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Expression of p73 and Its Relation to Histopathology and Prognosis in Hepatocellular Carcinoma

Andrea Tannapfel, Mark Wasner, Karen Krause, Felix Geissler, Alexander Katalinic, Johann Hauss, Joachim Mössner, Kurt Engeland, Christian Wittekind

Affiliations of authors: A. Tannapfel, C. Wittekind (Institute of Pathology), M. Wasner, K. Krause, J. Mössner, K. Engeland (Department of Internal Medicine II), F. Geissler, J. Hauss (Department of Surgery II), University of Leipzig, Germany. A. Katalinic, Institute of Cancer Epidemiology, University of Lübeck, Germany.

Correspondence to: Andrea Tannapfel Institute of Pathology, University of Leipzig, Liebigstrasse 26, 04103 Leipzig, Germany (e-mail: tana{at}medizin.uni-leipzig.de).


    ABSTRACT
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: The protein p73, the first identified homologue of the tumor suppressor gene p53 (also known as TP53), has been shown to induce apoptosis (programmed cell death), but its function in tumor development has not been established. This study was undertaken to investigate the expression of p73 in liver tissue of patients with hepatocellular carcinoma (HCC) and to determine whether this expression has any impact on prognosis. METHODS: In situ hybridization and immunohistochemistry for the detection of p73 RNA transcripts and protein, respectively, were performed in tissues from 193 patients with curatively (R0-) resected HCC. Patients receiving liver transplantation were excluded. The results obtained were analyzed with respect to their association with pathohistologic stage, Edmondson grade, p53 expression status and several histopathologic factors of possible prognostic value, and, finally, with patient survival. RESULTS: RNA transcripts encoding p73 were detected by in situ hybridization in tumor cells but not in stromal, endothelial, or inflammatory cells or in cholangiocytes. Transcripts were also found occasionally in non-neoplastic hepatocytes. By immunohistochemistry, we detected p73 protein in 61 (32%) of the 193 carcinomas examined. Positive immunohistochemical staining was confined to the cell nucleus. Univariate survival analysis showed that p73 expression status was statistically significantly related to prognosis (two-sided P<.0001). Patients with p73-positive tumors had a poorer prognosis than those with p73-negative carcinomas. Multivariate Cox survival analysis identified the age of the patient, p73 expression status, co-existing cirrhosis, and Edmondson grade as independent prognostic factors. CONCLUSION: The protein p73 is overexpressed by a subset of HCCs and could serve as a useful indicator of prognosis in patients with this disease.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
The tumor suppressor gene p53 (also known as TP53) has come to be known as a "master guardian of the genome" because of its role in regulating cell growth and death (1). The p53 protein is important in triggering apoptosis after exposure to genotoxic stimuli or arresting growth by G1 checkpoint control at transition from G1 to S phase in cells in which defects can still be repaired. A gene with strong homology to p53 was discovered and named p73 (2). This gene mapped to the short arm of chromosome 1. It also was reported that the p73 protein can—at least when overproduced—activate the transcription of p53-responsive genes and inhibit cell growth in a p53-like manner by inducing apoptosis. But, unlike p53, no mutation of p73 has been found in human tumors (2-4).

We performed in situ hybridization to identify the main site of p73 messenger RNA (mRNA) expression within hepatocellular carcinomas (HCCs) and surrounding non-neoplastic liver tissue. To examine the possible association of p73 with clinicopathologic characteristics of HCC and the prognostic value of such association, immunohistochemical analysis of a large series of tumors was performed.


    SUBJECTS AND METHODS
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
Patients and Tissue Samples

One hundred ninety-three patients with HCC undergoing partial hepatectomy (segmental or lobar resection) from January 1979 through December 1996 were included in this retrospective study. No patient received preoperative or adjuvant chemotherapy or radiotherapy. All patients underwent surgery with curative intent (R0 resections). Patients who received orthotopic liver transplantation were excluded from this study. Each tumor was re-evaluated with regard to typing, staging, and Edmondson grading (5). Tumor typing and staging were performed by use of criteria of the World Health Organization (5) and the International Union Against Cancer (UICC) (6), respectively. Maximum tumor diameter was measured macroscopically in fresh specimens. In addition, every tumor was examined macroscopically and microscopically for the presence of vascular invasion, capsule formation, satellites, multiplicity, inflammatory reaction, necrosis, bile production, presence of giant cells, and dysplasia in the surrounding liver tissue and cirrhosis. Multiplicity includes multiple nodules representing multiple, independent primary tumors as well as intrahepatic metastases from a single primary hepatic carcinoma. Satellites were defined as tumor nodules, smaller than the main nodule, and were located within a maximum distance of 2 cm. Vascular invasion included gross as well as histologic involvement. In all cases, slides prepared from four different paraffin blocks of tissue, sampled from different tumor areas, were examined. The patients and their pathohistologic data are summarized in Table 1.Go


