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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
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 cDNA (8), the p73
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
is the longer splice variant of
the two known forms, p73
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-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 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pathologic data are summarized in Table 1. 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, A and B). The transcripts were
found heterogeneously distributed within the tumor, with a predominance
in peripheral tumor regions (Fig. 1
, 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. 1
, 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.
|
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. 1, 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. 1
, 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 1; Fig. 2
).
|
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.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
We thank Martina Fügenschuh for her excellent technical assistance and Drs. Judith Roth and Matthias Dobbelstein for reagents.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1
Harris CC, Hollstein M. Clinical implications of the p53
tumor-suppressor gene. N Engl J Med 1993;329:1318-27.
2 Kaghad M, Bonnet H, Yang A, Creancier L, Biscan JC, Valent A, et al. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 1997;90:809-19.[Medline]
3 Wittekind C. Hepatocellular carcinoma. In: Hermanek P, Gospodarowicz MK, Henson DE, Hutter RV, Sobin LH, editors. Prognostic factors in cancer. Berlin (Germany): Springer-Verlag; 1995. p. 88-93.
4 Nomoto S, Haruki N, Kondo N, Konishi H, Takahashi T, Takahashi T, et al. Search for mutations and examination of allelic expression of the p73 gene at 1p36.33 in human lung cancers. Cancer Res 1998;58:1380-3.[Abstract]
5 World Health Organization. Histological typing of tumours of the liver. 2nd ed. In: Ishak T, Ishak KG, Anthony PP, Sobin LH, editors. Berlin (Germany): Springer-Verlag; 1994.
6 International Union Against Cancer: TNM classification of malignant tumors. 5th ed. In: Sobin LH, Wittekind CH, editors. New York (NY): Wiley-Liss; 1997.
7 Tannapfel A, Hahn HA, Katalinic A, Fietkau R, Kuhn R, Wittekind CH. Prognostic value of ploidy and proliferation markers in renal cell carcinoma. Cancer 1996;77:164-71.[Medline]
8
Roth J, Konig C, Wienzek S, Weigel S, Ristea S, Dobbelstein M.
Inactivation of p53 but not p73 by adenovirus type 5E1B 55-kilodalton and E4 34-kilodalton
oncoproteins. J Virol 1998;72:8510-6.
9
Bolognese F, Wasner M, Dohna CL, Gurtner A, Ronchi A,
Muller H, et al. The cyclin B2 promoter depends on NF-, a trimer whose CCAAT-binding
activity is cell-cycle regulated. Oncogene 1999;18:1845-53.[Medline]
10 Mai M, Yokomizo A, Quian C, Yang P, Tindall DJ, Smith DI, Liu W. Activation of p73 silent allele in lung cancer. Cancer Res 1998;58:2347-9.[Abstract]
11 Clurman B, Groudine M. Tumour-suppressor genes. Killer in search of a motive? [news].Nature 1997;389:122-3.[Medline]
12 Jost CA, Marin MC, Kaelin WG Jr. p73 is a human p53-related protein that can induce apoptosis. Nature 1997;389:191-4.[Medline]
13 Hui AM, Kanai Y, Sakamoto M, Tsuda H, Hirohashi S. Reduced p21 (WAF1/CIP1) expression and p53 mutation in HCC. Hepatology 1997;25:575-9.[Medline]
14 Takahashi H, Ichimiya Y, Watanabe M, Furusato M, Wakui S, Yatani R, et al. Mutation, allotyping, and transcription analyses of the p73 gene in prostatic carcinoma. Cancer Res 1998;58:2076-7.[Abstract]
Manuscript received August 17, 1998; revised April 28, 1999; accepted May 10, 1999.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |