Pathology – Research and Practice 210 (2014) 363–368
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Original Article
N-cadherin and P-cadherin are biomarkers for invasion, metastasis, and poor prognosis of gallbladder carcinomas Shengen Yi a , Zhu-lin Yang a,∗ , Xiongying Miao a , Qiong Zou b , Jinghe Li c , Lufeng Liang d , Guixiang Zeng e , Senlin Chen f a
Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, PR China Department of Pathology, Third Xiangya Hospital, Central South University, Changsha, Hunan 410011, PR China c Department of Pathology, Basic Medical Science College, Central South University, Changsha, Hunan 410011, PR China d Department of Hepatobiliary and Pancreatic Surgery, Hunan Provincial People’s Hospital, Changsha, Hunan 410007, PR China e Department of Pathology, Loudi Central Hospital, Loudi, Hunan 417011, PR China f Department of Pathology, Hunan Provincial Tumor Hospital, Changsha, Hunan 410013, PR China b
a r t i c l e
i n f o
Article history: Received 29 September 2013 Received in revised form 22 December 2013 Accepted 30 January 2014 Keywords: Gallbladder cancer Adenocarcinoma Squamous cell carcinoma Adenosquamous carcinoma N-cadherin P-cadherin Prognosis Metastasis
a b s t r a c t Gallbladder cancer (GBC) is a rare, but highly aggressive cancer. The most common type of gallbladder cancer is adenocarcinoma (AC), while squamous cell/adenosquamous carcinoma (SC/ASC) is a rare type of gallbladder cancer. The clinicopathologic and biological characteristics of SC/ASC have not been well documented. In this study, the protein expression of N-cadherin and P-cadherin in 46 SC/ASCs and 80 ACs was measured using immunohistochemistry. We demonstrated that positive N-cadherin and P-cadherin expression were significantly associated with large tumor size, invasion, and lymph node metastasis of both SC/ASC and AC. In contrast, positive N-cadherin and P-cadherin expression were significantly associated with differentiation and TNM stage in only AC. Univariate Kaplan–Meier analysis showed that positive N-cadherin and P-cadherin expression, differentiation, tumor size, TNM stage, invasion, lymph node metastasis, and surgical curability were significantly associated with overall survival in both SC/ASC and AC patients. Multivariate Cox regression analysis showed that positive N-cadherin and Pcadherin expression are independent poor-prognostic factors in both SC/ASC and AC patients. Our study suggested that positive N-cadherin and P-cadherin expression closely correlated with clinicopathological and biological behaviors, and poor-prognosis of gallbladder cancer. © 2014 Elsevier GmbH. All rights reserved.
Introduction Gallbladder cancer (GBC) is a relatively uncommon, but highly aggressive type of cancer [1]. The prognosis of GBC is extremely poor, and over 90% of GBC patients are diagnosed at an advanced, inoperable stage with serious invasion and metastasis [2]. Classic chemotherapy and radiotherapy are ineffective for these patients with advanced disease. Biological therapy of GBC is not clinically available due to a lack of specific biomarkers. Among various types of GBC, adenocarcinoma (AC, >95%) is the most common [3]. In contrast, squamous cell/adenosquamous carcinoma (SC/ASC) accounts for only 1–12% of various gallbladder cancers [4,5]. The clinicopathological characteristics of SC/ASC have not been well documented compared to that of AC [6]. Although progress has
∗ Corresponding author. Tel.: +86 731 88187376; fax: +86 731 84898168. E-mail addresses: [email protected], [email protected] (Z.-l. Yang). http://dx.doi.org/10.1016/j.prp.2014.01.014 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
been made in identifying biomarkers for the prognosis of AC, none of the proposed markers have reached clinical application [7]. In particular, few markers associated with the aggressive characteristics of GBC have been identified. The progress in identifying biomarkers for SC/ASC is much slower. The accumulated evidence suggests that alterations in the adhesion properties of tumor cells endow tumor cells with an invasive and migratory phenotype [8]. The alterations in adhesion properties are commonly associated with changes in the expression and function of cellular adhesion molecules, which have been widely reported in a variety of tumors [9]. The deregulation of cellular adhesion molecules’ expression has been linked to every step of tumor invasion and metastasis, including detachment of tumor cells from the primary site, intravasation into the blood stream, extravasation to distant target organs, and formation of secondary lesions [10]. Cadherins are important adhesion molecules, which mediate intercellular cell–cell adhesion. Dysfunction of intercellular cell–cell adhesion allows tumor cells to acquire a more motile
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and invasive phenotype [11]. The cadherin superfamily consists of classic cadherins, which are the main mediators of calcium dependent cell–cell adhesion, and nonclassic cadherins, including desmosomal cadherins and protocadherins [12]. The most extensively studied classic cadherins include E-(epithelial), N-(neural), and P-(placental) cadherin. It has been widely demonstrated that normal E-cadherin expression and function are important factors in the maintenance of epithelial integrity and polarity as well as cell–cell adhesion [13]. In most cancers of epithelial origin, Ecadherin-mediated cell–cell adhesion is lost concomitantly with the acquisition of an invasive and aggressive phenotype [10,12,14]. This is because loss of E-cadherin expression interferes with the integrity of adherens junctions, reduces intercellular adhesion, and increases cellular motility. Therefore, E-cadherin expression is downregulated in many types of cancer [15]. In contrast, Pcadherin expression is highly associated with undifferentiated