Human Pathology (2014) 45, 1031–1038
www.elsevier.com/locate/humpath
Original contribution
Cthrc1 overexpression is an independent prognostic marker in gastric cancer☆,☆☆ Lina Gu MD a,1 , Lei Liu MD b,⁎,1 , Lili Zhong MD c , Yuxian Bai MD b , Hong Sui MD b , Xiaoli Wei MD b , Wenjie Zhang MD b , Peng Huang MD b , Dandan Gao MD b , Ying Kong MD b , Ge Lou MD a,⁎,1 a
Department of Gynecology, The Affiliated Tumor Hospital, Harbin Medical University, Haping Road 150 of Nangang District, Harbin, Heilongjiang Province, 150081, China b Department of Internal Medicine, The Affiliated Tumor Hospital, Harbin Medical University, Haping Road 150 of Nangang District, Harbin, Heilongjiang Province, 150081, China c Department of Pathology, The Affiliated First Hospital, Heilongjiang University of Chinese Medicine, Heping Road 24 of Xiangfang District, Harbin, Heilongjiang Province, 150081, China Received 1 November 2013; revised 21 December 2013; accepted 27 December 2013
Keywords: Collagen triple helix repeat containing 1; Immunohistochemistry; Gastric carcinoma; Survival; Prognosis
Summary Collagen triple helix repeat containing 1 (CTHRC1) was identified as a novel gene expressed in the adventitia and neointima on arterial injury and was found to be overexpressed in several malignant tumors, such as breast cancer and malignant melanoma. However, the expression of Cthrc1 and its role in gastric cancer progression remain unknown. We investigated the expression of the Cthrc1 protein by immunohistochemistry in 30 normal tissues from the control subjects and 166 gastric carcinomas and analyzed its correlation with various clinicopathological features, including patient outcome. Cthrc1 immunoreactivity was overexpressed in gastric carcinoma cases compared with normal tissues (P b .001). High Cthrc1 expression was found in 108 (65.06%) of these 166 carcinomas and was positively correlated with the American Joint Committee on Cancer stage classification, depth of gastric wall invasion, lymph node metastasis, lymphovascular space involvement, and recurrence but not with age, tumor site, and carcinoembryonic antigen level. Patients with high Cthrc1 expression had significantly poorer overall survival and disease-free survival compared with patients with low expression of Cthrc1 (P = .001 and P = .002, respectively). Multivariate analysis showed that high Cthrc1 expression was an independent prognostic factor for both overall survival and disease-free survival of patients with gastric carcinoma (both P = .005). These results showed that high Cthrc1 expression was associated with progression and prognosis of gastric carcinoma. © 2014 Elsevier Inc. All rights reserved.
☆ Competing interests: None of the authors has any financial arrangement with any company whose product or competing product plays a role in the manuscript. ☆☆ Funding/Support: The work was sponsored by the Medical Science and Technology Program of Health Department of Heilongjiang Province, China (No. 2011-136); Science and Technology Program of Heilongjiang Province, China (No. GC09C405-1); and Research Fund of the Affiliated Tumor Hospital, Harbin Medical University (No. JJ2009-03). ⁎ Corresponding authors. L. Liu is to be contacted at Department of Internal Medicine, The Affiliated Tumor Hospital, Harbin Medical University, Harbin 150081, China. G. Lou: Department of Gynecology, The Affiliated Tumor Hospital, Harbin Medical, University, Haping Road 150 of Nangang District, Harbin, Heilongjiang Province, 150081, China. E-mail addresses: [email protected] (L. Liu), [email protected] (G. Lou). 1 These authors contributed equally to this work.
0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.12.020
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1. Introduction Gastric cancer (GC) is among the most common malignancies worldwide and represents the third leading cause of cancer-related death [1]. Although an improvement in treatment effect has been achieved through curative resection and adjuvant therapies for some highly selected patients with GC [2], long-term survival remains dismal [3]. In recent years, there have been major advances in the treatment of patients with GC. Moreover, many studies focused on genes and their products involved in the carcinogenesis of GC, such as p53 [4], KRAS [5], survivin [6,7], ERCC1 [8], and c-erbB2 [9,10]. Besides, some of these markers were shown to be of prognostic significance [6,11]. Obviously, these findings provided new clues for prognosis prediction of GC. Collagen triple helix repeat containing 1 (CTHRC1) was originally identified as an overexpressed gene in injured rat arteries, and it encodes a secreted glycoprotein with 12 repeats of the Gly-X-Y motif, which is characteristic of collagens [12]. Cthrc1 increases cellular motility to repair the injury by limiting the deposition of collagen matrix and promoting cell migration [12]. Aberrant expression of Cthrc1 is also observed in some metastatic melanoma cells [13]. Experiments in cancer research have shown that Cthrc1 expression is highly up-regulated in solid human tumors and seems to be associated with cancer tissue invasion and metastasis [13,14]. Recently, some studies that focused on digestive tract tumors found that Cthrc1 is associated with Barrett esophagus and esophageal adenocarcinoma [15]. The clinicopathological significance and biological role of Cthrc1 in gastric carcinoma, however, remain unclear. The aim of the present study was to investigate the expression of Cthrc1 in GC and to analyze its relationship to various clinicopathological features, including patient outcome.
