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Cancer Biomarkers 13 (2013) 351–357 DOI 10.3233/CBM-130370 IOS Press

Peripheral blood microvesicles are potential biomarkers for hepatocellular carcinoma Wenjie Wanga,b,1, Huiyu Lic,1 , Yan Zhoua and Shenghua Jiea,∗ a

Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China b Department of Infectious Diseases, Wuhan General Hospital of Guangzhou Military Command, Wuhan, Hubei, China c Center for Stem Cell Research and Application, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

Abstract. BACKGROUND: Microvesicles (MVs) are produced through the outward vesicles budding and fission from the cell surface. Recently, it was discovered that extracellular MVs circulate in bodily fluids of cancer patients and could serve as potential diagnostic biomarkers. However, the diagnostic and prognostic roles of peripheral circulating MVs for hepatocellular carcinoma (HCC) remain unclear. OBJECTIVE: The aim of this study was to investigate whether the peripheral blood MVs could serve as potential biomarkers for detection of HCC. METHODS: Peripheral blood samples were obtained prior to treatment from 55 patients with HCC, 40 patients with liver cirrhosis and 21 healthy controls. MVs were isolated from peripheral blood by centrifugation and measured by using bicinchoninic acid assay. RESULTS: Peripheral blood MVs levels were significantly elevated in HCC patients compared to those in liver cirrhosis (p < 0.001). Furthermore, MVs levels was correlated with the HCC tumor size, pathological classification and TNM stage (p < 0.01). Of note, MVs levels were significantly reduced in the 1 month post-operative blood samples when compared to those in the pre-operative samples in the 17 HCC cases tested. MVs levels did not relate to liver enzymes, AFP levels, alcohol drinking or smoking habits (p > 0.05). In contrast, serum MVs levels correlated with the age of patients, leukocytes, platelets and prothrombin time. The results of receiver operating characteristic (ROC) analysis indicated better performance of MVs than AFP for early detection of HCC. The areas under the ROC curve of MVs for discriminating patients with early (TNM stage I) and relatively early (TNM stage II) HCC from liver cirrhosis was 0.83 (95% CI: 0.74–0.93) and 0.94 (95% CI: 0.88–1.00), respectively. CONCLUSIONS: Peripheral blood MVs levels were increased in patients with HCC and associated with the progression of disease. Serum MVs might serve as novel biomarkers for the diagnosis of HCC at early stage. Keywords: HCC, MVs, AFP, diagnosis

1. Introduction Hepatocellular carcinoma (HCC) is the sixth most common human cancer worldwide, but the third lead∗ Corresponding author: Shenghua Jie, Department of Infectious Diseases, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei, China. Tel.: +86 027 8572 6135; Fax: +86 027 8572 9267; E-mail: abeycd@ 126.com. 1 These authors contributed equally to this paper.

ing cause of cancer death [1]. Despite recent progress in surgical and nonsurgical treatment, the prognosis for HCC remains poor, partially because it is usually diagnosed at an advanced stage when curative therapy is not possible [2]. Therefore, HCC screening of subjects at risk can facilitate tumor detection at an early stage, which in turn can reduce mortality [3]. Although imaging techniques, including ultrasound, computed tomography and magnetic resonance imaging have been recommended for HCC early detection, these approaches are highly operator’s experience-dependent,

