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From the Institute of Pathology, Medizinische Hochschule Hannover,Hannover, Germany
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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) during breast cancer progression. For this purpose
invasive and noninvasive tumor cell populations as well as hyperplastic
cell proliferations were isolated from a series of archival breast
tissue specimens (n = 57) using laser-assisted
microdissection. A new real-time polymerase chain reaction-based assay
was used for the sensitive and quantitative determination of the
cell-specific methylation status. We found that aberrant promoter
methylation was already prevalent in pure intraductal carcinoma with
different frequencies and different methylation levels for the four
genes analyzed. For RASSF1A and 14-3-3
promoter methylation was also demonstrated in epithelial hyperplasia
and intraductal papillomas. By contrast, aberrant methylation
of cyclinD2 and p16 was restricted to
cancerous epithelium. Increased methylation of the
cyclinD2 gene was significantly associated with a higher
van Nuys grade. Furthermore, when intraductal and invasive
tumor cells were compared, significant quantitative changes in
the methylation level were detected primarily within the
cyclinD2 gene. These results demonstrate that promoter
methylation is an early and frequent event in breast cancer
development, but displays great quantitative and gene-specific
differences, and changes in a gene-specific manner during tumor
progression.
Through the recruitment of histone modifying enzymes to the DNA the cytosine methylation initiates the formation of a closed chromatin conformation thereby repressing transcription.3,4 This methylation-mediated inhibition of gene expression is now widely recognized as an important mechanism for the inactivation of growth regulatory genes in the process of malignant transformation.5
To answer the question whether the aberrant hypermethylation of regulatory sequences is an early event during cancer development, small and precancerous lesions have to be analyzed. These lesions are almost exclusively available as formalin-fixed, paraffin-embedded biopsy specimens. Isolation of homogeneous and morphologically defined cell populations from tissue sections has now become possible by laser-assisted microdissection.6
A very sensitive and widely used method for the detection of cytosine methylation in small tissue samples is the methylation-specific polymerase chain reaction (PCR).7 However this method provides only qualitative data regarding the methylation status of the regulatory region analyzed. This may mask potential quantitative differences between samples from different patients and also dynamic changes in the extent of methylation during tumor evolution. These differences might also be important for the classification of lesions and might have greater prognostic significance. For these reasons several groups including our own have developed independently from each other assays for the detection and quantification of CpG methylation,8-13 most of which are real-time PCR based. This technology combines the advantages of high through-put with a superior sensitivity and accuracy for quantification.14
In breast cancer promoter hypermethylation has now been described for several genes covering all aspects of cellular function.15 But almost all of these studies have analyzed advanced invasive mammary carcinomas in a purely qualitative manner. Therefore we started the analysis of hyperplastic epithelia and intraductal as well as invasive breast cancer cells using a quantitative assay.
The aim of this study was to analyze the promoter hypermethylation of
four key growth regulatory genes (p16, RASSF1A, cyclinD2,
14-3-3) during breast cancer progression in a quantitative
manner in morphologically defined laser-microdissected archival tissue
specimens. The new real-time PCR-based methylation assay enables the
detection of quantitative changes that may precede or accompany
microscopically visible morphological alterations. To determine at
which point of the morphological spectrum ranging from ductal
hyperplasia to invasive carcinoma this epigenetic modification occurs,
different hyperplastic and malignant lesions were analyzed.
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Materials and Methods |
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A total of 40 cases of ductal carcinoma in situ (DCIS) were retrieved from the archive of the Institute of Pathology, Medizinische Hochschule Hannover, Hannover, Germany. In addition 7 specimens containing intraductal papillomas and 10 specimens with intraductal epithelial hyperplasia were retrieved. Two pathologists (FL and HK) have reviewed each case independently and classified the DCIS specimens according to the van Nuys classification system.16 For control 10 blood samples were collected after informed consent from healthy volunteers without any history of neoplastic malignancy. Cell lines were purchased from the American Type Culture Collection (Rockville, MD) and cultivated according to the supplier’s instructions given.
