Ann Surg Oncol (2014) 21:1099–1106 DOI 10.1245/s10434-013-3339-1
ORIGINAL ARTICLE – GASTROINTESTINAL ONCOLOGY
CD151 Overexpression is Associated with Poor Prognosis in Patients with pT3 Gastric Cancer Sang Yun Ha, MD1, In-Gu Do, MD, PhD1,2, Jeeyun Lee, MD, PhD3, Se Hoon Park, MD, PhD3, Joon Oh Park, MD, PhD3, Won Ki Kang, MD, PhD3, Min-Gew Choi, MD, PhD4, Jun Ho Lee, MD, PhD4, Jae Moon Bae, MD, PhD4, Sung Kim, MD, PhD4, Kyoung-Mee Kim, MD, PhD1, and Tae Sung Sohn, MD, PhD4 1
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; Samsung Genome Institute, Samsung Medical Center, Seoul, Korea; 3Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 4Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
2
ABSTRACT Introduction. CD151, a transmembrane protein of the tetraspanin family, is implicated in the regulation of cellsubstrate adhesion and cell migration. Overexpression of CD151 has been reported in several cancers and controls MET-dependent neoplastic growth by enhancing receptor signaling. However, association of CD151 overexpression with MET or tumor progression has not been reported in gastric cancer. Materials and Methods. We conducted immunohistochemical analysis of CD151 overexpression in 491 pT3 gastric carcinomas and analyzed the relationship with MET overexpression and prognostic significance. Results. CD151 was highly expressed in 119 gastric carcinomas (24.2 %) and was significantly associated with higher pN stages. Patients with CD151-positive gastric cancer showed shorter overall (p = 0.003) and disease-free survival (p = 0.001) compared with patients with CD151negative gastric carcinoma. CD151 overexpression was an independent prognostic factor for overall survival [hazard ration (HR) 1.335; 95 % CI 1.005–1.775; p = 0.046] and disease-free survival (HR 1.903; 95 % CI 1.348–2.685; p \ 0.001). Co-overexpression of CD151 and MET was observed in 30 (6.1 %) gastric cancers and was more frequent in advanced pN stages than in other groups. Ó Society of Surgical Oncology 2013 First Received: 21 April 2013; Published Online: 4 December 2013 K.-M. Kim, MD, PhD e-mail: [email protected] T. S. Sohn, MD, PhD e-mail: [email protected]
Moreover, co-overexpression of CD151 and MET was a strong independent prognostic factor for overall survival (HR 3.163; 95 % CI 1.958–5.108; p \ 0.001) and diseasefree survival (HR 3.834; 95 % CI 2.145–6.852; p \ 0.001). Conclusion. CD151 overexpression is an independent prognostic factor and could be a potential molecular therapeutic target in patients with advanced gastric cancers. Further studies are needed to establish the biological significance of CD151/MET co-overexpression and the potential of targeting both molecules as a therapeutic strategy.
Gastric cancer is the second most common cause of cancer-related death worldwide.1 After the demonstration that trastuzumab has a significant survival benefit in patients with HER2-positive gastric cancer, there has been growing interest in new molecular targets for gastric cancer.2 Tetraspanins are evolutionarily conserved membrane proteins that are thought to form complexes between themselves or with other transmembrane proteins, including various adhesion receptors, such as integrins, or receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) or MET.3–6 Through these interactions, tetraspanins are involved in tumor cell motility, invasiveness, and proliferation.7,8 CD151 was the first member of the tetraspanin family to be identified as a promoter of metastasis.9 Overexpression of CD151 and its association with poor prognosis was reported in cancers of lung, colorectum, pancreas, liver, esophagus, kidney, endometrium, and breast.3,6,10–18 CD151 is known to be involved in cancer progression by
1100
forming multimolecular signaling complexes with MET or members of the integrin family.6,13,19–21 However, the association of CD151 overexpression with MET and its role in prognosis has not been studied in gastric cancers. In this study, we performed correlative analyses on CD151 and MET overexpression in T3 gastric cancers to define their roles in cancer progression. MATERIALS AND METHODS
S. Y. Ha et al. TABLE 1 Patient overexpression Characteristics, n (%)
characteristics
according
to
CD151 expression
CD151 p value
Total 491 (100)
Low 372 (75.8)
High 119 (24.2)
323 (65.8) 168 (34.2)
240 (64.5) 132 (35.5)
83 (69.7) 36 (30.3)
0.29
0.12
Gender Male Female Age (years)
Study Design and Patients We used the gastric cancer cohorts described in our previous studies.22 All 495 patients had advanced pT3restricted gastric cancers and had undergone curative surgical resection and extensive (D2) lymph node dissection. This retrospective study was undertaken after obtaining approval from the SMC institutional review board. Clinicopathologic characteristics obtained from medical records included gender, age, tumor size, tumor location, histological type, Lauren classification, differentiation grade, surgical record, treatment record, recurrence, and vital status at the last follow-up date. The median follow up period was 82.2 (range 0–139) months. For tissue microarray construction, all H&E-stained slides were reviewed and representative areas were carefully selected and marked on paraffin blocks. Four tissue cores (0.6-mm diameter) were obtained from the invasive front, both lateral sides, and the luminal surface of the representative tumor block for each primary carcinoma. Immunohistochemistry Immunohistochemistry (IHC) studies were performed on formalin-fixed, paraffin-embedded, 4-lm-thick tissue sections using mouse monoclonal anti-CD151 (NCL-CD151, Novocastra, Newcastle upon Tyne, UK) as the primary antibody. Tissue sections were deparaffinized three times in xylene for a total of 15 min and subsequently rehydrated. Bond-max autoimmunostainer (Leica Biosystem, Melbourne, Australia) and BondTM Polymer refine detection DS9800 (Leica Biosystem) were used according to the manufacturers’ protocol. Two pathologists (Sang Yun Ha and Kyoung-Mee Kim) independently evaluated the IHC results with no prior knowledge of clinicopathological results. Interpretation of IHC for CD151 was scored according to recent HER2 assessment guidelines for gastric cancer as follows: 0, no reactivity; 1?, faint/barely perceptible membranous reactivity; 2?, weak to moderate complete or basolateral membranous reactivity; 3?, moderate to strong complete or basolateral membranous reactivity in [10 % of tumor cells.23 Cases with a score of
