J Mol Hist DOI 10.1007/s10735-014-9597-9

ORIGINAL PAPER

ATP5b and b2-microglobulin are predictive markers for the prognosis of patients with gallbladder cancer Jianning Sun • Zhu-lin Yang • Xiongying Miao Qiong Zou • Jinghe Li • Lufeng Liang • Guixiang Zeng • Senlin Chen



Received: 20 June 2014 / Accepted: 30 September 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The differences in clinical, pathological, and biological characteristics between adenocarcinoma (AC) and squamous cell/adenosquamous carcinoma (SC/ASC) of the gallbladder have not been well documented. This study investigates the clinical and pathological associations of ATP5B and b2M with benign and malignant lesions of the gallbladder. In this study, ATP5B and b2M expression in 46 SC/ASCs and 80 ACs were examined using immunohistochemistry. The rate of ATP5B positive expression was significantly lower, while the rate of b2M expression was significantly higher, in AC and SC/ASC than in gallbladder adenomas, gallbladder polyps, or gallbladder epithelium with stone (P \ 0.01). More SC/ASCs had larger tumor mass and good differentiation compared to ACs. Positive b2M and negative ATP5B expression were significantly associated with large tumor size, high TNM stage, lymph node metastasis, and invasion of SC/

ASCs and ACs. Univariate Kaplan–Meier analysis showed that positive b2M (P \ 0.05 or P \ 0.001) expression and negative ATP5B (P \ 0.001) expression were significantly associated with decreased overall survival in both SC/ASC and AC patients. Multivariate Cox regression analysis showed that negative ATP5B expression is an independentprognostic factor for poor prognosis in both SC/ASC (P \ 0.01) and AC (P \ 0.001) patients. Positive b2M expression is an independent-prognostic factor for poor prognosis in AC (P \ 0.05) patients. Our study suggested that positive b2M expression or loss of ATP5B expression in tumor tissues is closely related to the metastasis, invasion, and poor-prognosis of gallbladder cancer.

J. Sun Department of Radiology, Second Xiangya Hospital, Central South University, Changsha 410011, Hunan, People’s Republic of China

L. Liang Department of Hepatobiliary and Pancreatic Surgery, Hunan Provincial People’s Hospital, Changsha 410007, Hunan, People’s Republic of China

Z. Yang (&)  X. Miao Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha 410011, Hunan, People’s Republic of China e-mail: [email protected]; [email protected]

G. Zeng Department of Pathology, Loudi Central Hospital, Loudi 417011, Hunan, People’s Republic of China

Q. Zou Department of Pathology, Third Xiangya Hospital, Central South University, Changsha 410013, Hunan, People’s Republic of China

Keywords Gallbladder cancer  Adenocarcinoma  Squamous cell carcinoma  Adenosquamous carcinoma  ATP5B  b2M  Prognosis  Metastasis

S. Chen Department of Pathology, Hunan Provincial Tumor Hospital, Changsha 410013, Hunan, People’s Republic of China

J. Li Department of Pathology, Basic School of Medicine, Central South University, Changsha 410078, Hunan, People’s Republic of China

