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22
In Situ Simultaneous Detection of Hepatitis C Virus
RNA and Hepatitis C Virus?Related Antigens in
Hepatocellular Carcinoma
Domenico Sansonno, M.D.
Vito Cornacchiulo, M.D.
Vito Racanelli, M.D.
Franco Dammacco, M.D.
BACKGROUND. The overwhelming evidence that chronic infection with the hepatitis
C virus (HCV) is an important cause of hepatocellular carcinoma (HCC) is based
on epidemiologic, case-control, and cohort studies as well as laboratory investiga-
Department of Biomedical Sciences and Human
Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical
School, Bari, Italy.
tions. To address better the pathogenesis of HCV infection at the single-cell level,
the authors developed a specific reproducible method for the simultaneous detection of HCV specific sequences and antigens in liver tissue, using a combination
of nonradioactive in situ hybridization and immunohistochemistry.
METHODS. After immunohistochemical staining of the liver sections for E2/NS-1,
C22-3, C33c, C100-3, and NS-5 antigens with immunogold-silver technique, in situ
hybridization was performed on the same sections using digoxigenin-labeled HCV
5* NonCoding specific probes. The hybridization signal was detected by an antidigoxigenin, Fab fragment?alkaline phosphatase conjugate. This simultaneous detection permitted the subcellular localization of HCV RNA and antigens with excellent preservation of tissue morphology and absence of background staining. In
addition, the types and percentages of cells harboring HCV in tissue could be
determined.
RESULTS. The in situ detection of HCV showed positive signals in both cancerous
and noncancerous areas of liver tissue in six of six HCV-infected patients with
HCC and in none of four controls, including three HCV negative HCC patients
and one patient with epithelioid hemangioendothelioma. Two classes of infected
cells were distinguished throughout the liver: (1) cells containing large amounts
of negative-stranded HCV RNA, which were probably undergoing active viral replication; and (2) cells displaying positive-stranded HCV RNA only, with unpredictable levels of viral replication. Both types expressed core, envelope, and NS-3, -4,
and -5 proteins. HCV RNA and antigens were exclusively cytoplasmic. Detection
Supported in part by a grant from the Italian
Ministry for the Universities and Scientific and
Technological Research, Liver Cirrhosis and Viral Hepatitis Group, and by the Associazione
Italiana per la Ricerca sul Cancro (AIRC).
of viral proteins was highly predictive of the presence of large amounts of HCV
RNA in the same cell. Fewer HCV positive cells were consistently demonstrated in
the cancerous area.
CONCLUSIONS. These findings support the contention that HCV infects hepatocytes
and replicates in them, even after their malignant transformation. Cancer
1997;80:22?33. q 1997 American Cancer Society.
V.C. is supported by a fellowship from AIRC.
Address for reprints: Franco Dammacco, M.D.,
Department of Biomedical Sciences and Human
Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical
School, Policlinico 11, Piazza G. Cesare 70124,
Bari, Italy.
Received November 20, 1996; revision received
February 26, 1997; accepted February 26, 1997.
KEYWORDS: hepatitis C virus, hepatocellular carcinoma, in situ hybridization, immunohistochemistry.
H
epatocellular carcinoma (HCC) is one of the most common tumors
worldwide. The main risk factor for its occurrence has been found
to be cirrhosis.1,2 In areas where hepatitis B virus (HBV) infection is
nonendemic, i.e., with HBsAg carrier rates less than 1%, a large number of case-control studies have demonstrated a high prevalence of
hepatitis C virus (HCV) infection in patients with HCC, ranging from
q 1997 American Cancer Society
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HCV in Hepatocellular Carcinoma/Sansonno et al.
