Histopathology 2015, 66, 706–714. DOI: 10.1111/his.12584
Loss of CD155 expression predicts poor prognosis in hepatocellular carcinoma Ping Qu, Xiaojun Huang, Xingchun Zhou, Zhuomin L€ u,1 Fang’e Liu, Zhiyong Shi,2 Lin L€ u,3 Yanqiu Wu4 & Yibing Chen State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, 1Department of Pain Management, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, 2Department of Radioactive Intervention, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, 3Department of Oncology, Guangzhou First People’s Hospital, Guangzhou, Guangdong, and 4Department of Burns and Plastic Surgery, the First Affiliated Hospital of the General Hospital of PLA, Beijing, China Date of submission 2 April 2014 Accepted for publication 10 October 2014 Published online Article Accepted 15 October 2014
Qu P, Huang X, Zhou X, L€ u Z, Liu F, Shi Z, L€ u L, Wu Y & Chen Y (2015) Histopathology 66, 706–714. DOI: 10.1111/his.12584
Loss of CD155 expression predicts poor prognosis in hepatocellular carcinoma Aims: CD155 is an important ligand in triggering tumour rejection by immune cells. However, the expression of CD155 and its clinical significance in hepatocellular carcinoma (HCC) remains unknown. Methods and results: We examined the expression level of CD155 in 174 HCC tissue samples by immunohistochemical staining and in HCC cell lines by flow cytometry; 63.8% (111 of 174) of HCC tissue samples showed negative CD155 expression. When compared with adjacent peritumour tissues, HCC tissues exhibited a significantly lower expression of CD155 (P < 0.001). Flow cytometry analysis indicated that HCC cell lines had low levels of CD155 expression. Moreover, negative CD155 expression
was associated significantly with higher serum a-fetoprotein level (P = 0.016) and a higher incidence of portal vein tumour thrombus (P = 0.050). Importantly, patients with positive CD155 expression had better overall survival after surgery than those with negative CD155 expression (P = 0.005). Furthermore, Cox regression analyses showed that CD155 expression was an independent prognostic factor for HCC (P = 0.049). Conclusions: Our findings suggest that loss of CD155 expression may play an important role in the immune escape of HCC cells and thus CD155 may serve as a prognostic marker as well as a potential therapeutic target for HCC.
Keywords: CD155, hepatocellular carcinoma, immunohistochemistry, prognosis
Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies and the third leading cause of Address for correspondence: Y Chen MD, PhD, State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, Fourth Military Medical University, 169 West Changle Road, Xi’an 710032, China. e-mail: [email protected] and Y Wu MD, PhD, Department of Burns and Plastic Surgery, the First Affiliated Hospital of the General Hospital of PLA, 51 Fucheng Road, Beijing 100048, China. e-mail: [email protected] Ping Qu and Xiaojun Huang contributed equally to this work. © 2014 John Wiley & Sons Ltd.
cancer-related death worldwide.1 Although hepatectomy is one of the best methods to eliminate tumour burden, the high postoperative recurrence rate remains the major barrier against long survival of HCC patients.2 Although some clinicopathological features, including tumour size, vascular invasion, a-fetoprotein (AFP) level and histopathological grade, have been demonstrated to be effective prognosis indicators for surgical HCC patients, the majority of molecular biomarkers for prediction of HCC prognosis have not been well documented. Therefore, the identification of novel prognostic biomarkers for the improvement of treatment decision-making in HCC is urgently needed.