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Table 1. Patients and pathohistologic data*

 
In Situ Hybridization

Two segments of the p73 complementary DNA (cDNA) were cloned into HindIII and KpnI sites of the pSPT18 vector (Boehringer Mannheim GmbH, Mannheim, Germany) and were used as probes for the detection of different parts of p73 mRNA. Segments I and II were amplified from a human colon tumor cDNA preparation with the following primers: I-1 5'-TTTTTGGTACCGACTCATCTG TCATGGC-3', I-2 5'-TTTTTTAAGCTTTTTCTTCAAGAGCGGGGA-3', II-1 5'-AAAAAA GGTACCACAACCATGGCCACGCAG-3', II-2 5'-AAAAAAAAGCTTCTGACGAG GCTGGGGTC-3'. Constructs were confirmed by DNA sequencing. Northern blot analysis of HCC tissue with these probes revealed strong signals corresponding to a size of 4.4 kilobases. RNA probes were synthesized with the digoxigenin RNA labeling kit from Boehringer Mannheim GmbH, according to the instructions of the manufacturer. Probe I detects nucleotides 281-560 and probe II detects nucleotides 1471-1594 in the human p73 mRNA (GenBank Accession No. Y11416). The sense probe was prepared by use of the T7 promoter, and the antisense probe was transcribed with the help of the SP6 RNA polymerase.

In situ hybridization of mRNA was performed as described earlier (7) on deparaffinized and dried sections after pretreatment with proteinase K (Boehringer Mannheim GmbH; 10 mg/mL [in 50 mM Tris-HCl at pH 7.6 and 5 mM EDTA at pH 8.0]) for 30 minutes at 37 °C. Postfixation was performed with paraformaldehyde (4%). After the slides were dried, prehybridization was performed under stringent conditions covering the slides with EnhancedChemiLuminenscent (ECL)TM gold hybridization buffer solution (Amersham Life Science Inc., Braunschweig, Germany) for 30 minutes. After denaturation of the probe, the slides were covered with the hybridization solution containing digoxigenin-labeled probe (in ECLTM gold hybridization buffer solution) for 16 hours at 42 °C. After the hybridization reaction, the slides were incubated with ribonuclease (RNase) A at 37 °C, followed by stringent washing procedures (0.3 M NaCl and 30 mM sodium citrate [pH 7.0]; 0.1% sodium dodecyl sulfate at room temperature and also at 50 °C, 30 minutes each). After the slides were washed with 0.3% Triton X-100 for 10 minutes and covered with antidigoxigenin conjugates, BCIP/NBT solution (5-bromo-4-chloro-3-indolyle-phosphate, 4-toluidine-salt/nitrobluetretrazolium chloride) for detection was applied (Boehringer Mannheim GmbH). The slides were incubated at room temperature for 48 hours. Finally, the reaction was stopped and the slides were covered and mounted.

Hybridization signals were evaluated by two observers (A. Tannapfel and C. Wittekind) who were trained in HCC histology. For all probes, the signals were evaluated relative to background signal and RNase-treated control slides were hybridized with the same antisense probe. Sense probes detecting the noncoding strand were used as negative controls. Sense and antisense probes were applied to paired serial slides. As an additional negative control, one slide in each set was treated with RNase A prior to hybridization to deplete the sample of mRNA. No-probe slides were prepared as additional controls for every five slides.

