Background
Results
Patient material
Patient characteristics | SCC | AKd
| NSd
| Peripheral blood | ||||
---|---|---|---|---|---|---|---|---|
Patient | Gendera
| Ageb
| Transplanted organ | Drugsc
| location | location | location | Yes/No |
P-3 | M | 56 | Kidney | MMF, P | Upper leg | Forearm | Lower back | Yes |
P-5 | M | 72 | Kidney | Aza, P | Lower arm | Forearm | Lower back | Yes |
P-11 | F | 56 | Kidney | Aza, P | Tibia | Forearm | Lower back | Yes |
P-15 | M | 62 | Kidney | P | Lower arm | n.a. | n.a. | No |
P-18 | M | 51 | Kidney-pancreas | Aza, P | Dorsal hand | Forearm | Lower back | Yes |
P-24 | M | 74 | Kidney | Aza, P | Lower leg | Forearm | Lower back | Yes |
P-27 | F | 56 | Liver | Tacroli-mus, P | Shoulder | Forearm | Lower back | Yes |
P-33 | F | 66 | Kidney | Aza, P | Shoulder | Forearm | Lower back | Yes |
P-38 | F | 47 | Kidney | Aza, P | Shoulder | Forearm | Lower back | Yes |
P-39 | F | 65 | Kidney | Aza, P | Lower leg | Upper arm | Lower back | Yes |
P-40 | M | 60 | Kidney | MMF, P | Dorsal hand | Forearm | Lower back | Yes |
P-41 | M | 61 | Kidney | Aza, P | Dorsal hand | Forearm | Lower back | Yes |
P-42 | F | 48 | Kidney | Aza, P | Dorsal hand | Dorsal hand | n.a. | No |
P-44 | M | 62 | Kidney | Aza, P | Dorsal hand | Forearm | Lower back | Yes |
P-57 | F | 53 | Kidney | Aza, P | Tibia | Forearm | Lower back | Yes |
Genome-wide SNP analysis of SCCs and AKs
Type of aberration | Cytoband | Mb location | Size (Mb) | ||
---|---|---|---|---|---|
SCC_P-03 | Loss | 4q28.3 | 1:136,448,440 - 136,988,409 | 0.54 | * |
Gain | 5p | * | |||
Gain | 14q23.1-32.2 | 14:57,558,740 - 96,468,442 | 38.9 | * | |
SCC_P-11 | Gain | 9q | * | ||
SCC_P-18 | Gain | 8q24.22-24.23 | 8:132,122,430 - 139,467,653 | 7.3 | * |
Gain | 14q13.3 | 14:71,054,120 - 73,596,540 | 2.5 | ||
Gain | 9p23-tel | 9:1–12,039,121 | 12 | * | |
Loss | 9p23-cen | ~40 | * | ||
Loss | 9q | * | |||
Loss | 14q13.3-tel | ~70 | * | ||
cnLOH | 17 | * | |||
SCC_P-27 | Loss | 3p | |||
Gain | 3q | ||||
Loss | 4p12-tel | 4:1–46,377,946 | 46,4 | * | |
Gain | 5q25.3-tel | 5:83,159,010 - 100,338,915 | 17.1 | * | |
Loss | 6q13-14.1 | 6:75,600,000 - 76,627,715 | 1.0 | * | |
Loss | 9p | ||||
Gain | 9q | ||||
cnLOH | 18p | ||||
SCC_P-38 | cnLOH | 6p21.31-tel | 6:1–35,593,684 | 35.5 | |
