-
Cutoff values for iTILs, TILV and fTILs
According to the ROC curve, the areas under the curve (AUCs) of iTILs, TILV, and fTILs are 0.589 (95% CI 0.540-0.637, P=0.00319), 0.577 (95% CI 0.527-0.625, P=0.0308,) and 0.553 (95 % CI 0.503-0.602, P=0.0323) respectively, and the best cut-off values were 10%, 12% and 50% respectively (Fig.1).
A total of 336 men (81.6%) and 76 women (18.4%) were eligible for this study. Most subjects (72.3%) had a current or past history of smoking. As shown in Table 1, 52.2% of patients had supraglottic squamous cell carcinoma, and 47.8% of patients had glottal laryngeal squamous cell carcinoma. Most patients (65.3%) had localized early tumors (T1 or T2), most (59.0%) being moderately or poorly differentiated.
-
Survival analysis based on tumor inflammation markers
In our study, 330 patients had higher iTILs (80.1%, Figure 2A), 82 patients had lower iTILs (19.9%, Figure 2B). The 5-year OS rate was significantly higher in the high iTILs group (75.76%) than in the low iTILs group (59.76%, p < 0.05, Figure 3A). When the patients were stratified into high TILV group (137 patients or 33.3%, Figure 2C) and low TILV group (275 patients or 66.7%, Figure 2D), the 5-year OS rate is significantly higher in the high TILV group (81.20%) than in the low TILC group (68.36%, p < 0.05, Figure 3B). Finally, we stratified the patients again, according to fTILs. 240 patients had higher fTILs (58.3%, Figure 2E), and 172 patients had lower fTILs (41.7%%, Figure 2F). The 5-year OS rate of the high fTILs group (77.50%) was significantly higher than that of the low fTILs group (65.70%, P<0.05, Figure 3C). Therefore, the levels of iTILs, TILV, and fTILs are related to the patient’s survival. Furthermore, we analyzed the levels of iTILs, TILV, and fTILs and the recurrence of the disease and found no significant correlation (P>0.05, Figure 2.)
-
Single- and multiple-factor analyses
Clinicopathological parameters, including the levels of iTILs, TILV and fTILs, and OS and RFS, were subjected to univariate multivariate analyses to determine independent predictors of OS and RFS in LSCC patients. BMI<24, history of drinking, low levels of differentiation, supraglottic carcinoma, high T/N stage or TNM, and low iTILs/TILV/fTILs levels were identified as predictors of poor prognosis (Table 1). These factors are determined by the single-factor analysis. Next, we established multiple linear regression models to observe the main effects of iTILs, TILV, and fTILs. age, alcohol consumption, and T4 tumor stage showed statistical significance in these three models. More importantly, the three linear regression models showed that high iTILs (P=0.002, HR: 0.518, 95%CI 0.341-0.785), high TILV (P=0.026, HR: 0.604, 95%CI: 0.387-0.943) and high FTILs (P=0.011, HR: 0.605, 95%CI: 0.410-0.892) were significantly correlated with better OS (Table 2). Therefore, we believe that high levels of iTILs, TILV, and fTILs are independent predictors of good prognosis. In the Cox regression model analysis to determine the statistically significant factors related to RFS, the relationship between the three factors and the recurrence was not found to be statistically significant, in either single-factor or multivariate analyses (Table 1 & 2).
