Original Paper Received: June 27, 2014 Accepted after revision: November 30, 2014 Published online: January 22, 2015
Dig Surg 2014;31:468–478 DOI: 10.1159/000370252
Yes-Associated Protein Expression is a Predictive Marker for Recurrence of Hepatocellular Carcinoma after Liver Transplantation Hua Li Shouhua Wang Guoying Wang ZhiGang Zhang Xiaocai Wu Tong Zhang Binsheng Fu Guihua Chen Department of Hepatic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou,Guangdong, PR China
Key Words Hepatocellular carcinoma · Liver transplantation · Yes-associated protein YAP · Immunohistochemistry · Tumor recurrence
Abstract Purpose: To explore the expression of Yes-associated protein (YAP) in hepatocellular carcinoma (HCC) patients, and assess its prognostic value to recurrence of HCC after liver transplantation (LT). Methods: Collected data of 105 consecutive patients undergoing LT for HCC were analyzed retrospectively. The immunohistochemistry was used to detect the expression of YAP, Mst1, Lats1/2, pYAP, pLats1/2 and pMst1/2 in tumor tissues. Contingency table and χ2-test were used to investigate the correlation between expression of YAP, Mst1, Lats1/2 and clinical characteristics. Univariate survival analysis and Multivariate Cox regression analysis were also performed to analyze the correlation of clinical and pathological factors with tumor recurrence after LT. The Kaplan-Meier method and log-rank test were used to analyze HCC-specific disease-free survival (DFS) rate. Results: Forty patients fulfilled Milan criteria with 1-, 2-, 3- and 5-years DFS of 86.7, 84.6, 84, 84%, respectively. The positive rates of YAP, Lats1/2, Mst1 in HCC were 51.4, 45.7, 64.8%, respectively. YAP expression in HCC tumors was significantly associated with tumor size (p = 0.041), venous infiltration (p = 0.002), AJCC tumor stage (p = 0.027). Lats1/2 expression was sig-
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nificantly associated with tumor size (p = 0.001) and AJCC tumor stage (p = 0.019). Mst1 expression was also significantly associated with tumor size (p = 0.042). HCC-specific DFS was significantly longer for patients with YAP negative expression compared with patients with YAP positive expression (1-, 2-, 3- and 5-years DFS of 71.7, 65.3, 65.3, 65.3 vs. 42.5, 36.6, 32.5, 30.4%, respectively, log-rank = 12.89, p < 0.001). Multivariate Cox regression analysis indicated that YAP expression (HR = 2.011, p = 0.020) in HCC was an independent prognostic factor for HCC-specific DFS after liver transplantation. Conclusions: YAP is an independent prognostic marker for tumor recurrence for HCC patients after © 2015 S. Karger AG, Basel liver transplantation.
Introduction
Hepatocellular carcinoma (HCC) is the sixth most common malignancy and the third leading cause of cancer death worldwide [1]. Liver transplantation (LT) is the preferred treatment for selected patients with hepatocellular carcinoma (HCC). With liver transplantation development in recent years, the 5-year survival rate is significantly improved [2]. However, recurrence and metastasis
H.L. and S.W. contributed equally to this work.
Guihua Chen Department of Hepatic Surgery, Third Affiliated Hospital of Sun Yat-sen University No. 600 Tianhe Road, Tianhe District Guangzhou, Guangdong 510630 (PR China) E-Mail chenguihua1955 @ 163.com
of HCC after LT remains a formidable problem; several large centers have reported that the recurrence rate was approximately 20%, which is the rate-limiting factor for long-term survival [3]. So currently, prevention and treatment of tumor recurrence is one of the hot issues for the research for HCC patients after liver transplantation. The Hippo signaling pathway originally discovered in Drosophila consists of several negative growth regulators acting in a kinase cascade that ultimately phosphorylates and inactivates Yorkie (Yki), a transcriptional coactivator that positively regulates cell growth, survival, and proliferation. In mammals, the Hippo pathway is well conserved. Core components of the Hippo pathway include the Mst1/2, Lats1/2, and YAP, which consists of a kinase cascade in which Mst1/2 forms a complex with the adaptor protein WW45 and phosphorylates the kinases Lats1 and Lats2 as well as the adaptor protein Mob. A Lats/Mob complex then phosphorylates and represses the transcriptional coactivator YAP. YAP is phosphorylated and inhibited by the Lats tumor suppressor, and this phosphorylation results in its association with 14-3-3 binding motif and from the nucleus into the cytoplasm, where coactivator activity is lost [4–6]. Deregulation of the tumor suppressors in the Hippo pathway and overexpression of YAP during cancer development and progression is now clear. YAP, which was termed a candidate oncogene, is evident in several human cancers, including breast, pancreas, prostate, and liver [7, 8]. Recent studies have demonstrated YAP genes are important for adaptive liver enlargement and tumorigenesis in the liver [9]. Zhao et al. evaluated that among the 115 cases of HCC samples examined, 63 samples (54%) showed strong YAP staining, while 95% of normal liver tissue samples showed very weak staining, suggesting a significant difference in YAP protein levels between normal and cancerous tissues. Recent study has shown that the expression of YAP was significantly correlated with HCC prognosis and YAP was an independent prognostic marker for overall and disease-free survival for HCC patients after partial hepatectomy [10–12]. Moreover, increased expression of YAP in YAP activity and nuclear location in transgenic mice lead to HCC development, which also suggests a direct link between dysregulation of the Hippo pathway and liver tumorigenesis [13]. YAP has been reported to bind and regulate various human transcriptional regulators including p73 and p53-binding protein-2 [14]. Precise biological function and physiological regulation of YAP is unclear as of now. In this study, we analyzed the expression and correlation of YAP, and pYAP, pLats1/2, pMst1/2, Mst1, Lats1/2 YAP Expression Predicts HCC Recurrence after Liver Transplantation
in HCC by immunohistochemical staining. The relationship between YAP, Lats1/2, and Mst1 expression and clinicopathological features was also investigated to understand their role on recurrence of HCC after liver transplantation. The present study was an extension of the study mentioned earlier, and analyzed their clinical significance and relationship with recurrence of HCC after liver transplantation. Materials and Methods Patients and Clinical Samples One hundred and five cases of HCC patients who underwent liver transplantation at the Department of Hepatic Surgery in the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, Guangdong, China) between June 2004 and September 2009 were collected. The preoperative clinicopathological features of the patients were analyzed by the tumors’ maximum diameter, number of tumor lesions, vascular invasion (portal vein or hepatic venous system) and the level of alpha-fetoprotein (AFP) and postoperative pathological data. Diagnostic criteria included the following: (1) All of the cases were consistent with hepatocellular carcinoma (HCC) clinical diagnostic criteria. (2) All of the patients were histologically diagnosed with hepatocellular carcinoma. Inclusion criteria included the following: (1) All