cells in normal adult epithelial tissues as well as with poorly differentiated carcinomas [16]. N-cadherin is involved in the maintenance of microvessel stability and plays a role in blood vessel formation [17]. Therefore, it is not surprising that up-regulation of N-cadherin and P-cadherin expression has been observed in gallbladder cancer [18]. However, the association of N-cadherin and P-cadherin expression with AC and SC/ASC as well as their clinical importance in AC and SC/ASC have not been addressed. In this study, the protein expression of N-cadherin and Pcadherin in 80 AC and 46 SC/ASC tumor tissues was examined using immunohistochemistry. The correlations of N-cadherin and P-cadherin expression with clinicopathological characteristics and prognosis of AC and SC/ASC were comparatively evaluated. Significant correlations of positive N-cadherin and P-cadherin expression with invasion, lymph node metastasis, and poor prognosis have been observed. Materials and methods Case selection A total of 46 squamous cell/adenosquamous carcinoma (SC/ASC) and 80 adenocarcinoma (AC) samples were collected from January 1995 to December 2009. This study was pre-approved by The Ethics Committee for Human Research, Central South University. Among the 46 SC/ASC patients, 27 patients were female and 19 were male (F/M = 1.42) with an average age of 55.8 ± 9.6 years. Among the 80 AC patients, 54 patients were female and 26 patients were male (F/M = 2.08) with an average age of 53.8 ± 9.9 years. Surgery included radical resection for 14 SC/ASC and 26 AC patients, palliative surgery for 18 SC/ASC and 28 AC patients, and only biopsy for 14 SC/ASC and 26 AC patients. Survival information of patients was obtained through letters and phone calls, and patients were followed up for 2 years. Patients that survived longer than 2 years were included in the analysis as censored cases. Immunohistochemistry staining The rabbit anti-human N-cadherin and P-cadherin antibodies were purchased from Abgent Company (California, USA). The peroxidase-based EnVisionTM Detection kit was purchased from Dako Laboratories (CA, USA). Briefly, 4 M sections were cut from routinely paraffin-embedded tumor tissues. After the sections were deparaffinized and incubated with 3% H2 O2 for 15 min, they were incubated with N-cadherin (1:100 dilution) or P-cadherin (1:100 dilution) antibody for 1 hr at room temperature. After rinsing sections 3 times with PBS, sections were incubated with solution A containing HRP-conjugated secondary antibody for 30 min followed by addition of the substrate DAB, counter-staining with
hematoxylin, dehydration, and soaking the sections in xylene. The positive control sections were purchased from Beijing Zhongshan Biotechnology Company (Beijing, China) while the negative control was designed by replacing the primary antibody with 5% fetal bovine serum. The percentage of positive cells was calculated from 500 cells in 10 random fields. Cases with positive staining cells ≥25% were considered positive, while cases with positive staining cells 3 cm (p = 0.039) was significantly lower in ACs than in SC/ASCs, while the percentage of cases with poorlydifferentiated tumors (p = 0.014) was significantly higher in ACs than in SC/ASCs. No difference in invasion, lymph node metastasis, TNM stage, overall survival, and the percentage of positive Ncadherin and P-cadherin expression was observed between SC/ASC and AC patients. EnVision immunohistochemistry revealed that Ncadherin and P-cadherin positive staining were mainly localized in the cytoplasm of SC/ASC and AC cells (Fig. 1). The association of N-cadherin and P-cadherin expression with clinicopathologic characteristics of patients with SC/ASC and AC As shown in Table 1, the percentage of positive N-cadherin and P-cadherin expression was significantly higher in SC/ASC cases with large tumor size, invasion, and lymph node metastasis than in cases with small tumor size, no invasion, and no lymph node metastasis (p < 0.05). Neither N-cadherin nor P-cadherin was associated with sex, age, differentiation, TNM stage, or gallstones in SC/ASC. The percentage of positive N-cadherin and P-cadherin expression in AC tumors was significantly higher in cases with large tumor size, high TNM stage, poor differentiation, invasion, and lymph node metastasis compared to cases with small tumor size, low TNM stage, well-differentiated tumor, no invasion, and no lymph node metastasis (p < 0.05, p < 0.01, or p < 0.001). Neither N-cadherin nor P-cadherin was associated with sex, age, or gallstones in AC. The correlation between N-cadherin and P-cadherin expression with survival in patients with SC/ASC and AC Among the 46 SC/ASC patients, 33 patients survived shorter than 1 year, 13 patients survived longer than 1 year, and 4 patients survived longer than 2 years. Among the 80 AC patients, 57 patients survived shorter than 1 year, 23 patients survived longer than 1 year, and 9 patients survived longer than 2 years. No significant difference in post-operative survival time was observed between SC/ASC and AC patients. Kaplan–Meier survival analysis of SC/ASC patients revealed that the differentiation, tumor size, TNM stage, lymph node metastasis, invasion, and surgical curability were significantly associated
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Fig. 1. N-cadherin and P-cadherin expression in gallbladder cancer tissues. EnVision immunohistochemistry, original magnification ×200. N-cadherin and P-cadherin positive staining was mainly localized in the cytoplasm. (A) Positive N-cadherin expression in SC. (B) Positive N-cadherin expression in AC. (C) Positive P-cadherin expression in SC. (D) Positive P-cadherin expression in AC.
Table 1 The association of N-cadherin and P-cadherin expression with the clinicopathological characteristics of AC. Clinicopathological characteristics
Total No.