L. Gu et al. were determined during and after surgery. None of our study patients had received preoperative chemotherapy and/ or radiotherapy. Clinicopathological parameters of GCs were determined according to the AJCC classification. The follow-up duration for disease-free survival (DFS) was defined as the period between the operation date and the day of relapse, according to the patient’s chart. The institutional ethics committee approval for the project was achieved before the study was started.
2.2. Immunohistochemical staining Cthrc1 expression was detected using immunohistochemistry (IHC) for paraffin-embedded cancerous and noncancerous specimens. Four-micrometer-thick sections were stained by IHC using an anti-Cthrc1 antibody (rabbit polyclonal to Cthrc1, 1:100 dilution; Abcam, Cambridge MA). IHC staining was performed according to the avidin-biotin immunoperoxidase technique. After collection of paraffin-embedded tissue blocks of gastric specimens, all tissue blocks were cut in a microtome to the desired thickness (approximately 4 μm is ideal for IHC) and affixed onto the slide. Tissue sections were dewaxed in xylene and rehydrated through graded alcohol concentrations using standard procedures. After washing in phosphate-buffered saline (3 times for 5 minutes), endogenous peroxidase was blocked by incubation in 0.3% hydrogen peroxide for 10 minutes at room temperature. Then antigen retrieval with heating was performed for Cthrc1 (0.01 mol/L pH 6.0 citrate buffer, microwave oven, 95°C, 30 minutes). The primary antibody was used overnight at 4°C at a dilution of 1:100. The sections were incubated with biotin-labeled secondary antibody. After washing, sections were incubated with horseradish peroxidase-conjugated streptavidin. 3,3′-Diaminobenzidine tetrahydrochloride was added to visualize the reaction, followed by counterstaining with Mayer hematoxylin.
2. Materials and methods 2.3. Evaluation procedures 2.1. Patients and tissue samples Tissue samples were obtained from 166 patients with GC who underwent surgical resection in the Affiliated Tumor Hospital of Harbin Medical University from 2002 to 2005. Among them, 116 were males and 50 were females, with the median age of 59 (range, 33-81) years. Resected specimens were examined pathologically using the criteria of the American Joint Committee on Cancer (AJCC) pathologic TNM (pTNM) classification system [16]. Clinicopathological parameters included patient age and sex, tumor location and size, Lauren classification, wall invasion, tumor differentiation, lymph node metastasis, vascular invasion, lymphatic invasion, and perineural invasion. All cases in this study received radical total or subtotal gastrectomy with D1 or D2 lymph node dissection. Histologic type, differentiation grade, lymph node invasion, and TNM staging [17] of GC
The expression levels of Cthrc1 were classified semiquantitatively based on the total combined scores of the percentage of positively staining tumor cells together with the staining intensity. The immunostaining was evaluated according to the following standards: staining intensity was also divided into 4 subgroups (no staining = 0, weak = 1, moderate = 2, strong = 3), and the percentage of staining was designated 0 (b5%), 1 (5%-50%), or 2 (N50%). The final score of Cthrc1 expression was defined as low expression if the sum of the positive score and the corresponding intensity score lay in the range 0 to 2 and high expression if the sum ranged from 3 to 4. The scoring procedure was carried out twice by 2 independent pathologists who were experienced in evaluating IHC and had no knowledge of the clinicopathological information and corresponding hematoxylin-eosin slides.
Cthrc1 and gastric cancer
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2.4. Statistical analysis
gastric carcinoma expression (P = .005). One patient was lost to follow-up, without recurrence having been diagnosed.
The χ2 test was used to analyze the differences of categorical variables. Overall survival (OS) or DFS was calculated according to the Kaplan-Meier method and evaluated by the log-rank test. Cox regression (proportional hazard model) was performed for multivariate analysis of prognostic predictors. P b .05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software (SPSS, Chicago, IL).
3. Results 3.1. Cthrc1 expression Cthrc1 expression was overexpressed in gastric carcinoma cases compared with normal tissues, and the difference was statistically significant (P b .001). As demonstrated in Table 1, of the 166 specimens with gastric carcinoma examined, Cthrc1 expression was low in 58 (34.94%) of 166 patients with gastric carcinoma and high in 108 (65.06%) of 166 patients. As shown in Fig. 1A to D, the Cthrc1 expression was localized in the cytoplasm of tumor cells. Cthrc1 expression was significantly higher in cases assigned a high AJCC pTNM classification (P b .001), recurrence (P = .04), and poor tumor differentiation (P = .0233); it was also significantly increased in patients with cancers showing lymph node metastasis (P b .001), peritoneal seeding (P b .001), larger tumor size (P = .006), a deep gastric wall invasion (P b .001), and lymphovascular space involvement (P b .001). There was no significant association between Cthrc1 immunoreactivity and other clinicopathological factors, as shown in Table 2.
