Clinicopathologic Features
As reported in Table 1, which summarizes the clinical and pathological features of these 525 patients, the median age was 61 years with a wide age distribution ranging from 34 to 83 years. Four hundred twenty-nine patients (81.7%) were males. A history of tobacco smoking was observed in 203 (38.7%) patients. As the anatomic site, 5% cases were located in the upper esophagus, 44.8% in the middle and the other 45.5% in the lower with a mean of tumor size of 3.4 cm of the biggest axis. The histopathological diagnoses consisted of well-differentiated ESCC in 20 patients (3.8%), moderately- differentiated ESCC in 295 patients (56.2%) and poorly-differentiated ESCC in 210 patients (40.0%). The vessel and nerve invasion was observed in 22.3% and 34.5% patients. According to the T stage, we found that 51 patients (9.7%) were in T1 stage, 116 (22.1%) patients were in T2 stage, 357 (68.0%) patients were in T3 stage and 1 (0.2%) patients were in T4 stage. The lymph node metastases were detected in 243 (46.3%) patients. According to 8th TNM stage, 290 (55.2%) patients were in the stage I-II group, and 235 (44.8%) patients were in the stage III-IVa group.
Table 1
Correlation between p16 expression and clinicopathologic features
Variable
|
p16 IHC positive
|
|
p16 IHC overexpression
|
|
|
No
|
Yes
|
P value
|
No
|
Yes
|
P value
|
Age (years)
|
|
|
0.125
|
|
|
0.885
|
< 60
|
202
|
22
|
|
212
|
12
|
|
≥ 60
|
258
|
43
|
|
284
|
17
|
|
Sex
|
|
|
0.080
|
|
|
0.730
|
Female
|
79
|
17
|
|
90
|
6
|
|
Male
|
381
|
48
|
|
406
|
23
|
|
Smoking
|
|
|
0.095
|
|
|
0.634
|
No
|
276
|
46
|
|
303
|
19
|
|
Yes
|
184
|
19
|
|
193
|
10
|
|
Tumor Size
|
|
|
0.917
|
|
|
0.516
|
< 3.4
|
265
|
37
|
|
287
|
15
|
|
≥ 3.4
|
195
|
28
|
|
209
|
14
|
|
Tumor Location
|
|
|
0.281
|
|
|
0.323
|
Upper
|
23
|
3
|
|
24
|
2
|
|
Middle
|
199
|
36
|
|
219
|
16
|
|
Lower
|
214
|
25
|
|
229
|
10
|
|
Differentiation
|
|
|
0.743
|
|
|
0.377
|
Well
|
19
|
1
|
|
20
|
0
|
|
Middle
|
258
|
37
|
|
281
|
14
|
|
Poor
|
183
|
27
|
|
195
|
15
|
|
Vessel invasion
|
|
|
0.153
|
|
|
0.832
|
No
|
353
|
55
|
|
385
|
23
|
|
Yes
|
107
|
10
|
|
111
|
6
|
|
Nerve invasion
|
|
|
0.219
|
|
|
0.422
|
No
|
297
|
47
|
|
323
|
21
|
|
Yes
|
163
|
18
|
|
173
|
8
|
|
pT
|
|
|
0.574
|
|
|
0.337
|
T1
|
45
|
6
|
|
48
|
3
|
|
T2
|
98
|
18
|
|
113
|
3
|
|
T3
|
316
|
41
|
|
334
|
23
|
|
T4
|
1
|
0
|
|
1
|
0
|
|
Lymph node metastasis
|
|
|
0.611
|
|
|
0.079
|
No
|
249
|
33
|
|
271
|
11
|
|
Yes
|
211
|
32
|
|
225
|
18
|
|
pN
|
|
|
0.809
|
|
|
0.242
|
N0
|
249
|
33
|
|
271
|
11
|
|
N1
|
114
|
18
|
|
122
|
10
|
|
N2
|
73
|
12
|
|
78
|
7
|
|
N3
|
24
|
2
|
|
25
|
1
|
|
Clinical stage
|
|
|
0.612
|
|
|
0.123
|
I-II
|
256
|
34
|
|
278
|
12
|
|
III-IVa
|
204
|
31
|
|
218
|
17
|
|
Disease progression
|
|
|
0.057
|
|
|
0.792
|
No
|
204
|
37
|
|
227
|
14
|
|
Yes
|
256
|
28
|
|
269
|
15
|
|
Death
|
|
|
0.066
|
|
|
0.825
|
No
|
206
|
37
|
|
229
|
14
|
|
Yes
|
254
|
28
|
|
267
|
15
|
|
IHC, immunohistochemistry
|
p16 expression in ESCC
The levels of p16 expression in the tumor samples of all ESCC patients are presented in Table 2. When a cutoff value of > 10% was used, p16 positive was seen in 65 specimens (12.4%) and negative in 460 specimens (87.6%). When a cutoff value of > 70%++ was used, p16 overexpression was seen in 29 (5.5%) specimens. Among 65 ESCC with p16 IHC positive, 29 (5.5%) cases were p16 overexpression with diffuse and strong staining, and the other 36 (6.9%) cases showed focal and weak staining (Fig. 1).
