Combined detection of dickkopf-1 subtype classification autoantibodies as biomarkers for the diagnosis and prognosis of non-small cell lung cancer

Figures & data

Table 1 Relationship of serum DKK1 autoantibodies with clinicopathological characteristics of 206 NSCLC patients

Group	N (%)	Pep A	P-value	Pep B	P-value	Pep C	P-value	Pep D	P-value
		Median (IQR)		Median (IQR)		Median (IQR)		Median (IQR)
Age, years			0.267		0.064		0.142		0.715
>60	92 (45)	0.49 (0.35–0.74)		0.55 (0.43–0.94)		0.52 (0.36–0.72)		0.52 (0.39–0.92)
≤60	114 (55)	0.45 (0.32–0.67)		0.51 (0.35–0.78)		0.46 (0.31–0.64)		0.54 (0.38–0.84)
Gender			0.038		0.034		0.003		0.105
Male	144 (70)	0.50 (0.33–0.78)		0.57 (0.40–0.89)		0.52 (0.35–0.79)		0.56 (0.40–0.90)
Female	62 (30)	0.45 (0.28–0.59)		0.51 (0.35–0.69)		0.41 (0.29–0.57)		0.48 (0.34–0.83)
Smoking history			0.244		0.009		0.158		0.625
Yes	125 (61)	0.49 (0.33–0.75)		0.60 (0.42–0.89)		0.50 (0.34–0.72)		0.54 (0.39–0.85)
No	81 (39)	0.46 (0.29–0.60)		0.47 (0.35–0.69)		0.44 (0.30–0.68)		0.52 (0.38–0.85)
Histological subtype			0.072		0.053		0.029		0.005
A	119 (58)	0.45 (0.31–0.59)		0.51 (0.37–0.75)		0.45 (0.32–0.62)		0.49 (0.34–0.80)
S	87 (42)	0.51 (0.35–0.90)		0.65 (0.39–0.72)		0.54 (0.34–0.96)		0.57 (0.42–1.27)
TNM stage			0.209		0.001		0.001		0.002
I	32 (16)	0.43 (0.30–0.81)		0.53 (0.35–0.85)		0.51 (0.42–0.83)		0.69 (0.45–0.92)
II	25 (12)	0.41 (0.35–0.53)		0.57 (0.42–0.77)		0.43 (0.28–0.53)		0.51 (0.30–0.79)
III	84 (40)	0.51 (0.37–0.76)		0.60 (0.43–1.04)		0.56 (0.40–0.78)		0.59 (0.44–1.06)
IV	65 (32)	0.45 (0.29–0.59)		0.46 (0.33–0.69)		0.36 (0.30–0.68)		0.45 (0.30–0.59)
Lymph node			0.822		0.792		0.789		0.356
Yes	143 (69)	0.48 (0.32–0.63)		0.55 (0.37–0.83)		0.47 (0.32–0.68)		0.51 (0.38–0.85)
No	61 (30)	0.46 (0.33–0.83)		0.53 (0.37–0.88)		0.48 (0.34–0.68)		0.57 (0.40–0.94)
Unknown	2
Distant metastases			0.126		0.007		0.007		0.001
Yes	65 (32)	0.45 (0.29–0.59)		0.46 (0.33–0.69)		0.36 (0.29–0.57)		0.45 (0.30–0.59)
No	141 (68)	0.49 (0.35–0.76)		0.59 (0.40–0.90)		0.52 (0.38–0.76)		0.59 (0.42–0.95)

Abbreviations: A, adenocarcinoma; DKK1, dickkopf-1; IQR, inter-quartile range; NSCLC, non-small cell lung cancer; S, squamous cell carcinoma; TNM, tumor-node-metastasis.

Figure 1 Epitope mapping by peptide array-based immunological screening.

Notes: (A) Nine groups of pooled serum from NSCLC patients (n=72) and two groups from healthy controls (n=16) and commercial DKK1 monoclonal antibody were used to probe DKK1 microarrays, respectively. The spots A1 and D22 are the positive controls. (B) Plot of DKK1 peptides was in response to NSCLC and healthy serum samples. Samples with intensity value greater than half of the positive controls were considered as seropositive reaction. (C) Four peptides, shown in black box, were selected as highly responsive peptides.
Abbreviations: DKK1, dickkopf-1; NSCLC, non-small cell lung cancer.

