Amplification of D22S283 as a favorable prognostic indicator in liver fluke related cholangiocarcinoma

Table 1 Patients’ data including survival time

No.	Age(yr)	Sex	Staging	Histological grading	Invasion			Survival time (wk)
					Vessel	Nerve	Lymph
1	61	Female	IVa	Adenosquamous	Yes	No	Yes	16.85
2	50	Male	IVb	Well differentiated	Yes	Yes	Yes	27.28
3	56	Male	IVb	Poorly differentiated	Yes	No	Yes	4.85
4	54	Male	IVa	Well differentiated	Yes	No	No	56.57
5	46	Male	IVb	Adenosquamous	Yes	No	Yes	7.71
6	53	Male	IVa	Well differentiated	Yes	No	Yes	42.14
7	64	Male	IVa	Poorly differentiated	Yes	No	Yes	7.28
8	66	Male	IVb	Poorly differentiated	Yes	No	Yes	7.00
9	72	Male	IVb	Unclassified	No	No	Yes	20.14
10	64	Female	IVb	Unclassified	Yes	Yes	Yes	7.14
11	49	Male	IVb	Well differentiated	Yes	No	No	15.57
12	53	Male	III	Poorly differentiated	Yes	No	Yes	242.85
13	53	Female	IVa	Well differentiated	Yes	No	No	2.00
14	62	Male	IVb	Adenosquamous	Yes	Yes	Yes	3.00
15	56	Male	II	Poorly differentiated	Yes	No	Yes	30.71
16	55	Male	IVb	Moderately differentiated	Yes	No	Yes	72.57
17	68	Male	IVb	Moderately differentiated	Yes	Yes	Yes	32.00
18	72	Female	IVb	Poorly differentiated	Yes	No	No	18.57
19	65	Female	IVb	Well differentiated	Yes	Yes	Yes	20.85
20	52	Male	IVa	Moderately differentiated	Yes	Yes	Yes	36.71
21	53	Male	IVb	Unclassified	Yes	No	No	27.14
22	66	Male	IVb	Moderately differentiated	Yes	Yes	Yes	10.42
23	49	Male	IVb	Well differentiated	No	Yes	Yes	67.71
24	56	Male	IVb	Adenosquamous	Yes	No	Yes	10.00
25	41	Male	IVb	Moderately differentiated	No	Yes	No	47.14
26	67	Female	IVb	Poorly differentiated	No	No	Yes	118.28
27	63	Male	IVa	Unclassified	Yes	Yes	Yes	73.85
28	39	Female	IVa	Well differentiated	Yes	Yes	Yes	30.42
29	44	Male	IVb	Unclassified	Yes	Yes	Yes	18.71
30	60	Female	IVb	Poorly differentiated	Yes	No	Yes	75.71
31	40	Male	III	Poorly differentiated	Yes	No	Yes	26.57
32	62	Male	IVb	Unclassified	No	No	Yes	40.00
33	43	Female	IVb	Moderately differentiated	No	Yes	Yes	29.42
34	70	Male	IVa	Unclassified	Yes	No	Yes	79.42
35	63	Female	III	Unclassified	No	No	No	80.71
36	61	Male	IVb	Moderately differentiated	No	Yes	Yes	77.28
37	48	Male	III	Unclassified	No	No	No	94.00
38	55	Male	IVb	Moderately differentiated	Yes	Yes	Yes	12.14
39	50	Female	IVb	Moderately differentiated	No	Yes	Yes	40.14
40	39	Male	IVb	Adenosquamous	Yes	No	Yes	9.14
41	58	Male	IVb	Well differentiated	Yes	Yes	Yes	70.42
42	40	Female	III	Moderately differentiated	No	Yes	No	17.42
43	36	Female	IVb	Well differentiated	No	Yes	Yes	35.14
44	75	Male	IVb	Poorly differentiated	Yes	No	Yes	22.14
45	40	Male	III	Well differentiated	No	Yes	Yes	3.42
46	52	Male	IVa	Adenosquamous	No	Yes	Yes	49.00
47	49	Male	IVb	Moderately differentiated	No	Yes	Yes	27.42
48	63	Male	IVb	Well differentiated	Yes	Yes	Yes	18.28
49	50	Male	IVb	Adenosquamous	Yes	Yes	Yes	35.00
50	56	Male	III	Poorly differentiated	Yes	No	Yes	41.14
51	61	Male	IVb	Poorly differentiated	Yes	No	No	3.00
52	58	Male	IVb	Well differentiated	Yes	No	Yes	13.00
53	55	Female	IVb	Well differentiated	Yes	Yes	Yes	67.85
54	55	Female	IVb	Poorly differentiated	No	Yes	Yes	40.85
55	50	Male	nd	Well differentiated	Yes	No	Yes	10.71
56	38	Male	IVb	Well differentiated	Yes	Yes	Yes	27.85
57	46	Female	nd	Well differentiated	Yes	Yes	Yes	9.71
58	43	Male	IVb	Well differentiated	Yes	No	Yes	65.00
59	54	Male	IVa	Well differentiated	No	No	No	9.14
60	42	Male	IVb	Moderately differentiated	No	Yes	Yes	7.14
61	34	Male	IVa	Well differentiated	Yes	Yes	Yes	9.00
62	61	Female	IVa	Unclassified	No	No	Yes	89.14
63	62	Female	IVb	Poorly differentiated	Yes	Yes	Yes	45.28
64	51	Male	IVa	Well differentiated	Yes	Yes	Yes	55.42
65	46	Male	IVa	Poorly differentiated	Yes	Yes	Yes	18.71

