Postgraduate Medicine
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The Role of Liver Cancer Stem Cells in Donor Liver Allocation for Patients With Hepatocellular Carcinoma Jie Zhou MD, Zhenhua Hu MD, Zhiwei Li MD, Pengfei Yu MD, Jian Wu MD, PhD & ShuSen Zheng MD, PhD, FACS To cite this article: Jie Zhou MD, Zhenhua Hu MD, Zhiwei Li MD, Pengfei Yu MD, Jian Wu MD, PhD & ShuSen Zheng MD, PhD, FACS (2013) The Role of Liver Cancer Stem Cells in Donor Liver Allocation for Patients With Hepatocellular Carcinoma, Postgraduate Medicine, 125:6, 24-30 To link to this article: http://dx.doi.org/10.3810/pgm.2013.11.2709
Published online: 13 Mar 2015.
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C L I N I C A L F E AT U R E S
The Role of Liver Cancer Stem Cells in Donor Liver Allocation for Patients With Hepatocellular Carcinoma
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DOI: 10.3810/pgm.2013.11.2709
Jie Zhou, MD 1–3 Zhenhua Hu, MD 1–3 Zhiwei Li, MD 1–3 Pengfei Yu, MD 1–3 Jian Wu, MD, PhD 1–3 ShuSen Zheng, MD, PhD, FACS 1–3 Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China; 2Key Laboratory of Combined MultiOrgan Transplantation, Ministry of Public Health, First Affiliated Hospital of Zhejiang University, Hangzhou, China; 3Key Laboratory of Organ Transplantation, First Affiliated Hospital of Zhejiang University, Hangzhou, China 1
Abstract: Previous criteria for liver transplantation in patients with hepatocellular carcinoma (HCC) has predominantly emphasized the size and number of existent tumors; however, criteria have not included tumor biology, which may comprise a critical factor for predicting patient prognosis. This issue has been recognized in the Hangzhou criteria, which take tumor histopathologic grade and pre-transplant a-fetoprotein (AFP) level into consideration. However, neither histopathologic grading nor AFP level are precise enough to adequately represent tumor biology in patients with HCC. Recent research has indicated that the development and progression of HCC are driven by a subpopulation of liver cells with stem cell features (cancer stem cells, [CSCs]). Liver CSCs with cluster of differentiation (CD)133 antigen positivity show a high tumorigenic capacity, and the increase in the percentage of CD133+ liver cancer cells is consistent with increased levels of serum AFP. In addition, the number of CD90+ cells increases with the tumorigenicity of HCC, and a positive correlation between the number of circulating CD90+ liver CSCs and disease progression has been observed. As liver CSCs can be detected using the CD profile and could more accurately represent tumor biology in HCC, we hypothesized that liver CSCs with specific phenotypes could be used for modifying the Hangzhou criteria, thereby providing valuable guidance for the development of more accurate prognosis prediction algorithms for patients with HCC being considered for liver transplantation. We provide reliable evidence supporting this hypothesis, and offer proposals for future applications in transplant practice. Keywords: cluster of differentiation; CD133+; CD90+; hepatocellular carcinoma; liver transplantation; liver cancer stem cells
Introduction
Correspondence: ShuSen Zheng, MD, PhD, FACS, N0. 79 Qingchun Road, Hangzhou, 310003, China. Tel: 86-571-87236570 Fax: 86-571-87236616 E-mail: [email protected]
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Liver transplantation is considered the optimal treatment for patients with end-stage liver disease, including hepatocellular carcinoma (HCC), because the procedure results in the removal of all tumor cells and correction of liver dysfunction.1 However, with increasing candidates on the transplantation waiting list and organ resource shortage, wide application of liver transplantation for patients with HCC has been restricted. Understanding how to best allocate the limited number of available donor grafts has become an important issue in transplant practice. Criteria for selecting suitable candidates for liver transplantation have been established. The most used criteria sets are 1) the Milan criteria: solitary tumor with a diameter # 5 cm, or # 3 tumors with the largest # 3 cm; no vascular invasion; and 2) the University of California, San Francisco (UCSF) criteria: solitary tumor # 6.5 cm, or # 3 tumors, with the largest # 4.5 cm and accumulative diameters # 8 cm; without macrovascular invasion.2,3 Both sets of criteria, however, mainly focus on tumor morphology, which emphasizes the importance
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Liver CSCs in Donor Allocation for HCC
of the number and size of the tumors. Histologic grade of the tumor is not included, which may be a critical factor for predicting patient survival following liver transplantation.4 Several studies in the literature have observed the predictable value of histologic grading in patients with HCC5,6; therefore, we proposed the Hangzhou criteria: apart from the presence of macrovascular invasion, patients with HCC must fulfill 1 of 2 criteria: a) total tumor diameter # 8 cm; or b) total tumor diameter . 8 cm, with histopathologic grade I or II and preoperative a-fetoprotein (AFP) level simultaneously # 400 ng/mL.7 However, this approach is not precise enough to allow adequate grading, such as mild, moderate or severe, and therefore a more precise strategy for patient pre-transplant evaluation is needed. Research indicates that the development and progression of HCC is driven by a small population of cancer stem cells (CSCs), which share multiple characteristics of tissue stem cells and have extensive proliferative, self-renewal, and differential potential.8 The presence of CSCs in human liver with specific phenotypes has been repeatedly reported in recent years, with surface markers like cluster of differentiation (CD)133, CD90, and epithelial cell adhesion molecule (EpCAM) identified to classify liver CSC subpopulations. One study by Zeng et al9 revealed that the presence of high pre-transplant EpCAM staining in HCC liver explants is an independent predictor for tumor recurrence in recipients who underwent liver transplantation. Liver CSCs with specific phenotypes could serve as an important predictive factor in the setting of liver transplantation. We have increasingly recognized that liver CSCs might be the initiator cells for tumor formation and that they may serve to maintain tumor growth. They are also involved in the origin of metastases and recurrence, and may therefore accurately represent the tumor biology of HCC.