c 2013 – IOS Press and the authors. All rights reserved ISSN 1574-0153/13/$27.50 

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costly and radiation [4]. Thus, there is a need for sensitive serum markers for early detection of HCC. A major problem with HCC surveillance is the lack of reliable biomarkers for early detection of HCC. Alpha-fetoprotein (AFP) has been the most widely used tumor marker for the detection and monitoring of HCC. However, it has been reported that only one third of patients with HCC have higher AFP levels [5]. Des-gamma-carboxy prothrombin (DCP) is a complementary tumor marker to AFP, but elevated DCP levels are only present in 44–47% of patients with small HCC [6]. Other currently available tumor markers for HCC, including Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3), glypican-3, osteopontin are also not satisfactory markers in terms of sensitivity and specificity [7,8]. Therefore, developing new serum biomarkers will improve the efficiency of HCC detection. Microvesicles (MVs), are plasma membrane-derived particles which are released from cells by the outward budding and fission of the plasma membrane [9]. MVs are released by various cell types, including platelets, monocytes, endothelial cells and as well as tumor cells. Importantly, MVs are shed at low levels in normal and parental cells and more easily detectable when these cells acquired a tumorigenic phenotype [10]. The cargo of MVs consists of proteins, lipids, nucleic acids (RNA, microRNA, DNA) and others bioactive molecules. The function of MVs appears to be dependent on the cargo they carry [9, 11]. MVs can facilitate cell-to-cell interactions, transfer proteins and mRNA to cells, and induce cell signaling [12–15]. MVs play a role in many aspects of tumor progression, including angiogenesis, evasion of immune surveillance, extracellular matrix degradation and metastasis [16]. The release of metalloproteases and urokinase-typeplasminogen activator from MVs shed by tumor cells promotes tumor invasion and metastases [17]. MVs shed from tumor cells contain proangiogenic stimuli and induce endothelial cell proliferation and therefore angiogenesis [16,18,19]. In addition, MVs contribute to tumor cells immunoescape via carrying Fas ligand, which induces apoptosis in T-cells and prevents their cytotoxic effects on tumor cells [20–22]. MVs in cancer patients were first documented in 1978 with their identification in cultures of spleen nodules and lymph nodes from a patient with Hodgkin disease [23]. Recent studies have demonstrated that MVs into blood are increased and associated with disease progression in several cancers, such as breast, ovarian, lung, prostate, colorectal, and gas-

tric cancers [16,19]. However, the diagnostic and prognostic roles of peripheral circulating MVs for HCC remain unclear. In this present study, we aimed to test the hypothesis that MVs could serve as novel biomarkers for early detection of HCC.

2. Materials and methods 2.1. Subjects Blood samples were collected under informed consent from 55 HCC patients (44 male, 11 female; mean age, 52.73 years) in the infectious department of our hospital from 2009 to 2010 (Union Hospital, Wuhan, China). The diagnosis of HCC was based on typical findings in three-phase dynamic CT or MRI, and the diagnosis was confirmed by histopathology. For comparison, 40 with liver cirrhosis (31 males and 9 females; mean age, 50.43 years) we encountered during the same period were also included. Serum samples were obtained prior to initial treatment. Tumor stages were determined by the 2002 American Joint Committee on Cancer/International Union against Cancer tumor-node-metastasis (TNM) classification system, and tumor differentiation was graded by the grading system of Edmondson and Steiner [24]. In 17 cases, serum samples were obtained before and after chemoembolization therapy. Blood samples were also obtained from 21 apparently healthy subjects. This study was approved by the Institute Research Ethics Committee of Union Hospital, and informed consent was obtained from each patient according to the committee’s regulations. 2.2. Isolation of MVs MVs were isolated from blood according to the modified protocol proposed by Mrvar-Brecko et al. [25]. Briefly, after an overnight fast, 3 ml venous blood was collected into tubes containing 3.2% (0.109 M) trisodium citrate. Blood samples were centrifuged for 20 min at 1550 g and 20◦ C to remove cells. To sediment MVs, supernatant was taken and centrifuged for 1 h at 17570 g and 20◦ C. Supernatants was removed and MV pellets were washed in phosphate buffer saline (PBS)-citrate. MV pellets were resuspended in PBS and dissolved in RIPA cell lysis buffer (Santa Cruz Biotechnology, Santa Cruz, USA). MVs protein concentration was quantified by bicinchoninic acid assay (Thermo Pierce, Rockford, USA).

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Table 1 Clinical and biochemical characteristics of the study subjects groups HC (n = 21) 49.47 ± 12.65

LC (n = 40) 50.43 ± 11.38

HCC (n = 55) 52.73 ± 11.72

16(76.19%) 5(23.81%)

31(77.50%) 9(22.50%)

44(80.00%) 11(20.00%)

Smoking Yes No

8(38.09%) 13(61.91%)

15(37.50%) 25(62.50%)

21(38.18%) 34(61.82%)

Alcohol drinking Yes No

9(42.86%) 12(57.14%)

17(42.50%) 23(57.50%)

23(41.82%) 32(58.18%)

HBsAg Positive Negative

2(9.52%) 19(90.48%)

23(57.50%) 17(42.50%)

35(63.64%) 20(36.36%)

23.65 ± 4.78 20.39 ± 5.65 32.18 ± 10.79 10.65 ± 4.28 2.18 ± 1.09 6.37 ± 3.21

61.78 ± 27.13a 76.11 ± 48.87a 53.46 ± 37.74a 35.95 ± 14.82a 16.62 ± 10.58a 21.76 ± 24.18a