Figure 2 The serum autoantibodies levels in NSCLC patients and healthy volunteers.

Notes: (A) Differences in the levels of serum autoantibodies between NSCLC patients and healthy controls. (B) Levels of serum autoantibodies across the consecutive M stages in NSCLC patients. (C) To assess the diagnostic value of the four peptides, a set of ROC analyses were performed. Combination of the four peptides was also shown in the picture. (D) Competitive ELISA. The serum samples were incubated with recombinant DKK1 protein and the four peptides were used to coat a 96-well ELISA plate. Following incubation, the samples were used to probe the plate in triplicate. Influence of DKK1 protein preincubation with serum samples on the ELISA signals was calculated. Error bars represented SD of triplicate wells. **P<0.01; ***P<0.001; NS indicates P>0.05.
Abbreviations: DKK1, dickkopf-1; ELISA, enzyme-linked immunosorbent assay; NS, not significant; NSCLC, non-small cell lung cancer; ROC, receiver operating characteristic.

Figure 3 Kaplan–Meier analyses of overall survival (OS) and progression-free survival (PFS) according to the serum autoantibodies status.

Notes: Kaplan–Meier plots indicate the association between OS (A) or PFS (B) and either one of the four peptides. Samples with red line represent tumors with positive autoantibody and black line represent tumors with negative autoantibody.

Table 2 Cox regression analyses of overall survival (OS) and progession-free survival (PFS) in NSCLC patients

Parameters	OS			PFS
	Univariate analysis	Multivariate analysis		Univariate analysis	Multivariate analysis
	P-value	HR (95% CI)	P-value	P-value	HR (95% CI)	P-value
Age	0.011	2.133 (1.250–3.639)	0.005	0.528	0.948 (0.609–1.478)	0.815
Gender	0.797	0.877 (0.439–1.749)	0.709	0.124	0.676 (0.355–1.287)	0.233
Smoking status	0.640	1.239 (0.638–2.405)	0.527	0.096	1.291 (0.677–2.465)	0.438
Pathological type	0.033	0.977 (0.529–1.802)	0.940	0.001	1.322 (0.765–2.283)	0.317
N status	<0.001	1.847 (0.831–4.103)	0.132	<0.001	1.380 (0.735–2.591)	0.316
TNM	<0.001	1.809 (0.479–6.832)	0.392	<0.001	2.481 (0.807–7.627)	0.069
Pep A	0.379	1.600 (0.829–3.088)	0.162	0.289	1.137 (0.666–1.941)	0.638
Pep B	0.004	0.435 (0.236–0.802)	0.008	0.006	0.533 (0.322–0.950)	0.032
Pep C	0.065	0.616 (0.313–1.212)	0.161	0.141	1.131 (0.612–2.090)	0.695
Pep D	0.07	0.989 (0.474–2.065)	0.977	0.118	0.831 (0.425–1.626)	0.589

Abbreviations: HR, hazard ratio; NSCLC, non-small cell lung cancer; TNM, tumor-node-metastasis.

Table 3 Results for measurement of autoantibodies against the four peptides, or all the four peptides in the diagnosis of NSCLC patients

Index	AUC (95% CI)	Sensitivity (%)	Specificity (%)	PPR (%)	NPR (%)
NSCLC vs healthy
Pep A	0.744 (0.684–0.799)	46.4	90.1	91.6	41.8
Pep B	0.809 (0.756–0.854)	64.0	82.0	87.5	53.7
Pep C	0.740 (0.684–0.791)	66.0	69.4	82.9	48.0
Pep D	0.767 (0.712–0.814)	78.1	62.1	80.3	59.0
All peptides	0.821 (0.764–0.868)	58.1	85.3	81.0	79.6
Early-stage NSCLC4 vs healthy
Pep A	0.706 (0.611–0.790)	35.1	90.1	65.3	72.5
Pep B	0.808 (0.732–0.871)	68.1	84.3	89.8	83.2
Pep C	0.723 (0.640–0.796)	73.1	69.0	59.3	80.6
Pep D	0.780 (0.704–0.843)	70.4	74.7	61.3	81.5
All peptides	0.818 (0.719–0.894)	76.9	75.9	58.8	88.0

Note:

4 Defined as NSCLC clinical stage I and II.

Abbreviations: AUC, area under the receiver operating characteristic curve; NPR, negative predictive value; PPR, positive predictive value; NSCLC, non-small cell lung cancer.

