High Levels of Serum IgG for Opisthorchis viverrini and CD44 Expression Predict Worse Prognosis for Cholangiocarcinoma Patients after Curative Resection

Figures & data

Table 1 Patient Characteristics by Level of OV IgG

Variables	Overall	Level of OV IgG			p-value
		Negative	Low-Positive	High-Positive
Overall	176	54 (30.7%)	87 (49.4%)	35 (19.9%)
Age (mean, year)	59.6	59.1	60.1	59.4
Gender					0.10
Female	68 (38.6%)	23 (42.6%)	37 (42.5%)	8 (22.8%)
Male	108 (61.4%)	31 (57.4%)	50 (57.5%)	27 (77.2%)
Tumor site					0.57
Intrahepatic	123 (70%)	36 (66.7%)	64 (73.6%)	23 (65.7%)
Extrahepatic	53 (30%)	18 (33.3)	23 (26.4%)	12 (34.3%)
Histology					0.35
Papillary	97 (55.1%)	33 (61.1%)	45 (51.7%)	19 (54.3%)
Non-papillary	79 (44.9%)	21 (38.9%)	42 (48.3%)	10 (45.7%)
Stage					0.39
0	11 (6.3%)	7 (13%)	4 (4.6%)	0
I	27 (15.3%)	7 (13%)	15 (17.2%)	5 (14.3%)
II	27 (15.3%)	9 (16.6%)	14 (16.1%)	4 (11.4%)
III	103 (58.5%)	29 (53.7%)	50 (57.5%)	24 (68.5%)
IV	8 (4.6%)	2 (3.7%)	4 (4.6%)	2 (5.7%)
Lymph node metastasis					0.10
No	84 (47.7%)	31 (57.4%)	41 (47.1%)	12 (34.3%)
Yes	92 (52.3%)	23 (42.6%)	46 (52.9%)	23 (65.7%)
Surgical margin					0.48
Negative	96 (54.5%)	12 (48%)	22 (40%)	9 (56.3%)
Positive	80 (45.5%)	13 (52%)	33 (60%)	7 (43.7%)
Adjuvant chemotherapy					0.07
Yes	53 (30%)	13 (24%)	33 (37.9%)	7 (20%)
No	123 (70%)	41 (76%)	54 (62.1%)	28 (80%)

Table 2 The Expression of CD44s, CD44v6, CD44v8-10 and Nestin by Level of OV IgG

Variables	Overall	Level of OV IgG			p-value
		Negative	Low-Positive	High-Positive
Overall	176	54 (30.7%)	87 (49.4%)	35 (19.9%)
CD44	135	41	67	27	0.98
Negative	81 (60%)	25 (61%)	40 (59.7%)	16 (59.3%)
Positive	54 (40%)	16 (39%)	27 (40.3%)	11 (40.7%)
CD44v6	138	43	68	27	0.59
Low	42 (30.4%)	11 (25.6%)	21 (30.9%)	10 (37%)
High	96 (69.6%)	32 (74.4%)	47 (69.1%)	17 (63%)
CD44v8	137	43	67	27	0.37
Low	50 (36.5%)	12 (27.9%)	27 (40.3%)	11 (40.7%)
High	87 (63.5%)	31 (72.1%)	40 (59.7%)	16 (59.3%)
Nestin	130	40	64	26	0.65
Low	109 (83.9%)	32 (80%)	54 (84.4%)	23 (88.5%)
High	21 (16.1%)	8 (20%)	10 (15.6%)	3 (11.5%)

Figure 1 Representative IHC staining of CD44s, CD44v6, CD44v8-10 and Nestin in human CCA tissues (magnification 20X).