Laser Microdissection and DNA Isolation
All breast tissue samples used for methylation studies were prepared by laser-assisted microdissection from stained histological sections. Genomic DNA was isolated from fresh frozen biopsies, the peripheral blood mononuclear cell fraction, and from tissue culture cells using Proteinase K digestion and organic extractions according to standard procedures. Laser-microdissection of stained histological sections from breast tissue and subsequent isolation of DNA was performed essentially as described.17
Quantitative Methylation Analysis
Bisulfite treatment of genomic DNA isolated from microdissected
samples was performed as described.12
All primer and
hybridization probe sequences are listed in Table 1
. For real-time PCR up to 5 µl of the
DNA samples were analyzed in a total volume of 25 µl containing 20
mmol/L Tris-HCl (pH 8.4), 50 mmol/L KCl, 200 µmol/L dNTP, 240 nmol/L
of each primer, 125 nmol/L hybridization probe, 1 µmol/L ROX
(Tibuolbiol, Bolin, Germany), 1.5 to 4.5 mmol/L
MgCl2, and 0.625 U Platinum-Taq (Gibco
BRL, Life Technologies, Karlsrule, Germany). After an initial
denaturation step at 95°C for 5 minutes 45 cycles followed by 15
seconds at 95°C and 60 seconds at 62°C for annealing and extension.
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CT(target -
reference) values and the extent of methylation (Figure 1B)
. A region of the actin gene that
contains no CpG dinucleotides and therefore is independent from the
methylation status was used as a reference target.11
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Selection of the Primer/Probe Target Sequences
For the design of the primers and hybridization probes we took
into account all published data (and also our unpublished observations)
about the cytosine residues in the CpG island around the start site of
transcription that are actually methylated. For the p16 gene
we have chosen the region identified by Huschtscha and
colleagues18
and Wong and colleagues19
as the
one that is early and most heavily methylated in human mammary
epithelial cells (HMECs) during the process of cellular
transformation. For the RASSF1A gene we selected as a target
for the methylation-specific real-time PCR the residues identified by
Dammann and colleagues20
by genomic sequencing as the
critical residues methylated in lung and breast cancer samples. For the
cyclinD2 gene and the 14-3-3 gene the regions
identified by Evron and colleagues,21
Ferguson and
colleagues,22
and Iwata and colleagues,23
respectively, were selected for the development of the quantitative
methylation-specific PCR. For all CpG sites analyzed in this study a
close correlation between methylation and repression of transcription
has been documented by reverse transcriptase-PCR or Western blotting
analysis.19-23
Evaluation of Reaction Efficiencies
The quantitative methylation-specific PCR assay used in this study
is based on a relative quantification explained in detail
elsewhere.24
To use this relative quantification it has to
be proven that the reaction efficiencies for all PCR systems that will
be compared are equal. Figure 1A
clearly demonstrates as an example for
14-3-3 as the target gene and ACTß as the
reference gene very similar reaction efficiencies over a wide range of
template concentrations down to the threshold of detection indicated by
a constant difference of the CT values for these
two primer/probe systems.
Validation of Quantification
To validate the reliability of the real-time PCR-based
quantification we analyzed different mixtures of DNA isolated from cell
lines for which the methylation status of the gene under study was
known. Figure 1B
demonstrates as an example for the RASSF1A
gene a very good linear correlation (r = 0.985)
between the extent of methylation and the measured differences of the
CT values [CT (target
gene) - CT (reference gene)]. Similar
calibration curves were obtained for all genes analyzed in this study
(data not shown).
Promoter Methylation in Physiological and Hyperplastic Cell Proliferations of the Breast
To define whether promoter hypermethylation occurs already under
luteinizing hormone (LH) stimulation and in hyperplastic proliferations
such as epithelial hyperplasia or intraductal papilloma we analyzed two
specimens of lactating breast epithelium, 7 intraductal papillomas, and
10 cases of epithelial hyperplasia. In addition the methylation status
of the four genes under study was assessed in normal epithelium
(n = 6), breast stromal tissue
(n = 2), and one case of apocrine metaplasia
(Figure 2A)
a clearly benign alteration
of the mammary epithelium. The results are summarized in Figure 3
. Whereas for p16 and
cyclin D2 no methylation or only very low levels of
methylation could be detected, the RASSF1A and
14-3-3 gene are quite frequently and also in part heavily
methylated. RASSF1A was extensively methylated in cases of
epithelial hyperplasia and papilloma but not in normal epithelium,
apocrine metaplasia, and the highly proliferating lactating epithelium.
