Histopathology 2014, 65, 517–526. DOI: 10.1111/his.12413
TNFAIP8 overexpression is associated with lymph node metastasis and poor prognosis in intestinal-type gastric adenocarcinoma Ming Yang, Qi Zhao, Xiaoxia Wang,1 Tianbo Liu,2 Guodong Yao,3 Changjie Lou & Yanqiao Zhang Department of Gastrointestinal Medical Oncology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China, 1Department of Emergency, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China, 2Department of Gynecology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China, and 3Department of Pathology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China Date of submission 22 November 2013 Accepted for publication 8 March 2014 Published online Article Accepted 12 March 2014
Yang M, Zhao Q, Wang X, Liu T, Yao G, Lou C, Zhang Y (2014) Histopathology 65, 517–526
TNFAIP8 overexpression is associated with lymph node metastasis and poor prognosis in intestinal-type gastric adenocarcinoma Aims: Tumour necrosis factor alpha-induced protein 8 (TNFAIP8) is implicated in the progression of several human malignancies, but its role in gastric adenocarcinoma is unknown. TNFAIP8 expression and its correlation with clinical significance in gastric adenocarcinoma are evaluated in this study. Methods and results: The expression of TNFAIP8 was determined in primary gastric adenocarcinoma tissues using immunohistochemistry (IHC) and Western blotting analysis. TNFAIP8 expression was higher in gastric adenocarcinoma tissues. Elevated expression of TNFAIP8 in gastric adenocarcinoma was associated significantly with depth of invasion (P = 0.024),
lymph node metastasis (P = 0.038) and Lauren classification (P = 0.048). Patients with tumours showing high TNFAIP8 expression had a significantly poorer overall survival (OS) and disease-free survival (DFS) than those with low TNFAIP8 expression in intestinal-type gastric adenocarcinoma (IGA) (P = 0.001 for both). In the multivariate Cox analysis, TNFAIP8 expression was an independent prognostic marker for OS and DFS in IGA with P-values of 0.006 and 0.007, respectively. Conclusions: Our data suggest that TNFAIP8 overexpression may contribute to lymph node metastasis and poor prognosis in IGA.
Keywords: gastric adenocarcinoma, immunohistochemistry, prognosis, TNFAIP8
Introduction Gastric cancer is one of the most common malignant cancers worldwide. Nearly 989 600 new stomach cancer cases and 738 000 gastric cancer-related deaths occurred in 2008, accounting for 8% of total cancer cases and 10% of total cancer-related deaths, of which Address for correspondence: Y Zhang, Department of Gastrointestinal Medical Oncology, The Affiliated Tumour Hospital of Harbin Medical University, Harbin, Heilongjiang, China. e-mail:
[email protected] © 2014 John Wiley & Sons Ltd.
more than 70% of the new cases and deaths were recorded in developing countries.1 Thus, improving the clinical outcome of gastric cancer remains a challenging but urgent medical problem because of its high morbidity and mortality, especially in China. Gastric carcinogenesis is a multistep process, particularly for gastric cancer of intestinal-type according to the Lauren classification.2 Therefore, further understanding of the underlying molecular mechanisms of gastric cancer progression, and identification of biomarkers to provide novel therapeutic targets, are crucial for improving prognosis.
M Yang et al.
Tumour necrosis factor alpha-induced protein 8 (TNFAIP8), also known as SCC-S2, GG2-1 and MDC3.13, was found originally in human metastatic head and neck squamous cell carcinoma cell lines.3 This 21-kDa cytosolic protein was identified as a novel member of the FLICE (caspase-8)-inhibitory family,4 and is expressed in many malignancies, including head and neck squamous cell carcinoma,3 breast and renal cancers,5 colon cancer,6 non-small-cell lung cancer,7 oesophageal squamous cell carcinoma,8 epithelial ovarian cancer,9 cervical cancer10 and prostate cancer.11 Studies have indicated that TNFAIP8 is an apoptosis regulator, and so may be important in cancer cell growth, invasion, metastasis and apoptosis inhibition.12 However, the status of TNFAIP8 expression and its role in gastric adenocarcinoma is currently unknown. In this study, TNFAIP8 expression in gastric cancer was investigated by Western blotting and immunohistochemical staining, and its clinical significance in gastric adenocarcinoma was evaluated.
Materials and methods PATIENTS AND SPECIMENS
The study protocol was approved by the Medical Ethics Committee of the TumourHospital of Harbin Medical University. Specimens used for immunohistochemistry (IHC) were obtained from 138 patients diagnosed with gastric adenocarcinoma who underwent surgery in the Tumour Hospital of Harbin Medical University between December 2006 and December 2007. Thirty benign gastric tissues from patients with chronic gastritis who underwent endoscopic biopsy were also obtained from the Tumour Hospital of Harbin Medical University. In addition, 13 fresh gastric adenocarcinoma and paired adjacent non-neoplastic tissues were collected and frozen at 80°C until protein extraction. Follow-up information was obtained by review of the patients’ medical records. None of the patients received radiotherapy or chemotherapy before surgical resection. All 138 cases were reviewed and reclassified according to the Union International Control Cancer criteria13 and categorized as intestinal, diffuse or mixed, according to the Lauren classification.14 Tumours of mixed type were excluded from this study because of the small sample size. The remaining 123 cases were analysed using pathological parameters, including age, gender, clinical stage, depth of tumour invasion, lymph node metastasis, distant metastasis, differentiation, tumour location and Lauren classification.
