Background
Methods
Study subjects and DNA isolation
Characteristics | No. of patients (%) |
---|---|
Gender | |
Male | 66 (69) |
Female | 30 (31) |
Age (mean years ± S.D.) | 58.9 ± 9.2 |
≤ 60 | 56 (58) |
> 60 | 40 (42) |
Smoking history (pack-years) | |
0 | 30 (31) |
1-39 | 35 (37) |
≥ 40 | 31 (32) |
Tumor size (mean cm ± S.D.) | 3.9 ± 1.7 |
1-3 | 37 (38) |
3-5 | 44 (46) |
> 5 | 15 (16) |
Histologic type | |
Adenocarcinoma (ADC) | 30 (31) |
Brochoalveplar cell (BAC) | 6 (6) |
Adenocarcinoma (non-BAC) | 24 (25) |
Squamous (SCC) | 66 (69) |
Histologic stage | |
I | 54 (56) |
II | 32 (34) |
III | 10 (10) |
Lymph node metastasis | |
No | 70 (73) |
Yes | 26 (27) |
Pleural indentation | |
No | 75 (78) |
Yes | 21 (22) |
Invasion or Adhesion | |
No | 65 (68) |
Yes | 31 (32) |
Sodium bisulfite treatment
Quantitative methylation-specific PCR (Q-MSP) analysis
Genes | Forward primer sequence (5'→3') | Probe sequence (5'→3') | Reverse primer sequence (5'→3') |
---|---|---|---|
CALCA
| GTTTTGGAAGTATGAGGGTGACG | 6FAM-ATTCCGCCAATACACAACAACCAATAAACG-TAMRA | TTCCCGCCGCTATAAATCG |
CDH1
| AATTTTAGGTTAGAGGGTTATCGCGT | 6FAM-CGCCCACCCGACCTCGCAT-TAMRA | TCCCCAAAACGAAACTAACGAC |
DAPK1
| GGATAGTCGGATCGAGTTAACGTC | 6FAM-TTCGGTAATTCGTAGCGGTAGGGTTTGG-TAMRA | CCCTCCCAAACGCCGA |
IRX2
| GCGGGTCGTTTAGGTTAGTATTCG | 6FAM-CCCTCCATCCACGCCCGACCGAAA-TAMRA | CGCCGAACAACGAACAAAACG |
TIMP3
| GCGTCGGAGGTTAAGGTTGTT | 6FAM-AACTCGCTCGCCCGCCGAA-TAMRA | CTCTCCAAAATTACCGTACGCG |
PAX6
| ATATAGGACGGCGGTTTAGGTTG | 6FAM-CCCAAAATCCGACCGACTCCAACCCCTA-TAMRA | TTCCGACCGAACGAAAACCTAC |
β-Actin
| TGGTGATGGAGGAGGTTTAGTAAGT | 6FAM-ACCACCACCCAACACACAATAACAAACACA-TAMRA | AACCAATAAAACCTACTCCTCCCTTAA |
Statistical analysis
Results
Demographics
Frequent promoter methylation in NSCLC
Association of promoter methylaton with clinicopathological charactristics of NSCLC
Genes | Gender (n = 96) | Histologic type (n = 96) | Smoking history (n = 96) | ||||||
---|---|---|---|---|---|---|---|---|---|
Female | Male |
P
| SCC | ADC |
P
| Y | N |
P
| |
(n = 30) | (n = 66) | (n = 66) | (n = 30) | (n = 66) | (n = 30) | ||||
CALCA
| 145.0 ± 149.6 | 112.1 ± 106.5 | 0.28 | 152.1 ± 152.1 | 96.4 ± 90.6 | 0.07 | 155.2 ± 150.2 | 89.6 ± 92.7 | 0.03* |
CDH1
| 33.1 ± 18.7 | 31.5 ± 21.2 | 0.71 | 35.4 ± 19.2 | 26.3 ± 18.6 | 0.03* | 32.2 ± 18.7 | 33.4 ± 21.3 | 0.77 |
DAPK1
| 18.5 ± 59.6 | 23 ± 45.2 | 0.71 | 26.2 ± 65.8 | 5.9 ± 7.6 | 0.09 | 17.4 ± 59.6 | 25.4 ± 45.2 | 0.52 |
IRX2
| 96.4 ± 131.5 | 63.9 ± 34.8 | 0.19 | 92.3 ± 115.7 | 72.9 ± 103.6 | 0.43 | 95.3 ± 132.1 | 66.3 ± 36.1 | 0.24 |
TIMP3
| 60.5 ± 72.8 | 50.9 ± 39.1 | 0.50 | 64.9 ± 71.7 | 41.3 ± 39.8 | 0.09 | 61.5 ± 72.8 | 48.8 ± 38.9 | 0.37 |
PAX6
| 23.4 ± 15.1 | 24.2 ± 13.8 | 0.78 | 24.0 ± 15.5 | 23.1 ± 12.8 | 0.79 | 23.9 ± 15.6 | 23.3 ± 12.6 | 0.85 |
Genes
|
Lymph node metastasis (n = 96)
|
Pleural indentation (n = 96)
|
Invasion or Adhesion (n = 96)
| ||||||