Loss | 9p | ||||
Loss | 14q | ||||
SCC_P-41 | cnLOH | 22q12.1-tel | 22:24,348,300 - 49,554,696 | 25.2 | |
AK_P-03 | Loss | 4q | * | ||
Loss | 5q15-31.1 | 5:96,745,240 - 133,072,806 | 36.3 | * | |
cnLOH | 5q31.1-tel | 5:133,268,490 - 180,857,816 | 47.2 | * | |
cnLOH | 9q | * | |||
AK_P-05 | Loss | 5q | * | ||
Loss | 9p21.1-tel | 9:1–33,109,143 | * | ||
AK_P-11 | Loss | 2p22.2-25.1 | 2:11,690,000 - 36,950,000 | 25.3 | * |
cnLOH | 8p | * | |||
Loss | 13q | * | |||
Loss | 17p | * | |||
Loss | 18q12.1-tel | 18:26,013,440 - 76,117,152 | 50.1 | * | |
AK_P-33 | Loss | 6q22.31-tel | 6:122,757,319 - 170,975,699 | 48.2 | * |
AK_P-41 | Loss | 1p32.3 | 1:54,783,720 - 55,100,840 | 0.4 | * |
Loss | 3p | ||||
Gain | 3q | * | |||
Loss | 4q33-tel | 4:171,450,620 - 191,411,215 | 20.0 | ||
Gain | 5q31.2-tel | 5:138,387,890 - 180,857,851 | 42.5 | * | |
Loss | 8p12-tel | 8:1–38,377,990 | 38.4 | ||
Loss | 8q22.1 | 8:95,250,740 - 96,376,326 | 1.3 | ||
Loss | 9p22.2-cen | 9:17,230,530 - 50,549,240 | 33.3 | ||
Gain | 10p | 10:1–40,144,085 | 40.1 | * | |
Loss | 12q24.23 | 0.5 | |||
Loss | 14q | ||||
AK_P-44 | cnLOH | 8q33.3-tel | 8:126,444,040 - 137,979,818 | 11.4 |
Cluster and single-gene analyses of expression profiles of SCCs and AKs
SCC vs. NS | AK vs. NS | SCC vs. AK | ‘Progression’ | |
---|---|---|---|---|
# of samples | 15 vs. 13 | 14 vs. 13 | 15 vs. 14 | |
# of DEPs with FDR < 0.01 | 6820* (3333/3487) | 3734 (1554/2180) | 2131 (1088/1043) | 658 (262/396) |
# of DEPs with FDR < 0.01 and log2FC > = 0.5 | 2087 (1087/1009) | 977 (526/451) | 571 (289/282) | 180 (90/90) |
# of DEP with FDR < 0.01 and log2FC > = 1.0 | 639 (370/269) | 219 (167/52) | 145 (81/64) | 40 (32/8) |
Geneset enrichment analyses of expression of SCCs and AKs
TF | TF class | Target genes | Z-scorea
| Fisher scoreb
|
---|---|---|---|---|
SCC vs NS
| ||||
Up- and downregulated genes (1514 of 2036 genes included
c
)
| ||||
SRF | MADS | 107 | 12.25 | 7.38E-05 |
Hand1-Tcfe2a | bHLH | 1148 | 10.30 | 3.31E-06 |
RELA | REL | 576 | 8.01 | 1.21E-06 |
SP1 | ZN-FINGER, C2H2 | 1036 | 7.80 | 1.34E-09 |
SPIB | ETS | 1374 | 5.97 | 4.72E-06 |
Upregulated genes (736 of 1039 genes included)
| ||||
SRF | MADS | 62 | 16.93 | 5.02E-05 |
RELA | REL | 291 | 11.68 | 2.66E-04 |
Hand1-Tcfe2a | bHLH | 565 | 9.30 | 1.53E-02 |
