Vascular endothelial growth factor C (VEGF-C) and its receptor, VEGF receptor 3 (VEGFR-3), have been identified as the principal growth factors and the vital receptor for lymphangiogenesis in a variety of human malignancies, including gastric cancer [
CNTN-1 is a member of the contactin subgroup of the immunoglobulin superfamily which participates in various signal transduction pathways [
The purpose of the present study was to investigate the presence of CNTN-1 in patients with primary gastric cancer using RT-PCR and Western Blot. In addition, we analyzed the relationships between CNTN-1, VEGF-C, and VEGFR-3, and their correlations with clinicopathologic features, LVD, and survival time.
A total of 105 samples were selected from patients who underwent radical gastrectomy (D2 or D3) for primary gastric carcinoma at our hospital, from January 2004 to July 2009. This group of patients included 73 male and 32 female, and the median age of the patients was 62 years old (range 29~82 years old). Among them, fresh tissues of 33 cases from May 2008 to July 2009 were also assessed by RT-PCR for CNTN-1 mRNA and by Western Blot for CNTN-1 protein. None of them accepted any preoperative chemotherapy or radiotherapy. Preoperative informed consent was obtained from each patient included in the study in accordance with institutional guidance. Specimens from primary tumor and normal tissue, identified by pathological observation, at >5 cm distance adjacent to primary lesion were obtained. Samples for immunostaining were fixed in 10% buffered formalin and embedded in paraffin. Pathological and clinical records were reviewed and tumor staging was performed according to the International Union Against Cancer (UICC) classification 5th edition criteria [
Total RNA was extracted from the gastric carcinoma specimens with a RNeasy Mini Kit (Qiagen, CA) according to the manufacturer’s instructions. RT-PCR was performed with the isolated RNA, and the primers sequences for CNTN-1 were 5′-TGTTCAGCAAATTCATCCCA-3′ (forward) and 5′-TCTACCCACTCAGGGAATGC-3′ (reverse), and for
The protein extractions were performed on liquid nitrogen frozen tumor tissues and noncancerous gastric tissues. Protein concentrations were estimated with BCA Protein Assay Kit (Thermo, USA) using bovine serum albumin as the standard. Western Blot was performed as follows: proteins (20
Serial tissue sections with 4
VEGF-C and CNTN-1 were both observed almost in the cytoplasm of gastric tumor cells. VEGFR-3 was detected not only on tumor cells but also on lymphatic endothelium [
The entire tissue section was scanned to assign the scores. The staining intensity was scored as 0 (negative), 1 (weak), 2 (medium), and 3 (strong). Percentage of staining cells was scored as 0 (0%), 1 (1~25%), 2 (26~50%), 3 (51~75%), and 4 (76~100%), according to the percentages of the positive staining tumor cells in relation to the total tumor cells counted. The percentage of the positive staining cells was calculated as follows: the percentage of the positive staining cells = (the number of positive cells/1000 tumor cells counted) × 100%. The sum of the intensity score and the percentage score was used as the final staining score (0~7). For the purpose of statistical evaluation, tumors having a final staining score of ≥3 were considered to be positive [
All of the stained vessels with brown by D2-40 immunostaining were observed as typically positive lymphatic vessel in thin-walled and tube-like structures exhibiting a distinct inner cavity and devoid of red blood cells. LVD (lymphatic vessels density) were determined from the counts of D2-40-positive vessels [
All statistical analyses were performed with the software of SPSS 13.0 (SPSS, Chicago, IL, USA). Statistical analysis of RT-PCR and Western Blot was carried out using Student’s
We initially examined the expression of CNTN-1 mRNAs in gastric cancer by semiquantitative RT-PCR. Different expression level of CNTN-1 mRNA was detected in tumour samples and in noncancerous gastric samples. Tumor samples expressed higher level of CNTN-1 mRNA than that in noncancerous gastric samples.
Expression of CNTN-1 mRNA (a) and CNTN-1 protein (b) in gastric cancer. (a) Semiquantitative RT-PCR for expression of CNTN-1 mRNA in gastric cancer.
CNTN-1 protein expression was analyzed by Western Blot. Different expression level of CNTN-1 protein was detected in tumour samples and in noncancerous gastric samples. Tumor samples expressed higher level of CNTN-1 protein than that in noncancerous gastric samples.