39% to 77%.3 ? 7 In these studies, the mean relative risk
of HCC was higher than 5, supporting the notion that
HCV infection is a major factor for the development
of HCC worldwide.8 The mechanism of malignant
transformation, however, is not known. The lack of a
reliable tissue culture system for HCV has prevented
work in this area. The pathogenesis of hepatocyte
damage in HCV chronic infection also remains unclear, and a variety of specific and nonspecific immunologic mechanisms for cell damage have been proposed.9 ? 11
Polymerase chain reaction (PCR) of nucleic acid
extracts of tissue homogenates is a sensitive approach
for detecting virus nucleic acid sequences in which a
relatively homogeneous population of cells contain a
small number of virus genomes per cell.12 ? 15 This approach is well suited for studying the presence of plusand minus-strand HCV RNA in both neoplastic and
surrounding tissue of HCC, suggesting that HCV replicates in tumor cells and surrounding nonmalignant
hepatocytes.13 ? 15 The presence of the minus strand of
HCV, antigenomic RNA, as the PCR product has been
recently questioned by some authors16,17 due to the
possible false priming of the incorrect strand during
the cDNA step.
In situ hybridization (ISH) is a valuable approach
whenever a small proportion of cells in a population
may be infected. It can also provide a correlation with
histologic features, including subcellular structures
and distribution of viral nucleic acids.18 ? 22 The latter
method provided evidence of HCV infection in both
the neoplastic cells of patients with HCC and the surrounding nonmalignant hepatocytes. In addition, HCV
proteins, particularly core protein, were detected in
scattered HCC tumor cells, supporting the notion that
HCV persists in hepatocytes during and after malignant transformation.23,24
Simultaneous demonstration of HCV specific nucleotide sequences and antigens in HCC cells is a significant approach to a better understanding of the
pathogenesis of HCV infection at the single cell level.
In this study of six HCV-infected patients with HCC,
we present detailed evidence that in cells with significant amounts of cytoplasmic HCV RNA, much of this
is in negative polarity-stranded form. In addition, we
provide a description of different patterns of viral RNA
and antigen accumulation within different hepatocytes in the same chronically infected liver with HCC.
MATERIALS AND METHODS
Patients
Fourteen chronically HCV-infected patients with primary liver tumors underwent hepatectomy between
1990 and 1995. Six who stringently satisfied the follow-
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23
ing criteria were selected: (1) clear and unequivocal
separation of cancerous tissue from surrounding noncancerous liver, and (2) tissue preparation procedure
with appropriate protection against enzymatic degradation of nucleic acids. All patients lived in Italy, and
all but one had cirrhosis. For one patient, long term
alcohol intake was proved. Hemochromatosis, Wilson?s disease, a1-antitrypsin deficiency, and HIV infection were all ruled out.
The control group included 4 anti-HCV, HCV
RNA-negative patients with primary liver tumor from
whom wedge-shaped liver biopsies were taken. In two
of these patients, HCC developed in HBsAg-positive
cirrhosis; in one, alcoholic cirrhosis was diagnosed;
and in the fourth, an epithelioid hemangioendothelioma involving the entire left lobe was resected.
Tissue Preparation
All tissue samples from both HCV-infected patients
and controls were obtained from surgical specimens.
After extensive washings in cold phosphate-buffered
saline (PBS) containing 0.02% diethylpyrocarbonate
(DEPC, Sigma Chemical Company, St. Louis, MO), tissues were fixed in 4% paraformaldehyde (PFA) in PBS
containing 0.02% DEPC for 2 hours. Samples were
then mounted in tissue specimen embedding medium
(OCT, Miles Scientific, Naperville, IL) and frozen in
liquid nitrogen.
Because immunohistochemistry (IHC) preceded
ISH, appropriate protection against enzymatic degradation of target RNA in tissue was required. In this
context, the current protocol included the use of heparin (5 mg/mL) as an inhibitor of RNAase to dilute antiHCV antibodies and all other solutions employed for
IHC. Slides were extensively washed with PBS containing heparin (PBS-H) and incubated overnight with
murine monoclonal antibodies (MoAbs) directed
against structural and nonstructural HCV-related proteins. Immunochemical characteristics of these reagents have been described in detail elsewhere.22,25
MoAb anti-E2/NS-1 and anti-C22-3 proteins were
used at a working concentration of 1.2 mg/mL. AntiC100-3 antibodies comprised a mixture of four MoAbs
and were used at protein concentration in the working
mixture of 1.0 mg/mL each. Anti-C33c and anti-NS-5
proteins were used at concentrations of 1.5 mg/mL.
