Med Oncol (2015) 32:63 DOI 10.1007/s12032-014-0463-0
ORIGINAL PAPER
CD40 expression and its prognostic significance in human gastric carcinoma Jing Guo • Jia-Jia Xiao • Xiaoli Zhang Kai-Xi Fan
•
Received: 19 August 2014 / Accepted: 16 December 2014 Ó Springer Science+Business Media New York 2015
Abstract This study aimed to detect the relationship between CD40 (protein and mRNA) expression and human gastric cancer and to determine the prognostic significance of CD40 in gastric cancer patients. We collected 128 cases of gastric cancer specimens, and the expression of CD40 (protein and mRNA) was measured by immunohistochemistry and in situ hybridization. Our study indicated that CD40 is constitutively expressed in human gastric carcinoma tissues. Positive expression of CD40 (protein and mRNA) in gastric cancer tissues was closely related to the tumor TNM stage and the presence of distant metastasis, with CD40 mRNA also being correlated with the presence of lymphatic metastasis. Furthermore, the expression of CD40 (protein and mRNA) is closely related to the prognosis of gastric cancer patients. The expression of CD40 protein and mRNA is positively correlated with the presence of distant (for both protein and mRNA) and lymphatic (for mRNA only) metastasis, and an increased tumor TNM stage in gastric carcinoma. Patients who express low levels of CD40 may have a better prognosis than those who have higher levels of CD40.
J. Guo (&) K.-X. Fan The Affiliated Hospital of Shandong Academy of Medical Sciences, University of Jinan, No. 38 Wu Ying-Shan Road, Tianqiao District, Jinan 250031, Shandong Province, China e-mail: [email protected] J.-J. Xiao Shanghai First People’s Hospital, School of Medicine, Shanghai Jiao Tong University, No. 100 Haining Road, Hongkou District, Shanghai 200080, China X. Zhang Department of Oncology, Renmin Hospital of Wuhan University, No. 238 Jiefang Road, Wuchang District, Wuhan 430060, Hubei Province, China
Keywords Metastasis
CD40 Gastric cancer TNM stage
Introduction Gastric cancer is the second most frequent cause of cancerrelated mortality worldwide, especially in Eastern Asian and Eastern European countries [1–3]. CD40, a cell surface molecule, belongs to the tumor necrosis factor receptor superfamily (TNFRSF) [4], and it is broadly expressed by immune cells [5], particularly B cells, monocytes, and dendritic cells (DC), as well as other normal cells. Conversely, CD40 has abnormal expression in breast cancer, lung cancer, melanoma, and other epithelial tumors [6], and this abnormal expression is closely related to tumor invasion, metastasis, and other biological behaviors [7]. Many studies on CD40 expression and the CD40 pathway have been carried out owing to its role in the pathogenesis of cancer and the potential implications of that role for cancer immunotherapy. In hematologic malignancies, studies of CD40 have led to several therapeutic advancements, and some immune therapies targeting hematologic malignancies have entered the clinical trial stage [8–10]. As for gastric cancer, research into the role of CD40 has also been gradually carried out. Lo et al. [11] (for protein only) and Li et al. [12] (protein and mRNA) examined 32 and 73 cases of gastric cancer tissue specimens, respectively, and found that high CD40 expression is closely associated with the poor prognosis of gastric cancer. In the present study, we observed a larger number of patients (128 cases) and used more intuitive methods (immunohistochemistry and in situ hybridization) to determine whether there is a correlation between CD40 (protein and mRNA) expression and human gastric cancer. At the same time, we
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also combined the data from the patient follow-ups to investigate the relationship between CD40 and prognosis in patients with gastric cancer.
Materials and methods Patients The present work included primary gastric adenocarcinoma specimens from 128 patients (81 males and 47 females) who had received resection at the Shandong Cancer Hospital between January, 2007 and April, 2009. The age of the patients ranged from 30 to 74 years with an average of 48 years. All these patients were confirmed pathologically with gastric adenocarcinoma, and they had not received chemotherapy or radiotherapy before the operation. All these cases had complete clinical, pathological, and followup data. Additionally, 22 specimens of normal gastric mucosa (C5 cm) were taken from around the tumors of some of the patients and confirmed by pathology analysis. The tumor histologies were determined according to the criteria of the World Health Organization [13]. The TNM stage was assessed according to the unified international gastric cancer staging classification system, as incorporated in the Union for International Cancer Control (UICC) TNM classification manual, 7th edition [14]. Based on the status of differentiation, 16 cases were well differentiated, 28 were moderately differentiated, 66 were poorly differentiated, 10 were mucinous adenocarcinoma, and eight were signet-ring cell carcinoma. The clinical outcome of the patients was followed up from the date of surgery to either the date of death or April 20, 2014, resulting in a follow-up period of 1–60 months (mean, 40 months). The survival time of 40 cases was longer than 5 years, while the other 88 cases lived \5 years. This study was conducted in accordance with the declaration of Helsinki. The Ethics Committee of The Affiliated Hospital of Shandong Academy of Medical Sciences approved our experimental protocols. Informed consent was obtained from all individual participants included in the study. Immunohistochemistry Immunohistochemistry staining was performed as described previously [15]. Mouse anti-human CD40 antibody (1:200 dilution; BD Biosciences, San Jose, CA, USA) was used as the primary antibody and was detected with a biotin-labeled secondary antibody (DAKO Diagnostics, Glostrup, Denmark). Negative controls were stained following the same procedure, except that PBS was used in place of the primary antibody. The staining intensity and
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percentage of CD40-positive cells were simultaneously evaluated in a blinded manner by three pathologists, and a consensus was reached for each score. The cells positive for CD40 were those with a golden or brown plasma membrane and cytoplasm. We used the ratio of stained cells as a measure of the expression level. At least nine visual fields were observed for each section under a highpower lens. The staining intensity was judged based on the following standards: samples were considered ‘‘negative expression’’ if the percentage of CD40-positive cells was B10 %, and ‘‘positive expression’’ if it was [10 %. In situ hybridization In situ hybridization was performed as described previously [16]. The mRNA sequence of the CD40 gene was retrieved from the NCBI database. The oligonucleotide probe sequence was designed (using the software Primer 3) as the following: 50 -caggagatcaattttcccga-30 (Sangon biotech, Shanghai) and was marked by digoxin. Blank controls were operated following the same procedure in the absence of the probe. The positive signal for in situ hybridization is a clear yellow–brown granular material, which for CD40 was located on the cell membrane. The CD40 staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The percentage of CD40-positive cells was scored into four categories: 1 (0–10 %), 2 (11–30 %), 3 (31–60 %), and 4 (61–100 %). These two grades (staining intensity and percentage of CD40-positive cells) were summed to yield the following categories: negative (1–3), positive (4–5), and strongly positive (6–7). Statistical analysis The SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The levels of CD40 staining in specimens from patients with different stages of gastric cancer were compared using a Chi-squared test. A Kaplan–Meier survival curve and a log-rank test were used to analyze the correlation between CD40 expression and patient survival. The correlation analysis was performed using a Spearman’s correlation test, and the inspection level was a = 0.05. p \ 0.05 was considered statistically significant.
Results CD40 protein expression in gastric cancer tissue and its correlation with clinical pathology Positive staining of the CD40 protein was mainly located in the cytoplasm and plasma membrane. The staining was
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golden or brown in color (Fig. 1a). Occasionally, we observed a few cells with positive expression of CD40 protein in normal gastric mucosa tissues, but the overall evaluation for these samples was negative. The CD40 protein was positively expressed in gastric cancer tissues at a rate of 34 %, and the difference between the cancer and normal samples was statistically significant (v2 = 5.960, p = 0.014). The positive rate of CD40 in the superior differentiation group (including well-differentiated and moderately differentiated adenocarcinoma) was 36.4 %; however, the ratio was 33.3 % in the inferior differentiation group (containing poorly differentiated, mucinous adenocarcinoma, and signet-ring cell carcinoma), and the difference between these groups was not statistically significant (p [ 0.05). Similarly, there were no significant associations between CD40-positive expression and gender, age, tumor location, degree of gastric cancer differentiation, lymph node metastasis, or depth of invasion (p [ 0.05). Notably, CD40-positive expression was significantly correlated with the TNM stage and the presence of distant metastasis (p \ 0.05) (Table 1).
