Patient characteristics
The study cohort included 132 patients in this who were diagnosed with HPSCC, and the clinical characteristics of these patients are summarized in Table 1. The samples included 131 males and 1 female with a median age of 60 years (range: 40-76 years). Twenty-nine (22%) patients had higher pathological grading (grade III), and 103 (78%) patients had lower pathological grading (grades I and II). As described above, the HPSCC patients were divided into 2 groups based on their overall staging group according to the AJCC 7th (American Joint Committee on Cancer) edition cancer staging system: namely overall staging group III (35, patients, 26.5%) and IVA or IVB (97 patients, 73.5%). Patients smoked at least 20 packs of cigarettes per year as many as 115 (87.1%) and smoking less than 20 packs group was 17 (12.9%). Regarding alcohol consumption, 28 (21.2%) patients consumed less than 10-40 g/day, and 104 (78.8%) patients consumed at least 40 g/day. Most tumours were located in the pyriform sinus (PS).
Table 1. Clinicopathological characteristics of 132 patients
Characteristic
|
N (%)
|
Age at diagnosis
|
|
<60y
|
71 (53.8)
|
≥60y
|
61 (46.2)
|
Sex
|
|
Male
|
131 (99.2)
|
Female
|
1 (0.8)
|
Smoke history
|
|
No
|
17 (12.9)
|
Yes
|
115 (87.1)
|
Drink history
|
|
No
|
28 (21.2)
|
Yes
|
104 (78.8)
|
Site
|
|
Pyriform sinus
|
116 (87.9)
|
Not pyriform sinus
|
16 (12.1)
|
Grade
|
|
G1+G2
|
103 (78.0)
|
G3
|
29 (22.0)
|
Stage
|
|
III
|
35 (26.5)
|
IVA/IVB
|
97 (73.5)
|
T category
|
|
T1-3
|
55 (41.7)
|
T4a
|
77 (58.3)
|
N category
|
|
N0-1
|
55 (41.7)
|
N2-3
|
77 (58.3)
|
Laryngectomy
|
|
Total
|
77 (58.3)
|
Partial
|
55 (41.7)
|
Follow up
With a median follow-up of 28.4 months (interquartile range 20.9-39.1 months), the 3-year OS, DFS, DMFS and LRFS for the entire cohort were 68.2% (95% confidence interval [CI], 57.8% to 78.6%), 62.1% (95% CI, 52.1% to 72.1%), 72.6% (95% CI, 62.2% to 83.0%) and 79.7% (95% CI, 72.4% to 87.0%), respectively. During the follow-up period, 42 (31.8%) patients experienced treatment failure. A total of 16 (12.1%) and 17 (12.8%) patients had only locoregional recurrence or distant metastasis, respectively, and 9 (6.8%) patients had both.
Association among different variables
Regarding the correlations of the immune markers with clinicopathological characteristics, high levels of TILs (CD8, Foxp3 and CD4) showed evident correlations with lower histopathological grade. The CD8/Foxp3 ratio was associated with the expression of CD8 and Foxp3, and the CD8/CD4 ratio correlated with each subtype of CD8 and CD4 infiltrates (P < 0.05). Similarly, Foxp3+ TILs exhibited an association with both overall staging group and T category (P =0.048 and P= 0.046, respectively). We also found marked correlations among CD8, CD4 and Foxp3 using Pearson’s correlation coefficient (P<0.001, Fig. 2A-C). Other relationships between immune marker expression and clinicopathological parameters are summarized in Table 2.
Table 2. Associations between the clinicopathological factors of HPSCC with the status of CD8, CD4, and Foxp3 infiltration and the CD8/Foxp3 and CD8/CD4 ratios (N = 132).