ELF5 | ETS | 658 | 9.25 | 2.38E-05 |
REL | REL | 450 | 8.40 | 2.86E-05 |
Downregulated genes (758 of 1004 genes included)
| ||||
FOXF2 | FORKHEAD | 221 | 12.93 | 1.52E-06 |
SP1 | ZN-FINGER, C2H2 | 536 | 11.03 | 1.12E-08 |
FOXD1 | FORKHEAD | 459 | 8.409 | 2.16E-06 |
Foxq1 | FORKHEAD | 349 | 7.176 | 1.47E-06 |
TP53 | P53 | 3 | 6.687 | 1.04E-01 |
AK vs NS
| ||||
Up- and downregulated genes (695 of 960 genes included)
| ||||
SP1 | ZN-FINGER, C2H2 | 494 | 16.58 | 1.32E-08 |
MZF1_1-4 | ZN-FINGER, C2H2 | 644 | 14.28 | 2.43E-07 |
SRF | MADS | 56 | 13.50 | 1.32E-04 |
Cebpa | bZIP | 437 | 10.90 | 1.99E-05 |
TEAD1 | TEA | 217 | 10.80 | 8.74E-05 |
MZF1_5-13 | ZN-FINGER, C2H2 | 527 | 10.37 | 1.59E-08 |
Upregulated genes (364 of 515 genes included)
| ||||
SRF | MADS | 35 | 19.82 | 8.10E-05 |
ELF5 | ETS | 319 | 11.05 | 2.04E-04 |
RELA | REL | 150 | 10.03 | 1.60E-04 |
SPIB | ETS | 338 | 9.25 | 2.28E-04 |
TLX1-NFIC | HOMEO/CAAT | 32 | 9.00 | 5.07E-03 |
Downregulated genes (695 of 960 genes included)
| ||||
SP1 | ZN-FINGER, C2H2 | 248 | 17.04 | 1.63E-07 |
MZF1_1-4 | ZN-FINGER, C2H2 | 309 | 11.7 | 3.74E-04 |
Arnt-Ahr | bHLH | 289 | 11.49 | 2.25E-09 |
Cebpa | bZIP | 228 | 11.18 | 5.86E-07 |
Roaz | ZN-FINGER, C2H2 | 130 | 10.88 | 2.11E-05 |
SCC vs AK
| ||||
Up- and downregulated genes (412 of 553 genes included)
| ||||
RREB1 | ZN-FINGER, C2H2 | 38 | 11.29 | 1.24E-03 |
FOXF2 | FORKHEAD | 111 | 10.99 | 7.16E-03 |
SRF | MADS | 36 | 10.01 | 4.10E-04 |
RELA | REL | 163 | 9.239 | 8.26E-04 |
TEAD1 | TEA | 120 | 8.031 | 2.46E-02 |
Upregulated genes (209 of 283 genes included)
| ||||
SRF | MADS | 24 | 15.39 | 6.72E-05 |
RELA | REL | 85 | 11.99 | 5.32E-03 |
Fos | bZIP | 157 | 10.98 | 3.68E-04 |
NF-kappaB | REL | 108 | 8.39 | 2.22E-04 |
REL | REL | 129 | 7.91 | 1.93E-03 |
Downregulated genes (204 of 271 genes included)
| ||||
FOXF2 | FORKHEAD | 70 | 22.37 | 2.55E-05 |
FOXI1 | FORKHEAD | 126 | 10.74 | 7.22E-02 |
RREB1 | ZN-FINGER, C2H2 | 22 | 9.86 | 1.81E-03 |
T | T-BOX | 28 | 8.77 | 5.00E-03 |
Foxd3 | FORKHEAD | 129 | 7.91 | 5.37E-02 |
Progression genes
| ||||
Up- and downregulated genes (128 of 172 genes included)
| ||||
RELA | REL | 54 | 10.18 | 1.00E-02 |
SP1 | ZN-FINGER, C2H2 | 94 | 9.66 | 1.76E-03 |
FOXF2 | FORKHEAD | 36 | 7.30 | 5.18E-02 |