Table 2
Univariate and Multivariate Analyses of Prognostic Factors for Survival
|
DFS
|
OS
|
Univariate analysis
|
P value
|
HR (95% CI)
|
P value
|
HR (95% CI)
|
Sex
|
0.196
|
1.223 (0.901–1.660)
|
0.120
|
1.286 (0.937–1.766)
|
Age
|
0.856
|
0.978 (0.774–1.237)
|
0.754
|
0.963 (0.759–1.222)
|
Smoking
|
0.300
|
1.134 (0.894–1.438)
|
0.212
|
1.166 (0.916–1.483)
|
Tumor Size
|
0.222
|
1.158 (0.915–1.464)
|
0.169
|
1.182 (0.931–1.502)
|
Tumor Location
|
0.922
|
0.990 (0.809–1.211)
|
0.788
|
1.029 (0.837–1.265)
|
Differentiation
|
0.018
|
1.292 (1.045–1.597)
|
0.047
|
1.244 (1.003–1.544)
|
Vessel invasion
|
< 0.001
|
1.620 (1.255–2.091)
|
< 0.001
|
1.592 (1.225–2.068)
|
Nerve invasion
|
0.005
|
1.407 (1.108–1.785)
|
0.001
|
1.494 (1.173–1.903)
|
pT Stage
|
< 0.001
|
1.687 (1.369–2.078)
|
< 0.001
|
1.807 (1.449–2.252)
|
Lymph node metastasis
|
< 0.001
|
2.789 (2.190–3.553)
|
< 0.001
|
2.836 (2.217–3.627)
|
pN Stage
|
< 0.001
|
1.600 (1.429–1.791)
|
< 0.001
|
1.615 (1.440–1.812)
|
Clinical stage
|
< 0.001
|
2.831 (2.226–3.601)
|
< 0.001
|
2.863 (2.242–3.657)
|
p16 positive
|
|
|
|
|
p16 focal expression
|
0.046
|
0.567 (0.325–0.991)
|
0.059
|
0.584 (0.334–1.020)
|
p16 overexpression
|
0.784
|
0.930 (0.552–1.565)
|
0.834
|
0.946 (0.562–1.592)
|
Mutivarate analysis
|
|
|
|
|
Differentiation
|
0.346
|
1.110 (0.893–1.381)
|
0.601
|
1.060 (0.852–1.319)
|
Vessel invasion
|
0.508
|
1.096 (0.835–1.437)
|
0.631
|
1.069 (0.813–1.406)
|
Nerve invasion
|
0.303
|
1.137 (0.890–1.454)
|
0.098
|
1.230 (0.963–1.573)
|
Clinical stage
|
< 0.001
|
2.635 (2.043–3.398)
|
< 0.001
|
2.672 (2.070–3.448)
|
p16 positive
|
0.310
|
|
0.364
|
|
p16 focal expression
|
0.144
|
0.659 (0.376–1.153)
|
0.170
|
0.676 (0.386–1.183)
|
p16 overexpression
|
0.595
|
0.868 (0.514–1.464)
|
0.654
|
0.887 (0.526–1.497)
|
p16 expression and clinicopathological parameters were analyzed. No significant association was observed between p16 positive or overexpression and age, sex, tumor size and location, histological differentiation, vessel and nerve invasion, T stage and lymph node metastasis (P > 0.05, Table 2).
p16 expression and patients’ prognosis
The follow-up period ranged from 3 to 102 months, with a median of 31 months. Two hundred and eighty two (53.7%) patients died within a median OS time of 42.0 months (95% CI: 33.0–51.0 months). Two hundred and eighty four (54.1%) patients had tumor progression within a median DFS time of 36.0 months (95% CI: 25.8–46.2 months).
When these patients were divided into p16 positive group (n = 65) and negative group (n = 460) defined by a cut-off value of 10%, the positive group demonstrated a better outcome compared with the negative group, however, this did not reach statistical significance (DFS P = 0.088 and OS P = 0.115) (Fig. 2A and 2B). When these patients were divided into p16 overexpression group (n = 29) and non-overexpression group (n = 496) defined by a cut-off value of 80% ++, there was no difference concerning DFS (P = 0.888) and OS (P = 0.933) of patients with p16 overexpression compared to those without overexpression (Fig. 2C and 2D). When these patients were divided into p16 negative group (n = 460), focal expression group (n = 36) and overexpression group (n = 29) defined by the above mentioned cut-off values, the survival of focal expression group tended to be better compared with negative group (DFS P = 0.040 and OS P = 0.052) and overexpression group (DFS P = 0.201 and OS P = 0.258), and there was no survival difference between negative group and overexpression group (DFS P = 0.780 and OS P = 0.837) (Fig. 2E and 2F). The univariate analysis indicated a significant association between poor differentiation, vessel invasion, nerve invasion, higher clinical stage and poorer survival, and an association between p16 focal expression and favorite survival. Then multivariate analysis for OS and DFS was performed and included above mentioned factors, and only clinical stage was found to be a significantly independent prognostic factor (Table 3) (Fig. 3A and 3B).
