JMD 2001, Vol. 3, No. 2
Copyright © 2001 American Society for Investigative Pathology & Association for Molecular Pathology
Diagnostic Impact of Fluorescence in Situ Hybridization in the Differentiation of Hepatocellular Adenoma and Well-Differentiated Hepatocellular Carcinoma
Ludwig Wilkens*,
Martin Bredt*,
Peer Flemming*,
Yvonne Schwarze*,
Thomas Becker
,
Michael Mengel*,
Reinhard von Wasielewski*,
Juergen Klempnauer and
Hans Kreipe*
From the Departments of Pathology
*
and Visceral and Transplant Surgery,
Hanover Medical School, Hanover, Germany
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Abstract
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Histopathological differentiation between hepatocellular adenoma
and well differentiated hepatocellular carcinoma (HCC) may be a
difficult task in small biopsies and occasionally in resected tumor
specimens. Whether the analysis of chromosome aberrations can
contribute to a more precise discrimination has not been analyzed
systematically up to now. Therefore, fluorescence in
situ hybridization was applied to 28 cases of adenoma and well
differentiated carcinoma, using centromeric probes for
chromosomes 1, 6, 7, 8, and X. None of
14 adenomas revealed an aberrant count in the analyses performed. By
contrast, 13/14 carcinomas demonstrated aberrations for 2–5
chromosomes/case. Chromosome 1 was aberrant in 8/12 cases informative
for this probe (67%), chromosomes 6 and 7 were aberrant in
9/14 cases (64%), chromosome 8 was aberrant in 11/14 cases
(79%), and chromosome X in 7/14 cases (50%). Taking results
for chromosomes 1 and 8 together, 13/14 HCC revealed
aberrations for at least one of these chromosomes. Probes for
6, 7, and X revealed no additional aberrant
cases.Thus, FISH for chromosomes 1 and 8, extended by
probes for chromosomes 6, 7 and X, represents a
promising approach toward a more accurate differentiation between
hepatocellular adenoma and carcinoma.
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Introduction
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Hepatocellular carcinoma (HCC) represents the most frequent
malignant tumor of the liver1
and is associated with
different etiologies such as viral infection and toxic
agents.2
Despite the advances in sonographic and
radiological techniques, histological examination still remains the
gold standard in the diagnosis of HCC.3
Whereas the
identification of moderate and poorly differentiated HCC is easily
achieved by histopathology, identification of well differentiated HCC
is more difficult. Distinction from liver cell adenoma still remains a
diagnostic challenge, particularly in small biopsies.4, 5
Analysis of cytogenetic aberrations in HCC could provide a potential solution to
problematic histological queries. Conventional cytogenetics (CG) is not
useful for this purpose due to well-known difficulties in obtaining
metaphases necessary for karyotyping.6, 7, 8, 9
The alternative
to CG, comparative genomic hybridization (CGH), permits karyotyping
without the need for metaphase preparation.10
With this
technique, larger numbers of HCC have been analyzed, revealing typical
aberration patterns not only in moderate or poorly differentiated HCC
but also in well-differentiated samples.11, 12, 13
These
patterns with numerous aberrations were strikingly different from the
low number of aberrations detected in a CGH study analyzing
hepatocellular adenoma (HCA).14
However, CGH is based on
an elaborate and time-consuming procedure comparable to conventional CG
and is difficult to apply in small biopsies in daily routine.
In comparison to CGH, fluorescence in situ hybridization
(FISH) detects aberrations of defined chromosome loci in intact nuclei
and preserved histological architecture even in small specimens. Since
the main aberrations occurring in HCC are now known, based on the
previous CGH results, it seems appropriate to analyze HCC and HCA by
FISH, taking probes for those loci most often affected. For this
purpose, we analyzed histological samples of 28 cases of HCA and
well-differentiated HCC by FISH with a panel of 5 centromere-specific
probes.
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Materials and Methods
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Tissue samples from 28 patients at the Medizinische Hochschule
Hannover were analyzed. There were 14 patients in the group suffering
from HCA, 11 of them female and 3 male (Table 1)
. Ages ranged from 27 to 59 years with a mean of 37 years, as listed in
Table 1
. Fourteen patients were known to have a well-differentiated
HCC; 4 were female and 10 were male. Ages ranged from 41 to 76 years
with a mean of 63 years. Diagnosis of HCA and HCC was based on
hematoxylin and eosin (H&E)-stained sections and also periodic
acid-Schiff, Elastica van Gieson, Orcein, and iron stains. To ensure
accuracy of histological diagnoses, HCA was assumed only when
disease-free survival of the patients exceeded 4 years. Furthermore,
HCA samples were included only when the tumor was resected and analyzed
by multiple additional tissue samples. To confirm diagnoses of HCC,
samples were accepted when histological examination of the tumor was
possible, as in HCA. In patients not undergoing surgical resection, HCC
was assumed when obvious signs of malignancy, in particular metastases,
were detectable.
