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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
Expression, cellular distribution and prognostic relevance of Tumor Necrosis Factor–
Related Apoptosis inducing ligand (TRAIL) receptors in hepatocellular carcinoma
Lydia Kriegl1, Andreas Jung1, Jutta Engel2, Rene Jackstadt3, Alexander Gerbes4, Eike Gallmeier4,
Jana A. Reiche1, Heiko Hermeking3, Antonia Rizzani4, Christiane J. Bruns5,
Frank T. Kolligs4, Thomas Kirchner1, Burkhard Göke4, Enrico N. De Toni4
1 Institute of Pathology, University of Munich, Thalkirchnerstr. 36, 80337, Munich, Germany. 2 Munich Cancer Registry (MCR) of the Munich Cancer Centre (MCC) at the Department of
medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-University (LMU),
University Hospital Großhadern, Marchioninistraße 15, 81377 Munich, Germany. 3 Experimental and Molecular Pathology, Institute of Pathology, University of Munich,
Thalkirchnerstr. 36, 80337, Munich, Germany. 4 Department of Medicine 2, University Hospital Grosshadern, University of Munich,
Marchioninistr. 15, 81377, Munich, Germany. 5 Department of Surgery, University Hospital Grosshadern, University of Munich,
Marchioninistr. 15, 81377, Munich, Germany.
Running title: prognostic relevance of TRAIL-receptors in HCC
Key Words: TRAIL, apoptosis, hepatocellular carcinoma
Financial support: this work was supported by the Deutsche Forschungsgemeinschaft (DFG) with the grant TO 605/2-1 to EDT.
Corresponding author: Enrico N. De Toni, University of Munich, University Hospital
Grosshadern, Department of Medicine II, Liver center Munich, Research Lab B 5 E01 308,
Marchioninistrasse 15, D-81377 Munich, Germany, Tel.: +49-89-7095-3176, E-Mail:
enrico.detoni@med.uni-muenchen.de
Published OnlineFirst on October 1, 2010 as 10.1158/1078-0432.CCR-09-3403
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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
Competing interests: no authors have competing interests regarding this manuscript to
declare.
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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
STATEMENT OF TRANSLATIONAL RELEVANCE
Antibodies specifically targeting TRAIL-R1 or -2 are now being tested in clinical trials. We
show that loss of these receptors is a common feature of HCC with strong and additive
prognostic relevance. Therefore, tumors lacking a TRAIL-receptor are likely not to
respond to the administration of the corresponding antibody, but “double-positive” tumors
will probably profit more from the targeting of both receptors. TRAIL-receptors-negative
tumors might also profit from the administration of chemotherapeutics capable of
increasing membrane-bound TRAIL-receptors (histone-deacetylase inhibitors,
demethylating agents, topoisomerase inhibitors, or interferon) administered alone or in
combination with TRAIL.
In vitro studies suggested that liver diseases underlying HCC might sensitize non-tumor
liver cells to TRAIL by increasing membrane-bound TRAIL-receptors. Our data show no
correlation between TRAIL-receptors staining and liver disease and have a reassuring
meaning concerning the possibility that liver diseases might represent a limitation to the
administration of TRAIL for therapeutic purposes.
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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
Abstract
Purpose. After the advent of targeted therapies for hepatocellular carcinoma (HCC) much
work is being done to provide a comprehensive description of the different signalling
pathways contributing to cell survival and proliferation in this tumor. Apoptotic signalling
mediated by TRAIL represents an important mechanism of tumorsurveillance, but its
importance in the development of HCC is not known. We thus investigated the cellular
distribution and the prognostic importance of TRAIL-receptors in HCC.
Experimental Design. Immunohistochemical staining for TRAIL receptors was evaluated in
HCC tissues and in matched surrounding non tumor tissues of 157 HCC patients treated with
liver transplantation or partial hepatectomy. Survival was analyzed in 93 patients who
underwent partial hepatectomy.
Results. The fraction of HCC samples with positive membrane staining for TRAIL receptor 1
(TRAIL-R1) and 2 (TRAIL-R2) was 1.4 and 2.7 folds lower compared to that of hepatocytes
from surrounding tissues (p=0.01). Loss of either TRAIL-R1 or TRAIL-R2, as confirmed by a
multivariate analysis, significantly worsened 5 year survival of HCC patients (survival:27
vs.52% and 15 vs.43%; hazard ratio [HR]=2.3 [1.1-4.4] and 2.4 [1.1-5.2] respectively). Loss
of both TRAIL-receptors further decreased survival of patients (HR=5.72 [2.1-15.5] vs.
double-negative staining – p=0.001) indicating an additive effect on survival of TRAIL-R1 and
TRAIL-R2.