After extensive washings in PBS-H, the slides were
incubated with goat antimouse immunoglobulin G labeled with gold particles 5 nm in size (Amersham,
Bucks, UK; code RPN 451) for 3 hours at room temperature (r.t.). The slides were washed once with PBS-H,
followed by extensive washings with redistilled water
(500 mL) to remove phosphate ions. Detection of colloidal gold particles in tissue was performed by en-
W: Cancer
24
CANCER July 1, 1997 / Volume 80 / Number 1
TABLE 1
Clinical, Serologic, and Virologic Patterns of Patients and Controls
Participants
Patients
1.
2.
3.
4.
5.
6.
Controls
7.
8.
9.
10.
Age (yrs)/
gender
Duration of
liver disease
(yrs)
Anti-HCV status
(5-1-1/C100/C33c/C22-3)
Serum HCV
genotypes
ALT
(n.v. � IU/L)
a-Feto protein
(n.v. � ng/mL)
HBV status
(HBsAg/a-HBs/a-HBc/
HBeAg/a-HBe)
Serum
HBV DNA
60/M
62/M
70/M
71/M
66/M
59/M
18
21
26
19
28
22
2//3//3//4/
0/0/2//3/
0/0/3//4/
0/2//3//4/
0/2//2//4/
0/0/0/4/
1b
1b
1b
2a
1b / 2a
1b
256
167
129
136
99
102
46
37
83
578
51
39
0/////0/0
0/0///0//
0/////0/0
0/0///0//
0/////0/0
0/0/0/0/0
0
0
0
0
0
0
55/M
59/M
69/M
52/F
26
33
31
2
0/0/0/0
0/0/0/0
0/0/0/0
0/0/0/0
0
0
0
0
199
110
61
33
66
166
52
8
//0///0//
//0///0//
0/////0/0
0/0/0/0/0
0
/
0
0
HCV: hepatitis C virus; HBV: hepatitis B virus; ALT: alanine aminotransferase; n.v.: normal values.
TABLE 2
Histology and Polymerase Chain Reaction for HCV RNA and HBV DNA in Cancerous and Noncancerous Liver Tissue
Noncancerous areas
Participants
Patients
1
2
3
4
5
6
Controls
7
8
9
10
Cancerous areas
Histology
HCV RNA
HBV DNA
Histology
No. of nodules
Gradea
HCV RNA
HBV DNA
CH/CIR
CH
CH/CIR
CH/CIR
CH/CIR
CH/CIR
/
/
/
/
/
/
0
0
0
0
0
0
HCC
HCC
HCC
HCC
HCC
HCC
1
1
4
5
1
2
1
2
2
1
1
2
/
/
/
/
/
/
0
0
0
0
0
0
CH/CIR
CH/CIR
CIR
PF
0
0
0
0
/
/
0
0
HCC
HCC
HCC
EHE
4
2
5
1
2
2
1
0
0
0
0
0
/
/
0
0
CH: chronic hepatitis; CIR: cirrhosis; PF: portal fibrosis; HCC: hepatocellular carcinoma; EHE: epithelioid hemangioendothelioma.
a
Degree of tumor differentiation according to grading of Edmondson and Steiner.
hanced silver deposition (Amersham, code RPN 491).
Sections were washed in DEPC-treated distilled water
for 20 minutes and fixed in freshly prepared 4% PFA
for 10 minutes. They were then rinsed twice in PBSH for 5 minutes and digested with 12.5 mg/mL proteinase K (Boehringer, Mannheim, Germany) for 5 minutes. The slides were rinsed again in PBS and refixed
in 4% PFA for 10 minutes, dipped in DEPC-treated
distilled water, and acetylated in 0.01% acetic anhydride in 0.1 M triethanolamine for 10 minutes. After
washing in PBS-H and 0.85% saline for 5 minutes each
and holding at 95 7C for 15 minutes to denature nucleic
acids and abolish endogenous alkaline phosphatase
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activity, the sections were refixed in 4% PFA, rinsed in
PBS-H and 0.85% saline, dehydrated through a series
of ethanol baths (40 ? 100%), and allowed to dry for at
least 1 hour.