3.2. Survival analysis In univariate Kaplan-Meier analysis, high expression of Cthrc1 was related to a poor OS or DFS for patients with gastric carcinoma (Fig. 2A and B; Table 3; P = .001 and P = .002, respectively). Multivariate analysis showed that pTNM and tumor differentiation were the strongest independent prognostic factors for OS (P = .011 and P = .003, respectively); this was followed by high Cthrc1 expression (P = .005). As demonstrated in Table 4, tumor differentiation and pTNM stage (P = .018 and P = .003, respectively) were the strongest independent prognostic factors for DFS, followed by high Table 1
Expression of Cthrc1 in gastric specimens
Group
n
Cthrc1 expression High expression
Normal Gastric carcinoma
30 166
1 108
Gastric carcinoma versus normal, P b .001; χ2 test.
a
P % 3.33 65.06
b.001
4. Discussion In this study, we analyzed Cthrc1 expression by IHC in gastric carcinoma using 166 surgical specimens. We found that high Cthrc1 expression was associated with tumor differentiation, recurrence, metastasis, and unfavorable outcome, indicating that Cthrc1 may be an independent prognostic factor. To the best of our knowledge, this is the first study to demonstrate in detail an association of clinicopathological parameters and prognostic significance of Cthrc1 expression in GC. We analyzed the correlation between Cthrc1 expression and clinicopathological features in GC. High Cthrc1 expression was significantly correlated with pTNM stage, tumor differentiation, depth of tumor invasion, lymph node metastasis, recurrence, vascular/lymphatic invasion, tumor size, and peritoneal seeding. Correlation analysis further demonstrated that high Cthrc1 expression was associated with pTNM stage, tumor differentiation, and Cthrc1 expression. Moreover, our data also demonstrated that the patients with high Cthrc1 expression had significantly poorer OS and DFS by using the Kaplan-Meier method and log-rank test. Multivariate analysis suggests that Cthrc1 expression was an independent prognostic factor for both OS and DFS in patients with gastric carcinoma. Taken together, our findings in this study provided evidence that high Cthrc1 expression plays a critical role in the progression of GC and is significantly associated with a poor prognosis independently of other factors. This raises the possibility that Cthrc1 may be a prognostic parameter for gastric carcinoma and suggests the possibility to use Cthrc1 in individualization of both patient prognosis and therapy. Previous studies have found that many aberrantly expressed genes in gastric tumor can help classify the risk of patient outcome [16,18–21]. However, whether such markers are effective for clinical application as replacements for or in addition to the prognostic parameters currently in use is still unclear, and further investigation is called for to identify whether combined detection of Cthrc1 together with some of these other molecules would be valuable in improving prognostic effectiveness. Our results are consistent with the previous reports of the roles of Cthrc1 in tumor progression, including breast cancer [22], melanoma [23], colorectal cancer [24], gastrointestinal tract, lung, thyroid, ovarian, cervix, liver [13], and the pancreas [25] and support a relationship between high expression of Cthrc1 protein and unfavorable biological behavior in gastric carcinoma. All these findings suggest an important tumor biological role of Cthrc1 in carcinogenesis and tumor progression. It is possible that Cthrc1 produced by gastric cells promotes tumor invasion by regulating the surrounding microenvironment. Currently, there have been some clues that are
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Fig. 1 Immunohistochemical staining of Cthrc1 in gastric specimens. A and B, High expression of Cthrc1 in gastric carcinoma. C, High expression of Cthrc1 and lymphatic invasion in gastric carcinoma. D and E, Low expression of Cthrc1 in gastric carcinoma. F, Low expression of Cthrc1 and lymphatic invasion in gastric carcinoma. G, Low expression of Cthrc1 in a normal gastric specimen.
Cthrc1 and gastric cancer
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Table 2 Association analyses between the expression levels of Cthrc1 and the clinicopathological characteristics of gastric carcinoma Variables
Pa
Patients
Cthrc1 expression
n
Low
High
37 21
79 29
.814
25 33
51 57
.992
17 13 27 1
26 29 49 4
.401
31 27
26 82
.006
56 2
53 55
b.001
50 8
48 60
b.001
41 17
29 79
b.001
34 24
16 92
b.001
55 3
81 27
.040
31 27
63 45
.985
32 26
14 94
b.001
30 28
22 86
23 35
21 87
Sex Male 116 Female 50 Age (y) b60 76 ≥60 90 Tumor site Upper third 43 Middle third 42 Lower third 76 Whole 5 Lauren classification Intestinal 57 Diffuse/mixed 109 Peritoneal seeding No 109 Yes 57 Size b5 cm 98 ≥5 cm 68 Depth of invasion T1, 2 70 T3, 4 96 Lymph node metastasis No 50 Yes 116 Recurrence No 136 Yes 30 Serum CEA level b N5 ng/mL 94 ≤5 ng/mL 72 Pathologic stage Stages 1, 2 46 Stages 3, 4 120 Tumor differentiation Well differentiated 52 Poorly differentiated 114 Vascular/lymphatic invasion No 44 Yes 122
.0233
b.001
Abbreviation: CEA, Carcinoembryonic antigen. a χ2 test. b P b 0.01.
able to help explain its mechanisms. A previous report showed that Cthrc1 stromal expression was enhanced in triple-negative cases and also in patients with bone metastasis. Periostin expression was high in primary tumors, in particular triple-negative ones and also in their lymph node metastases. Cox regression analysis showed that, in patients with high periostin, the risk of bone metastases increased
Fig. 2 Kaplan-Meier analysis of OS and DFS relates to the expression of Cthrc1. Patients with high expression of Cthrc1 had a poorer prognosis than that of patients with low expression of Cthrc1. A, Overall survival curves of the 165 gastric carcinoma patients according to their Cthrc1 expression status, P = .001. B, DFS curves of the 165 gastric carcinoma patients according to their Cthrc1 expression status, P = .002.