VEGF-C, VEGFR-3, and CNTN-1 were widely expressed in the primary lesion of gastric cancer. Both VEGF-C and CNTN-1 were observed almost exclusively in the cytoplasms of gastric tumor cells. The expression of VEGFR-3 was detected in the tumor epithelium and surrounding lymphatic vessels [
The positive rate comparison between experimental group and control group (
Carcinoma tissues | Noncancerous gastric tissue |
| |
---|---|---|---|
VEGF-C | |||
(+) | 59/56.19 | 18/17.14 | 0.01 |
(−) | 46/43.81 | 87/82.86 | |
VEGFR-3 | |||
(+) | 68/64.76 | 23/15.23 | 0.01 |
(−) | 37/35.24 | 82/84.77 | |
CNTN-1 | |||
(+) | 61/58.09 | 16/21.90 | 0.01 |
(−) | 44/41.91 | 89/78.10 |
Correlation of VEGF-C, VEGFR-3, and CNTN-1 expression (
VEGFR-3 | CNTN-1 | |||||||
---|---|---|---|---|---|---|---|---|
(+) | (−) |
|
|
(+) | (−) |
|
|
|
VEGF-C | 0.155 | <0.001 | 0.143 | <0.001 | ||||
(+) | 48 | 11 | 44 | 15 | ||||
(−) | 20 | 26 | 17 | 29 | ||||
VEGFR-3 | 0.118 | 0.001 | ||||||
(+) | — | — | 48 | 20 | ||||
(−) | 13 | 24 |
Note: results were calculated by Fisher’s exact test.
Immunohistochemical labeling for VEGF-C, VEGFR-3, CNTN-1, and D2-40 in primary lesion of gastric carcinoma. (a) VEGF-C expression is indicated as brown precipitates in the cytoplasm in gastric cancer. (b) CNTN-1 expression is observed as a dark brown colour in the cytoplasm. (c) VEGFR-3 expression is observed as a dark brown colour in the cytoplasm. (d) Tumour cells were identified inside D2-40-positive lymphatic vessels in some cases.
Expression of VEGF-C, VEGFR-3, and CNTN-1 was all significantly correlated with TNM stage, lymphatic invasion, and lymph node metastasis, but not with age, gender, tumor size, tumor location, Lauren’s classification, vascular invasion, or serosa invasion, respectively. Furthermore, the higher metastatic rate of lymph node showed wider expressions of these three proteins. The correlations of VEGF-C, VEGFR-3, and CNTN-1 expressions with clinicopathological parameters are summarized in Table
Correlation of clinicopathologic parameters with VEGF-C, VEGFR-3, and CNTN-1 expression in gastric cancer (
Clinicopathological features | Total cases | VEGF-C | VEGFR-3 | CNTN-1 | ||||||
---|---|---|---|---|---|---|---|---|---|---|
(+) | (−) |
|
(+) | (−) |
|
(+) | (−) |
|
||
Age (ys) |
|
|
|
0.925 |
|
|
|
|
|
0.972 |
≧60 | 69/65.7 | 39/66.1 | 30/65.2 | 47/69.1 | 22/59.5 | 40/65.6 | 29/65.9 | |||
<60 | 36/34.3 | 20/33.9 | 16/34.8 | 21/30.9 | 16/40.5 | 21/34.4 | 15/34.1 | |||
Gender | 0.676 | 0.571 | 0.545 | |||||||
Male | 73/69.5 | 42/71.2 | 31/67.4 | 46/67.6 | 27/73.0 | 41/67.2 | 32/72.7 | |||
Female | 32/30.5 | 17/28.8 | 15/32.6 | 22/32.4 | 10/27.0 | 20/34.8 | 12/27.3 | |||
Tumor size | 0.177 | 0.317 | 0.035 | |||||||
≧5 cm | 58/55.2 | 36/61.0 | 22/47.8 | 40/58.8 | 18/48.6 | 39/63.9 | 19/43.2 | |||
<5 cm | 47/44.8 | 23/39.0 | 24/52.2 | 28/41.2 | 19/51.4 | 22/36.1 | 25/56.8 | |||
Tumor location | 0.218 | 0.320 | 0.098 | |||||||