Dehydrated sections were prehybridized for 1 hour
at r.t. with a mixture containing 50% deionized formamide, 11 Denhardt?s solution, 1 mM ethylenediamine tetraacetic acid (EDTA), 100 mg/mL denatured
salmon sperm DNA, 100 mg/mL yeast RNA, 250 mg polyadenylic acid, and 41 standard saline citrate (SSC). Hybridization was carried out for 24 hours at 40 7C in the
same mixture containing digoxigenin-labeled probe at
the final concentration of 100 ng/mL, which was boiled
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25
FIGURE 1.
Percentages of cells positive for HCV antigen in cancerous and noncancerous liver tissue are
shown.
for 5 minutes and quenched on ice. Freshly prepared
dithiothreitol was added to a final concentration of 10
mM. Sections were overlaid with 20 mL hybridization
mixture, covered with Parafilm and placed in a moist
chamber. After incubation, the Parafilm (American National Can., Greenwich, CT) was removed and the slides
were washed for 1 hour at r.t. in 21 SSC, then for 1 hour
in 11 SSC, 10 minutes at 50 7C in 0.51 SSC, and finally
for 1 hour at r.t. in 0.11 SSC. After a brief wash in PBS,
sections were incubated with the Fab fragment of sheep
antidigoxigenin antibody conjugated with calf intestinal
alkaline phosphatase (Boehringer) for 4 hours at r.t. Unbound antibody was washed off by immersing the slides
in 0.1 M Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl,
followed by 0.1 M Tris-HCl pH 9.5 containing 0.1 M NaCl
and 0.05 M MgCl2 . To develop the color reaction, fast
red was used as the alkaline phosphatase substrate. Two
mg of naphthol-As-Mx phosphate (Sigma) was dissolved
in 0.2 mL of dimethylformamide Tris-HCl buffer 0.1 M
(pH 8.2) containing fast red TR salt at 1 mg/mL, and
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levamisole at a final concentration of 1 mM was added.
After color development, the sections were rinsed in 0.01
M Tris-HCl (pH 7.5), 1 mM EDTA, and 0.9% NaCl, then
counterstained and mounted.
In the control experiments before hybridization, sections were treated with 20 mg/mL ribonuclease A (Boehringer), 80 U/mL ribonuclease T1 (Boehringer), and 10
mg/mL deoxyribonuclease (Boehringer). In competition
experiments, unlabeled specific probes in 500-fold excess were added to the hybridization mixture.
At least 5?10 sections were examined for each case.
Photodocumentation was carried out on a Leitz DM RB/
E microscope equipped with the Wild MPS 48/52 camera
system (Leica, Wetzlar, Germany). Percentage measurements of positive cells were made with the Quantimet
500 analyzing system (Leica).
Probe Preparation
The following synthetic oligonucleotide DNAs were
used for ISH detection:
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a1, antisense: 5*-TCCTGGAGGCTGCACGACACTCATACTAACGCCATGGCTAGACGC-3* (0223 � 0267)
A2, sense: 5*-GCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGA-3* (0267 � 0223),
complementary to bases of the 5* NonCoding (5*NC)
region of the HCV genome in apex 23. These probes
were purchased from Genset (Paris, France). Fully deprotected oligos were purified by high-performance liquid chromatography (HPLC), checked by polyacrylamide
gel electrophoresis, and labeled at the 3* end with digoxigenin-11-deoxyurindine triphosphate (dUTP). As control, digoxigenin-labeled oligomers specific for the coding region of the wild-type hepatitis A virus (HAV) HM175 were used:
antisense: 5*-CTAGGATCATCCACTGATGACTCCAAGTCT-3* (3082 ? 3053)
sense: 5*-AGACTTGGAGTCATCAGTGGATGATCCTAG-3*
(3053?3082).