By contrast, the 14-3-3 gene was clearly methylated under
physiological conditions in breast epithelium and also quite heavily in
stromal tissue. Hyperplastic specimens exhibit 14-3-3
gene methylation ranging from 5 to 85% (Figure 3)
.
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Because several groups have reported the sensitive detection of
hypermethylated alleles in the blood of cancer patients by
methylation-specific PCR as a marker of disease,25-27
we
tested whether the four genes analyzed in this study could also be used
for the early detection of breast cancer in blood samples. For this
purpose we analyzed the methylation status of these genes in the
peripheral blood mononuclear cell fraction of healthy individuals
(n = 10) without any history of neoplastic
malignancies to asses the normal level of methylation. As shown in
Figure 3
for RASSF1A no methylation at all could be detected
whereas for p16 and cyclinD2 only occasionally a
very weak methylation signal (<1%) could be seen. In contrast to
this, for 14-3-3 in all 10 samples a very high level of
methylation (>95%) was found. In addition to the real-time PCR-based
assay we confirmed the almost complete the methylation of the
14-3-3 gene around the start point of transcription in
the leukocytes of healthy individuals using a restriction enzyme-based
methodology28
(data not shown).
We also tested the DNA isolated from four resected lymph nodes without
any morphological alteration and found very similar results to those
observed with peripheral blood mononuclear cell fractions (data not
shown). This excludes the use of the detection of 14-3-3
hypermethylation in lymph nodes or peripheral blood for the screening
for circulation tumor cells or micrometastasis that would otherwise be
a promising approach because of the very high prevalence of
14-3-3 hypermethylation in breast cancer cells (see
Ferguson et al22
and our own results).
Promoter Methylation in DCIS of the Breast
Altogether 40 archival specimens of DCIS have been analyzed for
methylation after laser-assisted microdissection of the intraductal
tumor cells. The results are summarized in Figure 3
. Marked differences
between the four genes are clearly visible. Whereas the p16
gene is only very rarely methylated, the RASSF1A gene is
methylated in the vast majority of samples. In Figure 4
two subsets of samples are displayed in
more detail: low-grade carcinomas (van Nuys grade 1 and 2,
n = 9) and pure intraductal carcinomas without
accompanying invasive components (van Nuys grade 1 to 3,
n = 10).
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For 16 specimens intraductal and invasively growing tumor cells
could be isolated from the same tissue section using
laser-microdissection as shown in Figure 6
. For the majority of cases the
methylation of the p16 gene, the 14-3-3 gene
and the RASSF1A gene is very similar in the intraductal and
in the invasive component indicating that in most cases the epigenetic
inactivation takes place before invasive growth develops (Figure 7)
. Interestingly, the
cyclinD2 gene shows four different patterns of methylation
during tumor progression: no methylation at all, high methylation
already in intraductal tumor cells or a sharp increase as well as a
sharp decrease in methylation during progression resulting in two types
of invasive tumor cells (no methylation or nearly complete
methylation). We could not find a correlation between the methylation
level in the invasive components and the histological grade, the
proliferation status (Ki-67 labeling) or the hormone receptor status
(estrogen and progesterone receptor immunohistochemistry).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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For all CpG-rich regions analyzed in this study a close correlation between methylation and gene silencing has been shown.19-22 It has also been shown by bisulfite sequencing that the cytosine residues recognized by the primers and probes are actually methylated.
In comparison to conventional methylation-specific PCR,7 the real-time PCR-based assay for the detection of methylation has several advantages. First, the omission of all postamplification steps reduces the risk of contamination and increases the throughput of the system. Second, the assay is more stringent and more specific because in addition to the two PCR primers the fluorescent-labeled hybridization probe has to anneal correctly between the two primers. Third and most important, conventional methylation-specific PCR does not provide exact quantitative data.
