Detecting plasma SHOX2, HOXA9, SEPTIN9, and RASSF1A methylation and circulating cancer cells for cholangiocarcinoma clinical diagnosis and monitoring

Table 1 The clinical characteristics of the 156 patients with cholangiocarcinoma and 98 controls

Clinicopathological data	n	SHOX2 positive	SEPT9 positive	HOXA9 positive	RASSF1A positive	Comprehensive methylation Positive	CTC counts/5 mL
Tumor location
iCCA	33	24.24%	24.24%	42.42%	27.27%	57.58%	10
pCCA	83	21.69%	27.71%	56.63%	27.71%	66.27%	12
dCCA	40	27.5%	32.5%	50%	35%	67.5%	14
iCCA vs dCCA		0.795	0.604	0.638	0.614	0.467	0.342
Gender
Male	87	22.99%	34.48%	47.13%	32.18%	68.97%	13
Female	69	24.64%	20.29%	57.97%	26.09%	59.42%	10
Male vs female		0.851	0.702	0.199	0.481	0.24	0.760
Age at diagnosis
≤ 60 years	91	21.98%	26.37%	41.76%	26.37%	59.34%	11
> 60 years	65	26.15%	30.77%	66.15%	33.85%	72.31%	14
Median age (years)	57
Mean age (years)	59
≤ 60 years vs > 60 years		0.571	0.591	0.195	0.374	0.126	0.276
Tumor stage
I-II	11	9.09%	9.09%	18.18%	9.09%	36.36%	3
III-IV	145	24.83%	29.66%	54.48%	31.03%	66.21%	13
I-II vs III-IV		0.237	0.144	0.020	0.176	0.046	0.001
Lymphatic invasion
L0	105	25.71%	27.62%	48.57%	32.38%	62.86%	4
L1	51	19.61%	29.41%	58.82%	23.53%	68.63%	16
L0 vs L1		0.400	0.815	0.331	0.255	0.479	0.001
Follow-up
Follow up available	43
Median follow-up months	14
Mean follow-up months	17
Range (months)	0-58
Deceased	23
Censored	20
Healthy donors
Male	42	3/42	2	2	3	3	1
Female	28	1/28	1	1	1	1	0
≤ 60 years	51	1	1	0	1	1	0
> 60 years	19	2	2	2	2	2	1
Benign bile duct patients
Male	21	2/21	1	1	1	1	0
Female	7	0	0	0	1	1	0
≤ 60 years	23	2	0	1	2	2	0
> 60 years	5	1	1	1	1	1	0

CTC: Circulating tumor cell; CCA: Cholangiocarcinoma.

Table 2 Performance of biomarkers and their combinations for diagnosing cholangiocarcinoma

Biomarker or combination	Positive test, CCA No.		Negative test, CCA No.		Sensitivity (%)	Early-stage CCA (%)	Specificity (%)	PPV (%)
SEPTIN9	44	3	112	95	28.21	9.09	96.94	54.72
HOXA9	81	3	75	95	51.92	18.18	96.94	69.29
SHOX2	37	2	119	96	23.72	9.09	97.96	52.36
RASSF1A	46	2	110	96	29.49	9.09	97.96	55.91
CA199	80	12	47	86	51.28	9.09	87.76	65.35
SEPTIN9 + HOXA9	81	3	75	95	51.92	18.18	96.94	69.29
SEP + HOXA9 + SHO	88	3	68	95	56.41	27.27	96.94	72.05
SEP + HOXA9 + SHO + RASS	101	6	55	92	64.74	36.36	93.88	75.98
Sep + HOXA9 + SHO + RASS + CA199	107	12	49	86	68.59	45.45	87.76	75.98

CA199: Carbohydrate antigen 19-9; CCA: Cholangiocarcinoma; PPV: Positive predictive value.

Table 3 Results of univariate and multivariate survival analyses (Cox proportional hazard models)

Variables		Number of patients	Univariate		Multivariate
			HR (95%CI)	P value	HR (95%CI)	P value
Tumor location	(iCCA vs dCCA)	33	1.246 (0.495-3.138)	0.624
Gender	(male vs female)	43	1.536 (0.673-3.861)	0.284
Age at diagnosis	(≤ 60 vs > 60 years)	43	1.527 (0.673-3.467)	0.273
Tumor stage	(I-II vs III-IV)	43	3.13 (1.06-9.242)	0.039	2.871 (1.056-7.653)	0.03
Lymphatic invasion	(V0 vs V1)	38	2.902 (1.069-7.874)	0.037	3.425 (1.081-8.523)	0.035
CA199	≤ 41 U/mL vs > 41 U/mL)	43	2.41 (1.079-5.384)	0.028
SHOX2 methylation	(SHOX2- vs SHOX2+)	43	1.713 (0.623-4.713)	0.210
SEPT9 methylation	(SEPT9- vs SEPT9+)	43	1.757 (0.698-4.422)	0.166
HOXA9 methylation	(HOXA9- vs HOXA9+)	43	1.718 (0.743-3.975)	0.200
RASSF1A methylation	(RASSF1A- vs RASSF1A+)	43	2.684 (1.0-7.557)	0.013
CTC count > 10/5 mL	(> 10/5 mL vs ≤ 10/5 mL)	43	3.542 (1.354-9.266)	0.027	3.24 (1.465-7.562)	0.021

CTC: Circulating tumor cells; CA199: Carbohydrate antigen 19-9; CCA: Cholangiocarcinoma; HR: Hazard ratio.