Table 3 Median Survival Time and Multivariate Analyses of Clinicopathological Data and CSC Markers

Variables	N	IR/100	MST, Months (95% CI)	Crude HR (95% CI)	Adjusted HR (95% CI)	p-value
Overall	176	2.7	18.6 (15.8–28.9)
Level of OV IgG
Negative	54	1.9	29.2 (15.9–42.1)	1	1
Low-positive	87	2.9	18.6 (15.6–24.2)	1.45 (0.98–2.15)	1.50 (0.95–2.37)	0.08
High-positive	35	4.0	10.5 (4.8–17.4)	1.99 (1.24–3.19)	2.24 (1.27–3.93)	<0.01*
Gender
Female	68	2.8	16.3 (12.8–28.8)	1	–	–
Male	108	2.6	18.9 (15.6–24.2)	0.97 (0.69–1.36)
Tumor site
Intrahepatic	123	2.7	17.4 (13.1–23.6)	1	–	–
Extrahepatic	53	2.7	20.8 (15.3–29.5)	0.97 (0.68–1.39)
Histology
Non-papillary	79	4.1	13.0 (8.9–16.3)	1	1
Papillary	97	2.0	26.9 (19.7–33.8)	0.56 (0.40–0.68)	0.72 (0.49–1.06)	0.10
Stage
0, I, II	65	1.1	51.8 (35.7–123.9)	1	1
III, IV	111	5.5	12.7 (9.2–15.9)	4.21 (2.85–6.23)	2.78 (1.46–5.29)	<0.01*
Lymph node metastasis
No	84	1.4	41.5 (28.1–63.0)	1	1
Yes	92	6.2	12.5 (8.5–15.9)	3.73 (2.58–5.39)	1.53 (0.86–2.71)	0.14
Resection margin
Negative	96	1.9	30.2 (21.8–41.5)	1	1
Positive	80	4.5	12.7 (8.9–16.3)	2.28 (1.63–3.19)	1.15 (0.76–1.72)	0.50
Adjuvant chemotherapy
Yes	53	2.8	19.2 (13.7–25.9)	1	–	–
No	123	2.6	18.1 (10.1–24.2)	1.06 (0.75–1.5)
CD44
Negative	81	2.4	22.8 (15.6–29.5)	1	1
Positive	54	4.2	15.8 (8.9–18)	1.52 (1.04–2.23)	1.43 (0.97–2.11)	0.07
CD44v6
Low	42	3.1	19.7 (11.1–24)	1	–	–
High	96	2.7	17.0 (13.2–24.9)	0.95 (0.64–1.42)
CD44v8-10
Low	50	3.0	17.4 (12.5–24.9)	1	–	–
High	87	2.7	18.6 (13.2–24)	0.90 (0.62–1.32)
Nestin
Low	109	2.9	16.8 (12.9–21.8)	1	–	–
High	21	2.2	26.2 (9.2–35.9)	0.78 (0.46–1.33)

Note: *P value equal to or less than 0.05 was considered statistically significant.

Abbreviations: N, number of patients; HR, hazard ratio; IR/100, incidence rate per 100 persons-month; MST, median survival time.

Figure 2 Kaplan–Meier analysis of serum IgG for OV and CD44s expression in 176 CCA patients. (A) Survival by different levels of serum IgG for OV. (B) Survival by different levels of CD44s.

Table 4 Survival Analysis by Level of Serum OV IgG, Clinicopathological Factors and IHC Results (CD44s, CD44v6, CD44v8-10, Nestin) According to Tumor Stage