86.45 ± 68.19a,b 137.81 ± 152.86a,b 244.47 ± 235.72a,b 89.40 ± 53.55a,b 34.94 ± 22.63a,b 161.87 ± 281.17a,b

Variables Age (years) Gender Male Female

AST (U/l) ALT (U/l) γ-GT (U/l) Total bilirubin (umol/l) Direct bilirubin (umol/l) AFP (ng/ml) a Showed P

< 0.01 between HC and LC groups, or HCC groups. b Showed P < 0.01 between LC and HCC groups. HC, healthy control; LC, liver cirrhosis; HCC, hepatocellular carcinoma; HBsAg, hepatitis B surface antigen; AST, aspartate aminotransferase ALT, alanine aminotransferase; γ-GT, gamma-glutamyl transpeptidase; AFP, alpha-fetoprotein.

2.3. Statistical analysis Data were presented as the mean ± standard deviation (SD). Differences between groups were evaluated for significance by one way ANOVA analysis. Bivariate correlations were tested with Pearson correlation coefficient (CC). The overall diagnostic accuracy of MVs levels was evaluated by receiver-operating characteristic (ROC) analysis. We determined the turning point of the curve to the best cut-off value for the diagnosis, and it was also a maximal value at the sum of the sensitivity and specificity. A p-value below 0.05 was considered statistically significant.

3. Results 3.1. Characteristics of subjects The characteristics of all subjects enrolled in this study were summarized in Table 1. Among 55 HCC patients, 44 cases are male (80.00%), and 11 cases are female (20.00%). The average age of 55 patients was 52.73 years (range 28 to 72 years). Thirty-five (63.64%) patients were infected with hepatitis B virus (HBV). Age and gender showed no significant difference between groups. No significant difference also was found considering smoking habit, alcohol drinking

Fig. 1. Peripheral blood MVs levels in patients with HCC, liver cirrhosis, and healthy subjects. Blood MVs levels in HCC patients were significantly higher than those in liver cirrhosis patients (P < 0.001). *p < 0.01, **p < 0.001, significantly different from healthy subjects. # p < 0.001, significantly different from liver cirrhosis patients.

and HBV infection between liver cirrhosis and HCC groups. A significant difference has been shown among all studied groups with regard to measured liver function tests and AFP.

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W. Wang et al. / MVs are potential biomarkers for HCC Table 2 Relationship between blood MVs and pathological data HCC Total (n = 55) Tumor size  3 cm (n = 29) < 3 cm (n = 26)

MVs (μg/ml) 10.32 ± 4.36 12.11 ± 4.76 8.39 ± 2.71a

TNM stage I (n = 28) II (n = 20) III (n = 7)

7.56 ± 2.75b 10.61 ± 3.02c 16.08 ± 7.18d

Differentiation Low differentiated (n = 32) High differentiated (n = 23)

12.32 ± 4.34 7.70 ± 2.24e 4.57 ± 2.49

Liver cirrhosis

P < 0.01 between tumor size  3 and < 3 cm. b Showed P < 0.01 between early HCC patients (stage I ) and liver cirrhosis patients. c Showed P < 0.01 between relatively early HCC patients (stage II) and early HCC patients (stage I ). d Showed P < 0.01 between advanced HCC patients (stage III ) and early (TNM stage I) or relatively early stage (TNM stage II) HCC patients. e Showed P < 0.01 between low differentiated and high differentiated. HCC, hepatocellular carcinoma; MVs, Microvesicles; TNM, tumor-nodemetastasis a Showed

Fig. 2. Peripheral blood MVs levels before and after surgical operation. MVs levels were detected in blood samples obtained from just before and after surgery in 17 cases, respectively. The levels of MVs were significantly decreased 1 month after surgery (P < 0.01). *p < 0.01, significantly different from pre-operative levels.

