Tumor Biol. DOI 10.1007/s13277-015-3155-2
RESEARCH ARTICLE
Prognostic value of purinergic P2X7 receptor expression in patients with hepatocellular carcinoma after curative resection Haiou Liu & Weisi Liu & Zheng Liu & Yidong Liu & Weijuan Zhang & Le Xu & Jiejie Xu
Received: 7 October 2014 / Accepted: 26 January 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract The family of type 2 purinergic (P2) receptors, especially P2X7, is responsible for the direct tumor-killing functions of extracellular adenosine triphosphate (ATP), but the precise role of P2X7 in the progression of hepatocellular carcinoma (HCC) remains elusive. This study aims to evaluate prognostic value of P2X7 expression in HCC patients after surgical resection. Expression of P2X7 was assessed by immunohistochemistry in tissue microarrays containing paired tumor and peritumoral liver tissues from 273 patients with HCC who had undergone hepatectomy between 2006 and 2007. Prognostic value of P2X7 expression and clinical outcomes were evaluated. Peritumoral P2X7 expression was significantly higher than intratumoral P2X7 expression. No significant prognostic difference was observed for overall survival for intratumoral P2X7 density, whereas peritumoral P2X7 density indicates unfavorable overall survival in training set and BCLC stage 0-A subset. Besides, peritumoral P2X7 density, which correlated with tumor size, venous invasion, and
Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3155-2) contains supplementary material, which is available to authorized users. H. Liu : W. Liu : Z. Liu : Y. Liu : J. Xu (*) Key Laboratory of Glycoconjugate Research, MOH, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Mailbox 103, 138 Yixueyuan Road, Shanghai 200032, China e-mail: [email protected] W. Zhang Department of Immunology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China L. Xu Department of Urology, Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai 200032, China
BCLC stage, was identified as an independent poor prognosticator for overall survival and recurrence-free survival. The association was further validated in validation set. Peritumoral P2X7 is a potential unfavorable prognosticator for overall survival and recurrence free survival in HCC patients after surgical resection. Further external validation and functional analysis should be pursued to evaluate its potential prognostic value and therapeutic significance for HCC patients. Keywords Hepatocellular carcinoma . Purinergic P2X7 receptor . Prognosis biomarker . Overall survival . Recurrence free survival
Introduction Hepatocellular carcinoma (HCC) is one of the most common solid malignancies and represents the third leading cause of cancer-related mortality worldwide [1]. Despite available curative treatment options (e.g., liver resection and transplantation) for newly diagnosed HCC patients, 15–20 % of early stage tumors still present with dismal outcome as a result of a high rate of recurrence [2]. HCC poses a major challenge as the extreme variability of the clinical outcome, which makes it difficult to properly stage the disease and thereby estimate the prognosis. The biggest challenge is relative crude classification of early stage patients and inability to reflect the prognosis of underlying liver disease using the current stage system, such as Barcelona Clinic Liver Cancer stage (BCLC). This considerable heterogeneity of HCC presents at the molecular level and has a genetic predisposition to it. Therefore, molecular approaches for stratifying patients with HCC, through incorporation of molecular information into BCLC stage system, will improve current prognostic stratification and provide important clinical relevant insights into predicting which
Tumor Biol.
patients are prone to develop recurrence and mortality after surgery. Inflammation, sustained cycles of necrosis and regeneration, and oxidative stress are characteristic of HBV-, HCV-, and alcohol-induced hepatocarcinogenesis, suggesting that these processes contribute in fundamental ways to HCC development [3]. Exactly how hepatocyte necrosis promotes HCC development is not known, but it has proposed that necrotic hepatocytes release factors, the so-called damage-associated molecular patterns (DAMPs), such as adenosine 5′-triphosphate (ATP), leading to a Bsterile^ inflammatory response [4, 5]. Sterile inflammation is a key process in drug-induced liver injury, a major determinant of carcinogenesis [6]. Extracellular ATP signals via the family of P2 receptors which contain seven P2X and eight P2Y receptors, and among them, P2X7 is unique because it results in activation of the inflammasome and production of interleukin (IL)-1β [7, 8]. Activation of P2X7 could significantly augment apoptosis in cancer epithelial cells, resulting in a significant delay in tumor progression [9]. However, Elena et al. found that overexpression P2X7 in embryonic kidney cells exhibited a more tumorigenic and anaplastic phenotype compared with control cells [10]. Differences in P2X7 receptor expression in cancer versus normal epithelial cells have been documented [11]. The distribution of P2X7 in the HCC tissue and its prognostic value remains to be elucidated. Based on these findings, we hypothesized that decreased expression of P2X7 and the abrogated P2X7-mediated apoptosis predispose proliferating liver cells to the effects of carcinogens and may lead to the development of HCC. Furthermore, we also intended to investigate the distribution of P2X7 by comparing the density of P2X7 expressed in peritumoral liver tissue.