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Introduction Gallbladder cancers (GBCs) are highly aggressive tumors with poor prognosis (Jayaraman and Jarnagin 2010). The accurate incidence of GBC in China is not available. However, the incidence rate identified in Shanghai, China is 5.2/100, 000 women (Randi et al. 2009). Early stage GBCs are operable with relatively good prognosis. However, most patients are diagnosed with advanced stage GBC characterized by serious invasion and metastasis because there is a lack of specific symptoms for early detection of GBC (Reid et al. 2007; Hawkins et al. 2004). Patients with advanced stage GBC are inoperable. Radiotherapy and chemotherapy are the primary treatments for advanced GBC, but their clinical efficacy is very limited. Currently, molecular targeting therapy is still not available for GBC patients. Relative to other gastrointestinal tract cancers, the biological characteristics of GBC are not as well documented although identification of biomarkers for GBC has shown some progress recently (Zou et al. 2013; Cai et al. 2013; Li et al. 2014). GBCs include several histological subtypes with more than 90 % of GBCs being adenocarcinomas (ACs) (Ootani et al. 1992). Squamous cell/adenosquamous carcinoma (SC/ASC) is a rare subtype of GBC, representing 1–10 % of GBCs (Jayaraman and Jarnagin 2010; Roa et al. 2011). Due to a lack of cases of SC/ASC, the clinical and pathological characteristics of SC/ASC have not been well documented. Currently, most reports on SC/ ASC are individual case analyses of its clinical features without identification of its biological characteristics. Thus, the clinical, pathological, and biological characteristics of SC/ASC should be urgently analyzed using a large sample size. b1-F1-ATPase (ATP5B) is the b subunit of mitochondrial H?-ATP synthase, which is a rate-limiting enzyme of oxidative phosphorylation (Garcı´a 2011). Although the downregulation of ATP5B is generally thought to be a hallmark of most human carcinomas (Willers and Cuezva 2011), contradictory results have been commonly observed. b2-microglobulin (b2M) is an 11.6-kDa noncovalently bound light chain of major histocompatibility complex class I (MHC I), which interacts with and stabilizes the heavy chain of MHC I (Eichner and Radford 2011). Besides its roles in immunity, b2M also acts as a prototypical oncogenic factor capable of stimulating proliferation, apoptosis, and metastasis of cancers (Jiang et al. 2012). Elevated level of circulating b2M has also been demonstrated to be an important predictive factor of poor survival in multiple myeloma (Bataille et al. 1983), lymphomas (Cooper and Plesner 1980), and leukemias (Molica et al. 1999). However, the expression of ATP5B and b2M in GBC tumors has not been reported.

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In this study, the expression of ATP5B and b2M in 80 AC and 46 SC/ASC tissues were examined using immunohistochemistry. The correlations of ATP5B and b2M expression with clinical characteristics, pathological characteristics, and prognosis of AC and SC/ASC were evaluated.

Materials and methods Patients Tumor tissues, clinical and pathological characteristics, and survival information for 46 SC/ASC patients and 80 AC patients were collected from January 1995 to December 2009. AC and SC/ASC were diagnosed by pathologists at the Department of Pathology, Second Xiangya Hospital. No adjuvant or neoadjuvant treatment was given before surgery. TNM staging of gallbladder cancer was conducted according to standard TNM classification of malignant tumor 7th edition (International union against cancer). The diagnostic criteria of World Health Organization (WHO) were used for SC and ASC diagnosis. A gallbladder cancer sample was diagnosed as SC or ASC when most malignant cells are squamous cells but less than 10 % are adenocarcinoma cells, or the sample contains both squamous cells and adenocarcinoma cells with over 10 % adenocarcinoma or squamous cells. The average age of 80 AC patients was 53.8 ± 9.9 years. The average age of 46 SC/ASC patients was 55.8 ± 9.6 years. Surgery included radical resection (resection of the primary tumor, the surrounding invaded organs, and regional metastatic lymph nodes)for 14 SC/ ASC and 26 AC patients, palliative surgery (partial resection of the primary tumor, metastases, or involved organs, but still leaving some lesions or metastases) for 18 SC/ASC and 28 AC patients, and only biopsy (excised from primary tumor) for 14 SC/ASC and 26 AC patients. Survival information 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. Gallbladder tissues from 30 patients with gallbladder adenomas, 25 patients with gallbladder polyp, and 35 patients with gallstones were collected during the same period time. Of the 30 gallbladder adenomas, 5 were pathologically confirmed as normal mucosa, 10 were mild dysplasia, 9 were moderate dysplasia, and 6 were severe dysplasia. Among the 25 gallbladder polyps, the polyp diameter was 8–20 mm. The pathological examination confirmed that 17 polyps had normal to mild epithelial dysplasia, and 8 had moderate to severe dysplasia. Among the 35 tissues from patients with gallstones, the pathological examination confirmed that 26 had normal to mild epithelial dysplasia, and 9 had moderate to severe