of the patients first underwent liver transplantation. (2) Routine post-liver transplantation immunosuppressive regimen was used by tacrolimus-based individualized treatment plan. (3) The hormone therapy did not exceed 3 months, except acute rejection after liver transplantation. Postoperative follow-up included the postoperative follow-up routine in transplant specialist outpatient. All of the patients received standard post-liver transplantation care and follow-up of the experimental study deadline to December 2012. The means of follow-up were through outpatient visits, letters, and phone calls. Patients who were not contactable for 1 year were considered lost. Regular follow-up testing excluded tumor recurrence or metastasis. Patients were observed HCC recurrence as the deadline events. Disease-free survival (DFS) times were calculated from the date of curative surgery to HCC recurrence, death, or the last follow-up date. Patients who were lost to follow-up, due to death or because recurrence was not observed were defined as censored data during the follow-up period. The present study was approved by our institutional ethics committee. Informed consent was obtained from each patient and family members. The procedure met all applicable guidelines of our institute as well as governmental regulations concerning the ethical use of donated organs, and the latest version of the Declaration of Helsinki. Immunohistochemistry (IHC) Pathological data were obtained from the Department of Pathology at the Third Affiliated Hospital of Sun Yat-sen University. Paraffin-embedded tumor tissue blocks were sectioned (4 μm) for immunohistochemical staining. After antigen retrieval and peroxidase blocking, the sections were incubated with rabbit polyclonal antibody against human YAP (127), Phosph-YAP (127) (Cell Signaling, American), rabbit polyclonal antibody against human Mst1, Anti-Mst1/2 (phosphoT183) (Abcam, Hong Kong), rabbit
Dig Surg 2014;31:468–478 DOI: 10.1159/000370252
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Table 1. Site of hepatocellular carcinoma recurrence or metastasis after liver transplantation
YAP (positive) YAP (negative)
Cases of tumor recurrence
Liver
Lung
Intra-abdominal metastasis
Liver + lung
Liver + lung + bone
37 17
26 14
6 2
2 0
1 1
2 0
polyclonal antibody against human Lats1/2, Phosph-Lats1/2 (immunoway, Hong Kong), working dilution are respectively 1: 200, 1:200, 1:550, 1:60, 1:200, 1:200, at 4 ° C overnight. After 3 washings in sterile phosphate-buffered saline, sections were incubated with a horseradish peroxidase (HRP) conjugated antibody against rabbit or mouse immunoglobulin G (IgG) (Gene Tech, Shanghai, China). For negative controls, sections were treated with 0.01 mol/l phosphate-buffered saline instead of primary antibodies.
IHC Staining Evaluation YAP was specifically located in the cytoplasm and the nucleus of carcinoma cells, pYAP (127), Lat1/2, pLats1/2, Mst1, pMst1/2 (phosphoT183) was specifically located in the cytoplasm of carcinoma cells. Staining intensity (A) was classified as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The percentage of positive cells (B) examined in 500 cells were divided into 0 (75%). According to the product of A and B, the IHC result was classified as 0, negative (–); positive was classified as 1–4, weakly positive (+); 5–8, moderately positive (++) and 9–12, strongly positive (+++), proteins immunoreactivity was graded as negative (score 0) and positive (score weakly positive and above is positive) according to the previously reported procedures [15]. Statistical Analysis Statistical analysis was performed using SPSS 13.0 Package, Contingency table and χ2-test were used to investigate the correlation between expression of YAP, Mst1, Lats1/2 and clinical characteristics (the Student t test for continuous data). Univariate and Multivariate Cox regression analyses of YAP and other proteins in HCC were also performed to determine the association expression of YAP, pYAP, pLats1/2, Lats1/2, pMst1/2, Mst1 and individual clinicopathologic variables with DFS times after liver transplantation. The Kaplan-Meier method and log-rank test were used to generate curves for HCC-specific disease-free survival (DFS). The prognostic factors for this cohort (tumor size, venous infiltration, tumor stage [American Joint Committee on Cancer (AJCC)], and α-fetoprotein AFP, etc.) were subsequently subjected to the multivariate Cox regression analysis to determine the hazards ratios (HRs) and the independence of effects. p < 0.05 was considered statistically significant.
Results
Protein Expression in Hippo Signaling Pathway in Hepatocellular Carcinoma Tissues All 105 patients included 98 men (93.3%) and 7 women (6.7%). Age ranged 22–72 years with the median age of 46. 470
Dig Surg 2014;31:468–478 DOI: 10.1159/000370252
Follow-up time ranged 1–96 months with the median follow-up time of 13 months. Eight patients lost to followup. During follow-up, tumor recurrence was observed in 54 cases, that is, in 51.4% of patients. The most common site of recurrence was in the grafted liver for 40 cases, and lung metastasis for 8 cases, and intra-abdominal metastasis for 2 cases. Additionally, some patients were diagnosed with combined organ recurrence or metastasis to the liver, lung, or bone (liver + lung is 2 case, liver + lung + bone is 2 case) (table 1), and recurrence time was 1–41 months. Of 105 patients, 40 fulfilled the Milan criteria with 1-, 2-, 3- and 5-years DFS of 86.7, 84.6, 84, 84%, respectively. The 105 HCC cases were grouped according to the expression scores. The positive rate of YAP in HCC cases was 51.4% (54/105) (fig. 1). YAP was specifically located in the cytoplasm and nucleus of carcinoma cells (fig. 2a). pYAP, Lats1/2, pLats1/2, Mst1, and pMst1 were specifically located in the cytoplasm of carcinoma cells (fig. 2b, 3a, b, 4a, b). The positive rate of Lats1/2 in HCC cases was 45.7% (48/105). The positive rate of Mst1 in HCC cases was 64.8% (68/105). The positive rates of pYAP, pLats1/2, and pMst1/2 were 55.2% (58/105), 9.5% (10/105), and 21% (21/105), respectively (fig. 1). The Correlation of YAP, Mst1, Lats1/2 with Clinical Characteristics The clinical association analysis by Contingency table and χ2-test revealed that YAP expression in HCC was significantly associated with the tumor size (χ2 = 4.173, p = 0.041), venous infiltration (χ2 = 5.875, p = 0.002), and tumor stage (χ2 = 7.545, p = 0.027) (table 2). Lats1/2 expression in HCC tumors was significantly associated with tumor size (χ2 = 14.413, p = 0.001) and AJCC tumor stage (χ2 = 7.850, p = 0.019). Mst1 expression in HCC tumors was also significantly associated with tumor size (χ2 = 4.129, p = 0.042) (table 2). Association between Protein Expression and HCC-Specific Disease-Free Survival (DFS) We examined whether the correction of expression of YAP or other core components of Hippo pathway and Li/Wang/Wang/Zhang/Wu/Zhang/Fu/ Chen
100
90 (90.5%)
Positive Negative
84 (80%)
The number of the cases
80
60
58 57 54 (55.2%) (54.3%) 51 (51.4%) 48 47 (49.6%) (45.8%) (45.7%)
37 (36.2%)
40
21 (20%)
20
0
10 (9.5%)
YAP
pYAP
Lats1/2
pLats1/2
Mst1
pMst1/2
Color version available online
Fig. 1. Positive and negative expression cases and rates of YAP, pYAP, Lats1/2, pLats1/2, Mst1 and pMst1/2 in clinical samples of hepatocellular carcinoma (HCC).