CD155 expression and HCC prognosis
Cancer immune surveillance is considered to be an important protective mechanism by which the host can maintain cellular homeostasis and thus inhibit carcinogenesis.3 The central roles of immune effector cells such as B, T natural killer (NK) and natural killer T (NK T) cells have since been clarified in cancer immune surveillance. Among these, NK cells are important cytolytic and cytokine-producing effector cells in the innate immune system and possess the ability to lyse tumour cells without the need for presentation of tumour-specific antigens. Accumulation of intratumoral NK cells in solid tumours has been found to be associated with the improved survival rate of patients with cancers, including colorectal cancer,4 gastric cancer,5 squamous cell lung cancer6 and HCC.7 The function of NK cells is regulated by a series of surface receptors which are capable of transducing either inhibitory or activating signals.8,9 The inhibitory receptors that lead to NK cell inactivation represent the major fail-safe mechanism to prevent the killing of normal major histocompatibility complex (MHC) class I autologous cells.10 In the absence of efficient inhibitory interactions, target cells are susceptible to NK-mediated killing. The killing of target cells also depends upon the engagement of specific activating receptors and co-receptors expressed on the NK cell surface, together with surface ligands expressed on target cells. NKp46, NKp30 and NKp44 molecules, collectively termed natural cytotoxicity receptors (NCRs), as well as NKG2D, appear to play a predominant role in the process of NK-mediated killing of most target cells.11 Another surface molecule that has been shown to participate in the induction phase of NK cell activation is DNAX accessory molecule-1 [DNAM-1 (CD226)], which is expressed by all human NK cells, T cells and monocytes.12 DNAM-1 binds to CD155 (Necl-5 or PVR) or CD112 (nectin-2), both of which have been found to be up-regulated on several types of tumour cells. In-vitro studies have shown that DNAM1 triggers the NK cell-mediated killing of a range of tumour cells expressing CD155 and CD112.13–17 In addition, the DNAM-1–CD155 interaction is essential for NK cell-mediated killing of tumour cells and CD155 is a key ligand recognised by DNAM-1 in NK cell-mediated suppression of metastases.18 It has been demonstrated that the low expression levels of NK cell activation ligands such as major histocompatibilty complex (MHC) class I polypeptiderelated sequence A (MICA), MHC class I polypeptiderelated sequence B (MICB) and UL16 binding protein 1 (ULBP1) on tumour cells are associated with the progression of cancer and poor prognosis of patients.19,20 Moreover, previous studies have reported the expression © 2014 John Wiley & Sons Ltd, Histopathology, 66, 706–714.
707
of CD155 in several types of malignant cells, including lung adenocarcinoma, melanoma, soft tissue sarcoma and colorectal cancer.21–24 However, the expression of CD155 and its clinical significance in HCC cells have not been investigated. In this study, we evaluated the expression of CD155 using immunohistochemistry and determined its association with clinicopathological parameters of HCC patients. In addition, we explored the potential prognostic value of CD155 for the postresection survival of HCC patients.
Materials and methods STUDY POPULATION
A total of 174 HCC patients were enrolled from January 2009 to December 2011 at the Eastern Hepatobiliary Surgery Hospital, Second Military Medical University in Shanghai, China. Patients who met all the following criteria of eligibility were included into our study: (i) diagnosis of primary HCC identified by histopathological examination; (ii) treatment with radical resection; (iii) availability of complete followup data; (iv) no pre-operative anticancer treatment, such as chemotherapy and radiotherapy; (v) no history of familial malignancy or other synchronous malignancy; and (vi) no death within 3 months after operation. The histopathological type and grade were determined using the criteria of the World Health Organization (WHO) classification. All patients were staged according to the seventh-edition tumour– node–metastasis (TNM) staging system of the Union for International Cancer Control (UICC) and American Joint Committee on Cancer (AJCC). Overall survival (OS) was defined as the time from operation to HCC-specific death or last follow-up, whichever come first. Patients dying of other causes were censored for survival analysis. Relapse-free survival (RFS) is defined as the time elapsed from operation to the date of the recurrence or distant metastasis of HCC. In addition, another 10 cases diagnosed with dysplasia were enrolled, including six males and four females with a median age of 57 years (range 41–65 years). The study was approved by the Ethical Committee of Second Military Medical University, and written informed consent was obtained from each patient. All study procedures were carried out in accordance with the ethical standards of the Helsinki Declaration. HCC CELL LINES