Transfection of a p73 Plasmid Into NIH3T3 Cells

From a plasmid with cloned p73{alpha} cDNA (8), the p73{alpha} insert was cut and cloned between the BamHI and XbaI sites of the pcDNA 3.1/His C vector (Invitrogen Corp., Groningen, The Netherlands). p73{alpha} is the longer splice variant of the two known forms, p73{alpha} and p73ß. The insert used for the hybridization probe and the region coding for the immunoreactive peptide, however, were identical in both. The resulting plasmid expressing a protein with a histidine tag at the aminoterminal end was transfected by lipofection into NIH3T3 cells (a mouse fibroblast cell line) (9). Mock transfections with the plasmid vector without insert were used for negative control.

Immunohistochemical Analysis and Assessment

Two rabbit polyclonal p73 antibodies were raised against two 14 amino acid peptides in the N-terminal part of p73 whose sequence is different from that of p53. The amino acid sequences of the p73-specific peptides are as follows: 1) NH2-FHLEGMTTSVMAQF-COOH and 2) NH2-VKKRRHGDEDTYYL-COOH. The specificity of these antisera was shown in and found to be identical in mouse NIH3T3 fibroblasts prior to and after transfecting the p73{alpha}-expressing vector described above. The transfected cells show specific staining of the cell nuclei; mock-transfected NIH3T3 cells were uniformly negative (data not shown). For immunohistochemical analysis of p73 and p53, the material was routinely fixed in 4% formaldehyde solution and embedded in paraffin. Sections (4 µm thick) were cut, dewaxed in xylene, and then rehydrated. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol for 30 minutes. After a short rinse of phosphate-buffered saline, the sections were preincubated with avidin-biotin (Linaris Bioproducts, Wertheim, Germany) for 15 minutes to reduce nonspecific background staining. The sections were covered with normal goat serum for 20 minutes and then incubated with the primary antisera against p73 or the p53 antibody, respectively (p53: Clone DO-7, dilution 1 : 1000; DakoR, Hamburg, Germany). Thereafter, the sections were washed with phosphate-buffered saline, incubated with biotinylated goat anti-rabbit immunoglobulin G (BioGenexR, Hamburg, Germany) for 30 minutes, and covered with peroxidase-conjugated streptavidin (DakoR). The peroxidase reaction was allowed to proceed for 8 minutes, with 0.05% 3,3-diaminobenzidine tetrahydrochloride solution as substrate. Slides were counterstained with hematoxylin and finally mounted (7). Sections known to stain positively were included in each batch and negative controls were also prepared by replacing the primary antibody with mouse or goat ascites fluid (Sigma Chemical Co.-Aldrich BiochemicalsR, St. Louis, MO). The slides were examined and scored independently by three of us (A. Tannapfel, F. Geissler, and C. Wittekind) who were blinded to clinical and pathologic information.

Statistical Methods

Differences in frequencies between subgroups were analyzed by use of the Kruskal-Wallis test and the Mann-Whitney U test for unpaired samples. Correlation coefficients were calculated according to Pearson, and {chi}2 statistics were used for contingency tables. Overall observed survival functions and probabilities were estimated by use of the Kaplan-Meier method. The logrank test was used to detect differences between survival curves for stratified variables. Identification of relevant prognostic factors was performed by use of univariate and multivariate Cox regression analyses. The significance level was defined as two-sided P<.05. The median follow-up of our patients was 78 months (range, 12 days to 183 months). No patient was lost during follow-up. The medical records of all 193 patients were re-examined to assess the status of disease at the closing date of the study (October 31, 1997). At this time, one patient was still alive. All of the 192 patients who died during the follow-up period had intrahepatic and metastatic disease on their last visit to the oncologic outpatients clinic. We concluded that death in these patients was related to HCC.


    RESULTS
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
Histopathologic Features and Prognosis

The pathologic data are summarized in Table 1Go. A concomitant liver cirrhosis occurred in 104 (54%) of 193 patients, and dysplasia within the cirrhotic liver was diagnosed in 53 (27%) of 193 patients. One hundred eighteen patients (61%) had multiple HCC nodules. Satellite formation occurred in 108 (56%) of 193 patients. The overall observed 1-year disease-related survival rate of all patients was 44% (95% confidence interval [CI] = 37-52). The median survival of all 193 patients studied was 251 days (95% CI = 159-343 days), and the 5-year survival rate was 17.7% (95% CI = 11-21).