PCR for HBV DNA
DNA from neoplastic and nonneoplastic liver specimens as well as DNA obtained from plasma were amplified in a final volume of 75 mL containing 10 pmol
of each primer on S (5*-CCCAATACCACATCATCC-3*,
downstream: 760 ? 743 and 5*-TTCCTATGGGAGTGG3*, upstream: 637 ? 651) and X (5*-CTGGATCCTGCGCGGGACGTCCTT-3*, upstream: 1400 ? 1423 and 5*GTTCACGGTGGTCTCCAT-3*, downstream: 1627?1610)
regions of HBV genome, 200 mmol of each of the 4
deoxynucleotides per liter, the PCR buffer and 2.5 U
of Taq polymerase (Perkin-Elmer, Norwalk, CT). Fortyfive cycles (94 7C, 1 minute; 55 7C, 1 minute; 72 7C, 1
minute) were carried out. The amplification products
(10 mL)were analyzed by electrophoresis on 2% agarose gel. Specificity was confirmed by hybridization
with a 32P-labeled cloned HBV genome.26
PCR for HCV RNA
Tissue and sera were obtained from patients on the
same day. A two-stage PCR with nested primers was
used to amplify complementary DNA synthesized on
reverse transcription (RT), as described previously.27
Primers were selected from the 5*NC region of the
27
HCV genome. HCV positivity was confirmed by a
nested PCR amplification of the core region of HCV,
according to the method described by Okamoto et
al.,28 to characterize the HCV genotypes. Furthermore,
biotinylated universal primers referred to 5*NC of HCV
genome were employed to amplify and hybridize to
genotype specific probes (Line Probe assay, LiPA, Immunogenetics, Brussels, Belgium). Each sample was
tested in triplicate, and adequate positive and negative
controls were always included.
RESULTS
Clinical, serologic, and virologic data of patients and
controls are reported in Table 1. Five patients were
reactive for at least two HCV-related antigens, and all
were viremic in view of the demonstration of HCV
RNA sequences in serum by RT-PCR. One patient was
coinfected with genotypes 1b plus 2a. With regard to
markers of HBV infection, all patients were HBsAg negative, and PCR for HBV DNA amplification of S and X
regions failed to demonstrate viral sequences. Liver
tests of hepatocytolysis as well as serum a-fetoprotein
concentration were increased in all patients. All four
patients in the control group showed negative reactions for either anti-HCV antibodies or HCV RNA. Two
were HBV chronic carriers, and in one of them HBV
DNA could be demonstrated in the serum.
Pathologic features are described in Table 2. Cirrhosis with or without inflammatory activity was present in the resected liver of five patients, and chronic
hepatitis without obvious cirrhosis was diagnosed in
the sixth. Multinodular location of HCC was found in
3 patients, whereas a solitary lesion ranging in size
from 2.2 ? 5.9 cm was present in the other 3, and they
showed better cellular differentiation (Edmondson?s
Grades 1 and 2). A trabecular pattern was detected
in four patients. Nuclear crowding, irregular nuclear
margins, and anisokaryosis were particularly prominent in two cases. Heavy inflammatory infiltrates
within the tumor mass were noted in three cases.
RT-PCR demonstrated HCV RNA sequences in
both noncancerous and cancerous samples. In addition, analysis of HCV genotypes in liver and tumor
b
FIGURE 2.
Histology and immune staining of HCV-related antigens (Ags) is shown in noncancerous areas of liver tissue from Patient 3. (A) H & E
preparation shows a cirrhotic picture with well-differentiated parenchymal nodules separated by fibrous septa and heavy inflammatory infiltration (original
magnification 1100). (B) E2/NS-1-Ag was found in the hepatocytes throughout the section, with no special relation to vascular or biliary structures
(original magnification 1250). (C) NS-5-Ag is demonstrated in the hepatocytes. Cirrhotic nodules are completely stained by the specific immunoreaction
(original magnification 1250). (D) At higher magnification, cytoplasmic C22-3 immune reactants appear as homogeneous material. Positive hepatocytes
are within an area of piecemeal necrosis. Some C22-3-Ag positive cells resemble the morphology of inflammatory cells (arrows) (original magnification
1400).
