Background
Methods
Ethics statement
Biological specimen collection
Characteristic | Cases (%) |
---|---|
Age (years) | |
≤50 | 78 (56.93) |
>50 | 59 (43.07) |
Age at menarche | |
≤12 | 26 (18.98) |
>12 | 111 (81.02) |
Menopausal status | |
Premenopausal | 40 (29.20) |
Postmenopausal | 97 (70.80) |
Age at menopause | |
≤45 | 33 (34.02) |
>45 | 64 (65.98) |
ER status | |
Positive | 81 (59.12) |
Negative | 56 (40.88) |
PR status | |
Positive | 47 (34.31) |
Negative | 90 (65.69) |
Her2 status | |
Positive | 66 (48.18) |
Negative | 71 (51.82) |
Molecular subtypes of breast cancer | |
Luminal A | 46 (33.58) |
Luminal B | 38 (27.74) |
Her2-enriched | 28 (20.44) |
TNBC | 25 (18.25) |
Tumor size | |
≤5 | 55 (40.15) |
>5 | 82 (59.85) |
Lymph node status | |
Positive | 99 (72.26) |
Negative | 38 (27.74) |
Clinical stage | |
I+II | 45(32.85) |
III+IV | 92(67.15) |
Histological grade | |
I+II | 95 (69.34) |
III | 42 (30.66) |
Genomic DNA extraction
Automated DNA sequencing
Primer | Primer Sequence | PCR product size (base pair) | Annealing Temperatu-re (°C) |
Mutation primers | |||
YAP1 exon 1 (serine 61) | F 5’-AGGCAGAAGCCATGGATC-3’ | 338 | 56.6 |
R 5’-GGTTACCTGTCGGGAGTG-3’ | |||
YAP1 exon 2 (serine 109 and 127) | F 5’-GGCTGCAATTAAGCGCTGAC-3’ | 292 | 61.5 |
R 5’-TGCTGGCAGAGGTACATCATC-3’ | |||
YAP1 exon 2 (serine 164) | F 5’-CGAGCTCATTCCTCTCCAGC-3’ | 236 | 55.5 |
R 5’-AGATAACTGTCTCCCACC-3’ | |||
YAP1 exon 8 (serine 397) | F 5’-TTCAGACATTGCAGGACAGG-3’ | 248 | 58.8 |
R 5’-CCTGTATCCATCTCATCCACAC-3’ | |||
YAP1 exon 9 (lysine 494) | F 5’-CTCTGTGTGTTTCCACTAGG-3’ | 317 | 57.5 |
R 5’-CCGGTGCATGTGTCTCCTTAG-3’ | |||
Methylation primers | |||
YAP1 methylation | F 5’-AGTTCGTATAGGCGTTTCGTTC-3’ | 187 | 57.9 |
F 5’-CTTAACTACAAAAAATTCTTCCGCT-3’ | |||
YAP1 unmethylation | F 5’-AAGTTTGTATAGGTGTTTTGTTTGG-3’ | 188 | 57.9 |
F 5’-CTTAACTACAAAAAATTCTTCCACT-3’ | |||
Expression primers | |||
YAP1 | F 5’-AAGCTGCCCGACTCCTTCTTCAAG-3’ | 161 | 64.1 |
R 5’-GTCAGTGTCCCAGGAGAAACA-3’ | |||
GAPDH | F 5’-CACTGCCACCCAGAAGACTG-3’ | 150 | 61 |
R 5’-ATGCCAGTGAGCTTCCCGTT-3’ |
The Cancer Genome Atlas (TCGA)
Catalogue of Somatic Mutations in Cancer (COSMIC) Analysis for YAP mutations
Methylation-specific polymerase chain reaction (MS-PCR)
Real-time polymerase chain reaction
Immunohistochemistry
Statistical analysis
Results
Downregulated YAP mRNA expression in breast cancer tissue
