Characteristics of patients with lung cancer
Clinical characteristics of the patients with lung cancer, including age, gender, histological type, stage, T classification, N classification, M classification, radiation therapy, residual tumor, vital status, sample type, KLRK1 expression, were shown in Table 1. The percentage of two histological types, lung adenocarcinoma (50.74%) and lung squamous cell carcinoma (49.26%), was close. About half of the patients were in stage I (51.13%). As for T, N, M classification, T2 (56.04%), N0 (63.98%), and M0 (74.39%) were highest among each classification. Most patients (99.80%) were primary tumor.
Table 1
Clinical characteristics of the patients with lung cancer
Characteristics
|
Number (%)
|
Age
|
|
<55
|
109(10.70)
|
>=55
|
881(86.46)
|
NA
|
29(2.84)
|
Gender
|
|
Female
|
407(39.94)
|
Male
|
611(59.96)
|
NA
|
1(0.10)
|
Histological type
|
|
Lung adenocarcinoma
|
517(50.74)
|
Lung squamous cell carcinoma
|
502(49.26)
|
Stage
|
|
I
|
521(51.13)
|
II
|
284(27.87)
|
III
|
168(16.49)
|
IV
|
33(3.24)
|
NA
|
13(1.28)
|
T classification
|
|
T1
|
284(27.87)
|
T2
|
571(56.04)
|
T3
|
118(11.58)
|
T4
|
42(4.12)
|
TX
|
3(0.29)
|
NA
|
1(0.10)
|
N classification
|
|
N0
|
652(63.98)
|
N1
|
227(22.28)
|
N2
|
114(11.19)
|
N3
|
7(0.69)
|
NX
|
17(1.67)
|
NA
|
2(0.20)
|
M classification
|
|
M0
|
758(74.39)
|
M1
|
32(3.14)
|
MX
|
220(21.59)
|
NA
|
9(0.88)
|
Radiation therapy
|
|
NO
|
782(76.74)
|
YES
|
110(10.79)
|
NA
|
127(12.46)
|
Residual tumor
|
|
R0
|
743(72.91)
|
R1
|
25(2.45)
|
R2
|
8(0.79)
|
RX
|
48(4.71)
|
NA
|
195(19.14)
|
Vital status
|
|
Deceased
|
404(39.65)
|
Living
|
614(60.26)
|
NA
|
1(0.10)
|
Sample type
|
|
Primary tumor
|
1017(99.80)
|
Recurrent tumor
|
2(0.20)
|
KLRK1 expression
|
|
High
|
701(68.79)
|
Low
|
318(31.21)
|
Note: NA: not available; X represents uncertain. |
Low KLRK1 expression in tumor
As shown in Fig. 1, the expression of KLRK1 in tumor was lower than that in normal tissue (P<2.2e-16). The association of KLRK1 expression with clinical features, including histological type, sample type, age, gender, radiation therapy, was further evaluated. KLRK1 expression in lung squamous cell carcinoma was lower than that in lung adenocarcinoma (P=2.2e-05). KLRK1 expression in male was lower than that in female (P=3e-04). Other clinical features did not show statistical differences (P>0.05).
Association of KLRK1 expression with stage, T classification, N classification, M classification, residual tumor and vital status was evaluated (Fig. 2). The KLRK1 expression got decreased progressively with higher stages (P=0.0015) and T classification (P=2.6e-06). No significances were observed in N classification (P=0.200), M classification (P=0.091) and residual tumor (P=0.790). The KLRK1 expression in living patients was a little higher than that in deceased patients (P=0.011).
Diagnostic value of KLRK1 for lung cancer
As shown in Fig. 2, the ROC analysis was first performed in all lung cancer patients, indicating a modest diagnostic value with AUC of 0.789. Moreover, different stages of lung cancer were analyzed. It was suggested that the diagnostic value was increasing with stage getting higher from stage I (AUC=0.766) to stage II (AUC=0.797) to stage III (AUC=0.835) finally to stage IV (AUC=0.853) (Fig. 3).
Correlation of KLRK1 expression with clinical features
According to the threshold value ascertained by ROC analysis, patients were divided to two subgroups, high (n=701) and low (n=318) KLRK1 expression group. The relationship between the clinical features and KLRK1 expression in patients with lung cancer was shown in Table 2. KLRK1 expression was associated with gender (P=0.005), histologic grade (P=0.001), stage (P=0.001), T classification (P<0.001) and vital status (P=0.001). No correlation was found between KLRK1 expression and age (P=0.431), N classification (P=0.218), M classification (P=0.383), radiation therapy (P=0.399), residual tumor (P=0.336), and sample type (P=0.850).
