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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:

[email protected]

Published OnlineFirst on October 1, 2010 as 10.1158/1078-0432.CCR-09-3403

Research. on September 7, 2018. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 1, 2010; DOI: 10.1158/1078-0432.CCR-09-3403

http://clincancerres.aacrjournals.org/



Competing interests: no authors have competing interests regarding this manuscript to

declare.






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.






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.






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.






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).






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%.






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






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






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).






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.






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






[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






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.






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.






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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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




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




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




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




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




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




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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