N-cadherin Pos. No. (%)
P-cadherin 2
P
Pos. No. (%)
2
P
SC/ASC Differentiation Well Moderate Poor
16 24 6
8 (50.0) 12 (50.0) 4 (66.7)
0.46
0.787
7 (43.8) 12 (50.0) 4 (66.7)
0.92
0.613
Tumor size ≤3 cm >3 cm
20 26
7 (35.0) 17 (65.4)
4.18
0.046
6 (30.0) 17 (65.4)
5.66
0.021
TNM stage I + II III IV
12 20 14
4 (33.3) 10 (50.0) 10 (71.4)
3.82
0.145
4.09
0.128
Lymph metastasis No Yes
17 29
5 (29.4) 19 (65.5)
5.60
0.018
5 (29.4) 18 (62.1)
4.57
0.031
Invasion No Yes
16 30
5 (31.3) 19 (63.3)
4.30
0.038
4 (25.0) 19 (63.3)
6.13
0.014
AC Differentiation Well Moderate Poor
27 25 28
10 (37.0) 13 (52.0) 21 (75.0)
8.01
0.018
9 (33.3) 13 (52.0) 20 (71.4)
8.00
0.018
Tumor size ≤3 cm >3 cm
50 30
21 (42.0) 23 (76.7)
7.110
0.008
21 (42.0) 21 (70.0)
5.90
0.015
TNM stage I + II III IV
21 38 21
5 (23.8) 21 (55.3) 18 (85.7)
13.76
0.001
Lymph metastasis No Yes
30 50
10 (33.3) 34 (68.0)
4.301
10 (33.3) 32 (64.0)
7.07
0.008
Invasion No Yes
31 49
10 (32.3) 34 (69.4)
7.955
11 (35.5) 31 (63.2)
5.88
0.019
10.05
0.007
4 (33.3) 9 (45.0) 10 (71.4)
5 (23.8) 20 (52.6) 17 (80.9)
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Table 2 Relationship between N-cadherin and P-cadherin expression, clinicopathological characteristics and average survival of GBC patients. C.P. characteristics
Samples (n)
Average survival (month)
SC/ASC Differentiation Well Moderate Poor
16 24 6
13.81 (5–24) 8.92 (4–18) 5.83 (4–9)
Tumor mass size ≤3 cm >3 cm
20 26
14.35 (7–24) 7.04 (4–11)
TNM stage I + II III IV
12 20 14
17.00 (9–24) 9.20 (7–15) 5.86 (4–8)
Lymph node metastasis 17 No 29 Yes Invasion No Yes
16 30
Chi-square
P value
19.125
0.000
31.337
0.000
51.139
0.000
14.24 (4–24) 7.86 (4–15)
16.219
0.000
15.75 (9–24) 7.27 (4–12)
32.271
0.000
50.165
0.000
Operative procedure 14 Radical Palliative 18 14 Biopsy
16.64 (10–24) 8.50 (6–12) 6.00 (4–8)
N-cadherin − +
22 24
11.96 (4–24) 8.63 (4–24)
5.046
0.025
P-cadherin − +
23 23
11.83 (5–24) 8.61 (4–15)
4.220
0.040
AC Differentiation Well Moderate Poor
27 25 28
15.07 (5–24) 10.60 (4–24) 6.68 (3–14)
32.501
0.000
Tumor size ≤3 cm >3 cm
50 30
13.70 (6–24) 5.80 (3–10)
TNM stage I + II III IV
21 38 21
18.96 (5–24) 9.29 (6–15) 5.14 (3–7)
Lymph metastasis No Yes
30 50
Invasion No Yes
31 49
68.283
0.000
105.825
0.000
16.27 (4–24) 7.42 (3–14)
42.372
0.000
16.68 (7–24) 6.98 (3–11)
55.535
0.000
113.141
0.000
0.005
Operative procedure 26 Radical Palliative 28 26 Biopsy
18.31 (10–24) 8.64 (6–11) 5.42 (3–9)
N-cadherin − +
36 44
12.75 (4–24) 9.09 (3–24)
7.915
P-cadherin − +
38 42
12.18 (4–24) 9.43 (3–24)
4.790
Fig. 2. N-cadherin and P-cadherin expression and survival in patients with SC/ASC of gallbladder. (A) Kaplan–Meier plots of overall survival in patients with SC/ASC and with N-cadherin positive and negative expression. (B) Kaplan–Meier plots of overall survival in patients with SC/ASC and with P-cadherin positive and negative expression.
results as SC/ASC patients (Table 2). The average survival time of N-cadherin and P-cadherin positive AC patients was significantly lower than patients with negative N-cadherin (p = 0.005) and Pcadherin (p = 0.029) (Table 2 and Fig. 3). Cox multivariate analysis showed that positive N-cadherin and P-cadherin expression negatively correlated with overall survival in patients with SC/ASC and AC, suggesting that they are independent risk factors of SC/ASC and AC patients (Table 3). Positive N-cadherin and P-cadherin expression were negatively correlated with survival, suggesting that they are independent risk factors of SC/ASC and AC patients (Table 3). Discussion
0.029
C.P: clinicopathological.
with the average survival time (p < 0.001) (Table 2). The average survival time of N-cadherin and P-cadherin positive patients was significantly shorter than patients with negative N-cadherin (p = 0.025) and P-cadherin (p = 0.04) expression (Table 2 and Fig. 2). Kaplan–Meier survival analysis of AC patients revealed similar
Although the expression of N-cadherin and P-cadherin in gallbladder carcinoma has been previously reported, their associations with the invasive and metastatic phenotypes of gallbladder cancer as well as the prognosis of patients have not been addressed. Particularly, no study has compared N-cadherin and P-cadherin expression and clinical importance between AC and SC/ASC tumors. In this study, we used an extensive collection of gallbladder adenocarcinoma (AC) and the rare squamous cell/adenosquamous carcinoma (SC/ASCs) subtype to demonstrate that overexpression of N-cadherin and P-cadherin are important risk factors for the invasion, metastasis, and
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Table 3 Multivariate Cox regression analysis of survival rate in GBC patients. Groups
Factors
RC
SE
Wald
P
RR
SC/ASC Differentiation Tumor mass size TNM stage Lymph metastasis Invasion Surgery N-cadherin P-cadherin
Well/moderately/poorly ≤3 cm/>3 cm I + II/III/IV No/yes No/yes Radical/palliative/biopsy −/+ −/+
1.251 2.187 1.385 1.480 2.554 1.112 1.036 0.981
.412 .663 .574 .526 .741 .487 .472 .433
9.220 10.881 5.822 7.917 11.880 5.214 4.818 5.133
.002 001 .016 .005 .001 .022 .028 .023
AC Differentiation Tumor mass size TNM stage Lymph metastasis Invasion Surgery N-cadherin P-cadherin
Well/moderately/poorly ≤3 cm/>3 cm I + II/III/IV No/yes No/yes Radical/palliative/biopsy −/+ −/+
1.010 1.082 1.268 1.187 1.369 1.635 .980 .838
.457 .412 .413 .492 .520 .417 .320 .360
4.884 6.897 9.426 5.821 6.931 15.373 9.379 5.419
.027 .009 .002 .016 .008 .000 .002 .020
95% CI Lower
Upper
3.494 8.908 3.995 4.393 12.858 3.040 2.818 2.667
1.558 2.429 1.297 1.567 3.009 1.171 1.117 1.141
7.834 32.671 12.305 12.317 54.947 7.897 7.107 6.232
2.746 2.951 3.554 3.277 3.931 5.219 2.664 2.312
1.121 1.316 1.582 1.249 1.419 2.265 1.423 1.142
6.724 6.616 7.984 8.596 10.894 11.615 4.989 4.681
RC, regression coefficients; SE, standard error; RR, relative risk; CI, confidence interval.
poor-prognosis of AC and SC/ASC patients. Our study suggested that both N-cadherin and P-cadherin function as oncogenes involved in the invasion, metastasis, and prognosis of AC and SC/ASC.