p73 In Situ Hybridization

The hybridization with the antisense probe for p73 was informative in 74 cases. In 119 cases, a high degree of nonspecific binding to one or another component of the tissue section was observed after comparison of the signal with the background signal on RNase-treated control slides hybridized with the same antisense probe. Transcripts for p73 were detected in tumor cells of 25 (34%) of 74 carcinomas examined (Fig. 1,Go A and B). The transcripts were found heterogeneously distributed within the tumor, with a predominance in peripheral tumor regions (Fig. 1Go, B). Only a small number of grains were found in the surrounding non-neoplastic liver tissue. A specific hybridization with strong signals localized to the cytoplasm and nucleus was observed. The tumor stroma, endothelial cells, cholangiocytes, fibroblasts, or inflammatory cells were negative for p73 (Fig. 1Go, B). Transcripts were not observed within mitotic or apoptotic cells. Thus, in situ hybridization clearly localized neoplastic hepatocytes as the main site of p73 transcription.



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Fig. 1. In situ hybridization of p73 messenger RNA (mRNA) and immunohistochemical demonstration of p73 protein. A) mRNA transcripts (brown reaction product) within a hepatocellular carcinoma (HCC) nodule (right) and non-neoplastic surrounding hepatocytes and fibrous tissue without transcripts (original magnification x10). B) p73 mRNA expression within an HCC. Staining signal localized to the cytoplasm and also within tumor cell nuclei (original magnification x40). C) Immunohistochemical demonstration of p73 protein. Strong expression of p73 within the carcinoma cells (left); surrounding liver tissue (right) is negative for p73 (original magnification x10). D) Immunohistochemical demonstration of p73 protein within the tumor cell nuclei of HCC (original magnification x80).

 
The amount of transcripts showed no correlation to Edmondson grade and tumor stage or to p53 status. All 25 tumors with detectable p73 transcripts were immunohistochemically positive for p73. The 49 tumors without a specific signal for p73 mRNA were negative for p73 protein by immunohistochemistry.

p73 Immunohistochemistry

p73 immunostaining revealed p73 positivity in 61 (32%) of 193 cases. Within these tumors, we generally observed a strong immunoreactivity of the tumor cell nuclei (Fig. 1Go, C and D). With few exceptions, the cytoplasm was negative. In very few tumors with strong staining of nuclei, a faint intracytoplasmic immunoreactivity was occasionally observed. p73 did not stain all malignant cells within a tumor (Fig. 1Go, D). Furthermore, the staining intensity varied within the tumor with slightly more pronounced staining at the infiltrating margins. Mitotic, apoptotic, or necrotic cells were uniformly negative. In 132 HCCs, none or only very few positive cell nuclei (<1% of all tumor cells) were observed.

In the case of surrounding, non-neoplastic cirrhotic liver tissue, normal hepatocytes were seen to be occasionally positive. In contrast to the corresponding tumors, however, only very few nuclei (<1%) expressed p73 in nontumorous tissue. Bile duct epithelial cells were uniformly negative, as was the fibrovascular stroma within cirrhotic livers. We failed to find an association of p73 expression with any other histopathologic parameters examined or with expression of p53, concomitant cirrhosis, or Edmondson grade of tumor differentiation in a given case.

p53 Immunohistochemistry

We could detect positive nuclear staining of p53 antigen in 97 (50%) of 193 carcinomas examined (Table 1). Within the tumors, the amount of positive cells varied from 15% to 80%. In a very few cases, p53 immunoreactivity of surrounding, non-neoplastic cells was observed as well. However, in all of these cases, the positivity rate was less than 10%. There was no association between the status of p53 and p73. In 27 cases, the tumors were positive for p73 and also (mutant) p53.

Survival Rate

The survival analysis was performed on 193 patients and took into account the following variables: p73 and p53 positivity (defined by immunohistochemistry), UICC tumor stage (6), Edmondson grade, vascular invasion, capsule formation, multiplicity, satellites, dysplasia, inflammatory reaction, necrosis, bile production, presence of giant cells, co-existing cirrhosis, and patient's age. Consistent with published data, UICC stage, Edmondson grade, co-existing cirrhosis, and patient's age were valuable prognostic parameters. Survival was statistically significantly shorter in patients older than 60 years and in those with co-existing cirrhosis. Univariate analysis showed p73 to be a strong predictor of survival. There was a statistically significant difference in survival between patients with tumors showing p73 immunoreactivity and those whose tumors did not (P<.001, logrank test). The mean survival time for p73-positive tumors was 127 days versus 462 days for those without expression (Table 1Go; Fig. 2Go).