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Correlation between the CD40 protein expression and the prognosis of these gastric cancer patients In the present study, the survival time was over 5 years for 40 cases. Among them, six cases had positive expression of CD40 protein, accounting for 14 % of the total cases with positive expression of CD40, while the other 34 cases had no or little detectable CD40 protein, accounting for 41 % of the total cases with negative expression for CD40. The difference between these two groups was statistically significant (v2 = 4.841, p = 0.028). The statistical result from the Kaplan–Meier test indicated that the survival time of the group with positive expression of CD40 was significantly shorter than that of the negative group. A logrank test indicated that the difference between the two groups was statistically significant (v2 = 7.526, v = 1, p = 0.006). Thus, patients with negative expression of CD40 protein had a better prognosis than those with a positive expression of CD40 protein (Fig. 2). CD40 mRNA expression in gastric cancer tissue and its correlation with clinical pathology CD40 mRNA had different degrees of expression in the gastric tumor tissues. Its positive staining was mostly located on plasma membranes, although positive staining could be also seen in the cytoplasm of gastric tumor tissues (Fig. 3a). A total of 48.4 % of the specimens were positive for CD40 mRNA. Although positive staining of CD40 mRNA could occasionally be seen in normal gastric mucosa, this phenomenon was minimal and the score was determined to be negative. The difference in the ratio of CD40 mRNA expression between gastric tumor tissue and normal gastric mucosa was statistically significant (v2 = 11.999, p = 0.001). There was no significant correlation between positive expression of CD40 mRNA and gender, age, tumor location, degree of gastric cancer differentiation, or depth of invasion (p [ 0.05). Positive expression of CD40 mRNA in gastric cancer tissues is closely correlated with the TNM stage and the presence of lymph node metastasis and distant metastasis (p \ 0.05). Additionally, the rate of CD40 mRNA positive expression gradually increased with the progression of TNM stage (Table 2). Correlation between the CD40 mRNA expression and the prognosis of these gastric cancer patients
Fig. 1 CD40 protein expression in cancerous and normal gastric mucosa. a, b Immunohistochemistry analysis for CD40 expression in (a) gastric cancer tissue (9400) and (b) normal gastric mucosa (9200)
In this study, 40 patients survived more than 5 years. Among them, ten cases had positive expression of CD40 mRNA, accounting for 16.1 % of the total cases with positive expression of CD40 mRNA. The other 30 cases were negative for CD40 mRNA expression, accounting for
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Table 1 Correlation between CD40 protein expression and clinicopathologic indices of gastric cancer
Total (n)
CD40 protein (case) ?
p
-
Differentiation Superior differentiation
44
16
28
Inferior differentiation
84
28
56
0.732
Depth of invasion Mucosa and submucosa
16
6
10
Muscularis
48
16
32
Serous membrane from inner to outer
64
22
42
Yes
98
38
60
No
30
6
24
0.955
Lymphatic metastasis
Distant metastasis Yes
12
12
0
116
32
84
I
32
6
26
II
40
10
30
III
44
16
28
IV
12
12
0
No
0.058
0.000
TNM stage 0.000
Fig. 2 The relationship between CD40 protein expression levels and the survival of gastric cancer patients. Survival curve of CD40 protein expression in gastric cancer patients
45.5 % of the total number of CD40 mRNA negative cases. This difference was statistically significant (v2 = 5.106, p = 0.024). Additionally, a Kaplan–Meier test showed that the survival time of the group with positive expression of CD40 mRNA was significantly shorter than that of the negative group. A log-rank test indicated that the difference between the two groups was statistically significant (v2 = 9.722, v = 1, p = 0.002) (Fig. 4). These data demonstrated that patients with positive expression of CD40
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Fig. 3 CD40 mRNA expression in cancerous and normal gastric mucosa. a, b In situ hybridization analysis for CD40 mRNA expression in (a) gastric cancer tissue (9400) and (b) normal gastric mucosa (9100)
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Table 2 Correlation between CD40 mRNA expression and clinicopathologic indices of gastric cancer
Total (n)
CD40mRNA (case) ?
63
p
-
Differentiation Superior differentiation
44
22
22
Inferior differentiation
84
40
44
0.798
Depth of invasion Mucosa and submucosa
16
7
9
Muscularis
48
23
25
Serous membrane from inner to outer
64
32
32
Yes
98
56
42
No
30
6
24
0.901
Lymphatic metastasis
Distant metastasis Yes
12
12
0
116
50
66
I
32
10
22
II
40
16
24
III
44
24
20
IV
12
12
0
No
0.000
0.000
TNM stage
Fig. 4 The relationship between CD40 mRNA expression levels and the survival of gastric cancer patients. Survival curve of CD40 mRNA expression in gastric cancer patients
mRNA had a worse prognosis than the patients who were negative for CD40 mRNA expression.
