Aberrant gene methylation in the peritoneal fluid is a risk factor predicting peritoneal recurrence in gastric cancer

Table 1 Primer and probe sequence of the LightCycler system for q-MSP

Gene		Primer sequence	Ref.
CHFR	Forward	TTTCGTGATTCGTAGGCGAC	[33]
	Reverse	CGACAACTAAAACGAAACCGA
	Probe	5' FAM-CGCGAAAATAAACGCGTAAAAAACGCTCG-3'BHQ
p16	Forward	TGGAATTTTCGGTTGATTGGTT	[34]
	Reverse	AACAACGTCCGCACCTCCT
	Probe	5' FAM- ACCCGACCCCGAACCGCG -3'BHQ
RUNX3	Forward	CGTTCGATGGTGGACGTGT	[35]
	Reverse	GACGAACAACGTCTTATTACAACGC
	Probe	5' FAM-CGCACGAACTCGCCTACGTAATCCG-3'BHQ
E-cadherin	Forward	AATTTTAGGTTAGAGGGTTATCGCGT	[34,36]
	Reverse	TAACTAAAAATTCACCTACCGAC
	Probe	5' FAM-CGCCCACCCGACCTCGCAT-3'BHQ
hMLH1	Forward	CGTTATATATCGTTCGTAGTATTCGTGTTT	[34]
	Reverse	CTATCGCCGCCTCATCGT
	Probe	5' FAM-CGCGACGTCAAACGCCACTACG-3'BHQ
ABCG2	Forward	TTGGGTAATTTGTGCGTTA
	Reverse	CTACGAAAATCACCAAACGCTC
	Probe	5' FAM-TTAATCGCCGTCACTAACCGA-3'BHQ
BNIP3	Forward	TAGGATTCGTTTCGCGTACG	[37]
	Reverse	ACCGCGTCGCCCATTAACCGCG
	Probe	5' FAM-CGTAAAATACGTATAACACGTACGAC-3'BHQ
β-actin	Forward	TGGTGATGGAGGAGGTTTAGTAAGT	[34]
	Reverse	AACCAATAAAACCTACTCCTCCCTTAA
	Probe	5' FAM-ACCACCACCCAACACACAATAACAAACACA-3'BHQ

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)

tub: Tubular adenocarcinoma; por: Poorly differentiated adenocarcinoma; sig: Signet-ring cell carcinoma; muc: Mucinous adenocarcinoma. Tumor invasion of mucosa and/or muscularis mucosa (M), muscularis propia (MP) or subserosa (SS), tumor invasion of adjacent structures (SI). Cancer cells on peritoneal cytology (CY), peritoneal metastasis (P).

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

Abbreviations as in Table 1.

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