Hypotheses
We propose the hypothesis that liver CSCs with specific CD markers could be detected and selected as critical precise assessment tools for tumor biology in patients with HCC. The markers can be utilized for guiding liver transplantation for management of patients with HCC. An analysis of the quantity and localization of liver CSCs could reflect the tumor biology of HCC, which is closely related to patient prognosis following liver transplantation. The addition of such tumor biology analysis to modify our Hangzhou criteria could surmount existing simplistic, crude, and subjective assessments of histopathologic grading in patients with HCC. Based on inclusive, meticulous pre-transplant e valuation of tumor
biology, focusing on specific biomarkers as well as recipient prognosis, the modified Hangzhou criteria would allow the developments of more accurate prediction algorithms for post-transplant survival of HCC patients. We believe that the use of liver CSCs with specific CD markers in this context would be advantageous and could aid the achievement of more satisfactory outcomes.
New Elements for Assessing Candidacy for Liver Transplantation in Patients With HCC
Previously, applying measures from the 2 widely used c riteria sets, the Milan and UCSF criteria (both based on tumor morphology, emphasizing the importance of the number and size of the tumors), patients with HCC who met the selection criteria achieved excellent long-term survival outcomes. The 4-year overall patient survival rate has been 85%, and the patient tumor-free survival rate at 92% for recipients fulfilling the Milan criteria.2 The 1-year and 5-year overall survival rates have been 90% and 75.2%, respectively, for patients who fulfilled the UCSF criteria.3 A study to assess the predictability value of the Milan criteria compared with the UCSF criteria suggested the superiority of the UCSF criteria in predicting acceptable post-transplant patient outcomes.10 The suggested importance of tumor gross features was asserted in observations from a meta-analysis performed by Burroughs et al11—that assessment of the diameter of the largest tumor and total tumor diameters were the best predictors for patient post-transplant prognosis. However, tumor morphology alone may not provide the best possible index for patient outcomes when other biologic marker measures can be taken into account. Data from many studies have indicated that microvascular invasion is the most critical factor in determining the likelihood of HCC recurrence.12–18 Tumor differentiation has been proposed as a predictor for microscopic vascular invasion.5,19 Currently, more research is emphasizing the critical role of tumor histologic grading in predicting long-term prognosis in patient with HCC who are liver recipients.5,6 Previous clinical data revealed that patients with a single large mass after hepatic resection can have a good outcome, similar to the survival rate of patients with small HCC, and significantly better than that of patients with nodular HCC20; however, patients with a small mass can have an adverse outcome, e mphasizing the critical role of histologic grading in patients with smallmass HCC.21 The study by Merani et al22 investigated the predictive role of pre-transplant AFP levels in patients with HCC on outcomes following liver transplantation.
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Zhou et al
The researchers found that patients with pre-transplant AFP levels . 400 ng/mL, whose AFP levels were successfully reduced to , 400 ng/mL could achieve comparable intent-to-treat (ITT) and post-transplant survival rates as those patients with pre-transplant AFP levels , 400 ng/mL, and significantly superior to patients with AFP levels that remained . 400 ng/mL. Merani et al also demonstrated that latest patient pre-transplant AFP level was an independent predictor for both ITT and post-transplant survival rates and suggested use of the pre-transplant AFP level in combination with other morphological variables to assess suitability of candidates for liver transplantation. The result was in accordance with previous research, which had demonstrated the close relationship between pre-operative patient AFP level and patient long-term survival.23,24 In another study, Toso et al25 attempted to reassess recipient selection criteria for patients with HCC, and found the combination of patient total tumor volume and pre-transplant AFP level (cutoff at AFP level of 400 ng/mL) could most effectively predict recipient survival. So it appears that neither the number nor the size of the tumors alone is a determining factor for patient prognosis after transplantation. As a matter of fact, the number and size of tumors may change at different times in the disease course of patients with HCC. Images obtained through many modalities (including computed tomography [CT], magnetic resonance imaging [MRI], contrast ultrasound) could not demonstrate this dynamic process, but could only describe the 2 indexes at specific time-points of tumor growth. We recognized this limitation and therefore introduced histopathologic grade and pre-transplant AFP levels into the liver transplantation candidacy assessment for patients with HCC, using the Hangzhou criteria. Studies validated that recipients who met the H angzhou criteria achieved comparable survival rates to those who met the Milan criteria.7,26,27 Patient 1-, 3-, 5-year overall survival rates and tumor-free survival rates were 92.8%, 70.7%, 70.7%, and 83.7%, 65.6%, 62.4% respectively, in patients fulfilling the Hangzhou criteria, significantly better than those exceeding the Hangzhou criteria, with 49.9%, 27.0%, 18.9%, and 25.8%, 12.5%, 4.7%, respectively.7 In our study, survival rates were similar between recipients fulfilling the Milan criteria and the Hangzhou criteria, which was also confirmed by the research of Audet et al.7,27 We have found that 2 problems remain when using both tumor histopathology grading and patient pre-transplant AFP level to predict patient outcomes. First, the approach is not precise enough to allow grading as mild, moderate, or severe. It relies too heavily on the experience of the i ndividual 26
pathologist, which is relatively subjective. Secondly, recent data have suggested that patient AFP level measure is not sensitive or specific enough for general use as an effective diagnostic and surveillance assay for HCC as was once thought. Moreover, an elevated AFP level can also be observed in some patients with intrahepatic cholangiocarcinoma (ICC) and liver diseases originating from colon tumor metastasis.28,29 Other research has suggested that patients with a tumor diameter , 2 cm seldom have elevated AFP levels and that some patients with HCC (approximately 25%) have normal AFP levels.30,31 Therefore, patient pre-transplant AFP level may not offer an accurate predictor for post-transplant prognosis in patients with HCC. More recently, the relationship between gene signatures, as well as microRNA (miRNA) and epigenetic alterations, and prognostic prediction in patients with HCC have been increasingly recognized.32–35 Biomarkers, including G3, EpCAM, and miR-26, have been identified in patients with HCC.36 Such factors should be included in tumor biology assessment so that a more accurate patient prognosis evaluation algorithm and prediction of treatment response can be developed in liver transplantation.
