01.12.2017 | Research article | Ausgabe 1/2017 Open Access

DNA alterations in Cd133+ and Cd133- tumour cells enriched from intra-operative human colon tumour biopsies
- Zeitschrift:
- BMC Cancer > Ausgabe 1/2017
Electronic supplementary material
Background
Methods
Patients
Patients (n)
|
|
---|---|
Male/Female
|
11/15
|
Age at surgery
|
71.1 (44–94)
|
Tumour stage (I-IV)
|
I 5, II 10, III 9, IV 2
|
Tumour differentiation
|
Medium 23/High 2/Mucinous 1
|
Positive lymph nodes (analysed 5–52)
|
1 (0–6)
|
Tuymour location
|
Right 14 / Left 11/Multiple 1
|
Radical surgery Y/U/N
|
23/2/1
|
Recurrent disease (June 2015)
|
3
|
MACS tumour sample separation
Immunocytochemistry for CD133 detection
DNA and RNA isolation
Gene expression of stem cells markers
DNA Whole Genome Amplification
DNA Purification
Array CGH – DNA alterations in CD133+ and CD133−/EpCAM+ cell populations
Q-PCR – DNA alterations in tumour tissue
Results
Immuno-cytochemical detection of CD133 expression in the tumour cell fractions
Gene expression of stem cell markers
DNA alterations in CD133+ and CD133−/EpCAM+ cell populations
Specific alterations
|
Total alterations
|
|||||
---|---|---|---|---|---|---|
(N
° arrays)
|
CNV
CD133 (20)
|
CNV
EpCAM (20)
|
T-test
p value
|
CNV
CD133 (20)
|
CNV
EpCAM (20)
|
T-test
p value
|
CNV (Mbp/patient)
|
28.5 ± 13.6
|
24.8 ± 5.1
|
0.801
|
71.7 ± 22.2
|
68.6 ± 22.0
|
0.922
|
% of all
|
53
|
47
|
51
|
49
|
||
Amp (Mbp/patient)
|
12.3 ± 11.2
|
2.5 ± 1.5
|
0.392
|
14.7 ± 11.5
|
4.63 ± 1.52
|
0.391
|
Amp % of all
|
83
|
17
|
76
|
24
|
||
Del (Mbp/patient)
|
16.2 ± 5.8
|
22.3 ± 5.0
|
0.433
|
57.1 ± 19.4
|
64.0 ± 22.2
|
0.815
|
Del % of all
|
42
|
58
|
47
|
53
|
Amplifications
|
Deletions
|
|||||
---|---|---|---|---|---|---|
CD133
(20)
|
EpCAM
(20)
|
Shared
(20)
|
CD133
(20)
|
EpCAM
(20)
|
Shared
(20)
|
|
Chr 1
|
4
|
2
|
3
|
11
|
14
|
17
|
Chr 2
|
2
|
1
|
1
|
10
|
6
|
11
|
Chr 3
|
2
|
2
|
17
|
9
|
9
|
8
|
Chr 4
|
1
|
3
|
1
|
4
|
8
|
5
|
Chr 5
|
0
|
0
|
0
|
9
|
6
|
7
|
Chr 6
|
2
|
0
|
0
|
10
|
9
|
7
|
Chr 7
|
2
|
0
|
0
|
10
|
15
|
14
|
Chr 8
|
1
|
2
|
8
|
6
|
8
|
11
|
Chr 9
|
3
|
1
|
0
|
10
|
8
|
5
|
Chr 10
|
0
|
1
|
0
|
8
|
10
|
13
|
Chr 11
|
2
|
1
|
5
|
11
|
9
|
10
|
Chr 12
|
2
|
1
|
3
|
14
|
11
|
14
|
Chr 13
|
4
|
2
|
1
|
7
|
5
|
4
|
Chr 14
|
0
|
0
|
20
|
5
|
10
|
16
|
Chr 15
|
1
|
1
|
0
|
8
|
9
|
15
|
Chr 16
|
0
|
2
|
1
|
10
|
11
|
11
|
Chr 17
|
5
|
10
|
8
|
7
|
11
|
7
|
Chr 18
|
0
|
0
|
0
|
4
|
5
|
12
|
Chr 19
|
7
|
5
|
2
|
6
|
12
|
11
|
Chr 20
|
2
|
2
|
1
|
7
|
7
|
3
|
Chr 21
|
1
|
2
|
0
|
4
|
5
|
10
|
Chr 22
|
1
|
1
|
0
|
6
|
7
|
6
|
DNA alterations in CD133+ versus CD133−/EpCAM+ cell populations
Amplifications
|
Deletions
|
|||||
