Introduction
Materials and methods
Tumor and patient selection
Clinical factors | ER negative (n = 65) | ER positive (n = 65) |
---|---|---|
Menopausal status | ||
Premenopause | 35 | 35 |
Postmenopause | 30 | 30 |
T stage | ||
T1c | 24 | 24 |
T2 | 37 | 37 |
T3 | 4 | 4 |
N stage | ||
N0 | 38 | 38 |
N1 | 26 | 27 |
N2 | 1 | 0 |
M stage | ||
M0 | 65 | 65 |
M1 | 0 | 0 |
AJCC stage | ||
I | 17 | 17 |
IIa | 28 | 28 |
IIb | 16 | 16 |
IIIa | 4 | 4 |
IV | 0 | 0 |
HER2/neu | 14/51 (27%) | 15/56 (27%) |
Total | 65 | 65 |
Clinical factors | HER2/neu negative | HER2/neu positive | P valuea |
---|---|---|---|
T stage | |||
T1 | 29 | 10 | |
T2/3 | 49 | 16 | NS |
N stage | |||
N0 | 50 | 15 | |
N1 | 28 | 14 | NS |
Age (years) | 50.2 ± 11.3 | 50.8 ± 12.6 | NS |
DNA processing and methylation-specific PCR
Gene | Methylated/unmethylated | Direction | Sequence |
---|---|---|---|
RASSF1A
| Methylated | Forward | 5'-GTGTTAACGCGTTGCGTATC-3' |
Reverse | 5'-AACCCCGCGAACTAAAAACGA-3' | ||
Unmethylated | Forward | 5'-TTTGGTTGGAGTGTGTTAATGTG-3' | |
Reverse | 5'-CAAACCCCACAAACTAAAAACAA-3' | ||
CCND2
| Methylated | Forward | 5'-TACGTGTTAGGGTCGATCG-3' |
Reverse | 5'-CGAAAACATAAAACCTCCACG-3' | ||
Unmethylated | Forward | 5'-GTTATGTTATGTTTGTTGTATG-3' | |
Reverse | 5'-TAAAATCCACCAACACAATCA-3' | ||
GSTP1
| Methylated | Forward | 5'-TTCGGGGTGTAGCGGTCGTC-3' |
Reverse | 5'-GCCCCAATACTAAATCACGACG-3' | ||
Unmethylated | Forward | 5'-GATGTTTGGGGTGTAGTGGTTGTT-3' | |
Reverse | 5'-CCACCCCAATACTAAATCACAACA-3' | ||
APC
| Methylated | Forward | 5'-TATTGCGGAGTGCGGGTC-3' |
Reverse | 5'-TCGACGAACTCCCGACGA-3' | ||
Unmethylated | Forward | 5'-GTGTTTTATTGTGGAGTGTGGGTT-3' | |
Reverse | 5'-CCAATCACAAACTCCCAACAA-3' | ||
TWIST
| Methylated | Forward | 5'-TTTCGGATGGGGTTGTTATCG-3' |
Reverse | 5'-GACGAACGCGAAACGATTTC-3' | ||
Unmethylated | Forward | 5'-TTGGATGGGGTTGTTATTGT-3' | |
Reverse | 5'-ACCTTCCTCCAACAAACACA-3' | ||
NES1
| Methylated | Forward | 5'-TTCGAAGTTTATGGCGTTTC-3' |
Reverse | 5'-TTATTTCCGCAATACGCGAC-3' | ||
Unmethylated | Forward | 5'-TTGTAGAGGTGGTGTTGTTT-3' | |
Reverse | 5'-CACACAATAAAACAAAAAACCA-3' | ||
RARβ2
| Methylated | Forward | 5'-GAACGCGAGCGATTCGAGT-3' |
Reverse | 5'-GACCAATCCAACCGAAACG-3' | ||
Unmethylated | Forward | 5'-GGATTGGGATGTTGAGAATGT-3' | |
Reverse | 5'-CAACCAATCCAACCAAAACAA-3' | ||
CDH1
| Methylated | Forward | 5'-TTAGGTTAGAGGGTTATCGCGT-3' |
Reverse | 5'-TAACTAAAAATTCACCTACCGAC-3' | ||
Unmethylated | Forward | 5'-TAATTTTAGGTTAGAGGGTTATTGT-3' | |
Reverse | 5'-CACAACCAATCAACAAC ACA-3' |
Statistical analysis
Results
Methylation status of genes in ER-negative and ER-positive tumors
Gene | ER negative (n = 65) | ER positive (n = 65) | P valuea | ||
---|---|---|---|---|---|
n
| % |
n
| % | ||
RASSF1A
| 36 | 55% | 60 | 92% | <0.0001 |
CCND2
| 26 | 40% | 45 | 69% | 0.0008 |
GSTP1
| 4 | 6% | 19 | 29% | 0.0006 |
TWIST
| 17 | 26% | 32 | 49% | 0.0066 |
APC
| 20 | 31% | 31 | 48% | 0.048 |
NES1
| 13 | 20% | 22 | 34% | NS |
RARβ2
| 18 | 28% | 17 | 26% | NS |
CDH1
| 53 | 82% | 54 | 83% | NS |
Gene | Premenopause (n = 70) | Postmenopause (n = 60) | P valuea | ||