Upper | 17/16.2 | 11/18.6 | 6/13.0 | 13/19.1 | 4/10.8 | 6/9.8 | 11/25.0 | |||
Middle | 34/32.4 | 15/25.5 | 19/41.3 | 19/27.9 | 15/40.5 | 20/32.8 | 14/31.8 | |||
Lower | 54/51.4 | 33/55.9 | 21/45.7 | 36/52.9 | 18/58.7 | 35/57.4 | 19/43.2 | |||
Lauren’s classification | 0.071 | 0.885 | 0.328 | |||||||
Intestinal | 70/66.7 | 35/59.3 | 35/76.1 | 45/66.2 | 25/67.6 | 43/70.5 | 27/61.4 | |||
Diffuse | 35/33.3 | 24/40.7 | 11/23.9 | 23/33.8 | 12/32.4 | 18/29.5 | 17/38.6 | |||
TNM stage | 0.001 | <0.001 | 0.002 | |||||||
I + II | 41/39.0 | 15/25.4 | 26/56.5 | 18/26.5 | 23/62.2 | 16/26.2 | 25/56.8 | |||
III + IV | 64/61.0 | 44/74.6 | 20/43.5 | 50/73.5 | 14/37.8 | 45/73.8 | 19/43.2 | |||
Vascular invasion | 0.526 | 0.179 | 0.385 | |||||||
(+) | 26/24.8 | 16/32.2 | 10/15.2 | 14/20.6 | 12/32.4 | 17/27.9 | 9/20.5 | |||
(−) | 79/75.2 | 43/67.8 | 36/84.8 | 54/79.4 | 25/67.6 | 44/72.1 | 35/79.5 | |||
Serosa invasion | 0.363 | 0.084 | 0.148 | |||||||
(+) | 37/35.2 | 23/39.0 | 14/30.4 | 28/41.2 | 9/24.3 | 18/29.5 | 19/43.2 | |||
(−) | 68/64.8 | 36/61.0 | 32/69.6 | 40/58.8 | 28/75.7 | 43/70.5 | 25/56.8 | |||
Lymphatic invasion | <0.001 | <0.001 | 0.001 | |||||||
(+) | 66/62.9 | 49/83.1 | 17/37.0 | 54/79.4 | 12/32.4 | 47/77.0 | 19/43.2 | |||
(−) | 39/37.1 | 10/16.9 | 29/63.0 | 14/20.6 | 25/67.6 | 14/23.0 | 25/56.8 | |||
Lymph node metastasis | <0.001 | <0.001 | <0.001 | |||||||
(+) | 70/66.7 | 49/83.1 | 21/45.7 | 54/79.4 | 16/43.2 | 50/82.0 | 20/45.5 | |||
(−) | 35/33.3 | 10/16.9 | 25/54.3 | 14/20.6 | 21/56.8 | 11/18.0 | 24/54.5 |
Combined expressions of VEGF-C and CNTN-1 associated with clinicopathologic characteristics (
Characteristic | Both high expressiona |
Both non high expressionb ( |
|
---|---|---|---|
Age (ys) | 0.761 | ||
≧60 | 16/61.5 | 12/66.7 | |
<60 | 10/38.5 | 6/33.3 | |
Gender | 0.323 | ||
Male | 20/76.9 | 11/61.1 | |
Female | 6/23.1 | 7/38.9 | |
Tumor size | 0.234 | ||
≧5 cm | 17/65.4 | 8/47.1 | |
<5 cm | 9/34.6 | 9/52.9 | |
Tumor location | 0.292 | ||
Upper | 10/38.4 | 3/16.7 | |
Middle | 8/30.8 | 7/38.9 | |
Lower | 8/30.8 | 8/44.4 | |
Lauren’s classification | 0.395 | ||
Intestinal | 14/53.8 | 12/66.7 | |
Diffuse | 12/46.2 | 6/33.3 | |
TNM stage | 0.023 | ||
I + II | 5/19.2 | 10/55.6 | |
III + IV | 21/80.8 | 8/44.4 | |
Vascular invasion | 0.325 | ||
(+) | 15/57.7 | 13/72.2 | |
(−) | 11/42.3 | 5/27.8 | |
Serosa invasion | 0.014 | ||
(+) | 9/34.6 | 13/72.2 | |
(−) | 17/63.4 | 5/27.8 | |
Lymphatic invasion | 0.005 | ||
(+) | 25/96.2 | 11/61.1 | |
(−) | 1/3.8 | 7/38.9 | |
Lymph node metastasis | 0.013 | ||
(+) | 25/96.2 | 12/66.7 | |
(−) | 1/3.8 | 6/33.3 | |
Lymph node metastatic ratio | 0.395 | ||
≧20% | 12/46.2 | 6/33.3 | |
<20% | 14/53.8 | 12/66.7 | |
Average survival (mean ± SD) (months) | 16.8 ± 3.8 | 23.2 ± 5.0 | 0.032 |
Note: “a”: indicates that expression of both VEGF-C and CNTN-1 in tumor cells is high. “b”: indicates samples with combined expressions of VEGF-C and CNTN-1 except those of both high expressions.
