Background
Methods
Patients
Cell culture and DNA methylation inhibitor treatment
Cell transfection
Cell viability assay
Colony formation assay
Cell migration assay
TOP/FOP assay
Immunofluorescence staining
Immunoblot analysis
Quantitative real-time PCR (qRT-PCR)
Methylation analyses
Chromatin immunoprecipitation
Statistical analysis
Results
Promoter methylation causes SFRP1 silencing in human HB cell lines
Restored SFRP1 expression affects WNT signaling activity and HB tumor cell characteristics
SFRP1 DNA methylation correlates with the tumor type and a late onset of the disease
SFRP1 expression | High (n = 17) | Low (n = 28) | Total (n = 45) | p value |
---|---|---|---|---|
Gender | 0.848 | |||
f | 8 (47.1%) | 14 (50.0%) | 22 (48.9%) | |
m | 9 (52.9%) | 14 (50.0%) | 23 (51.1%) | |
Age at diagnosis in month | 0.386 | |||
nd | 0 | 1 | 1 | |
Mean (SD) | 67.867 (72.955) | 50.780 (55.905) | 57.382 (62.777) | |
Range | 1.874–199.435 | 0.000–184.179 | 0.000–199.435 | |
Outcome | 0.758 | |||
DOD | 3 (17.6%) | 6 (21.4%) | 9 (20.0%) | |
NED | 14 (82.4%) | 22 (78.6%) | 36 (80.0%) | |
Cause of death | 0.541 | |||
nd | 0 | 1 | 1 | |
Progressive | 2 (11.8%) | 2 (7.4%) | 4 (9.1%) | |
Recurrence | 2 (11.8%) | 3 (11.1%) | 5 (11.1%) | |
Alive | 13 (76.5%) | 22 (81.5%) | 35 (79.5%) | |
Tumor type | 0.170 | |||
HB | 10 (58.8%) | 20 (71.4%) | 30 (66.7%) | |
HCC/TLCT | 5 (29.4%) | 8 (28.6%) | 13 (28.9%) | |
NSET | 2 (11.8%) | 0 (0.0%) | 2 (4.4%) | |
Differentiation | 0.388 | |||
nd | 2 | 0 | 2 | |
Epithelial | 6 (40.0%) | 17 (60.7%) | 23 (53.5%) | |
Fibrolamellar | 3 (20.0%) | 1 (3.6%) | 4 (9.3%) | |
Well differentiated | 0 (0.0%) | 2 (7.1%) | 2 (4.4%) | |
Moderately differentiated | 1 (6.7%) | 2 (7.1%) | 3 (7.0%) | |
Mixed | 5 (33.3%) | 6 (21.4%) | 11 (25.6%) | |
Component | 0.332 | |||
na | 6 (35.3%) | 5 (17.9%) | 11 (24.4%) | |
E | 1 (5.9%) | 0 (0.0%) | 1 (2.2%) | |
E/F | 3 (17.6%) | 6 (21.4%) | 9 (20.0%) | |
E > F | 1 (5.9%) | 1 (3.6%) | 2 (4.4%) | |
F | 2 (11.8%) | 4 (14.3%) | 6 (13.3%) | |
F > E | 3 (17.6%) | 12 (42.9%) | 15 (33.3%) | |
Pure OS | 1 (5.9%) | 0 (0.0%) | 1 (2.2%) | |
Stage | 0.296 | |||
nd | 2 | 1 | 3 | |
I | 1 (6.7%) | 6 (22.2%) | 7 (16.7%) | |
II | 0 (0.0%) | 1 (3.7%) | 1 (2.4%) | |
III | 9 (60.0%) | 9 (33.3%) | 18 (42.9%) | |
IV | 5 (33.3%) | 11 (40.7%) | 16 (38.1%) | |
PRETEXT | 0.025 | |||
nd | 3 | 1 | 4 | |
1 | 2 (13.3%) | 0 (0.0%) | 2 (4.8%) | |
2 | 3 (20.0%) | 11 (40.7%) | 14 (33.3%) | |