with increased Cthrc1 expression. These data suggested that Cthrc1 and periostin contribute to the tumor microenvironment, with implications for lymph node and bone metastasis [26]. Wang et al [27] found that the up-regulated expression of Cthrc1 in gastric carcinogenesis contributes to tumor cell invasion and metastasis, and promoter demethylation and transforming growth factor β1 may coregulate the expression
1036 Table 3
L. Gu et al. Univariate survival analysis of OS and DFS in 165 patients with gastric carcinoma
Variables
n
OS, mo Mean ± SE
Sex Male Female Age (y) b60 ≥60 Peritoneal seeding No Yes Size b5 cm ≥5 cm Depth of invasion T1, 2 T3, 4 Lymph node metastasis No Yes Recurrence No Yes Tumor differentiation Well differentiated Poorly differentiated Vascular/lymphatic invasion Yes No Cthrc1 expression Low expression High expression
DFS, mo 95% CI
P
a
Mean ± SE
95%CI
Pa
116 49
55 ± 2 53 ± 2
51-58 50-56
.795
57 ± 2 54 ± 2
53-61 50-56
.803
76 89
57 ± 3 55 ± 1
52-61 52-58
.663
59 ± 3 57 ± 1
57-63 54-60
.691
108 57
59 ± 2 28 ± 2
57-62 25-31
b.001
59 ± 2 29 ± 2
57-62 26-33
b.001
98 67
60 ± 2 51 ± 3
57-63 46-53
.006
60 ± 2 52 ± 3
57-63 48-55
.007
70 95
61 ± 3 48 ± 2
57-66 45-52
.005
61 ± 3 51 ± 2
57-66 48-54
.007
49 116
64 ± 1 42 ± 4
62-67 37-47
b.001
64 ± 1 47 ± 4
62-67 42-52
b.001
136 29
60 ± 2 24 ± 7
55-64 15-32
b.001
60 ± 2 31 ± 5
55-64 24-38
b.001
52 113
59 ± 2 36 ± 3
56-62 33-40
b.001
60 ± 2 37 ± 3
57-62 32-41
b.001
121 44
57 ± 2 25 ± 2
54-60 22-28
b.001
59 ± 2 25 ± 2
55-62 22-28
b.001
58 107
40 ± 6 59 ± 3
33-47 55-63
b.001
40 ± 6 60 ± 3
33-47 56-64
b.001
Abbreviations: CI, confidence interval; DFS, disease-free survival; OS, overall survival. a Log-rank test.
of Cthrc1. Their research showed that Cthrc1 could activate and enhance the promoter demethylation and transforming growth factor β1 signaling pathways to improve a tumor cell invasion and metastasis, which results in the tumorigenesis. Moreover, recent data indicate an involvement of Cthrc1 in signal transduction pathways, thus enhancing cell proliferation or survival. Kelley [28] showed that Cthrc1 may play an important role in the Wnt-planar cell polarity pathway, which regulates invasion and metastasis [29]. Another study has Table 4
inferred that Cthrc1 can activate several key signaling molecules, including Src, focal adhesion kinase, paxillin, mitogenactivated protein kinase, extracellular signal–regulated kinase, and Rac1, which results in cancer progression and metastasis by regulating migration and adhesion activities of cancer cells [25]. These data provide important evidence to explicate the mechanism by which Cthrc1 expression contributes to carcinogenesis and tumor progression, although the role of Cthrc1 in signal transduction pathways in tumor cells is certainly worth further research.
Multivariate survival analysis of OS and DFS in 165 patients with gastric carcinoma
Variables
Cthrc1 pTNM Tumor differentiation
OS
DFS a
Exp(B)
95% CI
P
5.751 2.926 5.386
1.593-19.778 1.548-4.973 1.099-13.831
.005 .011 .003
Abbreviations: Exp(B), CI, confidence interval; DFS, disease-free survival; OS, overall survival. a Cox regression test.
Exp(B)
95% CI
Pa
5.534 2.988 5.659
1.512-17.662 1.562-5.299 1.652-13.751
.005 .018 .003
Cthrc1 and gastric cancer GC is one of the most common causes of cancer-related death worldwide [30], especially in East Asian countries such as China and Japan [31]. Therefore, many articles focused on the identification of its significant prognostic factors, including clinicopathological variables and specific markers [32–34]. The current study provided a novel candidate, Cthrc1, as a molecular marker of both biological behaviors and survival outcome of GC. The finding that Cthrc1 expression was closely correlated with late stage, unfavorable differentiation grade, and some invasion modes of breast cancer made its impact on postresectional survival easily understood, similar with that in gastric carcinoma [35]. Thus, the CTHRC1 gene may be focused the as a candidate of targets for inhibition against GC in the future because of its comprehensive roles in progression, invasion, and survival. In conclusion, we demonstrated that Cthrc1 is overexpressed in a great proportion of patients with gastric carcinoma and high Cthrc1 expression correlated with the disease progression and poor prognosis in gastric carcinoma. Furthermore, Cthrc1 might be an attractive target for GC therapy. However, these findings remain to be confirmed by a much larger study.
Acknowledgments The authors thank Falin Zhao for statistical analysis.