Conclusions. This pilot study suggests that loss of TRAIL receptors is a frequent feature of
hepatocellular carcinomas and an independent predictor of survival in patients undergoing
partial hepatectomy. Future therapeutic protocols are likely to profit from the characterization
of their expression and cellular distribution.
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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
Introduction
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and
shows a rising incidence. Unfortunately, while curative surgical or local ablative therapies are
feasible only in 30% of patients, at this time systemic chemotherapy alone does not provide a
curative option and HCC still represents the third most frequent cause of cancer-related
death with a survival averaging 10% at 5 years [1].
Recently, the clinical course of HCC patients has improved thanks to the employment
of the Raf/VEGF inhibitor Sorafenib [2]. This success has stimulated intensive research
intended to unveil the role played by other different signaling pathways likely to influence
survival and proliferation of liver cancer cells. It is expected that a comprehensive description
of these pathways will allow treatment schemata tailored on corresponding molecular targets
in individual tumors [3, 4].
Induction of apoptosis through the interaction of TRAIL with its receptors on the
surface of cancer cells is a well described mechanism of tumorsurveillance [5]. The in vivo
importance of loss of sensitivity to TRAIL-mediated apoptosis is shown by clinical studies
demonstrating a correlation between TRAIL receptors expression, poor prognosis and tumor
recurrence [6]; also, TRAIL-knock out mice exhibit enhanced metastases formation [7] and
we recently showed that expression of the TRAIL-binding soluble decoy receptor OPG
correlates with tumor stage and metastasis formation in patients affected by colon carcinoma
[8].
Following the recognition of the physiological importance of TRAIL signalling, growing
experimental evidence accumulated on the fact that TRAIL induces apoptosis in cancer cells
but not in normal cells [9]. For this reason TRAIL has been developed as a promising
alternative therapeutic strategy and many types of recombinant TRAIL or agonistic
antibodies targeting TRAIL-receptors have been made available for the clinic [5].
However, the role of the TRAIL system in pathogenesis of HCC is not clear and no study had
yet assessed the relevance of TRAIL-receptors expression as prognostic marker in this
tumor [10].
We found that loss of TRAIL receptors represents a common feature of HCC and an
independent prognostic marker identifying a subset of tumors which have lost sensitivity to
receptor-mediated apoptosis.
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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
Patients and Methods
Patients and pathological material. Patients with a diagnosed HCC who had been treated
with liver transplantation or partial hepatectomy at the university clinic Munich Großhadern
between 1985 – 2008 were considered for analysis. To avoid high selection bias due to
inclusion criteria for transplantation, for survival analyses patients who received a liver
transplantation were excluded. Survival data of patients were retrieved from the database of
the Munich Cancer Registry (MCR - www.tumorregister-muenchen.de). Tissue samples were
obtained from paraffin blocks archived at the institute of pathology of the University of
Munich. A tissue micro array containing tumor samples as well as matched surrounding non-
tumor tissue was established as previously described [11].
Immunohistochemical Staining. 5 μm sections of TMA blocks were used for
immunohistochemical staining. Anti-TRAIL R1 monoclonal goat antibody (Santa Cruz
Biotechnology Inc., Heidelberg, Germany) and Anti-TRAIL R2 monoclonal rabbit antibody
(Calbiochem, California, U.S.A.) were applied as primary antibody. For antigen retrieval
sections were pre-treated by boiling in a microwave oven 2 times at 15 min at 750 W in
Target Retrieval Solution (Dako, Hamburg, Germany). Endogenous peroxidase was blocked
by incubation in 7.5% hydrogen peroxide for 10 minutes. Vectastain ABC-Kit Elite Universal
(Vector Laboratories, CA, USA) kit was taken for antibody detection and AEC (Zytomed
Systems) was used as a chromogen. Slides were counterstained with hematoxylin (Vector).
Positive staining for TRAIL-R1 or TRAIL-R2 was categorized according to its cellular
distribution and regardless of the intensity of the signal as follows: positive staining in
cytoplasm only, positive staining on cells membranes only, positive staining in both cell
membrane and cytoplasm. According to the rationale that TRAIL-receptors are functionally
active if situated on the surface of cell membranes, for statistical analysis tumors exhibiting
TRAIL-receptors staining on cell membranes (“membrane staining” group) were compared to
tumors which altogether showed no TRAIL-receptors staining or in which TRAIL-receptors
were confined to the cytoplasm only (“no membrane staining” group). The efficacy of TRAIL
receptors evaluation by immunohistochemistry to discriminate between membrane and
cytoplasmatic staining was confirmed by co-staining of TRAIL-receptors and membrane-
bound E-Cadherin by using confocal microscopy (additional material). Additionally, we
conducted a semi-quantitative analysis of HCC samples by assigning a score ranging from 0
to 3+ to TRAIL-receptors staining. Immunohistochmical evaluation of TRAIL-receptors was
confirmed by qPCR analysis of 39 samples showing a significant correlation of TRAIL-
receptors staining and the intensity of their immunohistochemical signal (χ2 test: TRAIL-R1:
p=0.021; TRAIL-R2: p=0.01).