\60
306 (62.3)
239 (64.2)
67 (56.3)
C60
185 (37.7)
133 (35.8)
52 (43.7)
Tumor location Upper third
79 (16.1)
57 (15.3)
22 (18.5)
Middle third
198 (40.3)
156 (41.9)
42 (35.3)
Lower third
181 (36.9)
134 (36.0)
47 (39.5)
Whole stomach
33 (6.7)
25 (6.7)
8 (6.7)
0.6
Tumor size (cm) \4.5
111 (22.6)
87 (23.4)
24 (20.2)
C4.5
380 (77.4)
285 (76.6)
95 (79.8)
0.46
Borrmann type IV
58 (11.8)
Others 433 (88.2) Lauren classification
47 (12.6)
11 (9.2)
325 (87.4)
108 (90.8)
Diffuse
290 (59.1)
233 (62.5)
57 (47.9)
Intestinal
173 (35.2)
117 (31.5)
56 (47.1)
Mixed
28 (5.7)
22 (5.9)
6 (5)
Differentiated
163 (33.2)
114 (30.6)
49 (41.2)
Undifferentiated
328 (66.8)
258 (69.4)
70 (58.8)
Negative
246 (50.1)
196 (52.7)
50 (42)
Positive
245 (49.9)
176 (47.3)
69 (58)
Negative
437 (89.9)
329 (88.4)
108 (90.8)
Positive
54 (11)
43 (11.6)
11 (9.2)
341 (69.5)
253 (68)
88 (73.9)
150 (30.5)
119 (32)
31 (26.1)
N0
92 (18.7)
75 (20.2)
17 (14.3)
N1
96 (19.6)
85 (22.8)
11 (9.2)
N2
94 (19.1)
68 (18.3)
26 (21.8)
N3
209 (42.6)
144 (38.7)
65 (54.6)
Negative
481 (98.0)
364 (97.8)
117 (98.3)
Positive
10 (2.0)
8 (2.2)
2 (1.7)
0.31
0.008
Histology 0.034
Lymphatic invasion 0.043
Vascular invasion 0.48
Perineural invasion Negative Positive N stage
0.22
0.001
Metastasis 1.00a
Recurrence of disease Yes
160 (32.6)
106 (28.5)
54 (45.4)
No
331 (67.4)
266 (71.5)
65 (54.6)
0.001
CD151 in Gastric Cancers
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TABLE 1 continued Characteristics, n (%)
CD151 expression Total 491 (100)
Low 372 (75.8)
p value High 119 (24.2)
Died of disease
a
Yes
242 (49.3)
171 (46)
71 (59.7)
No
249 (50.7)
201 (54)
48 (40.3)
0.009
By Fisher’s exact test, otherwise by v2 test
2? or 3? were classified as CD151-positive group and those with a score of 0 or 1? were classified as CD151-negative. For IHC of MET, we used CONFIRM anti-total MET (SP44) rabbit monoclonal primary antibody (Ventana Medical Systems, Tucson, AZ, USA) in a Ventana BenchMark XT automated slide processing system according to the manufacturer’s protocol. Briefly, 4-lm tissue sections were deparaffinized and rehydrated, and antigens were retrieved for 40 min in a citrate buffer (pH 6.1) at 95 °C. Diaminobenzidine (DAB) was used as the chromogen, and the sections were counterstained with hematoxylin. For interpretation, membranous and cytoplasmic staining was scored as follows: 0, no reactivity or faint staining; 1?, faint or weak staining; 2?, moderate staining; 3?, strong staining in [10 % of tumor cells. Some of the results were published previously.24 Cases with a score of 2? or 3? were classified as MET-positive and those with a score of 0 or 1? were classified as MET-negative. Statistical Analysis For analysis of the relationship between clinicopathologic characteristics and CD151 overexpression, Pearson’s Chi square test, Fisher’s exact test, or linear-by-linear test were used as appropriate. Disease-free survival (DFS) and overall survival (OS) were determined using the Kaplan– Meier method, and survival curves were compared by logrank test. Survival was measured from the date of surgery. The Cox proportional hazard model was used to evaluate associations between clinicopathologic factors and survival. The hazard ratio (HR) and its 95 % confidence interval (CI) were assessed for each factor. All tests were two sided, and p values \0.05 were considered statistically significant. Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, IL, USA). RESULTS CD151 Overexpression Interpretation was impossible in four cases due to tissue loss; therefore, 491 cases were included in the final
analysis. CD151 overexpression was scored as 0 in 155 (31.6 %), 1? in 217 (44.2 %), 2? in 105 (21.4 %), and 3? in 14 (2.8 %) cases. Thus, 119 cases (24.2 %) with a score of 2? or 3? were considered CD151-positive. Patient characteristics and correlations with CD151 overexpression are summarized in Table 1. Intestinal type by Lauren classification and differentiated histologic type were more frequent in the CD151positive group than in the CD151-negative group. CD151positivity was associated with more frequent lymphatic invasion (p = 0.043) and advanced N stage (p = 0.001). Kaplan–Meier survival analysis showed that CD151positive patients had shorter OS and DFS than CD151-negative patients [OS, mean 71.9 months (95 % CI 61.9–82.0) vs. 89.5 months (95 % CI 83.8–95.1), p = 0.003; DFS, mean 79.1 months (95 % CI 67.8–90.5) vs. 103.1 months (95 % CI 97.3–108.9), p \ 0.001; Figs. 1, 2a, b]. On multivariate analysis with variables, including age, size, Lauren classification, Borrman type, N stage, M stage, and CD151 overexpression status, CD151-positivity was an independent prognostic factor for OS [HR 1.335; 95 % CI 1.005–1.775; p = 0.046] and DFS [HR 1.903; 95 % CI 1.348–2.685; p \ 0.001]. Co-overexpression of CD151 and MET We analyzed IHC results for CD151 and MET in the same cohort and subdivided the cases into four subgroups as follows: CD151-/MET- (n = 296, 60.3 %); CD151?/ MET- (n = 89, 18.1 %); CD151-/MET? (n = 76, 15.5 %); CD151?/MET? (n = 30, 6.1 %).24 Patient characteristics among these four groups are summarized in Table 2. Intestinal type by Lauren classification was more frequent in the CD151?/MET? group than in the CD151-/ MET- group (p = 0.014). Lymphatic invasion was more frequent in the CD151?/MET? group than the other groups (p = 0.023). The proportion of the CD151?/MET? subgroup tended to increase in higher N stages (p = 0.031). Patients with CD151?/MET? overexpression showed shorter OS (mean 52.7 months; 95 % CI 33.7–71.8) than those with CD151-/MET- overexpression (mean 92.1 months; 95 % CI 85.9–98.3; p \ 0.001), CD151?/ MET- overexpression (mean 73.7 months; 95 % CI 63.2– 84.1; p = 0.014), and CD151-/MET? overexpression (mean 77.0 months; 95 % CI 64.4–89.6; p = 0.026; Fig. 2c). Patients with CD151?/MET? overexpression showed shorter DFS (mean 69.0 months; 95 % CI 45.0– 93.0) than those with CD151-/MET- overexpression (mean 106.4; 95 % CI 100.1–112.6; p \ 0.001; Fig. 2d). Patients with CD151?/MET- overexpression and CD151-/MET? overexpression showed shorter DFS than them with CD151-/MET- overexpression (p = 0.001 and p = 0.025, respectively).