FISH analyses were performed either on biopsies taken in
vivo by fine needle aspiration (n = 15) or
on biopsies taken from tissue obtained after surgical removal of the
tumors (n = 13). Specimens were fixed for at
least 24 hours in formalin, embedded in paraffin, and sampled together
in a multi-tissue block (MultiBlock; Zytomed, Berlin, Germany).
FISH
FISH was performed on all samples with centromere-specific probes.
Centromeric regions contain highly repetitive sequences, that are much
easier to detect with FISH probes than low- or even single-copy
sequences of chromosomes. The probes used were chosen with regard to
CGH results in well differentiated HCC detected by our own group and
other authors. In these studies, the most often altered chromosomes
included 1, 4, 5, 6, 7, 8, 16, 17, and X with centromeric regions
frequently affected in chromosomes 1, 4, 6, 7, 8, and X. Because a
centromeric probe for chromosome 4 was not commercially available at
the time the experimental part of the study was performed, this
chromosome was excluded. The panel of probes applied to the specimens
therefore included centromeric probes for 1, 6, 7, 8, and X (all Oncor,
Heidelberg, Germany).
FISH was performed on 5-µm sections mounted on superfrost slides
(Omnilab, Gehrten, Germany). Tissue sections were baked overnight at
56°C and then deparaffinized by immersing in xylene for 20 minutes
and then in graded ethanol. Slides were placed in citric acid solution
(0.01 mol/L) and heated in a microwave oven at 900 and 600 W for 15
minutes each. Diluted RNase A (0.1%) was added to the sections for 10
minutes and then rinsed in PBD (Oncor). Incubation with 3%
H2O2 for 10 minutes was
carried out at room temperature, followed by washing in PBD. Afterward,
slides were washed in graded ethanol and air-dried for 5 minutes. A
total of 0.5 µl of the probe was added to 10 µl Hybrisol VI (Oncor)
and pipetted onto the slide, placed under a glass coverslip, sealed
with rubber cement, heated to 92°C for 12 minutes, and incubated
overnight at 37°C in a humifidied chamber. Detection started with
rinsing the sections in 0.25x SSC at 60°C for 5 minutes followed by
a short wash in PBD. Then 30 µl of horseradish peroxidase (HRP),
diluted 1:30, were added for 20 minutes at 37°C under a coverslip.
Washing in PBD followed. Thirty microliters of fluorescein
isothiocyanate-conjugated tyramine (DuPont NEN, Boston, MA) were added
and incubated for 20 minutes at 37°C. After rinsing in PBD,
counterstaining was done with 5 µl of propidium iodide (Oncor), and
the slides were placed under a coverslip.
Evaluation of signals was done using an epifluorescence microscope
(Axiophot; Zeiss, Oberkochen, Germany) equipped with a
fluorescein/rhodamine filter set and a 50 W mercury lamp. Only bright
signals not connected to a second signal were counted. At least 100
nuclei were evaluated in each case.
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Results
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In a first step, signal distribution was determined in
non-neoplastic liver tissues. For this purpose, 55 specimens of normal
liver were analyzed. As listed in Table 2
, the mean values for distribution of signals were 81% of nuclei
bearing two signals, 17% revealing one signal, and 2.3% showing three
or more signals. The standard deviations were 4.7, 4.2, and 2.6%,
respectively. Pursuant to the recommendations of Ward et
al,15
three standard deviations were added to the mean
values and defined as monosomy and trisomy at 29% and 10%,
respectively.
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Table 2. Mean, Standard Deviation (SD), and Threshold for Defining Monosomy and
Polysomy Based on the Evaluation of 55 Normal Specimens
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Hepatocellular Adenoma
One signal was seen in 4.3 to 24.6% of cells analyzed. Two
signals per nucleus were seen in 73.8 to 91.1% of nuclei (Table 3
, Figure 1
). Three or more signals were detectable in 0.0 to 7.5% of the cells.
None of these values passed the thresholds of 29% and 10% defining
monosomy and trisomy, respectively. In seven analyses of seven
different cases, an analysis was not informative due to lack of
specific probe binding.
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Figure 1. H&E-stained section of case HCA 12 reveals variations in nuclear size
and prominent nucleoli (a; original
magnification, x400). Silver stain demonstrates
a preserved delicate network of reticulin fibers around the tumor cells
(b; x400).
FISH for chromosome 6 gives a normal distribution of signals, as
indicated by one or two fluorescent signals in the nuclei
(c; x1000).
Due to cutting artifacts, not all nuclei are expected to bear two
signals. The number of signals shown in this photograph, however, is
too low, and varying the plane of focus for approximately 2 µm brings
up additional signals, as indicated for an exemplary cell by an
arrow (d;
x1000).
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Well-Differentiated Hepatocellular Carcinoma
One signal was seen in 1.7% to 76.2% of cells, two signals were
seen in 20.2% to 79.8%, and three or more signals occurred in 0% to
70.5% (Table 3
, Figure 2
). Chromosome 1 was found to be aberrant in 8 samples, chromosome 6 in 9
samples, chromosome 7 in 10 samples, chromosome 8 in 11, and chromosome
X in 8 samples. Five chromosomes were found to be aberrant in 2 cases,
4 chromosomes were aberrant in 4 samples, 3 chromosomes were aberrant
in 6 samples, 2 chromosomes were aberrant in 1 case, and in 1 sample no
aberrant count was seen (Table 4)
. In two analyses in two different cases, experiments were not
informative.