Characteristic | Total (N) | YAP1 mRNA expression relative to GAPDH | p value |
---|---|---|---|
Normal | 137 | 2.27 ± 1.65 | 0.0001 |
Tumor | 137 | 0.11 ± 5.60 | |
Age (years) | |||
≤50 | 78 (56.93) | 0.149 ± 0.00 | 0.464 |
>50 | 59 (43.07) | 0.053 ± 0.00 | |
Age at menarche | |||
≤12 | 26 (18.98) | 0.033 ± 0.00 | 0.524 |
>12 | 111 (81.02) | 0.125 ± 0.00 | |
Menopausal status | |||
Premenopausal | 40 (29.20) | 0.036 ± 0.00 | 0.462 |
Postmenopausal | 97 (70.80) | 0.137 ± 0.00 | |
Age at menopause | |||
≤45 | 33 (34.02) | 0.297 ± 1.09 | 0.281 |
>45 | 64 (65.98) | 0.055 ± 1.98 | |
ER status | |||
Positive | 81 (59.12) | 0.121 ± 0.00 | 0.373 |
Negative | 56 (40.88) | 0.088 ± 0.00 | |
PR status | |||
Positive | 47 (34.31) | 0.141 ± 0.00 | 0.741 |
Negative | 90 (65.69) | 0.090 ± 0.00 | |
Her2 status | |||
Positive | 66 (48.18) | 0.053 ± 0.00 | 0.506 |
Negative | 71 (51.82) | 0.159 ± 0.00 | |
Molecular subtypes of breast cancer | |||
Luminal A | 46 (33.58) | 0.149 ± 0.00 | 0.731 |
Luminal B | 38 (27.74) | 0.079 ± 0.00 | |
Her2-enriched | 28 (20.44) | 0.018 ± 0.00 | |
TNBC | 25 (18.25) | 0.178 ± 0.00 | |
Tumor size | |||
≤5 | 55 (40.15) | 0.183 ± 0.00 | 0.169 |
>5 | 82 (59.85) | 0.057 ± 0.00 | |
Lymph node status | |||
Positive | 99 (72.26) | 0.109 ± 0.00 | 0.203 |
Negative | 38 (27.74) | 0.103 ± 0.00 | |
Clinical stage | |||
I+II | 45(32.85) | 0.206 ± 0.00 | 0.038 |
III+IV | 92(67.15) | 0.06 ± 0.00 | |
Histological grade | |||
I+II | 95 (69.34) | 0.096 ± 0.00 | 0.869 |
III | 42 (30.66) | 0.135 ± 0.00 |
YAP protein expression is frequently absent in breast cancer
Characteristic | Total (N) | YAP1 absent | YAP1 present | p value |
---|---|---|---|---|
Age (years) | ||||
≤50 | 78 (56.93) | 64 (82.05) | 14 (17.95) | 0.3 |
>50 | 59 (43.07) | 44 (74.58) | 15 (25.42) | |
Age at menarche | ||||
≤12 | 26 (18.98) | 23 (88.46) | 3 (11.54) | 0.258 |
>12 | 111 (81.02) | 85 (76.58) | 26 (23.42) | |
Menopausal status | ||||
Premenopausal | 40 (29.20) | 32 (80) | 8 (20) | 1 |
Postmenopausal | 97 (70.80) | 76 (78.35) | 21 (21.65) | |
Age at menopause | ||||
≤45 | 33 (34.02) | 28 (84.85) | 5 (15.15) | 0.309 |
>45 | 64 (65.98) | 48 (75) | 16 (25) | |
ER status | ||||
Positive | 81 (59.12) | 58 (71.60) | 23 (28.4) | 0.018 |
Negative | 56 (40.88) | 50 (89.29) | 6 (10.71) | |
PR status | ||||
Positive | 47 (34.31) | 38 (80.85) | 9 (19.15) | 0.826 |
Negative | 90 (65.69) | 70 (77.78) | 20 (22.22) | |
Her2 status | ||||