Y
|
N
|
P
|
Y
|
N
|
P
|
Y
|
N
|
P
| |
(n = 26)
|
(n = 70)
|
(n = 21)
|
(n = 75)
|
(n = 31)
|
(n = 65)
| ||||
CALCA
| 175.7 ± 175.1 | 119.5 ± 119.1 | 0.08 | 129.9 ± 132.5 | 136 ± 140.2 | 0.86 | 174.5 ± 160 | 115.1 ± 122.5 | 0.048* |
CDH1
| 31.0 ± 13.9 | 33.1 ± 21.2 | 0.64 | 35.3 ± 21.2 | 31.8 ± 18.9 | 0.47 | 30.7 ± 16.6 | 33.5 ± 20.7 | 0.52 |
DAPK1
| 40.3 ± 100.5 | 12.3 ± 18.5 | 0.03* | 12.4 ± 11.9 | 21.9 ± 62.3 | 0.49 | 10.9 ± 10.6 | 24.2 ± 66.6 | 0.27 |
IRX2
| 42.7 ± 51 | 91.2 ± 127.2 | 0.47 | 73.1 ± 38.4 | 89.9 ± 124.9 | 0.54 | 71.8 ± 48.3 | 93.1 ± 131.6 | 0.38 |
TIMP3
| 69.3 ± 91 | 53.2 ± 51 | 0.28 | 53.9 ± 42.9 | 58.6 ± 69.2 | 0.77 | 52.7 ± 53.1 | 59.8 ± 69.1 | 0.61 |
PAX6
| 23.2 ± 14.2 | 23.9 ± 14.9 | 0.83 | 25.2 ± 13.9 | 23.3 ± 14.9 | 0.59 | 25.8 ± 15.7 | 22.7 ± 14.1 | 0.32 |
Genes | Male vs. Female | Age (per 10 years) | Smoking history | Pack-years1 | SCC vs. ADC |
---|---|---|---|---|---|
CALCA
| 0.73 (0.28-1.90) | 0.94 (0.58-1.52) | 1.43 (0.57-3.57) | 0.88 (0.51-1.51) | 2.21 (0.89-5.47) |
CDH1
| 2.13 (0.86-5.22) | 0.83 (0.53-1.30) | 1.41 (0.59-3.39) | 0.91 (0.55-1.50) | 2.63 (1.05-6.60)* |
DAPK1
| 1.45 (0.43-4.87) | 1.16 (0.60-2.24) | 2.11 (0.64-6.92) | 1.83 (0.84-3.98) | 6.64 (1.85-23.8)* |
IRX2
| 1.29 (0.54-3.06) | 0.76 (0.48-1.19) | 1.06 (0.45-2.52) | 0.91 (0.55-1.50) | 2.89 (1.17-7.13)* |
TIMP3
| 1.09 (0.46-2.60) | 1.09 (0.70-1.71) | 1.09 (0.46-2.60) | 1.27 (0.76-2.11) | 2.51 (1.01-6.21)* |
PAX6
| 1.04 (0.37-2.88) | 1.19 (0.70-2.03) | 0.78 (0.27-2.25) | 0.93 (0.51-1.69) | 1.04 (0.37-2.88) |
Genes
|
Tumor size
2
|
Lymph node metastasis
|
Pleural indentation
|
Invasion or Adhesion
|
Histologic stage
3
|
CALCA
| 1.01 (0.55-1.88) | 1.74 (0.62-4.91) | 1.18 (0.41-3.41) | 2.44 (0.88-6.79) | 2.13 (1.01-4.47)* |
CDH1
| 1.20 (0.67-2.13) | 1.02 (0.41-2.51) | 1.79 (0.67-4.76) | 0.96 (0.41-2.27) | 1.11 (0.62-2.02) |
DAPK1
| 1.81 (0.72-4.54) | 5.17 (0.64-41.95) | 1.63 (0.33-8.02) | 1.70 (0.43-6.67) | 1.24 (0.50-3.08) |
IRX2
| 1.57 (0.88-2.83) | 1.17 (0.47-2.88) | 1.37 (0.52-3.63) | 0.86 (0.36-2.01) | 1.14 (0.63-2.07) |
TIMP3
| 1.67 (0.92-3.01) | 2.05 (0.82-5.10) | 1.16 (0.44-3.05) | 0.69 (0.29-1.66) | 1.88 (1.01-3.49)* |
PAX6
| 0.78 (0.40-1.54) | 0.74 (0.26-2.09) | 2.04 (0.54-7.69) | 1.84 (0.61-5.56) | 1.76 (0.79-3.93) |
Genes | Smoking history | SCC vs. ADC | Lymph node metastasis | Pleural indentation |
---|---|---|---|---|
CALCA
| 1.07 (0.40-2.88) | 2.66 (0.85-8.32) | 1.43 (0.46-4.41) | 2.19 (0.61-7.79) |
CDH1
| 0.98 (0.37-2.58) | 6.77 (1.71-26.8)* | 0.78 (0.29-2.13) | 5.27 (1.27-21.9)* |
DAPK1
| 0.83 (0.19-3.73) | 10.7 (2.16-52.6)* | 3.07 (0.32-29.4) | 6.98 (1.06-45.8)* |
IRX2
| 0.69 (0.26-1.83) | 6.38 (1.77-23.0)* | 0.85 (0.31-2.33) | 3.89 (1.01-15.0)* |
TIMP3
| 0.78 (0.30-2.02) | 3.37 (1.03-11.0)* | 1.75 (0.65-4.75) | 2.66 (0.77-9.16) |
PAX6
| 0.67 (0.21-2.08) | 1.82 (0.50-6.57) | 0.77 (0.25-2.38) | 2.66 (0.58-12.3) |