Discussion In the present study, immunohistochemistry and in situ hybridization were used to examine the CD40 protein and mRNA expression in 128 cases of gastric cancer patients, and their positive expression rates in gastric cancer tissue were 34 and 48.4 %, respectively. We found that the differences in CD40 protein and mRNA expression between
0.000
gastric cancer tissue and normal gastric mucosa were statistically significant. Furthermore, CD40 mRNA was positively correlated to CD40 protein expression. Additionally, as the clinical stages increased, the positive expression rate of CD40 also increased gradually. This finding is consistent with previous reports that CD40 expression in gastric cancer tissues was significantly higher than that in normal tissue. Therefore, detection of the CD40 expression level may help in the early diagnosis of gastric cancer [7]. Yamaguchi et al. [17] reported that CD40-positive gastric cancer cells had a low apoptosis ratio when patients received chemotherapy. In other words, CD40-positive gastric cancer cells were less sensitive to chemotherapy, and thus, CD40-positive patients should be given more powerful treatments. Detecting the expression of CD40 in gastric tissue may help guide the choice of therapeutic strategies. Here, we found that positive expression of CD40 had a significant correlation with the presence of distant metastasis, indicating that CD40 could be an index to help determine the severity of the disease. The positive expression rate of CD40 between these four stages was statistically significant. We found that CD40 expression in gastric cancer tissues was closely associated with the tumor TNM stage and the presence of distant metastasis. These results suggest that CD40 is likely involved in the metastasis and invasion of gastric cancer, but the specific mechanism is still not clear.
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Several studies have investigated potential mechanisms for the role of CD40 in gastric cancer. The engagement of CD40 by CD40L on the surface of human endothelial cells was found to induce activation of the Ras-PI3 K signaling pathway. Activation of this pathway is known to up-regulate the expression of several angiogenic factors, including vascular endothelial growth factor (VEGF), and promote angiogenesis [18, 19]. Other studies have found that active CD40 can induce the expression of matrix metalloproteinase [20, 21]. Evidence is accumulating to suggest that low-level constitutive engagement of CD40 may contribute to tumor cell growth [22]. In some malignant tumors, CD40 ligation induces the expression of Bcl-2, A20, BclxL, and other survival proteins and protects the cells from apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL), genotoxic agents, IgM, or anti-Fas [23, 24]. Most of these survival proteins are regulated by the transcription factor NF-kB, which is rapidly activated following CD40 ligation. Additionally, the apoptosis signal transduced by CD40 can be suppressed by the P13K and ERK pathways. Some combination of these pathways is likely responsible for the role of CD40 in the progression of gastric cancer; however, future studies are needed to better understand how these pathways contribute to the progression of this disease. We also investigated the correlation between CD40 expression and the survival of these gastric cancer patients. Follow-up on these patients indicated that the difference in survival rate between the CD40-positive and CD40-negative expression groups was statistically significant, and that those with a positive expression of CD40 (protein and mRNA) had a worse prognosis than those with a negative expression of CD40 (protein and mRNA). In the present study, the immunohistochemistry results indicated that there was no significant correlation between the positive expression of CD40 protein and the presence of lymph node metastasis, which is not consistent with the results from the study by Li et al. [12]. This difference may be related to the number of cases, the constituent ratio, or specimen differences. Although the difference was not statistically significant, we did observe a similar trend in our results, in which the CD40 protein expression tended to correlate with the presence of lymph node metastasis. Unlike the results from the immunohistochemistry for CD40 protein, the in situ hybridization results indicated that the positive expression of CD40 mRNA in gastric cancer tissues did significantly correlate with the presence of lymph node metastasis. This difference may indicate that CD40 is not involved in the whole process of gastric cancer progression, or perhaps that the expression level of CD40 in gastric cancer tissues is regulated by other pathways during translation.
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CD40-CD40L (ligand of CD40) signaling is also involved in tumor immunity. It plays a key role in the IL12-dependent specific immune response mediated by cytotoxic T Lymphocytes (CTL) and Thl T cells. The interaction of CD40–CD40L may inhibit the growth of tumor cells by multiple mechanisms. It can mobilize the antitumor properties of the immune system and can induce neoplastic cells to undergo apoptosis. Due to the results from previous studies, CD40 has already become a targeted molecule for cancer treatment [25], and some anti-CD40 monoclonal antibodies have displayed promising anticancer effects. Three anti-CD40 monoclonal antibodies, CHIR-12.12, CP-870.893, and SGN40, have successfully entered the clinical trial stage [8–10, 26, 27]. Activation of CD40 by monoclonal antibodies may exert an anticancer effect by directly inhibiting CD40? tumor cells or by enhancing the response to tumor cells of the innate and adaptive immune systems. Either way, the CD40 pathway appears to be a promising target for the development of tumor immunity therapies. In summary, we have demonstrated that in gastric cancer, CD40 expression is significantly correlated with the TNM stage and the presence of distant metastasis. Patients with positive expression of CD40 had a poor 5-year survival rate. These results suggest that CD40 could be used as an auxiliary diagnostic index and an examination index before treatments are determined. Therefore, additional studies on the role of CD40 in cancer may lead to the development of promising antitumor treatments. Acknowledgments This research was supported by Natural Science Foundation of Shandong Province (No. Y2007C025). Conflict of interest work.