Positive | 66 (48.18) | 52 (78.79) | 14 (21.21) | 1 |
Negative | 71 (51.82) | 56 (78.87) | 15 (21.13) | |
Molecular subtypes of breast cancer | ||||
Luminal A | 46 (33.58) | 33 (71.74) | 13 (28.26) | 0.146 |
Luminal B | 38 (27.74) | 28 (73.68) | 10 (26.32) | |
Her2-enriched | 28 (20.44) | 24 (85.71) | 4 (14.29) | |
TNBC | 25 (18.25) | 23 (92) | 2 (8) | |
Tumor size | ||||
≤5 | 55 (40.15) | 47 (85.45) | 8 (14.55) | 0.139 |
>5 | 82 (59.85) | 61 (74.39) | 21 (25.61) | |
Lymph node status | ||||
Positive | 99 (72.26) | 77 (77.78) | 22 (22.22) | 0.816 |
Negative | 38 (27.74) | 31 (81.58) | 7 (18.42) | |
Clinical stage | ||||
I+II | 45(32.85) | 38 (84.44) | 7 (15.56) | 0.373 |
III+IV | 92(67.15) | 70 (76.09) | 22 (23.91) | |
Histological grade | ||||
I+II | 95 (69.34) | 76 (80) | 19 (20) | 0.653 |
III | 42 (30.66) | 32 (76.19) | 10 (23.81) |
Association between YAP promoter methylation and YAP protein expression in breast cancer
YAP1 protein expression | YAP Promoter | ||||
---|---|---|---|---|---|
Methylated (% within Protein expression) | Unmethylated (% within Protein expression) | Total | p value | OR (95% CI) | |
Present | 13 (44.83%) | 16 (55.17%) | |||
Absent | 84 (77.78%) | 24 (22.22%) | |||
Total (%) | 97 (70.80%) | 40 (29.20%) | 137 | 0.001 | 0.232 (0.098 - 0.549) |
YAP Promoter | YAP1 protein expression | ||||
Present | Absent | ||||
Methylated (% within methylation status) | 13 (13.4%) | 84 (86.6%) | |||
Unmethylated (% within unmethylation status) | 16 (40.0%) | 24 (60.0%) | |||
Total (%) | 29 (21.17%) | 108 (78.13%) | 137 |
Association between YAP promoter methylation and clinicopathological parameters in breast cancer
Characteristics | Cases (%) | Unmethylated (%) | Methylated (%) | p value |
---|---|---|---|---|
Age (years) | ||||
≤50 | 78 (56.93) | 21 (26.92) | 57 (73.08) | 0.571 |
>50 | 59 (43.07) | 19 (32.20) | 40 (67.80) | |
Age at menarche | ||||
≤12 | 26 (18.98) | 8 (30.77) | 18 (69.23) | 0.815 |
>12 | 111 (81.02) | 32 (28.83) | 79 (71.17) | |
Menopausal status | ||||
Premenopausal | 40 (29.20) | 12 (30) | 28 (70) | 1 |
Postmenopausal | 97 (70.80) | 28 (28.87) | 69 (71.13) | |
Age at menopause | ||||
≤45 | 33 (34.02) | 12 (36.36) | 21 (63.64) | 0.249 |
>45 | 64 (65.98) | 16 (25) | 48 (75) | |
ER status | ||||
Positive | 81 (59.12) | 27 (33.33) | 54 (66.67) | 0.252 |
Negative | 56 (40.88) | 13 (23.21) | 43 (76.79) | |
PR status | ||||