HCC cell lines SK-HEP-1, MHCC-97HH, MHCC-97L, BEL-7402 and HepG2 were obtained from the
P-value
0.007
0.518 (0.284–0.945)
0.607 (0.315–1.167)
2.859 (1.678–4.870)
Loss of CD155 expression predicts poor prognosis in hepatocellular carcinoma Ping Qu, Xiaojun Huang, Xingchun Zhou, Zhuomin L€ u,1 Fang’e Liu, Zhiyong Shi,2 Lin L€ u,3 Yanqiu Wu4 & Yibing Chen State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, 1Department of Pain Management, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, 2Department of Radioactive Intervention, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, 3Department of Oncology, Guangzhou First People’s Hospital, Guangzhou, Guangdong, and 4Department of Burns and Plastic Surgery, the First Affiliated Hospital of the General Hospital of PLA, Beijing, China Date of submission 2 April 2014 Accepted for publication 10 October 2014 Published online Article Accepted 15 October 2014
Qu P, Huang X, Zhou X, L€ u Z, Liu F, Shi Z, L€ u L, Wu Y & Chen Y (2015) Histopathology 66, 706–714. DOI: 10.1111/his.12584
Loss of CD155 expression predicts poor prognosis in hepatocellular carcinoma Aims: CD155 is an important ligand in triggering tumour rejection by immune cells. However, the expression of CD155 and its clinical significance in hepatocellular carcinoma (HCC) remains unknown. Methods and results: We examined the expression level of CD155 in 174 HCC tissue samples by immunohistochemical staining and in HCC cell lines by flow cytometry; 63.8% (111 of 174) of HCC tissue samples showed negative CD155 expression. When compared with adjacent peritumour tissues, HCC tissues exhibited a significantly lower expression of CD155 (P < 0.001). Flow cytometry analysis indicated that HCC cell lines had low levels of CD155 expression. Moreover, negative CD155 expression
was associated significantly with higher serum a-fetoprotein level (P = 0.016) and a higher incidence of portal vein tumour thrombus (P = 0.050). Importantly, patients with positive CD155 expression had better overall survival after surgery than those with negative CD155 expression (P = 0.005). Furthermore, Cox regression analyses showed that CD155 expression was an independent prognostic factor for HCC (P = 0.049). Conclusions: Our findings suggest that loss of CD155 expression may play an important role in the immune escape of HCC cells and thus CD155 may serve as a prognostic marker as well as a potential therapeutic target for HCC.
Keywords: CD155, hepatocellular carcinoma, immunohistochemistry, prognosis
Introduction Hepatocellular carcinoma (HCC) is one of the most common malignancies and the third leading cause of Address for correspondence: Y Chen MD, PhD, State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic Medicine, Fourth Military Medical University, 169 West Changle Road, Xi’an 710032, China. e-mail: [email protected] and Y Wu MD, PhD, Department of Burns and Plastic Surgery, the First Affiliated Hospital of the General Hospital of PLA, 51 Fucheng Road, Beijing 100048, China. e-mail: [email protected] Ping Qu and Xiaojun Huang contributed equally to this work. © 2014 John Wiley & Sons Ltd.
cancer-related death worldwide.1 Although hepatectomy is one of the best methods to eliminate tumour burden, the high postoperative recurrence rate remains the major barrier against long survival of HCC patients.2 Although some clinicopathological features, including tumour size, vascular invasion, a-fetoprotein (AFP) level and histopathological grade, have been demonstrated to be effective prognosis indicators for surgical HCC patients, the majority of molecular biomarkers for prediction of HCC prognosis have not been well documented. Therefore, the identification of novel prognostic biomarkers for the improvement of treatment decision-making in HCC is urgently needed.