Table 3
Univariate and Multivariate Analyses of Prognostic Factors for Survival in Stage I-II and III-IVa ESCC
|
DFS
|
OS
|
Univariate factor analysis
|
P value
|
HR (95% CI)
|
P value
|
HR (95% CI)
|
I-II Stage
|
|
|
|
|
Sex
|
0.764
|
0.937 (0.613–1.432)
|
0.850
|
0.959 (0.625–1.474)
|
Age
|
0.830
|
1.043 (0.712–1.528)
|
0.786
|
1.054 (0.719–1.547)
|
Smoking
|
0.469
|
0.859 (0.570–1.296)
|
0.626
|
0.904 (0.601–1.359)
|
Tumor Size
|
0.514
|
0.877 (0.591–1.301)
|
0.850
|
0.963 (0.651–1.423)
|
Tumor Location
|
0.677
|
0.935 (0.680–1.285)
|
0.744
|
0.947 (0.685–1.310)
|
Differentiation
|
0.104
|
1.321 (0.945–1.846)
|
0.191
|
1.251 (0.894–1.750)
|
Vessel invasion
|
0.350
|
1.288 (0.758–2.187)
|
0.488
|
1.213 (0.703–2.091)
|
Nerve invasion
|
0.916
|
1.023 (0.670–1.563)
|
0.400
|
1.195 (0.789–1.811)
|
p16 positive
|
0.045
|
|
0.052
|
|
p16 focal expression
|
0.025
|
0.270 (0.086–0.851)
|
0.029
|
0.277 (0.088–0.874)
|
p16 overexpression
|
0.247
|
0.508 (0.161-1.600)
|
0.266
|
0.521 (0.165–1.642)
|
III-IVa Stage
|
|
|
|
|
Sex
|
0.690
|
1.098 (0.695–1.733)
|
0.796
|
1.062 (0.673–1.677)
|
Age
|
0.464
|
1.118 (0.829–1.508)
|
0.238
|
1.198 (0.887–1.617)
|
Smoking
|
0.794
|
1.041 (0.772–1.403)
|
0.816
|
1.036 (0.768–1.398)
|
Tumor Size
|
0.270
|
1.183 (0.878–1.594)
|
0.252
|
1.191 (0.883–1.607)
|
Tumor Location
|
0.105
|
0.807 (0.623–1.046)
|
0.144
|
0.823 (0.634–1.069)
|
Differentiation
|
0.932
|
1.012 (0.764–1.341)
|
0.897
|
0.982 (0.740–1.302)
|
Vessel invasion
|
0.461
|
1.122 (0.827–1.522)
|
0.519
|
1.106 (0.813–1.505)
|
Nerve invasion
|
0.142
|
1.251 (0.928–1.689)
|
0.099
|
1.287 (0.953–1.739)
|
p16 positive
|
0.977
|
|
0.974
|
|
p16 focal expression
|
0.899
|
1.042 (0.549–1.977)
|
0.847
|
1.065 (0.561–2.021)
|
p16 overexpression
|
0.856
|
1.056 (0.586–1.901)
|
0.890
|
1.042 (0.578–1.878)
|
p16 expression and patients’ prognosis in I-II stage ESCC
Among 290 I-II stage patients, 110 (37.9%) patients died and 111 (38.3%) patients had tumor progression within a non-reached median OS and DFS time.
When these patients were divided into p16 negative group (n = 256), focal expression group (n = 22) and overexpression group (n = 12) defined by the above mentioned cut-off values, the survival of focal expression group was better compared with negative group (DFS P = 0.015 and OS P = 0.019), tended to be better compared with overexpression group (DFS P = 0.405 and OS P = 0.432). And p16 overexpression group tended to have better outcome compared with the negative group, however, this did not reach statistical significance (DFS P = 0.233 and OS P = 0.254) (Fig. 3C and 3D). The univariate analysis only indicated p16 focal expression was associated with favorite DFS and OS (Table 4).
p16 expression and patients’ prognosis in III-IVa stage ESCC
Among 235 III-IVa stage patients, 172 (73.2%) patients died within a median OS time of 25.0 months (95% CI: 22.9–27.1 months). 173 (73.6%) patients had tumor progression within a DFS mean time of 20.0 months (95% CI: 17.8–22.2 months).
When these patients were divided into p16 negative group (n = 204), focal expression group (n = 14) and overexpression group (n = 17) defined by the above mentioned cut-off values, there were no survival difference between focal expression group and negative group (DFS P = 0.899 and OS P = 0.848) or overexpression group (DFS P = 0.976 and OS P = 0.853) (Fig. 3E and 3F). In univariate statistical analysis, no prognostic factor was found in these patients (Table 4).