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Regular Articles |
From the Institutes of Pathology*
and
Radiobiology,
GSF-National Research
Center for Environment and Health, Neuherberg; the Institute of
Radiation Biology,
Ludwig Maximilian
University Munich, Munich; and the Institute of
Pathology§
and Department of
Surgery,¶
Technical University Munich,
Munich, Germany
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although numerous cytogenetic and molecular genetic studies have been performed on esophageal adenocarcinomas, fundamental data, especially pertaining to precursor lesions, which could substantially clarify our understanding of the tumorigenesis of BA are not available. Cytogenetic studies using G-banding, interphase fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH) have revealed a complex pattern of structural and numerical chromosomal aberrations in BA of the distal esophagus and gastric cardia.9-13 On the molecular genetic level, microsatellite analyses in previous studies have revealed frequent loss of heterozygosity (LOH) as well as allelic imbalances on chromosomes 5q, 17p, and 18q14 and on chromosomes 3q, 4q, 5q, 6q, 9p, 9q, 12p, 12q, 17p, and 18q.15 The latter studies have provided support for the proposed metaplasia-dysplasia-carcinoma sequence, in that they demonstrate a sequential accumulation of alterations and microsatellite changes in metaplasia and dysplasia. Other molecular studies of esophageal carcinoma have focused on alterations in specific candidate genes such as the fragile histidine triad (FHIT) gene on chromosome 3p14.216,17 or the DPC4 gene on chromosome 18q21.1.18 Despite the identification of molecular alterations in these chromosomal regions, FHIT and DPC4 do not seem to be important for the development of these carcinomas, and it appears that the genes which play key roles in carcinoma development in Barrett’s epithelium have not yet been identified.
To provide fundamental cytogenetic data in a large number of cases, we investigated 30 Barrett’s-associated adenocarcinomas, 25 premalignant lesions (6 IM, 8 LGD, 11 HGD), and 8 regional lymph node metastases by CGH. Using an approach that combined laser-assisted microdissection and CGH, we were able to identify recurrent chromosomal changes in the histologically proposed metaplasia-dysplasia-carcinoma sequence.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thirty patients (1 female, 29 males) with BA of the
distal esophagus diagnosed between 1990 and 1998 were studied.
Follow-up data were available for 28 cases (mean follow-up, 23 months;
range, 0–85 months). All patients underwent an esophagectomy without
preoperative radiotherapy or chemotherapy. Clinical and
histopathological data for the study group are summarized in Table 1
. The analyses were performed on
archival material from formalin-fixed tissues embedded in paraffin.
Hematoxylin and eosin-stained slides from the resection specimens were
re-evaluated independently by two pathologists for the identification
of intestinal metaplasia (IM), columnar epithelial dysplasia (low-grade
dysplasia - LGD or high-grade dysplasia - HGD), tumor differentiation
grade (well, moderate, poor), depth of tumor invasion, and regional
lymph node metastasis (LN). The adenocarcinomas (BA) were staged
according to the Union Internationale Contre le Cancer TNM
system.19
The most BA in our series were classified as pT1
and pT2, reflecting common surgical strategies which exclude advanced
cases from esophagectomy. Paraffin blocks of BA
(n = 30) were obtained from 30 resection
specimen including LN (n = 8). In addition, from
a subset of 14 resection specimen 25 premalignant lesions consisting of
HGD (n = 11), LGD (n =
8), and nondysplastic intestinal metaplasia IM
(n = 6) were selected for laser microdissection
and subsequent DNA extraction. Several samples were in close proximity
to each other (<0.5 cm) and are marked by a • in Table 2
.
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An UV laser microbeam (P.A.L.M, Wolfratshausen, Germany) was used to excise precisely defined tissue areas on unmounted H&E-stained 5-µm serial sections. At least 1 to 5 x 103 microdissected cells from 3 to 6 serial sections were sampled from normal squamous epithelium (control), IM, LGD, and HGD. From BA, 105 to 106 microdissected cells were sampled, representing thecomplete BA area of 1 to 3 serial sections. The cells were lysed in 50 µl of 100 mmol/L Tris-HCl (pH 7.5), 1 mg/ml proteinase K for 24 hours at 55°C.