Liver CSCs and Biologic Markers as Critical Factors for Determining Patient Post-Transplant Prognosis
The important role of liver CSCs in carcinogenesis has been increasingly recognized. Liver CSCs share many signaling pathways that exist in normal stem cells, such as Wnt proteins and transforming growth factor β (TGF β) pathways, which are mainly involved in the processes of cell proliferation, self-renewal, and differentiation, and would be deregulated in liver CSCs. The miRNA also plays a key role in the development of HCC, and recent literature suggests the important function of miRNA in liver CSCs. A study by Li et al37 utilized side population (SP) sorting to the culture of HCC cells and normal liver cells, and subsequently screened miRNA expression profiles between the 2 cell types. Distinct miRNA expression during hepatocarcinogenesis was observed, providing a useful understanding of miRNA function in liver CSCs. Increasing evidence suggests that liver CSCs with specific phenotypes play an important role in hepatocellular carcinogenesis. Commonly known as Prominin-1 (PROM1) in humans, CD133 has been widely recognized as an important stem cell surface marker in both normal and various cancer cells.38 Generally, CD133 is not expressed in normal adult liver cells but it could re-express in patients with HCC.
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Liver CSCs in Donor Allocation for HCC
Suetsugu et al39 first reported that CD133 antigen-positive (CD133+) cells marked a liver CSC subpopulation, which was subsequently confirmed by Yin et al.40 Liver CD133+ CSCs demonstrate high tumorigenic and self-renewal capacities,39–41 which may be partially explained by involvement in long interspersed element (LINE)-1 hypomethylation (one of most common molecular abnormalities during the process of carcinogenesis) and through the enhancement of interleukin (IL)-8 signaling pathway activity induced by network-identified neurotensin.42,43 Song et al44 found that an increase in the percentage of CD133+ liver cancer cells was consistent with increased serum AFP levels, relatively higher histopathologic grades, and a poor prognosis in patients with liver cancer. Furthermore, multivariate analyses identified elevated expression of CD133 to be an independent predictor for overall and tumor-free survival rates in patients with HCC. The study by Ma et al45 analyzed clinical samples from HCC and non-HCC tissues by flow cytometry and immunohistochemistry analysis and observed that CD133 occurred in only a small subpopulation in human HCC tissue. However, no expression of CD133 could be detected in normal liver tissue. Similarly, Ma et al found that the presence of CD133+ cells correlated with advanced stage of disease, higher tumor recurrence, and worse patient prognosis. A meta-analysis performed by Ma et al46 assessed 12 studies involving . 1300 patients to evaluate the presence of liver CSCs and CD133 expression as prognostic factors in patients with HCC. A significant correlation between the presence of liver CSCs and impaired overall and tumor-free survival were observed (HR 1.62 and 1.85, respectively) in patients, and CD133 was identified as having significant prognostic value in patients with HCC. The meta-analysis provided important implications for evaluating stem cell surface markers, like CD133, in liver CSCs in future clinical practice. In addition, CD90 may also be a marker for liver CSCs. A glycosylphosphatidylinositol (GPI)-anchored glycoprotein, CD90 (also known as Thy-1), expressed mainly in peripheral blood leukocytes, plays a role in cell-to-cell and cell-tomatrix interactions.47 Research by Yang et al48 on cell lines found that the number of CD90+ cells increased with HCC tumorigenicity. All clinical tumor tissue and most blood samples from patients with HCC contained the CD45+CD90+ population and there was also a positive relationship between CD90+ cells from HCC tissues and blood samples. Additionally, in their animal research, Yang et al used CD45-CD90+ cells separated from both tissue and blood samples to form tumor nodules with similar pathologic characteristics in immunodeficient mice, whereas the CD45-CD90- cells could
not generate such nodules. The study provided p rofound evidence of self-generation properties of CD90+ cells in patients with HCC, revealing that CD90+ is adequately sensitive and specific as a tumor marker for liver CSCs. In another study performed by his group, Yang found a positive correlation between the number of circulating CD90+ CSCs and disease progression in patients.49 A recent study on liver CSCs by Yamashita et al, using gene-expression analysis, observed that CD90+ cells have the features of vascular endothelial cells.50 When correlated with clinicopathologic characteristics, the researchers found that the expression of CD90+ cells was associated with a higher probability of distant metastasis. Moreover, a highly metastatic capacity was also observed when they implanted CD90+ cells derived from primary HCC into immunodeficient mice. All results suggest specific gene-expression patterns in liver CSCs with CD90+, and highlights the importance of CD90+ as a marker to help determine the prognosis of patients with HCC. Liver CSCs can be detected using the CD profile and can therefore more precisely represent the tumor biology in patients with HCC. Based on results from previous research in liver CSCs, specifically stem cell surface markers, we offer the following model and verification strategies: 1) Isotope labeling has been adopted to characterize specific CD profile expression (CD133+ and/or CD90+) targeted to liver CSCs. 2) Subsequently, we used emission computed tomography (ECT) imaging for local scanning. 