The end-points of the study were overall survival (OS) and disease-free survival (DFS). OS was defined as the period from the date of surgery until death or to the last date of follow-up; DFS was defined as the period from the date of surgery to the date of relapse or metastasis. Cases lost during follow-up and those ending in death from any cause other than gastric cancer were regarded as censored data in the analysis of survival rates. All 123 patients were followed-up for an interval of 2–69 months (median 33.00 months). WESTERN BLOT ANALYSIS
Total proteins from frozen tissues were extracted in RIPA buffer consisting of 1% protease inhibitor mixture and quantified using the Bradford method. Equivalent amounts of protein were separated on a sodium dodecyl sulphate-polyacrylamide gel and transferred to a polyvinylidene difluoride film (Roche Diagnostics, Mannheim, Germany). The membranes were blocked by 5% skimmed milk in 1 9 TBST (10 mM Tris, pH 7.4, 150 mM NaCl and 0.1% Tween20) at room temperature for 1 h and incubated overnight at 4°C with primary antibodies. Then, the film was incubated with horseradish peroxidase-labelled secondary antibody for 1 h at room temperature. Protein bands were visualized by chemiluminescent reagents (EZ-ECL; Biological Industries Israel Beit Haemek Ltd., Beit-Haemek, Israel), according to the manufacturer’s instructions. Two antibodies were used in this study: anti-TNFAIP8 (dilution 1:1000, ab64988; Abcam, Cambridge, MA, A
T1
T2
T3
T4
N1
N2
N3
N4 21 kDa
TNFAIP8 GAPDH B
2.5 Relative expressions of TNF AIP8
518
2 1.5 1 0.5 0
T
N
Figure 1. Elevated expression of TNFAIP8 in clinical gastric adenocarcinoma tissues (T = tumour; N = normal). A, TNFAIP8 expression was evaluated by Western blot; GAPDH was used as an internal control. B, Histogram of pooled data from representative gastric adenocarcinoma tissues and adjacent non-neoplastic mucosa. The data are presented as mean SD, P < 0.05. © 2014 John Wiley & Sons Ltd, Histopathology, 65, 517–526.
TNFAIP8 is a marker of poor prognostis in gastric cancer
USA) and anti-GAPDH (dilution 1:1000; Zhong Shan Golden Bridge Biological Technology Inc., Beijing, China). The experiment was repeated in triplicate. IMMUNOHISTOCHEMICAL STAINING
Surgically excised tumour specimens were fixed with 10% neutral formalin and embedded in paraffin, and 4-lm thick sections were prepared. Immunostaining was performed using the Two-Step IHC Detection Reagent (PV-6001) kit (Zhong Shan Golden Bridge Biological Technology Inc.), following the manufacturer’s instructions. Briefly, the sections were deparaffinized in xylene and rehydrated with graded alcohol. For antigen retrieval, slides were boiled in 0.01 M citrate buffer (pH 6.0) for 5 min in an autoclave. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity. Tissue sections were
519
then incubated overnight at 4°C with rabbit polyclonal TNFAIP8 antibody (1:150 dilution, ab64988; Abcam). After washing with phosphatebuffered saline, the sections were incubated with rabbit secondary antibody for 20 min. The 3,30 -diaminobenzidine tetrahydrochloride (Dako, Hamburg, Germany) staining reaction was then performed and followed by Meyer’s haematoxylin counterstaining. The sections were dehydrated in ethanol before mounting. The experiment was repeated in triplicate. IMMUNOHISTOCHEMICAL STAINING EVALUATION
The staining results were evaluated by two pathologists who were blinded to the clinicopathological information of the patients. Immunostaining of TNFAIP8 was scored by combining the proportion and intensity of positively stained immunoreactive cells, as described
A
C
E
B
D
F
Figure 2. Immunohistochemical staining of TNFAIP8 in gastric adenocarcinoma and benign gastric tissues. A, High expression in intestinaltype gastric adenocarcinoma. B, Low expression in intestinal-type gastric adenocarcinoma. C, High expression in diffuse-type gastric adenocarcinoma. D, Low expression in diffuse-type gastric adenocarcinoma. E, negative staining in gastric adenocarcinoma. F, low expression in non-neoplastic gastric tissue. © 2014 John Wiley & Sons Ltd, Histopathology, 65, 517–526.