Amplification and Labeling of Test DNA
Degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) was performed on DNA extracts from microdissected tissue according to a previously published method.20-23 DOP-PCR-amplified DNA as well as non-amplified DNA from tumor and control samples were labeled with biotin-16-dUTP (Boehringer Mannheim, Mannhein, Germany) using standard nick translation.
CGH and Image Analysis
CGH was performed on test DNA amplified by DOP-PCR according to
published procedures.21-23
For all CGH preparations 300
ng of labeled test DNA and SpectrumRed direct-labeled normal female or
male total human genomic DNA (Vysis, Inc., Downers Grove, IL), plus 25
µg CotIDNA were cohybridized to denatured metaphases for
72 hours at 37°C. After hybridization, biotin-labeled test DNA was
detected with Cy2-conjugated streptavidin (Dianova). For CGH analysis,
at least ten metaphases were imaged and karyotyped after visualization
with a Zeiss Axioplan 2 fluorescence microscope equipped with filter
sets (single-band excitation filters)
for 4'-6-diamidino-2-phenylindole, Cy2 and Texas Red. Averaged
profiles were generated by CGH analysis software (ISIS 3, V2.84;
MetaSystems, Altlussheim, Germany) from at least 10 to 15 homologous
chromosomes and interpreted according to published
criteria21,24
using statistical confidence limits based on
t-statistics. An overexpressed area was classified as a
high-level amplification when the CGH ratio exceeded a value of 1.5, or
when the Cy2 fluorescence showed a strong, distinct signal by visual
inspection and the corresponding ratio profile was diagnostic of
overrepresentation.25
Regions of high level amplification
are shown as thick bars in Figures 1 and 2
.
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DOP-PCR-amplified DNA obtained from morphologically normal-appearing esophageal squamous epithelium was hybridized with non-amplified male or female reference DNA (SpectrumRed) to metaphase preparations. In these experiments no chromosomal changes were detected except for chromosome region 1p34–36 and chromosome 19. These regions are known to show artifactual results by CGH.22,26,27 Therefore, chromosomes 1p and 19 were excluded from further analysis. In addition, three cases of BA were comprehensively analyzed using both DOP-PCR amplified and nonamplified DNA, with the same chromosomal changes being detected by both methods. CGH results were further validated by comparison with FISH analysis.
FISH Analysis
FISH analysis with specific probes was essential to complement the
CGH analysis both as validation of the CGH findings and as a control of
the degree of intratumor heterogeneity. Thirteen cases with known DNA
copy number changes on chromosomes 17q and 20q from CGH experiments
were selected to confirm these changes in the BA samples (Table 5)
.
Serial 5-µm sections of the tissue blocks were used for FISH analysis
which investigated areas corresponding to those examined by CGH. For
FISH analysis a PathVysion HER-2 DNA probe kit (Vysis, Inc.) was used
according to the manufacturer’s recommendations. The kit consists of
directly labeled, fluorescent DNA probes specific for the
HER-2/neu gene locus (17q11.2-q12) and a DNA probe specific
for the 
satellite DNA sequence at the centromeric region of
chromosome 17 (17p11.1-q11.1). DNA probes for the subchromosomal region
20q13.2 (Spectrum Orange-labeled, Vysis, Stuttgart, Germany) and for
the centromeric region of chromosome 20 (D20Z1, biotin-labeled, Oncor
Appligene, Heidelberg Germany) were used as described
elsewhere.28
Signals from 100 to 150 tumor cell nuclei
were counted using confocal laser scanning microscopy (Zeiss LSM 410).
According to published criteria,29
gene amplification was
detected if the ratio of locus-specific signals to centromeric signals
per cell was at least 3 in >10% of tumor cells or tight clusters of
>10 signals in multiple cells. Further controls consisted of adjacent,
phenotypically normal squamous epithelium and lymphocytes.