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Original contribution
Cthrc1 overexpression is an independent prognostic marker in gastric cancer☆,☆☆ Lina Gu MD a,1 , Lei Liu MD b,⁎,1 , Lili Zhong MD c , Yuxian Bai MD b , Hong Sui MD b , Xiaoli Wei MD b , Wenjie Zhang MD b , Peng Huang MD b , Dandan Gao MD b , Ying Kong MD b , Ge Lou MD a,⁎,1 a
Department of Gynecology, The Affiliated Tumor Hospital, Harbin Medical University, Haping Road 150 of Nangang District, Harbin, Heilongjiang Province, 150081, China b Department of Internal Medicine, The Affiliated Tumor Hospital, Harbin Medical University, Haping Road 150 of Nangang District, Harbin, Heilongjiang Province, 150081, China c Department of Pathology, The Affiliated First Hospital, Heilongjiang University of Chinese Medicine, Heping Road 24 of Xiangfang District, Harbin, Heilongjiang Province, 150081, China Received 1 November 2013; revised 21 December 2013; accepted 27 December 2013
Keywords: Collagen triple helix repeat containing 1; Immunohistochemistry; Gastric carcinoma; Survival; Prognosis
Summary Collagen triple helix repeat containing 1 (CTHRC1) was identified as a novel gene expressed in the adventitia and neointima on arterial injury and was found to be overexpressed in several malignant tumors, such as breast cancer and malignant melanoma. However, the expression of Cthrc1 and its role in gastric cancer progression remain unknown. We investigated the expression of the Cthrc1 protein by immunohistochemistry in 30 normal tissues from the control subjects and 166 gastric carcinomas and analyzed its correlation with various clinicopathological features, including patient outcome. Cthrc1 immunoreactivity was overexpressed in gastric carcinoma cases compared with normal tissues (P b .001). High Cthrc1 expression was found in 108 (65.06%) of these 166 carcinomas and was positively correlated with the American Joint Committee on Cancer stage classification, depth of gastric wall invasion, lymph node metastasis, lymphovascular space involvement, and recurrence but not with age, tumor site, and carcinoembryonic antigen level. Patients with high Cthrc1 expression had significantly poorer overall survival and disease-free survival compared with patients with low expression of Cthrc1 (P = .001 and P = .002, respectively). Multivariate analysis showed that high Cthrc1 expression was an independent prognostic factor for both overall survival and disease-free survival of patients with gastric carcinoma (both P = .005). These results showed that high Cthrc1 expression was associated with progression and prognosis of gastric carcinoma. © 2014 Elsevier Inc. All rights reserved.
☆ Competing interests: None of the authors has any financial arrangement with any company whose product or competing product plays a role in the manuscript. ☆☆ Funding/Support: The work was sponsored by the Medical Science and Technology Program of Health Department of Heilongjiang Province, China (No. 2011-136); Science and Technology Program of Heilongjiang Province, China (No. GC09C405-1); and Research Fund of the Affiliated Tumor Hospital, Harbin Medical University (No. JJ2009-03). ⁎ Corresponding authors. L. Liu is to be contacted at Department of Internal Medicine, The Affiliated Tumor Hospital, Harbin Medical University, Harbin 150081, China. G. Lou: Department of Gynecology, The Affiliated Tumor Hospital, Harbin Medical, University, Haping Road 150 of Nangang District, Harbin, Heilongjiang Province, 150081, China. E-mail addresses: [email protected] (L. Liu), [email protected] (G. Lou). 1 These authors contributed equally to this work.
0046-8177/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.humpath.2013.12.020
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1. Introduction Gastric cancer (GC) is among the most common malignancies worldwide and represents the third leading cause of cancer-related death [1]. Although an improvement in treatment effect has been achieved through curative resection and adjuvant therapies for some highly selected patients with GC [2], long-term survival remains dismal [3]. In recent years, there have been major advances in the treatment of patients with GC. Moreover, many studies focused on genes and their products involved in the carcinogenesis of GC, such as p53 [4], KRAS [5], survivin [6,7], ERCC1 [8], and c-erbB2 [9,10]. Besides, some of these markers were shown to be of prognostic significance [6,11]. Obviously, these findings provided new clues for prognosis prediction of GC. Collagen triple helix repeat containing 1 (CTHRC1) was originally identified as an overexpressed gene in injured rat arteries, and it encodes a secreted glycoprotein with 12 repeats of the Gly-X-Y motif, which is characteristic of collagens [12]. Cthrc1 increases cellular motility to repair the injury by limiting the deposition of collagen matrix and promoting cell migration [12]. Aberrant expression of Cthrc1 is also observed in some metastatic melanoma cells [13]. Experiments in cancer research have shown that Cthrc1 expression is highly up-regulated in solid human tumors and seems to be associated with cancer tissue invasion and metastasis [13,14]. Recently, some studies that focused on digestive tract tumors found that Cthrc1 is associated with Barrett esophagus and esophageal adenocarcinoma [15]. The clinicopathological significance and biological role of Cthrc1 in gastric carcinoma, however, remain unclear. The aim of the present study was to investigate the expression of Cthrc1 in GC and to analyze its relationship to various clinicopathological features, including patient outcome.