68 (64.8%)
a
b
c
d
Fig. 2. a YAP was specifically located in the cytoplasm and nucleus of carcinoma cells, and positive expression of YAP in clinical samples of hepatocellular carcinoma (HCC) is shown. b pYAP was specifically located in the cytoplasm and carcinoma cells, and positive expression of YAP in clinical samples of hepatocellular carci-
noma (HCC) is shown. c Negative expression of YAP in clinical samples of hepatocellular carcinoma (HCC) is shown. d Negative expression of pYAP in clinical samples of hepatocellular carcinoma (HCC) is shown (IHC, ×200).
YAP Expression Predicts HCC Recurrence after Liver Transplantation
Dig Surg 2014;31:468–478 DOI: 10.1159/000370252
471
Color version available online
a
b
c
d
Fig. 3. a Lats1/2 was specifically located in the cytoplasm of carci-
noma cells, and positive expression of Lats1/2 in clinical samples of hepatocellular carcinoma (HCC) is shown. b pLats1/2 was specifically located in the cytoplasm and carcinoma cells, and positive expression of pLats1/2 in clinical samples of hepatocellular carci-
clinicopathologic feature with HCC-specific DFS after liver transplantation. Univariate survival analysis indicated that the expression of YAP, Lats1/2, pMst1/2 and cellular differentiation, serum AFP level, venous infiltrationumor stage, and tumor size were associated with HCC-specific DFS after liver transplantation (p < 0.05) (table 3). Multivariate Cox regression analysis indicated that positive expression of YAP (HR = 2.011, p = 0.020), cellular differentiation (HR = 2.390, p = 0.001), tumor stage (HR = 1.574, p = 0.032), and tumor size (HR = 2.176, p = 0.021) were significantly associated with HCC-specific DFS after liver transplantation (table 4). Kaplan-Meier analysis also revealed HCC-specific DFS was significantly longer for patients with YAP negative expression compared with patients with YAP positive expression (1-, 2-, 3-, and 5-years DFS of 71.7, 65.3, 65.3, 65.3 vs. 42.5, 36.6, 32.5, 30.4%, respectively, log-rank = 12.89, p < 0.01) (fig. 5). Thus, our data indicated that YAP expression in HCC was a potential indicator for early disease recurrence. 472
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noma (HCC) is shown. c Negative expression of Lats1/2 in clinical samples of hepatocellular carcinoma (HCC) is shown. d Negative expression of p Lats1/2 in clinical samples of hepatocellular carcinoma (HCC) is shown (IHC, ×200).
Discussion
In the past decades, the Hippo pathway has been shown to have a critical role in controlling organ size by regulating both cell proliferation and apoptosis. Furthermore, the Hippo pathway was defined as a tumor suppressor pathway [5]. Some studies have put forth mechanisms involved in the deregulation of Hippo pathway during liver cancer pathogenesis in animals [16]. Our immunohistochemistry investigation found that the positive rates of YAP in HCC was approximately 51.4% (54/105). YAP expression in HCC was significantly associated with the tumor size, venous infiltration, and tumor stage. YAP, as a driving oncogene, is known to be amplified and nuclear location in multiple types of human cancers and strongly correlate with poor patient outcome, including ovarian cancers [17], lung cancers [18], esophageal squamous cell carcinoma [19] and prostate cancers [5]. In human HCC, elevated YAP expression Li/Wang/Wang/Zhang/Wu/Zhang/Fu/ Chen
Color version available online
a
b
c
d
Fig. 4. a Mst1 was specifically located in the cytoplasm of carci-
noma cells, and positive expression of Mst1 in clinical samples of hepatocellular carcinoma (HCC) is shown. b pMst1/2 was specifically located in the cytoplasm and carcinoma cells, and positive expression of pMst1/2 in clinical samples of hepatocellular carci-
noma (HCC) is shown. c Negative expression of Mst1 in clinical samples of hepatocellular carcinoma (HCC) is shown. d Negative expression of pMst1/2 in clinical samples of hepatocellular carcinoma (HCC) is shown (IHC, ×200).
and nuclear localization have been observed. Previous reports reveal that 54% of 115 HCC patients showed overexpression of YAP in a tissue microarray study in which the majority of normal liver demonstrated very weak staining [10]. However, as existing a tumor suppressor pathway, up to now, we have not found any association between the expression of YAP and tumor recurrence for HCC patients after liver transplantation. Multivariate Cox regression analysis in our study revealed the association of YAP overexpression with HCC-specific diseasefree survival, and YAP was found to be an independent indicator of tumor recurrence for HCC patients after liver transplantation. By analyzing 177 pairs of HCC patients and matched normal samples with complete clinical records, Xu et al. suggest that YAP was determined to be an independent prognostic marker for the overall survival and disease-free survival for HCC patients after hepatectomy [12]. Our findings were also consistent with their conclusion. Lamar et al. suggest that increased
YAP/TEAD activity promote the metastatic potential of breast cancer and melanoma cells, and YAP/TEAD activity plays a causal role in cancer metastasis [20]. We speculate that YAP may play an important role in tumor recurrence and metastasis of HCC after hepatectomy or liver transplantation, and future research is needed to prove this. Our study showed Lats1/2 expression in HCC tumors was significantly associated with the tumor size and AJCC tumor stage, and Mst1 expression was also significantly associated with the tumor size. Univariate survival analysis revealed the increased pMst1/2 expression and decreased Lats1/2 expression, where the association with HCC-specific DFS and Multivariate Cox regression analysis is not applicable. Upstream components of the Hippo pathway are involved in HCC via regulation of YAP expression. The MST1/2 regulated the activation states of LATS1 and LATS2, which directly phosphorylate LATS, and by MOB (Mats in Drosophila) family members, small
YAP Expression Predicts HCC Recurrence after Liver Transplantation
Dig Surg 2014;31:468–478 DOI: 10.1159/000370252
473
Table 2. The association of expression of YAP, Lats1/2 and Mst1 in clinical samples of hepatocellular carcinoma (HCC) with clinicapathologic features Clinicapathologic features
Classifies of features
Patients, n (%)
Sex
men women
98 (93.3) 7 (6.7)
χ2
YAP +
–
49 5
49 2
Age&
p value
1.201
0.273
1.837
0.069
Lats1/2 +
–
46 2
52 5
χ2
p value
0.888
0.346
0.983
0.328
Mst1 +
–
62 6
36 1
χ2
p value
1.443
0.230
0.751
0.454
Tumor size, cm
≤5 >5
51 (48.6) 54 (51.4)
21 33
30 21
4.173
0.041*
33 15
18 39
14.413
0.001*
38 30
13 24
4.129
0.042*
Tumor number
1 ≥2
55 (52.4) 50 (47.6)
25 29
30 21
1.650
0.199
26 22
29 28
0.113
0.737
34 34
21 16
0.439
0.737
Cellular differentiation
well differentiated moderately differentiated poorly differentiated
27 (25.7) 65 (61.9) 13 (12.4)
10 36 8
17 29 5
3.178
0.204
15 29 4
12 36 9
2.255
0.324
18 42 8
9 23 5
0.103
0.950
Serum AFP level, ng/ml 5
51 (15) 54 (39)
0.000*
3.559
1.921–6.595
Serum AFP level, ng/ml
0.05 no data is shown.