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dysplasia. This study was pre-approved by The Ethics Committee for Human Research of Second Xiangya Hospital, Central South University. Immunohistochemistry staining Rabbit anti-ATM5B and rabbit anti-b2M antibodies were purchased from Abgent Company (California, USA). EnVisionTM Detection kit was purchased from Dako Laboratories (CA, USA). Routinely paraffin-embedded AC and SC/ASC tumor tissues were sectioned. 5 lM sections were deparaffinized and incubated with 3 % H2O2 for 15 min. After rinsing with phosphate buffered saline (PBS), the sections were incubated with anti-ATM5B (1:50 dilution) or anti-b2M (1:50 dilution) antibody for 2 h at room temperature. After rinsing sections with PBS for 3 times, sections were incubated with HRP-conjugated secondary antibody for 30 min. DAB substrate was then added followed by hematoxylin counter-staining, dehydration, and soaking in xylene. The negative control was made using 5 % fetal bovine serum to replace the primary antibody. The positive control was the positive sections provided by Beijing Zhongshan Biotechnology Company (Beijing, China). The percentage of positive cells was calculated from 400 cells in five random fields. Cases with positive cells C25 % were considered positive, while cases with positive cells \25 % were considered negative (Jayaraman and Jarnagin 2010; Molica et al. 1999). Statistical analysis Data was analyzed using SPSS 13.0 (the statistical package for the Social Sciences Version 13.0). The inter-relationship of ATP5B or b2M expression with histological or clinical factors was analyzed using v2 or Fisher’s exact test. Kaplan–Meier test and Cox proportional hazards model were used for univariate survival analysis and to determine the 95 % confidence interval.

Table 1 Comparison of ATP5B and b2M expression between SC/ ASC and AC Gene

SC/ASC (n = 46)

AC (n = 80)

v2

P value

0.986

0.352

4.143

0.042

8.515

0.014

4.280

0.039

2.093

0.148

0.287

0.866

0.004

0.952

0.197

0.658

Sex Male

19 (41.3)

26 (32.5)

27 (58.7)

54 (67.5)

B45

3 (6.5)

16 (20.0)

[45

43 (93.5)

64 (80.0)

Well

16 (34.8)

27 (33.8)

Moderately

24 (52.2)

25 (31.3)

6 (13.0)

28 (35.0)

Female Age (years)

Differentiation

Poorly Tumor mass size B3 cm

20 (43.5)

50 (62.5)

[3 cm

26 (56.5)

30 (37.5)

No

18 (39.1)

42 (52.5)

Yes

28 (60.9)

38 (47.5)

I?II III

12 (26.1) 20 (33.5)

21 (26.3) 38 (47.5)

IV

14 (30.4)

21 (26.3)

No

17 (37.0)

30 (37.5)

Yes

29 (63.0)

50 (62.5)

No

16 (34.8)

31 (38.8)

Yes

30 (62.5)

49 (61.3)

Radical

14 (30.4)

26 (32.5)

Palliative Biopsy

18 (39.1) 14 (30.4)

28 (35.0) 26 (32.5)

0.215

0.898

10.07 (4–25)

10.34 (3–27)

0.014

0.906

-

25 (54.3)

45 (56.2)

0.043

0.892

?

21 (45.7)

35 (43.8)

-

17 (37.0)

27 (33.7)

0.132

0.714

?

27 (58.7)

44 (55.0)

Gallstones

TNM stage

Lymph node metastasis

Invasion

Surgery

Mean survival time(month) ATP5B

Results

b2M

Clinicopathological characteristics of SC/ASC and AC and the expression of ATP5B and b2M in benign and malignant lesions of gallbladder The clinicopathological characteristics of SC/ASC and AC as well as the expression of ATP5B and b2M in SC/ASC and AC were presented in Table 1. No significant differences in TNM stage, invasion, metastasis, and surgical method were observed between SC/ASCs and ACs (P [ 0.05). The percentage of cases with tumor mass [3 cm (P = 0.039), and well- or moderately-differentiated

tumors (P = 0.014) was significantly higher in SC/ASCs than in ACs. Immunohistochemistry revealed that ATP5B and b2M positive reactions were mainly localized in the cytoplasm of SC/ASCs (Fig. 1) and ACs (Fig. 2). No significant differences in the percentage of positive ATP5B and b2M expression were observed between SC/ASC and AC patients (Table 1). The rate of ATP5B positive

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J Mol Hist Fig. 1 ATP5B and b2M expression in SC/ASC. Immunohistochemistry, magnification 9200. Positive ATP5B and b2M reaction was mainly localized in the cytoplasm. a Positive ATP5B expression in moderately differentiated SC. b Negative ATP5B expression in poorly differentiated ASC. c Positive b2M expression in poorly differentiated SC. d Negative b2M expression in moderately differentiated ASC. Bar 100 lM