Characteristic
|
CD8
|
P
|
CD4
|
P
|
Foxp3
|
P
|
CD8/Foxp3
|
P
|
CD8/CD4
|
P
|
low
|
high
|
low
|
high
|
low
|
high
|
low
|
high
|
low
|
high
|
Age at diagnosis
|
|
|
0.033*
|
|
|
0.295
|
|
|
0.727
|
|
|
0.166
|
|
|
0.861
|
<60y
|
31
|
40
|
|
31
|
40
|
|
37
|
34
|
|
33
|
38
|
|
34
|
37
|
|
≥60y
|
38
|
23
|
|
33
|
28
|
|
29
|
32
|
|
36
|
25
|
|
31
|
30
|
|
Sex
|
|
|
1.000
|
|
|
1.000
|
|
|
1.000
|
|
|
1.000
|
|
|
0.492
|
Male
|
1
|
0
|
|
0
|
1
|
|
0
|
1
|
|
1
|
0
|
|
1
|
0
|
|
Female
|
68
|
63
|
|
64
|
67
|
|
66
|
65
|
|
68
|
63
|
|
64
|
67
|
|
Smoke history
|
|
|
0.124
|
|
|
0.120
|
|
|
0.604
|
|
|
0.124
|
|
|
0.072
|
No
|
12
|
5
|
|
5
|
12
|
|
7
|
10
|
|
12
|
5
|
|
12
|
5
|
|
Yes
|
57
|
58
|
|
59
|
56
|
|
59
|
56
|
|
57
|
58
|
|
53
|
62
|
|
Drink history
|
|
|
0.671
|
|
|
0.834
|
|
|
0.832
|
|
|
1.000
|
|
|
0.832
|
No
|
16
|
12
|
|
13
|
15
|
|
13
|
15
|
|
15
|
13
|
|
13
|
15
|
|
Yes
|
53
|
51
|
|
51
|
53
|
|
53
|
51
|
|
54
|
50
|
|
52
|
52
|
|
Site
|
|
|
0.064
|
|
|
0.290
|
|
|
0.059
|
|
|
0.433
|
|
|
0.948
|
Pyriform sinus
|
57
|
59
|
|
54
|
62
|
|
54
|
62
|
|
59
|
57
|
|
57
|
59
|
|
Not pyriform sinus
|
12
|
4
|
|
10
|
6
|
|
12
|
4
|
|
10
|
6
|
|
8
|
8
|
|
Grade
|
|
|
0.003*
|
|
|
0.037*
|
|
|
0.003*
|
|
|
0.834
|
|
|
0.835
|
G1+G2
|
61
|
42
|
|
55
|
48
|
|
59
|
44
|
|
53
|
50
|
|
50
|
53
|
|
G3
|
8
|
21
|
|
9
|
20
|
|
7
|
22
|
|
16
|
13
|
|
15
|
14
|
|
Stage
|
|
|
0.238
|
|
|
0.554
|
|
|
0.048*
|
|
|
0.609
|
|
|
1.000
|
III
|
15
|
20
|
|
15
|
20
|
|
12
|
23
|
|
17
|
18
|
|
17
|
18
|
|
IVA/IVB
|
54
|
43
|
|
49
|
48
|
|
54
|
43
|
|
52
|
45
|
|
48
|
49
|
|
T category
|
|
|
0.205
|
|
|
0.589
|
|
|
0.046*
|
|
|
0.449
|
|
|
0.718
|
T1-3
|
40
|
44
|
|
39
|
45
|
|
36
|
48
|
|
46
|
38
|
|
40
|
44
|
|
T4a
|
29
|
19
|
|
25
|
23
|
|
30
|
18
|
|
23
|
25
|
|
25
|
23
|
|
N category
|
|
|
1.000
|
|
|
0.599
|
|
|
0.289
|
|
|
0.536
|
|
|
0.860
|
N0-1
|
29
|
26
|
|
25
|
30
|
|
24
|
31
|
|
27
|
28
|
|
28
|
27
|
|
N2-3
|
40
|
37
|
|
39
|
38
|
|
42
|
35
|
|
42
|
35
|
|
37
|
40
|
|
Laryngectomy
|
|
|
0.379
|
|
|
0.599
|
|
|
0.480
|
|
|
0.725
|
|
|
0.295
|
Total
|
43
|
34
|
|
39
|
38
|
|
41
|
36
|
|
39
|
38
|
|
41
|
36
|
|
Partial
|
26
|
29
|
|
25
|
30
|
|
25
|
30
|
|
30
|
25
|
|
24
|
31
|
|
CD8 (cut off: 80)
|
|
|
|
|
|
0.001*
|
|
|
<0.001*
|
|
|
0.001*
|
|
|
0.039*
|
Low
|
|
|
|
43
|
26
|
|
50
|
19
|
|
46
|
23
|
|
40
|
29
|
|
High
|
|
|
|
21
|
42
|
|
16
|
47
|
|
23
|
40
|
|
25
|
38
|
|
CD4 (cut off: 30)
|
|
|
0.001*
|
|
|
|
|
|
<0.001*
|
|
|
0.163
|
|
|
<0.001*
|
Low
|
43
|
21
|
|
|
|
|
49
|
15
|
|
29
|
35
|
|
15
|
49
|
|
High
|
26
|
42
|
|
|
|
|
17
|
51
|
|
40
|
28
|
|
50
|
18
|
|
Foxp3 (cut off: 30)
|
|
|
<0.001*
|
|
<0.001*
|
|
|
|
|
|
0.014*
|
|
|
|
Low
|
50
|
16
|
|
49
|
17
|
|
|
|
|
27
|
39
|
|
29
|
37
|
0.296
|
High
|
19
|
47
|
|
15
|
51
|
|
|
|
|
42
|
24
|
|
36
|
30
|
|
Abbreviations: HPSCC, hypopharyngeal squamous cell carcinoma; G1, well differentiated; G2, moderately differentiated, G3, poorly differentiated.