Materials and methods Clinical specimens Two independent sets comprising 273 consecutive patients who underwent curative hepatectomy for HCC at Huashan Hospital of Fudan University (Shanghai, China) were enrolled in the study. Specimens of training cohort (n=150) were obtained in 2006, and specimens of validation set (n=123) were obtained in 2007. All cases were conformed to the following criteria: diagnosed by postoperative histopathology, complete follow-up data available, no extrahepatic metastasis, no other malignant diseases, and no preoperative anticancer therapy. Tumor differentiation was assessed according to the Edmonson and Steiner grading system. Data were censored at last follow-up for patients without recurrence or death. Overall survival (OS) and recurrence-free survival (RFS) were calculated from the date of surgery to the date of death or recurrence, respectively. None of these patients died from operative complications or from other factors. The last follow-up
was August 2012. HCC tissues and corresponding peritumoral tissue samples were obtained from 30 patients who underwent hepatectomy in Huashan Hospital of Fudan University between September 2013 and December 2013. Following surgical removal, the tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction. All subjects selected were required to provide written informed consent on the use of clinical specimens for Table 1 The clinicopathological characteristics of patients in the training and validation cohorts Characteristics
Training cohort N=150
Age (years) Gender Male Female Liver cirrhosis Absent Present Child-Pugh A B HBsAg Negative Positive ALT (U/I) ≤40 >40 AFP (ng/ml) ≤20 >20 Tumor size (cm)
Validation cohort %
51.0±11.71
N=123
P
%
49.0±11.07
130 20
86.7 13.3
103 20
83.7 16.3
0.496
15 135
10.0 90.0
14 109
11.4 88.6
0.712
144 6
96.0 4.0
116 7
94.3 5.7
0.514
21 129
14.0 86.0
16 107
13.0 87.0
0.812
69 81
46.0 54.0
54 69
43.9 56.1
0.729
47 103
31.3 68.7
33 90
26.8 73.2
0.416
≤5 83 >5 67 Tumor number Single 123 Multiple 27 Tumor encapsulation Complete 79 None 71 Tumor differentiation I–II 105 III–IV 45 Venous invasion Absent 77 Present 73 BCLC 0+A 81 B+C 69
55.3 44.7
64 59
52.0 48.0
0.586
82.0 18.0
111 12
90.2 9.8
0.053
52.7 47.3
65 58
52.8 47.2
0.977
70.0 30.0
107 16
87.0 13.0
0.001
51.3 48.7
68 55
55.3 44.7
0.515
54.0 46.0
62 61
50.4 49.6
0.554
Italicized values are statistically significant (P20 47 Tumor size (cm) ≤5 31 >5 42 Tumor number Single 61 Multiple 12 Tumor encapsulation Complete 34 None 39 Tumor differentiation I–II 51 III–IV 22 Venous invasion Absent 29 Present 44 BCLC 0+A 31 B+C 42
GAPDH sense 5′-ACAACAG CCTCAAGATCATCAG-3′, reverse 5′-GGTCCACCACTGACACGTTG-3′; P2X7 sense, 5′-ACTCCAGTAACTGCTGTCGC-3′, reverse 5′-CTCCAC AATGGACTCGCACT-3′. Tissue microarray and immunohistochemistry Tissue microarrays were constructed as previously described [12]. Briefly, two cores were taken from each representative tumor tissue and from liver tissue
Validation cohort P
High 65
58
51.8±11.2
0.542
49.5±11.7
50.1±10.5
0.794
67 10
0.911
55 10
48 10
0.973
8 69
0.913
6 59
8 50
0.609
76 1
0.188
61 4
55 3
0.877
12 65
0.735
7 58
9 49
0.608
38 39
0.495
30 35
24 34
0.594
21 56
0.355
16 49
17 41
0.702
52 25
0.004
25 40
39 19
0.003
62 15
0.786
55 10
56 2
0.055
45 32
0.197
32 33
33 25
0.503
54 23
0.887
55 10
52 6
0.575
48 29
0.009
20 45
48 10
RESEARCH ARTICLE
Prognostic value of purinergic P2X7 receptor expression in patients with hepatocellular carcinoma after curative resection Haiou Liu & Weisi Liu & Zheng Liu & Yidong Liu & Weijuan Zhang & Le Xu & Jiejie Xu
Received: 7 October 2014 / Accepted: 26 January 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract The family of type 2 purinergic (P2) receptors, especially P2X7, is responsible for the direct tumor-killing functions of extracellular adenosine triphosphate (ATP), but the precise role of P2X7 in the progression of hepatocellular carcinoma (HCC) remains elusive. This study aims to evaluate prognostic value of P2X7 expression in HCC patients after surgical resection. Expression of P2X7 was assessed by immunohistochemistry in tissue microarrays containing paired tumor and peritumoral liver tissues from 273 patients with HCC who had undergone hepatectomy between 2006 and 2007. Prognostic value of P2X7 expression and clinical outcomes were evaluated. Peritumoral P2X7 expression was significantly higher than intratumoral P2X7 expression. No significant prognostic difference was observed for overall survival for intratumoral P2X7 density, whereas peritumoral P2X7 density indicates unfavorable overall survival in training set and BCLC stage 0-A subset. Besides, peritumoral P2X7 density, which correlated with tumor size, venous invasion, and
Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-3155-2) contains supplementary material, which is available to authorized users. H. Liu : W. Liu : Z. Liu : Y. Liu : J. Xu (*) Key Laboratory of Glycoconjugate Research, MOH, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Mailbox 103, 138 Yixueyuan Road, Shanghai 200032, China e-mail: [email protected] W. Zhang Department of Immunology, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China L. Xu Department of Urology, Zhongshan Hospital, Shanghai Medical College of Fudan University, Shanghai 200032, China
BCLC stage, was identified as an independent poor prognosticator for overall survival and recurrence-free survival. The association was further validated in validation set. Peritumoral P2X7 is a potential unfavorable prognosticator for overall survival and recurrence free survival in HCC patients after surgical resection. Further external validation and functional analysis should be pursued to evaluate its potential prognostic value and therapeutic significance for HCC patients. Keywords Hepatocellular carcinoma . Purinergic P2X7 receptor . Prognosis biomarker . Overall survival . Recurrence free survival
Introduction Hepatocellular carcinoma (HCC) is one of the most common solid malignancies and represents the third leading cause of cancer-related mortality worldwide [1]. Despite available curative treatment options (e.g., liver resection and transplantation) for newly diagnosed HCC patients, 15–20 % of early stage tumors still present with dismal outcome as a result of a high rate of recurrence [2]. HCC poses a major challenge as the extreme variability of the clinical outcome, which makes it difficult to properly stage the disease and thereby estimate the prognosis. The biggest challenge is relative crude classification of early stage patients and inability to reflect the prognosis of underlying liver disease using the current stage system, such as Barcelona Clinic Liver Cancer stage (BCLC). This considerable heterogeneity of HCC presents at the molecular level and has a genetic predisposition to it. Therefore, molecular approaches for stratifying patients with HCC, through incorporation of molecular information into BCLC stage system, will improve current prognostic stratification and provide important clinical relevant insights into predicting which
Tumor Biol.
patients are prone to develop recurrence and mortality after surgery. Inflammation, sustained cycles of necrosis and regeneration, and oxidative stress are characteristic of HBV-, HCV-, and alcohol-induced hepatocarcinogenesis, suggesting that these processes contribute in fundamental ways to HCC development [3]. Exactly how hepatocyte necrosis promotes HCC development is not known, but it has proposed that necrotic hepatocytes release factors, the so-called damage-associated molecular patterns (DAMPs), such as adenosine 5′-triphosphate (ATP), leading to a Bsterile^ inflammatory response [4, 5]. Sterile inflammation is a key process in drug-induced liver injury, a major determinant of carcinogenesis [6]. Extracellular ATP signals via the family of P2 receptors which contain seven P2X and eight P2Y receptors, and among them, P2X7 is unique because it results in activation of the inflammasome and production of interleukin (IL)-1β [7, 8]. Activation of P2X7 could significantly augment apoptosis in cancer epithelial cells, resulting in a significant delay in tumor progression [9]. However, Elena et al. found that overexpression P2X7 in embryonic kidney cells exhibited a more tumorigenic and anaplastic phenotype compared with control cells [10]. Differences in P2X7 receptor expression in cancer versus normal epithelial cells have been documented [11]. The distribution of P2X7 in the HCC tissue and its prognostic value remains to be elucidated. Based on these findings, we hypothesized that decreased expression of P2X7 and the abrogated P2X7-mediated apoptosis predispose proliferating liver cells to the effects of carcinogens and may lead to the development of HCC. Furthermore, we also intended to investigate the distribution of P2X7 by comparing the density of P2X7 expressed in peritumoral liver tissue.