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extracts showed the same pattern as in each patient?s
serum. S and X gene sequences of the HBV genome
were not detectable, despite the high sensitivity of PCR
for HBV DNA (0.01 femtogram of viral nucleic acids).
Patients and controls were comparable in terms of
differentiation grade of histologic pictures, duration of
liver disease, and serum levels of transaminases and
a-fetoprotein. HCV RNA sequences were not demonstrated in tissues of the control group, whereas HBV
DNA sequences were found in two of them.
Sections from noncancerous and cancerous areas
were examined by IHC for E2/NS-1, C22-3, C33c,
C100-3, and NS-5 antigens (Ags) in progressive serial
sections and photographed. The fields previously photographed were located, rephotographed, and correlated for the presence of each antigen. Location of
HCV-Ags was solely cytoplasmic in both cancerous
and noncancerous areas. HCV-Ags usually coexisted in
the same area; indeed, areas negative for one marker
usually lacked the others.
As indicated in Figure 1, E2/NS-1-Ag was demonstrated in approximately 20 ? 80% of cells from 4
(66.7%) cases in noncancerous liver and in approximately 10% of cells of 3 (50%) cancerous samples. C223 protein was found in almost 10% of cells of all 6
noncancerous samples and in 1 ? 5% of the respective
cancerous areas. C33c was demonstrated in approximately 30 ? 60% of cells of noncancerous samples of 4
patients and in almost 10% of cells in the cancerous
counterpart in 1 patient. C100-3-Ag was demonstrable
in 10 ? 70% of cells from 2 nonneoplastic livers and
no samples from cancerous areas. Finally, the NS-5
protein was found in approximately 20 ? 80% of cells
from 4 noncancerous samples and in 10 ? 60% of cells
from 2 patients in the cancerous counterparts. The
extent of expression of HCV-related antigens in noncancerous areas was higher than in the corresponding
cancerous samples. None of the patients in the control
group showed evidence of immunodeposits.
As shown in Panels A ? D of Figure 2, which illustrates representative fields in noncancerous areas, the
number and intensity of signal-positive cells ranged
from a few faint cells to numerous strongly positive
cells distributed within large areas of negative cells.
Cirrhotic nodules were often completely stained and
29
gave frequent and peculiar pictures. Strong HCV-Ag
hepatocellular reaction was often observed within
areas of piecemeal necrosis. HCV immunodeposits
were never found in liver sinusoidal cells, bile duct
epithelium, or blood vessel walls and were rarely expressed in infiltrating inflammatory cells.
A significantly lower proportion of immunostained cells were counted in the cancerous counterpart. A positive reaction was identified in isolated cells
randomly distributed all over the section (Fig. 3B) or
in small clusters within large negative areas (Fig. 3C).
Their location had no apparent relation to vascular or
biliary structures. Cytologic preservation of the sections was adequate to allow correlation between histologic evidence of hepatocyte injury and detection of
HCV markers. No correlation was established between
HCV-Ags distribution and hepatocyte necrosis with
ballooning, hydropic, or eosinophilic degeneration. In
addition, no apparent correlation with the degree of
cell differentiation was detected, since small and large
anaplastic hepatocytes could be infected with HCV
(Fig. 3D).
Specificity of immunohistochemical reactions was
proved by different approaches. There was no staining
reaction in HBV-infected or uninfected liver samples
with any of the antibody preparations, nor in the liver
samples from the HCV-infected carriers after omission
of the primary antibody or substitution with irrelevant
probe (i.e., antihuman chorionic gonadotropin antibody). Preadsorption of E2/NS-1, C22-3, C33c, C1003, and NS-5 antisera with the relative recombinant
antigens before immunostaining abolished the positive staining; this was consistent with a parallel loss of
reactivity in enzyme-linked immunoadsorbent assay
(ELISA) or immunoblotting. In contrast, immunohistochemical, ELISA, or immunoblotting signals were retained after mock adsorption of these antisera against
the antigen diluent or unrelated antigens (i.e., HBsAg,
HBcAg, HAV-Ag).