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S. Y. Ha et al.
FIG. 1 Representative photomicrographs of CD151 overexpression interpreted as 0 (a), 1? (b), 2? (c), and 3? (d) in immunohistochemistry
On multivariate analyses, CD151?/MET? overexpression was an independent prognostic factor for OS (HR 3.163; 95 % CI 1.958–5.108; p \ 0.001) and DFS (HR 3.834; 95 % CI 2.145–6.852; p \ 0.001; Table 3). DISCUSSION In this study, we showed that CD151 was highly expressed in 24.2 % of advanced gastric carcinomas and that CD151 overexpression was associated with higher N stages and was an independent prognostic factor in OS and DFS in pT3 gastric carcinomas. Moreover, co-overexpression of CD151 and MET was a strong independent prognostic factor for OS and DFS. The prognostic significance of CD151 overexpression has been reported previously in cancers of lung, large intestine, pancreas, liver, esophagus, kidney, uterus, and breast.3,6,10–18 Knockdown of CD151 has been shown to decrease proliferation and invasion or migration in various cancer cell lines and
in vivo metastasis experiments.13,19–21,25 CD151 also is involved in tumor neoangiogenesis and epithelial-tomesenchymal transition in hepatocellular carcinoma cell lines.20,26 These findings explain why CD151 overexpression is associated with poor prognosis in cancer patients. In our study on advanced pT3-restricted gastric cancers, CD151 overexpression was associated with more frequent lymphatic invasion and advanced pN stages and was an independent prognostic factor. These results are consistent with previous studies on carcinomas in other locations.3,6,10–21 Further studies are needed to evaluate the functional mechanism of CD151 on tumor angiogenesis or epithelial-to-mesenchymal transition in gastric cancers. The most distinct feature of tetraspanin molecules is their ability to form multimolecular signaling complexes at the cell surface.13 The molecular partners of CD151 complexes are not fully understood. Devbhandari et al.13 characterized the molecular partners of CD151 by proteomic analysis in hepatocellular carcinoma cell lines and
CD151 in Gastric Cancers
1103
A
B
Overall survival probability
P = 0.003
Disease-free survival probability
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
CD151 negative CD151 positive 0
25
50
75
100
125
150
P < 0.001
CD151 negative CD151 positive 0
25
Time (months)
50
75
100
125
150
Time (months)
C
D
Overall survival probability
P < 0.001
1.0
CD151−/MET− CD151−/MET+ CD151+/MET− CD151+/MET+
0.8
Disease-free survival probability
0.8
0.6
0.6
0.4
0.4
CD151−/MET− CD151−/MET+ CD151+/MET− CD151+/MET+
0.2
0.2
0
25
50
75
100
125
150
Time (months)
P = 0.001
1.0
0
25
50
75
100
125
150
Time (months)
FIG. 2 Kaplan–Meier survival curves according to CD151 overexpression status for overall survival (a) and disease free survival (b). Kaplan– Meier survival curves according to combined CD151 overexpression and MET overexpression status for overall survival (c) and disease free survival (d)
identified 58 proteins that were mostly related to protein folding and cell migration. A few studies have strongly suggested MET and integrin family members as molecular partners of the CD151 complex. Klosek et al.21 showed that CD151 exhibited oncogenic activity through excessive activation of the HGF/MET signaling pathway by forming a structural and functional complex with MET and integrin a3/a6 in salivary gland cancer cells. Franco et al.19 showed that CD151 controlled MET-dependent neoplastic growth by enhancing receptor signaling through ß4 integrin-mediated pathways. To further explore the prognostic value of the complex, we included CD151 and MET overexpression in multivariate Cox regression analysis. Interestingly, patients with co-overexpression of CD151/MET showed significantly shorter survival, and CD151/MET
overexpression was an independent prognostic factor for unfavorable prognosis. These results suggest that crosstalk between CD151 and MET may be involved in cancer progression and might influence prognosis in advanced gastric cancers. The poor prognostic effect of co-overexpression of CD151 and MET has been previously been reported in hepatocellular carcinoma, pancreatic ductal adenocarcinoma,12 and intrahepatic cholangiocarcinoma.3,6 In conclusion, overexpression of CD151 is an independent prognostic factor and a potential molecular therapeutic target in advanced gastric cancers. Further studies are needed to establish the biological significance of CD151/ MET co-overexpression and targeting of both molecules could be considered when developing therapeutic strategies against gastric cancer.
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S. Y. Ha et al.