3) Active uptake foci were usually observed, and we then analyzed the quantity and localization of liver CSCs for accurate evaluation of HCC tumor biology. 4) Two conditions may indicate adverse tumor biology: (a) if the active uptake foci exceed the range of the tumor (Figure 1A); and/or (b) if the active uptake foci are limited to the tumor, but such foci are also detected in other parts of the liver (Figure 1B). Either condition may indicate that the tumor has infiltrated adjacent liver tissue or that intrahepatic metastases are present, which may be associated with defective tumor biology. Poor patient prognosis can therefore be predicted, despite liver transplantation. 5) In the presence of either or both of these 2 conditions, the patient is taken off the transplant waiting list and referred for other treatment, such as transarterial chemoembolization (TACE), radiofrequency ablation (RFA), or percutaneous ethanol injection (PEI). Only patients with active uptake foci limited to the tumor nodule (Figure 1C) are selected for liver transplantation. This is a novel but potentially effective strategy of choice for pre-transplant patient evaluation (Figure 2).
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Zhou et al Figure 1. A) Active uptake foci exceed the range of the tumor. B) Active uptake foci are limited to the tumor, but several are present in other parts of the liver. C) Active uptake foci are limited to the tumor range.
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Figure 2. Flow chart of steps post-selection for liver transplantation: a) Label isotope, CD133+ or CD90+. As seen on ECT, b) active uptake foci limited to the tumor range. [Proceed to transplantation.] c) Active uptake foci exceed the range of the tumor, or active uptake foci limited to the tumor but foci also detected in other parts of the liver. [Proceed to other treatment], such as d) transarterial chemoembolization (TACE), radiofrequency ablation (RFA), or percutaneous ethnol injection (PEI). 3DWLHQWVPHHW+DQJ]KRXFULWHULD ,QWUDYHQRXVLVRWRSHODEHOLQJ WRFKDUDFWHUL]H&'SURILOHD /LYHUVFDQQLQJE\(&7
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Abbreviations: CD, cluster of differentiation; ECT, emission computed tomography; PEI, percutaneous ethanol injection; RFA, radiofrequency ablation; TACE, transarterial chemoembolization.
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Conclusion
The Milan criteria and the UCSF criteria for determining patient candidacy for liver transplantation as a management strategy for HCC both emphasize the number and size of tumor masses, and do not include tumor biology. Although the Hangzhou criteria involve AFP level and tumor histopathologic grade in the selection of patients with HCC for liver transplantation, problems such as the crude assessment can occur. Thus, new strategies with inclusive criteria that can accurately reflect tumor biology using specific biomarkers are urgently needed for improving outcomes for patients undergoing liver transplantation in the management of HCC. Liver CSCs with specific CD profiles (CD133+ or CD90+) demonstrate a high tumorigenic capacity, and correlate with pathologic grading and a poor prognosis in patients with HCC. Isotope labeling has been adopted to characterize CD profiles, which can be detected by ECT. The quantity and localization of liver CSCs can be precisely identified, and early infiltration and intrahepatic metastases, which are not identified by other radiologic investigations, can be detected. As a result, patients with HCC can be selected for liver transplantation according to the amount and distribution of liver CSCs. Liver CSCs, which can be detected quantitatively and reflect tumor biology for HCC subpopulations, enhance guidance for the use of liver transplantation in the management of patients with HCC. However, the best method for detecting liver CSCs, not the specific CD profile, remains uncertain, and the possibility of combining the CD profile with other new biomarkers needs further investigation. The aim of our study was to investigate new factors that could usefully represent tumor biology and to modify criteria for the use of liver transplantation in patients with HCC. Liver CSCs appear to meet these requirements. Our hypothesis is a prompt for further investigations. Studies should be initialized to build more comprehensive models for the detection and the quantitative analysis of liver CSCs. There is also a need to evaluate the benefit of liver CSCs as a new criterion for the use of liver transplantation in the management of HCC.
Acknowledgment
We acknowledge Sunyi Ye for his excellent technical assistance.
Conflict of Interest Statement
Jie Zhou, MD, Zhenhua Hu, MD, Zhiwei Li, MD, Pengfei Yu, MD, Jian Wu, MD, PhD, and ShuSen Zheng, MD, PhD, FACS, declare no conflicts of interest.
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Liver CSCs in Donor Allocation for HCC
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45. Ma S, Tang KH, Chan YP, et al. miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1. Cell Stem Cell. 2010;7(6):694–707. 46. Ma YC, Yang JY, Yan LN. Relevant markers of cancer stem cells indicate a poor prognosis in hepatocellular carcinoma patients: a meta-analysis. Eur J Gastroenterol Hepatol. 2013. doi:10.1097/ MEG.0b013e32836019d8. 47. Rege TA, Hagood JS. Thy-1 as a regulator of cell-cell and cell-matrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, and fibrosis. FASEB J. 2006;20(8):1045–1054.
