Hiraki M, Kitajima Y, Sato S, Nakamura J, Hashiguchi K, Noshiro H, Miyazaki K. Aberrant gene methylation in the peritoneal fluid is a risk factor predicting peritoneal recurrence in gastric cancer. World J Gastroenterol 2010; 16(3): 330-338 [PMID: 20082478 DOI: 10.3748/wjg.v16.i3.330]
Corresponding Author of This Article
Dr. Kohji Miyazaki, MD, PhD, Professor, Department of Surgery, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-8501, Japan. miyazak2@cc.saga-u.ac.jp
Article-Type of This Article
Original Article
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Table 2 Primer and probe sequence of the LightCycler system for qRT-PCR
Gene
Primer sequence
CEA
Forward
AGTCTATGCAGAGCCACCCAA
Reverse
GGGAGGCTCTGATTATTTACCCA
Probe
5' FAM-ACCCTTCATCACCAGCAACAACTCCAA-3'BHQ
CK19
Forward
GACATGCGAAGCCAATATGA
Reverse
TCAGTAACCTCGGACCTGCT
Probe
5' FAM-CTGGTTCACCAGCCGGACTGAAGAATT-3'BHQ
β-actin
Forward
CGAGCGCGGCTACAGCTT
Reverse
TCCTTAATGTCACGCACGATTT
Probe
5' FAM-ACCACCACGGCCGAGCGG-3'BHQ
Table 3 Clinicopathological factors of the patients (n = 80)
Clinicopathological factors
n (%)
Gender
Female
28 (35.0)
Male
52 (65.0)
Age (yr)
65.9 ± 10.8
Range
39-88
Histological type
tub
36 (45.0)
por/sig/muc
44 (55.0)
Classification
Group A: M-MP with CY(-) and P(-)
35 (43.8)
Group B: SS-SI with CY(-) and P(-)
31 (38.8)
Group C: CY(+) or P(+)
14 (17.5)
Clinical stage at the sample collection
I
37 (46.3)
II
8 (10.0)
III
18 (22.5)
IV
15 (18.8)
Peritoneal recurrence
2 (2.5)
Table 4 Relationship between the q-MSP results and depth of cancer invasion, CY and P classification n (%)
Methylation status
Classification
P-value
Group A: M-MP with CY(-) and P(-)
Group B: SS-SI with CY(-) and P(-)
Group C: CY(+) or P(+)
CHFR
< 0.05
(-) 77 (96)
35 (100)
31 (100)
11 (79)
(+) 3 (4)
0 (0)
0 (0)
3 (21)
p16
0.821
(-) 33 (41)
14 (40)
14 (45)
5 (36)
(+) 47 (59)
21 (60)
17 (55)
9 (64)
RUNX3
0.304
(-) 36 (45)
19 (54)
11 (35)
6 (43)
(+) 44 (55)
16 (46)
20 (65)
8 (57)
E cadherin
< 0.05
(-) 52 (65)
28 (80)
17 (55)
7 (50)
(+) 28 (35)
7 (20)
14 (45)
7 (50)
hMLH1
0.861
(-) 43 (54)
20 (57)
16 (52)
7 (50)
(+) 37 (46)
15 (43)
15 (48)
7 (50)
ABCG2
0.244
(-) 30 (38)
12 (34)
10 (32)
8 (57)
(+) 50 (63)
23 (66)
21 (68)
6 (43)
BNIP3
< 0.05
(-) 50 (63)
26 (74)
20 (65)
4 (29)
(+) 30 (38)
9 (26)
11 (35)
10 (71)
Table 5 Correlation between the multigene methylation of the peritoneal wash specimens form gastric cancer and the depth of cancer invasion, CY and P classification n (%)
Multigene methylation (CHFR, E-cadherin, BNIP3)
P-value
Less than 2 genes
2 or more genes
Classification
Group A: M-MP with CY(-) and P(-)
32 (91)
3 (9)
< 0.001
Group B: SS-SI with CY(-) and P(-)
25 (81)
6 (19)
Group C: CY(+) or P(+)
6 (43)
8 (57)
Table 6 Relationship between the qRT-PCR results and depth of cancer invasion, CY and P classification n (%)
Methylation status
Classification
P-value
Group A: M-MP with CY(-) and P(-)
Group B: SS-SI with CY(-) and P(-)
Group C: CY(+) or P(+)
CEA
0.352
(-) 39 (62)
17 (61)
17 (71)
5 (45)
(+) 24 (38)
11 (39)
7 (29)
6 (55)
CK19
< 0.05
(-) 37 (59)
21 (75)
13 (54)
3 (27)
(+) 26 (41)
7 (25)
11 (46)
8 (73)
Table 7 Comparison of the methylation status between cancerous tissue and peritoneal fluid in group C
Sample No.
Pathology
CY
P
Examined tissue
Methylation status
Tissue
Peritoneal fluid
CHFR
E-cadherin
BNIP3
CHFR
E-cadherin
BNIP3
A-03
por
+
-
Primary cancer
Negative
Positive
Negative
Negative
Positive
Negative
A-09
por
+
-
Primary cancer
Negative
Positive
Positive
Negative
Positive
Positive
A-16
por
+
+
Primary cancer
Positive
Negative
Positive
Positive
Negative
Positive
A-29
tub
+
+
Dissemination nodule
Negative
Positive
Negative
Negative
Positive
Positive
A-51
por
+
-
Metastatic lymph node
Positive
Positive
Positive
Positive
Positive
Positive
A-59
sig
-
+
Primary cancer
Negative
Positive
Positive
Negative
Negative
Negative
A-65
por
-
+
Metastatic ovarian tumor
Negative
Positive
Positive
Negative
Positive
Positive
A-69
sig
+
+
Primary cancer
Negative
Positive
Positive
Negative
Positive
Positive
A-78
tub
+
-
Primary cancer
Negative
Positive
Positive
Negative
Positive
Positive
Table 8 Correlation between peritoneal recurrence and multigene methylation and also the CK19 expression results in group B
Multigene methylation (CHFR, E-cadherin, BNIP3)
P-value
CK19
P-value
Less than 2 genes
2 or more genes
Negative
Positive
Peritoneal recurrence
Positive
0
2
< 0.05
1
1
0.943
Negative
23
3
10
9
Citation: Hiraki M, Kitajima Y, Sato S, Nakamura J, Hashiguchi K, Noshiro H, Miyazaki K. Aberrant gene methylation in the peritoneal fluid is a risk factor predicting peritoneal recurrence in gastric cancer. World J Gastroenterol 2010; 16(3): 330-338