*The P value is significant
Correlation with prognosis
The Kaplan-Meier curves of 3-year OS, DFS, DMFS, LRFS for patients with TILs and the ratios are shown in Figs. 3-4. The 3-year OS, DFS, DMFS and LRFS rates according to high and low CD8+TIL density, were 80.9% vs 56.3%, 73.2% vs 51.4%, 80.4% vs 64.5% and 77.8% vs 82.1%, respectively. Significant differences were found between the high and low CD8+ TIL groups in 3-year OS, DFS and DMFS but not in LRFS (Fig. 3A-D). Similarly, a higher Foxp3+ TIL level was also strongly correlated with better OS, DFS and DMFS (P = 0.001, P = 0.028 and P = 0.009, respectively, Fig. 3E-H). Further analysis revealed that patients with a high CD8/Foxp3 ratio had significantly better DFS and DMFS (P = 0.013 and P = 0.029, respectively) (Fig. 4B-C), while a higher CD8/CD4 ratio evidently improved 3-year DFS and LRFS compared with a lower CD8/CD4 ratio (P = 0.021 and P = 0.033, respectively) (Fig. 4F, H). In contrast, no associations were observed between the status of the CD8/Foxp3 ratio or the CD8/CD4 ratio and OS (Fig. 4A, E). Both UVA and MVA were performed to determine the associations between prognosis and clinicopathological variables (Table 3-4). The results revealed that a high ratio of CD8/Foxp3 remained an independent favourable prognostic factor for DFS (HR=2.613; 95% CI, 1.203-5.673; P = 0.015) and DMFS (HR=3.606; 95% CI, 1.334-9.748; P = 0.011). Furthermore, the CD8/CD4 ratio was also an independent prognostic factor for LRFS (HR=2.418; 95% CI, 1.043-5.606; P = 0.040) in the MVA. In addition, Foxp3+ TIL, T category and site were found to be independent prognostic factors associated with DMFS, DFS and LRFS, respectively (Table 4).
Table 3. Univariate analyses of OS, DFS, DMFS and LRFS in the entire population (N = 132).
Variable
|
OS
|
|
DFS
|
|
DMFS
|
|
LRFS
|
|
HR
|
95%CI
|
P
|
|
HR
|
95%CI
|
P
|
|
HR
|
95%CI
|
P
|
|
HR
|
95%CI
|
P
|
Age, years (≥60y vs <60y)
|
0.521
|
0.254-1.073
|
0.077
|
|
0.637
|
0.341-1.188
|
0.156
|
|
0.787
|
0.361-1.715
|
0.547
|
|
0.594
|
0.263-1.346
|
0.212
|
Smoke (Yes vs No)
|
0.596
|
0.259-1.372
|
0.224
|
|
0.738
|
0.328-1.662
|
0.463
|
|
0.611
|
0.230-1.621
|
0.322
|
|
1.753
|
0.413-7.438
|
0.446
|
Drink history (Yes vs No)
|
0.748
|
0.348-1.607
|
0.457
|
|
0.945
|
0.452-1.976
|
0.881
|
|
0.822
|
0.330-2.050
|
0.675
|
|
1.991
|
0.596-6.653
|
0.263
|
Site (Not PS vs PS)
|
2.534
|
1.144-5.610
|
0.022*
|
|
2.524
|
1.206-5.284
|
0.014*
|
|
2.629
|
1.054-6.556
|
0.038*
|
|
2.600
|
1.038-6.513
|
0.041*
|
Grade (G3 vs G2+G1)
|
0.689
|
0.285-1.665
|
0.408
|
|
1.033
|
0.508-2.102
|
0.929
|
|
1.283
|
0.539-3.053
|
0.573
|
|
1.586
|
0.684-3.675
|
0.282
|
Stage (IVA/IVB vs III)
|
1.785
|
0.739-4.313
|
0.198
|
|
2.457
|
1.035-5.834
|
0.042*
|
|
0.208
|
0.049-0.880
|
0.033*
|
|
2.848
|
0.852-9.518
|
0.089
|
T category (T4a vs T1-3)
|
2.259
|
1.151-4.436
|
0.018*
|
|
2.508
|