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The Role of Liver Cancer Stem Cells in Donor Liver Allocation for Patients With Hepatocellular Carcinoma Jie Zhou MD, Zhenhua Hu MD, Zhiwei Li MD, Pengfei Yu MD, Jian Wu MD, PhD & ShuSen Zheng MD, PhD, FACS To cite this article: Jie Zhou MD, Zhenhua Hu MD, Zhiwei Li MD, Pengfei Yu MD, Jian Wu MD, PhD & ShuSen Zheng MD, PhD, FACS (2013) The Role of Liver Cancer Stem Cells in Donor Liver Allocation for Patients With Hepatocellular Carcinoma, Postgraduate Medicine, 125:6, 24-30 To link to this article: http://dx.doi.org/10.3810/pgm.2013.11.2709
Published online: 13 Mar 2015.
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C L I N I C A L F E AT U R E S
The Role of Liver Cancer Stem Cells in Donor Liver Allocation for Patients With Hepatocellular Carcinoma
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DOI: 10.3810/pgm.2013.11.2709
Jie Zhou, MD 1–3 Zhenhua Hu, MD 1–3 Zhiwei Li, MD 1–3 Pengfei Yu, MD 1–3 Jian Wu, MD, PhD 1–3 ShuSen Zheng, MD, PhD, FACS 1–3 Department of Hepatobiliary and Pancreatic Surgery, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China; 2Key Laboratory of Combined MultiOrgan Transplantation, Ministry of Public Health, First Affiliated Hospital of Zhejiang University, Hangzhou, China; 3Key Laboratory of Organ Transplantation, First Affiliated Hospital of Zhejiang University, Hangzhou, China 1
Abstract: Previous criteria for liver transplantation in patients with hepatocellular carcinoma (HCC) has predominantly emphasized the size and number of existent tumors; however, criteria have not included tumor biology, which may comprise a critical factor for predicting patient prognosis. This issue has been recognized in the Hangzhou criteria, which take tumor histopathologic grade and pre-transplant a-fetoprotein (AFP) level into consideration. However, neither histopathologic grading nor AFP level are precise enough to adequately represent tumor biology in patients with HCC. Recent research has indicated that the development and progression of HCC are driven by a subpopulation of liver cells with stem cell features (cancer stem cells, [CSCs]). Liver CSCs with cluster of differentiation (CD)133 antigen positivity show a high tumorigenic capacity, and the increase in the percentage of CD133+ liver cancer cells is consistent with increased levels of serum AFP. In addition, the number of CD90+ cells increases with the tumorigenicity of HCC, and a positive correlation between the number of circulating CD90+ liver CSCs and disease progression has been observed. As liver CSCs can be detected using the CD profile and could more accurately represent tumor biology in HCC, we hypothesized that liver CSCs with specific phenotypes could be used for modifying the Hangzhou criteria, thereby providing valuable guidance for the development of more accurate prognosis prediction algorithms for patients with HCC being considered for liver transplantation. We provide reliable evidence supporting this hypothesis, and offer proposals for future applications in transplant practice. Keywords: cluster of differentiation; CD133+; CD90+; hepatocellular carcinoma; liver transplantation; liver cancer stem cells
Introduction
Correspondence: ShuSen Zheng, MD, PhD, FACS, N0. 79 Qingchun Road, Hangzhou, 310003, China. Tel: 86-571-87236570 Fax: 86-571-87236616 E-mail: [email protected]
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Liver transplantation is considered the optimal treatment for patients with end-stage liver disease, including hepatocellular carcinoma (HCC), because the procedure results in the removal of all tumor cells and correction of liver dysfunction.1 However, with increasing candidates on the transplantation waiting list and organ resource shortage, wide application of liver transplantation for patients with HCC has been restricted. Understanding how to best allocate the limited number of available donor grafts has become an important issue in transplant practice. Criteria for selecting suitable candidates for liver transplantation have been established. The most used criteria sets are 1) the Milan criteria: solitary tumor with a diameter # 5 cm, or # 3 tumors with the largest # 3 cm; no vascular invasion; and 2) the University of California, San Francisco (UCSF) criteria: solitary tumor # 6.5 cm, or # 3 tumors, with the largest # 4.5 cm and accumulative diameters # 8 cm; without macrovascular invasion.2,3 Both sets of criteria, however, mainly focus on tumor morphology, which emphasizes the importance
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Liver CSCs in Donor Allocation for HCC
of the number and size of the tumors. Histologic grade of the tumor is not included, which may be a critical factor for predicting patient survival following liver transplantation.4 Several studies in the literature have observed the predictable value of histologic grading in patients with HCC5,6; therefore, we proposed the Hangzhou criteria: apart from the presence of macrovascular invasion, patients with HCC must fulfill 1 of 2 criteria: a) total tumor diameter # 8 cm; or b) total tumor diameter . 8 cm, with histopathologic grade I or II and preoperative a-fetoprotein (AFP) level simultaneously # 400 ng/mL.7 However, this approach is not precise enough to allow adequate grading, such as mild, moderate or severe, and therefore a more precise strategy for patient pre-transplant evaluation is needed. Research indicates that the development and progression of HCC is driven by a small population of cancer stem cells (CSCs), which share multiple characteristics of tissue stem cells and have extensive proliferative, self-renewal, and differential potential.8 The presence of CSCs in human liver with specific phenotypes has been repeatedly reported in recent years, with surface markers like cluster of differentiation (CD)133, CD90, and epithelial cell adhesion molecule (EpCAM) identified to classify liver CSC subpopulations. One study by Zeng et al9 revealed that the presence of high pre-transplant EpCAM staining in HCC liver explants is an independent predictor for tumor recurrence in recipients who underwent liver transplantation. Liver CSCs with specific phenotypes could serve as an important predictive factor in the setting of liver transplantation. We have increasingly recognized that liver CSCs might be the initiator cells for tumor formation and that they may serve to maintain tumor growth. They are also involved in the origin of metastases and recurrence, and may therefore accurately represent the tumor biology of HCC.