Materials and methods Clinical specimens Two independent sets comprising 273 consecutive patients who underwent curative hepatectomy for HCC at Huashan Hospital of Fudan University (Shanghai, China) were enrolled in the study. Specimens of training cohort (n=150) were obtained in 2006, and specimens of validation set (n=123) were obtained in 2007. All cases were conformed to the following criteria: diagnosed by postoperative histopathology, complete follow-up data available, no extrahepatic metastasis, no other malignant diseases, and no preoperative anticancer therapy. Tumor differentiation was assessed according to the Edmonson and Steiner grading system. Data were censored at last follow-up for patients without recurrence or death. Overall survival (OS) and recurrence-free survival (RFS) were calculated from the date of surgery to the date of death or recurrence, respectively. None of these patients died from operative complications or from other factors. The last follow-up
was August 2012. HCC tissues and corresponding peritumoral tissue samples were obtained from 30 patients who underwent hepatectomy in Huashan Hospital of Fudan University between September 2013 and December 2013. Following surgical removal, the tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction. All subjects selected were required to provide written informed consent on the use of clinical specimens for Table 1 The clinicopathological characteristics of patients in the training and validation cohorts Characteristics
Training cohort N=150
Age (years) Gender Male Female Liver cirrhosis Absent Present Child-Pugh A B HBsAg Negative Positive ALT (U/I) ≤40 >40 AFP (ng/ml) ≤20 >20 Tumor size (cm)
Validation cohort %
51.0±11.71
N=123
P
%
49.0±11.07
130 20
86.7 13.3
103 20
83.7 16.3
0.496
15 135
10.0 90.0
14 109
11.4 88.6
0.712
144 6
96.0 4.0
116 7
94.3 5.7
0.514
21 129
14.0 86.0
16 107
13.0 87.0
0.812
69 81
46.0 54.0
54 69
43.9 56.1
0.729
47 103
31.3 68.7
33 90
26.8 73.2
0.416
≤5 83 >5 67 Tumor number Single 123 Multiple 27 Tumor encapsulation Complete 79 None 71 Tumor differentiation I–II 105 III–IV 45 Venous invasion Absent 77 Present 73 BCLC 0+A 81 B+C 69
55.3 44.7
64 59
52.0 48.0
0.586
82.0 18.0
111 12
90.2 9.8
0.053
52.7 47.3
65 58
52.8 47.2
0.977
70.0 30.0
107 16
87.0 13.0
0.001
51.3 48.7
68 55
55.3 44.7
0.515
54.0 46.0
62 61
50.4 49.6
0.554
Italicized values are statistically significant (P20 47 Tumor size (cm) ≤5 31 >5 42 Tumor number Single 61 Multiple 12 Tumor encapsulation Complete 34 None 39 Tumor differentiation I–II 51 III–IV 22 Venous invasion Absent 29 Present 44 BCLC 0+A 31 B+C 42
GAPDH sense 5′-ACAACAG CCTCAAGATCATCAG-3′, reverse 5′-GGTCCACCACTGACACGTTG-3′; P2X7 sense, 5′-ACTCCAGTAACTGCTGTCGC-3′, reverse 5′-CTCCAC AATGGACTCGCACT-3′. Tissue microarray and immunohistochemistry Tissue microarrays were constructed as previously described [12]. Briefly, two cores were taken from each representative tumor tissue and from liver tissue
Validation cohort P
High 65
58
51.8±11.2
0.542
49.5±11.7
50.1±10.5
0.794
67 10
0.911
55 10
48 10
0.973
8 69
0.913
6 59
8 50
0.609
76 1
0.188
61 4
55 3
0.877
12 65
0.735
7 58
9 49
0.608
38 39
0.495
30 35
24 34
0.594
21 56
0.355
16 49
17 41
0.702
52 25
0.004
25 40
39 19
0.003
62 15
0.786
55 10
56 2
0.055
45 32
0.197
32 33
33 25
0.503
54 23
0.887
55 10
52 6
0.575
48 29
0.009
20 45
48 10