Table 1
Patient baseline characteristics and univariate analysis of OS and RFS
Items
|
No. (%)
|
Univariate
OS
|
Univariate
RFS
|
|
|
HR
|
95% CI
|
P-value
|
HR
|
95% CI
|
P-value
|
Gender
|
|
|
|
|
|
|
|
Male
|
336(81.6%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Female
|
76(18.4%)
|
0.980
|
0.610-1.574
|
0.932
|
0.893
|
0.492-1.618
|
0.708
|
Age (yr)
|
|
|
|
|
|
|
|
<60
|
223(54.1%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
≥60
|
189(45.9%)
|
1.367
|
0.945-1.978
|
0.097
|
1.120
|
0.720-1.742
|
0.615
|
BMI (kg/m2)
|
|
|
|
|
|
|
|
<24
|
273(66.3%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
≥24
|
139(33.7%)
|
0.577
|
0.374-0.889
|
0.013
|
0.754
|
0.464-1.226
|
0.255
|
Smoking
|
|
|
|
|
|
|
|
No
|
114(27.7%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Yes
|
298(72.3%)
|
1.305
|
0.847-2.011
|
0.228
|
1.615
|
0.933-2.797
|
0.087
|
Alcohol
|
|
|
|
|
|
|
|
No
|
226(54.9%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Yes
|
186(45.1%)
|
1.519
|
1.049-2.198
|
0.027
|
1.571
|
1.009-2.447
|
0.046
|
Initial Site
|
|
|
|
|
|
|
|
supraglottic
|
215(52.2%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
glottic larynx
|
197(47.8%)
|
0.611
|
0.417-0.895
|
0.011
|
0.681
|
0.435-1.068
|
0.094
|
Differentiation
|
|
|
|
|
|
|
|
Low-moderate
|
243(59.0%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
high
|
169(41.0%)
|
0.477
|
0.314-0.724
|
0.001
|
0.403
|
0.240-0.675
|
0.001
|
T-Stage
|
|
|
|
|
|
|
|
T1
|
135(32.3%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
T2
|
209(50.7%)
|
1.423
|
0.905-2.238
|
0.126
|
2.158
|
1.226-3.801
|
0.008
|
T3
|
52(12.6%)
|
2.204
|
1.251-3.883
|
0.006
|
1.820
|
0.826-4.012
|
0.137
|
T4
|
16(3.9%)
|
2.312
|
1.010-5.294
|
0.047
|
3.357
|
1.230-9.164
|
0.018
|
N-Stage
|
|
|
|
|
|
|
|
N0
|
312(75.7%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
N1
|
38(9.2%)
|
1.792
|
0.987-3.253
|
0.055
|
1.785
|
0.906-3.516
|
0.094
|
N2
|
62(15.1%)
|
3.941
|
2.609-5.954
|
<0.001
|
2.567
|
1.497-4.400
|
0.001
|
TNM Stage
|
|
|
|
|
|
|
|
1
|
119(28.9%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
2
|
150(36.4%)
|
1.166
|
0.668-2.037
|
0.589
|
2.013
|
1.053-3.848
|
0.034
|
3
|
71(17.2%)
|
2.042
|
1.129-3.691
|
0.018
|
2.327
|
1.119-4.838
|
0.024
|
4
|
72(17.5%)
|
4.416
|
2.594-7.516
|
<0.001
|
3.464
|
1.709-7.022
|
0.001
|
iTILs
|
|
|
|
|
|
|
|
Lower
|
82(19.9%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Higher
|
330(80.1%)
|
0.545
|
0.363-0.818
|
0.003
|
1.025
|
0.584-1.798
|
0.933
|
TILV
|
|
|
|
|
|
|
|
Lower
|
275(66.7%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Higher
|
137(33.3%)
|
0.548
|
0.354-0.850
|
0.007
|
1.179
|
0.748-1.857
|
0.478
|
fTILs
|
|
|
|
|
|
|
|
Lower
|
172(41.7%)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Higher
|
240(58.3%)
|
0.611
|
0.422-0.884
|
0.009
|
0.895
|
0.574-1.396
|
0.626
|
Table 2
Multi-model multi-factor analysis of factors related to LSCC OS
Items
|
Multivariate
iTILs
|
Multivariate
TILV
|
Multivariate
fTILs
|
|
HR
|
95% CI
|
P-value
|
HR
|
95% CI
|
P-value
|
HR
|
95% CI
|
P-value
|
Gender
|
|
|
|
|
|
|
|
|
|
Male(ref)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Female
|
1.065
|
0.618-1.836
|
0.820
|
1.057
|
0.613-1.820
|
0.843
|
1.249
|
0.719-2.169
|
0.430
|
Age (yr)
|
|
|
|
|
|
|
|
|
|
<60 (ref)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
≥60
|
1.500
|
1.029-2.186
|
0.035
|
1.500
|
1.029-2.188
|
0.035
|