Fig. 3. N-cadherin and P-cadherin expression and survival in patients with AC. (A) Kaplan–Meier plots of overall survival in patients with AC and with N-cadherin positive and negative expression. (B) Kaplan–Meier plots of overall survival in patients with AC and with P-cadherin positive and negative expression.
Previous studies demonstrated that N-cadherin promotes motility and induces cell migration, invasion and metastasis in breast cancer cells [19,20]. Moreover, soluble N-cadherin has been reported to stimulate tumor angiogenesis and invasion of cancer cells [21]. A previous study using a small sample of gallbladder adenocarcinomas showed that 66% of gallbladder adenocarcinomas express N-cadherin [22]. However, this study did not address the role of N-cadherin in the invasion and metastasis of gallbladder cancer. In our study, positive N-cadherin expression was identified in 52% of SC/ASC patients and 55% of AC patients. We first demonstrated that positive N-cadherin expression significantly correlated with invasion and lymph node metastasis in both AC and SC/ASC with a more significant correlation in AC. It has been revealed that the roles of P-cadherin in the invasion and metastasis of tumor cells are tissue type specific. For example, there is a progressive loss of normal P-cadherin expression in melanocytes [23,24]. Pcadherin functions as a pro-adhesive and anti-invasive molecule, inhibiting invasion and metastasis of melanocytes [25]. In contrast, P-cadherin is able to enhance cell invasion and tumor aggressiveness in breast cancer [26]. Riener et al. study reported positive P-cadherin expression in 64% of gallbladder carcinomas but provided no data for its association with invasion and metastasis [18]. In this our study, positive P-cadherin expression was identified in 50% of SC/ASC patients and 52.5% of AC patients. We further demonstrated that positive P-cadherin expression significantly correlated with invasion and lymph node metastasis in both AC and SC/ASC. Our study suggests that both N-cadherin and P-cadherin behave as tumor oncogenes in gallbladder cancer and are associated with a phenotype of high invasion and metastasis. Besides showing that N-cadherin and P-cadherin share similar roles in invasion and metastasis in both SC/ASC and AC tumors, our study also showed that these two cadherins share similar roles in SC/ASC and AC prognosis. Patients expressing these two molecules showed significantly shorter survival. In addition, positive expression of these two molecules is an independent predictive factor for poor prognosis in patients with SC/ASC and AC. However, the predictive role of N-cadherin and P-cadherin in the prognosis of SC/ASC and AC is not as effective as other clinicopathologic characteristics, such as tumor differentiation, tumor size, TNM stage, invasion, and lymph node metastasis. In addition, both N-cadherin and Pcadherin expression significantly correlated with large tumor size, but not with the differentiation of SC/ASC tumors or the TNM stage in SC/ASC patients. In contrast, both N-cadherin and P-cadherin
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expression significantly correlated with large tumor size and differentiation of AC tumors as well as with the TNM stage of AC patients. These observations suggest that: (1) N-cadherin and Pcadherin contributed more to cancer progression in AC than in SC/ASC; (2) N-cadherin and P-cadherin could be useful biomarkers for the pathological and clinical stage of AC. Previous studies suggested that squamous carcinoma is more aggressive than adenocarcinoma, but squamous tumors are less frequently present with lymph node metastasis [27,28]. In addition, a previous study reported that SC/ASC occurs predominantly in females (F/M = 3.8) [5]. In this study, our results revealed that squamous carcinoma is no more aggressive than adenocarcinoma because there were no differences in the occurrence of invasion and lymph node metastasis between AC and SC/ASC although more SC/ASC patients had large tumor size. In contrast, AC tumors exhibited significantly poorer differentiation than SC/ASC tumors. Furthermore, there were no significant differences in TNM stage and overall survival time between patients with AC and SC/ASC. Our study also showed that SC/ASC occurred with no significant difference between females and males (F/M ratio is only 1.4). Therefore, our study suggested that the clinicopathologic characteristics of SC/ASC did not seem to be significantly different from ordinary AC. The inconsistency in findings may be mainly caused by the limitation in study size. The conclusions of previous studies come mainly from individual case reports or analyses of small case series. In this study, the clinicopathologic characteristics were analyzed in a large sample (n = 46) of SC/ASC relative to previous reports seen in literature. Our study may provide more reliable information on the differences between the rare SC/ASC subtype and the common AC subtype of gallbladder cancer. In addition, N-cadherin and P-cadherin play different roles in differentiation and TNM stage between SC/ASC and AC with more roles in AC, suggesting possible biological differences. Our study demonstrated that high N-cadherin and P-cadherin expression are associated with an invasive and metastatic phenotype and poor prognosis of AC and SC/ASC. This study also suggested that N-cadherin and P-cadherin are positive regulators of tumor growth and could be used as diagnostic markers for AC. Conflict of interest All authors declared no conflict of interest. Acknowledgement This article was supported by The Innovation Fund Project for Doctoral Student of Hunan Province, No. is CX2012B098. References [1] K.M. Reid, A. Ramos-De la Medina, J.H. Donohue, Diagnosis and surgical management of gallbladder cancer: a review, J. Gastrointest. Surg. 11 (2007) 671–681. [2] W.G. Hawkins, R.P. DeMatteo, W.R. Jarnagin, L. Ben-Porat, L.H. Blumgart, Y. Fong, Jaundice predicts advanced disease and early mortality in patients with gallbladder cancer, Ann. Surg. Oncol. 11 (2004) 310–315.