Positive | 47 (34.31) | 12 (25.53) | 35 (74.47) | 0.556 |
Negative | 90 (65.69) | 28 (31.11) | 62 (68.89) | |
Her2 status | ||||
Positive | 66 (48.18) | 18 (27.27) | 48 (72.73) | 0.708 |
Negative | 71 (51.82) | 22 (30.99) | 49 (69.01) | |
Molecular subtypes of breast cancer | ||||
Luminal A | 46 (33.58) | 16 (34.78) | 30 (65.22) | 0.584 |
Luminal B | 38 (27.74) | 12 (31.58) | 26 (68.42) | |
Her2-enriched | 28 (20.44) | 6 (21.43) | 22 (78.57) | |
TNBC | 25 (18.25) | 6 (24) | 19 (76) | |
Tumor size | ||||
≤5 | 55 (40.15) | 15 (27.27) | 40 (72.73) | 0.707 |
>5 | 82 (59.85) | 25 (30.49) | 57 (69.51) | |
Lymph node status | ||||
Positive | 99 (72.26) | 29 (29.29) | 70 (70.71) | 1 |
Negative | 38 (27.74) | 11 (28.95) | 27 (71.05) | |
Clinical stage | ||||
I+II | 45(32.85) | 11 (24.44) | 34 (75.56) | 0.43 |
III+IV | 92(67.15) | 29 (31.52) | 63 (68.48) | |
Histological grade | ||||
I+II | 95 (69.34) | 27 (28.42) | 68 (71.58) | 0.839 |
III | 42 (30.66) | 13 (30.95) | 29 (69.05) |
Association between YAP protein expression and clinicopathological parameters in breast cancer
Characteristic | Total (N) | Methylated YAP1 | p value | Total (N) | YAP1 loss | p value | ||
---|---|---|---|---|---|---|---|---|
YAP1 Absent | YAP1 Present | Unmethylated YAP1 | Methylated YAP1 | |||||
Age (years) | ||||||||
≤50 | 57 | 50 | 7 | 0.767 | 64 | 14 | 50 | 1 |
>50 | 40 | 34 | 6 | 44 | 10 | 34 | ||
Age at menarche | ||||||||
≤12 | 18 | 17 | 1 | 0.451 | 23 | 6 | 17 | 0.585 |
>12 | 79 | 67 | 12 | 85 | 18 | 67 | ||
Menopausal status | ||||||||
Premenopausal | 28 | 23 | 5 | 0.512 | 32 | 9 | 23 | 0.447 |
Postmenopausal | 69 | 61 | 8 | 76 | 15 | 61 | ||
Age at menopause | ||||||||
≤45 | 21 | 21 | 0 | 0.095 | 28 | 7 | 21 | 0.388 |
>45 | 48 | 40 | 8 | 48 | 8 | 40 | ||
ER status | ||||||||
Positive | 54 | 45 | 9 | 0.375 | 58 | 13 | 45 | 1 |
Negative | 43 | 39 | 4 | 50 | 11 | 39 | ||
PR status | ||||||||
Positive | 35 | 30 | 5 | 1 | 38 | 8 | 30 | 1 |
Negative | 62 | 54 | 8 | 70 | 16 | 54 | ||
Her2 status | ||||||||
Positive | 48 | 42 | 6 | 1 | 52 | 10 | 42 | 0.497 |
Negative | 49 | 42 | 7 | 56 | 14 | 42 | ||
Molecular subtypes of breast cancer | ||||||||
Luminal A | 30 | 25 | 5 | 0.837 | 33 | 8 | 25 | 0.87 |
Luminal B | 26 | 22 | 4 | 28 | 6 | 22 | ||
Her2-enriched | 22 | 20 | 2 | 24 | 4 | 20 | ||
TNBC | 19 | 17 | 2 | 23 | 6 | 17 | ||
Tumor size | ||||||||
≤5 | 40 | 36 | 4 | 0.549 | 47 | 11 | 36 | 0.819 |