Table 2
Relationship between the clinical features and KLRK1 expression in patients with lung cancer
Clinical characteristics
|
Variable
|
Number of patients
|
KLRK1
|
χ2
|
P value
|
High
|
%
|
Low
|
%
|
Age
|
<55
|
109
|
71
|
(10.41)
|
38
|
(12.34)
|
0.620
|
0.431
|
|
>=55
|
881
|
611
|
(89.59)
|
270
|
(87.66)
|
|
|
Gender
|
Female
|
407
|
301
|
(42.94)
|
106
|
(33.44)
|
7.819
|
0.005
|
|
Male
|
611
|
400
|
(57.06)
|
211
|
(66.56)
|
|
|
Histological type
|
Lung adenocarcinoma
|
517
|
381
|
(54.35)
|
136
|
(42.77)
|
11.285
|
0.001
|
|
Lung squamous cell carcinoma
|
502
|
320
|
(45.65)
|
182
|
(57.23)
|
|
|
Stage
|
I
|
521
|
386
|
(55.86)
|
135
|
(42.86)
|
15.336
|
0.001
|
|
II
|
284
|
181
|
(26.19)
|
103
|
(32.70)
|
|
|
|
III
|
168
|
102
|
(14.76)
|
66
|
(20.95)
|
|
|
|
IV
|
33
|
22
|
(3.18)
|
11
|
(3.49)
|
|
|
T classification
|
T1
|
284
|
229
|
(32.67)
|
55
|
(17.35)
|
29.391
|
<0.001
|
|
T2
|
571
|
373
|
(53.21)
|
198
|
(62.46)
|
|
|
|
T3
|
118
|
69
|
(9.84)
|
49
|
(15.46)
|
|
|
|
T4
|
42
|
27
|
(3.85)
|
15
|
(4.73)
|
|
|
|
TX
|
3
|
3
|
(0.43)
|
0
|
(0)
|
|
|
N classification
|
N0
|
652
|
461
|
(65.86)
|
191
|
(60.25)
|
5.610
|
0.218
|
|
N1
|
227
|
146
|
(20.86)
|
81
|
(25.55)
|
|
|
|
N2
|
114
|
75
|
(10.71)
|
39
|
(12.30)
|
|
|
|
N3
|
7
|
4
|
(0.57)
|
3
|
(0.95)
|
|
|
|
NX
|
17
|
14
|
(2.00)
|
3
|
(0.95)
|
|
|
M classification
|
M0
|
758
|
516
|
(74.03)
|
242
|
(77.32)
|
1.904
|
0.383
|
|
M1
|
32
|
21
|
(3.01)
|
11
|
(3.51)
|
|
|
|
MX
|
220
|
160
|
(22.96)
|
60
|
(19.17)
|
|
|
Radiation therapy
|
NO
|
782
|
540
|
(88.38)
|
242
|
(86.12)
|
0.711
|
0.399
|
|
YES
|
110
|
71
|
(11.62)
|
39
|
(13.88)
|
|
|
Residual tumor
|
R0
|
743
|
513
|
(90.80)
|
230
|
(88.8)
|
3.305
|
0.336
|
|
R1
|
25
|
14
|
(2.48)
|
11
|
(4.25)
|
|
|
|
R2
|
8
|
4
|
(0.71)
|
4
|
(1.54)
|
|
|
|
RX
|
48
|
34
|
(6.02)
|
14
|
(5.41)
|
|
|
Vital status
|
Deceased
|
404
|
253
|
(36.09)
|
151
|
(47.63)
|
11.673
|
0.001
|
|
Living
|
614
|
448
|
(63.91)
|
166
|
(52.37)
|
|
|
Sample type
|
Primary tumor
|
1017
|
699
|
(99.71)
|
318
|
(100)
|
0.036
|
0.850
|
|
Recurrent tumor
|
2
|
2
|
(0.29)
|
0
|
(0)
|
|
|
Note: X represents uncertain. |
Overall survival and relapse free survival of KLRK1 for lung cancer
Given that KLRK1 was correlated with survival, the prognostic value of KLRK1 was further studied. As shown in Fig. 4, lung cancer patients with high KLRK1 expression presented an improved overall survival (P=0.0036) and relapse free survival (P=0.0031). As shown in Fig. 5, lung adenocarcinoma cancer patients with high KLRK1 expression presented an improved overall survival (P=0.015) and relapse free survival (P=0.0094). But no significances were found in lung squamous cell cancer patients with high KLRK1 expression.