3 | 9 (60.0%) | 10 (37.0%) | 19 (45.2%) | |
4 | 0 (0.0%) | 6 (22.2%) | 6 (14.3%) | |
Extrahepatic | 0.820 | |||
nd | 0 | 2 | 2 | |
No | 16 (94.1%) | 24 (92.3%) | 40 (93.0%) | |
Yes | 1 (5.9%) | 2 (7.7%) | 3 (7.0%) | |
Multifocal | 0.666 | |||
nd | 2 | 1 | 3 | |
No | 12 (80.0%) | 20 (74.1%) | 32 (76.2%) | |
Yes | 3 (20.0%) | 7 (25.9%) | 10 (23.8%) | |
Metastasis | 0.447 | |||
nd | 0 | 1 | 1 | |
No | 12 (70.6%) | 16 (59.3%) | 28 (63.6%) | |
Yes | 5 (29.4%) | 11 (40.7%) | 16 (36.4%) | |
Chemotherapy | 0.282 | |||
No | 2 (11.8%) | 7 (25.0%) | 9 (20.0%) | |
Yes | 15 (88.2%) | 21 (75.0%) | 36 (80.0%) | |
Resection margin | 0.911 | |||
Nd | 2 | 1 | 3 | |
R0 | 13 (81.2%) | 23 (82.1%) | 36 (81.8%) | |
R1 | 2 (12.5%) | 4 (14.3%) | 6 (13.6%) | |
16-gene signature | 0.172 | |||
na | 2 (11.8%) | 0 (0.0%) | 2 (4.4%) | |
C1 | 9 (52.9%) | 18 (64.3%) | 27 (60.0%) | |
C2 | 6 (35.3%) | 10 (35.7%) | 16 (35.6%) | |
CTNNB1 | 0.017 | |||
Mutant | 11 (64.7%) | 26 (92.9%) | 37 (82.2%) | |
Wildtype | 6 (35.3%) | 2 (7.1%) | 8 (17.8%) | |
NFE2L2 | 0.137 | |||
nd | 3 | 0 | 3 | |
Mutant | 0 (0.0%) | 4 (14.3%) | 4 (9.5%) | |
Wildtype | 14 (100.0%) | 24 (85.7%) | 38 (90.5%) | |
TERT | 0.620 | |||
nd | 1 | 0 | 1 | |
Mutant | 1 (6.2%) | 3 (10.7%) | 4 (9.1%) | |
Wildtype | 15 (93.8%) | 25 (89.3%) | 40 (90.9%) | |
SFRP1 methylation | 0.616 | |||
M | 6 (35.3%) | 12 (42.9%) | 18 (40.0%) | |
U | 11 (64.7%) | 16 (57.1%) | 27 (60.0%) |
SFRP1 methylation | M (n = 18) | U (n = 27) | Total (n = 45) | p value |
---|---|---|---|---|
Gender | 0.021 | |||
f | 5 (27.8%) | 17 (63.0%) | 22 (48.9%) | |
m | 13 (72.2%) | 10 (37.0%) | 23 (51.1%) | |
Age at diagnosis in month | < 0.001 | |||
nd | 1 | 0 | 1 | |
Mean (SD) | 98.48 (73.09) | 31.50 (37.82) | 57.38 (62.78) | |
Range | 7.37–199.44 | 0.00–156.33 | 0.00–199.44 | |
Outcome | 0.287 | |||
DOD | 5 (27.8%) | 4 (14.8%) | 9 (20.0%) | |
NED | 13 (72.2%) | 23 (85.2%) | 36 (80.0%) | |
Cause of death | 0.512 | |||
nd | 1 | 0 | 1 | |
Progressive | 2 (11.8%) | 2 (7.4%) | 4 (9.1%) | |
Recurrence | 3 (16.6%) | 2 (7.4%) | 5 (11.1%) | |
Alive | 12 (70.6%) | 23 (85.2%) | 35 (79.5%) | |
Tumor type | 0.004 | |||
HB | 7 (38.9%) | 23 (85.2%) | 30 (66.7%) | |
HCC/TLCT | 10 (55.6%) | 3 (11.1%) | 13 (28.9%) | |
NSET | 1 (5.6%) | 1 (3.7%) | 2 (4.4%) | |
Differentiation | 0.042 | |||
nd | 1 | 1 | 2 | |
Epithelial | 8 (47.1%) | 15 (57.7%) | 23 (53.5%) | |
Fibrolamellar | 4 (23.5%) | 0 (0.0%) | 4 (9.3%) | |