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Original Article
N-cadherin and P-cadherin are biomarkers for invasion, metastasis, and poor prognosis of gallbladder carcinomas Shengen Yi a , Zhu-lin Yang a,∗ , Xiongying Miao a , Qiong Zou b , Jinghe Li c , Lufeng Liang d , Guixiang Zeng e , Senlin Chen f a
Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, PR China Department of Pathology, Third Xiangya Hospital, Central South University, Changsha, Hunan 410011, PR China c Department of Pathology, Basic Medical Science College, Central South University, Changsha, Hunan 410011, PR China d Department of Hepatobiliary and Pancreatic Surgery, Hunan Provincial People’s Hospital, Changsha, Hunan 410007, PR China e Department of Pathology, Loudi Central Hospital, Loudi, Hunan 417011, PR China f Department of Pathology, Hunan Provincial Tumor Hospital, Changsha, Hunan 410013, PR China b
a r t i c l e
i n f o
Article history: Received 29 September 2013 Received in revised form 22 December 2013 Accepted 30 January 2014 Keywords: Gallbladder cancer Adenocarcinoma Squamous cell carcinoma Adenosquamous carcinoma N-cadherin P-cadherin Prognosis Metastasis
a b s t r a c t Gallbladder cancer (GBC) is a rare, but highly aggressive cancer. The most common type of gallbladder cancer is adenocarcinoma (AC), while squamous cell/adenosquamous carcinoma (SC/ASC) is a rare type of gallbladder cancer. The clinicopathologic and biological characteristics of SC/ASC have not been well documented. In this study, the protein expression of N-cadherin and P-cadherin in 46 SC/ASCs and 80 ACs was measured using immunohistochemistry. We demonstrated that positive N-cadherin and P-cadherin expression were significantly associated with large tumor size, invasion, and lymph node metastasis of both SC/ASC and AC. In contrast, positive N-cadherin and P-cadherin expression were significantly associated with differentiation and TNM stage in only AC. Univariate Kaplan–Meier analysis showed that positive N-cadherin and P-cadherin expression, differentiation, tumor size, TNM stage, invasion, lymph node metastasis, and surgical curability were significantly associated with overall survival in both SC/ASC and AC patients. Multivariate Cox regression analysis showed that positive N-cadherin and Pcadherin expression are independent poor-prognostic factors in both SC/ASC and AC patients. Our study suggested that positive N-cadherin and P-cadherin expression closely correlated with clinicopathological and biological behaviors, and poor-prognosis of gallbladder cancer. © 2014 Elsevier GmbH. All rights reserved.
Introduction Gallbladder cancer (GBC) is a relatively uncommon, but highly aggressive type of cancer [1]. The prognosis of GBC is extremely poor, and over 90% of GBC patients are diagnosed at an advanced, inoperable stage with serious invasion and metastasis [2]. Classic chemotherapy and radiotherapy are ineffective for these patients with advanced disease. Biological therapy of GBC is not clinically available due to a lack of specific biomarkers. Among various types of GBC, adenocarcinoma (AC, >95%) is the most common [3]. In contrast, squamous cell/adenosquamous carcinoma (SC/ASC) accounts for only 1–12% of various gallbladder cancers [4,5]. The clinicopathological characteristics of SC/ASC have not been well documented compared to that of AC [6]. Although progress has
∗ Corresponding author. Tel.: +86 731 88187376; fax: +86 731 84898168. E-mail addresses: [email protected], [email protected] (Z.-l. Yang). http://dx.doi.org/10.1016/j.prp.2014.01.014 0344-0338/© 2014 Elsevier GmbH. All rights reserved.
been made in identifying biomarkers for the prognosis of AC, none of the proposed markers have reached clinical application [7]. In particular, few markers associated with the aggressive characteristics of GBC have been identified. The progress in identifying biomarkers for SC/ASC is much slower. The accumulated evidence suggests that alterations in the adhesion properties of tumor cells endow tumor cells with an invasive and migratory phenotype [8]. The alterations in adhesion properties are commonly associated with changes in the expression and function of cellular adhesion molecules, which have been widely reported in a variety of tumors [9]. The deregulation of cellular adhesion molecules’ expression has been linked to every step of tumor invasion and metastasis, including detachment of tumor cells from the primary site, intravasation into the blood stream, extravasation to distant target organs, and formation of secondary lesions [10]. Cadherins are important adhesion molecules, which mediate intercellular cell–cell adhesion. Dysfunction of intercellular cell–cell adhesion allows tumor cells to acquire a more motile
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and invasive phenotype [11]. The cadherin superfamily consists of classic cadherins, which are the main mediators of calcium dependent cell–cell adhesion, and nonclassic cadherins, including desmosomal cadherins and protocadherins [12]. The most extensively studied classic cadherins include E-(epithelial), N-(neural), and P-(placental) cadherin. It has been widely demonstrated that normal E-cadherin expression and function are important factors in the maintenance of epithelial integrity and polarity as well as cell–cell adhesion [13]. In most cancers of epithelial origin, Ecadherin-mediated cell–cell adhesion is lost concomitantly with the acquisition of an invasive and aggressive phenotype [10,12,14]. This is because loss of E-cadherin expression interferes with the integrity of adherens junctions, reduces intercellular adhesion, and increases cellular motility. Therefore, E-cadherin expression is downregulated in many types of cancer [15]. In contrast, Pcadherin expression is highly associated with undifferentiated cells in normal adult