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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
Histological assessment of tumors and surrounding non-tumor tissue samples. For
assessment of tumors, tumor grade, vessels invasion, number of lesions and tumor size
were considered. Tumor grading was evaluated according to the WHO criteria [12] and was
based on the area showing the highest grade. Vascular invasion was defined according to
macroscopic or microscopic evidence of blood vessels invasion. Tumor size and number of
lesions were assessed by macroscopic pathological investigation of resected livers. Matched
non-tumor tissues were investigated for anatomopathological features describing underlying
liver disease (grade of fibrosis/cirrhosis, portal inflammation, piececemeal necrosis and
steatosis). Histological evaluation of tumors or surrounding non-tumor tissue was performed
on slides stained with hematoxilin/eosin (HE) and evaluated by a senior pathologist and two
of the authors (LK and EDT) who were blinded to tissues annotations and prognostic data.
Liver fibrosis, portal inflammation and piecemeal necrosis were assessed according to the
Ishak score for chronic hepatitis [13]. Steatosis and lobular infiltration were evaluated
according to the NAFLD activity score and staging system [14].
Statistical analysis. All statistical analyses were run using SPSS (version 17, SPSS inc.
Chicago – IL). For frequency data, exact chi square tests were used. Significance level was
adjusted using the Bonferroni correction for repeated tests. Observed (unadjusted overall)
survival was first estimated with the Kaplan-Meier method and tested with the log-rank
procedure. Relative survival was computed by the ratio of the observed survival rate to the
expected survival rate [15]. The expected survival time of age- and gender-matched
individuals was calculated from the life tables of the normal German population. Relative
survival was thus used as an estimate for disease specific survival. Survival was then
investigated with a Cox proportional hazards regression model. Hazard ratios (HR) and 95%
confidence intervals (CI) of these four Cox proportional hazards regressions are presented.
For survival analysis the significance level was set at 5%.
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Prognostic relevance of TRAIL receptors in HCC
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Results
Patients and clinico-pathological variables. 157 patients with a diagnosed hepatic cellular
cancer who underwent liver resection or liver transplantation due to HCC could be identified.
Of these patients, 55 were treated with liver transplantation whilst 102 underwent partial
hepatectomy and were considered for survival analysis. Of these patients, 101 were also
registered in the database of the Munich Cancer Registry (MCR) where survival was
available for 93 patients who underwent liver resection at the University Hospital
Grosshadern between 2001 and 2007. The demographic clinicopathological features of the
patients are listed in table 1. For patients who underwent liver resection who had been
considered for analysis of survival, the median follow up was 27 months. When stratified by
survival status, the median follow-up for survivors was 46 months, compared to 19 months
for patients who died. Almost all patients died due to tumor recurrence, as shown by the
overlap of the overall and relative survival of these patients (fig. 2A).
Immunohistochemical staining for TRAIL receptors 1 and 2 in hepatocellular
carcinoma cells and in hepatocytes from surrounding non tumor tissue. TRAIL-R1
stained positive in all non tumor tissues surrounding liver cancer lesions. In 8% of these
samples TRAIL-R1 stained positive in cytoplasm only. Membrane staining was positive in all
other tissue samples (92%). Instead, 3% of HCC samples stained altogether negative for
TRAIL-R1, 30% showed cytoplasmatic staining only and 66% stained positive on cell
membranes (fig. 1). When TRAIL-R2 was examined, no staining was detected in 13% of non
tumor tissues surrounding tumor lesions; 37% of non-tumor tissues showed cytoplasmatic
staining only, and 50% had a positive staining on cell membranes. In tumor tissues 46% of
samples showed no signal for TRAIL-R2, 36% of samples stained positive in cytoplasm and
only 18% of samples stained positive on cell membranes (fig. 1). The efficacy of
immunohistochemical staining to discriminate between membrane and cytoplasmatic staining
was confirmed by co-staining of TRAIL-receptors and membrane-bound E-Cadherin by using
confocal microscopy (additional material). Altogether, the fraction of samples exhibiting
receptor staining on cells membranes was remarkably lower in tumor tissues vs. that
detected in surrounding HCC lesions (TRAIL-R1: 92% in normal cells vs. 66% in HCC;
TRAIL-R2: 50% in non tumor cells vs. 18% in cancer cells; exact chi square test: TRAIL-R1:
p=0.0148, TRAIL-R2: p=0.0150). Similarly, 45% of non-tumor samples vs. 17.6% of tumor
samples exhibited positive membrane staining for both TRAIL receptors; conversely, only
2.8% of non tumor samples exhibited double negative staining on cell membrane for TRAIL-
recaeptors vs. 30.7% of tumor samples. Therefore, overall positive staining of TRAIL-R2 was
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Prognostic relevance of TRAIL receptors in HCC
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remarkably lower in tumor samples than in matched normal tissue; the fraction of tumor
samples exhibiting TRAIL-R1 or TRAIL-R2 staining on the surface of cell membranes was
significantly lower than that of matched surrounding non tumor tissues.