Subgroup analysis of KLRK1 for lung cancer`s overall survival
By overall survival analysis of subgroups (Fig. 6, S1-2), KLRK1 was found to have significant prognostic value in lung adenocarcinoma (P=0.015), stage I/II (P=0.03), older patients (P=0.0052), and male (P=0.0047). For lung adenocarcinoma cancer, KLRK1 was found to have significant prognostic value in stage I/II (P=0.049), older patients (P=0.0064), and male (P=0.013).
Subgroup analysis of KLRK1 for lung cancer`s relapse free survival
By relapse free survival analysis of subgroups (Fig. 7, S3-4), KLRK1 was found to have significant prognostic value in lung adenocarcinoma (P=0.0094), stage I/II (P=0.0076), older patients (P=0.0072), and male (P=0.0033). For lung adenocarcinoma cancer, KLRK1 was found to have significant prognostic value in stage I/II (P=0.0025), and older patients (P=0.012).
KLRK1 is an independent risk factor for lung cancer`s overall survival
The univariate Cox model of overall and relapse free survival in patients with lung cancer was established (Table 3-4, S1), and the multivariate analysis of overall and relapse free survival in patients with lung cancer was further performed (Table 5-6, S2).
As shown in Table 3, no obvious differences were observed in age (P=0.867), gender (P=0.201), histological type (P=0.282), M classification (P=0.220), and radiation therapy (P=0.983). T classification (P<0.001), N classification (P<0.001), residual tumor (P=0.016), stage (P<0.001) and KLRK1 expression (P=0.004) showed significant differences for lung cancer. As shown in Table 4, T classification (P<0.001), N classification (P<0.001), M classification (P=0.029), residual tumor (P=0.037), stage (P<0.001) and KLRK1 expression (P=0.016) showed significant differences for lung adenocarcinoma cancer. As shown in Table 5, T classification (HR=1.18; 95% CI: 1.02-1.36; P=0.025) and stage (HR=1.34; 95% CI: 1.14-1.57; P<0.001) were risk factors for lung cancer.
Table 3
Univariate analysis of overall and relapse free survival in patients with lung cancer
|
Overall survival
|
Relapse free survival
|
|
Hazard Ratio
|
95%CI
|
P value
|
Hazard Ratio
|
95%CI
|
P value
|
Age
|
1.03
|
0.74-1.43
|
0.867
|
0.95
|
0.63-1.43
|
0.819
|
Gender
|
1.14
|
0.93-1.40
|
0.201
|
0.94
|
0.73-1.20
|
0.613
|
Histological type
|
1.12
|
0.91-1.36
|
0.282
|
0.63
|
0.49-0.82
|
<0.001
|
T classification
|
1.42
|
1.25-1.61
|
<0.001
|
1.35
|
1.14-1.59
|
<0.001
|
N classification
|
1.39
|
1.22-1.57
|
<0.001
|
1.41
|
1.20-1.65
|
<0.001
|
M classification
|
1.16
|
0.91-1.49
|
0.220
|
0.83
|
0.62-1.11
|
0.210
|
Radiation therapy
|
1.00
|
0.79-1.27
|
0.983
|
1.76
|
1.32-2.35
|
<0.001
|
Residual tumor
|
1.17
|
1.03-1.33
|
0.016
|
1.29
|
1.11-1.49
|
0.001
|
Stage
|
1.48
|
1.33-1.64
|
<0.001
|
1.41
|
1.23-1.62
|
<0.001
|
KLRK1 expression
|
0.74
|
0.60-0.91
|
0.004
|
0.68
|
0.52-0.88
|
0.003
|
KLRK1 is an independent risk factor for lung cancer`s relapse free survival
As shown in Table 3, no obvious differences were observed in age (P=0.819), gender (P=0.613), and M classification (P=0.210). Histological type (P<0.001), T classification (P<0.001), N classification (P<0.001), radiation therapy (P<0.001), residual tumor (0.001), stage (P<0.001) and KLRK1 expression (P=0.003) showed significant differences. As shown in Table 4, stage (P<0.001) showed significant differences for lung adenocarcinoma cancer. As shown in Table 5, T classification (HR=1.22; 95% CI: 1.01-1.47; P=0.040), radiation therapy (HR=1.42; 95% CI: 1.06-1.91; P=0.018) and residual tumor (HR=1.23; 95% CI: 1.07-1.42; P=0.004) were risk factors for lung cancer.