epithelial tissues as well as with poorly differentiated carcinomas [16]. N-cadherin is involved in the maintenance of microvessel stability and plays a role in blood vessel formation [17]. Therefore, it is not surprising that up-regulation of N-cadherin and P-cadherin expression has been observed in gallbladder cancer [18]. However, the association of N-cadherin and P-cadherin expression with AC and SC/ASC as well as their clinical importance in AC and SC/ASC have not been addressed. In this study, the protein expression of N-cadherin and Pcadherin in 80 AC and 46 SC/ASC tumor tissues was examined using immunohistochemistry. The correlations of N-cadherin and P-cadherin expression with clinicopathological characteristics and prognosis of AC and SC/ASC were comparatively evaluated. Significant correlations of positive N-cadherin and P-cadherin expression with invasion, lymph node metastasis, and poor prognosis have been observed. Materials and methods Case selection A total of 46 squamous cell/adenosquamous carcinoma (SC/ASC) and 80 adenocarcinoma (AC) samples were collected from January 1995 to December 2009. This study was pre-approved by The Ethics Committee for Human Research, Central South University. Among the 46 SC/ASC patients, 27 patients were female and 19 were male (F/M = 1.42) with an average age of 55.8 ± 9.6 years. Among the 80 AC patients, 54 patients were female and 26 patients were male (F/M = 2.08) with an average age of 53.8 ± 9.9 years. Surgery included radical resection for 14 SC/ASC and 26 AC patients, palliative surgery for 18 SC/ASC and 28 AC patients, and only biopsy for 14 SC/ASC and 26 AC patients. Survival information of patients was obtained through letters and phone calls, and patients were followed up for 2 years. Patients that survived longer than 2 years were included in the analysis as censored cases. Immunohistochemistry staining The rabbit anti-human N-cadherin and P-cadherin antibodies were purchased from Abgent Company (California, USA). The peroxidase-based EnVisionTM Detection kit was purchased from Dako Laboratories (CA, USA). Briefly, 4 M sections were cut from routinely paraffin-embedded tumor tissues. After the sections were deparaffinized and incubated with 3% H2 O2 for 15 min, they were incubated with N-cadherin (1:100 dilution) or P-cadherin (1:100 dilution) antibody for 1 hr at room temperature. After rinsing sections 3 times with PBS, sections were incubated with solution A containing HRP-conjugated secondary antibody for 30 min followed by addition of the substrate DAB, counter-staining with
hematoxylin, dehydration, and soaking the sections in xylene. The positive control sections were purchased from Beijing Zhongshan Biotechnology Company (Beijing, China) while the negative control was designed by replacing the primary antibody with 5% fetal bovine serum. The percentage of positive cells was calculated from 500 cells in 10 random fields. Cases with positive staining cells ≥25% were considered positive, while cases with positive staining cells 3 cm (p = 0.039) was significantly lower in ACs than in SC/ASCs, while the percentage of cases with poorlydifferentiated tumors (p = 0.014) was significantly higher in ACs than in SC/ASCs. No difference in invasion, lymph node metastasis, TNM stage, overall survival, and the percentage of positive Ncadherin and P-cadherin expression was observed between SC/ASC and AC patients. EnVision immunohistochemistry revealed that Ncadherin and P-cadherin positive staining were mainly localized in the cytoplasm of SC/ASC and AC cells (Fig. 1). The association of N-cadherin and P-cadherin expression with clinicopathologic characteristics of patients with SC/ASC and AC As shown in Table 1, the percentage of positive N-cadherin and P-cadherin expression was significantly higher in SC/ASC cases with large tumor size, invasion, and lymph node metastasis than in cases with small tumor size, no invasion, and no lymph node metastasis (p < 0.05). Neither N-cadherin nor P-cadherin was associated with sex, age, differentiation, TNM stage, or gallstones in SC/ASC. The percentage of positive N-cadherin and P-cadherin expression in AC tumors was significantly higher in cases with large tumor size, high TNM stage, poor differentiation, invasion, and lymph node metastasis compared to cases with small tumor size, low TNM stage, well-differentiated tumor, no invasion, and no lymph node metastasis (p < 0.05, p < 0.01, or p < 0.001). Neither N-cadherin nor P-cadherin was associated with sex, age, or gallstones in AC. The correlation between N-cadherin and P-cadherin expression with survival in patients with SC/ASC and AC Among the 46 SC/ASC patients, 33 patients survived shorter than 1 year, 13 patients survived longer than 1 year, and 4 patients survived longer than 2 years. Among the 80 AC patients, 57 patients survived shorter than 1 year, 23 patients survived longer than 1 year, and 9 patients survived longer than 2 years. No significant difference in post-operative survival time was observed between SC/ASC and AC patients. Kaplan–Meier survival analysis of SC/ASC patients revealed that the differentiation, tumor size, TNM stage, lymph node metastasis, invasion, and surgical curability were significantly associated
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Fig. 1. N-cadherin and P-cadherin expression in gallbladder cancer tissues. EnVision immunohistochemistry, original magnification ×200. N-cadherin and P-cadherin positive staining was mainly localized in the cytoplasm. (A) Positive N-cadherin expression in SC. (B) Positive N-cadherin expression in AC. (C) Positive P-cadherin expression in SC. (D) Positive P-cadherin expression in AC.
Table 1 The association of N-cadherin and P-cadherin expression with the clinicopathological characteristics of AC. Clinicopathological characteristics
Total No.
