Two distinct pathways of p16 gene inactivation in gallbladder cancer

Table 1 The primer sequence of the p16 genes

Exon	Primer sequence	Size(bp)	Anneling temperature (°C)
Exon1α	F: 5'-GAGAGGGGGAGAGCAGGCAG-3'	250	58
	R: 5'-GCAAACTTCGTCCTCCAGAGT-3'
Exon2-1	F: 5'-AGCTTCCTTTCCGTCATGC-3'	203	56
	R: 5'-GCAGCACCACCAGCGTG-3'
Exon2-2	F: 5'-AGCCCAACTGCGCCGAC-3'	147	58
	R: 5'-CCAGGTCCACGGGCAGA-3'
Exon2-3	F: 5'-TGGACGTGCGCGATGC-3'	189	56
	R: 5'-GGAAGCTCTCAGGGTACAAATTC-3'
Exon3	F: 5'-CCGGTAGGGACGGCAAGAGA-3'	169	58
	R: 5'-CTGTAGGACCCTCGGTGACTGATGA-3'

Table 2 Association between p16 alterations and clinicopathological variables in gallbladder cancers

Clinicopathological variables	p16 protein expression			Methylation of p16INK4a			LOH at 9p21-22			Homozygous deletion of p16 gene
	Absent n	Present n	P	Absent n	Present n	P	Retension n	LOH n	P	Absent n	Present n	P
Age
< 65	13	7	0.789	5	15	0.753	10	10	0.427	13	6	0.481
> 65	19	12		9	22		12	19		24	7
Gender
Female	14	11	0.329	6	19	0.588	10	15	0.657	20	4	0.148
Male	18	8		8	18		12	14		17	9
Tumor type
Adenocarcinoma	28	18	0.401	14	32	0.148	19	27	0.423	35	10	0.068
Adenosquamous	4	1		0	5		3	2		2	3
Differentiation grade
Well-Moderate	22	17	0.144	12	27	0.907	18	21	0.115	29	10	0.16
Poor	6	1		2	5		1	6		6	0
Stage
0 and IA, IB	15	13	0.135	9	19	0.407	15	13	0.097	20	7	0.99
IIA and IIB	17	6		5	18		7	16		17	6
T factor
Tis and T1	6	6	0.296	3	9	0.828	8	4	0.060	11	1	0.11
T2 and T3	26	13		11	28		14	25		26	12
N factor
N0	21	16	0.150	9	28	0.416	19	18	0.054	28	8	0.329
N1	11	3		5	9		3	11		9	5

All P values were revealed by χ²-test.

Table 3 Association between p16 protein expression and 9p21-22

	p16 protein expression
	Negative	Positive	P
LOH (-)	12	10
LOH (+)	20	9	0.292

OH was estiomated by allelic status at D9S171, D9S1748, D9S942, D9S974, D9S1749. LOH: Loss of heterozygosity.

Table 4 Association between p16 protein expression and LOH at 9p21

	p16 protein expression
	Negative	Positive	P
LOH (-)	16	16
LOH (+)	16	3	0.0146

LOH was estiomated by allelic status at D9S1748, D9S942, D9S974.

Table 5 Association between p16 immunohistochemistry and p16 alterations in gallbladder cancers

	p16 immunohistochemistry
	Positive	Negative	P
Retension	3	3
Hypermethyaltion	9	6
LOH	1	3
LOH (multiple) + Hypermethylation	2	8
Multiple LOH	0	3
Homozygous deletion	4	9	P < 0.05

LOH: Loss of heterozygosity; Multiple LOH: LOH in 9p21 at more than two loci.