3.2. MVs levels in HCC patients Blood MVs levels in HCC patients, liver cirrhosis patients and healthy subjects were presented in Fig. 1. MVs levels were significantly elevated in HCC patients (10.32 ± 4.36 μg/ml) compared to those in liver cirrhosis patients (4.57 ± 2.49 μg/ml) (P < 0.001). Furthermore, In 17 cases, MVs levels were determined just before and after chemoembolization therapy. As shown in Fig. 2, MVs levels were only slightly decreased 1 day post-operative, but significantly decreased 1 month after surgery (P < 0.01) (Fig. 2). 3.3. Relationship between MVs levels and pathological characteristics of HCC patients As summarized in Table 2, blood MVs levels were significantly higher in patients with large HCC ( 3 cm) compared to those with small HCC (< 3 cm) (P < 0.01). Blood MVs levels in early (TNM stage I) HCC patients were significantly increased compared to those in liver cirrhosis patients (P < 0.01). Of note, blood MVs levels were associated with the TNM stage of HCC. HCC patients with advanced stage (TNM stage III) showed significantly higher MVs protein levels than early (TNM stage I) or relatively early stage (TNM stage II) HCC patients (P < 0.01). Patients

Table 3 Association between blood MVs levels and clinic data in patients with HCC Variables Age Smoking Alcohol drinking Bilirubin ALT AST γ-GT AFP PLT leucocytes PT

CC value 0.21 −0.12 −0.15 −0.13 −0.12 −0.12 −0.15 −0.14 0.17 −0.28 −0.19

P value < 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 > 0.05 < 0.05 < 0.05 < 0.05

HCC, hepatocellular carcinoma; MVs, Microvesicles; CC, correlation coefficient; ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GT, gamma-glutamyltranspeptidase; AFP, alpha-fetoprotein; PLT, latelet count; PT, prothrombin time.

were also divided into two categories according to the Edmondson classification, high differentiation (I, II, I– II) and low differentiation (II–III, III, IV). The statistical results revealed that MVs levels were significantly higher in HCC patients with low differentiation compared to those with high differentiation (P < 0.01). 3.4. Relationship between MVs levels and other clinic characteristics of HCC patients The correlations between MV levels and patient’s other clinical data were summarized in Table 3. MVs levels correlated with the age of patients (CC = 0.21,

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(a)

355

(b)

Fig. 3. ROC curve analysis using peripheral blood MVs for discriminating HCC patients from liver cirrhosis patients. ROC curves for MVs and AFP in early (TNM stage I) (a) and relatively early (TNM stage II) (b) patients, compared with liver cirrhosis patients. ROC curve analysis of blood MVs showed higher AUC compared to that of AFP (P < 0.001).

p < 0.05), platelet count (PLT) (CC = 0.17, p < 0.05), leukocyte count (CC = −0.28, p < 0.05) and prothrombin time (PT) (CC = −0.19, p < 0.05), respectively. There was no significant correlation of MVs protein levels with smoking, alcohol consumption, liver function tests (bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (γ-GT) and AFP levels (P > 0.05). 3.5. The diagnostic value of MVs for early detection of HCC To evaluate whether MVs in peripheral blood could be used as potential diagnostic markers for early detection of HCC, ROC curve analyses were performed. As shown in Fig. 3, the AUC (the areas under the ROC curve) of AFP for discriminating patients with early (TNM stage I) and relatively early (TNM stage II) HCC from liver cirrhosis was 0.61 (95% CI: 0.47– 0.74) and 0.78 (95% CI: 0.65–0.90), respectively. Of note, ROC curve analysis of blood MVs showed significantly higher AUC (TNM stage I: 0.83 (95% CI: 0.74–0.93), TNM stage II: 0.94 (95% CI: 0.88–1.00)) compared to that of AFP (P < 0.01). Taking both sensitivity and specificity into account, the cut-off point was selected according to maximum number of sensitivity and specificity. MVs at the cut-off value of 5.41 μg/ml showed 88.89% specificity and 62.5.8%

sensitivity. In contrast, AFP showed 85.71% specificity and 40.00% sensitivity at the cut-off value of 20 ng/ml. These findings suggested that MVs levels could be potential markers for discriminating HCC patients from liver cirrhosis. 4. Discussion MVs, constitutionally released by tumor cells, may interact with target cells by surface-expressed ligands and promote tumor progression by transferring membrane growth factor receptors, metastasis-related proteases and other bioactive molecules to adjacent or remote cells [12,26]. Furthermore, MVs, released into body fluids such as blood, urine and ascites, call attention to their promises as circulating biomarkers in the diagnosis, prognosis and surveillance of disease progression. MVs have also been detected in the circulation of patients with several cancers [16,19]. Mitchell et al. demonstrated that MVs levels were significantly increased in malignant effusions, serum and urine from prostate cancer patients [27]. MV levels were notably elevated in cancers with associated thromboses, such as colorectal carcinoma, breast cancer, and pancreatic adenocarcinoma [28,29]. Skog et al. demonstrated that MVs were detected in the serum of patients with glioblastoma. Of note, circulating MVs levels were decreased after removal of tumor [30]. In the present study, we demonstrated that the MVs