D2-40-positively stained vessels were observed typically as thin-walled and tube-like structures exhibiting a distinct inner cavity and devoid of red blood cells. Occasional invasion of the carcinoma cells into the lymph vessels was seen (Figure
The expression of LVD was significantly higher in patients with vascular invasion, lymphatic invasion, lymph node metastasis, and later TNM stage than that in patients without serosa invasion, lymphatic invasion, lymph node metastasis, and later TNM stage. Furthermore, the higher LVD could be detected in the subgroup of higher lymph node metastatic ratio. This study also revealed the close correlation of VEGF-C, VEGFR-3, or CNTN-1 expression with LVD. The patients with positive expression of VEGF-C, VEGFR-3, or CNTN-1 shared significantly higher LVD than that in the patients with negative expression (Table
Relationship between LVD and other clinicopathological features.
Clinicopathological features | Total cases | LVD (mean ± SD) |
|
---|---|---|---|
Age (ys) | |||
≧60 | 69 | 10.94 ± 8.27 | 0.261 |
<60 | 36 | 8.97 ± 8.79 | |
Gender | |||
Male | 73 | 9.71 ± 8.25 | 0.314 |
Female | 32 | 11.52 ± 8.92 | |
Tumor size | |||
≧5 cm | 58 | 11.05 ± 8.35 | 0.291 |
<5 | 47 | 9.29 ± 8.59 | |
Tumor location | |||
Upper | 17 | 11.62 ± 7.64 | 0.435 |
Middle | 34 | 8.78 ± 8.41 | |
Lower | 54 | 10.77 ± 8.75 | |
Lauren’s classification | |||
Intestinal | 70 | 10.21 ± 8.51 | 0.934 |
Diffuse | 35 | 10.36 ± 8.49 | |
TNM stage | |||
I + II | 41 | 7.41 ± 9.08 | 0.008 |
III + IV | 64 | 12.09 ± 7.56 | |
Vascular invasion | |||
(+) | 26 | 13.92 ± 6.70 | 0.004 |
(−) | 79 | 9.06 ± 8.67 | |
Serosa invasion | |||
(+) | 37 | 11.5 ± 7.82 | 0.257 |
(−) | 68 | 9.59 ± 8.77 | |
Lymphatic invasion | |||
(+) | 66 | 12.83 ± 7.46 | <0.001 |
(−) | 39 | 5.92 ± 8.37 | |
Lymph node metastasis | |||
(+) | 70 | 12.20 ± 7.87 | 0.001 |
(−) | 35 | 6.39 ± 8.37 | |
Lymph node metastatic ratio | |||
≧20% | 51 | 13.40 ± 7.63 | <0.001 |
<20% | 54 | 7.30 ± 8.19 | |
VEGF-C | |||
(+) | 59 | 12.63 ± 7.41 | 0.001 |
(−) | 46 | 7.23 ± 8.34 | |
VEGFR-3 | |||
(+) | 68 | 13.11 ± 7.01 | 0.011 |
(−) | 37 | 9.12 ± 8.13 | |
CNTN-1 | |||
(+) | 61 | 12.80 ± 7.95 | <0.001 |
(−) | 44 | 6.75 ± 7.96 |
Within a mean postoperative follow-up duration of
Multivariate survival analysis (Cox proportional hazards model).