YAP Expression Predicts HCC Recurrence after Liver Transplantation
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475
Table 4. Multivariate analysis was performed using Cox regression to analyze the association of protein expres-
sion with HCC-specific disease-free survival (DFS) after liver transplantation Variables
B
SE
p
Hazard risk (HR)
95% CI
YAP Mst1 pMst1/2 Lats1 Age Cellular differentiation Serum AFP level AJCC tumor stage Tumor size Venous infiltration
0.699 – – – – 0.871 – 0.454 0.777 –
0.301 – – – – 0.256 – 0.211 0.336 –
0.020* 0.767 0.615 0.730 0.178 0.001* 0.101 0.032* 0.021* 0.585
2.011 – – – – 2.390 – 1.574 2.176 –
1.115–3.628 – – – – 1.448–3.945 – 1.041–2.381 1.125–4.206 –
YAP expression in tumor YAP negative YAP positive 1-censored 2-censored
1.0
Disease-free survival rate
0.8
0.6
Color version available online
* Statistically significant. – = p > 0.05 no data is shown.
0.4
0.2
0 0
20
40
60
80
100
Months after liver transplantation
Fig. 5. Kaplan-Meier disease-free survival analyses of hepatocel-
lular carcinoma (HCC) patients based on Yes-associated protein (YAP) immunoreactivity are shown. YAP positive expression (n = 54) in tumors was found to be associated with poor disease-free survival (DFS) in 105 patients who underwent liver transplantation.
stream negative regulators of YAP/TAZ ( Mst1/2 and Lats 1/2) has such multiple mutations that would be required to inactivate the pathway in a tumor. The YAP-TAZTEAD complex is a downstream effector of the Hippo pathway, and in cooperation with the TEAD transcription factor, overexpression of YAP could aberrantly activate an array of target genes (e.g. CTGF, CCND1, ITGB2, 476
Dig Surg 2014;31:468–478 DOI: 10.1159/000370252
and BCL2L1) responsible for cell proliferation, antiapoptosis, survival, and migration. Among the four transcription factor TEADs proteins, TEAD1 and TEAD4 are most often associated with proliferation and cancer development [25–27]. The activation of YAP/TAZ could be the result of other growth-regulating pathways such as Ras, TGF-β, and Wnt, which exert suppressive pressure on Hippo signaling, such that deregulation of those pathways leads indirectly to the activation of YAP/TAZ [28, 29]. Factors affecting tumor recurrence of HCC after liver transplantation have been previously reported with preoperative AFP levels [30, 31], tumor size [32, 33], histologic grade [33, 34], and vascular invasion [33, 35]. Our experimental results suggest cellular differentiation (p = 0.001), tumor stage (p = 0.032), and tumor size (p = 0.021) in HCC were significantly associated with HCC-specific diseasefree survival after liver transplantation. Most patients after liver transplantation used tacrolimus-based individualized immunosuppressant strategies. The definition of the most appropriate immunosuppression combination, allowing decreased risk of tumor recurrence, is still a matter of debate. No single protocol has gained broad acceptance until now. Several reports have suggested a lower risk of tumor recurrence with the use of sirolimus-based immunosuppression and have effects on HCC patients that lead to a decreased risk of tumor recurrence and improved the over survival time after liver transplantation [36–38]. Based on the follow-up date, our result indicates that immunosuppressant strategies (FK506+RAPA group) were not associated with tumor recurrence significantly; an expanded sample is needed for this. Li/Wang/Wang/Zhang/Wu/Zhang/Fu/ Chen
So far, there is no effective and systemic means of diagnosis and treatment for preventing the recurrence of hepatocellular carcinoma after liver transplantation; only an early diagnosis can prevent and treat tumor recurrence and distant metastasis. Especially, some of the patients with recurrence and metastasis do not have obvious clinical symptoms; images did not detect the recurrence tumor site early enough for treatment; this resulted in the poor prognosis of patients after liver transplantation. This experimental study believes that the current study provides the clinical evidence that YAP is an independent prognostic marker for tumor recurrence time of HCC patients after liver transplantation. Thus, our findings may provide help for the assessment of tumor recurrence time and lead to the long-term survival of HCC patients after liver transplantation; they will also help in screening risk populations by theoretical and experimental basis for fur-
ther studying the mechanism of the Hippo signaling pathway. Of course, the retrospective study is a limitation; a prospective study will make more sense to develop strategies to implement in the future.
Acknowledgments We thank that Department of Pathology the Third Affiliated Hospital of Sun Yat-Sen University (Guangzhou, Guangdong, China) for pathological section and pathological diagnosis.
Financial Support This study is supported by China National Key Basic Research and Development Program (973 Program) (No. 2009CB522404) and China National Natural Science Foundation (No. 81172038).
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18 Wang Y, Zhao Q, Ma S, Li Z, Wang E, Qiu X: Overexpression of yes-associated protein contributes to progression and poor prognosis of non-small-cell lung cancer. Cancer Sci 2010;101:1279–1285. 19 Muramatsu T, Imoto I, Matsui T, Kozaki K, Haruki S, Sudol M, et al: YAP is a candidate oncogene for esophageal squamous cell carcinoma. Carcinogenesis 2011;32:389–398. 20 Lamar JM, Stern P, Liu H, Schindler JW, Jiang ZG, Hynes RO, et al: The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain. Proc Natl Acad Sci U S A 2012;109:E2441–E2450. 21 Zhou D, Conrad C, Xia F, Park JS, Payer B, Yin Y, et al: Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 2009; 16: 425–438. 22 Ji T, Liu D, Shao W, Yang W, Wu H, Bian X, et al: Decreased expression of LATS1 is correlated with the progression and prognosis of glioma. J Exp Clin Cancer Res 2012;31:67. 23 Takahashi Y, Miyoshi Y, Takahata C, Irahara N, Taguchi T, Tamaki Y, et al: Down-regulation of LATS1 and LATS2 mRNA expression by promoter hypermethylation and its association with biologically aggressive phenotype in human breast cancers. Clin Cancer Res 2005;11:1380–1385. 24 Li H, Wolfe A, Septer S, Edwards G, Zhong X, Abdulkarim AB, et al: Deregulation of Hippo kinase signalling in human hepatic malignancies. Liver Int 2012;32:38–47. 25 Dong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA, et al: Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 2007;130:1120–1133.