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levels in peripheral blood of HCC patients were significantly higher than those in liver cirrhosis, indicating that the number and protein content of MVs were increased in peripheral circulation of patients with HCC. Of note, MVs protein levels associated with the HCC tumor size, pathological classification and TNM stage, indicating that MVs levels related to the progression of HCC. Furthermore, MVs levels were significantly reduced in the 1 month post-operative blood samples when compared to those in the pre-operative samples. MVs protein levels were not significantly decreased 1 day after chemoembolization therapy. It is possibly because that chemoembolization could lead to ischemic necrosis of local tumor cells, and necrotic cells could also release a great quantity of MVs. MVs levels related to the severity of HCC, and did not show correlations with the liver damage markers, such as bilirubin, ALT, AST, and γ-GT, suggesting MVs may be serum biomarkers to indicate the progression of HCC, but not indicators of liver damage. In addition, MVs levels correlated with the age of patients. There were no significant correlations between MVs levels with smoking and alcohol drinking habits. Interesting, we found that the MVs levels associated with the number of leukocytes. Peripheral blood leukocytes mainly include neutrophil granulocytes and lymphocytes, which play import role in tumor immunity. It has been reported that MVs, released from tumor cell, can induce tumor-specific lymphocytes apoptosis via the apoptosis receptors, and promote tumor immune escape process [20–22]. Our observation found that the MVs levels negatively correlated with peripheral blood leucocytes, indicating that MVs might be involved in the tumor evasion from immune surveillance. In addition to tumor cells, platelets can also be triggered to release MVs [31]. In the present study, we demonstrated that MVs levels positively associated with PLT, suggesting that MVs were partially released from platelets. Meanwhile, MVs levels related to PT. MVs bearing tissue factor (TF) play an important role in hypercoagulation and related disorders in cancer patients [32]. TF is the initiator of the clotting cascade. Active TF-bearing MVs, mostly originated from the tumor cells, have been found in the circulation of cancer patients [33]. Additionally, TF-bearing MVs associated with an increased risk of thromboembolic disease in malignancy [34]. These observations support our finding that MVs levels associated with PT. To evaluate whether MVs in peripheral blood could be used as potential diagnostic markers for early detection of HCC, ROC curve analyses were performed. AFP has been most widely used as a serum marker for detection of HCC, but its sensitivity and specificity

are not satisfying [35]. In the present study, serum AFP yielded an AUC of 0.61 (95% CI: 0.47–0.74) and 0.78 (95% CI: 0.65–0.90) for discriminating early and relatively early HCC from liver cirrhosis, respectively. In contrast, the AUC of peripheral blood MVs for discriminating early and relatively early HCC patients from liver cirrhosis was 0.83 (95% CI: 0.74– 0.93) and 0.94 (95% CI: 0.88–1.00), respectively. We also found MVs levels did not correlate with AFP levels (p > 0.05). These findings suggested that MVs could be used as potential biomarkers for early detection of HCC. This study has some limitations. First, although we could get samples that were rich with MVs according to the published protocol [25], numerous platelets and leukocytes can be also found in the isolate [25]. To date, there are no validated MVs isolation protocols available. Additional effort should be put forward for improvement of the protocol for MVs isolation and observation. Second, we only determined the total amount of blood MVs, but not detected the specific MVs originated from tumor cells. Tumor cells are able to constitutively release large amounts of MVs bearing tumor-specific antigens, or others bioactive molecules. Thus, cancer-derived MVs may serve as novel and specific cancer biomarkers that may prove useful for screening and diagnosis [14]. Therefore, we intend to analyze HCC specific MVs in further studies. In conclusion, MV levels were significantly increased in HCC patients, might serve as novel and potential biomarkers for early detection of HCC. Further clinical investigations by using larger sample sizes are required to determine the diagnostic value of MVs.

Acknowledgements This study was supported by the National Natural Science Foundation of China (No: 81171638).

Conflict of interest The authors declare that there is no conflict of interest.

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