---|---|---|---|---|---|---|
CD133
(Mbp/patient)
|
EpCAM
(Mbp/patient)
|
T-test
p value
|
CD133
(Mbp/patient)
|
EpCAM
(Mbp/patient)
|
T-test
p value
|
|
Chr 1
|
0.17 ± 0.1
|
0.06 ± 0.05
|
0.37
|
0.68 ± 0.3
|
1.70 ± 0.4
|
0.03
|
Chr 2
|
0.22 ± 0.2
|
0.01 ± 0.01
|
0.24
|
0.50 ± 0.3
|
0.24 ± 0.1
|
0.39
|
Chr 3
|
0.01 ± 0.008
|
0.009 ± 0.006
|
0.77
|
0.56 ± 0.4
|
0.34 ± 0.2
|
0.62
|
Chr 4
|
0.06 ± 0.06
|
0.08 ± 0.07
|
0.86
|
0.06 ± 0.03
|
0.04 ± 0.02
|
0.44
|
Chr 5
|
0
|
0
|
-
|
2.13 ± 1.8
|
0.42 ± 0.4
|
0.37
|
Chr 6
|
0.02 ± 0.02
|
0
|
0.17
|
0.21 ± 0.1
|
0.92 ± 0.6
|
0.23
|
Chr 7
|
0.01 ± 0.01
|
0
|
0.25
|
0.76 ± 0.4
|
2.23 ± 0.6
|
0.05
|
Chr 8
|
0.06 ± 0.06
|
0.09 ± 0.08
|
0.75
|
0.38 ± 0.2
|
0.16 ± 0.07
|
0.33
|
Chr 9
|
0.03 ± 0.02
|
0.01 ± 0.01
|
0.35
|
0.70 ± 0.5
|
1.00 ± 0.6
|
0.68
|
Chr 10
|
0
|
0.12 ± 0.12
|
0.32
|
0.37 ± 0.2
|
1.99 ± 1.9
|
0.40
|
Chr 11
|
0.02 ± 0.02
|
0.002 ± 0.002
|
0.22
|
1.17 ± 0.6
|
1.09 ± 0.5
|
0.91
|
Chr 12
|
6.47 ± 6.4
|
0.002 ± 0.002
|
0.32
|
1.06 ± 0.4
|
2.15 ± 9
|
0.27
|
Chr 13
|
4.81 ± 4.77
|
0.01 ± 0.008
|
0.32
|
0.08 ± 0.04
|
0.06 ± 0.04
|
0.70
|
Chr 14
|
0
|
0
|
-
|
0.22 ± 0.2
|
0.37 ± 0.2
|
0.53
|
Chr 15
|
0.01 ± 0.01
|
0.02 ± 0.02
|
0.80
|
0.45 ± 0.2
|
0.16 ± 0.1
|
0.21
|
Chr 16
|
0
|
0.04 ± 0.03
|
0.28
|
1.45 ± 0.6
|
0.49 ± 0.2
|
0.13
|
Chr 17
|
0.16 ± 0.09
|
0.23 ± 0.08
|
0.60
|
3.24 ± 2.2
|
0.78 ± 0.4
|
0.28
|
Chr 18
|
0
|
0
|
-
|
0.06 ± 0.04
|
0.13 ± 0.06
|
0.31
|
Chr 19
|
0.15 ± 0.06
|
0.08 ± 0.05
|
0.35
|
1.54 ± 1.4
|
7.49 ± 2.7
|
0.05
|
Chr 20
|
0.04 ± 0.03
|
1.54 ± 1.54
|
0.33
|
0.12 ± 0.06
|
0.23 ± 0.1
|
0.48
|
Chr 21
|
0.01 ± 0.01
|
0.19 ± 0.13
|
0.18
|
0.03 ± 0.03
|
0.002 ± 0.001
|
0.28
|
Chr 22
|
0.01 ± 0.01
|
0.006 ± 0.006
|
0.70
|
0.42 ± 0.3
|
0.26 ± 0.1
|
0.68
|
Total
|
12.3 ± 11.2
|
2.5 ± 1.5
|
0.39
|
16.2 ± 5.8
|
22.3 ± 5.0
|
0.43
|
Chr location
|
CNV
|
N
o patients
CD133
(20)
|
N
o patients
EpCAM
(20)
|
N
o genes
|
Examples of genes in the region
|
---|---|---|---|---|---|
CD133
|
|||||
1q42.12
|
Del
|
7
|
7
|
7
|
LEFTY1/2, PYCR2, ACBD3
|
8p11.22
|
Del
|
5
|
5
|
2
|
ADAM5P, ADAM3A
|
12q13.13 - q14.1
|
Del
|
15
|
8
|
195
|
HOTAIR, GDF11, ERBB3, CDK2, CD63, PTGES3, MIR1228
|
EpCAM
|
|||||
8q24.3
|
Del
|
7
|
8
|
27
|
NAPRT, MAPK15, SCRIB, PUF60, MIR661, SHARPIN
|
11q12.2 - q13.3
|
Del
|
9
|
9
|
247
|
SYT7, MIR1908, MIR612, MIR192, MIR194–2, VEGFB, MALAT1, BAD, FOSL1, RELA
|
11q12.3
|
Amp
|
7
|
5
|
4
|
SLC22A9/10/24/25
|
11q13.4
|
Del
|
6
|
8
|
5
|
PDE2A, MIR139, ARAP1, STARD10, ATG16L2
|
11q23.3
|
Del
|
9
|
8
|
12
|
BCL9L, UPK2, FOXR1, TRAPPC4
|
17p13.2
|
Del
|
5
|
8
|
12
|
PFN1, INCA1, KIF1C
|
17q21.2
|
Amp
|
6
|
11
|
39
|
Keratins and keratin-associated proteins
|