Hypotheses
We propose the hypothesis that liver CSCs with specific CD markers could be detected and selected as critical precise assessment tools for tumor biology in patients with HCC. The markers can be utilized for guiding liver transplantation for management of patients with HCC. An analysis of the quantity and localization of liver CSCs could reflect the tumor biology of HCC, which is closely related to patient prognosis following liver transplantation. The addition of such tumor biology analysis to modify our Hangzhou criteria could surmount existing simplistic, crude, and subjective assessments of histopathologic grading in patients with HCC. Based on inclusive, meticulous pre-transplant e valuation of tumor
biology, focusing on specific biomarkers as well as recipient prognosis, the modified Hangzhou criteria would allow the developments of more accurate prediction algorithms for post-transplant survival of HCC patients. We believe that the use of liver CSCs with specific CD markers in this context would be advantageous and could aid the achievement of more satisfactory outcomes.
New Elements for Assessing Candidacy for Liver Transplantation in Patients With HCC
Previously, applying measures from the 2 widely used c riteria sets, the Milan and UCSF criteria (both based on tumor morphology, emphasizing the importance of the number and size of the tumors), patients with HCC who met the selection criteria achieved excellent long-term survival outcomes. The 4-year overall patient survival rate has been 85%, and the patient tumor-free survival rate at 92% for recipients fulfilling the Milan criteria.2 The 1-year and 5-year overall survival rates have been 90% and 75.2%, respectively, for patients who fulfilled the UCSF criteria.3 A study to assess the predictability value of the Milan criteria compared with the UCSF criteria suggested the superiority of the UCSF criteria in predicting acceptable post-transplant patient outcomes.10 The suggested importance of tumor gross features was asserted in observations from a meta-analysis performed by Burroughs et al11—that assessment of the diameter of the largest tumor and total tumor diameters were the best predictors for patient post-transplant prognosis. However, tumor morphology alone may not provide the best possible index for patient outcomes when other biologic marker measures can be taken into account. Data from many studies have indicated that microvascular invasion is the most critical factor in determining the likelihood of HCC recurrence.12–18 Tumor differentiation has been proposed as a predictor for microscopic vascular invasion.5,19 Currently, more research is emphasizing the critical role of tumor histologic grading in predicting long-term prognosis in patient with HCC who are liver recipients.5,6 Previous clinical data revealed that patients with a single large mass after hepatic resection can have a good outcome, similar to the survival rate of patients with small HCC, and significantly better than that of patients with nodular HCC20; however, patients with a small mass can have an adverse outcome, e mphasizing the critical role of histologic grading in patients with smallmass HCC.21 The study by Merani et al22 investigated the predictive role of pre-transplant AFP levels in patients with HCC on outcomes following liver transplantation.
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Zhou et al
The researchers found that patients with pre-transplant AFP levels . 400 ng/mL, whose AFP levels were successfully reduced to , 400 ng/mL could achieve comparable intent-to-treat (ITT) and post-transplant survival rates as those patients with pre-transplant AFP levels , 400 ng/mL, and significantly superior to patients with AFP levels that remained . 400 ng/mL. Merani et al also demonstrated that latest patient pre-transplant AFP level was an independent predictor for both ITT and post-transplant survival rates and suggested use of the pre-transplant AFP level in combination with other morphological variables to assess suitability of candidates for liver transplantation. The result was in accordance with previous research, which had demonstrated the close relationship between pre-operative patient AFP level and patient long-term survival.23,24 In another study, Toso et al25 attempted to reassess recipient selection criteria for patients with HCC, and found the combination of patient total tumor volume and pre-transplant AFP level (cutoff at AFP level of 400 ng/mL) could most effectively predict recipient survival. So it appears that neither the number nor the size of the tumors alone is a determining factor for patient prognosis after transplantation. As a matter of fact, the number and size of tumors may change at different times in the disease course of patients with HCC. Images obtained through many modalities (including computed tomography [CT], magnetic resonance imaging [MRI], contrast ultrasound) could not demonstrate this dynamic process, but could only describe the 2 indexes at specific time-points of tumor growth. We recognized this limitation and therefore introduced histopathologic grade and pre-transplant AFP levels into the liver transplantation candidacy assessment for patients with HCC, using the Hangzhou criteria. Studies validated that recipients who met the H angzhou criteria achieved comparable survival rates to those who met the Milan criteria.7,26,27 Patient 1-, 3-, 5-year overall survival rates and tumor-free survival rates were 92.8%, 70.7%, 70.7%, and 83.7%, 65.6%, 62.4% respectively, in patients fulfilling the Hangzhou criteria, significantly better than those exceeding the Hangzhou criteria, with 49.9%, 27.0%, 18.9%, and 25.8%, 12.5%, 4.7%, respectively.7 In our study, survival rates were similar between recipients fulfilling the Milan criteria and the Hangzhou criteria, which was also confirmed by the research of Audet et al.7,27 We have found that 2 problems remain when using both tumor histopathology grading and patient pre-transplant AFP level to predict patient outcomes. First, the approach is not precise enough to allow grading as mild, moderate, or severe. It relies too heavily on the experience of the i ndividual 26
pathologist, which is relatively subjective. Secondly, recent data have suggested that patient AFP level measure is not sensitive or specific enough for general use as an effective diagnostic and surveillance assay for HCC as was once thought. Moreover, an elevated AFP level can also be observed in some patients with intrahepatic cholangiocarcinoma (ICC) and liver diseases originating from colon tumor metastasis.28,29 Other research has suggested that patients with a tumor diameter , 2 cm seldom have elevated AFP levels and that some patients with HCC (approximately 25%) have normal AFP levels.30,31 Therefore, patient pre-transplant AFP level may not offer an accurate predictor for post-transplant prognosis in patients with HCC. More recently, the relationship between gene signatures, as well as microRNA (miRNA) and epigenetic alterations, and prognostic prediction in patients with HCC have been increasingly recognized.32–35 Biomarkers, including G3, EpCAM, and miR-26, have been identified in patients with HCC.36 Such factors should be included in tumor biology assessment so that a more accurate patient prognosis evaluation algorithm and prediction of treatment response can be developed in liver transplantation.