Well differentiated | 1 (5.9%) | 1 (3.8%) | 2 (4.4%) | |
Moderately differentiated | 2 (11.8%) | 1 (3.8%) | 3 (7.0%) | |
Mixed | 2 (11.8%) | 9 (34.6%) | 11 (25.6%) | |
Component | 0.260 | |||
na | 8 (44.4%) | 3 (11.1%) | 11 (24.4%) | |
E | 0 (0.0%) | 1 (3.7%) | 1 (2.2%) | |
E/F | 3 (16.7%) | 6 (22.2%) | 9 (20.0%) | |
E > F | 1 (5.6%) | 1 (3.7%) | 2 (4.4%) | |
F | 2 (11.1%) | 4 (14.8%) | 6 (13.3%) | |
F > E | 4 (22.2%) | 11 (40.7%) | 15 (33.3%) | |
pure OS | 0 (0.0%) | 1 (3.7%) | 1 (2.2%) | |
Stage | 0.078 | |||
nd | 1 | 2 | 3 | |
I | 5 (29.4%) | 2 (8.0%) | 7 (16.7%) | |
II | 1 (5.9%) | 0 (0.0%) | 1 (2.4%) | |
III | 4 (23.5%) | 14 (56.0%) | 18 (42.9%) | |
IV | 7 (41.2%) | 9 (36.0%) | 16 (38.1%) | |
PRETEXT | 0.552 | |||
nd | 4 | 0 | 4 | |
1 | 1 (6.7%) | 1 (3.7%) | 2 (4.8%) | |
2 | 6 (40.0%) | 8 (29.6%) | 14 (33.3%) | |
3 | 5 (33.3%) | 14 (51.9%) | 19 (45.2%) | |
4 | 2 (13.3%) | 4 (14.8%) | 6 (14.3%) | |
Extrahepatic | 0.020 | |||
nd | 2 | 0 | 2 | |
No | 13 (81.2%) | 27 (100.0%) | 40 (93.0%) | |
Yes | 3 (18.8%) | 0 (0.0%) | 3 (7.0%) | |
Multifocal | 0.280 | |||
nd | 3 | 0 | 3 | |
No | 10 (66.7%) | 22 (81.5%) | 32 (76.2%) | |
Yes | 5 (33.3%) | 5 (18.5%) | 10 (23.8%) | |
Metastasis | 0.598 | |||
nd | 1 | 0 | 1 | |
No | 10 (58.8%) | 18 (66.7%) | 28 (63.6%) | |
Yes | 7 (41.2%) | 9 (33.3%) | 16 (36.4%) | |
Chemotherapy | 0.761 | |||
No | 4 (22.2%) | 5 (18.5%) | 9 (20.0%) | |
Yes | 14 (77.8%) | 22 (81.5%) | 36 (80.0%) | |
Resection margin | 0.046 | |||
nd | 3 | 0 | 3 | |
R0 | 11 (64.7%) | 25 (92.6%) | 36 (81.8%) | |
R1 | 4 (23.5%) | 2 (7.4%) | 6 (13.6%) | |
16-gene signature | 0.871 | |||
na | 1 (5.6%) | 1 (3.7%) | 2 (4.4%) | |
C1 | 10 (55.6%) | 17 (63.0%) | 27 (60.0%) | |
C2 | 7 (38.9%) | 9 (33.3%) | 16 (35.6%) | |
CTNNB1 | 0.340 | |||
Mutant | 16 (88.9%) | 21 (77.8%) | 37 (82.2%) | |
Wildtype | 2 (11.1%) | 6 (22.2%) | 8 (17.8%) | |
NFE2L2 | 0.571 | |||
nd | 2 | 1 | 3 | |
Mutant | 1 (6.2%) | 3 (11.5%) | 4 (9.5%) | |
Wildtype | 15 (93.8%) | 23 (88.5%) | 38 (90.5%) | |
TERT | 0.008 | |||
nd | 1 | 0 | 1 | |
Mutant | 4 (23.5%) | 0 (0.0%) | 4 (9.1%) | |
Wildtype | 13 (76.5%) | 27 (100.0%) | 40 (90.9%) | |
SFRP1 expression | 0.908 | |||
Mean (SD) | 1.327 (1.956) | 1.265 (1.575) | 1.290 (1.716) | |
Range | 0.015–6.287 | 0.029–6.450 | 0.015–6.450 | |
SFRP1 exp. category | 0.616 | |||
High | 6 (33.3%) | 11 (40.7%) | 17 (37.8%) | |
Low | 12 (66.7%) | 16 (59.3%) | 28 (62.2%) |