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For comparison of average aberration frequencies in each entity,
the SEM was calculated. A pairwise comparison analysis was applied to
compare the occurrence of chromosomal imbalances in different stages of
the metaplasia-dysplasia-carcinoma sequence. For this purpose, 14 cases
were selected with available data for at least one precursor stage (IM,
LGD, or HGD) and the corresponding carcinoma. The data were compared
pairwise in Table 4
, indicating concordant results for each chromosome
aberration. Concordance was classified into two groups: 1–1
(aberration present in both lesions) and 0–0 (aberration absent from
both lesions). This comparison resulted in four pairs each for IM
versus LGD and IM versus HGD, six pairs each for
IM versus BA and LGD versus BA, eight pairs for
LGD versus BA, and eleven pairs HGD versus BA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. A statistical correlation between cytogenetic
and clinicopathological data could not be observed using Fisher’s
exact test (P > 0.05 for gender, survival, and
TNM stage). The detailed DNA gains and DNA losses for each of
the 63 samples investigated are presented in Table 2
. The chromosomal
imbalances which were found are presented in Figure 1
for the
adenocarcinomas and in Figure 2
for the areas of IM, LGD, and HGD. A
comparison of the results from FISH and CGH analysis is presented in
Table 5
. BA and LN
An average of 13.3 ± 1.4 chromosomal imbalances per case
were detected in the 30 BAs. The chromosomal alterations most often
identified were gains on 8q (80%), 20q (60%), 2p, 7p and 10q (47%
each), 6p (37%), 15q (33%), and 17q (30%). Losses were observed
predominantly on the Y-chromosome (76%), 4q (50%), 5q and 9p (43%
each), 18q (40%), 7q (33%), and 14q (30%) (Figure 1)
. High-level
amplifications were observed on 8q23-qter, 8p12-pter, 7p11-p14,
7q21–31, and 17q11-q23. In the eight LN, an average of 22.0 ±
1.2 chromosomal imbalances per case were detected, involving namely
gains on 8q and 20q (8/8), 7p and 17q (7/8 each), 2p, 3p, 6p, and 16p
(6/8 each), and 9q, 10p, 10q, and 16q (5/8 each) and losses on 4q
(7/8), 2q, 5q, and 13q (6/8 each), Y chromosome (4/8), 9p and 15q (4/8
each), and 18q, 14q, and 6q (3/8 each).
HGD, LGD, and IM
In the 11 samples taken from HGD areas, an average of 13.4 ± 1.1 chromosomal imbalances per case were detected, with the following recurrent alterations: gains on 8q (9/11), 20p and 20q (8/11), 2p (7/11), 7p (6/11), 10q (5/11), 15q (4/11), and 6p(4/11), and losses on the Y chromosome (10/11), 5q (6/11), 9p, 13q (5/11 each), 2q, 4q, 14q, and 18q (4/11 each), and 8p (3/11). In the 8 LGD areas, an average of 10.8 ± 2.2 chromosomal imbalances per case was detected, with gains on 2p (6/8), 8q (5/8), 3p (4/8), and 15q and 20q (3/8 each) and losses on the Y chromosome (8/8), 7q and 9q (4/8 each), and 13q (3/8). Among the 6 samples taken from IM areas without dysplasia, there was an average of 7.0 ± 1.7 chromosomal imbalances per case, with gains on 8q (4/6), 6p (3/6), and 8p and 10q (2/6), and losses on chromosome 13q (5/6), Y chromosome (3/6), and 9p and 12q (2/6 each) being present.
Comparison of Chromosomal Changes between IM, LGD, HGD, BA, and LN Areas
The average number of chromosomal imbalances increased steadily
from IM (7.0 ± 1.7) to LGD (10.8 ± 2.2) to HGD (13.4
± 1.1) and BA (13.3 ± 1.4), and again to LN areas (22 ±
1.2). The largest differences in copy number changes were seen i)
between BA and LN areas, in particular the frequency of DNA gain on 20q
and 17q and DNA loss on 4q, in LN (Table 3)
; ii) between LGD and HGD areas,
especially with DNA losses being more frequent in HGD, but without a
particular chromosomal change which distinguished between them; and
iii) between IM and LGD areas, with losses on 4q and 5q appearing first
in LGD and remaining in all subsequent stages. In contrast, the average
number and frequency of chromosome copy number changes were very
similar in BA and HGD. In addition, a pairwise comparison analysis of
14 cases with at least one precursor stage and the corresponding
carcinoma was performed to identify concordances for the occurrence of
chromosomal aberrations between IM versus LGD, IM
versus HGD, IM versus BA, LGD versus
HGD, LGD versus BA, and HGD versus BA. This
analysis of a subset of 14 cases revealed a stepwise occurrence of
chromosomal imbalances in each stage of the
metaplasia-dysplasia-carcinoma sequence (Table 4)
.