TABLE 2 Patient characteristics according to expression pattern of CD151 and MET Patient characteristics, n (%)
CD151-/ METn = 296 (60.3 %)
CD151?/ METn = 89 (18.1 %)
CD151-/ MET? n = 76 (32.9 %)
CD151?/ MET? n = 30 (6.1 %)
p value
189 (63.9) 107 (36.1)
61 (68.5) 28 (31.5)
51 (67.1) 25 (32.9)
22 (73.3) 8 (26.7)
0.66
\60
199 (67.2)
48 (53.9)
40 (52.6)
19 (63.3)
0.03
C60
97 (32.8)
41 (46.1)
36 (47.4)
11 (36.7)
45 (15.2)
18 (20.2)
12 (15.8)
4 (13.3)
Gender Male Female Age (years)
Tumor location Upper third Middle third
127 (42.9)
30 (33.7)
29 (38.2)
12 (40)
Lower third
104 (35.1)
35 (39.3)
30 (39.5)
12 (40)
Whole stomach
20 (6.8)
6 (6.7)
5 (6.6)
2 (6.7)
\4.5
69 (23.3)
15 (16.9)
18 (23.7)
9 (30)
C4.5
227 (76.7)
74 (83.1)
58 (76.3)
21 (70)
38 (12.8)
9 (10.1)
9 (11.8)
2 (6.7)
258 (87.2)
80 (89.9)
67 (88.2)
28 (93.3)
Diffuse
198 (66.9)
44 (49.4)
35 (46.1)
13 (43.3)
Intestinal
80 (27)
40 (44.9)
37 (48.7)
16 (53.3)
Mixed
18 (6.1)
5 (5.6)
4 (5.3)
1 (3.3)
0.64
Tumor size (cm) 0.43a
Borrmann type IV Others Lauren classification
0.81b
0.001b
Histology Differentiated
83 (28)
36 (40.4)
31 (40.8)
13 (43.3)
Undifferentiated
213 (72)
53 (59.6)
45 (59.2)
17 (56.7)
Negative
157 (53)
41 (46.1)
39 (51.3)
9 (30)
Positive
139 (47)
48 (53.9)
37 (48.7)
21 (70)
Negative
259 (87.5)
81 (91)
70 (92.1)
27 (90)
Positive
37 (12.5)
8 (9)
6 (7.9)
3 (10)
196 (66.2)
70 (78.7)
57 (75.0)
18 (60)
100 (33.8)
19 (21.3)
19 (25.0)
12 (40)
N0
63 (21.3)
14 (15.7)
12 (15.8)
3 (10)
N1
68 (23)
8 (9)
17 (22.4)
3 (10)
N2
53 (17.9)
18 (20.2)
15 (19.7)
8 (26.7)
N3
112 (37.8)
49 (55.1)
32 (42.1)
16 (53.3)
Negative
289 (97.6)
89 (100)
75 (98.7)
28 (93.3)
Positive
7 (2.4)
0 (0)
1 (1.3)
2 (6.7)
Yes
78 (26.4)
39 (43.8)
28 (36.8)
15 (50)
No
218 (73.6)
50 (56.2)
48 (63.2)
15 (50)
0.028
Lymphatic invasion 0.08*
Vacular invasion 0.68b
Perineural invasion Negative Positive N stage
0.06
0.031
Metastasis 0.13b
Recurrence of disease 0.002
CD151 in Gastric Cancers
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TABLE 2 continued Patient characteristics, n (%)
CD151-/ METn = 296 (60.3 %)
CD151?/ METn = 89 (18.1 %)
CD151-/ MET? n = 76 (32.9 %)
CD151?/ MET? n = 30 (6.1 %)
p value
Yes
131 (44.3)
49 (55.1)
40 (52.6)
22 (73.3)
0.009
No
165 (55.7)
40 (44.9)
36 (47.4)
8 (26.7)
Died of disease
a
Linear-by-linear test
b
By Fisher’s exact test, otherwise by v2 test
* CD151?/MET? versus others, p = 0.023
TABLE 3 Results of multivariate Cox proportional hazard model analyses Prognostic factors
Overall survival
Disease-free survival
HR
CI
p value
HR
CI
p value
1.413
1.086–1.839
0.01
1.296
0.934–1.798
0.121
1.527
1.052–2.217
0.026
1.55
0.995–2.416
0.053
1.282
0.952–1.726
0.102
1.615
1.112–2.345
0.012
2.134
1.520–2.996
\0.001
1.646–3.656
\0.001
N1 versus N0
1.453
0.876–2.409
\0.001
2.13
1.163–3.902
\0.001
N2 versus N0
1.422
0.858–2.356
1.494
0.802–2.783
N3 versus N0
3.281
2.156–4.994
2.965
1.749–5.028
2.23
1.122–4.432
0.022
2.143
0.857–5.355
0.103
CD151?/MET- versus CD151-/MET-
1.248
0.886–1.758
\0.001
1.974
1.324–2.945
\0.001
CD151-/MET? versus CD151-/MET-
1.497
1.038-2.159
1.98
1.271-3.085
CD151?/MET? versus CD151-/MET-
3.163
1.958–5.108
3.834
2.145-6.852
Age (years) 60 versus \60 Size (cm) C4.5 versus \4.5 Lauren classification Diffuse or mixed versus intestinal Borrmann type Type IV versus others pN stage
Metastasis Present versus absent Expression status of CD151 and MET
ACKNOWLEDGMENT Disclosure None of the authors have financial relationships relevant to this publication.
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4. Yunta M, Lazo PA. Tetraspanin proteins as organisers of membrane microdomains and signalling complexes. Cell Signal. 2003;15:559–64. 5. Kazarov AR, Yang X, Stipp CS, Sehgal B, Hemler ME. An extracellular site on tetraspanin CD151 determines alpha 3 and alpha 6 integrindependent cellular morphology. J Cell Biol. 2002;158:1299–309. 6. Ke AW, Shi GM, Zhou J, et al. Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma. Hepatology. 2009;49:491–503. 7. Hemler ME. Tetraspanin proteins mediate cellular penetration, invasion, and fusion events and define a novel type of membrane microdomain. Annu Rev Cell Dev Biol. 2003;19:397–422. 8. Romanska HM, Berditchevski F. Tetraspanins in human epithelial malignancies. J Pathol. 2011;223:4–14. 9. Testa JE, Brooks PC, Lin JM, Quigley JP. Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis. Cancer Res. 1999;59:3812–20.
1106 10. Tokuhara T, Hasegawa H, Hattori N, et al. Clinical significance of CD151 gene expression in non-small cell lung cancer. Clin Cancer Res. 2001;7:4109–14. 11. Hashida H, Takabayashi A, Tokuhara T, et al. Clinical significance of transmembrane 4 superfamily in colon cancer. Br J Cancer. 2003;89:158–67. 12. Zhu GH, Huang C, Qiu ZJ, et al. Expression and prognostic significance of CD151, c-Met, and integrin alpha3/alpha6 in pancreatic ductal adenocarcinoma. Dig Dis Sci. 2011;56:1090–8. 13. Devbhandari RP, Shi GM, Ke AW, et al. Profiling of the tetraspanin CD151 web and conspiracy of CD151/integrin beta1 complex in the progression of hepatocellular carcinoma. PLoS One. 2011;6:e24901. 14. Suzuki S, Miyazaki T, Tanaka N, et al. Prognostic significance of CD151 expression in esophageal squamous cell carcinoma with aggressive cell proliferation and invasiveness. Ann Surg Oncol. 2011;18:888–93. 15. Yoo SH, Lee K, Chae JY, Moon KC. CD151 expression can predict cancer progression in clear cell renal cell carcinoma. Histopathology. 2011;58:191–7. 16. Voss MA, Gordon N, Maloney S, et al. Tetraspanin CD151 is a novel prognostic marker in poor outcome endometrial cancer. Br J Cancer. 2011;104:1611–8. 17. Kwon MJ, Park S, Choi JY, et al. Clinical significance of CD151 overexpression in subtypes of invasive breast cancer. Br J Cancer. 2012;106:923–30. 18. Sadej R, Romanska H, Baldwin G, et al. CD151 regulates tumorigenesis by modulating the communication between tumor cells and endothelium. Mol Cancer Res. 2009;7:787–98.