Variables		Early Stage			Late Stage
	N	Crude HR (95% CI)	p-value	N	Crude HR (95% CI)	p-value
Gender (Ref: Female)	65			111
Male		0.82 (0.49–1.38)	0.447		1.03 (0.70–1.50)	0.901
Tumor site (Ref: Intrahepatic)	65			111
Extrahepatic		1.05 (0.59–1.56)	0.866		0.78 (0.52–1.16)	0.211
Histology (Ref: Non-papillary)	65			111
Papillary		0.55 (0.32–0.92)	0.024*		0.70 (0.48–1.03)	0.069
Resection margin (Ref: Negative)	65			111
Positive		0.97 (0.54–1.75)	0.929		1.59 (1.08–2.35)	0.018*
Adjuvant chemotherapy (Ref: Yes)	65			111
No		1.25 (0.69–2.26)	0.455		1.50 (1.00–2.24)	0.048*
Level of OV IgG (Ref: Negative)	65			111
Low-positive		1.41 (0.82–2.44)	0.212		1.12 (0.72–1.75)	0.607
High-positive		2.01 (0.91–4.46)	0.085		1.36 (0.79–2.32)	0.269
CD44s (Ref: Negative)	47			88
Positive		2.31 (1.25–4.26)	0.008*		1.20 (0.78–1.84)	0.416
CD44v6 (Ref: Low)	49			89
High		0.83 (0.45–1.54)	0.557		0.86 (0.55–1.36)	0.524
CD44v8-10 (Ref: Low)	49			88
High		1.41 (0.74–2.68)	0.292		1.00 (0.65–1.54)	0.989
Nestin (Ref: Low)	46			83
High		0.79 (0.76–2.29)	0.669		0.58 (0.32–1.04)	0.066
CD44s and OV IgG	47			88
Positive		2.45 (1.22–4.95)	0.012*		1.53 (0.96–2.46)	0.077
CD44v8-10 and OV IgG	49			88
High and Positive		2.10 (1.14–3.87)	0.018*		1.41 (0.91–2.19)	0.120
CD44s and CD44v8-10	47			88
Positive and High		3.27 (1.68–6.36)	<0.001*		1.27 (0.78–2.05)	0.336
OV IgG and CD44s and CD44v8-10	47			88
Positive and Positive and High		4.39 (1.90–10.14)	0.001*		1.74 (1.01–2.98)	0.044*

Notes: *P value equal to or less than 0.05 was considered statistically significant. Early stage, tumor stage 0–II; late stage, tumor stage III–IV.

Abbreviations: HR, hazard ratio.

Table 5 Multivariable Analysis of IHC Results in Early Stage CCA

Early Stage (0, I, II)
Variables	N	Adjusted HR (95% CI)	p-value
Model A
Histology	65
Papillary		0.51 (0.27–0.97)	0.040*
CD44s	47
Positive		2.13 (1.14–3.96)	0.017*
Model B
Histology	65
Papillary		0.47 (0.25–0.89)	0.021*
CD44s and OV IgG	47
Positive		2.38 (1.17–4.81)	0.016*
Model C
Histology	65
Papillary		0.42 (0.22–0.80)	0.008*
CD44v8-10 and OV IgG	49
High and Positive		2.37 (1.26–4.45)	0.007*
Model D
Histology	65
Papillary		0.44 (0.23–0.85)	0.014*
CD44s and CD44v8-10	47
Positive and High		3.34 (171–6.52)	<0.001*
Model E
Histology	65
Papillary		0.53 (0.28–1.03)	0.060
OV IgG and CD44s and CD44v8-10	47
Positive and Positive and High		3.75 (1.61–8.72)	0.002*

Note: *P value equal to or less than 0.05 was considered statistically significant.

Abbreviation: HR, hazard ratio.

Figure 3 Kaplan–Meier analysis of overall survival with a combination of serum IgG for OV and IHC results in early stage CCA. (A) Survival with a combination of CD44s and IgG for OV. (B) Survival with a combination of CD44v8-10 and IgG for OV. (C) Survival with a combination of CD44s and CD44v8-10. (D) Survival with a combination of CD44s, CD44v8-10 and IgG for OV.

Table 6 Multivariate Analyses of IHC Results in Late Stage CCA

Late Stage (III, IV)
Variables	N	Adjusted HR (95% CI)	p-value
Resection margin	111
Positive		1.54 (0.99–2.39)	0.056
Adjuvant chemotherapy	111
No		1.72 (1.08–2.76)	0.023*
OV IgG and CD44s and CD44v8-10	88
Positive and Positive and High		1.76 (1.02–3.03)	0.041*

Note: *P value equal to or less than 0.05 was considered statistically significant.

Abbreviation: HR, hazard ratio.

Figure 4 Kaplan–Meier analysis of overall survival with a combination of serum IgG for OV and IHC results in late stage CCA. (A) Survival with a combination of CD44s and IgG for OV. (B) Survival with a combination of CD44v8-10 and IgG for OV. (C) Survival with a combination of CD44s and CD44v8-10. (D) Survival with a combination of CD44s, CD44v8-10 and IgG for OV.