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A total of 17 FISH experiments on 13 BA were performed. A
comparison of the results from FISH and CGH analyses is presented in
Table 5
. Each of the tumors investigated
showed striking heterogeneity, with several areas displaying different
levels of Her-2/neu or 20q13.2 signals. Even in cases with
only lower levels of amplification (eg, cases 6 and 14 for
Her-2/neu) there were small subpopulations (<5%) with
tight clusters of signals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue microdissection is particularly important for BA and its precursor lesions since they usually contain a significant component of non-neoplastic cells. As an alternate microdissection technique to previous CGH studies of BA,11-13 we isolated histopathologically defined areas using laser assisted microdissection on each specimen. Using this technique, the chromosomal regions most frequently showing gains in the BA were 8q, 20q, 2p, 7p, 10q, 6p, 15q, and 17q. Losses were frequently observed on the Y chromosome, 4q, 5q, 9p, 18q, 7q, and 14q. These results are in agreement with previous CGH and LOH studies.11-15 Furthermore, we were able to identify recurrent DNA gains on two chromosomal areas, 2p and 10q, which have not been described previously in BA. Gains and high-level amplifications on 2p have been detected by CGH in several other neoplasms, including gastric adenocarcinoma,30 in which the amplification of the proto-oncogene NMYC located on 2p23–24 was described. NMYC amplification has been observed in multiple studies of neuroblastoma, in which NMYC amplification remains the most widely accepted predictive parameter of long-term, disease-free survival.31 However, we found no statistically significant correlation between DNA gain on 2p23–24 and survival (P > 0.05). The gain on 10q25–26 might indicate involvement of the K-SAM gene, which was previously identified in a gastric cancer cell line32,33 and is known to encode one of the heparin-binding growth factor receptors or fibroblast growth factor receptors. Recently, using immunohistochemistry, Hattori et al demonstrated that 20 of 38 undifferentiated type advanced gastric cancers overexpressed the K-SAM protein.34 The most frequent DNA gain we observed was on 8q. This amplification involves the minimal common chromosomal region 8q23–24, which contains the proto-oncogene CMYC, previously shown to be amplified by interphase FISH in 15.5% of gastric adenocarcinomas.35 In our investigation we demonstrated a higher frequency of 8q amplification than had been reported in the previous cytogenetic studies of BA11-13 or gastric carcinoma.30,32 Furthermore, amplification of 8q23–24 was already detectable in IM areas without dysplasia, ie, in morphological preneoplastic mucosa. Other frequent gains and losses detected in our study point to further gene alterations including the EGFR gene (chromosome 7p1236 ), AIB1, BTAK, SRC (chromosome 20q),37,38 CDKN2A (p16), (chromosome 9p21-pter39 ), DPC4 (chromosome 18q40 ) and, so far unknown, candidate genes on three discrete areas on chromosome 4q.41 LOH studies of several of these gene loci indicate that they are involved in esophageal adenocarcinoma. However, CGH is limited in its ability to detect DNA losses and amplifications, because the use of metaphase chromosomes limits detection of events involving small genomic regions (of <20 Mbp) and resolution of closely spaced aberrations. This may explain why our analysis failed to find some of the alterations, such as loss of heterozygosity on 17p, which are known to be very frequent in BA.2,42 It has been proposed that hybridization to high resolution arrays of mapped sequences rather than the metaphase spreads used in conventional CGH might overcome this problem.43 A further methodological problem may occur in the interpretation of telomeric regions by CGH.44 In our study some of the observed changes are located near telomeric regions, but for the most part they cover more than a single chromosomal band. Moreover, telomeric regions did not reveal chromosomal imbalances in our control hybridizations (DOP-PCR-amplified DNA obtained from histomorphologically normal esophageal squamous epithelium was hybridized with non-amplified male or female reference DNA). Thus, it seems unlikely that chromosomal imbalances near the telomeric regions have been misinterpreted.