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32 Yamamoto J, Kosuge T, Saiura A, Sakamoto Y, Shimada K, Sano T, et al: Effectiveness of hepatic resection for early-stage hepatocellular carcinoma in cirrhotic patients: subgroup analysis according to Milan criteria. Jpn J Clin Oncol 2007;37:287–295. 33 Dudek K, Kornasiewicz O, Remiszewski P, Kobryn K, Ziarkiewicz-Wroblewska B, Gornicka B, et al: Impact of tumor characteristic on the outcome of liver transplantation in patients with hepatocellular carcinoma. Transplant Proc 2009;41:3135–3137. 34 Margarit C, Charco R, Hidalgo E, Allende H, Castells L, Bilbao I, et al: Liver transplantation for malignant diseases: selection and pattern of recurrence. World J Surg 2002;26:257–263.
35 Perez Saborido B, Loinaz Segurola C, Gimeno Calvo A, Meneu Diaz JC, Abradelo De Usera M, Calvo Pulido J, et al: Liver transplantation for hepatocellular carcinoma: our experience from 1986. Transplant Proc 2003; 35: 1825– 1826. 36 Kneteman NM, Oberholzer J, Al Saghier M, Meeberg GA, Blitz M, Ma MM, et al: Sirolimus-based immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma. Liver Transpl 2004;10:1301–1311. 37 Toso C, Merani S, Bigam DL, Shapiro AM, Kneteman NM, et al: Sirolimus-based immunosuppression is associated with increased survival after liver transplantation for hepatocellular carcinoma. Hepatology 2010; 51: 1237–1243. 38 Zhou J, Wang Z, Wu ZQ, Qiu SJ, Yu Y, Huang XW, et al: Sirolimus-based immunosuppression therapy in liver transplantation for patients with hepatocellular carcinoma exceeding the Milan criteria. Transplant Proc 2008; 40:3548–3553.
Li/Wang/Wang/Zhang/Wu/Zhang/Fu/ Chen
Copyright: S. Karger AG, Basel 2015. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Dig Surg 2014;31:468–478 DOI: 10.1159/000370252
Yes-Associated Protein Expression is a Predictive Marker for Recurrence of Hepatocellular Carcinoma after Liver Transplantation Hua Li Shouhua Wang Guoying Wang ZhiGang Zhang Xiaocai Wu Tong Zhang Binsheng Fu Guihua Chen Department of Hepatic Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou,Guangdong, PR China
Key Words Hepatocellular carcinoma · Liver transplantation · Yes-associated protein YAP · Immunohistochemistry · Tumor recurrence
Abstract Purpose: To explore the expression of Yes-associated protein (YAP) in hepatocellular carcinoma (HCC) patients, and assess its prognostic value to recurrence of HCC after liver transplantation (LT). Methods: Collected data of 105 consecutive patients undergoing LT for HCC were analyzed retrospectively. The immunohistochemistry was used to detect the expression of YAP, Mst1, Lats1/2, pYAP, pLats1/2 and pMst1/2 in tumor tissues. Contingency table and χ2-test were used to investigate the correlation between expression of YAP, Mst1, Lats1/2 and clinical characteristics. Univariate survival analysis and Multivariate Cox regression analysis were also performed to analyze the correlation of clinical and pathological factors with tumor recurrence after LT. The Kaplan-Meier method and log-rank test were used to analyze HCC-specific disease-free survival (DFS) rate. Results: Forty patients fulfilled Milan criteria with 1-, 2-, 3- and 5-years DFS of 86.7, 84.6, 84, 84%, respectively. The positive rates of YAP, Lats1/2, Mst1 in HCC were 51.4, 45.7, 64.8%, respectively. YAP expression in HCC tumors was significantly associated with tumor size (p = 0.041), venous infiltration (p = 0.002), AJCC tumor stage (p = 0.027). Lats1/2 expression was sig-
© 2015 S. Karger AG, Basel 0253–4886/15/0316–0468$39.50/0 E-Mail [email protected] www.karger.com/dsu
nificantly associated with tumor size (p = 0.001) and AJCC tumor stage (p = 0.019). Mst1 expression was also significantly associated with tumor size (p = 0.042). HCC-specific DFS was significantly longer for patients with YAP negative expression compared with patients with YAP positive expression (1-, 2-, 3- and 5-years DFS of 71.7, 65.3, 65.3, 65.3 vs. 42.5, 36.6, 32.5, 30.4%, respectively, log-rank = 12.89, p < 0.001). Multivariate Cox regression analysis indicated that YAP expression (HR = 2.011, p = 0.020) in HCC was an independent prognostic factor for HCC-specific DFS after liver transplantation. Conclusions: YAP is an independent prognostic marker for tumor recurrence for HCC patients after © 2015 S. Karger AG, Basel liver transplantation.
Introduction
Hepatocellular carcinoma (HCC) is the sixth most common malignancy and the third leading cause of cancer death worldwide [1]. Liver transplantation (LT) is the preferred treatment for selected patients with hepatocellular carcinoma (HCC). With liver transplantation development in recent years, the 5-year survival rate is significantly improved [2]. However, recurrence and metastasis
H.L. and S.W. contributed equally to this work.