Liver CSCs and Biologic Markers as Critical Factors for Determining Patient Post-Transplant Prognosis
The important role of liver CSCs in carcinogenesis has been increasingly recognized. Liver CSCs share many signaling pathways that exist in normal stem cells, such as Wnt proteins and transforming growth factor β (TGF β) pathways, which are mainly involved in the processes of cell proliferation, self-renewal, and differentiation, and would be deregulated in liver CSCs. The miRNA also plays a key role in the development of HCC, and recent literature suggests the important function of miRNA in liver CSCs. A study by Li et al37 utilized side population (SP) sorting to the culture of HCC cells and normal liver cells, and subsequently screened miRNA expression profiles between the 2 cell types. Distinct miRNA expression during hepatocarcinogenesis was observed, providing a useful understanding of miRNA function in liver CSCs. Increasing evidence suggests that liver CSCs with specific phenotypes play an important role in hepatocellular carcinogenesis. Commonly known as Prominin-1 (PROM1) in humans, CD133 has been widely recognized as an important stem cell surface marker in both normal and various cancer cells.38 Generally, CD133 is not expressed in normal adult liver cells but it could re-express in patients with HCC.
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Liver CSCs in Donor Allocation for HCC
Suetsugu et al39 first reported that CD133 antigen-positive (CD133+) cells marked a liver CSC subpopulation, which was subsequently confirmed by Yin et al.40 Liver CD133+ CSCs demonstrate high tumorigenic and self-renewal capacities,39–41 which may be partially explained by involvement in long interspersed element (LINE)-1 hypomethylation (one of most common molecular abnormalities during the process of carcinogenesis) and through the enhancement of interleukin (IL)-8 signaling pathway activity induced by network-identified neurotensin.42,43 Song et al44 found that an increase in the percentage of CD133+ liver cancer cells was consistent with increased serum AFP levels, relatively higher histopathologic grades, and a poor prognosis in patients with liver cancer. Furthermore, multivariate analyses identified elevated expression of CD133 to be an independent predictor for overall and tumor-free survival rates in patients with HCC. The study by Ma et al45 analyzed clinical samples from HCC and non-HCC tissues by flow cytometry and immunohistochemistry analysis and observed that CD133 occurred in only a small subpopulation in human HCC tissue. However, no expression of CD133 could be detected in normal liver tissue. Similarly, Ma et al found that the presence of CD133+ cells correlated with advanced stage of disease, higher tumor recurrence, and worse patient prognosis. A meta-analysis performed by Ma et al46 assessed 12 studies involving . 1300 patients to evaluate the presence of liver CSCs and CD133 expression as prognostic factors in patients with HCC. A significant correlation between the presence of liver CSCs and impaired overall and tumor-free survival were observed (HR 1.62 and 1.85, respectively) in patients, and CD133 was identified as having significant prognostic value in patients with HCC. The meta-analysis provided important implications for evaluating stem cell surface markers, like CD133, in liver CSCs in future clinical practice. In addition, CD90 may also be a marker for liver CSCs. A glycosylphosphatidylinositol (GPI)-anchored glycoprotein, CD90 (also known as Thy-1), expressed mainly in peripheral blood leukocytes, plays a role in cell-to-cell and cell-tomatrix interactions.47 Research by Yang et al48 on cell lines found that the number of CD90+ cells increased with HCC tumorigenicity. All clinical tumor tissue and most blood samples from patients with HCC contained the CD45+CD90+ population and there was also a positive relationship between CD90+ cells from HCC tissues and blood samples. Additionally, in their animal research, Yang et al used CD45-CD90+ cells separated from both tissue and blood samples to form tumor nodules with similar pathologic characteristics in immunodeficient mice, whereas the CD45-CD90- cells could
not generate such nodules. The study provided p rofound evidence of self-generation properties of CD90+ cells in patients with HCC, revealing that CD90+ is adequately sensitive and specific as a tumor marker for liver CSCs. In another study performed by his group, Yang found a positive correlation between the number of circulating CD90+ CSCs and disease progression in patients.49 A recent study on liver CSCs by Yamashita et al, using gene-expression analysis, observed that CD90+ cells have the features of vascular endothelial cells.50 When correlated with clinicopathologic characteristics, the researchers found that the expression of CD90+ cells was associated with a higher probability of distant metastasis. Moreover, a highly metastatic capacity was also observed when they implanted CD90+ cells derived from primary HCC into immunodeficient mice. All results suggest specific gene-expression patterns in liver CSCs with CD90+, and highlights the importance of CD90+ as a marker to help determine the prognosis of patients with HCC. Liver CSCs can be detected using the CD profile and can therefore more precisely represent the tumor biology in patients with HCC. Based on results from previous research in liver CSCs, specifically stem cell surface markers, we offer the following model and verification strategies: 1) Isotope labeling has been adopted to characterize specific CD profile expression (CD133+ and/or CD90+) targeted to liver CSCs. 2) Subsequently, we used emission computed tomography (ECT) imaging for local scanning. 