---|---|---|---|---|---|
n
| % |
n
| % | ||
RASSF1A
| 52 | 74% | 44 | 73% | NS |
CCND2
| 40 | 57% | 31 | 52% | NS |
GSTP1
| 13 | 19% | 10 | 17% | NS |
TWIST
| 25 | 36% | 24 | 40% | NS |
APC
| 27 | 39% | 24 | 40% | NS |
Gene | T1 (n = 48) | T2/T3 (n = 82) | P valuea | ||
---|---|---|---|---|---|
n
| % |
n
| % | ||
RASSF1A
| 34 | 71% | 62 | 77% | NS |
CCND2
| 30 | 63% | 41 | 50% | NS |
GSTP1
| 9 | 19% | 14 | 17% | NS |
TWIST
| 18 | 38% | 31 | 38% | NS |
APC
| 17 | 35% | 34 | 41% | NS |
Gene | N0 (n = 76) | N1/N2 (n = 54) | P valuea | ||
---|---|---|---|---|---|
n
| % |
n
| % | ||
RASSF1A
| 54 | 71% | 42 | 78% | NS |
CCND2
| 42 | 55% | 29 | 54% | NS |
GSTP1
| 8 | 11% | 15 | 28% | 0.011 |
TWIST
| 28 | 37% | 21 | 39% | NS |
APC
| 30 | 39% | 21 | 39% | NS |
Methylation status of genes in ER-negative and ER-positive early-stage tumors
T1c (n = 48) | T2/T3 (n = 82) | |||||
---|---|---|---|---|---|---|
Gene | ER negative (n = 24) | ER positive (n = 24) | P valuea | ER negative (n = 41) | ER positive (n = 41) | P valuea |
RASSF1A
| 13 (54%) | 21 (88%) | 0.011 | 23 (56%) | 39 (95%) | <0.0001 |
CCND2
| 10 (42%) | 20 (83%) | 0.0029 | 16 (39%) | 25 (61%) | 0.0468 |
GSTP1
| 3 (13%) | 6 (25%) | NS | 1 (2%) | 13 (32%) | 0.0004 |
TWIST
| 6 (25%) | 12 (50%) | NS | 11 (27%) | 20 (49%) | 0.0404 |
APC
| 9 (38%) | 8 (33%) | NS | 11 (27%) | 23 (56%) | 0.0071 |
N0 (n = 76) | N1/N2 (n = 54) | |||||
---|---|---|---|---|---|---|
Genes | ER negative (n = 38) | ER positive (n = 38) | P valuea | ER negative (n = 27) | ER positive (n = 27) | P valuea |
RASSF1A
| 18 (47%) | 36 (95%) | <0.0001 | 18 (67%) | 24 (89%) | 0.0495 |
CCND2
| 16 (42%) | 26 (68%) | 0.021 | 10 (37%) | 19 (70%) | 0.014 |
GSTP1
| 1 (3%) | 7 (18%) | 0.025 | 3 (11%) | 12 (44%) | 0.0062 |
TWIST
| 11 (29%) | 17 (45%) | NS | 6 (22%) | 15 (56%) | 0.012 |
APC
| 11 (29%) | 19 (50%) | NS | 9 (33%) | 12 (44%) | NS |
Methylation status of tumor-related genes relevant to HER2/neu status
HER2/neu negative (n = 78) | HER2/neu positive (n = 29) | P valuea | |||
---|---|---|---|---|---|
Gene |
n
| % |
n
| % | |
RASSF1A
| 55 | 71% | 27 | 93% | 0.007 |
CCND2
| 44 | 56% | 15 | 52% | NS |
GSTP1
| 10 | 13% | 12 | 41% | 0.0019 |
TWIST
| 29 | 37% | 11 | 38% | NS |
APC
| 26 | 33% | 17 | 59% | 0.0185 |
NES1
| 22 | 28% | 7 | 24% | NS |
RARβ2
| 20 | 26% | 11 | 38% | NS |
CDH1
| 68 | 87% | 20 | 67% | 0.036 |
Gene | Double negative (n = 37) | Either ER positive or HER2/neu positive (n = 70) | P valuea | ||
---|---|---|---|---|---|
n
| % |
n
| % | ||
RASSF1A
| 18 | 47% | 64 | 91% | <0.0001 |
CCND2
| 16 | 43% | 43 | 61% | NS |
GSTP1
| 0 | 0% | 22 | 31% | <0.0001 |
TWIST
| 7 | 19% | 33 | 47% | 0.0031 |
APC
| 8 | 22% | 35 | 50% | 0.0035 |
NES1
| 8 | 22% | 21 | 30% | NS |
RARβ2
| 10 | 27% | 21 | 30% | NS |
CDH1
| 33 | 89% | 55 | 79% | NS |
Relation between the tumor-related genes with respect to methylation status
Genes | TWIST | RASSF1A | CCND2 | APC |
---|---|---|---|---|
GSTP1
| <0.0001 | 0.008 | 0.0006 | 0.0002 |
TWIST
| - | 0.047 | 0.003 | NS |
RASSF1A
| - | - | NS | 0.01 |
CCND2
| - | - | - | NS |