The authors report no conflicts of interest in this
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ORIGINAL PAPER
CD40 expression and its prognostic significance in human gastric carcinoma Jing Guo • Jia-Jia Xiao • Xiaoli Zhang Kai-Xi Fan
•
Received: 19 August 2014 / Accepted: 16 December 2014 Ó Springer Science+Business Media New York 2015
Abstract This study aimed to detect the relationship between CD40 (protein and mRNA) expression and human gastric cancer and to determine the prognostic significance of CD40 in gastric cancer patients. We collected 128 cases of gastric cancer specimens, and the expression of CD40 (protein and mRNA) was measured by immunohistochemistry and in situ hybridization. Our study indicated that CD40 is constitutively expressed in human gastric carcinoma tissues. Positive expression of CD40 (protein and mRNA) in gastric cancer tissues was closely related to the tumor TNM stage and the presence of distant metastasis, with CD40 mRNA also being correlated with the presence of lymphatic metastasis. Furthermore, the expression of CD40 (protein and mRNA) is closely related to the prognosis of gastric cancer patients. The expression of CD40 protein and mRNA is positively correlated with the presence of distant (for both protein and mRNA) and lymphatic (for mRNA only) metastasis, and an increased tumor TNM stage in gastric carcinoma. Patients who express low levels of CD40 may have a better prognosis than those who have higher levels of CD40.
J. Guo (&) K.-X. Fan The Affiliated Hospital of Shandong Academy of Medical Sciences, University of Jinan, No. 38 Wu Ying-Shan Road, Tianqiao District, Jinan 250031, Shandong Province, China e-mail: [email protected] J.-J. Xiao Shanghai First People’s Hospital, School of Medicine, Shanghai Jiao Tong University, No. 100 Haining Road, Hongkou District, Shanghai 200080, China X. Zhang Department of Oncology, Renmin Hospital of Wuhan University, No. 238 Jiefang Road, Wuchang District, Wuhan 430060, Hubei Province, China
Keywords Metastasis
CD40 Gastric cancer TNM stage
Introduction Gastric cancer is the second most frequent cause of cancerrelated mortality worldwide, especially in Eastern Asian and Eastern European countries [1–3]. CD40, a cell surface molecule, belongs to the tumor necrosis factor receptor superfamily (TNFRSF) [4], and it is broadly expressed by immune cells [5], particularly B cells, monocytes, and dendritic cells (DC), as well as other normal cells. Conversely, CD40 has abnormal expression in breast cancer, lung cancer, melanoma, and other epithelial tumors [6], and this abnormal expression is closely related to tumor invasion, metastasis, and other biological behaviors [7]. Many studies on CD40 expression and the CD40 pathway have been carried out owing to its role in the pathogenesis of cancer and the potential implications of that role for cancer immunotherapy. In hematologic malignancies, studies of CD40 have led to several therapeutic advancements, and some immune therapies targeting hematologic malignancies have entered the clinical trial stage [8–10]. As for gastric cancer, research into the role of CD40 has also been gradually carried out. Lo et al. [11] (for protein only) and Li et al. [12] (protein and mRNA) examined 32 and 73 cases of gastric cancer tissue specimens, respectively, and found that high CD40 expression is closely associated with the poor prognosis of gastric cancer. In the present study, we observed a larger number of patients (128 cases) and used more intuitive methods (immunohistochemistry and in situ hybridization) to determine whether there is a correlation between CD40 (protein and mRNA) expression and human gastric cancer. At the same time, we
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63
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also combined the data from the patient follow-ups to investigate the relationship between CD40 and prognosis in patients with gastric cancer.