Correlation of TRAIL-receptors staining with clinicopathological features of tumor
tissues and non tumor surrounding tissue. To assess whether TRAIL-receptors staining
in tumor samples or in matched non-tumor tissue samples could be associated to specific
clinicopathological features, membrane staining of TRAIL-receptors 1 and 2 was correlated
with clinicopathological parameters including age and gender of patients, etiology of the
underlying liver disease, presence of severe fibrosis or liver cirrhosis, as well as specific
pathological features of tumors such as grading, size of and number of lesions in the liver or
the presence of signs of microscopic or macroscopic blood vessels invasion. After adjusting
significance levels according to the Bonferroni correction for repeated tests, a significant
association between membrane staining for TRAIL-R1 in tumor samples and smaller tumors
(<5 cm, p=0.004) was found. Also, a border-line significance was found between higher
degree of liver fibrosis (Ishak score ≥ 5) and TRAIL-R1 staining on cell membrane (p=0.008).
No relevant correlation was found between TRAIL-receptors staining on cell membranes in
surrounding non tumor cells and these clinico-pathological variables (not shown).
Prognostic significance of TRAIL-receptors staining in patients undergoing liver
resection. To avoid selection bias due to inclusion criteria for transplantation, analyses of
survival were conducted in patients undergoing partial hepatectomy only. In these patients
(fig. 2A), the overall and relative survival curves showed a substantial coincidence.
Therefore, in further analyses overall survival was considered as representative for tumor-
related survival in these patients.
A Kaplan-Meier analysis showed vascular invasion (fig. 2B) and TRAIL-receptors staining on
cell membranes (fig. 2CD) as significant determinant of survival in these patients. Patients
showing TRAIL-R1 staining on cell membranes had a better prognosis vs. patients bearing
tumors without TRAIL-R1 membrane staining (five year overall survival was 52% vs. 27%
respectively). Overall 5 year survival of patients exhibiting TRAIL-R2 membrane staining was
43% whilst that of patients with no TRAIL-R2 staining was 15%. Instead, analysis of survival
conducted using semi quantitative criteria to assess TRAIL-R staining intensity (scoring
ranging from 0 – no signal – to 3+) failed to show any difference in survival, neither if the
overall staining regardless of the cellular distribution was considered, nor when the analysis
was conducted only on membrane-positive tumor samples. A difference in survival could be
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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
seen only if patients were stratified according altogether to the presence or the absence of
TRAIL-R1 staining in tumor samples (long rank test, p=0.02, data not shown).
A Cox-multivariate analysis taking into account age, gender, tumor grading, tumor size, the
presence of multifocal lesions or signs of blood vessel invasion showed that membrane
staining of TRAIL-receptors 1 and 2 independently influences the survival of HCC patients
after partial hepatectomy. Calculated Hazard Ratios (HR) for TRAIL-R1 and TRAIL-R2 were
2.3 (p=0.01) and 2.4 (p=0.02) respectively (tab. 3). In contrast, no correlation was found
between TRAIL-receptors staining in non-tumor tissues surrounding tumor lesions and
survival of patients (TRAIL-R1: p=0.89; TRAIL-R2: p=0.11). When survival of patients was
assessed according to the simultaneous staining of TRAIL-receptors on cell membranes,
patients with double-positive staining for TRAIL-R1 and TRAIL-R2 showed in the Cox-
multivariate analysis a better prognosis in comparison to patients with altogether no
membrane staining for TRAIL-receptors (HR=5.72 [CI: 2.10 – 15.5] p=0.001) and in
comparison to patients bearing tumors with positive membrane staining for one receptor only
(HR=2.85 [CI: 0.99 – 8.28] p=0.05 – fig. 3).