Table 2 Sequences of markers located at chromosomal region 22q12-qter including two housekeeping genes

Gene/marker	Band	Primer sequence (5’-3’)
D22S283	22q12.3	5’ ACC AAC CAG CAT CAT CAT 3’ 5’ AGC TCG GGA CTT TCT GAG 3’
D22S423	22q13.1-13.2	5’ TGC AAA CTC AGC CTG GA 3’ 5’ ACC AAC TGA CTC GTT TAG GTC AT 3’
D22S274	22q13.3	5’ GTC CAG GAG GTT GAT GC 3’ 5’ AGT GCC CAT TTC TCA AAA TA 3’
NF2	22q12.2	5’ AAG AGC AAG CAT CTG CAG GA 3’ 5’ TGG TAT TGT GCT TGC TGC TG 3’
TIMP3	22q12.3	5’ TGT CTC TGG ACC GAC ATG CT 3’ 5’ TGG CGC TCA GGG ATC TGT G 3’
ST13	22q13.2	5’ GTT ACA CTA TTT AAG AGC TGA AT 3’ 5’ GGT CTT CTA CTT AGA AAA ACC TA 3’
TOB2	22q13.2-q13.31	5’ GAA GAC ACC CCT TTG TGG AA 3’ 5’ TCT GTG GTC TTG GGT GCT C 3’
BIK	22q13.31	5’ ATG ACC ACT GCC CTG GAG 3’ 5’ CTA AAC ACA GGC CAC AGT TAA CC 3’
TP	22q13.33	5’ GGG GCT CAA GTC GCG AGG 3’ 5’ CCT GCG GGG ATG CCT GAC 3’
β-actin (Reference)	7p15-p12	5’ TCA CCC ACA CTG TGC CCA TCT ACG A 3’ 5’ CAG CGG AAC CGC TCA TTG CCA ATG G 3’
GAPDH (Reference)	12p13	5’ ACA GTC CAT GCC ATC ACT GCC 3’ 5’ GCC TGC TTC ACC ACC TTC TTG 3’

Table 3 Cox regression analysis using univariate and multivariate models in CCA patients

Variables	Categories	Univariate analysis		Multivariate analysis
		P-value	HR (95% CI)	P-value	HR (95% CI)
Stage	IVa + IVb vs II + III	NS	1.560 (0.660-3.690)
Histology	Poorly vs Unclassified	NS	1.152 (0.507-2.619)
	Moderate vs Unclassified	NS	0.994 (0.473-2.087)
	Well diff vs Unclassified	NS	0.929 (0.429-2.013)
Vessel invasion	Yes vs No	0.021	2.080 (1.120-3.877)	0.042	1.911 (1.022-3.571)
Nerve invasion	Yes vs No	NS	1.328 (0.770-2.293)
Lymphatic invasion	Yes vs No	NS	1.591 (0.716-3.536)
TP	Deletion vs Normal	NS	0.791 (0.314-1.997)
	Amplification vs Normal	NS	1.034 (0.576-1.857)
D22S274	Deletion vs Normal	NS	1.186 (0.625-2.250)
	Amplification vs Normal	NS	0.869 (0.439-1.718)
BIK	Deletion vs Normal	NS	1.271 (0.704-2.294)
	Amplification vs Normal	NS	1.714 (0.771-3.810)
TOB2	Deletion vs Normal	NS	1.176 (0.277-4.995)
	Amplification vs Normal	NS	0.945 (0.545-1.641)
ST13	Deletion vs Normal	NS	0.913 (0.466-1.788)
	Amplification vs Normal	NS	0.938 (0.500-1.761)
D22S423	Deletion vs Normal	NS	1.183 (0.667-2.097)
	Amplification vs Normal	NS	0.896 (0.366-2.192)
D22S283	Deletion vs Normal	NS	0.793 (0.397-1.585)
	Amplification vs Normal	0.003	0.384 (0.202-0.728)	0.006	0.411 (0.217-0.779 )
TIMP3	Deletion vs Normal	NS	1.758 (0.527-5.864)
	Amplification vs Normal	NS	0.736 (0.418-1.296)
NF2	Deletion vs Normal	NS	0.929 (0.420-2.055)
	Amplification vs Normal	NS	0.621 (0.344-1.122)