Guihua Chen Department of Hepatic Surgery, Third Affiliated Hospital of Sun Yat-sen University No. 600 Tianhe Road, Tianhe District Guangzhou, Guangdong 510630 (PR China) E-Mail chenguihua1955 @ 163.com
of HCC after LT remains a formidable problem; several large centers have reported that the recurrence rate was approximately 20%, which is the rate-limiting factor for long-term survival [3]. So currently, prevention and treatment of tumor recurrence is one of the hot issues for the research for HCC patients after liver transplantation. The Hippo signaling pathway originally discovered in Drosophila consists of several negative growth regulators acting in a kinase cascade that ultimately phosphorylates and inactivates Yorkie (Yki), a transcriptional coactivator that positively regulates cell growth, survival, and proliferation. In mammals, the Hippo pathway is well conserved. Core components of the Hippo pathway include the Mst1/2, Lats1/2, and YAP, which consists of a kinase cascade in which Mst1/2 forms a complex with the adaptor protein WW45 and phosphorylates the kinases Lats1 and Lats2 as well as the adaptor protein Mob. A Lats/Mob complex then phosphorylates and represses the transcriptional coactivator YAP. YAP is phosphorylated and inhibited by the Lats tumor suppressor, and this phosphorylation results in its association with 14-3-3 binding motif and from the nucleus into the cytoplasm, where coactivator activity is lost [4–6]. Deregulation of the tumor suppressors in the Hippo pathway and overexpression of YAP during cancer development and progression is now clear. YAP, which was termed a candidate oncogene, is evident in several human cancers, including breast, pancreas, prostate, and liver [7, 8]. Recent studies have demonstrated YAP genes are important for adaptive liver enlargement and tumorigenesis in the liver [9]. Zhao et al. evaluated that among the 115 cases of HCC samples examined, 63 samples (54%) showed strong YAP staining, while 95% of normal liver tissue samples showed very weak staining, suggesting a significant difference in YAP protein levels between normal and cancerous tissues. Recent study has shown that the expression of YAP was significantly correlated with HCC prognosis and YAP was an independent prognostic marker for overall and disease-free survival for HCC patients after partial hepatectomy [10–12]. Moreover, increased expression of YAP in YAP activity and nuclear location in transgenic mice lead to HCC development, which also suggests a direct link between dysregulation of the Hippo pathway and liver tumorigenesis [13]. YAP has been reported to bind and regulate various human transcriptional regulators including p73 and p53-binding protein-2 [14]. Precise biological function and physiological regulation of YAP is unclear as of now. In this study, we analyzed the expression and correlation of YAP, and pYAP, pLats1/2, pMst1/2, Mst1, Lats1/2 YAP Expression Predicts HCC Recurrence after Liver Transplantation
in HCC by immunohistochemical staining. The relationship between YAP, Lats1/2, and Mst1 expression and clinicopathological features was also investigated to understand their role on recurrence of HCC after liver transplantation. The present study was an extension of the study mentioned earlier, and analyzed their clinical significance and relationship with recurrence of HCC after liver transplantation. Materials and Methods Patients and Clinical Samples One hundred and five cases of HCC patients who underwent liver transplantation at the Department of Hepatic Surgery in the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, Guangdong, China) between June 2004 and September 2009 were collected. The preoperative clinicopathological features of the patients were analyzed by the tumors’ maximum diameter, number of tumor lesions, vascular invasion (portal vein or hepatic venous system) and the level of alpha-fetoprotein (AFP) and postoperative pathological data. Diagnostic criteria included the following: (1) All of the cases were consistent with hepatocellular carcinoma (HCC) clinical diagnostic criteria. (2) All of the patients were histologically diagnosed with hepatocellular carcinoma. Inclusion criteria included the following: (1) All of the patients first underwent liver transplantation. (2) Routine post-liver transplantation immunosuppressive regimen was used by tacrolimus-based individualized treatment plan. (3) The hormone therapy did not exceed 3 months, except acute rejection after liver transplantation. Postoperative follow-up included the postoperative follow-up routine in transplant specialist outpatient. All of the patients received standard post-liver transplantation care and follow-up of the experimental study deadline to December 2012. The means of follow-up were through outpatient visits, letters, and phone calls. Patients who were not contactable for 1 year were considered lost. Regular follow-up testing excluded tumor recurrence or metastasis. Patients were observed HCC recurrence as the deadline events. Disease-free survival (DFS) times were calculated from the date of curative surgery to HCC recurrence, death, or the last follow-up date. Patients who were lost to follow-up, due to death or because recurrence was not observed were defined as censored data during the follow-up period. The present study was approved by our institutional ethics committee. Informed consent was obtained from each patient and family members. The procedure met all applicable guidelines of our institute as well as governmental regulations concerning the ethical use of donated organs, and the latest version of the Declaration of Helsinki. Immunohistochemistry (IHC) Pathological data were obtained from the Department of Pathology at the Third Affiliated Hospital of Sun Yat-sen University. Paraffin-embedded tumor tissue blocks were sectioned (4 μm) for immunohistochemical staining. After antigen retrieval and peroxidase blocking, the sections were incubated with rabbit polyclonal antibody against human YAP (127), Phosph-YAP (127) (Cell Signaling, American), rabbit polyclonal antibody against human Mst1, Anti-Mst1/2 (phosphoT183) (Abcam, Hong Kong), rabbit
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Table 1. Site of hepatocellular carcinoma recurrence or metastasis after liver transplantation
YAP (positive) YAP (negative)
Cases of tumor recurrence
Liver
Lung
Intra-abdominal metastasis
Liver + lung
Liver + lung + bone
37 17
26 14
6 2
2 0
1 1
2 0
polyclonal antibody against human Lats1/2, Phosph-Lats1/2 (immunoway, Hong Kong), working dilution are respectively 1: 200, 1:200, 1:550, 1:60, 1:200, 1:200, at 4 ° C overnight. After 3 washings in sterile phosphate-buffered saline, sections were incubated with a horseradish peroxidase (HRP) conjugated antibody against rabbit or mouse immunoglobulin G (IgG) (Gene Tech, Shanghai, China). For negative controls, sections were treated with 0.01 mol/l phosphate-buffered saline instead of primary antibodies.
IHC Staining Evaluation YAP was specifically located in the cytoplasm and the nucleus of carcinoma cells, pYAP (127), Lat1/2, pLats1/2, Mst1, pMst1/2 (phosphoT183) was specifically located in the cytoplasm of carcinoma cells. Staining intensity (A) was classified as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The percentage of positive cells (B) examined in 500 cells were divided into 0 (75%). According to the product of A and B, the IHC result was classified as 0, negative (–); positive was classified as 1–4, weakly positive (+); 5–8, moderately positive (++) and 9–12, strongly positive (+++), proteins immunoreactivity was graded as negative (score 0) and positive (score weakly positive and above is positive) according to the previously reported procedures [15]. Statistical Analysis Statistical analysis was performed using SPSS 13.0 Package, Contingency table and χ2-test were used to investigate the correlation between expression of YAP, Mst1, Lats1/2 and clinical characteristics (the Student t test for continuous data). Univariate and Multivariate Cox regression analyses of YAP and other proteins in HCC were also performed to determine the association expression of YAP, pYAP, pLats1/2, Lats1/2, pMst1/2, Mst1 and individual clinicopathologic variables with DFS times after liver transplantation. The Kaplan-Meier method and log-rank test were used to generate curves for HCC-specific disease-free survival (DFS). The prognostic factors for this cohort (tumor size, venous infiltration, tumor stage [American Joint Committee on Cancer (AJCC)], and α-fetoprotein AFP, etc.) were subsequently subjected to the multivariate Cox regression analysis to determine the hazards ratios (HRs) and the independence of effects. p < 0.05 was considered statistically significant.