The results concerning the nearly complete hypermethylation of the
14-3-3 gene in normal lymphocytes (Figure 3)
demonstrate
the importance of microdissection that enables the isolation of pure
morphologically defined cell populations. Laser microdissection also
allows the direct comparison of neighboring intraductal and invasive
tumor cells as illustrated in Figure 6
. It cannot be excluded that the
traces of 14-3-3 gene methylation <1% found in a minor
portion of the microdissected samples could be because of single
lymphocytes occasionally present in otherwise homogenous tumor cell
populations.
During the preparation of this manuscript, Umbricht and
colleagues33
published results concerning the methylation
of one of the four genes analyzed in this study, namely
14-3-3, in epithelial hyperplasia of the mammary gland
and intraductal breast cancer cells that principally confirm the
results presented in this study for this particular gene.
Aberrant hypermethylation was found in benign hyperplastic lesions of
the mammary gland and in low-grade intraductal carcinomas. The most
frequently and most heavily methylated gene was the recently identified
putative tumor-suppressor gene RASSF1A.34
This
gene was methylated to a similar extent in intraductal papillomas and
epithelial hyperplasia, but never in normal breast epithelium,
lactating breast tissue, stromal cells, or lymphocytes (Figure 3)
. This
finding suggests that methylation of RASSF1A might be a new
marker for nonphysiological epithelial proliferation in the breast.
The observed dichotomy concerning the methylation of the
cyclinD2 gene in invasive carcinomas (Figure 7)
may be
explained by the opposing functions ascribed to the cyclin D2 protein
during cell-cycle progression. The protein functions as activator of
the cell cycle but also as inhibitor of proliferation and inducer of
senescence depending on the cellular context.35,36
This
has to be addressed in future studies.
The changes in methylation levels could be because of epigenetic variations in a given clone (eg, loss of methylation capacity). Alternatively, they could be caused by clonal selection during tumor progression.
During the immortalization of HMECs the p16 gene is heavily methylated.18,19 Also several established breast carcinoma cell lines show clear hypermethylation of the p16 gene.37 In contrast to these findings in cell culture experiments, the methylation of this tumor suppressor gene in intraductal breast cancer cells, which are clearly malignant transformed, is only a rare event. Therefore the epigenetic pathways contributing to the immortalization and transformation in vivo seem to be different from those observed in cell culture using HMEC lines. These results demonstrate the importance of in vivo studies to elucidate the role of methylation in the process of malignant transformation and may indicate that the widely used HMEC lines are not in every respect a suitable model for the study of the malignant transformation of luminal breast epithelial cells that are the precursor cells of the vast majority of mammary carcinomas. In this context it should be mentioned that large scale cDNA array-based expression studies have revealed that HMECs are more similar to basal epithelial cells of the mammary gland than to luminal epithelial cells.38 This might explain the difference concerning the epigenetic inactivation of p16 in cultured HMEC lines versus DCIS seen in this study.
The frequency of p16 gene hypermethylation in invasive growing tumor cells reported in this study is lower than in the majority of published studies.28,37,39-42 In contrast to previous studies performed predominantly on fairly large and advanced invasive tumors we have analyzed primarily intraductal lesions, part of them with accompanying small invasive lesions. Furthermore, it cannot be excluded that the comparably lower incidence of p16 methylation reflects regional differences in patient cohorts because another study on breast cancer from this area applying a completely different method yielded also a low incidence of p16 methylation not exceeding 5%.28
We could find absolutely no indication for a reduced methylation of the
p16 gene in breast cancer cells during tumor progression
(Figure 7)
or in comparison to normal breast epithelium (Figure 3)
, as
was reported by Van Zee and colleagues.43
This discrepancy
could be because of completely different methodology and to the fact
that in the present study pure tumor cell populations isolated by
laser-assisted microdissection were analyzed in contrast to the tumor
homogenates analyzed in the earlier study.
In conclusion, our results demonstrate that the epigenetic modification of key regulatory genes is an early and frequent event in the development of breast cancer. A quantitative assessment of promoter methylation clearly shows huge gene-specific differences for the extent of methylation and also specific alterations of methylation levels during tumor progression.
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Acknowledgements |
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Footnotes |
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Supported by grant Deutsche Forschungsgemeinschaft Fe 516/1-1.
Accepted for publication November 1, 2001.
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References |
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