reaction
1.72%
0/20
neg
(266)
2.71%
0/35
N/D
0.42%
N/D
pos
(69)
1.45%
1/63
N/D
3.67%
0/200
neg
(625)
0.06%
25/25
N/D
Day149
Frequency
QPCR
Cells/reaction
0.73%
neg
(84)
2.08%
neg
(2220)
0.03%
pos
(23)
Relapse
Frequency
FISH
QPCR
Cells/reaction
0.01%
N/D
neg
(82)
84.01%
98/103
pos
(8333)
6.15%
31/35
pos
(3000)
Diagnosis
Frequency
FISH
QPCR
Cells/reaction
0.03%
N/D
neg
(74)
6.43%
96/100
pos
(1725)
0.68%
92/98
pos
(1010)
5 Months
Frequency
QPCR
Cells/reaction
0.58%
neg
(753)
1.13%
neg
(1804)
0.002%
pos
(10)
Relapse
Frequency
FISH
QPCR
Cells/reaction
0.007%
N/D
pos
(31)
4.81%
98/100
pos
(1250)
0.52%
2/2
pos
(783)
Diagnosis
Frequency
FISH
QPCR
Cells/raction
0.34%
0/92
N/D
6.69%
N/D
pos
(907)
0.44%
2/2
N/D
Day15
Frequency
FISH
QPCR
Cells/reaction
0.048%
N/D
N/D
0.001%
N/D¥
N/D¥
0.008%#
N/D
N/D
0.21%
N/D
N/D
0.007%
N/D¥
N/D¥
0%
N/D¥
N/D¥
Day121
Frequency
FISH
QPCR
Cells/reaction
Patient 2
Patient 3
Day33
Frequency
FISH
QPCR
7
B
Patient 4
HSC
Stem/B
Patient 5
HSC
ProB
Stem/B
42.6%
95/104
13.9%
96/100
Diagnosis
Frequency
FISH
QPCR
0.04%
0/50
N/D
7.29%
101/106
N/D
0.73%
54/60
N/D
0.03%
0/5
N/D
0.24%
2/5
N/D
1.18%
56/80
N/D
Day15
Frequency
FISH
QPCR
0.013%
0/6
N/D
0.1%
91/105
N/D
0.02%
17/21
N/D
Day29c
Frequency
FISH
7.62%
0/10
0.01%
N/D¥
0.59%
14/15
Day36c
Frequency
FISH
QPCR
7.07%
0/2
N/D
0.007%
N/D¥
N/D¥
0.006%
N/D¥
N/D ¥
Day50c
Frequency
FISH
9.03%
0/10
0.96%
0/75
0%
N/D¥
Diagnosis
Frequency
FISH
0.07%
0/30
2.13%
28/28
0.21%
24/24
Diagnosis
Frequency
FISH
0.019%
0/1
Day15
Frequency
FISH
QPCR
ProB
Patient 6
Patient 7
Diagnosis
Frequency
FISH
QPCR
0.16%
1/30
N/D
49.57%
90/103
N/D
5.17%
56/60
N/D
Day 15
Frequency
FISH
QPCR
0.01%
0/30
N/D
0.02%
95/100
N/D
0.035%
42/67
N/D
Day15
Frequency
FISH
QPCR
0.21%
N/D
N/D
0.0017%*
N/D
N/D
0.011%#
N/D
N/D
Day 29
Frequency
FISH
QPCR
Cells/reaction
0.11%
0/14
neg
(64)
0.001%
0/1
N/D
0.008%#
N/D
N/D
Day147
Frequency
FISH
QPCR
0.33%
0/3
N/D
0.58%
0/50
N/D
0%
N/D¥
N/D¥
0.13%
2/56
20.34%
34/34
2.38%
30/30
0.04%
0/3
N/D
0.004%
N/D
pos
(10)
0.031%
1/4
N/D
0.1%
0/15
N/D
0.0003%
0/1
N/D
0.0007%
N/D¥
N/D¥
Patient 8
Diagnosis
Frequency
FISH
Day15
Frequency
FISH
QPCR
Cells/reaction
Day29
Frequency
FISH
8
Supplementary Table 1: FISH and RQ-PCR analysis of sorted cell populations from
cALL patients. A. Analysis of purified leukaemia subpopulations at diagnosis and different
follow-up time points after the start of chemotherapy in 3 cALL cases that eventually
relapsed. Frequencies of cell compartments (percentages of total BM MNC), frequencies of
FISH+ cells (out of total cells analysed) and results of BCR/ABL1 and TEL/AML1 RQ-PCR
analysis, indicating cell numbers used per reaction, are shown. B. FISH and RQ-PCR
analysis of TEL-AML1cases which remain in long-term remission. N/D; not determined. ¥No
cells could be sorted. #Aberrant phenotype. *ProB cells with unclear leukaemic status as PCR
failed. cCD34+ selected cells.
References
1.
2.
3.
A. Castor et al., Nat Med 11, 630 (Jun, 2005).
D. Hong et al., Science 319, 336 (Jan 18, 2008).
J. Gabert et al., Leukemia 17, 2318 (Dec, 2003).
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