As detailed in Figure 4, viral RNA location was
exclusively cytoplasmic. Approximately 80% of cells
positive for HCV RNA expressed viral proteins. HCV
RNA and Ags usually coexisted in the same cells; cells
negative for HCV RNA usually lacked viral proteins.
Distribution of HCV RNA-positive cells showed fea-
b
FIGURE 3.
Histology and HCV immune reactants are shown in the liver sections of the cancerous area from patient 3. (A) H & E section shows a
trabecular pattern of liver-cell plates with high degree of hepatocyte pleomorphism (original magnification 1250). (B) C22-3 immune reactants are found
in the hepatocytes scattered all over the section (original magnification 1150). (C) A small cluster of neoplastic hepatocytes demonstrates NS-5-Ag
deposits within a large area of negative cells (original magnification 1250). (D) Higher magnification demonstrates C22-3-Ag deposits in the cytoplasm
of an isolated atypical liver cell. Note the complete negativity in the nucleus (original magnification 1400).
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tures similar to those described for HCV-Ags. Again,
there was a significant decrease in the number of positive cells in cancerous tissue compared with the corresponding noncancerous areas. No HCV RNA signal
was observed in biliary cells or mesenchymal cells.
Rare HCV RNA positive cells were found in inflammatory mononuclear cells.
Simultaneous detection of HCV RNA and Ags displayed the same subcellular localization. Both viral
markers were cytoplasmic, and no nuclear or nucleolar
signals were demonstrated.
The following observations confirmed the specificity for HCV RNA: (1) no reaction was obtained with
uninfected liver sections or with probes complementary to coding region of the wild-type HAV, (2) the
reaction was unaffected by DNAase pretreatment but
was abolished by RNAase pretreatment, (3) omission
of specific probes resulted in negative staining, and
(4) competitive hybridization with 500-fold excess of
unlabeled specific probes abolished the signal. Finally,
denaturation of liver tissue prior to hybridization was
found to be an essential step. Its omission resulted in
poor quality and low-intensity signals. This suggests
that probe access to target sequences may be prevented by the state of viral genome, which may be
self-annealed, annealed to complementary replicative
intermediates, or complexed with cellular components.
Genomic and antigenomic HCV RNA were detected. We demonstrated plus-strand viral RNA in both
cancerous and noncancerous samples of all cases;
however, minus-strand viral RNA was detected in all
6 noncancerous samples and in 4 of the relative cancerous areas. Again, in terms of the number of minusstrand RNA-containing cells, cancerous areas showed
a significantly lower number of putative virus replicative patterns than the corresponding noncancerous
portions.
DISCUSSION
We have developed a nonradioactive double-labeling
method by applying immunogold-silver technique to
detect primary antibodies directed against HCV-Ags
31
and alkaline phosphatase ? linked Fab fragment antibody to digoxigenin to reveal digoxigenin-labeled oligonucleotide probes. The sequence of techniques in
this combined procedure is critical. Application of IHC
first avoids denaturing of the antigens during the long
and deeply stressed ISH procedure. In contrast, appropriate protection against enzymatic degradation of target RNA is necessary. A stringent protocol directed at
preserving tissue integrity and avoiding any possible
source of RNA degradation was therefore devised. The
argument that in this sequence hybridized RNA could
be lost from the sections during IHC, thus decreasing
the hybridization sensitivity, is valid only if a watersoluble reaction product is formed in the signal development. IHC signals detected by the immunogold-silver technique result in water-insoluble reaction products that are unaffected by the subsequent application
of the alkaline phosphatase technique.
After removal of DNAs by deoxyribonucleases and
heat denaturation of the tissue, we readily detected
HCV RNA in infected liver cells. The specificity of the
assay was carefully confirmed by several approaches.
Both viral RNA and HCV Ags were focally distributed in the liver. Basically, two main pictures of infected cells were established: (1) most infected hepatocytes (�%) with high levels of HCV replication showing minus-strand RNA component, and (2) cells with
unpredictable levels of viral replication in which only
plus-strand RNA component could be demonstrated.