S. Y. Ha et al. 19. Franco M, Muratori C, Corso S, et al. The tetraspanin CD151 is required for Met-dependent signaling and tumor cell growth. J Biol Chem. 2010;285:38756–64. 20. Ke AW, Shi GM, Zhou J, et al. CD151 amplifies signaling by integrin alpha6beta1 to PI3 K and induces the epithelial-mesenchymal transition in HCC cells. Gastroenterology. 2011;140:1629–41.e15. 21. Klosek SK, Nakashiro K, Hara S, Shintani S, Hasegawa H, Hamakawa H. CD151 forms a functional complex with c-Met in human salivary gland cancer cells. Biochem Biophys Res Commun. 2005;336:408–16. 22. An JY, Kim KM, Choi MG, Noh JH, Sohn TS, Bae JM, Kim S. Prognostic role of p-mTOR expression in cancer tissues and metastatic lymph nodes in pT2b gastric cancer. Int J Cancer. 2010;126:2904–13. 23. Hofmann M, Stoss O, Shi D, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology. 2008;52:797–805. 24. Ha SY, Lee J, Kang SY, et al. MET overexpression assessed by new interpretation method predicts gene amplification and poor survival in advanced gastric carcinomas. Mod Pathol. 2013. doi:10.1038/modpathol.2013.108. 25. Takeda Y, Li Q, Kazarov AR, Epardaud M, Elpek K, Turley SJ, Hemler ME. Diminished metastasis in tetraspanin CD151knockout mice. Blood. 2011;118:464–72. 26. Shi GM, Ke AW, Zhou J, et al. CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma. Hepatology. 2010;52:183–96.
ORIGINAL ARTICLE – GASTROINTESTINAL ONCOLOGY
CD151 Overexpression is Associated with Poor Prognosis in Patients with pT3 Gastric Cancer Sang Yun Ha, MD1, In-Gu Do, MD, PhD1,2, Jeeyun Lee, MD, PhD3, Se Hoon Park, MD, PhD3, Joon Oh Park, MD, PhD3, Won Ki Kang, MD, PhD3, Min-Gew Choi, MD, PhD4, Jun Ho Lee, MD, PhD4, Jae Moon Bae, MD, PhD4, Sung Kim, MD, PhD4, Kyoung-Mee Kim, MD, PhD1, and Tae Sung Sohn, MD, PhD4 1
Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; Samsung Genome Institute, Samsung Medical Center, Seoul, Korea; 3Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 4Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
2
ABSTRACT Introduction. CD151, a transmembrane protein of the tetraspanin family, is implicated in the regulation of cellsubstrate adhesion and cell migration. Overexpression of CD151 has been reported in several cancers and controls MET-dependent neoplastic growth by enhancing receptor signaling. However, association of CD151 overexpression with MET or tumor progression has not been reported in gastric cancer. Materials and Methods. We conducted immunohistochemical analysis of CD151 overexpression in 491 pT3 gastric carcinomas and analyzed the relationship with MET overexpression and prognostic significance. Results. CD151 was highly expressed in 119 gastric carcinomas (24.2 %) and was significantly associated with higher pN stages. Patients with CD151-positive gastric cancer showed shorter overall (p = 0.003) and disease-free survival (p = 0.001) compared with patients with CD151negative gastric carcinoma. CD151 overexpression was an independent prognostic factor for overall survival [hazard ration (HR) 1.335; 95 % CI 1.005–1.775; p = 0.046] and disease-free survival (HR 1.903; 95 % CI 1.348–2.685; p \ 0.001). Co-overexpression of CD151 and MET was observed in 30 (6.1 %) gastric cancers and was more frequent in advanced pN stages than in other groups. Ó Society of Surgical Oncology 2013 First Received: 21 April 2013; Published Online: 4 December 2013 K.-M. Kim, MD, PhD e-mail: [email protected] T. S. Sohn, MD, PhD e-mail: [email protected]
Moreover, co-overexpression of CD151 and MET was a strong independent prognostic factor for overall survival (HR 3.163; 95 % CI 1.958–5.108; p \ 0.001) and diseasefree survival (HR 3.834; 95 % CI 2.145–6.852; p \ 0.001). Conclusion. CD151 overexpression is an independent prognostic factor and could be a potential molecular therapeutic target in patients with advanced gastric cancers. Further studies are needed to establish the biological significance of CD151/MET co-overexpression and the potential of targeting both molecules as a therapeutic strategy.
