Development and Evaluation of Serum CST1 Detection for Early Diagnosis of Esophageal Squamous Cell Carcinoma

Figures & data

Table 1 Clinical Characteristics of the Early ESCC, EBL and HC Groups

Variable	Early ESCC (n = 112)			EBL (n = 107)			HC (n = 151)			P
	No.	Mean	%	No.	Mean	%	No.	Mean	%
Age (years):										>0.05
Mean		60.5			60.0			58.3
Standard deviation		9.5			11.1			8.6
Sex:										>0.05
Male	78		69.6	67		62.6	102		67.5
Female	34		30.4	40		37.4	49		32.5
Smoking history:	35		31.3	32		29.9	43		28.5	>0.05
Drinking history:	20		17.9	14		13.1	19		12.6	>0.05
Stage:
0	33		29.5
I	31		27.7
II	48		42.8
EBL:
Esophageal benign tumors				54		50.5
Benign ulcer of esophagus				8		7.5
Esophageal erosions				32		29.9
Reflux esophagitis				13		12.1

Figure 1 IHC staining of CST1 in ESCC tissue and paracancerous tissue (×400). (A) Strong expression of CST1 with 3+ staining in ESCC tissue. (B) Negative expression of CST1 in paracancerous tissue.

Table 2 Detection Limit of CLEIA for CST1

	RLU		RLU
Measured value 1	8124	Measured value 11	7986
Measured value 2	7913	Measured value 12	8278
Measured value 3	7873	Measured value 13	8501
Measured value 4	8032	Measured value 14	8224
Measured value 5	8206	Measured value 15	8146
Measured value 6	8169	Measured value 16	8004
Measured value 7	8043	Measured value 17	8206
Measured value 8	7884	Measured value 18	8004
Measured value 9	8263	Measured value 19	8231
Measured value 10	8237	Measured value 20	8023
$\overline{X}$= 8117
SD=157.7

Figure 2 Standard curve for CLEIA. The RLU value is the mean of each diluted level detected twice; C is the diluted level (pg/mL) of CST1 calibrator. The same base logarithms of RLU and C are taken as X and Y axis, respectively, to draw the curve.

Table 3 Accuracy Evaluation of CLEIA for CST1

CST1 (pg/mL)	Sample 1		Sample 2		Sample 3		Basic Sample
	1	2	1	2	1	2	1	2
Detection level	27.70	26.24	33.94	34.52	41.37	40.11	24.58	25.46
Mean	26.97		34.23		40.74		25.02
Recovery level	1.95		9.21		15.72
Additive level	1.76		10.12		14.81
Recovery rate (%)	110.80		91.01		106.14
Mean (%)	102.65

Table 4 Precision Evaluation of CLEIA for CST1

CST1 (pg/mL)	Intra-Batch Precision			Inter-Batch Precision
	SD (pg/mL)	CV (%)	1/4 TEa (%)	SD (pg/mL)	CV (%)	1/3 TEa (%)
28.25	1.252	4.43	6.25	1.291	1.39	8.33
92.92	1.805	1.94	6.25	1.770	1.90	8.33

Abbreviations: SD, standard deviation; CV, coefficient of variation; 1/4 TEa, 1/4 total error allowance; 1/3 TEa, 1/3 total error allowance.

Table 5 Evaluation of Anti-Interference Ability of CLEIA for CST1

Interferent	Sample 1			Sample 2
	Basic (pg/mL)	Test (pg/mL)	Bias (%)	Basic (pg/mL)	Test (pg/mL)	Bias (%)
Hb (2 g/L)	20.67	21.36	3.34	62.17	65.39	5.18
RF (750 IU/mL)	20.67	22.28	7.79	62.17	65.65	5.60
Bil (342 μmol/L)	20.67	22.63	9.48	62.17	66.04	6.22
TG (37 mmol/L)	20.67	22.52	8.95	62.17	66.14	6.39
AFP (5 mg/dL)	20.67	22.07	6.77	62.17	65.96	6.10
CEA (0.2 μg/mL)	20.67	22.34	8.08	62.17	67.54	8.64
VitC (12.3 mg/dL)	20.67	22.38	8.27	62.17	65.30	5.03
CST4 (800 pg/mL)	20.67	22.13	7.06	62.17	66.89	7.59

Figure 3 Line chart of quantitative detection results for low, medium and high levels of the serum samples stored at 4 °C and 25°C for eight time points. (A) The quantitative detection results of the low, medium and high levels of the serum samples stored at 4 °C for 0, 1, 2, 3, 4, 5, 6 and 7 d. (B) The quantitative detection results of the low, medium and high levels of the serum samples stored at 25 °C for 0, 1, 2, 3, 4, 5, 6 and 7 d.

Figure 4 Line chart of quantitative detection results for CST1 calibrators and serum samples by the reagents stored at 25 °C and 37 °C for five time points. The quantitative detection results of the reagents stored at 25 °C for 0, 1, 3, 5 and 7 d are presented in (A) and (B). The quantitative detection results of the reagents stored at 37 °C for 0, 1, 3, 5 and 7 d are presented in (C) and (D).

Figure 5 Histogram of quantitative detection results for CST1 by three batches reagents stored at 4 °C for four time points. The quantitative detection results for CST1 calibrator 2 (12.5 pg/mL), 3 (25 pg/mL), 4 (50 pg/mL), 5 (100 pg/mL), 6 (200 pg/mL), 7 (400 pg/mL) and serum sample by three batches reagents are presented in (A–G), respectively.

Figure 6 Diagnostic performances of serum CST1 and three traditional tumor markers CEA, CYFRA21-1, and SCC-Ag for early ESCC patients. Boxplot, scatter and ROC curve for CST1 (A) CEA (B) CYFRA21-1 (C) and SCC-Ag (D). (E) ROC curve for an optimal panel of CST1 with CEA and SCC-Ag. *Compared with the early ESCC group, P<0.05.

Table 6 Comparison of Different Combinations of Serum CST1 and Three Traditional Tumor Markers for Early Diagnosis of ESCC Patients

Index	AUC	SE	95% CI	Sensitivity (%)	Specificity (%)
CEA, CYFRA21-1	0.669	0.032	0.586–0.712	33.04	92.64
CEA, SCC-Ag	0.730^a	0.029	0.672–0.788	29.46	92.25
CEA, CST1	0.660	0.032	0.596–0.724	37.50	91.09
CYFRA21-1, SCC-Ag	0.705	0.031	0.644–0.766	39.29	91.09
CYFRA21-1, CST1	0.659	0.032	0.596–0.722	42.86	90.31
SCC-Ag, CST1	0.730^a	0.030	0.673–0.788	43.75	91.09
CEA, CYFRA21-1, SCC-Ag	0.732	0.030	0.674–0.790	40.18	89.53
CEA, CYFRA21-1, CST1	0.668	0.032	0.605–0.732	44.64	88.76
CEA, SCC-Ag, CST1	0.736^b	0.039	0.678–0.793	49.11	89.53
CYFRA21-1, SCC-Ag, CST1	0.728	0.030	0.670–0.786	48.21	89.15
CEA, CYFRA21-1, SCC-Ag, CST1	0.735	0.030	0.677–0.793	50.89	87.98

Notes: ^aThe highest AUC in the combined detection of any two indicators. ^bThe highest AUC in the combined detection of any three indicators.