N-cadherin Pos. No. (%)
P-cadherin 2
P
Pos. No. (%)
2
P
SC/ASC Differentiation Well Moderate Poor
16 24 6
8 (50.0) 12 (50.0) 4 (66.7)
0.46
0.787
7 (43.8) 12 (50.0) 4 (66.7)
0.92
0.613
Tumor size ≤3 cm >3 cm
20 26
7 (35.0) 17 (65.4)
4.18
0.046
6 (30.0) 17 (65.4)
5.66
0.021
TNM stage I + II III IV
12 20 14
4 (33.3) 10 (50.0) 10 (71.4)
3.82
0.145
4.09
0.128
Lymph metastasis No Yes
17 29
5 (29.4) 19 (65.5)
5.60
0.018
5 (29.4) 18 (62.1)
4.57
0.031
Invasion No Yes
16 30
5 (31.3) 19 (63.3)
4.30
0.038
4 (25.0) 19 (63.3)
6.13
0.014
AC Differentiation Well Moderate Poor
27 25 28
10 (37.0) 13 (52.0) 21 (75.0)
8.01
0.018
9 (33.3) 13 (52.0) 20 (71.4)
8.00
0.018
Tumor size ≤3 cm >3 cm
50 30
21 (42.0) 23 (76.7)
7.110
0.008
21 (42.0) 21 (70.0)
5.90
0.015
TNM stage I + II III IV
21 38 21
5 (23.8) 21 (55.3) 18 (85.7)
13.76
0.001
Lymph metastasis No Yes
30 50
10 (33.3) 34 (68.0)
4.301
10 (33.3) 32 (64.0)
7.07
0.008
Invasion No Yes
31 49
10 (32.3) 34 (69.4)
7.955
11 (35.5) 31 (63.2)
5.88
0.019
10.05
0.007
4 (33.3) 9 (45.0) 10 (71.4)
5 (23.8) 20 (52.6) 17 (80.9)
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Table 2 Relationship between N-cadherin and P-cadherin expression, clinicopathological characteristics and average survival of GBC patients. C.P. characteristics
Samples (n)
Average survival (month)
SC/ASC Differentiation Well Moderate Poor
16 24 6
13.81 (5–24) 8.92 (4–18) 5.83 (4–9)
Tumor mass size ≤3 cm >3 cm
20 26
14.35 (7–24) 7.04 (4–11)
TNM stage I + II III IV
12 20 14
17.00 (9–24) 9.20 (7–15) 5.86 (4–8)
Lymph node metastasis 17 No 29 Yes Invasion No Yes
16 30
Chi-square
P value
19.125
0.000
31.337
0.000
51.139
0.000
14.24 (4–24) 7.86 (4–15)
16.219
0.000
15.75 (9–24) 7.27 (4–12)
32.271
0.000
50.165
0.000
Operative procedure 14 Radical Palliative 18 14 Biopsy
16.64 (10–24) 8.50 (6–12) 6.00 (4–8)
N-cadherin − +
22 24
11.96 (4–24) 8.63 (4–24)
5.046
0.025
P-cadherin − +
23 23
11.83 (5–24) 8.61 (4–15)
4.220
0.040
AC Differentiation Well Moderate Poor
27 25 28
15.07 (5–24) 10.60 (4–24) 6.68 (3–14)
32.501
0.000
Tumor size ≤3 cm >3 cm
50 30
13.70 (6–24) 5.80 (3–10)
TNM stage I + II III IV
21 38 21
18.96 (5–24) 9.29 (6–15) 5.14 (3–7)
Lymph metastasis No Yes
30 50
Invasion No Yes
31 49
68.283
0.000
105.825
0.000
16.27 (4–24) 7.42 (3–14)
42.372
0.000
16.68 (7–24) 6.98 (3–11)
55.535
0.000
113.141
0.000
0.005
Operative procedure 26 Radical Palliative 28 26 Biopsy
18.31 (10–24) 8.64 (6–11) 5.42 (3–9)
N-cadherin − +
36 44
12.75 (4–24) 9.09 (3–24)
7.915
P-cadherin − +
38 42
12.18 (4–24) 9.43 (3–24)
4.790
Fig. 2. N-cadherin and P-cadherin expression and survival in patients with SC/ASC of gallbladder. (A) Kaplan–Meier plots of overall survival in patients with SC/ASC and with N-cadherin positive and negative expression. (B) Kaplan–Meier plots of overall survival in patients with SC/ASC and with P-cadherin positive and negative expression.
results as SC/ASC patients (Table 2). The average survival time of N-cadherin and P-cadherin positive AC patients was significantly lower than patients with negative N-cadherin (p = 0.005) and Pcadherin (p = 0.029) (Table 2 and Fig. 3). Cox multivariate analysis showed that positive N-cadherin and P-cadherin expression negatively correlated with overall survival in patients with SC/ASC and AC, suggesting that they are independent risk factors of SC/ASC and AC patients (Table 3). Positive N-cadherin and P-cadherin expression were negatively correlated with survival, suggesting that they are independent risk factors of SC/ASC and AC patients (Table 3). Discussion
0.029
C.P: clinicopathological.
with the average survival time (p < 0.001) (Table 2). The average survival time of N-cadherin and P-cadherin positive patients was significantly shorter than patients with negative N-cadherin (p = 0.025) and P-cadherin (p = 0.04) expression (Table 2 and Fig. 2). Kaplan–Meier survival analysis of AC patients revealed similar
Although the expression of N-cadherin and P-cadherin in gallbladder carcinoma has been previously reported, their associations with the invasive and metastatic phenotypes of gallbladder cancer as well as the prognosis of patients have not been addressed. Particularly, no study has compared N-cadherin and P-cadherin expression and clinical importance between AC and SC/ASC tumors. In this study, we used an extensive collection of gallbladder adenocarcinoma (AC) and the rare squamous cell/adenosquamous carcinoma (SC/ASCs) subtype to demonstrate that overexpression of N-cadherin and P-cadherin are important risk factors for the invasion, metastasis, and
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Table 3 Multivariate Cox regression analysis of survival rate in GBC patients. Groups
Factors
RC
SE
Wald
P
RR
SC/ASC Differentiation Tumor mass size TNM stage Lymph metastasis Invasion Surgery N-cadherin P-cadherin
Well/moderately/poorly ≤3 cm/>3 cm I + II/III/IV No/yes No/yes Radical/palliative/biopsy −/+ −/+
1.251 2.187 1.385 1.480 2.554 1.112 1.036 0.981
.412 .663 .574 .526 .741 .487 .472 .433
9.220 10.881 5.822 7.917 11.880 5.214 4.818 5.133
.002 001 .016 .005 .001 .022 .028 .023
AC Differentiation Tumor mass size TNM stage Lymph metastasis Invasion Surgery N-cadherin P-cadherin
Well/moderately/poorly ≤3 cm/>3 cm I + II/III/IV No/yes No/yes Radical/palliative/biopsy −/+ −/+
1.010 1.082 1.268 1.187 1.369 1.635 .980 .838
.457 .412 .413 .492 .520 .417 .320 .360
4.884 6.897 9.426 5.821 6.931 15.373 9.379 5.419
.027 .009 .002 .016 .008 .000 .002 .020
95% CI Lower
Upper
3.494 8.908 3.995 4.393 12.858 3.040 2.818 2.667
1.558 2.429 1.297 1.567 3.009 1.171 1.117 1.141
7.834 32.671 12.305 12.317 54.947 7.897 7.107 6.232
2.746 2.951 3.554 3.277 3.931 5.219 2.664 2.312
1.121 1.316 1.582 1.249 1.419 2.265 1.423 1.142
6.724 6.616 7.984 8.596 10.894 11.615 4.989 4.681
RC, regression coefficients; SE, standard error; RR, relative risk; CI, confidence interval.
poor-prognosis of AC and SC/ASC patients. Our study suggested that both N-cadherin and P-cadherin function as oncogenes involved in the invasion, metastasis, and prognosis of AC and SC/ASC.