Materials and methods Patients The present work included primary gastric adenocarcinoma specimens from 128 patients (81 males and 47 females) who had received resection at the Shandong Cancer Hospital between January, 2007 and April, 2009. The age of the patients ranged from 30 to 74 years with an average of 48 years. All these patients were confirmed pathologically with gastric adenocarcinoma, and they had not received chemotherapy or radiotherapy before the operation. All these cases had complete clinical, pathological, and followup data. Additionally, 22 specimens of normal gastric mucosa (C5 cm) were taken from around the tumors of some of the patients and confirmed by pathology analysis. The tumor histologies were determined according to the criteria of the World Health Organization [13]. The TNM stage was assessed according to the unified international gastric cancer staging classification system, as incorporated in the Union for International Cancer Control (UICC) TNM classification manual, 7th edition [14]. Based on the status of differentiation, 16 cases were well differentiated, 28 were moderately differentiated, 66 were poorly differentiated, 10 were mucinous adenocarcinoma, and eight were signet-ring cell carcinoma. The clinical outcome of the patients was followed up from the date of surgery to either the date of death or April 20, 2014, resulting in a follow-up period of 1–60 months (mean, 40 months). The survival time of 40 cases was longer than 5 years, while the other 88 cases lived \5 years. This study was conducted in accordance with the declaration of Helsinki. The Ethics Committee of The Affiliated Hospital of Shandong Academy of Medical Sciences approved our experimental protocols. Informed consent was obtained from all individual participants included in the study. Immunohistochemistry Immunohistochemistry staining was performed as described previously [15]. Mouse anti-human CD40 antibody (1:200 dilution; BD Biosciences, San Jose, CA, USA) was used as the primary antibody and was detected with a biotin-labeled secondary antibody (DAKO Diagnostics, Glostrup, Denmark). Negative controls were stained following the same procedure, except that PBS was used in place of the primary antibody. The staining intensity and
123
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percentage of CD40-positive cells were simultaneously evaluated in a blinded manner by three pathologists, and a consensus was reached for each score. The cells positive for CD40 were those with a golden or brown plasma membrane and cytoplasm. We used the ratio of stained cells as a measure of the expression level. At least nine visual fields were observed for each section under a highpower lens. The staining intensity was judged based on the following standards: samples were considered ‘‘negative expression’’ if the percentage of CD40-positive cells was B10 %, and ‘‘positive expression’’ if it was [10 %. In situ hybridization In situ hybridization was performed as described previously [16]. The mRNA sequence of the CD40 gene was retrieved from the NCBI database. The oligonucleotide probe sequence was designed (using the software Primer 3) as the following: 50 -caggagatcaattttcccga-30 (Sangon biotech, Shanghai) and was marked by digoxin. Blank controls were operated following the same procedure in the absence of the probe. The positive signal for in situ hybridization is a clear yellow–brown granular material, which for CD40 was located on the cell membrane. The CD40 staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The percentage of CD40-positive cells was scored into four categories: 1 (0–10 %), 2 (11–30 %), 3 (31–60 %), and 4 (61–100 %). These two grades (staining intensity and percentage of CD40-positive cells) were summed to yield the following categories: negative (1–3), positive (4–5), and strongly positive (6–7). Statistical analysis The SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The levels of CD40 staining in specimens from patients with different stages of gastric cancer were compared using a Chi-squared test. A Kaplan–Meier survival curve and a log-rank test were used to analyze the correlation between CD40 expression and patient survival. The correlation analysis was performed using a Spearman’s correlation test, and the inspection level was a = 0.05. p \ 0.05 was considered statistically significant.
Results CD40 protein expression in gastric cancer tissue and its correlation with clinical pathology Positive staining of the CD40 protein was mainly located in the cytoplasm and plasma membrane. The staining was
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golden or brown in color (Fig. 1a). Occasionally, we observed a few cells with positive expression of CD40 protein in normal gastric mucosa tissues, but the overall evaluation for these samples was negative. The CD40 protein was positively expressed in gastric cancer tissues at a rate of 34 %, and the difference between the cancer and normal samples was statistically significant (v2 = 5.960, p = 0.014). The positive rate of CD40 in the superior differentiation group (including well-differentiated and moderately differentiated adenocarcinoma) was 36.4 %; however, the ratio was 33.3 % in the inferior differentiation group (containing poorly differentiated, mucinous adenocarcinoma, and signet-ring cell carcinoma), and the difference between these groups was not statistically significant (p [ 0.05). Similarly, there were no significant associations between CD40-positive expression and gender, age, tumor location, degree of gastric cancer differentiation, lymph node metastasis, or depth of invasion (p [ 0.05). Notably, CD40-positive expression was significantly correlated with the TNM stage and the presence of distant metastasis (p \ 0.05) (Table 1).