Gastric cancer is the second most common cause of cancer-related death worldwide.1 After the demonstration that trastuzumab has a significant survival benefit in patients with HER2-positive gastric cancer, there has been growing interest in new molecular targets for gastric cancer.2 Tetraspanins are evolutionarily conserved membrane proteins that are thought to form complexes between themselves or with other transmembrane proteins, including various adhesion receptors, such as integrins, or receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) or MET.3–6 Through these interactions, tetraspanins are involved in tumor cell motility, invasiveness, and proliferation.7,8 CD151 was the first member of the tetraspanin family to be identified as a promoter of metastasis.9 Overexpression of CD151 and its association with poor prognosis was reported in cancers of lung, colorectum, pancreas, liver, esophagus, kidney, endometrium, and breast.3,6,10–18 CD151 is known to be involved in cancer progression by
1100
forming multimolecular signaling complexes with MET or members of the integrin family.6,13,19–21 However, the association of CD151 overexpression with MET and its role in prognosis has not been studied in gastric cancers. In this study, we performed correlative analyses on CD151 and MET overexpression in T3 gastric cancers to define their roles in cancer progression. MATERIALS AND METHODS
S. Y. Ha et al. TABLE 1 Patient overexpression Characteristics, n (%)
characteristics
according
to
CD151 expression
CD151 p value
Total 491 (100)
Low 372 (75.8)
High 119 (24.2)
323 (65.8) 168 (34.2)
240 (64.5) 132 (35.5)
83 (69.7) 36 (30.3)
0.29
0.12
Gender Male Female Age (years)
Study Design and Patients We used the gastric cancer cohorts described in our previous studies.22 All 495 patients had advanced pT3restricted gastric cancers and had undergone curative surgical resection and extensive (D2) lymph node dissection. This retrospective study was undertaken after obtaining approval from the SMC institutional review board. Clinicopathologic characteristics obtained from medical records included gender, age, tumor size, tumor location, histological type, Lauren classification, differentiation grade, surgical record, treatment record, recurrence, and vital status at the last follow-up date. The median follow up period was 82.2 (range 0–139) months. For tissue microarray construction, all H&E-stained slides were reviewed and representative areas were carefully selected and marked on paraffin blocks. Four tissue cores (0.6-mm diameter) were obtained from the invasive front, both lateral sides, and the luminal surface of the representative tumor block for each primary carcinoma. Immunohistochemistry Immunohistochemistry (IHC) studies were performed on formalin-fixed, paraffin-embedded, 4-lm-thick tissue sections using mouse monoclonal anti-CD151 (NCL-CD151, Novocastra, Newcastle upon Tyne, UK) as the primary antibody. Tissue sections were deparaffinized three times in xylene for a total of 15 min and subsequently rehydrated. Bond-max autoimmunostainer (Leica Biosystem, Melbourne, Australia) and BondTM Polymer refine detection DS9800 (Leica Biosystem) were used according to the manufacturers’ protocol. Two pathologists (Sang Yun Ha and Kyoung-Mee Kim) independently evaluated the IHC results with no prior knowledge of clinicopathological results. Interpretation of IHC for CD151 was scored according to recent HER2 assessment guidelines for gastric cancer as follows: 0, no reactivity; 1?, faint/barely perceptible membranous reactivity; 2?, weak to moderate complete or basolateral membranous reactivity; 3?, moderate to strong complete or basolateral membranous reactivity in [10 % of tumor cells.23 Cases with a score of
\60
306 (62.3)
239 (64.2)
67 (56.3)
C60
185 (37.7)
133 (35.8)
52 (43.7)
Tumor location Upper third
79 (16.1)
57 (15.3)
22 (18.5)
Middle third
198 (40.3)
156 (41.9)
42 (35.3)
Lower third
181 (36.9)
134 (36.0)
47 (39.5)
Whole stomach
33 (6.7)
25 (6.7)
8 (6.7)
0.6
Tumor size (cm) \4.5
111 (22.6)
87 (23.4)
24 (20.2)
C4.5
380 (77.4)
285 (76.6)
95 (79.8)
0.46
Borrmann type IV
58 (11.8)
Others 433 (88.2) Lauren classification
47 (12.6)
11 (9.2)
325 (87.4)
108 (90.8)
Diffuse
290 (59.1)
233 (62.5)
57 (47.9)
Intestinal
173 (35.2)
117 (31.5)
56 (47.1)
Mixed
28 (5.7)
22 (5.9)
6 (5)
Differentiated
163 (33.2)
114 (30.6)
49 (41.2)
Undifferentiated
328 (66.8)
258 (69.4)
70 (58.8)
Negative
246 (50.1)
196 (52.7)
50 (42)
Positive
245 (49.9)
176 (47.3)
69 (58)
Negative
437 (89.9)
329 (88.4)
108 (90.8)
Positive
54 (11)
43 (11.6)
11 (9.2)
341 (69.5)
253 (68)
88 (73.9)
150 (30.5)
119 (32)
31 (26.1)
N0
92 (18.7)
75 (20.2)
17 (14.3)
N1
96 (19.6)
85 (22.8)
11 (9.2)
N2
94 (19.1)
68 (18.3)
26 (21.8)
N3
209 (42.6)
144 (38.7)
65 (54.6)
Negative
481 (98.0)
364 (97.8)
117 (98.3)
Positive
10 (2.0)
8 (2.2)
2 (1.7)
0.31
0.008
Histology 0.034
Lymphatic invasion 0.043
Vascular invasion 0.48
Perineural invasion Negative Positive N stage
0.22
0.001
Metastasis 1.00a
Recurrence of disease Yes
160 (32.6)
106 (28.5)
54 (45.4)
No
331 (67.4)
266 (71.5)
65 (54.6)
0.001
CD151 in Gastric Cancers
1101
TABLE 1 continued Characteristics, n (%)
CD151 expression Total 491 (100)
Low 372 (75.8)
p value High 119 (24.2)
Died of disease
a
Yes
242 (49.3)
171 (46)
71 (59.7)
No
249 (50.7)
201 (54)
48 (40.3)
0.009
By Fisher’s exact test, otherwise by v2 test
2? or 3? were classified as CD151-positive group and those with a score of 0 or 1? were classified as CD151-negative. For IHC of MET, we used CONFIRM anti-total MET (SP44) rabbit monoclonal primary antibody (Ventana Medical Systems, Tucson, AZ, USA) in a Ventana BenchMark XT automated slide processing system according to the manufacturer’s protocol. Briefly, 4-lm tissue sections were deparaffinized and rehydrated, and antigens were retrieved for 40 min in a citrate buffer (pH 6.1) at 95 °C. Diaminobenzidine (DAB) was used as the chromogen, and the sections were counterstained with hematoxylin. For interpretation, membranous and cytoplasmic staining was scored as follows: 0, no reactivity or faint staining; 1?, faint or weak staining; 2?, moderate staining; 3?, strong staining in [10 % of tumor cells. Some of the results were published previously.24 Cases with a score of 2? or 3? were classified as MET-positive and those with a score of 0 or 1? were classified as MET-negative. Statistical Analysis For analysis of the relationship between clinicopathologic characteristics and CD151 overexpression, Pearson’s Chi square test, Fisher’s exact test, or linear-by-linear test were used as appropriate. Disease-free survival (DFS) and overall survival (OS) were determined using the Kaplan– Meier method, and survival curves were compared by logrank test. Survival was measured from the date of surgery. The Cox proportional hazard model was used to evaluate associations between clinicopathologic factors and survival. The hazard ratio (HR) and its 95 % confidence interval (CI) were assessed for each factor. All tests were two sided, and p values \0.05 were considered statistically significant. Statistical analysis was performed using SPSS software (SPSS Inc., Chicago, IL, USA). RESULTS CD151 Overexpression Interpretation was impossible in four cases due to tissue loss; therefore, 491 cases were included in the final