CD155 expression and HCC prognosis
Cancer immune surveillance is considered to be an important protective mechanism by which the host can maintain cellular homeostasis and thus inhibit carcinogenesis.3 The central roles of immune effector cells such as B, T natural killer (NK) and natural killer T (NK T) cells have since been clarified in cancer immune surveillance. Among these, NK cells are important cytolytic and cytokine-producing effector cells in the innate immune system and possess the ability to lyse tumour cells without the need for presentation of tumour-specific antigens. Accumulation of intratumoral NK cells in solid tumours has been found to be associated with the improved survival rate of patients with cancers, including colorectal cancer,4 gastric cancer,5 squamous cell lung cancer6 and HCC.7 The function of NK cells is regulated by a series of surface receptors which are capable of transducing either inhibitory or activating signals.8,9 The inhibitory receptors that lead to NK cell inactivation represent the major fail-safe mechanism to prevent the killing of normal major histocompatibility complex (MHC) class I autologous cells.10 In the absence of efficient inhibitory interactions, target cells are susceptible to NK-mediated killing. The killing of target cells also depends upon the engagement of specific activating receptors and co-receptors expressed on the NK cell surface, together with surface ligands expressed on target cells. NKp46, NKp30 and NKp44 molecules, collectively termed natural cytotoxicity receptors (NCRs), as well as NKG2D, appear to play a predominant role in the process of NK-mediated killing of most target cells.11 Another surface molecule that has been shown to participate in the induction phase of NK cell activation is DNAX accessory molecule-1 [DNAM-1 (CD226)], which is expressed by all human NK cells, T cells and monocytes.12 DNAM-1 binds to CD155 (Necl-5 or PVR) or CD112 (nectin-2), both of which have been found to be up-regulated on several types of tumour cells. In-vitro studies have shown that DNAM1 triggers the NK cell-mediated killing of a range of tumour cells expressing CD155 and CD112.13–17 In addition, the DNAM-1–CD155 interaction is essential for NK cell-mediated killing of tumour cells and CD155 is a key ligand recognised by DNAM-1 in NK cell-mediated suppression of metastases.18 It has been demonstrated that the low expression levels of NK cell activation ligands such as major histocompatibilty complex (MHC) class I polypeptiderelated sequence A (MICA), MHC class I polypeptiderelated sequence B (MICB) and UL16 binding protein 1 (ULBP1) on tumour cells are associated with the progression of cancer and poor prognosis of patients.19,20 Moreover, previous studies have reported the expression © 2014 John Wiley & Sons Ltd, Histopathology, 66, 706–714.
707
of CD155 in several types of malignant cells, including lung adenocarcinoma, melanoma, soft tissue sarcoma and colorectal cancer.21–24 However, the expression of CD155 and its clinical significance in HCC cells have not been investigated. In this study, we evaluated the expression of CD155 using immunohistochemistry and determined its association with clinicopathological parameters of HCC patients. In addition, we explored the potential prognostic value of CD155 for the postresection survival of HCC patients.
Materials and methods STUDY POPULATION
A total of 174 HCC patients were enrolled from January 2009 to December 2011 at the Eastern Hepatobiliary Surgery Hospital, Second Military Medical University in Shanghai, China. Patients who met all the following criteria of eligibility were included into our study: (i) diagnosis of primary HCC identified by histopathological examination; (ii) treatment with radical resection; (iii) availability of complete followup data; (iv) no pre-operative anticancer treatment, such as chemotherapy and radiotherapy; (v) no history of familial malignancy or other synchronous malignancy; and (vi) no death within 3 months after operation. The histopathological type and grade were determined using the criteria of the World Health Organization (WHO) classification. All patients were staged according to the seventh-edition tumour– node–metastasis (TNM) staging system of the Union for International Cancer Control (UICC) and American Joint Committee on Cancer (AJCC). Overall survival (OS) was defined as the time from operation to HCC-specific death or last follow-up, whichever come first. Patients dying of other causes were censored for survival analysis. Relapse-free survival (RFS) is defined as the time elapsed from operation to the date of the recurrence or distant metastasis of HCC. In addition, another 10 cases diagnosed with dysplasia were enrolled, including six males and four females with a median age of 57 years (range 41–65 years). The study was approved by the Ethical Committee of Second Military Medical University, and written informed consent was obtained from each patient. All study procedures were carried out in accordance with the ethical standards of the Helsinki Declaration. HCC CELL LINES
HCC cell lines SK-HEP-1, MHCC-97HH, MHCC-97L, BEL-7402 and HepG2 were obtained from the
P-value
0.007
0.518 (0.284–0.945)
0.607 (0.315–1.167)
2.859 (1.678–4.870)