Figure S1 Layout of DKK1 peptide microarrays.

Notes: DKK1 were cut into peptides which were 12-mers with 9aa overlap. All spots were organized as a 4×22 array. A1–D22 represent each peptide. The red dots (A1 and D22) in the figure are positive controls.

Abbreviation: DKK1, dickkopf-1.

Figure S2 Competitive ELISA.

Notes: The serum samples were incubated with BSA and the four peptides were used to coat a 96-well ELISA plate. Following incubation, the samples were used to probe the plate. Influence of BSA preincubation with serum samples on the ELISA signals was calculated.

Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.

Table S1 Peptide sequences corresponding to each dot on the microarray

No		Peptide sequence	aa
1	A1	FLAG
2	A2	M-M-A-L-G-A-A-G-A-T-R-V	1–12
3	A3	L-G-A-A-G-A-T-R-V-F-V-A	4–15
4	A4	A-G-A-T-R-V-F-V-A-M-V-A	7–18
5	A5	T-R-V-F-V-A-M-V-A-A-A-L	10–21
6	A6	F-V-A-M-V-A-A-A-L-G-G-H	13–24
7	A7	M-V-A-A-A-L-G-G-H-P-L-L	16–27
8	A8	A-A-L-G-G-H-P-L-L-G-V-S	19–30
9	A9	G-G-H-P-L-L-G-V-S-A-T-L	22–33
10	A10	P-L-L-G-V-S-A-T-L-N-S-V	25–36
11	A11	G-V-S-A-T-L-N-S-V-L-N-S	28–39
12	A12	A-T-L-N-S-V-L-N-S-N-A-I	31–42
13	A13	N-S-V-L-N-S-N-A-I-K-N-L	34–45
14	A14	L-N-S-N-A-I-K-N-L-P-P-P	37–48
15	A15	N-A-I-K-N-L-P-P-P-L-G-G	40–51
16	A16	K-N-L-P-P-P-L-G-G-A-A-G	43–54
17	A17	P-P-P-L-G-G-A-A-G-H-P-G	46–57
18	A18	L-G-G-A-A-G-H-P-G-S-A-V	49–60
19	A19	A-A-G-H-P-G-S-A-V-S-A-A	52–63
20	A20	H-P-G-S-A-V-S-A-A-P-G-I	55–66
21	A21	S-A-V-S-A-A-P-G-I-L-Y-P	55–66
22	A22	S-A-A-P-G-I-L-Y-P-G-G-N	61–72
23	B1	P-G-I-L-Y-P-G-G-N-K-Y-Q	64–75
24	B2	L-Y-P-G-G-N-K-Y-Q-T-I-D	67–78
25	B3	G-G-N-K-Y-Q-T-I-D-N-Y-Q	70–81
26	B4	K-Y-Q-T-I-D-N-Y-Q-P-Y-P	73–84
27	B5	I-L-Y-P-G-G-N-K-Y-Q-T-I	76–87
28	B6	N-Y-Q-P-Y-P-C-A-E-D-E-E	79–90
29	B7	P-Y-P-C-A-E-D-E-E-C-G-T	82–93
30	B8	C-A-E-D-E-E-C-G-T-D-E-Y	85–96
31	B9	D-E-E-C-G-T-D-E-Y-C-A-S	88–99
32	B10	C-G-T-D-E-Y-C-A-S-P-T-R	91–102
33	B11	D-E-Y-C-A-S-P-T-R-G-G-D	94–105
34	B12	C-A-S-P-T-R-G-G-D-A-G-V	97–108
35	B13	P-T-R-G-G-D-A-G-V-Q-I-C	100–111