analysis. CD151 overexpression was scored as 0 in 155 (31.6 %), 1? in 217 (44.2 %), 2? in 105 (21.4 %), and 3? in 14 (2.8 %) cases. Thus, 119 cases (24.2 %) with a score of 2? or 3? were considered CD151-positive. Patient characteristics and correlations with CD151 overexpression are summarized in Table 1. Intestinal type by Lauren classification and differentiated histologic type were more frequent in the CD151positive group than in the CD151-negative group. CD151positivity was associated with more frequent lymphatic invasion (p = 0.043) and advanced N stage (p = 0.001). Kaplan–Meier survival analysis showed that CD151positive patients had shorter OS and DFS than CD151-negative patients [OS, mean 71.9 months (95 % CI 61.9–82.0) vs. 89.5 months (95 % CI 83.8–95.1), p = 0.003; DFS, mean 79.1 months (95 % CI 67.8–90.5) vs. 103.1 months (95 % CI 97.3–108.9), p \ 0.001; Figs. 1, 2a, b]. On multivariate analysis with variables, including age, size, Lauren classification, Borrman type, N stage, M stage, and CD151 overexpression status, CD151-positivity was an independent prognostic factor for OS [HR 1.335; 95 % CI 1.005–1.775; p = 0.046] and DFS [HR 1.903; 95 % CI 1.348–2.685; p \ 0.001]. Co-overexpression of CD151 and MET We analyzed IHC results for CD151 and MET in the same cohort and subdivided the cases into four subgroups as follows: CD151-/MET- (n = 296, 60.3 %); CD151?/ MET- (n = 89, 18.1 %); CD151-/MET? (n = 76, 15.5 %); CD151?/MET? (n = 30, 6.1 %).24 Patient characteristics among these four groups are summarized in Table 2. Intestinal type by Lauren classification was more frequent in the CD151?/MET? group than in the CD151-/ MET- group (p = 0.014). Lymphatic invasion was more frequent in the CD151?/MET? group than the other groups (p = 0.023). The proportion of the CD151?/MET? subgroup tended to increase in higher N stages (p = 0.031). Patients with CD151?/MET? overexpression showed shorter OS (mean 52.7 months; 95 % CI 33.7–71.8) than those with CD151-/MET- overexpression (mean 92.1 months; 95 % CI 85.9–98.3; p \ 0.001), CD151?/ MET- overexpression (mean 73.7 months; 95 % CI 63.2– 84.1; p = 0.014), and CD151-/MET? overexpression (mean 77.0 months; 95 % CI 64.4–89.6; p = 0.026; Fig. 2c). Patients with CD151?/MET? overexpression showed shorter DFS (mean 69.0 months; 95 % CI 45.0– 93.0) than those with CD151-/MET- overexpression (mean 106.4; 95 % CI 100.1–112.6; p \ 0.001; Fig. 2d). Patients with CD151?/MET- overexpression and CD151-/MET? overexpression showed shorter DFS than them with CD151-/MET- overexpression (p = 0.001 and p = 0.025, respectively).
1102
S. Y. Ha et al.
FIG. 1 Representative photomicrographs of CD151 overexpression interpreted as 0 (a), 1? (b), 2? (c), and 3? (d) in immunohistochemistry
On multivariate analyses, CD151?/MET? overexpression was an independent prognostic factor for OS (HR 3.163; 95 % CI 1.958–5.108; p \ 0.001) and DFS (HR 3.834; 95 % CI 2.145–6.852; p \ 0.001; Table 3). DISCUSSION In this study, we showed that CD151 was highly expressed in 24.2 % of advanced gastric carcinomas and that CD151 overexpression was associated with higher N stages and was an independent prognostic factor in OS and DFS in pT3 gastric carcinomas. Moreover, co-overexpression of CD151 and MET was a strong independent prognostic factor for OS and DFS. The prognostic significance of CD151 overexpression has been reported previously in cancers of lung, large intestine, pancreas, liver, esophagus, kidney, uterus, and breast.3,6,10–18 Knockdown of CD151 has been shown to decrease proliferation and invasion or migration in various cancer cell lines and
in vivo metastasis experiments.13,19–21,25 CD151 also is involved in tumor neoangiogenesis and epithelial-tomesenchymal transition in hepatocellular carcinoma cell lines.20,26 These findings explain why CD151 overexpression is associated with poor prognosis in cancer patients. In our study on advanced pT3-restricted gastric cancers, CD151 overexpression was associated with more frequent lymphatic invasion and advanced pN stages and was an independent prognostic factor. These results are consistent with previous studies on carcinomas in other locations.3,6,10–21 Further studies are needed to evaluate the functional mechanism of CD151 on tumor angiogenesis or epithelial-to-mesenchymal transition in gastric cancers. The most distinct feature of tetraspanin molecules is their ability to form multimolecular signaling complexes at the cell surface.13 The molecular partners of CD151 complexes are not fully understood. Devbhandari et al.13 characterized the molecular partners of CD151 by proteomic analysis in hepatocellular carcinoma cell lines and
CD151 in Gastric Cancers
1103
A
B
Overall survival probability
P = 0.003
Disease-free survival probability
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
CD151 negative CD151 positive 0
25
50
75
100
125
150
P < 0.001
CD151 negative CD151 positive 0
25
Time (months)
50
75
100
125
150
Time (months)
C
D
Overall survival probability
P < 0.001
1.0
CD151−/MET− CD151−/MET+ CD151+/MET− CD151+/MET+
0.8
Disease-free survival probability
0.8
0.6
0.6
0.4
0.4
CD151−/MET− CD151−/MET+ CD151+/MET− CD151+/MET+
0.2
0.2
0
25
50
75
100
125
150
Time (months)
P = 0.001
1.0
0
25
50
75
100
125
150
Time (months)
FIG. 2 Kaplan–Meier survival curves according to CD151 overexpression status for overall survival (a) and disease free survival (b). Kaplan– Meier survival curves according to combined CD151 overexpression and MET overexpression status for overall survival (c) and disease free survival (d)
identified 58 proteins that were mostly related to protein folding and cell migration. A few studies have strongly suggested MET and integrin family members as molecular partners of the CD151 complex. Klosek et al.21 showed that CD151 exhibited oncogenic activity through excessive activation of the HGF/MET signaling pathway by forming a structural and functional complex with MET and integrin a3/a6 in salivary gland cancer cells. Franco et al.19 showed that CD151 controlled MET-dependent neoplastic growth by enhancing receptor signaling through ß4 integrin-mediated pathways. To further explore the prognostic value of the complex, we included CD151 and MET overexpression in multivariate Cox regression analysis. Interestingly, patients with co-overexpression of CD151/MET showed significantly shorter survival, and CD151/MET
overexpression was an independent prognostic factor for unfavorable prognosis. These results suggest that crosstalk between CD151 and MET may be involved in cancer progression and might influence prognosis in advanced gastric cancers. The poor prognostic effect of co-overexpression of CD151 and MET has been previously been reported in hepatocellular carcinoma, pancreatic ductal adenocarcinoma,12 and intrahepatic cholangiocarcinoma.3,6 In conclusion, overexpression of CD151 is an independent prognostic factor and a potential molecular therapeutic target in advanced gastric cancers. Further studies are needed to establish the biological significance of CD151/ MET co-overexpression and targeting of both molecules could be considered when developing therapeutic strategies against gastric cancer.