A particularly important aspect of our study was the ability to study
areas precisely corresponding to the metaplasia-dysplasia-carcinoma
sequence of Barrett’s esophagus using laser microdissection. The
detection of chromosomal changes in these areas allowed us to assess
clonal evolution in the precursor lesions of BA. In several samples we
found a high concordance between specific aberration patterns and the
steps of the metaplasia-dysplasia-carcinoma sequence (Table 2)
. The
observation of common molecular abnormalities in premalignant lesions
and adjacent carcinomas suggests that the process of clonal expansion
underlies the proposed histopathological tumorigenetic pathway in
Barrett’s esophagus. This is also supported by recent studies, one of
which reported two cases with a consistent pattern of X chromosome
inactivation, indicating clonal expansion from metaplasia to dysplasia
and carcinoma.45
This other study demonstrated in three
cases that dysplastic Barrett’s epithelium and adjacent BA displayed
the same pattern of microsatellite alleles at multiple
loci.15
Very recently, a case report of a multifocal BA
using CGH indicated that monoclonal evolution of the cancer cells was a
more likely mechanism than the concept of field
cancerization.46
The latter study also reported
shared DNA gains and losses of HGD adjacent to BA, indicating that the
HGD was a precursor of the BA. In our series of samples no substantial
difference in the pattern and frequency of chromosomal changes between
HGD and BA was observed. Thus, our data generally support the paradigm
of the clonal derivation of invasive cancer from HGD, with the BA
exhibiting the same genetic alterations seen in the HGD, as well as, in
some cases, additional changes not yet present in the HGD lesion. On
the other hand, a review of our data also shows that some HGD lesions
had genetic abnormalities that were not present in the corresponding
adjacent invasive cancer. This indicates that genetic divergence during
the clonal evolution of cancer, particularly at the time when HGD
progresses to invasive cancer, is also an important factor.
This phenomenon of genetic divergence is also illustrated by the fact
that a number of areas of IM and dysplasia exhibited chromosomal
changes that were not detected in the BA from the same resection
specimen. For example, the IM from cases 10 and 16 shared only a few
chromosomal changes with the corresponding BA sample. Either the
carcinoma in such cases did not arise from the clone of premalignant
tissue that was examined, or, during the process of clonal
expansion (an ongoing process in both the carcinoma and premalignant
lesions), new abnormalities arose in the premalignant areas that were
not present when the carcinoma first developed. This may be illustrated
by gains on chromosome 3p, which are more frequently found in precursor
lesions than in invasive carcinoma (Figures 1 and 2)
, reflecting the
clonal diversity and genetic complexity which cannot sufficiently be
explained by linear accumulation of alterations in Barrett’s
esophagus.
The development of divergent clones during the process of clonal
expansion is proposed be the explanation for tumor cell
heterogeneity.47
Such intratumoral heterogeneity was
demonstrated in our cases by the results of our FISH analysis. Each of
the investigated carcinomas was composed of several areas with
different centromeric 17/Her2-neu signals, indicating a high
prevalence of intratumor heterogeneity (Figure 3)
. Even if there were limited
concordance between areas of premalignancy and adjacent BA, this does
not necessarily conflict with the model of histological progression.
Barrett et al demonstrated, using Barrett’s esophagus cell lines, that
clonal evolution is more complex than predicted by linear
models.42
In addition, most premalignant human tissues do
not appear to progress to cancer, even when multiple somatic genetic
abnormalities are present.48-51
This is probably a
reflection of the existence of many divergent neoplastic subpopulations
and highlights one of the main problems associated with surveillance of
Barrett’s patients, namely sampling error.
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and Table 4
). Particularly
interesting was a remarkable increase in gains on chromosome 7q and
loss on chromosome 4q between precursor lesions (IM, LGD) and BA (Table 4)
, indicating that there is an important genetic event at this regions
during progression. At the other end of the sequence, ie, between BA
and LN, losses on 4q and 5q and gains of chromosomal regions 2p, 8q,
10q, and 20q were more frequent in LN than in the primary BA. These
late events are consistent with the model of clonal expansion of tumor
cell populations during tumor progression and may indicate chromosomal
areas involved in the development of metastatic potential. In conclusion, tumor development in Barrett’s esophagus is proposed to occur via a multistep pathway which is recognized histologically as a metaplasia-dysplasia-carcinoma sequence. Using an approach combining laser-assisted microdissection and CGH, we detected recurrent chromosomal changes in this proposed sequence. Furthermore, we identified novel amplified chromosomal regions on chromosomes 2p and 10q in both BA and premalignant lesions. Although the detection of common chromosomal alterations in premalignant lesions and adjacent BA suggests a process of clonal expansion, the occurrence of several chromosomal changes without specific order was also apparent and provides striking evidence that clonal evolution is more complex than predicted by linear models.
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Acknowledgements |
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Footnotes |
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Accepted for publication October 19, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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