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Correlation between the CD40 protein expression and the prognosis of these gastric cancer patients In the present study, the survival time was over 5 years for 40 cases. Among them, six cases had positive expression of CD40 protein, accounting for 14 % of the total cases with positive expression of CD40, while the other 34 cases had no or little detectable CD40 protein, accounting for 41 % of the total cases with negative expression for CD40. The difference between these two groups was statistically significant (v2 = 4.841, p = 0.028). The statistical result from the Kaplan–Meier test indicated that the survival time of the group with positive expression of CD40 was significantly shorter than that of the negative group. A logrank test indicated that the difference between the two groups was statistically significant (v2 = 7.526, v = 1, p = 0.006). Thus, patients with negative expression of CD40 protein had a better prognosis than those with a positive expression of CD40 protein (Fig. 2). CD40 mRNA expression in gastric cancer tissue and its correlation with clinical pathology CD40 mRNA had different degrees of expression in the gastric tumor tissues. Its positive staining was mostly located on plasma membranes, although positive staining could be also seen in the cytoplasm of gastric tumor tissues (Fig. 3a). A total of 48.4 % of the specimens were positive for CD40 mRNA. Although positive staining of CD40 mRNA could occasionally be seen in normal gastric mucosa, this phenomenon was minimal and the score was determined to be negative. The difference in the ratio of CD40 mRNA expression between gastric tumor tissue and normal gastric mucosa was statistically significant (v2 = 11.999, p = 0.001). There was no significant correlation between positive expression of CD40 mRNA and gender, age, tumor location, degree of gastric cancer differentiation, or depth of invasion (p [ 0.05). Positive expression of CD40 mRNA in gastric cancer tissues is closely correlated with the TNM stage and the presence of lymph node metastasis and distant metastasis (p \ 0.05). Additionally, the rate of CD40 mRNA positive expression gradually increased with the progression of TNM stage (Table 2). Correlation between the CD40 mRNA expression and the prognosis of these gastric cancer patients
Fig. 1 CD40 protein expression in cancerous and normal gastric mucosa. a, b Immunohistochemistry analysis for CD40 expression in (a) gastric cancer tissue (9400) and (b) normal gastric mucosa (9200)
In this study, 40 patients survived more than 5 years. Among them, ten cases had positive expression of CD40 mRNA, accounting for 16.1 % of the total cases with positive expression of CD40 mRNA. The other 30 cases were negative for CD40 mRNA expression, accounting for
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Table 1 Correlation between CD40 protein expression and clinicopathologic indices of gastric cancer
Total (n)
CD40 protein (case) ?
p
-
Differentiation Superior differentiation
44
16
28
Inferior differentiation
84
28
56
0.732
Depth of invasion Mucosa and submucosa
16
6
10
Muscularis
48
16
32
Serous membrane from inner to outer
64
22
42
Yes
98
38
60
No
30
6
24
0.955
Lymphatic metastasis
Distant metastasis Yes
12
12
0
116
32
84
I
32
6
26
II
40
10
30
III
44
16
28
IV
12
12
0
No
0.058
0.000
TNM stage 0.000
Fig. 2 The relationship between CD40 protein expression levels and the survival of gastric cancer patients. Survival curve of CD40 protein expression in gastric cancer patients
45.5 % of the total number of CD40 mRNA negative cases. This difference was statistically significant (v2 = 5.106, p = 0.024). Additionally, a Kaplan–Meier test showed that the survival time of the group with positive expression of CD40 mRNA was significantly shorter than that of the negative group. A log-rank test indicated that the difference between the two groups was statistically significant (v2 = 9.722, v = 1, p = 0.002) (Fig. 4). These data demonstrated that patients with positive expression of CD40
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Fig. 3 CD40 mRNA expression in cancerous and normal gastric mucosa. a, b In situ hybridization analysis for CD40 mRNA expression in (a) gastric cancer tissue (9400) and (b) normal gastric mucosa (9100)
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Table 2 Correlation between CD40 mRNA expression and clinicopathologic indices of gastric cancer
Total (n)
CD40mRNA (case) ?
63
p
-
Differentiation Superior differentiation
44
22
22
Inferior differentiation
84
40
44
0.798
Depth of invasion Mucosa and submucosa
16
7
9
Muscularis
48
23
25
Serous membrane from inner to outer
64
32
32
Yes
98
56
42
No
30
6
24
0.901
Lymphatic metastasis
Distant metastasis Yes
12
12
0
116
50
66
I
32
10
22
II
40
16
24
III
44
24
20
IV
12
12
0
No
0.000
0.000
TNM stage
Fig. 4 The relationship between CD40 mRNA expression levels and the survival of gastric cancer patients. Survival curve of CD40 mRNA expression in gastric cancer patients
mRNA had a worse prognosis than the patients who were negative for CD40 mRNA expression.