>5 | 57 | 48 | 9 | 61 | 13 | 48 | ||
Lymph node status | ||||||||
Positive | 70 | 61 | 9 | 0.751 | 77 | 16 | 61 | 0.613 |
Negative | 27 | 23 | 4 | 31 | 8 | 23 | ||
Clinical stage | ||||||||
I+II | 34 | 31 | 3 | 0.533 | 38 | 7 | 31 | 0.629 |
III+IV | 63 | 53 | 10 | 70 | 17 | 53 | ||
Histological grade | ||||||||
I+II | 68 | 59 | 9 | 1 | 76 | 17 | 59 | 1 |
III | 29 | 25 | 4 | 32 | 7 | 25 |
Characteristic | Total (N) | YAP1 methylation status | YAP1 expression | p value | |
---|---|---|---|---|---|
Absent | Present | ||||
Age (years) | |||||
≤50 | 78 (56.93) | M | 50 | 7 | 0.046 |
UM | 14 | 7 | |||
>50 | 59 (43.07) | M | 34 | 6 | 0.017 |
UM | 10 | 9 | |||
Age at menarche | |||||
≤12 | 26 (18.98) | M | 17 | 1 | 0.215 |
UM | 6 | 2 | |||
>12 | 111 (81.02) | M | 67 | 12 | 0.002 |
UM | 18 | 14 | |||
Menopausal status | |||||
Premenopausal | 40 (29.20) | M | 23 | 5 | 0.677 |
UM | 9 | 3 | |||
Postmenopausal | 97 (70.80) | M | 61 | 8 | <0.0007 |
UM | 15 | 13 | |||
Age at menopause | |||||
≤45 | 33 (34.02) | M | 21 | 0 | 0.003 |
UM | 7 | 5 | |||
>45 | 64 (65.98) | M | 40 | 8 | 0.016 |
UM | 8 | 8 | |||
ER status | |||||
Positive | 81 (59.12) | M | 45 | 9 | <0.002 |
UM | 13 | 14 | |||
Negative | 56 (40.88) | M | 39 | 4 | 0.615 |
UM | 11 | 2 | |||
PR status | |||||
Positive | 47 (34.31) | M | 30 | 5 | 0.205 |
UM | 8 | 4 | |||
Negative | 90 (65.69) | M | 54 | 8 | <0.003 |
UM | 16 | 12 | |||
Her2 status | |||||
Positive | 66 (48.18) | M | 42 | 6 | 0.014 |
UM | 10 | 8 | |||
Negative | 71 (51.82) | M | 42 | 7 | 0.057 |
UM | 14 | 8 | |||
Molecular subtypes of breast cancer | |||||
Luminal A | 46 (33.58) | M | 25 | 5 | 0.036 |
UM | 8 | 8 | |||
Luminal B | 38 (27.74) | M | 22 | 4 | 0.045 |
UM | 6 | 6 | |||
Her2-enriched | 28 (20.44) | M | 20 | 2 | 0.191 |
UM | 4 | 2 | |||
TNBC | 25 (18.25) | M | 17 | 2 | 1 |
UM | 6 | 0 | |||
Tumor size | |||||
≤5 | 55 (40.15) | M | 36 | 4 | 0.193 |
UM | 11 | 4 | |||
>5 | 82 (59.85) | M | 48 | 9 | <0.005 |
UM | 13 | 12 | |||
Lymph node status | |||||
Positive | 99 (72.26) | M | 61 | 9 | <0.002 |
UM | 16 | 13 | |||
Negative | 38 (27.74) | M | 23 | 4 | 0.39 |
UM | 8 | 3 | |||
Clinical stage | |||||
I+II | 45(32.85) | M | 31 | 3 | 0.05 |
UM | 7 | 4 | |||
III+IV | 92(67.15) | M | 53 | 10 | 0.016 |
UM | 17 | 12 | |||
Histological grade | |||||
I+II | 95 (69.34) | M | 59 | 9 | 0.02 |
UM | 17 | 10 | |||
III | 42 (30.66) | M | 25 | 4 | 0.05 |
UM | 7 | 6 |