1.520
|
1.042-2.219
|
0.030
|
BMI (kg/m2)
|
|
|
|
|
|
|
|
|
|
<24 (ref)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
≥24
|
0.667
|
0.430-1.033
|
0.070
|
0.670
|
0.432-1.040
|
0.074
|
0.665
|
0.429-1.031
|
0.068
|
Alcohol
|
|
|
|
|
|
|
|
|
|
No (ref)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Yes
|
1.533
|
1.018-2.308
|
0.041
|
1.525
|
1.014-2.292
|
0.043
|
1.632
|
1.080-2.467
|
0.020
|
Initial Site
|
|
|
|
|
|
|
|
|
|
supraglottic
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
glottic larynx
|
1.068
|
0.660-1.728
|
0.790
|
1.114
|
0.685-1.811
|
0.663
|
1.049
|
0.645-1.706
|
0.848
|
Differentiation
|
|
|
|
|
|
|
|
|
|
Low-moderate
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
high
|
0.581
|
0.365-0.925
|
0.022
|
0.660
|
0.411-1.060
|
0.086
|
0.619
|
0.388-0.987
|
0.044
|
TNM Stage
|
|
|
|
|
|
|
|
|
|
1
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
2
|
1.099
|
0.622-1.942
|
0.745
|
1.144
|
0.649-2.018
|
0.642
|
1.063
|
0.599-1.886
|
0.835
|
3
|
1.627
|
0.860-3.080
|
0.135
|
1.828
|
0.972-3.437
|
0.061
|
1.674
|
0.888-3.159
|
0.111
|
4
|
3.584
|
1.966-6.533
|
<0.001
|
3.730
|
2.043-6.811
|
<0.001
|
3.375
|
1.828-6.325
|
<0.001
|
iTILs
|
|
|
|
|
|
|
|
|
|
Lower
|
ref
|
ref
|
ref
|
|
|
|
|
|
|
Higher
|
0.518
|
0.341-0.785
|
0.002
|
|
|
|
|
|
|
TILV
|
|
|
|
|
|
|
|
|
|
Lower
|
|
|
|
ref
|
ref
|
ref
|
|
|
|
Higher
|
|
|
|
0.604
|
0.387-0.943
|
0.026
|
|
|
|
fTILs
|
|
|
|
|
|
|
|
|
|
Lower
|
|
|
|
|
|
|
ref
|
ref
|
ref
|
Higher
|
|
|
|
|
|
|
0.605
|
0.410-0.892
|
0.011
|
Table 3
Multi-model multi-factor analysis of factors related to LSCC RFS
Items
|
Multivariate
iTILs
|
Multivariate
TILV
|
Multivariate
fTILs
|
HR
|
95% CI
|
P-value
|
HR
|
95% CI
|
P-value
|
HR
|
95% CI
|
P-value
|
Gender
|
|
|
|
|
|
|
|
|
|
Male
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Female
|
0.930
|
0.478-1.809
|
0.831
|
0.953
|
0.490-1.853
|
0.887
|
0.946
|
0.482-1.856
|
0.871
|
Age (yr)
|
|
|
|
|
|
|
|
|
|
<60 (ref)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
≥60
|
1.215
|
0.774-1.907
|
0.396
|
1.209
|
0.771-1.896
|
0.409
|
1.221
|
0.777-1.919
|
0.386
|
BMI (kg/m2)
|
|
|
|
|
|
|
|
|
|
<24 (ref)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
≥24
|
0.778
|
0.475-1.274
|
0.318
|
0.772
|
0.472-1.263
|
0.302
|
0.782
|
0.477-1.282
|
0.329
|
Alcohol
|
|
|
|
|
|
|
|
|
|
No (ref)
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
Yes
|
1.521
|
0.925-2.501
|
0.098
|
1.532
|
0.933-2.517
|
0.092
|
1.529
|
0.928-2.519
|
0.095
|
Differentiation
|
|
|
|
|
|
|
|
|
|
Low-moderate
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
high
|
0.454
|
0.265-0.778
|
0.004
|
0.439
|
0.257-0.753
|
0.003
|
0.453
|
0.265-0.774
|
0.004
|
TNM Stage
|
|
|
|
|
|
|
|
|
|
1
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
ref
|
2
|
1.904
|
0.991-3.662
|
0.053
|
1.912
|
0.996-3.670
|
0.052
|
1.882
|
0.975-3.633
|
0.059
|
3
|
1.868
|
0.886-3.941
|
0.101
|
1.862
|
0.885-3.914
|
0.101
|
1.854
|
0.880-3.905
|
0.104
|
4
|
2.605
|
1.258-5.394
|
0.010
|
2.651
|
1.282-5.482
|
0.009
|
2.567
|
1.231-5.351
|
0.012
|
iTILs
|
|
|
|
|
|
|
|
|
|
Lower
|
ref
|
ref
|
ref
|
|
|
|
|
|
|
Higher
|
0.997
|
0.565-1.759
|
0.993
|
|
|
|
|
|
|
TILV
|
|
|
|
|
|
|
|
|
|
Lower
|
|
|
|
ref
|
ref
|
ref
|
|
|
|
Higher
|
|
|
|
1.324
|
0.835-2.097
|
0.233
|
|
|
|
fTILs
|
|
|
|
|
|
|
|
|
|
Lower
|
|
|
|
|
|
|
ref
|
ref
|
ref
|
Higher
|
|
|
|
|
|
|
0.933
|
0.591-1.473
|
0.766
|