Fig. 2 ATP5B and b2M expression in AC. Immunohistochemistry, magnification 9200. ATP5B and b2M positive reaction was mainly localized in the cytoplasm. a Positive ATP5B expression in well differentiated AC. b Negative ATP5B expression in moderately differentiated AC. c Positive b2M expression in moderately differentiated AC. d Negative b2M expression in well differentiated AC. Bar 100 lM

expression was significantly lower in AC and SC/ASC than that in the gallbladder adenomas, gallbladder polyps, or gallbladder epithelium with stone (P \ 0.01). In contrast, the rate of b2M expression was significantly higher in AC and SC/ASC than that in the gallbladder adenomas, gallbladder polyps, or gallbladder epithelium with stone (Table 2, P \ 0.01). The epithelium of benign gallbladder with negative ATP5B and/or positive b2M expression showed moderate to severe dysplasia, suggesting that both

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molecules could be regarded as markers to evaluate premalignant changes. The association of ATP5B and b2M expression with clinicopathological characteristics of patients with SC/ASC and AC As shown in Table 3, the percentage of positive b2M expression or negative ATP5B expression was significantly

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Fig. 3 ATP5B and b2M expression and survival in patients with SC/ ASC. a Kaplan–Meier plots of overall survival in patients with SC/ ASC and with positive and negative ATP5B expression. b Kaplan– Meier plots of overall survival in patients with SC/ASC and with positive and negative b2M expression

higher in SC/ASC cases with large tumor size, high TNM stage, invasion, and lymph node metastasis compared to the cases with small tumor size, low TNM stage, and no lymph metastasis (P \ 0.05). Positive ATP5B expression showed a tendency to be associated with differentiation of SC/ASC (P = 0.051). As shown in Table 4, the percentage of positive b2M and negative ATP5B expression in AC tumors was significantly higher in cases with poor differentiation, large tumor mass size, high TNM stage, lymph node metastasis, and invasion compared to cases with well differentiated tumor, small tumor size, low TNM stage, no lymph node metastasis, and no invasion.

Fig. 4 ATP5B and b2M expression and survival in patients with AC. a Kaplan–Meier plots of overall survival in patients with AC and with positive and negative ATP5B expression. b Kaplan–Meier plots of overall survival in patients with AC and with positive and negative b2M expression

Table 2 ATP5B and b2M expression in benign and malignant lesions of gallbladder Tissue types

Case no.

ATP5B positive (%)

b2M positive (%)

AC

80

35 (47.5)

53 (66.3) 29 (63.0)

SC/ASC

46

21 (45.6)

gallbladder adenomas

30

23 (76.8)*,#

6 (20.0)*,#

gallbladder polyp

25

21 (84.0)*,#

4 (16.0)*,#

gallbladder stone

35

31 (88.6)*,#

5 (14.3)*,#

*

#

P\0.01 versus AC;

P\0.01 versus SC/ASC

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J Mol Hist Table 3 The association of ATP5B and b2M expression with the clinicopathological characteristics of SC/ASC

CP characteristics

Total no.

b2M

ATP5B Pos no. (%)

2

v

P

2.936

0.087

Pos no. (%)

v2

P

18 (69.2)

0.983

0.322

4.331

0.115

4.954

0.026

7.668

0.023

8.912

0.003

3.920

0.048

Pathological type SC

26

9 (34.6)

ASC

20

12 (60.0)

11 (55.0)

Differentiation Well

16

9 (56.3)

Moderately

24

12 (50.0)

Poorly

6

5.947

0.051

10 (62.5) 13 (54.2)

0 (0)

6 (100.0)

Tumor size B 3 cm

20

13 (65.0)

[ 3 cm

26

8 (30.8)

TNM stage I?II

12

9 (66.7)

III

20

9 (45.0)

IV

14

3 (14.3)

5.339

0.021

9 (45.0) 20 (76.9) 4 (33.3)

7.480

0.024

13 (65.0) 2 (14.3)

Lymph node metastasis No

17

12 (70.6)

Yes

29

9 (31.0)

No

16

12 (75.0)

Yes

30

9 (30.0)

6.758

0.009

6 (35.3) 23 (79.3)

Invasion

Table 4 The association of ATP5B and b2M expression with the clinicopathological characteristics of AC

CP characteristics

Total no.