36	B14	G-G-D-A-G-V-Q-I-C-L-A-C	103–114
37	B15	A-G-V-Q-I-C-L-A-C-R-K-R	106–117
38	B16	Q-I-C-L-A-C-R-K-R-R-K-R	109–120
39	B17	L-A-C-R-K-R-R-K-R-C-M-R	112–123
40	B18	R-K-R-R-K-R-C-M-R-H-A-M	115–126
41	B19	R-K-R-C-M-R-H-A-M-C-C-P	118–129
42	B20	C-M-R-H-A-M-C-C-P-G-N-Y	121–132
43	B21	H-A-M-C-C-P-G-N-Y-C-K-N	124–135
44	B22	C-C-P-G-N-Y-C-K-N-G-I-C	127–138
45	C1	G-N-Y-C-K-N-G-I-C-V-S-S	130–141
46	C2	C-K-N-G-I-C-V-S-S-D-Q-N	133–144
47	C3	G-I-C-V-S-S-D-Q-N-H-F-R	136–147
48	C4	V-S-S-D-Q-N-H-F-R-G-E-I	139–150
49	C5	D-Q-N-H-F-R-G-E-I-E-E-T	142–153
50	C6	H-F-R-G-E-I-E-E-T-I-T-E	145–156
51	C7	G-E-I-E-E-T-I-T-E-S-F-G	148–159
52	C8	E-E-T-I-T-E-S-F-G-N-D-H	151–162
53	C9	I-T-E-S-F-G-N-D-H-S-T-L	154–165
54	C10	S-F-G-N-D-H-S-T-L-D-G-Y	157–168
55	C11	N-D-H-S-T-L-D-G-Y-S-R-R	160–171
56	C12	S-T-L-D-G-Y-S-R-R-T-T-L	163–174
57	C13	D-G-Y-S-R-R-T-T-L-S-S-K	166–177
58	C14	S-R-R-T-T-L-S-S-K-M-Y-H	169–180
59	C15	T-T-L-S-S-K-M-Y-H-T-K-G	172–183
60	C16	S-S-K-M-Y-H-T-K-G-Q-E-G	175–186
61	C17	M-Y-H-T-K-G-Q-E-G-S-V-C	178–189
62	C18	T-K-G-Q-E-G-S-V-C-L-R-S	181–192
63	C19	Q-E-G-S-V-C-L-R-S-S-D-C	184–195
64	C20	S-V-C-L-R-S-S-D-C-A-S-G	187–198
65	C21	L-R-S-S-D-C-A-S-G-L-C-C	190–201
66	C22	S-D-C-A-S-G-L-C-C-A-R-H	193–204
67	D1	A-S-G-L-C-C-A-R-H-F-W-S	196–207
68	D2	L-C-C-A-R-H-F-W-S-K-I-C	199–210
69	D3	A-R-H-F-W-S-K-I-C-K-P-V	202–213
70	D4	F-W-S-K-I-C-K-P-V-L-K-E	205–216
71	D5	K-I-C-K-P-V-L-K-E-G-Q-V	208–219
72	D6	K-P-V-L-K-E-G-Q-V-C-T-K	211–222
73	D7	L-K-E-G-Q-V-C-T-K-H-R-R	214–225
74	D8	G-Q-V-C-T-K-H-R-R-K-G-S	217–228
75	D9	C-T-K-H-R-R-K-G-S-H-G-L	220–231
76	D10	H-R-R-K-G-S-H-G-L-E-I-F	223–234
77	D11	K-G-SH-G-L-E-I-F-Q-R-C	226–237
78	D12	H-G-L-E-I-F-Q-R-C-Y-C-G	229–240
79	D13	E-I-F-Q-R-C-Y-C-G-E-G-L	232–243
80	D14	Q-R-C-Y-C-G-E-G-L-S-C-R	235–246
81	D15	Y-C-G-E-G-L-S-C-R-I-Q-K	238–249
82	D16	E-G-L-S-C-R-I-Q-K-D-H-H	241–252
83	D17	S-C-R-I-Q-K-D-H-H-Q-A-S	244–255
83	D18	I-Q-K-D-H-H-Q-A-S-N-S-S	247–258
85	D19	D-H-H-Q-A-S-N-S-S-R-L-H	250–261
86	D20	Q-A-S-N-S-S-R-L-H-T-C-Q	253–264
87	D21	N-S-S-R-L-H-T-C-Q-R-H	256–266
88	D22	FLAG