L. Gu et al. were determined during and after surgery. None of our study patients had received preoperative chemotherapy and/ or radiotherapy. Clinicopathological parameters of GCs were determined according to the AJCC classification. The follow-up duration for disease-free survival (DFS) was defined as the period between the operation date and the day of relapse, according to the patient’s chart. The institutional ethics committee approval for the project was achieved before the study was started.
2.2. Immunohistochemical staining Cthrc1 expression was detected using immunohistochemistry (IHC) for paraffin-embedded cancerous and noncancerous specimens. Four-micrometer-thick sections were stained by IHC using an anti-Cthrc1 antibody (rabbit polyclonal to Cthrc1, 1:100 dilution; Abcam, Cambridge MA). IHC staining was performed according to the avidin-biotin immunoperoxidase technique. After collection of paraffin-embedded tissue blocks of gastric specimens, all tissue blocks were cut in a microtome to the desired thickness (approximately 4 μm is ideal for IHC) and affixed onto the slide. Tissue sections were dewaxed in xylene and rehydrated through graded alcohol concentrations using standard procedures. After washing in phosphate-buffered saline (3 times for 5 minutes), endogenous peroxidase was blocked by incubation in 0.3% hydrogen peroxide for 10 minutes at room temperature. Then antigen retrieval with heating was performed for Cthrc1 (0.01 mol/L pH 6.0 citrate buffer, microwave oven, 95°C, 30 minutes). The primary antibody was used overnight at 4°C at a dilution of 1:100. The sections were incubated with biotin-labeled secondary antibody. After washing, sections were incubated with horseradish peroxidase-conjugated streptavidin. 3,3′-Diaminobenzidine tetrahydrochloride was added to visualize the reaction, followed by counterstaining with Mayer hematoxylin.
2. Materials and methods 2.3. Evaluation procedures 2.1. Patients and tissue samples Tissue samples were obtained from 166 patients with GC who underwent surgical resection in the Affiliated Tumor Hospital of Harbin Medical University from 2002 to 2005. Among them, 116 were males and 50 were females, with the median age of 59 (range, 33-81) years. Resected specimens were examined pathologically using the criteria of the American Joint Committee on Cancer (AJCC) pathologic TNM (pTNM) classification system [16]. Clinicopathological parameters included patient age and sex, tumor location and size, Lauren classification, wall invasion, tumor differentiation, lymph node metastasis, vascular invasion, lymphatic invasion, and perineural invasion. All cases in this study received radical total or subtotal gastrectomy with D1 or D2 lymph node dissection. Histologic type, differentiation grade, lymph node invasion, and TNM staging [17] of GC
The expression levels of Cthrc1 were classified semiquantitatively based on the total combined scores of the percentage of positively staining tumor cells together with the staining intensity. The immunostaining was evaluated according to the following standards: staining intensity was also divided into 4 subgroups (no staining = 0, weak = 1, moderate = 2, strong = 3), and the percentage of staining was designated 0 (b5%), 1 (5%-50%), or 2 (N50%). The final score of Cthrc1 expression was defined as low expression if the sum of the positive score and the corresponding intensity score lay in the range 0 to 2 and high expression if the sum ranged from 3 to 4. The scoring procedure was carried out twice by 2 independent pathologists who were experienced in evaluating IHC and had no knowledge of the clinicopathological information and corresponding hematoxylin-eosin slides.
Cthrc1 and gastric cancer
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2.4. Statistical analysis
gastric carcinoma expression (P = .005). One patient was lost to follow-up, without recurrence having been diagnosed.
The χ2 test was used to analyze the differences of categorical variables. Overall survival (OS) or DFS was calculated according to the Kaplan-Meier method and evaluated by the log-rank test. Cox regression (proportional hazard model) was performed for multivariate analysis of prognostic predictors. P b .05 was considered significant. Statistical analysis was carried out using SPSS 16.0 software (SPSS, Chicago, IL).
3. Results 3.1. Cthrc1 expression Cthrc1 expression was overexpressed in gastric carcinoma cases compared with normal tissues, and the difference was statistically significant (P b .001). As demonstrated in Table 1, of the 166 specimens with gastric carcinoma examined, Cthrc1 expression was low in 58 (34.94%) of 166 patients with gastric carcinoma and high in 108 (65.06%) of 166 patients. As shown in Fig. 1A to D, the Cthrc1 expression was localized in the cytoplasm of tumor cells. Cthrc1 expression was significantly higher in cases assigned a high AJCC pTNM classification (P b .001), recurrence (P = .04), and poor tumor differentiation (P = .0233); it was also significantly increased in patients with cancers showing lymph node metastasis (P b .001), peritoneal seeding (P b .001), larger tumor size (P = .006), a deep gastric wall invasion (P b .001), and lymphovascular space involvement (P b .001). There was no significant association between Cthrc1 immunoreactivity and other clinicopathological factors, as shown in Table 2.