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Fig. 2. Overall survival in patients with p73-positive and p73-negative tumors (p73+ = p73 present; p73- = p73 not detectable by immunohistochemistry). Numbers of patients at risk at 1095 days (3 years) and 2555 days (7 years) after curative tumor resection are shown below the x-axis. The survival probabilities at 1095 and 2555 days (plus 95% confidence intervals) are 32% (27%-36%) and 18% (14%-22%), respectively, for patients with p73-negative tumors and 13% (10%-16%) and 8% (5%-10%), respectively, for patients with p73-positive tumors.

 
Multiplicity, satellites, bile production, capsule formation, presence of giant cells, inflammatory infiltrate, and dysplasia within the nontumorous liver all lacked prognostic significance. We failed to identify p53 immunostaining as a valuable prognostic factor.

Multivariate Cox regression analysis of the ungrouped variables UICC tumor stage (6), Edmondson grade, co-existing cirrhosis, patient's age, and p73 identified p73 as an independent statistically significant prognostic indicator (Table 2). The odds ratio for p73 is 2.13. The risk of patients with p73-positive tumors to die within a specific time was twice as high than the risk of patients (to die within the same time course) with p73-negative tumors.


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Table 2. Results of multivariate Cox regression analysis*

 
In those 74 patients with informative results in in situ hybridization for p73, survival analysis was performed in univariate fashion. In concordance with the results obtained by immunohistochemical p73 assessment, p73, UICC tumor stage, Edmondson grade, cirrhosis, and patient's age were statistically significant prognostic factors. We did not perform multivariate survival analysis within these subsets of patients because of the small number of cases.


    DISCUSSION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
This study identifies p73 protein as an indicator of poor prognosis for patients with HCC treated by surgical tumor resection. Patients with tumors overexpressing p73 had a statistically significantly shorter survival time than those without detectable p73 protein. However, we failed to identify clear associations of p73 expression with other histopathologic variables, e.g., Edmondson grade or stage of disease. After comparing p73 with these other factors known to be of prognostic value in patients with curative resected HCC in a multivariate model, we identified p73 as a new and independent marker for assessing the prognosis of patients with this disease.

In addition to identifying higher p73 protein levels in tumor samples with poor patient survival prognosis, we also detected by in situ hybridization elevated amounts of p73 transcripts in the hepatocellular tumor cells, identifying these cells as the site with high levels of p73 transcription. However, in accordance with the literature (2,4,10), we detected low levels of p73 mRNA expression in non-neoplastic hepatocytes as well.

The remarkable homology between p73 and p53, together with the ability of p73 to induce the expression of cell cycle inhibitor p21WAF1/CIP1 (11,12), suggests that p73 acts as a transcription factor (12). Hui et al. (13) reported that, in the case of a detectable mutation of the p53 gene, the p21WAF/CIP1 expression was found to be reduced. p73 has, however, not yet been found to be mutated in tumors (4,10,14). Thus, the overexpressed protein is likely to be wild-type and should still be functional as a transcription factor and inducer of apoptosis. It is not clear if elevated p73 levels detected in the tumors presented in this study are sufficient to induce apoptosis or if p73 is even able to antagonize p53 function at these expression levels and thereby allow increased proliferation in tumors with a poor prognosis. Future work will have to clarify at which expression levels p73 and p53 cooperate or act antagonistically to lead cells into apoptosis or allow them to proliferate.

Our data, identifying high expression levels of p73 protein in tumors of patients with a poor survival prognosis, provides, to our knowledge, a first analysis of this protein as a prognostic factor in patients with HCC. The molecular basis of this finding remains to be elucidated.


    NOTES
 
Supported in part by the Bundesministerium für Bildung und Forschung through the Interdisziplinäres Zentrum für Klinische Forschung Leipzig (K. Engeland).

We thank Martina Fügenschuh for her excellent technical assistance and Drs. Judith Roth and Matthias Dobbelstein for reagents.


    REFERENCES
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 

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Manuscript received August 17, 1998; revised April 28, 1999; accepted May 10, 1999.


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