520
M Yang et al.
previously.9 Cytoplasmic immunostaining in tumour cells was considered positive staining. The proportion of cells exhibiting TNFAIP8 expression was categorized as follows: 0, absent; 1, 1–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%. The staining intensity was categorized as follows: 0, negative; 1, weak; 2, moderate; and 3, strong. The proportion and intensity scores were then added together to obtain a total score. To obtain final statistical results, a score 60
53 (43.1)
24 (44.4)
29 (42.0)
Gender Male
94 (76.4)
44 (81.5)
50 (72.5)
Female
29 (23.6)
10 (18.5)
19 (27.5)
TNM stage I + II
51 (41.5)
22 (40.7)
29 (42)
III + IV
72 (58.5)
32 (59.3)
40 (58)
27 (21.9)
17 (31.5)
10 (14.5)
96 (78.1)
37 (68.5)
59 (85.5)
36 (29.3)
21 (38.9)
15 (21.7)
87 (70.7)
33 (61.1)
54 (78.3)
109 (88.6)
49 (90.7)
60 (87)
14 (11.4)
5 (9.3)
9 (13)
52 (42.3)
23 (42.6)
29 (42.0)
Poor
71 (57.7)
31 (57.4)
40 (58.0)
Location Upper
21 (17.1)
9 (16.7)
12 (17.4)
Middle
29 (23.6)
9 (16.7)
20 (29.0)
Lower
73 (59.3)
36 (66.6)
37 (53.6)
78 (63.4)
29 (53.7)
49 (71.0)
45 (36.6)
25 (46.3)
20 (29.0)
Depth of invasion T1 + T2 T3 + T4 Lymph node metastasis Negative Positive Distant metastasis M0 M1 Differentiation Good/moderate
Lauren classification Intestinal Diffuse
P-value* 0.788
0.242
0.886
0.024
0.038
0.512
0.950
0.243
0.048
*Chi squared test. © 2014 John Wiley & Sons Ltd, Histopathology, 65, 517–526.
TNFAIP8 is a marker of poor prognostis in gastric cancer
squared test was used to compare the clinicopathological variables and TNFAIP8 expression. For continuous variables, Student’s t-test was performed. Cumulative survival was estimated by the Kaplan–Meier method, and comparisons among groups were made with a logrank test. A multivariate analysis was conducted to assess the influence of each variable on survival using the Cox proportional hazards regression model. All Pvalues were based on a two-sided statistical analysis, and P < 0.05 was considered statistically significant.
521
Results TNFAIP8 PROTEIN IS OVEREXPRESSED IN GASTRIC ADENOCARCINOMA
Western blotting was used to investigate the differences in expression of TNFAIP8 between tumours and adjacent non-neoplastic tissues. A 21-kDa TNFAIP8 protein was expressed in carcinoma and adjacent mucosa (Figure 1A). Expression of TNFAIP8 protein
Table 2. Association of TNFAIP8 protein expression with clinicopathological variables of intestinal/diffuse type gastric adenocarcinoma patients TNFAIP8 expression in intestinal type (n = 78)
Low
High
P*
14
11
0.947
11
9
19
15
6
5
6
14
19
6
4
4
21
16
4
6
21
14
24
19
1
1
4
7
21
13
6
2
13
3
7
26
16
11
Variables
Low
High
Age (years) ≤60
16
29
>60
13
20
Gender Male
25
35
Female
4
14
TNM stage I + II
16
15
III + IV
13
34
13
6
16
43
17
9
12
40
25
41
4
8
19
22
Poor
10
27
Location Upper
3
10
Middle
6
Lower
20
Depth of invasion T1 + T2 T3 + T4 Lymph node metastasis Negative Positive Distant metastasis M0 M1 Differentiation Good/moderate
*Chi squared test. †Fisher’s exact test. © 2014 John Wiley & Sons Ltd, Histopathology, 65, 517–526.
P*
TNFAIP8 expressionin diffuse type (n = 45)
0.729
0.134
0.032
0.001
0.05). Moreover, separately analysing the clinical data of intestinal-type gastric adenocarcinomas (IGA) and diffuse-type gastric adenocarcinomas (DGA), for both types overexpression of TNFAIP8 was associated with TNM stage (P = 0.032 and P = 0.002, respectively; Table 2). However, TNFAIP8 overexpression was associated with depth of invasion and lymph node metastasis in IGA (P = 0.001 and P < 0.001, respectively) but not in DGA. RELATIONSHIP BETWEEN TNFAIP8 PROTEIN
Table 3. Univariate survival analysis of overall survival in 123 patients with gastric adenocarcinoma Total cases
n (123)
Variables
TNFAIP8 expression Low 54
Mean
SE
45.678
3.157
High
69
34.361
3.280
Gender Male
94
39.078
2.779
Female
29
40.545
4.449
Age (years) ≤60
70
38.018
3.206
53
41.182
3.470
21
65.089
0.483
II
30
47.558
4.453
III
59
30.989
3.138
IV
13
13.923
2.995
Depth of invasion T1 9
63.875
0.695
>60 TNM stage I
T2
18
53.667
5.193
T3
33
36.799
4.579
T4
63
32.221
3.059
62.301
2.449
Lymph node metastasis N0 36 N1
21
42.476
5.178
N2
24
31.142
4.437
N3
42
21.548
3.019
Distant metastasis M0 109
42.618
2.508
14
15.429
3.010
3
54.667
7.621
Moderate
49
43.487
3.755
Poor
71
35.744
3.053
Location Upper
21
37.552
5.647
Middle
29
32.862
4.718
Lower
73
42.348
3.040
P* 0.088
0.965
0.463