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Prognostic relevance of TRAIL receptors in HCC
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Discussion
Targeted therapies and TRAIL-signaling in HCC. The clinical employment of
Sorafenib [2] has intensified the research of novel molecular targets for cancer therapy [16,
17]. Impairment of TRAIL-mediated apoptosis due to loss of TRAIL-receptors or inhibition of
the interaction with the respective ligands have been documented in several tumor entities
[18-20] and several antibodies specifically targeting TRAIL-receptors have been developed
as anticancer therapy [5]. Increasing evidence has shown also that TRAIL-receptors play a
role in the pathogenesis of several liver diseases [21, 22]. Yet, no systematic
immunohistochemical study has been conducted to assess the importance of the TRAIL
system and its prognostic value in HCC or in the diseased liver (reviewed by Herr et al. -
[21]).
Staining and localization of TRAIL-receptors in tumors versus surrounding non tumor
tissues. In our cohort almost all tumor samples had a positive staining for TRAIL-R1 whereas
46% of tumor samples stained negative for TRAILR2 (fig. 1). Loss of TRAIL-R2 in tumor cells
is in agreement with previous reports on other tumor entities [20, 23]. Instead, the only work
published so far investigating TRAIL-receptors in HCC samples reported overall positive
staining for both TRAIL-receptors in all 60 tumor samples examined [24]; the discrepancy is
unlikely to be due to suboptimal staining of our tissue samples, since the wide majority of the
co-stained samples from matched samples of surrounding non tumor tissue showed positive
staining for TRAIL-receptors (fig. 1). Differences in the size of the collective and the
underlying liver disease of patients recruited in these two cohorts might represent an
explanation for this difference. In particular, in this previous study 55 out of the 60 patients
were HBV positive whereas in our collective there was a predominant presence of patients
affected by a toxic-metabolic disease. This reflects the different epidemiology of liver
cirrhosis in China and in the region of Germany where the study was performed and might be
responsible for the differences observed. Future studies performed on a wider patient
collective will be needed to confirm these data and unveil possible effects of underlying liver
diseases in determining TRAIL-receptors expression. For statistical analysis we first
discriminated the fraction of tumors exhibiting TRAIL-receptors staining on cell membranes
with the rationale of assessing the fraction of receptors exposed to the action of circulating
TRAIL. According to this criterion, 44% of tumor samples showed negative membrane
staining for TRAIL-R1 and 92% were negative for TRAIL-R2 (fig. 1). This suggests that, as
recently shown in vitro [25], internalization of TRAIL-receptors in HCC might play a central
role in determining loss of the functional fraction of these receptors as distinctive feature of
cancer cells.
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Prognostic relevance of TRAIL receptors in HCC
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Prognostic relevance of TRAIL-receptors. To assess whether loss of membrane-
bound TRAIL-receptors might have a functional significance in the progression of HCC, we
assessed their effect on survival. Five-year survival of patients bearing tumors exhibiting
TRAIL-R1 or TRAIL-R2 staining on cell membranes doubled that of the other patients
undergoing the same treatment (fig. 2, tab. 3). Stratification based on TRAIL-R intensity
staining scores showed instead no statistical significance in the analysis of survival. A
significant survival advantage was shown only if patients were stratified on the basis of
overall TRAIL-R1 staining vs. the absence of immunoreactive products, this condition
possibly reflecting the absence of membrane-bound TRAIL-receptors in this subset. Patients
bearing tumors with double-positive membrane staining for both TRAIL-receptors survived
significantly longer not only in comparison to patients showing double-negative membrane
staining, but also in comparison to patients bearing tumors with positive membrane staining
for only one receptor. This suggests that membrane-bound TRAIL-R1 and TRAIL-R2 might
have an additive and favorable effect on the survival of these patients (fig. 3).
Clinical and pathological correlates of TRAIL-receptors staining and role of TRAIL-
receptors loss in carcinogenesis. In our cohort of patients membrane staining for TRAIL-R1
but not for TRAIL-R2 in tumor samples was associated with a smaller size of tumors,
possibly reflecting a favorable effect of TRAIL-R1 expression on tumor growth. However, this
couldn’t be observed for TRAIL-R2. Furthermore, no other statistically significant association
between membrane staining status of each receptor and the other pathological features of
tumors could be shown. To further investigate the role of TRAIL-receptors in the
pathogenesis of HCC we therefore subsequently assessed the correlation between TRAIL-
receptors membrane staining and clinicopathological variables related to the liver diseases
underlying hepatocellular carcinomas. Since most primary liver cancers arise on the ground
of liver diseases [1] we had hypothesized that TRAIL-receptors loss could represent a
progressive process related to the severity of liver fibrosis, the presence of steatosis,
leucocytes infiltration or to their etiologies. However, no significant correlation between
TRAIL-receptors status in non tumor tissues and these pathological features or patient
survival could be found. These data suggest that loss of TRAIL-receptors might not concur
with the underlying liver disease in the pathogenesis of HCC as pre-carcinogenetic condition,
but might occur later as additional feature of malignant cells. This is in agreement with two
recent studies showing that TRAIL-receptors loss does not play a critical role in tumor
development in APC mutant mice [26] and does not affect the formation of primary
squamous cell carcinomas [27], but had a decisive effect on metastasis formation [26, 27].