8.517

0.004

7 (43.8) 22 (73.3)

b2M

ATP5B 2

Pos no. (%)

v

P

Pos no. (%)

v2

P

13.574

0.001

12 (44.4)

12.443

0.002

6.265

0.012

11.392

0.003

8.232

0.004

10.066

0.007

Differentiation Well

27

18 (66.7)

Moderately

25

12 (48.0.0)

16 (64.0)

Poorly

28

5 (17.9)

25 (89.3)

B3 cm

50

29 (58.0)

[3 cm

30

6 (20)

I ? II

21

16 (76.2)

III

38

16 (42.1)

IV 21 Lymph node metastasis

3 (14.3)

Tumor size 11.001

0.003

28 (56.0) 25 (83.3)

TNM stage

No

30

20 (66.7)

Yes

50

15 (30.0)

No

31

20 (64.5)

Yes

49

15 (30.6)

8 (38.1) 16.424

0.000

27 (71.1) 18 (85.7)

10.243

0.002

14 (46.7) 39 (78.0)

Invasion

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6.868

0.009

14 (45.2) 39 (79.6)

J Mol Hist Table 5 Relationship between ATP5B and b2M expression and survival of SC/ASC patients C.P Characteristics

Samples (n)

Average survival (month)

Chi square

P value

25 28

13.86 (4–15) 8.00 (4–15)

25.283

0.000

-

17

12.82 (8–24)

5.904

0.015

?

29

8.69 (4–24)

5.148

0.000

48.257

0.000

SC/ASC ATP5B ? b2M

AC ATP5B -

45

7.09 (3–18)

?

35

15.43 (6–24)

-

27

17.31 (9–24)

?

53

7.44 (3–16)

b2M

C.P clinicopathological

The correlation of ATP5B or b2M expression with survival in patients with SC/ASC and AC Survival information was collected from all patients for 2 years. 33 SC/ASC and 57 AC patients survived \1 year.

13 SC/ASC patients survived C1 year (4 cases survived [2 years) with an average survival time of 10.07 ± 0.78 months. 23 AC patients survived C1 year (9 cases survived [2 years) with an average survival time of 10.34 ± 0.63 months. No significant difference in total survival time between SC/ASC and AC patients was observed. The Kaplan–Meier survival analysis in SC/ASC (Table 5; Fig. 3) and AC (Table 5; Fig. 4) patients revealed that the average survival time of patients with b2M positive expression or negative ATP5B expression was significantly shorter than patients having negative b2M and positive ATP5B expression. The differentiation, tumor size, T stage, N stage, lymph node metastasis, and invasion were also significantly associated with average survival time (P \ 0.001) (Data not shown). Cox multivariate analysis of SC/ASC patients’ survival showed that the differentiation, tumor size (C3 cm), TNM stage, invasion, lymph node metastasis and negative ATP5B expression were negatively correlated with overall survival (Table 6). ATP5B-negative expression is an independent risk factor for SC/ASC patients (Table 6). Cox multivariate analysis of AC patients revealed that differentiation, tumor size (C3 cm), TNM stage, invasion, lymph node metastasis, positive b2M positive and negative ATP5B expression were negatively correlated with overall survival (Table 6). b2M-positive and ATP5B-negative

Table 6 Multivariate Cox regression analysis of survival rate in SC/ASC and AC patients Groups

Factors

RC

SE

wald

P

RR

95 % confidence interval Lower

Upper

SC/ASC Pathological types

SC/ASC

0.365

0.327

1.246

0.264

1.441

0.759

2.734

Differentiation

Well/moderately/ poorly

0.861

0.449

3.677

0.055

2.366

0.981

5.703

Tumor mass size

B3 cm/[ 3 cm

2.127

0.782

7.398

0.007

8.390

1.812

38.850

TNM stage

I?II/III/IV

1.393

0.498

7.824

0.005

4.027

1.517

10.688

Lymph metastasis Invasion

No/Yes No/Yes

1.795 2.364

0.611 0.812

8.631 8.476

0.003 0.004

6.019 10.633

1.817 2.165

19.937 52.223

ATP5B

-/?

-2.466

0.832

8.785

0.003

0.085

0.017

0.434

b2M

-/?

0.935

0.495

3.568

0.059

2.547

0.965

6.721

Differentiation

Well/moderately/poorly

1.144

0.465

6.053

0.014

3.139

1.262

7.810

Tumor mass size

B3 cm/[ 3 cm

1.051

0.417

6.352

0.012

2.861

1.263

6.477

TNM stage

I?II/III/IV

1.238

0.453

7.469

0.006

3.449

1.419

8.380

Lymph metastasis

No/Yes

1.132

0.447

6.413

0.011

3.102

1.292

7.449

Invasion

No/Yes

1.434

0.516

7.723

0.005

4.195

1.526

11.535

ATP5B

-/?