Discussion In the present study, immunohistochemistry and in situ hybridization were used to examine the CD40 protein and mRNA expression in 128 cases of gastric cancer patients, and their positive expression rates in gastric cancer tissue were 34 and 48.4 %, respectively. We found that the differences in CD40 protein and mRNA expression between
0.000
gastric cancer tissue and normal gastric mucosa were statistically significant. Furthermore, CD40 mRNA was positively correlated to CD40 protein expression. Additionally, as the clinical stages increased, the positive expression rate of CD40 also increased gradually. This finding is consistent with previous reports that CD40 expression in gastric cancer tissues was significantly higher than that in normal tissue. Therefore, detection of the CD40 expression level may help in the early diagnosis of gastric cancer [7]. Yamaguchi et al. [17] reported that CD40-positive gastric cancer cells had a low apoptosis ratio when patients received chemotherapy. In other words, CD40-positive gastric cancer cells were less sensitive to chemotherapy, and thus, CD40-positive patients should be given more powerful treatments. Detecting the expression of CD40 in gastric tissue may help guide the choice of therapeutic strategies. Here, we found that positive expression of CD40 had a significant correlation with the presence of distant metastasis, indicating that CD40 could be an index to help determine the severity of the disease. The positive expression rate of CD40 between these four stages was statistically significant. We found that CD40 expression in gastric cancer tissues was closely associated with the tumor TNM stage and the presence of distant metastasis. These results suggest that CD40 is likely involved in the metastasis and invasion of gastric cancer, but the specific mechanism is still not clear.
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Several studies have investigated potential mechanisms for the role of CD40 in gastric cancer. The engagement of CD40 by CD40L on the surface of human endothelial cells was found to induce activation of the Ras-PI3 K signaling pathway. Activation of this pathway is known to up-regulate the expression of several angiogenic factors, including vascular endothelial growth factor (VEGF), and promote angiogenesis [18, 19]. Other studies have found that active CD40 can induce the expression of matrix metalloproteinase [20, 21]. Evidence is accumulating to suggest that low-level constitutive engagement of CD40 may contribute to tumor cell growth [22]. In some malignant tumors, CD40 ligation induces the expression of Bcl-2, A20, BclxL, and other survival proteins and protects the cells from apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL), genotoxic agents, IgM, or anti-Fas [23, 24]. Most of these survival proteins are regulated by the transcription factor NF-kB, which is rapidly activated following CD40 ligation. Additionally, the apoptosis signal transduced by CD40 can be suppressed by the P13K and ERK pathways. Some combination of these pathways is likely responsible for the role of CD40 in the progression of gastric cancer; however, future studies are needed to better understand how these pathways contribute to the progression of this disease. We also investigated the correlation between CD40 expression and the survival of these gastric cancer patients. Follow-up on these patients indicated that the difference in survival rate between the CD40-positive and CD40-negative expression groups was statistically significant, and that those with a positive expression of CD40 (protein and mRNA) had a worse prognosis than those with a negative expression of CD40 (protein and mRNA). In the present study, the immunohistochemistry results indicated that there was no significant correlation between the positive expression of CD40 protein and the presence of lymph node metastasis, which is not consistent with the results from the study by Li et al. [12]. This difference may be related to the number of cases, the constituent ratio, or specimen differences. Although the difference was not statistically significant, we did observe a similar trend in our results, in which the CD40 protein expression tended to correlate with the presence of lymph node metastasis. Unlike the results from the immunohistochemistry for CD40 protein, the in situ hybridization results indicated that the positive expression of CD40 mRNA in gastric cancer tissues did significantly correlate with the presence of lymph node metastasis. This difference may indicate that CD40 is not involved in the whole process of gastric cancer progression, or perhaps that the expression level of CD40 in gastric cancer tissues is regulated by other pathways during translation.
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CD40-CD40L (ligand of CD40) signaling is also involved in tumor immunity. It plays a key role in the IL12-dependent specific immune response mediated by cytotoxic T Lymphocytes (CTL) and Thl T cells. The interaction of CD40–CD40L may inhibit the growth of tumor cells by multiple mechanisms. It can mobilize the antitumor properties of the immune system and can induce neoplastic cells to undergo apoptosis. Due to the results from previous studies, CD40 has already become a targeted molecule for cancer treatment [25], and some anti-CD40 monoclonal antibodies have displayed promising anticancer effects. Three anti-CD40 monoclonal antibodies, CHIR-12.12, CP-870.893, and SGN40, have successfully entered the clinical trial stage [8–10, 26, 27]. Activation of CD40 by monoclonal antibodies may exert an anticancer effect by directly inhibiting CD40? tumor cells or by enhancing the response to tumor cells of the innate and adaptive immune systems. Either way, the CD40 pathway appears to be a promising target for the development of tumor immunity therapies. In summary, we have demonstrated that in gastric cancer, CD40 expression is significantly correlated with the TNM stage and the presence of distant metastasis. Patients with positive expression of CD40 had a poor 5-year survival rate. These results suggest that CD40 could be used as an auxiliary diagnostic index and an examination index before treatments are determined. Therefore, additional studies on the role of CD40 in cancer may lead to the development of promising antitumor treatments. Acknowledgments This research was supported by Natural Science Foundation of Shandong Province (No. Y2007C025). Conflict of interest work.
The authors report no conflicts of interest in this
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