1104
S. Y. Ha et al.
TABLE 2 Patient characteristics according to expression pattern of CD151 and MET Patient characteristics, n (%)
CD151-/ METn = 296 (60.3 %)
CD151?/ METn = 89 (18.1 %)
CD151-/ MET? n = 76 (32.9 %)
CD151?/ MET? n = 30 (6.1 %)
p value
189 (63.9) 107 (36.1)
61 (68.5) 28 (31.5)
51 (67.1) 25 (32.9)
22 (73.3) 8 (26.7)
0.66
\60
199 (67.2)
48 (53.9)
40 (52.6)
19 (63.3)
0.03
C60
97 (32.8)
41 (46.1)
36 (47.4)
11 (36.7)
45 (15.2)
18 (20.2)
12 (15.8)
4 (13.3)
Gender Male Female Age (years)
Tumor location Upper third Middle third
127 (42.9)
30 (33.7)
29 (38.2)
12 (40)
Lower third
104 (35.1)
35 (39.3)
30 (39.5)
12 (40)
Whole stomach
20 (6.8)
6 (6.7)
5 (6.6)
2 (6.7)
\4.5
69 (23.3)
15 (16.9)
18 (23.7)
9 (30)
C4.5
227 (76.7)
74 (83.1)
58 (76.3)
21 (70)
38 (12.8)
9 (10.1)
9 (11.8)
2 (6.7)
258 (87.2)
80 (89.9)
67 (88.2)
28 (93.3)
Diffuse
198 (66.9)
44 (49.4)
35 (46.1)
13 (43.3)
Intestinal
80 (27)
40 (44.9)
37 (48.7)
16 (53.3)
Mixed
18 (6.1)
5 (5.6)
4 (5.3)
1 (3.3)
0.64
Tumor size (cm) 0.43a
Borrmann type IV Others Lauren classification
0.81b
0.001b
Histology Differentiated
83 (28)
36 (40.4)
31 (40.8)
13 (43.3)
Undifferentiated
213 (72)
53 (59.6)
45 (59.2)
17 (56.7)
Negative
157 (53)
41 (46.1)
39 (51.3)
9 (30)
Positive
139 (47)
48 (53.9)
37 (48.7)
21 (70)
Negative
259 (87.5)
81 (91)
70 (92.1)
27 (90)
Positive
37 (12.5)
8 (9)
6 (7.9)
3 (10)
196 (66.2)
70 (78.7)
57 (75.0)
18 (60)
100 (33.8)
19 (21.3)
19 (25.0)
12 (40)
N0
63 (21.3)
14 (15.7)
12 (15.8)
3 (10)
N1
68 (23)
8 (9)
17 (22.4)
3 (10)
N2
53 (17.9)
18 (20.2)
15 (19.7)
8 (26.7)
N3
112 (37.8)
49 (55.1)
32 (42.1)
16 (53.3)
Negative
289 (97.6)
89 (100)
75 (98.7)
28 (93.3)
Positive
7 (2.4)
0 (0)
1 (1.3)
2 (6.7)
Yes
78 (26.4)
39 (43.8)
28 (36.8)
15 (50)
No
218 (73.6)
50 (56.2)
48 (63.2)
15 (50)
0.028
Lymphatic invasion 0.08*
Vacular invasion 0.68b
Perineural invasion Negative Positive N stage
0.06
0.031
Metastasis 0.13b
Recurrence of disease 0.002
CD151 in Gastric Cancers
1105
TABLE 2 continued Patient characteristics, n (%)
CD151-/ METn = 296 (60.3 %)
CD151?/ METn = 89 (18.1 %)
CD151-/ MET? n = 76 (32.9 %)
CD151?/ MET? n = 30 (6.1 %)
p value
Yes
131 (44.3)
49 (55.1)
40 (52.6)
22 (73.3)
0.009
No
165 (55.7)
40 (44.9)
36 (47.4)
8 (26.7)
Died of disease
a
Linear-by-linear test
b
By Fisher’s exact test, otherwise by v2 test
* CD151?/MET? versus others, p = 0.023
TABLE 3 Results of multivariate Cox proportional hazard model analyses Prognostic factors
Overall survival
Disease-free survival
HR
CI
p value
HR
CI
p value
1.413
1.086–1.839
0.01
1.296
0.934–1.798
0.121
1.527
1.052–2.217
0.026
1.55
0.995–2.416
0.053
1.282
0.952–1.726
0.102
1.615
1.112–2.345
0.012
2.134
1.520–2.996
\0.001
1.646–3.656
\0.001
N1 versus N0
1.453
0.876–2.409
\0.001
2.13
1.163–3.902
\0.001
N2 versus N0
1.422
0.858–2.356
1.494
0.802–2.783
N3 versus N0
3.281
2.156–4.994
2.965
1.749–5.028
2.23
1.122–4.432
0.022
2.143
0.857–5.355
0.103
CD151?/MET- versus CD151-/MET-
1.248
0.886–1.758
\0.001
1.974
1.324–2.945
\0.001
CD151-/MET? versus CD151-/MET-
1.497
1.038-2.159
1.98
1.271-3.085
CD151?/MET? versus CD151-/MET-
3.163
1.958–5.108
3.834
2.145-6.852
Age (years) 60 versus \60 Size (cm) C4.5 versus \4.5 Lauren classification Diffuse or mixed versus intestinal Borrmann type Type IV versus others pN stage
Metastasis Present versus absent Expression status of CD151 and MET
ACKNOWLEDGMENT Disclosure None of the authors have financial relationships relevant to this publication.
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