-2.966

0.832

12.709

0.000

0.052

0.010

0.263

b2M

-/?

3.235

0.893

13.123

0.000

25.406

4.414

146.245

AC

RC Regression coefficients, SE standard error, RR relative risk

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expression are independent risk factors for AC patients (Table 6).

Discussion Current understanding of the clinical and pathological characteristics of SC/ASC is mainly based on the analysis of individual cases or small case series. However, this study collected 46 SC/ASC samples, which is a relatively large sample size due to its very low incidence. With this case series, we have addressed the differences in the clinical and pathological characteristics between SC/ASC and ordinary AC tumors (Liu and Yang 2013). Highly similar features were observed between these two subtype tumors although SC/ASC patients had large tumor size. Interestingly, these two GBC subtypes also exhibited similar biological characteristics. However, identification of a biological marker to distinguish between these two subtypes of GBC is important for accurate diagnosis. In this study, we analyzed the protein expression of ATP5B and b2M in 80 AC and 46 SC/ASC by immunohistochemistry. Results revealed that positive b2M expression was observed in 55 % of AC and 58.7 % of SC/ ASC patients, while negative ATP5B expression was observed in 55.2 % of AC and 54.3 % of SC/ASC patients. Positive b2M expression and loss of ATP5B expression are closely related to the metastasis, invasion and poor-prognosis of gallbladder cancer. Besides the essential role of the mitochondria in the production of biological energy, it also exerts a crucial role in the execution of cell death (Wang 2001). H?-ATP synthase is a key player in mitochondria’s function in cell death (Santamaria et al. 2006). H?-ATP synthase exerts its role through the generation of reactive oxygen species (ROS), which in turn promote severe oxidative damage to cellular and mitochondrial proteins (Harris et al. 2000). High expression of ATP5B has been demonstrated to increase cell death in response to cytotoxic agents, whereas abrogated mitochondrial activity contributes to a diminished potential for ROS signaling and resistance to cytotoxic agents (Sanchez-Arago et al. 2010). Downregulation of ATP5B, the catalytic subunit of H?-ATP synthase, is commonly observed in most human carcinomas (Willers and Cuezva 2011). Loss of ATP5B expression was significantly associated with large tumor size, high TNM stage, invasion, and lymph node metastasis in both ACs and SC/ASCs. Importantly, negative ATP5B expression significantly correlated with shorter survival and is an independent risk factor for both AC and SC/ASC patients. As a light chain of the MHC 1 molecule, it is easy to understand the role of b2M in immunity. However, high b2M level has been observed to promote tumorigenesis and metastasis as an oncogene (Jiang et al. 2012). This may be

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associated with b2M’s ability to stimulate the proliferation and apoptosis of cancers cells (Jiang et al. 2012). The role of b2M in promoting tumor cell growth is related to the activation of PKA and enhancement of tumor angiogenesis (Nomura et al. 2006). In contrast, anti-b2M monoclonal antibody has been revealed to induce cell apoptosis in hematological malignant cells (Yang et al. 2006). b2M has also been revealed to be a biomarker in a variety of tumors. Elevated level of circulating b2M or high expression in tumor tissues has been demonstrated to be an important predictive factor of poor prognosis in cancer patients. In this study, positive b2M expression was observed in 55 % of AC and 58.7 % of SC/ASC tissues. Positive b2M expression correlated with large tumor mass size, high TNM stage, lymph node metastasis, and invasion in both AC and SC/ASC. However, positive b2M expression was significantly associated with poor differentiation in AC, but not in SC/ASC. Positive b2M expression significantly correlated with shorter survival in both AC and SC/ASC patients. However, b2M is an independent risk factor for AC, but not for SC/ASC patients. In conclusion, positive b2M expression and loss of ATP5B expression in tumor tissues are associated with the aggressive characteristics and poor prognosis of AC and SC/ASC. Also, these two molecules may be used as targets for gene therapy of AC and SC/ASC. Conflict of interest

All authors declared no conflict of interest.

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ATP5b and β2-microglobulin are predictive markers for the prognosis of patients with gallbladder cancer.

The differences in clinical, pathological, and biological characteristics between adenocarcinoma (AC) and squamous cell/adenosquamous carcinoma (SC/AS...
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