Fig. 3. N-cadherin and P-cadherin expression and survival in patients with AC. (A) Kaplan–Meier plots of overall survival in patients with AC and with N-cadherin positive and negative expression. (B) Kaplan–Meier plots of overall survival in patients with AC and with P-cadherin positive and negative expression.
Previous studies demonstrated that N-cadherin promotes motility and induces cell migration, invasion and metastasis in breast cancer cells [19,20]. Moreover, soluble N-cadherin has been reported to stimulate tumor angiogenesis and invasion of cancer cells [21]. A previous study using a small sample of gallbladder adenocarcinomas showed that 66% of gallbladder adenocarcinomas express N-cadherin [22]. However, this study did not address the role of N-cadherin in the invasion and metastasis of gallbladder cancer. In our study, positive N-cadherin expression was identified in 52% of SC/ASC patients and 55% of AC patients. We first demonstrated that positive N-cadherin expression significantly correlated with invasion and lymph node metastasis in both AC and SC/ASC with a more significant correlation in AC. It has been revealed that the roles of P-cadherin in the invasion and metastasis of tumor cells are tissue type specific. For example, there is a progressive loss of normal P-cadherin expression in melanocytes [23,24]. Pcadherin functions as a pro-adhesive and anti-invasive molecule, inhibiting invasion and metastasis of melanocytes [25]. In contrast, P-cadherin is able to enhance cell invasion and tumor aggressiveness in breast cancer [26]. Riener et al. study reported positive P-cadherin expression in 64% of gallbladder carcinomas but provided no data for its association with invasion and metastasis [18]. In this our study, positive P-cadherin expression was identified in 50% of SC/ASC patients and 52.5% of AC patients. We further demonstrated that positive P-cadherin expression significantly correlated with invasion and lymph node metastasis in both AC and SC/ASC. Our study suggests that both N-cadherin and P-cadherin behave as tumor oncogenes in gallbladder cancer and are associated with a phenotype of high invasion and metastasis. Besides showing that N-cadherin and P-cadherin share similar roles in invasion and metastasis in both SC/ASC and AC tumors, our study also showed that these two cadherins share similar roles in SC/ASC and AC prognosis. Patients expressing these two molecules showed significantly shorter survival. In addition, positive expression of these two molecules is an independent predictive factor for poor prognosis in patients with SC/ASC and AC. However, the predictive role of N-cadherin and P-cadherin in the prognosis of SC/ASC and AC is not as effective as other clinicopathologic characteristics, such as tumor differentiation, tumor size, TNM stage, invasion, and lymph node metastasis. In addition, both N-cadherin and Pcadherin expression significantly correlated with large tumor size, but not with the differentiation of SC/ASC tumors or the TNM stage in SC/ASC patients. In contrast, both N-cadherin and P-cadherin
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expression significantly correlated with large tumor size and differentiation of AC tumors as well as with the TNM stage of AC patients. These observations suggest that: (1) N-cadherin and Pcadherin contributed more to cancer progression in AC than in SC/ASC; (2) N-cadherin and P-cadherin could be useful biomarkers for the pathological and clinical stage of AC. Previous studies suggested that squamous carcinoma is more aggressive than adenocarcinoma, but squamous tumors are less frequently present with lymph node metastasis [27,28]. In addition, a previous study reported that SC/ASC occurs predominantly in females (F/M = 3.8) [5]. In this study, our results revealed that squamous carcinoma is no more aggressive than adenocarcinoma because there were no differences in the occurrence of invasion and lymph node metastasis between AC and SC/ASC although more SC/ASC patients had large tumor size. In contrast, AC tumors exhibited significantly poorer differentiation than SC/ASC tumors. Furthermore, there were no significant differences in TNM stage and overall survival time between patients with AC and SC/ASC. Our study also showed that SC/ASC occurred with no significant difference between females and males (F/M ratio is only 1.4). Therefore, our study suggested that the clinicopathologic characteristics of SC/ASC did not seem to be significantly different from ordinary AC. The inconsistency in findings may be mainly caused by the limitation in study size. The conclusions of previous studies come mainly from individual case reports or analyses of small case series. In this study, the clinicopathologic characteristics were analyzed in a large sample (n = 46) of SC/ASC relative to previous reports seen in literature. Our study may provide more reliable information on the differences between the rare SC/ASC subtype and the common AC subtype of gallbladder cancer. In addition, N-cadherin and P-cadherin play different roles in differentiation and TNM stage between SC/ASC and AC with more roles in AC, suggesting possible biological differences. Our study demonstrated that high N-cadherin and P-cadherin expression are associated with an invasive and metastatic phenotype and poor prognosis of AC and SC/ASC. This study also suggested that N-cadherin and P-cadherin are positive regulators of tumor growth and could be used as diagnostic markers for AC. Conflict of interest All authors declared no conflict of interest. Acknowledgement This article was supported by The Innovation Fund Project for Doctoral Student of Hunan Province, No. is CX2012B098. References [1] K.M. Reid, A. Ramos-De la Medina, J.H. Donohue, Diagnosis and surgical management of gallbladder cancer: a review, J. Gastrointest. Surg. 11 (2007) 671–681. [2] W.G. Hawkins, R.P. DeMatteo, W.R. Jarnagin, L. Ben-Porat, L.H. Blumgart, Y. Fong, Jaundice predicts advanced disease and early mortality in patients with gallbladder cancer, Ann. Surg. Oncol. 11 (2004) 310–315.
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