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Figure 2. Variations of nuclear size and prominent nucleoli are also seen in case
HCC 7 (a; original magnification,
x400). Reticulin fibers are seen in a part of
the tumor, whereas in some areas the network is interrupted
(b; x400).
FISH for chromosome 8 demonstrates polyploidy for this chromosome
(c; x1000).
Varying the plane of focus again brings up additional signals
(d; x1000).
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Statistical Evaluation
The number of aberrations found in HCA and HCC samples was
significantly different (P < 0.01, Mann-Whitney
U test). Specificity of FISH in detecting HCC was 100%, and
sensitivity was 92.3%. Positive predictive value was 100%, and
negative predictive value reached 93.3%.
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Discussion
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Knowledge about cytogenetic alterations in HCC has increased over
the past few years due to the application of new molecular techniques
such as CGH. Larger numbers of HCC have now been analyzed, and
recurrent patterns of chromosomal imbalances have been
identified.11, 12, 13
In particular, imbalances of chromosomes
1, 4, 6, 7, 8, and X, including total and partial gains and losses,
have been demonstrated. Although not all of these aberrations were
detectable in every case analyzed, at least some of them were found in
varying combinations in all HCC cases described. By contrast, HCA
demonstrated a much lower number and different chromosomes affected
compared to HCC.14
Based on these findings we used FISH as
an alternative method to CGH to analyze HCA and HCC. The main reason
for this approach was that FISH is easier to perform and much easier to
evaluate than CGH. Whereas CGH requires a karyotype analysis similar to
conventional cytogenetics, FISH requires only the counting of single
signal spots in the nuclei. Therefore, the correct identification of
chromosomes, which requires a lot of experience, is not mandatory in
FISH.
Obvious differences in signal distributions of the particular
chromosomes were seen between HCA and HCC. Whereas none of the HCA
revealed aberrant counts for any of the chromosomes analyzed, HCC
demonstrated aberrations for at least two chromosomes in 13/14 cases.
In one HCC sample, probes for chromosomes 6, 7, 8, and X demonstrated
normal distribution of signals and the probe for chromosome 1 did not
give sufficient results. In this patient, partial hepatectomy was
performed after diagnosis of HCC and the tumor was analyzed by CGH.
Beside aberrations for 5q, 6p, 8p, 17p, and 17q, a gain of 8q was
detectable, confirming a trisomic count seen in FISH using a
locus-specific probe for 8q21. However, this probe failed to give
reliable results in many cases examined. Therefore, it cannot be
recommended for routine purposes and was consequently excluded from
this study.
The restriction to five centromeric probes for FISH analysis is based
on aspects of availability and applicability in daily routine.
Centromeric probes most often give brighter signals than probes
localized on the arms of the chromosomes. Evaluation of the signals can
be done by epifluorescence microscopy with a standard filter set
without the need for sophisticated technical equipment. All centromeric
probes as well as the detection system are commercially available and
can be processed by the same protocol. For these reasons, chromosome 4,
also often reported to be altered in HCC,11, 12, 16
was not
analyzed in this study, since a digoxigenin-labeled probe was not
available at the time our study was performed. This does not seem to
hamper FISH analysis, since results obtained for chromosomes 1 and 8
already highlighted 13/14 cases as aberrant. No additional case was
detected by analysis of chromosomes 6,7, and X. Therefore, it may be
useful to apply probes for chromosomes 1 and 8 at first and, if one or
both of these probes fail to give any results, to add the probes
specific for chromosomes 6, 7, and X.
The aneuploidy found by the panel of probes is seen not only in HCC but
also in a variety of other malignant tumors affecting the same
chromosomes in similar patterns, as summarized by Mitelman et
al.17
Lengauer et al18
discussed these
findings as an increased genetic instability, based on the inability of
the aberrant cell to control chromosomal alterations. This assumption
is underscored by the observation that the chromosome changes found in
distinct carcinomas are not always identical for all chromosomes. The
mechanisms responsible for this genetic instability are not yet known
and require further investigation. Nevertheless, they are
diagnostically helpful, as shown here for HCA and HCC.
In conclusion, FISH for chromosomes 1 and 8, extended by probes
specific for chromosomes 6, 7, and X, enables the differentiation of
HCA and well-differentiated HCC to be performed with a high degree of
accuracy. Particularly with regard to small biopsies, FISH represents a
promising adjunct to classical histology in the differentiation between
hepatocellular adenoma and carcinoma.
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Footnotes
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Address reprint requests to Dr. Ludwig Wilkens, Institut für Pathologie der Medizinischen Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail: wilkens.ludwig{at}mh-hannover.de
Accepted for publication March 1, 2001.
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Abstract
Introduction