NFKB1 | REL | 35 | 6.21 | 1.09E-02 |
MZF1_1-4 | ZN-FINGER, C2H2 | 118 | 6.11 | 3.15E-02 |
Upregulated genes (53 of 85 genes included)
| ||||
RELA | REL | 23 | 14.0 | 5.41E-02 |
NFKB1 | REL | 19 | 9.78 | 2.48E-03 |
SRF | MADS | 6 | 8.09 | 3.82E-02 |
TLX1-NFIC | HOMEO/CAAT | 7 | 7.65 | 2.27E-02 |
MIZF | ZN-FINGER, C2H2 | 11 | 7.11 | 3.21E-02 |
Downregulated genes (75 of 87 genes included)
| ||||
FOXF2 | FORKHEAD | 29 | 14.63 | 6.46E-04 |
FOXI1 | FORKHEAD | 48 | 11.42 | 1.14E-01 |
T | T-BOX | 13 | 8.14 | 7.45E-03 |
Foxa2 | FORKHEAD | 50 | 8.02 | 4.40E-02 |
SP1 | ZN-FINGER, C2H2 | 56 | 7.60 | 7.94E-03 |
Comparison of genome-wide SNP analysis and GWEA
Validation of the GWEA with QPCR
QPCR validation experiment | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Normalized relative expressiona
| Fold change and P-value Student’sT-testb
| |||||||||||
NS (n = 10) | AK (n = 13) | SSC (n = 15) | SCC vs NS | AK vs NS | SCC vs AK | |||||||
Gene | Median | SE | Median | SE | Median | SE | log2(FC) | p-value | log2(FC) | p-value | log2(FC) | p-value |
CCL27 | 18,45 | 2,53 | 2,46 | 0,91 | 0,11 | 0,14 | −7,34 |
5,48E-05
| −2,91 |
1,80E-04
| −4,43 |
3,30E-03
|
KRT17 | 0,12 | 0,03 | 1,19 | 0,41 | 5,20 | 0,97 | 5,39 |
7,13E-05
| 3,26 |
7,07E-03
| 2,13 |
1,13E-03
|
MMP1 | 0,01 | 0,01 | 0,28 | 1,16 | 47,19 | 35,2 | 12,18 | 5,68E-02 | 4,80 |
2,32E-01
| 7,38 | 6,14E-02 |
MMP3 | 0,01 | 0,01 | 0,36 | 0,37 | 17,91 | 4,95 | 10,99 |
3,03E-03
| 5,36 | 4,07E-02 | 5,63 |
4,31E-03
|
MMP9 | 0,07 | 0,01 | 1,83 | 0,74 | 4,49 | 4,21 | 6,07 |
1,82E-02
| 4,77 |
4,42E-03
| 1,30 | 6,07E-02 |
MMP10 | 0,02 | 0,03 | 0,95 | 1,17 | 17,70 | 8,24 | 9,59 |
2,69E-03
| 5,38 | 4,53E-02 | 4,21 |
5,16E-03
|
PI3 | 0,02 | 0,01 | 2,01 | 1,67 | 10,62 | 6,23 | 8,95 |
5,46E-03
| 6,55 |
7,71E-03
| 2,40 |
3,25E-02
|
SERPINB4 | 0,004 | 0,002 | 1,99 | 6,2 | 18,79 | 15,7 | 12,33 |
2,35E-02
| 9,09 |
3,96E-02
| 3,24 | 1,46E-01 |
TUBB3 | 0,51 | 0,08 | 0,64 | 0,17 | 3,68 | 0,94 | 2,85 |
1,17E-03
| 0,32 |
3,20E-01
| 2,53 |
1,78E-03
|
Genome-wide expression analysis
| ||||||||||||
Normalized expression
c
|
Results limma analysis
| |||||||||||
NS (n = 13)
|
AK (n = 14)
|
SSC (n = 15)
|
SCC vs NS
|
AK vs NS
|
SCC vs AK
| |||||||
Gene
|
Median
|
SE
|
Median
|
SE
|
Median
|
SE
|
log
2
FC
|