3.2. Survival analysis In univariate Kaplan-Meier analysis, high expression of Cthrc1 was related to a poor OS or DFS for patients with gastric carcinoma (Fig. 2A and B; Table 3; P = .001 and P = .002, respectively). Multivariate analysis showed that pTNM and tumor differentiation were the strongest independent prognostic factors for OS (P = .011 and P = .003, respectively); this was followed by high Cthrc1 expression (P = .005). As demonstrated in Table 4, tumor differentiation and pTNM stage (P = .018 and P = .003, respectively) were the strongest independent prognostic factors for DFS, followed by high Table 1
Expression of Cthrc1 in gastric specimens
Group
n
Cthrc1 expression High expression
Normal Gastric carcinoma
30 166
1 108
Gastric carcinoma versus normal, P b .001; χ2 test.
a
P % 3.33 65.06
b.001
4. Discussion In this study, we analyzed Cthrc1 expression by IHC in gastric carcinoma using 166 surgical specimens. We found that high Cthrc1 expression was associated with tumor differentiation, recurrence, metastasis, and unfavorable outcome, indicating that Cthrc1 may be an independent prognostic factor. To the best of our knowledge, this is the first study to demonstrate in detail an association of clinicopathological parameters and prognostic significance of Cthrc1 expression in GC. We analyzed the correlation between Cthrc1 expression and clinicopathological features in GC. High Cthrc1 expression was significantly correlated with pTNM stage, tumor differentiation, depth of tumor invasion, lymph node metastasis, recurrence, vascular/lymphatic invasion, tumor size, and peritoneal seeding. Correlation analysis further demonstrated that high Cthrc1 expression was associated with pTNM stage, tumor differentiation, and Cthrc1 expression. Moreover, our data also demonstrated that the patients with high Cthrc1 expression had significantly poorer OS and DFS by using the Kaplan-Meier method and log-rank test. Multivariate analysis suggests that Cthrc1 expression was an independent prognostic factor for both OS and DFS in patients with gastric carcinoma. Taken together, our findings in this study provided evidence that high Cthrc1 expression plays a critical role in the progression of GC and is significantly associated with a poor prognosis independently of other factors. This raises the possibility that Cthrc1 may be a prognostic parameter for gastric carcinoma and suggests the possibility to use Cthrc1 in individualization of both patient prognosis and therapy. Previous studies have found that many aberrantly expressed genes in gastric tumor can help classify the risk of patient outcome [16,18–21]. However, whether such markers are effective for clinical application as replacements for or in addition to the prognostic parameters currently in use is still unclear, and further investigation is called for to identify whether combined detection of Cthrc1 together with some of these other molecules would be valuable in improving prognostic effectiveness. Our results are consistent with the previous reports of the roles of Cthrc1 in tumor progression, including breast cancer [22], melanoma [23], colorectal cancer [24], gastrointestinal tract, lung, thyroid, ovarian, cervix, liver [13], and the pancreas [25] and support a relationship between high expression of Cthrc1 protein and unfavorable biological behavior in gastric carcinoma. All these findings suggest an important tumor biological role of Cthrc1 in carcinogenesis and tumor progression. It is possible that Cthrc1 produced by gastric cells promotes tumor invasion by regulating the surrounding microenvironment. Currently, there have been some clues that are
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Fig. 1 Immunohistochemical staining of Cthrc1 in gastric specimens. A and B, High expression of Cthrc1 in gastric carcinoma. C, High expression of Cthrc1 and lymphatic invasion in gastric carcinoma. D and E, Low expression of Cthrc1 in gastric carcinoma. F, Low expression of Cthrc1 and lymphatic invasion in gastric carcinoma. G, Low expression of Cthrc1 in a normal gastric specimen.
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Table 2 Association analyses between the expression levels of Cthrc1 and the clinicopathological characteristics of gastric carcinoma Variables
Pa
Patients
Cthrc1 expression
n
Low
High
37 21
79 29
.814
25 33
51 57
.992
17 13 27 1
26 29 49 4
.401
31 27
26 82
.006
56 2
53 55
b.001
50 8
48 60
b.001
41 17
29 79
b.001
34 24
16 92
b.001
55 3
81 27
.040
31 27
63 45
.985
32 26
14 94
b.001
30 28
22 86
23 35
21 87
Sex Male 116 Female 50 Age (y) b60 76 ≥60 90 Tumor site Upper third 43 Middle third 42 Lower third 76 Whole 5 Lauren classification Intestinal 57 Diffuse/mixed 109 Peritoneal seeding No 109 Yes 57 Size b5 cm 98 ≥5 cm 68 Depth of invasion T1, 2 70 T3, 4 96 Lymph node metastasis No 50 Yes 116 Recurrence No 136 Yes 30 Serum CEA level b N5 ng/mL 94 ≤5 ng/mL 72 Pathologic stage Stages 1, 2 46 Stages 3, 4 120 Tumor differentiation Well differentiated 52 Poorly differentiated 114 Vascular/lymphatic invasion No 44 Yes 122
.0233
b.001
Abbreviation: CEA, Carcinoembryonic antigen. a χ2 test. b P b 0.01.
able to help explain its mechanisms. A previous report showed that Cthrc1 stromal expression was enhanced in triple-negative cases and also in patients with bone metastasis. Periostin expression was high in primary tumors, in particular triple-negative ones and also in their lymph node metastases. Cox regression analysis showed that, in patients with high periostin, the risk of bone metastases increased
Fig. 2 Kaplan-Meier analysis of OS and DFS relates to the expression of Cthrc1. Patients with high expression of Cthrc1 had a poorer prognosis than that of patients with low expression of Cthrc1. A, Overall survival curves of the 165 gastric carcinoma patients according to their Cthrc1 expression status, P = .001. B, DFS curves of the 165 gastric carcinoma patients according to their Cthrc1 expression status, P = .002.