Recently Takeda and colleagues have shown that adoptive transfer of TRAIL-expressing
natural killer cells prevents recurrence of hepatocellular carcinoma after partial hepatectomy
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Prognostic relevance of TRAIL receptors in HCC
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[28, 29]. Together with these studies our data support the idea that, rather than influencing
tumor formation, loss of TRAIL-receptors might affect tumor progression at a later stage due
to the selection of cell clones resistant to circulating TRAIL and to immune-mediated
mechanisms controlling clearance of metastatic cells.
Clinical consequences of TRAIL-receptors loss in liver cancer cells. Although the
mechanism by which TRAIL-receptors are lost in tumor cells is not fully understood, recent
evidence showed that genetic loss or mutation of TRAIL-receptors is a rare event in cancer
cells, averaging 1% in hepatocellular carcinoma [18, 30]. Furthermore, several compounds
[25, 31-34] proved to increase TRAIL-receptors expression and synergize with TRAIL
administration to trigger apoptosis [35], suggesting that epigenetic gene silencing or TRAIL-
receptors internalization is a potentially reversible cause for the loss of functional TRAIL-
receptors. Our data showing a correlation between membrane staining and prognosis,
represent a clinical correlate of these studies. Therefore, patients bearing tumors without
membrane staining for TRAIL-receptors might profit from the administration of agents
capable of increasing their expression or functional localization on cell membrane and
eventually restoring the efficacy of endogenous TRAIL [29]. Interestingly, it has been shown
that Sorafenib synergizes with TRAIL to induce apoptosis in cancer cells [36]. This suggests
that the association of these agents to Sorafenib might represent a profitable strategy in the
treatment of hepatocellular carcinoma.
Another obvious consequence of the frequent functional loss of TRAIL-receptors in HCC is
that many liver tumors might not respond to administration of the specific agonistic antibodies
recently made available for the clinic targeting either TRAIL-R1 such as Mapatumumab or
TRAIL-R2 such as Lexatumumab [5]. However, since loss of these receptors had a strong
and additive prognostic relevance, patients lacking a TRAIL-receptor are likely not to respond
to the administration of the corresponding antibody, but “double-positive” patients will
probably profit more from the targeting of both receptors.
Furthermore, agonistic antibodies targeting TRAIL-receptors might synergize to induce
apoptosis in combination with agents capable of increasing TRAIL-receptors expression [35]
and this combination could be used for therapeutic purposes.
Of clinical relevance is the lack of association between TRAIL-receptors staining and liver
disease which could be observed in our analysis. Concerns on the safety of a therapeutic
use of TRAIL have originated from in vitro reports showing an increased sensitivity to TRAIL
in the diseased liver, likely mediated by enhanced expression of TRAIL-receptors [37]. Our
data have a reassuring meaning related to the possibility that enhanced TRAIL-receptors
expression might represent a cause for increased susceptibility of normal cells to apoptosis
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Prognostic relevance of TRAIL receptors in HCC
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mediated by TRAIL administered for therapeutic purposes. Future research should be
directed to prospectively assessing the significance of TRAIL-receptors in cancer cells.
Importantly, the effect of several drugs which are now undergoing investigation in clinical
trials, including sorafenib and histone-deacetylase inhibitors should be examined concerning
the possibility that their effect might be correlated to their influence on TRAIL-R expression.
Summary. In summary, loss of TRAIL-receptors is a common feature of HCC. Their
localization on cell membranes appears to be a determinant of survival. Future therapeutic
protocols might profit from the characterization TRAIL-receptors staining and cellular
distribution.
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Prognostic relevance of TRAIL receptors in HCC
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Legend to figures
Fig. 1. A: TRAIL receptor-1 and TRAIL receptor-2 staining in HCC cells vs. surrounding non
tumor cells. Percentage of samples showing no staining (none), cytoplasmatic staining
(cytoplasm only), membrane staining (membrane) or both (m+c) are shown. B, C:
representative typical positive staining of TRAIL-R2 in normal tissue (B) and negative
staining (C) in tumor tissue (magnification 400 x). D-F: at higher magnification (1000 x) the
intracellular distribution of TRAIL-R2 with prevalent staining in cytoplasm (D), cell
membranes (E), or in both (F) is shown.