Variables | Coefficient |
Standard error | Chi-squared |
|
Relative risk ratio | 95% Confidence interval |
---|---|---|---|---|---|---|
VEGF-C expression | 0.907 | 0.377 | 5.795 | 0.016 | 0.404 | 0.193~0.845 |
VEGFR-3 expression | 0.207 | 0.383 | 0.291 | 0.590 | 1.230 | 0.580~2.605 |
CNTN-1 expression | 0.812 | 0.382 | 4.524 | 0.033 | 0.444 | 0.210~0.938 |
Lymphatic invasion | 1.389 | 0.614 | 5.117 | 0.024 | 0.249 | 0.075~0.831 |
Lymph node metastasis | 0.264 | 0.580 | 0.207 | 0.649 | 1.302 | 0.418~4.057 |
Serosa invasion | 1.245 | 0.421 | 8.732 | 0.003 | 0.288 | 0.126~0.658 |
TNM stage | 0.426 | 0.491 | 0.753 | 0.385 | 0.653 | 0.250~1.709 |
Kaplan-Meier survival curves of patients with gastric cancer according to VEGF-C, VEGFR-3, and CNTN-1 protein expression. Correlation between overall survival of the patients and VEGF-C expression was found to be statistically significant (log rank:
Recent researches showed that VEGF-C played an important role in the dissemination of many solid tumors [
CNTN-1 is a glycosylphosphatidylinositol (GPI)-anchored 135-kDa cell surface protein that belongs to a family of immunoglobulin (lg) domain-containing cell adhesion molecules (CAMs) that also includes N-CAM, L1, and Nr-CAM [
It is now widely accepted that malignant tumors contain heterogeneous populations of cells of varying metastatic potential [
As well known, it had been demonstrated that CNTN-1 might be an adjusting factor in downstream of VEGF-C/VEGFR-3 axis to promote lymph node metastasis. Theoretically, the high expression of VEGF-C occurs unanimously with the high expression of CNTN-1. However, the scores calculated by the sum of positivity intensity and positivity percentage are unavoidable to have some individual deviation, especially as the number of samples is not big enough in this study of ours. On the other hand, for some other proper explanations to this unanimous phenomenon, another adjusting mechanism may exist which should be probed furthermore.
In this study of ours, 26 cases shared the high expressions of both VEGF-C and CNTN-1, and 18 cases shared the high expression of ether VEGF-C or CNTN-1. After comparing the differences in clinicopathological outcomes and prognosis between these two groups, the patients with the high expressions of both VEGF-C and CNTN-1 possessed later TNM stage, easier to result in serosa infiltration, lymphatic vessel invasion, and lymph node metastasis. Much more important, the patients with the high expressions of both VEGF-C and CNTN-1 possessed poorer survival.
We further evaluated the prognostic value of VEGF-C, VEGFR-3, and CNTN-1 expression. Patients with tumors expressing VEGF-C, VEGFR-3, or CNTN-1 had a poorer prognosis as compared with those with expression-negative tumors. On Cox regression analysis, VEGF-C expression, CNTN-1 expression, lymphatic invasion, and serosa invasion were shown to be of statistical significance. A multiple central study with larger samples would be necessary to confirm the prognostic relevance of VEGFR-3, lymph node metastasis, and TNM stage in gastric carcinoma. Our data suggests that the detection of VEGF-C expression, CNTN-1 expression, lymphatic invasion, and serosa invasion may be a useful indicator of poorer prognosis in gastric cancer, respectively.
The expression of CNTN-1 correlated with the expression of VEGF-C and VEGFR-3. All of them correlated with the presence of lymphatic invasion and prognosis in gastric carcinoma. This phenomenon may raise the possibility that intrinsic relationship of VEGF-C and CNTN-1 overexpression might play an important role in the lymphatic invasion in patients with gastric cancer. Also, the predictive ability for mortality of lymphatic metastasis can be improved with the combined evaluation of the immunohistochemical expression of these three proteins.
All the authors declared that they had no conflict of interests in this work.
S.-H. Wu contributed in study concepts, paper preparation and paper editing and carried out study design, definition of intellectual content, literature research, experimental studies, data acquisition, data analysis, and statistical analysis. J.-W. Yu contributed in study concepts, paper preparation, and paper editing. L.-H. Zheng, J.-W. Yu, J.-G. Wu, X.-C. Ni, and B.-J. Jiang contributed in clinical managements. X.-Q. Li and G.-Y. Du contributed in pathological studies. R.-Q. Lu, G.-C. Zhou, and H.-G. Jiang contributed in detection of Western Blot. B.-J. Jiang and J.-W. Yu contributed in guarantor of integrity of the entire study, study concepts, study design, and paper review. J.-W. Yu and S.-H. Wu contributed equally to this work.
This research is supported by Grants of Shanghai Committee of Science and Technology (Grant no. 09411962300) as well as Science Research Foundations of Shanghai Jiao-Tong University School of Medicine (Grant nos. 2007XY032; 2009XJ21037). The authors appreciate Dr. Xiao-Juan Zhu for the excellent supports in the laboratory work. Written consent for publication was obtained from the patient or their relative. All authors read and approved the paper.