Table 4
Univariate analysis of overall and relapse free survival in patients with lung adenocarcinoma cancer
|
Overall survival
|
Relapse free survival
|
|
Hazard Ratio
|
95%CI
|
P value
|
Hazard Ratio
|
95%CI
|
P value
|
Age
|
0.82
|
0.55-1.22
|
0.322
|
-
|
-
|
-
|
Gender
|
1.05
|
0.78-1.4
|
0.760
|
-
|
-
|
-
|
T classification
|
1.52
|
1.26-1.82
|
<0.001
|
1.16
|
0.96-1.4
|
0.132
|
N classification
|
1.67
|
1.41-1.97
|
<0.001
|
1.15
|
0.92-1.44
|
0.228
|
M classification
|
1.4
|
1.04-1.9
|
0.029
|
0.93
|
0.68-1.28
|
0.662
|
Radiation therapy
|
1.25
|
0.89-1.75
|
0.199
|
-
|
-
|
-
|
Residual tumor
|
1.2
|
1.01-1.42
|
0.037
|
1.14
|
0.96-1.35
|
0.150
|
Stage
|
1.68
|
1.47-1.93
|
<0.001
|
1.51
|
1.21-1.89
|
<0.001
|
KLRK1
|
0.69
|
0.51-0.93
|
0.016
|
0.78
|
0.57-1.07
|
0.129
|
KLRK1 inhibits lung cancer cell proliferation and migration in vitro
We further studied the effect of KLRK1 on cells by overexpressing KLRK1 in lung cancer A549 cell lines. The CCK-8 assay showed that KLRK1 upregulation significantly inhibited cell proliferation (P < 0.05; Fig. 8A). Compared with control group, KLRK1 increased the percentage of dead cells by live/dead staining (Fig. 8B). KLRK1 decreased the migration distance of cancer cell (P < 0.05; Fig. 8C and 8D).
Table 5
Multivariate analysis of overall survival and relapse free survival in patients with lung cancer
|
Overall survival
|
Relapse-free survival
|
|
Hazard Ratio
|
95%CI
|
P value
|
Hazard Ratio
|
95%CI
|
P value
|
Histological type
|
-
|
-
|
-
|
0.58
|
0.45-0.76
|
<0.001
|
T classification
|
1.18
|
1.02-1.36
|
0.025
|
1.22
|
1.01-1.47
|
0.040
|
N classification
|
-
|
-
|
-
|
1.11
|
0.89-1.4
|
0.343
|
Radiation therapy
|
-
|
-
|
-
|
1.42
|
1.06-1.91
|
0.018
|
Residual tumor
|
1.13
|
0.99-1.28
|
0.067
|
1.23
|
1.07-1.42
|
0.004
|
Stage
|
1.34
|
1.14-1.57
|
<0.001
|
1.16
|
0.93-1.43
|
0.183
|
KLRK1
|
0.79
|
0.64-0.97
|
0.025
|
0.74
|
0.57-0.96
|
0.022
|
Table 6
Multivariate analysis of overall survival and relapse free survival in patients with lung adenocarcinoma cancer
|
Overall survival
|
Relapse free survival
|
|
Hazard Ratio
|
95%CI
|
P value
|
Hazard Ratio
|
95%CI
|
P value
|
T classification
|
1.39
|
1.13-1.71
|
0.002
|
1.18
|
0.94-1.49
|
0.155
|
N classification
|
1.45
|
1.2-1.76
|
<0.001
|
1.12
|
0.85-1.48
|
0.410
|
Radiation therapy
|
1.97
|
1.37-2.82
|
<0.001
|
1.62
|
1.12-2.35
|
0.010
|
Residual tumor
|
1.20
|
1.01-1.43
|
0.035
|
1.16
|
0.98-1.38
|
0.088
|
Stage
|
1.44
|
1.23-1.7
|
<0.001
|
1.17
|
0.91-1.51
|
0.209
|
KLRK1
|
0.64
|
0.45-0.9
|
0.010
|
0.74
|
0.52-1.06
|
0.099
|