Fig. 2. Survival curves showing: (A) expected survival of an age and sex-matched population
(dotted line), relative (dashed line) and overall survival (continous line) in HCC patients
treated by partial hepatectomy. (B) overall survival according to the presence or absence of
histological evidence of vessels invasion of patients treated by partial hepatectomy. (C,D):
Overall survival of patients treated with partial hepatectomy according to membrane staining
for TRAIL receptor-1 (C) and TRAIL receptor-2 (D). In graphs censored cases are indicated
by a cross.
Fig. 3. Survival of patients according to membrane staining status of both TRAIL receptors.
Kaplan-Meier curves represent overall survival related to membrane staining of TRAIL
receptor 1 and 2 vs. patients bearing tumors staining negative for both TRAIL receptors (A)
and of patients bearing tumors staining positive for either TRAIL-R1 or TRAIL-R2 vs. patients
bearing tumors exhibiting double-positive staining for TRAIL receptors (B). In graphs
censored cases are indicated by a cross.
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Prognostic relevance of TRAIL receptors in HCC
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Acknowledgments: The authors are thankful to Mrs. Dominique Schühmann for retrieving
pathological material and contributing the establishment of the tissue micro array used for
these experiments.
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Prognostic relevance of TRAIL receptors in HCC
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Prognostic relevance of TRAIL receptors in HCC
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research
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Prognostic relevance of TRAIL receptors in HCC
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Table 1. Summary of clinical and pathologic features
Feature Feature
n % n %
Age at diagnosis Lobular inflammation
< 60 y. 70 44.6 0-1 113 72.0
≥ 60 y. 87 55.4 2-3 39 24.8
not available 5 3.2
Sex Tumor size
male 122 78.2 < 5cm 96 61.2
female 35 21.8 ≥ 5cm 61 38.9
Etiology Extrahepatic metastasis
HCV 40 25.5 yes 6 3.8
HBV 16 10.2 no 151 96.2
toxic/metabolic 92 58.6
unknown 9 5.7
Severe fibrosis/cirrhosis Grading
< 5 64 40.8 1 29 18.5
≥ 5 88 56.1 2 81 51.6
not available 5 3.2 3 33 21.0
not available 14 8.9
Steatosis Blood vessels invasion
0 65 41.4 no 122 77.1
1-3 87 55.4 yes 35 22.9
not available 5 3.2
Portal inflammation Multifocal lesions
0-2 71 45.2 no 106 67.5
3-4 81 51.6 yes 47 29.9
not available 5 3.2 not available 4 2.5
Piecemeal necrosis Treatment
0-2 119 75.8 liver transplantation 55 35.0
3-4 33 21.0 partial hepatectomy 102 65.0
not available 5 3.2
patients count patients count
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Table 2. Liver cancer cells: TRAIL-receptors membrane staining and clinicopathological data
Feature
p p
negative positive negative positive
n (%) n (%) n (%) n (%)
Age at diagnosis 0.978 0.609
< 60 y. 22 (14.3) 46 (29.9) 56 (36.4) 12 (7.8)
≥ 60 y. 28 (18.2) 58 (37.7) 68 (44.2) 18 (11.7)
Sex 0.015 0.744
male 33 (21.6) 86 (56.2) 95 (62.1) 24 (15.7)
female 17 (11.1) 17 (11.1) 28 (18.3) 6 (3.9)
Etiology 0.269 0.729
HCV 10 (6.9) 28 (19.3) 30 (20.7) 8 (5.5)
HBV 3 (2.1) 13 (9.0) 14 (9.7) 2 (1.4)
toxic/metabolic 33 (22.8) 58 (40.0) 72 (49.7) 19 (13.1)
Severe fibrosis/cirrhosis 0.008 0.620
< 5 28 (18.7) 36 (24.0) 50 (33.3) 14 (9.3)
≥ 5 20 (13.3) 66 (44.0) 70 (46.7) 16 (10.7)
Steatosis 0.681 0.160