with increased Cthrc1 expression. These data suggested that Cthrc1 and periostin contribute to the tumor microenvironment, with implications for lymph node and bone metastasis [26]. Wang et al [27] found that the up-regulated expression of Cthrc1 in gastric carcinogenesis contributes to tumor cell invasion and metastasis, and promoter demethylation and transforming growth factor β1 may coregulate the expression
1036 Table 3
L. Gu et al. Univariate survival analysis of OS and DFS in 165 patients with gastric carcinoma
Variables
n
OS, mo Mean ± SE
Sex Male Female Age (y) b60 ≥60 Peritoneal seeding No Yes Size b5 cm ≥5 cm Depth of invasion T1, 2 T3, 4 Lymph node metastasis No Yes Recurrence No Yes Tumor differentiation Well differentiated Poorly differentiated Vascular/lymphatic invasion Yes No Cthrc1 expression Low expression High expression
DFS, mo 95% CI
P
a
Mean ± SE
95%CI
Pa
116 49
55 ± 2 53 ± 2
51-58 50-56
.795
57 ± 2 54 ± 2
53-61 50-56
.803
76 89
57 ± 3 55 ± 1
52-61 52-58
.663
59 ± 3 57 ± 1
57-63 54-60
.691
108 57
59 ± 2 28 ± 2
57-62 25-31
b.001
59 ± 2 29 ± 2
57-62 26-33
b.001
98 67
60 ± 2 51 ± 3
57-63 46-53
.006
60 ± 2 52 ± 3
57-63 48-55
.007
70 95
61 ± 3 48 ± 2
57-66 45-52
.005
61 ± 3 51 ± 2
57-66 48-54
.007
49 116
64 ± 1 42 ± 4
62-67 37-47
b.001
64 ± 1 47 ± 4
62-67 42-52
b.001
136 29
60 ± 2 24 ± 7
55-64 15-32
b.001
60 ± 2 31 ± 5
55-64 24-38
b.001
52 113
59 ± 2 36 ± 3
56-62 33-40
b.001
60 ± 2 37 ± 3
57-62 32-41
b.001
121 44
57 ± 2 25 ± 2
54-60 22-28
b.001
59 ± 2 25 ± 2
55-62 22-28
b.001
58 107
40 ± 6 59 ± 3
33-47 55-63
b.001
40 ± 6 60 ± 3
33-47 56-64
b.001
Abbreviations: CI, confidence interval; DFS, disease-free survival; OS, overall survival. a Log-rank test.
of Cthrc1. Their research showed that Cthrc1 could activate and enhance the promoter demethylation and transforming growth factor β1 signaling pathways to improve a tumor cell invasion and metastasis, which results in the tumorigenesis. Moreover, recent data indicate an involvement of Cthrc1 in signal transduction pathways, thus enhancing cell proliferation or survival. Kelley [28] showed that Cthrc1 may play an important role in the Wnt-planar cell polarity pathway, which regulates invasion and metastasis [29]. Another study has Table 4
inferred that Cthrc1 can activate several key signaling molecules, including Src, focal adhesion kinase, paxillin, mitogenactivated protein kinase, extracellular signal–regulated kinase, and Rac1, which results in cancer progression and metastasis by regulating migration and adhesion activities of cancer cells [25]. These data provide important evidence to explicate the mechanism by which Cthrc1 expression contributes to carcinogenesis and tumor progression, although the role of Cthrc1 in signal transduction pathways in tumor cells is certainly worth further research.
Multivariate survival analysis of OS and DFS in 165 patients with gastric carcinoma
Variables
Cthrc1 pTNM Tumor differentiation
OS
DFS a
Exp(B)
95% CI
P
5.751 2.926 5.386
1.593-19.778 1.548-4.973 1.099-13.831
.005 .011 .003
Abbreviations: Exp(B), CI, confidence interval; DFS, disease-free survival; OS, overall survival. a Cox regression test.
Exp(B)
95% CI
Pa
5.534 2.988 5.659
1.512-17.662 1.562-5.299 1.652-13.751
.005 .018 .003
Cthrc1 and gastric cancer GC is one of the most common causes of cancer-related death worldwide [30], especially in East Asian countries such as China and Japan [31]. Therefore, many articles focused on the identification of its significant prognostic factors, including clinicopathological variables and specific markers [32–34]. The current study provided a novel candidate, Cthrc1, as a molecular marker of both biological behaviors and survival outcome of GC. The finding that Cthrc1 expression was closely correlated with late stage, unfavorable differentiation grade, and some invasion modes of breast cancer made its impact on postresectional survival easily understood, similar with that in gastric carcinoma [35]. Thus, the CTHRC1 gene may be focused the as a candidate of targets for inhibition against GC in the future because of its comprehensive roles in progression, invasion, and survival. In conclusion, we demonstrated that Cthrc1 is overexpressed in a great proportion of patients with gastric carcinoma and high Cthrc1 expression correlated with the disease progression and poor prognosis in gastric carcinoma. Furthermore, Cthrc1 might be an attractive target for GC therapy. However, these findings remain to be confirmed by a much larger study.
Acknowledgments The authors thank Falin Zhao for statistical analysis.
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