1.365-4.610
|
0.003*
|
|
2.681
|
1.231-5.842
|
0.013*
|
|
2.069
|
0.944-4.538
|
0.070
|
N category (N2-3 vs N0-1)
|
1.319
|
0.652-2.666
|
0.441
|
|
1.682
|
0.874-3.236
|
0.120
|
|
1.681
|
0.731-3.868
|
0.222
|
|
2.361
|
0.943-5.915
|
0.067
|
Laryngectomy (Total vs Partial)
|
1.896
|
0.853-4.215
|
0.117
|
|
1.860
|
0.951-3.638
|
0.070
|
|
2.233
|
0.894-5.578
|
0.085
|
|
2.370
|
0.946-5.938
|
0.066
|
CD8 (Low vs High)
|
2.324
|
1.127-4.795
|
0.022*
|
|
1.892
|
1.005-3.560
|
0.048*
|
|
2.391
|
1.038-5.505
|
0.040*
|
|
0.900
|
0.411-1.974
|
0.793
|
CD4 (Low vs High)
|
1.174
|
0.593-2.323
|
0.645
|
|
1.119
|
0.610-2.052
|
0.717
|
|
1.656
|
0.749-3.661
|
0.212
|
|
0.968
|
0.442-2.123
|
0.936
|
Foxp3 (Low vs High)
|
3.253
|
1.511-7.001
|
0.003*
|
|
1.999
|
1.063-3.760
|
0.032*
|
|
3.006
|
1.263-7.153
|
0.013*
|
|
1.615
|
0.726-3.596
|
0.240
|
CD8/Foxp3 (Low vs High)
|
1.490
|
0.752-2.953
|
0.253
|
|
2.205
|
1.159-4.195
|
0.016*
|
|
2.463
|
1.069-5.674
|
0.034*
|
|
1.522
|
0.683-3.391
|
0.305
|
CD8/CD4 (Low vs High)
|
1.857
|
0.930-3.705
|
0.079
|
|
2.058
|
1.099-3.851
|
0.024*
|
|
1.947
|
0.878-4.317
|
0.101
|
|
2.424
|
1.046-5.621
|
0.039*
|
Abbreviations: PS, pyriform sinus; OS, overall survival; DFS, disease-free survival; DMFS, distant metastasis-free survival; LRFS, local relapse-free survival;
G1, well differentiated; G2, moderately differentiated, G3, poorly differentiated.
*The P value is significant
Table 4. Multivariate analyses of OS, DFS, DMFS and LRFS in the entire population (N = 132).
Variable
|
OS
|
|
DFS
|
|
DMFS
|
|
LRFS
|
|
HR
|
95%CI
|
P
|
|
HR
|
95%CI
|
P
|
|
HR
|
95%CI
|
P
|
|
HR
|
95%CI
|
P
|
Site (Not PS vs PS)
|
1.621
|
0.706-3.726
|
0.255
|
|
1.756
|
0.810-3.807
|
0.154
|
|
1.549
|
0.614-3.909
|
0.354
|
|
2.590
|
1.034-6.491
|
0.042*
|
Stage (IVA/IVB vs III)
|
|
|
|
|
1.496
|
0.563-3.979
|
0.419
|
|
2.489
|
0.530-11.688
|
0.248
|
|
|
|
|
T category (T4a vs T1-3)
|
1.880
|
0.947-3.735
|
0.071
|
|
2.196
|
1.077-4.476
|
0.030*
|
|
2.115
|
0.911-4.911
|
0.081
|
|
|
|
|
CD8 (Low vs High)
|
1.231
|
0.509-2.977
|
0.644
|
|
0.716
|
0.300-1.709
|
0.452
|
|
0.698
|
0.217-2.242
|
0.546
|
|
|
|
|
Foxp3 (Low vs High)
|
2.387
|
0.932-6.111
|
0.070
|
|
2.066
|
0.885-4.826
|
0.094
|
|
3.253
|
1.038-10.196
|
0.043*
|
|
|
|
|
CD8/Foxp3 (Low vs High)
|
|
|
|
|
2.613
|
1.203-5.673
|
0.015*
|
|
3.606
|
1.334-9.748
|
0.011*
|
|
|
|
|
CD8/CD4 (Low vs High)
|
|
|
|
|
1.708
|
0.871-3.349
|
0.119
|
|
|
|
|
|
2.418
|
1.043-5.606
|
0.040*
|
Multivariate cox regression analyses were performed for all variables that were significantly associated with survival in univariate analysis
Abbreviations: PS, pyriform sinus; OS, overall survival; DFS, disease-free survival; DMFS, distant metastasis-free survival; LRFS, local relapse-free survival;
G1, well differentiated; G2, moderately differentiated, G3, poorly differentiated.
*The P value is significant