0 19 (12.7) 44 (29.3) 47 (31.3) 16 (10.7)
1-3 29 (19.3) 58 (38.7) 73 (48.7) 14 (9.3)
Portal inflammation 0.207 0.413
0-2 26 (17.3) 44 (29.3) 54 (36.4) 16 (10.7)
3-4 22 (14.7) 58 (38.7) 66 (44.0) 14 (9.3)
Piecemeal necrosis 0.279 0.767
0-2 40 (26.7) 77 (51.3) 93 (62.0) 24 (16.0)
3-4 8 (5.3) 25 (16.7) 27 (18.0) 6 (4.0)
Lobular inflammation 0.071 0.926
0-1 31 (20.7) 80 (53.3) 89 (59.3) 22 (14.7)
2-3 17 (11.3) 22 (14.7) 31 (20.7) 8 (5.3)
Tumor size 0.004 0.713
< 5cm 22 (14.3) 71 (46.1) 74 (48.1) 19 (12.3)
≥ 5cm 28 (18.2) 33 (21.4) 50 (32.5) 11 (7.1)
Grading 0.064 0.170
G1 5 (3.3) 23 (15.3) 20 (13.3) 8 (5.3)
G2 or G3 44 (29.3) 78 (52.0) 101 (67.3) 21 (14.0)
Blood vessels invasion 0.575 0.122
no 40 (26.0) 79 (51.3) 99 (64.3) 20 (13.0)
yes 10 (6.5) 25 (16.2) 25 (16.2) 10 (6.5)
Multifocal lesions 0.779 0.708
no 33 (21.9) 71 (47.0) 84 (55.6) 21 (13.9)
yes 16 (10.6) 31 (20.5) 38 (25.2) 8 (5.3)
Note: significance level adjusted acc. to Bonferroni's correction for repeated measures = 0.0041
TRAIL-R1 TRAIL-R2
membrane staining membrane staining
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Table 3. Cox regression model for TRAIL receptors staining on cell membranes of tumor tissues samples
Lower Upper Lower Upper
<60 y.
≥60 y.
male
female
G1
G2-G3
<5 cm
≥5 cm
single lesion
multiple lesions
yes
no
membrane staining
no membrane staining
0.82
1.07 0.59 1.93
1.10 5.29
0.84 0.43 1.66
0.42 0.21
0.42 1.63
0.85 0.44 1.64
0.59 2.56
2.3 1.17 4.49
0.83
1.12
2.41
0.99 0.49 2.00
0.3 1.44 0.62
0.86 0.42 1.77
1.23 0.68 2.12
0.88 0.45 1.72
0.988
0.627
0.692
0.483
1.2 0.57 2.50
0.001
0.015
0.601
0.636
0.580
0.812
0.634
0.012
0.027
0.724
TRAIL receptor 1
Sig.HRHR 95.0% CI
TRAIL receptor 2
Sig.(HR)HR 95.0% CI
Blood vessels invasion
TRAIL-Receptor status
Sex
Age
Grading
Size of tumor
Multifocal lesions
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TRAIL R1 non tumor cellsA
Figure 1
TRAIL R1 HCCTRAIL-R1 - non tumor cells
none0%
cytoplasm only8%
m+c55%
cytoplasm only30%
none3%m+c
36%
TRAIL-R1 - HCC
membrane36% membrane
30%
none13%
m+c10% membrane
15%
m+c3%
TRAIL-R2 - non tumor cells TRAIL-R2 - HCC
cytoplasm only37%
membrane39%
none46%
cytoplasm only36%
B C
D E F
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Figure 2%
90100
Tumor-related deaths
A
102030405060708090 Tumor related deaths
overall
relative
expected
p=n.s.
1 3 5 7 9 11 13 15010
Years
p n.s.
vessels invasion
no
%
708090100B
non=73
yesn=20
0 1 2 3 4 5 6 7 8 9 100102030405060
P<0.001
0 1 2 3 4 5 6 7 8 9 10Years%
5060708090100
CTRAIL-R1membrane staining
positiven=58
01020304050
0 1 2 3 4 5 6 7 8 9 10Years
negativen=35
p=0.047
D %
30405060708090100 TRAIL-R2
membrane staining
positiven=18
negativen=75
0 1 2 3 4 5 6 7 8 9 100102030
Years
n=75
p=0.016
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Figure 3
%A
30405060708090100
ATRAIL-R 1 and 2membrane staining
both positiven=17
n 34both negative
0102030
0 1 2 3 4 5 6 7 8 9 10Years
n=34
p=0.015
%100
BTRAIL-R 1 and 2
2030405060708090
moth positiven=17
mixed statusn=42
0 032
membrane staining
0 1 2 3 4 5 6 7 8 9 1001020
Years
p=0.032
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Published OnlineFirst October 1, 2010.Clin Cancer Res Lydia Kriegl, Andreas Jung, Jutta Engel, et al. (TRAIL) receptors in hepatocellular carcinomaTumour Necrosis Factor-Related Apoptosis inducing ligand Expression, cellular distribution and prognostic relevance of
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