3) Active uptake foci were usually observed, and we then analyzed the quantity and localization of liver CSCs for accurate evaluation of HCC tumor biology. 4) Two conditions may indicate adverse tumor biology: (a) if the active uptake foci exceed the range of the tumor (Figure 1A); and/or (b) if the active uptake foci are limited to the tumor, but such foci are also detected in other parts of the liver (Figure 1B). Either condition may indicate that the tumor has infiltrated adjacent liver tissue or that intrahepatic metastases are present, which may be associated with defective tumor biology. Poor patient prognosis can therefore be predicted, despite liver transplantation. 5) In the presence of either or both of these 2 conditions, the patient is taken off the transplant waiting list and referred for other treatment, such as transarterial chemoembolization (TACE), radiofrequency ablation (RFA), or percutaneous ethanol injection (PEI). Only patients with active uptake foci limited to the tumor nodule (Figure 1C) are selected for liver transplantation. This is a novel but potentially effective strategy of choice for pre-transplant patient evaluation (Figure 2).
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Zhou et al Figure 1. A) Active uptake foci exceed the range of the tumor. B) Active uptake foci are limited to the tumor, but several are present in other parts of the liver. C) Active uptake foci are limited to the tumor range.
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Figure 2. Flow chart of steps post-selection for liver transplantation: a) Label isotope, CD133+ or CD90+. As seen on ECT, b) active uptake foci limited to the tumor range. [Proceed to transplantation.] c) Active uptake foci exceed the range of the tumor, or active uptake foci limited to the tumor but foci also detected in other parts of the liver. [Proceed to other treatment], such as d) transarterial chemoembolization (TACE), radiofrequency ablation (RFA), or percutaneous ethnol injection (PEI). 3DWLHQWVPHHW+DQJ]KRXFULWHULD ,QWUDYHQRXVLVRWRSHODEHOLQJ WRFKDUDFWHUL]H&'SURILOHD /LYHUVFDQQLQJE\(&7
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Abbreviations: CD, cluster of differentiation; ECT, emission computed tomography; PEI, percutaneous ethanol injection; RFA, radiofrequency ablation; TACE, transarterial chemoembolization.
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Conclusion
The Milan criteria and the UCSF criteria for determining patient candidacy for liver transplantation as a management strategy for HCC both emphasize the number and size of tumor masses, and do not include tumor biology. Although the Hangzhou criteria involve AFP level and tumor histopathologic grade in the selection of patients with HCC for liver transplantation, problems such as the crude assessment can occur. Thus, new strategies with inclusive criteria that can accurately reflect tumor biology using specific biomarkers are urgently needed for improving outcomes for patients undergoing liver transplantation in the management of HCC. Liver CSCs with specific CD profiles (CD133+ or CD90+) demonstrate a high tumorigenic capacity, and correlate with pathologic grading and a poor prognosis in patients with HCC. Isotope labeling has been adopted to characterize CD profiles, which can be detected by ECT. The quantity and localization of liver CSCs can be precisely identified, and early infiltration and intrahepatic metastases, which are not identified by other radiologic investigations, can be detected. As a result, patients with HCC can be selected for liver transplantation according to the amount and distribution of liver CSCs. Liver CSCs, which can be detected quantitatively and reflect tumor biology for HCC subpopulations, enhance guidance for the use of liver transplantation in the management of patients with HCC. However, the best method for detecting liver CSCs, not the specific CD profile, remains uncertain, and the possibility of combining the CD profile with other new biomarkers needs further investigation. The aim of our study was to investigate new factors that could usefully represent tumor biology and to modify criteria for the use of liver transplantation in patients with HCC. Liver CSCs appear to meet these requirements. Our hypothesis is a prompt for further investigations. Studies should be initialized to build more comprehensive models for the detection and the quantitative analysis of liver CSCs. There is also a need to evaluate the benefit of liver CSCs as a new criterion for the use of liver transplantation in the management of HCC.
Acknowledgment
We acknowledge Sunyi Ye for his excellent technical assistance.
Conflict of Interest Statement
Jie Zhou, MD, Zhenhua Hu, MD, Zhiwei Li, MD, Pengfei Yu, MD, Jian Wu, MD, PhD, and ShuSen Zheng, MD, PhD, FACS, declare no conflicts of interest.
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© Postgraduate Medicine, Volume 125, Issue 6, November 2013, ISSN – 0032-5481, e-ISSN – 1941-9260 ResearchSHARE®: www.research-share.com • Permissions: [email protected] • Reprints: [email protected] Warning: No duplication rights exist for this journal. Only JTE Multimedia, LLC holds rights to this publication. Please contact the publisher directly with any queries.