Differential pictures in the distribution of minus or
plus-RNA strands suggest the existence of a regulatory
mechanism favoring the production of plus-strand
over minus-strand RNA, as previously demonstrated
for other flavivirus components, in that one obtains
a minimum plus-to-minus ratio of 10:1.29 This may
suggest that only the plus-polarity genomic RNA is
synthesized late in HCV infection.
The current data and previous results from our
own laboratory and others25,30 ? 34 indicate that the cytoplasm is the primary subcellular compartment of
both viral genome and proteins. Indeed, extranuclear
localization of HCV was the main picture even in neoplastic cells. Some data that conflict with these have
b
FIGURE 4.
Simultaneous detection of HCV RNA and HCV antigens (Ags) by combination of ISH and IHC in liver cancerous tissue, obtained from
Patient 1. The red signal indicates the presence of HCV RNA, whereas the dark brown signal indicates that of C22-3-Ag. Both positive signals are defined
in the cytoplasm of the hepatocytes. Nuclei display completely negative response to sense (A) and antisense (B) digoxigenin-labeled HCV specific probes.
The sections were counterstained with Mayer?s hematoxylin (original magnification 1400). Higher magnification (C) shows that, in addition to cells
containing both (0) polarity stranded form of HCV RNA and C22-3-Ag, there are cells demonstrating HCV RNA only (arrows) (original magnification
1600). (D) The (/) polarity stranded form of HCV RNA and C22-3-Ag is demonstrated in the section adjacent to that detailed in (C) (original magnification
1600).
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CANCER July 1, 1997 / Volume 80 / Number 1
shown nuclear localization of the core protein.35,36 Nuclear staining of the core protein was also observed
in rat embryo fibroblasts transfected with HCV core
gene,37 The reason for this discrepancy is not clear at
this time; however, results obtained with in vitro models of established cell lines for the study of HCV replication are not ideal in this respect. Using HPBMa102, a human T-cell line,38 and Daudi lymphoblastoid
cells, which belong to a human B-cell line,39 morphologic evidence of nuclear involvement during viral
morphogenesis was not found. Electron micrographs
and immunodetection of these cultured cells revealed
a large number of viruslike particles assembled within
vescicles in the cytoplasmic compartment.
We have demonstrated that all HCCs efficiently
expressed HCV core protein, though fewer than in the
noncancerous counterpart. This suggests that core
translation is retained even after neoplastic transformation, possibly through a multistep process. Whether
the transcriptional regulation of cellular proto-oncogenes of core protein plays a role in the transforming
activity remains to be elucidated. In addition to core
protein, other HCV-related proteins have been demonstrated in neoplastic hepatocytes. E2/NS1, C33c,
and NS-5 antigens were present in different proportions in our patients. Transformation of NIH3T3 cells
to tumorigenic phenotype by NS-3-encoded product
has been recently reported.40 Proteinase activity associated with this protein has been proposed as a further
potentially transforming factor.
It is noteworthy that many more HCV-infected
cells were always found in nonneoplastic areas as
compared with their neoplastic counterparts. This
suggests that cell dedifferentiation may change the affinity of neoplastic cells for HCV and indicates that
transformed cells are no longer permissive to HCV replication. Neoplastic cells have been noted to dedifferentiate as HCC grows, and hepatocytes may retain affinity for HCV in the early stages of malignant transformation.41 These observations imply that HCV is not a
proliferating stimulus in the late stages and that other
factors contribute to ongoing liver disease.
Molecular biology techniques have been used to
demonstrate low-level HBV and/or variant infections
in individuals with chronic liver disease previously
thought to be unrelated to HBV infection.42 HBsAg
negative patients with HCC have been shown to harbor occult HBV infection, and the integration of HBV
into the chromosomes of infected hepatocytes probably plays a key role in malignant transformation. In
the current series, however, latent infection with HBV
was reasonably ruled out by the failure of a very sensitive PCR technique to detect the HBV DNA amplifiable
sequences in serum and tissue.
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The current findings indicate that HCV replicates
and expresses several viral proteins in HCC. The possible mechanisms of HCV-related hepatocarcinogenesis
must await further studies.
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