Results
Protein Expression in Hippo Signaling Pathway in Hepatocellular Carcinoma Tissues All 105 patients included 98 men (93.3%) and 7 women (6.7%). Age ranged 22–72 years with the median age of 46. 470
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Follow-up time ranged 1–96 months with the median follow-up time of 13 months. Eight patients lost to followup. During follow-up, tumor recurrence was observed in 54 cases, that is, in 51.4% of patients. The most common site of recurrence was in the grafted liver for 40 cases, and lung metastasis for 8 cases, and intra-abdominal metastasis for 2 cases. Additionally, some patients were diagnosed with combined organ recurrence or metastasis to the liver, lung, or bone (liver + lung is 2 case, liver + lung + bone is 2 case) (table 1), and recurrence time was 1–41 months. Of 105 patients, 40 fulfilled the Milan criteria with 1-, 2-, 3- and 5-years DFS of 86.7, 84.6, 84, 84%, respectively. The 105 HCC cases were grouped according to the expression scores. The positive rate of YAP in HCC cases was 51.4% (54/105) (fig. 1). YAP was specifically located in the cytoplasm and nucleus of carcinoma cells (fig. 2a). pYAP, Lats1/2, pLats1/2, Mst1, and pMst1 were specifically located in the cytoplasm of carcinoma cells (fig. 2b, 3a, b, 4a, b). The positive rate of Lats1/2 in HCC cases was 45.7% (48/105). The positive rate of Mst1 in HCC cases was 64.8% (68/105). The positive rates of pYAP, pLats1/2, and pMst1/2 were 55.2% (58/105), 9.5% (10/105), and 21% (21/105), respectively (fig. 1). The Correlation of YAP, Mst1, Lats1/2 with Clinical Characteristics The clinical association analysis by Contingency table and χ2-test revealed that YAP expression in HCC was significantly associated with the tumor size (χ2 = 4.173, p = 0.041), venous infiltration (χ2 = 5.875, p = 0.002), and tumor stage (χ2 = 7.545, p = 0.027) (table 2). Lats1/2 expression in HCC tumors was significantly associated with tumor size (χ2 = 14.413, p = 0.001) and AJCC tumor stage (χ2 = 7.850, p = 0.019). Mst1 expression in HCC tumors was also significantly associated with tumor size (χ2 = 4.129, p = 0.042) (table 2). Association between Protein Expression and HCC-Specific Disease-Free Survival (DFS) We examined whether the correction of expression of YAP or other core components of Hippo pathway and Li/Wang/Wang/Zhang/Wu/Zhang/Fu/ Chen
100
90 (90.5%)
Positive Negative
84 (80%)
The number of the cases
80
60
58 57 54 (55.2%) (54.3%) 51 (51.4%) 48 47 (49.6%) (45.8%) (45.7%)
37 (36.2%)
40
21 (20%)
20
0
10 (9.5%)
YAP
pYAP
Lats1/2
pLats1/2
Mst1
pMst1/2
Color version available online
Fig. 1. Positive and negative expression cases and rates of YAP, pYAP, Lats1/2, pLats1/2, Mst1 and pMst1/2 in clinical samples of hepatocellular carcinoma (HCC).
68 (64.8%)
a
b
c
d
Fig. 2. a YAP was specifically located in the cytoplasm and nucleus of carcinoma cells, and positive expression of YAP in clinical samples of hepatocellular carcinoma (HCC) is shown. b pYAP was specifically located in the cytoplasm and carcinoma cells, and positive expression of YAP in clinical samples of hepatocellular carci-
noma (HCC) is shown. c Negative expression of YAP in clinical samples of hepatocellular carcinoma (HCC) is shown. d Negative expression of pYAP in clinical samples of hepatocellular carcinoma (HCC) is shown (IHC, ×200).
YAP Expression Predicts HCC Recurrence after Liver Transplantation
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471
Color version available online
a
b
c
d
Fig. 3. a Lats1/2 was specifically located in the cytoplasm of carci-
noma cells, and positive expression of Lats1/2 in clinical samples of hepatocellular carcinoma (HCC) is shown. b pLats1/2 was specifically located in the cytoplasm and carcinoma cells, and positive expression of pLats1/2 in clinical samples of hepatocellular carci-
clinicopathologic feature with HCC-specific DFS after liver transplantation. Univariate survival analysis indicated that the expression of YAP, Lats1/2, pMst1/2 and cellular differentiation, serum AFP level, venous infiltrationumor stage, and tumor size were associated with HCC-specific DFS after liver transplantation (p < 0.05) (table 3). Multivariate Cox regression analysis indicated that positive expression of YAP (HR = 2.011, p = 0.020), cellular differentiation (HR = 2.390, p = 0.001), tumor stage (HR = 1.574, p = 0.032), and tumor size (HR = 2.176, p = 0.021) were significantly associated with HCC-specific DFS after liver transplantation (table 4). Kaplan-Meier analysis also revealed HCC-specific DFS was significantly longer for patients with YAP negative expression compared with patients with YAP positive expression (1-, 2-, 3-, and 5-years DFS of 71.7, 65.3, 65.3, 65.3 vs. 42.5, 36.6, 32.5, 30.4%, respectively, log-rank = 12.89, p < 0.01) (fig. 5). Thus, our data indicated that YAP expression in HCC was a potential indicator for early disease recurrence. 472
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noma (HCC) is shown. c Negative expression of Lats1/2 in clinical samples of hepatocellular carcinoma (HCC) is shown. d Negative expression of p Lats1/2 in clinical samples of hepatocellular carcinoma (HCC) is shown (IHC, ×200).
Discussion
In the past decades, the Hippo pathway has been shown to have a critical role in controlling organ size by regulating both cell proliferation and apoptosis. Furthermore, the Hippo pathway was defined as a tumor suppressor pathway [5]. Some studies have put forth mechanisms involved in the deregulation of Hippo pathway during liver cancer pathogenesis in animals [16]. Our immunohistochemistry investigation found that the positive rates of YAP in HCC was approximately 51.4% (54/105). YAP expression in HCC was significantly associated with the tumor size, venous infiltration, and tumor stage. YAP, as a driving oncogene, is known to be amplified and nuclear location in multiple types of human cancers and strongly correlate with poor patient outcome, including ovarian cancers [17], lung cancers [18], esophageal squamous cell carcinoma [19] and prostate cancers [5]. In human HCC, elevated YAP expression Li/Wang/Wang/Zhang/Wu/Zhang/Fu/ Chen
Color version available online
a
b
c
d
Fig. 4. a Mst1 was specifically located in the cytoplasm of carci-
noma cells, and positive expression of Mst1 in clinical samples of hepatocellular carcinoma (HCC) is shown. b pMst1/2 was specifically located in the cytoplasm and carcinoma cells, and positive expression of pMst1/2 in clinical samples of hepatocellular carci-
noma (HCC) is shown. c Negative expression of Mst1 in clinical samples of hepatocellular carcinoma (HCC) is shown. d Negative expression of pMst1/2 in clinical samples of hepatocellular carcinoma (HCC) is shown (IHC, ×200).
and nuclear localization have been observed. Previous reports reveal that 54% of 115 HCC patients showed overexpression of YAP in a tissue microarray study in which the majority of normal liver demonstrated very weak staining [10]. However, as existing a tumor suppressor pathway, up to now, we have not found any association between the expression of YAP and tumor recurrence for HCC patients after liver transplantation. Multivariate Cox regression analysis in our study revealed the association of YAP overexpression with HCC-specific diseasefree survival, and YAP was found to be an independent indicator of tumor recurrence for HCC patients after liver transplantation. By analyzing 177 pairs of HCC patients and matched normal samples with complete clinical records, Xu et al. suggest that YAP was determined to be an independent prognostic marker for the overall survival and disease-free survival for HCC patients after hepatectomy [12]. Our findings were also consistent with their conclusion. Lamar et al. suggest that increased
YAP/TEAD activity promote the metastatic potential of breast cancer and melanoma cells, and YAP/TEAD activity plays a causal role in cancer metastasis [20]. We speculate that YAP may play an important role in tumor recurrence and metastasis of HCC after hepatectomy or liver transplantation, and future research is needed to prove this. Our study showed Lats1/2 expression in HCC tumors was significantly associated with the tumor size and AJCC tumor stage, and Mst1 expression was also significantly associated with the tumor size. Univariate survival analysis revealed the increased pMst1/2 expression and decreased Lats1/2 expression, where the association with HCC-specific DFS and Multivariate Cox regression analysis is not applicable. Upstream components of the Hippo pathway are involved in HCC via regulation of YAP expression. The MST1/2 regulated the activation states of LATS1 and LATS2, which directly phosphorylate LATS, and by MOB (Mats in Drosophila) family members, small
YAP Expression Predicts HCC Recurrence after Liver Transplantation
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Table 2. The association of expression of YAP, Lats1/2 and Mst1 in clinical samples of hepatocellular carcinoma (HCC) with clinicapathologic features Clinicapathologic features
Classifies of features
Patients, n (%)
Sex
men women
98 (93.3) 7 (6.7)
χ2
YAP +
–
49 5
49 2
Age&
p value
1.201
0.273
1.837
0.069
Lats1/2 +
–
46 2
52 5
χ2
p value
0.888
0.346
0.983
0.328
Mst1 +
–
62 6
36 1
χ2
p value
1.443
0.230
0.751
0.454
Tumor size, cm
≤5 >5
51 (48.6) 54 (51.4)
21 33
30 21
4.173
0.041*
33 15
18 39
14.413
0.001*
38 30
13 24
4.129
0.042*
Tumor number
1 ≥2
55 (52.4) 50 (47.6)
25 29
30 21
1.650
0.199
26 22
29 28
0.113
0.737
34 34
21 16
0.439
0.737
Cellular differentiation
well differentiated moderately differentiated poorly differentiated
27 (25.7) 65 (61.9) 13 (12.4)
10 36 8
17 29 5
3.178
0.204
15 29 4
12 36 9
2.255
0.324
18 42 8
9 23 5
0.103
0.950
Serum AFP level, ng/ml 5
51 (15) 54 (39)
0.000*
3.559
1.921–6.595
Serum AFP level, ng/ml
0.05 no data is shown.
YAP Expression Predicts HCC Recurrence after Liver Transplantation
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Table 4. Multivariate analysis was performed using Cox regression to analyze the association of protein expres-
sion with HCC-specific disease-free survival (DFS) after liver transplantation Variables
B
SE
p
Hazard risk (HR)
95% CI
YAP Mst1 pMst1/2 Lats1 Age Cellular differentiation Serum AFP level AJCC tumor stage Tumor size Venous infiltration
0.699 – – – – 0.871 – 0.454 0.777 –
0.301 – – – – 0.256 – 0.211 0.336 –
0.020* 0.767 0.615 0.730 0.178 0.001* 0.101 0.032* 0.021* 0.585
2.011 – – – – 2.390 – 1.574 2.176 –
1.115–3.628 – – – – 1.448–3.945 – 1.041–2.381 1.125–4.206 –
YAP expression in tumor YAP negative YAP positive 1-censored 2-censored
1.0
Disease-free survival rate
0.8
0.6
Color version available online
* Statistically significant. – = p > 0.05 no data is shown.
0.4
0.2
0 0
20
40
60
80
100
Months after liver transplantation
Fig. 5. Kaplan-Meier disease-free survival analyses of hepatocel-
lular carcinoma (HCC) patients based on Yes-associated protein (YAP) immunoreactivity are shown. YAP positive expression (n = 54) in tumors was found to be associated with poor disease-free survival (DFS) in 105 patients who underwent liver transplantation.
stream negative regulators of YAP/TAZ ( Mst1/2 and Lats 1/2) has such multiple mutations that would be required to inactivate the pathway in a tumor. The YAP-TAZTEAD complex is a downstream effector of the Hippo pathway, and in cooperation with the TEAD transcription factor, overexpression of YAP could aberrantly activate an array of target genes (e.g. CTGF, CCND1, ITGB2, 476
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and BCL2L1) responsible for cell proliferation, antiapoptosis, survival, and migration. Among the four transcription factor TEADs proteins, TEAD1 and TEAD4 are most often associated with proliferation and cancer development [25–27]. The activation of YAP/TAZ could be the result of other growth-regulating pathways such as Ras, TGF-β, and Wnt, which exert suppressive pressure on Hippo signaling, such that deregulation of those pathways leads indirectly to the activation of YAP/TAZ [28, 29]. Factors affecting tumor recurrence of HCC after liver transplantation have been previously reported with preoperative AFP levels [30, 31], tumor size [32, 33], histologic grade [33, 34], and vascular invasion [33, 35]. Our experimental results suggest cellular differentiation (p = 0.001), tumor stage (p = 0.032), and tumor size (p = 0.021) in HCC were significantly associated with HCC-specific diseasefree survival after liver transplantation. Most patients after liver transplantation used tacrolimus-based individualized immunosuppressant strategies. The definition of the most appropriate immunosuppression combination, allowing decreased risk of tumor recurrence, is still a matter of debate. No single protocol has gained broad acceptance until now. Several reports have suggested a lower risk of tumor recurrence with the use of sirolimus-based immunosuppression and have effects on HCC patients that lead to a decreased risk of tumor recurrence and improved the over survival time after liver transplantation [36–38]. Based on the follow-up date, our result indicates that immunosuppressant strategies (FK506+RAPA group) were not associated with tumor recurrence significantly; an expanded sample is needed for this. Li/Wang/Wang/Zhang/Wu/Zhang/Fu/ Chen
So far, there is no effective and systemic means of diagnosis and treatment for preventing the recurrence of hepatocellular carcinoma after liver transplantation; only an early diagnosis can prevent and treat tumor recurrence and distant metastasis. Especially, some of the patients with recurrence and metastasis do not have obvious clinical symptoms; images did not detect the recurrence tumor site early enough for treatment; this resulted in the poor prognosis of patients after liver transplantation. This experimental study believes that the current study provides the clinical evidence that YAP is an independent prognostic marker for tumor recurrence time of HCC patients after liver transplantation. Thus, our findings may provide help for the assessment of tumor recurrence time and lead to the long-term survival of HCC patients after liver transplantation; they will also help in screening risk populations by theoretical and experimental basis for fur-
ther studying the mechanism of the Hippo signaling pathway. Of course, the retrospective study is a limitation; a prospective study will make more sense to develop strategies to implement in the future.
Acknowledgments We thank that Department of Pathology the Third Affiliated Hospital of Sun Yat-Sen University (Guangzhou, Guangdong, China) for pathological section and pathological diagnosis.
Financial Support This study is supported by China National Key Basic Research and Development Program (973 Program) (No. 2009CB522404) and China National Natural Science Foundation (No. 81172038).
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