Adj. p-val
|
log
2
FC
|
Adj. p-val
|
log
2
FC
|
Adj. p-val
|
CCL27 | 12,92 | 0,02 | 10,74 | 0,10 | 8,60 | 0,03 | −4,24 | 2,03E-15 | −2,24 | 9,64E-08 | −2,00 | 1,65E-06 |
KRT17 | 10,58 | 0,07 | 14,43 | 0,10 | 15,56 | 0,01 | 4,84 | 1,28E-14 | 3,16 | 5,56E-09 | 1,68 | 3,66E-04 |
MMP1 | 8,32 | 0,00 | 8,39 | 0,05 | 10,80 | 0,08 | 2,41 | 5,70E-10 | 0,22 | 5,65E-01 | 2,20 | 1,61E-07 |
MMP3 | 8,33 | 0,01 | 8,55 | 0,06 | 10,59 | 0,08 | 2,44 | 1,16E-09 | 0,50 | 1,59E-01 | 1,84 | 6,86E-06 |
MMP9 | 8,48 | 0,01 | 9,29 | 0,06 | 10,50 | 0,09 | 2,22 | 1,37E-07 | 1,05 | 8,50E-03 | 1,93 | 2,02E-06 |
MMP10 | 8,31 | 0,00 | 8,33 | 0,02 | 10,02 | 0,10 | 1,93 | 3,07E-07 | 0,09 | 8,41E-01 | 1,16 | 4,61E-03 |
PI3 | 9,16 | 0,05 | 13,60 | 0,14 | 15,27 | 0,04 | 5,68 | 1,33E-13 | 3,96 | 1,09E-08 | 1,72 | 3,44E-03 |
SERPINB4 | 8,34 | 0,02 | 10,22 | 0,13 | 12,04 | 0,10 | 3,81 | 5,15E-09 | 2,16 | 3,64E-04 | 1,65 | 6,04E-03 |
TUBB3 | 8,84 | 0,01 | 8,86 | 0,05 | 10,45 | 0,05 | 1,91 | 5,45E-11 | 0,23 | 3,66E-01 | 1,67 | 5,28E-08 |
Discussion
Conclusions
Methods
Patient material
RNA and DNA isolation
Genome-wide SNP analysis
Genome-wide expression analysis (GWEA)
Quantitative RT-PCR (QPCR)
Gene | Accession number | Forward primer 5′ > 3′
| Reverse primer 5′ > 3′
| Amplicon size (bp) |
---|---|---|---|---|
Genes of interest
| ||||
CCL27 | NM_006664 | GACTGTCACCTCCAGGCTTT | TCTCTTGGTGCTCAAACCAC | 100 |
K17 | NM_000422 | CTGGAGCAGGAGATTGCCAC | GGGTGGTCACCGGTTCTTTC | 88 |
MMP1 | NM_002421 | AGGTCTCTGAGGGTCAAGCA | CTGGTTGAAAAGCATGAGCA | 111 |
MMP3 | NM_002422 | TGCTTTGTCCTTTGATGCTG | GGAAGAGATGGCCAAAATGA | 135 |
MMP9 | NM_004994 | CCTGGAGACCTGAGAACCAA | ATTTCGACTCTCCACGCATC | 103 |
MMP10 | NM_002425 | GTGGAGTTCCTGACGTTGGT | TCAATGGCAGAATCAACAGC | 130 |
PI3 | NM_002638 | GACTGCCCAGGAATCAAGAA | CAGCAGGGACTTAGGACCAG | 148 |
SERPINB4 | NM_002974 | CAAAGGGCAGTGGGAGAATA | CCTCCAGCAAGGCAAAATTA | 131 |
TUBB3 | NM_006086 | CTCAGGGGCCTTTGGACATC | CCCTCCGTGTAGTGACCCTT | 96 |
Reference genes
| ||||
ARPC2 | NM_005731 | TCCGGGACTACCTGCACTAC | GGTTCAGCACCTTGAGGAAG | 96 |
BAT3 | NM_004639 | AAGAGACGCAAGACGATGCAG | TGTAGCTCTCCTGAACCTCTGG | 151 |
RPS29 | NM_001032 | TATGTGCCGCCAGTGTTTCC | TGCCCCGGATAATCCTCTGA | 92 |
ZNF410 | NM_021188 | GCTGTGGTAAGCAGTTTACTACAG | CTTGGGCTTCACAAAGGAAAGG | 90 |